Methods for detecting cancer

Abstract

The present invention relates to a bodily fluid test method for detecting cancer using a combined biomarker panel comprising at least one cytokine molecule and at least one cell-free chromatin fraction.

Description

Technical Field

[0001] The present invention relates to a bodily fluid test method for detecting cancer using a combined biomarker panel comprising at least one cytokine molecule and at least one cell-free chromatin fraction. Background Art

[0002] Cancer is a common disease with a high mortality rate. The biology of this disease is understood to relate to the progress from precancerous state to I, II, III phase and final IV phase cancer. For most cancer diseases, the mortality rate varies greatly, and this depends on whether the disease is detected in the early local stage (when effective treatment options are available), or when the disease may have been in the infected organ or beyond the late stage of the infected organ spread (when treatment is more difficult). Late stage cancer symptoms are different, including visible blood in the stool, hematuria, coughing up blood, vaginal discharge of blood, unexplained weight loss, persistent unexplained lump (for example, in the mammary gland), dyspepsia, dysphagia, the change of warts or moles and many other possible symptoms depending on the cancer type. However, most of the cancers diagnosed due to these symptoms will be in the late stage and difficult to treat. Most cancers are asymptomatic or have nonspecific symptoms that do not help diagnosis in the early stages. Therefore, ideally, cancer testing should be used to detect cancer as early as possible.

[0003] The cancer with the highest mortality rate in developed countries is lung cancer. For the case detected when the disease is still confined to the lung, the 5-year survival rate of lung cancer>50%, but is only 5% when the disease has spread to other organs. Unfortunately, most lung cancer cases are diagnosed (57%) when transferred, and only have 16% diagnosed in the early stages. For the patient who detects the disease in the I phase, the 5-year survival rate of breast cancer is about 85%, but for the patient who detects metastatic disease in the IV phase, only about 10%. Similarly, when detecting in the I phase, the 5-year survival rate of colorectal cancer (CRC)>90%, but for the patient who detects metastatic disease in the IV phase, only about 10%. Many other cancer diseases follow similar patterns, and for this reason, many countries have screening programs to identify the individuality of cancerous or precancerous conditions. The most commonly screened cancers are breast cancer via mammography, cervical cancer via Pap smear testing for HPV and / or abnormal cervical cells, colorectal cancer (CRC) via fecal immunochemical testing (FIT) and / or colonoscopy, and, more recently, lung cancer via low-dose computed tomography (LDCT). Blood measurement of prostate-specific antigen (PSA), although not FDA-approved as a screening test for prostate cancer, is also often performed on healthy men.

[0004] Some cancer cases (such as breast cancer or testicular cancer) can be detected by palpation of the inappropriate lumps, nodules or masses of the body.Any such lumps may or may not be cancerous in nature, and further research may be needed to determine whether the lumps are malignant or benign in nature. However, palpation of internal organs (such as lungs, colon or pancreas) is impossible, and other cancer tests are needed. Most cancer tests can be roughly classified as (i) scanning to visualize the nodules, masses or lumps in the body, (ii) biopsy to find the abnormal cells in the target organ or (iii) body fluid tests to the material released by cancer or related or surrounding tissues. All current cancer screening methods have shortcomings. Scanning allows the visualization detection of lumps or nodules, but, like palpation, can not usually distinguish between malignant nodules and inert or non-malignant (such as fibrous) masses, causing poor specificity and / or overdiagnosis. Biopsy relates to the high traumatic surgery or puncture biopsy of most tissues (such as lungs, liver, kidneys, prostate). Even relatively accessible tissue, such as cervical tissue, requires an invasive and traumatic biopsy procedure. Blood and other body fluid tests are low-cost and non-invasive, but are rare.

[0005] The Pap smear test is a long-established cancer screening method. It has been shown to be effective in preventing disease in individual women and reducing the prevalence of disease in the general population because (i) it detects precancerous cervical tissue, which can be removed before it develops into cancer, and (ii) cervical cancer affects younger women, so prevention can maintain a high quality of life for many years. The Pap smear test involves taking a scraping sample of cells from the surface of the cervix and examining it for any abnormal cancerous or precancerous cells. The scraping can be examined by cytological examination of the cells or by examining the cells for the incorporation of human papillomavirus (HPV) DNA. Cytological examination of the sample provides correct results in 70%-80% of women tested, while HPV testing of the sample provides correct results in 90%-95% of women tested. However, Pap smear sampling is invasive and traumatic, which affects patient acceptance of the test. Compliance varies with the woman's age but is generally approximately 80%. Currently, there is no commonly used blood test for cervical cancer.

[0006] The main screening method for CRC adopted in the United States is colonoscopy. This inspection is accurate for CRC detection and has also identified most colorectal polyps or adenomas, which are potential precancerous stages that may develop into CRC. Removing precancerous colorectal polyps leads to a favorable prognosis for the patient. However, colonoscopy is expensive and costs >$1000 in the United States. This inspection is also traumatic and invasive, requiring surgical admission and may occasionally cause damage (e.g., due to intestinal tearing). The procedure is usually performed under anesthesia and requires the patient to make unpleasant preparations in advance, wherein the intestine is thoroughly flushed. In addition, due to the low incidence of CRC, the disease is only detected in approximately 0.5% of screening colonoscopies, so the vast majority of screened people undergo surgical procedures with almost no benefit. All of these shortcomings affect the acceptance of the inspection, and the compliance of CRC screening by colonoscopy is poor in the United States, approximately 60% of the screening age group. Due to its shortcomings, colonoscopy is not used as a front-line CRC detection or screening method in most countries in the world.

[0007] Some healthcare providers use a related procedure called sigmoidoscopy, in which a shorter scope is used to examine only the descending colon. Although this method misses two-thirds of the colon, it does examine the area where cancer is most often seen. The disadvantages of sigmoidoscopy are similar to those of colonoscopy, and for similar reasons, it is not usually used as a first-line test. Virtual colonoscopy or computed tomography (CT) colonography is also used. This procedure uses a combination of X-rays and computer technology to create images of the rectum and colon to detect colorectal tumors and adenomas.

[0008] The most commonly used CRC detection and screening approach involves a two-stage procedure in which the screening-age population is first screened with a noninvasive, frontline stool test to identify subgroups of the screening population at higher risk for CRC. People who test positive on the stool test are referred for a subsequent colonoscopy, and approximately 5% of these people are typically found to have CRC. Most people have a negative stool screening result, so the two-stage approach prevents unnecessary colonoscopies in the majority of people who do not have lesions.

[0009] The rationale behind current stool tests for CRC is to detect bleeding in the colon or rectum. For example, when the colon or rectum is partially blocked by an invading cancerous or precancerous growth, the movement of stool past the obstruction can cause damage and bleeding. This bleeding is detected by testing stool samples for the presence of hemoglobin. Because the extent of bleeding can vary greatly from day to day, the test may need to be performed several times on different days.

[0010] All current stool CRC tests are designed to detect fecal hemoglobin. The guaiac fecal occult blood test (FOBT or gFOBT) is a chemical test for hemoglobin in which the patient or operator typically smears a small amount of stool onto α-guaiac acid-coated paper or other substrate. If blood is present in the stool, hydrogen peroxide is added to the paper, which produces a rapid color change by oxidizing α-guaiac acid to a blue quinone in a reaction catalyzed by heme (a component of hemoglobin). Consumption of meat (and therefore heme) and some vegetables (containing other catalyst molecules that behave like heme in the test) can lead to false-positive results. Similarly, some substances (including vitamin C) can lead to false-negative results, so dietary restrictions are often recommended before the test. The guaiac FOBT test can have high clinical specificity, depending on the cutoff value used, and has a sensitivity of 60-70% for the detection of CRC. Detection of precancerous adenomas is poor. The chemical FOBT method was the method of choice in the past and, although still widely used, is being replaced by the FIT method.

[0011] The FIT method (also known as iFOBT or FOBTi) is essentially an immunoassay for human hemoglobin in stool samples. The FIT method is less susceptible to false positive and false negative results caused by dietary factors and can detect smaller amounts of blood in the stool. These tests detect approximately 72% of CRC cases with a specificity of 95%, and therefore detect slightly more clinically relevant cancer lesions than the gFOBT, which has similar specificity. Detection of adenomas is very poor. The FIT and gFOBT tests are non-invasive and low-cost, but require the patient to manipulate the stool, which is unpleasant to perform and leads to low compliance. In the most compliant European countries with national screening programs, patient compliance with the FIT and FOBT methods is 60%-70%, but in many countries it is as low as 10%-20%. In addition, the tests are not completely reliable. FIT misses approximately 30% of CRC cases, and in addition, most FIT-positive subjects do not have cancer and therefore undergo subsequent invasive colonoscopy with little or no benefit. The Cologuard stool test for CRC, produced by Exact Sciences, uses several stool DNA measurements in addition to fecal hemoglobin measurements, increasing the test's accuracy to 92% sensitivity at 87% specificity. Blood tests for CRC detection are not used clinically, primarily because they lack accuracy. For example, the only blood test currently approved by the FDA for CRC is the Epi Procolon test, which detects 68% of CRC cases with 80% specificity (Potter et al., 2014).

[0012] When treatment options have better results, breast cancer screening is usually performed by mammography for early detection of the disease. Mammography uses low-dose X-rays to visualize any lumps in the breast before they can be felt, or to show tiny calcium clusters called microcalcifications. It is necessary to perform histological confirmation of cancer by biopsy because lumps or spots may be caused by other conditions such as fat cells or cysts, and up to 50% of positive findings are false positives. In addition, some true positive results lead to overdiagnosis, in which small lumps are inert in nature and will not develop into life-threatening diseases. False positives and overdiagnosis can lead to unnecessary invasive procedures and exposure to further X-rays. These shortcomings and the inherent dangers of X-ray exposure lead to reduced patient compliance. Current blood tests for breast cancer detection are not accurate enough for routine clinical use. For example, CA15-3 (the most commonly used tumor marker for breast cancer) detects 19% of breast cancer cases with 95% specificity (Wojtacki et al., 1994).

[0013] LDCT has recently been recommended for lung cancer screening as a screening test for early detection of the disease in high-risk subjects (e.g., long-term heavy smokers). LDCT uses low-dose X-rays to visualize early-stage small masses or nodules in the lungs. However, as with other scanning methods, any mass observed may or may not be cancerous in nature, and it is necessary to perform histological confirmation of the cancer by biopsy. Up to 40% of positive results on LDCT are false positives, where the lesion detected is not cancerous. In addition, many nodules of unknown etiology are found, where the nodules may or may not be malignant in nature but are too small to be biopsied. False-positive results can lead to unnecessary invasive procedures, and nodules of unknown etiology can lead to repeated follow-up scans to monitor the mass through repeated exposure to further X-rays. Blood tests for lung cancer detection are not used clinically, primarily because they lack accuracy. For example, a miRNA test that detected 21% of lung cancer cases with a specificity of 76% has been proposed as a frontline screening test, while the Early CDT-Lung test, which detected 41% of lung cancer cases with a specificity of 87%, is being evaluated as a primary screening test (Midthun, 2016).

[0014] A major failing of current screening methods is low patient compliance, as failure to screen can lead to early mortality and increase the burden of expensive late-stage cancer treatment on healthcare providers. Compliance with colonoscopy for CRC screening in the United States is poor, at approximately 60% for individuals over 50 years of age. The remaining unscreened individuals are at significantly increased risk of CRC. Compliance with the European FIT screening program for CRC is similarly poor, at 60%-70%. Mammography and LDCT examinations involve exposure to damaging X-rays, and frequent or repeated examinations have the potential to cause cancer. At the time of writing, LDCT is a recent screening development, but early experience suggests poor compliance, perhaps as low as 20%. This may include the need for repeated scans every 3-6 months for nodules of unknown etiology, exposing subjects to repeated X-ray doses that may accelerate cancer progression, while also potentially slowing down the growth of growing nodules. The U.S. Preventive Services Task Force (USPSTF) has identified the need for biomarkers to accurately distinguish benign from malignant nodules identified on LDCT scans (Moyer, 2014). All current cancer screening methods suffer from a combination of poor accuracy, overdiagnosis, high cost, high invasiveness, exposure to X-rays, and poor patient compliance.

[0015] Most commonly occurring cancers are not screened for, including, for example, lymphoma, kidney cancer, bladder cancer, pancreatic cancer, uterine cancer, myeloma, thyroid cancer, ovarian cancer, or liver cancer. This reflects the lack of good cancer blood tests for these diseases. An exception is prostate cancer; although there is no approved or recommended screening test, the PSA test is often performed on healthy men, and a positive result leads to follow-up testing for suspected prostate cancer. The main advantage of the PSA test stems not from its accuracy but from its nature as a blood test. It is a low-cost, non-invasive test that can be included in routine health examinations, eliminating most compliance issues because no hospital visit is required, no special patient preparation is required, and the small amount of blood required (<100 µL) means that no specialized blood draw is required. Therefore, the test can be ordered by the physician as part of a routine health examination, along with other routine tests (such as cholesterol, blood sugar, and liver enzymes).

[0016] To address the need for a simple, routine cancer blood test, a number of blood-borne biomarkers have been investigated as potential cancer tests, including carcinoembryonic antigen (CEA) for CRC, alpha-fetoprotein (AFP) for liver cancer, CA125 for ovarian cancer, CA19-9 for pancreatic cancer, CA 15-3 for breast cancer, and PSA for prostate cancer. However, their clinical accuracy is too low for routine diagnostic use, and they are considered better for patient monitoring.

[0017] Recently, the use of hypermethylation of specific gene sequences as diagnostic biomarkers for cancer in the blood has been studied. For example, the DNA methylation status of specific genes or loci has been studied by selective bisulfite deamination of cytosine rather than 5-methylcytosine to uracil, resulting in primary DNA sequence changes that can be detected by sequencing or other means (Yang et al., 2004). One such test for hypermethylation of the SEPTIN-9 gene is currently the only blood test approved by the U.S. Food and Drug Administration (FDA) for CRC detection. It was found that this test detected 68% of CRC cases with 80% specificity. Similarly, there is also great interest in using circulating tumor DNA (ctDNA) as the basis for cancer detection in blood tests. However, although ctDNA tests identify late-stage cancers, they do not detect early-stage cancers. In addition, ctDNA tests are expensive and require a large amount of blood. These shortcomings mean that it is unlikely to be used as a conventional cancer screening method.

[0018] Workers in the field have also investigated many other biomarkers for detecting cancer, including circulating cell-free nucleosomes themselves (Holdenrieder et al., 2001) and inflammatory molecules such as TNFα, interleukin-6 (IL-6), and interleukin-8 (IL-8) (Chadha et al., 2014).

[0019] Although it is well known that the circulating levels of cell-free nucleosomes themselves may be elevated in a variety of cancer conditions, cell-free nucleosome measurements have not yet been used clinically to detect cancer or for any other clinical purpose (Holdenrieder et al., 2001). The main drawback of measuring cell-free nucleosomes themselves in clinical applications is that elevated levels are nonspecific indicators of cell death, and elevated levels have been reported for a variety of conditions, including gynecological diseases, autoimmune diseases, inflammatory diseases, stroke, heart disease, sepsis, graft-versus-host disease, and burns, trauma after surgery or exercise (Holdenrieder et al., 2005 and Holdenrieder and Stieber, 2009). Therefore, measuring elevated levels of nucleosomes themselves is considered a nonspecific indicator of disease and cannot be used in oncology.

[0020] Circulating cell-free nucleosomes containing specific epigenetic signals, including specific post-translational modifications, histone isoforms, modified nucleotides, and non-histone chromatin proteins, have also been investigated as cancer markers (as cited in WO2005019826, WO2013030577, WO2013030579, and WO2013084002).

[0021] It has also been reported that the circulating levels of cytokine inflammatory molecules (including many interleukin proteins) change in cancer. There are more than 50 kinds of coded interleukins and related proteins in the human genome. These are called interleukin 1 (IL-1), interleukin 2 (IL-2) etc. Many cytokines with elevated levels have been reported in cancer, including, for example, but not limited to IL-1, IL-2, IL-6, IL-7, IL-8, IL-11, IL-12, TNF-α and CRP (Lipitz and Harris, 2016 and Allin et al., 2011). For example, the effect of IL-6 in tumorigenesis has been studied in a wide range of human cancers, including CRC and lymphoma, glioma, melanoma, breast cancer, ovarian cancer, renal cancer and pancreatic cancer (Wang and Sun, 2014). Elevated circulating IL-6 levels are associated with tumorigenesis, disease progression, and poor prognosis in many cancers, including colorectal, prostate, skin, breast, lung, esophageal, liver, pancreatic, gastric, gynecological, renal, bladder, and hematological cancers (Taniguchi and Karin, 2014). Similarly, circulating IL-8 levels are increased in many cancers, including colorectal, gastric, melanoma, ovarian, pancreatic, prostate, and breast cancers.

[0022] However, cytokine markers are not typically used as tests for cancer. The main drawback of these markers is that they are nonspecific markers for a wide range of conditions, including obesity, diabetes, autoimmune diseases, inflammatory diseases, and infections.

[0023] Despite recent advances, there are generally no blood tests for cancer screening, with the possible exception of the use of PSA for prostate cancer detection (although there are recommendations against it). There is a need to develop non-invasive blood tests for various cancers and for general cancer diagnosis, either for use as general cancer screening, or as a potential diagnosis in symptomatic patients or as an adjunct to other cancer detection methods to confirm or exclude cancer. Summary of the Invention

[0024] According to a first aspect of the present invention, there is provided use of a biomarker panel in a body fluid sample for diagnosing and / or monitoring cancer, wherein the biomarkers comprise at least one cell-free chromatin fraction and at least one cytokine molecule.

[0025] According to another aspect of the present invention, there is provided a method for diagnosing cancer in a patient, comprising:

[0026] detecting or measuring at least one cytokine molecule and at least one cell-free chromatin fraction in a bodily fluid sample obtained from the patient; and

[0027] The levels or concentrations of cytokine molecules and cell-free chromatin fragments detected in a bodily fluid sample are used to determine whether a patient has cancer.

[0028] According to another aspect of the present invention, there is provided a method of assessing a patient's suitability for cancer research, comprising:

[0029] detecting or measuring at least one cytokine molecule and at least one cell-free chromatin fraction in a bodily fluid sample obtained from the patient; and

[0030] The levels or concentrations of cytokine molecules and cell-free chromatin fragments detected in the body fluid sample are used to determine whether the patient requires further cancer research.

[0031] According to another aspect of the present invention, there is provided a method of treating cancer in a patient, comprising:

[0032] (i) detecting or measuring at least one cytokine molecule and at least one cell-free chromatin fraction in a body fluid sample obtained from a patient;

[0033] (ii) using the levels or concentrations of cytokine molecules and cell-free chromatin fragments detected in a body fluid sample to determine whether the patient has cancer; and

[0034] (iii) if it is determined in step (ii) that the patient has cancer, administering treatment to the patient.

[0035] According to another aspect of the present invention, a kit comprising reagents for detecting at least one cytokine molecule and at least one cell-free chromatin fragment in a body fluid sample is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Figure 2: Box plots and receiver operating characteristic (ROC) curves for a dual assay panel containing nucleosomes containing histone H3.1 and IL-6, trained to optimize the distinction between lung cancer patients and normal donors in a 144-subject lung cancer cohort. Box plots represent the probability of cancer for any given subject calculated by logistic regression analysis.

[0037] Figure 2 Figure 3: Box plots and ROC curves for a three-assay panel containing measurements of nucleosomes themselves, nucleosomes containing histone isoform H3.1, and IL-6, trained to optimize the distinction between colorectal cancer (CRC) patients and patients with symptoms but no findings on colonoscopy in a 100-subject CRC cohort. Box plots represent the probability of cancer for any given subject calculated by logistic regression analysis.

[0038] Figure 3Figure 3: Box plots and ROC curves for a three-assay panel containing measurements of nucleosomes themselves, nucleosomes containing histone isoform H3.1, and IL-6, trained to optimize the discrimination between patients with CRC compared to subjects with no findings on colonoscopy and patients with non-malignant benign colon or intestinal diseases in a 100-subject CRC cohort. Box plots represent the probability of cancer for any given subject calculated by logistic regression analysis.

[0039] Figure 4 Figure 2: Box plots and ROC curves of a dual assay model (lung / CRC combined model) comprising nucleosomes containing histone isoform H3.1 and IL-6, trained to optimize the discrimination between patients with CRC or lung cancer compared to normal donors and subjects without findings on colonoscopy, applied to a 70-subject validation cohort, including 30 lung cancer patients, 30 normal donor subjects, and 10 subjects with chronic obstructive pulmonary disease (COPD). Box plots represent the model / algorithm output for each subject calculated by logistic regression analysis.

[0040] Figure 5 Figure 2: Box plots and ROC curves of a dual-assay model (lung / CRC combined model) containing nucleosomes containing histone isoform H3.1 and IL-6, trained to optimize the distinction between patients with CRC or lung cancer compared to normal donors and subjects without findings on colonoscopy, applied to a 63-subject validation cohort, including 30 patients with various cancer diseases and 33 normal donor subjects. The box plots represent the model / algorithm output for each subject calculated by logistic regression analysis. The upper and lower dashed lines represent the cutoff values ​​at 90% specificity and 80% specificity, respectively.

[0041] Figure 6 Figure 3: Box plots and receiver operating characteristic (ROC) curves for a dual-assay panel comprising nucleosomes containing histone isoform H3.1 and IL-6, trained on a 100-subject CRC cohort to optimize discrimination between patients with CRC and those with symptoms but no findings on colonoscopy. The panel was applied to a 63-subject validation cohort, including 30 patients with various cancer diseases and 33 normal donor subjects. The box plots represent the model / algorithm output for each subject, calculated by logistic regression analysis. The upper and lower dashed lines indicate the cutoff values ​​at 90% and 80% specificity, respectively.

[0042] Figure 7Figure 3: Box plots and receiver operating characteristic (ROC) curves for a three-assay panel containing nucleosomes alone, nucleosomes containing histone isoform H3.1, and IL-6. The panel was trained on a 100-subject CRC cohort to optimize discrimination between CRC patients and patients with symptoms but no findings on colonoscopy. The panel was applied to a 63-subject validation cohort, including 30 patients with various cancer diseases and 33 normal donor subjects. The box plots represent the model / algorithm output for each subject calculated by logistic regression analysis. The upper and lower dashed lines represent the cutoff values ​​at 90% and 80% specificity, respectively. DETAILED DESCRIPTION

[0043] According to a first aspect of the present invention, there is provided use of a biomarker panel in a body fluid sample for diagnosing and / or monitoring cancer, wherein the biomarkers comprise at least one cell-free chromatin fraction and at least one cytokine molecule.

[0044] In the Examples provided herein, we have demonstrated that blood levels of several interleukins are increased in cancer patients compared to levels observed in people without cancer. We have demonstrated this for a variety of cancer diseases. We have also demonstrated that circulating levels of nucleosomes themselves are increased in cancer patients compared to levels observed in people without cancer. We have also demonstrated that circulating levels of nucleosomes containing specific histone isoforms are increased in cancer patients compared to levels observed in people without cancer. We have also demonstrated that measurement of interleukin molecules and nucleosome fractions as a combined biomarker panel is highly accurate for detecting cancer diseases, both in the proportion of cancer patients and in the breadth of cancer disease types that can be detected.

[0045] It will be clear to those skilled in the art that the methods of the present invention will detect all or most cancer types and all common cancer types tested. As shown in the Examples, the exemplified 2-assay panel and 3-assay panel embodiments detect most cancer cases and all or most types of cancer. It will also be clear to those skilled in the art that further nucleosome and / or cytokine assays can be added to these panels to further improve either or both the sensitivity of detection and the breadth of cancers detected. In other embodiments, other non-nucleosome or non-cytokine assays can be added to these panels to improve their performance. For example, but not limited to, CA19-9 can be added to improve the performance of pancreatic cancer, CEA can be added for CRC, AFP can be added for liver cancer, CA125 can be added for ovarian cancer, PSA can be added for prostate cancer, and the like.

[0046] We have shown that a combination of assays including cytokine and nucleosome assays yields improved results for distinguishing cancer patients from normal (healthy) subjects, both in terms of the accuracy of cancer detection and the diversity of cancer diseases detected. We have also shown that such assay panels are effective when the nucleosome assays are directed against nucleosomes themselves or against nucleosomes containing histone isoforms or variants. We now report the development of a non-invasive blood test to predict the presence of cancer in a subject.

[0047] The present invention comprises cell-free chromatin fragments as biomarkers. The chromatin fragments can be detected as circulating nucleosomes in a body fluid sample (e.g., a blood, serum, or plasma sample), i.e., they are cell-free nucleosomes. In one embodiment, the cell-free chromatin fragments are cell-free nucleosomes or components thereof. Thus, there is provided the use of at least one cytokine fraction and at least one nucleosome fraction as biomarkers in a body fluid sample for diagnosing and / or monitoring and / or assessing cancer.

[0048] As used herein, the term "chromatin fragment" refers to a complex of proteins and nucleic acids, the source of which is located in the chromosomes of the cell. A fragment of chromatin may contain any of a variety of non-histone chromatin-associated proteins in a nucleosome and / or associated DNA and / or polyprotein-nucleic acid complex. Some examples of non-histone chromatin-associated proteins (i.e., protein adducts) include transcription factors, cofactors, coactivators, co-repressors, RNA polymerase moieties, elongation factors, chromatin remodeling factors, mediators, STAT moieties, upstream binding factors (UBFs), and the like.

[0049] The nucleosome is the basic unit of chromatin structure and is composed of a protein complex of eight highly conserved core histones (including one pair of each of histones H2A, H2B, H3, and H4). Around this complex are wound approximately 146 base pairs of DNA. Another histone (H1 or H5) acts as a linker and participates in chromatin compaction. DNA is wrapped around successive nucleosomes in a structure often described as resembling "beads on a string," which forms the basic structure of open or euchromatin. In compacted or heterochromatin, this string is coiled and super-coiled into a closed and complex structure (Herranz and Esteller (2007)).

[0050] When detected in a bodily fluid sample, reference to "nucleosomes" may refer to "cell-free nucleosomes". It should be understood that the term "cell-free nucleosomes" as used throughout this document is intended to include any cell-free chromatin fragment containing one or more nucleosomes. The epigenetic signaling structures / characteristics of cell-free nucleosomes as referred to herein may include, but are not limited to, one or more histone post-translational modifications, histone isoforms / variants, modified nucleotides, and / or proteins that bind to nucleosomes as nucleosome-protein adducts.

[0051] As used herein, the term "component thereof" refers to a portion of a nucleosome, i.e., the entire nucleosome does not need to be detected. In one embodiment, the component of the cell-free nucleosome is selected from: a histone protein (i.e., histone H1, H2A, H2B, H3, or H4), a histone post-translational modification, a histone variant or isoform, a protein bound to a nucleosome (i.e., a nucleosome-protein adduct), a DNA fragment associated with a nucleosome, and / or a modified nucleotide associated with a nucleosome. For example, in one embodiment, a component thereof is histone (isoform) H3.1 or histone H1. Therefore, in one embodiment, the use includes nucleosomes containing histone H3.1 and / or nucleosomes containing histone H1 as biomarkers.

[0052] In one embodiment, cell-free nucleosomes are measured as a measure of the nucleosome itself. Mention of "nucleosome itself" refers to the total nucleosome level or concentration present in the sample, regardless of any epigenetic features that the nucleosome may include or not include. This type of determination is often also referred to as a total nucleosome assay, and generally involves detecting histones common to all nucleosomes, such as histone H4 or histone H3. Therefore, in one embodiment, nucleosomes themselves are measured by detecting core histones such as histone H3. As described herein, histones form structural units called nucleosomes, which are used to package DNA in eukaryotic cells. In one embodiment, the histone is a core histone, such as H2A, H2B, H3 or H4. As previously reported in WO 2016067029 (incorporated herein by reference), specific histone variants (such as histone H3.1, H3.2 or H3t) can be used to separate cell-free nucleosomes derived from tumor cells. Therefore, the level of cell-free nucleosomes of tumor origin can be detected.

[0053] Total cell-free nucleosomes or nucleosomes themselves can also be measured by quantifying their DNA fragment content. Circulating cell-free DNA (ccfDNA) in blood consists of DNA fragments less than 200 base pairs in length that circulate in the form of chromatin fragments, particularly nucleosomes. Blood measurements of ccfDNA using PicoGreen nucleic acid staining have been shown to have a 95% correlation with ELISA measurements of cell-free nucleosomes (Bjorkman et al., 2003). Therefore, ccfDNA measurements can be considered equivalent to or an alternative to measuring total nucleosome or total chromatin fragment levels. Typical methods for quantifying ccfDNA as an alternative to nucleosomes include, but are not limited to, the use of nucleic acid dyes (e.g., PicoGreen, SYBR Green, SYBER Gold, oxazole yellow, and thiazole orange) or quantification by polymerase chain reaction (PCR) or other methods for amplifying and measuring repetitive DNA sequences of single-copy gene sequences. Therefore, in one embodiment, cell-free chromatin fragments are measured (or quantified) by detecting ccfDNA. In another embodiment, ccfDNA is measured using nucleic acid dyes. In another embodiment, ccfDNA is measured by PCR. Furthermore, according to another aspect of the present invention, there is provided use of a biomarker panel in a body fluid sample for diagnosing and / or monitoring cancer, wherein the biomarkers include measurement of at least one cytokine molecule and ccfDNA.

[0054] Normal cell turnover in adults involves the production of approximately 10 11 The death of a similar number of cells is mainly through apoptosis. During apoptosis, chromatin is broken down into mononucleosomes and oligonucleosomes that are released from the cell. It is reported that under normal conditions, the level of circulating nucleosomes found in healthy subjects is low. Elevated levels have been found in subjects with a variety of conditions (including many cancers, autoimmune diseases, inflammatory conditions, stroke and myocardial infarction) (Holdenrieder and Stieber, 2009).

[0055] Mononucleosomes and oligonucleosomes can be detected by enzyme-linked immunosorbent assay (ELISA), and several methods have been reported (Salgame et al., 1997; Holdenrieder et al., 2001; van Nieuwenhuijze et al., 2003; WO2005019826; WO2013030577; WO2013030579; and WO2013084002, all of which are incorporated herein by reference). These assays typically use anti-histone antibodies (e.g., anti-H2B, anti-H3, or anti-H1, H2A, H2B, H3, and H4) as capture antibodies and detection antibodies (which vary depending on the moiety to be detected), or anti-histone antibodies as capture antibodies and anti-DNA antibodies as detection antibodies. In one embodiment, the anti-histone antibodies include anti-H3 antibodies or anti-H1 antibodies.

[0056] Circulating nucleosomes are not homogeneous groups of protein-nucleic acid complexes. On the contrary, they are heterogeneous groups of chromatin fragments derived from the digestion of chromatin when cells die, and include a wide variety of epigenetic structures, including specific histone isoforms (or variants), post-translational histone modifications, nucleotides or modified nucleotides, and protein adducts. It will be clear to those skilled in the art that the rise in nucleosome levels will be relevant to the rise in some circulating nucleosome subpopulations containing specific epigenetic signals, including nucleosomes containing specific histone isoforms (or variants), containing specific post-translational histone modifications, containing specific nucleotides or modified nucleotides and containing specific protein adducts. The determination of these types of chromatin fragments is known in the art (e.g., referring to WO2005019826, WO2013030579, WO2013030578, WO2013084002, which are incorporated herein by reference).

[0057] Thus, in an alternative embodiment, the cell-free nucleosomes contain epigenetic features. In another embodiment, the epigenetic features are selected from histone post-translational modifications, histone isoforms, modified nucleotides and / or proteins that bind to nucleosomes (i.e., as nucleosome-protein adducts). It should be understood that the terms "epigenetic signal structure" and "epigenetic features" are used interchangeably herein. They refer to specific features of nucleosomes that can be detected.

[0058] In one embodiment, the cell-free nucleosomes comprise histone isoforms. The nucleosome portion measured as part of a biomarker panel can be a circulating cell-free nucleosome containing one or more specific or specified histone isoforms. Many histone isoforms are known in the art. Nucleotide sequences of a large number of histone isoforms are publicly available, for example, in the National Human Genome Research Institute NHGRI Histone Database (Mariño-Ramírez, L., Levine, KM, Morales, M., Zhang, S., Moreland, RT, Baxevanis, AD and Landsman, D. The Histone Database: an integrated resource for histones and histone fold-containing proteins. Database Vol. 2011), GenBank (NIH gene sequence) database, EMBL nucleotide sequence database and the DNA database of Japan (DDBJ). In a preferred embodiment, the cell-free nucleosomes comprise histone isoforms of histone H3, for example, histone isoforms selected from H3.1, H3.2 and H3t.

[0059] In another embodiment, the cell-free nucleosome comprises one or more specific or specified post-translational histone modifications. The structure of the nucleosome can be changed according to the post-translational modification (PTM) of the histone. The PTM of the histone usually occurs in the tail of the core histone, and common modifications include acetylation, methylation or ubiquitination of lysine residues and methylation of arginine residues and phosphorylation of serine residues and many other modifications. Many histone modifications are known in the art, and with the identification of new modifications, the number is increasing (Zhao and Garcia, 2015).

[0060] In one embodiment, a group or class of related histone (post-translational) modifications (rather than a single modification) is detected. Typical examples of this embodiment involve, but are not limited to, a two-site immunoassay using an antibody or other selective binding agent that is directed to bind to nucleosomes and an antibody or other selective binding agent that is directed to bind to the group of histone modifications. Examples of such antibodies that are directed to bind to a group of histone modifications include (for illustrative purposes, but not limited to) anti-panacetylation antibodies (e.g., panacetyl H4 antibodies), anti-citrullination antibodies, or anti-ubiquitin antibodies.

[0061] In one embodiment, the cell-free nucleosomes include one or more DNA modifications (i.e., modified nucleotides). In addition to the epigenetic signals mediated by nucleosomal histone isoforms and histone post-translational modification compositions, nucleosomes are also different in their nucleotides and modified nucleotide compositions. Overall DNA hypomethylation is a hallmark of cancer cells, and some nucleosomes may include more 5-methylcytosine residues (or 5-hydroxymethylcytosine residues or other nucleotides or modified nucleotides) than other nucleosomes. For example, 5-hydroxymethylation can be detected in the CpG islands of the genome. In one embodiment, the DNA modification is selected from 5-methylcytosine or 5-hydroxymethylcytosine.

[0062] In another embodiment, the cell-free nucleosomes comprise protein adducts, i.e., nucleosomes and another non-histone protein adducted to the nucleosomes or chromatin fragments. Such adducts may include any protein containing or comprising a DNA binding domain or a nucleosome binding domain or a histone binding domain. Examples include transcription factors, structural chromatin proteins, CpG methyl-CpG binding domain proteins, high mobility group box proteins (e.g., HMGB1), epigenetic enzymes (e.g., histone acetyltransferases, histone methyltransferases, histone deacetylases, DNA methyltransferases), PARP (poly ADP ribose polymerase) binders, and many other proteins.

[0063] In one embodiment, the protein that binds to nucleosomes (and therefore can be used as a biomarker) is selected from the group consisting of: a transcription factor, a high mobility group protein, or a chromatin modifying enzyme. Reference to a "transcription factor" refers to a protein that binds to DNA and regulates gene expression by promoting (i.e., activators) or repressing (i.e., repressors) transcription. Transcription factors contain one or more DNA binding domains (DBDs) that attach to specific sequences of DNA adjacent to the genes they regulate. All circulating nucleosomes and nucleosome fractions, types, or subpopulations described herein can be used in the present invention.

[0064] In one embodiment, cytokine molecules are interleukin molecules. Interleukin (IL) is a group of cytokines, usually secreted by leukocytes, which serve as signaling molecules. They play a key role in stimulating immune response and inflammation. They were first identified in the 1970s, and as more interleukin types are found, they are indicated with numbers. The example of interleukin includes but is not limited to: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14 and IL-15.

[0065] In one embodiment, the interleukin molecule comprises interleukin-6. Interleukin-6 (IL-6) is a cytokine with a variety of biological functions. It is a potent inducer of fever and acute phase reactions. The sequence of human IL-6 is known in the art and is described under UniProt accession number P05231.

[0066] In one embodiment, the use includes interleukin-8 as a biomarker. Interleukin-8 (IL-8, also known as CXCL8) is a chemokine that attracts immune cells such as neutrophils, basophils, and T cells. It is released from several cell types in response to inflammatory stimuli. The sequence of human IL-8 is known in the art and is described by UniProt accession number P10145.

[0067] In one embodiment, the use includes interleukin-10 as a biomarker. Interleukin-10 (IL-10) is an anti-inflammatory cytokine with a variety of biological functions. The sequence of human IL-10 is known in the art and is described by UniProt accession number P22301.

[0068] In a preferred embodiment of the present invention, the biomarker comprises IL-6, IL-8, IL-10 or a combination thereof. In another embodiment, the at least one cytokine molecule is IL-6.

[0069] In one embodiment, the at least one cell-free chromatin fragment comprises histone isoform H3.1 and IL-6. In another embodiment, the biomarker comprises histone isoform H3.1, nucleosomes themselves (i.e., the total level of cell-free nucleosomes in the sample), and IL-6. In an alternative embodiment, the biomarker consists of histone isoform H3.1, IL-6, and optionally nucleosomes themselves. As described above, there are various methods for detecting the level of nucleosomes themselves, for example, by using reagents to detect core histone proteins (e.g., histone H3).

[0070] It will be clear to those skilled in the art that additional biomarkers (in addition to the nucleosome portion and the cytokine portion) can be used in a biomarker panel to detect cancer and / or identify organs affected by the disease. In one embodiment, the use further includes one or more biomarkers selected from the group consisting of ferritin, carcinoembryonic antigen (CEA), CYFRA 21-1 (cytokeratin 19 fragment), cancer antigen 125 (CA 125), carbohydrate antigen 19-9 (CA 19-9), carbohydrate antigen 15-3 (CA 15-3), alpha-fetoprotein (AFP), prolactin, human chorionic gonadotropin (HCG), prostate specific antigen (PSA), and C-reactive protein (CRP).

[0071] The sample can be any biological fluid (or body fluid) sample taken from the subject, including but not limited to cerebrospinal fluid (CSF), whole blood, serum, plasma, menstrual blood, endometrial fluid, urine, saliva or other body fluids (feces, tears, synovial fluid, sputum), breath (e.g., as concentrated breath), or extracts or purified products thereof, or diluents thereof. Biological samples also include samples from living subjects or collected posthumously. Samples can be prepared, for example, under appropriate dilution or concentration, and stored in a conventional manner. It should be understood that the methods and uses of the present invention are particularly useful for obtaining blood, serum or plasma samples from patients. In one embodiment, the sample is a blood or plasma sample. In another embodiment, the sample is a serum sample. In another embodiment, both serum and plasma samples are used to measure different members of the assay group.

[0072] In one embodiment, biomarkers are used to diagnose the stage of cancer. Cancer can be divided into Phase I, Phase II, Phase III, and Phase IV. The stage definition varies with different cancer diseases and is known in the art. Generally, when the cancer is small and locally confined to the tissue of origin, it is classified as Phase I. When the cancer grows larger and exceeds its origin into the nearby tissues in the organ or reaches nearby lymph nodes, it is classified as Phase II. When the cancer has grown into the nearby tissues beyond the organ of origin, but has not spread to other more distant parts of the body, it is classified as Phase III. When the cancer has spread to one or more distant parts of the body (such as the liver or lungs), it is classified as Phase IV.

[0073] In one embodiment, the cancer is a stage I (e.g., stage IA or stage IB), stage II (e.g., stage IIA or stage IIB), stage III (e.g., stage IIIA, stage IIIB, or stage IIIC), or stage IV (e.g., stage IVA or stage IVB) cancer. The present invention can be used to detect early-stage cancers, particularly stage I and stage II cancers. Thus, in one embodiment, the cancer is stage I, stage II, or stage III. In another embodiment, the cancer is stage I or stage II. In an alternative embodiment, the cancer is stage II or stage III. The present invention can also be used to detect late-stage cancers, particularly stage III and stage IV cancers. Thus, in one embodiment, the cancer is stage III or stage IV. In another embodiment, the cancer is stage IV.

[0074] In one embodiment, the cancer is selected from the group consisting of lung cancer, colon cancer, rectal cancer, stomach cancer, kidney cancer, skin cancer, prostate cancer, cervical cancer, breast cancer, pharyngeal cancer, laryngeal cancer, ovarian cancer, esophageal cancer, oral cancer, pancreatic cancer, and bladder cancer. In another embodiment, the cancer is selected from the group consisting of lung cancer, colorectal cancer, ovarian cancer, and prostate cancer, particularly lung cancer and colorectal cancer. In yet another embodiment, the cancer is lung cancer (e.g., non-small cell lung cancer or small cell lung cancer).

[0075] According to another aspect of the present invention, there is provided use of a cell-free chromatin fragment binder and a cytokine molecule binder in the manufacture of a kit for diagnosing and / or monitoring cancer in a body fluid sample.

[0076] Diagnostic methods

[0077] According to another aspect of the present invention, there is provided a method for diagnosing cancer in a patient, comprising:

[0078] detecting or measuring at least one cytokine molecule and at least one cell-free chromatin fraction in a bodily fluid sample obtained from the patient; and

[0079] The levels or concentrations of cytokine molecules and cell-free chromatin fragments are used to determine whether a patient has cancer.

[0080] In one embodiment, the cytokine molecule measured is an interleukin molecule. In a preferred embodiment, the cytokine molecule measured is any one or all of IL-6, IL-8 and IL-10, including combinations thereof. In another embodiment, the interleukin molecule is IL-6.

[0081] In another embodiment, the method of the present invention is implemented to identify subjects who are at high risk for developing cancer and therefore require further investigation (i.e., further cancer research), particularly to identify the organ location of the cancer. Further investigation may involve one or more endoscopic or scanning methods, including, for example, whole body scans, MRI scans, ultrasound scans, LDCT, mammography, computed tomography (CT) colonoscopy, or other scanning methods.

[0082] Therefore, according to another aspect of the present invention, there is provided a method for detecting cancer and studying organs affected by cancer, comprising:

[0083] detecting or measuring the levels of at least one cytokine molecule and at least one cell-free chromatin fragment in a bodily fluid sample obtained from the patient;

[0084] using measured cytokine and cell-free chromatin fragment levels as an indicator of the presence of cancer in vivo; and

[0085] The location of the cancer (or tumor) in the body is detected by endoscopy or scanning.

[0086] In addition to their use as stand-alone tests, cancer determination or exclusion blood tests can be used as adjunctive methods for other screening modalities, including, for example, in people who are LDCT positive, mammography positive, PSA positive, or FIT positive. All of these tests are nonspecific and therefore may be enhanced when used in conjunction with the methods described herein. LDCT positive patients have masses or nodules in their lungs, but the nodules may not be malignant, and LDCT has a specificity of approximately 60%. Similarly, mammography positive patients have masses or nodules in their breasts, but the nodules may not be malignant either. The specificity of the PSA test is also low, at approximately 60-70%. In the case of asymptomatic subjects screened by FIT, only approximately 5% of those found to be positive for fecal hemoglobin at the time of screening were actually found to have CRC at the time of subsequent colonoscopy. Therefore, many of the screening colonoscopies performed are hindsight and are unnecessary.

[0087] In addition to screening colonoscopy, a large number of monitoring or surveillance colonoscopies are performed on patients who have previously been diagnosed with CRC or precancerous adenomas (which may have been treated or surgically removed) to monitor any disease recurrence or progression. The FIT test can also be used for monitoring or surveillance to select subjects for colonoscopy. Colonoscopy is also performed to study patients who show symptoms consistent with possible CRC. Similarly, the FIT test can be used to select subjects for colonoscopy. In all these cases, most of the colonoscopies performed do not find cancer, and there are many harmful consequences for patients and health care providers, including: (i) a large number of unnecessary invasive medical colonoscopy procedures are performed on people without colonic lesions, (ii) a large amount of health care expenditure for expensive and unnecessary colonoscopies, (iii) due to historical underinvestment in colonoscopy infrastructure, the colonoscopy capacity of health care providers (particularly in European CRC screening programs) is currently insufficient to meet medical needs, resulting in an increase in the backlog of colonoscopies not performed and an increase in the colonoscopy waiting time for people with FIT positives, and (iv) this waiting time increase has resulted in a potential fatal delay in the start of CRC treatment for those patients who do suffer from CRC. By identifying that colorectal bleeding is most likely owing to those FIT positive people that cancer causes, to classify those FIT positive people that most need urgent referral to carry out colonoscopy, determine or exclude blood test and will overcome most in these problems.Similarly, the experimenter with potential cancerous nodule or other pathological changes of lung or mammary gland identified by LDCT or mammography can use determine or exclude blood test to test, to classify those patients that pathological change is most likely to be pernicious in nature.This will avoid unnecessary biopsy and potential danger repeatedly being exposed to X-ray radiation.The men with the PSA level of rising can use determine or exclude blood test to test, to classify those patients that are most likely to be pernicious in nature the reason that PSA level rises.Equally, this can avoid the men being in the prostate disease active monitoring to carry out some unnecessary repeated biopsies.Therefore, in one embodiment, the patient using method test of the present invention is FIT positive, LDCT positive, mammography positive or PSA positive.

[0088] We have shown that using circulating nucleosome levels and / or cytokine levels together with a numerical FIT score can be used to identify FIT-positive subjects who had no lesions found on colonoscopy (i.e., true negatives). Thus, the present invention can be used to determine whether a patient who tests positive for fecal hemoglobin does not have malignant colorectal lesions (i.e., the patient does not have cancer). Thus, the present invention can be used to assess a patient's suitability for colonoscopy.

[0089] According to another aspect of the present invention, there is provided a method for assessing a patient's suitability for colonoscopy, comprising:

[0090] (i) detecting or measuring the level of fecal hemoglobin in a stool sample obtained from a patient;

[0091] (ii) detecting or measuring the level of at least one cytokine molecule, optionally including the level of at least one cell-free chromatin fraction, in a bodily fluid sample obtained from the patient; and

[0092] (iii) using measured fecal hemoglobin and cytokine levels, optionally in combination with cell-free chromatin fragment levels, as an indication of the patient's suitability for colonoscopy.

[0093] Fecal hemoglobin tests are well known in the art. It will be appreciated that this aspect of the invention can be used in conjunction with patients who have a positive fecal hemoglobin test, i.e., for whom fecal hemoglobin levels have been measured. Thus, in one embodiment, step (i) can involve identifying a patient who has a positive fecal hemoglobin test. In one embodiment, a patient's fecal hemoglobin test is considered positive if the patient's fecal hemoglobin level is greater than about 20 μg hemoglobin / g stool (equivalent to 100 ng / ml in the diluted sample used in the OC-sensor FIT test).

[0094] In one embodiment, the cytokine molecule is selected from IL-6 and / or IL-8. In another embodiment, the cell-free chromatin fragment is histone isoform H3.1.

[0095] In one embodiment, the method further comprises measuring the CRP level (i.e., circulating level of CRP) in a body fluid sample obtained from the patient. In one embodiment, fecal hemoglobin levels and circulating CRP levels, and optionally also one or more nucleosome fractions and / or one or more interleukin levels, are used as an indicator of the absence of cancer in the body.

[0096] In one embodiment, the method further comprises measuring one or more tumor markers to investigate the organ location of the cancer. Such tumor markers include CEA (indicating CRC or lung cancer or pancreatic cancer), CYFRA 21-1 (indicating CRC), CA 125 (indicating ovarian cancer), CA 19-9 (indicating pancreatic cancer), CA 15-3 (indicating breast cancer), AFP (indicating liver cancer), prolactin (indicating pituitary tumor), HCG (indicating ovarian cancer), PSA (indicating prostate cancer).

[0097] Therefore, according to another aspect of the present invention, there is provided a method for detecting cancer and studying organs affected by cancer, comprising:

[0098] detecting or measuring the levels of at least one cytokine molecule and at least one cell-free chromatin fragment in a bodily fluid sample obtained from the patient;

[0099] using measured cytokine and cell-free chromatin fragment levels as an indicator of the presence of cancer in vivo; and

[0100] The levels of one or more tumor markers are detected or measured to determine the location of the cancer.

[0101] It will be understood that the levels of tumor markers need only be detected or measured once it has first been determined / indicated that a patient has cancer (ie, after testing the biomarker panel described herein).

[0102] In another embodiment, circulating tumor (ctDNA) or cell-free DNA (cfDNA) measurements are performed in addition to or instead of tumor marker measurements. Analysis of ctDNA or cfDNA can reveal the location of the tumor, for example, by methylated DNA sequencing or analysis, mutation sequencing or analysis, or nucleosome occupancy pattern sequencing or analysis. Thus, in one embodiment, the method comprises analyzing cfDNA or ctDNA associated with cell-free chromatin fragments to determine the location of the cancer.

[0103] Therefore, according to another aspect of the present invention, there is provided a method for detecting cancer and studying organs affected by cancer, comprising:

[0104] detecting or measuring the levels of at least one cytokine molecule and at least one cell-free chromatin fragment in a bodily fluid sample obtained from the patient;

[0105] using measured cytokine and cell-free chromatin fragment levels as an indicator of the presence of cancer in vivo; and

[0106] cfDNA or ctDNA in a sample of body fluid is analyzed to detect the location of cancer.

[0107] Furthermore, it should be understood that analysis of cfDNA or ctDNA is only necessary once it is first determined / indicated that a patient has cancer (ie, after testing the biomarker panel described herein).

[0108] In one embodiment, the method further comprises determining at least one clinical parameter of the patient. This parameter can be used in the interpretation of the results. The clinical parameter can include any relevant clinical information, such as, but not limited to, gender, weight, body mass index (BMI), smoking status, and dietary habits. Thus, in one embodiment, the clinical parameter is selected from: age, gender, and body mass index (BMI). In one embodiment, the method is only used for patients who are above an age-dependent cutoff value, such as patients who are above 50 years of age.

[0109] In one embodiment, higher levels of cytokine molecules and / or higher levels of cell-free chromatin fragments compared to a control indicate the presence and / or progression of cancer.

[0110] The data obtained by the methods of the present invention can be analyzed using appropriate algorithms, such as those listed in Table 1.

[0111] Table 1. Example Models or Algorithms for Interpreting Assay Panel Results

[0112] Group score = a[IL-6] + b[H3.1-nucleosome] Group score = a[IL-6] + b[nucleosome itself] Group score = a[IL-6] + b[nucleosome itself] + c[H3.1-nucleosome]

[0113] In one embodiment, the measuring step comprises using the algorithms listed in Table 1. Methods for obtaining models or algorithms such as those in Table 1 are well known in the art, and suitable software packages are available. Typical software tools used for this purpose include SPSS (Statistical Package for the Social Sciences) and "R". These software packages provide linear and nonlinear data modeling of clinical data.

[0114] Other methods of analyzing results can also be used for the methods of the present invention. In one embodiment, an artificial intelligence model is used. In one embodiment, a separate assay cutoff level is used, and if the separate group assay result is higher than (or lower than, if applicable) the assay cutoff level for all or the minimum number of group assays (e.g., one of two, two of two, two of three, etc.), the patient is considered to be positive in the group test. In one embodiment of the invention, a decision tree model or algorithm is used to analyze the results.

[0115] It will be clear to those skilled in the art that any combination of the biomarkers disclosed herein can be used in panels and algorithms for detecting cancer, and that additional markers can be added to panels containing these markers.

[0116] It will be clear to those skilled in the art that the sensitivity of the assay for patients with cancer or pre-cancer can be further improved by including nucleosome assays and cytokine assays as part of a larger (blood) panel of assays for cancer.

[0117] In a preferred embodiment, the panel detects the level or concentration of IL-6 and the level or concentration of nucleosomes containing the histone isoform H3.1.

[0118] According to another aspect of the present invention, there is provided a use of two or more binding agents in the manufacture of a kit for a method of diagnosing cancer in a bodily fluid sample, wherein one of the binding agents is specific for at least one cytokine molecule and the binding agent is specific for at least one cell-free chromatin fragment. The method comprises detecting or measuring the concentration or level of the cytokine molecules and the cell-free chromatin fragments in a bodily fluid sample obtained from a patient; and determining whether the patient has cancer using the levels or concentrations of the cytokine molecules and the cell-free chromatin fragments detected in the bodily fluid sample.

[0119] According to another aspect of the present invention, there is provided a use of two or more binding agents in the manufacture of a kit for a method for assessing a patient's suitability for cancer research, wherein one of the binding agents is specific for at least one cytokine molecule and the binding agent is specific for at least one cell-free chromatin fragment. The method comprises detecting or measuring the concentration or level of the cytokine molecule and the cell-free chromatin fragment in a bodily fluid sample obtained from the patient; and using the levels or concentrations of the cytokine molecule and the cell-free chromatin fragment detected in the bodily fluid sample to determine whether the patient requires further cancer research.

[0120] Differential diagnosis methods

[0121] Another advantage of the biomarkers of the present invention is that they can be used in differential diagnosis methods. Therefore, according to another aspect of the present invention, there is provided a method for differential diagnosis of a patient suspected of having cancer, comprising:

[0122] (i) detecting or measuring the level of at least one cytokine molecule and at least one cell-free chromatin fragment in a bodily fluid sample obtained from a patient; and

[0123] (ii) comparing the levels obtained in step (i) with the levels of at least one cytokine molecule and at least one cell-free chromatin fragment in a bodily fluid sample obtained from a patient with a non-cancerous disease,

[0124] wherein a difference in the levels compared in step (ii) indicates that the patient has cancer.

[0125] Reference to "non-cancerous diseases" refers to diseases that are non-cancerous, e.g., do not lead to the development of malignant tumors. They are sometimes referred to as "benign" diseases. The present invention has found particular use in differential diagnosis methods, where the suspected cancer and the non-cancerous disease being compared thereto are located in the same organ and / or have similar symptoms. For example, the differential diagnosis of suspected lung cancer compared to a non-cancerous lung disease. Non-cancerous lung diseases include, but are not limited to, asthma, bronchitis, chronic cough, chronic obstructive pulmonary disease (COPD), cryptococcosis, pneumonia, sarcoidosis, and tuberculosis. The present invention has particular use in diagnosing patients suspected of having lung cancer (e.g., due to symptoms and / or identification of a mass in the lungs) because the test distinguishes between patients with lung cancer and other non-cancerous lung diseases. Therefore, in one embodiment, a non-cancerous lung disease is a disease with signs and / or symptoms similar to lung cancer, such as COPD.

[0126] As shown in the Examples presented herein, the biomarker panels of the present invention are able to differentiate between lung cancer patients (small cell and non-small cell lung cancer) and COPD patients and healthy patients (see Figure 1 and 4 ).

[0127] As another example, a differential diagnosis can be made for a patient with suspected colorectal cancer compared to a noncancerous colon or intestinal disease. Noncancerous colon and intestinal diseases include, but are not limited to, polyps, Crohn's disease, colitis, inflammatory bowel disease, ulcerative colitis, and diverticular disease. The present invention has particular use in diagnosing a patient with suspected colorectal cancer (e.g., due to symptoms and / or identification of blood in the stool) because the test distinguishes between patients with CRC and other noncancerous diseases. Thus, in one embodiment, a noncancerous colon or intestinal disease is a disease with signs and / or symptoms similar to colorectal cancer, such as diverticular disease.

[0128] As shown in the Examples presented herein, the biomarker panels of the present invention are able to distinguish patients with colorectal cancer from patients with various non-cancerous colon and intestinal diseases and healthy patients (see Figure 2 and 3 For example, using a three-assay panel including measurements of nucleosomes themselves, nucleosomes containing histone isoform H3.1, and IL-6, with optimized models and cutoff values ​​to distinguish CRC patients from subjects with no findings on colonoscopy and patients with non-malignant benign colon or intestinal diseases, the method of the present invention was able to identify 50% of CRC cases with 90% specificity in all other patients (affected and nonaffected) with clear disease stage dependency, as shown in Figure 2. Figure 3 Thus, this embodiment of the invention can be used to examine humans with symptoms of colorectal disease to identify patients with CRC from those with other non-malignant conditions or who are disease-free.

[0129] As another example, a differential diagnosis can be made for a patient suspected of having ovarian cancer compared to a non-cancerous condition. A woman may have a pelvic mass of unknown etiology. This mass may be malignant, but may also be cystic or fibroid in nature due to a variety of other reasons. Non-cancerous conditions of the ovary include, but are not limited to, endometriosis, ovarian cysts, and polycystic ovary syndrome. The present invention can be used to diagnose a patient suspected of having ovarian cancer (e.g., due to symptoms) because the test can distinguish between patients with ovarian cancer and other non-cancerous conditions. Thus, in one embodiment, the non-cancerous condition is a pelvic mass or a non-cancerous condition of the ovary with signs and / or symptoms similar to those of ovarian cancer.

[0130] As another example, can be compared with non-cancerous prostate disease and carry out differential diagnosis to suspected prostate cancer patient.Prostatic non-cancerous disease includes but is not limited to: benign prostatic hyperplasia or prostatitis.The present invention can be used for diagnosing suspected prostate cancer patient (for example, due to symptom), because this test can distinguish the patient who suffers from prostate cancer and other non-cancerous diseases.Therefore, in one embodiment, non-cancerous prostate disease is the disease with the sign and / or symptom similar to prostate cancer.

[0131] Treatment

[0132] According to another aspect of the present invention, there is provided a method of treating cancer in a patient, comprising:

[0133] (i) detecting or measuring at least one cytokine molecule and at least one cell-free chromatin fraction in a body fluid sample obtained from a patient;

[0134] (ii) using the levels or concentrations of the cytokine molecules and the cell-free chromatin fragments detected in the body fluid sample to determine whether the patient has cancer; and

[0135] (iii) If it is determined in step (ii) that the patient has cancer, administering treatment to the patient.

[0136] In one embodiment, the method further comprises measuring the level of one or more tumor markers to detect the location of the cancer (eg, prior to step (iii)).

[0137] In one embodiment, the method further comprises an endoscopic procedure to detect the location of the cancer (e.g. prior to step (iii)).

[0138] In one embodiment, the method further comprises analyzing DNA associated with the cell-free chromatin fragments (e.g., prior to step (iii)). This embodiment may include analyzing the subject's circulating tumor DNA (ctDNA) or cell-free DNA (cfDNA) to detect the location of cancer (e.g., prior to step (iii)).

[0139] In this aspect of the invention, there are a number of alternative ctDNA or cfDNA analyses that can be used alone or in combination, including, for example, DNA sequence mutation analysis, methylated DNA sequence analysis (e.g., as previously described for the SEPTIN-9 gene), and nucleosome positioning or "fragmentomics" analysis as described by Snyder et al., 2016 (incorporated herein by reference).

[0140] In one embodiment, the method further comprises performing one or more scanning methods on the subject (eg, prior to step (iii)). Scanning methods can be used to detect the location of cancer.

[0141] In this aspect of the invention, there are many alternative scanning methods that can be used alone or in combination, including, for example, whole body scans, MRI scans, ultrasound scans, LDCT, mammography, computed tomography (CT) colonoscopy or other scanning methods.

[0142] Available cancer treatments include surgery (including biopsy), radiation therapy (including brachytherapy), hormone therapy, immunotherapy, and various drug treatments for chemotherapy. In one embodiment, the one or more treatments administered are selected from: surgery, radiation therapy, hormone therapy, immunotherapy, and / or chemotherapy.

[0143] According to another aspect of the present invention, a method for treating cancer is provided, comprising identifying a patient in need of treatment for cancer using a panel assay of the present invention, wherein the panel assay comprises reagents for detecting at least one cytokine molecule and at least one cell-free chromatin fragment in a bodily fluid sample obtained from the patient, and providing said treatment. In one embodiment, a patient is at high risk for cancer if the patient has elevated levels of the cytokine and / or cell-free chromatin fragment compared to a control.

[0144] In one embodiment, the control comprises a healthy subject, a disease-free subject, and / or a cancer-free subject. In one embodiment, the method comprises comparing the amount of a biomarker present in a body fluid sample obtained from a subject with the amount of a biomarker present in a body fluid sample obtained from a normal subject. It should be understood that a "normal" subject refers to a healthy / disease-free subject.

[0145] In one embodiment, the control includes a subject suffering from a non-cancerous disease. The method of the present invention can distinguish between a subject suffering from cancer and a subject suffering from a non-cancerous disease such as COPD (when compared with lung cancer) and colitis or diverticular disease (when compared with CRC), as described in the method of the differential diagnosis section provided herein. Therefore, in one aspect, diagnosis includes differential diagnosis of cancer from non-cancerous diseases.

[0146] Methods of patient assessment

[0147] The present invention has particular utility in assessing whether a patient needs further research for cancer (e.g., for diagnosis and / or identification of organ location). These procedures, including colonoscopy, other endoscopic methods, mammography, X-rays, LDCT scans, other scans, and biopsies are invasive or potentially dangerous and relatively expensive for healthcare providers. Therefore, it is desirable to reduce the number of patients sent for unnecessary research. For example, this aspect of the present invention can be used to assess people who are FIT or LDCT positive and need colonoscopy or biopsy. Therefore, according to another aspect of the present invention, a method for assessing the suitability of a patient for cancer research (i.e., determining whether a patient needs further cancer research trials) is provided, comprising:

[0148] detecting or measuring at least one cytokine molecule and at least one cell-free chromatin fraction in a bodily fluid sample obtained from the patient; and

[0149] The levels or concentrations of the measured cytokine molecules and cell-free chromatin fragments are used to determine whether the patient requires further cancer research.

[0150] As shown in the examples provided herein, the present invention has application as a CRC blood test for detecting CRC in asymptomatic subjects, in patients who are non-compliant with FIT, or in addition to FIT to obtain a lower combined false positive rate. A positive result in the test of the present invention, like FIT, indicates the need for referral for colonoscopy.

[0151] According to another aspect of the present invention, a method for identifying a patient requiring a colonoscopy is provided, the method comprising applying a body fluid sample obtained from the patient to a panel test as defined herein, and using the results obtained from the panel test to identify whether the patient requires a colonoscopy.

[0152] According to another aspect of the present invention, there is provided a method of identifying a patient requiring LDCT, mammography or other scan, the method comprising applying a body fluid sample obtained from the patient to a panel of tests as defined herein, and using the results obtained from the panel of tests to identify whether the patient requires a scan.

[0153] In one embodiment, the methods described herein are repeated at multiple times. This embodiment provides the advantage of allowing the test results to be monitored over a period of time. Such an arrangement will provide the benefit of monitoring or assessing the therapeutic efficacy of a disease state. This monitoring method of the present invention can be used to monitor onset, progression, stabilization, improvement, relapse, and / or remission.

[0154] Therefore, the present invention also provides a method for monitoring the therapeutic efficacy of the morbid state of a subject suspected of having this disease, including detecting and / or quantifying the biomarkers (e.g., biomarker panels as described herein) present in the biological sample from the subject. In monitoring methods, test specimens can be collected at two or more opportunities. The method may further include comparing the level of the biomarker present in the test specimen with one or more controls and / or with one or more previous test specimens previously taken from the same test subject (e.g., before therapy begins) and / or taken from the same test subject in the early stages of therapy. The method may include detecting the property of the biomarker in the test specimen obtained at different opportunities or the change in amount.

[0155] Thus, according to another aspect of the present invention, there is provided a method for monitoring the efficacy of a therapy for a disease state in a human or animal subject, comprising:

[0156] (a) quantifying a panel of biomarkers as defined herein; and

[0157] (b) comparing the group results in the test sample with the group results in one or more controls and / or one or more previous test samples taken at an earlier time from the same test subject.

[0158] A change in the biomarker results in a test sample relative to the levels in a previous test sample taken earlier from the same test subject can indicate a beneficial effect of the therapy on the condition or suspected condition, such as stabilization or improvement. In addition, once treatment is completed, the methods of the present invention can be repeated periodically to monitor for recurrence of the disease.

[0159] Methods for monitoring therapeutic efficacy can be used to monitor the therapeutic effectiveness of existing and new therapies in human subjects and non-human animals (e.g., animal models). These monitoring methods can be incorporated into the screening of new drug substances and combinations of substances.

[0160] In another embodiment, monitoring for more rapid changes due to fast-acting therapies can be performed over shorter time intervals of hours or days.

[0161] Group test

[0162] The combinations of markers described herein can be used to prepare panels of tests, in particular for diagnosing cancer and / or monitoring patients with cancer or suspected cancer.

[0163] Thus, according to another aspect of the present invention, a panel assay comprising reagents for detecting at least one cytokine molecule and at least one cell-free chromatin fraction is provided. The panel assay described herein can be used to diagnose cancer, such as lung cancer, colorectal cancer, ovarian cancer, and / or prostate cancer.

[0164] In one embodiment, the at least one cytokine molecule is selected from IL-6, IL-8, and / or IL-10. In one embodiment, the at least one cell-free chromatin fragment is selected from cell-free nucleosomes and histone isoform H3.1. Thus, according to another aspect of the present invention, a panel is provided comprising reagents for detecting IL-6, histone H3.1, and optionally one or more biomarkers selected from IL-8, IL-10, nucleosomes or components thereof, and epigenetic signatures of nucleosomes.

[0165] According to another aspect of the present invention, a panel is provided comprising reagents for detecting IL-6, total nucleosome levels and optionally one or more biomarkers selected from the group consisting of IL-8, IL-10 and epigenetic signatures of nucleosomes (e.g., histone H3.1).

[0166] In a preferred embodiment, the panel assay comprises (especially optionally) reagents for detecting IL-6 and histone H3.1. In other embodiments, the panel may comprise reagents for detecting total nucleosome levels and / or H1-nucleosome levels and / or may comprise other nucleosome measurements. Thus, in another embodiment, the panel assay comprises (especially optionally) reagents for detecting the (total) level of IL-6, histone H3.1, and cell-free nucleosomes in a sample. In an alternative embodiment, the panel assay comprises reagents for detecting the (total) level of IL-6 and cell-free nucleosomes (i.e., nucleosomes themselves) in a sample.

[0167] In one embodiment, the panel assay further comprises reagents for detecting one or more biomarkers selected from the group consisting of: IL-8, IL-10, nucleosomes or components thereof, and epigenetic signatures of nucleosomes. In one embodiment, the panel assay is used on a bodily fluid sample obtained from a patient.

[0168] As shown in the Examples provided herein, a panel assay comprising the measurement of nucleosomes containing histone isoform H3.1 and IL-6 was able to detect 77% of lung cancer cases from normal donors, as shown in Table 2, and also detected lung cancer cases from those patients with COPD, as shown in Table 2. Figure 1 As shown in . Therefore, these panel assays have high sensitivity and specificity and are used as lung cancer tests in high-risk groups (e.g., long-term heavy smokers), or in patients who are not compliant with low-dose computed tomography (LDCT), or in place of repeated LDCT scans for monitoring patients with nodules of unknown etiology to avoid repeated dangerous X-ray exposure, or as adjunctive tests to LDCT to help investigate false-positive results that result from LDCT screening due to low specificity. Using a panel that includes measuring nucleosomes themselves, nucleosomes containing the histone isoform H3.1, and IL-6, it was possible to identify 80% of CRC cases with a specificity of 89%, as Figure 2 This accuracy is comparable to that of the FIT CRC screening test, which has a sensitivity of approximately 72% and a specificity of 95%, and can be applied to detect CRC.

[0169] In one embodiment, the panel test further comprises a reagent for measuring fecal hemoglobin levels. As described in the Examples, the present invention can be used in conjunction with fecal hemoglobin levels to increase the specificity of the FIT test.

[0170] In one embodiment, the panel assay comprises (particularly optionally) reagents for detecting IL-10 and histone H3.1. In other embodiments, the panel may comprise reagents for detecting total nucleosome levels and / or H1-nucleosome levels and / or may include other nucleosome measurements. Thus, in an alternative embodiment, the panel assay comprises reagents for detecting (total) levels of IL-10 and cell-free nucleosomes (i.e., nucleosomes themselves) in a sample.

[0171] According to another aspect of the present invention there is provided the use of a panel assay as defined herein for identifying a patient in need of cancer treatment.

[0172] According to another aspect of the present invention, there is provided the use of a panel test as defined herein for monitoring cancer progression (e.g., further growth of a tumor or progression to a different stage of cancer) in a patient. Embodiments of this aspect include the use of a panel test as defined herein for detecting disease progression in watchful waiting, active monitoring, and monitoring for recurrence after surgery or other treatment.

[0173] According to another aspect of the present invention there is provided the use of a panel assay as defined herein for evaluating the effectiveness of a cancer treatment in a patient.

[0174] According to another aspect of the present invention there is provided the use of a panel assay as defined herein for selecting a treatment for a cancer patient.

[0175] In other embodiments, the panel may include reagents for detecting total nucleosome levels and / or H1-nucleosome levels and / or may include other nucleosome measurements, such as epigenetic features of nucleosomes (e.g., histone H3.1 levels). Some additional embodiments are listed in Table 1 as example algorithms, but are not limited thereto.

[0176] Measurement method

[0177] In one embodiment, the levels or concentrations of the detected cytokine molecules and cell-free chromatin fragments are compared to a control. It will be clear to those skilled in the art that a control subject can be selected on a variety of bases, which may include, for example, a subject known to be free of disease or a subject with a different disease (e.g., for use in differential diagnostic studies). A "control" may include healthy subjects, disease-free subjects, and / or cancer-free subjects. A control may also be a subject at a different stage of cancer, such as stage I, II, III, or IV cancer. Comparisons to controls are well known in the diagnostic arts.

[0178] It should be understood that it is not necessary to measure healthy / disease-free controls for comparison purposes at every opportunity, because once a "normal range" is established, it can be used as a benchmark for all subsequent tests. A normal range can be established by obtaining samples from multiple control subjects who do not have cancer and testing the levels of biomarkers. The results (i.e., biomarker levels) of subjects suspected of having cancer can then be checked to see if they fall within or outside their respective normal ranges. Using a "normal range" is standard practice for detecting disease.

[0179] If the subject is determined not to have cancer, the present invention can still be used for the purpose of monitoring disease progression. For example, if the use includes a blood, serum, or plasma sample from a subject determined not to have cancer, the biomarker level measurement can be repeated at another time point to determine whether the biomarker level has changed.

[0180] References to "subject" or "patient" are used interchangeably herein. In one embodiment, the patient is a human patient. In one embodiment, the patient is a (non-human) animal. The uses, compositions and methods described herein can be performed in vitro, in vivo or ex vivo.

[0181] In one embodiment, the detection or measurement of the cytokine and the cell-free chromatin fraction comprises an immunoassay, an immunochemical method, a mass spectrometry method, a chromatography method, a chromatin immunoprecipitation method, or a biosensor method.

[0182] In one embodiment, the detection or measurement comprises an immunoassay. In a preferred embodiment of the invention, a 2-site immunoassay of a cytokine and / or nucleosome portion is provided. In particular, this method is preferably used for in situ measurement of nucleosomes or nucleosomes incorporating epigenetic features, using a combination of two anti-nucleosome binders or anti-nucleosome binders with anti-histone modification or anti-histone variant or anti-DNA modification or anti-adduct protein detection binding agents. In another embodiment of the invention, a 2-site immunoassay using a labeled anti-nucleosome detection binding agent in combination with a fixed anti-histone modification or anti-histone variant or anti-DNA modification or anti-adduct protein binding agent is provided.

[0183] Detecting or measuring the level of a biomarker can be performed using one or more reagents (e.g., suitable binding agents). In one embodiment, the one or more binding agents comprise a ligand or binding agent specific for a desired biomarker (e.g., IL-8, IL-6, IL-10, a nucleosome or a component thereof, an epigenetic signature of a nucleosome, or a structure / shape mimetic of a nucleosome or a component thereof). The term "biomarker" as defined herein includes any single biomarker moiety or combination of separate biomarker moieties in a biomarker panel.

[0184] It will be clear to those skilled in the art that the terms "antibody," "binding agent," or "ligand" with respect to any aspect of the present invention are not limiting, but are intended to include any binding agent capable of binding to a specific molecule or entity, and any suitable binding agent may be used in the methods of the present invention. It will also be clear that the term "nucleosome" is intended to include mononucleosomes and oligonucleosomes, as well as any protein-DNA chromatin fragment that can be analyzed in a fluid medium.

[0185] Methods for detecting biomarkers are known in the art. In one embodiment, the reagent comprises one or more ligands or binding agents. In one embodiment, the ligands or binding agents of the present invention comprise naturally occurring or chemically synthesized compounds that are capable of specifically binding to the desired target. The ligand or binding agent may comprise a peptide, antibody, or fragment thereof, or a synthetic ligand such as a plastic antibody, or an aptamer or oligonucleotide that is capable of specifically binding to the desired target. The antibody may be a monoclonal antibody or a fragment thereof. It will be understood that if an antibody fragment is used, it retains the ability to bind to the biomarker so that the biomarker can be detected (according to the present invention). The ligand / binding agent may be labeled with a detectable marker, such as a luminescent marker, a fluorescent marker, an enzyme marker, or a radioactive marker; alternatively or additionally, the ligand according to the present invention may be labeled with an affinity tag, such as biotin, avidin, streptavidin, or a His (e.g., hexa-His) tag. Alternatively, ligand binding may be determined using a label-free technique, such as the technology of ForteBio Inc.

[0186] Diagnostic or monitoring kits (or sets) are provided for performing the methods of the invention. Such kits will suitably comprise one or more ligands for detecting and / or quantifying biomarkers according to the invention, and / or biosensors, and / or arrays as described herein, optionally together with instructions for use of the kit.

[0187] Another aspect of the present invention is a kit for detecting the presence of a disease state, comprising a biosensor capable of detecting and / or quantifying one or more biomarkers as defined herein. As used herein, the term "biosensor" refers to any substance capable of detecting the presence of a biomarker. Examples of biosensors are described herein. The biosensor can include a ligand binding agent or ligand as described herein that can specifically bind to a biomarker. Such a biosensor can be used to detect and / or quantify the biomarkers of the present invention.

[0188] Suitably, biosensors for detecting one or more biomarkers of the present invention combine biomolecular recognition with suitable means to detect or quantify the presence of a biomarker in a sample and convert it into a signal. Biosensors can be used in "point-of-care" diagnostic assays, such as in hospital rooms, clinics, surgical procedures, at home, in the field, and in the workplace. Biosensors for detecting one or more biomarkers of the present invention include acoustic sensors, plasmon resonance sensors, holographic sensors, biolayer interferometry (BLI) sensors, and microengineered sensors. Imprinted recognition elements, thin film transistor technology, magnetoacoustic resonator devices, and other novel acoustoelectric systems can be used in biosensors to detect one or more biomarkers of the present invention.

[0189] Biomarkers used to detect the presence of disease are essential targets for discovering new targets and drug molecules that slow or halt disease progression. Because the results of a biomarker or biomarker panel are indicative of disease and drug response, biomarkers can be used to identify new therapeutic compounds in in vitro and / or in vivo assays. The biomarkers and biomarker panels of the present invention can be used in methods to screen for compounds that modulate the activity of a biomarker.

[0190] Therefore, in another aspect of the present invention, there is provided the use of a binding agent or ligand as described, which may be a peptide, antibody or fragment thereof or aptamer or oligonucleotide directed against a biomarker according to the present invention; or the use of a biosensor or array or kit according to the present invention for identifying substances that can promote and / or inhibit the production of a biomarker.

[0191] The term "biomarker" refers to a unique biological or biologically derived indicator of a process, event, or condition. Biomarkers can be used in diagnostic (e.g., clinical screening) and prognostic assessment methods and can be used to monitor therapy outcomes, can be used to identify subjects most likely to respond to a specific therapeutic treatment, and can be used in drug screening and development. Biomarkers and their uses are valuable for identifying new drug therapies and discovering new targets for drug therapies.

[0192] As used herein, the terms "detection" or "diagnosis" encompass the identification, confirmation, and / or characterization of a disease state. Detection, monitoring, and diagnostic methods according to the present invention can be used to confirm the presence of a disease, monitor the development of a disease by assessing the onset and progression of the disease, or assess improvement or regression of the disease. Detection, monitoring, and diagnostic methods can also be used to assess clinical screening, prognosis, therapy selection, and methods for evaluating therapeutic benefit (i.e., for drug screening and drug development).

[0193] Identification and / or quantification can be carried out by any method suitable for identifying the presence and / or amount of a specific protein in a biological sample or a purified product or extract of a biological sample or its dilution from a subject. In the method of the present invention, quantification can be carried out by measuring the concentration of the target in one or more samples. The biological samples that can be tested in the method of the present invention include those as defined above. Samples can be prepared, for example, under suitable dilution or concentration, and stored in a conventional manner.

[0194] Identification and / or quantification of biomarkers can be performed by detecting biomarkers or fragments thereof (e.g., fragments with C-terminal truncations or N-terminal truncations). Fragments are suitably greater than 4 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. Of particular note, peptides identical to or related to histone tail sequences are particularly useful histone fragments.

[0195] For example, detection and / or quantification can be performed using immunological methods such as immunoassays. Immunoassays include any method using one or more antibodies or other specific binding agents that are directed to bind to the biomarkers defined herein. Immunoassays include 2-site immunoassays or immunometric assays (e.g., ELISAs) using enzyme detection methods, fluorescently labeled immunoassays, time-resolved fluorescently labeled immunoassays, chemiluminescent immunoassays, immunoturbidimetric assays, microparticle-labeled immunoassays, and immunoradiometric assays, as well as single-site immunoassays, reagent-limited immunoassays, competitive immunoassays (comprising labeled antigens and labeled antibodies), and single antibody immunoassays with various labeling types (including radioactivity, enzymes, fluorescence, time-resolved fluorescence, and microparticle labels). All of the immunoassays are well known in the art, including for cytokines and for nucleosomes.

[0196] In another example, detection and / or quantification can be carried out by one or more methods selected from: SELDI (-TOF), MALDI (-TOF), analysis based on 1-D gel, analysis based on 2-D gel, mass spectrometry (MS), reversed phase (RP) LC, size permeation (gel filtration), ion exchange, affinity, HPLC, UPLC and other technology based on LC or LC-MS. Suitable LC MS technology includes ICAT (Applied Biosystems, CA, USA) or iTRAQ (Applied Biosystems, CA, USA). Liquid chromatography (such as high pressure liquid chromatography (HPLC) or low pressure liquid chromatography (LPLC)), thin layer chromatography, NMR (nuclear magnetic resonance) spectroscopy can also be used.

[0197] Methods involving identification and / or quantification of one or more biomarkers of the present invention can be performed on a benchtop instrument or can be incorporated into a disposable, diagnostic or monitoring platform that can be used in a non-laboratory setting, such as in a physician's office or at a subject's bedside. Suitable biosensors for performing the methods of the present invention include "credit" cards with optical or acoustic readers. Biosensors can be configured to allow the collected data to be electronically transmitted to a physician for interpretation and thus can form the basis of electronic medicine.

[0198] The identification of biomarkers for disease states allows the integration of diagnostic procedures and treatment regimens. The detection of the biomarkers of the present invention can be used to screen subjects before they participate in clinical trials. Biomarkers provide a means of indicating treatment response, response failure, adverse side effect profile, drug compliance level, and reaching sufficient serum drug levels. Biomarkers can be used to provide warnings of adverse drug reactions. Biomarkers can be used to develop personalized therapies because the assessment of the response can be used to fine-tune the dosage, minimize the number of prescribed drugs, reduce delays in obtaining effective therapies, and avoid adverse drug reactions. Therefore, by monitoring the biomarkers of the present invention, subject care can be accurately customized to match the needs determined by the subject's condition and drug genome map, so biomarkers can be used to titrate optimal doses, predict positive treatment responses, and identify those subjects at high risk of serious side effects.

[0199] Biomarker-based tests provide a first-line assessment of “new” subjects and offer an objective measure of accurate and rapid diagnosis, which is not achievable using current measures.

[0200] Biomarker monitoring methods, biosensors, and kits are also crucial as subject monitoring tools, enabling physicians to determine whether a relapse is due to a worsening of the condition. If pharmacological treatment is assessed as inadequate, therapy can be resumed or increased; if appropriate, therapy can be changed. Because biomarkers are sensitive to the condition's state, they provide an indication of the impact of drug therapy.

[0201] It will be appreciated that the embodiments described herein are applicable to all aspects of the invention, ie embodiments described with respect to uses are equally applicable to the claimed methods etc.

[0202] The invention will now be illustrated with reference to the following non-limiting examples. Example

[0203] Example 1

[0204] Blood (plasma) samples were obtained from 144 individuals, including 47 patients with lung cancer (small cell lung cancer and non-small cell lung cancer), 43 age-matched normal donors, and 54 patients with chronic obstructive pulmonary disease (COPD). Nucleosomes containing histone isoform H3.1 and IL-10 were measured by ELISA. Briefly, nucleosomes containing histone isoform H3.1 were measured as follows: 80 µl of assay buffer and 20 µl of plasma sample or standard nucleosome preparation were added to microtiter wells coated with an antibody specifically binding to histone H3.1. The microtiter plate was covered and incubated at room temperature with gentle shaking for 2.5 hours. The contents of the microtiter wells were discarded. The wells were washed three times with 200 µl of wash solution, and 100 µl of biotinylated anti-nucleosome antibody was added. The microtiter plate was covered again and incubated at room temperature with gentle shaking for 1.5 hours. The contents of the microtiter wells were discarded. Wash the wells three times with 200 µl of wash solution and add 100 µl of streptavidin-HRP solution. Cover the microtiter plate again and incubate at room temperature with gentle shaking for 0.5 hour. Discard the contents of the microtiter wells. Wash the wells three times with 200 µl of wash solution and add 100 µl of HRP (horseradish peroxidase) substrate solution. Cover the microtiter plate and incubate at room temperature in the dark with gentle shaking for 20 minutes. Measure the absorbance (OD) of the wells at 405 nm. Use the OD level directly or interpolate the plasma levels of nucleosomes containing histone H3.1 from a standard curve. Plasma IL-10 levels were measured using a commercially available ELISA method.

[0205] We modeled the assay results using logistic regression analysis to train a model or algorithm with the highest AUC for comparing lung cancer patients with normal donors. The results showed that IL-10 results could be combined with results for nucleosomes containing histone H3.1 to form an effective assay panel with an associated algorithm for cancer detection. This algorithm was able to distinguish 68% of lung cancer patients from normal donors with 93% specificity, as shown in Table 2.

[0206] Example 2

[0207] Plasma samples from the same 144 individuals as described in Example 1 were assayed for nucleosomes containing the histone isoform H3.1 as described in Example 1, and for IL-6 and IL-8 using a commercially available ELISA method. The assay results were modeled by logistic regression analysis to train a model or algorithm with the highest AUC for comparing lung cancer patients with normal donors. The results showed that the IL-6 results could be combined with the results for nucleosomes containing histone H3.1 as an effective set of assays with an associated algorithm for lung cancer detection. The algorithm was able to detect 77% of lung cancer cases from normal donors with a specificity of 90%, as shown in Table 2, and was also able to detect lung cancer cases from those with COPD, as shown in Table 2. Figure 1 shown.

[0208] Table 2. Accuracy of individual biomarkers and biomarker groups for lung cancer detection (relative to lung cancer patients) normal donors)

[0209]

[0210] Example 3

[0211] Blood (plasma) samples were obtained from 100 people, including 20 patients with colorectal cancer (CRC), 62 patients with various non-malignant colon diseases (including colitis, Crohn's disease and diverticular disease) and 18 patients with gastrointestinal symptoms but no findings on colonoscopy. We used commercially available ELISA methods to measure nucleosomes containing the histone isoform H3.1 as described in Example 1 and IL-6, IL-8 and IL-10. We also used a similar method as described above for nucleosomes containing histone H3.1, but used anti-H3 antibodies coated on microtiter wells to measure the nucleosomes themselves. We modeled the assay results by logistic regression analysis to train a model or algorithm with the highest AUC for comparing CRC patients with patients with no findings on colonoscopy. The results showed that IL-6 results can be combined with results for nucleosomes themselves and nucleosomes containing histone H3.1 as an effective assay group with a related algorithm for cancer detection. As shown in Tables 3 and Figure 2 As shown, the algorithm was able to detect 80% of CRC cancer cases from patients without findings with 89% specificity, with clear disease stage dependence.

[0212] Table 3. Individual and panel biomarker results for accuracy of CRC detection (CRC vs. Symptomatic subjects with no findings on colonoscopy)

[0213]

[0214] Example 4

[0215] We used the same three-assay panel containing three measurements of nucleosomes themselves, nucleosomes containing histone isoform H3.1, and IL-6 as described in Example 3 on the same 100 patients as described in Example 3, but we modeled the assay results by logistic regression analysis to train a model or algorithm with the highest AUC for comparing CRC patients with patients who had no findings on colonoscopy and patients with non-malignant benign colon or intestinal diseases. Figure 3 As shown, the method of the present invention is able to identify 50% of CRC cases with 90% specificity among all other patients (affected and non-affected) with a clear disease stage dependency. The embodiment of the present invention described in Example 4 herein can be used to examine humans with symptoms of colorectal disease to identify CRC patients from those with other non-malignant conditions or without disease.

[0216] The results of Examples 3 and 4 show that the accuracy of the sensitivity and specificity of the methods of the present invention can be tailored to maximize the sensitivity of the test for asymptomatic subjects, or to maximize the specificity of the test for symptomatic patients, to avoid false positive cancer diagnoses in patients with benign diseases by alternative model training techniques. This convenience allows the method to be tailored for symptomatic or asymptomatic patients or other applications.

[0217] Example 5

[0218] We hypothesized that a panel blood test that includes levels of both cytokine interleukin molecules and nucleosome fractions could be used as a marker not only for individual lung cancer or colorectal cancer, but also for cancer in general. To test this hypothesis, we measured nucleosomes containing histone isoform H3.1 and IL-6 by ELISA as described above in the 244 people described in Examples 1 and 3 (combination of a 144-person group for lung cancer and a 100-person group for CRC). We then modeled the assay results using the combined data from the two groups by logistic regression analysis to train a model or algorithm with the highest AUC for comparing lung cancer or CRC patients with patients who had no findings on colonoscopy and normal donors. This training produced a relevant regression model that gave a combined accuracy for detecting lung cancer or colorectal cancer of 75% sensitivity at a specificity of 90% or 81% sensitivity at a specificity of 80%.

[0219] We also tested the efficacy of this method of the present invention in detecting CRC and lung cancer separately in each of two separate CRC and lung cancer (training) cohorts. In the lung cancer cohort, the result for detecting lung cancer alone was 77% sensitivity at 90% specificity, or 83% sensitivity at 80% specificity (see Table 2). In the CRC cohort, the result for detecting CRC alone was 45% sensitivity at 90% specificity, or 80% sensitivity at 80% specificity (see Table 3). Therefore, the method of the present invention trained on CRC and lung cancer in combination is as effective as the method trained on each disease separately for detecting either disease separately.

[0220] Example 6

[0221] To further test the hypothesis that a panel blood test that includes levels of both cytokine interleukin molecules and nucleosome fractions can be used generally as a blood test for detecting cancer, we applied the combined model developed in the two training cohorts to two additional validation patient cohorts collected in a different country independently of the training cohorts. First, we measured nucleosomes containing histone isoform H3.1 and IL-6 by ELISA in another independently collected 70-person lung cancer validation cohort (including 30 lung cancer patients (small cell and non-small cell lung cancer), 30 normal donor subjects, and 10 COPD patients). We then calculated the algorithm score for these 70 people using the algorithm developed for the combined 244-person lung cancer and CRC training cohorts in Example 5. As Figure 4 As shown, algorithmic scoring of 70 individuals demonstrated that the two-assay combination panel was able to detect 93% of lung cancers in normal donors with 93% specificity and 100% of lung cancers in the validation cohort with 80% specificity. Surprisingly, the accuracy of the method of the present invention observed in the validation cohort was higher than that observed in the training set. This may be related to the cancer stage and the mix of patients with small cell and non-small cell disease in both cohorts. These results demonstrate the effectiveness and utility of the method of the present invention.

[0222] Example 7

[0223] We measured nucleosomes containing histone isoform H3.1 and IL-6 by ELISA in an independently collected 63-person multi-cancer validation cohort, including 30 patients with various cancers (including lung, colon, rectal, gastric, renal, prostate, breast, pharyngeal, laryngeal, ovarian, esophageal, and bladder cancers) and 33 normal donor subjects. This was done to determine whether the two-assay combination model could detect a variety of different cancer diseases in addition to CRC and lung cancer. We then used the algorithm developed in Example 5 for the combined lung cancer and CRC training cohorts of 244 people to calculate the algorithm scores for these 63 people. The algorithm scores for the 63 people showed that the two-assay combination panel was able to detect 47% of cancers with 90% specificity, including renal cancer (1 of 2 cases or 1 / 2), laryngeal cancer (2 / 3), lung cancer (1 / 5), esophageal cancer (1 / 1), ovarian cancer (6 / 8), prostate cancer (2 / 4), and rectal cancer (1 / 2). At 80% specificity, the 2-assay combination panel was able to detect 67% of cancers overall and every cancer type tested except gastric cancer, including bladder cancer (1 / 1), breast cancer (1 / 1), kidney cancer (1 / 2), laryngeal cancer (2 / 3), lung cancer (2 / 5), esophageal cancer (1 / 1), ovarian cancer (7 / 8), pharyngeal cancer (1 / 2), prostate cancer (3 / 4), rectal cancer (1 / 2), and gastric cancer (0 / 1). Only one gastric cancer patient was included in the cohort, so it is expected that inclusion of additional gastric cancer patients will result in an increase in detection rate. The results are shown in Figure 5 and demonstrates the breadth of utility of the methods of the invention for detecting multiple cancer types, with utility as a test for cancer itself.

[0224] Example 8

[0225] We measured nucleosomes themselves and IL-6 in a 100-person CRC cohort described in Example 3, which included 20 CRC patients, 62 patients with a variety of non-malignant colon diseases (including colitis, Crohn's disease, and diverticular disease), and 18 patients with gastrointestinal symptoms but no colonoscopy findings. We modeled the assay results by logistic regression analysis to train a model or algorithm with the highest AUC for comparing CRC patients with patients with no colonoscopy findings. The method of the present invention was able to identify 45% of CRC cases in patients with no colonoscopy findings with a specificity of 90%. This embodiment of the present invention is useful for detecting people with CRC from people without the disease.

[0226] In a manner similar to the experiments described in Example 7, the model was then applied to a validation cohort that included patients with a variety of cancer diseases. The 2-assay panel, which included IL-6 and the nucleosome itself, was able to detect 60% of cancers with 90% specificity, including kidney cancer (1 / 2), laryngeal cancer (2 / 3), lung cancer (1 / 5), esophageal cancer (1 / 1), ovarian cancer (8 / 8), pharyngeal cancer (1 / 2), prostate cancer (3 / 4), and rectal cancer (1 / 2). At 80% specificity, the 2-assay panel detected 73% of all various cancer cases and every cancer type tested except gastric cancer, including bladder cancer (1 / 1), breast cancer (1 / 1), kidney cancer (1 / 2), laryngeal cancer (2 / 3), lung cancer (2 / 5), esophageal cancer (1 / 1), ovarian cancer (8 / 8), pharyngeal cancer (1 / 2), prostate cancer (4 / 4), rectal cancer (1 / 2), and gastric cancer (0 / 1). The results are shown in Figure 6 and demonstrates the breadth of utility of the methods of the invention for detecting multiple cancer types, with utility as a test for cancer itself.

[0227] Example 9

[0228] To further demonstrate the method of the present invention, we trained another three-assay panel model on the results of the CRC training cohort described in Examples 3 and 8, including measurements of nucleosomes themselves, nucleosomes containing histone isoform H3.1, and IL-6. This model has an accuracy of 80% sensitivity at 89% specificity or 80% sensitivity at 80% specificity for CRC detection (Table 3). In a manner similar to the experiment described in Example 7, the model was then applied to a validation cohort including patients with a variety of cancer diseases. This 3-assay panel was able to detect 37% of cancers with a specificity of 90%, including kidney cancer (1 / 2), lung cancer (2 / 5), ovarian cancer (5 / 8), prostate cancer (1 / 4), rectal cancer (1 / 2), and gastric cancer (1 / 1). At 80% specificity, the 3-assay panel detected 57% of all cancer cases and every cancer type tested except breast cancer, including bladder cancer (1 / 1), kidney cancer (2 / 2), laryngeal cancer (1 / 3), lung cancer (3 / 5), esophageal cancer (1 / 1), ovarian cancer (6 / 8), pharyngeal cancer (2 / 2), prostate cancer (2 / 4), rectal cancer (1 / 2), and gastric cancer (1 / 1). The results were shown in Figure 7 and demonstrates the breadth of utility of the methods of the invention for detecting multiple cancer types, with utility as a test for cancer itself.

[0229] Example 10

[0230] To demonstrate the methods of the present invention using simple cutoffs, rather than models or algorithms based on regression analysis of results, we reanalyzed the combined data from 144 individuals, including the 47 lung cancer patients described in Example 1, plus 100 individuals, including the 20 colorectal cancer (CRC) patients described in Example 3, and 70 individuals, including the 30 lung cancer patients described in Example 6 (a total of 314 subjects, including 97 cancer patients), using simple positive and negative cutoffs for a two-assay panel including IL-6 and nucleosomes containing histone isoform H3.1. The cutoff for IL-6 was set at ≥4 pg / ml, while the cutoff for nucleosomes containing histone isoform H3.1 was set at an assay response of ≥1.2 OD units (equivalent to approximately ≥±200 ng / ml). Any sample with at least one positive result (above the respective threshold cutoffs) was considered positive. The analysis detected 71% of cancer cases with 93% specificity. This is very close to the accuracy of the FIT test, which is widely used as a first-line screening test for (colorectal) cancer.

[0231] Advantages of using simple cutoff values ​​in group tests include the ease with which clinicians can understand the test and the elimination of any need for software or other aids in interpreting the test results.

[0232] Example 11

[0233] Plasma samples were collected from 135 patients who were referred for colonoscopy after a positive FIT test using the OC sensor FIT test (fecal hemoglobin level ≥100 ng / ml). Patients included asymptomatic screening patients, symptomatic patients, and patients under surveillance for CRC recurrence or disease progression. Of the 135 patients, 41 were found to have no lesions at the time of colonoscopy, 37 had one or more non-advanced adenomas, 35 had one or more advanced adenomas, and 22 had CRC, of ​​which 5 were diagnosed with stage I disease, 2 were diagnosed with stage II, 8 were diagnosed with stage III, 6 were diagnosed with stage IV, and 1 was diagnosed with unknown stage disease.

[0234] We tested the levels of nucleosomes containing histone isoform H3.1, IL-6, IL-8, IL-10, CRP and HMGB1 in plasma samples. Numerical FIT levels (ng / ml) and plasma results were analyzed using ROC analysis of CRC relative to people with no findings on colonoscopy to determine the specificity of the assay or model when the sensitivity reached 100% (i.e., the proportion of people who correctly identified as negative in people who did not find CRC or adenoma on colonoscopy, while correctly identifying all 22 CRC patients as positive). This provides a potential reduction in the number of colonoscopies, which can be achieved while prioritizing all patients with cancer for colonoscopy by the classification blood test of the present invention. All individual assays performed had a positive AUC and were able to correctly identify some patients who did not have colorectal lesions and the CRC false negative rate was zero. Table 4 shows the specificity of the biomarker for CRC at 100% sensitivity. This is a measure of the proportion (%) of patients with no findings on colonoscopy who were correctly predicted not to have CRC and no cancer cases were missed.

[0235] Table 4. Performance of Individual Assays in Categorizing FIT-Positive Subjects

[0236] Determination AUC (%) Specificity at 100% sensitivity (%) FIT 86 32 H3.1-nucleosome 68 5 IL-6 59 5 1L-8 66 3 IL-10 53 3 CRP 71 12 HMGB1 53 8

[0237] The blood test results were then combined with the FIT results to generate an algorithm, through regression analysis, for identifying people who do not have CRC while maintaining a false negative rate for CRC of zero. Some of the outcome models or algorithms developed are shown in Table 5.

[0238] Table 5. Performance of the Combined FIT / Blood Assay Model for Classifying FIT-Positive Subjects

[0239]

[0240] The FIT test alone identified 32% of patients who had blood in their stool but no lesions on colonoscopy. However, using the method of the present invention, this was increased to as high as 58%. When the derived model was applied to symptomatic and asymptomatic subjects in the test (no monitoring subjects), the proportion of people without colorectal lesions who were correctly identified as negative further increased to as high as 68%.

[0241] References

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[0247] Holdenrieder et al., Int J Cancer, 95: 114–20, 2001

[0248] Holdenrieder et al., Clin Chem, 51(6): 1026-1029, 2005

[0249] Holdenrieder and Stieber, Crit Rev Clin Lab Sci, 46(1): 1–24, 2009

[0250] Lipitz and Harris, Oncoimmunol, 5: e1093722, 2016

[0251] Madej-Michniewicz et al., Nat Sci Rep, 5: 14382, 2015

[0252] Midthun, F1000Res, 5: F1000 Faculty Rev-739, 2016

[0253] Moyer, Ann Intern Med, 160(5): 330-338, 2014

[0254] Potter et al., Clin Chem, 60(9): 1183–1191, 2014

[0255] Salgame et al., Nucleic Acids Res, 25(3): 680-681, 1997

[0256] Snyder et al., Cell, 164: 57–68, 2016

[0257] Taniguchi and Karin, Semin Immunol, 26: 54–74, 2014

[0258] van Nieuwenhuijze et al., Ann Rheum Dis, 62: 10–14, 2003

[0259] Wang and Sun, Int J Onocology, 44: 1032-1040, 2014

[0260] Wojtacki et al., Neoplasma, 41(4): 213-6, 1994

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[0264] Zhao and Garcia, Cold Spring Harb Perspective Biol, 7: a025064, 2015.

Claims

1. Use of a histone isoform H3.1 binder and an IL-6 binder in the preparation of a kit for diagnosing and / or monitoring cancer in a body fluid sample, wherein the cancer is selected from the group consisting of lung cancer, colon cancer, rectal cancer, gastric cancer, kidney cancer, prostate cancer, breast cancer, pharyngeal cancer, laryngeal cancer, ovarian cancer, esophageal cancer and bladder cancer.

2. The use according to claim 1, wherein the kit further comprises an IL-8 binding agent and / or an IL-10 binding agent.

3. The use according to any one of claims 1 to 2, wherein the body fluid sample is a blood, serum or plasma sample.

4. The use according to any one of claims 1 to 2, wherein the histone isoform H3.1 and IL-6 are measured by immunoassay or mass spectrometry.

5. Use of a histone isoform H3.1 binder and an IL-6 binder in the preparation of a kit for use in a method for diagnosing cancer in a patient, the method comprising: detecting or measuring IL-6 and histone isoform H3.1 in a bodily fluid sample obtained from the patient; and using the levels or concentrations of IL-6 and the histone isoform H3.1 detected in the body fluid sample to determine whether the patient has cancer, The cancer is selected from the group consisting of lung cancer, colon cancer, rectal cancer, stomach cancer, kidney cancer, prostate cancer, breast cancer, pharyngeal cancer, laryngeal cancer, ovarian cancer, esophageal cancer and bladder cancer.

6. Use of a histone isoform H3.1 binder and an IL-6 binder in the preparation of a kit for use in a method for assessing the suitability of a patient for cancer research, the method comprising: detecting or measuring IL-6 and histone isoform H3.1 in a bodily fluid sample obtained from the patient; and using the levels or concentrations of IL-6 and the histone isoform H3.1 detected in the body fluid sample to determine whether the patient requires further cancer research, The cancer is selected from the group consisting of lung cancer, colon cancer, rectal cancer, stomach cancer, kidney cancer, prostate cancer, breast cancer, pharyngeal cancer, laryngeal cancer, ovarian cancer, esophageal cancer and bladder cancer.

7. Use according to claim 5 or claim 6, wherein the method further comprises determining at least one clinical parameter of the patient.

8. The use according to claim 7, wherein the clinical parameter is selected from the group consisting of: age, sex and body mass index (BMI).

9. Use according to any one of claims 5 to 6 and 8, wherein the method further comprises measuring IL-8 and / or IL-10.

10. Use according to any one of claims 5 to 6 and 8, wherein the levels or concentrations of IL-6 and the histone isoform H3.1 detected are compared with a control.

11. The use according to any one of claims 5 to 6 and 8, wherein the body fluid sample is a blood, serum or plasma sample.

12. Use according to any one of claims 5 to 6 and 8, wherein the detecting or measuring is performed using an immunoassay or mass spectrometry.

13. Use according to any one of claims 5 to 6 and 8, wherein the method further comprises isolating DNA associated with the histone isoform H3.1 detected in the body fluid sample and optionally sequencing the DNA.

14. Use of a histone isoform H3.1 binder and an IL-6 binder in the preparation of a kit for use in a method for assessing a patient's suitability for colonoscopy, the method comprising: (i) detecting or measuring the level of fecal hemoglobin in a stool sample obtained from the patient; (ii) detecting or measuring the level of IL-6 and the level of histone isoform H3.1 in a body fluid sample obtained from the patient; and (iii) using said measured fecal haemoglobin, IL-6 and histone isoform H3.1 levels as an indication of said patient's suitability for colonoscopy.

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