PCR method for detecting CTCF occupied cfdna fragments

Abstract

The present invention provides a method for detecting the presence of CCCTC-binding factor (CTCF) occupied cell free DNA (cfDNA) fragments originating from a pre- determined genomic location, in a body fluid sample obtained from a subject, which comprises the steps of: (i) contacting the body fluid sample with a binding agent which binds to cfDNA-CTCF nucleoproteins; (ii) isolating cfDNA-CTCF nucleoproteins bound in step (i); (iii) contacting cfDNA isolated in step (ii) with one or more pairs of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs or equal to or less than 80 base pairs that comprises a CTCF binding site located at the pre-determined genomic location; and (iv) determining the presence, absence or quantity of DNA sequences of equal to or less than 100 base pairs or equal to or less than 80 base pairs in length produced in step (iii), as an indicator of the presence of CTCF-occupied cfDNA fragments originating from the pre-determined genomic location in the body fluid sample.

Description

[0001] PCR METHOD FOR DETECTING CTCF OCCUPIED CFDNA FRAGMENTS

[0002] FIELD OF THE INVENTION

[0003] The invention relates to methods for preparing a sample and / or for analysis of a sample to determine the presence, absence or amount of cell free DNA fragments associated with a CCCTC-binding factor (CTCF), and to PCR methods for detecting cancer in a subject by means of a minimally invasive body fluid test. In particular, the invention relates to methods and panels for detecting, analysing or measuring cell free DNA fragments associated with CTCF.

[0004] BACKGROUND OF THE INVENTION

[0005] Detection of cancer at stage I or II disease enables early treatment and leads to improved patient outcomes. Analysis of circulating tumour DNA (ctDNA) holds great promise for early cancer detection in symptomatic or even asymptomatic subjects by means of a blood test. However, whilst ctDNA analysis is becoming commonplace in clinical practice for the management of cancer patients to aid in monitoring, prognosis and treatment selection decisions, their utility for early detection has been limited by their accuracy, complexity and cost.

[0006] Most blood tests involve detection of the presence of a circulating biomarker above a low threshold background level. For example the detection of COVID-19 DNA in a blood sample by PCR is indicative of a COVID-19 infection. The presence of ctDNA in a blood sample is similarly indicative of a cancer condition, but detection of the presence of early stage cancer derived ctDNA in blood samples is difficult because (unlike other blood tests) the background DNA level is not low but may be more than a thousand fold higher than the ctDNA level to be measured. The primary challenge faced by workers in the liquid biopsy field is therefore to identify any tumour derived ctDNA which may be present in a plasma sample that also contains a much higher level of non-tumour derived cell free DNA (cfDNA) of near identical nucleotide sequence.

[0007] To address this problem, liquid biopsy methods for ctDNA analysis involve extraction of cfDNA from a patient plasma sample for analysis by next generation sequencing (NGS). Differentiation of tumour derived and non-tumour derived background plasma cfDNA fragments is then performed in silico by bioinformatic analysis of NGS data. Cancer- derived and healthy or wild type cfDNA fragment sequences may be differentiated on the basis of cancer associated mutations or cfDNA methylation patterns, as well as by fragmentomics analysis of cancer associated changes in cfDNA fragment size, genome location and nucleosome positioning patterns.

[0008] We have previously described the use of a chromatin fragment containing a tissue specific transcription factor as a plasma biomarker for the detection or diagnosis of a cancer in a subject (WO2017162755) and methods for the detection of circulating cell free chromatin fragments containing a transcription factor-DNA complex (WO2022144407).

[0009] Detection of circulating transcription factors can provide valuable information during the diagnosis of disease, however current methods rely on expensive, specialist protocols with complicated bioinformatics analysis.

[0010] There is a need in the art to provide low cost, rapid, cancer tests that can be used in any hospital worldwide without specialist equipment.

[0011] SUMMARY OF THE INVENTION

[0012] According to a first aspect of the present invention, there is provided a PCR method for detecting the presence of CCCTC-binding factor (CTCF) occupied cell free DNA (cfDNA) fragments originating from a pre-determined genomic location, in a body fluid sample obtained from a subject, which comprises the steps of:

[0013] (i) contacting the body fluid sample with a binding agent which binds to cfDNA-CTCF nucleoproteins;

[0014] (ii) isolating cfDNA-CTCF nucleoproteins bound in step (i);

[0015] (iii) contacting cfDNA isolated in step (ii) with one or more pairs of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs (e.g. equal to or less than 80 base pairs) that comprises a CTCF binding site located at the predetermined genomic location; and

[0016] (iv) determining the presence, absence or quantity of DNA sequences of equal to or less than 100 base pairs in length (e.g. equal to or less than 80 base pairs) produced in step (iii), as an indicator of the presence of CTCF-occupied cfDNA fragments originating from the pre-determined genomic location in the body fluid sample.

[0017] According to a further aspect of the present invention, there is provided a PCR method for detecting the presence of CCCTC-binding factor (CTCF) occupied cell free DNA (cfDNA) fragments originating from a pre-determined genomic location, in a body fluid sample obtained from a subject, which comprises the steps of: (i) contacting the body fluid sample with a binding agent which binds to cfDNA-CTCF nucleoproteins;

[0018] (ii) isolating cfDNA-CTCF nucleoproteins bound in step (i);

[0019] (iii) contacting cfDNA isolated in step (ii) with one or more pairs of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs (e.g. equal to or less than 80 base pairs) that comprises a CTCF binding site located at the predetermined genomic location; and

[0020] (iv) determining the presence, absence or quantity of DNA sequences of equal to or less than 80 base pairs in length produced in step (iii), as an indicator of the presence of CTCF-occupied cfDNA fragments originating from the pre-determined genomic location in the body fluid sample.

[0021] According to a further aspect of the present invention, there is provided a method comprising:

[0022] (i) contacting a body fluid sample with an antibody which binds a cfDNA- CTCF nucleoprotein complex;

[0023] (ii) isolating antibody-bound complexes formed in step (i) and extracting cfDNA from the isolated complexes;

[0024] (iii) contacting the extracted cfDNA with one or more pairs of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs (e.g. equal to or less than 80 base pairs) that comprises a CTCF binding site located at a pre-determined genomic location to produce DNA amplicons of equal to or less than 100 base pairs (e.g. equal to or less than 80 base pairs) in length; and

[0025] (iv) determining the presence, absence or quantity of the DNA amplicons in the body fluid sample.

[0026] In an embodiment, step (i) comprises contacting a first portion of a body fluid sample with an antibody that specifically binds a cfDNA-nucleoprotein complex and contacting a second portion of the body fluid sample with a binding agent that does not specifically bind the cfDNA-nucleoprotein complex.

[0027] In an embodiment, prior to step (i), the method comprises treating a body fluid sample with a reagent to crosslink nucleoprotein and / or cfDNA in the sample. In an embodiment, the body fluid sample is a plasma sample, and the treating further comprises treating with a quenching agent. In an embodiment, said contacting in step (iii) comprises contacting the extracted cfDNA with one or more pairs of PCR primers configured to produce amplicons of equal to or less than 100 base pairs or equal to or less than 80 base pairs in length from predetermined genomic locations comprising a CTCF binding site listed in Table 1 , Table 2, or Table 3, or Table 10 or Table 11. In one embodiment, the pre-determined genomic locations comprising a CTCF binding site are listed in Table 1 , Table 10 or Table 11. In one embodiment, the pre-determined genomic locations comprising a CTCF binding site are listed in Table 1. In one embodiment, the pre-determined genomic locations comprising a CTCF binding site are listed in Table 10 or Table 11. In one embodiment, the pre-determined genomic locations comprising a CTCF binding site are listed in Table 10.

[0028] According to a further aspect of the invention, there is provided a PCR method for detecting the presence of CTCF occupied cfDNA fragments originating from a predetermined genomic location, in an isolated CTCF-cfDNA preparation produced from a body fluid sample obtained from a subject, which comprises the steps of:

[0029] (i) contacting a first sample of the body fluid with a candidate binding agent which binds to cfDNA-CTCF nucleoproteins, and contacting a second sample of the body fluid with a control binding agent which does not specifically bind to cfDNA-CTCF nucleoproteins;

[0030] (ii) isolating cfDNA from the first sample bound by the candidate binding agent and from the second sample bound by the control binding agent in step (i);

[0031] (iii) contacting isolated cfDNA from the first sample and the second sample with one or more pairs of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs (e.g. equal to or less than 80 base pairs) that includes a CTCF binding site located at the pre-determined genomic location;

[0032] (iv) determining the presence, absence or quantity of DNA sequences of equal to or less than 100 base pairs (e.g. equal to or less than 80 base pairs) in length produced in step (iii) by subtracting the quantity of DNA in the second sample from the first sample; and

[0033] (v) using the presence, absence or quantity of DNA determined in step (iv), as an indicator of whether, or how many, CTCF-occupied cfDNA fragments originating from the pre-determined genomic location were present in the body fluid sample.

[0034] According to a further aspect of the invention, there is provided a method for determining a genomic location comprising a CTCF binding site that is selectively bound by CTCF during a cancer, comprising: (i) contacting a first body fluid sample obtained from a subject diagnosed with the cancer with a binding agent which binds to cfDNA-CTCF nucleoproteins, and contacting a second body fluid sample obtained from a subject with no known cancer or a different cancer with a binding agent which binds to cfDNA-CTCF nucleoproteins;

[0035] (ii) isolating cfDNA-CTCF nucleoproteins from the first and second sample bound in step (i);

[0036] (iii) sequencing cfDNA isolated from the first and second sample in step (ii) to determine genomic locations that are selectively occupied by CTCF in the first sample but not in the second sample, to determine genomic locations comprising a CTCF binding site that is selectively bound by CTCF during the cancer.

[0037] According to a further aspect of the invention, there is provided a method of detecting cancer in a subject which comprises the steps of:

[0038] (i) detecting the presence of CTCF occupied cfDNA fragments using a method as described herein; and

[0039] (ii) using the presence of the CTCF occupied cfDNA fragments as an indicator of the presence of cancer in the subject.

[0040] According to a further aspect of the invention, there is provided a method for the assessment of subject for suitability for a cancer treatment which comprises the steps of:

[0041] (i) detecting the presence of CTCF occupied cfDNA fragments using a method as described herein; and

[0042] (ii) using the CTCF occupied cfDNA fragments detected in step (i) as a parameter for the selection of a suitable treatment for the subject.

[0043] According to a further aspect of the invention, there is provided a method for monitoring a treatment of a subject diagnosed with cancer which comprises the steps of:

[0044] (i) detecting the presence of CTCF occupied cfDNA fragments using a method as described herein;

[0045] (ii) repeating the detection of step (i) in a body fluid sample obtained from the subject on one or more occasions; and

[0046] (iii) using any changes in the presence of CTCF occupied cfDNA fragments determined in step (i) compared to step (ii) as a parameter for any changes in the condition of the subject.

[0047] According to a further aspect of the invention, there is provided a kit for the detection of a cell free chromatin fragment comprising CTCF and a DNA fragment as a combination biomarker which comprises a binding agent for CTCF together with reagents for detecting the presence of CTCF occupied cfDNA fragments using a method as described herein.

[0048] According to a further aspect of the invention, there is provided a method of treating cancer in a subject in need thereof, wherein said method comprises the following steps:

[0049] (i) contacting a body fluid sample obtained from the subject with a binding agent which binds to cfDNA-CTCF nucleoproteins;

[0050] (ii) isolating cfDNA-CTCF nucleoproteins bound in step (i);

[0051] (iii) contacting cfDNA isolated in step (ii) with one or more pairs of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs (e.g. equal to or less than 80 base pairs) that comprises a CTCF binding site located at a predetermined genomic location;

[0052] (iv) determining the presence, absence or quantity of DNA sequences of equal to or less than 100 base pairs (e.g. equal to or less than 80 base pairs) in length produced in step (iii), as an indicator of the presence of cancer in the subject; and

[0053] (v) administering a treatment if the subject is determined to have cancer.

[0054] According to a further aspect of the invention, there is provided a method of treating cancer in a subject in need thereof, wherein said method comprises the following steps:

[0055] (i) contacting a body fluid sample obtained from the subject with a binding agent which binds to cfDNA-CTCF nucleoproteins;

[0056] (ii) isolating cfDNA-CTCF nucleoproteins bound in step (i);

[0057] (iii) contacting cfDNA isolated in step (ii) with one or more pairs of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs (e.g. equal to or less than 80 base pairs) that comprises a CTCF binding site located at a predetermined genomic location;

[0058] (iv) determining the presence, absence or quantity of DNA sequences of equal to or less than 100 base pairs (e.g. equal to or less than 80 base pairs) in length produced in step (iii), as an indicator of the presence of cancer in the subject; and

[0059] (v) administering a treatment if the subject is determined to have cancer.

[0060] According to a further aspect of the invention, there is provided a pair of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs or equal to or less than 80 base pairs that comprises a CTCF binding site located at a pre-determined genomic location. According to a further aspect of the invention, there is provided a pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a 30-100 bp or 30-80 bp region of the human genome comprising a CCCTC- binding factor (CTCF) binding site or part thereof.

[0061] According to a further aspect of the invention, there is provided a kit for detecting the presence of CCCTC-binding factor (CTCF) occupied cell free DNA (cfDNA) fragments originating from a pre-determined genomic location, in a body fluid sample obtained from a subject, wherein the kit comprises a pair of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs or equal to or less than 80 base pairs that comprises a CTCF binding site located at a pre-determined genomic location and an anti- CTCF antibody.

[0062] According to a further aspect of the invention, there is provided a kit for detecting the presence of CCCTC-binding factor (CTCF) occupied cell free DNA (cfDNA) fragments originating from a pre-determined genomic location, in a body fluid sample obtained from a subject, wherein the kit comprises a pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a 30- 100 bp or 30-80 bp region of the human genome comprising a CCCTC-binding factor (CTCF) binding site or part thereof and an anti-CTCF antibody.

[0063] According to a further aspect of the invention, there is provided a kit for detecting the presence of CCCTC-binding factor (CTCF) occupied cell free DNA (cfDNA) fragments originating from a pre-determined genomic location, in a body fluid sample obtained from a subject, wherein the kit comprises:

[0064] (a) a first pair of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs that comprises a CTCF binding site located at a first pre-determined genomic location; and

[0065] (b) a second pair of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs that comprises a CTCF binding site located at a second predetermined genomic location.

[0066] According to a further aspect of the invention, there is provided a kit for detecting the presence of CCCTC-binding factor (CTCF) occupied cell free DNA (cfDNA) fragments originating from a pre-determined genomic location, in a body fluid sample obtained from a subject, wherein the kit comprises: (a) a first pair of PCR primers directed to a DNA sequence of equal to or less than 80 base pairs that comprises a CTCF binding site located at a first pre-determined genomic location; and

[0067] (b) a second pair of PCR primers directed to a DNA sequence of equal to or less than 80 base pairs that comprises a CTCF binding site located at a second predetermined genomic location.

[0068] According to a further aspect of the invention, there is provided a composition comprising:

[0069] (a) a first pair of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs that comprises a CTCF binding site located at a first pre-determined genomic location; and

[0070] (b) a second pair of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs that comprises a CTCF binding site located at a second predetermined genomic location.

[0071] According to a further aspect of the invention, there is provided a composition comprising:

[0072] (a) a first pair of PCR primers directed to a DNA sequence of equal to or less than 80 base pairs that comprises a CTCF binding site located at a first pre-determined genomic location; and

[0073] (b) a second pair of PCR primers directed to a DNA sequence of equal to or less than 80 base pairs that comprises a CTCF binding site located at a second predetermined genomic location.

[0074] According to a further aspect of the invention, there is provided a kit for detecting the presence of CCCTC-binding factor (CTCF) occupied cell free DNA (cfDNA) fragments comprising:

[0075] (a) a first pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a first 30-100 bp region of the human genome comprising a CTCF binding site or part thereof; and

[0076] (b) a second pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a second 30-100 bp region of the human genome comprising a CTCF binding site or part thereof.

[0077] According to a further aspect of the invention, there is provided a kit for detecting the presence of CCCTC-binding factor (CTCF) occupied cell free DNA (cfDNA) fragments comprising: (a) a first pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a first 30-80 bp region of the human genome comprising a CTCF binding site or part thereof; and

[0078] (b) a second pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a second 30-80 bp region of the human genome comprising a CTCF binding site or part thereof.

[0079] According to a further aspect of the invention, there is provided a composition comprising:

[0080] (a) a first pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a first 30-100 bp region of the human genome comprising a CTCF binding site or part thereof; and

[0081] (b) a second pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a second 30-100 bp region of the human genome comprising a CTCF binding site or part thereof.

[0082] According to a further aspect of the invention, there is provided a composition comprising:

[0083] (a) a first pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a first 30-80 bp region of the human genome comprising a CTCF binding site or part thereof; and

[0084] (b) a second pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a second 30-80 bp region of the human genome comprising a CTCF binding site or part thereof.

[0085] According to a further aspect of the invention, there is provided a biomarker panel of genomic locations comprising CTCF binding sites associated with five or more of the following genes: CATSPERE, CTNNA2, NOL10, DIAPH1 , TMEM260, GALC, OSBPL6, PI15, ANO4, FAM41AY1 , LINC02787, NR_038432.1 , GIPC2, CACNA1A, GNPD1 , TEX33, TRAV4, HEYL, FAM124B, COL21A1 , PLEKHG7, EPHA5, FAM149A, TFAP2B, LINC02934, SKAP1 , CDH5, ENSG00000229727, SNORA70, TBC1 D21 , TGIF2LX.

[0086] According to a further aspect of the invention there is provided use of a CTCF gain of occupancy site as a biomarker for cancer.

[0087] According to another aspect of the invention, there is provided a method of determining the cel l-of-orig i n or tissue-of-origin for cancer of unknown primary in a subject, the method comprising: (i) contacting a body fluid sample obtained from the subject with a binding agent which binds to cfDNA-CTCF nucleoproteins;

[0088] (ii) isolating cfDNA-CTCF nucleoproteins bound in step (i);

[0089] (iii) detecting the cfDNA isolated from the body fluid sample in step (ii) to determine genomic locations that are selectively occupied by CTCF in the body fluid sample; and

[0090] (iv) using the genomic locations determined in step (iii) to determine the cel I- of-origin or tissue-of-origin for the cancer of unknown primary.

[0091] According to another aspect of the invention, there is provided a method of identifying cancer-associated mutations in CTCF-binding sites, the method comprising:

[0092] (i) contacting a body fluid sample obtained from a subject with cancer with a binding agent which binds to cfDNA-CTCF nucleoproteins;

[0093] (ii) isolating cfDNA-CTCF nucleoproteins bound in step (i);

[0094] (iii) sequencing the cfDNA isolated from the body fluid sample in step (ii) to determine the sequence of CTCF binding sites that are occupied by CTCF in the body fluid sample; and

[0095] (iv) comparing the sequences of the CTCF binding sites determined in step (iii) with the sequences of corresponding CTCF binding sites from a reference genome, to identify CTCF binding sites which are occupied by CTCF in the body fluid sample and which comprise one or more mutations.

[0096] According to another aspect of the invention, there is provided a biomarker for cancer, the biomarker consisting of a DNA sequence of equal to or less than 100 base pairs and comprising a CTCF binding site sequence which is selectively occupied in cancer. The DNA sequence may be a cell free DNA fragment.

[0097] BRIEF DESCRIPTION OF THE FIGURES

[0098] Figure 1 : A schematic representation of an embodiment of a method of the present invention.

[0099] Figure 2: A graphical representation showing pairs of CTCF binding sites being bound to form regulatory chromatin loops with multiple folds which mediate long-range chromatin interactions by bringing linearly distant gene promoters and their enhancers into close proximity in the form of topologically associated domains (TADs). Figure 3: Western Blot analysis of chromatin fragments immunoprecipitated from 4 pooled STRECK EDTA plasma samples taken from patients diagnosed with CRC by chromatin immunoprecipitation (ChIP) using a mouse anti-CTCF antibody immobilized on magnetic polystyrene beads washed using the strong 1 .2% detergent mix wash buffer. All 4 plasma samples showed a band at around 140kD corresponding to CTCF protein (Anti CTCF: lanes 3, 5, 7 and 9). Negative control experiments using non-specific mouse IgG (NS-IgG) showed no band corresponding to CTCF (NS-IgG: lanes 2, 4, 6 and 8). The experiment demonstrated that CTCF protein was isolated from the plasma samples and that use of a strong wash buffer led to a relatively pure CTCF extract from plasma.

[0100] Figure 4: Normalised coverage of 9780 published CTCF TFBS loci by transcription factor bound (35-80bp) or nucleosome bound (135-155bp or 156-180bp) cfDNA fragments, (a) Specific CTCF coverage by a cfDNA sequence library obtained for a CRC patient STRECK EDTA plasma sample, (b) Non-specific coverage by a cfDNA sequence library obtained from chromatin fragments bound non-specifically to mouse IgG coated particles. The results show that the peak of specific cfDNA coverage originating from plasma circulating CTCF-DNA complexes correlates with published CTCF TFBS loci. The expected oscillating coverage pattern due to nucleosome binding is minimal across the 5kb span investigated. In the control sample no peak cfDNA coverage at the CTCF binding loci was observed.

[0101] Figure 5: Normalised coverage of 1041 published CTCF TFBS loci selectively occupied more frequently by CTCF in cancer cells for the same CRC patient STRECK EDTA plasma sample described in Figure 4. Coverage is shown for transcription factor bound (35-80bp) or nucleosome bound (135-155bp or 156-180bp) cfDNA fragments, (a) CTCF occupation of cancer associated loci by a cfDNA sequence library obtained for a CRC patient. The results show coverage in the 35-80bp size range confirming CTCF occupancy of some or all of these 1041 sites and are therefore indicative of cancer in the subject from whom the sample was taken, (b) There was no CTCF occupancy peak observed in a non-specific control experiment.

[0102] Figure 6: NGS generated cfDNA fragment length profiles of CTCF-associated DNA isolated by ChIP from: (a) EDTA plasma spiked with formaldehyde cross-linked digested MCF7 breast cancer cell chromatin extract; (b) EDTA plasma spiked with native MCF7 chromatin extract; (c) STRECK plasma sample from a patient diagnosed with AML (1969 ng / ml H3.1 -nucleosomes); (d) STRECK plasma sample from a patient diagnosed with elevated inflammatory markers (5687 ng / ml H3.1 -nucleosomes); (e) size distribution profile of the subset of AML cfDNA fragments shown in Figure 6(d) that include 9,780 annotated CTCF binding sites, compared with the range of cfDNA fragment sizes detected by NGS using conventional double-stranded DNA library preparation (Snyder et al. 2016).

[0103] Figure ?: Example V-plot analysis of CTCF binding motif within cfDNA fragment sequence reads for the forward and reverse motif sequences. The sequence data for a data for pooled CRC patient was analysed for the size of fragment including a CTCF binding motif and the position of the binding motif within the fragment for (a) CTCF- associated DNA isolated by specific anti-CTCF ChIP; (b) material isolated by non-specific ChIP using mouse IgG coated magnetic beads; (c) material isolated by ChIP using antinucleosome antibody coated magnetic beads.

[0104] Figure 8: Example Bioanalyzer generated fragment length profiles and NGS generated normalised coverage analysis of published CTCF TFBS loci by transcription factor bound (35-80bp) or nucleosome bound (135-155bp or 156-180bp) cfDNA fragments for specific and non-specific CTCF coverage for (a) a single AML patient; (b) a single subject with elevated levels of circulating inflammatory markers and (c) a single healthy subject.

[0105] Figure 9: Example forward and reverse motif sequence V-plots showing multiple spots with discrete sizes that include a centrally located CTCF binding motif for plasma samples collected from (a) a single AML patient; (b) a single subject with elevated levels of circulating inflammatory markers and (c) a single healthy subject.

[0106] Figure 10: Summary of the bioinformatic process used for cfDNA sequence biomarker discovery.

[0107] Figure 11 : Sequencing results obtained for CTCF associated cfDNA fragments isolated by ChIP from plasma samples collected from a liver cancer patient: Normalized coverage of CTCF binding site loci sequences by 35-80bp cfDNA fragments and nucleosome size fragments (135-180bp), after (a) specific CTCF immunoprecipitation using a mouse anti- CTCF antibody and (b) unspecific background precipitation with non-specific mouse IgG; (c) Volcano plot showing CTCF-DNA binding sites associated with the presence of liver cancer compared to healthy patients.

[0108] Figure 12: Sequencing results obtained for CTCF associated cfDNA fragments isolated by ChIP from plasma samples collected from a prostate cancer patient: Normalized coverage of CTCF binding site loci sequences by 35-80bp cfDNA fragments and nucleosome size fragments (135-180bp), after (a) specific CTCF immunoprecipitation using a mouse anti-CTCF antibody and (b) unspecific background precipitation with nonspecific mouse IgG; (c) Volcano plot showing CTCF-DNA binding sites associated with the presence of prostate cancer compared to healthy patients.

[0109] Figure 13: Sequencing results obtained for CTCF associated cfDNA fragments isolated by ChIP from plasma samples collected from a colorectal cancer patient: Normalized coverage of CTCF binding site loci sequences by 35-80bp cfDNA fragments and nucleosome size fragments (135-180bp), after (a) specific CTCF immunoprecipitation using a mouse anti-CTCF antibody and (b) unspecific background precipitation with nonspecific mouse IgG; (c) Volcano plot showing CTCF-DNA binding sites associated with the presence of CRC compared to healthy patients.

[0110] Figure 14: Sequencing results obtained for CTCF associated cfDNA fragments isolated by ChIP from plasma samples collected from a lung cancer patient: Normalized coverage of CTCF binding site loci sequences by 35-80bp cfDNA fragments and nucleosome size fragments (135-180bp), after (a) specific CTCF immunoprecipitation using a mouse anti- CTCF antibody and (b) unspecific background precipitation with non-specific mouse IgG; (c) Volcano plot showing CTCF-DNA binding sites associated with the presence of lung cancer compared to healthy patients.

[0111] Figure 15: Sequencing results obtained for CTCF associated cfDNA fragments isolated by ChIP from plasma samples collected from a breast cancer patient: Normalized coverage of CTCF binding site loci sequences by 35-80bp cfDNA fragments and nucleosome size fragments (135-180bp), after (a) specific CTCF immunoprecipitation using a mouse anti-CTCF antibody and (b) unspecific background precipitation with nonspecific mouse IgG; (c) Volcano plot showing CTCF-DNA binding sites associated with the presence of breast cancer compared to healthy patients.

[0112] Figure 16: Western Blot analysis of CTCF isolated from canine cell line or human cell lines developed using a human anti-CTCF antibody coated to magnetic particles.

[0113] DETAILED DESCRIPTION OF THE INVENTION

[0114] The present invention provides a method for the detection, including early detection, of cancer. Tumour detection by ctDNA liquid biopsy begins with discovering the difference between cfDNA in blood samples collected from patients with or without a tumour. The present invention provides methods that can accurately and quickly differentiate between these.

[0115] The workflow of conventional next generation sequencing based ctDNA analysis essentially involves extraction of cfDNA from a patient plasma sample, amplification of all the cfDNA extracted and preparation of a DNA library for sequencing, followed by sequencing of the library using a next generation sequencing method and analysis of the sequence method by bioinformatics to determine whether or not any tumour derived ctDNA was present in the sample. Whilst the accuracy of liquid biopsy methods for ctDNA analysis have improved over recent years due to improvements in each of these steps, the basic workflow has remained unchanged. We now report a novel liquid biopsy method for ctDNA analysis that does not involve preparation of a DNA library, next generation sequencing or bioinformatics. We also now report a novel liquid biopsy method for ctDNA analysis that does not involve amplification of the extracted cfDNA, preparation of a DNA library, next generation sequencing or bioinformatics.

[0116] In preferred embodiments, the method of the invention does not comprise a step of sequencing all or substantially all of the cfDNA in a sample. In this context, the term “substantially all” may refer to 80% or more, 90% or more, 95% or more, or 99% or more of the cfDNA in a sample. Preferably, sequencing is not used to detect the presence of CCCTC-binding factor (CTCF) occupied cell free DNA (cfDNA) fragments originating from a pre-determined genomic location.

[0117] In more preferred embodiments, the method of the invention does not comprise a step of sequencing the cfDNA.

[0118] This method for the first time physically isolates ctDNA from non-tumour derived background cfDNA of the same sequence, thereby removing the background cfDNA problem of liquid biopsy.

[0119] Blood samples collected from patients diagnosed with early stage cancer may contain 1 ,000-10,000 times more background cfDNA of non-tumour origin than tumour derived ctDNA (Jamshidi et al, 2022). Liquid biopsy methods of the art involve extracting all the cfDNA (including all the background cfDNA) and use of sophisticated technical and bioinformatic processes to identify whether a particular extracted cfDNA sample does, or does not, contain any ctDNA amongst the large cfDNA background. The present invention avoids this problem by removing the background before testing for ctDNA. The solution described herein provides a method for isolating tumour derived ctDNA with complete, or near complete, separation from all background cfDNA of the same sequence. The sample can then be tested for the presence of the ctDNA sequence by any method known in the art (for example, without limitation, PCR, qPCR, RT-PCR or digital PCR) to establish whether the ctDNA sequence is present without interference from a high background signal. The liquid biopsy test then becomes similar in principle to, for example, a hepatitis DNA test, where the simple presence of the hepatitis DNA sequence (above a low background level) is sufficient to detect disease.

[0120] The elimination of the cfDNA background to facilitate simple PCR cancer detection was achieved by combining two disparate elements. One element is a method for the complete, or near complete, separation of a circulating cell free chromatin form from all other circulating chromatin forms. The other is a biomarker discovery element for DNA sequences which occur in that chromatin form exclusively, or nearly exclusively, in the circulation of cancer patients and is absent from the circulation (in that form) of subjects with no cancer. Such sequences can be utilised as biomarker cfDNA sequences. When these two elements are combined, it becomes possible to isolate the chromatin form which contains the DNA sequence exclusively during cancer, thereby separating it from all non-cancer cfDNA of the same sequence.

[0121] For clarity, the isolated chromatin form preparation need not, and will not, be entirely of tumour origin, but the isolated chromatin form comprising DNA fragments of the specified biomarker ctDNA sequence will be entirely of tumour origin. The simple presence of this ctDNA biomarker sequence is then indicative of cancer in a subject. It will be clear to those skilled in the art that cfDNA sequence biomarkers do not have exclusive application in cancer but may be applied to any disease based on the methods described herein.

[0122] Current liquid biopsy methods use cfDNA extracted from plasma as a sample matrix for sequence analysis. However, physical isolation of extracted ctDNA fragments from other extracted cfDNA fragments comprising the same DNA sequence is not possible by any method known in the art. It will be clear to those skilled in the art that extraction of cfDNA from plasma involves the loss or destruction of all epigenetic information present in the protein components of chromatin, or jointly in the protein and DNA components, leaving only information present entirely in the DNA alone remaining. Free double-helix DNA has a very short-life in the circulation. The cfDNA fragments analysed in liquid biopsy are therefore all protected from metabolism by binding to proteins in the form of nucleoproteins. Most circulating nucleoproteins are nucleosomes but others include circulating transcription factor bound DNA complexes, including circulating CTCF-DNA nucleoproteins.

[0123] A cfDNA fragment including a CTCF binding site may be occupied (and protected from metabolism) by binding to CTCF. Herein an occupied CTCF binding site. Alternatively, it may not be occupied by CTCF, but protected instead by binding to histone proteins in the form of a nucleosome. Herein an unoccupied CTCF binding site. The present invention employs differences in the epigenetic forms of circulating cell free chromatin fragments based on the occupation of CTCF binding sites in cfDNA derived from cancer versus healthy subjects.

[0124] The present invention is based on the principle that, although CTCF can bind to tens of thousands of specific CTCF DNA binding sites in the human genome, the binding patterns of sites actually occupied in a particular cell are cancer and tissue specific. The occupancy patterns of CTCF DNA binding sites are altered in cancer, including both gain and loss of occupied sites. Some gain of occupation sites occur selectively in transformed cells. In addition, some sites are specifically occupied in particular tissue cell types. Hence, if you can find the occupied CTCF binding sites you can both detect cancer and determine the tissue of origin of the cancer. This difference is used as the basis for physical isolation of CTCF occupied ctDNA fragments of tumour origin bearing cancer associated gain of occupancy binding site sequences, from non-tumour derived unoccupied background cfDNA of the same sequences (protected by histones or other proteins), thereby removing the background cfDNA problem of liquid biopsy.

[0125] In preferred embodiments, the method of the invention is used for a human subject. However, the methods of the invention may also be used for species other than humans. In some embodiments, the method of the invention is used for an animal subject, for example selected from a rodent (e.g. mouse, rat, hamster, gerbil or chipmunk), feline (i.e. a cat), canine (i.e. a dog), equine (i.e. a horse), porcine (i.e. a pig) or bovine (i.e. a cow) subject. For species in which the CTCF protein has a high degree of homology with that of human CTCF (e.g. canine CTCF), the same binding agents and protocols may be used. The CTCF TFBS target sequences may each be positive for multiple cancer types. It may be that a sequence is not uniquely positive only for one type of cancer. In this case, the different target sequences can be chosen which are selectively positive for different cancers or combinations of cancers, i.e. a panel of sequences may be used.

[0126] The CTCF-TFBS occupancy patterns vary with tumour type. Thus analysis of CTCF gain of binding patterns in larger numbers of cancer patient samples may reveal cell-of-origin binding patterns facilitating tumour cell-of-origin analysis.

[0127] Therefore in one aspect of the present invention the methods of the invention can be used to determine the cell of origin of a cancer.

[0128] This cancer detection can be achieved by a cfDNA (or ctDNA) biomarker sequence discovery process where CTCF gain of occupancy sites in cancer are identified such that the ctDNA biomarker sequence only occurs in CTCF-cfDNA fragments derived from cancer. This will mean that in the cfDNA of a healthy subject, the gain of occupancy sequence will only occur in nucleosome associated cfDNA fragments. In contrast, the blood of a cancer patient will contain both nucleosome associated cfDNA fragments (derived from non-cancer cells) and CTCF-bound cfDNA fragments (derived from cancer cells) containing the binding site of interest (the cfDNA or ctDNA biomarker sequence). Those skilled in the art will recognise that all, human or animal, genome sequences will be found in blood samples obtained from that human or animal and that a (total) cfDNA extract from a plasma sample, as performed for current liquid biopsy methods, will therefore always contain cfDNA fragments comprising the sequence of interest whether the sample was from a cancer patient or a healthy subject. Therefore, CTCF occupancy knowledge is not useful for analysis by PCR unless the background nucleosome fragments containing the sequence can be removed before cfDNA is extracted. We therefore isolated CTCF-DNA complexes from patient samples before cfDNA extraction, removing all nucleosomes and leaving only CTCF-DNA nucleoprotein complexes including cancer gain of occupancy CTCF binding site sequences in the extracted cfDNA.

[0129] It is the loci of gain of occupancy sites in cancer that are of primary interest in the present invention. In particular, loci of CTCF binding sites that are exclusively occupied in cancer are potential ctDNA biomarker sequences. However, hundreds or thousands of CTCF binding sites are described as having cancer selective binding (Liu et al, 2017), but very few of these will be exclusively bound in cancer. Moreover, even if exclusively bound in living cancer cells, they may not be detectable in ctDNA. We have identified exclusively bound ctDNA biomarker sequences that are detectable as ctDNA by performing a novel biomarker discovery process directly in plasma. This biomarker discovery process was possible only because we were able to effectively completely isolate CTCF-cfDNA complexes from other chromatin components and particularly from nucleosomes.

[0130] Cell free DNA (cfDNA) extracts from plasma predominantly comprise approximately 150- 200bp free DNA fragments. However, prior to extraction, DNA does not circulate predominantly as free double helix, but in the form of mononucleosomes including a 150- 200bp DNA fragment. Subnucleosomal sized cfDNA fragments in the 30-80bp range have also been reported to occur in cfDNA extracts, but these smaller sub-1 OObp fragments are invisible to conventional liquid biopsy methods (Snyder eta / , 2016). These smaller cfDNA fragments may include transcription factor (TF) binding site sequences and originate from circulating TF-DNA nucleoprotein complexes. The TF occupancy of a genomic TF binding site (TFBS) in a cfDNA library may be determined by the sizes of cfDNA fragments that incorporate a TFBS DNA sequence and its flanking sequences. The finding of a TFBS sequence in a cfDNA fragment of nucleosomal size in cfDNA extracted from a patient blood sample indicates that the TFBS was nucleosome occupied in the plasma sample before extraction. Alternatively, a disruption of nucleosome positioning near transcription start sites and / or the finding of a TFBS sequence in a cfDNA fragment in the 30-80bp size range indicates that the TFBS was TF occupied in the blood sample before extraction. As TF occupancy patterns are altered in cancer, combinations of cfDNA size and sequence data obtained for NGS data of cfDNA extracted from plasma may be used to identify samples containing ctDNA, indicating a probable cancer in the patient from whom the sample was taken. Moreover, TFBS occupancy patterns are tissue specific so this analysis, may also identify the cancer tissue of origin. This principle has been exploited in fragmentomics bioinformatic analysis of cfDNA. Fragmentomics liquid biopsy methods, involve the extraction of all cfDNA (including any ctDNA if present) from a patient plasma sample, followed by amplification of (all) extracted cfDNA, library preparation and NGS. The NGS sequencing data is then analysed in silico using bioinformatics for the fragmentation pattern of the cfDNA to identify binding site occupancy.

[0131] In accordance with the present invention physical separation of tumour derived and nontumour derived cfDNA nucleoprotein complexes may in principle obviate the need for in silico separation and hence obviate the need for next generation sequencing and bioinformatic analysis of cfDNA sequence data. Physical separation may therefore enable lower cost, simpler, faster ctDNA analysis methods and preferably automation. We now report the physical isolation of CCCTC-binding factor-DNA (CTCF-DNA) nucleoprotein complexes from plasma by immunoprecipitation (IP), identification of individual CTCF-TFBSs that are selectively occupied in cancers including lung, colorectal, prostate, breast, blood and liver cancer and testing for disease selective CTCF-TFBS occupancy in plasma samples by qPCR by the method summarized in Figure 1.

[0132] We have also been able to identify ctDNA associated with early-stage cancer.

[0133] Such methods of the invention may offer the potential for automation and are capable of being supplied as low-cost products for testing in routine hospital laboratories worldwide.

[0134] Thus, advantages provided by the present invention include:

[0135] • Minimally invasive;

[0136] • Short timeframe to at least an indication of disease state;

[0137] • Highly sensitive;

[0138] • Low cost of sample isolations;

[0139] • Ability to monitor continuous disease evolutions;

[0140] • Real time monitoring of drug response;

[0141] • Feasibility of automation; and

[0142] • Feasibility of community screening.

[0143] According to one aspect of the present invention, there is provided a method for isolating disease derived cfDNA fragments from a body fluid sample with complete, or near complete, separation from all non-disease derived cfDNA of the same sequence or sequences.

[0144] According to another aspect of the invention, there is provided a method for isolating cfDNA fragments originating from diseased cells, from a body fluid sample with complete, or near complete, separation from all cfDNA of the same sequence or sequences that originate from other cell types.

[0145] According to another aspect of the invention, there is provided a method for the detection or diagnosis of a disease in a body fluid sample obtained from a subject, which comprises the steps of: (i) isolating a disease derived cfDNA fragment of predetermined sequence from the sample with complete, or near complete, separation from all non-disease derived cfDNA of the same sequence;

[0146] (ii) testing the isolate prepared in step (i) for the presence or amount of DNA fragments comprising the predetermined sequence; and

[0147] (iii) using the presence or amount of the fragments comprising the predetermined sequence, or sequences, as an indicator of the presence of a disease condition in the subject.

[0148] As described herein, the disease derived cfDNA fragments are physically separated from the body fluid sample and non-disease derived cfDNA of the same sequence, by virtue of binding to a chromatin structure (e.g. a transcription factor protein) distinct to that disease state. Therefore, according to these aspects of the invention, the isolating step is performed by contacting the sample with a binding agent that binds to a chromatin structure that is present in the disease state and not present in the non-disease state.

[0149] In a sample only 0.1 % or less of the total cfDNA may be associated with the disease state therefore the method uses complete separation or near complete separation of the cfDNA associated with the disease state at a level of at least 99% from the remaining cfDNA of the same sequence.

[0150] Polypeptide or polynucleotide sequences are said to be the “same” as or “identical” to other polypeptide or polynucleotide sequences, if they share 100% sequence identity. Therefore, references herein to “same sequence” refer to sequences that share 100% sequence identity, e.g. between the disease derived cfDNA fragment and the non- disease derived cfDNA of the same sequence. The disease and non-disease derived cfDNA may be the same sequence if they share the same core sequence, particularly at the binding site involved in the distinguishing chromatin structure. However, it will be understood that the same sequence (i.e. non-disease derived cfDNA) may be in a different context and therefore may be present as a slightly longer or shorter length than the target sequence. Single base changes (e.g. which may occur due to mutation or to sequencing errors) may still fall within the scope of “same sequence”. It will also be appreciated that the CTCF-bound cfDNA fragments detected will have a wide variety of lengths, starting points and end points. For example, where the CTCF binding site is approximately 20bp in length, a variety of cfDNA fragments of varying lengths between 30-1 OObp may occur that all contain the core 20bp CTCF binding site sequence but also contain additional different, longer or shorter, bp lengths at either end of the binding site sequence.

[0151] According to another aspect of the invention, there is provided a method for identifying a cfDNA fragment comprising a DNA sequence for use as a biomarker for a disease condition, comprising the steps of:

[0152] (i) obtaining body fluid samples or cells from subjects with or without the disease condition;

[0153] (ii) isolating chromatin fragments comprising a predetermined chromatin structure from the samples or cells;

[0154] (iii) sequencing the cfDNA associated with the chromatin fragments isolated in step (ii);

[0155] (iv) identifying a DNA sequence that occurs in the sequence data obtained for samples or cells from subjects who have the disease condition, that does not occur in the data obtained for samples or cells from subjects who do not have the disease condition; and

[0156] (v) using the cfDNA sequence identified in step (iv) as a biomarker sequence as described herein.

[0157] The predetermined chromatin structure may be an epigenetic feature such as a transcription factor (TF)-DNA nucleoprotein or TF-binding site.

[0158] In one embodiment, the disease derived cfDNA fragments are ctDNA fragments. In one embodiment the disease is a cancer or tumour.

[0159] According to another aspect of the present invention, there is provided a PCR method for detecting the presence of CCCTC-binding factor (CTCF) occupied cell free DNA (cfDNA) fragments originating from a pre-determined genomic location, in a body fluid sample obtained from a subject, which comprises the steps of:

[0160] (i) contacting the body fluid sample with a binding agent which binds to cfDNA-CTCF nucleoproteins;

[0161] (ii) isolating cfDNA-CTCF nucleoproteins bound in step (i);

[0162] (iii) contacting cfDNA isolated in step (ii) with one or more pairs of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs or equal to or less than 80 base pairs that comprises a CTCF binding site located at the predetermined genomic location; and (iv) determining the presence, absence or quantity of DNA sequences of equal to or less than 100 base pairs or equal to or less than 80 base pairs in length produced in step (iii), as an indicator of the presence of CTCF-occupied cfDNA fragments originating from the pre-determined genomic location in the body fluid sample.

[0163] The present invention utilises a pre-determined genomic location (preferably a panel of such locations) to provide a simplified cfDNA detection method which can identify selectively occupied CTCF binding sites by PCR. This information can be used to detect diseases, such as cancer.

[0164] The present invention may employ a panel of pre-determined genomic locations which are cancer specific or a panel which includes pre-determined genomic locations which are specific for a particular cancer or a range of cancers. Thus, the present invention can be envisaged as a specific cancer test or as a multi-cancer test. In one embodiment the panel comprises two or more, such as three or more, four or more, or five or more predetermined genomic regions comprising CTCF binding sites. In one embodiment the panel comprises 30 or fewer, 25 or fewer, 20 or fewer, 15 or fewer, or 10 or fewer predetermined genomic regions comprising CTCF binding sites. In one embodiment the panel comprises from 5 to 30 pre-determined genomic regions comprising CTCF binding sites.

[0165] According to another aspect of the invention, there is provided a method of detecting, analysing or measuring a cell free chromatin fragment comprising CTCF and a DNA fragment in a body fluid sample obtained from a subject, which comprises the steps of:

[0166] (i) contacting the body fluid sample with a binding agent which binds to CTCF;

[0167] (ii) isolating the CTCF bound in step (i); and

[0168] (iii) analysing DNA fragments associated with the bound CTCF; wherein the DNA fragments are analysed by detecting a panel of two or more (preferably five or more, such as between 5 and 30) pre-determined genomic regions comprising CTCF binding sites.

[0169] We observed single nucleotide changes in cfCTCF-DNA isolates from some samples which may represent cancer-associated mutations. Mutations to CTCF binding sites have been observed in cancer cells, and can completely suppress CTCF function or induce changes to CTCF binding and functionality (Wang et al, 2012). Therefore, in one aspect the method of the present invention can be used to detect CTCF binding site mutations. The number of occupied CTCF sites in the chromatin of human cells is reported to be approximately 40,000-60,000 (Wang et al, 2012), and CTCF-associated DNA fragments are approximately 50-1 OObp in length, both in cfDNA and in digested human cellular chromatin. On this basis, we estimate that occupied CTCF binding sites comprise approximately 0.1 % of the human genome in cellular chromatin. Not surprisingly, the amounts of CTCF-bound cfDNA isolated from plasma samples were small. Despite this, target CTCF TFBS sequences were detectable by qPCR.

[0170] In one embodiment, panels of up to three PCR assays for cancer detection are used, but the small volume required per PCR demonstrates that larger panels can be used on CTCF-DNA isolates even from 1mL of plasma.

[0171] Thus, in one embodiment the present invention employs equal to or more than 1 mL of a body fluid, such as plasma.

[0172] To demonstrate short cfDNA CTCF gain of occupancy biomarkers, we tested in patient samples diagnosed with a variety of cancers using qPCR sequence targets derived from analysis of AML patient samples. We observed positive biomarker results for all cancer types tested including positive results for samples from early Stage I and II cancer patients.

[0173] We then performed further target sequence analysis using samples obtained from solid cancer patients that yielded at least hundreds of potential CTCF gain of occupancy sequence targets. Further testing of the qPCR assays in a larger number of plasma samples collected from patients with colorectal cancer, Crohn’s disease, and colitis revealed selectivity of the markers for colorectal cancer over inflammatory diseases of the colon.

[0174] In summary, we have described a new class of body fluid biomarkers, such as plasma biomarkers, as well as a novel technology for their analysis by isolation of plasma cfCTCF-DNA nucleoproteins using ChIP and PCR analysis of tumor-associated CTCF gain of occupancy binding site sequences. This strategy provides access to analysis of pure ctDNA without NGS, and may provide the basis for rapid, low-cost, PCR liquid biopsy detection methods for cancer.

[0175] Cancer-associated, CTCF-bound sub-100 bp cfDNA fragments represent a novel class of biomarkers that have not been reported previously. This is because they are not detectable by current ctDNA assay methods and isolation of TF-bound plasma cfDNA nucleoproteins by chromatin immunoprecipitation (ChIP) has not previously been reported. We describe the identification of CTCF binding site sequences selectively occupied in cancer as a new class of oncology plasma biomarkers, and their use in a novel, wet chemistry process for direct measurement of ctDNA by polymerase chain reaction (PCR). Physical isolation of a class of ctDNA fragments from all background cfDNA fragments of the same sequences may enable direct, rapid, low-cost Capture- PCR liquid biopsy by removing the need for DNA library preparation, NGS, and bioinformatic analysis of results (Figure 1).

[0176] Thus in one embodiment of the present invention there is provided a body fluid biomarker for cancer comprising a CTCF sequence which is selectively occupied in cancer and which has a DNA sequence of equal to or less than 100 base pairs. This sequence will be one which is not occupied in a healthy subject or a subject with an inflammatory condition. Non-limiting examples of such sequences are provided herein. These selectively occupied sequences have not previously been described because they are effectively invisible to current NGS liquid biopsy methods, or because the sequences have not been investigated with respect to selective occupation in subject with cancer as opposed to healthy subjects.

[0177] Current liquid biopsy methods involve the sequencing of DNA libraries produced using conventional double-stranded DNA library preparation methods. As shown in Figure 6(e), these DNA libraries do not include small DNA fragments of less than approximately 100bp (Snyder et a / , 2016).

[0178] In one embodiment, ChlP-isolated cfDNA fragments were considered to be CTCF-cfDNA derived if they met four criteria, i.e. , (i) they were present in the specific anti-CTCF ChIP isolate but not present in the non-specific IgG control isolate; (ii) they included an annotated genomic CTCF binding site with flanking DNA sequences; (iii) they were sub- 100bp in length; and (iv) the CTCF binding site sequence was located centrally within the cfDNA fragment.

[0179] CTCF

[0180] CTCF (also called CCCTC-binding factor) is a highly conserved 11 zinc finger regulatory and structural chromatin protein with diverse functions including as a transcriptional activator, repressor, insulator and in the 3-dimensional organization of chromosome structure in the nucleus through the formation of topologically associated domains (TADs). TADs are formed by pairs of CTCF binding sites that delineate regulatory chromatin loops with multiple folds which mediate long-range chromatin interactions by bringing linearly distant gene promoters and their enhancers into close proximity. Chromosomal architecture is organised through differential binding of CTCF to the tens of thousands of CTCF binding sites present in the genome to form some 2000-5000 TADs present in the nuclei of human cells. (Figure 2)

[0181] Experimentally determined DNA binding times for most TFs are short and lie within seconds to minutes. The intra-nuclear occupancy of binding sites with short residence times is therefore driven by a dynamic equilibrium determined by local nuclear concentrations of free and DNA bound TF and unoccupied DNA binding sites. We surmise that cancer derived intact short-lived TF-DNA nucleoprotein fragments are unlikely to be detectable at significant levels in plasma because once a TF-DNA complex becomes disassociated, the dilution of free TF and free DNA in the circulation will prevent re-association for kinetic reasons. In contrast, the half-life of complexes of recombinant CTCF bound to oligonucleotides comprising CTCF TFBS sequences has been measured experimentally at 22 hours (Quitschke et al. 2000). This suggests that at least some CTCF-DNA complexes may be sufficiently stable to be detectable in the circulation and is consistent with reports of direct plasma cfDNA foot-printing of CTCF occupied DNA binding sites.

[0182] Without wishing to be bound by any theory the mechanism for release of tumour-derived CTCF-DNA nucleoproteins into the circulation may derive from cancer-associated CTCF gain of occupancy, but may also originate from functional co-factor interactions leading to altered CTCF chromatin binding affinity that, in turn, may affect their stability and release into the circulation. Oncogenic gene expression may also be modulated by functional interactions between CTCF and other oncogenic TFs. Recent reports indicate that CTCF chromatin binding affinities may be increased by interaction with other proteins. In prostate cancer, the oncogenic TF MYC binds with CTCF at MYC / CTCF common binding regions, resulting in stabilization of CTCF chromatin and three- dimensional reorganization of chromatin in cancer (Wei et al.2023). The evidence suggests a role for co-factors, in concert with CTCF, in cancer-associated reprogramming of transcription control.

[0183] The CTCF-TFBS occupancy and epigenetic regulation of gene transcription through TADs is tissue specific and is altered in cancer. We hypothesised that isolation and analysis of CTCF-DNA nucleoprotein fragments in plasma may facilitate the identification of fragments with a cancer origin and also identify the tissue of origin.

[0184] Wild type human CTCF is identified by UniProt ID: P49711 , and is encoded by the CTCF gene, identified by Ensembl Gene ID: ENSG00000102974. The protein CTCF plays among others a role of repressing the insulin-like growth factor 2 gene, by binding to the H-19 imprinting control region (ICR) along with Differentially-methylated Region-1 (DMR1) and MAR3.

[0185] An investigation of CTCF binding sites in the human genome identified 77,811 distinct binding sites across 19 different cell types (Wang et al, 2012). 27,662 of the 77,811 binding sites were found to be occupied in all 19 cell types investigated. CTCF binding of the remaining 50,149 binding sites exhibited tissue specificity. The 19 cell types investigated included 12 normal cell types and 7 cancer or EBV-immortalised cell lines representing colorectal cancer (Caco-2), cervical cancer (HeLa-S3), hepatocellular cancer (HepG2), neuroblastoma (SK-N-SH_RA), retinoblastoma (WERI-RB-1) and EBV- transformed lymphoplastoid (GM06990). CTCF binding at 1 ,236 binding sites was found to be specific to cancer cell lines, and occupancy of these binding sites distinguished immortal and cancer cell lines from normal cells including epithelia, fibroblasts and endothelia (Liu et al, 2017).

[0186] CHROMATIN IMMUNOPRECIPITATION (ChIP) OF ENDOGENOUS CTCF-DNA NUCLEOPROTEINS

[0187] We used a mouse anti-CTCF antibody to immunoprecipitate endogenous CTCF-DNA from 4 pooled cross-linked EDTA plasma samples (collected in STRECK cfDNA BCTs) collected from 18 subjects diagnosed as suffering from cancer. We performed Western blot analysis of the protein isolated on the solid phase support. The results in Figure 3 show that a protein band corresponding to CTCF at a molecular weight of approximately 140kD was present in all 4 pooled samples (but not in control experiments employing non-specific mouse IgG in place of the anti-CTCF antibody). The band at approximately 50kD corresponds to binding of the labelled anti-mouse IgG antibody used for Western blot to the heavy chain of the mouse anti-CTCF antibody employed for ChIP.

[0188] To check that the endogenous cfDNA fragments bound to CTCF were in the 35-80bp size range and included characterised CTCF binding site sequences, the amplified cfDNA library isolated using anti-CTCF immunoprecipitation was sequenced by Next Generation sequencing methods. Results for an amplified library prepared from a cross-linked EDTA plasma sample (collected in STRECK cfDNA BCTs) collected from a patient diagnosed with CRC are shown in Figure 4. We observed enrichment of small cfDNA fragment binding of 9780 published CTCF TFBS sequences (Kelly et al, 2012). By contrast, we observed little or no CTCF occupied nucleosome sized cfDNA fragments (135-155bp or 156-180bp). The cfDNA library obtained for binding to non-specific mouse IgG showed no enrichment.

[0189] We then reanalysed the data for binding to a subset of CTCF TFBS that have been reported to be selectively bound more frequently in cancer tissues to see if these TFBS were occupied in the samples obtained from CRC patients. These sites were also occupied by 35-80bp cfDNA fragments but not by nucleosomes sized cfDNA fragments. The non-specific binding was again low for all fragments sizes. The results are shown in Figure 5.

[0190] We then repeated the ChIP method to immunoprecipitate endogenous CTCF-DNA complexes using cross-linked EDTA plasma samples (collected in STRECK cfDNA BCTs) collected from a subject diagnosed as suffering from breast cancer. We extracted cfDNA fragments from the solid phase support, ligated the extracted DNA fragments to adapter oligonucleotides and amplified the cfDNA present. The amplified cfDNA library was analysed by electrophoresis and the resulting electropherograms showed that the library contained small fragments in the 35-80bp range with a specific signal of 1000 fluorescence units (Fll). The signal for the background peak obtained with non-specific mouse IgG coated solid support was 80 Fll.

[0191] The method of the present invention allows for enrichment of the less than 100bp cfDNA fragments up to at least 250 times that of the level of such fragments in body fluids which have not been subjected to the method of the invention. Another way of looking at this is that the purity of the short ctDNA isolated by the ChIP and analysed by the PCR can be equal to or greater than 80%, 85%, 90%, 95%, or 98%, or 100%. This has been confirmed by conducting sequencing to determine the long versus short reads which are analysed by the PCR as if the ChIP removes all ora substantial portion of the nucleosome bound DNA, then only the short fragments (or a substantial proportion of them) should remain. When tested, we have found that the detectable remaining nucleosome associated DNA is zero in more than half of some cancer samples which have been subject to the CTCF ChIP method in accordance with the present invention. CTCF BINDING AGENTS

[0192] The term “binding agent” as used herein refers to ligands or binders, such as naturally occurring or chemically synthesized compounds, capable of specific binding to a biomarker (i.e. to a specific transcription factor). A ligand or binder according to the invention may comprise a peptide, an antibody or a fragment thereof, or a synthetic ligand such as a plastic antibody, or an aptamer or oligonucleotide or a molecular imprinted surface or device, capable of specific binding to the biomarker. The antibody can be a monoclonal antibody or a fragment thereof capable of specific binding to the target. A ligand or binder according to the invention may be labelled with a detectable marker, such as a luminescent, fluorescent, enzyme or radioactive marker; alternatively or additionally a ligand according to the invention may be labelled with an affinity tag, e.g. a biotin, avidin, streptavidin or His (e.g. hexa-His) tag. The binding agent (e.g. antibody, ligand or binder) may be bound to a solid support, for example a magnetic bead. In one embodiment, the binding agent is selected from: an antibody, an antibody fragment or an aptamer. In a further embodiment, the binding agent used is an antibody. The terms “antibody”, “binding agent” or “binder” are used interchangeably herein.

[0193] In one embodiment, the binding agent binds to cfDNA-CTCF nucleoproteins. In a further embodiment, the binding agent specifically binds to CTCF. It will be understood that the CTCF may be present in a complex, i.e. CTCF occupied cfDNA fragments bound by the binding agent may be present in a nucleoprotein complex. The specificity of a binding agent (e.g. an antibody) is the ability of the binding agent to recognize a particular antigen as a unique molecular entity and distinguish it from another. A binding agent that “specifically binds” to an antigen or an epitope is a term well understood in the art. A binding agent is said to exhibit “specific binding” if it reacts more frequently, more rapidly, with greater duration and / or with greater affinity with a particular target antigen or epitope, than it does with alternative targets. An antibody “specifically binds” to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances.

[0194] Preferred binding agents have high affinity for CTCF, so that binding will occur at low concentrations, as well as high specificity for binding of CTCF, so that non-specific binding of other proteins is minimal.

[0195] The binding agent is used to isolate nucleoproteins comprising CTCF occupied cfDNA fragments, in particular by specifically binding to CTCF. Therefore, in one embodiment, the binding agent selectively binds to cfDNA-CTCF nucleoproteins. A selective antibody does not cross-react with any off-target protein that is present in the assay and thus it exclusively binds to the designated target, i.e. nucleoproteins comprising CTCF occupied cfDNA fragments.

[0196] In preferred embodiments, the binding agent used is an antibody directed to bind to CTCF. In one embodiment, the binding agent which binds to CTCF is an antibody or a fragment (i.e. a binding fragment) thereof.

[0197] The present invention also encompasses use of a binding agent which binds to a nucleoprotein containing a CTCF binding site sequence. Transcription factors do not typically bind to a TFBS alone in isolation, but as one of a number of co-factors bound together in a larger regulatory complex. The length of DNA protected by binding to CTCF alone has been experimentally determined at 39 base pairs (Quitschke et al. 2000). As the CTCF associated cfDNA fragments described herein are of sizes up to 100bp (Figure 9), the CTCF nucleoproteins isolated by ChIP contain multiple proteins. It will be clear to those skilled in the art that a ChIP method using a binder directed to bind to another protein present in a CTCF nucleoprotein complex can be used for methods of the invention described herein.

[0198] Without limitation, one protein complex that co-binds with CTCF is cohesin as shown in Figure 2. Cohesin is a protein complex with multiple subunits including Smc1 , Smc3, SA1 / 2 and Rad21. Indeed, 97% of Rad21 binding loci identified by ChlP-seq experiments using cellular chromatin are co-located with CTCF binding loci (Hansen etal, 2017). It is clear that such proteins may be used as ChIP targets for methods of the invention.

[0199] Therefore, in one aspect of the invention, cfDNA fragments are isolated by a ChIP method using a binder directed to bind to a (non-CTCF) protein present in a CTCF nucleoprotein complex.

[0200] In one embodiment, the non-CTCF protein is cohesin, Smc1 , Smc3, SA1 / 2 or Rad21.

[0201] It will also be appreciated that in one embodiment the binding agent is a co-factor of the transcription factor.

[0202] In one embodiment, the binding agent is immobilised on a solid phase, i.e. to isolate CTCF from the sample. Use of a solid phase facilitates isolation of antibody bound transcription factor-DNA complexes or chromatin fragments. The binding agent may be coated on a solid support, such as sepharose, sephadex, plastic, agarose or magnetic beads. Preferably, the solid support is a paramagnetic or magnetic bead. Magnetic beads may be purchased from Dynabead. In some embodiments the support is a protein A / G bead. The reagent may be conjugated to the physical support before contacting. In some embodiments, the conjugating is a covalent linkage such as by epoxy chemistry.

[0203] In one embodiment, said solid support comprises a porous material. In another embodiment, the binding agent is derivatized to include a tag or linker which can be used to attach the binding agent to a suitable support which has been derivatized to bind to the tag. Many such tags and supports are known in the art (e.g. Sortag, Click Chemistry, biotin / streptavidin, his-tag / nickel or cobalt, GST-tag / GSH, antibody / epitope tags and many more).

[0204] Isolation of the binding agent may then be performed prior to, concurrently with, or following the reaction of the binding agent with a transcription factor. For ease of use, the coated support may be included within a device, for example a microfluidic device.

[0205] In other embodiments the binding agent is added in solution and isolated by cross-linking and precipitating the bound transcription factors with a precipitation agent such as polyethylene glycol (PEG). The precipitated pellet can then be isolated as a separate phase, for example by centrifugation or filtration. Many immunoprecipitation methods are known in the art and any such methods may be useful in methods of the invention.

[0206] Commercially available antibodies directed to bind to transcription factors are available. In one embodiment, the antibody binds to CTCF derived from human, mouse, rat, monkey, bovine, canine, feline and equine samples. In one embodiment, the antibody binds to CTCF derived from human samples.

[0207] For ChIP we immobilized the antibodies on commercially available magnetic polystyrene particles. Therefore, in a preferred embodiment of the invention the binding agent which binds to CTCF is a solid phase anti-CTCF antibody (or a part thereof) immobilized on a magnetic polystyrene particle. CTCF BINDING SITES

[0208] There are many known CTCF binding sites. See, e.g. the CTCFBSDB (CTCF Binding Site Database), available on the Internet at insulatordb.uthsc.edu / (Ziebarth et al, 2013). A CTCF binding motif is described in Kim, T.H., et al., 2007. Cell, 128(6), pp.1231-1245. A CTCF binding site may comprise or consist of the consensus sequence CC(A / G / T)(C / G)NNG(A / G)NGG(A / C / T)(A / G / T)(C / G) (SEQ ID NO: 50). For example, CTCF may bind to the consensus sequence CCGCGNGGNGGCAG (SEQ ID NO: 1).

[0209] In an embodiment, a CTCF binding site comprises or consists of a nucleotide sequence having up to three, up to two, or up to one nucleotide substitution, addition or deletion compared to the consensus sequence of SEQ ID NO: 50 or SEQ ID NO: 1. In an embodiment, a CTCF binding site comprises or consists of a nucleotide sequence having up to three nucleotide substitutions compared to the consensus sequence of SEQ ID NO:

[0210] 50 or SEQ ID NO: 1 . In an embodiment, a CTCF binding site comprises or consists of a nucleotide sequence having up to two nucleotide substitutions compared to the consensus sequence of SEQ ID NO: 50 or SEQ ID NO: 1. In an embodiment, a CTCF binding site comprises or consists of a nucleotide sequence having up to one nucleotide substitution compared to the consensus sequence of SEQ ID NO: 50 or SEQ ID NO: 1.

[0211] CTCF may bind to the sense strand or anti-sense strand of a DNA molecule. A sense strand sequence which corresponds to antisense CTCF binding site may comprise or consist of the consensus sequence (C / G)(A / C / T)(A / G / T)CCN(C / T)CNN(C / G)(A / C / T)GG (SEQ ID NO: 51). For example, a sense strand sequence which corresponds to an antisense CTCF binding site having the sequence of CCGCGNGGNGGCAG (SEQ ID NO: 1) is CTGCCNCCNCGCGG (SEQ ID NO: 52).

[0212] In an embodiment, a CTCF binding site comprises or consists of a nucleotide sequence having up to three, up to two, or up to one nucleotide substitution, addition or deletion compared to the consensus sequence of SEQ ID NO: 51 or SEQ ID NO: 52. In an embodiment, a CTCF binding site comprises or consists of a nucleotide sequence having up to three nucleotide substitutions compared to the consensus sequence of SEQ ID NO:

[0213] 51 or SEQ ID NO: 52. In an embodiment, a CTCF binding site comprises or consists of a nucleotide sequence having up to two nucleotide substitutions compared to the consensus sequence of SEQ ID NO: 51 or SEQ ID NO: 52. In an embodiment, a CTCF binding site comprises or consists of a nucleotide sequence having up to one nucleotide substitution compared to the consensus sequence of SEQ ID NO: 51 or SEQ ID NO: 52. Accordingly, the skilled person is able to perform methods of the invention by detecting pre-determined genomic regions comprising CTCF binding sites. In preferred embodiments, the pre-determined genomic location is selectively occupied by CTCF in a disease state. In more preferred embodiments, the pre-determined genomic location is selectively occupied by CTCF in a cancer, for example a cancer selected from the group consisting of: lung cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, blood cancer (such as leukaemia or lymphoma) and skin cancer (such as melanoma).

[0214] In one embodiment, the pre-determined genomic locations are selectively occupied by CTCF in blood cancer. Example blood cancer-specific CTCF binding sites are provided in Tables 1 and 3.

[0215] In one embodiment, the pre-determined genomic locations are selectively occupied by CTCF in lung cancer. Example lung cancer-specific CTCF binding sites are provided in Table 3.

[0216] In one embodiment, the pre-determined genomic locations are selectively occupied by CTCF in colorectal cancer. Example colorectal cancer-specific CTCF binding sites are provided in Table 3. In one embodiment a pre-determined genomic location selectively occupied by CTCF in colorectal cancer is associated with the PLXNA4 gene. In one embodiment a pre-determined genomic location selectively occupied by CTCF in colorectal cancer is chr12:92570492-92570605.

[0217] In one embodiment, the pre-determined genomic locations are selectively occupied by CTCF in prostate cancer. Example prostate cancer-specific CTCF binding sites are provided in Table 3.

[0218] In one embodiment, the pre-determined genomic locations are selectively occupied by CTCF in breast cancer. Example breast cancer-specific CTCF binding sites are provided in Table 3.

[0219] Any suitable assay may be used to determine selectively occupied sites, for example by anti-CTCF ChlP-seq or any of the methods described herein. In preferred embodiments, the pre-determined genomic location is present in CTCF-occupied cfDNA fragments isolated from subjects with a disease. In more preferred embodiments, the predetermined genomic location is present in CTCF-occupied cfDNA fragments isolated from subjects with cancer, for example a cancer selected from the group consisting of: lung cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, blood cancer (such as leukaemia or lymphoma) and skin cancer (such as melanoma). In a further embodiment, the pre-determined genomic location is not present in CTCF-occupied cfDNA fragments isolated from healthy subjects.

[0220] In some embodiments, the panel comprises at least 2, 3, 4, 5, 10, 20, 30, 50, 70, 100, 200, or 500 CTCF binding sites, or 5-10, 10-20, 20-30, 30-50, 50-100, 100-200 CTCF binding sites, e.g., such as CTCF binding regions described in the CTCFBSDB. In some embodiments, the panel comprises two or more, three or more, four or more, or five or more pre-determined genomic regions comprising CTCF binding sites. In some embodiments, the panel comprises 30 or fewer, 25 or fewer, 20 or fewer, 15 or fewer, or 10 or fewer pre-determined genomic regions comprising CTCF binding sites. In a further embodiment, the method comprises detecting a panel of between 5 and 30 predetermined genomic regions comprising CTCF binding sites. In a yet further embodiment, the method comprises detecting a panel of 10 pre-determined genomic regions comprising CTCF binding sites.

[0221] In preferred embodiments, the CTCF binding sites are disease-specific. In more preferred embodiments, the CTCF binding sites are cancer-specific, for example a cancer selected from the group consisting of: lung cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, blood cancer (such as leukaemia or lymphoma) and skin cancer (such as melanoma). Any suitable assay may be used to determine whether a CTCF binding site is disease-specific. For example, the CTCF binding sites may have a higher occupancy score and / or a higher binding level in diseased cells compared to corresponding normal tissues. Hundreds or thousands of CTCF binding sites are described as having cancer selective binding (Liu et al, 2017).

[0222] In preferred embodiments, the CTCF binding sites are present in CTCF-occupied cfDNA fragments isolated from subjects with a disease. In more preferred embodiments, the CTCF binding sites are present in CTCF-occupied cfDNA fragments isolated from subjects with cancer, for example a cancer selected from the group consisting of: lung cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, blood cancer (such as leukaemia or lymphoma) and skin cancer (such as melanoma). In a further embodiment, the CTCF binding sites are not present in CTCF-occupied cfDNA fragments isolated from healthy subjects. Example cancer-specific CTCF binding sites are listed in Table 2 (from Wang et al, 2012) and Table 3 (from Fang, C., et al., 2020. Genome biology, 21 , pp.1-30). In some embodiments, the method comprises detecting one or more, two or more, three or more, four or more, five or more, or 10 or more of the cancer-specific CTCF binding sites listed in Table 1 , Table 2 and / or Table 3. In some embodiments, the method comprises detecting one or more, two or more, three or more, four or more, five or more, or 10 or more of the lung cancer-specific CTCF binding sites listed in Table 3. In some embodiments, the method comprises detecting one or more, two or more, three or more, four or more, five or more, or 10 or more of the colorectal cancer-specific CTCF binding sites listed in Table 3. In some embodiments, the method comprises detecting one or more, two or more, three or more, four or more, five or more, or 10 or more of the breast cancer-specific CTCF binding sites listed in Table 3. In some embodiments, the method comprises detecting one or more, two or more, three or more, four or more, five or more, or 10 or more of the prostate cancer-specific CTCF binding sites listed in Table 3. In some embodiments, the method comprises detecting one or more, two or more, three or more, four or more, five or more, or 10 or more of the blood cancer-specific CTCF binding sites listed in Table 1 or Table 3.

[0223] Table 2: Example cancer-specific CTCF binding sites

[0224] Cancer-specific CTCF binding sites (in GRCh37 / hg19) chr1 :1905824-1905958, chr1 :2703304-2703438, chr1 :2704131 -2704265, chr1 :2718300-2718434, chr1 :2738668-2738802, chr1 :2838710-2838844, chr1 :3347940-3348074, chr1 :3830996-3831130, chr1 :4233339-4233473, chr1 :4554744-4554878, chr1 :4654521 -4654655, chr1 :4754720-4754854, chr1 :5542124-5542258, chr1 :5890950-5891084, chr1 :6438271 -6438405, chr1 :7058504-7058638, chr17347227-7347361 , chr1 :9664310-9664444, chr1 :11193098-11193232, chr1 :12167022-12167156, chr1 :12820451 -12820585, chr1 : 14277274-14277408, chr1 :14761887-14762021 , chr1 : 18246482-18246616, chr1 :18311122-18311256, chr1 :18573164-18573298, chr1 :18629436-18629570, chr1 :18700512-18700646, chr1 :18857549-18857683, chr1 :19054531 -19054665, chr1 :32327117-32327251 , chr1 :34090659-34090793, chr1 :36010751 -36010885, chr1 :36912052-36912186, chr1 :37197426-37197560, chr1 :37329915-37330049, chr1 :37351246-37351380, chr1 :37562275-37562409, chr1 :38231823-38231957, chr1 :41995856-41995990, chr1 :42192553-42192687, chr1 :44460579-44460713, chr1 :47935744-47935878, chr1 :51807721-51807855, chr1 :53370648-53370782, chr1 :73190711-73190845, chr1 :142639262-142639396, chr1 :147313165- 147313299, chr1 :151108066-151 108200, chr1 :151337612-151337746, chr1 :151688658-151688792, chr1 :153744501 -153744635, chr1 :154714438-154714572, chr1 :156389801 -156389935, chr1 :156554738- 156554872, chr1 :156931634-156931768, chr1 :157281041 -157281175, chr1 :157743621-157743755, chr1 :159791398-159791532, chr1 :160807403-160807537, chr1 :165986770-165986904, chr1 : 166227431 - 166227565, chr1 :179038369-179038503, chr1 :180218583-180218717, chr1 :181473544-181473678, chr1 :194312127-194312261 , chr1 :199923956-199924090, chr1 :200948827-200948961 , chr1 :201278742- 201278876, chr1 :202091860-202091994, chr1 :202128451 -202128585, chr1 :202206343-202206477, chr1 :202625319-202625453, chr1 :202629231 -202629365, chr1 :203053757-203053891 , chr1 :203413594- 203413728, chr1 :204593231 -204593365, chr1 :204684008-204684142, chr1 :204796217-204796351 , chr1 :204943560-204943694, chr1 :204998222-204998356, chr1 :207080950-207081084, chr1 :207106798- 207106932, chr1 :207108953-207109087, chr1 :207134447-207134581 , chr1 :208364258-208364392, chr1 :213094388-213094522, chr1 :219491224-219491358, chr1 :220259636-220259770, chr1 :225133775- 225133909, chr1 :226150197-226150331 , chr1 :230717587-230717721 , chr1 :234225261 -234225395, chr1 :234917205-234917339, chr1 :237288293-237288427, chr1 :241432550-241432684, chr1 :242121667- 242121801 , chr1 :243100173-243100307, chr1 :247282380-247282514, chr1 :248020913-248021047, chr1 :248850280-248850414, chr1 :248859671 -248859805, chr2:172206-172340, chr2:1136222-1136356, chr2:1901022-1901156, chr2:1904644-1904778, chr2:3066408-3066542, chr2:3104922-3105056, chr2:6931903-6932037, chr2:8570890-8571024, chr2:8739813-8739947, chr2:8767945-8768079, chr2:9126912-9127046, chr2:9516296-9516430, chr2:10148708-10148842, chr2:10457912-10458046, chr2:12188809-12188943, chr2: 15805909-15806043, chr2:20792409-20792543, chr2:20876236-20876370, chr2:21618374-21618508, chr2:21645055-21645189, chr2:23536012-23536146, chr2:24244366-24244500, chr2:30420513-30420647, chr2:38750992-38751126, chr2:42282065-42282199, chr2:42997813-42997947, chr2:43021749-43021883, chr2:43435149-43435283, chr2:47008086-47008220, chr2:47140845-47140979, chr2:71899355-71899489, chr2:73263497-73263631 , chr2:74505431 -74505565, chr2:74734375-74734509, chr2:74990007-74990141 , chr2:78769267-78769401 , chr2:86477209-86477343, chr2:90108259-90108393, chr2:90144104-90144238, chr2:91799452-91799586, chr2:95536209-95536343, chr2:95540057-95540191 , chr2:97351563-97351697, chr2:99497267-99497401 , chr2:1 13403139-113403273, chr2:113932109- 113932243, chr2: 120963948-120964082, chr2:121679142-121679276, chr2: 127240535-127240669, chr2:127526784-127526918, chr2:127761 148-127761282, chr2:145198821-145198955, chr2:147170318- 147170452, chr2:218666066-218666200, chr2:231188048-231 188182, chr2:231794066-231794200, chr2:231807175-231807309, chr2:232794810-232794944, chr2:234292786-234292920, chr2:239991765- 239991899, chr2:240653377-24065351 1 , chr2:241543845-241543979, chr2:241796207-241796341 , chr3:13068903-13069037, chr3:13082716-13082850, chr3:13503206-13503340, chr3:13922193-13922327, chr3:15568603-15568737, chr3:16875848-16875982, chr3:17839574-17839708, chr3:38669575-38669709, chr3:50259363-50259497, chr3:52119094-521 19228, chr3:128309907-128310041 , chr3:150497502- 150497636, chr3:163340329-163340463, chr3:167211 180-167211314, chr4:1784193-1784327, chr4:2443570-2443704, chr4:3454308-3454442, chr4:3609951 -3610085, chr4:3785603-3785737, chr4:3788029-3788163, chr4:3794665-3794799, chr4:4329460-4329594, chr4:4869303-4869437, chr4:5965449-5965583, chr4:5981851 -5981985, chr4:6046657-6046791 , chr4:6078247-6078381 , chr4:6136438-6136572, chr4:6364099-6364233, chr4:6649739-6649873, chr4:7652481 -7652615, chr4:8690015-8690149, chr4:9167861 -9167995, chr4:85165711-85165845, chr4:144502860-144502994, chr4:148989316-148989450, chr4:185064992-185065126, chr4:187810334-187810468, chr4: 188592814- 188592948, chr5:13212-13346, chr5:982696-982830, chr5:1010373-1010507, chr5:1026036-1026170, chr5:1107939-1108073, chr5:1235625-1235759, chr5:1245249-1245383, chr5:1494971-1495105, chr5:2015369-2015503, chr5:2208192-2208326, chr5:2701901 -2702035, chr5:4942513-4942647, chr5:5881595-5881729, chr5:6202879-6203013, chr5:6526196-6526330, chr5:6781026-6781160, chr5:6837752-6837886, chr5:7519439-7519573, chr5:8012543-8012677, chr5:10305199-10305333, chr5:11075197-11075331 , chr5:16453847-16453981 , chr5:20250710-20250844, chr5:25858254-25858388, chr5:31664896-31665030, chr5:33588987-33589121 , chr5:62048907-62049041 , chr5:65790402-65790536, chr5:71047458-71047592, chr5:75182165-75182299, chr5:79500988-79501122, chr5:92221773-92221907, chr5:101426605-101426739, chr5:1 14369704-114369838, chr5:132583663-132583797, ch r5 : 140221302- 140221436, chr5:149540847-149540981 , chr5:149666548-149666682, chr5:173472554-173472688, chr5:175044581 -175044715, chr5:175123675-175123809, chr5:180038280-180038414, chr6:217182- 217316, chr6:1515725-1515859, chr6:2596613-2596747, chr6:3196237-3196371 , chr6:6225314-6225448, chr6:7339453-7339587, chr6:7814079-7814213, chr6:14281 117-14281251 , chr6:14816858-14816992, chr6:19211576-19211710, chr6:31442795-31442929, chr6:31553418-31553552, chr6:32053727-32053861 , chr6:33059871 -33060005, chr6:33935892-33936026, chr6:34458287-34458421 , chr6:41159435-41159569, chr6:42277603-42277737, chr6:43254917-43255051 , chr6:45468783-45468917, chr6:50152852-50152986, chr6:53016591 -53016725, chr6:66348073-66348207, chr6:91207273-91207407, chr6:97164253-97164387, chr6:109557731 -109557865, chr6:1 10699869-110700003, chr6:1 18730150-118730284, chr6: 126080362- 126080496, chr6:126871617-126871751 , chr6:131406288-131406422, chr6:134138708-134138842, chr6:151149411-151 149545, chr6:155473494-155473628, chr6:156681 187-156681321 , chr6:158368336- 158368470, chr6:159961324-159961458, chr6:161260164-161260298, chr6: 166826469-166826603, chr6:167493377-167493511 , chr6:167528680-167528814, chr6:167533681-167533815, chr6:168098117- 168098251 , chr6:168168118-168168252, chr6:168445822-168445956, chr6:168472419-168472553, chr6:169391902-169392036, chr7:307130-307264, chr7:1408787-1408921 , chr7:1633268-1633402, chr7:1798321-1798455, chr7:1807302-1807436, chr7:4266161 -4266295, chr7:4512529-4512663, chr7:4855322-4855456, chr7:4869815-4869949, chr7:4885625-4885759, chr7:8473318-8473452, chr7:30830850-30830984, chr7:31100977-3110111 1 , chr7:35764937-35765071 , chr7:37392599-37392733, chr7:44325793-44325927, chr7:45019186-45019320, chr7:45118147-451 18281 , chr7:45186494-45186628, chr7:46169555-46169689, chr7:51519931 -51520065, chr7:51688579-51688713, chr7:63643609-63643743, chr7:64696118-64696252, chr7:64942366-64942500, chr7:71390761-71390895, chr7:7771 1331-77711465, chr7:86227757-86227891 , chr7:86689096-86689230, chr7:98384560-98384694, chr7:100198124- 100198258, chr7:102092136-102092270, chr7:103982131 -103982265, chr7:104056306-104056440, chr7:104503288-104503422, chr7:106689070-106689204, chr7:131229757-131229891 , chr7:131231486- 131231620, chr7:131798636-131798770, chr7:134917596-134917730, chr7:139778898-139779032, chr7:140345060-140345194, chr7:143317262-143317396, chr7:148308919-148309053, chr7:149497019- 149497153, chr7:149558560-149558694, chr7:150451360-150451494, chr7:150663219-150663353, chr7:150706524-150706658, chr7:151560611-151560745, chr7:151681721-151681855, chr7:152591588- 152591722, chr7:152810407-152810541 , chr7:152819469-152819603, chr7:153078762-153078896, chr7:154432317-154432451 , chr7:154536056-154536190, chr7:154581257-154581391 , chr7:154624407- 154624541 , chr7: 154650070-154650204, chr7:155191798-155191932, chr7:155205471-155205605, chr7:155867791 -155867925, chr7:156352898-156353032, chr7:157030912-157031046, chr7:157091229- 157091363, chr7:157092521-157092655, chr7:157267419-157267553, chr7:157449052-157449186, chr7:157668280-157668414, chr7:157669278-157669412, chr7:157847918-157848052, chr8:702229- 702363, chr8:807698-807832, chr8:991983-992117, chr8:1273463-1273597, chr8:2368397-2368531 , chr8:21313601 -21313735, chr8:37642185-37642319, chr8:41369830-41369964, chr8:41669869-41670003, chr8:47829391 -47829525, chr8:48091921 -48092055, chr8:58122180-58122314, chr8:108767909- 108768043, chr8:125660048-125660182, chr8:139126673-139126807, chr8:139197227-139197361 , chr8:139512780-139512914, chr8:139926767-139926901 , chr8:140659242-140659376, chr8: 142637009- 142637143, chr8: 142639628-142639762, chr8:143070020-143070154, chr8:143084368-143084502, chr8:143210297-143210431 , chr8:143230803-143230937, chr8:143379542-143379676, chr8: 143632597- 143632731 , chr8:144461931-144462065, chr9:4117868-4118002, chr9:68455415-68455549, chr9:96060183-96060317, chr9:96582989-96583123, chr9:98336588-98336722, chr9:99820058-99820192, chr9:102338369-102338503, chr9:104219421-104219555, chr9:122871616-122871750, chr9: 123087438- 123087572, chr9:124397383-124397517, chr9:124623875-124624009, chr9: 127209242-127209376, chr9:127237571 -127237705, chr9:128982219-128982353, chr9:129679336-129679470, chr9:131944980- 131945114, chr9:132483587-132483721 , chr9:133928675-133928809, chr9:134150840-134150974, chr9:135674475-135674609, chr9:136454667-136454801 , chr9:136634136-136634270, chr9:137109747- 137109881 , chr9:137731389-137731523, chr9:137875064-137875198, chr9:138113996-138114130, chr9:138133674-138133808, chr9:138173097-138173231 , chr9:138235917-138236051 , chr9:138457362- 138457496, chr9:138508268-138508402, chr9:139652937-139653071 , chr9:139962780-139962914, chr9:140862945-140863079, chr9:140995541 -140995675, chr10:572865-572999, chr10:1402325-1402459, chr10:1411165-141 1299, chr10:1758046-1758180, chr10:3777412-3777546, chr10:13394652-13394786, chr10:43186133-43186267, chr10:43471209-43471343, chr10:44525752-44525886, chr10:44667003- 44667137, chr10:45420745-45420879, chr10:45651370-45651504, chr10:48478626-48478760, chr10:501 10337-501 10471 , chr10:50430420-50430554, chr10:50508649-50508783, chr10:65661 120- 65661254, chr10:70824811-70824945, chr10:72083711-72083845, chr10:72716221 -72716355, chr10:73407433-73407567, chr10:76736431 -76736565, chr10:77686676-77686810, chr10:80019945- 80020079, chr10:81368652-81368786, chr10:85885794-85885928, chr10:87870288-87870422, chr10:87898929-87899063, chr10:87938288-87938422, chr10:88109347-88109481 , chr10:88352207- 88352341 , chr10:102214555-102214689, chr10:105236214-105236348, chr10:108623425-108623559, chr10:112902018-112902152, chr10:129772320-129772454, chr10:130122456-130122590, chr10:130538358-130538492, chr10:132139708-132139842, chr10:132485084-132485218, chr10:132699676-132699810, chr10:132882729-132882863, chr10:132886903-132887037, chr10:1331 10867-133111001 , chr10:133194508-133194642, chr10:133368238-133368372, chr10:133521506-133521640, chr10:133672596-133672730, chr10:133915107-133915241 , chr10:134707387-134707521 , chr10:134717867-134718001 , chr11 :1409309-1409443, chr11 :1712877- 1713011 , chr1 1 :4744179-4744313, chr11 :6291314-6291448, chr11 :9117134-9117268, chr11 :9142614- 9142748, chr1 1 :17495080-17495214, chr11 :20894726-20894860, chr11 :32395883-32396017, chr11 :44449091-44449225, chr11 :48406365-48406499, chr11 :60253535-60253669, chr11 :61345329- 61345463, chr11 :61504716-61504850, chr11 :62784358-62784492, chr11 :63275633-63275767, chr11 :63374046-63374180, chr11 :64332958-64333092, chr11 :64424404-64424538, chr11 :66104214- 66104348, chr11 :67415716-67415850, chr11 :69523961 -69524095, chr11 :72044908-72045042, chr11 :72264923-72265057, chr11 :75063401 -75063535, chr11 :75207424-75207558, chr11 :75479156- 75479290, chr11 :83422280-83422414, chr11 :86282696-86282830, chr11 :113561828-113561962, chr11 :114717308-114717442, chr11 :114780717-114780851 , chr11 :114823889-114824023, chr11 :115860738-115860872, chr11 :115943630-115943764, chr11 :117939431-117939565, chr11 :119026949-119027083, chr11 :119664419-119664553, chr11 :133701316-133701450, chr11 :134263576-134263710, chr11 :134449410-134449544, chr11 :134597355-134597489, chr11 :134689062-134689196, chr11 :134731514-134731648, chr11 :134809985-134810119, chr12:1909907-1910041 , chr12:2566780-2566914, chr12:4492566-4492700, chr12:6022238-6022372, chr12:14115802-14115936, chr12:14809906-14810040, chr12:38447572-38447706, chr12:48836354- 48836488, chr12:53086160-53086294, chr12:53605679-53605813, chr12:54342830-54342964, chr12:54378697-54378831 , chr12:55445131 -55445265, chr12:62415729-62415863, chr12:65002109- 65002243, chr12:66627842-66627976, chr12:117301160-117301294, chr12:117840345-117840479, chr12:119458676-119458810, chr12:119882385-119882519, chr12:122791073-122791207, chr12:123749278-123749412, chr12:125235415-125235549, chr12:125721732-125721866, chr12: 126034510-126034644, chr12:126168424-126168558, chr12:127459495-127459629, chr12:129068116-129068250, chr12:129098454-129098588, chr12:129328915-129329049, chr12:129454054-129454188, chr12:129460745-129460879, chr12:131146554-131146688, chr12:131617067-131617201 , chr12:131714017-131714151 , chr12:131812384-131812518, chr12:131937278-131937412, chr12:131939379-131939513, chr12:132703638-132703772, chr12:132918942-132919076, chr13:19172863-19172997, chr13:19280803-19280937, chr13:19663585- 19663719, chr13:19728353-19728487, chr13:53424594-53424728, chr13:61047830-61047964, chr13:76593629-76593763, chr13:92299738-92299872, chr13:95878889-95879023, chr13:98283585- 98283719, chr13:99083491 -99083625, chr13:99425500-99425634, chr13:100252630-100252764, chr13:112157578-112157712, chr13: 113778204-113778338, chr13:114059896-114060030, chr14:19614770-19614904, chr14:20264812-20264946, chr14:20612204-20612338, chr14:21133679- 21133813, chr14:23523744-23523878, chr14:23815350-23815484, chr14:24105013-24105147, chr14:25501774-25501908, chr14:29637469-29637603, chr14:60795470-60795604, chr14:72883692- 72883826, chr14:73698699-73698833, chr14:75040174-75040308, chr14:97405368-97405502, chr14:100114861-100114995, chr14:100886327-100886461 , chr14:101006933-101007067, chr14:103522653-103522787, chr14: 104690883-104691017, chr14:104693365-104693499, chr14:104760595-104760729, chr14:104798314-104798448, chr14:104808412-104808546, chr14:104836698-104836832, chr14:104976118-104976252, chr14:106172416-106172550, chr14:106397817-106397951 , chr14:106410931 -106411065, chr14:106411900-106412034, chr14:106441136-106441270, chr14:106465459-106465593, chr14:106465952-106466086, chr14:106472278-106472412, chr14:106477949-106478083, chr14:106501574-106501708, chr14:106518262-106518396, chr14:106627021-106627155, chr14:106642624-106642758, chr14:106653229-106653363, chr14:106667437-106667571 , chr14:106714225-106714359, chr14:106725056-106725190, chr14:106734220-106734354, chr14:106743427-106743561 , chr14:106765288-106765422, chr14:106775122-106775256, chr14:106805048-106805182, chr14:106829433-106829567, chr14:106834258-106834392, chr14:106855049-106855183, chr14:106866265-106866399, chr14:106873636-106873770, chr14:106877458-106877592, chr14:106898907-106899041 , chr14:106933749-106933883, chr14:106974365-106974499, chr14:106986510-106986644, chr14:106986972-106987106, chr14:106993677-106993811 , chr14:107012801 -107012935, chr14:107020162-107020296, chr14:107042226-107042360, chr14:107061967-107062101 , chr14:107074625-107074759, chr14:107083095-107083229, chr14:107094965-107095099, chr14:107113598-107113732, chr14:107117767-107117901 , chr14:107119874-107120008, chr14:107120204-107120338, chr14:107130891-107131025, chr14:107147677-107147811 , chr14:107148322-107148456, chr14:107150636-107150770, chr14:107150931-107151065, chr14:107161867-107162001 , chr14:107183261-107183395, chr14:107210795-107210929, chr14:107218539-107218673, chr14:107238948-107239082, chr14:107239601 -107239735, chr15:20179100-20179234, chr15:20192774-20192908, chr15:20398401- 20398535, chr15:20561743-20561877, chr15:22387614-22387748, chr15:22436155-22436289, chr15:22461145-22461279, chr15:22461361 -22461495, chr15:22461679-22461813, chr15:22462203- 22462337, chr15:22467122-22467256, chr15:22472759-22472893, chr15:22509269-22509403, chr15:22511182-22511316, chr15:22512251 -22512385, chr15:22803038-22803172, chr15:24068796- 24068930, chr15:29906226-29906360, chr15:32396783-32396917, chr15:34781370-34781504, chr15:40629096-40629230, chr15:50554353-50554487, chr15:66880983-66881117, chr15:70726032- 70726166, chr15:75437906-75438040, chr15:77876720-77876854, chr15:78596372-78596506, chr15:93595861 -93595995, chr15:100332460-100332594, chr15:101693146-101693280, chr16:1124049- 1124183, chr16:1588904-1589038, chr16:3000896-3001030, chr16:3137276-3137410, chr16:3541125- 3541259, chr16:5060986-5061120, chr16:5462841 -5462975, chr16:5478995-5479129, chr16:5535734- 5535868, chr16:5540698-5540832, chr16:5552491 -5552625, chr16:5666229-5666363, chr16:6395243- 6395377, chr16:6623765-6623899, chr16:7136983-7137117, chr16:8244922-8245056, chr16:8963624- 8963758, chr16:9319514-9319648, chr16:9391471-9391605, chr16:17090818-17090952, chr16:17305732- 17305866, chr16:17352784-17352918, chr16:18015570-18015704, chr16:18064970-18065104, chr16:20016429-20016563, chr16:20371457-20371591 , chr16:20636540-20636674, chr16:22195312- 22195446, chr16:23848401 -23848535, chr16:24082321 -24082455, chr16:26581256-26581390, chr16:26657077-26657211 , chr16:27870476-27870610, chr16:27985075-27985209, chr16:28519476- 28519610, chr16:30123544-30123678, chr16:30124168-30124302, chr16:31489304-31489438, chr16:31967630-31967764, chr16:32007773-32007907, chr16:32876772-32876906, chr16:32909254- 32909388, chr16:32951302-32951436, chr16:32993842-32993976, chr16:33020945-33021079, chr16:33618907-33619041 , chr16:33619138-33619272, chr16:33630128-33630262, chr16:33647112- 33647246, chr16:33740669-33740803, chr16:33764149-33764283, chr16:34256767-34256901 , chr16:34337274-34337408, chr16:34666569-34666703, chr16:34726762-34726896, chr16:48151487- 48151621 , chr16:50644304-50644438, chr16:54278165-54278299, chr16:56366697-56366831 , chr16:56903567-56903701 , chr16:57694704-57694838, chr16:58131231 -58131365, chr16:66623441- 66623575, chr16:71040563-71040697, chr16:71474672-71474806, chr16:72229598-72229732, chr16:79295518-79295652, chr16:79368257-79368391 , chr16:84984683-84984817, chr16:84992784- 84992918, chr16:85090981 -85091115, chr16:85474781 -85474915, chr16:85524724-85524858, chr16:88321003-88321137, chr16:88555048-88555182, chr16:88560980-88561114, chr16:88825680- 88825814, chr16:88905594-88905728, chr16:89002538-89002672, chr16:89119944-89120078, chr17:159264-159398, chr17:2461958-2462092, chr17:8736074-8736208, chr17: 14262598-14262732, chr17:17289169-17289303, chr17:21837330-21837464, chr17:22007303-22007437, chr17:22012563- 22012697, chr17:22194085-22194219, chr17:22202677-22202811 , chr17:22203231 -22203365, chr17:25365869-25366003, chr17:25708146-25708280, chr17:26697343-26697477, chr17:30614961- 30615095, chr17:32276295-32276429, chr17:32846432-32846566, chr17:33220016-33220150, chr17:34309184-34309318, chr17:36666736-36666870, chr17:36762703-36762837, chr17:37248666- 37248800, chr17:39643157-39643291 , chr17:40315184-40315318, chr17:43799652-43799786, chr17:43972931 -43973065, chr17:45820116-45820250, chr17:46306214-46306348, chr17:47076553- 47076687, chr17:48680843-48680977, chr17:49513130-49513264, chr17:52621634-52621768, chr17:52831970-52832104, chr17:55536649-55536783, chr17:59436303-59436437, chr17:62294182- 62294316, chr17:63181915-63182049, chr17:64447738-64447872, chr17:65012654-65012788, chr17:65624696-65624830, chr17:66630624-66630758, chr17:66737063-66737197, chr17:66770499- 66770633, chr17:71475266-71475400, chr17:71779303-71779437, chr17:71952852-71952986, chr17:72004434-72004568, chr17:72350322-72350456, chr17:72777140-72777274, chr17:73285725- 73285859, chr17:73611645-73611779, chr17:73749881 -73750015, chr17:74185989-74186123, chr17:75396774-75396908, chr17:75724448-75724582, chr17:75768579-75768713, chr17:75888579- 75888713, chr17:76134148-76134282, chr17:76848887-76849021 , chr17:77199244-77199378, chr17:77297701 -77297835, chr17:77434295-77434429, chr17:77632819-77632953, chr17:77696292- 77696426, chr17:77879736-77879870, chr17:77898299-77898433, chr17:77944632-77944766, chr17:78226818-78226952, chr17:78623961 -78624095, chr17:78932792-78932926, chr17:79163549- 79163683, chr17:79407865-79407999, chr17:79766914-79767048, chr17:80060869-80061003, chr17:80308269-80308403, chr17:80637560-80637694, chr17:81153631 -81153765, chr18:11722-11856, chr18:82913-83047, chr18:9792685-9792819, chr18:9811083-9811217, chr18:10490847-10490981 , chr18:11387995-11388129, chr18:13796665-13796799, chr18:13982259-13982393, chr18:33357118- 33357252, chr18:43973323-43973457, chr18:44787735-44787869, chr18:54719548-54719682, chr18:71639094-71639228, chr18:72837820-72837954, chr18:73838470-73838604, chr18:76419882- 76420016, chr18:76639129-76639263, chr18:77693918-77694052, chr18:77694628-77694762, chr18:77958241 -77958375, chr19:384004-384138, chr19:605730-605864, chr19:2243545-2243679, chr19:2550770-2550904, chr19:3783734-3783868, chr19:3849244-3849378, chr19:7838345-7838479, chr19:8082817-8082951 , chr19:8804751 -8804885, chr19:8808591 -8808725, chr19:11489057-11489191 , chr19:11662866-11663000, chr19:12095852-12095986, chr19:13306630-13306764, chr19:13345506- 13345640, chr19:14429818-14429952, chr19:14897216-14897350, chr19:15272527-15272661 , chr19:15870909-15871043, chr19:17757417-17757551 , chr19:19005897-19006031 , chr19:28503378- 28503512, chr19:29434688-29434822, chr19:29969664-29969798, chr19:30236323-30236457, chr19:34242957-34243091 , chr19:36282082-36282216, chr19:36468438-36468572, chr19:38565088- 38565222, chr19:38643740-38643874, chr19:38969299-38969433, chr19:40372274-40372408, chr19:46282550-46282684, chr19:50758505-50758639, chr19:50762288-50762422, chr19:50982495- 50982629, chr19:51373872-51374006, chr19:51798079-51798213, chr19:52068133-52068267, chr19:54096296-54096430, chr19:54416006-54416140, chr19:54865630-54865764, chr19:55392849- 55392983, chr19:55395554-55395688, chr19:55723940-55724074, chr19:56289063-56289197, Table 3: Example lung cancer-specific, colorectal cancer-specific, breast cancer-specific, prostate cancer-specific, and blood-cancer specific CTCF binding sites

[0225] Exemplary genomic regions comprising CTCF binding sites are listed in Table 1. In one embodiment, the method comprises detecting the genomic regions listed in Table 1. In a further embodiment, the method comprises detecting 2 or more, 3 or more, 4 or more, 5 or more, or 10 or more of the genomic regions listed in T able 1 .

[0226] As described hereinbefore, CTCF can affect the 3D structure of chromatin, thereby influencing the expression of genes located upstream or downstream of the binding site. Therefore, the CTCF binding site may not necessarily be located in the gene affected by the CTCF binding (the “target gene”). As such, the CTCF binding sites may be described in relation to the nearest (“associated”) genes which are likely to be the genes regulated by CTCF binding. In one embodiment, the pre-determined genomic regions are associated with the following genes: CATSPERE, CTNNA2, NOL10, DIAPH1 , TMEM260, GALC, OSBPL6, PI15, ANO4, FAM41AY1 , LINC02787, NR_038432.1 , GIPC2, CACNA1A, GNPD1 , TEX33, TRAV4, HEYL, FAM124B, COL21A1 , PLEKHG7, EPHA5, FAM149A, TFAP2B, LINC02934, SKAP1, CDH5, ENSG00000229727, SNORA70, TBC1 D21 , TGIF2LX. In a further embodiment, the pre-determined genomic regions are associated with the following genes: CATSPERE, CTNNA2, NOL10, DIAPH1 , TMEM260, GALC, PI15, ANO4, LINC02787, NR_038432.1 , GIPC2, CACNA1A, GNPD1 , TEX33, TRAV4, HEYL, FAM124B, COL21A1 , PLEKHG7, EPHA5, TFAP2B, LINC02934, SKAP1 , ENSG00000229727, TBC1 D21 , TGIF2LX. In a further embodiment, the pre-determined genomic regions are associated with the following genes: CATSPERE, TMEM260, LINC02787, NR_038432.1 , NOL10, CTNNA2, DIAPH1 , GALC, P115, ANO4, . In a yet further embodiment, the pre-determined genomic regions are associated with the following genes: CATSPERE, CTNNA2, TMEM260, NOL10, PI15, ANO4, LINC02787 and NR_038432.1. In a yet further embodiment, the pre-determined genomic regions are associated with the following genes: CTNNA2, TMEM260, ANO4, LINC02787 and NR_038432.1. In a yet further embodiment, the pre-determined genomic regions are associated with TMEM260 and LINC02787. In an alternative further embodiment, the pre-determined genomic regions are associated with TMEM260 and P115. In a yet further embodiment, the pre-determined genomic regions are associated with TMEM260, P115 and LINC02787. In one embodiment, the pre-determined genomic regions are selected from the following regions: chr1 :244603555-244603648; chr2: 10668571 -10668663; chr2:79219540- 79219628; chr5:141567670-141567748; chr14:56491464-56491546; chr14: 87896549- 87896634; chr2: 178280431 -178280518; chr8:74853546-74853641 ; chr12:100741271- 100741352; chrY: 17430722-17430795; chr1 : 90579362-90579440; chr11 :34926223- 34926312; chr1 :78130901 -78130992; chr19: 13262535-13262621 ; chr5: 141992498- 141992587; chr22:36986359-36986452; chr14:21734605-21734701 ; chr1 : 39609752- 39609824; chr2:224369183-224369264; chr6:55980780-55980855; chr12:92570523- 92570593; chr4:64713399-64713474; chr4:186150099-186150166; chr6:50792057- 50792142; chr2:65573278-65573362; chr17:48336036-48336115; chr16:66275978-

[0227] 66276056; chr2:7427441 -7427517; chr8: 5062480-5062559; chr15:73874049-73874135; and chrX:89757930-89758000 (in genome assembly GRCh38 / hg38). In a further embodiment, the pre-determined genomic regions are selected from the following regions: chr1 :244603555-244603648, chr14:56491464-56491546, chr1 : 90579362- 90579440, chr11 :34926223-34926312, chr1 :78130901 -78130992, chr5: 141992498- 141992587, chr22:36986359-36986452, chr14:21734605-21734701 , chr6: 55980780- 55980855, chr4:64713399-64713474, chr17:48336036-48336115, chr2:7427441- 7427517, chr15:73874049-73874135, chrX: 89757930-89758000, chr2: 10668571 - 10668663, chr2:79219540-79219628, chr5:141567670-141567748, chr14: 87896549- 87896634, chr8:74853546-74853641 , chr12:100741271-100741352, chr19: 13262535- 13262621 , chr22:36986359-36986452, chr1 : 39609752-39609824, chr2:224369183- 224369264, chr12:92570523-92570593, chr6:50792057-50792142, chr2:65573278- 65573362 and chr15:73874049-73874135 (in genome assembly GRCh38 / hg38). In a yet further embodiment, the pre-determined genomic regions are selected from the following regions: chr1 :244603555-244603648, chr14:56491464-56491546, chr1 : 90579362- 90579440, chr11 :34926223-34926312, chr2: 10668571 -10668663, chr2:79219540- 79219628, chr5:141567670-141567748, chr14:87896549-87896634, chr8: 74853546- 74853641 , and chr12:100741271-100741352 (in genome assembly GRCh38 / hg38). In a yet further embodiment, the pre-determined genomic regions are chr14:56491464- 56491546 and chr1 :90579362-90579440 (in genome assembly GRCh38 / hg38). In an alternative further embodiment, the pre-determined genomic regions are chr14:56491464-56491546 and chr8:74853546-74853641 (in genome assembly GRCh38 / hg38). In a yet further embodiment, the pre-determined genomic regions are chr14:56491464-56491546, chr1 :90579362-90579440, and chr8:74853546-74853641 (in genome assembly GRCh38 / hg38). In one embodiment, the CTCF binding sites are selected from the following: chr1 :244603599-244603619, chr14: 56491500-56491520, chr1 : 90579403-90579423, chr11 :34926268-34926288, chr1 :78130945-78130965, chr5:141992536-141992556, chr22:36986402-36986422, chr14:21734657-21734677, chr6:55980809-55980829, chr4:64713438-64713458, chr17:48336072-48336092, chr2:7427475-7427495, chr15:73874089-73874109, chrX: 89757963-89757983, chr2: 10668604-10668624, chr2:79219569-79219589, chr5:141567693-141567713, chr14:87896582-87896602, chr8:74853585-74853605, chr12:100741297-100741317, chr19: 13262563-13262583, chr22:36986399-36986419, chr1 : 39609779-39609799, chr2:224369205-224369225, chr12:92570541-92570561 , chr6: 50792087-50792107, chr2:65573310-65573330, and chr15:73874086-73874106 (in genome assembly GRCh38 / hg38). In a further embodiment, the CTCF binding sites are selected from the following: chr1 :244603599- 244603619, chr14:56491500-56491520, chr1 : 90579403-90579423, chr11 :34926268-

[0228] 34926288, chr2: 10668604-10668624, chr2:79219569-79219589, chr5: 141567693- 141567713, chr14:87896582-87896602, chr8:74853585-74853605, and chr12:100741297-100741317 (in genome assembly GRCh38 / hg38). In a yet further embodiment, the CTCF binding sites are chr14:56491500-56491520 and chr1 :90579403-90579423 (in genome assembly GRCh38 / hg38). In an alternative further embodiment, the pre-determined genomic regions are chr14:56491500-56491520 and chr8:74853585-74853605 (in genome assembly GRCh38 / hg38). In a yet further embodiment, the CTCF binding sites are chr14:56491500-56491520, chr1 :90579403- 90579423, and chr8:74853585-74853605 (in genome assembly GRCh38 / hg38).

[0229] able 1: Exemplary genomic regions comprising CTCF binding sites

[0230]

[0231]

[0232] Other exemplary genomic regions comprising CTCF binding sites are listed in Table 10 and Table 11. In one embodiment, the method comprises detecting the genomic regions listed in Table 10 or Table 11. In a further embodiment, the method comprises detecting 2 or more, 3 or more, 4 or more, 5 or more, or 10 or more of the genomic regions listed in Table 10 orTable 11.

[0233] In one embodiment, the pre-determined genomic regions are associated with one or more of the following genes: CTNNA2, GCA, TMEM44, ENSG00000286456, SLC10A5P1, TRAV4, ENSG00000283445, UST, DMRTA1, GALNT8, FAM174B, SHLD1, NAP1L4P2, MMADHC, PTPRG, BASP1, PDE4D, SPMIP4, CLNS1A, SKAP1, IKBKE, ARHGAP15, DPP4, ZNF622, MINDY3, TMEM236, SMPD1, ENSG00000260945, NLRP1, ARSH, ROBO1, ALCAM, MTCO3P27, EDIL3, EXOC2, IFNGR1, DLD, MKX, QSER1, CTBP2P6, ZDHHC17, MIPOL1, LINC01418, TERF2IP, CORO2B, GIPC2, ENSG00000230472, PHGDH, CATSPERE, ARMCX4, OSBPL6, CAST, PHEX, ZNF157, TIMM50, LINC02583, PTMA, HEPACAM2, IFNWP4, LINC02572, PARD3, GRIK5, IFIH1, ADAM28, LINC00589, ENSG00000228566, ELF5, PPFIBP1, WIPF1, MCCC1, DNAH10, CREG1, TPRG1, GNPDA1, MTND2P8, LINC02446, CPS1, ERBB4, OR7E43P, TMEM260, DUT, ENSG00000226530, TANC1, MAPK13, ENSG00000232436, AMBRA1, CENPSCORT, TMEM182, LHFPL3AS1, JAK2, TECTA, CLMP, NAA16, SDK2, VRK3, YTHDF1, SBF2, MSCAS1, GAPDH, LINC02389, FCGR2A, ENSG00000287730, TP5BI11, SINHCAF, ENSG00000275393, FCGRT, PROSER2, LINC02535, LPAL2, ENSG00000230649, TPPP2, LRP8, CYP1B1, KCNIP4, ANK2, PDE4D, KRT18P54, MRPL15, GDF6, MPP7, SCGB1A1, LINC00937, SLAMF7, LILRP1, SDE2, NID1, FGFBP2, SPOCK3, RNF14, OR4S1, SNX20, RGS9, SPTLC3, NOL10, DPPA3P2, CCDC8, TLX1, FERMT1, GPR27, SIRT6, LINC01307, IFI6, CCNA1, UNC119, TGM6, IFI16, MTARC1, MY03BAS1, DDX3ILA1, NKAIN1, LINC02810, SERINC2, ENSG00000236069, PREP, CEACAM1, VAV3, ENSG00000235819, PLXNA4, NPM1P46, RN7SL361P, ENSG00000266573, GLDC, SWAP7Q, QOQ8A, ENSG00000225620, RPL7AP47, SMUG1, TTLL11, ENSG00000228852, LRRC7, VAV3, GPD1L, SPICE1, CEP63, IL20RB, FAUP2, LINC02357, LINC02503, TICAM2, PKHD1, ENSG00000218813, SYTL3, PACRG, HAP1, PLXNA4, RN7SL135P, NDUFB9P3, ENSG00000252852, DSCC1, ENSG00000235819, ENSG00000279503, SLC16A12, HECTD2, PLEKHG7, LINC02409, LINC02300, SLC25A21, LCTL, PHLPP1, PHLPP1, ZFP64, MTOR, GLIS1, RN7SKP272, ENSG00000229727, RN7SL361P, POU3F3, CHCHD5, ITGB6, NPM1P46, FAM124B, ZNF876P, UCHL1, ENSG00000266573, ENSG00000225620, Q0Q8A, TFAP2B, GLDC, RPL7AP47, SWAP7Q,

[0234] ENSG00000228852, TTLL11 , SMUG1 , AN04, EPHA5, and NR_038432.1.

[0235] In one embodiment, the pre-determined genomic regions are selected from the following regions: chr2:79219519-79219648, chr2: 162301295- 162301441 , chr3: 194594813- 194594949, chr7:64625243-64625355, chr8:81641590-81641720, chr14:21734606- 21734734, chr1 :58739989-58740101 , chr6: 149028960-149029063, chr9:22452551- 22452664, chr12:4849148-4849254, chr15:92797259-92797393, chr20:5788487- 5788587, chrX: 106481411-106481533, chr2:149561424-149561553, chr3:61381935- 61382023, chr5: 17190626-17190742, chr5:59390194-59390288, chr7:25160122- 25160245, chr11 :77619731-77619846, chr17:48336001-48336147, chr1 :206461901-

[0236] 206462094, chr2:143110966-143111104, chr2:162123970-162124051 , chr5: 16456356-

[0237] 16456495, chr10:15902711-15902826, chr10:17742984-17743122, chr11 :6379394- 6379487, chr16: 17579398-17579529, chr17:5592340-5592644, chrX:3035865- 3036000, chr3:78659532-78659619, chr3: 105518948-105519094, chr4:64585094- 64585216, chr5:83927264-83927382, chr6:593261 -593392, chr6: 137314444-

[0238] 137314535, chr7: 107874746-107874889, chr10:27656752-27656912, chr11 :32960716-

[0239] 32960828, chr11 :43567527-43567641 , chr12:76791108-76791220, chr14:37230722- 37230848, chr15:81939739-81939846, chr16:75789293-75789426, chr15:68554138- 68554266, chr1 :78130884-78131005, chr6: 50602320-50602449, chr1 : 119675667- 119675836, chr1 :244603541 -244603674, chrX:101415625-101415728 chr2: 178280411-178280526, chr5: 96752586-96752688, chrX:22081630-22081732 chrX:47384626-47384769, chr19: 39474211 -39474377, chr2:46464602-46464705 chr2:231715363-231715468, chr7:93228366-93228499, chr9:21111090-21111213 chr2:129849717-129849817, chr10:34638577-34638699, chr19:42028327-42028524 chr2: 162298074-162298189, chr8:24229478-24229598, chr8:29647427-29647523 chr10:63720138-63720300, chr11 : 34492959-34493085, chr12:27652288-27652426 chr2 : 174700116-174700251 , chr3 : 183122469- 183122581 , chr 12 : 123905436-

[0240] 123905607, chr1 : 167540493-167540653, chr3: 189148131 -189148251 , chr5: 141992479- 141992603, chr9: 78821242-78821375, chr12: 10562452- 10562563, chr2:210577647-210577793, chr2:212567753-212567856, chr4:4183014-4183121 , chr14:56491434-56491551 , chr15:48352569-48352712, chrX:51461172-51461282, chr2: 159118703-159118816, chr6: 36095298-36095473, chr1 :221435546-221435673, chr11 :46537952-46538113, chr1 : 10428186-10428296, chr2: 102840031-102840180, chr7: 104848545- 104848654, chr9:4988029-4988170, chr11 : 121194847-121194979, chr11 : 123247327-123247457, chr13:41353128-41353267, chr17:73369977-73370136, chr19:49983756-49983882, chr20:63223466-63223579, chr11 :10077000-10077143, chr8:71975358-71975486, chr12:6537982-6538105, chr12:64891000-64891169, chr1 :161487131-161487247, chr4: 155442425-155442520, chr11 :44911428-44911580, chr12:31272008-31272161 , chr16:85717990-85718123, chr19:49547082-49547241 , chr10:11819934-11820065, chr6:85355152-85355270, chr6:160519981-160520091 , chr7: 136009627-136009724, chr14:21014774-21014934, chr1: 53393434-53393577, chr2:38115355-38115496, chr4:21789365-21789486, chr4: 113394834-113394942, chr5:59673572-59673710, chr6: 85323564-85323677, chr8:54169437-54169546, chr8:96180035-96180344, chr10:28241944-28242032, chr11 :62380869-62381016, chr12 : 8396766-8396964, chr1 : 160726939- 160727057, chr19: 54676361 -54676503, chr1 :225976081 -225976216, chr1 :236103745-236103862, chr4:16026149-16026413, chr4: 167021693- 167021794, chr5:141960806-141960987, chr11 :48307888-48308019, chr16:50665497-50665627, chr17:65198881 -65199032, chr20:13119634-13119763, chr2: 10668563-10668699, chr14:36374168-36374282, chr19:46402330-46402434, chr10:101161791-101161998, chr20:6142524-6142649, chr3:71729971 -71730106, chr19:4204720-4204856, chr1 : 101288544-101288669, chr1 :27669254-27669382, chr13:36420493-36420628, chr17:28540839-28540954, chr20:2351923-2352109, chr1 : 158998804-158998962, chr1 :220810250-220810376, chr2: 170409173-

[0241] 170409293, chr7: 78040335-78040434, chr1 :31162784-31162912, chr1 : 17661594- 17661807, chr1 :31422842-31422978, chr1 : 192445984-192446109, chr6: 105424291 - 105424437, chr19:42529383-42529506, chr1 : 107782340-107782444, chr9:86414420- 86414532, chr7:132188991-132189067, chr2:197374043-197374122, chr2:60642442- 60642560, chr18:24736101-24736161 , chr9:6594697-6594768, ch r11 :9675318- 9675421 , chr1 :226944700-226944781 , chr1 :202640729-202640819, chr9: 14216485- 14216587, chr12:54188268-54188338, chr9:121838761-121838825, chr1 : 95264973- 95265040, chr1 :69737726-69737836, chr1 : 107782340-107782444, chr3:32144255- 32144365, chr3:113494238-113494353, chr3: 134474064-134474175, chr3: 137105390- 137105526, chr3: 181326780-181326885, chr4:26082030-26082146, chr4: 103934283- 103934454, chr5:115586654-115586772, chr6: 52049619-52049757, chr6:63764478- 63764585, ch r6: 158691470- 158691594, chr6:162908032-162908121 , chr7: 130832002- 130832124, chr7: 132188991 -132189067, chr8:63262036-63262139, chr8:110830607- 110830727, chr8:117329067-117329210, chr8:119867220-119867390, ch r9: 86414420- 86414532, chr9:90142700-90142839, chr10:89563998-89564154, chr10:91389742- 91389869, chr12:92570492-92570605, chr12:97308762-97308849, chr14:28635549- 28635670, chr14:37149849-37149969, chr15:66567130-66567354, chr18:62958013- 62958123, chr18:62982806-62982908, chr20:52312263-52312398, chr1 :11241826- 11241943, chr1 :53754832-53754948, chr1 :89987314-89987463, chr2:7427425-

[0242] 7427538, chr2:60642442-60642560, chr2: 104849680-104849792, chr2: 112582400- 112582523, chr2:160110087-160110199, chr2:197374043-197374122, chr2:224369161-224369299, chr4:242369-242480, chr4:41281166-41281273, chr4: 122758089- 122758199, chr4: 166165962- 166166073, chr4: 169810464-

[0243] 169810607, chr5:32489802-32489952, chr5: 123371732-123371827, chr5: 172370813- 172370900, chr6:33123829-33123955, chr6:49849713-49849822, chr6:110942102- 110942167, chr6: 113909396-113909512, chr6: 159008718-159008830, chr10:25700952-25701067, chr10:92885025-92885143, chr10:94630132-94630263, chr11 :30346226-30346339, chr11 :110433891-110434020, chr11 : 124586369-

[0244] 124586468, chr12:102702668-102702830, chr12:111451647-111451929, chr13:33228694-33228791 , chr13:50085114-50085230, chr13:53250379-53250572, chr13:57627719-57627850, chr13: 100722041-100722178, chr14:20327405-20327535, chr14:24735042-24735188, chr15:31974953-31975090, chr18:24736101-24736161 , chr19:54674154-54674274, chr22:22201213-22201344, chrX:22136053-22136155, chrX: 129754802-129754877, chrX: 130079923-130080041 , chr1 : 19918128-19918255, chr1 :24587331 -24587473, chr1 :39720138-39720288, chr1 :65958784-65959095, chr1 : 74689941-74690070, chr1 :88877501 -88877633, chr1 :161227460-161227587, chr1 :202640729-202640819, chr1 :226944700-226944781 , chr2: 17646148-17646327, chr2:227862361 -227862483, chr2:235594168-235594292, chr2:238462941-

[0245] 238463069, chr3: 124079474-124079582, chr3: 156541160-156541317, chr3: 194404789- 194404904, chr3: 195522387- 195522501 , chr4: 16794985- 16795080, chr4:27048448-27048583, chr4:52026152-52026244, chr4:52376236-52376358, chr4: 140999943-141000112, chr4: 146104133-146104288, chr4: 158850959-

[0246] 158851065, chr4: 165464923-165465025, chr5:18314314-18314440, chr5:28014922- 28015045, chr5: 74648924-74649026, chr5: 102223966-102224084, chr5: 129664478- 129664599, chr5: 155139754-155139857, chr6:12893990-12894132, chr6: 30380603- 30380684, chr6: 50792043-50792155, chr6:51855414-51855525, chr6: 123415992- 123416085, chr7:10412547-10412686, chr7:25180362-25180528, chr7: 38374563- 38374659, chr7:91830142-91830274, chr7:94395571 -94395689, chr8:4833976-

[0247] 4834088, chr8:95758783-95758908, chr8:119070591-119070729, chr9:6354233- 6354348, chr9:6594697-6594768, chr9:14216485-14216587, chr9: 17063671 -17063809, chr9: 19028989-19029108, chr10:3192966-3193102, chr10: 16828259-16828398, chr10:25726026-25726156, chr10:87656598-87656695, chr10:87735375-87735496, chr10: 125098674-125098794, chr11 :9675318-9675421 , chr11 : 17230210-17230310, chr11 :32323655-32323771 , chr11 : 72667585-72667696, chr11 : 124402455-124402570, chr12:20946875-20946995, chr12:53791868-53791960, chr12:55619731-55619974, chr12:125403356-125403461 , chr12:126069664-126069777, chr13:85828223-

[0248] 85828338, chr14:45426816-45426941 , chr14:93655215-93655320, chr15:44288413- 44288574, chr15:90956673-90956821 , chr16:24253220-24253372, chr16:63361142- 63361267, chr16:66728261-66728409, chr17:35312798-35312956, chr17:78013974- 78014108, chr18:43571212-43571318, chr18:65781879-65782020, chr20: 36466920- 36467064, chr22:25093742-25093869, chrX:51614499-51614639, chrX:110156310- 110156388, chr22_KI270733v1_random: 136322-136475, chr1 : 32272584-32272646, chr1 : 86742093-86742193, chr1 :88998835-88998937, chr1 :95264973-95265040, chr1 : 163410082- 163410225, chr1 : 169055544- 169055615, chr1 :236452124-

[0249] 236452243, chr1 :240149603-240149760, chr1 :240189505-240189629, ch r2: 18378 ISO- 18378279, chr2:38581666-38581810, chr2: 152861493- 152861578, chr2:166539491-

[0250] 166539609, chr2:176814897-176815015, chr2: 182186287-182186395, chr2:227890621-227890778, chr3:87298250-87298349, chr3:89211716-89211800, chr3: 109732032- 109732122, chr3: 134492629- 134492738, chr3: 156509828-

[0251] 156509965, chr3: 157137568-157137691 , chr3: 161273765-161273866, chr3:166013225-166013311 , chr4:23135919-23136011 , chr4:48087272-48087368, chr4:70183776-70183887, chr4:73019582-73019720, chr4:86044122-86044254, chr4: 174365159- 174365240, chr5:5141805-5141917, chr5:80083642-80083758, chr5:81299634-81299801 , chr5:112175136-112175244, chr5: 146662279-146662392, chr5: 148733715-148733808, chr5: 154157507-154157598, chr6:35449508-35449669, chr6: 52021110-52021230, chr6: 56902964-56903131 , chr6:70706662-70706775, chr6:74091490-74091627, chr6:112180232-112180339, chr6: 135451738-135451874, chr7: 148504024- 148504134, chr7: 156582102- 156582204, chr8: 12866970- 12867090, chr8:69663836-69663985, chr8:75257410-75257540, chr8:80612676-80612794, chr8:90948403-90948519, chr8:132651571-132651698, chr9: 13427664-13427772, chr9: 15873723-15873819, chr9: 17462998-17463108, chr9: 121400273-121400386, chr9:121838761-121838825, chr10:61284412-61284550, chr10:93352770-93352892, chr10:116490211-116490321 , chr10:125982446-125982551 , chr11 :6738008-6738119, chr11 :84384764-84384893, chr11 :88092327-88092414, chr11 :94519623-94519738, chr11 :96464846-96464973, chr11 :115117490-115117553, chr11 : 128414446

[0252] 128414512, chr12:7642988-7643098, chr12:21680506-21680612, chr12:29519728 29519832, chr12:34034045-34034152, chr12:54188268-54188338, chr12:58346176 58346282, chr12:100741240-100741368, chr12:119160250-119160324 chr13:97132337-97132465, chr14: 19878197-19878283, chr14:25400748-25400891 , chr14:53055029-53055173, chr14:64303608-64303708, chr14:82000025-82000111 , chr14:98138734-98138826, chr15:85505208-85505330, chr15: 98881743-98881866, chr16: 16449419- 16449513, chr17:700647-700751 , chr17: 16258067- 16258156, chr18:24489020-24489155, chr18:31819747-31819885, chr19:28325490-28325587, chr19:47125320-47125481 , chrX:11144386-11144529, chrX: 87299704-87299805, chrX: 133670537-133670648, chrX: 152758817-152758894, chr1 :2091761 -2091844, chr1 : 5225260-5225331 , chr1 : 110555394-110555522, chr1 : 158036123-158036245, chr1 : 176559961 -176560058, chr1 :207405060-207405185, chr1 :238188421 -

[0253] 238188505, chr2:39059461 -39059589, chr2:60707803-60707914, chr2:62412204- 62412318, chr2: 106791905-106792003, chr2:151437690-151437807, chr2: 158010423- 158010526, chr2:164717151-164717267, chr2: 179440792-179440892, chr2: 181715178-181715317, chr2: 197707735-197707844, chr2:232270870-

[0254] 232270944, chr2:241078520-241078659, chr3: 13600010-13600082, chr3:26712570- 26712658, chr3:65785206-65785301 , chr3: 98039424-98039547, chr3: 146065649- 146065763, chr3: 147191901 -147192041 , chr3: 173584256-173584361 , ch r4 : 38856812- 38856976, chr4:64713379-64713507, chr4:67746823-67746964, chr4: 147097166- 147097267, chr4:163484959-163485038, chr4: 185634052-185634304, chr5: 19469171- 19469243, chr5:36870575-36870732, chr5: 52657324-52657444, chr5:66307768- 66307915, chr5: 103335135-103335205, chr6:47631424-47631547, chr6:52945168- 52945233, chr6:75893429-75893588, chr6: 93664709-93664784, chr6:115244749- 115244826, chr6: 116287793-116287931 , chr6: 158605406-158605520, chr6: 170561934-170561998, chr7:26198200-26198319, chr7:50142454-50142556, chr7:81482732-81482834, chr7: 154504103-154504239, chr8:9831026-9831238, chr9: 8086869-8086940, chr10: 117940922- 117940998, chr11 : 34926239-34926356, chr11 :82682336-82682444, chr11 : 121765670-121765767, chr12: 12376782-12376859, chr12:20950374-20950501 , chr12:23949721-23949794, chr12:24095121 -24095194, chr12:29889866-29889994, chr12:72987449-72987521 , chr12:73391018-73391090, chr12:78146406-78146532, chr12:90959676-90959826, chr13:43908534-43908635, chr13:67238415-67238557, chr13:84896266-84896371 , chr13: 102141733-102141898, chr13: 110839093-110839188, chr14:78082948-78083033, chr14: 100624900-

[0255] 100625009, chr15:24585660-24585745, chr15:31880238-31880317, chr15:41031918- 41032105, chr15:69322673-69322785, chr15:80419036-80419163, chr15: 99704486- 99704631 , chr17:31216272-31216386, chr17:48728857-48728929, chr17:74612182- 74612276, chr17:79893762-79893854, chr18:25853208-25853305, chr18:64037936- 64038067, and chr19:54080140-54080207 (in genome assembly GRCh38 / hg38).

[0256] In one embodiment, the method comprises detecting the genomic regions listed in Table 10. In a further embodiment, the method comprises detecting 2 or more, 3 or more, 4 or more, 5 or more, or 10 or more of the genomic regions listed in Table 10.

[0257] In one embodiment, the pre-determined genomic regions are associated with one or more of the following genes: CTNNA2, GCA, TMEM44, ENSG00000286456, SLC10A5P1, TRAV4, ENSG00000283445, UST, DMRTA1 , GALNT8, FAM174B, SHLD1 , NAP1 L4P2, MMADHC, PTPRG, BASP1 , PDE4D, SPMIP4, CLNS1A, SKAP1 , IKBKE, ARHGAP15, DPP4, ZNF622, MINDY3, TMEM236, SMPD1 , ENSG00000260945, NLRP1 , ARSH, R0B01 , ALCAM, MTCO3P27, EDIL3, EX0C2, IFNGR1 , DLD, MKX, QSER1 , CTBP2P6, ZDHHC17, MIP0L1 , LINC01418, TERF2IP, C0R02B, GIPC2, ENSG00000230472, PHGDH, CATSPERE, ARMCX4, 0SBPL6, CAST, PHEX, ZNF157, TIMM50, LINC02583, PTMA, HEPACAM2, IFNWP4, LINC02572, PARD3, GRIK5, IFIH1 , ADAM28, LINC00589, ENSG00000228566, ELF5, PPFIBP1 , WIPF1 , MCCC1 , DNAH10, CREG1 , TPRG1 , GNPDA1 , MTND2P8, LINC02446, CPS1 , ERBB4, OR7E43P, TMEM260, DUT, ENSG00000226530, TANC1 , MAPK13, ENSG00000232436, AMBRA1 , CENPSCORT, TMEM182, LHFPL3AS1 , JAK2, TECTA, CLMP, NAA16, SDK2, VRK3, YTHDF1 , SBF2, MSCAS1 , GAPDH, LINC02389, FCGR2A, ENSG00000287730, TP5BI11 , SINHCAF, ENSG00000275393, FCGRT, PROSER2, LINC02535, LPAL2, ENSG00000230649, TPPP2, LRP8, CYP1 B1 , KCNIP4, ANK2, PDE4D, KRT18P54, MRPL15, GDF6, MPP7, SCGB1A1 , LINC00937, SLAMF7, LILRP1 , SDE2, NID1 , FGFBP2, SPOCK3, RNF14, OR4S1 , SNX20, RGS9, SPTLC3, NOL10, DPPA3P2, CCDC8, TLX1 , FERMT1 , GPR27, SIRT6, LINC01307, IFI6, CCNA1 , UNC119, TGM6, IFI16, MTARC1 , MY03BAS1 , DDX3ILA1 , NKAIN1 , LINC02810, SERINC2, ENSG00000236069, PREP, CEACAM1 , VAV3, ENSG00000235819, PLXNA4, NPM1 P46, RN7SL361 P, ENSG00000266573, GLDC, SWAP7Q, QOQ8A, ENSG00000225620, RPL7AP47, SMUG1 , TTLL11 , and ENSG00000228852.

[0258] In one embodiment, the pre-determined genomic regions are selected from the following regions: chr2:79219519-79219648, chr2: 162301295- 162301441 , chr3: 194594813- 194594949, chr7:64625243-64625355, chr8:81641590-81641720, chr14:21734606- 21734734, chr1 :58739989-58740101 , chr6: 149028960-149029063, chr9:22452551- 22452664, chr12:4849148-4849254, chr15:92797259-92797393, chr20:5788487- 5788587, chrX: 106481411-106481533, chr2:149561424-149561553, chr3:61381935- 61382023, chr5: 17190626-17190742, chr5:59390194-59390288, chr7:25160122- 25160245, chr11 :77619731-77619846, chr17:48336001-48336147, chr1 :206461901- 206462094, chr2:143110966-143111104, chr2:162123970-162124051 , ch r5: 16456356- 16456495, chr10:15902711-15902826, chr10:17742984-17743122, chr11 :6379394- 6379487, chr16: 17579398-17579529, chr17:5592340-5592644, chrX:3035865- 3036000, chr3:78659532-78659619, chr3: 105518948-105519094, chr4:64585094- 64585216, chr5:83927264-83927382, chr6:593261 -593392, chr6: 137314444- 137314535, chr7: 107874746-107874889, chr10:27656752-27656912, chr11 :32960716- 32960828, chr11 :43567527-43567641 , chr12:76791108-76791220, chr14:37230722- 37230848, chr15:81939739-81939846, chr16:75789293-75789426, chr15:68554138-

[0259] 68554266, chr1 :78130884-78131005, chr6: 50602320-50602449, ch r1 :119675667-

[0260] 119675836, chr1 :244603541 -244603674, chrX: 101415625- 101415728, chr2: 178280411-178280526, chr5: 96752586-96752688, chrX:22081630-22081732 chrX:47384626-47384769, chr19: 39474211 -39474377, chr2:46464602-46464705 chr2:231715363-231715468, chr7:93228366-93228499, chr9:21111090-21111213 chr2: 129849717-129849817, chr10:34638577-34638699, chr19:42028327-42028524 chr2: 162298074-162298189, chr8:24229478-24229598, chr8:29647427-29647523 chr10:63720138-63720300, chr11 : 34492959-34493085, chr12:27652288-27652426 chr2 : 174700116-174700251 , chr3: 183122469- 183122581 , chr12 : 123905436-

[0261] 123905607, chr1 : 167540493-167540653, chr3: 189148131 -189148251 , chr5: 141992479- 141992603, chr9: 78821242-78821375, chr12: 10562452- 10562563, chr2:210577647-210577793, chr2:212567753-212567856, chr4:4183014-4183121 , chr14:56491434-56491551 , chr15:48352569-48352712, chrX:51461172-51461282, chr2: 159118703-159118816, chr6: 36095298-36095473, chr1 :221435546-221435673, chr11 :46537952-46538113, chr1 : 10428186-10428296, chr2: 102840031-102840180, chr7: 104848545-104848654, chr9:4988029-4988170, chr11 :121194847-121194979, chr11 : 123247327-123247457, chr13:41353128-41353267, chr17:73369977-73370136, chr19:49983756-49983882, chr20:63223466-63223579, chr11 :10077000-10077143, chr8:71975358-71975486, chr12:6537982-6538105, chr12:64891000-64891169, chr1 :161487131-161487247, chr4: 155442425-155442520, chr11 :44911428-44911580, chr12:31272008-31272161 , chr16:85717990-85718123, chr19:49547082-49547241 , chr10:11819934-11820065, chr6:85355152-85355270, chr6:160519981-160520091 , chr7: 136009627-136009724, chr14:21014774-21014934, chr1: 53393434-53393577, chr2:38115355-38115496, chr4:21789365-21789486, chr4: 113394834-113394942, chr5:59673572-59673710, chr6: 85323564-85323677, chr8:54169437-54169546, chr8:96180035-96180344, chr10:28241944-28242032, chr11 :62380869-62381016, chr12 : 8396766-8396964, chr1 : 160726939- 160727057, chr19: 54676361 -54676503, chr1 :225976081 -225976216, chr1 :236103745-236103862, chr4:16026149-16026413, chr4: 167021693- 167021794, chr5: 141960806-141960987, chr11 :48307888-48308019, chr16:50665497-50665627, chr17:65198881 -65199032, chr20:13119634-13119763, chr2: 10668563-10668699, chr14:36374168-36374282, chr19:46402330-46402434, chr10:101161791-101161998, chr20:6142524-6142649, chr3:71729971 -71730106, chr19:4204720-4204856, chr1 : 101288544-101288669, chr1 :27669254-27669382, chr13:36420493-36420628, chr17:28540839-28540954, chr20:2351923-2352109, chr1 : 158998804-158998962, chr1 :220810250-220810376, chr2: 170409173- 170409293, chr7: 78040335-78040434, chr1 :31162784-31162912, chr1 : 17661594- 17661807, chr1 :31422842-31422978, chr1 : 192445984-192446109, chr6: 105424291 - 105424437, chr19:42529383-42529506, chr1 : 107782340-107782444, chr9:86414420- 86414532, chr7:132188991-132189067, chr2:197374043-197374122, chr2:60642442- 60642560, chr18:24736101-24736161 , chr9:6594697-6594768, chr11 :9675318-

[0262] 9675421 , chr1 :226944700-226944781 , chr1 :202640729-202640819, chr9: 14216485- 14216587, chr12:54188268-54188338, chr9:121838761-121838825, and chr1 :95264973-95265040 (in genome assembly GRCh38 / hg38).

[0263] In one embodiment, the pre-determined genomic regions are associated with one or more of the following genes: CTNNA2, GCA, TMEM44, ENSG00000286456, SLC10A5P1, TRAV4, ENSG00000283445, UST, DMRTA1 , GALNT8, FAM174B, SHLD1 , NAP1 L4P2, MMADHC, PTPRG, BASP1 , PDE4D, SPMIP4, CLNS1A, SKAP1 , IKBKE, ARHGAP15, DPP4, ZNF622, MINDY3, TMEM236, SMPD1 , ENSG00000260945, NLRP1 , ARSH, ROBO1 , ALCAM, MTCO3P27, EDIL3, EXOC2, IFNGR1 , DLD, MKX, QSER1 , CTBP2P6, ZDHHC17, MIPOL1 , LINC01418, and TERF2IP. In one embodiment, the pre-determined genomic regions are associated with one or more of the following genes: CTNNA2, GCA, TMEM44, ENSG00000286456, SLC10A5P1 , TRAV4, ENSG00000283445, UST, DMRTA1 , GALNT8, FAM174B, SHLD1 , NAP1 L4P2, MMADHC, PTPRG, BASP1 , PDE4D, SPMIP4, CLNS1A, and SKAP1. In one embodiment, the pre-determined genomic region is associated with the CTNNA2 gene.

[0264] In one embodiment, the pre-determined genomic regions are selected from the following regions: chr2:79219519-79219648, chr2: 162301295- 162301441 , chr3: 194594813- 194594949, chr7:64625243-64625355, chr8:81641590-81641720, chr14:21734606- 21734734, chr1 :58739989-58740101 , chr6: 149028960-149029063, chr9:22452551- 22452664, chr12:4849148-4849254, chr15:92797259-92797393, chr20:5788487- 5788587, chrX: 106481411-106481533, chr2:149561424-149561553, chr3:61381935- 61382023, chr5: 17190626-17190742, chr5:59390194-59390288, chr7:25160122- 25160245, chr11 :77619731-77619846, chr17:48336001-48336147, chr1 :206461901- 206462094, chr2:143110966-143111104, chr2:162123970-162124051 , ch r5: 16456356- 16456495, chr10:15902711-15902826, chr10:17742984-17743122, chr11 :6379394- 6379487, chr16: 17579398-17579529, chr17:5592340-5592644, chrX:3035865- 3036000, chr3:78659532-78659619, chr3: 105518948-105519094, chr4:64585094- 64585216, chr5:83927264-83927382, chr6:593261 -593392, chr6: 137314444- 137314535, chr7: 107874746-107874889, chr10:27656752-27656912, chr11 :32960716- 32960828, chr11 :43567527-43567641 , chr12:76791108-76791220, chr14:37230722- 37230848, chr15:81939739-81939846, and chr16:75789293-75789426 (in genome assembly GRCh38 / hg38). In one embodiment, the pre-determined genomic regions are selected from the following regions: chr2:79219519-79219648, chr2: 162301295- 162301441 , chr3: 194594813-194594949, chr7:64625243-64625355, chr8:81641590- 81641720, chr14:21734606-21734734, chr1 :58739989-58740101 , chr6: 149028960- 149029063, chr9:22452551 -22452664, chr12:4849148-4849254, chr15:92797259- 92797393, chr20:5788487-5788587, chrX:106481411-106481533, ch r2: 149561424- 149561553, chr3:61381935-61382023, chr5: 17190626-17190742, chr5:59390194- 59390288, chr7:25160122-25160245, chr11 :77619731-77619846, and chr17:48336001- 48336147 (in genome assembly GRCh38 / hg38). In one embodiment, the pre-determined genomic region is chr2:79219519-79219648.

[0265] PRE-DETERMINED GENOMIC LOCATION AND BIOMARKER DISCOVERY

[0266] The present invention is based on the concept of physical separation of at least some of the ctDNA from cancer from cfDNA that is not derived from cancer, e.g. normal cfDNA or cfDNA arising as a result of an inflammatory response. Therefore it is not necessary to determine whether the detected cfDNA is from cancer because it is already separated using the method of the invention. In other words, the present invention employs the concept of detecting the presence, following separation, of CTCF occupied cfDNA fragments originating from a pre-determined genomic location. The occupation states of these pre-determined genomic locations have previously been determined to be associated with a disease state, such as cancer.

[0267] It will be understood that reference to “pre-determined genomic location” is used interchangeably with “pre-determined genomic region” herein.

[0268] The pre-determined locations may be chosen by analysing all of the CTCF occupied cfDNA in healthy subjects. Additionally, the CTCF occupied cfDNA in subjects with a disease state, such as cancer, may be analysed. Thus, the CTCF binding sites that are occupied in the disease state but which are not occupied in the healthy subjects can be identified.

[0269] Therefore, according to a further aspect of the invention, there is provided a method for determining a genomic location comprising a CTCF binding site that is selectively bound by CTCF during a cancer, comprising:

[0270] (i) contacting a first body fluid sample obtained from a subject diagnosed with the cancer with a binding agent which binds to cfDNA-CTCF nucleoproteins, and contacting a second body fluid sample obtained from a subject with no known cancer or a different cancer with a binding agent which binds to cfDNA-CTCF nucleoproteins;

[0271] (ii) isolating cfDNA-CTCF nucleoproteins from the first and second sample bound in step (i);

[0272] (iii) sequencing cfDNA isolated from the first and second sample in step (ii) to determine genomic locations that are selectively occupied by CTCF in the first sample but not in the second sample, to determine genomic locations comprising a CTCF binding site that is selectively bound by CTCF during the cancer.

[0273] It will be understood that references to “during a cancer” indicate that the CTCF binding site is selectively bound by CTCF in a cancerous cell (i.e. a cell present in or derived from a tumour).

[0274] The pre-determined location can be selected based on the disease state to be assessed. For example, if a specific type of cancer is to be detected, then a CTCF binding site that is selectively bound by CTCF during said type of cancer may be selected. Alternatively, if a more general disease state is to be detected (e.g. all types of cancer; a “pan-cancer” test), then a range of CTCF binding sites that are selectively bound by CTCF during the general disease state (e.g. in a range of cancer types) can be selected. Bioinformatic methods and artificial intelligence programs can enable the analysis of large data sets to determine appropriate CTCF binding sites. Once identified in this way, pre-determined cfDNA sequences that are selectively occupied in disease are effective biomarkers for the presence of the disease or condition for which they were developed. In one embodiment, these cfDNA biomarkers are equal to or less than 100bp, or equal to or less than 80bp. In one embodiment, the cfDNA biomarkers have a minimum length of equal to or greater than 20bp, or equal to or greater than 22bp.

[0275] As described in the Examples provided herein, DNA fragments associated with CTCF were isolated from plasma by immunoprecipitation. The extracted cfDNA was sequenced by next generation sequencing and the data was analysed for coverage of 12,424 previously characterized CTCF binding sites in the human genome (disclosed in the ENCODE database, see Ziebarth et al. 2013). We used bioinformatics to identify CTCF binding site sequences that were present in the cfDNA fragments isolated from the 4 cancer (AML) samples but were not present in the 5 healthy or 5 hospitalized patients. This identified 30 genomic sites that were selectively CTCF bound in AML. We selected pre-determined genomic locations as biomarkers if they fulfilled the following criteria: (i) they include a genomic CTCF binding site plus flanking DNA sequences characterized in the ENCODE database, (ii) they are present in an anti-CTCF IP isolate but not present in a non-specific (e.g. mouse IgG) control isolate, (iii) they are 30-80bp in length and (iv) the CTCF binding site sequence is located centrally within the fragment.

[0276] Pre-determined genomic locations may also be identified by applying one, two, three or all four of these criteria to sequences identified by sequencing all CTCF associated cfDNA fragments in a sample. For example, using fragmentomics analysis.

[0277] In one embodiment, the pre-determined genomic locations include a genomic CTCF binding site plus flanking DNA sequences and are 30-1 OObp (e.g. 30-80bp) in length.

[0278] In one embodiment, a plurality of pre-determined genomic locations are detected, such as via multiplex PCR or multiple single PCR assays. In one embodiment, two or more, three or more, four or more, or five or more pre-determined genomic regions are detected. In some embodiments, 30 or fewer, 25 or fewer, 20 or fewer, 15 or fewer, or 10 or fewer pre-determined genomic regions are detected. In a further embodiment, from 5 to 30 predetermined genomic regions are detected.

[0279] Sequencing methods are well known in the art. There are many methods known in the art to analyse, quantify or identify a DNA sequence and any DNA analysis method may be employed for methods of the current invention including, without limitation, next generation sequencing methods, isothermal DNA amplification, cold PCR (coamplification at lower denaturation temperature- PCR), MAP (MIDI-Activated Pyrophosphorolysis), PARE (personalized analysis of rearranged ends), DNA hybridization methods (including gene chip methods and in situ hybridization methods). In addition, the gene sequence may also be analysed for epigenetically altered DNA sequences by epigenetic DNA sequencing analysis (e.g. for sequences containing 5- methylcytosine using bisulfite conversion of unmodified cytosine to uracil). Therefore, in one embodiment, the associated DNA is analysed using DNA sequencing, for example a sequencing method selected from Next Generation Sequencing (targeted or whole genome) and methylated DNA sequencing analysis, BEAMing, PCR including digital PCR and cold PCR (co-amplification at lower denaturation temperature-PCR), isothermal amplification, hybridization, MIDI-Activated Pyrophosphorolysis (MAP) or Personalized Analysis of Re-arranged Ends (PARE). In some embodiments, the sequencing is NGS. NGS is also known as high-throughput sequencing or massively parallel sequencing. Such sequencing is well known in the art and can make use of Illumina arrays, pore and nanopore sequencers and ion torrent. In some embodiments only a portion of the genome is sequenced. In some embodiments, a chip or array relating to only a portion of the genome is used.

[0280] In some embodiment the method further uses methylation sensitive sequencing. In some embodiments, the method comprises bisulfite conversion before sequencing. In performing sequencing, the methylation status of the DNA can also be discerned, in this way the protein-DNA association data can also be combined with the DNA methylation data.

[0281] In one embodiment, the pre-determined genomic location is within, or associated with, a gene associated with cancer, such as an oncogene or a proto-oncogene. For example, the pre-determined genomic location may be within an oncogene or a proto-oncogene, or within an oncogene or proto-oncogene regulatory region (e.g. an oncogene or protooncogene promoter region).

[0282] In another aspect of the invention there is provided a computer program product for use in identifying CTCF-binding sites which can be used to differentiate between a disease state and a healthy state comprising using genome occupancy in a disease and a healthy state.

[0283] SAMPLE PREPARATION

[0284] In one embodiment, the sample is a body fluid (which is used interchangeably with the term “biological fluid” herein). Any body fluid sample type may be used for the invention including without limitation blood, plasma, serum, menstrual blood, endometrial fluid, faeces, urine, saliva, mucous, semen and breath, e.g. as condensed breath, or an extract or purification therefrom, or dilution thereof. Biological samples also include specimens from a live subject or taken post-mortem. The samples can be prepared, for example where appropriate diluted or concentrated, and stored in the usual manner. In a preferred embodiment, the biological fluid sample is selected from: blood, serum or plasma. It will be clear to those skilled in the art that the detection of chromatin fragments in a body fluid has the advantage of being a minimally invasive method that does not require biopsy. In one embodiment, the samples are processed within 1 , 2, 6, 24 or 48 hours of collection. In one embodiment, the sample are stored at or between 4°C or room temperature prior to processing. In one embodiment, physical agitation of the samples is minimised prior to processing. Ideally, the samples are processed as quickly as possible and / or the samples are stored in a fridge before being analysed.

[0285] The preferred sample type for analysis of cfDNA, ctDNA or nucleosomes is EDTA plasma. The function of EDTA or citrate in a plasma blood collection tube is to chelate and sequester calcium ions in the blood to prevent clotting (the clotting cascade in blood requires the presence of calcium ions). Centrifugation of the tube separates the cellular component of the blood from the plasma supernatant, which can be removed and used as a sample matrix for many clinical diagnostic purposes.

[0286] Where blood samples are used for methods of the invention this may be whole blood, a serum sample or a plasma sample. Whole blood or serum samples may be used as substrates for the analysis of any (stably-bound) transcription factor-DNA chromatin fragment involving a transcription factor with any DNA binding domain (DBD) type.

[0287] Plasma samples, such as EDTA plasma samples may also be used in methods of the invention. In a typical plasma sample collection method, the whole blood is collected into a citrate or EDTA blood collection tube and centrifuged within 2 hours. The resulting supernatant plasma may be used fresh or may be frozen until analysed. However, calcium ion sequestrators used as additives to blood collection tubes to produce plasma, cause disassociation of circulating zinc finger transcription factor-DNA complexes. Zinc finger transcription factors are the most common class of transcription factors that includes CTCF.

[0288] There are a number of ways to overcome this difficulty including, without limitation: (i) to use serum samples, (ii) to use heparin plasma or other plasma sample types not involving calcium sequestration or (iii) to prevent the disassociation of transcription factor-DNA complexes, for example by cross-linking the proteins and / or DNA in the chromatin fragment in a blood sample.

[0289] In one embodiment, the body fluid sample is a serum sample. Serum is thought to contain contaminating chromatin material derived from white blood cells (e.g. neutrophil extracellular traps (NETs)). This contamination interferes in the analysis of cfDNA and therefore plasma is the sample matrix most commonly used for ctDNA methods. However, the isolation of a chromatin fragment containing CTCF from other chromatin material present prior to DNA analysis removes such interference. Moreover, the contamination of serum with chromatin material is a result of the formation of NETs by neutrophil cells in the blood sample triggered by coagulation (a known inducer of NETosis). Provided the serum sample collection tube containing whole blood is processed in a timely manner, for example 15-60 minutes following venipuncture, the contaminating NETs material will be large chromatin rather than small chromatin fragments and will not interfere in the analysis of small transcription factor-DNA complexes. Therefore, widening the sample type that may be used is a further advantage of the methods of the invention.

[0290] The presence of contaminating NETs in serum may be further minimized or eliminated by the addition of an inhibitor of NETosis to the serum blood collection tube. This prevents NETosis and hence minimizes the level of background chromatin present in a serum sample. Many inhibitors of NETosis are known in the art. Preferred inhibitors include the anthracycline class of drugs, in particular doxorubicin. Therefore, in one embodiment, the method comprises contacting the sample with an inhibitor of NETosis.

[0291] The binding of zinc finger transcription factors to their DNA TFBS is dependent on the presence of zinc ions. However, the calcium chelators used in plasma blood collection tubes also chelate zinc ions. Chelation and removal of zinc ions from a zinc finger transcription factor results in a loss of transcription factor binding to DNA (Ralston, 2008). The interaction of zinc chelating agents and zinc finger transcription factors means this family of transcription factors behave differently in EDTA plasma than transcription factors that bind DNA through other DBD types.

[0292] In one embodiment, the body fluid sample is any plasma sample including a plasma sample produced using a calcium sequestrator such as EDTA plasma or citrate plasma wherein the plasma sample is obtained by contacting a whole blood sample with a crosslinking agent. The cross-linking agent may be contacted with whole blood in a first step of a process involving: (1) contacting a whole blood sample with a cross-linking agent; (2) contacting the cross-linked sample with a calcium ion chelating agent; and (3) isolating plasma from the sample.

[0293] Cross-linking is a well-known technique in the art. The most commonly used cross-linking reagent is formaldehyde which binds protein molecules to each other and to DNA. However, excess cross-linking may lead to changes in the structure of antibody binding epitopes in transcription factors (and hence to loss of antibody binding) and even the cross-linking of transcription factors to separate protein molecules or complexes. To prevent this, cross-linking is often quenched shortly after adding formaldehyde, for example by addition of excess glycine or tris(hydroxymethyl)aminomethane (TRIS), to stop further cross-linking. Cross-linking may be used with any sample, such as serum as well as plasma.

[0294] Therefore, in one embodiment, the method comprises contacting the sample with a crosslinking agent and optionally adding a quenching agent to stop further cross-linking. The method may additionally comprise contacting the sample with a calcium ion chelating agent.

[0295] In a preferred embodiment, formaldehyde, paraformaldehyde or a formaldehyde releasing agent is used as a cross-linking agent. In one embodiment, EDTA is used as a chelator of calcium ions to prevent coagulation. In preferred embodiments the formaldehyde is added to whole blood immediately following the collection of the whole blood sample, for example by adding the whole blood sample to a tube already containing formaldehyde. The tube is left for sufficient time for the cross-linking reaction to proceed and then the reaction is stopped by the addition of a quencher to prevent excess crosslinking of plasma components. The quencher is typically an amine compound such as glycine or TRIS that reacts with formaldehyde. The quencher may be added with the EDTA, for example by addition of a solution of glycine and EDTA in TRIS buffer. The whole blood sample is then centrifuged and the plasma containing cross-linked transcription factor bound DNA complexes, is isolated for analysis by methods of the invention.

[0296] As discussed above, the transcription factors present in the circulation are most likely those that are stably bound to DNA rather than those which associate transiently with DNA and disassociate in a dynamic manner. For most stably bound DNA circulating chromatin fragments including transcription factors, cross-linking with formaldehyde in whole cultured cells or tissue samples is rapid and takes place in approximately 5 seconds (Poorey et al; 2013). We reasoned that, whilst 5 seconds may be required for diffusion and entry of formaldehyde into a cell, followed by entry into the nucleus, followed by cross-linking of chromatin, this time may be reduced in a whole blood context where the chromatin fragments are free in solution and immediately accessible to cross-linking. The cross-linking reagent used may be formaldehyde or be a formaldehyde releasing agent (also called a formaldehyde releaser, formaldehyde donor or formaldehyde releasing preservative). A formaldehyde releasing agent is a moiety that slowly releases formaldehyde or a moiety that releases small amounts of formaldehyde into solution to maintain a low formaldehyde concentration. Many formaldehyde releasing agents are known in the art and are commonly used as antimicrobial preservatives in the cosmetics industry, for example in skin care and hair care products where high levels of formaldehyde are avoided due to toxicity but low protective levels are maintained by release. Therefore, in one embodiment the cross-linking agent is a formaldehyde releasing agent.

[0297] In one embodiment of the invention, the cross-linking reagent may be added simultaneously with the calcium ion chelator. Blood collection tubes (BCT) containing both EDTA and a formaldehyde releasing agent are available commercially, for example the Cell-Free DNA BCT available from STRECK Inc. Whole blood added to such tubes is exposed simultaneously to EDTA and a cross-linking agent.

[0298] It will be appreciated that cross-linking may be reversed for subsequent analysis of DNA. Formaldehyde cross-linking is conventionally reversed by heating.

[0299] REMOVAL OF CELL FREE NUCLEOSOMES

[0300] The sample preparation may optionally also involve a pre-purification step to remove most of the nucleosomes and nucleosome bound DNA from the sample prior to analysis. This reduces background signal, improves the efficiency of isolation of the transcription factor bound DNA fragments of interest and may improve the analytical and clinical sensitivity of the methods of the invention. Therefore, in one embodiment, the method additionally comprises removing cell free nucleosomes from the body fluid sample. The chromatin fragments comprising a nucleosome may be removed from the sample (optionally to be analysed separately) prior to the employment of the methods of the invention described herein. The purpose of this preparative step is to remove the bulk of the DNA fragments from the sample to lower any background signal they may create in the analysis. This may be done for example, without limitation, by contacting the sample with a binding agent which binds to nucleosomes, such as a solid phase anti-nucleosome binder including, for example an antibody or a nucleosome binding protein such as the proteins described in WO2021038010. The antibody may selectively bind to a histone protein, for example a core histone protein such as H2A, H2B, H3 or H4, or a linker histone protein such as H1. In preferred embodiments, the nucleosome binding agent is directed to bind to a nucleosome core epitope. The core histones H2A, H2B, H3 and H4 all feature core domains as well as histone tails of approximately 20-30 amino acids in length. In one embodiment, the antibody is directed to bind to histone H3 at an epitope located between amino acids 1-21. In one embodiment, antibodies are directed to histone H3 epitopes, such as H3.1 epitopes, located at amino acids 30-33.

[0301] All or most of nucleosomal material present in the sample may be adsorbed (e.g. onto the solid phase) and hence removed from the sample. Therefore, in one embodiment, the method comprises contacting the body fluid sample with a binding agent which binds to nucleosomes or a component thereof, and removing the sample bound to the binding agent prior to contacting the sample with a transcription factor binding agent.

[0302] In a preferred embodiment, magnetic or paramagnetic beads are used as a solid phase support, but any suitable material may be used such as agarose beads. Magnetic beads may be purchased from Dynabead. In some embodiments the support is aa protein A / G bead. The reagent may be conjugated to the physical support before contacting. In some embodiments, the conjugating is a covalent linkage such as by epoxy chemistry. Similarly, any of the methods for nucleosome binding methods described by in W02016067029, WO2017068371 , W02021038010 and WO2022144408 as a method of removing nucleosomes may be used. Therefore, in one embodiment, the sample used in methods of the invention does not comprise nucleosomes. In a further embodiment, the cell free chromatin fragment detected by the methods of the present invention consists of a transcription factor and a DNA fragment.

[0303] IMMUNOPRECIPITATION OF TRANSCRIPTION FACTOR-DNA COMPLEXES

[0304] Physical separation of tumour derived and non-tumour derived cfDNA nucleoprotein complexes may in principle obviate the need for in silico separation and hence obviate the need for DNA library preparation, next generation sequencing and bioinformatic analysis of cfDNA sequence data. Physical separation may therefore enable lower cost, simpler, faster ctDNA analysis methods and automation. We now report the physical isolation of CCCTC-binding factor-DNA (CTCF-DNA) nucleoprotein complexes from plasma by immunoprecipitation (IP).

[0305] Thus, the present invention involves a chromatin immunoprecipitation sequencing (ChlP- Seq) method involving the direct isolation and analysis of DNA associated with CTCF sequences. Immunoprecipitation is in principle a simple process. In a typical method, an antibody directed to bind specifically to a protein of interest is coated to a solid support and exposed to a biological sample containing the protein. The protein of interest is bound by the antibody and hence adsorbed to the surface of the solid phase, whilst other proteins and other substances remain in solution. The solid phase is isolated from the sample and washed leaving a pure sample of the protein of interest attached to the solid support.

[0306] Cell and tissue-based ChlP-Seq methods are well described in the art. It is usual to use 20-30 pg of digested or sonicated chromatin extracted from tissue or cultured cells as the substrate material. As chromatin consists of approximately 40% DNA this represents some 8-24 pg of substrate DNA. However, the concentration of circulating cfDNA is low and has been measured at 30±14 ng / ml in healthy human subjects and 71±55 ng / ml in gastric cancer patients. Therefore, a 1 ml plasma sample will yield approximately 200- 500 fold less chromatin material than normally used in ChlP-Seq.

[0307] As the majority of circulating cell free chromatin is comprised of nucleosomes, the available circulating cell free transcription factor-DNA chromatin fragment material is extremely small. Moreover, the available circulating cell free transcription factor-DNA chromatin fragment material will comprise thousands of transcription factors. Therefore, the available substrate material for analysis by methods of the invention represented by a single transcription factor will be a small fraction of the small amount of circulating cell free transcription factor-DNA material present in the circulation.

[0308] In addition, chromatin extracts from cells are relatively pure chromatin material. In contrast body fluids, such as blood, serum or plasma contain a small amount of chromatin but higher concentrations of a huge number of proteins and other compounds any of which may interfere in methods of the invention by adhering non-specifically to the solid phase transcription factor antibody or other binding agent used. An extra complexity for the immunoprecipitation of circulating transcription factor DNA complexes from blood, serum or plasma is that the background non-specific binding is therefore high in relation to the small amount of target transcription factor bound to a specific binder on a solid phase support and may obscure its detection. Despite these challenges we have demonstrated that the method can be successfully achieved with effective separation of pure CTCF-DNA complexes from nucleosomes comprising DNA fragments of the same sequence. In particular, one aspect of the invention involves subtracting background effects (or the control) from the results of binding with the candidate agent (or true binding agent). Thus, in one embodiment, a background level of non-specific cfDNA isolated during the binding step is determined and subtracted from the presence, absence or quantity of DNA sequences determined in methods of the invention. Background subtraction is a common aspect of many analytical methods and well known in the art.

[0309] Accordingly, in one aspect of the present invention there is provided a PCR method for detecting the presence of CTCF occupied cfDNA fragments originating from a predetermined genomic location, in an isolated CTCF-cfDNA preparation produced from a body fluid sample obtained from a subject, which comprises the steps of:

[0310] (i) contacting a first sample of the body fluid with a candidate binding agent which binds to cfDNA-CTCF nucleoproteins, and contacting a second sample of the body fluid with a control binding agent which does not specifically bind to cfDNA-CTCF nucleoproteins;

[0311] (ii) isolating cfDNA from the first sample bound by the candidate binding agent and from the second sample bound by the control binding agent in step (i);

[0312] (iii) contacting isolated cfDNA from the first sample and the second sample with one or more pairs of PCR primers directed to a DNA sequence of equal to or less than 80 base pairs that includes a CTCF binding site located at the pre-determined genomic location;

[0313] (iv) determining the presence, absence or quantity of DNA sequences of equal to or less than 100 base pairs (e.g. equal to or less than 80 base pairs) in length produced in step (iii) by subtracting the quantity of DNA in the second sample from the first sample; and

[0314] (v) using the presence, absence or quantity of DNA determined in step (iv), as an indicator of whether, or how many, CTCF-occupied cfDNA fragments originating from the pre-determined genomic location were present in the body fluid sample.

[0315] Methods of the invention have also been developed to address the previous difficulties associated with (ChlP-Seq) methods.

[0316] In one embodiment, the antibody bound transcription factor-DNA complex may be washed with a strong (e.g. a concentration of at least 1 %, for example 1.2%) detergent or mix of detergents prior to extraction of the transcription factor associated DNA. In one embodiment, the transcription factor bound by the binding agent in step (i) is washed with a buffer solution containing at least 1 % concentration of detergent, prior to detection of the associated DNA fragment. There are a very large number of detergents that may be used for this purpose. A few common examples include, without limitation, Triton detergents (e.g. Triton X-100), Tween detergents (e.g. Tween 20 and Tween 80), sodium deoxycholate, sodium dodecyl sulfate, octylphenoxypolyethoxyethanol (IGEPAL CA- 630), tricosaethylene glycol dodecyl ether (Brij), n-dodecyl-beta-maltososide, octyl-beta- glucoside, octylthio glucoside, 3-((3-cholamidopropyl) dimethylammonio)-1- propanesulfonate (CHAPS) and many more.

[0317] We used magnetic polystyrene microbeads and washed repeatedly (5 washes) with a wash solution containing a mixture of 1% octylphenoxypolyethoxyethanol, 0.1 % sodium deoxycholate and 0.1% sodium dodecyl sulfate.

[0318] In a preferred embodiment of the invention the solid-phase support is a polystyrene particle, for example a magnetic polystyrene particle. The antibody (or other binder of a transcription factor) used may be directly or indirectly attached to the support.

[0319] In a preferred embodiment of the invention the solid phase bound transcription factor- DNA complex isolated on the solid-phase support is washed with a solution containing at least 0.25%, or at least 0.5% or at least 1% of detergent or surfactant. The detergent used may consist of a single detergent or of a mixture of detergents as described herein. The efficacy of these methods is demonstrated by the purity of CTCF cfDNA fragments illustrated in Figure 6(e).

[0320] DNA SEPARATION

[0321] The method comprises isolating the transcription factor from the remaining body fluid sample, prior to detection of the associated DNA fragment. For example, a wash buffer may be applied to the CTCF in the sample bound to the (solid phase) binding agent to remove the remaining sample which is not bound to the binding agent.

[0322] In one embodiment the method comprises extracting the DNA fragment associated with CTCF. In a further embodiment, the method comprises amplification of the extracted DNA fragment. In another embodiment, the method does not involve extracting the DNA fragment associated with the CTCF before analysis by PCR. In other words, after immunoprecipitation, the DNA fragment does not need to be eluted from the solid phase (usually beads) and this solid phase can be directly used in the PCR (e.g. where the proteins binding cfDNA are denatured). PCR

[0323] Conventional prior art approaches to analysing ctDNA take the approach of extraction of cfDNA from a patient sample, amplification of (all) the extracted cfDNA, library preparation and NGS DNA sequencing. The present invention takes a different approach. Rather than using complex bioinformatic analysis methods to differentiate tumour and non-tumour derived cfDNA, the present involved uses a wet chemical detection method, namely PCR. Thus, in the present invention, PCR is not used as an intermediate step simply for amplification of (all) cfDNA prior to DNA sequencing by for example NGS (targeted or whole genome) or methylated DNA sequencing analysis, rather PCR is used as the analysis tool per se to identify the presence or absence of pre-determined ctDNA fragments in a sample. As a result, the present invention allows for low cost, simple, rapid ctDNA analysis that has the potential to be easily accessed by hospitals and in other health care settings.

[0324] The present invention can be distinguished from known methods which employ amplification of (all) extracted cfDNA to prepare a library for subsequent sequencing e.g. by NGS. In such known methods every cfDNA sequence is sought to be amplified. The general strategy involves creating a library of DNA molecules, often large in number and diverse, with common flanking sequences containing binding sites for universal sequencing primers. The present invention involves the selection and isolation of a particular sequence and amplification of only that selected sequence. The present invention involves the use of primers directed to selected sequences, rather than universal sequences. Once the selected sequence has been identified, the present invention does not involve amplification followed by library preparation, nor does the method involve ligating sequencing adapters to the selected DNA as would be the case if PCR was being used to generate sequence-ready libraries for a large number of DNA sequences for further investigation (e.g. by sequencing). In the present invention the PCR is directed to a single sequence to identify whether or not it is present and can be seen as the final step in the process.

[0325] There are many polymerase chain reaction (PCR) methods known in the art including, without limitation, quantitative PCR, real time PCR, reverse transcriptase PCR, nested PCR, digital PCR, multiplex PCR, arbitrary primed PCR, cold PCR (co-amplification at lower denaturation temperature-PCR). In some embodiments the method includes DNA quantification. In some embodiments, the DNA fragment associated with CTCF is detected and / or measured by quantitative PCR. In the context of the present invention, the means for detecting the level of DNA fragments associated with CTCF, are preferably oligonucleotides for PCR, and more particularly oligonucleotides for chain amplification by quantitative polymerization in real time (RT- qPCR). Such amplification by RT-qPCR can be carried out by several different techniques, including the SYBR Green technique and the techniques using fluorescent probes (e.g. “TaqMan”, “FRET”, “Beacon”, etc.). The term “probe” refers to any molecule that is capable of selectively binding to a specifically intended target biomolecule, for example, a nucleotide transcript. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labelled, e.g. fluorescently labelled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0326] In the case of detection based on the use of fluorescent probes, the method may comprise a sense primer, an antisense primer, and a fluorescent probe (“TaqMan probe”, “FRET probe”, “Beacon Probe”, etc.). Suitable oligonucleotides can readily be selected and constructed by those skilled in the art depending on the technique chosen for performing RT-qPCR. Alternatively, suitable oligonucleotides and I or microfluidic plates containing appropriate oligonucleotides can be ordered from suppliers such as Applied Biosystem (Carlsbad, California, USA). By “microfluidic plate” is meant any plate suitable for use in a high-throughput platform to perform chemical and I or biological reactions such as RT-qPCR.

[0327] Analytical sensitivity is important for circulating cell free chromatin fragments containing transcription factors that occur at low levels, near to, or below, the limits of detection by immunoassay. The analytical limit of detection of immunoassays varies with the design of the assay and with the affinity of the binder used (usually an antibody) but may be in the picomolar concentration range. However, the analytical sensitivity of the PCR detection of DNA is orders of magnitude lower. PCR may detect concentrations as low as a few individual molecules per sample. Therefore, use of a method for the detection of the DNA associated with a transcription factor, rather than by use of an antibody directed to bind to DNA (or to a nucleosome epitope) allows the detection of circulating chromatin fragments containing a transcription factor at extremely low levels.

[0328] As well as increased sensitivity through use of PCR for detection, analysis of chromatin fragments containing transcription factors based on their associated DNA content also leads to high analytical specificity for CTCF occupation of each particular (predetermined) sequence location in the genome.

[0329] According to some embodiments, detecting or measuring the DNA fragment is performed by amplification of the DNA fragment, for example using a quantitative method to determine the presence and / or amount of DNA fragment.

[0330] Using PCR, specific sequences within a DNA or cDNA template can be copied, or “amplified”, many thousand- to a million-fold using sequence specific oligonucleotides, heat stable DNA polymerase, and thermal cycling. In traditional (end-point) PCR, detection and quantification of the amplified sequence are performed at the end of the reaction after the last PCR cycle and involve post-PCR analysis such as gel electrophoresis and image analysis. In real-time quantitative PCR, the PCR product is measured at each cycle. By monitoring reactions during the exponential amplification phase of the reaction, users can determine the initial quantity of a target with great precision. PCR theoretically amplifies DNA exponentially, doubling the number of target molecules with each amplification cycle. To address the need for robust quantification, the technique of RT-qPCR was developed and end-point PCR is used mostly to amplify specific DNA for sequencing, cloning, and use in other molecular biology techniques. In real-time PCR, the amount of DNA is measured after each cycle via fluorescent dyes that yield increasing fluorescent signal in direct proportion to the number of PCR product molecules (amplicons) generated.

[0331] Fluorescent reporters used in real time PCR include double-stranded DNA (dsDNA)- binding dyes, or dye molecules attached to PCR primers or probes that hybridize with PCR product during amplification. The change in fluorescence over the course of the reaction is measured by an instrument that combines thermal cycling with fluorescent dye scanning capability. By plotting fluorescence against the cycle number, the real-time PCR instrument generates an amplification plot that represents the accumulation of product over the duration of the entire PCR reaction. The advantages of real-time PCR include: the ability to monitor the progress of the PCR reaction as it occurs in real time; the ability to precisely measure the amount of amplicon at each cycle, which allows highly accurate quantification of the amount of starting material in samples; an increased dynamic range of detection; and amplification and detection occurs in a single tube, eliminating post- PCR manipulations. The present invention can employ the concept of relative fluorescence vs. cycle number. In this technique, amplification plots are created when the fluorescent signal from each sample is plotted against cycle number; therefore, amplification plots represent the accumulation of product over the duration of the real-time PCR experiment. The samples used to create the plots are a dilution series of the target DNA sequence.

[0332] In more detail, there are three major steps that make up each cycle in a real-time PCR reaction. Reactions are generally run for 40 cycles.

[0333] 1. Denaturation: High temperature incubation is used to “melt” double-stranded DNA into single strands and loosen secondary structure in single-stranded DNA. The highest temperature that the DNA polymerase can withstand is typically used (usually 95°C). The denaturation time can be increased if template GC content is high.

[0334] 2. Annealing: During annealing, complementary sequences have an opportunity to hybridize, so an appropriate temperature is used that is based on the calculated melting temperature (Tm) of the primers (5°C below the Tm of the primer).

[0335] 3. Extension: At 70-72°C, the activity of the DNA polymerase is optimal, and primer extension occurs at rates of up to 100 bases per second. When an amplicon in real time PCR is small, this step is often combined with the annealing step using 60°C as the temperature.

[0336] The present invention may advantageously make use of automation. In this respect different models of real time PCR instruments are available. Each model must have an excitation source, which excites the fluorescent dyes, and a detector to detect the fluorescent emissions. In addition, each model must have a thermal cycler. The thermal block may be either fixed, as in the StepOnePlus ® system or user interchangeable, as in the ViiA™ 7 system and QuantStudio™ 12K Flex system. Blocks are available to accept a variety of PCR reaction vessels: 48-well plates, 96-well plates, 384-well plates, 384-microwell cards, 3072-through-hole plates, etc. All real time PCR instruments also come with software for data collection and analysis.

[0337] In one embodiment, detecting or measuring the DNA fragment is performed by digital PCR. Digital PCR employs the same primer sets, fluorescent labels, and enzymatic reagents as traditional qPCR; both TaqMan Assay and SYBR Green dye-based approaches are compatible with digital PCR. The big difference is that in digital PCR, reactions are partitioned into tens to thousands of picoliter-scale compartments for amplification. QuantStudio™ Digital PCR Plates, with precisely-sized reaction compartments and numerous configuration options, are ideal for this application. The goal of digital PCR is for each mini-reaction to contain zero or one target molecule; experiments typically include many replicates of several dilutions to accomplish this. In a digital PCR experiment, reactions are thermal cycled and data can be collected either in real time (as in traditional qPCR) or as an endpoint read. Some systems for digital PCR do not have the ability to read results during the thermal cycling process and only endpoint data can be collected. For applications that require maximum sensitivity, reviewing amplification plots helps differentiate true positive results from false positives and can provide information on the number of target copies present in each reaction. After thermal cycling and data collection, the ratio of positive to negative mini-reactions can be used to calculate the absolute number of target molecules in the original sample.

[0338] PCR primer design

[0339] The invention finds particular use in analysing small DNA fragments bound by transcription factors, usually in the size range of 30-100 base pairs, 35-100 base pairs, 30-80 base pairs, or 35-80 base pairs (bp). Therefore, in one embodiment the cfDNA that is detected is a small DNA fragment, such as DNA fragments comprising less than about 100bp, such as less than about 80bp, in particular less than about 60bp. In a further embodiment, the DNA fragment comprises equal to or less than about 80bp.

[0340] In one embodiment, the cfDNA that is detected has a length of 30 bp or more, 35 bp or more, or 40 bp or more. In one embodiment, the cfDNA that is detected has a length of 100 bp or less, 95 bp or less, 90 bp or less, 85 bp or less, 80 bp or less, 75 bp or less, 70 bp or less, 65 bp or less, or 60 bp or less. In one embodiment, the cfDNA that is detected has a length of from 30 bp to 100 bp, from 30 bp to 95 bp, from 30 bp to 90 bp, from 30 bp to 85 bp, from 30 bp to 80 bp, from 30 bp to 75 bp, or from 30 bp to 70 bp. In one embodiment, the cfDNA that is detected has a length of from 35 bp to 100 bp, from 35 bp to 95 bp, from 35 bp to 90 bp, from 35 bp to 85 bp, from 35 bp to 80 bp, from 35 bp to 75 bp, or from 35 bp to 70 bp.

[0341] Methods of the invention utilise one or more pairs of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs or equal to or less than 80 base pairs that comprises a CTCF binding site located at the pre-determined genomic location. “Primers” as used herein refer to oligonucleotides that can be used in the methods described herein. In a typical PCR, at least one set / pair of primers, one forward primer and one reverse primer (i.e. a “pair” of PCR primers), are needed to amplify a target polynucleotide sequence. Conventionally, when a target DNA sequence consisting of a (+) strand and a (-) strand is amplified, a forward primer is an oligonucleotide that can hybridize to the 3' end of the (-) strand under the reaction condition and can therefore initiate the polymerization of a new (+) strand; whereas a reverse primer is an oligonucleotide that can hybridize to the 3' end of the (+) strand under the reaction condition and can therefore initiate the polymerization of a new (-) strand. As an example, a forward primer may have the same sequence as the 5' end of the (+) strand, and a reverse primer may have the same sequence as the 5' end of the (-) strand.

[0342] PCR primers may be designed in accordance with methods well known in the art. Well known PCR primer design programs are available and include: Primer-BLAST, Primer3, Primer3Plus, PrimerQuest, OligoPerfect, PeriPrimer, OLIGO, GenScript Real-time PCT (TaqMan) Primer Design, GenScript Online PCR Primers Designs Tool, AutoPrimer, RexPrimer, BatchPrimer3, Eurofins Genomics’ Primer Design Tools.

[0343] In one embodiment, primers for use in the invention may be approximately 30 to 70 nucleotides in length to be compatible for the detection of short CTCF associated 30-100 bp, 35-1 OObp or 35-80bp ctDNA fragments. In other embodiments, the primers are from 15 to 40 nucleotides in length, 15 to 35 nucleotides in length, from 15 to 30 nucleotides in length, or from 15 to 25 nucleotides in length. The length of ctDNA fragments comprising the sequence of any particular gene locus will not be constant, but will also vary in length with slightly different degrees of digestion so the primer design should include core sequences common to all or most CTCF associated ctDNA fragments. The optimum length of primers vary for different target binding site loci.

[0344] The primers may amplify a target polynucleotide sequence that is from 30 to 100 nucleotides in length, 35 to 100 nucleotides in length, 30 to 80 nucleotides in length or 35 to 80 nucleotides in length. In some embodiments, the primers amplify a target polynucleotide sequence that is at least 30 nucleotides in length, at least 35 nucleotides in length, or at least 40 nucleotides in length. In some embodiments, the primers amplify a target polynucleotide sequence that is 100 or fewer nucleotides in length, 90 or fewer nucleotides in length, 80 or fewer nucleotides in length, 75 or fewer nucleotides in length, or 70 or fewer nucleotides in length. Thus according to the present invention the PCR primers are directed to amplify a DNA sequence that are equal to or less than 100 base pairs, or equal to or less than 80 base pairs.

[0345] The target polynucleotide sequence may comprise all or part of a CTCF binding site. In some embodiments, the target polynucleotide sequence comprises at least 5 nucleotides of a CTCF binding site. In some embodiments, the target polynucleotide sequence comprises CC(A / G / T)(C / G)N; GG(A / C / T)(A / G / T)(C / G); (C / G)(A / C / T)(A / G / T)CC;

[0346] N(C / G)(A / C / T)GG; or variants thereof having one or two nucleotide substitutions. For example, the target polynucleotide sequence may comprise (N)xCC(A / G / T)(C / G)N; GG(A / C / T)(A / G / T)(C / G)(N)x; (N)x(C / G)(A / C / T)(A / G / T)CC; N(C / G)(A / C / T)GG(N)x; or variants thereof having one or two nucleotide substitutions, wherein x is from 25 to 75, from 30 to 70, or from 35 to 65.

[0347] In some embodiments, the target polynucleotide sequence comprises at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, or at least 14 nucleotides of a CTCF binding site. In some embodiments, the target polynucleotide sequence comprises a CTCF binding site.

[0348] In other embodiments, the target polynucleotide sequence does not comprise any part of a CTCF binding site. For example, the target polynucleotide sequence may consist of a region of the human genome flanking a CTCF-binding site. In one embodiment, the region is 40 bp or less, 35 bp or less, 30 bp or less, 25 bp or less, 20 bp or less, 15 bp or less, 10 bp or less, 5 bp or less, 4 bp or less, 3 bp or less, 2 bp or less, or 1 bp upstream or downstream of the CTCF-binding site.

[0349] The primers useful in the present invention will be designed according to standard PCR guidelines. They should be specific for the target sequence and be free of internal secondary structure. Primers should avoid stretches of homopolymer sequences (e.g., poly (dG)) or repeating motifs, as these can hybridize inappropriately.

[0350] Primer pairs should have compatible melting temperatures (within 5°C) and contain approximately 50% GC content. Primers with a high GC content can form stable imperfect hybrids. Conversely, a high AT content depresses the Tm of perfectly matched hybrids. If possible, the 3’ end of the primer should be GC rich (GC clamp) to enhance annealing of the end that will be extended. Ideally, primer pair sequences should be designed to avoid complementarity and hybridization between primers (i.e. to avoid the formation of primer-dimers).

[0351] For qRT-PCR, primers are designed to anneal to exons on both sides of an intron (or span an exon / exon boundary of the mRNA) to allow differentiation between amplification of cDNA and potential contaminating genomic DNA by melting curve analysis. To confirm the specificity of primers, a BLAST search can be conducted against public databases to be sure that the primers only recognize the target of interest. In one embodiment, the pre-determined genomic regions are analysed using one or more of the primer pairs. Preferred primers designed using the Primer3 software are shown below in Table 4.

[0352] able 4: Exemplary primer sequences

[0353] PCR PRIMER PAIRS

[0354] In one aspect, the present invention provides a pair of PCR primers directed to a DNA sequence of equal to or less than 100 or equal to or less than 80 base pairs that comprises a CTCF binding site located at a pre-determined genomic location.

[0355] In some embodiments, the primers are from 15 to 40 nucleotides in length, from 15 to 35 nucleotides in length, from 15 to 30 nucleotides in length, or from 15 to 25 nucleotides in length. In some embodiments, one or both primers are fluorescently labelled. In some embodiments, neither primer comprises a barcoding sequence.

[0356] In preferred embodiments, the pre-determined genomic location is selectively occupied by CTCF in a disease state. In more preferred embodiments, the pre-determined genomic location is selectively occupied by CTCF in a cancer, for example a cancer selected from the group consisting of: lung cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, blood cancer (such as leukaemia or lymphoma) and skin cancer (such as melanoma). In one embodiment, the pre-determined genomic location is selectively occupied by CTCF in blood cancer. In one embodiment, the pre-determined genomic location is selectively occupied by CTCF in lung cancer. In one embodiment, the predetermined genomic location is selectively occupied by CTCF in colorectal cancer. In one embodiment, the pre-determined genomic location is selectively occupied by CTCF in breast cancer. In one embodiment, the pre-determined genomic location is selectively occupied by CTCF in prostate cancer.

[0357] In preferred embodiments, the pre-determined genomic location is present in CTCF- occupied cfDNA fragments isolated from subjects with a disease. In more preferred embodiments, the pre-determined genomic location is present in CTCF-occupied cfDNA fragments isolated from subjects with cancer, for example a cancer selected from the group consisting of: lung cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, blood cancer (such as leukaemia or lymphoma) and skin cancer (such as melanoma). In one embodiment, the pre-determined genomic location is present in CTCF-occupied cfDNA fragments isolated from subjects with blood cancer. In one embodiment, the pre-determined genomic location is present in CTCF-occupied cfDNA fragments isolated from subjects with lung cancer. In one embodiment, the predetermined genomic location is present in CTCF-occupied cfDNA fragments isolated from subjects with colorectal cancer. In one embodiment, the pre-determined genomic location is present in CTCF-occupied cfDNA fragments isolated from subjects with breast cancer. In one embodiment, the pre-determined genomic location is present in CTCF- occupied cfDNA fragments isolated from subjects with prostate cancer. In a further embodiment, the pre-determined genomic location is not present in CTCF-occupied cfDNA fragments isolated from healthy subjects.

[0358] In some embodiments, the pre-determined genomic region is selected from the following: chr1 :244603555-244603648; chr2: 10668571 -10668663; chr2:79219540-79219628; chr5: 141567670- 141567748; chr14: 56491464-56491546; chr14: 87896549-87896634; chr2: 178280431 -178280518; chr8: 74853546-74853641 ; chr12:100741271-100741352; chrY: 17430722-17430795; chr1 : 90579362-90579440; chr11 :34926223-34926312; chr1 : 78130901 -78130992; chr19: 13262535- 13262621 ; chr5: 141992498-141992587; chr22:36986359-36986452; chr14:21734605-21734701 ; chr1 :39609752-39609824; chr2:224369183-224369264; chr6:55980780-55980855; chr12:92570523-92570593; chr4:64713399-64713474; chr4:186150099-186150166; chr6:50792057-50792142; chr2:65573278-65573362; chr17:48336036-48336115; chr16:66275978-66276056; chr2:7427441 -7427517; chr8:5062480-5062559; chr15:73874049-73874135; and chrX:89757930-89758000 (in genome assembly GRCh38 / hg38). In a further embodiment, the pre-determined genomic regions is selected from the following: chr1 :244603555-244603648, chr14: 56491464-56491546, chr1 : 90579362-90579440, chr11 :34926223-34926312, chr1 :78130901 -78130992, chr5:141992498-141992587, chr22:36986359-36986452, chr14:21734605-21734701 , chr6:55980780-55980855, chr4:64713399-64713474, chr17:48336036-48336115, chr2:7427441 -7427517, chr15:73874049-73874135, chrX: 89757930-89758000, chr2: 10668571-10668663, chr2:79219540-79219628, chr5:141567670-141567748, chr14:87896549-87896634, chr8:74853546-74853641 , chr12:100741271-100741352, chr19: 13262535-13262621 , chr22:36986359-36986452, chr1 : 39609752-39609824, chr2:224369183-224369264, chr12:92570523-92570593, chr6:50792057-50792142, chr2:65573278-65573362 and chr15:73874049-73874135 (in genome assembly GRCh38 / hg38). In a yet further embodiment, the pre-determined genomic region is selected from the following region: chr1 :244603555-244603648, chr14: 56491464-56491546, chr1 : 90579362-90579440, chr11 :34926223-34926312, chr2: 10668571 -10668663, chr2:79219540-79219628, chr5: 141567670- 141567748, chr14: 87896549-87896634, chr8: 74853546-74853641 , and chr12: 100741271 -100741352 (in genome assembly GRCh38 / hg38).

[0359] In some embodiments, the pre-determined genomic region is selected from the following: chr2:79219519-79219648, chr2: 162301295- 162301441 , chr3: 194594813- 194594949, chr7:64625243-64625355, chr8:81641590-81641720, chr14:21734606-21734734, chr1 :58739989-58740101 , chr6: 149028960- 149029063, chr9:22452551 -22452664, chr12:4849148-4849254, chr15:92797259-92797393, chr20:5788487-5788587, chrX: 106481411-106481533, chr2: 149561424-149561553, chr3:61381935-61382023, chr5: 17190626-17190742, chr5:59390194-59390288, chr7:25160122-25160245, chr11 7619731 -77619846, chr17:48336001 -48336147, chr1 :206461901 -206462094, chr2:143110966-143111104, chr2: 162123970- 162124051 , chr5:16456356-16456495, chr10: 15902711-15902826, chr10: 17742984-17743122, chr11 :6379394-6379487, chr16: 17579398-17579529, chr17:5592340-5592644, chrX: 3035865-3036000, chr3:78659532-78659619, chr3: 105518948-105519094, chr4:64585094-64585216, chr5:83927264-83927382, chr6:593261 -593392, chr6:137314444-137314535, chr7: 107874746-107874889, chr10:27656752-27656912, chr11 :32960716-32960828, chr11 :43567527-43567641 , chr12:76791108-76791220, chr14:37230722-37230848, chr15:81939739-81939846, chr16:75789293-75789426, chr15:68554138-68554266, chr1 :78130884-78131005, chr6:50602320-50602449, chr1 : 119675667-119675836, chr1 :244603541 -244603674, chrX: 101415625- 101415728, chr2 : 178280411 -

[0360] 178280526, chr5: 96752586-96752688, chrX:22081630-22081732, chrX: 47384626- 47384769, chr19: 39474211-39474377, chr2:46464602-46464705, chr2:231715363- 231715468, chr7: 93228366-93228499, chr9:21111090-21111213, chr2 : 129849717- 129849817, chr10:34638577-34638699, chr19:42028327-42028524, chr2: 162298074- 162298189, chr8:24229478-24229598, chr8:29647427-29647523, chr10:63720138- 63720300, chr11 :34492959-34493085, chr12:27652288-27652426, chr2:174700116- 174700251 , chr3:183122469-183122581 , chr12:123905436-123905607, chr1 : 167540493-167540653, chr3: 189148131-189148251 , chr5: 141992479-

[0361] 141992603, chr9: 78821242-78821375, chr12: 10562452-10562563, chr2:210577647- 210577793, chr2:212567753-212567856, chr4:4183014-4183121 , chr14:56491434- 56491551 , chr15:48352569-48352712, chrX:51461172-51461282, chr2: 159118703- 159118816, chr6: 36095298-36095473, chr1 :221435546-221435673, chr11 :46537952- 46538113, chr1 : 10428186-10428296, chr2: 102840031 -102840180, chr7: 104848545- 104848654, chr9:4988029-4988170, chr11 :121194847-121194979, chr11 :123247327- 123247457, chr13:41353128-41353267, chr17:73369977-73370136, chr19:49983756- 49983882, chr20:63223466-63223579, chr11 :10077000-10077143, chr8:71975358- 71975486, chr12:6537982-6538105, chr12:64891000-64891169, chr1 :161487131- 161487247, chr4: 155442425-155442520, chr11 :44911428-44911580, chr12:31272008- 31272161 , chr16:85717990-85718123, chr19:49547082-49547241 , chr10:11819934- 11820065, chr6:85355152-85355270, chr6:160519981-160520091 , chr7: 136009627- 136009724, chr14:21014774-21014934, chr1 :53393434-53393577, chr2:38115355- 38115496, chr4:21789365-21789486, chr4:113394834-113394942, chr5:59673572- 59673710, chr6: 85323564-85323677, chr8: 54169437-54169546, chr8: 96180035- 96180344, chr10:28241944-28242032, chr11 :62380869-62381016, chr12:8396766- 8396964, chr1 : 160726939-160727057, chr19:54676361 -54676503, chr1 :225976081- 225976216, chr1 :236103745-236103862, chr4:16026149-16026413, chr4: 167021693- 167021794, chr5: 141960806-141960987, chr11 :48307888-48308019, chr16:50665497- 50665627, chr17:65198881 -65199032, chr20: 13119634-13119763, chr2: 10668563- 10668699, chr14:36374168-36374282, chr19:46402330-46402434, chr10:101161791- 101161998, chr20:6142524-6142649, chr3:71729971 -71730106, chr19:4204720- 4204856, chr1 :101288544-101288669, chr1 :27669254-27669382, chr13: 36420493- 36420628, chr17:28540839-28540954, chr20:2351923-2352109, chr1 : 158998804-

[0362] 158998962, chr1 :220810250-220810376, chr2: 170409173-170409293, chr7:78040335- 78040434, chr1 :31162784-31162912, chr1 : 17661594-17661807, chr1 :31422842- 31422978, chr1 : 192445984-192446109, chr6: 105424291 -105424437, chr19:42529383- 42529506, chr1 : 107782340-107782444, chr9:86414420-86414532, chr7: 132188991 - 132189067, chr2:197374043-197374122, chr2:60642442-60642560, chr18:24736101- 24736161 , chr9:6594697-6594768, chr11 :9675318-9675421 , chr1 :226944700-

[0363] 226944781 , chr1 :202640729-202640819, chr9:14216485-14216587, chr12:54188268- 54188338, chr9:121838761-121838825, chr1 :95264973-95265040, chr1 :69737726- 69737836, chr1 : 107782340-107782444, chr3: 32144255-32144365, chr3:113494238- 113494353, chr3: 134474064-134474175, chr3: 137105390-137105526, chr3: 181326780- 181326885, chr4:26082030-26082146, chr4: 103934283- 103934454, chr5:115586654-115586772, ch r6: 52049619-52049757, chr6:63764478-63764585, chr6: 158691470- 158691594, chr6: 162908032- 162908121 , chr7: 130832002-

[0364] 130832124, chr7: 132188991 -132189067, chr8:63262036-63262139, chr8:110830607- 110830727, chr8:117329067-117329210, chr8:119867220-119867390, ch r9: 86414420- 86414532, chr9:90142700-90142839, chr10:89563998-89564154, chr10:91389742- 91389869, chr12:92570492-92570605, chr12:97308762-97308849, chr14:28635549- 28635670, chr14:37149849-37149969, chr15:66567130-66567354, chr18:62958013- 62958123, chr18:62982806-62982908, chr20:52312263-52312398, chr1 :11241826- 11241943, chr1 :53754832-53754948, chr1 :89987314-89987463, chr2:7427425-

[0365] 7427538, chr2:60642442-60642560, chr2: 104849680-104849792, chr2:112582400- 112582523, chr2:160110087-160110199, chr2: 197374043-197374122, chr2:224369161-224369299, chr4:242369-242480, chr4:41281166-41281273, chr4: 122758089- 122758199, chr4: 166165962- 166166073, chr4: 169810464-

[0366] 169810607, chr5:32489802-32489952, chr5: 123371732-123371827, chr5: 172370813- 172370900, chr6:33123829-33123955, chr6:49849713-49849822, chr6:110942102- 110942167, chr6: 113909396-113909512, chr6: 159008718-159008830, chr10:25700952-25701067, chr10:92885025-92885143, chr10:94630132-94630263, ch r 11 :30346226-30346339, chr11 :110433891-110434020, chr11 : 124586369-

[0367] 124586468, chr12: 102702668-102702830, chr12: 111451647-111451929, chr13:33228694-33228791 , chr13:50085114-50085230, chr13:53250379-53250572, chr13:57627719-57627850, chr13: 100722041-100722178, chr14:20327405-20327535, chr14:24735042-24735188, chr15:31974953-31975090, chr18:24736101-24736161 , chr19:54674154-54674274, chr22:22201213-22201344, chrX:22136053-22136155, chrX: 129754802-129754877, chrX: 130079923-130080041 , chr1 : 19918128-19918255, chr1 :24587331 -24587473, chr1 :39720138-39720288, chr1 :65958784-65959095, chr1 : 74689941-74690070, chr1 :88877501 -88877633, chr1 :161227460-161227587, chr1 :202640729-202640819, chr1 :226944700-226944781 , chr2: 17646148-17646327, chr2:227862361 -227862483, chr2:235594168-235594292, chr2:238462941-

[0368] 238463069, chr3: 124079474-124079582, chr3: 156541160-156541317, chr3: 194404789- 194404904, chr3: 195522387- 195522501 , chr4: 16794985- 16795080, chr4:27048448-27048583, chr4:52026152-52026244, chr4:52376236-52376358, chr4: 140999943-141000112, chr4: 146104133-146104288, chr4: 158850959-

[0369] 158851065, chr4: 165464923-165465025, chr5:18314314-18314440, chr5:28014922- 28015045, chr5: 74648924-74649026, chr5: 102223966-102224084, chr5: 129664478- 129664599, chr5: 155139754-155139857, chr6:12893990-12894132, chr6: 30380603- 30380684, chr6: 50792043-50792155, chr6:51855414-51855525, chr6: 123415992- 123416085, chr7:10412547-10412686, chr7:25180362-25180528, chr7: 38374563- 38374659, chr7:91830142-91830274, chr7:94395571 -94395689, chr8:4833976-

[0370] 4834088, chr8:95758783-95758908, chr8:119070591-119070729, chr9:6354233- 6354348, chr9:6594697-6594768, chr9:14216485-14216587, chr9: 17063671 -17063809, chr9: 19028989-19029108, chr10:3192966-3193102, chr10: 16828259-16828398, chr10:25726026-25726156, chr10:87656598-87656695, chr10:87735375-87735496, chr10: 125098674-125098794, chr11 :9675318-9675421 , chr11 : 17230210-17230310, chr11 :32323655-32323771 , chr11 : 72667585-72667696, chr11 : 124402455-124402570, chr12:20946875-20946995, chr12:53791868-53791960, chr12:55619731-55619974, chr12:125403356-125403461 , chr12:126069664-126069777, chr13:85828223-

[0371] 85828338, chr14:45426816-45426941 , chr14:93655215-93655320, chr15:44288413- 44288574, chr15:90956673-90956821 , chr16:24253220-24253372, chr16:63361142- 63361267, chr16:66728261-66728409, chr17:35312798-35312956, chr17:78013974- 78014108, chr18:43571212-43571318, chr18:65781879-65782020, chr20: 36466920- 36467064, chr22:25093742-25093869, chrX:51614499-51614639, chrX: 110156310- 110156388, chr22_KI270733v1_random: 136322-136475, chr1 : 32272584-32272646, chr1 : 86742093-86742193, chr1 :88998835-88998937, chr1 :95264973-95265040, chr1 : 163410082-163410225, chr1 : 169055544-169055615, chr1 :236452124- 236452243, chr1 :240149603-240149760, chr1 :240189505-240189629, ch r2: 18378 ISO- 18378279, chr2:38581666-38581810, chr2: 152861493- 152861578, chr2:166539491- 166539609, chr2: 176814897-176815015, chr2: 182186287-182186395, chr2:227890621-227890778, chr3:87298250-87298349, chr3:89211716-89211800, chr3: 109732032-109732122, chr3: 134492629-134492738, chr3: 156509828- 156509965, chr3: 157137568-157137691 , chr3: 161273765-161273866, chr3: 166013225- 166013311 , chr4:23135919-23136011 , chr4:48087272-48087368, chr4:70183776-70183887, chr4:73019582-73019720, chr4:86044122-86044254, chr4: 174365159- 174365240, chr5:5141805-5141917, chr5:80083642-80083758, chr5:81299634-81299801 , chr5: 112175136-112175244, chr5: 146662279-146662392, chr5: 148733715-148733808, chr5: 154157507-154157598, chr6:35449508-35449669, chr6:52021110-52021230, chr6: 56902964-56903131 , chr6:70706662-70706775, chr6:74091490-74091627, chr6:112180232-112180339, chr6: 135451738-135451874, chr7: 148504024- 148504134, chr7: 156582102-156582204, chr8: 12866970- 12867090, chr8:69663836-69663985, chr8:75257410-75257540, chr8:80612676-80612794, chr8:90948403-90948519, chr8:132651571-132651698, chr9: 13427664-13427772, chr9: 15873723-15873819, chr9: 17462998-17463108, chr9: 121400273- 121400386, chr9: 121838761-121838825, chr10:61284412-61284550, chr10:93352770-93352892, chr10:116490211-116490321 , chr10:125982446-125982551 , chr11 :6738008-6738119, chr11 :84384764-84384893, chr11 :88092327-88092414, chr11 :94519623-94519738, chr11 :96464846-96464973, chr11 :115117490-115117553, chr11 : 128414446- 128414512, chr12:7642988-7643098, chr12:21680506-21680612, chr12:29519728- 29519832, chr12:34034045-34034152, chr12:54188268-54188338, chr12:58346176- 58346282, chr12 : 100741240- 100741368, chr12:119160250-119160324, chr13:97132337-97132465, chr14: 19878197-19878283 , chr14:25400748-25400891 , chr14:53055029-53055173, chr14:64303608-64303708 , chr14:82000025-82000111 , chr14:98138734-98138826, chr15:85505208-85505330 , chr15:98881743-98881866, chr16:16449419-16449513, chr17:700647-700751 , chr17: 16258067- 16258156, chr18:24489020-24489155, chr18:31819747-31819885, chr19:28325490-28325587, chr19:47125320-47125481 , chrX:11144386-11144529, chrX: 87299704-87299805, chrX: 133670537-133670648, chrX: 152758817-152758894, chr1 :2091761 -2091844, chr1: 5225260-5225331, chr1 : 110555394-110555522, chr1 : 158036123-158036245, chr1 : 176559961 -176560058, chr1 :207405060-207405185, chr1 :238188421-

[0372] 238188505, chr2:39059461 -39059589, chr2:60707803-60707914, chr2:62412204- 62412318, chr2: 106791905-106792003, chr2:151437690-151437807, chr2: 158010423- 158010526, chr2:164717151-164717267, chr2: 179440792-179440892, chr2: 181715178-181715317, chr2: 197707735-197707844, chr2:232270870- 232270944, chr2:241078520-241078659, chr3: 13600010-13600082, chr3:26712570- 26712658, chr3:65785206-65785301 , chr3: 98039424-98039547, chr3: 146065649- 146065763, chr3:147191901-147192041 , chr3: 173584256-173584361 , ch r4 : 38856812- 38856976, chr4:64713379-64713507, chr4:67746823-67746964, chr4: 147097166- 147097267, chr4:163484959-163485038, chr4: 185634052-185634304, chr5: 19469171- 19469243, chr5:36870575-36870732, chr5: 52657324-52657444, chr5:66307768- 66307915, chr5: 103335135-103335205, chr6:47631424-47631547, chr6:52945168- 52945233, chr6:75893429-75893588, chr6: 93664709-93664784, chr6:115244749- 115244826, chr6: 116287793-116287931 , chr6: 158605406-158605520, chr6: 170561934-170561998, chr7:26198200-26198319, chr7:50142454-50142556, chr7:81482732-81482834, chr7: 154504103-154504239, chr8:9831026-9831238, chr9: 8086869-8086940, chr10: 117940922- 117940998, chr11 : 34926239-34926356, chr11 :82682336-82682444, chr11 : 121765670-121765767, chr12: 12376782-12376859, chr12:20950374-20950501 , chr12:23949721-23949794, chr12:24095121 -24095194, chr12:29889866-29889994, chr12:72987449-72987521 , chr12:73391018-73391090, chr12:78146406-78146532, chr12:90959676-90959826, chr13:43908534-43908635, chr13:67238415-67238557, chr13:84896266-84896371 , chr13: 102141733-102141898, chr13: 110839093-110839188, chr14:78082948-78083033, chr14: 100624900-

[0373] 100625009, chr15:24585660-24585745, chr15:31880238-31880317, chr15:41031918- 41032105, chr15:69322673-69322785, chr15:80419036-80419163, chr15: 99704486- 99704631 , chr17:31216272-31216386, chr17:48728857-48728929, chr17:74612182- 74612276, chr17:79893762-79893854, chr18:25853208-25853305, chr18:64037936- 64038067, and chr19:54080140-54080207 (in genome assembly GRCh38 / hg38).

[0374] In some embodiments, the pre-determined genomic region is selected from the following: chr2: 79219519-79219648, chr2 : 162301295-162301441 , chr3: 194594813- 194594949, chr7:64625243-64625355, chr8:81641590-81641720, chr14:21734606-21734734, chr1 :58739989-58740101 , chr6:149028960-149029063, chr9:22452551 -22452664, chr12:4849148-4849254, chr15:92797259-92797393, chr20:5788487-5788587, chrX: 106481411-106481533, chr2:149561424-149561553, chr3:61381935-61382023, chr5: 17190626-17190742, chr5:59390194-59390288, chr7:25160122-25160245, chr1177619731 -77619846, chr17:48336001 -48336147, chr1 :206461901 -206462094, chr2: 143110966- 143111104, chr2 : 162123970- 162124051 , chr5: 16456356- 16456495, chr10:15902711-15902826, chr10:17742984-17743122, chr11 :6379394-6379487, chr16: 17579398-17579529, chr17:5592340-5592644, chrX: 3035865-3036000, chr3:78659532-78659619, chr3: 105518948-105519094, chr4:64585094-64585216, chr5:83927264-83927382, chr6:593261 -593392, chr6:137314444-137314535, chr7: 107874746-107874889, chr10:27656752-27656912, chr11:32960716-32960828, chr11 :43567527-43567641 , chr12:76791108-76791220, chr14:37230722-37230848, chr15:81939739-81939846, chr16:75789293-75789426, chr15:68554138-68554266, chr1 :78130884-78131005, chr6:50602320-50602449, chr1 : 119675667-119675836, chr1 :244603541 -244603674, chrX: 101415625- 101415728, chr2 : 178280411 -

[0375] 178280526, chr5: 96752586-96752688, chrX:22081630-22081732, chrX: 47384626- 47384769, chr19: 39474211-39474377, chr2:46464602-46464705, chr2:231715363- 231715468, chr7: 93228366-93228499, chr9:21111090-21111213, chr2 : 129849717- 129849817, chr10:34638577-34638699, chr19:42028327-42028524, chr2: 162298074- 162298189, chr8:24229478-24229598, chr8:29647427-29647523, chr10:63720138- 63720300, chr11 :34492959-34493085, chr12:27652288-27652426, chr2:174700116- 174700251, chr3:183122469-183122581, chr12:123905436-123905607, chr1 : 167540493-167540653, chr3: 189148131-189148251 , chr5: 141992479-

[0376] 141992603, chr9: 78821242-78821375, chr12:10562452-10562563, chr2:210577647- 210577793, chr2:212567753-212567856, chr4:4183014-4183121, chr14:56491434- 56491551, chr15:48352569-48352712, chrX:51461172-51461282, chr2: 159118703- 159118816, chr6: 36095298-36095473, chr1:221435546-221435673, chr11:46537952- 46538113, chr1: 10428186-10428296, chr2: 102840031 -102840180, chr7: 104848545- 104848654, chr9:4988029-4988170, chr11 :121194847-121194979, chr11 :123247327- 123247457, chr13:41353128-41353267, chr17:73369977-73370136, chr19:49983756- 49983882, chr20:63223466-63223579, chr11 : 10077000- 10077143, chr8:71975358- 71975486, chr12:6537982-6538105, chr12:64891000-64891169, chr1 :161487131-

[0377] 161487247, chr4: 155442425-155442520, chr11 :44911428-44911580, chr12:31272008- 31272161, chr16:85717990-85718123, chr19:49547082-49547241, chr10:11819934- 11820065, chr6:85355152-85355270, chr6:160519981-160520091, chr7: 136009627- 136009724, chr14:21014774-21014934, chr1 :53393434-53393577, chr2:38115355- 38115496, chr4:21789365-21789486, chr4:113394834-113394942, chr5:59673572- 59673710, chr6: 85323564-85323677, chr8: 54169437-54169546, chr8: 96180035- 96180344, chr10:28241944-28242032, chr11:62380869-62381016, chr12:8396766- 8396964, chr1 : 160726939-160727057, chr19:54676361 -54676503, chr1 :225976081- 225976216, chr1:236103745-236103862, chr4:16026149-16026413, ch r4: 167021693- 167021794, chr5: 141960806-141960987, chr11 :48307888-48308019, chr16:50665497- 50665627, chr17:65198881 -65199032, chr20: 13119634-13119763, chr2: 10668563- 10668699, chr14:36374168-36374282, chr19:46402330-46402434, chr10:101161791- 101161998, chr20:6142524-6142649, chr3:71729971 -71730106, chr19:4204720- 4204856, chr1:101288544-101288669, chr1:27669254-27669382, chr13: 36420493- 36420628, chr17:28540839-28540954, chr20:2351923-2352109, chr1 : 158998804- 158998962, chr1 :220810250-220810376, chr2: 170409173-170409293, chr7:78040335- 78040434, chr1 :31162784-31162912, chr1 : 17661594-17661807, chr1 :31422842- 31422978, chr1 : 192445984-192446109, chr6: 105424291 -105424437, chr19:42529383- 42529506, chr1 : 107782340-107782444, chr9:86414420-86414532, chr7: 132188991 - 132189067, chr2:197374043-197374122, chr2:60642442-60642560, chr18:24736101- 24736161 , chr9:6594697-6594768, chr11 :9675318-9675421 , chr1 :226944700-

[0378] 226944781 , chr1 :202640729-202640819, chr9: 14216485- 14216587, chr12:54188268- 54188338, chr9:121838761-121838825, and chr1 :95264973-95265040 (in genome assembly GRCh38 / hg38).

[0379] In another aspect, the present invention provides a pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a 30-100 bp or 30-80 bp region of the human genome comprising a CCCTC-binding factor (CTCF) binding site or part thereof or a region of the human genome flanking a CTCF- binding site.

[0380] In some embodiments, the primers are from 15 to 40 nucleotides in length, from 15 to 35 nucleotides in length, from 15 to 30 nucleotides in length, or from 15 to 25 nucleotides in length. In some embodiments, neither primer comprises a barcoding sequence.

[0381] In some embodiments, the target polynucleotide sequence consists of a region of the human genome that is at least 35 bp in length or at least 40 bp in length. In some embodiments, the target polynucleotide sequence consists of a region of the human genome that is 75 bp or less in length, or 70 bp or less in length. In some embodiments, the target polynucleotide sequence consists of a region of the human genome that is 35- 75 bp in length or 40-70 bp in length.

[0382] In some embodiments, the target polynucleotide sequence consists of a 30-100 bp region or a 30-80 bp region of the human genome comprising a CCCTC-binding factor (CTCF) binding site or part thereof. In some embodiments, the target polynucleotide sequence comprises at least 5 bp, at least 6 bp, at least 7 bp, at least 8 bp, at least 9 bp, at least 10 bp, at least 11 bp, at least 12 bp, at least 13 bp of a CTCF binding site, or a 14bp CTCF binding site.

[0383] In some embodiments, the target polynucleotide sequence comprises at least 5 bp of the CTCF binding site. In some embodiments, the target polynucleotide sequence comprises CC(A / G / T)(C / G)N; GG(A / C / T)(A / G / T)(C / G); (C / G)(A / C / T)(A / G / T)CC; N(C / G)(A / C / T)GG; or variants thereof having one or two nucleotide substitutions. For example, the target polynucleotide sequence may comprise (N)xCC(A / G / T)(C / G)N; GG(A / C / T)(A / G / T)(C / G)(N)x; (N)x(C / G)(A / C / T)(A / G / T)CC; N(C / G)(A / C / T)GG(N)x; or variants thereof having one or two nucleotide substitutions, wherein x is from 25 to 95, from 30 to 95, from 25 to 75, from 30 to 70, or from 35 to 65. (N)x may consist of a region of the human genome flanking the CTCF-binding site.

[0384] In some embodiments, the target polynucleotide sequence comprises at least 6 bp of the CTCF binding site. In some embodiments, the target polynucleotide sequence comprises CC(A / G / T)(C / G)NN; NGG(A / C / T)(A / G / T)(C / G); (C / G)(A / C / T)(A / G / T)CCN;

[0385] NN(C / G)(A / C / T)GG; or variants thereof having one or two nucleotide substitutions. For example, the target polynucleotide sequence may comprise (N)xCC(A / G / T)(C / G)NN; NGG(A / C / T)(A / G / T)(C / G)(N)x; (N)x(C / G)(A / C / T)(A / G / T)CCN; NN(C / G)(A / C / T)GG(N)x; or variants thereof having one or two nucleotide substitutions, wherein x is from 24 to 94, from 29 to 94, from 24 to 74, from 29 to 69, or from 34 to 64. (N)x may consist of a region of the human genome flanking the CTCF-binding site.

[0386] In some embodiments, the target polynucleotide sequence comprises at least 7 bp of the CTCF binding site. In some embodiments, the target polynucleotide sequence comprises CC(A / G / T)(C / G)NNG; (A / G)NGG(A / C / T)(A / G / T)(C / G); (C / G)(A / C / T)(A / G / T)CCN(C / T); CNN(C / G)(A / C / T)GG; or variants thereof having one or two nucleotide substitutions. For example, the target polynucleotide sequence may comprise (N)xCC(A / G / T)(C / G)NNG; (A / G)NGG(A / C / T)(A / G / T)(C / G)(N)x; (N)x(C / G)(A / C / T)(A / G / T)CCN(C / T);

[0387] CNN(C / G)(A / C / T)GG(N)x; or variants thereof having one or two nucleotide substitutions, wherein x is from 23 to 93, from 28 to 93, from 23 to 73, from 28 to 68, or from 33 to 63. (N)x may consist of a region of the human genome flanking the CTCF-binding site.

[0388] In some embodiments, the target polynucleotide sequence comprises at least 8 bp of the CTCF binding site. In some embodiments, the target polynucleotide sequence comprises CC(A / G / T)(C / G)NNG(A / G); G(A / G)NGG(A / C / T)(A / G / T)(C / G);

[0389] (C / G)(A / C / T)(A / G / T)CCN(C / T)C; (C / T)CNN(C / G)(A / C / T)GG; or variants thereof having one or two nucleotide substitutions. For example, the target polynucleotide sequence may comprise (N)xCC(A / G / T)(C / G)NNG(A / G); G(A / G)NGG(A / C / T)(A / G / T)(C / G)(N)x; (N)x(C / G)(A / C / T)(A / G / T)CCN(C / T)C; (C / T)CNN(C / G)(A / C / T)GG(N)x; or variants thereof having one or two nucleotide substitutions, wherein x is from 22 to 92, from 27 to 92, from 22 to 72, from 27 to 67, or from 32 to 62. (N)x may consist of a region of the human genome flanking the CTCF-binding site. In some embodiments, the target polynucleotide sequence comprises at least 9 bp of the CTCF binding site. In some embodiments, the target polynucleotide sequence comprises CC(A / G / T)(C / G)NNG(A / G)N; NG(A / G)NGG(A / C / T)(A / G / T)(C / G);

[0390] (C / G)(A / C / T)(A / G / T)CCN(C / T)CN; N(C / T)CNN(C / G)(A / C / T)GG; or variants thereof having one or two nucleotide substitutions. For example, the target polynucleotide sequence may comprise (N)xCC(A / G / T)(C / G)NNG(A / G)N;

[0391] NG(A / G)NGG(A / C / T)(A / G / T)(C / G)(N)x; (N)x(C / G)(A / C / T)(A / G / T)CCN(C / T)CN;

[0392] N(C / T)CNN(C / G)(A / C / T)GG(N)x; or variants thereof having one or two nucleotide substitutions, wherein x is from 21 to 91 , from 26 to 91 , from 21 to 71 , from 26 to 66, or from 31 to 61. (N)x may consist of a region of the human genome flanking the CTCF- binding site.

[0393] In some embodiments, the target polynucleotide sequence comprises at least 10 bp of the CTCF binding site. In some embodiments, the target polynucleotide sequence comprises CC(A / G / T)(C / G)NNG(A / G)NG; NNG(A / G)NGG(A / C / T)(A / G / T)(C / G); (C / G)(A / C / T)(A / G / T)CCN(C / T)CNN; CN(C / T)CNN(C / G)(A / C / T)GG; or variants thereof having one or two nucleotide substitutions. For example, the target polynucleotide sequence may comprise (N)xCC(A / G / T)(C / G)NNG(A / G)NG; NNG(A / G)NGG(A / C / T)(A / G / T)(C / G)(N)x; (N)x(C / G)(A / C / T)(A / G / T)CCN(C / T)CNN; CN(C / T)CNN(C / G)(A / C / T)GG(N)x; or variants thereof having one or two nucleotide substitutions, wherein x is from 20 to 90, from 25 to 90, from 20 to 70, from 25 to 65, or from 30 to 60. (N)x may consist of a region of the human genome flanking the CTCF- binding site.

[0394] In some embodiments, the target polynucleotide sequence comprises at least 11 bp of the CTCF binding site. In some embodiments, the target polynucleotide sequence comprises CC(A / G / T)(C / G)NNG(A / G)NGG; (C / G)NNG(A / G)NGG(A / C / T)(A / G / T)(C / G); (C / G)(A / C / T)(A / G / T)CCN(C / T)CNN(C / G); CCN(C / T)CNN(C / G)(A / C / T)GG; or variants thereof having one or two nucleotide substitutions. For example, the target polynucleotide sequence may comprise (N)xCC(A / G / T)(C / G)NNG(A / G)NGG; (C / G)NNG(A / G)NGG(A / C / T)(A / G / T)(C / G)(N)x;

[0395] (N)x(C / G)(A / C / T)(A / G / T)CCN(C / T)CNN(C / G); CCN(C / T)CNN(C / G)(A / C / T)GG(N)x; or variants thereof having one or two nucleotide substitutions, wherein x is from 19 to 89, from 24 to 89, from 19 to 69, from 24 to 64, or from 29 to 59. (N)x may consist of a region of the human genome flanking the CTCF-binding site. In some embodiments, the target polynucleotide sequence comprises at least 12 bp of the CTCF binding site. In some embodiments, the target polynucleotide sequence comprises CC(A / G / T)(C / G)NNG(A / G)NGG(A / C / T);

[0396] (A / G / T)(C / G)NNG(A / G)NGG(A / C / T)(A / G / T)(C / G); (C / G)(A / C / T)(A / G / T)CCN(C / T)CNN(C / G)(A / C / T);

[0397] (A / G / T)CCN(C / T)CNN(C / G)(A / C / T)GG; or variants thereof having one or two nucleotide substitutions. For example, the target polynucleotide sequence may comprise (N)xCC(A / G / T)(C / G)NNG(A / G)NGG(A / C / T);

[0398] (A / G / T)(C / G)NNG(A / G)NGG(A / C / T)(A / G / T)(C / G)(N)x; (N)x(C / G)(A / C / T)(A / G / T)CCN(C / T)CNN(C / G)(A / C / T);

[0399] (A / G / T)CCN(C / T)CNN(C / G)(A / C / T)GG(N)x; or variants thereof having one or two nucleotide substitutions, wherein x is from 18 to 88, from 23 to 88, from 18 to 68, from 23 to 63, or from 28 to 58. (N)x may consist of a region of the human genome flanking the CTCF-binding site.

[0400] In some embodiments, the target polynucleotide sequence comprises at least 13 bp of the CTCF binding site. In some embodiments, the target polynucleotide sequence comprises CC(A / G / T)(C / G)NNG(A / G)NGG(A / C / T)(A / G / T) (SEQ ID NO: 53); C(A / G / T)(C / G)NNG(A / G)NGG(A / C / T)(A / G / T)(C / G) (SEQ ID NO: 54);

[0401] (C / G)(A / C / T)(A / G / T)CCN(C / T)CNN(C / G)(A / C / T)G (SEQ ID NO: 55);

[0402] (A / C / T)(A / G / T)CCN(C / T)CNN(C / G)(A / C / T)GG (SEQ ID NO: 56); or variants thereof having one or two nucleotide substitutions. For example, the target polynucleotide sequence may comprise (N)xCC(A / G / T)(C / G)NNG(A / G)NGG(A / C / T)(A / G / T) (SEQ ID NO: 57); C(A / G / T)(C / G)NNG(A / G)NGG(A / C / T)(A / G / T)(C / G)(N)x (SEQ ID NO: 58); (N)x(C / G)(A / C / T)(A / G / T)CCN(C / T)CNN(C / G)(A / C / T)G (SEQ ID NO: 59); (A / C / T)(A / G / T)CCN(C / T)CNN(C / G)(A / C / T)GG(N)x (SEQ ID NO: 60); or variants thereof having one or two nucleotide substitutions, wherein x is from 17 to 87, from 22 to 87, from 17 to 67, from 22 to 62, or from 27 to 57. (N)x may consist of a region of the human genome flanking the CTCF-binding site.

[0403] In some embodiments, the target polynucleotide sequence comprises a CTCF binding site, for example a 14 bp CTCF binding site. In some embodiments, the target polynucleotide sequence consists of CTCF binding site flanked each side by about 0 to about 40 bp. For example, the target polynucleotide sequence may comprise (N)xCC(A / G / T)(C / G)NNG(A / G)NGG(A / C / T)(A / G / T)(C / G)(N)y (SEQ ID NO: 61); (N)x(C / G)(A / C / T)(A / G / T)CCN(C / T)CNN(C / G)(A / C / T)GG(N)y (SEQ ID NO: 62); or variants thereof having one or two nucleotide substitutions, wherein x and y are each independently from 0 to 86 and x+y is from 16 to 86, or x and y are each independently from 0 to 86 and x+y is from 21 to 86, or wherein x and y are each independently from 0 to 66 and x+y is from 16 to 66, x and y are each independently from 0 to 61 and x+y is from 21 to 61 , or x and y are each independently from 0 to 56 and x+y is from 26 to 56. (N)x and (N)y may each consist of a region of the human genome flanking the CTCF- binding site.

[0404] In other embodiments, the target polynucleotide sequence does not comprise any part of a CTCF binding site. For example, the target polynucleotide sequence may consist of a region of the human genome flanking a CTCF-binding site. In one embodiment, the region is 40 bp or less, 35 bp or less, 30 bp or less, 25 bp or less, 20 bp or less, 15 bp or less, 10 bp or less, 5 bp or less, 4 bp or less, 3 bp or less, 2 bp or less, or 1 bp upstream or downstream of the CTCF-binding site.

[0405] In preferred embodiments, the CTCF binding site is selectively occupied by CTCF in a disease state. In more preferred embodiments, the CTCF binding site is selectively occupied by CTCF in a cancer, for example a cancer selected from the group consisting of: lung cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, blood cancer (such as leukaemia or lymphoma) and skin cancer (such as melanoma). In one embodiment, the CTCF binding site is selectively occupied by CTCF in blood cancer. In one embodiment, the CTCF binding site is selectively occupied by CTCF in lung cancer. In one embodiment, the CTCF binding site is selectively occupied by CTCF in colorectal cancer. In one embodiment, the CTCF binding site is selectively occupied by CTCF in breast cancer. In one embodiment, the CTCF binding site is selectively occupied by CTCF in prostate cancer.

[0406] In preferred embodiments, the CTCF binding site location is present in CTCF-occupied cfDNA fragments isolated from subjects with a disease. In more preferred embodiments, the CTCF binding site is present in CTCF-occupied cfDNA fragments isolated from subjects with cancer, for example a cancer selected from the group consisting of: lung cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, blood cancer (such as leukaemia or lymphoma) and skin cancer (such as melanoma). In one embodiment, the CTCF binding site location is present in CTCF-occupied cfDNA fragments isolated from subjects with blood cancer. In one embodiment, the CTCF binding site location is present in CTCF-occupied cfDNA fragments isolated from subjects with lung cancer. In one embodiment, the CTCF binding site location is present in CTCF- occupied cfDNA fragments isolated from subjects with colorectal cancer. In one embodiment, the CTCF binding site location is present in CTCF-occupied cfDNA fragments isolated from subjects with breast cancer. In one embodiment, the CTCF binding site location is present in CTCF-occupied cfDNA fragments isolated from subjects with prostate cancer. In further embodiments, the CTCF binding site is not present in CTCF-occupied cfDNA fragments isolated from healthy subjects.

[0407] In some embodiments, the CTCF binding site is selected from the cancer-specific CTCF binding sites listed in Table 1 , Table 2, or Table 3. In some embodiments, the CTCF binding site is selected from the lung cancer-specific CTCF binding sites listed in Table 3. In some embodiments, the CTCF binding site is selected from the colorectal cancerspecific CTCF binding sites listed in Table 3. In some embodiments, the CTCF binding site is selected from the breast cancer-specific CTCF binding sites listed in Table 3. In some embodiments, the CTCF binding site is selected from the prostate cancer-specific CTCF binding sites listed in Table 3. In some embodiments, the CTCF binding site is selected from the blood cancer-specific CTCF binding sites listed in Table 1 or Table 3. In some embodiments, the CTCF binding site is selected from the cancer-specific CTCF binding sites listed in Table 1.

[0408] In some embodiments, the CTCF binding site is selected from the following: chr1 :244603599-244603619, chr14: 56491500-56491520, chr1 : 90579403-90579423, chr11 :34926268-34926288, chr1 :78130945-78130965, chr5:141992536-141992556, chr22:36986402-36986422, chr14:21734657-21734677, chr6:55980809-55980829, chr4:64713438-64713458, chr17:48336072-48336092, chr2:7427475-7427495, chr15:73874089-73874109, chrX: 89757963-89757983, chr2: 10668604-10668624, chr2:79219569-79219589, chr5:141567693-141567713, chr14:87896582-87896602, chr8:74853585-74853605, chr12:100741297-100741317, chr19: 13262563-13262583, chr22:36986399-36986419, chr1 : 39609779-39609799, chr2:224369205-224369225, chr12:92570541-92570561 , chr6: 50792087-50792107, chr2:65573310-65573330, and chr15:73874086-73874106 (in genome assembly GRCh38 / hg38). In a further embodiment, the CTCF binding site is selected from the following: chr1 :244603599- 244603619, chr14:56491500-56491520, chr1 : 90579403-90579423, chr11 :34926268- 34926288, chr2: 10668604-10668624, chr2:79219569-79219589, chr5: 141567693- 141567713, chr14:87896582-87896602, chr8:74853585-74853605, and chr12:100741297-100741317. PCR PRIMER KITS

[0409] In one aspect, the present invention provides a kit comprising one or more pairs of PCR primers according to the present invention. The kit may be for detecting the presence of CCCTC-binding factor (CTCF) occupied cell free DNA (cfDNA) fragments originating from a pre-determined genomic location, in a body fluid sample obtained from a subject.

[0410] In some embodiments, the kit comprises two or more pairs of PCR primers. In some embodiments, the kit comprises three or more pairs of PCR primers, four or more pairs of PCR primers, five or more pairs of PCR primers. In some embodiments, the kit comprises six or more pairs of PCR primers, seven or more pairs of PCR primers, eight or more pairs of PCR primers, nine or more pairs of PCR primers, or ten or more pairs of PCR primers. In some embodiments, the kit comprises five or more pairs of PCR primers. In some embodiments, the kit comprises thirty or fewer pairs of PCR primers.

[0411] In some embodiments, each of the two or more PCR primers are fluorescently labelled. In some embodiments, the fluorophores are chosen, such that the emission spectra do not overlap. For example, fluorophores suitable for multiplex qPCR may be selected.

[0412] Each of the two or more pairs of PCR primers may be directed to a different genomic location. In some embodiments, the kit comprises two or more pairs of PCR primers which are directed to a different chromosomal region. In some embodiments, the kit comprises two or more pairs of PCR primers which are directed to a different chromosome.

[0413] For example, in some embodiments, the kit comprises:

[0414] (a) a first pair of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs that comprises a CTCF binding site located at a first predetermined genomic location; and

[0415] (b) a second pair of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs that comprises a CTCF binding site located at a second predetermined genomic location;

[0416] (c) optionally, a third pair of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs that comprises a CTCF binding site located at a third pre-determined genomic location;

[0417] (d) optionally, a fourth pair of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs that comprises a CTCF binding site located at a fourth pre-determined genomic location; and (e) optionally, a fifth pair of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs that comprises a CTCF binding site located at a fifth pre-determined genomic location.

[0418] For example, in some embodiments, the kit comprises:

[0419] (a) a first pair of PCR primers directed to a DNA sequence of equal to or less than 80 base pairs that comprises a CTCF binding site located at a first pre-determined genomic location; and

[0420] (b) a second pair of PCR primers directed to a DNA sequence of equal to or less than 80 base pairs that comprises a CTCF binding site located at a second predetermined genomic location;

[0421] (c) optionally, a third pair of PCR primers directed to a DNA sequence of equal to or less than 80 base pairs that comprises a CTCF binding site located at a third pre-determined genomic location;

[0422] (d) optionally, a fourth pair of PCR primers directed to a DNA sequence of equal to or less than 80 base pairs that comprises a CTCF binding site located at a fourth pre-determined genomic location; and

[0423] (e) optionally, a fifth pair of PCR primers directed to a DNA sequence of equal to or less than 80 base pairs that comprises a CTCF binding site located at a fifth pre-determined genomic location.

[0424] In some embodiments, the first pre-determined genomic location is at a different chromosomal region to the second pre-determined genomic location. In some embodiments, each of the pre-determined genomic locations are at a different chromosomal region. In some embodiments, the first pre-determined genomic location is on a different chromosome to the second pre-determined genomic location. In some embodiments, each of the pre-determined genomic locations are on a different chromosome.

[0425] For example, in some embodiments, the kit comprises:

[0426] (a) a first pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a first 30-100 bp region of the human genome comprising a CCCTC-binding factor (CTCF) binding site or part thereof; and (b) a second pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a second 30-100 bp region of the human genome comprising a CCCTC-binding factor (CTCF) binding site or part thereof;

[0427] (c) optionally, a third pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a third 30-100 bp region of the human genome comprising a CCCTC-binding factor (CTCF) binding site or part thereof;

[0428] (d) optionally, a fourth pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a fourth 30- 100 bp region of the human genome comprising a CCCTC-binding factor (CTCF) binding site or part thereof; and

[0429] (e) optionally, a fifth pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a fifth 30-100 bp region of the human genome comprising a CCCTC-binding factor (CTCF) binding site or part thereof.

[0430] For example, in some embodiments, the kit comprises:

[0431] (a) a first pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a first 30-80 bp region of the human genome comprising a CCCTC-binding factor (CTCF) binding site or part thereof; and

[0432] (b) a second pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a second 30-80 bp region of the human genome comprising a CCCTC-binding factor (CTCF) binding site or part thereof;

[0433] (c) optionally, a third pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a third 30-80 bp region of the human genome comprising a CCCTC-binding factor (CTCF) binding site or part thereof;

[0434] (d) optionally, a fourth pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a fourth 30-80 bp region of the human genome comprising a CCCTC-binding factor (CTCF) binding site or part thereof; and

[0435] (e) optionally, a fifth pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a fifth 30-80 bp region of the human genome comprising a CCCTC-binding factor (CTCF) binding site or part thereof.

[0436] In some embodiments, the first region of the human genome is at a different chromosomal region to the second region of the human genome. In some embodiments, each of the regions of the human genome are at a different chromosomal region. In some embodiments, the first region of the human genome is on a different chromosome to the second region of the human genome. In some embodiments, each of the regions of the human genome are on a different chromosome.

[0437] In some embodiments, the kit further comprises an anti-CTCF antibody. In some embodiments, kit further comprises magnetic particles coated with an anti-CTCF antibody.

[0438] For example, in some embodiments, the kit comprises: (a) a pair of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs or equal to or less than 80 base pairs that comprises a CTCF binding site located at a pre-determined genomic location; and (b) an anti-CTCF antibody, optionally in the form of magnetic particles coated with the anti-CTCF antibody. In some embodiments the kit comprises two or more, three or more, four or more, or five or more of said pair of PCR primers.

[0439] For example, in some embodiments, the kit comprises: (a) a pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a second 30-100 bp, 35-1 OObp, 35-80 bp or a 30-80 bp region of the human genome comprising a CCCTC-binding factor (CTCF) binding site or part thereof; and (b) an anti-CTCF antibody, optionally in the form of magnetic particles coated with the anti- CTCF antibody. In some embodiments the kit comprises two or more, three or more, four or more, or five or more of said pair of PCR primers.

[0440] The kit may comprise any other suitable reagents for use in a PCR for detecting the presence of CCCTC-binding factor (CTCF) occupied cell free DNA (cfDNA) fragments.

[0441] PCR PRIMER COMPOSITIONS

[0442] In one aspect, the present invention provides a composition comprising two or more pairs of PCR primers according to the present invention. The composition may be for detecting the presence of CCCTC-binding factor (CTCF) occupied cell free DNA (cfDNA) fragments originating from a pre-determined genomic location, in a body fluid sample obtained from a subject. In some embodiments, the composition comprises three or more pairs of PCR primers, four or more pairs of PCR primers, five or more pairs of PCR primers. In some embodiments, the composition comprises six or more pairs of PCR primers, seven or more pairs of PCR primers, eight or more pairs of PCR primers, nine or more pairs of PCR primers, or ten or more pairs of PCR primers. In some embodiments, the composition comprises five or more pairs of PCR primers. In some embodiments, the composition comprises thirty or fewer pairs of PCR primers.

[0443] In preferred embodiments, each of the two or more PCR primers are fluorescently labelled. In some embodiments, the emission spectra do not overlap. For example, fluorophores suitable for multiplex qPCR may be selected based on those known in the art.

[0444] Each of the two or more pairs of PCR primers may be directed to a different genomic location. In some embodiments, the composition comprises two or more pairs of PCR primers which are directed to a different chromosomal region. In some embodiments, the composition comprises two or more pairs of PCR primers which are directed to a different chromosome.

[0445] The composition may be provided in any suitable form. In some embodiments, the composition is in liquid form. In some embodiments, the composition is in lyophilized form. In some embodiments, the composition is in a tube.

[0446] COMBINATION BIOMARKERS

[0447] In one embodiment, the cell free chromatin fragment comprising CTCF and a DNA fragment (i.e. the DNA associated with the cell free chromatin fragment comprising CTCF) is detected or measured as one of a panel of measurements. For example, in combination with the other cell free chromatin transcription factor markers, or with any other biomarkers.

[0448] PANELS

[0449] According to a further aspect of the invention, there is provided a biomarker panel of genomic locations comprising CTCF binding sites associated with two or more, preferably five or more, of the following genes: CATSPERE, CTNNA2, NOL10, DIAPH1 , TMEM260, GALC, OSBPL6, PI15, ANO4, FAM41AY1 , LINC02787, NR_038432.1 , GIPC2, CACNA1A, GNPD1 , TEX33, TRAV4, HEYL, FAM124B, COL21A1 , PLEKHG7, EPHA5, FAM149A, TFAP2B, LINC02934, SKAP1, CDH5, ENSG00000229727, SNORA70, TBC1 D21 and TGIF2LX. In a further embodiment, the biomarker panel of genomic locations comprising CTCF binding sites associated with two or more, preferably five or more, more preferably all, of the following genes: CATSPERE, TMEM260, LINC02787, NR_038432.1 , NOL10, CTNNA2, DIAPH1 , GALC, PI15 and ANO4. In a further embodiment, the biomarker panel of genomic locations comprising CTCF binding sites associated with TMEM260, P115 and LINC02787.

[0450] In one embodiment, the genomic locations are selected from two or more, preferably five or more, of the following regions (in genome assembly GRCh38 / hg38): ch r1 : 244603555- 244603648; chr2: 10668571 -10668663; chr2:79219540-79219628; chr5: 141567670- 141567748; chr14:56491464-56491546; chr14:87896549-87896634; chr2: 178280431 - 178280518; chr8: 74853546-74853641 ; chr12:100741271-100741352; chrY: 17430722- 17430795; chr1 : 90579362-90579440; chr11 :34926223-34926312; chr1 :78130901- 78130992; chr19: 13262535- 13262621 ; chr5:141992498-141992587; chr22:36986359- 36986452; chr14:21734605-21734701 ; chr1 : 39609752-39609824; chr2:224369183- 224369264; chr6:55980780-55980855; chr12:92570523-92570593; chr4:64713399- 64713474; chr4:186150099-186150166; chr6:50792057-50792142; chr2:65573278- 65573362; chr17:48336036-48336115; chr16:66275978-66276056; chr2:7427441- 7427517; chr8:5062480-5062559; chr15:73874049-73874135; and chrX: 89757930- 89758000. In a further embodiment, the genomic locations are selected from two or more, preferably five or more, more preferably all, of the following regions (in genome assembly G RCh38 / hg38) : chr1 :244603555-244603648, chr14: 56491464-56491546, chr1 : 90579362-90579440, chr11 :34926223-34926312, chr2: 10668571 -10668663, chr2:79219540-79219628, chr5:141567670-141567748, chr14:87896549-87896634, chr8:74853546-74853641 , and chr12:100741271-100741352.

[0451] According to a further aspect of the invention, there is provided a biomarker panel of genomic locations comprising CTCF binding sites associated with two or more, preferably five or more, of the following genes: CTNNA2, GCA, TMEM44, ENSG00000286456, SLC10A5P1 , TRAV4, ENSG00000283445, UST, DMRTA1 , GALNT8, FAM174B, SHLD1 , NAP1 L4P2, MMADHC, PTPRG, BASP1 , PDE4D, SPMIP4, CLNS1A, SKAP1 , IKBKE, ARHGAP15, DPP4, ZNF622, MINDY3, TMEM236, SMPD1 , ENSG00000260945, NLRP1 , ARSH, ROBO1 , ALCAM, MTCO3P27, EDIL3, EXOC2, IFNGR1 , DLD, MKX, QSER1, CTBP2P6, ZDHHC17, MIPOL1 , LINC01418, TERF2IP, CORO2B, GIPC2, ENSG00000230472, PHGDH, CATSPERE, ARMCX4, OSBPL6, CAST, PHEX, ZNF157, TIMM50, LINC02583, PTMA, HEPACAM2, IFNWP4, LINC02572, PARD3, GRIK5, IFIH1 , ADAM28, LINC00589, ENSG00000228566, ELF5, PPFIBP1 , WIPF1, MCCC1 , DNAH10, CREG1 , TPRG1 , GNPDA1 , MTND2P8, LINC02446, CPS1 , ERBB4, OR7E43P, TMEM260, DUT, ENSG00000226530, TANC1 , MAPK13, ENSG00000232436, AMBRA1 , CENPSCORT, TMEM182, LHFPL3AS1 , JAK2, TECTA, CLMP, NAA16, SDK2, VRK3, YTHDF1 , SBF2, MSCAS1 , GAPDH, LINC02389, FCGR2A, ENSG00000287730, TP5BI11 , SINHCAF, ENSG00000275393, FCGRT, PROSER2, LINC02535, LPAL2, ENSG00000230649, TPPP2, LRP8, CYP1 B1 , KCNIP4, ANK2, PDE4D, KRT18P54, MRPL15, GDF6, MPP7, SCGB1A1 , LINC00937, SLAMF7, LILRP1 , SDE2, NID1 , FGFBP2, SPOCK3, RNF14, OR4S1 , SNX20, RGS9, SPTLC3, NOL10, DPPA3P2, CCDC8, TLX1 , FERMT1 , GPR27, SIRT6, LINC01307, IFI6, CCNA1 , UNC119, TGM6, IFI16, MTARC1 , MY03BAS1 , DDX3ILA1 , NKAIN1 , LINC02810, SERINC2, ENSG00000236069, PREP, CEACAM1 , VAV3, ENSG00000235819, PLXNA4, NPM1 P46, RN7SL361 P, ENSG00000266573, GLDC, SWAP7Q, QOQ8A, ENSG00000225620, RPL7AP47, SMUG1 , TTLL11 , ENSG00000228852, LRRC7, VAV3, GPD1 L, SPICE1 , CEP63, IL20RB, FAUP2, LINC02357, LINC02503, TICAM2, PKHD1 , ENSG00000218813, SYTL3, PACRG, HAP1 , PLXNA4, RN7SL135P, NDUFB9P3, ENSG00000252852, DSCC1 , ENSG00000235819, ENSG00000279503, SLC16A12, HECTD2, PLEKHG7,

[0452] LINC02409, LINC02300, SLC25A21 , LCTL, PHLPP1 , PHLPP1 , ZFP64, MTOR, GLIS1 , RN7SKP272, ENSG00000229727, RN7SL361 P, POU3F3, CHCHD5, ITGB6, NPM1 P46, FAM124B, ZNF876P, UCHL1 , ENSG00000266573, ENSG00000225620, QOQ8A, TFAP2B, GLDC, RPL7AP47, SWAP7Q, ENSG00000228852, TTLL11 , SMUG1 , ANO4, EPHA5, and NR_038432.1.

[0453] In one embodiment, the genomic locations are selected from two or more, preferably five or more, of the following regions (in genome assembly GRCh38 / hg38): chr2:79219519- 79219648, chr2:162301295-162301441 , chr3: 194594813-194594949, chr7:64625243- 64625355, chr8:81641590-81641720, chr14:21734606-21734734, chr1 : 58739989- 58740101 , chr6: 149028960-149029063, chr9:22452551 -22452664, chr12:4849148- 4849254, chr15:92797259-92797393, chr20: 5788487-5788587, chrX: 106481411- 106481533, chr2: 149561424-149561553, chr3:61381935-61382023, chr5: 17190626- 17190742, chr5:59390194-59390288, chr7:25160122-25160245, chr11 :77619731- 77619846, chr17:48336001-48336147, chr1 :206461901 -206462094, ch r2 : 143110966- 143111104, chr2:162123970-162124051 , chr5: 16456356- 16456495, chr10:15902711- 15902826, chr10:17742984-17743122, chr11 :6379394-6379487, chr16: 17579398- 17579529, chr17:5592340-5592644, chrX: 3035865-3036000, chr3: 78659532- 78659619, chr3: 105518948-105519094, chr4:64585094-64585216, chr5:83927264- 83927382, chr6:593261 -593392, chr6:137314444-137314535, chr7: 107874746-

[0454] 107874889, chr10:27656752-27656912, chr11 :32960716-32960828, chr11 :43567527- 43567641 , chr12:76791108-76791220, chr14:37230722-37230848, chr15:81939739- 81939846, chr16:75789293-75789426, chr15:68554138-68554266, chr1 :78130884- 78131005, chr6: 50602320-50602449, chr1 :119675667-119675836, chr1 :244603541-

[0455] 244603674, chrX:101415625-101415728, chr2: 178280411-178280526, chr5:96752586-

[0456] 96752688, chrX:22081630-22081732, chrX: 47384626-47384769, chr19:39474211- 39474377, chr2:46464602-46464705, chr2:231715363-231715468, chr7:93228366- 93228499, chr9:21111090-21111213, chr2: 129849717- 129849817, chr10:34638577- 34638699, chr19:42028327-42028524, chr2:162298074-162298189, chr8:24229478- 24229598, chr8:29647427-29647523, chr10:63720138-63720300, chr11 :34492959- 34493085, chr12:27652288-27652426, chr2: 174700116-174700251 , chr3:183122469-

[0457] 183122581 , chr12: 123905436- 123905607, chr1 : 167540493-167540653, chr3: 189148131 -189148251, chr5:141992479-141992603, chr9:78821242-78821375, chr12 : 10562452- 10562563, chr2 :210577647-210577793, chr2:212567753-212567856, chr4:4183014-4183121 , chr14:56491434-56491551 , chr15:48352569-48352712, chrX:51461172-51461282, chr2:159118703-159118816, chr6: 36095298-36095473, chr1 :221435546-221435673, chr11 :46537952-46538113, chr1 : 10428186-10428296, chr2: 102840031-102840180, chr7: 104848545-104848654, chr9:4988029-4988170, chr11 : 121194847-121194979, chr11 : 123247327- 123247457, chr13:41353128-

[0458] 41353267, chr17:73369977-73370136, chr19:49983756-49983882, ch r20:63223466- 63223579, chr11 :10077000-10077143, chr8:71975358-71975486, chr12:6537982- 6538105, chr12:64891000-64891169, chr1 :161487131-161487247, chr4: 155442425- 155442520, chr11 :44911428-44911580, chr12:31272008-31272161 , chr16:85717990- 85718123, chr19:49547082-49547241 , chr10:11819934-11820065, chr6:85355152- 85355270, chr6: 160519981-160520091 , chr7: 136009627-136009724, chr14:21014774- 21014934, chr1: 53393434-53393577, chr2:38115355-38115496, chr4:21789365- 21789486, chr4:113394834-113394942, chr5:59673572-59673710, chr6:85323564- 85323677, chr8:54169437-54169546, chr8:96180035-96180344, chr10:28241944- 28242032, chr11 :62380869-62381016, chr12:8396766-8396964, chr1 : 160726939- 160727057, chr19:54676361 -54676503, chr1 :225976081-225976216, chr1 :236103745- 236103862, chr4:16026149-16026413, chr4: 167021693- 167021794, chr5: 141960806- 141960987, chr11 :48307888-48308019, chr16:50665497-50665627, chr17:65198881- 65199032, chr20:13119634-13119763, chr2: 10668563-10668699, chr14:36374168- 36374282, chr19:46402330-46402434, chr10:101161791-101161998, chr20:6142524- 6142649, chr3:71729971 -71730106, chr19:4204720-4204856, chr1 : 101288544-

[0459] 101288669, chr1 :27669254-27669382, chr13:36420493-36420628, chr17:28540839- 28540954, chr20:2351923-2352109, chr1 : 158998804-158998962, chr1 :220810250- 220810376, chr2: 170409173-170409293, chr7: 78040335-78040434, chr1 :31162784- 31162912, chr1 : 17661594-17661807, chr1 :31422842-31422978, chr1 : 192445984- 192446109, chr6: 105424291 -105424437, chr19:42529383-42529506, chr1 : 107782340- 107782444, chr9:86414420-86414532, chr7: 132188991 -132189067, chr2: 197374043- 197374122, chr2:60642442-60642560, chr18:24736101-24736161 , chr9:6594697- 6594768, chr11 :9675318-9675421 , chr1 :226944700-226944781 , chr1 :202640729- 202640819, chr9:14216485-14216587, chr12:54188268-54188338, chr9:121838761- 121838825, chr1 :95264973-95265040, chr1 :69737726-69737836, chr1 : 107782340- 107782444, chr3: 32144255-32144365, chr3:113494238-113494353, chr3: 134474064- 134474175, chr3: 137105390-137105526, chr3: 181326780-181326885, chr4:26082030- 26082146, chr4: 103934283-103934454, chr5:115586654-115586772, chr6: 52049619- 52049757, chr6:63764478-63764585, chr6:158691470-158691594, chr6: 162908032- 162908121 , chr7: 130832002-130832124, chr7:132188991-132189067, chr8:63262036- 63262139, chr8:110830607-110830727, chr8:117329067-117329210, chr8:119867220- 119867390, chr9:86414420-86414532, chr9:90142700-90142839, chr10:89563998- 89564154, chr10:91389742-91389869, chr12:92570492-92570605, chr12:97308762- 97308849, chr14:28635549-28635670, chr14:37149849-37149969, chr15:665671 SO- 66567354, chr18:62958013-62958123, chr18:62982806-62982908, chr20:52312263- 52312398, chr1 :11241826-11241943, chr1 : 53754832-53754948, chr1 : 89987314- 89987463, chr2:7427425-7427538, chr2:60642442-60642560, chr2: 104849680-

[0460] 104849792, chr2:112582400-112582523, chr2:160110087-160110199, chr2:197374043-197374122, chr2:224369161 -224369299, chr4:242369-242480, chr4:41281166-41281273, chr4:122758089-122758199, chr4:166165962-166166073, chr4: 169810464- 169810607, chr5:32489802-32489952, chr5:123371732-123371827, chr5: 172370813-172370900, chr6:33123829-33123955, chr6:49849713-49849822, chr6: 110942102-110942167, chr6: 113909396-113909512, chr6: 159008718-

[0461] 159008830, chr10:25700952-25701067, chr10:92885025-92885143, chr10:94630132- 94630263, chr11 :30346226-30346339, chr11 :110433891-110434020, chr11 : 124586369-124586468, chr12 : 102702668- 102702830, chr12 : 111451647- 111451929, chr13:33228694-33228791 , chr13:50085114-50085230, chr13:53250379- 53250572, chr13:57627719-57627850, chr13: 100722041 -100722178, chr14:20327405- 20327535, chr14:24735042-24735188, chr15:31974953-31975090, chr18:24736101- 24736161 , chr19:54674154-54674274, chr22:22201213-22201344, chrX:22136053- 22136155, chrX: 129754802-129754877, chrX: 130079923-130080041 , chr1 : 19918128- 19918255, chr1 :24587331 -24587473, chr1 :39720138-39720288, chr1 :65958784- 65959095, chr1 : 74689941-74690070, chr1 :88877501 -88877633, chr1 : 161227460- 161227587, chr1 :202640729-202640819, chr1 :226944700-226944781 , ch r2: 17646148- 17646327, chr2:227862361 -227862483, chr2:235594168-235594292, chr2:238462941- 238463069, chr3: 124079474-124079582, chr3: 156541160-156541317, chr3: 194404789- 194404904, chr3: 195522387- 195522501 , chr4: 16794985- 16795080, chr4:27048448-27048583, chr4:52026152-52026244, chr4:52376236-52376358, chr4: 140999943-141000112, chr4: 146104133-146104288, chr4: 158850959-

[0462] 158851065, chr4: 165464923-165465025, chr5:18314314-18314440, chr5:28014922- 28015045, chr5: 74648924-74649026, chr5: 102223966-102224084, chr5: 129664478- 129664599, chr5: 155139754-155139857, chr6:12893990-12894132, chr6: 30380603- 30380684, chr6: 50792043-50792155, chr6:51855414-51855525, chr6: 123415992- 123416085, chr7:10412547-10412686, chr7:25180362-25180528, chr7: 38374563- 38374659, chr7:91830142-91830274, chr7:94395571 -94395689, chr8:4833976-

[0463] 4834088, chr8:95758783-95758908, chr8:119070591-119070729, chr9:6354233- 6354348, chr9:6594697-6594768, chr9:14216485-14216587, chr9: 17063671 -17063809, chr9: 19028989-19029108, chr10:3192966-3193102, chr10: 16828259-16828398, chr10:25726026-25726156, chr10:87656598-87656695, chr10:87735375-87735496, chr10: 125098674-125098794, chr11 :9675318-9675421 , chr11 : 17230210-17230310, chr11 :32323655-32323771 , chr11 : 72667585-72667696, chr11 : 124402455-124402570, chr12:20946875-20946995, chr12:53791868-53791960, chr12:55619731-55619974, chr12:125403356-125403461 , chr12:126069664-126069777, chr13:85828223-

[0464] 85828338, chr14:45426816-45426941 , chr14:93655215-93655320, chr15:44288413- 44288574, chr15:90956673-90956821 , chr16:24253220-24253372, chr16:63361142- 63361267, chr16:66728261-66728409, chr17:35312798-35312956, chr17:78013974- 78014108, chr18:43571212-43571318, chr18:65781879-65782020, chr20: 36466920- 36467064, chr22:25093742-25093869, chrX:51614499-51614639, chrX:110156310- 110156388, chr22_KI270733v1_random: 136322-136475, chr1 : 32272584-32272646, chr1 : 86742093-86742193, chr1 :88998835-88998937, chr1 :95264973-95265040, chr1 : 163410082-163410225, chr1 : 169055544-169055615, chr1 :236452124-

[0465] 236452243, chr1 :240149603-240149760, chr1 :240189505-240189629, ch r2: 18378 ISO- 18378279, chr2:38581666-38581810, chr2: 152861493- 152861578, chr2:166539491- 166539609, chr2:176814897-176815015, chr2: 182186287-182186395, chr2:227890621-227890778, chr3:87298250-87298349, chr3:89211716-89211800, chr3: 109732032- 109732122, chr3: 134492629- 134492738, chr3: 156509828-

[0466] 156509965, chr3: 157137568-157137691 , chr3: 161273765-161273866, chr3:166013225-166013311 , chr4:23135919-23136011 , chr4:48087272-48087368, chr4:70183776-70183887, chr4:73019582-73019720, chr4:86044122-86044254, chr4: 174365159- 174365240, chr5:5141805-5141917, chr5:80083642-80083758, chr5:81299634-81299801 , chr5:112175136-112175244, chr5: 146662279-146662392, chr5: 148733715-148733808, chr5: 154157507-154157598, chr6:35449508-35449669, chr6: 52021110-52021230, chr6: 56902964-56903131 , chr6:70706662-70706775, chr6:74091490-74091627, chr6:112180232-112180339, chr6: 135451738-135451874, chr7: 148504024- 148504134, chr7: 156582102- 156582204, chr8: 12866970- 12867090, chr8:69663836-69663985, chr8:75257410-75257540, chr8:80612676-80612794, chr8:90948403-90948519, chr8:132651571-132651698, chr9: 13427664-13427772, chr9: 15873723-15873819, chr9: 17462998-17463108, chr9: 121400273-121400386, chr9:121838761-121838825, chr10:61284412-61284550, chr10:93352770-93352892, chr10:116490211-116490321 , chr10:125982446-125982551 , chr11 :6738008-6738119, chr11 :84384764-84384893, chr11 :88092327-88092414, chr11 :94519623-94519738, chr11 :96464846-96464973, chr11 :115117490-115117553, chr11 : 128414446-

[0467] 128414512, chr12:7642988-7643098, chr12:21680506-21680612, chr12:29519728-

[0468] 29519832, chr12:34034045-34034152, chr12:54188268-54188338, chr12:58346176-

[0469] 58346282, chr12 : 100741240- 100741368, chr12:119160250-119160324, chr13:97132337-97132465, chr14: 19878197-19878283, chr14:25400748-25400891 chr14:53055029-53055173, chr14:64303608-64303708, chr14:82000025-82000111 chr14:98138734-98138826, chr15:85505208-85505330, chr15: 98881743-98881866 chr16:16449419-16449513, chr17:700647-700751 , chr17: 16258067- 16258156 chr18:24489020-24489155, chr18:31819747-31819885, chr19:28325490-28325587 chr19:47125320-47125481 , chrX:11144386-11144529, chrX: 87299704-87299805 chrX: 133670537-133670648, chrX: 152758817-152758894, chr1 :2091761 -2091844 chr1 : 5225260-5225331 , chr1 : 110555394-110555522, chr1 : 158036123-158036245, chr1 : 176559961 -176560058, chr1 :207405060-207405185, chr1 :238188421 -

[0470] 238188505, chr2:39059461 -39059589, chr2:60707803-60707914, chr2:62412204- 62412318, chr2: 106791905-106792003, chr2:151437690-151437807, chr2: 158010423- 158010526, chr2:164717151-164717267, chr2: 179440792-179440892, chr2: 181715178-181715317, chr2: 197707735-197707844, chr2:232270870-

[0471] 232270944, chr2:241078520-241078659, chr3: 13600010-13600082, chr3:26712570- 26712658, chr3:65785206-65785301 , chr3: 98039424-98039547, chr3: 146065649- 146065763, chr3:147191901-147192041 , chr3: 173584256-173584361 , ch r4 : 38856812- 38856976, chr4:64713379-64713507, chr4:67746823-67746964, chr4: 147097166- 147097267, chr4: 163484959-163485038, chr4: 185634052-185634304, chr5: 19469171- 19469243, chr5:36870575-36870732, chr5: 52657324-52657444, chr5:66307768- 66307915, chr5: 103335135-103335205, chr6:47631424-47631547, chr6:52945168- 52945233, chr6:75893429-75893588, chr6: 93664709-93664784, chr6:115244749- 115244826, chr6: 116287793-116287931 , chr6: 158605406-158605520, chr6: 170561934-170561998, chr7:26198200-26198319, chr7:50142454-50142556, chr7:81482732-81482834, chr7: 154504103-154504239, chr8:9831026-9831238, chr9: 8086869-8086940, chr10: 117940922- 117940998, chr11 : 34926239-34926356, chr11 :82682336-82682444, chr11 : 121765670-121765767, chr12: 12376782-12376859, chr12:20950374-20950501 , chr12:23949721-23949794, chr12:24095121 -24095194, chr12:29889866-29889994, chr12:72987449-72987521 , chr12:73391018-73391090, chr12:78146406-78146532, chr12:90959676-90959826, chr13:43908534-43908635, chr13:67238415-67238557, chr13:84896266-84896371 , chr13: 102141733-102141898, chr13: 110839093-110839188, chr14:78082948-78083033, chr14: 100624900-

[0472] 100625009, chr15:24585660-24585745, chr15:31880238-31880317, chr15:41031918- 41032105, chr15:69322673-69322785, chr15:80419036-80419163, chr15: 99704486- 99704631 , chr17:31216272-31216386, chr17:48728857-48728929, chr17:74612182- 74612276, chr17:79893762-79893854, chr18:25853208-25853305, chr18:64037936- 64038067, and chr19:54080140-54080207.

[0473] In a further embodiment, the biomarker panel of genomic locations comprising CTCF binding sites associated with two or more, preferably five or more, more preferably all, of the following genes: CTNNA2, GCA, TMEM44, ENSG00000286456, SLC10A5P1, TRAV4, ENSG00000283445, UST, DMRTA1 , GALNT8, FAM174B, SHLD1 , NAP1 L4P2, MMADHC, PTPRG, BASP1 , PDE4D, SPMIP4, CLNS1A, SKAP1 , IKBKE, ARHGAP15, DPP4, ZNF622, MINDY3, TMEM236, SMPD1 , ENSG00000260945, NLRP1 , ARSH, ROBO1 , ALCAM, MTCO3P27, EDIL3, EXOC2, IFNGR1 , DLD, MKX, QSER1 , CTBP2P6, ZDHHC17, MIPOL1 , LINC01418, TERF2IP, CORO2B, GIPC2, ENSG00000230472, PHGDH, CATSPERE, ARMCX4, OSBPL6, CAST, PHEX, ZNF157, TIMM50, LINC02583, PTMA, HEPACAM2, IFNWP4, LINC02572, PARD3, GRIK5, IFIH1 , ADAM28, LINC00589, ENSG00000228566, ELF5, PPFIBP1 , WIPF1 , MCCC1 , DNAH10, CREG1 , TPRG1 , GNPDA1 , MTND2P8, LINC02446, CPS1 , ERBB4, OR7E43P, TMEM260, DUT, ENSG00000226530, TANC1 , MAPK13, ENSG00000232436, AMBRA1 , CENPSCORT, TMEM182, LHFPL3AS1 , JAK2, TECTA, CLMP, NAA16, SDK2, VRK3, YTHDF1 , SBF2, MSCAS1 , GAPDH, LINC02389, FCGR2A, ENSG00000287730, TP5BI11 , SINHCAF, ENSG00000275393, FCGRT, PROSER2, LINC02535, LPAL2, ENSG00000230649, TPPP2, LRP8, CYP1 B1 , KCNIP4, ANK2, PDE4D, KRT18P54, MRPL15, GDF6, MPP7, SCGB1A1 , LINC00937, SLAMF7, LILRP1 , SDE2, NID1 , FGFBP2, SPOCK3, RNF14, OR4S1 , SNX20, RGS9, SPTLC3, NOL10, DPPA3P2, CCDC8, TLX1 , FERMT1 , GPR27, SIRT6, LINC01307, IFI6, CCNA1 , UNC119, TGM6, IFI16, MTARC1 , MY03BAS1 , DDX3ILA1 , NKAIN1 , LINC02810, SERINC2, ENSG00000236069, PREP, CEACAM1 , VAV3, ENSG00000235819, PLXNA4, NPM1 P46, RN7SL361 P, ENSG00000266573, GLDC, SWAP7Q, Q0Q8A, ENSG00000225620, RPL7AP47, SMUG1 , TTLL11 , and ENSG00000228852.

[0474] In one embodiment, the genomic locations are selected from two or more, preferably five or more, of the following regions (in genome assembly GRCh38 / hg38): chr2:79219519- 79219648, chr2:162301295-162301441 , chr3: 194594813-194594949, chr7:64625243- 64625355, chr8:81641590-81641720, chr14:21734606-21734734, chr1 : 58739989- 58740101 , chr6: 149028960-149029063, chr9:22452551 -22452664, chr12:4849148- 4849254, chr15:92797259-92797393, chr20: 5788487-5788587, chrX: 106481411- 106481533, chr2: 149561424-149561553, chr3:61381935-61382023, chr5: 17190626- 17190742, chr5:59390194-59390288, chr7:25160122-25160245, chr11 :77619731- 77619846, chr17:48336001-48336147, chr1 :206461901 -206462094, ch r2 : 143110966- 143111104, chr2:162123970-162124051 , chr5: 16456356- 16456495, chr10:15902711- 15902826, chr10:17742984-17743122, chr11 :6379394-6379487, chr16: 17579398- 17579529, chr17:5592340-5592644, chrX: 3035865-3036000, chr3: 78659532- 78659619, chr3: 105518948-105519094, chr4:64585094-64585216, chr5:83927264- 83927382, chr6:593261 -593392, chr6:137314444-137314535, chr7: 107874746-

[0475] 107874889, chr10:27656752-27656912, chr11 :32960716-32960828, chr11 :43567527- 43567641 , chr12:76791108-76791220, chr14:37230722-37230848, chr15:81939739- 81939846, chr16:75789293-75789426, chr15:68554138-68554266, chr1 :78130884- 78131005, chr6: 50602320-50602449, chr1 :119675667-119675836, chr1 :244603541-

[0476] 244603674, chrX:101415625-101415728, chr2: 178280411-178280526, chr5:96752586- 96752688, chrX:22081630-22081732, chrX: 47384626-47384769, chr19:39474211- 39474377, chr2:46464602-46464705, chr2:231715363-231715468, chr7:93228366- 93228499, chr9:21111090-21111213, chr2: 129849717- 129849817, chr10:34638577- 34638699, chr19:42028327-42028524, chr2:162298074-162298189, chr8:24229478- 24229598, chr8:29647427-29647523, chr10:63720138-63720300, chr11 :34492959- 34493085, chr12:27652288-27652426, chr2: 174700116-174700251 , chr3: 183122469- 183122581 , chr12:123905436-123905607, chr1 : 167540493-167540653, chr3: 189148131 -189148251, chr5:141992479-141992603, chr9:78821242-78821375, chr12 : 10562452- 10562563, chr2 :210577647-210577793, chr2:212567753-212567856, chr4:4183014-4183121 , chr14:56491434-56491551 , chr15:48352569-48352712, chrX:51461172-51461282, chr2:159118703-159118816, chr6: 36095298-36095473, chr1 :221435546-221435673, chr11 :46537952-46538113, chr1 : 10428186-10428296, chr2: 102840031-102840180, chr7: 104848545-104848654, chr9:4988029-4988170, chr11 : 121194847-121194979, chr11 : 123247327- 123247457, chr13:41353128-

[0477] 41353267, chr17:73369977-73370136, chr19:49983756-49983882, ch r20:63223466- 63223579, chr11 :10077000-10077143, chr8:71975358-71975486, chr12:6537982- 6538105, chr12:64891000-64891169, chr1 :161487131-161487247, chr4: 155442425- 155442520, chr11 :44911428-44911580, chr12:31272008-31272161 , chr16:85717990- 85718123, chr19:49547082-49547241 , chr10:11819934-11820065, chr6:85355152- 85355270, chr6: 160519981-160520091 , chr7: 136009627-136009724, chr14:21014774- 21014934, chr1: 53393434-53393577, chr2:38115355-38115496, chr4:21789365- 21789486, chr4:113394834-113394942, chr5:59673572-59673710, chr6:85323564- 85323677, chr8:54169437-54169546, chr8:96180035-96180344, chr10:28241944- 28242032, chr11 :62380869-62381016, chr12:8396766-8396964, chr1 : 160726939- 160727057, chr19:54676361 -54676503, chr1 :225976081-225976216, chr1 :236103745- 236103862, chr4:16026149-16026413, chr4: 167021693- 167021794, chr5: 141960806- 141960987, chr11 :48307888-48308019, chr16:50665497-50665627, chr17:65198881- 65199032, chr20:13119634-13119763, chr2: 10668563-10668699, chr14:36374168- 36374282, chr19:46402330-46402434, chr10:101161791-101161998, chr20:6142524- 6142649, chr3:71729971 -71730106, chr19:4204720-4204856, chr1 : 101288544- 101288669, chr1 :27669254-27669382, chr13:36420493-36420628, chr17:28540839- 28540954, chr20:2351923-2352109, chr1 : 158998804-158998962, chr1 :220810250- 220810376, chr2: 170409173-170409293, chr7: 78040335-78040434, chr1 :31162784- 31162912, chr1 : 17661594-17661807, chr1 :31422842-31422978, chr1 : 192445984- 192446109, chr6: 105424291 -105424437, chr19:42529383-42529506, chr1 : 107782340- 107782444, chr9:86414420-86414532, chr7: 132188991 -132189067, chr2: 197374043- 197374122, chr2:60642442-60642560, chr18:24736101-24736161 , chr9:6594697- 6594768, chr11 :9675318-9675421 , chr1 :226944700-226944781 , chr1 :202640729- 202640819, chr9:14216485-14216587, chr12:54188268-54188338, chr9:121838761- 121838825, and chr1 : 95264973-95265040 (in genome assembly GRCh38 / hg38).

[0478] In one embodiment, the genomic locations are selected from two or more, preferably five or more, such as about ten, of the genomic locations of Table 1 , Table 10, or Table 11. In one embodiment, the genomic locations are selected from two or more, preferably five or more, such as about ten, of the genomic locations of Table 1 or Table 10. In one embodiment, the genomic locations are selected from two or more, preferably five or more, such as about ten, of the genomic locations of Table 1. In one embodiment, the genomic locations are selected from two or more, preferably five or more, such as about ten, of the genomic locations of Table 10 or Table 11 . In one embodiment, the genomic locations are selected from two or more, preferably five or more, such as about ten, of the genomic locations of Table 10. In one embodiment, the biomarker panel is a qPCR assay panel.

[0479] According to a further aspect of the invention, there is provided the use of the biomarker panel described herein for the detection or diagnosis of cancer.

[0480] METHODS OF DISEASE DETECTION AND MONITORING

[0481] In one embodiment, the method comprises detecting a panel of sequences of DNA fragments associated with CTCF as a combined biomarker for indicating the presence of the disease in the subject. The term “biomarker” means a distinctive biological or biologically derived indicator of a process, event, or condition. Biomarkers can be used in methods of diagnosis, e.g. clinical screening, and prognosis assessment and in monitoring the results of therapy, identifying subjects most likely to respond to a particular therapeutic treatment, drug screening and development. Such biomarkers include, for example, the presence (e.g. sequence), level, concentration or amount of DNA associated with a transcription factor. References herein to a “combined biomarker” refer to a biomarker which involves more than one biological, or biologically derived, indicator, e.g. in particular the level, concentration or amount of CTCF associated with a particular sequence, or sequences, of DNA.

[0482] Determination of the disease state of a subject may include, for example, the detection, diagnosis, treatment selection, monitoring or prognosis of or for a disease.

[0483] In preferred embodiments, the disease is cancer. In a further embodiment, the cancer is selected from: lung cancer (such as non-small lung cell cancer), colorectal cancer, prostate cancer, breast cancer, blood cancer (such as leukaemia, in particular Acute Myeloid Lymphoma, or lymphoma in particular Non-Hodgkin Lymphoma), hepatic cancer, bile duct cancer (such as extrahepatic and intrahepatic bile duct cancer) and cervical cancer.

[0484] In one embodiment, the cancer is blood cancer. Examples of blood cancer include leukaemia (in particular Acute Myeloid Lymphoma) and lymphoma (in particular NonHodgkin Lymphoma). In one embodiment, the cancer is leukaemia. In one embodiment, the cancer is Acute Myeloid Lymphoma (AML). In one embodiment, the cancer is lymphoma. In one embodiment, the cancer is Non-Hodgkin Lymphoma.

[0485] In one embodiment, the cancer is lung cancer. In one embodiment, the cancer is non- small lung cell cancer. In one embodiment, the cancer is colorectal cancer.

[0486] In one embodiment, the cancer is prostate cancer.

[0487] In one embodiment, the cancer is breast cancer.

[0488] In one embodiment, methods of the invention are for use in diagnosing at all stages of cancer. Cancer may be assigned as stage 0, stage I, stage II, stage III or stage IV. Stage definition varies with different cancer diseases and is known in the art. Typically, stage I is classified as when the cancer is small and confined locally to the tissue of origin. Stage II is classified as when the cancer has grown larger and beyond its origin into nearby tissues within the organ or to nearby lymph nodes. Stage III is classified as when the cancer has grown into nearby tissues beyond the organ of origin but has not spread to other more distant parts of the body. Stage IV is classified as when the cancer has spread to one or more distant parts of the body, such as the liver or lungs. The cancer stages can be divided into groups depending upon the size of the tumour, whether the cancer has spread to the lymph nodes, surrounding tissue or a distant part of the body. Stage I can be divided into Stage IA and Stage IB. Stage II can be divided into Stage IIA and Stage IIB. Stage III can be divided into Stage IIIA, Stage IIIB and Stage NIC. Stage IV can be divided into Stage IVA and Stage IVB. As shown in the Examples, methods of the invention were able to be used to detect cancer at an early stage. Early stage cancer generally includes stages 0, I and II. Late stage cancer generally includes stages III and IV. The present invention may also be useful for diagnosing pre-cancer.

[0489] According to a further aspect of the invention there is provided a method for detecting or diagnosing cancer in a subject which comprises the steps of:

[0490] (i) detecting the presence of CTCF occupied cfDNA fragments using a method as described herein; and

[0491] (ii) using the presence of the CTCF occupied cfDNA fragments as an indicator of the presence of cancer in the subject.

[0492] In one embodiment of the invention, the presence of a cell free chromatin fragment comprising CTCF and the DNA fragment (i.e. CTCF occupied cfDNA fragments) in a sample is used to determine the optimal treatment regime for a subject in need of such treatment. According to a further aspect of the invention there is provided a method for assessment of a subject for suitability for a cancer treatment which comprises the steps of:

[0493] (i) detecting the presence of CTCF occupied cfDNA fragments using a method as described herein; and

[0494] (ii) using the CTCF occupied cfDNA fragments detected in step (i) as a parameter for the selection of a suitable treatment for the subject.

[0495] According to a further aspect of the invention there is provided a method for monitoring a treatment of a subject diagnosed with cancer which comprises the steps of:

[0496] (i) detecting the presence of CTCF occupied cfDNA fragments using a method as described herein;

[0497] (ii) repeating the detection of step (i) in a body fluid sample obtained from the subject on one or more occasions; and

[0498] (iii) using any changes in the presence of CTCF occupied cfDNA fragments determined in step (i) compared to step (ii) as a parameter for any changes in the condition of the subject.

[0499] A change in the level of the analysed DNA fragments associated with CTCF in the test sample relative to the level detected in a previous test sample taken earlier from the same test subject may be indicative of a beneficial effect, e.g. stabilization or improvement, of said therapy on the disorder or suspected disorder. Furthermore, once treatment has been completed, the method of the invention may be periodically repeated in order to monitor for the recurrence of a disease.

[0500] It will be understood that measurements or assays performed by methods of the invention may include the use of a reference material as a calibrant or positive control to provide a standard against which the output of the assay can be compared or calibrated and / or to confirm or monitor the correct functioning of the chemistry of the assay. Suitable reference materials may include biologically sourced chromatin fragments containing transcription factors or recombinant chromatin fragments including without limitation recombinant transcription factor-DNA complexes.

[0501] The terms “detecting” and “diagnosing” as used herein encompass identification, confirmation, and / or characterization of a disease state. Methods of detecting, monitoring and of diagnosis according to the invention are useful to confirm the existence of a disease, to monitor development of the disease by assessing onset and progression, or to assess amelioration or regression of the disease, including to detect or monitor minimal residual disease if it is present. Methods of detecting, monitoring and of diagnosis are also useful in methods for assessment of clinical screening, prognosis, choice of therapy, evaluation of therapeutic benefit, i.e. for drug screening and drug development.

[0502] Efficient diagnosis and monitoring methods provide very powerful “patient solutions” with the potential for improved prognosis, by establishing the correct diagnosis, allowing rapid identification of the most appropriate treatment (thus lessening unnecessary exposure to harmful drug side effects), and reducing relapse rates.

[0503] It will be understood that identifying and / or quantifying can be performed by any method suitable to identify the presence and / or amount of a specific protein or DNA fragment in a biological sample from a patient or a purification or extract of a biological sample or a dilution thereof. Biological samples that may be tested in a method of the invention include those as defined hereinbefore. The samples can be prepared, for example where appropriate diluted or concentrated, and stored in the usual manner.

[0504] Identifying and / or quantifying the analyte biomarkers may be performed using an immunological method, involving an antibody, or a fragment thereof capable of specific binding to the biomarker.

[0505] Diagnostic or monitoring kits are provided herein for performing methods of the invention. Such kits for detection and / or quantification of the biomarker or combined biomarker will suitably comprise a ligand or binder for the transcription factor and optionally reagents for use in a method to detect DNA associated with said transcription factor and optionally a ligand or binder for nucleosomes, optionally together with instructions for use of the kit. Therefore, according to a further aspect of the invention, there is provided a kit for the detection of a cell free chromatin fragment comprising CTCF and a DNA fragment as a combination biomarker which comprises a binding agent for CTCF together with reagents for identifying the presence of CTCF occupied cfDNA fragments using a method as described herein (e.g. reagents for performing PCR, such as the one or more pairs of PCR primers used to amplify the pre-determined genomic location).

[0506] Biomarker monitoring methods, biosensors and kits are also vital as patient monitoring tools, to enable the physician to determine whether relapse is due to worsening of the disorder. If pharmacological treatment is assessed to be inadequate, then therapy can be reinstated or increased; a change in therapy can be given if appropriate. As the biomarkers are sensitive to the state of the disorder, they provide an indication of the impact of drug therapy.

[0507] According to a further aspect, there is provided a method of treating cancer in a subject in need thereof, wherein said method comprises the following steps:

[0508] (i) contacting a body fluid sample obtained from the subject with a binding agent which binds to cfDNA-CTCF nucleoproteins;

[0509] (ii) isolating cfDNA-CTCF nucleoproteins bound in step (i);

[0510] (iii) contacting cfDNA isolated in step (ii) with one or more pairs of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs that comprises a CTCF binding site located at a pre-determined genomic location;

[0511] (iv) determining the presence, absence or quantity of DNA sequences of equal to or less than 100 base pairs in length produced in step (iii), as an indicator of the presence of cancer in the subject; and

[0512] (v) administering a treatment if the subject is determined to have cancer.

[0513] According to a further aspect, there is provided a method of treating cancer in a subject in need thereof, wherein said method comprises the following steps:

[0514] (i) contacting a body fluid sample obtained from the subject with a binding agent which binds to cfDNA-CTCF nucleoproteins;

[0515] (ii) isolating cfDNA-CTCF nucleoproteins bound in step (i);

[0516] (iii) contacting cfDNA isolated in step (ii) with one or more pairs of PCR primers directed to a DNA sequence of equal to or less than 80 base pairs that comprises a CTCF binding site located at a pre-determined genomic location;

[0517] (iv) determining the presence, absence or quantity of DNA sequences of equal to or less than 80 base pairs in length produced in step (iii), as an indicator of the presence of cancer in the subject; and

[0518] (v) administering a treatment if the subject is determined to have cancer.

[0519] In one embodiment, the treatment administered is selected from: surgery, radiotherapy, chemotherapy, immunotherapy, hormone therapy and biological therapy.

[0520] According to a further aspect of the invention, there is provided a method of treating cancer in a subject in need thereof, wherein said method comprises the following steps:

[0521] (i) detecting or diagnosing cancer in the subject according to a method described herein; followed by (ii) administering an anti-cancer therapy, surgery or medicament to said subject.

[0522] In one embodiment, the subject is a mammalian subject. In a further embodiment, the subject is selected from a human or animal (such as a companion animal or a mouse) subject. In a yet further embodiment, the subject is a human subject. In one embodiment, the human subject is a non-embryonic subject (i.e. a human at any stage of development, other than an embryo). In a further embodiment, the human subject is an adult subject, i.e. greater than 16 years or age, such as greater than 18, 21 or 25 years of age. In an alternative embodiment, the subject is an animal subject. In a further embodiment, the animal subject is selected from a rodent (e.g. mouse, rat, hamster, gerbil or chipmunk), feline (i.e. a cat), canine (i.e. a dog), equine (i.e. a horse), porcine (i.e. a pig) or bovine (i.e. a cow) subject.

[0523] It will be understood that the uses and methods of the invention may be performed in vitro or ex vivo.

[0524] METHODS OF DETERMINING CANCER ORIGIN

[0525] As described above, the occupation of CTCF binding sites may vary across different tumour types, and therefore the methods of the present invention may be used to determine the cell-of-origin or tissue-of-origin for cancer.

[0526] The cancer may be cancer of unknown primary. Cancers of unknown primary (CLIPs) are histologically confirmed, metastatic malignancies with a primary tumor site that is unidentifiable on the basis of standard evaluation and imaging studies. CUP comprises 2-5% of all diagnosed cancers worldwide and is characterized by early and aggressive metastasis (see Lee, M.S. and Sanoff, H.K., 2020. Bmj, 371).

[0527] The present invention provides a method of determining the cell-of-origin or tissue-of- origin for cancer. The method may comprise the steps of:

[0528] (i) detecting the presence of CTCF occupied cfDNA fragments using a method as described herein; and

[0529] (ii) using the presence of the CTCF occupied cfDNA fragments to determine the cell-of-origin or tissue-of-origin for the cancer.

[0530] The genomic locations used to determine the cell-of-origin or tissue-of-origin may be any described herein. For example, described herein are example cancer-specific CTCF binding sites, which may be used to determine the cell-of-origin or tissue-of-origin. Analysis of CTCF gain of binding patterns in larger numbers of cancer patients samples may reveal further cell-of-origin binding patterns facilitating tumor cell-of-origin analysis.

[0531] According to another aspect of the invention, there is provided a method of determining the cell-of-origin or tissue-of-origin for cancer in a subject, the method comprising:

[0532] (i) contacting a body fluid sample obtained from the subject with a binding agent which binds to cfDNA-CTCF nucleoproteins;

[0533] (ii) isolating cfDNA-CTCF nucleoproteins bound in step (i);

[0534] (iii) detecting the cfDNA isolated from the body fluid sample in step (ii) to determine genomic locations that are selectively occupied by CTCF in the body fluid sample; and

[0535] (iv) using the genomic locations determined in step (iii) to determine the cell- of-origin or tissue-of-origin for the cancer.

[0536] In one embodiment, the cfDNA is detected by sequencing (e.g. by next generation sequencing). The cfDNA may be amplified prior to sequencing, for example, following ligation of adapter oligonucleotides to the DNA fragment. Adapter oligonucleotides may include primer sequences to facilitate amplification of DNA fragments by PCR or primer sequences may be added subsequently. Methods involving adapter oligonucleotides are well known in the art and routinely used to prepare libraries for next generation sequencing. In one embodiment, the cfDNA is not detected by PCR.

[0537] In one embodiment, the cfDNA is detected by PCR (e.g. qPCR, RT-PCR or digital PCR). Suitable panels of primers for determining the cell-of-origin or tissue-of-origin may be designed to detect cancer-specific CTCF binding sites. The primer panels may be any described herein. In one embodiment, the cfDNA is not detected by sequencing.

[0538] In one embodiment, the cell-of-origin is determined to be a lung, colorectal, prostate, breast, blood, hepatic, bile duct, or cervical cell. In one embodiment, the tissue-of-origin is determined to be lung, colorectal, prostate, breast, blood, hepatic, bile duct, or cervical. In one embodiment, the cancer is selected from: lung cancer (such as non-small lung cell cancer), colorectal cancer, prostate cancer, breast cancer, blood cancer (such as leukaemia, in particular Acute Myeloid Lymphoma, or lymphoma in particular NonHodgkin Lymphoma), hepatic cancer, bile duct cancer (such as extrahepatic and intrahepatic bile duct cancer) and cervical cancer. In one embodiment, the cell-of-origin is determined to be a blood cell. In one embodiment, the tissue-of-origin is determined to be blood. In one embodiment, the cancer is blood cancer. In one embodiment, the cancer is leukaemia. In one embodiment, the cancer is Acute Myeloid Lymphoma (AML). In one embodiment, the cancer is lymphoma. In one embodiment, the cancer is Non-Hodgkin Lymphoma.

[0539] In one embodiment, the cell-of-origin is determined to be a lung cell. In one embodiment, the tissue-of-origin is determined to be lung. In one embodiment, the cancer is lung cancer. In one embodiment, the cancer is non-small lung cell cancer.

[0540] In one embodiment, the cell-of-origin is determined to be a colorectal cell. In one embodiment, the tissue-of-origin is determined to be colorectal. In one embodiment, the cancer is colorectal cancer.

[0541] In one embodiment, the cell-of-origin is determined to be a prostate cell. In one embodiment, the tissue-of-origin is determined to be prostate. In one embodiment, the cancer is prostate cancer.

[0542] In one embodiment, the cell-of-origin is determined to be a breast cell. In one embodiment, the tissue-of-origin is determined to be breast. In one embodiment, the cancer is breast cancer.

[0543] In one embodiment, the subject is a mammalian subject. In a further embodiment, the subject is selected from a human or animal (such as a companion animal or a mouse) subject. In a yet further embodiment, the subject is a human subject.

[0544] METHODS OF IDENTIFYING CANCER-ASSOCIATED MUTATIONS

[0545] As described above, mutations to CTCF binding sites have been observed in cancer cells, and can completely suppress CTCF function or induce changes to CTCF binding and functionality.

[0546] The present invention provides a method of identifying cancer-associated mutations in CTCF-binding sites, the method comprising:

[0547] (i) sequencing CTCF occupied cfDNA fragments in a body fluid sample obtained from a subject with cancer using a method as described herein; and

[0548] (ii) comparing the sequences of the CTCF binding sites determined in step (i) with the sequences of corresponding CTCF binding sites from a reference genome, to identify CTCF binding sites which are occupied by CTCF in subject with cancer and which comprise one or more mutations.

[0549] In one embodiment, the cfDNA is sequenced by next generation sequencing. The cfDNA may be amplified prior to sequencing, for example, following ligation of adapter oligonucleotides.

[0550] The reference genome may be any suitable reference genome. For example, the latest genome assembly of the human genome from the Human Genome Project (e.g. GRCh38.p14). The sequences may be compared with the aid of any suitable programme or tool. For example, the sequences of the cfDNA fragments may be aligned with the reference genome to identify any mutations in the CTCF binding sites.

[0551] In one embodiment, the one or more mutations consist of one or more nucleotide substitutions. In one embodiment, the one or more mutations are a single nucleotide change.

[0552] In one embodiment, the cancer is selected from: lung cancer (such as non-small lung cell cancer), colorectal cancer, prostate cancer, breast cancer, blood cancer (such as leukaemia, in particular Acute Myeloid Lymphoma, or lymphoma in particular NonHodgkin Lymphoma), hepatic cancer, bile duct cancer (such as extrahepatic and intrahepatic bile duct cancer) and cervical cancer. In one embodiment, the cancer is blood cancer. In one embodiment, the cancer is leukaemia. In one embodiment, the cancer is Acute Myeloid Lymphoma (AML). In one embodiment, the cancer is lymphoma. In one embodiment, the cancer is Non-Hodgkin Lymphoma. In one embodiment, the cancer is lung cancer. In one embodiment, the cancer is non-small lung cell cancer. In one embodiment, the cancer is colorectal cancer. In one embodiment, the cancer is prostate cancer. In one embodiment, the cancer is breast cancer.

[0553] BODY FLUID BIOMARKERS

[0554] As described above, the present invention provides a new class of body fluid biomarkers for cancer. These biomarkers can be detected by isolating cfDNA-CTCF nucleoproteins and subsequent detection by PCR (e.g. qPCR, RT-PCR or digital PCR).

[0555] The present invention provides a biomarker for cancer, the biomarker consisting of a DNA sequence which comprises a CTCF sequence which is selectively occupied in cancer. The DNA sequence will be one which is not occupied in a healthy subject or a subject with an inflammatory condition. Non-limiting examples of such DNA sequences are provided herein. These selectively occupied sequences have not previously been described because current liquid biopsy methods use double-stranded DNA library preparation methods that are insensitive to small DNA fragments, or these sequences have not been investigated with respect to selective occupation in subject with cancer as opposed to healthy subjects.

[0556] In one embodiment, the DNA sequence comprises the consensus sequence of SEQ ID NO: 50 or 51 , or a nucleotide sequence having one or two nucleotide substitutions compared to the consensus sequence of SEQ ID NO: 50 or 51. In one embodiment, the DNA sequence comprises the consensus sequence of SEQ ID NO: 50 or 51 , or a nucleotide sequence having one nucleotide substitution compared to the consensus sequence of SEQ ID NO: 50 or 51. In one embodiment, the DNA sequence comprises the consensus sequence of SEQ ID NO: 50 or 51.

[0557] In one embodiment, the DNA sequence comprises less than about 100bp, such as less than about 80bp, in particular less than about 60bp. In a further embodiment, the DNA sequence comprises equal to or less than about 80bp. In one embodiment, the DNA sequence has a length of 30 bp or more, 35 bp or more, or 40 bp or more. In one embodiment, the DNA sequence has a length of 100 bp or less, 95 bp or less, 90 bp or less, 85 bp or less, 80 bp or less, 75 bp or less, 70 bp or less, 65 bp or less, or 60 bp or less. In one embodiment, the DNA sequence has a length of from 30 bp to 100 bp, from 30 bp to 95 bp, from 30 bp to 90 bp, from 30 bp to 85 bp, from 30 bp to 80 bp, from 30 bp to 75 bp, or from 30 bp to 70 bp. In one embodiment, the DNA sequence has a length of from 35 bp to 100 bp, from 35 bp to 95 bp, from 35 bp to 90 bp, from 35 bp to 85 bp, from 35 bp to 80 bp, from 35 bp to 75 bp, or from 35 bp to 70 bp.

[0558] In one embodiment, the DNA sequence comprises or consists of (N)xCC(A / G / T)(C / G)NNG(A / G)NGG(A / C / T)(A / G / T)(C / G)(N)y (SEQ ID NO: 61); (N)x(C / G)(A / C / T)(A / G / T)CCN(C / T)CNN(C / G)(A / C / T)GG(N)y (SEQ ID NO: 62); or variants thereof having one or two nucleotide substitutions, wherein x and y are each independently from 0 to 86 and x+y is from 16 to 86. In one embodiment, x and y are each independently from 0 to 86 and x+y is from 21 to 86; x and y are each independently from 0 to 66 and x+y is from 16 to 66; x and y are each independently from 0 to 61 and x+y is from 21 to 61 ; or x and y are each independently from 0 to 56 and x+y is from 26 to 56. (N)x and (N)y may each consist of a region of the human genome flanking a CTCF- binding site.

[0559] In one embodiment, the DNA sequence comprises or consists of any of the following, or variants thereof having one or two nucleotide substitutions:

[0560] In one embodiment, the CTCF binding site comprises one or more mutations compared to the corresponding CTCF binding site in a reference genome. Any method described herein may be used to identify such CTCF binding sites.

[0561] Any suitable methods may be used to determine the presence and / or amount of the biomarker in a body fluid sample, for example any of the methods described herein may be used. In one embodiment, the biomarker is detected by a method comprising:

[0562] (i) contacting a body fluid sample with a binding agent which binds to cfDNA- CTCF nucleoproteins;

[0563] (ii) isolating cfDNA-CTCF nucleoproteins bound in step (i); and

[0564] (iii) detecting the presence or amount of the biomarker in the isolated cfDNA- CTCF nucleoproteins. In one embodiment, the biomarker is detected by PCR (e.g. qPCR, RT-PCR or digital PCR). Suitable primers may be designed to detect the biomarker. The primers may be any described herein. In one embodiment, the biomarker is not detected by sequencing. In one embodiment, the biomarker is detected by sequencing (e.g. by next generation sequencing). The biomarker may be amplified prior to sequencing, for example, following ligation of adapter oligonucleotides. In one embodiment, the biomarker is not detected by PCR.

[0565] The biomarker may be used to detect or diagnose cancer in a subject. For example, any of the method described herein may be used to detect cancer using the biomarker. In one embodiment, the presence and / or amount of the biomarker in a body fluid sample is used to detect cancer. In one embodiment, the present invention provides a method for detecting or diagnosing cancer in a subject, the method comprising the steps of: (i) detecting the presence or amount of the biomarker in a body fluid sample obtained from the subject, optionally using a method as described herein; and (ii) using the presence or amount of the biomarker to detect or diagnose cancer in the subject.

[0566] In one embodiment, the cancer is selected from: lung cancer (such as non-small lung cell cancer), colorectal cancer, prostate cancer, breast cancer, blood cancer (such as leukaemia, in particular Acute Myeloid Lymphoma, or lymphoma in particular NonHodgkin Lymphoma), hepatic cancer, bile duct cancer (such as extrahepatic and intrahepatic bile duct cancer) and cervical cancer. In one embodiment, the cancer is blood cancer. In one embodiment, the cancer is leukaemia. In one embodiment, the cancer is Acute Myeloid Lymphoma (AML). In one embodiment, the cancer is lymphoma. In one embodiment, the cancer is Non-Hodgkin Lymphoma. In one embodiment, the cancer is lung cancer. In one embodiment, the cancer is non-small lung cell cancer. In one embodiment, the cancer is colorectal cancer. In one embodiment, the cancer is prostate cancer. In one embodiment, the cancer is breast cancer.

[0567] CLAUSES

[0568] A set of clauses defining the invention and its preferred aspects is as follows:

[0569] Clause 1. A PCR method for detecting the presence of CCCTC-binding factor (CTCF) occupied cell free DNA (cfDNA) fragments originating from a pre-determined genomic location, in a body fluid sample obtained from a subject, which comprises the steps of: (i) contacting the body fluid sample with a binding agent which binds to cfDNA-CTCF nucleoproteins;

[0570] (ii) isolating cfDNA-CTCF nucleoproteins bound in step (i);

[0571] (iii) contacting cfDNA isolated in step (ii) with one or more pairs of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs or equal to or less than 80 base pairs that comprises a CTCF binding site located at the predetermined genomic location; and

[0572] (iv) determining the presence, absence or quantity of DNA sequences of equal to or less than 80 base pairs in length produced in step (iii), as an indicator of the presence of CTCF-occupied cfDNA fragments originating from the pre-determined genomic location in the body fluid sample.

[0573] Clause 2. The PCR method of clause 1 , wherein the DNA is extracted from the cfDNA-CTCF nucleoproteins isolated in step (ii).

[0574] Clause 3. The PCR method of clause 1 or clause 2, wherein a background level of non-specific cfDNA isolated in step (ii) is determined and subtracted from the presence, absence or quantity of DNA sequences determined in step (iv).

[0575] Clause 4. The PCR method of any one of clauses 1 to 3, wherein a plurality of predetermined genomic locations are detected, such as via multiplex PCR or multiple single PCR assays.

[0576] Clause 5. The PCR method of any one of clauses 1 to 4, wherein the method comprises detecting the presence of CTCF occupied cfDNA fragments originating from a panel of 5 or more pre-determined genomic locations, such as a panel of between 5 and 30 pre-determined genomic locations.

[0577] Clause 6. The PCR method of any one of clauses 1 to 5, wherein the PCR method is quantitative PCR (qPCR) or real-time PCR (rtPCR).

[0578] Clause 7. The PCR method of any one of clauses 1 to 6, wherein the PCR method is digital PCR.

[0579] Clause 8. The PCR method of any one of clauses 1 to 7, wherein the one or more pairs of PCR primers are directed to a DNA sequence comprising less than 70, 60 or 50 base pairs. Clause 9. The PCR method of any one of clauses 1 to 8, wherein the pre-determined genomic location is selectively occupied by CTCF in a disease state.

[0580] Clause 10. The PCR method of clause 9, wherein the disease state is cancer.

[0581] Clause 11. The PCR method of clause 10, wherein the cancer is selected from the group consisting of: lung cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, blood cancer (such as leukaemia or lymphoma) and skin cancer (such as melanoma).

[0582] Clause 12. The PCR method of any one of clauses 1 to 11 , wherein the cfDNA-CTCF nucleoproteins bound by the binding agent in step (i) are washed with a buffer solution containing at least 1 % concentration of detergent.

[0583] Clause 13. The PCR method of any one of clauses 1 to 12, wherein the binding agent which binds to cfDNA-CTCF nucleoproteins is an antibody or an antigen binding fragment thereof.

[0584] Clause 14. The PCR method of any one of clauses 1 to 13, wherein the binding agent which binds to cfDNA-CTCF nucleoproteins is immobilised on a solid support, in particular a magnetic bead.

[0585] Clause 15. The PCR method of any one of clauses 1 to 14, wherein the body fluid sample is a blood, serum or plasma sample.

[0586] Clause 16. The PCR method of clause 15, wherein the body fluid sample contains a cfDNA-CTCF crosslinked nucleoprotein, in particular wherein the body fluid sample is an EDTA plasma sample wherein the cfDNA-CTCF nucleoprotein is cross-linked.

[0587] Clause 17. The PCR method of any one of clauses 1 to 16, wherein the predetermined genomic location is associated with one or more of the following genes: CATSPERE, CTNNA2, NOL10, DIAPH1 , TMEM260, GALC, OSBPL6, PI15, ANO4, FAM41AY1 , LINC02787, NR_038432.1 , GIPC2, CACNA1A, GNPD1 , TEX33, TRAV4, HEYL, FAM124B, COL21A1 , PLEKHG7, EPHA5, FAM149A, TFAP2B, LINC02934, SKAP1 , CDH5, ENSG00000229727, SNORA70, TBC1 D21 , TGIF2LX. Clause 18. The PCR method of clause 17, wherein the pre-determined genomic location is associated with one or more of the following genes: CATSPERE, TMEM260, LINC02787, NR_038432.1 , NOL10, CTNNA2, DIAPH1 , GALC, PI15 and ANO4.

[0588] Clause 19. The PCR method of any one of clauses 1 to 16, wherein the predetermined genomic locations is selected from one or more of the following regions (in genome assembly GRCh38 / hg38): chr1 :244603555-244603648; chr2: 10668571 - 10668663; chr2:79219540-79219628; chr5:141567670-141567748; chr14:56491464- 56491546; chr14:87896549-87896634; chr2: 178280431 -178280518; chr8: 74853546- 74853641 ; chr12:100741271-100741352; chrY: 17430722-17430795; chr1 : 90579362- 90579440; chr11 :34926223-34926312; chr1 :78130901 -78130992; chr19: 13262535- 13262621 ; chr5:141992498-141992587; chr22:36986359-36986452; chr14:21734605- 21734701 ; chr1 : 39609752-39609824; chr2:224369183-224369264; chr6:55980780- 55980855; chr12:92570523-92570593; chr4:64713399-64713474; chr4: 186150099- 186150166; chr6:50792057-50792142; chr2:65573278-65573362; chr17:48336036- 48336115; chr16:66275978-66276056; chr2:7427441 -7427517; chr8: 5062480-5062559; chr15:73874049-73874135; and chrX:89757930-89758000.

[0589] Clause 20. The PCR method of clause 19, wherein the pre-determined genomic location is selected from one or more of the following regions (in genome assembly GRCh38 / hg38): chr1 :244603555-244603648, chr14:56491464-56491546, chr1 : 90579362-90579440, chr11 :34926223-34926312, chr2: 10668571 -10668663, chr2:79219540-79219628, chr5:141567670-141567748, chr14:87896549-87896634, chr8:74853546-74853641 , and chr12:100741271-100741352.

[0590] Clause 21. The PCR method of any one of clauses 1 to 20, wherein the CTCF occupied cfDNA fragments are detected or measured as one of a panel of measurements.

[0591] Clause 22. The PCR method of any one of clauses 1 to 21 , wherein the subject is a human or an animal subject.

[0592] Clause 23. A PCR method for detecting the presence of CTCF occupied cfDNA fragments originating from a pre-determined genomic location, in an isolated CTCF- cfDNA preparation produced from a body fluid sample obtained from a subject, which comprises the steps of: (i) contacting a first sample of the body fluid with a candidate binding agent which binds to cfDNA-CTCF nucleoproteins, and contacting a second sample of the body fluid with a control binding agent which does not specifically bind to cfDNA-CTCF nucleoproteins;

[0593] (ii) isolating cfDNA from the first sample bound by the candidate binding agent and from the second sample bound by the control binding agent in step (i);

[0594] (iii) contacting isolated cfDNA from the first sample and the second sample with one or more pairs of PCR primers directed to a DNA sequence of less than 80 base pairs that includes a CTCF binding site located at the pre-determined genomic location;

[0595] (iv) determining the presence, absence or quantity of DNA sequences of equal to or less than 100 base pairs or equal to or less than 80 base pairs in length produced in step (iii) by subtracting the quantity of DNA in the second sample from the first sample; and

[0596] (v) using the presence, absence or quantity of DNA determined in step (iv), as an indicator of whether, or how many, CTCF-occupied cfDNA fragments originating from the pre-determined genomic location were present in the body fluid sample.

[0597] Clause 24. A method for determining a genomic location comprising a CTCF binding site that is selectively bound by CTCF during a cancer, comprising:

[0598] (i) contacting a first body fluid sample obtained from a subject diagnosed with the cancer with a binding agent which binds to cfDNA-CTCF nucleoproteins, and contacting a second body fluid sample obtained from a subject with no known cancer or a different cancer with a binding agent which binds to cfDNA-CTCF nucleoproteins;

[0599] (ii) isolating cfDNA-CTCF nucleoproteins from the first and second sample bound in step (i);

[0600] (iii) sequencing cfDNA isolated from the first and second sample in step (ii) to determine genomic locations that are selectively occupied by CTCF in the first sample but not in the second sample, to determine genomic locations comprising a CTCF binding site that is selectively bound by CTCF during the cancer.

[0601] Clause 25. A method of detecting cancer in a subject which comprises the steps of:

[0602] (i) detecting the presence of CTCF occupied cfDNA fragments using the PCR method of any one of clauses 1 to 22; and

[0603] (ii) using the presence of the CTCF occupied cfDNA fragments as an indicator of the presence of cancer in the subject. Clause 26. A method for the assessment of subject for suitability for a cancer treatment which comprises the steps of:

[0604] (i) detecting the presence of CTCF occupied cfDNA fragments using the PCR method of any one of clauses 1 to 22; and

[0605] (ii) using the CTCF occupied cfDNA fragments detected in step (i) as a parameter for the selection of a suitable treatment for the subject.

[0606] Clause 27. A method for monitoring a treatment of a subject diagnosed with cancer which comprises the steps of:

[0607] (i) detecting the presence of CTCF occupied cfDNA fragments using the PCR method of any one of clauses 1 to 22;

[0608] (ii) repeating the detection of step (i) in a body fluid sample obtained from the subject on one or more occasions; and

[0609] (iii) using any changes in the presence of CTCF occupied cfDNA fragments determined in step (i) compared to step (ii) as a parameter for any changes in the condition of the subject.

[0610] Clause 28. A kit for the detection of a cell free chromatin fragment comprising CTCF and a DNA fragment as a combination biomarker which comprises a binding agent for CTCF together with reagents for detecting the presence of CTCF occupied cfDNA fragments using the PCR method of any one of clauses 1 to 22.

[0611] Clause 29. A method of treating cancer in a subject in need thereof, wherein said method comprises the following steps:

[0612] (i) contacting a body fluid sample obtained from the subject with a binding agent which binds to cfDNA-CTCF nucleoproteins;

[0613] (ii) isolating cfDNA-CTCF nucleoproteins bound in step (i);

[0614] (iii) contacting cfDNA isolated in step (ii) with one or more pairs of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs or equal to or less than 80 base pairs that comprises a CTCF binding site located at a pre-determined genomic location;

[0615] (iv) determining the presence, absence or quantity of DNA sequences of equal to or less than 100 base pairs or equal to or less than 80 base pairs in length produced in step (iii), as an indicator of the presence of cancer in the subject; and

[0616] (v) administering a treatment if the subject is determined to have cancer. Clause 30. The method of clause 29, wherein the cancer is selected from the group consisting of: lung cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, blood cancer (such as leukaemia or lymphoma) and skin cancer (such as melanoma).

[0617] Clause 31 . The method of clause 30, wherein the treatment is selected from: surgery, radiotherapy, chemotherapy, immunotherapy, hormone therapy and biological therapy.

[0618] Clause 32. A biomarker panel of genomic locations comprising CTCF binding sites associated with five or more of the following genes: CATSPERE, CTNNA2, NOL10, DIAPH1, TMEM260, GALC, OSBPL6, PI15, ANO4, FAM41AY1 , LINC02787, NR_038432.1 , GIPC2, CACNA1A, GNPD1 , TEX33, TRAV4, HEYL, FAM124B, COL21A1 , PLEKHG7, EPHA5, FAM149A, TFAP2B, LINC02934, SKAP1 , CDH5, ENSG00000229727, SNORA70, TBC1 D21 , TGIF2LX.

[0619] Clause 33. The biomarker panel of clause 32, wherein the genomic locations are selected from five or more of the following regions (in genome assembly GRCh38 / hg38): chr1 :244603555-244603648; chr2: 10668571-10668663; chr2:79219540-79219628; chr5: 141567670- 141567748; chr14: 56491464-56491546; chr14: 87896549-87896634; chr2: 178280431 -178280518; chr8: 74853546-74853641 ; chr12:100741271-100741352; chrY: 17430722-17430795; chr1 : 90579362-90579440; chr11 :34926223-34926312; chr1 : 78130901 -78130992; chr19: 13262535- 13262621 ; chr5: 141992498-141992587; chr22:36986359-36986452; chr14:21734605-21734701 ; chr1 : 39609752-39609824; chr2:224369183-224369264; chr6:55980780-55980855; chr12:92570523-92570593; chr4:64713399-64713474; chr4: 186150099-186150166; chr6:50792057-50792142; chr2:65573278-65573362; chr17:48336036-48336115; chr16:66275978-66276056; chr2:7427441 -7427517; chr8:5062480-5062559; chr15:73874049-73874135; and chrX:89757930-89758000.

[0620] Clause ...

Claims

CLAIMS1. A PCR method for detecting the presence of CCCTC-binding factor (CTCF) occupied cell free DNA (cfDNA) fragments originating from a pre-determined genomic location, in a body fluid sample obtained from a subject, which comprises the steps of:(i) contacting the body fluid sample with a binding agent which binds to cfDNA-CTCF nucleoproteins;(ii) isolating cfDNA-CTCF nucleoproteins bound in step (i);(iii) contacting cfDNA isolated in step (ii) with one or more pairs of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs or equal to or less than 80 base pairs that comprises a CTCF binding site located at the predetermined genomic location; and(iv) determining the presence, absence or quantity of DNA sequences of equal to or less than 100 base pairs or equal to or less than 80 base pairs in length produced in step (iii), as an indicator of the presence of CTCF-occupied cfDNA fragments originating from the pre-determined genomic location in the body fluid sample.

2. The PCR method of claim 1 , wherein the DNA is extracted from the cfDNA-CTCF nucleoproteins isolated in step (ii).

3. The PCR method of claim 1 or claim 2, wherein a background level of non-specific cfDNA isolated in step (ii) is determined and subtracted from the presence, absence or quantity of DNA sequences determined in step (iv).

4. The PCR method of any one of claims 1 to 3, wherein the method comprises detecting the presence of CTCF occupied cfDNA fragments originating from a panel of 5 or more pre-determined genomic locations, such as a panel of between 5 and 30 predetermined genomic locations.

5. The PCR method of any one of claims 1 to 4, wherein the PCR method is quantitative PCR (qPCR), real-time PCR (rtPCR) or digital PCR.

6. The PCR method of any one of claims 1 to 5, wherein the one or more pairs of PCR primers are directed to a DNA sequence comprising less than 70, 60 or 50 base pairs.

7. The PCR method of any one of claims 1 to 6, wherein the pre-determined genomic location is selectively occupied by CTCF in a disease state.

8. The PCR method of claim 7, wherein the disease state is cancer.

9. The PCR method of claim 8, wherein the cancer is selected from the group consisting of: lung cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, blood cancer (such as leukaemia or lymphoma) and skin cancer (such as melanoma).

10. The PCR method of any one of claims 1 to 9, wherein the cfDNA-CTCF nucleoproteins bound by the binding agent in step (i) are washed with a buffer solution containing at least 1 % concentration of detergent.

11. The PCR method of any one of claims 1 to 10, wherein the binding agent which binds to cfDNA-CTCF nucleoproteins is an antibody or an antigen binding fragment thereof.

12. The PCR method of any one of claims 1 to 11 , wherein the binding agent which binds to cfDNA-CTCF nucleoproteins is immobilised on a solid support, in particular a magnetic bead.

13. The PCR method of any one of claims 1 to 12, wherein the body fluid sample is a blood, serum or plasma sample.

14. The PCR method of claims 1 to 13, wherein the body fluid sample contains a cfDNA-CTCF crosslinked nucleoprotein, in particular wherein the body fluid sample is an EDTA plasma sample wherein the cfDNA-CTCF nucleoprotein is cross-linked.

15. The PCR method of any one of claims 1 to 14, wherein the pre-determined genomic location is associated with one or more of the following genes: (a) CATSPERE, CTNNA2, NOL10, DIAPH1 , TMEM260, GALC, OSBPL6, PI15, ANO4, FAM41AY1 , LINC02787, NR_038432.1 , GIPC2, CACNA1A, GNPD1 , TEX33, TRAV4, HEYL, FAM124B, COL21A1 , PLEKHG7, EPHA5, FAM149A, TFAP2B, LINC02934, SKAP1 , CDH5, ENSG00000229727, SNORA70, TBC1 D21 , TGIF2LX; and / or (b) CTNNA2, GCA, TMEM44, ENSG00000286456, SLC10A5P1 , TRAV4, ENSG00000283445, UST, DMRTA1 , GALNT8, FAM174B, SHLD1 , NAP1 L4P2, MMADHC, PTPRG, BASP1 , PDE4D, SPMIP4, CLNS1A, SKAP1 , IKBKE, ARHGAP15, DPP4, ZNF622, MINDY3,TMEM236, SMPD1, ENSG00000260945, NLRP1, ARSH, R0B01, ALCAM, MTCO3P27, EDIL3, EX0C2, IFNGR1, DLD, MKX, QSER1, CTBP2P6, ZDHHC17, MIP0L1, LINC01418, TERF2IP, C0R02B, GIPC2, ENSG00000230472, PHGDH, CATSPERE, ARMCX4, 0SBPL6, CAST, PHEX, ZNF157, TIMM50, LINC02583, PTMA, HEPACAM2, IFNWP4, LINC02572, PARD3, GRIK5, IFIH1, ADAM28, LINC00589, ENSG00000228566, ELF5, PPFIBP1, WIPF1, MCCC1, DNAH10, CREG1, TPRG1, GNPDA1, MTND2P8, LINC02446, CPS1, ERBB4, OR7E43P, TMEM260, DUT, ENSG00000226530, TANC1, MAPK13, ENSG00000232436, AMBRA1, CENPSCORT, TMEM182, LHFPL3AS1, JAK2, TECTA, CLMP, NAA16, SDK2, VRK3, YTHDF1, SBF2, MSCAS1, GAPDH, LINC02389, FCGR2A, ENSG00000287730, TP5BI11, SINHCAF, ENSG00000275393, FCGRT, PROSER2, LINC02535, LPAL2, ENSG00000230649, TPPP2, LRP8, CYP1B1, KCNIP4, ANK2, PDE4D, KRT18P54, MRPL15, GDF6, MPP7, SCGB1A1, LINC00937, SLAMF7, LILRP1, SDE2, NID1, FGFBP2, SPOCK3, RNF14, OR4S1, SNX20, RGS9, SPTLC3, NOL10, DPPA3P2, CCDC8, TLX1, FERMT1, GPR27, SIRT6, LINC01307, IFI6, CCNA1, UNC119, TGM6, IFI16, MTARC1, MY03BAS1, DDX3ILA1, NKAIN1, LINC02810, SERINC2, ENSG00000236069, PREP, CEACAM1, VAV3, ENSG00000235819, PLXNA4, NPM1P46, RN7SL361P, ENSG00000266573, GLDC, SWAP7Q, QOQ8A, ENSG00000225620, RPL7AP47, SMUG1, TTLL11, ENSG00000228852, LRRC7, VAV3, GPD1L, SPICE1, CEP63, IL20RB, FAUP2, LINC02357, LINC02503, TICAM2, PKHD1, ENSG00000218813, SYTL3, PACRG, HAP1, PLXNA4, RN7SL135P, NDUFB9P3, ENSG00000252852, DSCC1, ENSG00000235819, ENSG00000279503, SLC16A12, HECTD2, PLEKHG7,LINC02409, LINC02300, SLC25A21, LCTL, PHLPP1, PHLPP1, ZFP64, MTOR, GLIS1, RN7SKP272, ENSG00000229727, RN7SL361P, POU3F3, CHCHD5, ITGB6, NPM1P46, FAM124B, ZNF876P, UCHL1, ENSG00000266573, ENSG00000225620, QOQ8A, TFAP2B, GLDC, RPL7AP47, SWAP7Q, ENSG00000228852, TTLL11, SMUG1, ANO4, EPHA5, NR_038432.1.

16. The PCR method of any one of claims 1 to 14, wherein the pre-determined genomic locations is selected from one or more of the following regions: (a) chr1:244603555-244603648; chr2: 10668571 -10668663; chr2:79219540-79219628; chr5: 141567670- 141567748; chr14: 56491464-56491546; chr14: 87896549-87896634; chr2: 178280431 -178280518; chr8:74853546-74853641; chr12:100741271-100741352; chrY: 17430722-17430795; chr1: 90579362-90579440; chr11:34926223-34926312; chr1:78130901 -78130992; chr19: 13262535-13262621; chr5:141992498-141992587; chr22:36986359-36986452; chr14:21734605-21734701 ; chr1 : 39609752-39609824; chr2:224369183-224369264; chr6:55980780-55980855; chr12:92570523-92570593;chr4:64713399-64713474; chr4:186150099-186150166; chr6:50792057-50792142; chr2:65573278-65573362; chr17:48336036-48336115; chr16:66275978-66276056; chr2:7427441 -7427517; chr8:5062480-5062559; chr15:73874049-73874135; and chrX:89757930-89758000; and / or (b) chr2:79219519-79219648, chr2: 162301295- 162301441 , chr3: 194594813-194594949, chr7:64625243-64625355, chr8:81641590- 81641720, chr14:21734606-21734734, chr1 :58739989-58740101 , chr6: 149028960- 149029063, chr9:22452551 -22452664, chr12:4849148-4849254, chr15:92797259- 92797393, chr20:5788487-5788587, chrX:106481411-106481533, chr2: 149561424- 149561553, chr3:61381935-61382023, chr5: 17190626-17190742, chr5:59390194- 59390288, chr7:25160122-25160245, chr1177619731 -77619846, chr17:48336001-48336147, chr1 :206461901 -206462094, chr2:143110966-143111104, chr2:162123970- 162124051 , chr5: 16456356-16456495, chr10:15902711-15902826, chr10:17742984- 17743122, chr11 :6379394-6379487, chr16: 17579398-17579529, chr17:5592340- 5592644, chrX: 3035865-3036000, chr3:78659532-78659619, chr3: 105518948-105519094, chr4:64585094-64585216, chr5:83927264-83927382, chr6:593261-593392, chr6:137314444-137314535, chr7: 107874746-107874889, chr10:27656752- 27656912, chr11 :32960716-32960828, chr11 :43567527-43567641 , chr12:76791108- 76791220, chr14:37230722-37230848, chr15:81939739-81939846, chr16:75789293- 75789426, chr15:68554138-68554266, chr1 :78130884-78131005, chr6: 50602320- 50602449, chr1 : 119675667-119675836, chr1 :244603541 -244603674, chrX: 101415625- 101415728, chr2: 178280411-178280526, chr5: 96752586-96752688, chrX:22081630- 22081732, chrX: 47384626-47384769, chr19: 39474211-39474377, chr2:46464602- 46464705, chr2:231715363-231715468, chr7: 93228366-93228499, chr9:21111090- 21111213, chr2:129849717-129849817, chr10:34638577-34638699, chr19:42028327- 42028524, chr2:162298074-162298189, chr8:24229478-24229598, chr8:29647427- 29647523, chr10:63720138-63720300, chr11 :34492959-34493085, chr12:27652288- 27652426, chr2:174700116-174700251 , chr3: 183122469-183122581 , chr12: 123905436-123905607, chr1 : 167540493-167540653, chr3: 189148131-189148251 , chr5:141992479-141992603, chr9:78821242-78821375, chr12: 10562452- 10562563, chr2:210577647-210577793, chr2:212567753-212567856, chr4:4183014- 4183121 , chr14:56491434-56491551 , chr15:48352569-48352712, chrX:51461172- 51461282, chr2:159118703-159118816, chr6: 36095298-36095473, chr1 :221435546- 221435673, chr11 :46537952-46538113, chr1 : 10428186-10428296, chr2: 102840031 - 102840180, chr7: 104848545-104848654, chr9:4988029-4988170, chr11 :121194847- 121194979, chr11 :123247327-123247457, chr13:41353128-41353267, chr17:73369977-73370136, chr19:49983756-49983882, chr20:63223466-63223579, chr11 :10077000-10077143, chr8:71975358-71975486, chr12:6537982-6538105,chr12:64891000-64891169, chr1 :161487131-161487247, chr4: 155442425-155442520, chr11 :44911428-44911580, chr12:31272008-31272161, chr16:85717990-85718123, chr19:49547082-49547241 , chr10: 11819934-11820065, chr6:85355152-85355270, chr6: 160519981 -160520091 , chr7: 136009627-136009724, chr14:21014774-21014934, chr1 :53393434-53393577, chr2:38115355-38115496, chr4:21789365-21789486, chr4:113394834-113394942, chr5: 59673572-59673710, chr6: 85323564-85323677, chr8:54169437-54169546, chr8:96180035-96180344, chr10:28241944-28242032, chr11 :62380869-62381016, chr12:8396766-8396964, chr1 : 160726939-160727057, chr19:54676361 -54676503, chr1 :225976081 -225976216, chr1 :236103745-236103862, chr4:16026149-16026413, chr4: 167021693-167021794, chr5:141960806-141960987, chr11 :48307888-48308019, chr16:50665497-50665627, chr17:65198881-65199032, chr20: 13119634-13119763, chr2: 10668563-10668699, chr14:36374168-36374282, chr19:46402330-46402434, chr10: 101161791-101161998, chr20:6142524-6142649, chr3:71729971 -71730106, chr19:4204720-4204856, chr1 :101288544-101288669, chr1 :27669254-27669382, chr13:36420493-36420628, chr17:28540839-28540954, chr20:2351923-2352109, chr1 : 158998804-158998962, chr1 :220810250-220810376, chr2: 170409173-170409293, chr7: 78040335-78040434, chr1 :31162784-31162912, chr1 : 17661594- 17661807, chr1 : 31422842-31422978, chr1 : 192445984- 192446109, chr6: 105424291-105424437, chr19:42529383-42529506, chr1 : 107782340-107782444, chr9:86414420-86414532, chr7:132188991-132189067, chr2:197374043-197374122, chr2:60642442-60642560, chr18:24736101-24736161 , chr9:6594697-6594768, chr11 :9675318-9675421 , chr1 :226944700-226944781 , chr1 :202640729-202640819, chr9:14216485-14216587, chr12:54188268-54188338, chr9:121838761-121838825, chr1 : 95264973-95265040, chr1 :69737726-69737836, chr1 : 107782340-107782444, chr3: 32144255-32144365, chr3: 113494238- 113494353, chr3: 134474064- 134474175, chr3: 137105390-137105526, chr3: 181326780-181326885, chr4:26082030-26082146, chr4: 103934283- 103934454, chr5: 115586654- 115586772, chr6: 52049619-52049757, chr6:63764478-63764585, chr6:158691470-158691594, chr6: 162908032-162908121 , chr7: 130832002- 130832124, chr7: 132188991 - 132189067, chr8:63262036-63262139, chr8: 110830607-110830727, chr8: 117329067-117329210, chr8: 119867220-119867390, chr9:86414420-86414532, chr9:90142700-90142839, chr10:89563998- 89564154, chr10:91389742-91389869, chr12:92570492-92570605, chr12:97308762- 97308849, chr14:28635549-28635670, chr14:37149849-37149969, chr15:665671 SO- 66567354, chr18:62958013-62958123, chr18:62982806-62982908, chr20:52312263- 52312398, chr1 :11241826-11241943, chr1 : 53754832-53754948, chr1 : 89987314- 89987463, chr2:7427425-7427538, chr2:60642442-60642560, chr2: 104849680-104849792, chr2:112582400-112582523, chr2:160110087-160110199,chr2: 197374043-197374122, chr2:224369161 -224369299, chr4:242369-242480, chr4:41281166-41281273, chr4:122758089-122758199, chr4:166165962-166166073, chr4:169810464-169810607, chr5:32489802-32489952, chr5:123371732-123371827, chr5: 172370813-172370900, chr6:33123829-33123955, chr6:49849713-49849822, chr6: 110942102-110942167, chr6: 113909396-113909512, chr6: 159008718-159008830, chr10:25700952-25701067, chr10:92885025-92885143, chr10:94630132- 94630263, chr11 :30346226-30346339, chr11 :110433891-110434020, chr11 : 124586369-124586468, chr12 : 102702668- 102702830, chr12: 111451647- 111451929, chr13:33228694-33228791 , chr13:50085114-50085230, chr13:53250379- 53250572, chr13:57627719-57627850, chr13: 100722041 -100722178, chr14:20327405- 20327535, chr14:24735042-24735188, chr15:31974953-31975090, chr18:24736101- 24736161 , chr19:54674154-54674274, chr22:22201213-22201344, chrX:22136053- 22136155, chrX: 129754802-129754877, chrX: 130079923-130080041 , chr1 : 19918128- 19918255, chr1 :24587331 -24587473, chr1 :39720138-39720288, chr1 :65958784- 65959095, chr1 :74689941 -74690070, chr1 :88877501 -88877633, chr1 : 161227460- 161227587, chr1 :202640729-202640819, chr1 :226944700-226944781 , ch r2: 17646148- 17646327, ch r2: 227862361 -227862483, chr2:235594168-235594292, chr2:238462941- 238463069, chr3: 124079474-124079582, chr3:156541160-156541317, chr3: 194404789- 194404904, chr3: 195522387- 195522501 , chr4: 16794985- 16795080, chr4:27048448-27048583, chr4:52026152-52026244, chr4: 52376236-52376358, chr4: 140999943-141000112, chr4: 146104133-146104288, chr4: 158850959-158851065, chr4: 165464923-165465025, chr5:18314314-18314440, chr5:28014922- 28015045, chr5: 74648924-74649026, chr5: 102223966-102224084, chr5: 129664478- 129664599, chr5: 155139754-155139857, chr6: 12893990-12894132, chr6: 30380603- 30380684, chr6: 50792043-50792155, chr6:51855414-51855525, chr6: 123415992- 123416085, chr7:10412547-10412686, chr7:25180362-25180528, chr7: 38374563- 38374659, chr7:91830142-91830274, chr7:94395571 -94395689, chr8:4833976-4834088, chr8:95758783-95758908, chr8:119070591-119070729, chr9:6354233- 6354348, chr9:6594697-6594768, chr9:14216485-14216587, chr9: 17063671 -17063809, chr9: 19028989-19029108, chr10:3192966-3193102, chr10:16828259- 16828398, chr10:25726026-25726156, chr10:87656598-87656695, chr10:87735375- 87735496, chr10:125098674-125098794, chr11 :9675318-9675421 , chr11 :17230210- 17230310, chr11 :32323655-32323771 , chr11 :72667585-72667696, chr11 : 124402455- 124402570, chr12:20946875-20946995, chr12:53791868-53791960, chr12:55619731- 55619974, chr12:125403356-125403461 , chr12: 126069664-126069777, chr13:85828223-85828338, chr14:45426816-45426941 , chr14:93655215-93655320, chr15:44288413-44288574, chr15:90956673-90956821 , chr16:24253220-24253372,chr16:63361142-63361267, chr16:66728261 -66728409, chr17:35312798-35312956, chr17:78013974-78014108, chr18:43571212-43571318, chr18:65781879-65782020, chr20:36466920-36467064, chr22:25093742-25093869, chrX:51614499-51614639, chrX: 110156310-110156388, chr22_KI270733v1_random: 136322-136475, chr1 : 32272584-32272646, chr1 :86742093-86742193, chr1 :88998835-88998937, chr1 : 95264973-95265040, chr1 : 163410082- 163410225, chr1 : 169055544- 169055615, chr1 :236452124-236452243, chr1 :240149603-240149760, chr1 :240189505- 240189629, chr2: 18378180-18378279, chr2:38581666-38581810, chr2 : 152861493- 152861578, chr2:166539491-166539609, chr2:176814897-176815015, chr2:182186287-182186395, chr2:227890621 -227890778, chr3:87298250-87298349, chr3:89211716-89211800, chr3: 109732032-109732122, chr3: 134492629-134492738, chr3: 156509828- 156509965, chr3: 157137568- 157137691 , chr3: 161273765-161273866, chr3:166013225-166013311 , chr4:23135919-23136011 , chr4:48087272- 48087368, chr4:70183776-70183887, chr4:73019582-73019720, chr4:86044122- 86044254, chr4: 174365159-174365240, chr5:5141805-5141917, chr5: 80083642- 80083758, chr5:81299634-81299801 , chr5:112175136-112175244, chr5: 146662279- 146662392, chr5:148733715-148733808, chr5: 154157507-154157598, ch r6: 35449508- 35449669, chr6:52021110-52021230, chr6:56902964-56903131 , chr6: 70706662- 70706775, chr6: 74091490-74091627, chr6:112180232-112180339, chr6: 135451738- 135451874, ch r7: 148504024- 148504134, chr7:156582102-156582204, chr8: 12866970- 12867090, chr8:69663836-69663985, chr8:75257410-75257540, chr8:80612676- 80612794, chr8:90948403-90948519, chr8: 132651571 -132651698, ch r9: 13427664- 13427772, chr9: 15873723-15873819, chr9: 17462998-17463108, chr9: 121400273- 121400386, chr9:121838761-121838825, chr10:61284412-61284550, chr10:93352770- 93352892, chr10:116490211 -116490321 , chr10:125982446-125982551 , chr11 :6738008-6738119, chr11 :84384764-84384893, chr11 :88092327-88092414, chr11 :94519623-94519738, chr11 :96464846-96464973, chr11 :115117490-115117553, chr11 :128414446-128414512, chr12:7642988-7643098, chr12:21680506-21680612, chr12:29519728-29519832, chr12:34034045-34034152, chr12:54188268-54188338, chr12:58346176-58346282, chr12:100741240-100741368, chr12:119160250-119160324, chr13:97132337-97132465, chr14: 19878197-19878283, chr14:25400748- 25400891 , chr14:53055029-53055173, chr14:64303608-64303708, chr14:82000025- 82000111 , chr14:98138734-98138826, chr15:85505208-85505330, chr15:98881743- 98881866, chr16:16449419-16449513, chr17:700647-700751 , chr17: 16258067- 16258156, chr18:24489020-24489155, chr18:31819747-31819885, chr19:28325490- 28325587, chr19:47125320-47125481 , chrX:11144386-11144529, chrX: 87299704- 87299805, chrX: 133670537-133670648, chrX: 152758817-152758894, chr1 :2091761-2091844, chr1 :5225260-5225331, chr1 :110555394-110555522, chr1 : 158036123- 158036245, chr1 : 176559961 -176560058, chr1:207405060-207405185, chr1:238188421 -238188505, chr2:39059461 -39059589, chr2:60707803-60707914, chr2:62412204-62412318, chr2: 106791905-106792003, chr2:151437690-151437807, chr2: 158010423-158010526, chr2:164717151-164717267, chr2: 179440792-179440892, chr2: 181715178-181715317, chr2: 197707735-197707844, chr2:232270870-232270944, chr2:241078520-241078659, chr3: 13600010-13600082, chr3:26712570-26712658, chr3:65785206-65785301 , chr3: 98039424-98039547, chr3: 146065649- 146065763, chr3: 147191901 - 147192041 , chr3: 173584256-173584361, chr4:38856812-38856976, chr4:64713379-64713507, chr4:67746823- 67746964, chr4:147097166-147097267, chr4:163484959-163485038, chr4: 185634052- 185634304, chr5: 19469171 -19469243, chr5:36870575-36870732, chr5: 52657324- 52657444, chr5:66307768-66307915, chr5: 103335135-103335205, chr6:47631424- 47631547, chr6:52945168-52945233, chr6:75893429-75893588, chr6: 93664709- 93664784, chr6:115244749-115244826, chr6:116287793-116287931 , chr6: 158605406- 158605520, chr6: 170561934-170561998, chr7:26198200-26198319, chr7: 50142454- 50142556, chr7:81482732-81482834, chr7: 154504103-154504239, chr8:9831026- 9831238, chr9:8086869-8086940, chr10:117940922-117940998, chr11 :34926239- 34926356, chr11 :82682336-82682444, chr11 :121765670-121765767, chr12:12376782- 12376859, chr12:20950374-20950501, chr12:23949721 -23949794, chr12:24095121- 24095194, chr12:29889866-29889994, chr12:72987449-72987521, chr12:73391018- 73391090, chr12:78146406-78146532, chr12:90959676-90959826, chr13:43908534- 43908635, chr13:67238415-67238557, chr13:84896266-84896371, chr13:102141733- 102141898, chr13:110839093-110839188, chr14:78082948-78083033, chr14: 100624900-100625009, chr15:24585660-24585745, chr15:31880238-31880317, chr15:41031918-41032105, chr15:69322673-69322785, chr15:80419036-80419163, chr15:99704486-99704631, chr17:31216272-31216386, chr17:48728857-48728929, chr17:74612182-74612276, chr17:79893762-79893854, chr18:25853208-25853305, chr18:64037936-64038067, and chr19:54080140-54080207.

17. A method, comprising:(i) contacting a body fluid sample with an antibody which binds a cfDNA- CTCF nucleoprotein complex;(ii) isolating the antibody-bound complexes formed in step (i) and extracting cfDNA from the isolated complexes;(iii) contacting the extracted cfDNA with one or more pairs of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs or equal to or lessthan 80 base pairs that comprises a CTCF binding site located at a pre-determined genomic location to produce DNA amplicons of equal to or less than 100 base pairs or equal to or less than 80 base pairs in length; and(iv) determining the presence, absence or quantity of the DNA amplicons in the body fluid sample.

18. The method of claim 17, wherein the antibody specifically binds CTCF in the CTCF nucleoprotein complex.

19. The method of claim 17 or 18, wherein said contacting in step (i) comprises contacting the body fluid sample with an antibody bound to a solid support.

20. The method of claim 19, wherein the solid support is a bead and the antibody is associated with the bead.

21. The method of any one of claims 17 to 20, wherein said contacting in step (i) comprises contacting a first portion of a body fluid sample with an antibody that specifically binds a cfDNA-nucleoprotein complex and contacting a second portion of the body fluid sample with a binding agent that does not specifically bind the cfDNA- nucleoprotein complex.

22. The method of any one of claims 17 to 21 , wherein said isolating in step (ii) comprises capturing the antibody-bound complexes on a solid support.

23. The method of any one of claims 17 to 22, wherein said extracting in step (ii) comprises contacting the isolated antibody-bound complexes with a reagent to extract cfDNA from the complexes.

24. The method of any one of claims 17 to 23, wherein said contacting in step (iii) comprises contacting the extracted cfDNA with one or more pairs of PCR primers configured to produce amplicons of equal to or less than 100 base pairs or equal to or less than 80 base pairs in length from pre-determined genomic locations that are each selectively occupied by CTCF in cancer.

25. The method of any one of claims 17 to 24, wherein said contacting in step (iii) comprises contacting the extracted cfDNA with one or more pairs of PCR primers configured to produce amplicons of equal to or less than 100 base pairs or equal to or less than 80 base pairs in length from pre-determined genomic locations that are each selectively occupied by CTCF in blood cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, and / or skin cancer.

26. The method of any one of claims 17 to 25, wherein said contacting in step (iii) comprises contacting the extracted cfDNA with one or more pairs of PCR primers configured to produce amplicons of equal to or less than 100 base pairs or equal to or less than 80 base pairs in length from pre-determined genomic locations comprising a CTCF binding site listed in Table 1 , Table 2, or Table 3, or Table 10 or Table 11 , preferably from pre-determined genomic locations comprising a CTCF binding site listed in Table 1 , Table 10 or Table 11.

27. The method of any one of claims 17 to 26, wherein said contacting in step (iii) comprises contacting the extracted cfDNA with one or more pairs of PCR primers configured to produce amplicons of equal to or less than 100 base pairs or equal to or less than 80 base pairs in length from pre-determined genomic locations selected from the group consisting of: (a) chr1:244603555-244603648; chr2: 10668571 -10668663; chr2:79219540-79219628; chr5:141567670-141567748; chr14:56491464-56491546; chr14:87896549-87896634; chr2: 178280431 -178280518; chr8: 74853546-74853641; chr12: 100741271-100741352; chrY: 17430722-17430795; chr1 : 90579362-90579440; chr11 :34926223-34926312; chr1 :78130901 -78130992; chr19: 13262535-13262621 ; chr5: 141992498-141992587; chr22:36986359-36986452; chr14:21734605-21734701 ; chr1 : 39609752-39609824; chr2:224369183-224369264; chr6:55980780-55980855; chr12:92570523-92570593; chr4:64713399-64713474; chr4:186150099-186150166; chr6:50792057-50792142; chr2:65573278-65573362; chr17:48336036-48336115; chr16:66275978-66276056; chr2:7427441 -7427517; chr8:5062480-5062559; chr15:73874049-73874135; and chrX:89757930-89758000; and / or (b) chr2:79219519- 79219648, chr2:162301295-162301441, chr3: 194594813-194594949, chr7:64625243- 64625355, chr8:81641590-81641720, chr14:21734606-21734734, chr1 : 58739989- 58740101 , chr6: 149028960-149029063, chr9:22452551 -22452664, chr12:4849148- 4849254, chr15:92797259-92797393, chr20:5788487-5788587, chrX: 106481411- 106481533, chr2: 149561424-149561553, chr3:61381935-61382023, chr5: 17190626- 17190742, chr5:59390194-59390288, chr7:25160122-25160245, chr11:77619731- 77619846, chr17:48336001-48336147, chr1 :206461901 -206462094, chr2: 143110966- 143111104, chr2: 162123970- 162124051, chr5: 16456356-16456495, chr10:15902711- 15902826, chr10:17742984-17743122, chr11 :6379394-6379487, chr16: 17579398- 17579529, chr17:5592340-5592644, chrX: 3035865-3036000, chr3: 78659532- 78659619, chr3: 105518948-105519094, chr4:64585094-64585216, chr5: 83927264- 83927382, chr6:593261 -593392, chr6: 137314444- 137314535, chr7: 107874746-107874889, chr10:27656752-27656912, chr11:32960716-32960828, chr11 :43567527- 43567641, chr12:76791108-76791220, chr14:37230722-37230848, chr15:81939739-81939846, chr16:75789293-75789426, chr15:68554138-68554266, chr1: 78130884- 78131005, chr6: 50602320-50602449, chr1 : 119675667-119675836, chr1 :244603541- 244603674, chrX:101415625-101415728, chr2: 178280411-178280526, chr5:96752586- 96752688, chrX:22081630-22081732, chrX:47384626-47384769, chr19:39474211- 39474377, chr2:46464602-46464705, chr2:231715363-231715468, chr7: 93228366- 93228499, chr9:21111090-21111213, chr2:129849717-129849817, chr10:34638577- 34638699, chr19:42028327-42028524, chr2: 162298074- 162298189, chr8:24229478- 24229598, chr8:29647427-29647523, chr10:63720138-63720300, chr11 :34492959- 34493085, chr12:27652288-27652426, chr2:174700116-174700251 , chr3: 183122469-183122581 , chr12:123905436-123905607, chr1 : 167540493-167540653, chr3:189148131-189148251 , chr5:141992479-141992603, chr9: 78821242-78821375, chr12 : 10562452- 10562563, chr2:210577647-210577793, chr2 :212567753-212567856, chr4:4183014-4183121 , chr14:56491434-56491551 , chr15:48352569-48352712, chrX:51461172-51461282, chr2:159118703-159118816, chr6:36095298-36095473, chr1 :221435546-221435673, chr11 :46537952-46538113, chr1 : 10428186-10428296, chr2: 102840031 - 102840180, chr7: 104848545- 104848654, chr9:4988029-4988170, chr11 : 121194847-121194979, chr11 :123247327- 123247457, chr13:41353128-41353267, chr17:73369977-73370136, chr19:49983756-49983882, chr20:63223466- 63223579, chr11 :10077000-10077143, chr8:71975358-71975486, chr12:6537982- 6538105, chr12:64891000-64891169, chr1 :161487131-161487247, chr4: 155442425- 155442520, chr11 :44911428-44911580, chr12:31272008-31272161 , chr16:85717990- 85718123, chr19:49547082-49547241 , chr10:11819934-11820065, chr6:85355152- 85355270, chr6: 160519981-160520091 , chr7: 136009627-136009724, chr14:21014774- 21014934, chr1 :53393434-53393577, chr2:38115355-38115496, chr4:21789365- 21789486, chr4:113394834-113394942, chr5:59673572-59673710, chr6: 85323564- 85323677, chr8: 54169437-54169546, chr8:96180035-96180344, chr10:28241944- 28242032, chr11 :62380869-62381016, chr12:8396766-8396964, chr1 : 160726939-160727057, chr19:54676361 -54676503, chr1 :225976081-225976216, chr1 :236103745- 236103862, chr4:16026149-16026413, chr4: 167021693-167021794, chr5: 141960806- 141960987, chr11 :48307888-48308019, chr16:50665497-50665627, chr17:65198881 - 65199032, chr20:13119634-13119763, chr2: 10668563-10668699, chr14:36374168- 36374282, chr19:46402330-46402434, chr10:101161791-101161998, chr20:6142524- 6142649, chr3:71729971 -71730106, chr19:4204720-4204856, chr1 : 101288544- 101288669, chr1 :27669254-27669382, chr13:36420493-36420628, chr17:28540839- 28540954, chr20:2351923-2352109, chr1 : 158998804-158998962, chr1 :220810250- 220810376, chr2: 170409173-170409293, chr7:78040335-78040434, chr1 :31162784- 31162912, chr1 : 17661594-17661807, chr1 :31422842-31422978, chr1 : 192445984-192446109, chr6: 105424291 -105424437, chr19:42529383-42529506, chr1 : 107782340- 107782444, chr9:86414420-86414532, chr7:132188991-132189067, chr2: 197374043- 197374122, chr2:60642442-60642560, chr18:24736101 -24736161 , chr9:6594697- 6594768, chr11 :9675318-9675421 , chr1 :226944700-226944781 , chr1 :202640729- 202640819, chr9:14216485-14216587, chr12:54188268-54188338, chr9: 121838761 - 121838825, chr1 : 95264973-95265040, chr1 :69737726-69737836, chr1 : 107782340- 107782444, chr3: 32144255-32144365, chr3:113494238-113494353, chr3: 134474064- 134474175, chr3: 137105390-137105526, chr3: 181326780-181326885, chr4:26082030- 26082146, chr4: 103934283-103934454, chr5:115586654-115586772, chr6: 52049619- 52049757, chr6:63764478-63764585, chr6:158691470-158691594, chr6: 162908032- 162908121 , chr7: 130832002-130832124, chr7:132188991-132189067, chr8:63262036- 63262139, chr8:110830607-110830727, chr8:117329067-117329210, chr8:119867220- 119867390, chr9:86414420-86414532, chr9:90142700-90142839, chr10:89563998- 89564154, chr10:91389742-91389869, chr12:92570492-92570605, chr12:97308762- 97308849, chr14:28635549-28635670, chr14:37149849-37149969, chr15:665671 SO- 66567354, chr18:62958013-62958123, chr18:62982806-62982908, chr20:52312263- 52312398, chr1 :11241826-11241943, chr1 : 53754832-53754948, chr1 : 89987314- 89987463, chr2:7427425-7427538, chr2:60642442-60642560, chr2: 104849680-104849792, chr2:112582400-112582523, chr2:160110087-160110199, chr2: 197374043-197374122, chr2:224369161 -224369299, chr4:242369-242480, chr4:41281166-41281273, chr4:122758089-122758199, chr4:166165962-166166073, chr4:169810464-169810607, chr5:32489802-32489952, chr5:123371732-123371827, chr5: 172370813-172370900, chr6:33123829-33123955, chr6:49849713-49849822, chr6: 110942102-110942167, chr6: 113909396-113909512, chr6: 159008718-159008830, chr10:25700952-25701067, chr10:92885025-92885143, chr10:94630132- 94630263, chr11 :30346226-30346339, chr11 :110433891-110434020, chr11 : 124586369-124586468, chr12: 102702668-102702830, chr12: 111451647- 111451929, chr13:33228694-33228791 , chr13:50085114-50085230, chr13:53250379- 53250572, chr13:57627719-57627850, chr13: 100722041 -100722178, chr14:20327405- 20327535, chr14:24735042-24735188, chr15:31974953-31975090, chr18:24736101- 24736161 , chr19:54674154-54674274, chr22:22201213-22201344, chrX:22136053- 22136155, chrX: 129754802-129754877, chrX: 130079923-130080041 , chr1 : 19918128- 19918255, chr1 :24587331 -24587473, chr1 :39720138-39720288, chr1 :65958784- 65959095, chr1 :74689941 -74690070, chr1 :88877501 -88877633, chr1 : 161227460- 161227587, chr1 :202640729-202640819, chr1 :226944700-226944781 , ch r2: 17646148- 17646327, ch r2: 227862361 -227862483, chr2:235594168-235594292, chr2:238462941- 238463069, chr3: 124079474-124079582, chr3:156541160-156541317,chr3: 194404789- 194404904, chr3: 195522387- 195522501 , chr4: 16794985- 16795080, chr4:27048448-27048583, chr4:52026152-52026244, chr4: 52376236-52376358, chr4: 140999943-141000112, chr4: 146104133-146104288, chr4: 158850959-158851065, chr4: 165464923-165465025, chr5:18314314-18314440, chr5:28014922- 28015045, chr5: 74648924-74649026, chr5: 102223966-102224084, chr5: 129664478- 129664599, chr5: 155139754-155139857, chr6: 12893990-12894132, chr6: 30380603- 30380684, chr6: 50792043-50792155, chr6:51855414-51855525, chr6: 123415992- 123416085, chr7:10412547-10412686, chr7:25180362-25180528, chr7: 38374563- 38374659, chr7:91830142-91830274, chr7:94395571 -94395689, chr8:4833976-4834088, chr8:95758783-95758908, chr8:119070591-119070729, chr9:6354233- 6354348, chr9:6594697-6594768, chr9:14216485-14216587, chr9: 17063671 -17063809, chr9: 19028989-19029108, chr10:3192966-3193102, chr10:16828259- 16828398, chr10:25726026-25726156, chr10:87656598-87656695, chr10:87735375- 87735496, chr10:125098674-125098794, chr11 :9675318-9675421 , chr11 :17230210- 17230310, chr11 :32323655-32323771 , chr11 :72667585-72667696, chr11 :124402455-124402570, chr12:20946875-20946995, chr12:53791868-53791960, chr12:55619731-55619974, chr12:125403356-125403461 , chr12: 126069664-126069777, chr13:85828223-85828338, chr14:45426816-45426941 , chr14:93655215-93655320 chr15:44288413-44288574, chr15:90956673-90956821 , chr16:24253220-24253372 chr16:63361142-63361267, chr16:66728261-66728409, chr17:35312798-35312956 chr17:78013974-78014108, chr18:43571212-43571318, chr18:65781879-65782020 chr20:36466920-36467064, chr22:25093742-25093869, chrX:51614499-51614639 chrX: 110156310-110156388, chr22_KI270733v1_random: 136322-136475, chr1 : 32272584-32272646, chr1 :86742093-86742193, chr1 :88998835-88998937, chr1 : 95264973-95265040, chr1 : 163410082- 163410225, chr1 : 169055544- 169055615, chr1 :236452124-236452243, chr1 :240149603-240149760, chr1 :240189505-240189629, chr2: 18378180-18378279, chr2:38581666-38581810, chr2 : 152861493-152861578, chr2:166539491-166539609, chr2:176814897-176815015, chr2:182186287-182186395, chr2:227890621 -227890778, chr3:87298250-87298349, chr3:89211716-89211800, chr3: 109732032-109732122, chr3: 134492629-134492738, chr3: 156509828- 156509965, chr3: 157137568- 157137691 , chr3: 161273765-161273866, chr3:166013225-166013311 , chr4:23135919-23136011 , chr4:48087272- 48087368, chr4:70183776-70183887, chr4:73019582-73019720, chr4:86044122- 86044254, chr4: 174365159-174365240, chr5:5141805-5141917, chr5: 80083642- 80083758, chr5:81299634-81299801 , chr5:112175136-112175244, chr5: 146662279- 146662392, chr5:148733715-148733808, chr5: 154157507-154157598, ch r6: 35449508- 35449669, chr6:52021110-52021230, chr6:56902964-56903131 , chr6: 70706662-70706775, chr6: 74091490-74091627, chr6:112180232-112180339, chr6: 135451738- 135451874, ch r7: 148504024- 148504134, chr7:156582102-156582204, chr8: 12866970- 12867090, chr8:69663836-69663985, chr8:75257410-75257540, chr8:80612676- 80612794, chr8:90948403-90948519, chr8: 132651571 -132651698, ch r9: 13427664- 13427772, chr9: 15873723-15873819, chr9: 17462998-17463108, chr9: 121400273- 121400386, chr9:121838761-121838825, chr10:61284412-61284550, chr10:93352770- 93352892, chr10:116490211 -116490321 , chr10:125982446-125982551 , chr11 :6738008-6738119, chr11 :84384764-84384893, chr11 :88092327-88092414, chr11 :94519623-94519738, chr11 :96464846-96464973, chr11 :115117490-115117553, chr11 :128414446-128414512, chr12:7642988-7643098, chr12:21680506-21680612, chr12:29519728-29519832, chr12:34034045-34034152, chr12:54188268-54188338, chr12:58346176-58346282, chr12:100741240-100741368, chr12:119160250-119160324, chr13:97132337-97132465, chr14:19878197-19878283, chr14:25400748- 25400891 , chr14:53055029-53055173, chr14:64303608-64303708, chr14:82000025- 82000111 , chr14:98138734-98138826, chr15:85505208-85505330, chr15:98881743- 98881866, chr16:16449419-16449513, chr17:700647-700751 , chr17: 16258067- 16258156, chr18:24489020-24489155, chr18:31819747-31819885, chr19:28325490- 28325587, chr19:47125320-47125481 , chrX:11144386-11144529, chrX: 87299704- 87299805, chrX: 133670537-133670648, chrX: 152758817-152758894, chr1 :2091761- 2091844, chr1 :5225260-5225331 , chr1 :110555394-110555522, chr1 : 158036123- 158036245, chr1 : 176559961 -176560058, chr1 :207405060-207405185, chr1 :238188421 -238188505, chr2:39059461 -39059589, chr2:60707803-60707914, chr2:62412204-62412318, chr2: 106791905-106792003, chr2:151437690-151437807, chr2: 158010423-158010526, chr2:164717151-164717267, chr2: 179440792-179440892, chr2: 181715178-181715317, chr2: 197707735-197707844, chr2:232270870-232270944, chr2:241078520-241078659, chr3: 13600010-13600082, chr3:26712570-26712658, chr3:65785206-65785301 , chr3:98039424-98039547, chr3: 146065649- 146065763, chr3: 147191901 - 147192041 , chr3: 173584256-173584361 , chr4:38856812-38856976, chr4:64713379-64713507, chr4:67746823- 67746964, chr4:147097166-147097267, chr4:163484959-163485038, chr4: 185634052- 185634304, chr5: 19469171 -19469243, chr5:36870575-36870732, chr5: 52657324- 52657444, chr5:66307768-66307915, chr5: 103335135-103335205, chr6:47631424- 47631547, chr6:52945168-52945233, chr6:75893429-75893588, chr6: 93664709- 93664784, chr6:115244749-115244826, chr6:116287793-116287931 , chr6: 158605406- 158605520, chr6: 170561934-170561998, chr7:26198200-26198319, chr7: 50142454- 50142556, chr7:81482732-81482834, chr7: 154504103-154504239, chr8:9831026- 9831238, chr9:8086869-8086940, chr10:117940922-117940998, chr11 :34926239-34926356, chr11 : 82682336-82682444, chr11 :121765670-121765767, chr12:12376782- 12376859, chr12:20950374-20950501 , chr12:23949721 -23949794, chr12:24095121- 24095194, chr12:29889866-29889994, chr12:72987449-72987521 , chr12:73391018- 73391090, chr12:78146406-78146532, chr12:90959676-90959826, chr13:43908534- 43908635, chr13:67238415-67238557, chr13:84896266-84896371 , chr13:102141733- 102141898, chr13:110839093-110839188, chr14:78082948-78083033, chr14: 100624900-100625009, chr15:24585660-24585745, chr15:31880238-31880317, chr15:41031918-41032105, chr15:69322673-69322785, chr15:80419036-80419163, chr15:99704486-99704631 , chr17:31216272-31216386, chr17:48728857-48728929, chr17:74612182-74612276, chr17:79893762-79893854, chr18:25853208-25853305, chr18:64037936-64038067, and chr19:54080140-54080207.

28. The method of any one of claims 17 to 27, wherein prior to said contacting in step (iii), the method comprises treating a body fluid sample with a reagent to crosslink nucleoprotein and / or cfDNA in the sample.

29. The method of claim 28, wherein the body fluid sample is a plasma sample, and said treating further comprises treating with a quenching agent.

30. A PCR method for detecting the presence of CTCF occupied cfDNA fragments originating from a pre-determined genomic location, in an isolated CTCF-cfDNA preparation produced from a body fluid sample obtained from a subject, which comprises the steps of:(i) contacting a first sample of the body fluid with a candidate binding agent which binds to cfDNA-CTCF nucleoproteins, and contacting a second sample of the body fluid with a control binding agent which does not specifically bind to cfDNA-CTCF nucleoproteins;(ii) isolating cfDNA from the first sample bound by the candidate binding agent and from the second sample bound by the control binding agent in step (i);(iii) contacting isolated cfDNA from the first sample and the second sample with one or more pairs of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs or equal to or less than 80 base pairs that includes a CTCF binding site located at the pre-determined genomic location;(iv) determining the presence, absence or quantity of DNA sequences of equal to or less than 100 base pairs or equal to or less than 80 base pairs in length produced in step (iii) by subtracting the quantity of DNA in the second sample from the first sample; and(v) using the presence, absence or quantity of DNA determined in step (iv), as an indicator of whether, or how many, CTCF-occupied cfDNA fragments originating from the pre-determined genomic location were present in the body fluid sample.31 . A method for determining a genomic location comprising a CTCF binding site that is selectively bound by CTCF during a cancer, comprising:(i) contacting a first body fluid sample obtained from a subject diagnosed with the cancer with a binding agent which binds to cfDNA-CTCF nucleoproteins, and contacting a second body fluid sample obtained from a subject with no known cancer or a different cancer with a binding agent which binds to cfDNA-CTCF nucleoproteins;(ii) isolating cfDNA-CTCF nucleoproteins from the first and second sample bound in step (i);(iii) sequencing cfDNA isolated from the first and second sample in step (ii) to determine genomic locations that are selectively occupied by CTCF in the first sample but not in the second sample, to determine genomic locations comprising a CTCF binding site that is selectively bound by CTCF during the cancer.

32. The method of claim 30 or claim 31 , wherein contacting a first body fluid sample comprises contacting a first portion of a body fluid sample and contacting a second body fluid sample comprises contacting a second portion of the body fluid sample.

33. A method of detecting cancer in a subject which comprises the steps of:(i) detecting the presence of CTCF occupied cfDNA fragments using the method of any one of claims 1 to 29; and(ii) using the presence of the CTCF occupied cfDNA fragments as an indicator of the presence of cancer in the subject.

34. A method for the assessment of subject for suitability for a cancer treatment which comprises the steps of:(i) detecting the presence of CTCF occupied cfDNA fragments using the method of any one of claims 1 to 29; and(ii) using the CTCF occupied cfDNA fragments detected in step (i) as a parameter for the selection of a suitable treatment for the subject.

35. A method for monitoring a treatment of a subject diagnosed with cancer which comprises the steps of:(i) detecting the presence of CTCF occupied cfDNA fragments using the PCR method of any one of claims 1 to 29;(ii) repeating the detection of step (i) in a body fluid sample obtained from the subject on one or more occasions; and(iii) using any changes in the presence of CTCF occupied cfDNA fragments determined in step (i) compared to step (ii) as a parameter for any changes in the condition of the subject.

36. A kit for the detection of a cell free chromatin fragment comprising CTCF and a DNA fragment as a combination biomarker which comprises a binding agent for CTCF together with reagents for detecting the presence of CTCF occupied cfDNA fragments using the method of any one of claims 1 to 29.

37. A kit for detecting the presence of CCCTC-binding factor (CTCF) occupied cell free DNA (cfDNA) fragments originating from a pre-determined genomic location, in a body fluid sample obtained from a subject, wherein the kit comprises:(a) a first pair of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs or equal to or less than 80 base pairs that comprises a CTCF binding site located at a first pre-determined genomic location; and(b) a second pair of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs or equal to or less than 80 base pairs that comprises a CTCF binding site located at a second pre-determined genomic location.

38. A composition comprising:(a) a first pair of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs or equal to or less than 80 base pairs that comprises a CTCF binding site located at a first pre-determined genomic location; and(b) a second pair of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs or equal to or less than 80 base pairs that comprises a CTCF binding site located at a second pre-determined genomic location.

39. The kit of claim 37 or composition of claim 38, wherein the kit or composition further comprises:(c) a third pair of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs or equal to or less than 80 base pairs that comprises a CTCF binding site located at a third pre-determined genomic location;(d) a fourth pair of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs or equal to or less than 80 base pairs that comprises a CTCF binding site located at a fourth pre-determined genomic location; and(e) a fifth pair of PCR primers directed to a DNA sequence of equal to or less than 100 base pairs or equal to or less than 80 base pairs that comprises a CTCF binding site located at a fifth pre-determined genomic location.

40. The kit of claim 37 or 39 or composition of claim 38 or 39, wherein the predetermined genomic locations are each selectively occupied by CTCF in a disease state, optionally wherein the disease state is cancer, for example a cancer selected from the group consisting of: lung cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, blood cancer (such as leukaemia or lymphoma) and skin cancer (such as melanoma).41 . The kit of any one of claims 37, 39 or 40 or the composition of any one of claims 38-40, wherein the pre-determined genomic locations are each selected from the following: (a) chr1 :244603555-244603648; chr2: 10668571 -10668663; chr2:79219540- 79219628; chr5:141567670-141567748; chr14:56491464-56491546; chr14:87896549- 87896634; chr2: 178280431 -178280518; chr8: 74853546-74853641 ; chr12:100741271- 100741352; chrY: 17430722-17430795; chr1 : 90579362-90579440; chr11 :34926223- 34926312; chr178130901 -78130992; chr19: 13262535-13262621 ; chr5: 141992498- 141992587; chr22:36986359-36986452; chr14:21734605-21734701 ; chr1 : 39609752- 39609824; chr2:224369183-224369264; chr6:55980780-55980855; chr12:92570523- 92570593; chr4:64713399-64713474; chr4:186150099-186150166; chr6: 50792057- 50792142; chr2:65573278-65573362; chr17:48336036-48336115; chr16:66275978- 66276056; chr2:7427441 -7427517; chr8:5062480-5062559; chr15:73874049-73874135; and chrX: 89757930-89758000; and / or (b) chr2:79219519-79219648, chr2:162301295-162301441 , chr3: 194594813-194594949, chr7:64625243-64625355, chr8: 81641590-81641720, chr14:21734606-21734734, chr1 : 58739989-58740101 , chr6: 149028960-149029063, chr9:22452551 -22452664, chr12:4849148-4849254, chr15:92797259-92797393, chr20:5788487-5788587, chrX: 106481411-106481533, chr2:149561424-149561553, chr3:61381935-61382023, chr5: 17190626-17190742, chr5:59390194-59390288, chr7:25160122-25160245, chr1177619731 -77619846, chr17:48336001 -48336147, chr1 :206461901 -206462094, chr2:143110966-143111104, chr2:162123970-162124051 , chr5: 16456356-16456495, chr10:15902711-15902826, chr10: 17742984-17743122, chr11 :6379394-6379487, chr16: 17579398-17579529, chr17: 5592340-5592644, chrX: 3035865-3036000, chr3: 78659532-78659619,chr3: 105518948-105519094, chr4:64585094-64585216, chr5:83927264-83927382, chr6: 593261 -593392 , chr6: 137314444- 137314535, chr7: 107874746- 107874889, chr10:27656752-27656912, chr11 :32960716-32960828, chr11 :43567527-43567641 , chr12:76791108-76791220, chr14:37230722-37230848, chr15:81939739-81939846, chr16:75789293-75789426, chr15:68554138-68554266, chr1 :78130884-78131005, chr6:50602320-50602449, chr1 :119675667-119675836, chr1 :244603541 -244603674, chrX: 101415625- 101415728, chr2 : 178280411-178280526, chr5:96752586-96752688, chrX:22081630-22081732, chrX: 47384626-47384769, chr19:39474211-39474377, chr2:46464602-46464705, chr2:231715363-231715468, chr7: 93228366-93228499, chr9:21111090-21111213, chr2: 129849717-129849817, chr10:34638577-34638699, chr19:42028327-42028524, chr2: 162298074- 162298189, chr8:24229478-24229598, chr8:29647427-29647523, chr10:63720138-63720300, chr11 :34492959-34493085, chr12 :27652288-27652426, chr2: 174700116-174700251 , chr3: 183122469- 183122581 , chr12: 123905436-123905607, chr1 : 167540493-167540653, chr3: 189148131-189148251 , chr5:141992479-141992603, chr9:78821242-78821375, chr12: 10562452- 10562563, chr2:210577647-210577793, chr2:212567753-212567856, chr4:4183014- 4183121 , chr14:56491434-56491551 , chr15:48352569-48352712, chrX:51461172- 51461282, chr2:159118703-159118816, chr6: 36095298-36095473, chr1 :221435546- 221435673, chr11 :46537952-46538113, chr1 : 10428186-10428296, chr2: 102840031 - 102840180, chr7: 104848545-104848654, chr9:4988029-4988170, chr11 :121194847- 121194979, chr11 :123247327-123247457, chr13:41353128-41353267, chr17:73369977-73370136, chr19:49983756-49983882, chr20:63223466-63223579, chr11 :10077000-10077143, chr8:71975358-71975486, chr12:6537982-6538105, chr12:64891000-64891169, chr1 :161487131-161487247, chr4: 155442425-155442520, chr11 :44911428-44911580, chr12:31272008-31272161 , chr16:85717990-85718123, chr19:49547082-49547241 , chr10: 11819934-11820065, chr6:85355152-85355270, chr6: 160519981 -160520091 , chr7: 136009627-136009724, chr14:21014774-21014934, chr1 :53393434-53393577, chr2:38115355-38115496, chr4:21789365-21789486, chr4:113394834-113394942, chr5: 59673572-59673710, chr6: 85323564-85323677, chr8:54169437-54169546, chr8:96180035-96180344, chr10:28241944-28242032, chr11 :62380869-62381016, chr12:8396766-8396964, chr1 : 160726939-160727057, chr19:54676361 -54676503, chr1 :225976081 -225976216, chr1 :236103745-236103862, chr4:16026149-16026413, chr4: 167021693-167021794, chr5:141960806-141960987, chr11 :48307888-48308019, chr16:50665497-50665627, chr17:65198881-65199032, chr20: 13119634-13119763, chr2: 10668563-10668699, chr14:36374168-36374282, chr19:46402330-46402434, chr10: 101161791-101161998, chr20:6142524-6142649, chr3:71729971 -71730106, chr19:4204720-4204856, chr1 :101288544-101288669,ch r1 :27669254-27669382, chr13:36420493-36420628, chr17:28540839-28540954, chr20:2351923-2352109, chr1 : 158998804-158998962, chr1 :220810250-220810376, chr2: 170409173-170409293, chr7: 78040335-78040434, chr1 :31162784-31162912, chr1 : 17661594- 17661807, chr1 : 31422842-31422978, chr1 : 192445984- 192446109, chr6: 105424291 - 105424437, chr19:42529383-42529506, chr1 : 107782340- 107782444, chr9:86414420-86414532, chr7:132188991-132189067, chr2:197374043-197374122, chr2:60642442-60642560, chr18:24736101-24736161 , chr9:6594697-6594768, chr11 :9675318-9675421 , chr1 :226944700-226944781 , chr1 :202640729-202640819, chr9:14216485-14216587, chr12:54188268-54188338, chr9:121838761-121838825, chr1 : 95264973-95265040, chr1 :69737726-69737836, chr1 : 107782340-107782444, chr3: 32144255-32144365, chr3: 113494238- 113494353, chr3: 134474064- 134474175, chr3: 137105390-137105526, chr3: 181326780-181326885, chr4:26082030-26082146, chr4: 103934283- 103934454, chr5: 115586654- 115586772, chr6: 52049619-52049757, chr6:63764478-63764585, chr6:158691470-158691594, chr6: 162908032-162908121 , chr7: 130832002- 130832124, chr7: 132188991 - 132189067, chr8:63262036-63262139, chr8: 110830607-110830727, chr8: 117329067-117329210, chr8: 119867220-119867390, chr9:86414420-86414532, chr9:90142700-90142839, chr10:89563998- 89564154, chr10:91389742-91389869, chr12:92570492-92570605, chr12:97308762- 97308849, chr14:28635549-28635670, chr14:37149849-37149969, chr15:665671 SO- 66567354, chr18:62958013-62958123, chr18:62982806-62982908, chr20:52312263- 52312398, chr1 :11241826-11241943, chr1 : 53754832-53754948, chr1 : 89987314- 89987463, chr2:7427425-7427538, chr2:60642442-60642560, chr2: 104849680-104849792, chr2:112582400-112582523, chr2:160110087-160110199, chr2: 197374043-197374122, chr2:224369161 -224369299, chr4:242369-242480, chr4:41281166-41281273, chr4:122758089-122758199, chr4:166165962-166166073, chr4:169810464-169810607, chr5:32489802-32489952, chr5:123371732-123371827, chr5: 172370813-172370900, chr6:33123829-33123955, chr6:49849713-49849822, chr6: 110942102-110942167, chr6: 113909396-113909512, chr6: 159008718-159008830, chr10:25700952-25701067, chr10:92885025-92885143, chr10:94630132- 94630263, chr11 :30346226-30346339, chr11 :110433891-110434020, chr11 : 124586369-124586468, chr12 : 102702668- 102702830, chr12: 111451647- 111451929, chr13:33228694-33228791 , chr13:50085114-50085230, chr13:53250379- 53250572, chr13:57627719-57627850, chr13: 100722041 -100722178, chr14:20327405- 20327535, chr14:24735042-24735188, chr15:31974953-31975090, chr18:24736101- 24736161 , chr19:54674154-54674274, chr22:22201213-22201344, chrX:22136053- 22136155, chrX: 129754802-129754877, chrX: 130079923-130080041 , chr1 : 19918128- 19918255, chr1 :24587331 -24587473, chr1 :39720138-39720288, chr1 :65958784-65959095, chr1 :74689941 -74690070, chr1 :88877501 -88877633, chr1 : 161227460-161227587, chr1 :202640729-202640819, chr1 :226944700-226944781 , ch r2: 17646148-17646327, ch r2: 227862361 -227862483, chr2:235594168-235594292, chr2:238462941-238463069, chr3: 124079474-124079582, chr3:156541160-156541317, chr3: 194404789- 194404904, chr3: 195522387- 195522501 , chr4: 16794985- 16795080, chr4:27048448-27048583, chr4:52026152-52026244, chr4: 52376236-52376358, chr4: 140999943-141000112, chr4: 146104133-146104288, chr4: 158850959-158851065, chr4: 165464923-165465025, chr5:18314314-18314440, chr5:28014922- 28015045, chr5: 74648924-74649026, chr5: 102223966-102224084, chr5: 129664478- 129664599, chr5: 155139754-155139857, chr6: 12893990-12894132, chr6: 30380603- 30380684, chr6: 50792043-50792155, chr6:51855414-51855525, chr6: 123415992- 123416085, chr7:10412547-10412686, chr7:25180362-25180528, chr7: 38374563- 38374659, chr7:91830142-91830274, chr7:94395571 -94395689, chr8:4833976-4834088, chr8:95758783-95758908, chr8:119070591-119070729, chr9:6354233- 6354348, chr9:6594697-6594768, chr9:14216485-14216587, chr9: 17063671 -17063809, chr9: 19028989-19029108, chr10:3192966-3193102, chr10:16828259- 16828398, chr10:25726026-25726156, chr10:87656598-87656695, chr10:87735375- 87735496, chr10:125098674-125098794, chr11 :9675318-9675421 , chr11 :17230210- 17230310, chr11 :32323655-32323771 , chr11 :72667585-72667696, chr11 :124402455-124402570, chr12:20946875-20946995, chr12:53791868-53791960, chr12:55619731-55619974, chr12:125403356-125403461 , chr12: 126069664-126069777, chr13:85828223-85828338, chr14:45426816-45426941 , chr14:93655215-93655320 chr15:44288413-44288574, chr15:90956673-90956821 , chr16:24253220-24253372 chr16:63361142-63361267, chr16:66728261-66728409, chr17:35312798-35312956 chr17:78013974-78014108, chr18:43571212-43571318, chr18:65781879-65782020 chr20:36466920-36467064, chr22:25093742-25093869, chrX:51614499-51614639 chrX:110156310-110156388, chr22_KI270733v1_random: 136322-136475 chr1 : 32272584-32272646, chr1 : 86742093-86742193, chr1 : 88998835-88998937 chr1 : 95264973-95265040, chr1 : 163410082- 163410225, chr1 : 169055544- 169055615, chr1 :236452124-236452243, chr1 :240149603-240149760, chr1 :240189505- 240189629, chr2: 18378180-18378279, chr2:38581666-38581810, chr2 : 152861493- 152861578, chr2:166539491-166539609, chr2: 176814897-176815015, chr2:182186287-182186395, chr2:227890621 -227890778, chr3:87298250-87298349, chr3:89211716-89211800, chr3: 109732032-109732122, chr3: 134492629-134492738, chr3: 156509828- 156509965, chr3: 157137568- 157137691 , chr3: 161273765-161273866, chr3:166013225-166013311 , chr4:23135919-23136011 , chr4:48087272- 48087368, chr4:70183776-70183887, chr4:73019582-73019720, chr4:86044122-86044254, chr4: 174365159-174365240, chr5:5141805-5141917, chr5: 80083642- 80083758, chr5:81299634-81299801 , chr5:112175136-112175244, chr5: 146662279- 146662392, chr5:148733715-148733808, chr5: 154157507-154157598, ch r6: 35449508- 35449669, chr6:52021110-52021230, chr6:56902964-56903131 , chr6: 70706662- 70706775, chr6: 74091490-74091627, chr6:112180232-112180339, chr6: 135451738- 135451874, chr7: 148504024-148504134, chr7:156582102-156582204, chr8: 12866970- 12867090, chr8:69663836-69663985, chr8:75257410-75257540, chr8:80612676- 80612794, chr8:90948403-90948519, chr8: 132651571 -132651698, ch r9: 13427664- 13427772, chr9: 15873723-15873819, chr9: 17462998-17463108, chr9: 121400273-121400386, chr9:121838761-121838825, chr10:61284412-61284550, chr10:93352770- 93352892, chr10:116490211 -116490321 , chr10:125982446-125982551 , chr11 :6738008-6738119, chr11 :84384764-84384893, chr11 :88092327-88092414, chr11 :94519623-94519738, chr11 :96464846-96464973, chr11 :115117490-115117553, chr11 :128414446-128414512, chr12:7642988-7643098, chr12:21680506-21680612, chr12:29519728-29519832, chr12:34034045-34034152, chr12:54188268-54188338, chr12:58346176-58346282, chr12:100741240-100741368, chr12:119160250-119160324, chr13:97132337-97132465, chr14: 19878197-19878283, chr14:25400748- 25400891 , chr14:53055029-53055173, chr14:64303608-64303708, chr14:82000025- 82000111 , chr14:98138734-98138826, chr15:85505208-85505330, chr15:98881743- 98881866, chr16:16449419-16449513, chr17:700647-700751 , chr17: 16258067- 16258156, chr18:24489020-24489155, chr18:31819747-31819885, chr19:28325490- 28325587, chr19:47125320-47125481 , chrX:11144386-11144529, chrX: 87299704- 87299805, chrX: 133670537-133670648, chrX: 152758817-152758894, chr1 :2091761- 2091844, chr1 :5225260-5225331 , chr1 :110555394-110555522, chr1 : 158036123- 158036245, chr1 : 176559961 -176560058, chr1 :207405060-207405185, chr1 :238188421 -238188505, chr2:39059461 -39059589, chr2:60707803-60707914, chr2:62412204-62412318, chr2: 106791905-106792003, chr2:151437690-151437807, chr2: 158010423-158010526, chr2:164717151-164717267, chr2: 179440792-179440892, chr2: 181715178-181715317, chr2: 197707735-197707844, chr2:232270870-232270944, chr2:241078520-241078659, chr3: 13600010-13600082, chr3:26712570-26712658, chr3:65785206-65785301 , chr3: 98039424-98039547, chr3: 146065649- 146065763, chr3: 147191901 - 147192041 , chr3: 173584256-173584361 , chr4:38856812-38856976, chr4:64713379-64713507, chr4:67746823- 67746964, chr4:147097166-147097267, chr4: 163484959-163485038, chr4: 185634052- 185634304, chr5: 19469171 -19469243, chr5:36870575-36870732, chr5: 52657324- 52657444, chr5:66307768-66307915, chr5: 103335135-103335205, chr6:47631424- 47631547, chr6:52945168-52945233, chr6:75893429-75893588, chr6:93664709-93664784, chr6:115244749-115244826, chr6:116287793-116287931 , chr6: 158605406- 158605520, chr6: 170561934-170561998, chr7:26198200-26198319, chr7: 50142454- 50142556, chr7:81482732-81482834, chr7: 154504103-154504239, chr8:9831026- 9831238, chr9:8086869-8086940, chr10:117940922-117940998, chr11 :34926239- 34926356, chr11 :82682336-82682444, chr11 :121765670-121765767, chr12:12376782- 12376859, chr12:20950374-20950501 , chr12:23949721 -23949794, chr12:24095121- 24095194, chr12:29889866-29889994, chr12:72987449-72987521 , chr12:73391018- 73391090, chr12:78146406-78146532, chr12:90959676-90959826, chr13:43908534- 43908635, chr13:67238415-67238557, chr13:84896266-84896371 , chr13:102141733- 102141898, chr13:110839093-110839188, chr14:78082948-78083033, chr14: 100624900-100625009, chr15:24585660-24585745, chr15:31880238-31880317, chr15:41031918-41032105, chr15:69322673-69322785, chr15:80419036-80419163, chr15:99704486-99704631 , chr17:31216272-31216386, chr17:48728857-48728929, chr17:74612182-74612276, chr17:79893762-79893854, chr18:25853208-25853305, chr18:64037936-64038067, and chr19:54080140-54080207.

42. The kit of any one of claims 37 or 39-41 or the composition of any one of claims 38-41 , wherein at least two of the pre-determined genomic locations are on different chromosomes.

43. A kit for detecting the presence of CCCTC-binding factor (CTCF) occupied cell free DNA (cfDNA) fragments comprising:(a) a first pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a first 30-100 bp or 30-80 bp region of the human genome comprising a CTCF binding site or part thereof; and(b) a second pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a second 30-100 bp or 30-80 bp region of the human genome comprising a CTCF binding site or part thereof.

44. A composition comprising:(a) a first pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a first 30-100 bp or 30-80 bp region of the human genome comprising a CTCF binding site or part thereof; and(b) a second pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a second 30-100 bp or 30-80 bp region of the human genome comprising a CTCF binding site or part thereof.

45. The kit of claim 43 or composition of claim 44, wherein the kit or composition further comprises:(c) a third pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a third 30-100 bp or 30-80 bp region of the human genome comprising a CTCF binding site or part thereof;(d) a fourth pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a fourth 30-100 bp or 30-80 bp region of the human genome comprising a CTCF binding site or part thereof; and(e) a fifth pair of PCR primers for amplifying a target polynucleotide sequence, wherein the target polynucleotide sequence consists of a fifth 30-100 bp or 30-80 bp region of the human genome comprising a CTCF binding site or part thereof.

46. The kit of claim 43 or 45 or the composition of claim 44 or 45, wherein the CTCF binding sites are each selectively occupied by CTCF in a disease state, optionally wherein the disease state is cancer, for example a cancer selected from the group consisting of: lung cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, blood cancer (such as leukaemia or lymphoma) and skin cancer (such as melanoma).

47. The kit of any one of claims 43, 45 or 46 or the composition of any one of claims 44-46, wherein the CTCF binding sites are each selected from the cancer-specific CTCF binding sites listed in Table 1 , Table 2, or Table 3, optionally wherein the CTCF binding sites are each selected from the cancer-specific CTCF binding sites listed in Table 1.

48. The kit of any one of claims 43 or 45-47 or the composition of any one of claims 44-47, wherein the CTCF binding sites are each selected from the colorectal cancerspecific CTCF binding sites listed in Table 3, the breast cancer-specific CTCF binding sites listed in Table 3, the prostate cancer-specific CTCF binding sites listed in Table 3, or the blood cancer-specific CTCF binding sites listed in Table 1 or Table 3.

49. The kit of any one of claims 43 or 45-48 or the composition of any one of claims 44-48, wherein the CTCF binding sites are each selected from the following: chr1 :244603599-244603619, chr14:56491500-56491520, chr1 : 90579403-90579423, chr11 :34926268-34926288, chr1 :78130945-78130965, chr5:141992536-141992556, chr22:36986402-36986422, chr14:21734657-21734677, chr6:55980809-55980829, chr4:64713438-64713458, chr17:48336072-48336092, chr2:7427475-7427495, chr15:73874089-73874109, chrX: 89757963-89757983, chr2: 10668604-10668624, chr2:79219569-79219589, chr5:141567693-141567713, chr14:87896582-87896602,chr8:74853585-74853605, chr12:100741297-100741317, chr19: 13262563-13262583, chr22:36986399-36986419, chr1 : 39609779-39609799, chr2:224369205-224369225, chr12:92570541-92570561, chr6:50792087-50792107, chr2:65573310-65573330, and chr15:73874086-73874106.

50. The kit of any one of claims 37, 39-43, or 45-49 or the composition of any one of claims 38-42 or 44-49, wherein each of the pair of PCR primers comprises a fluorescently-labelled primer, wherein the emission spectra do not overlap.

51. The kit of any one of claims 37, 39-43, or 45-50, wherein the kit further comprises an anti-CTCF antibody, optionally wherein the kit further comprises magnetic particles coated with an anti-CTCF antibody.

52. A biomarker panel of genomic locations comprising CTCF binding sites associated with five or more of the following genes: (a) CATSPERE, CTNNA2, NOL10, DIAPH1, TMEM260, GALC, OSBPL6, PI15, ANO4, FAM41AY1, LINC02787, NR_038432.1, GIPC2, CACNA1A, GNPD1, TEX33, TRAV4, HEYL, FAM124B, COL21A1 , PLEKHG7, EPHA5, FAM149A, TFAP2B, LINC02934, SKAP1, CDH5, ENSG00000229727, SNORA70, TBC1D21, TGIF2LX; and / or (b) CTNNA2, GCA, TMEM44, ENSG00000286456, SLC10A5P1, TRAV4, ENSG00000283445, UST, DMRTA1, GALNT8, FAM174B, SHLD1, NAP1 L4P2, MMADHC, PTPRG, BASP1, PDE4D, SPMIP4, CLNS1A, SKAP1, IKBKE, ARHGAP15, DPP4, ZNF622, MINDY3, TMEM236, SMPD1, ENSG00000260945, NLRP1, ARSH, ROBO1, ALCAM, MTCO3P27, EDIL3, EXOC2, IFNGR1, DLD, MKX, QSER1, CTBP2P6, ZDHHC17, MIPOL1 , LINC01418, TERF2IP, CORO2B, GIPC2, ENSG00000230472, PHGDH, CATSPERE, ARMCX4, OSBPL6, CAST, PHEX, ZNF157, TIMM50, LINC02583, PTMA, HEPACAM2, IFNWP4, LINC02572, PARD3, GRIK5, IFIH1, ADAM28, LINC00589, ENSG00000228566, ELF5, PPFIBP1, WIPF1, MCCC1, DNAH10, CREG1, TPRG1, GNPDA1, MTND2P8, LINC02446, CPS1, ERBB4, OR7E43P, TMEM260, DUT, ENSG00000226530, TANC1, MAPK13, ENSG00000232436, AMBRA1, CENPSCORT, TMEM182, LHFPL3AS1 , JAK2, TECTA, CLMP, NAA16, SDK2, VRK3, YTHDF1 , SBF2, MSCAS1, GAPDH, LINC02389, FCGR2A, ENSG00000287730, TP5BI11, SINHCAF, ENSG00000275393, FCGRT, PROSER2, LINC02535, LPAL2, ENSG00000230649, TPPP2, LRP8, CYP1B1, KCNIP4, ANK2, PDE4D, KRT18P54, MRPL15, GDF6, MPP7, SCGB1A1, LINC00937, SLAMF7, LILRP1, SDE2, NID1, FGFBP2, SPOCK3, RNF14, OR4S1 , SNX20, RGS9, SPTLC3, NOL10, DPPA3P2, CCDC8, TLX1, FERMT1, GPR27, SIRT6, LINC01307, IFI6, CCNA1, UNC119, TGM6, IFI16, MTARC1, MY03BAS1,DDX3ILA1, NKAIN1 , LINC02810, SERINC2, ENSG00000236069, PREP, CEACAM1, VAV3, ENSG00000235819, PLXNA4, NPM1P46, RN7SL361 P, ENSG00000266573, GLDC, SWAP7Q, Q0Q8A, ENSG00000225620, RPL7AP47, SMUG1 , TTLL11 , ENSG00000228852, LRRC7, VAV3, GPD1 L, SPICE1, CEP63, IL20RB, FAUP2, LINC02357, LINC02503, TICAM2, PKHD1, ENSG00000218813, SYTL3, PACRG, HAP1, PLXNA4, RN7SL135P, NDUFB9P3, ENSG00000252852, DSCC1, ENSG00000235819, ENSG00000279503, SLC16A12, HECTD2, PLEKHG7,LINC02409, LINC02300, SLC25A21 , LCTL, PHLPP1, PHLPP1 , ZFP64, MTOR, GLIS1, RN7SKP272, ENSG00000229727, RN7SL361P, POU3F3, CHCHD5, ITGB6, NPM1 P46, FAM124B, ZNF876P, UCHL1, ENSG00000266573, ENSG00000225620, QOQ8A, TFAP2B, GLDC, RPL7AP47, SWAP7Q, ENSG00000228852, TTLL11 , SMUG1, ANO4, EPHA5, NR_038432.1.

53. The biomarker panel of claim 52, wherein the genomic locations are selected from five or more of the following regions: (a) chr1 :244603555-244603648; chr2: 10668571 - 10668663; chr2:79219540-79219628; chr5:141567670-141567748; chr14:56491464- 56491546; chr14:87896549-87896634; chr2: 178280431 -178280518; chr8: 74853546- 74853641; chr12:100741271-100741352; chrY: 17430722-17430795; chr1 : 90579362- 90579440; chr11:34926223-34926312; chr1:78130901 -78130992; chr19: 13262535- 13262621 ; chr5:141992498-141992587; chr22:36986359-36986452; chr14:21734605- 21734701 ; chr1 :39609752-39609824; chr2:224369183-224369264; chr6: 55980780- 55980855; chr12:92570523-92570593; chr4:64713399-64713474; chr4: 186150099- 186150166; chr6:50792057-50792142; chr2:65573278-65573362; chr17:48336036- 48336115; chr16:66275978-66276056; chr2:7427441 -7427517; chr8:5062480-5062559; chr15:73874049-73874135; and chrX:89757930-89758000; and / or (b) chr2:79219519-79219648, chr2:162301295-162301441, chr3: 194594813-194594949, chr7:64625243-64625355, chr8:81641590-81641720, chr14:21734606-21734734, chr1:58739989-58740101, chr6: 149028960-149029063, chr9:22452551 -22452664, chr12:4849148-4849254, chr15:92797259-92797393, chr20:5788487-5788587, chrX: 106481411-106481533, chr2:149561424-149561553, chr3:61381935-61382023, chr5: 17190626-17190742, chr5:59390194-59390288, chr7:25160122-25160245, chr1177619731 -77619846, chr17:48336001 -48336147, chr1 :206461901 -206462094, chr2: 143110966- 143111104, chr2 : 162123970- 162124051 , chr5: 16456356- 16456495, chr10: 15902711-15902826, chr10: 17742984-17743122, chr11 :6379394-6379487, chr16:17579398-17579529, chr17:5592340-5592644, chrX: 3035865-3036000, chr3:78659532-78659619, chr3: 105518948-105519094, chr4:64585094-64585216, chr5:83927264-83927382, chr6:593261 -593392, chr6:137314444-137314535,chr7: 107874746-107874889, chr10:27656752-27656912, chr11 :32960716-32960828, chr11 :43567527-43567641 , chr12:76791108-76791220, chr14:37230722-37230848, chr15:81939739-81939846, chr16:75789293-75789426, chr15:68554138-68554266, chr1 : 78130884-78131005, chr6: 50602320-50602449, chr1 : 119675667- 119675836, chr1 :244603541 -244603674, chrX: 101415625-101415728, chr2: 178280411 - 178280526, chr5: 96752586-96752688, chrX:22081630-22081732, chrX: 47384626- 47384769, chr19: 39474211-39474377, chr2:46464602-46464705, chr2:231715363- 231715468, chr7: 93228366-93228499, chr9:21111090-21111213, chr2 : 129849717- 129849817, chr10:34638577-34638699, chr19:42028327-42028524, chr2: 162298074- 162298189, chr8:24229478-24229598, chr8:29647427-29647523, chr10:63720138- 63720300, chr11 :34492959-34493085, chr12:27652288-27652426, chr2: 174700116- 174700251 , chr3:183122469-183122581 , chr12: 123905436-123905607, chr1 : 167540493-167540653, chr3: 189148131-189148251 , chr5: 141992479-141992603, chr9:78821242-78821375, chr12: 10562452-10562563, chr2:210577647- 210577793, chr2:212567753-212567856, chr4:4183014-4183121 , chr14:56491434- 56491551 , chr15:48352569-48352712, chrX:51461172-51461282, chr2: 159118703- 159118816, chr6: 36095298-36095473, chr1 :221435546-221435673, chr11 :46537952- 46538113, chr1 : 10428186-10428296, chr2: 102840031 -102840180, chr7: 104848545- 104848654, chr9:4988029-4988170, chr11 :121194847-121194979, chr11 :123247327- 123247457, chr13:41353128-41353267, chr17:73369977-73370136, chr19:49983756- 49983882, chr20:63223466-63223579, chr11 :10077000-10077143, chr8:71975358- 71975486, chr12:6537982-6538105, chr12:64891000-64891169, chr1 :161487131-161487247, chr4: 155442425-155442520, chr11 :44911428-44911580, chr12:31272008-31272161 , chr16:85717990-85718123, chr19:49547082-49547241 , chr10:11819934- 11820065, chr6:85355152-85355270, chr6:160519981-160520091 , chr7: 136009627- 136009724, chr14:21014774-21014934, chr1 : 53393434-53393577, chr2:38115355- 38115496, chr4:21789365-21789486, chr4:113394834-113394942, chr5: 59673572- 59673710, chr6:85323564-85323677, chr8:54169437-54169546, chr8: 96180035- 96180344, chr10:28241944-28242032, chr11 :62380869-62381016, chr12:8396766- 8396964, chr1 : 160726939-160727057, chr19:54676361 -54676503, chr1 :225976081-225976216, chr1 :236103745-236103862, chr4:16026149-16026413, chr4: 167021693- 167021794, chr5: 141960806-141960987, chr11 :48307888-48308019, chr16:50665497- 50665627, chr17:65198881 -65199032, chr20: 13119634-13119763, chr2: 10668563- 10668699, chr14:36374168-36374282, chr19:46402330-46402434, chr10:101161791- 101161998, chr20:6142524-6142649, chr3:71729971 -71730106, chr19:4204720- 4204856, chr1 : 101288544-101288669, chr1 :27669254-27669382, chr13: 36420493- 36420628, chr17:28540839-28540954, chr20:2351923-2352109, chr1 : 158998804-158998962, chr1 :220810250-220810376, chr2: 170409173-170409293, chr7:78040335- 78040434, chr1 :31162784-31162912, chr1 : 17661594-17661807, chr1 :31422842- 31422978, chr1 : 192445984-192446109, chr6: 105424291 -105424437, chr19:42529383- 42529506, chr1 : 107782340-107782444, chr9:86414420-86414532, chr7: 132188991- 132189067, chr2: 197374043-197374122, chr2:60642442-60642560, chr18:24736101- 24736161 , chr9:6594697-6594768, chr11 :9675318-9675421 , chr1 :226944700-226944781 , chr1 :202640729-202640819, chr9:14216485-14216587, chr12:54188268- 54188338, chr9:121838761-121838825, chr1 : 95264973-95265040, chr1 :69737726- 69737836, chr1 : 107782340-107782444, chr3: 32144255-32144365, chr3:113494238- 113494353, chr3: 134474064-134474175, chr3: 137105390-137105526, chr3: 181326780-181326885, chr4:26082030-26082146, chr4: 103934283-103934454, chr5:115586654-115586772, chr6: 52049619-52049757, chr6:63764478-63764585, chr6: 158691470- 158691594, chr6: 162908032- 162908121 , chr7: 130832002-130832124, chr7:132188991-132189067, chr8:63262036-63262139, chr8:110830607- 110830727, chr8: 117329067-117329210, chr8:119867220-119867390, ch r9: 86414420- 86414532, chr9:90142700-90142839, chr10:89563998-89564154, chr10:91389742- 91389869, chr12:92570492-92570605, chr12:97308762-97308849, chr14:28635549- 28635670, chr14:37149849-37149969, chr15:66567130-66567354, chr18:62958013- 62958123, chr18:62982806-62982908, chr20:52312263-52312398, chr1 : 11241826- 11241943, chr1 : 53754832-53754948, chr1 : 89987314-89987463, chr2:7427425-7427538, chr2:60642442-60642560, chr2: 104849680-104849792, chr2:112582400- 112582523, chr2:160110087-160110199, chr2: 197374043-197374122, chr2:224369161-224369299, chr4:242369-242480, chr4:41281166-41281273, chr4:122758089-122758199, chr4:166165962-166166073, chr4: 169810464-169810607, chr5:32489802-32489952, chr5:123371732-123371827, chr5: 172370813- 172370900, chr6:33123829-33123955, chr6:49849713-49849822, chr6:110942102- 110942167, chr6:113909396-113909512, chr6: 159008718-159008830, chr10:25700952-25701067, chr10:92885025-92885143, chr10:94630132-94630263, chr11 :30346226-30346339, chr11 :110433891-110434020, chr11 : 124586369-124586468, chr12:102702668-102702830, chr12:111451647-111451929, chr13:33228694-33228791 , chr13:50085114-50085230, chr13:53250379-53250572, chr13:57627719-57627850, chr13: 100722041-100722178, chr14:20327405-20327535, chr14:24735042-24735188, chr15:31974953-31975090, chr18:24736101-24736161 , chr19:54674154-54674274, chr22:22201213-22201344, chrX:22136053-22136155, chrX: 129754802-129754877, chrX: 130079923-130080041 , chr1 : 19918128-19918255, chr1 :24587331 -24587473, chr1 :39720138-39720288, chr1 :65958784-65959095, chr1 :74689941 -74690070, chr1 :88877501 -88877633, chr1 :161227460-161227587,chr1 :202640729-202640819, chr1 :226944700-226944781 , chr2: 17646148-17646327, chr2:227862361 -227862483, chr2:235594168-235594292, chr2:238462941-238463069, chr3: 124079474- 124079582, chr3:156541160-156541317, chr3: 194404789- 194404904, chr3: 195522387- 195522501 , chr4: 16794985- 16795080, chr4:27048448-27048583, chr4:52026152-52026244, chr4: 52376236-52376358, chr4: 140999943-141000112, chr4: 146104133-146104288, chr4: 158850959-158851065, chr4: 165464923-165465025, chr5:18314314-18314440, chr5:28014922- 28015045, chr5: 74648924-74649026, chr5: 102223966-102224084, chr5: 129664478- 129664599, chr5: 155139754-155139857, chr6: 12893990-12894132, chr6: 30380603- 30380684, chr6: 50792043-50792155, chr6:51855414-51855525, chr6: 123415992- 123416085, chr7:10412547-10412686, chr7:25180362-25180528, chr7: 38374563- 38374659, chr7:91830142-91830274, chr7:94395571 -94395689, chr8:4833976-4834088, chr8:95758783-95758908, chr8:119070591-119070729, chr9:6354233- 6354348, chr9:6594697-6594768, chr9:14216485-14216587, chr9: 17063671 -17063809, chr9: 19028989-19029108, chr10:3192966-3193102, chr10:16828259- 16828398, chr10:25726026-25726156, chr10:87656598-87656695, chr10:87735375- 87735496, chr10:125098674-125098794, chr11 :9675318-9675421 , chr11 :17230210- 17230310, chr11 :32323655-32323771 , chr11 :72667585-72667696, chr11 :124402455-124402570, chr12:20946875-20946995, chr12:53791868-53791960, chr12:55619731-55619974, chr12: 125403356- 125403461 , ch r12 : 126069664- 126069777, chr13:85828223-85828338, chr14:45426816-45426941 , chr14:93655215-93655320 chr15:44288413-44288574, chr15:90956673-90956821 , chr16:24253220-24253372 chr16:63361142-63361267, chr16:66728261-66728409, chr17:35312798-35312956 chr17:78013974-78014108, chr18:43571212-43571318, chr18:65781879-65782020 chr20:36466920-36467064, chr22:25093742-25093869, chrX:51614499-51614639 chrX: 110156310-110156388, chr22_KI270733v1_random: 136322-136475, chr1 : 32272584-32272646, chr1 :86742093-86742193, chr1 :88998835-88998937, chr1 : 95264973-95265040, chr1 : 163410082- 163410225, chr1 : 169055544- 169055615, chr1 :236452124-236452243, chr1 :240149603-240149760, chr1 :240189505-240189629, chr2: 18378180-18378279, chr2:38581666-38581810, chr2 : 152861493- 152861578, chr2:166539491-166539609, chr2:176814897-176815015, chr2:182186287-182186395, chr2:227890621 -227890778, chr3:87298250-87298349, chr3:89211716-89211800, chr3: 109732032-109732122, chr3: 134492629-134492738, chr3: 156509828- 156509965, chr3: 157137568- 157137691 , chr3: 161273765-161273866, chr3:166013225-166013311 , chr4:23135919-23136011 , chr4:48087272- 48087368, chr4:70183776-70183887, chr4:73019582-73019720, chr4:86044122- 86044254, chr4: 174365159-174365240, chr5:5141805-5141917, chr5: 80083642-80083758, chr5:81299634-81299801 , chr5:112175136-112175244, chr5: 146662279- 146662392, chr5:148733715-148733808, chr5: 154157507-154157598, ch r6: 35449508- 35449669, chr6:52021110-52021230, chr6:56902964-56903131 , chr6: 70706662- 70706775, chr6: 74091490-74091627, chr6:112180232-112180339, chr6: 135451738- 135451874, chr7:148504024-148504134, chr7:156582102-156582204, chr8: 12866970- 12867090, chr8:69663836-69663985, chr8:75257410-75257540, chr8:80612676- 80612794, chr8:90948403-90948519, chr8: 132651571 -132651698, ch r9: 13427664- 13427772, chr9: 15873723-15873819, chr9: 17462998-17463108, chr9: 121400273- 121400386, chr9:121838761-121838825, chr10:61284412-61284550, chr10:93352770-93352892, chr10:116490211 -116490321 , chr10:125982446-125982551 , chr11 :6738008-6738119, chr11 :84384764-84384893, chr11 :88092327-88092414, chr11 :94519623-94519738, chr11 :96464846-96464973, chr11 :115117490-115117553, chr11 :128414446-128414512, chr12:7642988-7643098, chr12:21680506-21680612, chr12:29519728-29519832, chr12:34034045-34034152, chr12:54188268-54188338, chr12:58346176-58346282, chr12: 100741240-100741368, chr12: 119160250-119160324, chr13:97132337-97132465, chr14: 19878197-19878283, chr14:25400748- 25400891 , chr14:53055029-53055173, chr14:64303608-64303708, chr14:82000025- 82000111 , chr14:98138734-98138826, chr15:85505208-85505330, chr15:98881743- 98881866, chr16:16449419-16449513, chr17:700647-700751 , chr17: 16258067- 16258156, chr18:24489020-24489155, chr18:31819747-31819885, chr19:28325490- 28325587, chr19:47125320-47125481 , chrX:11144386-11144529, chrX: 87299704- 87299805, chrX: 133670537-133670648, chrX: 152758817-152758894, chr1 :2091761- 2091844, chr1 :5225260-5225331 , chr1 :110555394-110555522, chr1 : 158036123- 158036245, chr1 : 176559961 -176560058, chr1 :207405060-207405185, chr1 :238188421 -238188505, chr2:39059461 -39059589, chr2:60707803-60707914, chr2:62412204-62412318, chr2: 106791905-106792003, chr2:151437690-151437807, chr2: 158010423-158010526, chr2:164717151-164717267, chr2: 179440792-179440892, chr2: 181715178-181715317, chr2: 197707735-197707844, chr2:232270870-232270944, chr2:241078520-241078659, chr3: 13600010-13600082, chr3:26712570-26712658, chr3:65785206-65785301 , chr3: 98039424-98039547, chr3: 146065649- 146065763, chr3: 147191901 - 147192041 , chr3: 173584256-173584361 , chr4:38856812-38856976, chr4:64713379-64713507, chr4:67746823- 67746964, chr4: 147097166-147097267, chr4:163484959-163485038, chr4: 185634052- 185634304, chr5: 19469171 -19469243, chr5:36870575-36870732, chr5: 52657324- 52657444, chr5:66307768-66307915, chr5: 103335135-103335205, chr6:47631424- 47631547, chr6:52945168-52945233, chr6:75893429-75893588, chr6: 93664709- 93664784, chr6:115244749-115244826, chr6:116287793-116287931 , chr6: 158605406-158605520, chr6: 170561934-170561998, chr7:26198200-26198319, chr7: 50142454- 50142556, chr7:81482732-81482834, chr7: 154504103-154504239, chr8:9831026- 9831238, chr9:8086869-8086940, chr10:117940922-117940998, chr11 :34926239- 34926356, chr11 :82682336-82682444, chr11 :121765670-121765767, chr12:12376782- 12376859, chr12:20950374-20950501, chr12:23949721 -23949794, chr12:24095121- 24095194, chr12:29889866-29889994, chr12:72987449-72987521, chr12:73391018- 73391090, chr12:78146406-78146532, chr12:90959676-90959826, chr13:43908534- 43908635, chr13:67238415-67238557, chr13:84896266-84896371, chr13:102141733- 102141898, chr13:110839093-110839188, chr14:78082948-78083033, chr14: 100624900-100625009, chr15:24585660-24585745, chr15:31880238-31880317, chr15:41031918-41032105, chr15:69322673-69322785, chr15:80419036-80419163, chr15:99704486-99704631 , chr17:31216272-31216386, chr17:48728857-48728929, chr17:74612182-74612276, chr17:79893762-79893854, chr18:25853208-25853305, chr18:64037936-64038067, and chr19:54080140-54080207.

54. The biomarker panel of claim 52 or claim 53, wherein the biomarker panel is a quantitative PCT (qPCR) array.

55. A method of determining the cell-of-origin or tissue-of-origin for cancer of unknown primary in a subject, the method comprising:(i) contacting a body fluid sample obtained from the subject with a binding agent which binds to cfDNA-CTCF nucleoproteins;(ii) isolating cfDNA-CTCF nucleoproteins bound in step (i);(iii) detecting the cfDNA isolated from the body fluid sample in step (ii) to determine genomic locations that are selectively occupied by CTCF in the body fluid sample; and(iv) using the genomic locations determined in step (iii) to determine the cell- of-origin or tissue-of-origin for the cancer of unknown primary.

56. A method of identifying cancer-associated mutations in CTCF-binding sites, the method comprising:(i) contacting a body fluid sample obtained from a subject with cancer with a binding agent which binds to cfDNA-CTCF nucleoproteins;(ii) isolating cfDNA-CTCF nucleoproteins bound in step (i);(iii) sequencing the cfDNA isolated from the body fluid sample in step (ii) to determine the sequence of CTCF binding sites that are occupied by CTCF in the body fluid sample; and(iv) comparing the sequences of the CTCF binding sites determined in step (iii) with the sequences of corresponding CTCF binding sites from a reference genome, to identify CTCF binding sites which are occupied by CTCF in the body fluid sample and which comprise one or more mutations.

57. A biomarker for cancer, the biomarker consisting of a DNA sequence of equal to or less than 100 base pairs and comprising a CTCF sequence which is selectively occupied in cancer.

58. Use of the biomarker of claim 57 in a method of detecting or diagnosing cancer.

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