Methods and kits for detecting clonal hematopoiesis or cancer or for prenatal screening

Abstract

The present invention relates to detection of pathologies, such as clonal hematopoiesis and cancer, and fetal screening, by analysis of thrombocytes for disease-associated markers. The method includes the steps of providing a biological sample containing thrombocytes; extracting nucleic acid from said biological sample; analyzing said nucleic acid to identify the presence of one or more clonal hematopoiesis-associated mutations or cancer-associated nucleic acid fragments; and indicating the presence or prognosis of clonal hematopoiesis or cancer based on the presence of one or more clonal hematopoiesis-associated mutations or cancer-associated nucleic acid fragments; or analyzing the nucleic acid for genetic information associated with a fetus.

Description

[Technical field]

[0001] The present invention relates to the detection of pathologies, such as clonal hematopoiesis and cancer, by analysis of thrombocytes for disease-associated markers. [Background technology]

[0002] All blood cells originate from hematopoietic stem cells (HSCs), which are multipotent and self-renewing progenitors. HSCs generate lymphoid and myeloid progenitors. The latter can differentiate into granulocytes, monocytes, erythrocytes, and megakaryocytes. Platelets (also called thrombocytes), "blood cell fragments" produced by megakaryocytes, are the second most abundant cell type circulating in peripheral blood. They are packaged with a rich protein cargo and, although they do not contain a nucleus, they contain all the machinery necessary to receive a diverse repertoire of RNA molecules from the "parent" megakaryocyte and to process and translate them for protein synthesis. Platelets also have a high capacity for intracellular trafficking and endocytosis. In the peripheral circulation, platelets can actively internalize and decode the biomolecules they encounter, including proteins and nucleic acids released during tissue injury or by viral pathogens, and interpret and respond to signals in their immediate environment. Thus, in addition to their important role in blood clotting and vascular maintenance, platelets function as key players in innate immunity and tumor immune surveillance, acting as "vascular vacuum cleaners" and sensing tissue injury, transformation and infection.

[0003] Therefore, platelet analysis can be used to detect certain diseases, such as clonal hematopoiesis (CH) and cancer.

[0004] CH occurs when hematopoietic stem cells start to generate cells with the same genetic mutation, resulting in the over-emergence of a single clone of blood cells. CH is a pre-cancerous condition that is common in the elderly and detectable in >10% of people >70 years of age by analysis of granulocyte DNA, and is associated with an approximately 10-fold increased risk of developing blood cancers, an approximately 2-fold increased risk of cardiovascular disease, a similar increased risk of venous thromboembolism, and a significant increase in total mortality. Studies have further linked CH to a variety of other disorders, including degenerative diseases, diabetes, and autoimmune diseases. The degree of risk depends on the specific mutant allele driving the clonal expansion, the number of mutations, the mutant allele burden, and concurrent non-genetic risk factors, such as hypertension or smoking.

[0005] Identifying those with CH is important for early detection and intervention may lower the risk of complications. The cardiovascular risks associated with CH are more significant than the relatively rare tumor progression. Anti-inflammatory approaches may help prevent cardiac events and even reduce lung cancer cases (e.g., CANTOS study, see "Product type, therapeutic area and indication(s)" for details). Currently, the presence of CH is based on identifying the presence of a clone present at a frequency of 2% based on the lower limit of detection of most commonly used assays.

[0006] Furthermore, platelets contain mRNA transcripts and active splicing machinery mostly derived from their parent megakaryocytes, although they may pick up and carry nucleic acids derived from tumor cells. Other groups have focused on studying gene expression signatures in the platelet transcriptome. However, the platelet transcriptome alone is probably not specific enough for cancer and is difficult to distinguish from non-malignant inflammatory conditions. Currently, the main approach for liquid biopsy is the analysis of cell-free DNA (cfDNA) from tumor cells, and a major problem is the low content of cfDNA, resulting in low sensitivity, especially for early stage tumors. Therefore, novel methods are needed to increase the availability of cfDNA from tumor cells for analysis via liquid biopsy approaches. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012) Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, there is a need to develop additional methods for the detection of CH and cancer. [Means for solving the problem]

[0009] Platelets are small (2-5 μm) multifunctional cells derived from megakaryocytes in the bone marrow and lungs. Platelets are anucleate but contain RNA derived from parent megakaryocytes and the translation machinery required for protein synthesis. During cell death and abnormal mitosis, nucleated cells release chromosomal DNA, which rapidly fragments, resulting in "cell-free" DNA (cfDNA) in plasma. Excess cfDNA is harmful. Given their ability to sense and internalize pathogen-derived nucleic acids, we hypothesized that platelets may play a key role in the clearance of endogenous cfDNA. Here, we show that platelets, despite the absence of a nucleus, contain a repertoire of DNA fragments located across the nuclear genome, in addition to mitochondrial DNA. We show that this DNA is derived from non-megakaryocytic lineage cells and demonstrate the presence of fetal DNA in maternal platelets, as well as cancer cell-derived DNA in platelets from patients with premalignant lesions and overt solid cancers. This study establishes a role for platelets in isolating cfDNA, an aspect of platelet biology that has not been emphasized before, with the wide availability of minimally invasive liquid biopsies. Platelets are ideal "sentinels" of genetic perturbations because they are easily isolated and circulate continuously through tissues.

[0010] Platelets are fundamental to hemostasis and vascular maintenance, and contribute to innate and adaptive immunity, including induction of inflammatory responses via sensing pathogen-derived nucleic acids. As part of antiviral immunity, platelets internalize DNA and RNA viruses, and interestingly, it was recently reported that nucleic acids from pine pollen were detectable in human platelets, indicating that platelets sequester exogenous nucleic acids encountered in the circulation. While platelet RNA has been well studied and has novel utility as a liquid biopsy approach for hematological and solid malignancies, whether platelets contain DNA, and if so, its cellular origin, has not been widely investigated.

[0011] Analysis of cfDNA in plasma is rapidly being implemented in a wide range of clinical settings, including cancer care pathways and prenatal genetic testing. A major obstacle to the utility of cfDNA for cancer surveillance is the low content of circulating tumor-derived DNA (ctDNA) in standard cfDNA preparations, including isolation of DNA from platelet-depleted plasma. Recent efforts to overcome this have focused on increasing sequencing depth or capturing cancer-associated genetic abnormalities more broadly via whole genome sequencing (WGS) or epigenetic analysis. However, improved preanalytical methods that increase cfDNA capture would be highly beneficial in many diagnostic settings. Given their role in sensing pathogen-derived nucleic acids, we hypothesized that platelets could clear cfDNA from plasma and that important insights could be derived from analysis of genetic material in platelets derived from cell types encountered in their peripheral circulation.

[0012] The inventors have developed a method for isolating platelets from blood and the subsequent extraction of nucleic acids, RNA and DNA from the platelets. The group has demonstrated the identification of disease-associated genetic mutations in the isolated nucleic acids. Analysis of patient samples shows that mutations that are not detectable in other blood components are frequently detectable in platelets from patients. Thus, the method significantly increases the sensitivity of mutation detection. Using this approach, the inventors have demonstrated the utility of analysis of platelet-derived nucleic acids in the detection of pre-malignant hematological disorders, hematological cancers, and solid tumors.

[0013] A first aspect of the present invention provides a method for detecting or predicting clonal hematopoiesis, comprising the steps of: providing a biological sample containing thrombocytes; extracting nucleic acid from the biological sample; analyzing the nucleic acid to identify the presence of one or more clonal hematopoietic mutations; and indicating the presence or prognosis of clonal hematopoiesis based on the presence of one or more clonal hematopoiesis-associated mutations. The present invention relates to a method comprising the steps of:

[0014] One aspect of the invention is a method for detecting or predicting cancer, comprising the steps of: providing a biological sample containing thrombocytes; extracting nucleic acid from the biological sample; In certain embodiments, analyzing the nucleic acid to identify the presence of one or more cancer-associated nucleic acid fragments, including cancer-associated mutations and methylation profiles; and Indicating the presence or prognosis of cancer based on the presence of the cancer-associated nucleic acid fragment. The present invention relates to a method comprising the steps of:

[0015] One aspect of the invention is a method for determining a treatment for a subject, comprising: A detection or prediction method as described herein; and Determining the appropriate course of action The present invention relates to a method comprising the steps of:

[0016] One aspect of the invention relates to a kit comprising reagents for extracting nucleic acid from platelets and a panel of reagents that specifically bind and / or amplify one or more clonal hematopoietic associated mutations, and optionally instructions for use.

[0017] One aspect of the invention relates to a kit comprising a panel of reagents for extracting nucleic acids from platelets and reagents that specifically bind to and / or amplify one or more cancer-associated modifications or cancer-specific mutations.

[0018] One aspect of the invention is a method of treating a subject having cancer, comprising: providing a biological sample containing thrombocytes; extracting nucleic acid from the biological sample; analyzing the nucleic acid to identify the presence of one or more cancer-associated nucleic acid fragments; selecting a treatment; and Administering the Treatment The present invention relates to a method comprising the steps of:

[0019] One aspect of the present invention is a method for preparing a nucleic acid fraction, comprising the steps of: providing a biological sample containing thrombocytes; extracting nucleic acid from the biological sample to form a nucleic acid sample; enriching said nucleic acid sample for one or more cancer-associated nucleic acid fragments and / or clonal hematopoietic-associated fragments. The present invention relates to a method comprising the steps of:

[0020] One aspect of the invention is a method for genotyping a sample of thrombocytes, comprising the steps of: providing a biological sample containing thrombocytes; extracting RNA from the biological sample; converting the RNA into cDNA; analyzing said cDNA to identify the presence of one or more clonal hematopoietic mutations, thereby genotyping the sample. The present invention relates to a method comprising the steps of:

[0021] One aspect of the invention is a method for genotyping a sample of thrombocytes, comprising the steps of: providing a biological sample containing thrombocytes; extracting nucleic acid from the biological sample; analyzing said nucleic acid to identify the presence of one or more cancer-associated nucleic acid fragments, thereby genotyping the sample. The present invention relates to a method comprising the steps of:

[0022] One aspect of the present invention is a method for prenatal screening of fetal genetic information, comprising the steps of: Providing a biological sample containing thrombocytes obtained from a pregnant female; extracting nucleic acid from the biological sample; analyzing said nucleic acid for genetic information associated with the fetus. The present invention relates to a method comprising the steps of: [Brief description of the drawings]

[0023] [Figure 1]Overview of platelet and cfDNA isolation and purity assessment. a, (i) Methods for co-extraction of DNA (pDNA) from platelet pellets and DNA (cfDNA) from platelet-depleted plasma derived from peripheral blood; (ii) methods for platelet isolation and cryopreservation. Platelet freezing medium (0.9% (w / v) NaCl with 27% DMSO diluted in platelet-rich plasma to a final DMSO concentration of 6%). b, Platelet counts after cryopreservation. Absolute platelet counts in whole blood (WB), platelet-rich plasma (PRP), and after cryopreservation and thawing assessed using an automated cell counter. c, Flow cytometric analysis of platelet pellets confirms high yield of platelets with minimal leukocyte and erythrocyte contamination. Analysis of platelet pellets shown in size scatter plots of leukocytes (top) and platelets (bottom) using a threshold of 5000 for trapping leukocytes and erythrocytes and a lower threshold of 1000 for trapping platelets. Percentage of contaminating cells in total events is shown. d, Gene expression analysis of platelet pellets shows minimal expression of red blood cell (RBC) and white blood cell (WBC) associated genes. e, Quantification of leukocyte contamination in cytospins and platelet pellets using Wright-Giemsa staining for a representative sample. Figure 1A and 1B generated by BioRender.com. CH, clonal hematopoiesis; ddPCR, droplet digital PCR; DMSO, dimethyl sulfoxide; NGS, next generation sequencing; PLT, platelets; PRP, platelet-rich plasma; RBC, red blood cells; WB, whole blood; WBC, white blood cells. [Diagram 2]Detection of platelet-biased JAK2V617F-associated clonal hematopoiesis. a, JAK2V617F ddPCR assay accuracy study. Measurement error studies showed that both (gDNA and cDNA) assays were both accurate and precise. (i) Results of cDNA assay repeatability study for serial dilutions up to 0.1% final expected abundance ratio. Repeatability was evaluated in three independent ddPCR runs. To evaluate the performance of the assay for low DNA inputs, repeatability studies for different (ii) gDNA and (ii) cDNA inputs (up to 2.5ng and 10ng, respectively) were performed. b, JAK2V617F ddPCR assay QC. Experimental controls consisting of cell line-derived DNA (left panel) and granulocyte DNA from patients (right panel) were used to optimize the ddPCR assay and validate the highly specific detection of mutant JAK. Lower plot - ddPCR analysis of two representative donors showing JAK2V617F-CH detected exclusively in platelets (PLT cDNA) but not in granulocytes (GRA gDNA). Red boxes highlight positive mutant FAM signals. (c) Analysis of 151 donors suggests that testing of platelets substantially increases the sensitivity of CH detection. Bar graph (left panel) shows the number of JAK2V617F-associated CH cases detected in the cohort and its distribution between GRA gDNA and PLT cDNA samples. Shaded bars indicate cases fulfilling the criteria for CHIP with an abundance ratio of ≥2%. Scatter plot (right panel) shows a comparison of JAK2V6167F abundance ratios in granulocyte- and platelet-derived cDNA relative to the total number of CH, revealing a significantly higher abundance ratio in platelet cDNA (Mann-Whitney test; p=0.0383). Bars and error bars indicate median and 95% CI, respectively. Red grid lines correspond to 2% abundance. CH, clonal hematopoiesis; CHIP, clonal hematopoiesis of indeterminate potential; ddH2O, double distilled water; ddPCR, droplet digital PCR; Ctrl, control; GRA, granulocytes; MNC, mononuclear cells; PLT, platelets. [Diagram 3]Detection of platelet-biased clonal hematopoiesis in genes other than JAK2. a, Custom-made hybridization capture panel for parallel sequencing of gDNA and cDNA to detect common CH-associated gene mutations. b, Schematic diagram of the panel design showing the probes for JAK2. Exonic regions are shown in blue. Target regions (which are exons 12 and 14) are shown in green. Considering that exon 12 is 128 bp long, a single center-aligned 120 nucleotide-long probe (shown in yellow) was designed for use in variant detection in both gDNA and cDNA. However, for exon 14, which is 88 bp, a 120 nucleotide-long probe (shown in grey) spanning the adjacent intronic region was designed for gDNA capture, and an additional exon-exon probe (shown in orange) including exon 14 and part of exon 15 was designed for cDNA capture, allowing for simultaneous analysis of gDNA and cDNA templates. c, Oncoplot showing NGS-detected variants with a VAF >2% per gene (each row = gene) in (i) GRA gDNA and (ii) PLT cDNA for each individual (each column = separate donor). CI, confidence interval; GRA, granulocytes; NGS, next generation sequencing; PLT, platelets. [Figure 4] Detection of JAK2V617F in pDNA, cfDNA, and granulocyte paired genomic DNA. a, Results of ddPCR analysis for detection and quantification of JAK2V617F in genomic DNA extracted from platelets, plasma, and granulocytes from a representative patient diagnosed with JAK2 mutant myeloproliferative neoplasms. FAM channel positive events on the y-axis correspond to JAK2V617F positivity, and HEX channel positive events on the x-axis correspond to JAK2 WT events. b, Analysis showing allele frequency of JA2V617F variants in genomic DNA extracted from platelets, plasma, and granulocytes for five donors. [Diagram 5]Platelets sequester DNA during circulation. a, DNA abundance in cfDNA and pDNA calculated as genome equivalents per microliter (μl) of plasma. Graphs show median ± 95% CI, pDNA vs. cfDNA paired Wilcoxon signed rank test ****p<0.0001 for non-cancer controls (n=14), patients with premalignant colonic serrated lesions (SSL, n=29), and patients with known adenocarcinoma (n=21). b, Representative electrophoresis of DNA extracted from cfDNA and pDNA, showing a peak at approximately 150 base pairs (bp) in pDNA, and some platelet samples showing nucleosome footprints similar to cfDNA. All platelet samples showed larger DNA fragments ranging from approximately 12,000 to 16,000 bp, also observed in pDNA but not in cfDNA. c, Effect of DNase treatment on detection of JAK2 mutant alleles in pDNA and cfDNA. Data represent the mean ± SD of three independent experiments. ns p>0.05 and **p<0.01 by paired Wilcoxon signed rank test. d, Healthy donor human platelets identified by CD42-488 (cyan) show the intrinsic fluorescent signal of NUCLEAR-ID (magenta), a cell-permeant dye that intercalates into double-stranded DNA. White boxes indicate magnified areas and scale bar represents 2 μm. e, Time course of DNA uptake by platelets. [Figure 6] Extracellular DNA fragments carrying cancer-associated gene mutations are sequestered by platelets. a, Number of mutant alleles per μl of pDNA extracted from platelets incubated with (+) or without (-) colorectal cell lines (LS180, COLO205, and HCT116) and an erythroleukemia cell line (HEL). Mean ± SD, n = 3 independent experiments, *p < 0.05 calculated using paired Wilcoxon signed rank test. b, Representative droplet digital PCR analysis showing quantification of wild-type BRAF and BRAFV600E alleles in pDNA isolated from healthy donor platelets before (top) and after (bottom) co-incubation with BRAFV600E-positive COLO205 colorectal cancer cells. [Figure 7] Platelets contain a repertoire of DNA fragments located across the human nuclear genome, including tumor-derived DNA in patients with active malignancies. a, Chromosomal mapping of s-pDNA and paired cfDNA for a representative patient with pancreatic adenocarcinoma (sample 6). b, Percentage of fragments mapping to the mitochondrial genome from cell-free DNA (cfDNA), short (>100 and <600 bp) and long (>600 bp) platelet DNA (pDNA) fragments (s-pDNA and l-pDNA). Median ± individual data points are shown. c, Deviations from median coverage in 100 kilobase (kb) windows across all chromosomes for cfDNA (top) and s-pDNA (bottom) for sample 6 reveal chromosomal abnormalities in chromosomes 2, 6 and 7 (copy number gains and amplifications in red, deletions in green). d, Distribution of read depth per transcription start site for cfDNA mononucleosomal reads (top) and s-pDNA mononucleosomal reads (bottom) for genes highly (TPM>15, purple) or lowly (TPM≦15, blue) expressed in peripheral blood mononuclear cells (PBMCs). Depth per position per sample was normalized to the median read depth across all genes in that expression category. MNC, mononuclear cell; pos, position; TSS, transcription start site. [Figure 8] KRASG12D copies are more abundant in pDNA than in cfDNA in mice with localized and metastatic colorectal adenocarcinoma. a, Waterfall plot showing the fold difference in KRASG12D copy numbers detected per μl of DNA in pDNA vs. cfDNA. b and c, Representative droplet digital PCR plots showing higher amounts of KRASG12D (blue) in pDNA than in cfDNA in two representative mice. b, KPN mouse, and c, KP mouse. CRC, colorectal cancer; WT, wild type. [Figure 9]BRAFV600E in platelets from patients with premalignant lesions detected by colonoscopic screening. a, Pie graph showing detection of BRAFV600E in patients (n=29) with high-risk premalignant colonic lesions (serrated lesions, SSL) and in control individuals (n=14) who underwent colonoscopic screening and no lesions or malignancies were detected. Mutant BRAF was detected in 17.2% (5 / 29) of SSL patients and 0% (0 / 14) of controls. b, Relative copy number of BRAFV600E in pDNA compared to cfDNA in five patients with SSL in whom mutant BRAF was detectable in either cfDNA or pDNA. Data were transformed to binary logarithms with a pseudocount of 1. c, Droplet digital PCR plots showing BRAFV600E (blue) in pDNA (upper plot) and cfDNA (lower plot) in two SSL patients. SSL, serrated lesion; WT, wild type. [Figure 10] Proof-of-principle of the utility of pDNA analysis for prenatal genetic screening. a, Fluorescent in situ hybridization chromosome paint and droplet digital PCR (ddPCR) showing detection of the Y chromosome gene SRY in platelets but not mononuclear cells (MNC) of mothers pregnant with boys. Maternal blood was sampled before delivery. Platelets and MNC were counterstained with β-tubulin (blue) and imaged using a ZEISS LSM900, 63x magnification. Representative images are shown. b, Detection of SRY gene copies by ddPCR of DNA isolated from maternal platelets 24 and 48 hours after delivery of boys by Caesarean section (at delivery: n=9, 24 hours after delivery: n=5, and 48 hours after delivery: n=2). Mean values ​​± SD are shown. [Figure 11]Platelets sequester DNA during circulation. a, Healthy donor human platelets identified by CD42-488 (blue) show the internal fluorescent signal of NUCLEAR-ID (magenta), a cell-permeant dye that intercalates into double-stranded DNA. White box indicates the magnified area and scale bar represents 2 μm. b, Method for co-extraction of DNA (pDNA and cfDNA) from platelet pellet and platelet-depleted plasma from peripheral blood. c, DNA abundance in cfDNA and pDNA calculated as genome equivalents per microliter (μl) of plasma. Graph shows median ± 95% CI, ****p<0.0001 by paired Wilcoxon signed rank test for pDNA vs. cfDNA for non-cancer controls (n=14), patients with premalignant colonic serrated lesions (SSL, n=29), and patients with known adenocarcinoma (n=21). d, Fluorescent in situ hybridization chromosome paint and droplet digital PCR (ddPCR) showing detection of the Y chromosome gene SRY in maternal platelets, but not in mononuclear cells (MNCs), sampled from mothers prior to delivery of newborn boys. Platelets and MNCs were counterstained with β-tubulin (blue) and imaged using a ZEISS LSM900, 63x magnification. Representative images are shown. e, Detection of SRY gene copies by ddPCR on DNA isolated from maternal platelets 24 and 48 hours after delivery of boys by Caesarean section (at delivery: n=9, 24 hours after delivery: n=5, and 48 hours after delivery: n=2). Mean values ​​± SD are shown. f, Rapid clearance of platelets followed by an increase in cfDNA in healthy mice after administration of antiplatelet antibodies (n=20 for each of the isotype control and antiplatelet antibody groups). Plots show the mean fold change (±SEM) at days 1, 3, and 5 compared to baseline (day 0, untreated mice). [Figure 12]Platelets rapidly internalize DNA released by nucleated cells via uptake of DNA-loaded extracellular vesicles. a, CD42-488 (cyan)-labeled platelets from a healthy donor before (left) and after (middle and right) co-incubation with NUCLEAR-ID-labeled COLO205 cells, allowing tracking of DNA uptake (magenta). White boxes indicate magnified areas and the scale bar represents 2 μm. White arrows highlight COLO25 DNA uptake by platelets. The middle and right images are the same, and the bright field view shown on the right highlights the membrane of a COLO205 cell. b, Live cell imaging time-lapse showing internalization of fluorescently labeled DNA by platelets. The scale bar represents 3 μm. c, Quantification of platelet DNA fluorescence intensity over time (mean ± SD) (min). To detect any variations in background signal, AF647 signal was also measured in background areas of each image. ****p<0.0001 by paired Wilcoxon signed rank test. Traces shown for platelets incubated with control medium (n=168 platelets) or medium conditioned by NUCLEAR-ID labeled COLO205 cells (n=173 platelets). d, 3D reconstruction of clusters of extracellular vesicles (EVs) released by apoptotic BL2 cells labeled with an amine-reactive succinimidyl ester (SE) membrane dye (CF658, red) and DNA staining (DAPI, yellow) reveals internalized DNA. Imaged using a ZEISS LSM900, 63x magnification. e, After incubation with labeled EVs, platelets were stained with CD42-488 (cyan). EVs (red) were visualized within the platelets, confirming platelet uptake of apo-EVs. 3D modeling shows a 90° rotation of the platelet. f, 3D reconstruction of platelet internalization of DNA (yellow) loaded apo-EVs (red). Two representative platelets are shown. g, Droplet digital PCR quantification of the Y chromosome gene SRY in platelet-derived pDNA of a female donor after incubation with EVs derived from apoptotic BL2 cells (apo-EVs) versus EVs from apoptotic-resistant BL2 cells (non-apoEVs).h, Fluorescence in-situ hybridization micrographs and 3D rendering (right panel) demonstrating fragments of X and Y chromosomes present in female donor platelets after exposure to male BL2 cells. Platelets counterstained with β-tubulin (blue). Representative images are shown. Imaged using a ZEISS LSM900, 63x magnification. Images analyzed using ImageJ and ImarisViewer. Abbreviations: arbitrary intensity units (AIU); apoptosis (apo); extracellular vesicles (EV); Burkitt's lymphoma (BL2) cells; succinimidyl ester (SE); clockwise (CW); counterclockwise (CCW). [Figure 13]Extracellular DNA fragments carrying cancer-associated gene mutations are sequestered by platelets and protected from degradation. a, Platelets from healthy donors were co-incubated with malignant cells separated by a 1 μm membrane insert, allowing exchange of extracellular biomolecules and small EVs between the compartments, but not cells. After co-incubation, platelets were removed and washed three times before DNA extraction. Image generated by BioRender.com. b, Number of mutant alleles per μl of pDNA extracted from platelets incubated with (+) or without (-) colorectal cell lines (LS180, COLO205 and HCT116) and an erythroleukemia cell line (HEL). Mean ± SD, n = 3 independent experiments, *p < 0.05 calculated using paired Wilcoxon signed rank test. c, Representative droplet digital PCR analysis showing quantification of wild-type BRAF and BRAFV600E alleles in pDNA isolated from healthy donor platelets before (top) and after (bottom) co-incubation with BRAFV600E-positive COLO205 colorectal cancer cells. d, Quantification of BRAFV600E in red blood cells (RBCs), mononuclear cells (MNCs), and platelets (PLTs) after incubation in medium conditioned by COLO205 cells. Data represent the mean ± SD of three independent experiments. *p<0.05 using Mann-Whitney U test. e, Effect of DNase treatment on detection of JAK2 mutant alleles in pDNA and cfDNA. Data represent the mean ± SD of three independent experiments. ns p>0.05 and **p<0.01 by paired Wilcoxon signed rank test. Abbreviation: mononuclear cell (MNC); platelet (PLT); red blood cell (RBC); wild type (WT) [Figure 14]Platelets contain a repertoire of DNA fragments located across the human nuclear genome, including tumor-derived DNA from patients with active malignancies. a, Percentage of fragments mapping to the mitochondrial genome from cell-free DNA (cfDNA), short (>100 and <600 bp) and long (>600 bp) platelet DNA (pDNA) fragments (s-pDNA and l-pDNA). Median ± individual data points are shown. b, Plot showing significantly higher frequency of dinucleosomal fragments (fragments >250 bp long) in s-pDNA than in cfDNA (standard error of the mean shown in grey, p<0.0005 by chi-squared test of independence). c, Fragment length distribution of paired and aligned reads for cfDNA (top) and s-pDNA samples (bottom). Samples 1-5 have a peak fragment length of approximately 165 bp, representing mononucleosomal fragments, and a second small peak of approximately 325 bp (shown in the inset plot) represents dinucleosomal fragments. The mononucleosomal and dinucleosomal fragment lengths from sample 6 (red line), a patient with untreated pancreatic adenocarcinoma, are significantly shorter than samples 1-5 (from patients following anticancer therapy). d, Deviations from median coverage in 100 kilobase (kb) windows spanning all chromosomes for cfDNA (top) and s-pDNA (bottom) for sample 6 reveal chromosomal abnormalities in chromosomes 2, 6, and 7 (copy number gains and amplifications shown in red, deletions shown in green). e, Read depth distribution per transcription start site for cfDNA mononucleosomal reads (top) and s-pDNA mononucleosomal reads (bottom) for genes highly (TPM>15, purple) or lowly (TPM≦15, blue) expressed in peripheral blood mononuclear cells (PBMCs). Depth per position per sample was normalized to the median read depth across all genes in that expression category. Abbreviations: peripheral blood mononuclear cells (PBMNC); position (pos); transcription start site (TSS). [Figure 15]KRASG12D copies are more abundant in pDNA than in cfDNA in mice with localized and metastatic colorectal adenocarcinoma. a, Schematic showing isolation of platelet DNA (pDNA) and cell-free (cfDNA) from C57BL / 6 mice expressing KRASG12D and TP53 (KP) mutations through the villin promoter, causing locally invasive colorectal adenocarcinoma, and mice with aggressive metastatic disease harboring KRASG12D, TP53 and NOTCH (KPN) mutations. Image created by BioRender.com. b, Waterfall plot showing the fold difference in the number of KRASG12D copies detected per μl of DNA in pDNA vs. cfDNA. c and d, Representative droplet digital PCR plots showing higher amounts of KRASG12D (blue) in pDNA than in cfDNA in two representative mice. c, KPN mice, and d, KP mice. Abbreviations: colorectal cancer (CRC); wild type (WT). [Figure 16] BRAFV600E in platelets from patients with premalignant lesions detected by colonoscopic screening. a, Pie graph showing detection of BRAFV600E in patients (n=29) with high-risk premalignant colonic lesions (serrated lesions, SSL) and in control individuals (n=14) who underwent colonoscopic screening and no lesions or malignancies were detected. Mutant BRAF was detected in 17.2% (5 / 29) of SSL patients and 0% (0 / 14) of controls. b, Relative copy number of BRAFV600E in pDNA compared to cfDNA in five patients with SSL in whom mutant BRAF was detectable in either cfDNA or pDNA. Data were transformed to binary logarithms with a pseudocount of 1. c, Droplet digital PCR plots showing BRAFV600E (blue) in pDNA (upper plot) and cfDNA (lower plot) in two SSL patients. Abbreviations: serrated lesions (SSL); wild type (WT). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Embodiments of the present invention will now be further described. In the following sections, different embodiments are described. Each aspect so defined can be combined with any other aspect, unless expressly stated otherwise.

[0025] Generally, the terminology used in the context of cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization, and the techniques thereof described herein, are well known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification, unless otherwise specified. See, for example, Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012).

[0026] The present invention is based on the discovery that mutations associated with clonal hematopoiesis (CH) can be detected in anucleated cells, such as thrombocytes. The standard method for detecting CH is by analysis of whole blood or white blood cells. Although thrombocytes are not typically analyzed by standard approaches applied to the detection of cancer-associated gene mutations, growing evidence demonstrates that many long-term hematopoietic stem cells (HSCs) exclusively produce cells of the platelet lineage. To date, all CH studies are based on whole blood or granulocyte DNA, and therefore 1) they do not evaluate platelet-restricted clones, and 2) the amount of ribonucleic acid contained in platelets is diluted and mutations would be missed by sequencing nucleic acid from whole blood samples. However, the inventors have demonstrated that analysis of nucleic acid, especially from isolated thrombocytes, is much more sensitive than previous methods of analyzing whole blood or white blood cells.

[0027] Therefore, one aspect of the present invention is a method for detecting or predicting hematopoiesis, comprising the steps of: providing a biological sample containing thrombocytes; extracting nucleic acid from the biological sample; analyzing the nucleic acid to identify the presence of one or more clonal hematopoietic mutations; and indicating the presence or prognosis of hematopoiesis based on the presence of one or more clonal hematopoiesis-associated mutations. The present invention relates to a method comprising the steps of:

[0028] Clonal hematopoiesis (CH) occurs when hematopoietic stem cells start producing cells with the same genetic mutation. CH is a condition in which blood cancer-associated mutations are detectable in blood cells of people with normal blood cell parameters. This condition is common in individuals over 70 years of age (detectable in >10%). CH also increases the risk of blood cancer and cardiovascular disease. CH can be considered a pre-cancerous state, i.e., a state in which patients are identified as at risk for developing further pathologies. Detection of CH is important because it can be used as a biomarker for early detection of blood cancer, as well as the risk of blood clots and cardiovascular disease, which opens up opportunities for preventive intervention. Therefore, detection of CH can lead to the detection of subjects at high risk of developing blood cancer, blood clots, and cardiovascular disease.

[0029] As used herein, the terms "platelet" and "thrombocyte" are used interchangeably and refer to anucleated "blood cell fragments" produced by bone marrow megakaryocytes. Platelets are the second most abundant cell circulating in peripheral blood and play a major role in preventing bleeding and maintaining homeostasis. Although platelets do not have a nucleus, they can be packaged by the "parent" megakaryocyte with RNA molecules and translate them for protein synthesis.

[0030] The method of the invention includes a step of extracting nucleic acid from thrombocytes, which is then analyzed for the presence of CH-associated mutations. The extracted nucleic acid can be RNA or DNA, or RNA and DNA extracted simultaneously. RNA can be extracted from the biological sample without parallel extraction of DNA. DNA can be extracted from the biological sample without parallel extraction of RNA. If RNA is extracted from thrombocytes, the RNA is then converted to cDNA for analysis. The DNA extracted from thrombocytes can be genomic DNA (gDNA). If a combination of DNA and RNA is extracted from thrombocytes, the method can include parallel analysis of cDNA and gDNA. Analysis of cDNA and gDNA can be performed simultaneously, sequentially, or separately to detect CH-associated mutations. In one embodiment, RNA and gDNA are extracted from thrombocytes and analyzed in parallel for the presence of CH-associated mutations. In one embodiment, RNA and gDNA are extracted from thrombocytes and analyzed separately for the presence of CH-associated mutations. In one embodiment, RNA is extracted from thrombocytes and analyzed for the presence of CH-associated mutations. In one embodiment, gDNA is extracted from thrombocytes and analyzed for the presence of CH-associated mutations.

[0031] Conversion of RNA to cDNA can be performed using any suitable method known in the art, for example, extracted RNA is converted to cDNA via reverse transcription. Reverse transcriptase can be used to convert RNA to cDNA. Reverse transcriptase, also known as RNA-dependent DNA polymerase, is an enzyme used to generate complementary DNA (cDNA) from an RNA template. Specifically, this enzyme is a DNA polymerase enzyme that transcribes single-stranded RNA into DNA. This enzyme can synthesize double-stranded DNA once RNA is reverse transcribed into single-stranded DNA in the first step. RNA can be reverse transcribed into cDNA using RNA-dependent DNA polymerase, e.g., viral, retrotransposon, bacterial, etc. reverse transcriptase. These enzymes may have RNase H activity, or reverse transcriptases that are mutated such that the RNase H activity of the reverse transcriptase is limited or absent (e.g., MMLV-RT RNase H) can be used. Suitable reverse transcriptases include, but are not limited to, AMV reverse transcriptase, MMLV reverse transcriptase, engineered MMLV reverse transcriptase. RNA-dependent DNA synthesis (reverse transcription) can also be performed by enzymes that exhibit altered nucleic acid dependency due to mutations or modified reaction conditions, thus acquiring the function of RNA-dependent DNA polymerase. Commercial kits are available for reverse transcribing RNA into cDNA. Once RNA is reverse transcribed into cDNA, the DNA sequence can be analyzed for the presence of specific mutations or expression profiles associated with pathology. Expression profiles can be determined using selective nucleic acid hybridization, as described above. Such techniques are well known in the art and can include selective amplification using amplification primers that are specific for the mutation to be detected, or selective hybridization to a nucleic acid array using an mRNA-specific probe. Alternatively, DNA containing suspected mutations can be amplified using general primers, and then the mutations can be detected in the amplicons by selective nucleic acid hybridization using a probe that is specific for the mutation.

[0032] The term "clonal hematopoiesis-associated mutation" refers to any mutation indicative of CH. Mutations indicative of or associated with CH can be identified by comparing samples obtained from subjects known to have CH with samples obtained from healthy subjects. CH-associated mutations are those found in disease samples. The method can detect one or more, two or more, three or more, four or more, five or more, or ten or more CH-associated mutations. For example, the method can include detecting a panel of CH-associated mutations. In one embodiment, the CH-associated mutation is in one of the following genes: JAK2 (Ensembl ID: ENSG00000096968), CALR (Ensembl ID: ENSG00000179218), MPL (Ensembl ID: ENSG00000117400), CBL (Ensembl ID: ENSG00000110395), KRAS (Ensembl ID: ENSG00000133703), GNB1 (Ensembl ID: ENSG00000078369), DNMT3A (Ensembl ID: ENSG00000119772), TET2 (Ensembl ID: ENSG00000168769), ASXL1 (Ensembl ID: ENSG00000171456), IDH2 (Ensembl ID: ENSG0000017217), IDH3 (Ensembl ID: ENSG00000171613), IDH4 (Ensembl ID: ENSG00000171511), IDH5 (Ensembl ID: ENSG0000017172), IDH6 (Ensembl ID: ENSG0000017182), IDH7 (Ensembl ID: ENSG00000171613), IDH8 (Ensembl ID: ENSG0000017162), IDH9 (Ensembl ID: ENSG0000017163), IDH1 (Ensembl ID: ENSG0000017164), IDH2 (Ensembl ID: ENSG0000017165), IDH1 (Ensembl ID: ENSG0000017166), I In one embodiment, the one or more CH-associated mutations may be present in one or more of JAK2 (Ensembl ID: ENSG00000182054), SF3B1 (Ensembl ID: ENSG00000115524), SRSF2 (Ensembl ID: ENSG00000161547), U2AF1 (Ensembl ID: ENSG00000160201), PPM1D (Ensembl ID: ENSG00000170836), TP53 (Ensembl ID: ENSG00000141510). V617F , JAK2 exon 12, CALR exon 9, MPL S5050 , M.P.L. W515 , CBL exon 8, CBL exon 9, KRAS exon 2, KRAS exon 3, GNB1 exon 5, DNMT3A all exons, TET2 all exons, ASXL1 exon 12, IDH2 exon 4, SF3B1 exon 14, SF3B1 exon 15, SRSF2 P95, U2AF1 exon 2, U2AF1 exon 6, PPM1D exon 6, and all TP53 exons.

[0033] The method may further comprise analyzing the nucleic acid to identify the presence of one or more clonal hematopoietic markers. These CH markers may include mutations or other genetic abnormalities or expression profiles associated with CH.

[0034] The method may be combined with analysis of white blood cells to detect CH. Thus, in one embodiment, the method comprises: providing a biological sample containing granulocytes; extracting nucleic acid from the biological sample; and analyzing the nucleic acid to identify the presence of one or more clonal hematopoietic mutations. Further includes.

[0035] The analysis of thrombocytes and granulocytes can be performed in parallel in the same analysis or in separate analyses. The nucleic acid extracted from the granulocytes can include RNA and / or DNA. If RNA is extracted from the granulocytes, the RNA is subsequently converted to cDNA for analysis. The DNA extracted from the granulocytes can be genomic DNA (gDNA).

[0036] Because CH may be considered a pre-symptomatic state that indicates an elevated risk of developing other conditions, such as, but not limited to, blood cancers, blood clots, and cardiovascular disease, the method may further include selecting subjects identified as having CH for further monitoring. Further monitoring may include follow-up studies to monitor subjects over time to allow for early detection of subsequent development of other conditions.

[0037] In one embodiment, a subject identified as having clonal hematopoiesis may be further identified as having an elevated risk of a disease selected from one or more of cardiovascular disease, heart failure, diabetes, autoimmune disease, and / or myeloid hematologic cancer. In particular, a subject with clonal hematopoiesis may be further identified as having an elevated risk of hematologic cancer, e.g., myelodysplastic syndrome and acute myeloid leukemia. If a subject is identified as having an elevated risk of a condition, the subject may be selected for preventive treatment, e.g., measures taken to prevent disease. Preventive treatment may include environmental, lifestyle, and / or behavioral changes that may reduce the subject's risk of developing a condition.

[0038] In one aspect, the present invention provides a method for determining a treatment for a subject, comprising: A detection or prediction method as described herein; and Determining the appropriate course of action The present invention relates to a method comprising the steps of:

[0039] In one embodiment, the method for determining a treatment for a subject comprises: A method for detecting or predicting clonal hematopoiesis as described herein; determining subjects at high risk for a disease selected from one or more of cardiovascular disease, heart failure, diabetes, autoimmune disease, and / or myeloid hematological cancer, and determining an appropriate treatment; Includes.

[0040] There are several methods known in the art that can be used to identify one or more CH-associated mutations and / or markers.In one embodiment, the presence of one or more CH-associated mutations is identified through droplet digital PCR (ddPCR), next generation sequencing, allele-specific polymerase chain reaction (PCR), high resolution melting curve analysis, genomic sequencing, fluorescence in situ hybridization (FISH); comparative genomic hybridization (CGH), restriction fragment length polymorphism (RELP), amplification resistant mutation system (ARMS), reverse transcriptase PCR (RT-PCR), real-time PCR, multiplex ligation-dependent probe amplification (MLPA), denaturing gradient gel electrophoresis (DGGE), single-strand conformation polymorphism (SSCP), chemical cleavage of mismatches (CCM), protein truncation test (PTT), pyrosequencing, HPLC (high performance liquid chromatography), or oligonucleotide ligation assay (OLA).In a preferred embodiment, ddPCR is used to identify one or more CH-associated mutations.

[0041] The inventors further identified that thrombocytes take up disease-specific nucleic acid fragments that can be isolated and detected from thrombocytes. In particular, thrombocytes take up cell-free DNA fragments released by solid tumor cells. The inventors have shown that it is possible to detect tumor cell-specific gene mutations in DNA contained within thrombocytes isolated from peripheral blood. The ability to take up tumor cell-derived DNA fragments appears to be unique to platelets and does not occur in red or white blood cells. Previous platelet-based approaches to cancer detection have used platelet gene expression profiles to detect cancer. Because platelets lack nuclei, the platelet transcriptome is determined by (i) the mRNAs "inherited" from parent megakaryocytes, (ii) environmental influences on circulating platelets that alter mRNA splicing, and (iii) the mRNA molecules that are taken up by circulating platelets. Thus, detection of tumor-specific gene mutations at the RNA level in platelets requires that the mutations are expressed at a high enough level with respect to the mRNAs released by tumor cells and are stable enough to be transferred to circulating platelets. This approach is probably insufficiently sensitive and lacks the specificity to distinguish between malignant and non-malignant pathologies, such as wound healing.Other approaches to cancer detection aim to identify cancer-associated cell-free DNA from plasma samples.In contrast, this approach detects cell-free DNA fragments released by solid tumor cells that are taken up by circulating platelets.This approach extracts said cell-free DNA fragments from platelets, and as demonstrated herein, allows the isolation of significantly more DNA than from standard approaches using plasma, increasing the sensitivity of detection.

[0042] In one aspect, the present invention provides a method for detecting or predicting cancer, comprising: providing a biological sample containing thrombocytes; extracting nucleic acid from the biological sample; analyzing the nucleic acid to identify the presence of one or more cancer-associated nucleic acid fragments; and Indicating the presence or prognosis of cancer based on the presence of the cancer-associated nucleic acid fragment. The present invention relates to a method comprising the steps of:

[0043] In one embodiment, the nucleic acid extracted from the biological sample may be DNA and / or RNA. The extracted nucleic acid may be RNA or DNA, or RNA and DNA extracted simultaneously. RNA may be extracted from the biological sample without parallel extraction of DNA. DNA may be extracted from the biological sample without parallel extraction of RNA. If RNA is extracted from the thrombocytes, the RNA is subsequently converted to cDNA for analysis. The DNA extracted from the thrombocytes may be genomic DNA (gDNA). If a combination of DNA and RNA is extracted from the thrombocytes, the method may include parallel analysis of cDNA and gDNA. Analysis of cDNA and gDNA may be performed simultaneously, sequentially, or separately to detect CH-associated mutations. In one embodiment, RNA and gDNA are extracted from the thrombocytes and analyzed in parallel for the presence of cancer-associated nucleic acid fragments. In one embodiment, RNA and gDNA are extracted from the thrombocytes and analyzed separately for the presence of cancer-associated nucleic acid fragments. In one embodiment, RNA is extracted from the thrombocytes and analyzed for the presence of cancer-associated nucleic acid fragments. In one embodiment, gDNA is extracted from thrombocytes and analyzed for the presence of cancer-associated nucleic acid fragments.

[0044] The term "cancer-associated nucleic acid fragment" refers to a fragment of a nucleic acid indicative of cancer. In certain embodiments, a cancer-associated nucleic acid fragment is a fragment of DNA or RNA that contains a mutation associated with cancer. The presence of a cancer-associated nucleic acid fragment indicates the presence of a mutated gene present in the cancer cells of a subject, the cancer-associated nucleic acid fragment having an altered nucleic acid sequence relative to the normal gene of a healthy control. The term "cancer-associated nucleic acid fragment" may further refer to a nucleic acid that is produced, expressed, or present in a cancer cell, but not in a healthy non-diseased cell. In one embodiment, the term "cancer-associated nucleic acid fragment" may refer to a nucleic acid that has an altered expression level (enhanced or reduced) by or in a cancer cell compared to a healthy non-diseased cell. In one embodiment, the term "cancer-associated nucleic acid fragment" may refer to a nucleic acid that is produced, expressed, or present in a normal cell, but not produced, expressed, or present in a cancer cell. According to the present invention, a cancer-associated nucleic acid fragment is a cell-free nucleic acid fragment released by a cancer and taken up by a thrombocyte. The nucleic acid fragments are not part of the platelet transcriptome, but are cell-free fragments taken up by thrombocytes. The cancer-associated nucleic acid fragments can be DNA and / or RNA. In a preferred embodiment, the cancer-associated nucleic acid fragments are DNA. For example, the nucleic acid fragments are cell-free fragments of DNA released by nucleated cells, e.g., cancer cells, and taken up by thrombocytes.

[0045] Those skilled in the art will appreciate that cancer-associated nucleic acid fragments can be identified using a variety of methods and by a variety of characteristics, for example, the fragments may include a fragment length indicative of DNA fragments released by cancer cells, and / or a nucleosome footprint typical of DNA fragments released by cancer cells. As used herein, the term "nucleosome footprint" refers to gene expression information from the tissue of origin from which the fragments present in the nucleic acid fragments originate. The inventors have shown herein that platelets take up a variety of cell-free nucleic acid fragments. Two different populations of nucleic acid fragments taken up by platelets have been analyzed, the first population containing long nucleic acid fragments >10,000 base pairs (bp) and the second population containing short nucleic acid fragments <600 bp. Although both populations have fragments located in the nuclear genome, the long nucleic acid fragments have been shown to contain more fragments located in the mitochondrial genome, and the short fragments have been shown to enrich for the tumor-derived fraction. The cancer-associated nucleic acid fragments may have a fragment length of between 20bp and 500bp, between 20bp and 400bp, between 20bp and 300bp, between 20bp and 200bp, between 20bp and 150bp, between 50bp and 500bp, between 50bp and 400bp, between 50bp and 300bp, between 50bp and 200bp, between 50bp and 150bp, between 100bp and 500bp, between 100bp and 400bp, between 100bp and 300bp, between 100bp and 200bp, or between 100bp and 150bp. In a preferred embodiment, the fragment length is between 50bp and 250bp, or between 100bp and 200bp. In a preferred embodiment, the fragment length is approximately 150bp.In one embodiment, the method for detecting or predicting cancer comprises enriching the nucleic acid sample for short nucleic acids, for example enriching the nucleic acid sample for fragments having a length between 20bp and 500bp, between 20bp and 400bp, between 20bp and 300bp, between 20bp and 200bp, between 20bp and 150bp, between 50bp and 500bp, between 50bp and 400bp, between 50bp and 300bp, between 50bp and 200bp, between 50bp and 150bp, between 100bp and 500bp, between 100bp and 400bp, between 100bp and 300bp, between 100bp and 200bp, or between 100bp and 150bp. In a preferred embodiment, the nucleic acid sample is enriched for fragment lengths between 50bp and 250bp, or between 100bp and 200bp.

[0046] The cancer-associated nucleic acid fragments can comprise one or more cancer markers. The cancer markers can be cancer-associated modifications, cancer-specific mutations, cancer-specific methylation patterns, cancer-specific genetic abnormalities, and / or cancer-specific fragmentation patterns.

[0047] In one embodiment, the cancer-associated nucleic acid fragment is selected from a nucleic acid fragment that contains one or more mutations associated with cancer. Non-limiting examples of mutations include, for example, BRAFV600E, KRASG12D, PIKCAH1047R, TP53R273H.

[0048] There are several methods known in the art for detecting cancer-associated nucleic acid fragments, and as such, any suitable method can be used for detection.In one embodiment, cancer-associated nucleic acid fragments are identified through droplet digital PCR, which is next-generation sequencing, allele-specific polymerase chain reaction (PCR), high-resolution melting curve analysis, genomic sequencing fluorescence in situ hybridization (FISH); comparative genomic hybridization (CGH), restriction fragment length polymorphism (RELP), amplification resistant mutation system (ARMS), reverse transcriptase PCR (RT-PCR), real-time PCR, multiplex ligation-dependent probe amplification (MLPA), denaturing gradient gel electrophoresis (DGGE), single-strand conformation polymorphism (SSCP), chemical cleavage of mismatches (CCM), protein truncation test (PTT), or oligonucleotide ligation assay (OLA), methylation analysis, fragmentation pattern analysis.

[0049] In one embodiment, the cancer associated nucleic acid fragments can be a variety of different sizes, for example, the nucleic acid fragments can be between 10 and 1500 nucleotides, between 10 and 1400 nucleotides, between 10 and 1300 nucleotides, between 10 and 1200 nucleotides, between 10 and 1100 nucleotides, between 10 and 1000 nucleotides, between 10 and 900 nucleotides, between 10 and 800 nucleotides, between 10 and 700 nucleotides, between 10 and 600 nucleotides, between 10 and 500 nucleotides, between 10 and 400 nucleotides, between 10 and 300 nucleotides, between 10 and 200 nucleotides, between 10 and 100 nucleotides, The nucleic acid fragment may comprise between 50 and 1500 nucleotides, between 100 and 1500 nucleotides, between 200 and 1500 nucleotides, between 300 and 1500 nucleotides, between 400 and 1500 nucleotides, between 500 and 1500 nucleotides, between 600 and 1500 nucleotides, between 700 and 1500 nucleotides, between 800 and 1500 nucleotides, between 900 and 1500 nucleotides, between 1000 and 1500 nucleotides, between 1100 and 1500 nucleotides, between 1200 and 1500 nucleotides, between 1300 and 1500 nucleotides, or between 1400 and 1500 nucleotides. In one embodiment, the cancer associated nucleic acid fragment comprises between 300 and 500 nucleotides, or between 400 and 500 nucleotides. In one embodiment, the cancer associated nucleic acid fragment comprises between 800 and 1500 nucleotides.The cancer-associated nucleic acid fragments may comprise 20 nucleotides to 500 nucleotides, 20 nucleotides to 400 nucleotides, 20 nucleotides to 300 nucleotides, 20 nucleotides to 200 nucleotides, 20 nucleotides to 150 nucleotides, 50 nucleotides to 500 nucleotides, 50 nucleotides to 400 nucleotides, 50 nucleotides to 300 nucleotides, 50 nucleotides to 200 nucleotides, 50 nucleotides to 150 nucleotides, 100 nucleotides to 500 nucleotides, 100 nucleotides to 400 nucleotides, 100 nucleotides to 300 nucleotides, 100 nucleotides to 200 nucleotides, or 100 nucleotides to 150 nucleotides. In a preferred embodiment, the fragment length is between 50 nucleotides and 250 nucleotides, or between 100 nucleotides and 200 nucleotides. In a preferred embodiment, the fragment length is approximately 150 nucleotides. When multiple cancer-associated nucleic acid fragments are detected, the fragments may be in different size ranges, i.e., the fragments may each comprise a different number of nucleotides.

[0050] In one embodiment, the method may include separating the cancer-associated nucleic acid fragments from other nucleic acids extracted from the thrombocytes based on size, the method may include separating the cancer-associated nucleic acid fragments based on size, wherein a plurality of different sizes of cancer-associated nucleic acid fragments are detected.

[0051] The cancer-associated nucleic acid fragments may include DNA or RNA. Multiple nucleic acid fragments may be detected in the method, and the fragments may be DNA and / or RNA. If a combination of DNA and RNA fragments is detected, the RNA fragments may first be converted to cDNA. Thus, the method may include extracting RNA from a biological sample containing thrombocytes, converting the RNA to cDNA, and analyzing the cDNA to identify the presence of one or more cancer-associated nucleic acid fragments. The conversion of RNA to cDNA may be performed via reverse transcription as described herein. If a combination of DNA and RNA fragments is detected, the method may include parallel analysis of cDNA and gDNA. The analysis of cDNA and gDNA may be performed simultaneously, sequentially, or separately to detect cancer-associated nucleic acid fragments.

[0052] The cancer-associated nucleic acid fragment may be associated with a solid tumor. Types of solid tumors include sarcoma, carcinoma, and lymphoma. In one embodiment, the cancer-associated nucleic acid fragment is associated with a cancer selected from sarcoma, carcinoma, and / or lymphoma.

[0053] In one embodiment, the cancer-associated nucleic acid fragment is associated with a cancer selected from gastric cancer, lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bone cancer, pancreatic cancer, colon cancer, colorectal cancer, skin cancer, head and neck cancer, head and neck squamous cell carcinoma, melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, breast cancer, brain cancer, hepatocellular carcinoma, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, kidney cancer, soft tissue sarcoma, urethral cancer, bladder cancer, renal cancer, thymoma, urothelial carcinoma, leukemia, prostate cancer, prostate adenocarcinoma, mesothelioma, adrenocortical carcinoma, lymphoma, e.g., Hodgkin's disease, non-Hodgkin's disease, and multiple myeloma.

[0054] The method described herein relates to the analysis of a sample of thrombocytes obtained from a biological sample. The method may be performed on any suitable body sample containing thrombocytes, such as a tissue sample, including blood. In a preferred embodiment, the sample is a blood sample, such as a whole blood sample, also known as a peripheral blood sample. The blood sample may be a fresh blood sample or a stored sample, such as the sample may have been previously frozen or cryopreserved. Methods for obtaining blood or tissue samples are known in the art, for example, blood samples may be obtained via venous extraction. Tissue samples may be obtained via biopsy.

[0055] Various steps may be applied to the biological sample to isolate thrombocytes from the sample and also to improve the extraction of nucleic acids from said thrombocytes. Such steps are described more fully below with respect to the method of preparing a nucleic acid fraction. The biological sample may be processed to prepare an isolated sample of thrombocytes.

[0056] In one aspect, the present invention provides a method for determining a treatment for a subject, comprising: A detection or prediction method as described herein; and Determining the appropriate course of action The present invention relates to a method comprising the steps of:

[0057] In one embodiment, the method for determining a treatment for a subject comprises: A method for detecting or predicting cancer as described herein; and Determining the appropriate course of action Includes.

[0058] The term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment specifically directed to ameliorating a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment directed to removing the cause of the associated disease, pathological condition, or disorder. Furthermore, this term includes palliative treatment, i.e., treatment for the relief of symptoms rather than curing the disease, pathological condition, or disorder; preventive treatment, i.e., treatment directed to minimizing or partially or completely suppressing the occurrence of the associated disease, pathological condition, or disorder; and supportive treatment, i.e., treatment used to supplement another specific therapy directed to ameliorating the associated disease, pathological condition, or disorder.

[0059] Appropriate treatments may be selected based on the mutations identified through the detection or prediction methods. When the detection or prediction methods are directed to cancer, appropriate treatments may include therapeutic agents or radiation therapy, including gene therapy, viral therapy, RNA therapy, bone marrow transplantation, nanotherapy, targeted anti-cancer therapy, or oncolytic drugs. Examples of other therapeutic agents include checkpoint inhibitors, anti-tumor agents, immunogenic agents, attenuated cancer cells, tumor antigens, antigen-presenting cells, such as dendritic cells pulsed with tumor-derived antigens or nucleic acids, immune stimulating cytokines (e.g., IL-2, IFNa2, GM-CSF), targeted small molecules and biomolecules (e.g., agents that bind to tumor-specific antigens, including components of signal transduction pathways, such as modulators of tyrosine kinases, and inhibitors of receptor tyrosine kinases, and EGFR antagonists), anti-inflammatory agents, cytotoxic agents, radiotoxic agents, or immunosuppressants, and cells transfected with genes encoding immune stimulating cytokines (e.g., GM-CSF), chemotherapy. In one embodiment, the appropriate therapy may be an immunomodulatory agent, specifically an immune checkpoint inhibitor, examples of which include, but are not limited to, inhibitors of immune checkpoint proteins selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, TIM3, LAG-3, B7-H3, B7-H4, B7-H6, A2aR, BTLA, GAL9, and IDO. In one embodiment, the appropriate treatment may be surgery. In one embodiment, a combination therapy may be used, for example, the combination may include one or more of the therapies listed herein.

[0060] The term "subject" as used herein includes, but is not limited to, mammals, including, for example, humans, non-human primates, mice, pigs, cows, goats, cats, rabbits, rats, guinea pigs, hamsters, degus, horses, monkeys, sheep, or other non-human mammals; and non-mammalian animals, including, for example, non-mammalian vertebrates, such as birds (e.g., chickens or ducks), or fish, and invertebrates. The subject may be a healthy animal or human subject undergoing a routine check-up. Alternatively, the subject may be at risk of having a disease, such as a genetically predisposed subject, a subject with a medical and / or family history of cancer, or a subject that has been exposed. According to another embodiment, the subject may be a patient diagnosed with a disease, undergoing a routine check-up, or intermediate treatment.

[0061] In one aspect, the invention relates to a kit comprising reagents for extracting nucleic acids from platelets and a panel of reagents for specifically detecting and / or amplifying one or more clonal hematopoietic associated mutations, and optionally instructions for use.

[0062] The panel of reagents includes JAK2 V617F , JAK2 exon 12, CALR exon 9, MPL S5050 , M.P.L. W515 , CBL exon 8, CBL exon 9, KRAS exon 2, KRAS exon 3, GNB1 exon 5, DNMT3A all exons, TET2 all exons, ASXL1 exon 12, IDH2 exon 4, SF3B1 exon 14, SF3B1 exon 15, SRSF2 P95 The present invention may specifically detect one or more of CH-associated mutations selected from U2AF1 exon 2, U2AF1 exon 6, PPM1D exon 6, and TP53 all exons.

[0063] In one aspect, the invention relates to a kit comprising a panel of reagents for extracting nucleic acids from platelets and reagents for specifically detecting and / or amplifying one or more cancer-associated modifications or cancer-specific mutations.

[0064] The panel of reagents may specifically detect one or more of the following cancer-specific mutations: BRAFV600E, KRASG12D, PIKCAH1047R, TP53R273H.

[0065] CH-associated or cancer-associated mutations can be detected using targeted gene sequencing panels, next-generation sequencing, primers or probes designed to detect specific mutations.

[0066] In one aspect, the invention provides a method of treating a subject having cancer, comprising: providing a biological sample containing thrombocytes; extracting nucleic acid from the biological sample; analyzing the nucleic acid to identify the presence of one or more cancer-associated nucleic acid fragments; selecting a treatment; and Administering the Treatment The present invention relates to a method comprising the steps of:

[0067] In one embodiment, the methods for detecting or predicting cancer and CH described herein can be combined to provide a combined method for detecting cancer and CH, which can include any of the features described herein.

[0068] In one aspect, the present invention provides a method for preparing a nucleic acid fraction, comprising the steps of: providing a biological sample containing thrombocytes; extracting nucleic acid from the biological sample to form a nucleic acid sample; enriching said nucleic acid sample for one or more cancer-associated nucleic acid fragments and / or clonal hematopoietic-associated mutations. The present invention relates to a method comprising the steps of:

[0069] Various steps may be applied to the biological sample to isolate thrombocytes from the sample and also to improve the extraction of nucleic acids from said thrombocytes. These steps may be applied to any of the methods described herein. In one embodiment, the biological sample may be treated to prepare the sample for analysis. For example, the biological sample may be purified or degraded, or specific compounds may be extracted therefrom. Depending on the method to characterize the nucleic acids present in the thrombocytes in the biological sample, the thrombocytes may be extracted from the sample by methods known to those skilled in the art and transferred to any suitable medium for the extraction of nucleic acids. To prevent premature destruction of nucleic acids, the biological sample may be treated to remove large amounts of nucleolytic enzymes (e.g., RNase, DNase) from the sample.

[0070] In one embodiment, the peripheral blood sample is collected in either an EDTA tube or a Streck tube or a lithium heparin tube. In an embodiment, the peripheral blood sample is collected in either an EDTA tube or a Streck tube. Centrifugation may be used to allow isolation of pure fractions of granulocytes, platelets, and cell / platelet-depleted plasma. In one embodiment, the protocol for obtaining platelets from the peripheral blood sample may be optimized to improve the purity of the platelets. Optimization may be performed by modifying the centrifugation protocol (e.g., centrifugation speed and brake settings) and modifying the buffers used to isolate the platelets. For example, buffers that prevent platelet activation may be used in the methods of the present invention.

[0071] The purity of the platelets for analysis may be greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, or greater than 99.9%, with the purity being assessed in terms of the amount of other blood cell types relative to the platelets. Having a high purity platelet sample for analysis reduces contamination of the sample with other cell types. In one embodiment, the protocol for obtaining platelets from a peripheral blood sample may be optimized to improve the purity of the platelets and / or to reduce contamination with white blood cells and / or red blood cells.

[0072] In embodiments where the method includes centrifuging the biological sample, the method may further include isolating an upper percentage of the platelet-rich plasma following centrifugation. For example, the method may include isolating the upper 95%, 90%, 85%, 80%, 85%, 70% of the platelet-rich plasma following centrifugation. Such an isolation step may improve platelet purity and reduce contamination with other cell types.

[0073] In embodiments, the method may include the step of depleting leukocytes and enriching platelets using CD45 and / or CD42 beads.

[0074] In embodiments, the method may include using a leukofilter to reduce leukocyte contamination.

[0075] In one embodiment, the method for preparing a nucleic acid fraction comprises enriching a nucleic acid sample for short nucleic acids, and in particular, this step may be used when preparing a nucleic acid fraction enriched for one or more cancer-associated nucleic acids. For example, the method may comprise enriching a nucleic acid sample for fragments having a length between 20bp and 500bp, between 20bp and 400bp, between 20bp and 300bp, between 20bp and 200bp, between 20bp and 150bp, between 50bp and 500bp, between 50bp and 400bp, between 50bp and 300bp, between 50bp and 200bp, between 50bp and 150bp, between 100bp and 500bp, between 100bp and 400bp, between 100bp and 300bp, between 100bp and 200bp, or between 100bp and 150bp. In a preferred embodiment, the nucleic acid sample is enriched for fragment lengths between 50 bp and 250 bp, or between 100 bp and 200 bp.

[0076] One aspect of the invention is a method for genotyping a sample of thrombocytes, comprising the steps of: providing a biological sample containing thrombocytes; extracting RNA from the biological sample; converting the RNA into cDNA; analyzing said cDNA to identify the presence of one or more clonal hematopoietic mutations, thereby genotyping the sample. The present invention relates to a method comprising the steps of:

[0077] One aspect of the invention is a method for genotyping a sample of thrombocytes, comprising the steps of: providing a biological sample containing thrombocytes; extracting nucleic acid from the biological sample; analyzing said nucleic acid to identify the presence of one or more cancer-associated nucleic acid fragments, thereby genotyping the sample. The present invention relates to a method comprising the steps of:

[0078] The term "genotyping", also called genotyping, refers to the detection of differences in nucleic acid present in a cell, i.e., a thrombocyte, compared to a control. The control can be a healthy non-diseased thrombocyte. The differences in nucleic acid can be the presence or absence of a mutation, the up-regulation or down-regulation of a particular nucleic acid, and / or the presence or absence of a particular nucleic acid fragment.

[0079] Genotyping involves analyzing the nucleic acid to identify the presence of one or more cancer-associated nucleic acid fragments, which are cell-free nucleic acid fragments released by solid tumors and taken up by thrombocytes. In one embodiment, thrombocytes are genotyped for the presence of one or more cancer-associated nucleic acid fragments, which may include one or more of the following mutations: BRAFV600E, KRASG12D, PIKCAH1047R, TP53R273H.

[0080] In one embodiment, thrombocytes are identified by the following genes: JAK2 (Ensembl ID: ENSG00000096968), CALR (Ensembl ID: ENSG00000179218), MPL (Ensembl ID: ENSG00000117400), CBL (Ensembl ID: ENSG00000110395), KRAS (Ensembl ID: ENSG00000133703), GNB1 (Ensembl ID: ENSG00000078369), DNMT3A (Ensembl ID: ENSG00000119772), TET2 (Ensembl ID: ENSG00000168769), ASXL1 (Ensembl ID: ENSG00000171456), IDH2 (Ensembl ID: ENSG0000017217), IDH3 (Ensembl ID: ENSG00000171613), IDH4 (Ensembl ID: ENSG00000171511), IDH5 (Ensembl ID: ENSG00000171621), IDH6 (Ensembl ID: ENSG0000017173), IDH7 (Ensembl ID: ENSG0000017184), IDH8 (Ensembl ID: ENSG00000171636), IDH9 (Ensembl ID: ENSG0000017164), IDH1 (Ensembl ID: ENSG0000017165), IDH2 (Ensembl ID: ENSG0000017166), IDH1 (Ensembl ID: ENSG0000017167), IDH In one embodiment, the thrombocytes are genotyped by the presence of one or more CH-associated mutations present in one or more of the following mutations: JAK2 , SF3B1 (Ensembl ID: ENSG000001 15524), SRSF2 (Ensembl ID: ENSG00000161547), U2AF1 (Ensembl ID: ENSG00000160201), PPM1D (Ensembl ID: ENSG00000170836), TP53 (Ensembl ID: ENSG00000141510). V617F , JAK2 exon 12, CALR exon 9, MPL S5050 , M.P.L. W515 , CBL exon 8, CBL exon 9, KRAS exon 2, KRAS exon 3, GNB1 exon 5, DNMT3A all exons, TET2 all exons, ASXL1 exon 12, IDH2 exon 4, SF3B1 exon 14, SF3B1 exon 15, SRSF2 P95 The genotype is identified by the presence of one or more of U2AF1 exon 2, U2AF1 exon 6, PPM1D exon 6, and all TP53 exons.

[0081] The inventors have further determined that analysis of maternal platelet nucleic acid can be used to identify the genetic information of the child, and therefore platelet analysis may be useful in prenatal screening.

[0082] One aspect of the present invention is a method for prenatal screening of fetal genetic information, comprising the steps of: Providing a biological sample containing thrombocytes obtained from a pregnant female; extracting nucleic acid from the biological sample; analyzing said nucleic acid for genetic information associated with the fetus. The present invention relates to a method comprising the steps of:

[0083] The method of the invention comprises the step of extracting nucleic acid from thrombocytes obtained from a pregnant woman or a woman who has recently given birth. Nucleic acid is then extracted from said thrombocytes. The nucleic acid is then analyzed for genetic information related to the fetus of the pregnant woman or the child of the woman who has recently given birth. The extracted nucleic acid may be RNA and / or DNA. If RNA is extracted from the thrombocytes, the RNA is then converted to cDNA for analysis. The DNA extracted from the thrombocytes may be genomic DNA (gDNA). If a combination of DNA and RNA is extracted from the thrombocytes, the method may comprise parallel analysis of the cDNA and gDNA. The analysis of the cDNA and gDNA may be performed simultaneously, sequentially or separately to detect genetic information related to the fetus of the pregnant woman or the child of the woman who has recently given birth. In one embodiment, RNA and gDNA are extracted from the thrombocytes and analyzed in parallel for the presence of genetic information related to the fetus of the pregnant woman or the child of the woman who has recently given birth. In one embodiment, RNA and gDNA are extracted from the thrombocytes and analyzed separately for the presence of genetic information related to the fetus of the pregnant woman or the child of the woman who has recently given birth. In one embodiment, RNA is extracted from the thrombocytes and analyzed for the presence of genetic information relating to a fetus of a pregnant woman or a child of a recently born woman. In one embodiment, gDNA is extracted from the thrombocytes and analyzed for the presence of genetic information relating to a fetus of a pregnant woman or a child of a recently born woman.

[0084] The genetic information may relate to the sex of the fetus or child, genetic conditions such as Down's syndrome, Edwards' syndrome, Patau's syndrome, cystic fibrosis, spina bifida, sickle cell anemia, thalasaemia.

[0085] Genetic information associated with the fetus or child can be identified by analysis of the nucleic acid for specific markers or genes, for example, if one wishes to identify the sex of the fetus or child, one can screen for a fragment of the Y chromosome, in particular a fragment of the SRY gene.

[0086] Biological samples may be obtained from the pregnant female at specific times throughout the pregnancy or immediately after delivery. Biological samples may be obtained at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 weeks of pregnancy. Biological samples may be obtained 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days after delivery.

[0087] Unless otherwise defined herein, scientific and technical terms used in the context of this disclosure shall have the same meaning as commonly understood by those skilled in the art. The foregoing disclosure provides a general description of the subject matter falling within the scope of this disclosure, including the methods of making and using the disclosure, and the best mode thereof, while the following examples are provided to further enable those skilled in the art to practice the disclosure. However, those skilled in the art will understand that the details of these examples should not be read as limitations on the invention, the scope of which should be understood from the claims appended to this disclosure and their equivalents. In light of the present disclosure, further various aspects and embodiments of the present disclosure will become apparent to those skilled in the art.

[0088] All documents referred to herein, including references to gene accession numbers, scientific publications, and patent publications, are hereby incorporated by reference in their entirety.

[0089] "And / or," as used herein, should be considered a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" should be considered a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were individually presented herein. Unless the context dictates otherwise, the above feature descriptions and definitions are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described.

[0090] The terms "comprising" or "comprises," as used herein, mean including the specified components, but not to the exclusion of the presence of other components. The terms "consisting essentially of" or "consists essentially of" mean including the specified components, but excluding other components other than materials present as impurities, unavoidable materials present as a result of the process used to prepare the component, and the like.

[0091] The terms "consisting of" or "consists of" mean the inclusion of the specified components but excluding other components.

[0092] Where appropriate by context, use of the terms "comprises" or "comprising" may be taken to include the meanings "consists essentially of" or "consisting essentially of," as well as "consists of" or "consisting of."

[0093] Any features presented herein may be used either individually or in combination with one another, where appropriate, and may be combined as long as they are specifically presented in the appended claims. Any features presented herein with respect to each aspect or exemplary embodiment are also applicable, where appropriate, to all other aspects or exemplary embodiments of the invention. In other words, a person skilled in the art reading this specification will consider any features with respect to each aspect or exemplary embodiment of the invention to be interchangeable and usable between different aspects and exemplary embodiments.

[0094] The present invention is further illustrated in the following non-limiting examples. EXAMPLES

[0095] Materials and Methods Blood collection Samples were collected by the BRC Oxford Gl Biobank and the Oxford Radcliffe Biobank (ORB). All procedures were approved by the Yorkshire & The Humber - Sheffield Research Ethics Committee (REC reference number: 16 / YH / 0247), the ORB Research Tissue Bank Ethics (REC reference number: 19 / SC / 0173), and the INForMeD Study (REC: 16 / LO / 1376 (199833)). Participants signed informed consent where appropriate. 10-20 ml of peripheral blood was collected from patients with solid tumours presenting with colorectal, esophageal or pancreatic cancer at the time of their pre-operative evaluation into EDTA-coated blood collection tubes (Fisher Scientific, Cat. No. 367839) or cell-free DNA blood collection tubes (Streck, Cat. No. 218997). 10 ml of peripheral blood was also collected from patients with serrated adenomatosis. Healthy controls received fully anonymized blood samples from the Oxford Biomedical Research Laboratory (Clinical Diagnostic Lab). Samples were taken from individuals in either hospital or outpatient care and were in excess of clinical need. In accordance with HTA requirements, no clinical information was recorded. Mouse blood was purified using KPN (villinCre ER Kras G12D / + Trp53 fl / fl R26 N1icd / + ) and KP(villinCre ER Kras G12D / + Trp53 fl / fl ) mice via cardiac puncture into EDTA-coated microvette collection tubes (Sarstedt, catalogue no. 20.1288).

[0096] All procedures were performed in accordance with the ethical standards of the Declaration of Helsinki. Patient samples were collected by the Oxford Translational Gastroenterology Unit (TGU) and the Oxford Radcliffe Biobank (ORB). All patients provided informed consent and the procedures were approved by the Yorkshire & The Humber - Sheffield Research Ethics Committee (REC reference number: 16 / YH / 0247) and the ORB Research Tissue Bank Ethics (REC reference number: 19 / SC / 0173). 10-20 ml of peripheral blood was collected from patients presenting with colorectal, esophageal or pancreatic cancer in EDTA-coated blood collection tubes (Fisher Scientific, Cat. No. 367839) or cell-free DNA blood collection tubes (Streck, Cat. No. 218997). 10 ml of peripheral blood was collected from patients with serrated lesions and from patients on the bowel cancer screening pathway or from patients with inflammatory bowel disease undergoing colonoscopy. Healthy controls received fully anonymized blood samples from the Oxford Biomedical Research Laboratory (Clinical Diagnostic Lab) under the INForMeD Study (REC reference: 16 / LO / 1376). The use and collection of healthy human donor blood samples in Edinburgh was approved under project 21-EMREC-041 - The Role of Inflammation in Human Immunity.

[0097] Obstetric samples included 10 ml of blood collected from mothers at Caesarean section and after birth, 24 and 48 hours after delivery, into Streck blood collection tubes. Samples were collected at the Department of Women's and Reproductive Health, John Radcliffe Hospital Oxford, and approved by the Oxfordshire Research Ethics Committee (07 / H0607 / 74). The sex of the newborns was confirmed after delivery and all mothers had provided written informed consent.

[0098] animal All mice were bred and maintained in accordance with UK Home Office regulations. All experiments were performed under project licenses P2FF90EE8 and P0B63BC4D and approved by the University of Oxford Animal Welfare and Ethics Committee. All mice were housed in specific pathogen-free (SPF) facilities in the animal units of either the Functional Genetics Facility (Wellcome Centre for Human Genetics, University of Oxford) or the Biomedical Services Facility at John Radcliffe Hospital (The MRC Weatherall Institute of Molecular Medicine, University of Oxford), in individually ventilated cages with unlimited access to food and water, and were naive to any previous treatments.

[0099] For the immune thrombocytopenia model, CD45.2 female wild type mice were injected intravenously with either an anti-platelet antibody (Emfret, Cat. No. R300) to reduce platelet counts, or an IgG control (Emfret, Cat. No. C301), which has no cytotoxic effect on platelets in mice. Mice were humanely culled and then bled via cardiac puncture into an EDTA-coated microvette (Sarstedt, Cat. No. 20.1288) at 24, 72, and 120 hours after treatment. Platelet counts were determined using an automated blood cell counter. cfDNA was extracted from plasma and quantified. For the colorectal cancer (CRC) model, mouse blood was collected using KPN (villinCre ER Kras G12D / + Trp53 fl / fl R26N1 icd / + ) and KP(villinCre ER Kras G12D / + Trp53 fl / fl) mice via cardiac puncture into an EDTA-coated microvette (Sarstedt, Cat. No. 20.1288). All strains used in this model were maintained on a C57BL / 6J background. Both male and female KP and KPN mice were used.

[0100] Isolation of human platelets and cfDNA Human platelets were isolated within 4 or 24 hours of blood collection (depending on blood tube). Whole blood was supplemented with acid citrate-dextrose solution (ACD) (Sigma, Cat. No. C3821-50ML) and centrifuged at 180-200xg for 10-20 minutes at room temperature. 80% of the supernatant was removed, diluted with platelet washing buffer, and centrifuged at 600-1200xg for 10-20 minutes at room temperature. The resulting platelet pellet was resuspended in pre-warmed HEPES modified Tyrode's buffer. The remaining platelet poor plasma was centrifuged at 16000xg for 10 minutes at 4°C to ensure pelleting of all cellular components and isolation of pure cfDNA. Platelet counts in both samples were determined using an automated blood cell counter (Horiba, Pentra ES 60 Cell Counter).

[0101] Mouse platelet isolation Mouse platelets were also isolated within 4 hours of collection. Whole blood was supplemented with ACD and centrifuged at 100xg for 10 minutes at room temperature. Plasma was removed and collected in a clean Eppendorf tube. Plasma was then diluted with platelet washing buffer and centrifuged at 3500xg for 11 minutes to pellet platelets. Platelet pellets were resuspended in pre-warmed HEPES modified Tyrode's buffer. Platelet poor plasma was removed and centrifuged at either 1300xg or 16000xg for 10 minutes at 4°C depending on the experiment to remove all cells, and the final supernatant was collected for cfDNA isolation. Platelet counts were determined using an automated blood cell counter.

[0102] Blood tubes were kept at room temperature and handled gently to avoid platelet activation. For human samples, platelets were isolated within 4 or 24 hours (depending on blood tube) of blood collection using a modified protocol previously published. Briefly, whole blood was supplemented with acid citrate-dextrose solution (ACD) (Sigma Aldrich, Cat. No. C3821-50ML) and centrifuged at 180xg for 20 minutes at room temperature. 80% of the supernatant was removed, diluted with platelet washing buffer, and centrifuged at 600xg for 20 minutes at room temperature. The resulting platelet pellet was resuspended in pre-warmed HEPES modified Tyrode's buffer. The remaining platelet poor plasma was centrifuged at 16000xg for 10 minutes at 4°C to ensure pelleting of all cellular components and isolation of pure cfDNA. Platelet counts in both samples were determined using an automated blood cell counter (Horiba, Pentra ES 60 Cell Counter).

[0103] Isolation of human granulocytic cells Human granulocytes were isolated from freshly drawn venous blood in EDTA-coated tubes. Whole blood was supplemented with ACD and centrifuged at 180xg for 20 minutes at room temperature with low brake. The supernatant was removed and the remaining layer was diluted with wash buffer. The diluted blood was layered on top of density gradient medium (GE Healthcare, Cat. No. 5442-03) and centrifuged at 600xg for 20 minutes with low brake. The supernatant was removed and the remaining RBC, granulocyte pellet was resuspended in chilled RBC lysis buffer and incubated at 4°C for 5 minutes. The lysed sample was centrifuged at 1000xg for 5 minutes, the supernatant was discarded and the granulocyte pellet was diluted with wash buffer. If necessary, the lysis process was repeated with additional RBC lysis buffer to ensure efficient removal of contaminating RBCs. The remaining white granulocyte pellet was washed, centrifuged and stored at -80°C until required.

[0104] Platelet Purity Assessment Leukocyte contamination in the platelet pellet was assessed by flow cytometry using an LSR Fortessa X20. Single-color staining controls and fluorescence minus one (FMO) controls were used for all experiments. For analysis, washed human platelets were stained with antibodies for 20 min in the dark at room temperature, then washed and resuspended in FACS buffer (phenol-free IMDM + 10% FCS). Gates were set using FMO controls and the negative population. Viability was assessed using DAPI. Analysis was performed using FACSDiva v8.1 (BD Biosciences) and FlowJo v10.7.1 software. Two gating strategies were utilized to ensure accurate counting of platelets and contaminating leukocytes. Nuclear cell contamination in the platelet pellet was found to be 0.003%. RNA was extracted from the platelet pellet and gene expression was measured by RT-PCR to confirm expression of platelet-specific genes.

[0105] The purity of the human platelet pellet was assessed by flow cytometry using an LSR Fortessa X20 flow cytometer (Becton, Dickinson and Company). Single-color staining controls and fluorescence minus one (FMO) controls were used for all experiments. Washed human platelets were stained with antibodies for 20 minutes in the dark at room temperature, then washed and resuspended in FACS buffer (phenol-free IMDM + 10% FCS). Gates were set using the FMO controls and the negative population.

[0106] DNA extraction from platelets, cell-free plasma, and nucleated cells DNA was extracted from equal volumes of fresh platelets (resuspended in HEPES modified Tyrode's buffer) and "platelet poor plasma" using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Cat. No. 55114) according to the manufacturer's instructions. Genomic DNA was extracted from nucleated cells using the DNeasy Blood and Tissue Kit (Qiagen, Cat. No. 51104). Extracted DNA was quantified using a Qubit fluorometer (Thermofisher Scientific) and stored at -80°C until required.

[0107] Extraction and quantification of PA-DNA, cfDNA and gDNA DNA was extracted from equal volumes of fresh platelets and "platelet poor plasma" using the QIAamp Circulating Nucleic Acid Kit (Qiagen) according to the manufacturer's instructions. Genomic DNA was extracted from granulocytes using the DNeasy Blood and Tissue Kit (Qiagen). Extracted DNA was quantified using a Qubit fluorometer (Thermofisher Scientific) and stored at -80°C until required.

[0108] cfDNA and PA-DNA fragmentation analysis The fragmentation profiles of PA-DNA and paired cfDNA from solid tumor patients were analyzed using the Agilent Tapestation 2200 system (Agilent Technologies). cfDNA, HS D5000 and genomic DNA reagents, and screen tapes (Agilent, Cat. Nos. 5067-5630, 5067-5592, and 5067-5365) were used for the analysis. Electrophoretic data were analyzed using Tapestation analysis software.

[0109] cell line The following cell lines were used as positive and negative controls for common cancer-associated mutations: HCT116 (CVCL S744) PIK3CAH1047R, LS180 (CVCL 0397) KRSG12D, COLO205 (CVCL 0218) BRAFV600E, and HEL (CVCL_0001) JAK2V617F. All cell lines were purchased from the American Type Culture Collection (catalog numbers CCL-247, CL-187, CCL-222, and TIB-180 for HCT116, LS180, COLO205, and HEL cells, respectively). All cells were cultured according to the recommendations of the American Type Culture Collection and tested monthly for mycoplasma contamination.

[0110] The human Burkitt's lymphoma cell line BL2 (CVCL 1966, catalog number CRL-2959) was used to generate tumor cell-derived extracellular vesicles (EVs). To obtain apoptotic EVs (apo-EVs) from BL2 cells and non-apoptotic EVs from apoptosis-resistant BL2-Bcl-2 cells, both standard BL2 cells and BL2 cells stably transfected with the apoptosis-suppressing gene bcl-2 were used. Protein expression was routinely tested by flow cytometry, confirming that 98% of BL2-bcl-2 cells expressed Bcl-2. Both cell lines were maintained at 37°C and 5% CO2 in 50% X-VIVO medium (50% Gibco RPMI-1640, 50% X-VIVO-20 medium; Lonza, Basel, Switzerland) supplemented with 50 U / ml penicillin and 50 μg / ml streptomycin.

[0111] In vitro co-culture All co-culture experiments of hemocytes and tumor cells were performed using 3 × 10 5 Platelets / μl, 0.075 x 10 5 MNCs / µl, 3 x 10 5 RBCs / µL, and 0.03 x 10 5Tumor cells / μl were used. All cells were resuspended in Tyrode's salt (Sigma Aldrich, Cat. No. T2397-100ML).

[0112] Blood cell-tumor cell incubation Platelets, MNCs, and RBCs from healthy donors were added to individual wells in a 24-well plate, and 1.0 μm TC inserts (Sartstedt, Cat. No. 83.3932.101) were placed in the wells. Cancer cells were carefully placed in the inserts, and the plate was incubated at 37° C. for approximately 8 hours. Control samples were incubated simultaneously without the addition of tumor cells. After incubation, the inserts were removed and the blood cells were transferred to an Eppendorf tube. To ensure complete removal of any contaminating cfDNA in the medium, all cells / platelets were washed and centrifuged at 600×g for 10 minutes. The supernatant was removed and the pelleted blood cells were resuspended in fresh buffer. Cells were washed a total of three times and finally resuspended in 1 ml fresh pre-warmed HEPES modified Tyrode's buffer before DNA extraction using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Cat. No. 55114).

[0113] DNase treatment of platelets and conditioned medium After incubation with HEL erythroleukemia cells, platelets were treated with DNase (Ambion DNA-free kit, catalogue number AM1906) according to the manufacturer's protocol. DNase was also added to the conditioned medium to confirm the catalytic activity of the enzyme. After DNase treatment, platelet samples were washed with 500 μl of 5 mM EDTA and centrifuged at 600×g for 10 min. The supernatant was removed and the wash repeated. After the second centrifugation, the supernatant was discarded and the platelet pellet was resuspended in 1 ml of fresh pre-warmed HEPES modified Tyrode's buffer. After DNase inactivation of the conditioned medium, 900 μl of HEPES buffer was added to a final volume of 1 ml in preparation for DNA extraction. To the control sample, no DNase was added and 10 μl of 1 M tris-HCL was added instead of the 10X DNase I buffer for the platelet samples.

[0114] Generation of extracellular vesicles (EVs) from BL2 and BL2bcl2 cells BL2 and BL2-Bcl-2 cells were cultured at 20 × 10 in 50% X-vivo 20 filtered with 0.1 μm. 6 Cells were cultured at 1000 x 1000 cells / ml and exposed to six doses of 50 mJ / cm2 UV light for a total of 300 mJ / cm2 to induce apoptosis. Apoptosis was monitored hourly by Annexin V and Sytox Blue staining. To isolate EVs, culture supernatants were centrifuged at 25xg for 1 h and then filtered sequentially through a 5 μm mesh filter and a 1.2 μm syringe filter. EV concentrations were measured by nanoparticle tracking analysis on a Nanosight LM14.

[0115] EV purification and labeling EVs were purified from soluble factors and proteins derived from cell culture by size-exclusion chromatography using in-house prepared Sepharose columns (Thermofisher, Cat. No. 45-000-067 and Sigma Aldrich, Cat. No. CL6B200-100ML). Purified EVs suspended in 0.1 μm filtered HBSS were stained with Biotium CF568 NHS ester (Biotium, Cat. No. NC1542764) by covalent attachment of the dye on EV proteins according to the manufacturer's protocol. To remove unbound dye, EVs were again purified by size-exclusion chromatography through a Sepharose column.

[0116] EV-platelet incubation and DNA extraction 50 x 10 resuspended in Tyrode's buffer 8 0.1 x 10 labeled purified EVs 81000×g for 3 h at 37° C. with gentle inversion every 30 min. After incubation, samples were centrifuged at 800×g for 20 min. The supernatant was discarded and platelets were resuspended in 300 μl of phosphate-buffered saline (PBS) medium (Life Technologies, Cat. No. 10010-056). EV and platelet pellets were treated with DNase before DNA extraction according to the manufacturer's protocol (Sigma Aldrich, Cat. No. AMPD1). DNA extraction was performed using the QiaAMP DNA Blood Kit according to the manufacturer's protocol (Qiagen, Cat. No. 51104), samples were eluted in 30 μl of elution buffer, and DNA concentration was measured using a Qubit fluorometer.

[0117] cell culture Platelet-tumor cell incubation To confirm that specific cancer-associated gene rearrangements are detectable in platelets, platelets were incubated with various colorectal cancer cell lines. Platelets from healthy donors were added to individual wells in a 24-well plate, and 1.0 μm TC inserts (Sartstedt, Cat. No. 83.3932.101) were placed in the same wells. Tumor cells were carefully placed inside the inserts, and the plates were incubated at 37° C. for approximately 8 hours. Note that control samples were incubated without the addition of tumor cells. After incubation, the inserts were removed and the platelets were transferred to Eppendorf tubes. To maximize platelet collection, the wells were washed with pre-warmed HEPES modified Tyrode's buffer. All platelets were washed and centrifuged to ensure removal of cfDNA that may have been contaminating the medium. The supernatant was removed, and the pelleted platelets were resuspended in fresh buffer. Platelets were washed a total of three times and finally resuspended in 1 ml of fresh prewarmed HEPES modified Tyrode's buffer and prepared for DNA extraction using the QIAamp Circulating Nucleic Acid Kit (Qiagen).

[0118] DNase treatment of platelets and conditioned medium To determine whether platelets internalize cfDNA, platelets incubated with tumor cells were treated with DNase (Ambion DNA-free kit, catalog number AM1906). To confirm the catalytic activity of the enzyme, DNase was also added to the conditioned medium as described in the manufacturer's protocol. After treatment, platelet samples were then washed with 500 μl of 5 mM EDTA and pelleted. The supernatant was removed and the wash was repeated. After a second centrifugation, the supernatant was discarded and the platelet pellet was resuspended in 1 ml of fresh pre-warmed HEPES modified Tyrode's buffer. To the control sample, no DNase was added and 10 μl of 1 M tris-HCL was added instead of the 10X DNase I buffer for the platelet sample.

[0119] Digital Droplet (dd) PCR Human ddPCR To investigate the possibility that platelets acquire specific mutations through the incorporation of DNA fragments, ddPCR assays were ordered from Bio-Rad. All ddPCR assays were performed on a QX200 droplet digital PCR system using the manufacturer's recommended protocols and reagents. Positive and negative controls were included in every assay. Analysis was performed using QuantaSoft software (Bio-Rad Laboratories, Watford UK).

[0120] KRAS G12D Mouse ddPCR KRAS G12DA ddPCR assay was designed for the detection of the KRAS p.G12D c.35G>A point mutation in PA-DNA and cfDNA isolated from mice. To detect mouse KRASG12D, a ddPCR assay was designed for the detection of the KRAS p.G12D c.35G>A point mutation. The primers for detection of KRAS were as follows: forward 5'-GCCTGCTGAAAATGACTGAG-3' (SEQ ID NO: 1) and reverse 5'-CGTAGGGTCATACTCATCCAC-3' (SEQ ID NO: 2). Two dual-labeled probes were also used to target the wild-type and mutant sequences: wild-type 5'-HEX-ACGCC[+A][+C]CAG[+C]TCCAA-BHQ1-3' (SEQ ID NO: 3) and mutant 5'-6FAM-AC[+G]CC[+A][+T][+C]AG[+C]TCCAA-BHQ1-3' (SEQ ID NO: 4). Square brackets indicate LNA (locked nucleic acid) bases (Merck Life Sciences).

[0121] Immunofluorescence microscopy Platelet-tumor cell interactions Human platelets were isolated from healthy donors as described above and stained with CD42b / AF488 (Biolegend, Cat. No. 303914) for 30 minutes at room temperature. Separately, the highly specific live cell DNA stain SiR-DNA (Spirochrome, Cat. No. CHF260.00) was used to label COLO205 cells. The stained COLO205 cells were then washed twice with MEM Alpha 1X (phenol-free) supplemented with 5% FCS (Gibco, Cat. No. 41061-029) and added onto a microscope slide (Greiner Bio-One, Cat. No. 543079). The stained platelets were added to the wells containing the tumor cells immediately before imaging. To analyze endogenous wild-type DNA in healthy donor platelets, platelets were co-stained with anti-CD42 / AF488 and NUCLEAR-ID® Red DNA and imaged separately.

[0122] Platelet uptake of tumor-derived cfDNA The nuclear DNA of COLO205 cells was labeled using NUCLEAR-ID® Red DNA stain. After 45 minutes of incubation at 37°C, the cells were washed with MEM Alpha 5% FCS, pelleted, and the supernatant was removed. To ensure complete removal of the DNA stain, the cells were washed two more times and the COLO205 cells were resuspended in 200 μl of fresh MEM Alpha 5% FCS. The cells were incubated at 37°C and vortexed periodically to promote cell death and DNA release. After approximately 2 hours, the COLO205 cells were centrifuged at 16000xg for 10 minutes at 4°C. The supernatant containing the cfDNA was removed from the pelleted cells and transferred to a fresh Eppendorf tube, and the COLO205 cells were discarded. Healthy donor platelets were stained with anti-CD42 / AF488 as described above and added to wells of poly-L-lysine coated slides (Ibidi, Cat. No. 81201). DNA-labeled COLO205 cell conditioned medium was added to the platelets immediately prior to imaging. All cells were resolved by fluorescence microscopy using a Zeiss spinning disk confocal with a 63x oil immersion objective.

[0123] Imaging of apo-EVs BL2 cell-derived apo-EVs were isolated, labeled with an amine-reactive fluorescent dye (Biotium, Cat. No. 92131) and frozen as previously described. Prior to imaging, EVs were thawed and co-stained with DAPI for 30 min at room temperature. EVs were washed twice, pelleted at 20000xg for 30 min at 4°C and resuspended in HBSS buffer (Thermofisher, Cat. No. 88284). Labeled EVs were then added to poly-L-lysine coated chamber slides (Ibidi, Cat. No. 81201) and incubated overnight at 4°C. After incubation, the supernatant was carefully removed and EVs were fixed with 4% formaldehyde for 30 min at room temperature. The fixative was then removed and EVs were gently washed three times with PBS. To confirm imaging of EVs and not autofluorescent debris, PBS was stained with DAPI, incubated overnight and fixed on slides alongside the EV preparation. Slides were mounted and EV and PBS controls were imaged under a fluorescent microscope using a Zeiss LSM900 with a 63x oil immersion objective.

[0124] Platelet apo-EV incubation Human platelets were isolated from healthy donors as previously described and resuspended in 1 ml of HBSS buffer. BL2 cell-derived EVs labeled with an amine-reactive fluorescent dye (Biotium, Cat. No. 92131) were thawed and diluted at 5 × 10 9 EVs, 10 x 10 6100ml PBS. The platelets were added to 100 ml of PBS and incubated at 37°C for 3 hours. The cells were gently inverted every 30 minutes to ensure adequate mixing. After incubation, the cells were centrifuged for 20 minutes at 800xg with low brake. The supernatant (containing EVs) was removed and the platelets were resuspended in 100 ml of PBS. The platelets were centrifuged for 20 minutes at 600xg onto poly-L-lysine coated chamber slides (Ibidi, Cat. No. 81201) and fixed in 2% formaldehyde / PBS for 10 minutes at room temperature. The fixative was then removed and the platelets were washed twice with PBS. The platelets were then stained with anti-CD42 / AF488 (Biolegend, Cat. No. 303914) for 20 minutes at room temperature. After labeling, slides were washed, mounted with ProLong Gold antifade mounting medium (Thermofisher, Cat. No. P36930) and imaged using a Zeiss LSM900.

[0125] Fluorescence in Situ Hybridization (FISH) Platelets in suspension were centrifuged at 600xg for 20 minutes onto poly-L-lysine coated chamber slides (Ibidi, Cat. No. 81201) and fixed in 2% formaldehyde / PBS for 10 minutes at room temperature. Platelets were then permeabilized in 0.5% Triton X-100 / PBS for 10 minutes at room temperature and washed twice in 0.05% Triton-X-100 / PBS. Platelets were washed in 0.02% Tween20 / PBS (PBST) and incubated with anti-β-tubulin primary antibody (Sigma Aldrich, Cat. No. T5201) diluted in blocking buffer for 30 minutes at 37°C in a humidified box. Following primary labeling, platelets were washed in PBST and incubated with donkey anti-mouse IgG H&L AF405 secondary antibody (Abcam, Cat. No. ab175658) diluted in blocking buffer for 30 minutes at 37°C. The labeled platelets were then washed with PBST, fixed, and washed with PBS at room temperature for 10 min. FISH staining was performed according to the manufacturer's protocol. Briefly, slides were incubated in 0.1N HCL, washed with 2xSSC, and dehydrated in 70% ethanol for 3 min. Equal volumes of X and Y chromosome probes (Metasystems, Cat. Nos. D-0323-050-FI and D-0324-100-OR) were added to the slides and covered with a 22x22mm2 coverslip. Slides were incubated at 85°C for 5 min, sealed with rubber cement, and incubated overnight at 37°C in a humidified chamber. After hybridization, the rubber seal was removed and slides were washed in 0.1xSSC at 60°C. Slides were mounted and imaged with a Zeiss LSM900 with a 63x oil immersion objective. Control cells (HEL cells and MNCs) were fixed on slides, permeabilized, and labeled with X and Y chromosome paints as described above. After overnight hybridization, cells were washed and counterstained with DAPI.

[0126] Image Analysis Software Images were analyzed using ImageJ v2.1.0 (National Institute of Health, US-MD) and 3D reconstructions were generated with Imaris Viewer v9.7.0 (Oxford Instruments, Abingdon). CellProfiler v4.0.7 (Broad Institute, US-MA) was used to quantitatively analyze platelet-tumor cell interactions. Briefly, cells were segmented using the “Identify Primary / Secondary Object” module. A thresholding strategy was utilized to accurately mask cells for segmentation. Platelets were tracked using the “Track Objects” module. Finally, AF647 intensity was measured using “Measure Object Intensity” and data were “Exported to Spreadsheet”. This image analysis pipeline, and others, can be recreated in the Cell Profiler tool (freely available from the Broad Institute at www.cellprofiler.org).

[0127] Fragmentation analysis of cfDNA, pDNA and gDNA Fragmentation profiles of 803 pDNA and paired cfDNA were analyzed using an Agilent Tapestation 2200 system (Agilent Technologies). cfDNA, HS D5000 and genomic DNA reagents and screen tapes (Agilent, catalog numbers 5067-5630, 5067-5592, and 5067-5365, respectively) were used for analysis. Electrophoretic data was analyzed using Tapestation analysis software (Agilent).

[0128] Whole Genome Sequencing (WGS) Whole genome sequencing (WGS) was performed on paired pDNA and cfDNA isolated simultaneously from the same peripheral blood samples of six patients with recently diagnosed gastrointestinal adenocarcinoma. Libraries were prepared using an Agilent Tapestation from samples that were confirmed to have short DNA fragments of approximately 160 bp in addition to a longer peak of approximately 12,000 bp.

[0129] gDNA fragmentation 15 μl of DNA diluted in TE buffer (1 mM Tris-HCI, pH 8.0, 0.1 mM EDTA) was added to a Covaris microtube-15 (Covaris, Cat. No. 520145) and briefly centrifuged for 1 min at 3000×g. The microtube was loaded onto a Covaris ME220 Focused-sonicator and the protocol was run using the processing conditions required for the 150 base pair (bp) target peak. Fragment size was determined using an Agilent Tapestation 2200 system (Agilent Technologies) and sonication was repeated if DNA fragments were >150 bp.

[0130] Library preparation DNA-Seq libraries were prepared using the NEBNext Ultra II DNA Sample Preparation Kit for Illumina (New England BioLabs, Ipswich, MA, USA) according to the manufacturer's protocol. pDNA was size-selected into two groups: short fragments (s-pDNA; less than 600 bp) and long fragments (l-pDNA; more than 600 bp). The short fragments were further cleaned to remove fragments of <100 bp, and the platelet long fragments were fragmented via sonication as previously described. Following end-repair and adapter ligation, the adapter-ligated DNA fragments were amplified with enrichment PCR to generate the final libraries. The cleaned-up libraries were then multiplexed and 1.5 pM libraries were analyzed on an Illumina NextSeq 500 (300 cycle PE) at low pass (0.1X) for all samples and 10X for cfDNA and s-pDNA, with 4 lanes for each sample using 4% PhiX to monitor sequencing performance. This amounted to 240 fastq files.

[0131] Bioinformatics analysis Quality control of all fastq files was performed using FastQC vO.11.8 66 The run was led by Trim Galore. 67 The reads were adaptor trimmed and quality trimmed using bwa-mem vO.6.5 and aligned to the GATK Genome Reference Consortium Human Build 38 (GRCh38)68 using bwa-mem vO.7.1769. Reads were aligned to alternative contigs to represent common complex variants using bwa_postalt.js (https: / / github.com / lh3 / bwa / blob / master / bwakit / README.md)69. The resulting BAM files were analyzed using Samtools v1.13.0 70 So I merged, sorted and indexed it.

[0132] The GATK toolkit v4.1.7.0 was used to mark optical and PCR duplicates, estimate library complexity, and calculate summary metrics of insert size. Mapping rates were assessed with samtools flagstat, and the number of reads mapped to each chromosome and mitochondrial genome was assessed with idxstats. Repetitive elements in the ENCODE blacklist were analyzed using bedtools intersect v2.30.0. 73 was excluded using

[0133] Insert Size Distribution For all uniquely mapped, non-duplicate and well-matched read pairs, the frequency of insert size values ​​was counted across all autosomes and chrX using Rsamtools. The distribution was plotted using ggplot2. 74 was used to plot.

[0134] Read depth per transcription start site The transcription start site is refTSS v3.3 75 We defined TSSs as highly expressed by the refTSS entry. We used the closest refTSS entry for the 5' coding sequence of all protein-coding genes. Per-base coverage in a 2 kb region per each TSS was extracted by Rsamtools, which counts only uniquely mapped, non-duplicate, correctly paired reads with insert sizes between 150 bp and 170 bp ("mononucleosome") or between 280 bp and 350 bp ("dinucleosome"). PBMC gene expression values ​​were obtained from Blueprint epigenome experiment EGAX00001327129 (http: / / dcc.blueprint epigenome.eu / # / experiments / ERX1123729). Briefly, genes with a posterior mean estimate of transcripts per million >15 were defined as "highly expressed" in PBMCs.

[0135] Dinucleotide peak difference quantification To examine the relationship between DNA species (cfDNA and s-pDNA) and the ratio of dinucleotide fragments, a chi-square test of independence was performed in R with simulated p-values. We divided fragments into (i) fragments less than 250bp, which represent mononucleotide fragments; and (ii) fragments greater than 250bp, which represent dinucleotide fragments. The ratio of dinucleotide fragments was higher in s-pDNA than in cfDNA in all samples (p<0.0005).

[0136] Chromosome coverage Coverage was calculated using samtools bedcov using bed files of 10 kb non-overlapping windows for all major chromosomes, excluding reads with mapQ<1. Positions with the lowest 8th percentile of mappability (as defined by umap: https: / / bismap.hoffmanlab.org / ) were removed. Coverage was then corrected for mappability and GC content bias using a linear regression model and visualized with karyoplotR77 878 ​​(Gel B, Serra E, 2017).

[0137] Gini Coefficient Coverage was calculated using Samtools depth after first filtering pairwise, uniquely mapped, properly pairwise mapped, primary alignments, and non-duplicate reads (Samtools view parameters: -f 2 -F 3972 -q 1). Coverage uniformity was assessed by calculating the Gini coefficient for short pDNA and cfDNA coverage pre-binned into contiguous 10 Kbp bins.

[0138] ichorCNA Copy number alterations in 10X and low-pass WGS were assessed using ichorCNA vO.2.041 (github: https: / / qithub.com / broadinstitute / ichorCNA / releases / tag / v0.2.0).

[0139] statistical analysis When comparisons were made between the same groups and the data visually appeared to follow a normal distribution, a parametric test, e.g., Student's paired t-test, was performed. When the data were paired but not normally distributed, a non-parametric test, e.g., Wilcoxon signed-rank test, was used. On the other hand, when comparisons were made between different groups, the Mann-Whitney U test was used to determine statistical significance.

[0140] Experimental outline: detection of clonal hematopoiesis To test the utility of analyzing platelet-associated nucleic acids in the detection of CH and solid tumors, we first optimized a method to isolate purified platelets along with blood fractions currently used in liquid biopsies at each disease site - namely, granulocytes for CH detection and cell-free DNA for solid tumors (Figure 1A). The isolated platelets showed good recovery after cryopreservation and thawing (Figure 1b), and the platelet isolates were confirmed to have very high purity (>99.96%) with minimal contamination of other blood cell types by flow cytometry, gene expression analysis, and cell morphology (cytospin) (Figures 1C-1E). Further studies are being conducted to further optimize the custom collection method for platelet-associated nucleic acids.

[0141] To date, all CH studies have been based on whole blood or granulocyte DNA, and therefore, they do not evaluate platelet-restricted clones. Recent publications have shown that a proportion of blood stem cells give rise exclusively to megakaryocytes and platelets, and that the proportion of these "megakaryocyte-primed" stem cells increases with age, leading us to hypothesize that testing platelets in older individuals may increase the sensitivity of CH detection. The team developed a method to detect CH in platelet cDNA compared to granulocyte nucleic acid (gDNA and cDNA).

[0142] Digital droplet PCR (ddPCR) and next-generation sequencing panels were used to detect CH-associated mutations. ddPCR is a sensitive and specific method to detect and study genetic targets. Both assays were optimized using experimental controls and demonstrated very high specificity (Figures 2a and 2b). Paired samples of platelet cDNA and granulocyte gDNA (i.e., isolated from the same sample) were analyzed to compare the detection rate of JAK2V617F-driven CH in granulocytes versus platelets. In 151 samples analyzed, the abundance ratio of JAK2V617F mutations was significantly higher in platelets than in granulocytes (Figure 2c). In 6 / 151 samples (3.9%), the mutation was detected only in granulocytes, and in all cases was below the 2% cutoff typically used by diagnostic laboratories. In 13 / 151 samples (8.6%), it was detected only in platelets, with abundance ratios above 5% in some cases. Only two samples had detectable CH in both granulocytes and platelets (1.3%; Fig. 2c).

[0143] These findings confirm the existence of JAK2-V617F platelet-biased CH in humans, with nearly 10% of detected cases that would have been missed by testing granulocytes alone. Because an important component of CH is increased vascular disease, platelet-biased CH may be particularly important in identifying patients at elevated risk for cardiovascular events.

[0144] We further applied a custom hybridization capture panel that allows for parallel sequencing of both gDNA and cDNA (Figure 3a).CH was found in 42 samples, in 15 of them (36%) by analysis of granulocytes and in 67% of the samples by analysis of platelets, again indicating that many instances of CH are only detected when platelet nucleic acid is analyzed.

[0145] In addition to analysis of cDNA, CH and myeloid malignancies-associated mutations can be detected by analysis of platelet DNA, as shown in FIG. 4 for detection of the JAK2V617F mutation in a patient with known myeloproliferative neoplasm.

[0146] Experimental Overview - Detection of Non-Hematologic Cancer The second application is the early detection of solid tumors. Based on literature suggesting that platelets have many nucleic acid receptors and function as part of the innate immune system to "sense" viral RNA, and recent observations showing that non-human RNA from pollen (possibly obtained via pulmonary circulation) is detectable inside platelets, the inventors hypothesized that platelets may also take up nucleic acid fragments released by tumor cells. This study focused on DNA rather than RNA, because DNA is more stable and not all cancer-associated mutations can be easily detected at the RNA level.

[0147] Because platelets are anucleated cells and the association of DNA in platelets has not been reported previously, we first isolated DNA from a standard cell-free DNA preparation and compared the amount of DNA obtained with that isolated from platelet pellets. This demonstrated that DNA was substantially more abundant in platelets than free in plasma (Fig. 5a). Furthermore, in some donors, the fragmentation profile of platelet-associated DNA (pDNA) showed small fragments of approximately 150-160 bp with nucleosomal footprints, similar to those detected in cfDNA (Fig. 5b), while other donors showed DNA fragment lengths of approximately 10,000-15,000 bp. Notably, DNA detected in platelets was persistent after treatment of platelet pellets with DNAse, indicating that DNA was likely contained within the platelets and protected from degradation (Fig. 5c). Live cell imaging of platelets co-incubated with colorectal cancer cell lines showed that DNA fragments were detectable (Fig. 5d) within 2 and up to 10 min of co-culture (Fig. 5e).

[0148] We next sought to confirm that specific cancer-associated gene rearrangements were detectable in platelets. After in vitro coculture of platelets from healthy donors with various cancer cell lines (Figure 6A), we confirmed that multiple cancer-specific gene mutations were detectable in p-DNA but not in platelets from healthy donors (Figure 6B). These included BRAF, PIK3CA, TP53, and KRAS mutations from colon, ovarian, and hematopoietic cancer cell lines (Figure 6).

[0149] To further characterize platelet DNA, whole genome sequencing was performed, showing that pDNA contains fragments located throughout the human nuclear genome (Figure 7a) and mitochondrial genome (Figure 7b). The majority of both long and short DNA fragments in pDNA mapped to the nuclear genome, but a higher percentage of long DNA fragments (about 40%) than fragments <200 bp mapped to the mitochondrial genome (Figure 7b). In patients with active malignancies, copy number changes present in cell-free DNA were also detectable in pDNA (Figure 7c), and fragmentation analysis showed a reduction in reads per transcription start site, confirming that analysis of DNA can provide insight into gene expression from the cellular origin of DNA.

[0150] To investigate the detection of cancer cell-derived DNA fragments in in vivo pDNA, a mouse model of colorectal cancer was used (mice expressing KRAS, TP53+ / -NRAS mutations, FIG. 8A). Mutant KRAS G12D was easily detectable in mice with colorectal cancer (FIG. 8B), and in 13 / 20 mice, the abundance of mutant KRAS copy number was greater in platelets than in cfDNA, indicating that in some cases p-DNA may be more sensitive than cfDNA (FIG. 8).

[0151] To address whether this occurs in the setting of human cancer, samples were taken from patients with GI tumors and patients with high-risk premalignant serrated polyps. Mutant BRAF was indeed confirmed to be detectable in 17% (5 / 29) of patients tested so far in patients with high-risk polyps, and the abundance of mutant BRAF alleles was higher in pDNA than in cfDNA in 3 / 5 cases (Figure 9A). Notably, this indicates that pDNA can be a sensitive test for early-stage malignancies, adding considerable value over standard cfDNA methods (Figure 9).

[0152] Detailed Example Example 1 Platelets contain DNA To determine whether DNA was detectable in platelets isolated from peripheral blood of human donors, platelets were labeled with NUCLEAR-ID Red DNA, a highly specific cell-permeant dye that intercalates with double-stranded DNA. A percentage of platelets, identified by their positivity for the platelet cell surface integrin CD42b, showed a clear fluorescent signal for the DNA probe (Fig. 11a). To compare the relative abundance of DNA (pDNA) in platelets to that in platelet-depleted plasma, a protocol was developed to simultaneously isolate purified platelets and cfDNA from the same blood sample by sequential centrifugation (Fig. 11b). A cohort of 64 donors was studied, including individuals who had undergone colonoscopy for cancer surveillance; patients with inflammatory bowel disease (Crohn's disease or ulcerative colitis) (n=43), and patients with known gastrointestinal cancer (n=21, including colorectal cancer [CRC], esophageal cancer, and pancreatic cancer). In all cases, DNA was obtained from the platelet pellet, and in the majority of samples (55 / 64, 86%), the platelet pellet yielded more DNA (6.4+7.4-fold in genomic copies) than platelet-depleted plasma (cfDNA, P<0.0001, Fig. 11c). The high purity of the platelet pellet was confirmed by flow cytometric analysis and cytospin preparations, which confirmed that less than 2 per 10,000 cells were non-platelet cell types.

[0153] To determine whether the DNA detected in platelets was acquired in the peripheral circulation or simply derived from parental megakaryocytes, prenatal blood samples were taken to look for Y chromosome fragments in the platelets of mothers carrying boys. Using a Y chromosome Fluorescence in Situ Hybridization (FISH) probe and droplet digital PCR (ddPCR), Y chromosome fragments were visualized in the platelets of mothers carrying boys (Figure 11d). Using ddPCR, fetal sex was correctly confirmed by the presence or absence of the SRY allele in maternal platelets in 100% (10 / 10) of samples. 24 and 48 hours after delivery of the infant, SRY was still detectable in platelets (Figure 11e) and not in mononuclear cells (Figure 11d) or red blood cells from the same blood samples, indicating that sequestration of free fetal DNA by blood cells is specific to platelets and not due to cfDNA "contaminating" in the platelet pellet.

[0154] We reasoned that if platelets cleared cfDNA from plasma, the abundance of cell / platelet-free DNA in plasma would increase substantially if platelet counts were reduced. To test this, we induced rapid and specific removal of platelets in mice by treatment with anti-CD42b antiplatelet antibodies. Consistent with our hypothesis, induction of immune thrombocytopenia led to a rapid increase in cfDNA in plasma (Fig. 11f). Overall, these data support a role for platelets in sequestration and clearance of cfDNA from plasma.

[0155] Example 2 Platelets capture DNA from nucleated cells To confirm that platelets sequester chromosome fragments released from nucleated cells, in vitro DNA uptake was visualized using live cell imaging. Colorectal adenocarcinoma cells (COLO205) were labeled with cell-permeant fluorescent DNA probes that either irreversibly intercalate into double-stranded DNA (NUCLEAR ID) or covalently bind to DNA (SiR-DNA), washed 2x to remove non-internalized probe, and then co-incubated with platelets. Platelet acquisition of labeled DNA from COLO205 cells was observed (Figure 12a). To assess the time course of DNA capture, platelets were added to medium conditioned by DNA-labeled COLO205 cells, and platelet uptake of fluorescently tagged DNA was measured over a 10-minute time course at 16-second intervals. DNA was rapidly visible in platelets within minutes (Figure 12b), and the mean fluorescence intensity of imaged platelets reached a plateau at approximately 6 minutes, suggesting a possible saturation of DNA uptake (Figure 12c). Similar results were observed when the fluorescent signal of randomly selected individual platelets was followed over time after addition of platelets to COLO205 conditioned medium. Overall, these data demonstrate that DNA fragments released by nucleated cells are internalized by platelets and that uptake is not dependent on physical contact between platelets and nucleated cells.

[0156] Example 3 Nucleic acid transfer via DNA-loaded EVs One mechanism by which cells remove excess cytoplasmic DNA produced during abnormal mitotic cycles and chromosomal instability is through the release of DNA-loaded extracellular vesicles (EVs), which are also released during cell apoptosis. Platelets can internalize EVs via the open canalicular system (OCS), and platelet uptake of tumor cell-derived EVs has previously been reported to mediate the transfer of cancer cell-derived protein cargo and mRNA transcripts. However, whether EVs mediate the transfer of DNA fragments from nucleated cells to platelets has not previously been investigated. To determine whether EVs are mediators of DNA transfer into platelets and the role of tumor cell apoptosis in DNA release, platelets were isolated from female donors and incubated for 3 h with EVs isolated from BL2 cells - a human B cell lymphoma cell line derived from a male donor - or isogenic BL2 cells engineered to express the apoptosis-suppressing gene Bcl-2 (BL2-Bcl-2 cells). EVs were isolated from BL2 and BL2-Bcl-2 cells treated with UV irradiation to generate apoptotic EVs (apo-EVs from BL2 cells) and non-apoptotic (non-apoEVs) from BL2-Bcl-2 cells, and treated with DNase to remove any non-internalized DNA. Imaging of EVs confirmed internalized DNA within EVs (Fig. 12d), and following co-incubation, DNA-loaded EVs were visible attached to and within platelets (Fig. 12e and 12f). To track DNA transfer more specifically, ddPCR was used to quantitate SRY alleles. SRY copies were detectable in platelets from female donors only after incubation with BL2-derived EVs, and were significantly more abundant after co-incubation with apo-EVs than after co-incubation with non-apoEVs (Fig. 12g). Similarly, after co-incubation with HEL cells, fragments of both X and Y chromosomes were visualized in a subfraction of female platelets using X and Y whole chromosome FISH probes (FIG. 12h).

[0157] Example 4 Detecting cancer mutations in platelets We then sought to determine whether DNA fragments carrying specific cancer-associated gene mutations could be detected in platelets. Platelets from healthy donors were incubated with various human cancer cell lines containing common cancer-associated gene mutations, including COLO205, HCT116, and LS180 colorectal cells, and HEL erythroleukemia cells (Figure 13a). Cancer cell lines were separated from platelets by inserts with 1 μm pores, which facilitate the exchange of biomolecules and small EVs between the compartments but prevent the entry of nucleated cells into the lower compartment. ddPCR of DNA isolated from platelets after co-incubation with cancer cell lines detected all relevant oncogenic mutations, including KRASG12D, BRAFV600E, PIK3CAH1047R, and JAK2V617F (Figures 13b and 13c). No cancer-specific gene mutations were identified in healthy donor platelets incubated alone, but wild-type alleles were detected as expected (Figure 13c), likely indicating sequestration of DNA from non-transformed cells encountered by circulating platelets prior to blood collection. To determine whether the ability to capture extracellular DNA is unique to platelets or shared by other blood cell types, we tested whether BRAFV600E from COLO205 cells was sequestered by those cells after adding platelets, peripheral blood mononuclear cells (MNCs), and red blood cells (RBCs) to medium conditioned by COLO205 cells for 12 hours. After incubation of platelets in COLO205 conditioned medium and after direct platelet-COLO205 cell co-culture, BRAFV600E was readily detectable in pDNA (Fig. 13d), whereas very low copies of mutant BRAF were detected in MNCs (p=0.0152) and RBCs (p=0.0022) (Fig. 13d), indicating a substantially higher efficiency of tumor cell-derived DNA detection in platelets than in other blood cell types and also confirming that the mutant isoforms detected in platelets were not due to "contamination" of residual tumor cell conditioned medium.

[0158] Example 5 pDNA is protected from nucleases Our imaging data (Fig. S12a-c) suggested that pDNA was largely internalized rather than attached to the outer membrane. To confirm this and to examine whether extracellular DNA sequestered by platelets is protected from exonuclease degradation by the platelet membrane, we treated conditioned medium and platelets with DNase after conditioning with HEL cells harboring the JAK2V617F mutation. Detection of JAK2V617F in HEL cell conditioned medium was completely abolished by DNase treatment (Fig. S13e). In contrast, there was no reduction in the abundance of JAK2V617F in platelets after DNase treatment (Fig. S13e), indicating that the majority of the DNA is encapsulated by the platelet outer membrane and protected from enzymatic degradation.

[0159] Example 6 Cellular origin of pDNA fragments We then sought to confirm the cellular origin and genomic distribution of platelet-derived DNA. Electrophoretic analysis showed that pDNA contained fragments of approximately 12,000-16,000 base pairs (bp), and a percentage of platelet samples also contained short fragments of approximately 120-160 bp with nucleosome footprints reflecting periodicity, similar to the fragmentation profile typically observed in cfDNA. We selected six samples from donors recently diagnosed with gastrointestinal cancer that contained both short and long fragment length peaks, and performed whole genome sequencing (WGS) on pDNA and cfDNA isolated simultaneously from the same peripheral blood sample. Size selection was performed on pDNA to separate short fragments (<600 bp, "s-pDNA") from long fragments ("l-pDNA"). To determine the cellular origin, low-pass WGS (average target coverage 1x) was first performed on s-pDNA, l-pDNA, and paired cfDNA. Platelets contain a small number of mitochondria, estimated at 4-6 mitochondria per cell. Because the mitochondrial genome is approximately 16,500 bp, we reasoned that the l-pDNA fragments could be mitochondrial DNA. Indeed, a significantly larger subfraction of l-pDNA (0.9-38%) mapped to the mitochondrial genome compared with <0.001-0.36% of the short fragments (Fig. 14a). However, the majority of fragments from both s- and l-pDNA mapped to the nuclear genome (98.6-99.9% for s-pDNA and 62-99.1% for l-pDNA, Fig. 14a).

[0160] Example 7 Genome coverage of pDNA mirrors that of cfDNA We then obtained deeper sequencing of paired cfDNA and s-pDNA (average sequencing depth 15x±3 / 12x±1 for cfDNA and s-pDNA, respectively). Except for a higher estimated PCR overlap rate (cfDNA: 3.2%±0.6%, pDNA: 11.3%±7.2%), no significant differences in sequence quality were detected between s-pDNA and cfDNA (base quality, 3' bias, mapping rate), indicating a lower complexity of short pDNA libraries, possibly as a result of size selection.

[0161] Genome-wide mapping revealed that fragments in platelets encompassed the entire nuclear genome, mirroring those found in cfDNA, demonstrating that sampling of pDNA will enable detection of oncogenic changes in genes across chromosomes. s-pDNA fragments showed more uniform coverage compared to cfDNA, as indicated by the significantly lower Gini coefficient (0.22 ± 0.03 for s-pDNA vs. 0.27 ± 0.03 for cfDNA).

[0162] Nucleosome-bound cfDNA is more protected from degradation than nucleosome-free DNA. This results in a characteristic fragment length distribution of cfDNA, with mononucleosome and dinucleosome length peaks at approximately 167 bp and 320 bp. The peak average fragment length of s-pDNA was 166 bp ± 0.8, with a second dinucleosome peak at approximately 328 bp. In all six cases sequenced, the dinucleotide peak was partially but significantly larger in s-pDNA than in cfDNA (Figure 14b). One explanation for this could be improved protection of DNA fragments in platelets from nuclease degradation, as observed in our in vitro experiments (Figure 13e).

[0163] Some studies have shown that cfDNA fragments originating from tumor cells are shorter in length than wild-type fragments. Consistent with this, we observed that both mononucleotide and dinucleotide fragment lengths were significantly shorter in both pDNA and cfDNA in sample 6 from a patient with an inoperable pancreatic tumor than in the other 5 samples from individuals who had been treated with surgery and / or chemo-irradiation before blood collection (Figure 14c), indicating that fragmentome analysis can be applied to platelet DNA to detect the presence of cancer.

[0164] To validate platelet capture of circulating tumor cell-derived DNA (ctDNA), iChorCNA was used to detect copy number alterations (CNAs) and estimate the fraction of tumor-derived DNA in the samples. Clonal amplifications and deletions in chromosomes 6 and 7, and subclonal amplification in chromosome 2, were detected in both cfDNA and spDNA from sample 6 at an estimated tumor fraction of 25% (Figure 14d). No CNAs were observed in either cfDNA or s-pDNA in the other five samples. CNAs detected in s-pDNA reflected those present in cfDNA, and the estimated fraction of tumor-derived DNA was highly correlated between s-pDNA and cfDNA. Selection of DNA fragments smaller than 150 bp increased the estimated tumor content of s-pDNA from 25% to 30%, paralleling the increase seen in cfDNA. Notably, no CNAs were detected in l-pDNA from sample 6, in line with previous reports that ctDNA is enriched in short fragments, indicating that the long and short fragments of pDNA may have different cellular and / or tissue origins.

[0165] Enrichment of nucleosome-bound DNA in cfDNA has been used previously to gain insight into gene expression patterns and cellular origin, and previous reports have shown that read reduction occurs periodically across transcription start sites (TSSs) and in coverage to TSSs. Furthermore, DNA methylation studies have shown that the majority (>90%) of cfDNA in healthy individuals is of hematopoietic origin, including neutrophils, monocytes, erythroblasts, and megakaryocytes, with minor contributions from endothelial cells and hepatocytes. We found very similar reduction in sequencing coverage and periodicity around TSSs for s-pDNA and cfDNA of genes highly expressed in peripheral blood mononuclear cells (PBMNCs) previously reported for cfDNA, suggesting a shared cellular origin of pDNA and cfDNA (Figure 14e). Notably, the reduction in coverage of highly expressed PBMNC genes was less pronounced for sample 6, which had an estimated tumor fraction of 25%, than for the other samples (Figure 14e).

[0166] Example 8 KRASG12D alleles are more abundant in platelets than in cfDNA in CRC mice Having demonstrated that platelets contain tumor cell-derived DNA, we hypothesized that analysis of pDNA could be useful for cancer screening, and investigated the utility of this phenomenon towards the detection of cancer genetic abnormalities as an in vivo liquid biopsy approach. We used a mouse model of colorectal cancer in which expression of KRASG12D and TP53 (KP) mutations was induced via the villin promoter, resulting in locally invasive colorectal adenocarcinoma with a low incidence of metastasis, and mice with KRASG12D / +, TP53fl / fl, and NOTCHfl / + (KPN) that have a more aggressive metastatic disease (Fig. 15a). To determine the relative sensitivity of cfDNA and pDNA for the detection of tumor-derived mutant alleles, pDNA and cfDNA were simultaneously extracted from the same blood samples (Fig. 15a) and mutant KRASG12D alleles were detected using ddPCR. Similar to our observations in human peripheral blood, DNA was more abundant in platelets than in platelet-depleted plasma in mouse samples. In mice, KRASG12D was readily detectable in both cfDNA and platelets of mice with colorectal cancer. Notably, in just over half of the mice (13 / 20, 65%), the copy number of KRASG12D per μL of DNA was greater in pDNA than in cfDNA in both locally invasive (KP) and metastatic (KPN) disease mice (Figures 15b-d). These data suggest that inclusion of platelet DNA analysis in liquid biopsy procedures may improve the sensitivity of cancer screening in many cases, and that current liquid biopsy approaches that use only platelet-poor plasma for analysis of cfDNA lack the substantial genetic information contained within platelets.

[0167] Example 9 BRAFV600E in platelets from patients with precancer Improving the sensitivity of liquid biopsy screening is particularly important in settings with low tumor burden disease and in patients with premalignant lesions with low abundance of ctDNA. Serrated lesions (SSLs) are high-risk premalignant polyps that account for approximately one-quarter of colorectal cancers. The majority (>75%) are driven by mutations in BRAF, typically BRAFV600E, and detection of BRAFV600E in cfDNA has been investigated as a screening tool for SSLs, with high specificity (100%) but low sensitivity (16.9%). To investigate the utility of pDNA for the detection of premalignant lesions, platelets and cfDNA were collected from patients found to have SSLs at colonoscopy (n=29). Control samples (n=14) were collected from patients with inflammatory bowel disease and those invited to colonoscopy by the UK Bowel Cancer Screening Programme in whom no polyps or cancerous lesions were detected.

[0168] Using ddPCR, BRAFV600E was detected in cfDNA and / or pDNA in 17.2% of patients with serrated polyps (5 / 29, Fig. 16a), a detection rate consistent with previous reports of the frequency of detectable ctDNA in this patient cohort. Mirroring our findings in the mouse model, approximately half (3 / 5) of patients with detectable ctDNA had higher copy numbers of BRAFV600E in pDNA than in cfDNA (Fig. 16b and Fig. 16c). BRAFV600E was not detected in any of the pDNA samples from 14 controls (Fig. 16a), demonstrating the high specificity of targeted mutation analysis of platelet DNA in this setting. Collectively, these data reveal a novel biological role for platelets in isolating cfDNA from plasma and indicate that analysis of pDNA may be useful for detecting genetic abnormalities in some clinical settings.

[0169] Consideration Genomic material is continuously released into human biological fluids by cell death, abnormal mitotic cycles, or regulated DNA extrusion. cfDNA release is increased in malignancies, inflammation, and following tissue injury, and several physiological mechanisms exist to limit its abundance because excess extrachromosomal DNA in the cytosol and plasma is proinflammatory. Similar to red blood cells, platelets express nucleic acid-sensing receptors, and their capture of pathogen-derived nucleic acids is a key component of innate immunity. Here, we present data showing a previously unappreciated role for platelets as scavengers of endogenous extracellular cfDNA, including tumor-derived and free fetal DNA. cfDNA detection and analysis are rapidly being implemented in several clinical settings, including cancer screening, molecular profiling, and treatment response monitoring, as well as in prenatal diagnosis. Such liquid biopsy approaches are minimally invasive, allow access to tumors at hard-to-reach biopsy sites, and capture intratumor and intertumor heterogeneity more reliably than traditional tissue sampling. Currently, a major limitation in the field of cancer diagnostics is the insufficient sensitivity for low tumor burden disease, making preanalytical approaches to enhance ctDNA capture of great interest. Our data suggest that a substantial proportion of ctDNA is contained within platelets and is currently missed by standard sampling protocols that extract cfDNA from platelet-depleted plasma. Furthermore, we show that pDNA is more protected from nuclease degradation than cfDNA outside platelets. Platelets have long been associated with carcinogenesis and metastasis, contributing to tumor angiogenesis, tumor cell extravasation, and shielding cancer cells from immune surveillance during metastasis. Previous studies have also highlighted the role of platelets in cancer diagnostics. The unique biophysical properties of platelets include a surface-connected open canalicular system that allows rapid transport of molecules into and out of the platelet interior, for example, internalization of extracellular vesicles containing a cargo of tumor biomarkers. Platelets are highly abundant and easy to isolate, making them ideally suited as “sentinels” for genetic perturbations in tissues and for use in liquid biopsy approaches.Studies on platelet nucleic acids have previously shown that the platelet transcriptome is altered in the presence of cancer by altered splicing of the platelet mRNA repertoire derived from parental megakaryocytes, as well as by uptake of mRNA transcripts from tumor cells. However, because mRNA is less stable than DNA, and the platelet transcriptome and spliceosome are also altered in non-malignant pathologies, this is likely less specific than screening for oncogenic abnormalities in platelet DNA. Although we have shown that pDNA contains similar information to standard cfDNA isolated from platelet-poor plasma, we have not directly tested whether combined analysis of cfDNA plus pDNA would increase the sensitivity of the screening method, nor whether integration of DNA or protein cargo with an mRNA signature in platelets could be of added value. We have also not yet investigated whether a portion of the DNA cargo in platelets is derived from their parental megakaryocytes. Taken together, however, our data highlight a novel aspect of platelet biology and demonstrate the utility of pDNA analysis for liquid biopsy genetic screening in multiple clinical settings. This study paves the way for future studies to further establish the role of platelets in cfDNA clearance and homeostasis and the utility of this phenomenon in clinical diagnostic settings.

Claims

1. A method for detecting or predicting clonal hematopoiesis, A process for preparing a biological sample containing a plug bulb; A step of extracting nucleic acids from the aforementioned biological sample; A step of analyzing the nucleic acid to identify the presence of one or more clonal hematopoietic mutations; and A process to indicate the presence or prognosis of clonal hematopoiesis based on the presence of one or more clonal hematopoiesis-related mutations. Methods that include...

2. One or more clonal hematopoietic-associated mutations are present in JAK2 V617F JAK2 Exxon-12, CALR Exxon-9, MPL S5050 MPL W515 CBL exon 8, CBL exon 9, KRAS exon 2, KRAS exon 3, GNB1 exon 5, DNMT3A all exons, TET2 all exons, ASXL1 exon 12, IDH2 exon 4, SF3B1 exon 14, SF3B1 exon 15, SRSF2 P95 The method according to claim 1, wherein the exon is selected from U2AF1 exon 2, U2AF1 exon 6, PPM1D exon 6, and TP53 all exons.

3. The process includes extracting DNA and / or RNA from the biological sample, and optionally, The method according to claim 1 or 2, comprising the step of converting the RNA to cDNA.

4. A process for preparing a biological sample containing granulocytes; A step of extracting nucleic acids from the biological sample; and A step of analyzing the nucleic acid to identify the presence of one or more clonal hematopoietic mutations. This also includes, in some cases, The method according to claim 1 or 2, wherein RNA is converted to cDNA via reverse transcription.

5. Patients diagnosed with clonal hematopoiesis are selected for further monitoring, and in some cases, The method according to claim 1 or 2, wherein a subject diagnosed with clonal hematopoiesis is identified as being at high risk for cardiovascular disease, heart failure, diabetes, autoimmune disease, and / or myeloid hematological cancer.

6. The method according to claim 1 or 2, wherein the presence of one or more clonal hematopoietic-associated mutations is identified via next-generation sequencing such as droplet digital PCR, allele-specific polymerase chain reaction (PCR), high-resolution melting curve analysis, genome sequencing fluorescence in situ hybridization (FISH); comparative genome hybridization (CGH), restriction fragment length polymorphism (RELP), amplification-resistant mutation system (ARMS), reverse transcriptase PCR (RT-PCR), real-time PCR, multiple ligation-dependent probe amplification (MLPA), denaturant concentration gradient gel electrophoresis (DGGE), single-strand higher-order structure polymorphism (SSCP), chemical cleavage of mismatches (CCM), protein truncation test (PTT), pyrosequencing, HPLC (high-performance liquid chromatography), or oligonucleotide ligation assay (OLA).

7. A method for detecting or predicting cancer, A process for preparing a biological sample containing a plug bulb; A step of extracting nucleic acids from the aforementioned biological sample; A step of analyzing the nucleic acid to identify the presence of one or more cancer-related nucleic acid fragments; and A process of indicating the presence or prognosis of cancer based on the presence of one or more cancer-related nucleic acid fragments. Methods that include...

8. The method according to claim 7, wherein the cancer-related nucleic acid fragment is DNA and / or RNA.

9. Cancer-related nucleic acid fragments are cell-free nucleic acid fragments released from cancer and taken up by the stent globules, and in some cases, The method according to claim 7 or 8, wherein the cancer-related nucleic acid fragment comprises cancer-related modifications, or cancer-specific mutations, methylation patterns, genetic abnormalities and / or fragmentation patterns.

10. Cancer-related nucleic acid fragments include the following mutations, BRAF V600E KRAS G12D PIKCA H1047R TP53 R273H A nucleic acid fragment is selected from one or more of the following, and in some cases, Cancer-related nucleic acid fragments are identified via next-generation sequencing methods such as droplet digital PCR, allele-specific polymerase chain reaction (PCR), high-resolution melting curve analysis, genome sequencing fluorescence in situ hybridization (FISH); comparative genome hybridization (CGH), restriction fragment length polymorphism (RELP), amplification-resistant mutation systems (ARMS), reverse transcriptase PCR (RT-PCR), real-time PCR, multiple ligation-dependent probe amplification (MLPA), denaturant concentration gradient gel electrophoresis (DGGE), single-strand higher-order structure polymorphism (SSCP), chemical cleavage of mismatches (COM), protein truncation tests (PTT), or oligonucleotide ligation assays (OLA), methylation analysis, and fragmentation pattern analysis, and in some cases, The method according to claim 7 or 8, wherein the cancer-related nucleic acid fragment comprises 10 to 1500 nucleotides.

11. Cancer-related nucleic acid fragments are associated with solid tumors, and in some cases, Cancer-related nucleic acid fragments are associated with sarcomas, carcinomas, and / or lymphomas, and in some cases, The method according to claim 7 or 8, wherein the cancer-related nucleic acid fragment is associated with gastric cancer, lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bone cancer, pancreatic cancer, colon cancer, colorectal cancer, skin cancer, head and neck cancer, head and neck squamous cell carcinoma, melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, breast cancer, brain cancer, hepatocellular carcinoma, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, kidney cancer, soft tissue sarcoma, urethral cancer, bladder cancer, renal cancer, thymoma, urothelial carcinoma, leukemia, prostate cancer, prostate adenocarcinoma, mesothelioma, adrenocortical carcinoma, lymphoma, for example, Hodgkin's disease, non-Hodgkin's disease, and multiple myeloma.

12. A method for determining the appropriate treatment for a target group, The detection or prediction method according to claim 7 or 8; and The process of determining the appropriate treatment. Methods that include...

13. The biological sample is processed to prepare an isolation sample of the plug sphere, and in some cases, The method according to any one of claims 1 to 12, wherein the biological sample is a blood sample.

14. A panel of reagents for extracting nucleic acids from platelets, and reagents for specifically binding to and / or amplifying one or more clonal hematopoietic mutations, and optionally including instructions for use, optionally, The reagent is for parallel analysis of cDNA and gDNA containing one or more clonal hematopoietic or cancer-related mutations, and may, A kit containing reagents for detecting clonal hematopoietic-associated mutations in one or more of the following: JACK2, CALR, MPL, CBL, KRAS, GNB1, DNMT3A, TET2, ASXL1, IDH2, SF3B1, SRSF2, U2AF1, PPM1D, and TP53.

15. A kit comprising reagents for extracting nucleic acids from platelets, and a panel of reagents for specifically binding to and / or amplifying one or more cancer-related modifications or cancer-specific mutations.

16. A method for preparing nucleic acid fractions, The process of preparing a biological sample including a stopper bulb, A step of extracting nucleic acids from the aforementioned biological sample to form a nucleic acid sample, The process of concentrating the nucleic acid sample with respect to one or more cancer-related nucleic acid fragments and / or clonal hematopoietic-related fragments. Methods that include...

17. A method for prenatal screening, A process for preparing a biological sample containing plug globules obtained from a pregnant woman; A step of extracting nucleic acids from the aforementioned biological sample; A process of analyzing the nucleic acids with respect to the genetic information related to the fetus. Methods that include...

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