US20150141286A1 - Method and apparatus for determining a probability of colorectal cancer in a subject - Google Patents

Method and apparatus for determining a probability of colorectal cancer in a subject Download PDF

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US20150141286A1
US20150141286A1 US14/546,433 US201414546433A US2015141286A1 US 20150141286 A1 US20150141286 A1 US 20150141286A1 US 201414546433 A US201414546433 A US 201414546433A US 2015141286 A1 US2015141286 A1 US 2015141286A1
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Choong-Chin Liew
Samuel Chao
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Genenews Inc
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    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407Specifically defined cancers
    • G01N33/57419Specifically defined cancers of colon
    • G06F19/12
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
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    • C12Q2600/00Oligonucleotides characterized by their use
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    • C12Q2600/00Oligonucleotides characterized by their use
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    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/166Oligonucleotides used as internal standards, controls or normalisation probes
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/50Determining the risk of developing a disease
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/56Staging of a disease; Further complications associated with the disease
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding

Abstract

A method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer is disclosed. The method comprises, for each gene of a set of one or more genes selected from the group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1: determining a level of RNA encoded by the gene in blood of the test subject, thereby generating test data; providing positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and determining a probability that the test data corresponds to the positive control data and not to the negative control data, where the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.

Description

  • This application is a continuation application of U.S. non-provisional application Ser. No. 12/384,914, filed Apr. 10, 2009, which itself claims the benefit of U.S. provisional application 61/123,798, filed Apr. 10, 2008 and of U.S. provisional application 61/123,831, filed Apr. 11, 2008. The entire contents of the non-provisional application and both provisional applications are incorporated by reference herein.
  • TECHNICAL FIELD
  • The disclosure relates to apparatuses, kits and methods for determining a probability of colorectal cancer in a test subject. More particularly, the disclosure relates to apparatuses, kits and methods for diagnosing colorectal cancer in a test subject by measuring a level of one or more gene products in blood of the test subject.
  • BACKGROUND
  • Colorectal cancer causes 655,000 deaths worldwide per year, making it the second-leading cause of cancer-related deaths. It is the third most frequently diagnosed cancer in men and women in the United States and carries an overall population lifetime risk of 6%. (American Cancer Society. Cancer Facts and Figures. 2008. Atlanta: American Cancer Society). The American Cancer Society estimates that about 108,070 new cases of colon cancer (53,760 in men and 54,310 in women) and 40,740 new cases of rectal cancer (23,490 in men and 17,250 in women) will be diagnosed in 2008. Of those diagnosed, nearly half are expected to die within five years. In the United States in 2008 an estimated 50,000 men and women will die of cancer of the colon and rectum. (American Cancer Society 2008). This high mortality rate is due at least in part to the fact that a large proportion of cancers are detected at relatively late stages, such as following onset of overt symptoms, when the cancer is more difficult to treat. In addition, identification of colorectal cancer at later stages concomitantly necessitates harsher treatment, such as radical colostomy. It has been shown that the identification and treatment of colorectal cancer at earlier stages significantly reduces the risk of developing more advanced disease, and hence risk of death from the disease. Stage at detection is critically related to patient survival. Localized cancers (Dukes's Stage A or B) have an excellent prognosis of 82%-93% at five years. Regional (Dukes's Stage C) patients have a five year survival rates of 55% to 60%; and only 5% to 8% of patients with late stage cancer will survive the five year span. (O'Connell J B, Maggard M, Ko C Y. Colon cancer survival rates with the new American Joint Committee on cancer sixth edition staging. JNCI. 2004; 96: 1420-1425). Therefore, a test to screen for colorectal cancer so as to allow earlier treatment should markedly reduce the incidence of advanced-stage colorectal cancer (Ransohoff D F. Colorectal cancer screening in 2005: status and challenges. Gastroenterology. 2005 May; 128(6):1685-95) and decrease the current costs to the medical system. Thus, the American Cancer Society recommends that all Americans age 50 and older be screened regularly for colorectal cancer. Unfortunately, only a small fraction of the population at risk is screened for the disease (Mitka M. Colorectal cancer screening rates still fall far short of recommended levels. JAMA. 2008 Feb. 13; 299(6):622), as currently available screening methods require insufficiently available and/or costly resources, are associated with unacceptably low patient compliance, and/or are associated with significant health risks.
  • Currently utilized screening technologies to test for colorectal cancer include fecal occult blood test (FOBT), flexible sigmoidoscopy, double contrast barium enema (DCBE), and colonoscopy. The current recommended standards for screening for colorectal cancer in individuals over the age of 50 and who are considered part of an average risk population include: an FOBT yearly, a sigmoidoscopy every five years, a colonoscopy every ten years and a DCBE every five years. For a high risk population where one or more family members have had colorectal cancer, a colonoscopy is recommended every two years as a follow up to FOBT or sigmoidoscopy. Each of these tests suffers significant disadvantages. Fecal occult blood testing suffers from low sensitivity, requires significant dietary and other restrictions prior to testing and is associated with poor patient compliance. Sigmoidoscopy and colonoscopy are more sensitive than the other standard methods since they involve direct visualization of the lumen of the colon, however these methods are also associated with various significant disadvantages. Sigmoidoscopy and colonoscopy are both highly invasive procedures which cause significant levels of discomfort, causing many individuals to opt not to undergo these recommended screening procedures. Sigmoidoscopy only allows visualization of the distal part of the colon and hence cannot detect a relatively large fraction of cancers, and colonoscopy, despite allowing examination essentially along the entire length of the colon, is associated with a significant failure rate for detection of colorectal cancer. In addition, sigmoidoscopy and colonoscopy are costly, are insufficiently available, and may result in potentially lethal complications, such as accidental intestinal perforation.
  • Various approaches have been proposed in the prior art for colorectal cancer testing using identification and analysis of markers of this disease in blood (reviewed in Hundt S. et al. Blood markers for early detection of colorectal cancer: a systematic review. Cancer Epidemiol Biomarkers Prey. 2007 October; 16(10):1935-53). Such approaches, if successful, would have the advantage of circumventing critical disadvantages of the standard prior art methods, by virtue, for example, of being relatively non-invasive, minimally cumbersome, essentially risk-free and hence likely to be associated with increased patient screening compliance rates. However, none of these approaches has demonstrated an optimal capacity for diagnosing colorectal cancer.
  • Thus, there is a longstanding and urgent need for an improved method of determining a probability of colorectal cancer in a subject based on analysis of blood markers.
  • SUMMARY
  • The invention discloses novel methods, apparatuses and kits for determining a probability of colorectal cancer in a subject, based on novel blood markers of colorectal cancer. This use can be effected in a variety of ways as further described and exemplified herein.
  • According to one aspect of the invention there is provided a method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, the method comprising, for each gene of a set of one or more genes selected from the group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1: (a) determining a level of RNA encoded by the gene in blood of the test subject, thereby generating test data; (b) providing positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) determining a probability that the test data corresponds to the positive control data and not to the negative control data, wherein the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • According to another aspect of the invention there is provided a method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, the method comprising: (a) determining a level of RNA encoded by a ANXA3 gene in blood of the test subject, thereby generating test data; (b) providing positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) determining a probability that the test data corresponds to the positive control data and not to the negative control data, wherein the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • According to yet another aspect of the invention there is provided a method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, the method comprising: (a) determining a level of RNA encoded by a CLEC4D gene in blood of the test subject, thereby generating test data; (b) providing positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) determining a probability that the test data corresponds to the positive control data and not to the negative control data, wherein the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • According to one aspect of the invention there is provided a method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, the method comprising: (a) determining a level of RNA encoded by a IL2RB gene in blood of the test subject, thereby generating test data; (b) providing positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) determining a probability that the test data corresponds to the positive control data and not to the negative control data, wherein the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • According to still another aspect of the invention there is provided a method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, the method comprising: (a) determining a level of RNA encoded by a LMNB1 gene in blood of the test subject, thereby generating test data; (b) providing positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) determining a probability that the test data corresponds to the positive control data and not to the negative control data, wherein the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • According to a further aspect of the invention there is provided a method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, the method comprising: (a) determining a level of RNA encoded by a PRRG4 gene in blood of the test subject, thereby generating test data; (b) providing positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) determining a probability that the test data corresponds to the positive control data and not to the negative control data, wherein the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • According to yet a further aspect of the invention there is provided a method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, the method comprising: (a) determining a level of RNA encoded by a TNFAIP6 gene in blood of the test subject, thereby generating test data; (b) providing positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) determining a probability that the test data corresponds to the positive control data and not to the negative control data, wherein the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • According to still a further aspect of the invention there is provided a method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, the method comprising: (a) determining a level of RNA encoded by a VNN1 gene in blood of the test subject, thereby generating test data; (b) providing positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) determining a probability that the test data corresponds to the positive control data and not to the negative control data, wherein the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • According to further features of the invention described below, the determining of the level of RNA encoded by the gene in blood of the test subject is effected by determining the level of RNA encoded by the gene in a blood sample isolated from the test subject.
  • According to further features of the invention described below, the method of determining the probability that the human test subject has colorectal cancer as opposed to not having colorectal cancer further comprises determining levels of RNA encoded by the gene in blood of a population of human subjects having colorectal cancer, thereby providing the positive control data representing the levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and determining levels of RNA encoded by the gene in blood of a population of human subjects not having colorectal cancer, thereby providing the negative control data representing the levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer.
  • According to further features of the invention described below, the determining of the probability that the test data corresponds to the positive control data and not to the negative control data is effected by applying to the test data a mathematical model derived from the positive control data and from the negative control data, wherein the mathematical model is for determining the probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data.
  • According to another aspect of the invention there is provided a computer-based method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, from test data representing a level of RNA encoded by a ANXA3 gene in blood of the test subject, the method comprising, for each gene of a set of one or more genes selected from the group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 computer-implemented steps of: (a) applying to the test data a mathematical model derived from positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and from negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, wherein the mathematical model is for determining a probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data; and (b) outputting the probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data, wherein the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • According to another aspect of the invention there is provided a computer-based method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, from test data representing a level of RNA encoded by a ANXA3 gene in blood of the test subject, the method comprising computer-implemented steps of: (a) applying to the test data a mathematical model derived from positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and from negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, wherein the mathematical model is for determining a probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data; and (b) outputting the probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data, wherein the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • According to another aspect of the invention there is provided a computer-based method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, from test data representing a level of RNA encoded by a ANXA3 gene in blood of the test subject, the method comprising computer-implemented steps of: (a) applying to the test data a mathematical model derived from positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and from negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, wherein the mathematical model is for determining a probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data; and (b) outputting the probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data, wherein the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • According to another aspect of the invention there is provided a computer-based method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, from test data representing a level of RNA encoded by a ANXA3 gene in blood of the test subject, the method comprising computer-implemented steps of: (a) applying to the test data a mathematical model derived from positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and from negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, wherein the mathematical model is for determining a probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data; and (b) outputting the probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data, wherein the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • According to another aspect of the invention there is provided a computer-based method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, from test data representing a level of RNA encoded by a ANXA3 gene in blood of the test subject, the method comprising computer-implemented steps of: (a) applying to the test data a mathematical model derived from positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and from negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, wherein the mathematical model is for determining a probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data; and (b) outputting the probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data, wherein the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • According to another aspect of the invention there is provided a computer-based method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, from test data representing a level of RNA encoded by a ANXA3 gene in blood of the test subject, the method comprising computer-implemented steps of: (a) applying to the test data a mathematical model derived from positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and from negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, wherein the mathematical model is for determining a probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data; and (b) outputting the probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data, wherein the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • According to another aspect of the invention there is provided a computer-based method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, from test data representing a level of RNA encoded by a ANXA3 gene in blood of the test subject, the method comprising computer-implemented steps of: (a) applying to the test data a mathematical model derived from positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and from negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, wherein the mathematical model is for determining a probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data; and (b) outputting the probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data, wherein the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • According to another aspect of the invention there is provided a computer-based method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, from test data representing a level of RNA encoded by a ANXA3 gene in blood of the test subject, the method comprising computer-implemented steps of: applying to the test data a mathematical model derived from positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and from negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, wherein the mathematical model is for determining a probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data; and (b) outputting the probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data, wherein the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • According to another aspect of the present invention there is provided a method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising: (a) determining a level of RNA encoded by a ANXA3 gene in blood of the test subject, thereby generating test data; (b) providing negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) applying to the test data and to the negative control data a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, wherein an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • According to another aspect of the present invention there is provided a method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising: (a) determining a level of RNA encoded by a CLEC4D gene in blood of the test subject, thereby generating test data; (b) providing negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) applying to the test data and to the negative control data a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, wherein an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • According to yet another aspect of the present invention there is provided a method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising: (a) determining a level of RNA encoded by a IL2RB gene in blood of the test subject, thereby generating test data; (b) providing negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) applying to the test data and to the negative control data a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, wherein an indication by the value that the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • According to still another aspect of the present invention there is provided a method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising: (a) determining a level of RNA encoded by a LMNB1 gene in blood of the test subject, thereby generating test data; (b) providing negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) applying to the test data and to the negative control data a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, wherein an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • According to a further aspect of the present invention there is provided a method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising: (a) determining a level of RNA encoded by a PRRG4 gene in blood of the test subject, thereby generating test data; (b) providing negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) applying to the test data and to the negative control data a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, wherein an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • According to yet a further aspect of the present invention there is provided a method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising: (a) determining a level of RNA encoded by a TNFAIP6 gene in blood of the test subject, thereby generating test data; (b) providing negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) applying to the test data and to the negative control data a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, wherein an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • According to still a further aspect of the present invention there is provided a method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising: (a) determining a level of RNA encoded by a VNN1 gene in blood of the test subject, thereby generating test data; (b) providing negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) applying to the test data and to the negative control data a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, wherein an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • According to an additional aspect of the present invention there is provided a computer-based method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising computer-implemented steps of: (a) applying to test data representing a level of RNA encoded by a ANXA3 gene in blood of the test subject, and to negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (b) outputting the value, wherein an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • According to yet an additional aspect of the present invention there is provided a computer-based method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising computer-implemented steps of: (a) applying to test data representing a level of RNA encoded by a CLEC4D gene in blood of the test subject, and to negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (b) outputting the value, wherein an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • According to still an additional aspect of the present invention there is provided a computer-based method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising computer-implemented steps of: (a) applying to test data representing a level of RNA encoded by a IL2RB gene in blood of the test subject, and to negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (b) outputting the value, wherein an indication by the value that the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • According to yet still an additional aspect of the present invention there is provided a computer-based method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising computer-implemented steps of: (a) applying to test data representing a level of RNA encoded by a LMNB1 gene in blood of the test subject, and to negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (b) outputting the value, wherein an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • According to another aspect of the present invention there is provided a computer-based method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising computer-implemented steps of: (a) applying to test data representing a level of RNA encoded by a PRRG4 gene in blood of the test subject, and to negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (b) outputting the value, wherein an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer. According to yet another aspect of the present invention there is provided a computer-based method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising computer-implemented steps of: (a) applying to test data representing a level of RNA encoded by a TNFAIP6 gene in blood of the test subject, and to negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (b) outputting the value, wherein an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • According to still another aspect of the present invention there is provided a computer-based method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising computer-implemented steps of: (a) applying to test data representing a level of RNA encoded by a VNN1 gene in blood of the test subject, and to negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (b) outputting the value, wherein an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • According to a further aspect of the present invention there is provided a method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising, for each gene of a set of one or more genes selected from the group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1: (a) determining a level of RNA encoded by the gene in blood of the test subject, thereby generating test data; (b) providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) applying to the test data and to the negative control dataa mathematical formula for generating a value indicating, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, and indicating, for IL2RB, whether the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, wherein, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer, and wherein, for IL2RB, an indication by the value that the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • According to yet a further aspect of the present invention there is provided a computer-based method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising, for each gene of a set of one or more genes selected from the group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, computer-implemented steps of: applying to test data representing a level of RNA encoded by the gene in blood of the test subject, and to negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, a formula for calculating a value indicating, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, and indicating, for IL2RB, whether the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, wherein, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer, and wherein, for IL2RB, an indication by the value that the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • According to one aspect of the invention there is provided a method of diagnosing colorectal cancer in a test subject, the method comprising, for each gene of a set of one or more genes selected from the group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1: (a) determining a level of RNA encoded by the gene in blood of the test subject, thereby generating test data; (b) providing positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) determining a probability that the test data corresponds to the positive control data and not to the negative control data, wherein a determination that the test data corresponds to the positive control data and not to the negative control data provides an indication of colorectal cancer in the test subject.
  • According to a further aspect of the present invention there is provided a method of diagnosing colorectal cancer in a test subject, the method comprising, for each gene of a set of one or more genes selected from the group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1: (a) determining a level of RNA encoded by the gene in blood of the test subject, thereby generating test data; (b) providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) applying to the test data and to the negative control data a mathematical formula for generating a value indicating, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, and indicating, for IL2RB, whether the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, wherein, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer provides an indication of colorectal cancer in the test subject, and wherein, for IL2RB, an indication by the value that the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer provides an indication of colorectal cancer in the test subject.
  • According to another aspect of the present invention there is provided a method of determining whether a test subject is at an increased risk of having colorectal cancer relative to the general population, comprising: a) obtaining a test sample of blood from the subject; and i) determining a level of RNA encoded by a annexin A3 (ANXA3) gene in the test sample of blood, ii) comparing the level of RNA encoded by the gene as determined in step (i) with the level of the RNA encoded by the gene in control samples of blood; and b) concluding that the subject is at an increased risk of having colorectal cancer relative to the general population if the level of RNA encoded by the gene in the test sample of blood is higher than in the control samples of blood.
  • According to yet another aspect of the present invention there is provided a method of determining whether a test subject is at an increased risk of having colorectal cancer relative to the general population, comprising: a) obtaining a test sample of blood from the subject; and i) determining a level of RNA encoded by a C-type lectin domain family 4, member D (CLEC4D) gene in the test sample of blood, ii) comparing the level of RNA encoded by the gene as determined in step (i) with the level of the RNA encoded by the gene in control samples of blood; and b) concluding that the subject is at an increased risk of having colorectal cancer relative to the general population if the level of RNA encoded by the gene in the test sample of blood is higher than in the control samples of blood.
  • According to still another aspect of the present invention there is provided a method of determining whether a test subject is at an increased risk of having colorectal cancer relative to the general population, comprising: a) obtaining a test sample of blood from the subject; and i) determining a level of RNA encoded by a interleukin 2 receptor, beta (IL2RB) gene in the test sample of blood, ii) comparing the level of RNA encoded by the gene as determined in step (i) with the level of the RNA encoded by the gene in control samples of blood; and b) concluding that the subject is at an increased risk of having colorectal cancer relative to the general population if the level of RNA encoded by the gene in the test sample of blood is lower than in the control samples of blood.
  • According to a further aspect of the present invention there is provided a method of determining whether a test subject is at an increased risk of having colorectal cancer relative to the general population, comprising: a) obtaining a test sample of blood from the subject; and i) determining a level of RNA encoded by a lamin B1 (LMNB1) gene in the test sample of blood, ii) comparing the level of RNA encoded by the gene as determined in step (i) with the level of the RNA encoded by the gene in control samples of blood; and b) concluding that the subject is at an increased risk of having colorectal cancer relative to the general population if the level of RNA encoded by the gene in the test sample of blood is higher than in the control samples of blood.
  • According to yet a further aspect of the present invention there is provided a method of determining whether a test subject is at an increased risk of having colorectal cancer relative to the general population, comprising: a) obtaining a test sample of blood from the subject; and i) determining a level of RNA encoded by a proline rich Gla (G carboxyglutamic acid) 4 (transmembrane) (PRRG4) gene in the test sample of blood, ii) comparing the level of RNA encoded by the gene as determined in step (i) with the level of the RNA encoded by the gene in control samples of blood; and b) concluding that the subject is at an increased risk of having colorectal cancer relative to the general population if the level of RNA encoded by the gene in the test sample of blood is higher than in the control samples of blood.
  • According to still a further aspect of the present invention there is provided a method of determining whether a test subject is at an increased risk of having colorectal cancer relative to the general population, comprising: a) obtaining a test sample of blood from the subject; and i) determining a level of RNA encoded by a tumor necrosis factor, alpha induced protein 6 (TNFAIP6) gene in the test sample of blood, ii) comparing the level of RNA encoded by as determined in step (i) with the level of the RNA encoded by the gene in control samples of blood; and b) concluding that the subject is at an increased risk of having colorectal cancer relative to the general population if the level of RNA encoded by the gene in the test sample of blood is higher than in the control samples of blood.
  • According to still a further aspect of the present invention there is provided a method of determining whether a test subject is at an increased risk of having colorectal cancer relative to the general population, comprising: a) obtaining a test sample of blood from the subject; and i) determining a level of RNA encoded by a vanin 1 (VNN1) gene in the test sample of blood, ii) comparing the level of RNA encoded by the gene as determined in step (i) with the level of the RNA encoded by the gene in control samples of blood; and b) concluding that the subject is at an increased risk of having colorectal cancer relative to the general population if the level of RNA encoded by the gene in the test sample of blood is higher than in the control samples of blood.
  • According to an additional aspect of the present invention there is provided a method of determining whether a test subject is at an increased risk of having colorectal cancer relative to the general population, comprising: a) obtaining a test sample of blood from the subject; and for each gene of a set of genes selected from the group consisting of: ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, i) determining a level of RNA encoded by the gene in the test sample of blood, ii) comparing the level of RNA encoded by the gene of the set as determined in step (i) with the level of RNA encoded by the gene in one or more control samples of blood; and b) concluding that the subject is at an increased risk of having colorectal cancer relative to the general population if, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, the level of RNA encoded by the gene in the test sample of blood is higher than in the control samples of blood, and concluding that the subject is at an increased risk of having colorectal cancer relative to the general population if, for IL2RB, the level of RNA encoded by the gene in the test sample of blood is lower than in the control samples of blood.
  • According to an additional aspect of the present invention there is provided a method of diagnosing colorectal cancer in a test subject, comprising: a) obtaining a test sample of blood from the subject; and for each gene of a set of genes selected from the group consisting of: ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, i) determining a level of RNA encoded by the gene in the test sample of blood, and ii) applying to the level of RNA encoded by the gene of the set as determined in step (i) and to the level of RNA encoded by the gene in one or more control samples of blood a mathematical formula for generating a value indicating whether, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, the level of RNA encoded by the gene in the test sample of blood is higher than in the control samples of blood, and, for IL2RB, the level of RNA encoded by the gene in the test sample of blood is lower than in the control samples of blood; and b) concluding that there is an indication of colorectal cancer in the test subject, if, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, the value indicates that the level of RNA encoded by the gene in the test sample of blood is higher than in the control samples of blood, and concluding that there is an indication of colorectal cancer in the test subject if, for IL2RB, the value indicates that the level of RNA encoded by the gene in the test sample of blood is lower than in the control samples of blood.
  • According to further features of the invention described below, the control samples are from individuals who have been diagnosed as not having colorectal cancer.
  • According to still another aspect of the invention there is provided a kit comprising packaging and containing, for each gene of a set of two or more genes selected from the group consisting of ACTB, ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, a primer set capable of generating an amplification product of a polynucleotide complementary to RNA encoded, in a human subject, only by the gene.
  • According to further features of the invention described below, the kit further contains two or more components selected from the group consisting of a thermostable polymerase, a reverse transcriptase, deoxynucleotide triphosphates, nucleotide triphosphates and enzyme buffer.
  • According to further features of the invention described below, the kit further contains at least one labeled probe capable of selectively hybridizing to either a sense or an antisense strand of the amplification product.
  • According to further features of the invention described below, the kit further contains a computer-readable medium having instructions stored thereon that are operable when executed by a computer for applying a mathematical model to test data representing a level of RNA encoded by the gene in blood of a human test subject, wherein the mathematical model is derived from positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and from negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, wherein the mathematical model is for determining a probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data, and wherein the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • According to further features of the invention described below, the kit further contains a computer-readable medium having instructions stored thereon that are operable when executed by a computer for applying, to test data representing a level of RNA encoded by the gene in blood of a human test subject, and to negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, a mathematical formula for generating a value indicating, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, and, for IL2RB, whether the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, wherein, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer, and wherein, for IL2RB, an indication by the value that the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • According to further features of the invention described below, the set of one or more genes consists of ACTB and one or more genes selected from the group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1.
  • According to further features of the invention described below, the set of one or more genes consists of ACTB and ANXA3.
  • According to further features of the invention described below, the set of one or more genes consists of ACTB and CLEC4D.
  • According to further features of the invention described below, the set of one or more genes consists of ACTB and IL2RB.
  • According to further features of the invention described below, the set of one or more genes consists of ACTB and LMNB1.
  • According to further features of the invention described below, the set of one or more genes consists of ACTB and PRRG4.
  • According to further features of the invention described below, the set of one or more genes consists of TNFAIP6 and PRRG4.
  • According to further features of the invention described below, the set of one or more genes consists of ACTB and VNN1.
  • According to further features of the invention described below, the level of RNA encoded by the gene in blood of the test subject is determined via quantitative reverse transcriptase-polymerase chain reaction analysis.
  • According to further features of the invention described below, the level of RNA encoded by the gene in blood of the test subject and the levels of RNA encoded by the gene in blood of the control subjects are determined via the same method.
  • According to further features of the invention described below, the set of one or more genes is a set of one or more genes selected from the group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, wherein the level of RNA encoded by the gene in blood of the test subject is determined as a ratio to a level of RNA encoded by ACTB in blood of the test subject.
  • According to further features of the invention described below, the level of RNA encoded by the gene in blood of the test subject and the level of RNA encoded by ACTB in blood of the test subject are determined via duplex quantitative reverse transcriptase-polymerase chain reaction analysis of RNA encoded by the gene and of RNA encoded by ACTB.
  • According to further features of the invention described below, the set of one or more genes consists of IL2RB and one or more genes selected from the group consisting of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1.
  • According to further features of the invention described below, the set of one or more genes is a set of one or more genes selected from the group consisting of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, and wherein the level of RNA encoded by the gene in blood of the test subject is determined as a ratio to a level of RNA encoded by IL2RB in blood of the test subject.
  • According to further features of the invention described below, the level of RNA encoded by the gene in blood of the test subject and the level of RNA encoded by IL2RB in blood of the test subject are determined via duplex quantitative reverse transcriptase-polymerase chain reaction analysis of RNA encoded by the gene and of RNA encoded by IL2RB.
  • According to further features of the invention described below, the set of one or more genes consists of ANXA3.
  • According to further features of the invention described below, the set of one or more genes consists of CLEC4D.
  • According to further features of the invention described below, the set of one or more genes consists of IL2RB.
  • According to further features of the invention described below, the set of one or more genes consists of LMNB1.
  • According to further features of the invention described below, the set of one or more genes consists of PRRG4.
  • According to further features of the invention described below, the set of one or more genes consists of TNFAIP6.
  • According to further features of the invention described below, the set of one or more genes consists of VNN1.
  • According to further features of the invention described below, the set of one or more genes consists of IL2RB and ANXA3.
  • According to further features of the invention described below, the set of one or more genes consists of IL2RB and CLEC4D.
  • According to further features of the invention described below, the set of one or more genes consists of IL2RB and LMNB1.
  • According to further features of the invention described below, the set of one or more genes consists of IL2RB and PRRG4.
  • According to further features of the invention described below, the set of one or more genes consists of IL2RB and TNFAIP6.
  • According to further features of the invention described below, the set of one or more genes consists of IL2RB and VNN1.
  • DEFINITIONS
  • As will become apparent, preferred features and characteristics of one aspect of the invention are applicable to any other aspect of the invention. It should be noted that, as used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.
  • “Encode” A polynucleotide, including a gene, is said the to “encode” a RNA and/or polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof. The anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced there from.
  • The term “label” refers to a composition capable of producing a detectable signal indicative of the presence of the target polynucleotide in an assay sample. Suitable labels include radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • As used herein, a “sample” refers to a sample of tissue or fluid isolated from an individual, including but not limited to, for example, blood, plasma, serum, tumor biopsy, urine, stool, sputum, spinal fluid, pleural fluid, nipple aspirates, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, cells (including but not limited to blood cells), organs, and also samples of in vitro cell culture constituent.
  • Examples of amplification techniques include strand displacement amplification, as disclosed in U.S. Pat. No. 5,744,311; transcription-free isothermal amplification, as disclosed in U.S. Pat. No. 6,033,881; repair chain reaction amplification, as disclosed in WO 90/01069; ligase chain reaction amplification, as disclosed in European Patent Appl. 320 308; gap filling ligase chain reaction amplification, as disclosed in U.S. Pat. No. 5,427,930; and RNA transcription-free amplification, as disclosed in U.S. Pat. No. 6,025,134.
  • Examples of a primer of the invention include an oligonucleotide which is capable of acting as a point of initiation of polynucleotide synthesis along a complementary strand when placed under conditions in which synthesis of a primer extension product which is complementary to a polynucleotide is catalyzed. Such conditions include the presence of four different nucleotide triphosphates or nucleoside analogs and one or more agents for polymerization such as DNA polymerase and/or reverse transcriptase, in an appropriate buffer (“buffer” includes substituents which are cofactors, or which affect pH, ionic strength, etc.), and at a suitable temperature. A primer must be sufficiently long to prime the synthesis of extension products in the presence of an agent for polymerase. A typical primer contains at least about 5 nucleotides in length of a sequence substantially complementary to the target sequence, but somewhat longer primers are preferred.
  • The terms “complementary” or “complement thereof”, as used herein, refer to sequences of polynucleotides which are capable of forming Watson & Crick base pairing with another specified polynucleotide throughout the entirety of the complementary region. This term is applied to pairs of polynucleotides based solely upon their sequences and does not refer to any specific conditions under which the two polynucleotides would actually bind.
  • A primer will always contain a sequence substantially complementary to the target sequence, that is the specific sequence to be amplified, to which it can anneal.
  • In the context of this invention, the term “probe” refers to a molecule which can detectably distinguish between target molecules differing in structure, such as allelic variants. Detection can be accomplished in a variety of different ways but preferably is based on detection of specific binding. Examples of such specific binding include antibody binding and nucleic acid probe hybridization.
  • The term “gene” as used herein is a polynucleotide which may include coding sequences, intervening sequences and regulatory elements controlling transcription and/or translation. Genes of the invention include normal alleles of the gene encoding polymorphisms, including silent alleles having no effect on the amino acid sequence of the gene's encoded polypeptide as well as alleles leading to amino acid sequence variants of the encoded polypeptide that do not substantially affect its function. These terms also may otpyiosnlly include alleles having one or more mutations which affect the function of the encoded polypeptide's function.
  • The polynucleotide compositions, such as primers of the invention, of this invention include RNA, cDNA, DNA complementary to target cDNA of this invention or portion thereof, genomic DNA, unspliced RNA, spliced RNA, alternately spliced RNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
  • Where nucleic acid according to the invention includes RNA, reference to the sequence shown should be construed as reference to the RNA equivalent, with U substituted for T.
  • The term “amount” or “level” of RNA encoded by a gene of the invention, preferably a colorectal cancer biomarker gene described herein, or a housekeeping gene, encompasses the absolute amount of the RNA, the relative amount or concentration of the RNA, as well as any value or parameter which correlates thereto.
  • The methods of nucleic acid isolation, amplification and analysis are routine for one skilled in the art and examples of protocols can be found, for example, in the Molecular Cloning: A Laboratory Manual (3-Volume Set) Ed. Joseph Sambrook, David W. Russel, and Joe Sambrook, Cold Spring Harbor Laboratory; 3rd edition (Jan. 15, 2001), ISBN: 0879695773. Particularly useful protocol source for methods used in PCR amplification is PCR (Basics: From Background to Bench) by M. J. McPherson, S. G. Moller, R. Beynon, C. Howe, Springer Verlag; 1st edition (Oct. 15, 2000), ISBN: 0387916008.
  • “Kit” refers to a combination of physical elements, e.g., probes, including without limitation specific primers, labeled nucleic acid probes, antibodies, protein-capture agent(s), reagent(s), instruction sheet(s) and other elements useful to practice the invention, in particular to identify the levels of particular RNA molecules in a sample. These physical elements can be arranged in any way suitable for carrying out the invention. For example, probes and/or primers can be provided in one or more containers or in an array or microarray device.
  • Colorectal cancer, also called colon cancer or rectal cancer or colorectal carcinoma, is cancer that forms in either the colon or the rectum.
  • The present invention is useful in a diagnostic product or method to detect the level of RNA of genes of interest, in particular, the colorectal biomarkers of the present invention. Accordingly, the invention encompasses the use of diagnostic kits based on a variety of methodologies, e.g., PCR, reverse transcriptase-PCR, quantitative PCR, microarray, chip, mass-spectroscopy, which are capable of detecting RNA levels in a sample. The invention also provides an article of manufacturing comprising packaging material and an analytical agent contained within the packaging material, wherein the analytical agent can be used for determining and/or comparing the levels of RNA encoded by one or more target genes of the invention, and wherein the packaging material comprises a label or package insert which indicates that the analytical agent can be used to identify levels of RNA that correspond to a probability that a test subject has colorectal cancer, such as a probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • The present invention therefore provides kits comprising degenerate primers to amplify polymorphic alleles or variants of target genes of the invention, and instructions comprising an amplification protocol and analysis of the results. The kit may alternatively also comprise buffers, enzymes, and containers for performing the amplification and analysis of the amplification products. The kit may also be a component of a screening or prognostic kit comprising other tools such as DNA micro arrays. The kit may also provides one or more control templates, such as nucleic acids isolated from sample of patientss without colorectal cancer, and/or nucleic acids isolated from ssamples of patients with colorectal cancer.
  • The kit may also include instructions for use of the kit to amplify specific targets on a solid support. Where the kit contains a prepared solid support having a set of primers already fixed on the solid support, e.g. for amplifying a particular set of target polynucleotides, the kit also includes reagents necessary for conducting a PCR on a solid support, for example using an in situ-type or solid phase type PCR procedure where the support is capable of PCR amplification using an in situ-type PCR machine. The PCR reagents, included in the kit, include the usual PCR buffers, a thermostable polymerase (e.g. Taq DNA polymerase), nucleotides (e.g. dNTPs), and other components and labeling molecules (e.g. for direct or indirect labeling). The kits can be assembled to support practice of the PCR amplification method using immobilized primers alone or, alternatively, together with solution phase primers.
  • In one embodiment, the kit provides one or more primer pairs, each pair capable of amplifying RNA encoded by a target gene of the invention, thereby providing a kit for analysis of RNA expression of several different target genes of the invention in a biological sample in one reaction or several parallel reactions. Primers in the kits may be labeled, for example fluorescently labeled, to facilitate detection of the amplification products and consequent analysis of the RNA levels.
  • In one embodiment, levels of RNA encoded by more than one target gene can be determined in one analysis. A combination kit may therefore include primers capable of amplifying cDNA derived from RNA encoded by different target genes. The primers may be differentially labeled, for example using different fluorescent labels, so as to differentiate between RNA from different target genes.
  • Multiplex, such as duplex, real-time RT-PCR enables simultaneous quantification of 2 targets in the same reaction, which saves time, reduces costs, and conserves samples. These advantages of multiplex, real-time RT-PCR make the technique well-suited for high-throughput gene expression analysis. Multiplex qPCR assay in a real-time format facilitates quantitative measurements and minimizes the risk of false-negative results. It is essential that multiplex PCR is optimized so that amplicons of all samples are compared insub-plateau phase of PCR. Yun, Z., I. Lewensohn-Fuchs, P. Ljungman, L. Ringholm, J. Jonsson, and J. Albert. 2003. A real-time TaqMan PCR for routine quantitation of cytomegalovirus DNA in crude leukocyte lysates from stem cell transplant patients. J. Viol. Methods 110:73-79. [PubMed]. Yun, Z., I. Lewensohn-Fuchs, P. Ljungman, and A. Vahlne 2000. Real-time monitoring of cytomegalovirus infections after stem cell transplantation using the TaqMan polymerase chain reaction assays. Transplantation 69:1733-1736. [PubMed]. Simultaneous quantification of up to 2, 3, 4, 5, 6, 7, and 8 or more targets may be useful.
  • The primers and probes contained within the kit may include those listed in 19, and various subcombinations thereof.
  • A “control population” refers to a defined group of individuals or a group of individuals with or without colorectal cancer, and may optionally be further identified by, but not limited to geographic, ethnic, race, gender, one or more other conditions or diseases, and/or cultural indices. In most cases a control population may encompass at least 10, 50, 100, 1000, or more individuals.
  • “Positive control data” encompasses data representing levels of RNA encoded by a target gene of the invention in each of one or more subjects having colorectal cancer of the invention, and encompasses a single data point representing an average level of RNA encoded by a target gene of the invention in a plurality of subjects having colorectal cancer of the invention.
  • “Negative control data” encompasses data representing levels of RNA encoded by a target gene of the invention in each of one or more subjects not having colorectal cancer of the invention, and encompasses a single data point representing an average level of RNA encoded by a target gene of the invention in a plurality of subjects having colorectal cancer of the invention.
  • The probability that test data of the invention “corresponds” to positive control data or negative control data of the invention refers to the probability that the test data is more likely to be characteristic of data obtained in subjects having colorectal cancer than in subjects not having any colorectal pathology, or is more likely to be characteristic of data obtained in subjects not having any colorectal pathology than in subjects having colorectal cancer, respectively.
  • A primer which “selectively hybridizes” to a target polynucleotide is a primer which is capable of hybridizing only, or mostly, with a single target polynucleotide in a mixture of polynucleotides consisting of RNA of human blood, or consisting of DNA complementary to RNA of human blood.
  • A gene expression profile of the invention for colorectal cancer found in blood at the RNA level of one or more genes comprising, but preferably not limited to, an ANXA3 gene, a CLEC4D gene, an IL2RB gene, an LMNB1 gene, a PRRG4 gene, a TNFAIP6 gene and a VNN1 gene, can be identified or confirmed using many techniques, including but preferably not limited to PCR methods, as for example discussed further in the working examples herein, Northern analyses and and the microarray technique. This gene expression profile can be measured in a bodily sample, such as blood, using microarray technology. In an embodiment of this method, fluorescently labeled cDNA probes may be generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from blood. Labeled cDNA probes applied to the chip hybridize with specificity to each spot of DNA on the array. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance. For example, with dual color fluorescence, separately labeled cDNA probes generated from two sources of RNA are hybridized pair wise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously. Such methods have been shown to have the sensitivity required to detect rare transcripts, which are expressed at a few copies per cell, and to reproducibly detect at least approximately two-fold differences in the expression levels (Schena et al., Proc. Natl. Acad. Sci. USA 93(2):106-149 (1996)). Microarray analysis can be performed by commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GenChip technology, or Incyte's micro array technology.
  • Other features and advantages of the invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • The invention will now be described in relation to the drawings in which:
  • FIGS. 1A-H are sequence diagrams depicting the nucleotide sequences of the following genes: ACTB, ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, respectively.
  • FIG. 2 is a schematic depicting an exemplary computer system for practicing certain of the methods described herein.
  • DETAILED DESCRIPTION
  • The invention is of methods, kits, computer systems and computer-readable media for determining a probability that a human subject has colorectal cancer. Specifically, the invention can be used to determine such a probability via analysis of novel markers of colorectal cancer in blood which are disclosed herein.
  • Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
  • Effective methods of testing for colorectal cancer via analysis of blood markers would overcome critical disadvantages of prior art methods, which are excessively invasive, cumbersome, risky, unavailable and/or associated with low patient screening compliance rates. While various approaches have been proposed in the prior art for colorectal cancer testing via analysis of markers of this disease in blood (reviewed in Hundt S. et al. Blood markers for early detection of colorectal cancer: a systematic review. Cancer Epidemiol Biomarkers Prey. 2007 October; 16(10):1935-53), none of these approaches, however, has demonstrated a capacity to satisfactorily enable determination of the probability that a test subject has colorectal cancer as opposed to not having colorectal cancer.
  • Thus, the prior art fails to provide an effective method of testing a subject for colorectal cancer via analysis in a blood sample of levels of RNA encoded by one or more of the genes ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 blood markers.
  • While reducing the invention to practice it was surprisingly uncovered that levels of RNA encoded by the genes ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 are significantly higher in blood of subjects having colorectal cancer than in blood of subjects not having any colorectal pathology, and that levels of RNA encoded by IL2RB are significantly lower in blood of subjects having colorectal cancer than in blood of subjects not having any colorectal pathology (Example 2). While further reducing the invention to practice, it was surprisingly uncovered that mathematical models based on levels of RNA encoded by the 127 possible combinations of the colorectal cancer marker genes ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 in blood of a test subject could be derived capable of discriminating between subjects having colorectal cancer and subjects not having any colorectal pathology (Example 2). While further reducing the invention to practice, it was surprisingly uncovered that mathematical models based on levels of RNA encoded by the 63 possible combinations of the colorectal cancer marker genes ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6, and VNN1 in blood of a test subject, when normalized against levels of RNA encoded by IL2RB, could be derived capable of discriminating between subjects having colorectal cancer and subjects not having any colorectal pathology (Example 3). It will be appreciated that application of such mathematical models to test data representing blood levels in a test subject of RNA encoded by the aforementioned novel colorectal cancer marker genes disclosed herein can be used to provide the probability that the test subject has colorectal cancer as opposed to not having any colorectal pathology.
  • While reducing the invention to practice, fold changes of blood levels of RNA encoded by ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, including fold-changes of levels normalized to IL2RB, in subjects having colorectal cancer relative to subjects not having any colorectal pathology were surprisingly uncovered (Example 2, Example 3 and Example 6).
  • Thus, according to one aspect of the invention there is provided a method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer. In a first step, the method is effected by determining, for each gene of a set of one or more of the colorectal cancer marker genes: ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1; a level of RNA encoded by the gene in blood of the test subject, thereby generating test data. In a second step, the method is effected by determining the probability that the test data corresponds to positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer and not to negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer. The probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • Thus, according to an aspect of the invention, there is provided a method of classifying a test subject as being more likely to have colorectal cancer than to not have colorectal cancer. The method of classifying is effected by determining a level of RNA encoded by one or more of the set of genes consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and/or VNN1 in blood of the test subject, to thereby generate test data and applying to the test data, and to negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, a mathematical formula for generating a value indicating, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, and indicating, for IL2RB, whether the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer. For ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, and indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer; and where, for IL2RB, an indication by the value that the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • Determining whether the level of RNA encoded by ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 or VNN1 in blood of the test subject is higher than the level of RNA encoded by the gene in blood of control subjects not having colorectal cancer may be effected by determining whether there is a fold-change in the level between the test subject and the control subjects not having colorectal cancer which is higher than a minimum fold-change and/or which is within a range of fold-changes.
  • Determining whether the level of RNA encoded by IL2RB in blood of the test subject is lower than the level of RNA encoded by the gene in blood of control subjects not having colorectal cancer may be effected by determining whether there is a fold-change in the level between the test subject and the control subjects not having colorectal cancer which is lower than a maximum fold-change and/or which is within a range of fold-changes.
  • Examples of suitable fold-changes and ranges of fold-changes for classifying a test subject according to the invention are provided in Example 2, Example 3 and Example 6, below, and include the following ones.
  • For levels of RNA encoded by ANXA3, a suitable minimum fold-change is about 1.6 fold, and a suitable range of fold-changes is about 1.6 to about 11.5 fold, relative to an average level of RNA encoded by the housekeeping gene in blood of subjects not having any colorectal pathology.
  • For levels of RNA encoded by CLEC4D, a suitable minimum fold-change is which is about 1.4 fold, and a suitable range of fold-changes is which is about 1.4 to about 15.9 fold, relative to an average level of RNA encoded by the housekeeping gene in blood of subjects not having any colorectal pathology.
  • For levels of RNA encoded by LMNB1, a suitable minimum fold-change is about 1.3 fold, and a suitable range of fold-changes is about 1.3 to about 7.0 fold, relative to an average level of RNA encoded by the housekeeping gene in blood of subjects not having any colorectal pathology.
  • For levels of RNA encoded by PRRG4, a suitable minimum fold-change is about 1.5 fold, and a suitable range of fold-changes is about 1.5 to about 6.3 fold, relative to an average level of RNA encoded by the housekeeping gene in blood of subjects not having any colorectal pathology.
  • For levels of RNA encoded by TNFAIP6, a suitable minimum fold-change is about 1.4 fold, and a suitable range of fold-changes is about 1.45 to about 16.8 fold, relative to an average level of RNA encoded by the housekeeping gene in blood of subjects not having any colorectal pathology.
  • For levels of RNA encoded by VNN1, a suitable minimum fold-change is about 1.5 fold, and a suitable range of fold-changes is about 1.45 to about 23.6 fold, relative to an average level of RNA encoded by the housekeeping gene in blood of subjects not having any colorectal pathology.
  • For levels of RNA encoded by IL2RB, a suitable maximum fold-change is about 0.8 fold, and a suitable range of fold-changes is about 0.8 to about 0.1 fold, relative to an average level of RNA encoded by the housekeeping gene in blood of subjects not having any colorectal pathology.
  • For levels of RNA encoded by ANXA3 normalized to IL2RB, a suitable minimum fold-change is about 1.7 fold, and a suitable range of fold-changes is about 1.7 to about 20.7 fold, relative to an average level of RNA encoded by IL2RB in blood of subjects not having any colorectal pathology.
  • For levels of RNA encoded by CLEC4D normalized to IL2RB, a suitable minimum fold-change is which is about 1.5 fold, and a suitable range of fold-changes is which is about 1.5 to about 12.0 fold, relative to an average level of RNA encoded by IL2RB in blood of subjects not having any colorectal pathology.
  • For levels of RNA encoded by LMNB1 normalized to IL2RB, a suitable minimum fold-change is about 1.5 fold, and a suitable range of fold-changes is about 1.5 to about 10.6 fold, relative to an average level of RNA encoded by IL2RB in blood of subjects not having any colorectal pathology.
  • For levels of RNA encoded by PRRG4 normalized to IL2RB, a suitable minimum fold-change is about 1.3 fold, and a suitable range of fold-changes is about 1.3 to about 13.1 fold, relative to an average level of RNA encoded by IL2RB in blood of subjects not having any colorectal pathology.
  • For levels of RNA encoded by TNFAIP6 normalized to IL2RB, a suitable minimum fold-change is about 1.5 fold, and a suitable range of fold-changes is about 1.5 to about 16.4 fold, relative to an average level of RNA encoded by IL2RB in blood of subjects not having any colorectal pathology.
  • For levels of RNA encoded by VNN1 normalized to IL2RB, a suitable minimum fold-change is about 1.3 fold, and a suitable range of fold-changes is about 1.3 to about 11.9 fold, relative to an average level of RNA encoded by IL2RB in blood of subjects not having any colorectal pathology.
  • As used herein, the term “about” refers to a variability of plus or minus 10 percent. Thus, a test subject of the invention is classified as being more likely to have colorectal cancer than to not have colorectal cancer if, for each marker gene of the particular set of marker genes of the invention used to practice the method of classifying of the invention, the fold-change in level of RNA encoded by that gene in blood of the test subject relative to blood of the control subjects not having any colorectal cancer pathology classifies, according to the teachings of the invention, the test subject as being more likely to have colorectal cancer than to not have colorectal cancer.
  • Conversely, a test subject of the invention is classified as being more likely to not have colorectal cancer than to have colorectal cancer if, for each marker gene of the particular set of marker genes of the invention used to practice the method of classifying of the invention, the fold-change in level of RNA encoded by that gene in blood of the test subject relative to blood of the control subjects not having any colorectal cancer pathology does not classify, according to the teachings of the invention, the test subject as being more likely to have colorectal cancer than to not have colorectal cancer.
  • In one aspect of the invention, the set of one or more colorectal cancer marker genes may consist of any one of the possible combinations of one or more of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 (indicated in Table 6, where each logistic regression model is based on one particular gene combination, and each gene of the combination is assigned a logistic regression coefficient value).
  • Sets of marker genes of the invention which consist of one or more of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 which can be used to practice the invention include: ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6, VNN1; ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, VNN1; ANXA3, CLEC4D, IL2RB, PRRG4; ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4; ANXA3, CLEC4D, IL2RB, PRRG4, VNN1; ANXA3, IL2RB, LMNB1, PRRG4, VNN1; ANXA3, CLEC4D, IL2RB, PRRG4, TNFAIP6; ANXA3, IL2RB, LMNB1, PRRG4, TNFAIP6; ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6; ANXA3, CLEC4D, IL2RB, PRRG4, TNFAIP6, VNN1; ANXA3, IL2RB, LMNB1, PRRG4, TNFAIP6, VNN1; ANXA3, IL2RB, LMNB1, PRRG4; IL2RB, PRRG4, VNN1; ANXA3, IL2RB, PRRG4, VNN1; CLEC4D, IL2RB, PRRG4, VNN1; IL2RB, LMNB1, PRRG4, VNN1; CLEC4D, IL2RB, LMNB1, PRRG4, VNN1; ANXA3, IL2RB, PRRG4, TNFAIP6; IL2RB, PRRG4, TNFAIP6, VNN1; ANXA3, IL2RB, PRRG4, TNFAIP6, VNN1; CLEC4D, IL2RB, PRRG4, TNFAIP6, VNN1; IL2RB, LMNB1, PRRG4, TNFAIP6, VNN1; CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6, VNN1; IL2RB, PRRG4; ANXA3, IL2RB, PRRG4; CLEC4D, IL2RB, PRRG4; IL2RB, LMNB1, PRRG4; CLEC4D, IL2RB, LMNB1, PRRG4; IL2RB, PRRG4, TNFAIP6; CLEC4D, IL2RB, PRRG4, TNFAIP6; IL2RB, LMNB1, PRRG4, TNFAIP6; CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6; ANXA3, IL2RB, VNN1; ANXA3, CLEC4D, IL2RB, VNN1; ANXA3, IL2RB, LMNB1, VNN1; ANXA3, CLEC4D, IL2RB, LMNB1, VNN1; ANXA3, CLEC4D, LMNB1, PRRG4, VNN1; ANXA3, IL2RB, TNFAIP6, VNN1; ANXA3, CLEC4D, IL2RB, TNFAIP6, VNN1; ANXA3, IL2RB, LMNB1, TNFAIP6, VNN1; ANXA3, CLEC4D, IL2RB, LMNB1, TNFAIP6, VNN1; ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6, VNN1; ANXA3, IL2RB; ANXA3, CLEC4D, IL2RB; ANXA3, IL2RB, LMNB1; ANXA3, CLEC4D, IL2RB, LMNB1; ANXA3, CLEC4D, LMNB1, PRRG4; CLEC4D, IL2RB, LMNB1, VNN1; ANXA3, IL2RB, TNFAIP6; ANXA3, CLEC4D, IL2RB, TNFAIP6; ANXA3, IL2RB, LMNB1, TNFAIP6; ANXA3, CLEC4D, IL2RB, LMNB1, TNFAIP6; ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6; IL2RB, LMNB1, TNFAIP6, VNN1; CLEC4D, IL2RB, LMNB1, TNFAIP6, VNN1; IL2RB, LMNB1, VNN1; ANXA3, LMNB1, PRRG4, VNN1; ANXA3, LMNB1, PRRG4, TNFAIP6, VNN1; ANXA3, CLEC4D, PRRG4; ANXA3, LMNB1, PRRG4; CLEC4D, IL2RB, VNN1; ANXA3, CLEC4D, PRRG4, VNN1; IL2RB, LMNB1, TNFAIP6; CLEC4D, IL2RB, LMNB1, TNFAIP6; ANXA3, CLEC4D, PRRG4, TNFAIP6; ANXA3, LMNB1, PRRG4, TNFAIP6; IL2RB, TNFAIP6, VNN1; CLEC4D, IL2RB, TNFAIP6, VNN1; ANXA3, CLEC4D, PRRG4, TNFAIP6, VNN1; IL2RB, LMNB1; CLEC4D, IL2RB, LMNB1; IL2RB, VNN1; ANXA3, CLEC4D, LMNB1, VNN1; ANXA3, CLEC4D, LMNB1, TNFAIP6, VNN1; ANXA3, CLEC4D, LMNB1; ANXA3, PRRG4; ANXA3, CLEC4D, VNN1; ANXA3, LMNB1, VNN1; ANXA3, PRRG4, VNN1; ANXA3, CLEC4D, LMNB1, TNFAIP6; ANXA3, PRRG4, TNFAIP6; ANXA3, CLEC4D, TNFAIP6, VNN1; ANXA3, LMNB1, TNFAIP6, VNN1; ANXA3, PRRG4, TNFAIP6, VNN1; ANXA3; ANXA3, CLEC4D; ANXA3, LMNB1; ANXA3, VNN1; ANXA3, TNFAIP6; ANXA3, CLEC4D, TNFAIP6; IL2RB, TNFAIP6; CLEC4D, IL2RB, TNFAIP6; ANXA3, LMNB1, TNFAIP6; ANXA3, TNFAIP6, VNN1; CLEC4D, IL2RB; PRRG4, VNN1; CLEC4D, PRRG4, VNN1; LMNB1, PRRG4, VNN1; CLEC4D, LMNB1, PRRG4, VNN1; PRRG4, TNFAIP6, VNN1; CLEC4D, PRRG4, TNFAIP6, VNN1; LMNB1, PRRG4, TNFAIP6, VNN1; CLEC4D, LMNB1, PRRG4, TNFAIP6, VNN1; PRRG4; CLEC4D, PRRG4; LMNB1, PRRG4; CLEC4D, LMNB1, PRRG4; PRRG4, TNFAIP6; CLEC4D, PRRG4, TNFAIP6; LMNB1, PRRG4, TNFAIP6; CLEC4D, LMNB1, PRRG4, TNFAIP6; LMNB1, TNFAIP6, VNN1; CLEC4D, VNN1; LMNB1, VNN1; CLEC4D, LMNB1, VNN1; LMNB1, TNFAIP6; LMNB1, TNFAIP6; TNFAIP6, VNN1; CLEC4D, TNFAIP6, VNN1; CLEC4D, LMNB1, TNFAIP6, VNN1; LMNB1; CLEC4D, LMNB1; VNN1; CLEC4D, TNFAIP6; TNFAIP6; CLEC4D; and IL2RB.
  • According to the aspect of the invention where the set of one or more colorectal cancer marker genes consists of any one of the 127 possible combinations of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, the level of RNA encoded by a gene of the invention in blood of a subject of the invention may be determined as a ratio to a level of RNA encoded by a housekeeping gene in blood of the subject. It will be appreciated that such measurement of a level or RNA encoded by a gene relative to that of a housekeeping gene within individual samples can be used to control for sample to sample variability.
  • The housekeeping gene may be any one of various genes expressed in blood known to the ordinarily skilled artisan. In one aspect of the method, the housekeeping gene is ACTB. Alternately, the housekeeping gene may encode 18S rRNA.
  • Nucleotide sequences of target genes of the invention (ACTB, ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1) are described in Figures lA-H and in Table 1, below.
  • In another aspect of the invention, the set of one or more colorectal cancer marker genes may consist of any one of the possible combinations of one or more of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 (indicated in Table 5, where each logistic regression model is based on one particular gene combination, and each gene of the combination is assigned a logistic regression coefficient value).
  • The possible combinations of one or more of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 which can be used to practice the invention include: ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1; ANXA3, LMNB1, PRRG4, TNFAIP6 and VNN1; ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6; ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1; ANXA3, PRRG4, TNFAIP6 and VNN1; CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1; ANXA3, PRRG4 and TNFAIP6; CLEC4D, LMNB1, PRRG4 and TNFAIP6; ANXA3, CLEC4D, PRRG4, TNFAIP6 and VNN1; ANXA3, CLEC4D, PRRG4 and TNFAIP6; ANXA3, LMNB1, PRRG4 and VNN1; ANXA3, LMNB1, PRRG4 and TNFAIP6; CLEC4D, LMNB1, PRRG4 and VNN1; ANXA3, CLEC4D, LMNB1, PRRG4; ANXA3, CLEC4D, PRRG4 and VNN1; LMNB1, PRRG4 and VNN1; LMNB1, PRRG4, TNFAIP6 and VNN1; LMNB1, PRRG4 and TNFAIP6; ANXA3, CLEC4D and PRRG4; ANXA3, LMNB1 and PRRG4; ANXA3 and PRRG4; ANXA3, PRRG4 and VNN1; CLEC4D, LMNB1 and PRRG4; LMNB1 and PRRG4; CLEC4D, PRRG4, TNFAIP6 and VNN1; CLEC4D, PRRG4 and TNFAIP6; CLEC4D, PRRG4 and VNN1; CLEC4D and PRRG4; PRRG4, TNFAIP6 and VNN1; PRRG4 and VNN1; PRRG4 and TNFAIP6; PRRG4; TNFAIP6 and VNN1; VNN1; ANXA3, TNFAIP6 and VNN1; ANXA3, LMNB1, TNFAIP6 and VNN1; LMNB1, TNFAIP6 and VNN1; CLEC4D, TNFAIP6 and VNN1; ANXA3, CLEC4D, TNFAIP6 and VNN1; ANXA3, CLEC4D, LMNB1, TNFAIP6 and VNN1; CLEC4D, LMNB1, TNFAIP6 and VNN1; ANXA3 and VNN1; ANXA3, CLEC4D, LMNB1 and TNFAIP6; CLEC4D, LMNB1 and TNFAIP6; CLEC4D and VNN1; LMNB1 and VNN1; ANXA3, CLEC4D and VNN1; ANXA3, LMNB1 and VNN1; ANXA3, LMNB1 and TNFAIP6; LMNB1 and TNFAIP6; CLEC4D, LMNB1 and VNN1; ANXA3, CLEC4D, LMNB1 and VNN1; ANXA3, CLEC4D and TNFAIP6; CLEC4D and TNFAIP6; CLEC4D, LMNB1; ANXA3, CLEC4D and LMNB1; LMNB1; ANXA3 and TNFAIP6; ANXA3 and LMNB1; TNFAIP6; ANXA3 and CLEC4D; CLEC4D; and ANXA3.
  • According to the aspect of the invention where the set of one or more colorectal cancer marker genes consists of any one of the 63 possible combinations of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, the level of RNA encoded by a gene of the invention in blood of a subject of the invention may be determined as a ratio to a level of RNA encoded by IL2RB in blood of the subject.
  • It will be appreciated that data representing levels of RNA encoded by a set of genes of the invention may be combined with data representing levels of gene products of other genes which are differently expressed in blood in subjects having colorectal cancer relative to subjects not having any colorectal pathology so as to determine a probability that a test subject has colorectal cancer versus not having any colorectal pathology.
  • In another aspect, the method further comprises determining levels of RNA encoded by the gene in blood of a population of control human subjects having colorectal cancer, and/or in blood of a population of human control subjects not having colorectal cancer, to thereby provide the positive control data and/or the negative control data, respectively. Alternately, it is envisaged that the level of RNA encoded by a gene of the invention in control subjects of the invention could be provided by prior art data corresponding to control data of the invention.
  • The method of the invention may be practiced using any one of various types of control subjects.
  • In an aspect of the method of the invention, the control subjects not having colon cancer are subjects having been diagnosed as not having any colorectal pathology as a result of colonoscopic examination. As is described in the Examples section which follows, the method of the invention may be practiced using subjects not having any colorectal pathology as the control subjects not having colorectal cancer.
  • In an aspect of the method of the invention, the control subjects having colorectal cancer are subjects having been diagnosed as having colorectal cancer as a result of colonoscopic examination. As is described in the Examples section which follows, the method of the invention may be practiced using subjects diagnosed as not having any colorectal pathology as the control subjects not having colorectal cancer.
  • The method of the invention may furthermore be practiced using any one of various numbers of control subjects. One of ordinary skill in the art will possess the necessary expertise to select a sufficient number of control subjects so as to obtain control data having a desired statistical significance for practicing the method of the invention with a desired level of reliability.
  • For example, the method of the invention can be practiced using 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, or 200 or more of control subjects having colorectal cancer and/or of control subjects not having colorectal cancer.
  • In one aspect of the invention, the level of RNA encoded by a gene of the invention in blood of the test subject and the levels of RNA encoded by the gene in blood of the control subjects are determined via the same method. As is described in the Examples section, below, the method can be practiced where the level of RNA encoded by a gene of the invention in blood of the test subject and the levels of RNA encoded by the gene in blood of the control subjects are determined via the same method. Alternately, it is envisaged that the level of a gene of the invention in blood of a test subject of the invention and in blood of control subjects of the invention could be determined using different methods. It will be appreciated that use of the same method to determine the levels of RNA encoded by a gene of the invention in a test subject and in control subjects of the invention can be used to avoid method-to-method calibration to minimize any variability which might arise from use of different methods.
  • In one aspect of the method, determining of the level of RNA encoded by a gene of the invention in blood of a subject of the invention is effected by determining the level of RNA encoded by the gene in a blood sample isolated from the subject. Alternately, it is envisaged that determining of the level of RNA encoded by the gene in blood of a subject of the invention could be effected by determining the level of RNA encoded by the gene in an in-vivo sample using a suitable method for such a purpose.
  • In one aspect of the method, the level of RNA encoded by a gene of the invention in blood of a subject of the invention is determined in a sample of RNA isolated from blood of the subject. Alternately, it is envisaged that the level of RNA of a gene of the invention in blood of a subject of the invention could be determined in a sample which includes RNA of blood of the subject but from which RNA has not been isolated therefrom, using a suitable method for such a purpose.
  • Any one of various methods routinely employed in the art for isolating RNA from blood may be used to isolate RNA from blood of a subject of the invention, so as to enable practicing of the method of the invention.
  • In one aspect of the method, the level of RNA encoded by a gene of the invention in blood of a subject of the invention is determined in RNA of a sample of whole blood. Any one of various methods routinely employed in the art for isolating RNA from whole blood may be employed for practicing the method.
  • Alternately, it is envisaged that the level of RNA encoded by a gene of the invention in blood of a subject of the invention could be determined in RNA of a sample of fraction of blood which expresses the gene sufficiently specifically so as to enable the method. Examples of such blood fractions include preparations of isolated types of leukocytes, preparations of isolated peripheral blood mononuclear cells, preparations of isolated granulocytes, preparations of isolated whole leukocytes, preparations of isolated specific types of leukocytes, plasma-depleted blood, preparations of isolated lymphocytes, and the plasma fraction of blood.
  • In one aspect of the method, isolation of RNA from whole blood of a subject of the invention is effected by using a PAXgene Blood RNA Tube (obtainable from PreAnalytiX) in accordance with the instructions of the PAXgene Blood RNA Kit protocol. As is described in the Examples section below, the method of the invention may be practiced by determining a level of a gene of the invention in RNA isolated from blood from test and control subjects of the invention using PAXgene Blood RNA Tubes.
  • Determining of a level of RNA encoded by a gene of the invention in a sample of the invention may be effected in any one of various ways routinely practiced in the art.
  • For example, the level of RNA encoded by a gene of the invention in a sample of the invention may be determined via any one of various methods based on quantitative polynucleotide amplification which are routinely employed in the art for determining a level of RNA encoded by a gene in a sample.
  • Alternately, the level of RNA encoded by a gene of the invention may be determined via any one of various methods based on quantitative polynucleotide hybridization to an immobilized probe which are routinely employed in the art for determining a level of RNA encoded by a gene in a sample.
  • In one aspect of the method of the invention, the method based on quantitative polynucleotide amplification used to determine the level of RNA encoded by a gene of the invention is quantitative reverse transcriptase-polymerase chain reaction (PCR) analysis. Any one of various types of quantitative reverse transcriptase-PCR analyses routinely employed in the art to determine the level of RNA encoded by a gene in a sample may be used to practice the invention. For example, any one of various sets of primers may be used to perform quantitative reverse transcriptase-PCR analysis so as to practice the method of the invention.
  • In one aspect of the method of the invention, the quantitative reverse transcriptase-PCR analysis used to determine the level of RNA encoded by a gene of the invention is quantitative real-time PCR analysis of DNA complementary to RNA encoded by the gene using a labeled probe capable of specifically binding amplification product of DNA complementary to RNA encoded by the gene. For example, quantitative real-time PCR analysis may be performed using a labeled probe which comprises a polynucleotide capable of selectively hybridizing with a sense or antisense strand of amplification product of DNA complementary to RNA encoded by the gene. Labeled probes comprising a polynucleotide having any one of various nucleic acid sequences capable of specifically hybridizing with amplification product of DNA complementary to RNA encoded by the gene may be used to practice the method of the invention.
  • Quantitative real-time PCR analysis of a level of RNA encoded by a gene of the invention may be performed in any one of various ways routinely employed in the art.
  • In one aspect of the method of the invention, quantitative real-time PCR analysis is performed by analyzing complementary DNA prepared from RNA of blood a subject of the invention, using the QuantiTect™ Probe RT-PCR system (Qiagen, Valencia, Calif.; Product Number 204345), a TaqMan dual labelled probe, and a Real-Time PCR System 7500 instrument (Applied Biosystems). As is described in the Examples section which follows, such quantitative real-time PCR analysis may be used to practice the method of the invention.
  • As specified above, the level of RNA encoded by a gene of the invention may be determined via a method based on quantitative polynucleotide hybridization to an immobilized probe.
  • In one aspect, determining of the level of RNA encoded by a gene of the invention via a method based on quantitative polynucleotide hybridization is effected using a microarray, such as an Affymetrix U133Plus 2.0 GeneChip oligonucleotide array (Affymetrix; Santa Clara, Calif.).
  • As specified above, the level of RNA encoded by a gene of the invention in a sample of the invention may be determined via quantitative reverse transcriptase-PCR analysis using any one of various sets of primers and labeled probes to amplify and quantitate DNA complementary to RNA encoded by a marker gene of the invention produced during such analysis. Examples of suitable primers for use in quantitative reverse transcriptase-PCR analysis of the level of RNA encoded by a target gene of the invention are listed in Table 19. This table further lists examples of suitable polynucleotides comprised in labeled probes for practicing quantitative real-time PCR analysis according to the method of the invention.
  • TABLE 19
    PCR primers and matching polynucleotides of labeled probes for quantitative PCR analysis.
    Gene
    encoding Primer/
    amplified Assay Nucleic acid sequences of PCR primers and matching probe Amplicon
    cDNA reagent polynucleotides comprised in labeled probes position size (bp)
    ACTB 5′ 5′-CACCACACCTTCTACAATGAGCTG-3′ (SEQ ID NO: 1) 259 158
    primer 5′-ACAGCCTGGATAGCAACGTACA-3′ (SEQ ID NO: 2) 416
    3′ 5′-AACCGCGAGAAGATGACCCAGATCAT-3′ (SEQ ID NO: 3) 343
    primer
    probe
    5′ 5′-ACCTTCTACAATGAGCTGCG-3′ (SEQ ID NO: 4) 337 114
    primer 5′-GGTCTCAAACATGATCTGGGTC-3′ (SEQ ID NO: 5) 450
    3′ 5′-AAGGCCAACCGCGAGAAGAT-3′ (SEQ ID NO: 6) 409
    primer
    probe
    5′ 5′-CACCCAGCACAATGAAGATC-3′ (SEQ ID NO: 7) 1034 119
    primer 5′-CTGCTTGCTGATCCACATCT-3′ (SEQ ID NO: 8) 1152
    3′ 5′-ATCATTGCTCCTCCTGAGCG-3′ (SEQ ID NO: 9) 1057
    primer
    probe
    ANXA3 5′ 5′-GAAACATCTGGTGACTTCCG-3′ (SEQ ID NO: 10) 748 103
    primer 5′-TCTGGGCATCTTGTTTGG-3 ′ (SEQ ID NO: 11) 850
    3′ 5′-TTGACTTTGGCAGATGGCAGA-3′ (SEQ ID NO: 12) 778
    primer
    probe
    5′ 5′-GGAACAAACGAAGATGCCTTG-3′ (SEQ ID NO: 13) 628 137
    primer 5′-AAGTCACCAGATGTTTCGGA-3′ (SEQ ID NO: 14) 764
    3′ 5′-ATCTTAACTACCAGGACAAGCAGGCA-3′ (SEQ ID NO: 15) 655
    primer
    probe
    5′ 5′-CTACCAGGACAAGCAGGCAA-3′ (SEQ ID NO: 16) 662 138
    primer 5′-TTCTGCCATCTGCCAAAGT-3′ (SEQ ID NO: 17) 799
    3′ 5′-TCCGAAACATCTGGTGACTTCC-3′ (SEQ ID NO: 18) 745
    primer
    probe
    CLEC4D 5′ 5′-CCATTTAACCCACGCAGAG-3′ (SEQ ID NO: 19) 673 101
    primer 5′-CAGGCCCATTTATCTTGGTT-3′ (SEQ ID NO: 20) 773
    3′ 5′-CTGGCATAAGAATGAACCCGACA-3′ (SEQ ID NO: 21) 696
    primer
    probe
    5′ 5′-TCCGAAACATCTGGTGACTTCC-3′ (SEQ ID NO: 22) 406 118
    primer 5′-TCCTTTCACTCTCAGCCCAC-3′ (SEQ ID NO: 23) 523
    3′ 5′-ATGACCATCAGCACGGAAGC-3′ (SEQ ID NO: 24) 550
    primer
    probe
    5′ 5′-GGGCTGAGAGTGAAAGGAAC-3′ (SEQ ID NO: 25) 506 149
    primer 5′-CCACTGACCTTTGGCATTC-3′ (SEQ ID NO: 26) 654
    3′ 5′-ATGACCATCAGCACGGAAGC-3′ (SEQ ID NO: 27) 550
    primer
    probe
    IL2RB 5′ 5′-AAATCTCCCAAGCCTCCCA-3′ (SEQ ID NO: 28) 588 127
    primer 5′-AGGCAGATCCATTCCTGCT-3′ (SEQ ID NO: 29) 714
    3′ 5′-TTGAAAGACACCTGGAGTTCG-3 ′ (SEQ ID NO: 30) 612
    primer
    probe
    5′ 5′-GACCCACAGATGCAACATAAG-3′ (SEQ ID NO: 31) 562 137
    primer 5′-GCTTCTGCTTGAGAGTCAGC-3′ (SEQ ID NO: 32) 698
    3′ 5′-AAATCTCCCAAGCCTCCCAC-3′ (SEQ ID NO: 33) 588
    primer
    probe
    5′ 5′-TGGAGACCCACAGATGCAA-3′ (SEQ ID NO: 34) 558 141
    primer 5′-GCTTCTGCTTGAGAGTCAGC-3′ (SEQ ID NO: 35) 698
    3′ 5′-AAATCTCCCAAGCCTCCCAC-3′ (SEQ ID NO: 36) 588
    primer
    probe
    LMNB1 5′ 5′-GGAGTGGTTGTTGAGGAAGAA-3′ (SEQ ID NO: 37) 2051 151
    primer 5′-CTGAGAAGGCTCTGCACTGTA-3 ′ (SEQ ID NO: 38) 2201
    3′ 5′-AACCCCAAGAGCATCCAATAG-3′ (SEQ ID NO: 39) 2089
    primer
    probe
    5′ 5′-CTGGCGAAGATGTGAAGGT-3′ (SEQ ID NO: 40) 1935 135
    primer 5′-CTTCCTCAACAACCACTCCA-3′ (SEQ ID NO: 41) 2069
    3′ 5′-AATTCTCAGGGAGAGGAGGTTG-3′ (SEQ ID NO: 42) 1964
    primer
    probe
    5′ 5′-AGGCGAAGAAGAGAGGTTGAAG-3′ (SEQ ID NO: 43) 1513 103
    primer 5′-CCGCTTTCCTCTAGTTGTACG-3′ (SEQ ID NO: 44) 1615
    3′ 5′-TGTCTCCAAGCCCTTCTTCC-3′ (SEQ ID NO: 45) 1536
    primer
    probe
    PRRG4 5′ 5′-ATGCGGGAGAAGAAGTGTTTAC-3′ (SEQ ID NO: 46) 341 153
    primer 5′-CTCTGGCTTCCTCATAATTGC-3′ (SEQ ID NO: 47) 493
    3′ 5′-CTCTTCACTCCCGGCAACCTAGAA-3′ (SEQ ID NO: 48) 427
    primer
    probe
    5′ 5′-TGCTGCTGGAGTATTTTTGG-3′ (SEQ ID NO: 49) 618 130
    primer 5′-AATGATGGAGGGAGTGTGC-3′ (SEQ ID NO: 50) 747
    3′ 5′-AACATCCATGCTCTTCAGCC-3′ (SEQ ID NO: 51) 693
    primer
    probe
    5′ 5′-ACTCCCGGCAACCTAGAAAG-3′ (SEQ ID NO: 52) 433 176
    primer 5′-GTCAGAAGGCCCATAACATCTA-3′ (SEQ ID NO: 53) 608
    3′ 5′-AACGATTGCATTTTGGCAGG-3′ (SEQ ID NO: 54) 517
    primer
    probe
    TNFAIP6 5′ 5′-GCCTATTGCTACAACCCACA-3′ (SEQ ID NO: 55) 448 84
    primer 5′-TGGGAAGCCTGGAGATTTA-3′ (SEQ ID NO: 56) 531
    3′ 5′-AAGGAGTGTGGTGGCGTCTTTAC-3′ (SEQ ID NO: 57) 472
    primer
    probe
    5′ 5′-CAGGTTGCTTGGCTGATTATG-3 ′ (SEQ ID NO: 58) 632 172
    primer 5′-TTGATTTGGAAACCTCCAGC-3′ (SEQ ID NO: 59) 803
    3′ 5′-TGGCTTTGTGGGAAGATACTGTGG-3′ (SEQ ID NO: 60) 684
    primer
    probe
    5′ 5′-CATTAGACTCAAGTATGGTCAGCG-3′ (SEQ ID NO: 61) 567 142
    primer 5′-TCCACAGTATCTTCCCACAAAG-3′ (SEQ ID NO: 62) 708
    3′ 5′-CAGGTTGCTTGGCTGATTATGT-3′ (SEQ ID NO: 63) 632
    primer
    probe
    VNN1 5′ 5′-TGACAGGAAGTGGCATCTAT-3′ (SEQ ID NO: 64) 835 147
    primer 5′-TACTGCTGGCATAGGAAGTC-3′ (SEQ ID NO: 65) 981
    3′ 5′-AGAAGAGGGAAAACTCCTCCTCTCG-3′ (SEQ ID NO: 66) 896
    primer
    probe
    5′ 5′-CTGGAGAATTTCAGGTGTCA-3′ (SEQ ID NO: 67) 1360 111
    primer 5′-ATGCCCAGTCCTTCTCATAC-3′ (SEQ ID NO: 68) 1470
    3′ 5′-ACTGACGGACGCTTGTTTAGTCTGA-3′ (SEQ ID NO: 69) 1380
    primer
    probe
    5′ 5′-GTATTCCCAACAGCTTGGAT-3′ (SEQ ID NO: 70) 711 144
    primer 5′-ATAGATGCCACTTCCTGTCA-3′ (SEQ ID NO: 71) 854
    3′ 5′-CATGAGGGTCAATTTCCTTGCATC-3′ (SEQ ID NO: 72) 785
    primer
    probe
  • Determining the level of RNA encoded by the marker gene of the invention as a ratio to a housekeeping gene may be effected in any one of various ways routinely employed in the art for determining a ratio of a level of RNA encoded by one gene to a level of RNA encoded by a housekeeping gene, such as ACTB.
  • In one aspect of the method, determining the level of RNA encoded by the gene of the invention as a ratio to the housekeeping gene is effected via duplex quantitative reverse transcriptase-PCR analysis of RNA encoded by the gene and of RNA encoded by the housekeeping gene in a sample of the invention. Such “duplex quantitative reverse transcriptase PCR analysis” refers to quantitative reverse transcriptase-PCR analysis where DNA complementary to RNA encoded by the gene of the invention and DNA complementary to RNA encoded by the housekeeping gene are co-amplified in the same sample/reaction mixture.
  • DNA complementary to RNA encoded by the housekeeping gene may be amplified via quantitative reverse transcriptase-PCR analysis using any one of various suitable primers.
  • In one aspect, the primers may be selected so as to include a primer having a nucleotide sequence which is complementary to a region of a target cDNA template, where the region spans a splice junction joining a pair of exons. It will be appreciated that such a primer can be used to facilitate amplification of DNA complementary to messenger RNA, i.e. mature spliced RNA.
  • In one aspect of the method, where the housekeeping gene is ACTB, the primers used to amplify DNA complementary to RNA encoded by the housekeeping gene may include a primer having a nucleotide sequence identified as SEQ ID NO: 1, a primer having a nucleotide sequence identified as SEQ ID NO: 2, or both primers.
  • In another aspect of the method, the level of RNA encoded by the housekeeping gene in blood of the test subject is determined via quantitative reverse transcriptase-PCR analysis, using a labeled probe which comprises a polynucleotide capable of hybridizing to a sense or antisense strand of the amplification product of the DNA complementary to RNA encoded by the housekeeping gene.
  • In one aspect of the method where the housekeeping gene is ACTB and where the level of RNA encoded by the housekeeping gene in blood of the test subject is determined via quantitative reverse transcriptase-PCR analysis using a primer having a nucleotide sequence identified as SEQ ID NO: 1, and a primer having a nucleotide sequence identified as SEQ ID NO: 2, and a labeled probe, the probe comprises a polynucleotide having a nucleic acid sequence identified as SEQ ID NO: 3.
  • As is demonstrated in Example 2 of the Examples section which follows, the method of the invention can be practiced by determining the level of RNA encoded by any one of the marker genes ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 as a ratio to a level of RNA encoded by ACTB in blood of a subject of the invention, where the level is determined via duplex quantitative reverse transcriptase-PCR analysis using a primer having a nucleotide sequence identified as SEQ ID NO: 1, a primer having a nucleotide sequence identified as SEQ ID NO: 2, and a labeled probe which comprises a polynucleotide having a nucleic acid sequence identified as SEQ ID NO: 3.
  • Determining the level of RNA encoded by ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 or VNN1 as a ratio to IL2RB may be effected in any one of various ways.
  • In one aspect of the method, determining the level of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 or VNN1 as a ratio to a level of RNA encoded by IL2RB in a sample of the invention is effected via duplex quantitative reverse transcriptase-PCR analysis of RNA encoded by ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 or VNN1 and of RNA encoded by IL2RB in the sample. Such “duplex quantitative reverse transcriptase PCR analysis” refers to quantitative reverse transcriptase-PCR analysis where DNA complementary to RNA encoded by ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 or VNN1 and DNA complementary to RNA encoded by IL2RB are co-amplified in the same sample/reaction mixture.
  • As described above, following the step of obtaining the test data, the method of the invention comprises the step of determining the probability that the test data corresponds to the positive control data and not to the negative control data.
  • It will be appreciated that the probability that the test subject does not have any colorectal pathology as opposed to having colorectal cancer can be readily determined from the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer. For example, when expressing the probability that the test subject has colorectal cancer as a percentage probability, the probability that the test subject does not have any colorectal pathology as opposed to having colorectal cancer corresponds to 100 percent minus the probability that the test subject does not have any colorectal pathology as opposed to having colorectal cancer.
  • Determining the probability that the test data corresponds to the positive control data and not to the negative control data may be effected in any one of various ways known to the ordinarily skilled artisan for determining the probability that a gene expression profile of a test subject corresponds to a gene expression profile of of subjects having a pathology and not to a gene expression profile of subjects not having the pathology, where the gene expression profiles of the subjects having the pathology and the subjects not having the pathology are significantly different.
  • In one aspect of the method, determining the probability that the test data corresponds to the positive control data and not to the negative control data is effected by applying to the test data a mathematical model derived from the positive control data and from the negative control data.
  • Various suitable mathematical models which are well known in the art of medical diagnosis using disease markers may be employed to classify a test subject as more likely to have colorectal cancer than to not have colorectal cancer, to determine a probability that a test subject is likely to have colorectal cancer as opposed to not having colorectal cancer, or to diagnose a test subject as having colorectal cancer according to the teachings of the invention. Generally these mathematical models can be unsupervised methods performing a clustering whilst supervised methods are more suited to classification of datasets. (refer, for example, to: Dreiseitl S, Ohno-Machado L. Logistic regression and artificial neural network classification models: a methodology review. J Biomed Inform. 2002 October-December; 35(5-6):352-9; Pepe M S. The Statistical Evaluation of Medical Tests for Classification and Prediction. Oxford, England: Oxford University Press; 2003; Dupont W D. Statistical Modeling for Biomedical Researchers. Cambridge, England: Cambridge University Press; 2002; Pampel F C. Logistic regression: A Primer. Publication #07-132, Sage Publications: Thousand Oaks, Calif. 2000; King E N, Ryan T P. A preliminary investigation of maximum likelihood logistic regression versus exact logistic regression. Am Statistician 2002; 56:163-170; Metz C E. Basic principles of ROC analysis. Semin Nucl Med 1978; 8:283-98; Swets J A. Measuring the accuracy of diagnostic systems. Science 1988; 240:1285-93; Zweig M H, Campbell G. Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine. Clin Chem 1993; 39:561-77; Witten I H, Frank Eibe. Data Mining: Practical Machine Learning Tools and Techniques (second edition). Morgan Kaufman 2005; Deutsch J M. Evolutionary algorithms for finding optimal gene sets in microarray prediction. Bioinformatics 2003; 19:45-52; Niels Landwehr, Mark Hall and Eibe Frank (2003) Logistic Model Trees. pp 241-252 in Machine Learning: ECML 2003: 14th European Conference on Machine Learning, Cavtat-Dubrovnik, Croatia, September 22-26, 2003, Proceedings Publisher: Springer-Verlag GmbH, ISSN: 0302-9743). Examples of such mathematical models, related to learning machine, include: Random Forests methods, logistic regression methods, neural network methods, k-means methods, principal component analysis methods, nearest neighbour classifier analysis methods, linear discriminant analysis, methods, quadratic discriminant analysis methods, support vector machine methods, decision tree methods, genetic algorithm methods, classifier optimization using bagging methods, classifier optimization using boosting methods, classifier optimization using the Random Subspace methods, projection pursuit methods, genetic programming and weighted voting methods.
  • In one aspect of the invention, the model used is a logistic regression model. As is described in the Examples section below, logistic regression models can be used according to the method of the invention to determine the probability a test subject of the invention has colorectal cancer as opposed to not having any colorectal pathology. Logistic regression models may also be referred to in the art as “logistic models”, and “logit models”.
  • Any one of various particular cases of logistic regression models may be used, for any given set of genes of the invention, for determining the probability that the test data corresponds to the positive control data and not to the negative control data.
  • In one aspect of the method, determining the probability that the test data corresponds to the positive control data and not to the negative control data is effected by using one or more of the logistic regression models disclosed in Example 2, Example 3 and Example 6.
  • It will be appreciated that a computer may be used for determining the probability that the test subject has colorectal cancer using a mathematical model, according to the method of the invention.
  • One of skill in the art will know of suitable mathematical formulas for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher or lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer.
  • For example, a suitable formula, is one which generates a value representing the ratio of the level of RNA encoded by the gene in blood of the test subject to the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer. A ratio of greater than 1 indicates that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, and a ratio of less than 1 indicates that the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer. A formula for generating such a ratio value may have the form:

  • Value=[level of RNA encoded by the gene in blood of the test subject]/[level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer]
  • Alternately, a suitable formula is one which subtracts the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer from the level of RNA encoded by the gene in blood of the test subject, to generate a value representing the difference between the level of RNA encoded by the gene in blood of the test subject from the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer. A difference having a positive value indicates that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, and a difference having a negative value indicates that the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer. A formula for generating such a difference value may have the form:

  • Value=[level of RNA encoded by the gene in blood of the test subject]−[level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer]
  • Thus, according to another aspect of the invention there is provided a computer-based method of determining the probability that a test subject has colorectal cancer as opposed to not having colorectal cancer. The method is effected by causing a computer to apply to the test data a mathematical model according to the invention, and to output the probability, to thereby enable a determination of the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • Application of computers for determining a probability that a test subject has a disease as opposed to not having the disease, so as to enable the method of the invention, is routinely practiced in the art using computer systems, and optionally computer-readable media, routinely used in the art.
  • Thus, according to a further aspect of the invention there is provided a computer system for providing the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer. The computer system comprises a processor; and a memory configured with instructions that cause the processor to provide a user with the probability, where the instructions comprise applying a mathematical model of the invention to test data of the invention, to thereby determine the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • The instructions may be provided to the computer in any one of various ways routinely employed in the art. In one aspect, the instructions are provided to the computer using a computer-readable medium.
  • Thus, according to yet another aspect of the invention there is provided a computer-readable medium having instructions stored thereon that are operable when executed by a computer for applying a mathematical model of the invention to test data of the invention from, thereby determine the probability that a test subject has colorectal cancer as opposed to not having colorectal cancer.
  • As described above, following the step of obtaining the test data, the method of classifying of the invention comprises the step of comparing test data representing a level of RNA encoded by a marker gene of the invention to negative control data representing a level of RNA encoded by the gene in subjects not having any colorectal pathology, and determining the fold-change between the levels.
  • It will be appreciated that a computer may be used for comparing test data representing a level of RNA encoded by a marker gene of the invention to negative control data representing a level of RNA encoded by the gene in subjects not having any colorectal pathology, and determining the fold-change between the levels, according to methods of the invention. Thus, according to another aspect of the invention there is provided a computer-based method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer. The method is effected by using a computer to apply to test data from a test subject according to the invention, and to negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, or lower, for IL2RB, than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer. For ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer, and where, for IL2RB, an indication by the value that the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • Application of computers for provide a classification of a test subject as more likely to have a disease than to not have the disease, so as to enable the method of the invention, is routinely practiced in the art using computer systems, and optionally computer-readable media, routinely used in the art.
  • Thus, according to a further aspect of the invention there is provided a computer system for providing a classification that a test subject is more likely to have colorectal cancer than to not have colorectal cancer. The computer system comprises a processor; and a memory configured with instructions that cause the processor to provide a user with the classification, where the instructions comprise causing the processor to apply to test data, and to negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, a mathematical formula for generating a value representing a fold-change between the level of RNA encoded by the gene in blood of the test subject and the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer where, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, a value indicating that the level of RNA encoded by the gene in blood of the test subject is higher, for example within a range of suitable fold-changes taught herein, than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer, and where, for IL2RB, a value indicating that the level of RNA encoded by the gene in blood of the test subject is lower, for example within a range of suitable fold-changes disclosed herein, than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • The instructions may be provided to the computer in any one of various ways routinely employed in the art. In one aspect, the instructions are provided to the computer using a computer-readable medium.
  • Thus, according to yet another aspect of the invention there is provided a computer-readable medium having instructions stored thereon that are operable when executed by a computer for applying to test data and to negative control data representing a level of RNA encoded by a marker gene of the invention in blood of human control subjects not having colorectal cancer, a mathematical formula for generating a value representing the fold-change between the level of RNA encoded by the gene in blood of the test subject and the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, where, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, a value indicating that the level of RNA encoded by the gene in blood of the test subject is higher, for example, within a suitable range of fold-changes disclosed herein, than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer, and where, for IL2RB, a value indicating that the level of RNA encoded by the gene in blood of the test subject is lower, for example, within a suitable range of fold-changes disclosed herein, than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • Thus, according to still yet another aspect of the invention there is provided a computer-readable medium having instructions stored thereon that are operable when executed by a computer for applying, to test data representing a level of RNA encoded by the gene in blood of a human test subject, and to negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, a mathematical formula for generating a value indicating, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, and, for IL2RB, whether the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, where, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer, and where, for IL2RB, an indication by the value that the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • An exemplary computer system for practicing certain of the methods described herein is described in FIG. 1.
  • FIG. 1 shows a schematic of a general-purpose computer system 100 suitable for practicing the methods described herein. The computer system 100, shown as a self-contained unit but not necessarily so limited, comprises at least one data processing unit (CPU) 102, a memory 104, which will typically include both high speed random access memory as well as non-volatile memory (such as one or more magnetic disk drives) but may be simply flash memory, a user interface 108, optionally a disk 110 controlled by a disk controller 112, and at least one optional network or other communication interface card 114 for communicating with other computers as well as other devices. At least the CPU 102, memory 104, user interface 108, disk controller where present, and network interface card, communicate with one another via at least one communication bus 106.
  • Memory 104 stores procedures and data, typically including: an operating system 140 for providing basic system services; application programs 152 such as user level programs for viewing and manipulating data, evaluating formulae for the purpose of diagnosing a test subject; authoring tools for assisting with the writing of computer programs; a file system 142, a user interface controller 144 for handling communications with a user via user interface 108, and optionally one or more databases 146 for storing data of the invention and other information, optionally a graphics controller 148 for controlling display of data, and optionally a floating point coprocessor 150 dedicated to carrying out mathematical operations. The methods of the invention may also draw upon functions contained in one or more dynamically linked libraries, not shown in FIG. 1, but stored either in Memory 104, or on disk 110, or accessible via network interface connection 114.
  • User interface 108 may comprise a display 128, a mouse 126, and a keyboard 130. Although shown as separate components in FIG. 1, one or more of these user interface components can be integrated with one another in embodiments such as handheld computers. Display 128 may be a cathode ray tube (CRT), or flat-screen display such as an LCD based on active matrix or TFT embodiments, or may be an electroluminescent display, based on light emitting organic molecules such as conjugated small molecules or polymers. Other embodiments of a user interface not shown in FIG. 1 include, e.g., several buttons on a keypad, a card-reader, a touch-screen with or without a dedicated touching device, a trackpad, a trackball, or a microphone used in conjunction with voice-recognition software, or any combination thereof, or a security-device such as a fingerprint sensor or a retinal scanner that prohibits an unauthorized user from accessing data and programs stored in system 100.
  • System 100 may also be connected to an output device such as a printer (not shown), either directly through a dedicated printer cable connected to a serial or USB port, or wirelessly, or via a network connection.
  • The database 146 may instead, optionally, be stored on disk 110 in circumstances where the amount of data in the database is too great to be efficiently stored in memory 104. The database may also instead, or in part, be stored on one or more remote computers that communicate with computer system 100 through network interface connection 114.
  • The network interface 134 may be a connection to the internet or to a local area network via a cable and modem, or ethernet, firewire, or USB connectivity, or a digital subscriber line. Preferably the computer network connection is wireless, e.g., utilizing CDMA, GSM, or GPRS, or bluetooth, or standards such as 802.11a, 802.11b, or 802.11g.
  • It would be understood that various embodiments and configurations and distributions of the components of system 10 across different devices and locations are consistent with practice of the methods described herein. For example, a user may use a handheld embodiment that accepts data from a test subject, and transmits that data across a network connection to another device or location where the data is analyzed according to a formulae described herein. A result of such an analysis can be stored at the other location and/or additionally transmitted back to the handheld embodiment. In such a configuration, the act of accepting data from a test subject can include the act of a user inputting the information. The network connection can include a web-based interface to a remote site at, for example, a healthcare provider. Alternatively, system 10 can be a device such as a handheld device that accepts data from the test subject, analyzes the data, such as by inputting the data into a formula as further described herein, and generating a result that is displayed to the user. The result can then be, optionally, transmitted back to a remote location via a network interface such as a wireless interface. System 100 may further be configured to permit a user to transmit by e-mail results of an analysis directly to some other party, such as a healthcare provider, or a diagnostic facility, or a patient.
  • In one aspect of the invention there is provided a method of determining whether a subject is at an increased risk of having colorectal cancer relative to the general population. The method comprises obtaining a test biological sample of blood from the subject; for each of a set of genes selected from the group consisting of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, determining the amount of RNA encoded by the gene in the test biological sample; comparing the determined amount of RNA for each these genes with the amount in one or more control biological samples of blood; and concluding or determining that the subject is at increased risk, average risk or decreased risk of having colorectal cancer relative to the general population if the amount of RNA encoded by each gene in the test biological sample is higher than in the control biological samples for genes ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, and lower for IL2RB.
  • A test subject would be considered as being at “increased risk” of having or developing colorectal cancer if the amount of RNA encoded by ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and/or VNN1 present in the test biological sample is higher than that seen in the control samples to an approximate extent (plus or minus 10%) seen in the working examples herein. A test subject would be considered as being at “increased risk” of having or developing colorectal cancer if the amount of RNA encoded by IL2RB present in the test biological sample is lower than that seen in the control samples to an approximate extent (plus or minus 10%) seen in the working examples herein.
  • A combination of marker genes of the invention, such as ANXA3, CLEC4D, LMNB1, PRRG4, VNN1, and IL2RB, can be used together with the known CRC prevalence rate to determine useful thresholds for stratifying the probability of having colorectal cancer in an average risk population. Using the combined training/blind set (IL2RB duplex) described in the Examples, an increased probability threshold can be selected to identify a sub-population with a colorectal cancer occurrence rate of 1.5%, a 3-fold increase over the base disease prevalence rate; this threshold reflects the same relative risk associated with having a first degree relative with colorectal cancer. A decreased probability threshold reflecting a sensitivity for colorectal cancer detection of, for example, 80%, 75%, 70%, 65%, can be selected to identify a lower-than-average probability sub-population. This approach can be used to stratify patients into an increased probability group, a decreased probability, and an average probability group.
  • One of ordinary skill in the art will be able to determine directly from the literature, or will be able to calculate from available statistical data, a suitable prevalence rate of colorectal cancer for practicing embodiments of the invention. For example, the prevalence rate for colorectal cancer in the average risk population over 50 years of age has been determined to be 0.7% (see for example Imperiale T F. et al., 2004. Colorectal Cancer Study Group. Fecal DNA versus fecal occult blood for colorectal-cancer screening in an average-risk population. New Engl J Med 351:2704-14).
  • It will be appreciated that components for practicing quantitative PCR according to the method of the invention may be assembled in a kit.
  • Thus, according to still another aspect of the invention there is provided a kit. The kit comprises packaging and contains, for each gene of a set of two or more of the following target genes of the invention: ACTB, ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1; a primer set capable of generating an amplification product of DNA complementary to RNA encoded, in a human subject, only by the gene.
  • In various aspects of the kit of the invention, the set of genes may be any combination of two or more of the target genes of the invention, as described hereinabove and in the Examples section, below.
  • In one aspect of the invention, the kit further contains two or more of the following components: a thermostable polymerase, a reverse transcriptase, deoxynucleotide triphosphates, nucleotide triphosphates and enzyme buffer.
  • In another aspect of the invention, the kit further contains at least one labeled probe capable of selectively hybridizing to either a sense or an antisense strand of the amplification product.
  • In yet another aspect of the invention, the kit further contains a computer-readable medium of the invention.
  • In one aspect, the kit is identified in print in or on the packaging as being for determining a probability that a test subject has colorectal cancer, for example, a probability that a test subject has colorectal cancer as opposed to not having colorectal cancer.
  • In another aspect, the kit is identified in print in or on the packaging as being for classifying a test subject as being more likely to have colorectal cancer than to not have colorectal cancer, and/or as being more likely to not have colorectal cancer than to have colorectal cancer.
  • In a further aspect, the kit is identified in print in or on the packaging as being for determining whether a test subject is at an increased risk of having colorectal cancer relative to the general population
  • In various aspects of the kit of the invention, the set of genes may be any combination of two or more of the target genes of the invention.
  • Sets of genes of the invention which consist of two or more of ACTB, ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 include: ACTB, ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6, VNN1; ACTB, ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, VNN1; ACTB, ANXA3, CLEC4D, IL2RB, PRRG4; ACTB, ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4; ACTB, ANXA3, CLEC4D, IL2RB, PRRG4, VNN1; ACTB, ANXA3, IL2RB, LMNB1, PRRG4, VNN1; ACTB, ANXA3, CLEC4D, IL2RB, PRRG4, TNFAIP6; ACTB, ANXA3, IL2RB, LMNB1, PRRG4, TNFAIP6; ACTB, ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6; ACTB, ANXA3, CLEC4D, IL2RB, PRRG4, TNFAIP6, VNN1; ACTB, ANXA3, IL2RB, LMNB1, PRRG4, TNFAIP6, VNN1; ACTB, ANXA3, IL2RB, LMNB1, PRRG4; ACTB, IL2RB, PRRG4, VNN1; ACTB, ANXA3, IL2RB, PRRG4, VNN1; ACTB, CLEC4D, IL2RB, PRRG4, VNN1; ACTB, IL2RB, LMNB1, PRRG4, VNN1; ACTB, CLEC4D, IL2RB, LMNB1, PRRG4, VNN1; ACTB, ANXA3, IL2RB, PRRG4, TNFAIP6; ACTB, IL2RB, PRRG4, TNFAIP6, VNN1; ACTB, ANXA3, IL2RB, PRRG4, TNFAIP6, VNN1; ACTB, CLEC4D, IL2RB, PRRG4, TNFAIP6, VNN1; ACTB, IL2RB, LMNB1, PRRG4, TNFAIP6, VNN1; ACTB, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6, VNN1; ACTB, IL2RB, PRRG4; ACTB, ANXA3, IL2RB, PRRG4; ACTB, CLEC4D, IL2RB, PRRG4; ACTB, IL2RB, LMNB1, PRRG4; ACTB, CLEC4D, IL2RB, LMNB1, PRRG4; ACTB, IL2RB, PRRG4, TNFAIP6; ACTB, CLEC4D, IL2RB, PRRG4, TNFAIP6; ACTB, IL2RB, LMNB1, PRRG4, TNFAIP6; ACTB, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6; ACTB, ANXA3, IL2RB, VNN1; ACTB, ANXA3, CLEC4D, IL2RB, VNN1; ACTB, ANXA3, IL2RB, LMNB1, VNN1; ACTB, ANXA3, CLEC4D, IL2RB, LMNB1, VNN1; ACTB, ANXA3, CLEC4D, LMNB1, PRRG4, VNN1; ACTB, ANXA3, IL2RB, TNFAIP6, VNN1; ACTB, ANXA3, CLEC4D, IL2RB, TNFAIP6, VNN1; ACTB, ANXA3, IL2RB, LMNB1, TNFAIP6, VNN1; ACTB, ANXA3, CLEC4D, IL2RB, LMNB1, TNFAIP6, VNN1; ACTB, ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6, VNN1; ACTB, ANXA3, IL2RB; ACTB, ANXA3, CLEC4D, IL2RB; ACTB, ANXA3, IL2RB, LMNB1; ACTB, ANXA3, CLEC4D, IL2RB, LMNB1; ACTB, ANXA3, CLEC4D, LMNB1, PRRG4; ACTB, CLEC4D, IL2RB, LMNB1, VNN1; ACTB, ANXA3, IL2RB, TNFAIP6; ACTB, ANXA3, CLEC4D, IL2RB, TNFAIP6; ACTB, ANXA3, IL2RB, LMNB1, TNFAIP6; ACTB, ANXA3, CLEC4D, IL2RB, LMNB1, TNFAIP6; ACTB, ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6; ACTB, IL2RB, LMNB1, TNFAIP6, VNN1; ACTB, CLEC4D, IL2RB, LMNB1, TNFAIP6, VNN1; ACTB, IL2RB, LMNB1, VNN1; ACTB, ANXA3, LMNB1, PRRG4, VNN1; ACTB, ANXA3, LMNB1, PRRG4, TNFAIP6, VNN1; ACTB, ANXA3, CLEC4D, PRRG4; ACTB, ANXA3, LMNB1, PRRG4; ACTB, CLEC4D, IL2RB, VNN1; ACTB, ANXA3, CLEC4D, PRRG4, VNN1; ACTB, IL2RB, LMNB1, TNFAIP6; ACTB, CLEC4D, IL2RB, LMNB1, TNFAIP6; ACTB, ANXA3, CLEC4D, PRRG4, TNFAIP6; ACTB, ANXA3, LMNB1, PRRG4, TNFAIP6; ACTB, IL2RB, TNFAIP6, VNN1; ACTB, CLEC4D, IL2RB, TNFAIP6, VNN1; ACTB, ANXA3, CLEC4D, PRRG4, TNFAIP6, VNN1; ACTB, IL2RB, LMNB1; ACTB, CLEC4D, IL2RB, LMNB1; ACTB, IL2RB, VNN1; ACTB, ANXA3, CLEC4D, LMNB1, VNN1; ACTB, ANXA3, CLEC4D, LMNB1, TNFAIP6, VNN1; ACTB, ANXA3, CLEC4D, LMNB1; ACTB, ANXA3, PRRG4; ACTB, ANXA3, CLEC4D, VNN1; ACTB, ANXA3, LMNB1, VNN1; ACTB, ANXA3, PRRG4, VNN1; ACTB, ANXA3, CLEC4D, LMNB1, TNFAIP6; ACTB, ANXA3, PRRG4, TNFAIP6; ACTB, ANXA3, CLEC4D, TNFAIP6, VNN1; ACTB, ANXA3, LMNB1, TNFAIP6, VNN1; ACTB, ANXA3, PRRG4, TNFAIP6, VNN1; ACTB, ANXA3; ACTB, ANXA3, CLEC4D; ACTB, ANXA3, LMNB1; ACTB, ANXA3, VNN1; ACTB, ANXA3, TNFAIP6; ACTB, ANXA3, CLEC4D, TNFAIP6; ACTB, IL2RB, TNFAIP6; ACTB, CLEC4D, IL2RB, TNFAIP6; ACTB, ANXA3, LMNB1, TNFAIP6; ACTB, ANXA3, TNFAIP6, VNN1; ACTB, CLEC4D, IL2RB; ACTB, PRRG4, VNN1; ACTB, CLEC4D, PRRG4, VNN1; ACTB, LMNB1, PRRG4, VNN1; ACTB, CLEC4D, LMNB1, PRRG4, VNN1; ACTB, PRRG4, TNFAIP6, VNN1; ACTB, CLEC4D, PRRG4, TNFAIP6, VNN1; ACTB, LMNB1, PRRG4, TNFAIP6, VNN1; ACTB, CLEC4D, LMNB1, PRRG4, TNFAIP6, VNN1; ACTB, PRRG4; ACTB, CLEC4D, PRRG4; ACTB, LMNB1, PRRG4; ACTB, CLEC4D, LMNB1, PRRG4; ACTB, PRRG4, TNFAIP6; ACTB, CLEC4D, PRRG4, TNFAIP6; ACTB, LMNB1, PRRG4, TNFAIP6; ACTB, CLEC4D, LMNB1, PRRG4, TNFAIP6; ACTB, LMNB1, TNFAIP6, VNN1; ACTB, CLEC4D, VNN1; ACTB, LMNB1, VNN1; ACTB, CLEC4D, LMNB1, VNN1; ACTB, LMNB1, TNFAIP6; ACTB, LMNB1, TNFAIP6; ACTB, TNFAIP6, VNN1; ACTB, CLEC4D, TNFAIP6, VNN1; ACTB, CLEC4D, LMNB1, TNFAIP6, VNN1; ACTB, LMNB1; ACTB, CLEC4D, LMNB1; ACTB, VNN1; ACTB, CLEC4D, TNFAIP6; ACTB, TNFAIP6; ACTB, CLEC4D; and ACTB, IL2RB.
  • In one aspect of the kit of the invention, the set of one or more genes consists of a housekeeping gene such as ACTB, and one or more of the colorectal cancer marker genes: ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1.
  • In one aspect of the kit of the invention, the set of one or more genes consists of ACTB and ANXA3.
  • In one aspect of the kit of the invention, the set of one or more genes consists of ACTB and CLEC4D.
  • In one aspect of the kit of the invention, the set of one or more genes consists of ACTB and IL2RB.
  • In one aspect of the kit of the invention, the set of one or more genes consists of ACTB and LMNB1.
  • In one aspect of the kit of the invention, the set of one or more genes consists of ACTB and PRRG4.
  • In one aspect of the kit of the invention, the set of one or more genes consists of ACTB and TNFAIP6.
  • In one aspect of the kit of the invention, the set of one or more genes consists of ACTB and VNN1.
  • In another aspect of the kit of the invention, the set of one or more genes consists of IL2RB, and one or more of the colorectal cancer marker genes: ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1.
  • In one aspect of the kit of the invention, the set of one or more genes consists of IL2RB and ANXA3.
  • In one aspect of the kit of the invention, the set of one or more genes consists of IL2RB and CLEC4D.
  • In one aspect of the kit of the invention, the set of one or more genes consists of IL2RB and LMNB1.
  • In one aspect of the kit of the invention, the set of one or more genes consists of IL2RB and PRRG4.
  • In one aspect of the kit of the invention, the set of one or more genes consists of IL2RB and TNFAIP6.
  • In one aspect of the kit of the invention, the set of one or more genes consists of IL2RB and VNN1.
  • In one aspect of the invention, the kit contains a primer having a nucleotide sequence identified as SEQ ID NO: 1, and a primer having a nucleotide sequence identified as SEQ ID NO: 2.
  • In one aspect of the invention, the kit contains a primer having a nucleotide sequence identified as SEQ ID NO: 1, and a primer having a nucleotide sequence identified as SEQ ID NO: 2 and the kit further contains a labeled probe which comprises a polynucleotide having a nucleic acid sequence identified as SEQ ID NO: 3.
  • In one aspect of the invention, the kit contains a primer having a nucleotide sequence identified as SEQ ID NO: 10, and a primer having a nucleotide sequence identified as SEQ ID NO: 11.
  • In one aspect of the invention, the kit contains a primer having a nucleotide sequence identified as SEQ ID NO: 10, and a primer having a nucleotide sequence identified as SEQ ID NO: 11, and the kit further contains a labeled probe which comprises a polynucleotide having a nucleic acid sequence identified as SEQ ID NO: 12.
  • In one aspect of the invention, the kit contains a primer having a nucleotide sequence identified as SEQ ID NO: 19, and a primer having a nucleotide sequence identified as SEQ ID NO: 20.
  • In one aspect of the invention, the kit contains a primer having a nucleotide sequence identified as SEQ ID NO: 19, and a primer having a nucleotide sequence identified as SEQ ID NO: 20 and the kit further contains a labeled probe which comprises a polynucleotide having a nucleic acid sequence identified as SEQ ID NO: 21.
  • In one aspect of the invention, for example, the kit contains a primer having a nucleotide sequence identified as SEQ ID NO: 28, and a primer having a nucleotide sequence identified as SEQ ID NO: 29.
  • In one aspect of the invention, for example, the kit contains a primer having a nucleotide sequence identified as SEQ ID NO: 28, and a primer having a nucleotide sequence identified as SEQ ID NO: 29 and the kit further contains a labeled probe which comprises a polynucleotide having a nucleic acid sequence identified as SEQ ID NO: 30.
  • In one aspect of the invention, the kit contains a primer having a nucleotide sequence identified as SEQ ID NO: 37, and a primer having a nucleotide sequence identified as SEQ ID NO: 38.
  • In one aspect of the invention, the kit contains a primer having a nucleotide sequence identified as SEQ ID NO: 37, and a primer having a nucleotide sequence identified as SEQ ID NO: 38 and the kit further contains a labeled probe which comprises a polynucleotide having a nucleic acid sequence identified as SEQ ID NO: 39.
  • In one aspect of the invention, the kit contains a primer having a nucleotide sequence identified as SEQ ID NO: 46, and a primer having a nucleotide sequence identified as SEQ ID NO: 47.
  • In one aspect of the invention, the kit contains a primer having a nucleotide sequence identified as SEQ ID NO: 46, and a primer having a nucleotide sequence identified as SEQ ID NO: 47, and the kit further contains a labeled probe which comprises a polynucleotide having a nucleic acid sequence identified as SEQ ID NO: 48.
  • In one aspect of the invention, the kit contains a primer having a nucleotide sequence identified as SEQ ID NO: 55, and a primer having a nucleotide sequence identified as SEQ ID NO: 56.
  • In one aspect of the invention, the kit contains a primer having a nucleotide sequence identified as SEQ ID NO: 55, and a primer having a nucleotide sequence identified as SEQ ID NO: 56 and the kit further contains a labeled probe which comprises a polynucleotide having a nucleic acid sequence identified as SEQ ID NO: 57.
  • Further, non-limiting, specific aspects of the invention include the following:
  • One aspect of the invention disclosed herein is a method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, the method comprising: the steps of (a) determining a level of RNA encoded by a ANXA3 gene in blood of the test subject, thereby generating test data; (b) providing positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) determining a probability that the test data corresponds to the positive control data and not to the negative control data, where the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer. Another aspect of the invention disclosed herein is a method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, the method comprising the steps of (a) determining a level of RNA encoded by a CLEC4D gene in blood of the test subject, thereby generating test data; (b) providing positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) determining a probability that the test data corresponds to the positive control data and not to the negative control data, where the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer. Another aspect of the invention disclosed herein is a method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, the method comprising the steps of (a) determining a level of RNA encoded by a IL2RB gene in blood of the test subject, thereby generating test data; (b) providing positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) determining a probability that the test data corresponds to the positive control data and not to the negative control data, where the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer. Another aspect of the invention disclosed herein is a method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, the method comprising the steps of: (a) determining a level of RNA encoded by a LMNB1 gene in blood of the test subject, thereby generating test data; (b) providing positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) determining a probability that the test data corresponds to the positive control data and not to the negative control data, where the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer. Another aspect of the invention disclosed herein is a method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, the method comprising the steps of: (a) determining a level of RNA encoded by a PRRG4 gene in blood of the test subject, thereby generating test data; (b) providing positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) determining a probability that the test data corresponds to the positive control data and not to the negative control data, where the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer. Another aspect of the invention disclosed herein is a method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, the method comprising the steps of: (a) determining a level of RNA encoded by a TNFAIP6 gene in blood of the test subject, thereby generating test data; (b) providing positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) determining a probability that the test data corresponds to the positive control data and not to the negative control data, where the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer. Another aspect of the invention disclosed herein is a method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, the method comprising the steps of: (a) determining a level of RNA encoded by a VNN1 gene in blood of the test subject, thereby generating test data; (b) providing positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) determining a probability that the test data corresponds to the positive control data and not to the negative control data, where the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • An embodiment of aspects of the invention disclosed herein includes that the determining of the level of RNA encoded by the gene in blood of the test subject be effected by determining the level of RNA encoded by the gene in a blood sample isolated from the test subject. An embodiment of aspects of the invention disclosed herein includes the further step of determining the levels of RNA encoded by the gene in blood of a population of human subjects having colorectal cancer, thereby providing the positive control data representing the levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and determining levels of RNA encoded by the gene in blood of a population of human subjects not having colorectal cancer, thereby providing the negative control data representing the levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer. An embodiment of aspects of the invention disclosed herein includes that the level of RNA encoded by the gene in blood of the test subject is determined via quantitative reverse transcriptase-polymerase chain reaction analysis. An embodiment of aspects of the invention disclosed herein includes that the level of RNA encoded by the gene in blood of the test subject and the levels of RNA encoded by the gene in blood of the control subjects are determined via the same method. An embodiment of aspects of the invention disclosed herein includes that the determining of the probability that the test data corresponds to the positive control data and not to the negative control data is effected by applying to the test data a mathematical model derived from the positive control data and from the negative control data, and where the mathematical model is for determining the probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data. An embodiment of aspects of the invention disclosed herein includes that the level of RNA encoded by the gene in blood of the test subject is determined as a ratio to a level of RNA encoded by ACTB in blood of the test subject. An aspect of this latter embodiment includes that the level of RNA encoded by the gene in blood of the test subject and the level of RNA encoded by ACTB in blood of the test subject are determined via duplex quantitative reverse transcriptase-polymerase chain reaction analysis of RNA encoded by the gene and of RNA encoded by ACTB. An embodiment of aspects of the invention disclosed herein includes that the level of RNA encoded by the gene in blood of the test subject is determined as a ratio to a level of RNA encoded by IL2RB in blood of the test subject. An aspect of this latter embodiment includes that the level of RNA encoded by the gene in blood of the test subject and the level of RNA encoded by IL2RB in blood of the test subject are determined via duplex quantitative reverse transcriptase-polymerase chain reaction analysis of RNA encoded by the gene and of RNA encoded by IL2RB.
  • An aspect of the invention disclosed herein is a computer-based method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, from test data representing a level of RNA encoded by a ANXA3 gene in blood of the test subject, the method comprising computer-implemented steps of: (a) applying to the test data a mathematical model derived from positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and from negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, where the mathematical model is for determining a probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data; and (b) outputting the probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data, where the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer. Another aspect of the invention disclosed herein is a computer-based method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, from test data representing a level of RNA encoded by a ANXA3 gene in blood of the test subject, the method comprising computer-implemented steps of: inputting, to a computer, test data representing a level of RNA encoded by a CLEC4D gene in blood of the test subject; and causing the computer to apply to the test data a mathematical model derived from positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and from negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, where the mathematical model is for determining a probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data; and (b) outputting the probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data, where the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer. Another aspect of the invention disclosed herein is a computer-based method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, from test data representing a level of RNA encoded by a ANXA3 gene in blood of the test subject, the method comprising computer-implemented steps of: inputting, to a computer, test data representing a level of RNA encoded by a IL2RB gene in blood of the test subject; and causing the computer to apply to the test data a mathematical model derived from positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and from negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, where the mathematical model is for determining a probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data; and (b) outputting the probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data, where the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer. Another aspect of the invention disclosed herein is a computer-based method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, from test data representing a level of RNA encoded by a ANXA3 gene in blood of the test subject, the method comprising computer-implemented steps of: (a) applying to the test data a mathematical model derived from positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and from negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, where the mathematical model is for determining a probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data; and (b) outputting the probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data, where the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer. Another aspect of the invention disclosed herein is a computer-based method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, from test data representing a level of RNA encoded by a ANXA3 gene in blood of the test subject, the method comprising computer-implemented steps of: (a) applying to the test data a mathematical model derived from positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and from negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, where the mathematical model is for determining a probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data; and (b) outputting the probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data, where the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer. Another aspect of the invention disclosed herein is a computer-based method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, from test data representing a level of RNA encoded by a ANXA3 gene in blood of the test subject, the method comprising computer-implemented steps of: (a) applying to the test data a mathematical model derived from positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and from negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, where the mathematical model is for determining a probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data; and (b) outputting the probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data, where the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer. Another aspect of the invention disclosed herein is a computer-based method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, from test data representing a level of RNA encoded by a ANXA3 gene in blood of the test subject, the method comprising computer-implemented steps of: (a) applying to the test data a mathematical model derived from positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and from negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, where the mathematical model is for determining a probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data; and (b) outputting the probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data, where the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.
  • An embodiment of the invention's computer based methods includes where the level of RNA encoded by the gene in blood of the test subject is determined via quantitative reverse transcriptase-polymerase chain reaction analysis. An embodiment of computer based methods of the invention includes where the level of RNA encoded by the gene in blood of the test subject and the levels of RNA encoded by the gene in blood of the control subjects are determined via the same method. An embodiment of each of computer based methods of the invention includes where the level of RNA encoded by the gene in blood of the test subject is determined as a ratio to a level of RNA encoded by ACTB in blood of the test subject. An embodiment of computer based methods of the invention includes where the level of RNA encoded by the gene in blood of the test subject and the level of RNA encoded by ACTB in blood of the test subject are determined via duplex quantitative reverse transcriptase-polymerase chain reaction analysis of RNA encoded by the gene and of RNA encoded by ACTB. An embodiment of each of the computer based methods of the invention includes where the level of RNA encoded by the gene in blood of the test subject is determined as a ratio to a level of RNA encoded by IL2RB in blood of the test subject. In a further embodiment the level of RNA encoded by the gene in blood of the test subject and the level of RNA encoded by IL2RB in blood of the test subject are determined via duplex quantitative reverse transcriptase-polymerase chain reaction analysis of RNA encoded by the gene and of RNA encoded by IL2RB.
  • Another aspect of the invention disclosed herein is a method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, the method comprising, for each gene of a set of one or more genes selected from the group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1: comprising the steps of: (a) determining a level of RNA encoded by the gene in blood of the test subject, thereby generating test data; (b) providing positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) determining a probability that the test data corresponds to the positive control data and not to the negative control data, where the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer. An embodiment of this aspect of the invention disclosed herein is where the determining of the level of RNA encoded by the gene in blood of the test subject is effected by determining the level of RNA encoded by the gene in a blood sample isolated from the test subject. An embodiment of this aspect of the invention disclosed herein further comprises determining levels of RNA encoded by the gene in blood of a population of human subjects having colorectal cancer, thereby providing the positive control data representing the levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and determining levels of RNA encoded by the gene in blood of a population of human subjects not having colorectal cancer, thereby providing the negative control data representing the levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer. An embodiment of this aspect of the invention disclosed herein is where the level of RNA encoded by the gene in blood of the test subject is determined via quantitative reverse transcriptase-polymerase chain reaction analysis. An embodiment of this aspect of the invention disclosed herein is where the level of RNA encoded by the gene in blood of the test subject and the levels of RNA encoded by the gene in blood of the control subjects are determined via the same method. An embodiment of this aspect of the invention disclosed herein is where the determining of the probability that the test data corresponds to the positive control data and not to the negative control data is effected by applying to the test data a mathematical model derived from the positive control data and from the negative control data, and where the mathematical model is for determining the probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data. An embodiment of this aspect of the invention disclosed herein is where the level of RNA encoded by the gene in blood of the test subject is determined as a ratio to a level of RNA encoded by ACTB in blood of the test subject. In a further embodiment, the level of RNA encoded by the gene in blood of the test subject and the level of RNA encoded by ACTB in blood of the test subject are determined via duplex quantitative reverse transcriptase-polymerase chain reaction analysis of RNA encoded by the gene and of RNA encoded by ACTB. An embodiment of this aspect of the invention disclosed herein is where the set of one or more genes is a set of one or more genes selected from the group consisting of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, and where the level of RNA encoded by the gene in blood of the test subject is determined as a ratio to a level of RNA encoded by IL2RB in blood of the test subject. In a further embodiment, the level of RNA encoded by the gene in blood of the test subject and the level of RNA encoded by IL2RB in blood of the test subject are determined via duplex quantitative reverse transcriptase-polymerase chain reaction analysis of RNA encoded by the gene and of RNA encoded by IL2RB.
  • Another aspect of the invention disclosed herein is a computer-based method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer, from test data representing a level of RNA encoded by the gene in blood of the test subject, the method comprising, for each gene of a set of one or more genes selected from the group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, computer-implemented steps of: (a) applying to the test data a mathematical model derived from positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and from negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, where the mathematical model is for determining a probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data; and (b) outputting the probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data, where the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer. In an embodiment of this aspect of the invention disclosed herein is where the level of RNA encoded by the gene in blood of the test subject is determined via quantitative reverse transcriptase-polymerase chain reaction analysis. In an embodiment of this aspect of the invention disclosed herein is where the level of RNA encoded by the gene in blood of the test subject and the levels of RNA encoded by the gene in blood of the control subjects are determined via the same method. In an embodiment of this aspect of the invention disclosed herein is where the level of RNA encoded by the gene in blood of the test subject is determined as a ratio to a level of RNA encoded by ACTB in blood of the test subject. In a further embodiment of this aspect of the invention disclosed herein is where the level of RNA encoded by the gene in blood of the test subject and the level of RNA encoded by ACTB in blood of the test subject are determined via duplex quantitative reverse transcriptase-polymerase chain reaction analysis of RNA encoded by the gene and of RNA encoded by ACTB. In an embodiment of this aspect of the invention disclosed herein is where the set of one or more genes is a set of one or more genes selected from the group consisting of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, and where the level of RNA encoded by the gene in blood of the test subject is determined as a ratio to a level of RNA encoded by IL2RB in blood of the test subject. In a further embodiment of this aspect of the invention disclosed herein, the level of RNA encoded by the gene in blood of the test subject and the level of RNA encoded by IL2RB in blood of the test subject are determined via duplex quantitative reverse transcriptase-polymerase chain reaction analysis of RNA encoded by the gene and of RNA encoded by IL2RB. In an embodiment of this aspect of the invention disclosed herein is where the set of one or more genes consists of PRRG4. In an embodiment of this aspect of the invention disclosed herein is where the set of one or more genes consists of IL2RB and PRRG4.
  • Another aspect of the invention disclosed herein is a kit comprising packaging and containing, for each gene of a set of two or more genes selected from the group consisting of ACTB, ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, a primer set capable of generating an amplification product of DNA complementary to RNA encoded, in a human subject, only by the gene. An embodiment of this aspect of the invention disclosed herein is where the kit further contains two or more components selected from the group consisting of a thermostable polymerase, a reverse transcriptase, deoxynucleotide triphosphates, nucleotide triphosphates and enzyme buffer. An embodiment of this aspect of the invention disclosed herein is where the kit further contains at least one labelled probe capable of selectively hybridizing to either a sense or an antisense strand of the amplification product. An embodiment of this aspect of the invention disclosed herein is where the kit further contains a computer-readable medium having instructions stored thereon that are operable when executed by a computer for applying a mathematical model to test data representing a level of RNA encoded by the gene in blood of a human test subject, where the mathematical model is derived from positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and from negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, where the mathematical model is for determining a probability that data representing a level of RNA encoded by the gene corresponds to the positive control data and not to the negative control data, and where the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer. An embodiment of this aspect of the invention disclosed herein is where the set of one or more genes of the kit consists of ACTB and one or more genes selected from the group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1. An embodiment of this aspect of the invention disclosed herein is where the set of one or more genes of one or more genes of the kit consists of ACTB and ANXA3. An embodiment of this aspect of the invention disclosed herein is where the set of one or more genes of one or more genes of the kit consists of ACTB and CLEC4D. An embodiment of this aspect of the invention disclosed herein is where the set of one or more genes of one or more genes of the kit consists of ACTB and IL2RB. An embodiment of this aspect of the invention disclosed herein is where the set of one or more genes of one or more genes of the kit consists of ACTB and LMNB1. An embodiment of this aspect of the invention disclosed herein is where the set of one or more genes of one or more genes of the kit consists of ACTB and PRRG4. An embodiment of this aspect of the invention disclosed herein is where the set of one or more genes of one or more genes of the kit consists of ACTB and TNFAIP6. An embodiment of this aspect of the invention disclosed herein is where the set of one or more genes of one or more genes of the kit consists of ACTB and VNN1. An embodiment of this aspect of the invention disclosed herein is where the set of one or more genes of one or more genes of the kit consists of IL2RB and one or more genes selected from the group consisting of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1. An embodiment of this aspect of the invention disclosed herein is where the set of one or more genes of one or more genes of the kit consists of IL2RB and ANXA3. An embodiment of this aspect of the invention disclosed herein is where the set of one or more genes of one or more genes of the kit consists of \ IL2RB and CLEC4D. An embodiment of this aspect of the invention disclosed herein is where the set of one or more genes of one or more genes of the kit consists of IL2RB and LMNB1. An embodiment of this aspect of the invention disclosed herein is where the set of one or more genes of one or more genes of the kit consists of IL2RB and PRRG4. An embodiment of this aspect of the invention disclosed herein is where the set of one or more genes of one or more genes of the kit consists of IL2RB and TNFAIP6. An embodiment of this aspect of the invention disclosed herein is where the set of one or more genes of one or more genes of the kit consists of IL2RB and VNN1.
  • Another aspect of the invention disclosed herein is a method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising: (a) determining a level of RNA encoded by a ANXA3 gene in blood of the test subject, thereby generating test data; (b) providing negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) applying to the test data and to the negative control data a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, where an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer. Another aspect of the invention disclosed herein is a method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising: (a) determining a level of RNA encoded by a CLEC4D gene in blood of the test subject, thereby generating test data; (b) providing negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) applying to the test data and to the negative control data a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, where an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer. Another aspect of the invention disclosed herein is a method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising: (a) determining a level of RNA encoded by a IL2RB gene in blood of the test subject, thereby generating test data; (b) providing negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) applying to the test data and to the negative control data a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, where an indication by the value that the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer. Another aspect of the invention disclosed herein is a method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising: (a) determining a level of RNA encoded by a LMNB1 gene in blood of the test subject, thereby generating test data; (b) providing negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) applying to the test data and to the negative control data a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, where an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer. Another aspect of the invention disclosed herein is a method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising: (a) determining a level of RNA encoded by a PRRG4 gene in blood of the test subject, thereby generating test data; (b) providing negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) applying to the test data and to the negative control data a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, where an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer. Another aspect of the invention disclosed herein is a method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising: (a) determining a level of RNA encoded by a TNFAIP6 gene in blood of the test subject, thereby generating test data; (b) providing negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) applying to the test data and to the negative control data a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, where an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer. Another aspect of the invention disclosed herein is a method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising: (a) determining a level of RNA encoded by a VNN1 gene in blood of the test subject, thereby generating test data; (b) providing negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) applying to the test data and to the negative control data a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, where an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • An embodiment of the methods of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer of the invention includes determining of the level of RNA encoded by the gene in blood of the test subject is effected by determining the level of RNA encoded by the gene in a blood sample isolated from the test subject. An embodiment of the invention's methods of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer of the invention includes further determining levels of RNA encoded by the gene in blood of a population of human subjects not having colorectal cancer, thereby providing the negative control data representing the levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer. An embodiment of the invention's methods of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer of the invention includes where the level of RNA encoded by the gene in blood of the test subject is determined via quantitative reverse transcriptase-polymerase chain reaction analysis. An embodiment of the invention's methods of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer of the invention includes where the level of RNA encoded by the gene in blood of the test subject and the levels of RNA encoded by the gene in blood of the control subjects are determined via the same method. An embodiment of the invention's methods of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer of the invention includes where the level of RNA encoded by the gene in blood of the test subject is determined as a ratio to a level of RNA encoded by ACTB in blood of the test subject. In an aspect of this embodiment, the level of RNA encoded by the gene in blood of the test subject and the level of RNA encoded by ACTB in blood of the test subject are determined via duplex quantitative reverse transcriptase-polymerase chain reaction analysis of RNA encoded by the gene and of RNA encoded by ACTB. An embodiment of the invention's methods of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer includes where the level of RNA encoded by the gene in blood of the test subject is determined as a ratio to a level of RNA encoded by IL2RB in blood of the test subject, and/or where the level of RNA encoded by the gene in blood of the test subject and the level of RNA encoded by IL2RB in blood of the test subject are determined via duplex quantitative reverse transcriptase-polymerase chain reaction analysis of RNA encoded by the gene and of RNA encoded by IL2RB.
  • Another aspect of the invention disclosed herein is a computer-based method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising computer-implemented steps of: (a) applying to test data representing a level of RNA encoded by a ANXA3 gene in blood of the test subject and to negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (b) outputting the value, where an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer. Another aspect of the invention disclosed herein is a computer-based method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising computer-implemented steps of: (a) applying to test data representing a level of RNA encoded by a CLEC4D gene in blood of the test subject and to negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (b) outputting the value, where an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer. Another aspect of the invention disclosed herein is a computer-based method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising computer-implemented steps of: (a) applying to test data representing a level of RNA encoded by a IL2RB gene in blood of the test subject and to negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (b) outputting the value, where an indication by the value that the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer. Another aspect of the invention disclosed herein is a computer-based method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising computer-implemented steps of: (a) applying to test data representing a level of RNA encoded by a LMNB1 gene in blood of the test subject and to negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (b) outputting the value, where an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer. Another aspect of the invention disclosed herein is a computer-based method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising computer-implemented steps of: (a) applying to test data representing a level of RNA encoded by a PRRG4 gene in blood of the test subject and to negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (b) outputting the value, where an indication by the value that the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer. Another aspect of the invention disclosed herein is a computer-based method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising computer-implemented steps of: (a) applying to test data representing a level of RNA encoded by a TNFAIP6 gene in blood of the test subject and to negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (b) outputting the value, where an indication by the value that the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer. Another aspect of the invention disclosed herein is a computer-based method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising computer-implemented steps of: (a) applying to test data representing a level of RNA encoded by a VNN1 gene in blood of the test subject and to negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer a mathematical formula for generating a value indicating whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (b) outputting the value, where an indication by the value that the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer.
  • An embodiment of the invention's computer-based methods of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, includes where the level of RNA encoded by the gene in blood of the test subject is determined via quantitative reverse transcriptase-polymerase chain reaction analysis. An embodiment of the invention's computer-based methods of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, includes where the level of RNA encoded by the gene in blood of the test subject and the levels of RNA encoded by the gene in blood of the control subjects are determined via the same method. An embodiment of the invention's computer-based methods of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, includes where the level of RNA encoded by the gene in blood of the test subject is determined as a ratio to a level of RNA encoded by ACTB in blood of the test subject. An aspect of this embodiment includes where the level of RNA encoded by the gene in blood of the test subject and the level of RNA encoded by ACTB in blood of the test subject are determined via duplex quantitative reverse transcriptase-polymerase chain reaction analysis of RNA encoded by the gene and of RNA encoded by ACTB. An embodiment of the invention's computer-based methods of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, includes where the level of RNA encoded by the gene in blood of the test subject is determined as a ratio to a level of RNA encoded by IL2RB in blood of the test subject. An aspect of this embodiment includes where the level of RNA encoded by the gene in blood of the test subject and the level of RNA encoded by IL2RB in blood of the test subject are determined via duplex quantitative reverse transcriptase-polymerase chain reaction analysis of RNA encoded by the gene and of RNA encoded by IL2RB.
  • Another aspect of the invention disclosed herein is a method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising, for each gene of a set of one or more genes selected from the group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1: (a) determining a level of RNA encoded by the gene in blood of the test subject, thereby generating test data; (b) providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and (c) applying to the test data and to the negative control data a mathematical formula for generating a value indicating, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, and indicating, for IL2RB, whether the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, where, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, an indication by the value that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer, and where, for IL2RB, an indication by the value that the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer. An embodiment of this aspect includes determining of the level of RNA encoded by the gene in blood of the test subject is effected by determining the level of RNA encoded by the gene in a blood sample isolated from the test subject. Another embodiment of this aspect includes further comprising determining levels of RNA encoded by the gene in blood of a population of human subjects having colorectal cancer, thereby providing the positive control data representing the levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and determining levels of RNA encoded by the gene in blood of a population of human subjects not having colorectal cancer, thereby providing the negative control data representing the levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer. Anther embodiment of this aspect includes where the level of RNA encoded by the gene in blood of the test subject is determined via quantitative reverse transcriptase-polymerase chain reaction analysis. An embodiment of this aspect includes where the level of RNA encoded by the gene in blood of the test subject and the levels of RNA encoded by the gene in blood of the control subjects are determined via the same method. An embodiment of this aspect includes where the level of RNA encoded by the gene in blood of the test subject is determined as a ratio to a level of RNA encoded by ACTB in blood of the test subject. An further embodiment includes where the level of RNA encoded by the gene in blood of the test subject and the level of RNA encoded by ACTB in blood of the test subject are determined via duplex quantitative reverse transcriptase-polymerase chain reaction analysis of RNA encoded by the gene and of RNA encoded by ACTB. An embodiment of this aspect includes where the set of one or more genes is a set of one or more genes selected from the group consisting of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, and where the level of RNA encoded by the gene in blood of the test subject is determined as a ratio to a level of RNA encoded by IL2RB in blood of the test subject. An embodiment of this aspect includes where the level of RNA encoded by the gene in blood of the test subject and the level of RNA encoded by IL2RB in blood of the test subject are determined via duplex quantitative reverse transcriptase-polymerase chain reaction analysis of RNA encoded by the gene and of RNA encoded by IL2RB.
  • Another aspect of the invention disclosed herein is a computer-based method of classifying a human test subject as more likely to have colorectal cancer than to not have colorectal cancer, the method comprising, for each gene of a set of one or more genes selected from the group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, computer-implemented steps of: (a) applying to test data representing a level of RNA encoded by the gene in blood of the test subject and to negative control data representing a level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, a formula for calculating a value indicating, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, whether the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, and indicating, for IL2RB, whether the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer, where, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, an indication that the level of RNA encoded by the gene in blood of the test subject is higher than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer, and where, for IL2RB, an indication that the level of RNA encoded by the gene in blood of the test subject is lower than the level of RNA encoded by the gene in blood of human control subjects not having colorectal cancer classifies the test subject as more likely to have colorectal cancer than to not have colorectal cancer. An embodiment of this aspect of the invention disclosed herein includes where the level of RNA encoded by the gene in blood of the test subject is determined via quantitative reverse transcriptase-polymerase chain reaction analysis. Another embodiment of this aspect of the invention disclosed herein includes where the level of RNA encoded by the gene in blood of the test subject and the levels of RNA encoded by the gene in blood of the control subjects are determined via the same method. Another embodiment of this aspect of the invention disclosed herein includes where the level of RNA encoded by the gene in blood of the test subject is determined as a ratio to a level of RNA encoded by ACTB in blood of the test subject. In a further embodiment of this embodiment of the invention as disclosed herein is where the level of RNA encoded by the gene in blood of the test subject and the level of RNA encoded by ACTB in blood of the test subject are determined via duplex quantitative reverse transcriptase-polymerase chain reaction analysis of RNA encoded by the gene and of RNA encoded by ACTB. Another embodiment of this aspect of the invention disclosed herein includes where the set of one or more genes is a set of one or more genes selected from the group consisting of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, and where the level of RNA encoded by the gene in blood of the test subject is determined as a ratio to a level of RNA encoded by IL2RB in blood of the test subject. In a further embodiment of this embodiment of the invention as disclosed herein, the level of RNA encoded by the gene in blood of the test subject and the level of RNA encoded by IL2RB in blood of the test subject are determined via duplex quantitative reverse transcriptase-polymerase chain reaction analysis of RNA encoded by the gene and of RNA encoded by IL2RB. In a further embodiment of this embodiment of the invention as disclosed herein, the set of one or more genes consists of PRRG4. In a further embodiment of this embodiment of the invention as disclosed herein, the set of one or more genes consists of IL2RB and PRRG4.
  • Another aspect of the invention disclosed herein is a kit comprising packaging and containing, for each gene of a set of two or more genes selected from the group consisting of ACTB, ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, a primer set capable of generating an amplification product of DNA complementary to RNA encoded, in a human subject, only by the gene. In an embodiment of this aspect of the invention disclosed herein, the kit further containing two or more components selected from the group consisting of a thermostable polymerase, a reverse transcriptase, deoxynucleotide triphosphates, nucleotide triphosphates and enzyme buffer. In another embodiment of this aspect of the invention disclosed herein, the kit further contains at least one labelled probe capable of selectively hybridizing to either a sense or an antisense strand of the amplification product. In another embodiment of this aspect of the invention disclosed herein, the set of one or more genes of the kit consists of ACTB and one or more genes selected from the group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1. In another embodiment of this aspect of the invention disclosed herein, the set of one or more genes of the kit consists of ACTB and ANXA3. In another embodiment of this aspect of the invention disclosed herein, the set of one or more genes of the kit consists of ACTB and CLEC4D. In another embodiment of this aspect of the invention disclosed herein, the set of one or more genes of the kit consists of ACTB and IL2RB. In another embodiment of this aspect of the invention disclosed herein, the set of one or more genes of the kit consists of ACTB and LMNB1. In another embodiment of this aspect of the invention disclosed herein, the set of one or more genes of the kit consists of ACTB and PRRG4. In another embodiment of this aspect of the invention disclosed herein, the set of one or more genes of the kit consists of ACTB and TNFAIP6. In another embodiment of this aspect of the invention disclosed herein, the set of one or more genes of the kit consists of ACTB and VNN1. In another embodiment of this aspect of the invention disclosed herein, the set of one or more genes of the kit consists of IL2RB and one or more genes selected from the group consisting of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1. In another embodiment of this aspect of the invention disclosed herein, the set of one or more genes of the kit consists IL2RB and ANXA3. In another embodiment of this aspect of the invention disclosed herein, the set of one or more genes of the kit consists of IL2RB and CLEC4D. In another embodiment of this aspect of the invention disclosed herein, the set of one or more genes of the kit consists of IL2RB and LMNB1. In another embodiment of this aspect of the invention disclosed herein, the set of one or more genes of the kit consists of IL2RB and PRRG4. In another embodiment of this aspect of the invention disclosed herein, the set of one or more genes of the kit consists of IL2RB and TNFAIP6. In another embodiment of this aspect of the invention disclosed herein, the set of one or more genes of the kit consists of IL2RB and VNN1.
  • Another aspect of the invention disclosed herein is a method of determining whether a test subject is at an increased risk of having colorectal cancer relative to the general population, comprising: (a) obtaining a test sample of blood from the subject; and (i) determining a level of RNA encoded by a annexin A3 (ANXA3) gene in the test sample of blood, (ii) comparing the level of RNA encoded by ANXA3 as determined in step (i) with a level of the RNA encoded by the gene in control samples of blood; and (b) concluding that the subject is at an increased risk of having colorectal cancer relative to the general population if the level of RNA encoded by the gene in the test sample of blood is higher than in the control samples of blood. Another aspect of the invention disclosed herein is a method of determining whether a test subject is at an increased risk of having colorectal cancer relative to the general population, comprising: (a) obtaining a test sample of blood from the subject; and (i) determining a level of RNA encoded by a C-type lectin domain family 4, member D (CLEC4D) gene in the test sample of blood, (ii) comparing the level of RNA encoded by the gene as determined in step (i) with the level of the RNA encoded by the gene in control samples of blood; and (b) concluding that the subject is at an increased risk of having colorectal cancer relative to the general population if the level of RNA encoded by the gene in the test sample of blood is higher than in the control samples of blood. Another aspect of the invention disclosed herein is a method of determining whether a test subject is at an increased risk of having colorectal cancer relative to the general population, comprising: (a) obtaining a test sample of blood from the subject; and (i) determining a level of RNA encoded by a interleukin 2 receptor, beta (IL2RB) gene in the test sample of blood, (ii) comparing the level of RNA encoded by the gene as determined in step (i) with the level of the RNA encoded by the gene in control samples of blood; and (b) concluding that the subject is at an increased risk of having colorectal cancer relative to the general population if the level of RNA encoded by the gene in the test sample of blood is lower than in the control samples of blood. Another aspect of the invention disclosed herein is a method of determining whether a test subject is at an increased risk of having colorectal cancer relative to the general population, comprising: (a) obtaining a test sample of blood from the subject; and (i) determining a level of RNA encoded by a lamin B1 (LMNB1) gene in the test sample of blood, (ii) comparing the level of RNA encoded by the gene as determined in step (i) with the level of the RNA encoded by the gene in control samples of blood; and (b) concluding that the subject is at an increased risk of having colorectal cancer relative to the general population if the level of RNA encoded by the gene in the test sample of blood is higher than in the control samples of blood. Another aspect of the invention disclosed herein is a method of determining whether a test subject is at an increased risk of having colorectal cancer relative to the general population, comprising: (a) obtaining a test sample of blood from the subject; and (i) determining a level of RNA encoded by a proline rich Gla (G carboxyglutamic acid) 4 (transmembrane) (PRRG4) gene in the test sample of blood, (ii) comparing the level of RNA encoded by the gene as determined in step (i) with the level of the RNA encoded by the gene in control samples of blood; and (b) concluding that the subject is at an increased risk of having colorectal cancer relative to the general population if the level of RNA encoded by the gene in the test sample of blood is higher than in the control samples of blood. Another aspect of the invention disclosed herein is a method of determining whether a test subject is at an increased risk of having colorectal cancer relative to the general population, comprising: (a) obtaining a test sample of blood from the subject; and (i) determining a level of RNA encoded by a tumor necrosis factor, alpha induced protein 6 gene (TNFAIP6) in the test sample of blood, (ii) comparing the level of RNA encoded by the gene as determined in step (i) with the level of the RNA encoded by the gene in control samples of blood; and (b) concluding that the subject is at an increased risk of having colorectal cancer relative to the general population if the level of RNA encoded by the gene in the test sample of blood is higher than in the control samples of blood. Another aspect of the invention disclosed herein is a method of determining whether a test subject is at an increased risk of having colorectal cancer relative to the general population, comprising: (a) obtaining a test sample of blood from the subject; and (i) determining a level of RNA encoded by a vanin 1 (VNN1) gene in the test sample of blood, (ii) comparing the level of RNA encoded by the gene as determined in step (i) with the level of the RNA encoded by the gene in control samples of blood; and (b) concluding that the subject is at an increased risk of having colorectal cancer relative to the general population if the level of RNA encoded by the gene in the test sample of blood is higher than in the control samples of blood. In an embodiment of any one of these eight aspects these methods of determining whether a test subject is at an increased risk of having colorectal cancer relative to the general population, the control samples are from individuals who have been diagnosed as not having colorectal cancer.
  • Another aspect of the invention disclosed herein is a method of determining whether a test subject is at an increased risk of having colorectal cancer relative to the general population, comprising: (a) obtaining a test sample of blood from the subject; and for each gene of a set of genes selected from the group consisting of: ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, (i) determining a level of RNA encoded by the gene in the test sample of blood, thereby generating test data; and (ii) applying to the test data and to control data representing a level of RNA encoded by the gene in one or more control samples of blood a mathematical formula for generating a value indicating, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, whether the level of RNA encoded by the gene in the test sample of blood is higher than in the control samples of blood, and, for IL2RB, whether the level of RNA encoded by the gene in the test sample of blood is lower than in the control samples of blood; and (b) concluding that the subject is at an increased risk of having colorectal cancer relative to the general population if, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, the value indicates that the level of RNA encoded by the gene in the test sample of blood is higher than in the control samples of blood, and concluding that the subject is at an increased risk of having colorectal cancer relative to the general population if, for IL2RB, the value indicates that the level of RNA encoded by the gene in the test sample of blood is lower than in the control samples of blood. Another aspect of the invention disclosed herein is an isolated composition comprising a blood sample from a test subject and a nucleic acid molecule selected from one or more of the group consisting of RNA encoded by an ANXA3 gene, cDNA complementary to the RNA, an oligonucleotide which specifically hybridizes to the cDNA or the RNA under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA, and an amplification product of the cDNA. One embodiment of this composition further comprises a nucleic acid molecule selected from one or more of the group consisting of RNA encoded by one or more genes selected from the group of genes consisting of CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, cDNA complementary to the RNA of the group of genes, an oligonucleotide which specifically hybridizes to the cDNA complementary to the RNA of the group of genes or to the RNA of the group of genes under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA of the group of genes, and an amplification product of the cDNA of the RNA of the group of genes. Another aspect of the invention disclosed herein is an isolated composition comprising a blood sample from a test subject and a nucleic acid molecule selected from one or more of the group consisting of RNA encoded by a CLEC4D gene, cDNA complementary to the RNA, an oligonucleotide which specifically hybridizes to the cDNA or the RNA under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA, and an amplification product of the cDNA. One embodiment of this composition further comprises a nucleic acid molecule selected from one or more of the group consisting of RNA encoded by one or more genes selected from the group of genes consisting of ANXA3, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, cDNA complementary to the RNA of the group of genes, an oligonucleotide which specifically hybridizes to the cDNA complementary to the RNA of the group of genes or to the RNA of the group of genes under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA of the group of genes, and an amplification product of the cDNA of the RNA of the group of genes. Another aspect of the invention disclosed herein is an isolated composition comprising a blood sample from a test subject and a nucleic acid molecule selected from one or more of the group consisting of RNA encoded by a IL2RB gene, cDNA complementary to the RNA, an oligonucleotide which specifically hybridizes to the cDNA or the RNA under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA, and an amplification product of the cDNA. One embodiment of this composition further comprises a nucleic acid molecule selected from one or more of the group consisting of RNA encoded by one or more genes selected from the group of genes consisting of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, cDNA complementary to the RNA of the group of genes, an oligonucleotide which specifically hybridizes to the cDNA complementary to the RNA of the group of genes or to the RNA of the group of genes under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA of the group of genes, and an amplification product of the cDNA of the RNA of the group of genes. Another aspect of the invention disclosed herein is an isolated composition comprising a blood sample from a test subject and a nucleic acid molecule selected from one or more of the group consisting of RNA encoded by a LMNB1 gene, cDNA complementary to the RNA, an oligonucleotide which specifically hybridizes to the cDNA or the RNA under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA, and an amplification product of the cDNA. One embodiment of this composition further comprises a nucleic acid molecule selected from one or more of the group consisting of RNA encoded by one or more genes selected from the group of genes consisting of ANXA3, CLEC4D, IL2RB, PRRG4, TNFAIP6 and VNN1, cDNA complementary to the RNA of the group of genes, an oligonucleotide which specifically hybridizes to the cDNA complementary to the RNA of the group of genes or to the RNA of the group of genes under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA of the group of genes, and an amplification product of the cDNA of the RNA of the group of genes. Another aspect of the invention disclosed herein is an isolated composition comprising a blood sample from a test subject and a nucleic acid molecule selected from one or more of the group consisting of RNA encoded by a PRRG4 gene, cDNA complementary to the RNA, an oligonucleotide which specifically hybridizes to the cDNA or the RNA under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA, and an amplification product of the cDNA. One embodiment of this composition further comprises nucleic acid molecule selected from one or more of the group consisting of RNA encoded by one or more genes selected from the group of genes consisting of ANXA3, CLEC4D, IL2RB, LMNB1, TNFAIP6 and VNN1, cDNA complementary to the RNA of the group of genes, an oligonucleotide which specifically hybridizes to the cDNA complementary to the RNA of the group of genes or to the RNA of the group of genes under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA of the group of genes, and an amplification product of the cDNA of the RNA of the group of genes. Another aspect of the invention disclosed herein is an isolated composition comprising a blood sample from a test subject and a nucleic acid molecule selected from one or more of the group consisting of RNA encoded by a TNFAIP6 gene, cDNA complementary to the RNA, an oligonucleotide which specifically hybridizes to the cDNA or the RNA under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA, and an amplification product of the cDNA. One embodiment of this composition further comprises a nucleic acid molecule selected from one or more of the group consisting of RNA encoded by one or more genes selected from the group of genes consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, and VNN1, cDNA complementary to the RNA of the group of genes, an oligonucleotide which specifically hybridizes to the cDNA complementary to the RNA of the group of genes or to the RNA of the group of genes under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA of the group of genes, and an amplification product of the cDNA of the RNA of the group of genes. Another aspect of the invention disclosed herein is an isolated composition comprising a blood sample from a test subject and a nucleic acid molecule selected from one or more of the group consisting of RNA encoded by a VNN1 gene, cDNA complementary to the RNA, an oligonucleotide which specifically hybridizes to the cDNA or the RNA under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA, and an amplification product of the cDNA. One embodiment of this composition further comprises a nucleic acid molecule selected from one or more of the group consisting of RNA encoded by one or more genes selected from the group of genes consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, and TNFAIP6, cDNA complementary to the RNA of the group of genes, an oligonucleotide which specifically hybridizes to the cDNA complementary to the RNA of the group of genes or to the RNA of the group of genes under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA of the group of genes, and an amplification product of the cDNA of the RNA of the group of genes.
  • Another aspect of the invention disclosed herein is an isolated composition comprising an isolated nucleic acid molecule of a blood sample from a test subject, where the nucleic acid molecule is selected from one or more of the group consisting of RNA encoded by an ANXA3 gene, cDNA complementary to the RNA, an oligonucleotide which specifically hybridizes to the cDNA or the RNA under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA, and an amplification product of the cDNA. One embodiment of this composition further comprises a nucleic acid molecule selected from one or more of the group consisting of RNA encoded by one or more genes selected from the group of genes consisting of CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, cDNA complementary to the RNA of the group of genes or the complement thereof, an oligonucleotide which specifically hybridizes to the cDNA complementary to the RNA of the group of genes or to the RNA of the group of genes under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA of the group of genes, and an amplification product of the cDNA of the RNA of the group of genes. Another aspect of the invention disclosed herein is an isolated composition comprising an isolated nucleic acid molecule of a blood sample from a test subject, where the nucleic acid molecule is selected from one or more of the group consisting of RNA encoded by an CLEC4D gene, cDNA complementary to the RNA, an oligonucleotide which specifically hybridizes to the cDNA or the RNA under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA, and an amplification product of the cDNA. One embodiment of this composition further comprises a nucleic acid molecule selected from one or more of the group consisting of RNA encoded by one or more genes selected from the group of genes consisting of ANXA3, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, cDNA complementary to the RNA of the group of genes or the complement thereof, an oligonucleotide which specifically hybridizes to the cDNA complementary to the RNA of the group of genes or to the RNA of the group of genes under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA of the group of genes, and an amplification product of the cDNA of the RNA of the group of genes. Another aspect of the invention disclosed herein is an isolated composition comprising an isolated nucleic acid molecule of a blood sample from a test subject, where the nucleic acid molecule is selected from one or more of the group consisting of RNA encoded by an IL2RB gene, cDNA complementary to the RNA, an oligonucleotide which specifically hybridizes to the cDNA or the RNA under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA, and an amplification product of the cDNA. One embodiment of this composition further comprises a nucleic acid molecule selected from one or more of the group consisting of RNA encoded by one or more genes selected from the group of genes consisting of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, cDNA complementary to the RNA of the group of genes or the complement thereof, an oligonucleotide which specifically hybridizes to the cDNA complementary to the RNA of the group of genes or to the RNA of the group of genes under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA of the group of genes, and an amplification product of the cDNA of the RNA of the group of genes. Another aspect of the invention disclosed herein is an isolated composition comprising an isolated nucleic acid molecule of a blood sample from a test subject, where the nucleic acid molecule is selected from one or more of the group consisting of RNA encoded by an LMNB1 gene, cDNA complementary to the RNA, an oligonucleotide which specifically hybridizes to the cDNA or the RNA under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA, and an amplification product of the cDNA. One embodiment of this composition further comprises a nucleic acid molecule selected from one or more of the group consisting of RNA encoded by one or more genes selected from the group of genes consisting of ANXA3, CLEC4D, IL2RB, PRRG4, TNFAIP6 and VNN1, cDNA complementary to the RNA of the group of genes or the complement thereof, an oligonucleotide which specifically hybridizes to the cDNA complementary to the RNA of the group of genes or to the RNA of the group of genes under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA of the group of genes, and an amplification product of the cDNA of the RNA of the group of genes. Another aspect of the invention disclosed herein is an isolated composition comprising an isolated nucleic acid molecule of a blood sample from a test subject, where the nucleic acid molecule is selected from one or more of the group consisting of RNA encoded by a PRRG4, gene, cDNA complementary to the RNA, an oligonucleotide which specifically hybridizes to the cDNA or the RNA under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA, and an amplification product of the cDNA. One embodiment of this composition further comprises a nucleic acid molecule selected from one or more of the group consisting of RNA encoded by one or more genes selected from the group of genes consisting of ANXA3, CLEC4D, IL2RB, LMNB1, TNFAIP6 and VNN1, cDNA complementary to the RNA of the group of genes or the complement thereof, an oligonucleotide which specifically hybridizes to the cDNA complementary to the RNA of the group of genes or to the RNA of the group of genes under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA of the group of genes, and an amplification product of the cDNA of the RNA of the group of genes. Another aspect of the invention disclosed herein is an isolated composition comprising an isolated nucleic acid molecule of a blood sample from a test subject, where the nucleic acid molecule is selected from one or more of the group consisting of RNA encoded by a TNFAIP6 gene, cDNA complementary to the RNA, an oligonucleotide which specifically hybridizes to the cDNA or the RNA under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA, and an amplification product of the cDNA. One embodiment of this composition further comprises a nucleic acid molecule selected from one or more of the group consisting of RNA encoded by one or more genes selected from the group of genes consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, and VNN1, cDNA complementary to the RNA of the group of genes or the complement thereof, an oligonucleotide which specifically hybridizes to the cDNA complementary to the RNA of the group of genes or to the RNA of the group of genes under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA of the group of genes, and an amplification product of the cDNA of the RNA of the group of genes. An isolated composition comprising a blood sample from a test subject and a nucleic acid molecule selected from one or more of the group consisting of RNA encoded by a VNN1 gene, cDNA complementary to the RNA, an oligonucleotide which specifically hybridizes to the cDNA or the RNA under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA, and an amplification product of the cDNA. One embodiment of this composition further comprises a nucleic acid molecule selected from one or more of the group consisting of RNA encoded by one or more genes selected from the group of genes consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, and TNFAIP6, cDNA complementary to the RNA of the group of genes or the complement thereof, an oligonucleotide which specifically hybridizes to the cDNA complementary to the RNA of the group of genes or to the RNA of the group of genes under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA of the group of genes, and an amplification product of the cDNA of the RNA of the group of genes.
  • Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by an ANXA3 gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a VNN1 gene, or composition thereof. Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by an ANXA3 gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a TNFAIP6 gene, or composition thereof. Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by an ANXA3 gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a PRRG4 gene, or composition thereof. Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by an ANXA3 gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a PRRG4 gene, or composition thereof. Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by an ANXA3 gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a LMNB1 gene, or composition thereof. Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by an ANXA3 gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by an IL2RB gene, or composition thereof. Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by an ANXA3 gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a CLEC4D gene, or composition thereof. Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by an CLEC4D gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a VNN1 gene, or composition thereof. Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by an CLEC4D gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a TNFAIP6 gene, or composition thereof. Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by an CLEC4D gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a PRRG4 gene, or composition thereof. Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by an CLEC4D gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a PRRG4 gene, or composition thereof. Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by an CLEC4D gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a LMNB1 gene, or composition thereof. Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by an CLEC4D gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by an IL2RB gene, or composition thereof. Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by an IL2RB gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a TNFAIP6 gene, or composition thereof. Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by an IL2RB gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a PRRG4 gene, or composition thereof. Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by an IL2RB gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a LMNB1 gene, or composition thereof. Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by an IL2RB gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a VNN1 gene, or composition thereof. Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a LMNB1 gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a PRRG4 gene, or composition thereof. Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a LMNB1 gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a TNFAIP6 gene, or composition thereof. Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by an LMNB1 gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a VNN1 gene, or composition thereof. Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a PRRG4 gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a VNN1 gene, or composition thereof. Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a PRRG4 gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a TNFAIP6 gene, or composition thereof. Another aspect of the invention disclosed herein is a primer set comprising a first primer, where the first primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a VNN1 gene, and a second primer, where the second primer is one of a set of primers capable of generating an amplification product of cDNA complementary to RNA of encoded by a TNFAIP6 gene, or composition thereof.
  • Another aspect of the invention disclosed herein is test system comprising: a) two or more blood samples where each blood sample is from a different test subject, and b) an isolated nucleic acid molecule of each the blood sample from a test subject, where the nucleic acid molecule is selected from one or more of the group consisting of RNA encoded by an ANXA3 gene, cDNA complementary to the RNA, an oligonucleotide which specifically hybridizes to the cDNA or complement thereof, or the RNA under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA, and an amplification product of the cDNA. Another aspect of the invention disclosed herein is a test system comprising: a) two or more blood samples where each blood sample is from a different test subject, and b) an isolated nucleic acid molecule of each the blood sample from a test subject, where the nucleic acid molecule is selected from one or more of the group consisting of RNA encoded by a CLEC4D, gene, cDNA complementary to the RNA, an oligonucleotide which specifically hybridizes to the cDNA or complement thereof, or the RNA under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA, and an amplification product of the cDNA Another aspect of the invention disclosed herein is a test system comprising: a) two or more blood samples where each blood sample is from a different test subject, and b) an isolated nucleic acid molecule of each the blood sample from a test subject, where the nucleic acid molecule is selected from one or more of the group consisting of RNA encoded by an IL2RB gene, cDNA complementary to the RNA, an oligonucleotide which specifically hybridizes to the cDNA or complement thereof, or the RNA under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA, and an amplification product of the cDNA. Another aspect of the invention disclosed herein is a test system comprising: a) two or more blood samples where each blood sample is from a different test subject, and b) an isolated nucleic acid molecule of each the blood sample from a test subject, where the nucleic acid molecule is selected from one or more of the group consisting of RNA encoded by an LMNB1 gene, cDNA complementary to the RNA, an oligonucleotide which specifically hybridizes to the cDNA or complement thereof, or the RNA under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA, and an amplification product of the cDNA. Another aspect of the invention disclosed herein is a test system comprising: a) two or more blood samples where each blood sample is from a different test subject, and b) an isolated nucleic acid molecule of each the blood sample from a test subject, where the nucleic acid molecule is selected from one or more of the group consisting of RNA encoded by a PRRG4 gene, cDNA complementary to the RNA, an oligonucleotide which specifically hybridizes to the cDNA or complement thereof, or the RNA under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA, and an amplification product of the cDNA. Another aspect of the invention disclosed herein is a test system comprising: a) two or more blood samples where each blood sample is from a different test subject, and b) an isolated nucleic acid molecule of each the blood sample from a test subject, where the nucleic acid molecule is selected from one or more of the group consisting of RNA encoded by a TNFAIP6 gene, cDNA complementary to the RNA, an oligonucleotide which specifically hybridizes to the cDNA or complement thereof, or the RNA under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA, and an amplification product of the cDNA. Another aspect of the invention disclosed herein is a test system comprising: a) two or more blood samples where each blood sample is from a different test subject, and b) an isolated nucleic acid molecule of each the blood sample from a test subject, where the nucleic acid molecule is selected from one or more of the group consisting of RNA encoded by a VNN1 gene, cDNA complementary to the RNA, an oligonucleotide which specifically hybridizes to the cDNA or complement thereof, or the RNA under stringent conditions, a primer set capable of generating an amplification product of the cDNA complementary to RNA, and an amplification product of the cDNA. An embodiment of any of the test systems described in this paragraph includes where the test subject is being screened for colorectal cancer.
  • The following non-limiting examples are illustrative of the invention:
  • EXAMPLES Example 1 General Materials and Methods
  • Introduction:
  • The following materials and methods describe experiments performed to demonstrate that analysis of blood for levels of RNA encoded by genes surprisingly identified by the present inventors as colorectal cancer marker genes in blood via array hybridization analysis using an Affymetrix U133Plus 2.0 GeneChip oligonucleotide array (Affymetrix; Santa Clara, Calif.) (data not shown), can also serve as blood markers for diagnosing colorectal cancer via quantitative reverse-transcriptase PCR analysis.
  • Blood Sample Collection:
  • Samples of 2.5 ml whole blood were collected into PAXgene Blood RNA Tubes (PreAnalytiX) from human subjects not having any colorectal pathology and from human subjects having colorectal cancer. Samples were obtained from subjects enrolled in colorectal cancer studies conducted by GeneNews Corp. and collaborating institutions. Blood samples from subjects having colorectal cancer were collected prior to tumor resection, and cancer stage and histology were determined by institutional pathologists. Blood samples from subjects not having any colorectal pathology were collected from subjects presenting for endoscopy screening. Informed consent was obtained according to the research protocols approved by the research ethical boards of the institutions involved. Experimental group sample pairs were selected with an effort to match gender, age, body mass index (BMI), ethnicity and medical history. Samples were divided into training and test sets.
  • RNA Isolation:
  • A sample of 2.5 ml whole blood was collected into PAXgene Blood RNA tubes (PreAnalytiX) and processed in accordance with the instructions of the PAXgene Blood RNA Kit protocol. In brief, after storing the blood in the PAXgene tube for at least 2 hours, the blood sample was centrifuged and the supernatant discarded. To the remaining sample, 350 microliters of the supplied Buffer BR1 was added, and the sample was pipetted into the spin column and centrifuged, washed and finally eluted as isolated RNA and stored.
  • Reverse Transcription:
  • Reverse transcription of blood sample-derived RNA into single-stranded complementary DNA was performed using the High Capacity cDNA Reverse Transcription Kit from (Applied Biosystems; Foster City, Calif.; Product number 4368814), according to the manufacturer's instructions. Specifically, 1 microgram of isolated RNA was incubated with reverse transcriptase buffer, dNTPs, random primers and reverse transcriptase and incubated at 25° C. for 10 minutes and subsequently at 37° C. for two hours.
  • Quantitative Real Time RT-PCR:
  • Quantitative real-time PCR analysis to measure levels of RNA encoded by the genes listed in Table 1 was performed on cDNA samples using the QuantiTect™ Probe RT-PCR system (Qiagen; Valencia, Calif.; Product No. 204345), using the primers listed in Table 2 for amplification of cDNA template corresponding to the indicated gene, and TaqMan dual labeled probes comprising the polynucleotides listed in Table 3 for measuring levels of amplicon corresponding to the indicated gene. The TaqMan probe and primers were ordered from Applied Biosystems Assays-On-Demand, or from IDT (Integrated DNA Technologies, Coralville, Iowa), or from Biosearch Technologies (Novato, Calif.). Amplicon levels were measured in real time using a RealTime PCR System 7500 instrument (Applied Biosystems). Specifically, 20 nanograms of cDNA resulting from reverse transcription was added to the QuantiTect Probe PCR Master Mix as provided and no adjustments were made for magnesium concentration. Uracil-N-Glycosylase was not added. Both forward primer and reverse primer (Table 1) specific to the target genes were added to a concentration of 5 micromolar, and the resultant 25 microliter reaction volume was incubated as follows: 50 degrees centigrade for 2 minutes, followed by 95 degrees centigrade for 15 minutes, followed by 40 cycles of: [94 degrees centigrade for 15 seconds, followed by 55 degrees centigrade for 35 seconds, followed by 72 degrees centigrade for 30 seconds]. Amplification data was collected during each of the 40 incubations at 55 degrees centigrade. All quantitative reverse transcriptase-PCR analyses were performed as duplex amplifications of a target gene and a reference gene (either ACTB or IL2RB, as indicated) in the same reaction mixture. Serial dilution measurements for target and duplex partner genes were assayed, to ensure that the values were within linear range and that the amplification efficiencies were approximately equal. Examination via polyacrylamide gel electrophoresis provided confirmation of specific PCR amplification and the lack of primer-dimer formation in each reaction well.
  • TABLE 1
    Genes encoding target RNAs for determining colorectal cancer
    probability versus absence of colorectal pathology.
    Gene GenBank
    Symbol Accession Gene Description
    ACTB NM_001101 beta-actin
    ANXA3 NM_005139 annexin A3
    CLEC4D NM_080387 C-type lectin domain family 4, member D
    IL2RB NM_000878 interleukin 2 receptor, beta
    LMNB1 NM_005573 lamin B1
    PRRG4 NM_024081 proline rich Gla (G-carboxyglutamic acid)
    4 (transmembrane)
    TNFAIP6 NM_007115 tumor necrosis factor, alpha-induced protein 6
    VNN1 NM_004666 vanin 1
  • TABLE 2
    Primers used for quantitative PCR analysis.
    Gene
    encoding
    amplified Primer Amplicon
    cDNA Primer Primer pair sequences position size (bp)
    ACTB 5′ 5′-CACCACACCTTCTACAATGAGCTG-3′ (SEQ ID NO: 1) 259 158
    3′ 5′-ACAGCCTGGATAGCAACGTACA-3′ (SEQ ID NO: 2) 416
    ANXA3 5′ 5′-GAAACATCTGGTGACTTCCG-3′ (SEQ ID NO: 10) 748 103
    3′ 5′-TCTGGGCATCTTGTTTGG-3 ′ (SEQ ID NO: 11) 850
    CLEC4D 5′ 5′-CCATTTAACCCACGCAGAG-3′ (SEQ ID NO: 19) 673 101
    3′ 5′-CAGGCCCATTTATCTTGGTT-3′ (SEQ ID NO: 20) 773
    IL2RB 5′ 5′-AAATCTCCCAAGCCTCCCA-3′ (SEQ ID NO: 28) 588 127
    3′ 5′-AGGCAGATCCATTCCTGCT-3′ (SEQ ID NO: 29) 714
    LMNB1 5′ 5′-GGAGTGGTTGTTGAGGAAGAA-3′ (SEQ ID NO: 37) 2051 151
    3′ 5′-CTGAGAAGGCTCTGCACTGTA-3′ (SEQ ID NO: 38) 2201
    PRRG4 5′ 5′-ATGCGGGAGAAGAAGTGTTTAC-3′ (SEQ ID NO: 46) 341 153
    3′ 5′-CTCTGGCTTCCTCATAATTGC-3′ (SEQ ID NO: 47) 493
    TNFAIP6 5′ 5′-GCCTATTGCTACAACCCACA-3′ (SEQ ID NO: 55) 448 84
    3′ 5′-TGGGAAGCCTGGAGATTTA-3′ (SEQ ID NO: 56) 531
    VNN1 5′ 5′-TGACAGGAAGTGGCATCTAT-3′ (SEQ ID NO: 64) 835 147
    3′ 5′-TACTGCTGGCATAGGAAGTC-3′ (SEQ ID NO: 65) 981
  • TABLE 3
    TaqMan ® probes used for quantitative PCR analysis.
    Gene encoding Probe
    amplicon Taqman probe base sequence position
    ACTB 5′-AACCGCGAGAAGATGACCCAGATCAT-3′ (SEQ ID NO: 3) 343
    ANXA3 5′-TTGACTTTGGCAGATGGCAGA-3′ (SEQ ID NO: 12) 778
    CLEC4D 5′-CTGGCATAAGAATGAACCCGACA-3′ (SEQ ID NO: 21) 696
    IL2RB 5′-TTGAAAGACACCTGGAGTTCG-3 ′ (SEQ ID NO: 30) 612
    LMNB1 5′-AACCCCAAGAGCATCCAATAG-3′ (SEQ ID NO: 39) 2089
    PRRG4 5′-CTCTTCACTCCCGGCAACCTAGAA-3′ (SEQ ID NO: 48) 427
    TNFAIP6 5′-AAGGAGTGTGGTGGCGTCTTTAC-3′ (SEQ ID NO: 57) 472
    VNN1 5′-AGAAGAGGGAAAACTCCTCCTCTCG-3′ (SEQ ID NO: 66) 896
  • Determination of Observed Range of Fold-Changes in Levels of RNA Encoded by Marker Genes in Blood of Subjects Having Colorectal Cancer Relative to Subjects not Having any Colorectal Pathology:
  • For each of the sample training and sample test sets, average fold-change in levels of RNA encoded by marker genes, normalized to either ACTB or IL2RB, were calculated as the ratio of average levels of RNA encoded by marker genes in blood of subjects having colorectal cancer to average levels of RNA encoded by marker genes in blood of subjects not having any colorectal pathology. The statistical significance of the fold-changes were confirmed by a p-value of less than 0.05. Maximum observed directional fold-changes in normalized levels of RNA encoded by marker genes found to be higher in blood of subjects having colorectal cancer than in blood of subjects not having any colorectal pathology were further calculated, for each marker gene, as the ratio of the highest level observed in any single sample from a subject having colorectal cancer to the average level in subjects not having any colorectal pathology. Similarly, maximum observed directional fold-changes in normalized levels of RNA encoded by marker genes found to be lower in blood of subjects having colorectal cancer than in blood of subjects not having any colorectal pathology were further calculated, for each marker gene, as the ratio of the lowest level observed in any single sample from a subject having colorectal cancer to the average level in subjects not having any colorectal pathology. In this way, observed ranges of fold-changes, ranging from average fold-change to maximal observed directional fold-change, in levels of RNA encoded by marker genes in blood of subjects having colorectal cancer relative to subjects not having any colorectal pathology were determined.
  • Formulation of Mathematical Models for Determining Probability of Colorectal Cancer Versus Absence of Colorectal Pathology:
  • Logistic regression was used to formulate mathematical models for determining the probability that a test subject has colorectal cancer as opposed to not having any colorectal pathology. Levels of RNA encoded by colorectal cancer marker genes and of reference genes determined via duplex quantitative reverse transcriptase PCR in blood of positive and negative control subjects were analyzed via logistic regression so as to generate models having the general form:

  • P={1+ê−[K 0 +K 1 L 1 +K 2 L 2 +K 3 L 3 . . . +K n L n]}̂−1,
      • where P is the probability that a test subject has colorectal cancer as opposed to not having any colorectal pathology; K0 is a constant; K1 is a coefficient specific to a first marker gene; L1 is a ratio of a level of RNA encoded by the first gene to a level of RNA encoded by a reference gene in blood of the test subject; K2 is a coefficient specific to a second marker gene; L2 is a ratio of a level of RNA encoded by the second gene to a level of RNA encoded by the reference gene in blood of the test subject; K3 is a coefficient specific to a third marker gene; L3 is a ratio of a level of RNA encoded by the third gene to a level of RNA encoded by the reference gene in blood of the test subject; Kn is a coefficient specific to an nth marker gene; and Ln is a ratio of a level of RNA encoded by the nth gene to a level of RNA encoded by the reference gene in blood of the test subject. The ratio of the level of RNA encoded by a marker gene to the level of RNA encoded by a reference gene was calculated as the change (ΔCt) in the cycle number (Ct) at which the increase in fluorescence is exponential between the marker gene and the reference gene according to the equation: ΔCt=Ct (marker gene)−Ct (reference gene). The caret symbol “̂” is used herein to denote that a value preceding the caret is raised to a power corresponding to the value following the caret.
    Example 2 Measurement of Blood Levels of RNA Encoded by any Combination of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and/or VNN1 can be Used to Determine a Probability that a Test Subject has Colorectal Cancer as Opposed to not Having any Colorectal Pathology
  • Materials and Methods:
  • Refer to “General materials and methods”, above.
  • Experimental Results:
  • Sample Training Set:
  • Discovery of Significantly Different Levels of RNA Encoded by ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 and in Blood of Subjects Having Colorectal Cancer Relative to Subjects not Having any Colorectal Pathology:
  • Quantitative reverse transcriptase PCR analysis of gene expression in a training set of blood samples from 117 subjects having colorectal cancer and 130 subjects not having any colorectal pathology, using the housekeeping gene ACTB as duplex partner for normalization of gene expression levels was performed. The normalized RNA levels measured are shown in Table 4.
  • TABLE 4
    Sample training set levels of RNA encoded by ANXA3, CLEC4D, IL2RB, LMNB1,
    PRRG4, TNFAIP6 and VNN1 in blood of subjects having colorectal cancer (Group
    1) and subjects not having any colorectal pathology (Group 0), normalized
    to levels of RNA encoded by ACTB. Levels shown correspond to ΔCt.
    Gene
    Sample ID Group ANXA3 CLEC4D IL2RB LMNB1 PRRG4 TNFAIP6 VNN1
    CD0011pax 0 6.523 7.755 5.195 7.310 7.860 8.501 8.991
    CD0012pax 0 7.878 8.595 5.250 7.525 8.183 8.439 9.878
    CD0030pax 0 6.411 6.420 4.173 6.220 6.973 5.901 7.101
    CD0063pax 0 7.103 8.545 4.203 7.165 7.795 8.499 8.628
    CD0077pax 0 4.808 6.185 5.098 5.405 6.710 6.524 6.533
    CD0078pax 0 5.946 7.000 3.553 5.820 5.570 7.476 6.488
    CD0085pax 0 5.543 7.700 5.003 6.210 7.460 8.149 6.678
    CD0117pax 0 6.021 8.170 4.463 5.685 8.020 8.166 6.573
    CD0146pax 0 5.396 6.335 4.468 5.320 5.735 6.691 6.253
    CD0167pax 0 3.501 4.893 4.480 4.978 5.590 6.469 5.173
    CD0249pax 0 4.443 4.855 4.878 4.803 6.043 5.556 6.471
    CD0279pax 0 5.503 7.095 4.270 5.395 6.098 6.694 7.043
    CD0286pax 0 4.791 6.928 4.350 5.383 5.960 5.714 5.598
    CD0297pax 0 5.861 6.670 5.083 6.565 6.405 6.186 7.118
    CD0323pax 0 6.966 7.773 4.645 5.723 7.470 8.149 8.738
    CD0445pax 0 6.458 7.420 4.448 6.103 6.448 7.216 7.411
    CD0463pax 0 4.391 6.485 4.203 5.605 6.583 6.161 7.036
    CD0491pax 0 5.093 6.370 4.928 6.123 6.978 7.171 6.511
    CD0496pax 0 6.058 8.270 4.670 6.355 7.783 6.434 6.623
    CD0501pax 0 6.326 7.725 4.613 6.270 7.215 8.581 6.978
    CD0504pax 0 4.023 5.060 4.858 5.920 7.530 5.289 6.113
    CD0573pax 0 6.791 6.140 4.248 6.160 6.713 7.646 7.286
    CD0578pax 0 6.328 6.670 4.128 5.128 6.033 6.411 7.081
    CD0620pax 0 2.361 3.628 6.120 3.873 6.120 5.274 5.613
    CD0639pax 0 5.611 7.013 4.980 6.258 5.630 6.689 7.528
    CD0645pax 0 4.596 5.868 5.190 4.908 5.475 6.099 5.608
    CD0679pax 0 5.611 7.808 5.070 5.633 6.150 7.974 7.748
    CD0685pax 0 5.796 8.150 4.358 6.050 7.155 7.606 6.673
    CD0716pax 0 6.961 8.193 4.090 5.648 5.750 6.669 7.388
    CD0749pax 0 5.208 6.970 4.520 5.565 6.430 7.176 6.316
    CD0760pax 0 2.868 5.020 5.603 3.605 5.790 4.679 5.663
    CD0811pax 0 7.188 8.065 3.275 6.500 7.305 6.021 6.141
    CD0846pax 0 4.626 6.488 4.730 5.308 5.645 4.864 7.813
    CD0848pax 0 5.113 6.235 3.380 5.915 6.388 6.409 7.263
    CD0924pax 0 5.731 6.370 5.238 6.095 6.248 5.791 6.131
    CD1066pax 0 5.346 5.900 4.903 5.990 6.868 7.466 6.211
    CD1073pax 0 5.681 5.813 5.150 5.858 6.350 6.674 6.663
    CD1075pax 0 5.128 7.010 4.535 6.075 8.228 7.264 6.563
    CD1089pax 0 5.081 7.225 5.733 5.550 6.395 6.546 6.668
    CD1116pax 0 4.188 5.500 5.023 5.295 6.180 5.764 6.378
    CD1120pax 0 3.663 5.495 5.203 4.780 6.555 6.864 5.118
    CD1198pax 0 5.398 6.210 4.155 6.055 6.095 6.086 5.881
    PB1179pax 0 6.401 7.293 4.700 6.563 7.080 8.169 7.418
    PB1277pax 0 6.403 7.520 4.860 6.260 7.185 7.586 6.351
    PB1301pax 0 4.733 6.200 6.268 5.608 6.863 5.891 5.531
    PB1315pax 0 3.898 6.165 5.438 4.998 6.008 5.206 7.161
    PB1345pax 0 5.246 6.280 4.598 6.410 6.775 6.051 7.663
    PB1518pax 0 6.806 7.803 5.055 5.818 7.230 7.134 7.328
    PB1520pax 0 4.283 5.560 3.250 4.660 3.793 5.214 6.108
    PB1574pax 0 7.538 8.685 5.583 7.453 7.483 7.166 8.226
    PB1783pax 0 8.056 9.095 5.453 7.650 8.535 9.011 8.448
    PB1799pax 0 7.338 8.760 5.765 7.435 8.855 9.181 7.801
    PB1811pax 0 6.848 8.115 5.080 7.140 8.390 7.416 7.471
    PB1830pax 0 5.788 7.385 5.923 6.440 6.870 7.444 9.253
    PB1833pax 0 5.943 7.620 5.488 6.615 7.620 6.829 8.098
    PB1843pax 0 7.218 7.870 5.780 6.770 8.338 5.739 8.708
    PB1851pax 0 7.468 8.045 6.050 7.795 8.325 10.181 9.261
    PB1919pax 0 8.271 9.525 5.233 7.165 8.975 9.051 9.108
    PB1922pax 0 6.788 8.280 6.028 7.363 8.348 9.281 8.306
    PB1924pax 0 7.748 8.655 6.758 7.628 8.303 8.816 9.401
    PB1937pax 0 7.178 9.085 5.118 7.665 9.740 8.754 8.798
    PB1964pax 0 5.491 7.820 4.463 6.065 7.843 7.726 8.496
    PB2027pax 0 5.463 6.525 5.150 6.300 7.360 7.401 7.546
    PB2029pax 0 5.793 7.060 5.050 6.375 6.973 7.624 7.758
    PB2073pax 0 6.021 6.335 4.873 6.290 7.933 6.991 6.846
    PB2086pax 0 5.048 5.665 4.208 5.098 6.453 5.286 5.991
    PB2099pax 0 4.808 5.360 5.388 5.910 6.680 5.604 7.003
    PB2100pax 0 6.353 6.960 4.465 6.085 7.625 7.351 6.826
    PB2132pax 0 5.693 7.235 4.438 5.745 6.995 7.649 7.518
    PB2168pax 0 6.776 7.593 4.605 6.168 7.125 8.284 6.993
    PB2192pax 0 6.701 7.845 5.483 6.230 6.965 7.751 8.378
    PB2196pax 0 6.061 7.655 4.573 6.195 7.985 7.781 6.668
    PB2200pax 0 6.706 7.640 5.158 6.025 7.103 6.826 6.276
    PB2213pax 0 6.898 7.435 4.773 6.380 6.630 8.614 7.448
    PB2224pax 0 4.841 5.385 4.743 5.210 6.448 7.546 6.851
    PB2228pax 0 6.511 6.915 3.953 6.450 7.373 8.906 8.086
    PB2229pax 0 5.771 6.440 5.588 6.030 5.865 7.091 6.528
    PB2277pax 0 6.348 6.685 4.290 5.705 5.930 5.986 7.351
    PB2297pax 0 5.886 6.785 4.703 5.835 6.533 6.451 5.696
    PB2312pax 0 5.533 6.530 3.773 6.098 5.978 6.651 6.746
    PB2398pax 0 4.711 5.390 5.033 5.440 5.748 5.136 7.111
    PB2409pax 0 5.946 7.195 4.933 5.835 6.950 6.811 7.683
    PB2414pax 0 7.843 7.790 3.955 6.380 7.745 9.026 7.101
    PB2467pax 0 5.773 6.935 4.260 5.955 6.525 7.596 6.756
    PB2473pax 0 6.818 8.275 5.530 7.375 8.405 7.586 8.561
    PB2512pax 0 5.603 7.355 4.340 6.215 6.345 6.926 7.511
    PB2568pax 0 5.326 5.850 5.303 5.710 7.178 7.561 7.331
    PB2571pax 0 5.561 5.995 4.523 6.060 6.173 7.396 7.791
    PB2603pax 0 5.778 6.480 3.953 5.903 6.313 7.021 5.986
    PB2624pax 0 5.383 5.465 3.948 5.498 6.838 6.146 6.741
    PB2824pax 0 5.781 6.748 4.675 5.758 6.830 7.154 7.223
    PB2880pax 0 5.906 6.090 4.728 6.160 5.218 7.361 7.386
    PB3088pax 0 6.601 6.760 3.858 5.725 6.395 8.456 7.378
    RC0882pax 0 5.043 6.540 4.533 5.708 6.083 7.351 7.151
    RC0888pax 0 4.726 5.740 4.948 5.330 6.775 6.516 6.168
    RC0968pax 0 3.238 3.590 4.008 4.303 6.118 4.716 7.086
    RC2114pax 0 4.473 5.900 4.768 5.168 6.028 5.216 7.446
    RC2238pax 0 7.318 8.785 5.878 7.175 8.665 9.209 9.903
    RC2681pax 0 6.331 7.515 5.623 6.995 7.833 6.471 7.536
    RC2703pax 0 8.093 8.360 5.973 7.053 8.253 7.801 8.091
    RC2749pax 0 6.448 8.695 5.895 7.205 7.905 7.726 8.666
    RC2750pax 0 5.578 6.650 6.753 6.428 7.993 7.761 7.111
    RC2756pax 0 7.478 8.135 5.340 7.095 8.420 7.781 8.671
    RC2771pax 0 5.848 8.345 6.240 6.440 7.648 8.449 9.303
    RC2790pax 0 8.086 9.228 6.880 7.573 8.475 8.679 8.948
    RC2792pax 0 7.956 8.058 5.850 7.068 8.320 9.219 8.448
    RC2808pax 0 6.556 8.790 5.233 6.605 7.815 7.096 7.653
    RC2822pax 0 7.921 9.163 6.255 7.193 8.075 10.284 7.718
    RC2834pax 0 6.588 8.535 6.520 6.810 7.920 9.946 9.651
    RC2871pax 0 5.443 6.530 4.563 6.280 7.165 7.479 8.158
    RC2879pax 0 6.266 8.105 5.978 6.620 8.465 7.971 7.298
    RC2892pax 0 6.086 7.423 5.185 6.163 7.185 8.559 7.748
    RC2895pax 0 6.148 6.900 4.378 6.270 7.450 6.109 7.473
    RC2921pax 0 6.846 7.623 4.720 6.758 7.520 7.954 8.073
    RC2958pax 0 6.581 6.735 4.863 6.185 6.638 5.851 7.616
    RC3022pax 0 6.401 6.660 4.888 6.685 7.925 7.776 6.578
    RC3112pax 0 6.938 8.095 5.408 6.435 7.475 8.629 7.713
    RC3146pax 0 6.018 6.655 5.340 5.905 6.855 7.491 7.451
    RC3184pax 0 6.998 7.910 4.398 6.370 7.395 7.329 7.598
    RC3232pax 0 5.021 6.460 5.003 5.030 7.008 4.941 8.671
    RC3324pax 0 5.158 6.220 5.203 5.055 6.935 6.869 5.713
    RC3327pax 0 5.238 5.225 4.708 5.313 5.253 6.381 7.541
    RC3334pax 0 5.953 7.670 4.710 6.135 6.850 6.816 8.081
    RC3355pax 0 5.871 7.253 5.620 5.358 6.325 7.699 6.648
    RC3380pax 0 5.418 6.395 5.363 5.453 6.423 6.786 6.481
    RC3392pax 0 6.378 8.025 4.445 6.170 7.265 8.546 7.756
    RC3413pax 0 6.176 7.940 4.768 6.100 6.545 7.051 8.858
    RC3421pax 0 5.661 5.515 5.088 5.900 6.483 5.191 5.961
    RC3468pax 0 5.831 5.890 5.443 5.905 6.523 7.171 6.636
    RC3498pax 0 5.553 5.515 5.593 6.145 7.200 7.484 6.468
    CD0157pax 1 6.223 6.530 4.198 6.115 6.930 7.654 8.168
    CD0164pax 1 4.726 5.395 4.083 5.660 10.733 7.791 6.696
    CD0256pax 1 4.833 6.295 4.735 6.045 6.785 7.141 6.911
    CD0322pax 1 5.153 7.050 6.308 5.820 7.325 7.239 8.603
    CD0356pax 1 5.243 5.555 6.038 5.925 6.580 6.239 5.823
    CD0371pax 1 5.643 7.110 5.073 6.045 6.245 6.394 5.968
    CD0629pax 1 4.453 5.995 4.503 5.555 7.380 5.114 6.563
    CD1050pax 1 6.238 5.930 4.943 6.150 7.105 6.969 7.243
    MH0001pax 1 7.266 8.375 5.103 7.770 8.568 9.266 8.706
    MH0009pax 1 6.078 7.150 5.990 6.325 7.185 6.131 6.426
    MH0011pax 1 2.393 4.420 8.808 4.258 6.888 3.846 5.756
    MH0012pax 1 4.673 6.965 5.368 5.970 7.680 6.659 7.043
    MH0014pax 1 6.266 8.155 5.003 6.395 6.995 9.436 7.983
    MH0016pax 1 5.408 6.770 6.225 6.050 6.635 6.181 6.561
    MH0017pax 1 6.071 8.290 5.323 6.710 6.750 8.231 8.433
    MH0018pax 1 6.856 7.175 5.093 6.250 7.358 8.451 6.791
    MH0021pax 1 6.948 6.675 5.263 5.483 6.398 8.236 8.111
    MH0022pax 1 6.471 7.508 5.280 6.228 7.030 7.344 7.548
    MH0024pax 1 5.016 5.640 4.488 5.340 5.793 5.211 6.241
    MH0026pax 1 4.351 6.775 5.558 5.440 6.840 5.861 6.028
    MH0028pax 1 6.183 6.815 5.818 5.918 5.883 5.986 6.176
    MH0029pax 1 5.388 6.360 5.015 6.255 5.925 6.846 6.831
    MH0035pax 1 6.111 8.575 4.708 6.645 7.460 7.051 7.638
    MH0037pax 1 5.441 7.063 5.375 5.578 6.325 7.089 7.948
    MH0038pax 1 7.206 7.463 5.020 6.748 7.635 8.089 8.113
    MH0039pax 1 4.036 5.113 5.110 5.298 5.110 5.394 6.383
    MH0042pax 1 4.643 5.560 4.425 5.900 5.785 4.876 5.901
    MH0050pax 1 3.763 6.495 4.908 4.718 5.698 6.641 7.721
    MH0051pax 1 4.941 5.693 6.225 5.818 4.795 4.044 6.338
    PB1829pax 1 7.363 9.380 6.678 7.073 7.428 8.841 9.241
    PB1842pax 1 7.483 8.295 6.188 7.488 7.173 8.786 8.011
    PB1872pax 1 7.051 8.525 6.318 7.410 7.175 8.486 7.533
    PB2857pax 1 4.268 6.600 4.810 5.300 6.470 5.266 6.976
    RC2919pax 1 7.106 7.488 4.420 6.463 8.350 8.399 9.318
    RC3062pax 1 5.006 6.200 4.513 5.195 6.340 5.271 5.538
    RC3277pax 1 5.068 6.360 4.770 5.360 6.205 5.701 6.136
    RC3297pax 1 5.748 6.970 4.728 5.843 6.493 8.451 7.206
    RC3445pax 1 5.503 6.560 5.290 6.295 6.560 6.711 7.021
    RC3467pax 1 6.893 8.640 3.945 6.670 7.040 8.251 7.771
    CC0003pax 1 4.281 5.963 5.500 4.608 5.205 7.009 4.853
    DC0001pax 1 5.713 5.580 5.378 5.868 6.408 6.201 6.541
    DC0002pax 1 6.323 6.125 5.423 6.380 6.410 7.374 7.273
    DS0003pax 1 4.816 6.663 6.615 5.248 7.380 6.849 6.398
    FC0005pax 1 5.953 6.735 5.618 6.605 6.980 6.649 8.698
    FC0011pax 1 6.458 7.550 7.063 6.915 7.950 8.539 8.338
    FC0012pax 1 3.868 6.850 7.138 5.285 6.375 6.849 7.063
    JGA0001pax 1 5.426 6.250 7.448 6.125 8.173 6.586 7.096
    JGA0008pax 1 5.448 7.600 6.018 6.793 7.403 7.366 7.841
    JH0002pax 1 6.108 7.335 5.633 6.725 6.950 7.484 7.128
    JH0003pax 1 6.053 6.635 5.090 5.905 6.145 5.701 7.081
    JH0004pax 1 5.373 5.985 5.265 5.935 6.570 7.371 6.961
    JH0005pax 1 5.341 6.565 5.008 5.780 6.688 7.356 7.091
    JH0006pax 1 4.771 5.840 5.073 5.525 6.198 6.731 5.551
    JH0007pax 1 2.956 3.035 5.353 4.815 6.483 5.661 4.916
    JH0008pax 1 5.876 8.435 5.173 6.265 7.573 7.811 6.711
    JH0009pax 1 4.101 3.770 4.793 5.540 5.293 4.636 5.876
    JH0010pax 1 5.026 5.810 4.958 6.105 7.018 6.046 5.526
    JH0012pax 1 4.981 5.435 4.718 5.965 6.318 5.801 6.511
    JH0013pax 1 5.501 6.610 5.268 5.905 7.048 8.861 7.191
    JH0014pax 1 5.053 5.235 4.253 4.735 4.860 6.259 7.488
    JH0016pax 1 5.596 6.390 4.438 5.980 6.338 5.566 6.191
    JH0018pax 1 4.401 5.770 5.248 5.765 6.348 5.696 6.446
    JH0019pax 1 5.751 6.775 5.468 6.055 6.693 5.926 6.656
    JH0020pax 1 5.001 7.450 4.563 5.875 5.618 5.756 6.706
    JH0021pax 1 5.726 7.650 6.058 5.730 6.105 6.611 6.808
    JH0023pax 1 4.696 6.805 3.873 5.020 6.960 5.951 6.968
    JH0024pax 1 6.008 7.895 5.113 6.865 7.620 9.099 8.698
    JH0025pax 1 4.796 5.780 5.398 5.150 5.225 5.626 6.423
    JH0026pax 1 4.491 6.940 5.363 5.800 6.725 7.646 7.493
    JH0027pax 1 3.111 4.720 6.498 4.280 5.225 4.486 5.288
    JH0028pax 1 4.416 5.680 3.858 5.395 5.820 7.196 6.378
    JH0029pax 1 4.176 5.950 5.063 5.055 5.850 4.971 6.963
    JH0031pax 1 5.371 7.385 5.613 6.040 6.595 7.096 7.213
    JH0032pax 1 7.326 8.430 4.933 6.075 6.925 8.231 8.408
    JH0033pax 1 5.258 8.160 5.380 5.560 6.455 8.126 6.181
    JH0034pax 1 4.061 6.560 4.468 5.725 6.135 6.721 6.703
    JH0035pax 1 4.051 6.625 3.703 4.690 4.400 6.551 5.523
    JH0036pax 1 5.348 6.260 4.608 5.480 6.030 6.904 6.618
    JH0038pax 1 4.538 6.195 4.878 5.025 5.945 7.049 5.673
    JH0039pax 1 5.458 6.595 4.165 5.525 6.950 8.691 5.141
    JH0040pax 1 5.458 6.555 4.773 6.140 6.580 6.144 6.658
    JH0041pax 1 6.038 7.940 5.033 6.335 6.760 7.489 7.178
    JH0042pax 1 3.191 4.985 5.398 4.500 5.978 4.401 4.221
    JH0043pax 1 5.263 6.670 6.073 6.110 5.955 5.779 9.668
    JH0046pax 1 5.161 5.360 4.448 5.885 5.808 5.951 5.811
    JH0047pax 1 4.396 6.385 4.078 5.625 6.828 6.501 6.836
    JH0051pax 1 4.881 6.370 5.158 5.290 5.130 5.676 5.388
    JH0052pax 1 5.066 7.240 5.528 5.510 5.625 5.616 5.703
    JH0053pax 1 4.653 6.375 5.483 5.258 6.578 5.701 6.571
    JH0057pax 1 4.201 7.330 4.208 4.755 6.140 6.661 5.968
    JH0059pax 1 3.698 4.950 4.243 4.478 4.668 4.896 5.166
    JH0060pax 1 4.733 6.230 5.303 5.833 6.448 6.646 6.861
    JH0061pax 1 5.063 7.300 4.298 5.063 6.208 8.041 5.466
    JH0063pax 1 4.923 6.845 4.748 5.433 5.178 6.021 7.756
    JH0065pax 1 3.263 5.220 5.660 4.510 5.355 4.816 4.301
    JH0066pax 1 5.703 7.575 6.638 5.988 5.818 5.851 6.791
    JH0068pax 1 5.536 6.448 4.895 5.473 5.925 7.239 7.708
    JH0069pax 1 3.723 5.435 4.460 4.800 4.955 4.436 4.061
    JH0071pax 1 3.748 4.580 6.050 4.785 5.850 5.096 6.261
    JH0072pax 1 5.863 6.185 5.185 6.015 6.035 7.306 6.386
    JH0077pax 1 4.473 5.810 5.193 5.635 6.020 5.959 7.278
    JH0078pax 1 5.591 5.693 3.685 5.818 4.875 5.419 5.778
    JH0080pax 1 2.903 4.470 5.033 4.158 5.093 3.921 6.031
    JH0082pax 1 4.611 5.398 4.800 5.108 5.465 5.364 6.683
    JH0083pax 1 3.903 6.445 5.398 5.333 4.473 6.006 6.606
    JH0086pax 1 5.633 5.850 5.063 5.288 4.978 5.936 7.021
    JH0092pax 1 5.241 8.328 5.350 6.113 6.540 6.349 7.663
    MIP0004pax 1 3.201 3.098 5.340 3.873 4.510 4.459 2.743
    MP0013Apax 1 7.028 9.300 6.195 7.105 8.385 8.561 7.406
    MP0014Bpax 1 6.418 8.420 6.623 6.633 8.008 8.746 7.586
    MP0018Apax 1 6.003 7.900 7.005 6.740 6.635 7.146 6.281
    MP0019Bpax 1 6.283 7.090 6.653 6.528 7.058 6.696 8.656
    MP0024pax 1 5.436 7.823 6.375 6.383 7.655 8.939 8.013
    NK2001pax 1 5.061 6.843 6.230 5.613 4.680 6.349 7.873
    NK2002pax 1 5.516 5.903 5.210 5.568 5.585 6.809 6.753
    NK2003pax 1 4.986 6.388 5.590 5.588 6.345 7.404 5.723
    NK2004pax 1 4.626 6.648 5.435 5.048 4.945 6.319 5.318
  • Surprisingly, analysis of the data showed that RNA encoded by ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 is present on average at a significantly higher level (p-value less than 0.05) in blood of subjects having colorectal cancer relative to subjects having no colorectal pathology, and that RNA encoded by IL2RB is present on average at a significantly lower level (p-value less than 0.05) in blood of subjects having colorectal cancer relative to subjects having no colorectal pathology (Table 5). The ranges of fold-change in the levels of RNA encoded by these genes normalized to levels of RNA encoded by ACTB in blood of the training set subjects having colorectal cancer relative to the training set subjects not having any colorectal pathology are shown in Table 5.
  • TABLE 5
    Sample training set ranges of fold-changes in levels of RNA encoded by
    ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 normalized
    to levels of RNA encoded by ACTB in blood of subjects having colorectal cancer
    relative to subjects not having any colorectal pathology.
    Gene
    ANXA3 CLEC4D IL2RB LMNB1 PRRG4 TNFAIP6 VNN1
    Average normalized RNA 5.21 6.58 5.28 5.76 6.41 6.64 6.77
    level in subjects having
    colorectal cancer (ΔCt)
    Average normalized RNA 5.92 7.02 4.95 6.09 7.00 17.19 7.31
    level in subjects not having
    any colorectal pathology
    (ΔCt)
    Average RNA level fold- 1.63 1.36 0.80 1.26 1.51 1.46 1.45
    change
    p-value for average RNA level 5.0E−07 2.6E−03 1.1E−03 5.4E−04 2.3E−06 7.0E−04 1.4E−04
    fold-change
    Maximum observed RNA 11.53 15.86 0.07 4.66 6.07 10.12 23.63
    level directional fold-change
  • As can be seen in Table 5, a test subject having a blood level of RNA encoded by ANXA3 which is 1.6 to 11.5 fold higher than the average level of RNA encoded by this gene in blood of subjects not having any colorectal pathology is more likely to have colorectal cancer than to not have any colorectal pathology.
  • As can be seen in Table 5, a test subject having a blood level of RNA encoded by CLEC4D which is 1.4 to 15.9 fold higher than the average level of RNA encoded by this gene in blood of subjects not having any colorectal pathology is more likely to have colorectal cancer than to not have any colorectal pathology.
  • As can be seen in Table 5, a test subject having a blood level of RNA encoded by LMNB1 which is 1.3 to 4.7 fold higher than the average level of RNA encoded by this gene in blood of subjects not having any colorectal pathology is more likely to have colorectal cancer than to not have any colorectal pathology.
  • As can be seen in Table 5, a test subject having a blood level of RNA encoded by PRRG4 which is 1.5 to 6.1 fold higher than the average level of RNA encoded by this gene in blood of subjects not having any colorectal pathology is more likely to have colorectal cancer than to not have any colorectal pathology.
  • As can be seen in Table 5, a test subject having a blood level of RNA encoded by TNFAIP6 which is 1.46 to 10.12 fold higher than the average level of RNA encoded by this gene in blood of subjects not having any colorectal pathology is more likely to have colorectal cancer than to not have any colorectal pathology.
  • As can be seen in Table 5, a test subject having a blood level of RNA encoded by VNN1 which is 1.45 to 23.63 fold higher than the average level of RNA encoded by this gene in blood of subjects not having any colorectal pathology is more likely to have colorectal cancer than to not have any colorectal pathology.
  • As can be seen in Table 5, a test subject having a blood level of RNA encoded by IL2RB which is 0.8 to 0.1 fold that of the average level of RNA encoded by this gene in blood of subjects not having any colorectal pathology is more likely to have colorectal cancer than to not have any colorectal pathology.
  • Generation of Logistic Regression Models for Determining the Probability that a Test Subject has Colorectal Cancer Versus not Having any Colorectal Pathology Via Measurement of Levels of RNA Encoded by ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 Normalized to Levels of RNA Encoded by ACTB:
  • Linear regression analysis of levels of RNA encoded by ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 surprisingly showed that logistic regression models based on blood expression levels of all 127 possible combinations of one or more of these genes determined in the sample training set could be generated, for discriminating, with a receiver-operating characteristic (ROC) area under the curve (AUC) of at least 0.61, between subjects having colorectal cancer and subjects not having any colorectal pathology. Examples of these logistic regression models are shown in Table 6. A model based on ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 (Table 6, Model #1) was surprisingly found to enable discrimination with a ROC AUC of 0.79 between subjects having colorectal cancer and subjects not having any colorectal pathology.
  • By way of example, Model #1 of Table 6 corresponds to:

  • P={1+ê−[0.684+(−0.916)(L ANXA3)+(0.353)(L CLEC4D)+(0.871)(L IL2RB)+(0.907)(L LMNB1)+(−0.968)(L PRRG4)+(0.154)(L TNFAIP6)+(−0.355)(L VNN1)]}̂−1,
      • where P is the probability that a test subject has colorectal cancer as opposed to not having any colorectal pathology, LANXA3 is a ratio of a level of RNA encoded by ANXA3 to a level of RNA encoded by ACTB in blood of the test subject, LCLEC4D is a ratio of a level of RNA encoded by CLEC4D to a level of RNA encoded by ACTB in blood of the test subject, LIL2RB is a ratio of a level of RNA encoded by IL2RB to a level of RNA encoded by ACTB in blood of the test subject, LLMNB1 is a ratio of a level of RNA encoded by LMNB1 to a level of RNA encoded by ACTB in blood of the test subject, LPRRG4 is a ratio of a level of RNA encoded by PRRG4 to a level of RNA encoded by ACTB in blood of the test subject, LTNFAIP6 is a ratio of a level of RNA encoded by TNFAIP6 to a level of RNA encoded by ACTB in blood of the test subject, and LVNN1 is a ratio of a level of RNA encoded by VNN1 to a level of RNA encoded by ACTB in blood of the test subject.
  • Further by way of example, Model #104 of Table 6 corresponds to:

  • P={1+ê−[4.311+(−0.659)(L PRRG4)]}̂−1,
      • where P is the probability that a test subject has colorectal cancer as opposed to not having any colorectal pathology, and LPRRG4 is a ratio of a level of RNA encoded by PRRG4 to a level of RNA encoded by ACTB in blood of the test subject.
  • TABLE 6
    Logistic regression models based on blood expression levels of any possible combination of one or more
    of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 for determining the probability that a test
    subject has colorectal cancer as opposed to not having colorectal cancer. ROC AUC values for the models are
    shown for the sample training set used to generate the models, as well as for an independent blind sample test set
    used to test the models. The models, listed in order of decreasing ROC AUC value for the training set, are based
    on expression levels determined via quantitative reverse transcriptase-PCR analysis using ACTB as duplex partner
    for normalization. The form of these models is: P = {1 + e{circumflex over ( )}−[K0 + K1L1 + K2L2 + K3L3 . . . + KnLn]}{circumflex over ( )}−1, where P is
    the probability that a test subject has colorectal cancer as opposed to not having any colorectal pathology; K0 is a
    constant; K1 is a coefficient specific to a first gene; L1 is a ratio of a level of RNA encoded by the first gene to a
    level of RNA encoded by ACTB in blood of the test subject; K2 is a coefficient specific to a second gene; L2 is a
    ratio of a level of RNA encoded by the second gene to a level of RNA encoded by ACTB in blood of the test
    subject; K3 is a coefficient specific to a third gene; L3 is a ratio of a level of RNA encoded by the third gene to a
    level of RNA encoded by ACTB in blood of the test subject; Kn is a coefficient specific to an nth gene; and Ln is a
    ratio of a level of RNA encoded by the nth gene to a level of RNA encoded by ACTB in blood of the test subject.
    No regression coefficients are specified for genes which are not included in the gene combination (indicated by “—”)
    on which a given logistic regression model is based.
    No. of ROC AUC
    Logistic genes Blind Gene-specific regression coefficient
    regression in Training Test Constant (Kn)
    model # model Set Set (K0) ANXA3 CLEC4D IL2RB LMNB1 PRRG4 TNFAIP6 VNN1
    1 7 0.79 0.79 0.684 −0.916 0.353 0.871 0.907 −0.968   0.154 −0.355
    2 6 0.79 0.79 0.743 −0.859 0.402 0.870 0.893 −0.916 −0.341
    3 4 0.78 0.78 1.321 −0.614 0.358 0.898 −0.749
    4 5 0.78 0.79 0.343 −0.907 0.322 0.829 0.737 −0.925
    5 5 0.78 0.78 1.814 −0.527 0.424 0.943 −0.715 −0.267
    6 5 0.78 0.82 0.830 −0.641 0.883 0.935 −0.824 −0.270
    7 5 0.78 0.79 1.264 −0.658 0.318 0.898 −0.788   0.120
    8 5 0.78 0.82 0.359 −0.825 0.845 0.794 −0.927   0.189
    9 6 0.78 0.80 0.282 −0.953 0.280 0.829 0.740 −0.965   0.123
    10 6 0.78 0.78 1.772 −0.575 0.380 0.944 −0.760   0.141 −0.277
    11 6 0.78 0.82 0.727 −0.768 0.883 0.948 −0.918   0.230 −0.304
    12 4 0.77 0.82 0.477 −0.716 0.848 0.803 −0.850
    13 3 0.77 0.80 1.969 1.001 −0.752 −0.305
    14 4 0.77 0.81 1.950 −0.280 0.966 −0.610 −0.189
    15 4 0.77 0.78 1.915 0.161 1.009 −0.840 −0.374
    16 4 0.77 0.80 1.686 0.995 0.238 −0.855 −0.363
    17 5 0.77 0.79 1.748 0.130 1.005 0.147 −0.887 −0.397
    18 4 0.77 0.82 1.436 −0.487 0.925 −0.731   0.195
    19 4 0.77 0.80 1.934 1.008 −0.808   0.084 −0.334
    20 5 0.77 0.81 1.861 −0.398 0.965 −0.697   0.223 −0.218
    21 5 0.77 0.79 1.907 0.147 1.011 −0.852   0.029 −0.378
    22 5 0.77 0.79 1.697 1.001 0.207 −0.880   0.058 −0.376
    23 6 0.77 0.79 1.748 0.121 1.006 0.142 −0.894   0.021 −0.399
    24 2 0.76 0.79 1.225 0.957 −0.928
    25 3 0.76 0.81 1.570 −0.371 0.930 −0.649
    26 3 0.76 0.79 1.233 −0.006   0.957 −0.924
    27 3 0.76 0.79 1.328 0.960 −0.058   −0.894
    28 4 0.76 0.79 1.332 0.015 0.960 −0.072   −0.898
    29 3 0.76 0.79 1.249 0.956 −0.912 −0.019
    30 4 0.76 0.79 1.244 0.006 0.956 −0.914 −0.022
    31 4 0.76 0.79 1.328 0.959 −0.052   −0.890 −0.009
    32 5 0.76 0.79 1.333 0.022 0.960 −0.067   −0.893 −0.016
    33 3 0.75 0.80 0.744 −0.544 0.820 −0.282
    34 4 0.75 0.78 0.536 −0.717 0.252 0.784 −0.333
    35 4 0.75 0.80 0.324 −0.670 0.780 0.260 −0.313
    36 5 0.75 0.78 0.241 −0.798 0.236 0.757 0.189 −0.353
    37 5 0.75 0.75 2.890 −1.124 0.436 1.167 −0.723 −0.254
    38 4 0.75 0.80 0.701 −0.567 0.818   0.033 −0.288
    39 5 0.75 0.78 0.571 −0.702 0.266 0.785 −0.036 −0.330
    40 5 0.75 0.80 0.308 −0.680 0.779 0.254   0.019 −0.316
    41 6 0.75 0.78 0.271 −0.784 0.253 0.757 0.198 −0.043 −0.349
    42 6 0.75 0.76 2.851 −1.176 0.395 1.177 −0.767   0.134 −0.266
    43 2 0.74 0.80 0.043 −0.713 0.751
    44 3 0.74 0.79 −0.169 −0.843 0.160 0.721
    45 3 0.74 0.80 −0.101 −0.755 0.738 0.076
    46 4 0.74 0.79 −0.190 −0.848 0.159 0.719 0.011
    47 4 0.74 0.75 2.528 −1.146 0.378 1.036 −0.741
    48 4 0.74 0.77 1.234 −0.009   0.883 −0.489   −0.407
    49 3 0.74 0.80 0.070 −0.701 0.753 −0.015
    50 4 0.74 0.78 −0.082 −0.811 0.189 0.723 −0.068
    51 4 0.74 0.80 −0.079 −0.742 0.738 0.084 −0.021
    52 5 0.74 0.78 −0.130 −0.824 0.187 0.719 0.028 −0.069
    53 5 0.74 0.76 2.481 −1.189 0.342 1.038 −0.778   0.110
    54 4 0.74 0.77 1.245 0.878 −0.387   −0.120 −0.382
    55 5 0.74 0.77 1.272 0.059 0.877 −0.424   −0.139 −0.392
    56 3 0.73 0.77 1.238 0.883 −0.498   −0.409
    57 4 0.73 0.80 3.012 −0.891 1.247 −0.638 −0.182
    58 5 0.73 0.80 2.927 −1.011 1.251 −0.721   0.217 −0.212
    59 3 0.72 0.73 4.212 −0.733 0.451 −0.493
    60 3 0.72 0.80 2.728 −0.930 1.141 −0.660
    61 3 0.72 0.77 0.438 −0.175   0.826 −0.505
    62 4 0.72 0.73 4.547 −0.690 0.489 −0.466 −0.144
    63 3 0.72 0.76 0.855 0.835 −0.659   −0.188
    64 4 0.72 0.77 0.847 −0.031   0.836 −0.636   −0.177
    65 4 0.72 0.74 4.171 −0.772 0.418 −0.527   0.104
    66 4 0.72 0.80 2.614 −1.040 1.128 −0.735   0.188
    67 3 0.72 0.76 0.608 0.823 −0.217 −0.482
    68 4 0.72 0.77 0.637 −0.049   0.828 −0.194 −0.466
    69 5 0.72 0.74 4.526 −0.731 0.455 −0.504   0.117 −0.154
    70 2 0.71 0.76 0.786 0.840 −0.871  
    71 3 0.71 0.78 0.770 −0.122   0.844 −0.733  
    72 2 0.71 0.74 0.162 0.799 −0.616
    73 4 0.71 0.74 2.134 −1.028 0.310 0.571 −0.286
    74 5 0.71 0.74 2.164 −1.014 0.327 0.580 −0.044 −0.282
    75 3 0.70 0.75 1.725 −1.054 0.242 0.404
    76 2 0.70 0.80 4.710 −0.417 −0.372
    77 3 0.70 0.73 3.283 −0.791 0.371 −0.214
    78 3 0.70 0.79 2.309 −0.868 0.682 −0.231
    79 3 0.70 0.80 4.842 −0.393 −0.360 −0.050
    80 4 0.70 0.75 1.785 −1.031 0.269 0.421 −0.069
    81 3 0.70 0.80 4.558 −0.539 −0.455   0.200
    82 4 0.70 0.73 3.305 −0.783 0.378 −0.020 −0.211
    83 4 0.70 0.79 2.279 −0.886 0.671   0.034 −0.237
    84 4 0.70 0.81 4.759 −0.506 −0.440   0.211 −0.079
    85 1 0.69 0.79 3.324 −0.616
    86 2 0.69 0.74 2.681 −0.868 0.301
    87 2 0.69 0.79 1.931 −0.919 0.520
    88 2 0.69 0.79 3.768 −0.536 −0.126
    89 2 0.69 0.79 3.214 −0.654   0.047
    90 3 0.69 0.73 2.748 −0.847 0.321 −0.046
    91 2 0.69 0.73 −0.674 0.701 −0.435
    92 3 0.69 0.76 −0.326 −0.240   0.745 −0.281
    93 3 0.69 0.79 1.931 −0.919 0.520   0.000
    94 3 0.69 0.80 3.648 −0.587   0.075 −0.143
    95 2 0.68 0.75 −0.764 −0.455   0.736
    96 2 0.68 0.78 4.977 −0.524 −0.223
    97 3 0.68 0.76 4.939 0.113 −0.579 −0.275
    98 3 0.68 0.77 4.670 0.238 −0.620 −0.289
    99 4 0.68 0.76 4.716 0.067 0.185 −0.631 −0.304
    100 3 0.68 0.78 4.975 −0.531   0.012 −0.228
    101 4 0.68 0.76 4.941 0.134 −0.564 −0.042 −0.268
    102 4 0.68 0.77 4.655 0.255 −0.610 −0.028 −0.283
    103 5 0.68 0.76 4.702 0.091 0.200 −0.615 −0.055 −0.299
    104 1 0.67 0.76 4.311 −0.659
    105 2 0.67 0.77 4.327 −0.010   −0.652
    106 2 0.67 0.76 4.314 −0.001   −0.658
    107 3 0.67 0.77 4.309 −0.014   0.012 −0.656
    108 2 0.67 0.77 4.391 −0.607 −0.062
    109 3 0.67 0.76 4.356 0.035 −0.619 −0.079
    110 3 0.67 0.76 4.292 0.056 −0.629 −0.074
    111 4 0.67 0.76 4.299 0.024 0.038 −0.630 −0.082
    112 3 0.65 0.74 3.919 −0.142   −0.152 −0.303
    113 2 0.64 0.74 3.402 −0.105   −0.397
    114 2 0.64 0.75 3.927 −0.277   −0.340
    115 3 0.64 0.75 3.922 −0.010   −0.268   −0.337
    116 2 0.64 0.73 3.528 −0.373   −0.206
    117 3 0.64 0.73 3.528 −0.373   −0.206
    118 2 0.64 0.73 3.628 −0.189 −0.345
    119 3 0.64 0.73 3.610 0.020 −0.199 −0.352
    120 4 0.64 0.73 3.951 0.071 −0.188   −0.174 −0.316
    121 1 0.63 0.75 3.466 −0.603  
    122 2 0.63 0.78 3.457 −0.104   −0.482  
    123 1 0.63 0.71 3.191 −0.468
    124 2 0.63 0.74 2.657 −0.133   −0.269
    125 1 0.62 0.69 2.363 −0.357
    126 1 0.61 0.73 2.197 −0.338  
    127 1 0.61 0.64 −2.973 0.561
  • Blind Sample Test Set:
  • Quantitative RT-PCR analysis of gene expression in an independent blind test set of blood samples from 76 subjects having colorectal cancer and 77 subjects not having any colorectal pathology was performed as described above for the training set. The normalized RNA levels measured are shown in Table 7.
  • TABLE 7
    Sample test set levels of RNA encoded by ANXA3, CLEC4D, IL2RB, LMNB1,
    PRRG4, TNFAIP6 and VNN1 in blood of subjects having colorectal cancer (Group 1)
    and subjects not having any colorectal pathology (Group 0), normalized to levels of
    RNA encoded by ACTB. Levels shown correspond to ΔCt.
    Gene
    Sample ID Group ANXA3 CLEC4D IL2RB LMNB1 PRRG4 TNFAIP6 VNN1
    CD0214pax 0 5.56 6.17 4.50 6.44 7.07 5.78 6.69
    CD0242pax 0 5.87 7.32 4.54 5.43 5.19 5.87 7.38
    RC2897pax 0 6.67 8.37 5.49 5.95 7.18 7.80 7.96
    CD0670pax 0 7.79 5.80 3.25 6.35 7.10 8.40 7.80
    CD1401pax 0 5.84 6.39 5.42 5.85 7.30 6.55 7.12
    PB2924pax 0 5.56 7.17 4.12 5.97 6.51 5.90 7.12
    CD0482pax 0 7.65 8.34 3.43 6.91 7.02 8.94 8.48
    PB1275pax 0 5.26 6.13 5.18 5.54 7.09 5.89 8.78
    CD0148pax 0 5.90 7.83 3.98 5.95 7.60 7.35 7.20
    CD0122pax 0 6.55 8.40 5.02 6.38 7.07 7.96 8.83
    PB2272pax 0 7.30 8.30 3.95 6.57 7.42 9.42 7.49
    CD1708pax 0 6.28 9.11 4.07 5.74 6.01 7.48 7.13
    CD0354pax 0 6.55 8.20 4.93 6.30 8.06 8.28 7.81
    PB2634pax 0 5.76 7.74 5.28 6.80 7.20 9.18 7.39
    CD0204pax 0 5.90 6.53 4.83 5.56 6.25 7.29 6.87
    PB1336pax 0 7.60 9.00 6.87 6.99 7.13 8.92 9.71
    RC2699pax 0 7.72 9.42 6.03 7.12 7.99 8.67 8.57
    CD1278pax 0 5.95 7.57 4.20 5.72 6.31 6.83 7.36
    PB2062pax 0 7.31 7.76 4.31 6.50 7.29 8.38 7.78
    PB2464pax 0 5.95 8.33 4.73 5.52 5.52 6.39 8.43
    CD0053pax 0 6.17 7.90 4.09 6.56 7.18 8.25 7.44
    CD0192pax 0 5.49 7.60 4.19 5.66 7.38 5.48 6.03
    CD0244pax 0 6.32 7.31 5.26 6.21 7.56 7.63 6.89
    CD0833pax 0 5.52 8.65 4.72 5.55 6.33 6.60 5.47
    CD1719pax 0 6.19 7.94 5.40 5.93 6.39 6.50 7.51
    CD0036pax 0 5.20 5.95 4.60 4.95 4.51 5.99 5.82
    PB2015pax 0 6.62 7.13 3.85 5.99 7.62 8.53 7.13
    PB0662pax 0 6.09 7.17 5.33 6.49 7.40 7.43 6.60
    PB2024pax 0 6.16 6.10 4.49 5.94 6.86 8.23 7.44
    RC2565pax 0 6.63 8.81 5.94 6.33 8.38 7.11 8.75
    CD1561pax 0 7.29 6.78 4.73 6.48 7.49 8.18 8.21
    CD1728pax 0 7.13 6.89 4.30 6.52 7.76 7.08 8.63
    CD0238pax 0 5.47 7.06 4.85 6.10 7.13 7.38 7.14
    PB2342pax 0 7.65 4.19 6.21 7.29 8.02 8.00
    CD0800pax 0 7.42 8.19 3.99 6.61 7.78 8.79 8.44
    CD0437pax 0 4.74 5.92 5.05 4.90 5.58 6.27 6.17
    RC3214pax 0 6.35 8.13 4.96 6.22 7.02 8.19 7.01
    CD1487pax 0 5.24 7.83 4.51 5.99 7.26 7.36 6.48
    PB1763pax 0 7.83 9.12 5.21 7.40 8.78 9.20 8.37
    CD0580pax 0 5.14 5.72 3.17 4.54 4.78 6.28 5.28
    CD0840pax 0 4.94 6.63 4.96 5.38 5.60 6.63 6.10
    PB2757pax 0 5.32 7.54 4.98 5.51 6.48 6.77 8.12
    PB2184pax 0 5.11 7.34 5.73 5.68 6.63 6.30 8.26
    PB2179pax 0 5.57 6.84 4.80 5.59 5.75 5.74 6.56
    PB1324pax 0 4.77 9.26 4.24 5.57 6.78 7.16 7.37
    CD0237pax 0 6.46 8.06 5.42 5.89 6.89 7.51 7.26
    CD1329pax 0 6.03 6.72 4.93 5.93 6.06 7.37 7.38
    PB2005pax 0 7.51 7.37 4.66 6.41 7.63 9.46 8.15
    PB3227pax 0 5.08 6.46 4.44 5.74 6.46 7.62 7.51
    PB3163pax 0 4.51 6.23 4.17 5.32 6.10 7.12 6.54
    PB3481pax 0 5.66 7.99 5.16 5.95 8.33 6.48 8.26
    CD1320pax 0 4.16 5.93 4.79 4.60 5.55 5.10 6.16
    RC3191pax 0 7.15 7.64 4.92 6.94 7.20 8.20 8.30
    CD0583pax 0 4.29 7.00 4.44 4.16 3.96 8.82 3.97
    PB3032pax 0 5.12 6.51 5.09 5.96 6.08 6.91 7.87
    CD0367pax 0 5.75 6.68 4.34 5.49 6.25 7.12 7.06
    PB2889pax 0 6.03 8.25 4.75 6.39 7.27 7.04 7.68
    PB3524pax 0 6.66 6.73 5.40 6.83 7.72 6.07 7.38
    RC2986pax 0 6.90 7.96 5.36 6.14 7.22 8.87 7.75
    CD1428pax 0 6.64 9.25 3.67 5.82 7.04 7.12 6.09
    RC2236pax 0 6.82 6.87 5.24 6.21 7.41 7.27 8.02
    PB1918pax 0 8.72 7.41 5.20 7.28 8.29 10.44 9.02
    CD0277pax 0 6.42 8.60 5.37 6.63 7.61 8.57 7.04
    CD0667pax 0 5.15 6.09 5.57 5.26 6.33 6.27 6.41
    CD1741pax 0 5.46 6.18 5.57 5.76 6.50 7.24 7.18
    PB1973pax 0 8.07 9.15 4.68 7.12 8.89 9.13 10.01
    PB1222pax 0 5.80 7.45 5.57 5.95 5.81 6.15 7.61
    RC2683pax 0 8.25 9.29 5.83 6.98 8.07 9.46 9.66
    PB3200pax 0 5.00 6.73 4.23 5.42 6.59 6.31 7.35
    PB2130pax 0 6.31 7.61 4.91 5.50 5.79 6.48 6.81
    PB3097pax 0 6.33 3.94 5.77 6.08 6.72 7.78 7.51
    CD0571pax 0 5.28 6.04 3.76 5.54 6.60 6.99 5.82
    CD0676pax 0 5.50 6.68 5.78 5.79 5.91 6.91 7.18
    PB1514pax 0 7.57 5.96 5.35 7.38 8.09 7.63 8.37
    CD0547pax 0 4.62 7.13 6.05 5.85 7.44 6.70 6.88
    CD1068pax 0 5.79 6.49 4.84 6.23 7.14 8.55 6.32
    CD0715pax 0 2.97 4.03 4.74 4.51 4.94 5.38 6.46
    JH0130pax 1 5.32 5.90 6.01 5.47 5.77 5.93 6.90
    MH0079pax 1 6.41 6.17 5.06 6.19 6.42 6.61 7.57
    MH0082pax 1 7.06 4.93 5.26 6.82 6.74 8.27 7.32
    AN0013pax 1 4.94 7.96 3.89 5.60 5.49 7.86 7.95
    NK2005pax 1 4.20 5.83 5.59 5.08 5.65 4.02 6.98
    CD1111pax 1 6.62 6.64 4.17 6.17 7.32 7.62 7.72
    JH0105pax 1 5.31 7.29 4.84 5.39 5.34 7.13 8.28
    MIP1007pax 1 3.42 7.30 4.89 4.27 6.00 5.13 3.62
    DC0011pax 1 4.33 5.63 4.17 5.45 5.74 6.41 7.06
    MH0073pax 1 4.68 6.28 5.84 4.57 5.52 6.41 5.86
    DC0003pax 1 6.24 7.65 5.02 6.05 6.64 7.05 8.46
    DC1002pax 1 4.62 4.35 5.41 5.26 6.40 7.24 6.90
    JH0096pax 1 3.45 5.49 5.18 4.79 5.74 3.34 7.61
    KW0002pax 1 4.26 6.89 4.62 4.71 4.76 5.78 6.53
    JH0120pax 1 5.89 4.91 5.53 6.38 5.56 8.45 6.44
    MIP1008pax 1 4.84 7.74 5.47 5.41 5.63 5.36 6.93
    MIP0002pax 1 3.76 6.96 5.63 4.85 6.55 5.74 6.87
    MIP1011pax 1 4.70 6.28 5.12 4.63 4.60 5.68 4.55
    MH0074pax 1 5.11 5.73 5.75 5.68 6.74 7.59 6.00
    DC0008pax 1 4.97 6.33 4.16 5.44 4.95 6.54 5.87
    AN4014pax 1 3.93 5.76 4.25 4.58 5.76 5.20 7.74
    DC0012pax 1 4.25 6.07 5.15 5.08 7.00 4.86 6.37
    MIP2002pax 1 5.54 5.71 4.26 5.54 5.77 6.57 6.33
    NK1005pax 1 5.76 8.79 5.22 7.02 7.38 8.60 7.55
    MIP0007pax 1 2.91 3.55 6.69 3.21 4.38 3.48 4.85
    JH0118pax 1 5.72 7.53 4.77 5.56 5.85 6.56 6.04
    JH0089pax 1 5.11 7.30 4.99 5.68 5.58 6.54 7.38
    MH0057pax 1 5.80 6.60 6.01 6.35 6.36 7.44 6.44
    DC0005pax 1 5.98 7.66 4.72 6.38 6.39 7.47 7.19
    MH0067pax 1 4.75 6.06 6.11 4.96 5.30 6.78 5.54
    JH0085pax 1 5.19 6.48 5.92 5.99 5.76 6.65 7.12
    JH0127pax 1 5.48 6.43 5.18 6.13 7.59 6.63 7.04
    MIP1013pax 1 4.86 5.89 4.56 5.29 5.21 5.89 6.51
    JH0126pax 1 4.21 7.08 5.35 5.68 5.40 5.50 7.07
    AN4013pax 1 5.15 5.66 4.15 5.19 5.38 5.94 6.91
    MH0053pax 1 7.07 8.10 4.16 6.42 5.83 8.33 7.84
    JH0115pax 1 3.82 7.54 5.74 4.75 6.03 6.27 7.36
    CC0004pax 1 3.83 5.81 4.59 4.67 5.46 5.50 5.84
    JH0091pax 1 5.26 5.92 4.74 5.31 7.34 6.70 6.91
    NK1004pax 1 5.76 7.32 6.16 5.78 6.58 7.99 7.77
    NK1008pax 1 4.70 7.52 5.56 5.57 6.80 6.58 7.52
    MIP2006pax 1 4.25 6.38 4.28 4.74 5.69 5.36 5.38
    AN0020pax 1 4.12 6.61 5.57 4.81 4.61 4.45 6.07
    JH0117pax 1 3.36 8.59 4.90 4.29 4.99 5.12 5.76
    JH0100pax 1 5.54 6.69 4.86 5.66 6.57 7.23 7.26
    MH0066pax 1 5.12 5.25 6.28 5.59 6.61 6.27 6.13
    NK2009pax 1 5.30 5.93 4.95 5.76 5.27 7.71 5.77
    NK2008pax 1 4.14 5.57 5.88 4.93 5.45 6.59 5.66
    PB3067-2pax 1 5.84 8.25 4.70 6.20 6.51 7.78 6.81
    NK1003pax 1 4.41 6.99 5.48 5.30 6.08 6.48 7.13
    MIP1009pax 1 3.22 8.02 6.45 4.25 5.14 5.47 5.21
    DC2006pax 1 6.06 5.91 4.81 5.88 6.05 7.24 7.34
    JH0131pax 1 5.19 6.71 4.98 5.21 5.15 4.75 6.43
    DC0015pax 1 4.95 6.98 5.01 5.46 6.53 6.02 6.17
    AN0001pax 1 4.36 6.54 5.97 5.60 5.83 6.72 7.13
    JH0111pax 1 5.04 6.83 4.22 5.09 6.96 7.14 5.78
    MIP0005pax 1 3.74 5.56 5.98 4.46 5.80 5.74 5.33
    MH0065pax 1 4.50 5.37 5.63 5.51 6.30 6.09 5.82
    JH0136pax 1 3.39 4.63 6.12 4.86 5.19 7.76 4.59
    CD1351pax 1 6.86 7.94 5.55 6.06 6.52 8.46 7.62
    MH0075pax 1 6.05 7.17 5.57 5.77 6.75 7.32 7.27
    MH0078pax 1 4.60 6.70 5.49 5.26 4.21 5.25 7.54
    MH0068pax 1 7.58 5.49 4.48 6.43 7.14 8.00 6.36
    MIP2003pax 1 4.24 5.60 4.30 4.93 6.05 6.85 5.97
    NK2015pax 1 5.42 5.52 5.54 6.08 6.40 8.40 8.09
    MH0070pax 1 5.40 7.11 4.66 5.74 6.55 7.09 7.08
    JH0093pax 1 5.22 7.62 4.66 5.29 5.39 7.00 7.15
    JH0135pax 1 4.40 4.64 5.52 4.62 5.38 4.31 4.62
    CD1571pax 1 5.45 6.48 4.41 5.65 7.26 7.88 7.17
    MH0061pax 1 4.99 5.15 5.47 5.72 6.73 7.65 7.76
    NK2007pax 1 5.98 7.25 5.17 5.76 7.02 7.32 6.38
    JH0132pax 1 5.29 7.18 5.29 5.76 6.96 6.66 6.83
    MH0062pax 1 4.40 6.01 4.86 5.42 5.50 6.26 6.45
    JH0114pax 1 4.78 6.93 7.12 5.58 7.50 5.62 5.26
    CD1260pax 1 5.28 5.92 5.24 5.73 5.86 7.18 7.37
    JH0022pax 1 5.00 5.94 4.73 5.49 6.17 5.66 6.80
  • Analysis of the test set results confirmed the surprising finding based on the training set that ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 each express RNA on average at a significantly higher level (p-value less than 0.05) in blood of subjects having colorectal cancer relative to subjects having no colorectal pathology, and that IL2RB expresses RNA on average at a significantly lower level (p-value less than 0.05) in blood of subjects having colorectal cancer relative to subjects having no colorectal pathology (Table 8). The ranges of fold-change in the levels of RNA encoded by ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 normalized to levels of RNA encoded by ACTB in blood of the test set subjects having colorectal cancer relative to the test set subjects not having any colorectal pathology are also shown in Table 8.
  • TABLE 8
    Sample test set ranges of fold-change in levels of RNA encoded by
    ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6, VNN1
    normalized to levels of RNA encoded by ACTB in blood of subjects
    having colorectal cancer relative to subjects not having any colorectal pathology.
    Gene
    ANXA3 CLEC4D IL2RB LMNB1 PRRG4 TNFAIP6 VNN1
    Average normalized 4.98 6.45 5.18 5.42 6.01 6.51 6.63
    RNA level in subjects
    having colorectal
    cancer (ΔCt)
    Average normalized 6.10 7.32 4.83 6.01 6.86 7.41 7.40
    RNA level in subjects
    not having any
    colorectal pathology
    (ΔCt)
    Average RNA level 2.17 1.82 0.78 1.50 1.80 1.87 1.70
    fold-change
    p-value for average 1.7E−10 1.9E−06 1.4E−03 1.3E−07 1.6E−08 3.8E−06 4.4E−06
    RNA level fold-
    change
    Maximum observed 9.13 13.66 0.20 6.98 6.26 16.78 13.78
    RNA level directional
    fold-change
  • As can be seen in Table 8, a test subject having a blood level of RNA encoded by ANXA3 which is 2.2 to 9.1 fold higher than the average level of RNA encoded by this gene in blood of subjects not having any colorectal pathology is more likely to have colorectal cancer than to not have any colorectal pathology.
  • As can be seen in Table 8, a test subject having a blood level of RNA encoded by CLEC4D which is 1.8 to 13.7 fold higher than the average level of RNA encoded by this gene in blood of subjects not having any colorectal pathology is more likely to have colorectal cancer than to not have any colorectal pathology.
  • As can be seen in Table 8, a test subject having a blood level of RNA encoded by LMNB1 which is 1.5 to 7.0 fold higher than the average level of RNA encoded by this gene in blood of subjects not having any colorectal pathology is more likely to have colorectal cancer than to not have any colorectal pathology.
  • As can be seen in Table 8, a test subject having a blood level of RNA encoded by PRRG4 which is 1.8 to 6.3 fold higher than the average level of RNA encoded by this gene in blood of subjects not having any colorectal pathology is more likely to have colorectal cancer than to not have any colorectal pathology.
  • As can be seen in Table 8, a test subject having a blood level of RNA encoded by TNFAIP6 which is 1.9 to 16.8 fold higher than the average level of RNA encoded by this gene in blood of subjects not having any colorectal pathology is more likely to have colorectal cancer than to not have any colorectal pathology.
  • As can be seen in Table 8, a test subject having a blood level of RNA encoded by VNN1 which is 1.7 to 13.8 fold higher than the average level of RNA encoded by this gene in blood of subjects not having any colorectal pathology is more likely to have colorectal cancer than to not have any colorectal pathology.
  • As can be seen in Table 8, a test subject having a blood level of RNA encoded by IL2RB which is 0.8 to 0.2 fold that of the average level of RNA encoded by this gene in blood of subjects not having any colorectal pathology is more likely to have colorectal cancer than to not have any colorectal pathology.
  • Furthermore, the test set results confirmed the surprising finding based on the training set that logistic regression models based on blood expression levels for any of the 127 possible combinations of one or more of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, each of which normalized against expression levels of ACTB, can be used to discriminate, with a ROC AUC of at least 0.64 (Table 6), between subjects having colorectal cancer and subjects not having any colorectal pathology. As such, the novel logistic regression models listed in Table 6 can be used to determine the probability that a test subject has colorectal cancer as opposed to not having any colorectal pathology, based on blood levels of expression of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and/or VNN1.
  • Example 3 Measurement of Blood Levels of RNA Encoded by any Combination of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and/or VNN1 Relative to the Level of RNA Encoded by IL2RB can be Used to Determine the Probability that a Test Subject has Colorectal Cancer as Opposed to not Having any Colorectal Pathology
  • Materials and Methods:
  • Refer to “General materials and methods”, above.
  • Experimental Results:
  • Sample Training Set:
  • Discovery of Significantly Different Levels of RNA Encoded by ANXA3, CLEC4D, LMNB1, PRRG4, VNN1, TNFAIP6 Normalized to IL2RB in Blood of Subjects Having Colorectal Cancer Relative to Subjects not Having any Colorectal Pathology:
  • Quantitative reverse transcriptase-PCR analysis of gene expression in a training set of blood samples from 116 subjects having colorectal cancer and 127 subjects not having any colorectal pathology, using IL2RB as duplex partner for normalization of gene expression levels was performed. The normalized RNA levels measured are shown in Table 9
  • TABLE 9
    Sample training set levels of RNA encoded by ANXA3, CLEC4D, IL2RB,
    LMNB1, PRRG4, TNFAIP6 and VNN1 in blood of subjects having colorectal
    cancer (Group 1) and subjects not having any colorectal pathology (Group 0),
    normalized to levels of RNA encoded by IL2RB. Levels shown correspond
    to ΔCt.
    Gene
    Sample ID Group ANXA3 CLEC4D LMNB1 PRRG4 TNFAIP6 VNN1
    CD0011pax 0 0.8303 1.3467 1.2008 0.8909 2.1665 2.6036
    CD0012pax 0 1.2503 1.4917 0.8258 0.9309 1.9115 2.9086
    CD0030pax 0 1.2878 1.2957 0.7182 1.1632 0.7535 1.7757
    CD0063pax 0 2.4078 3.0807 1.6332 1.8532 3.4685 3.0157
    CD0077pax 0 −0.0047 1.2417 0.7058 0.7009 0.8465 1.7436
    CD0078pax 0 1.6928 2.4057 0.9432 0.9332 2.6735 1.4807
    CD0085pax 0 0.4428 1.5007 0.4032 0.8232 2.4335 0.4457
    CD0117pax 0 0.6028 2.2057 0.7632 2.0182 2.2585 0.6557
    CD0146pax 0 0.0353 0.7117 0.6708 0.1659 1.3215 0.7236
    CD0167pax 0 −1.3147 −0.1483 0.0358 0.2609 1.3315 0.0786
    CD0249pax 0 −0.7797 −0.6233 −0.8142 0.0609 −0.0085 0.2486
    CD0279pax 0 0.8278 1.9907 0.7632 0.7382 1.6185 1.4957
    CD0286pax 0 0.0753 1.5217 0.7058 0.6659 0.7215 0.7286
    CD0297pax 0 −0.1797 0.2867 0.1958 −0.3291 0.0665 0.4836
    CD0323pax 0 1.7303 1.6817 0.5408 1.2359 2.2865 3.0636
    CD0445pax 0 0.9878 1.2107 0.6682 0.6932 1.8085 1.6907
    CD0463pax 0 −0.3647 1.2267 0.3058 0.5559 1.2365 1.0136
    CD0491pax 0 −0.3972 0.3507 0.4482 0.7032 1.1785 0.2907
    CD0496pax 0 0.8753 2.4167 0.6958 1.4509 1.0515 0.6436
    CD0501pax 0 0.8753 1.7517 0.9558 0.9109 2.7615 1.0986
    CD0504pax 0 −0.4947 0.2367 0.2358 1.0209 0.3715 0.7936
    CD0573pax 0 1.9978 1.3107 0.9582 0.9982 2.4435 1.9357
    CD0578pax 0 1.6103 1.7317 0.6408 0.6259 1.6865 1.7136
    CD0639pax 0 0.1028 0.6657 0.6232 −0.2618 0.9985 1.5857
    CD0645pax 0 −0.8347 −0.1883 −0.4342 −0.4441 0.4965 0.0986
    CD0679pax 0 0.4703 1.7767 0.6658 0.5659 1.9465 2.2636
    CD0685pax 0 0.8753 2.3317 0.8008 1.3759 2.1915 1.4836
    CD0716pax 0 1.9978 2.4057 0.9182 0.4432 1.5835 2.0457
    CD0749pax 0 0.3303 1.3717 0.4058 0.5509 2.0465 1.0336
    CD0760pax 0 −2.5747 −1.3583 −1.9442 −1.1491 −1.2335 −0.8714
    CD0811pax 0 2.2353 2.8417 1.1858 1.3609 1.3865 1.3286
    CD0846pax 0 −0.6522 0.3407 −0.6418 −0.3468 −0.6315 1.5657
    CD0848pax 0 0.7278 1.2057 0.9832 1.0232 1.5785 1.8307
    CD0924pax 0 0.1428 0.5357 −0.0768 −0.4418 0.2935 −0.0793
    CD1066pax 0 0.1753 0.6667 0.2658 0.2059 1.9715 0.5786
    CD1073pax 0 0.1053 −0.0633 −0.0092 −0.1341 0.8015 1.0886
    CD1075pax 0 −0.3322 0.4757 −0.0118 1.1582 1.2385 0.1957
    CD1089pax 0 −1.1072 0.1657 −0.7818 −0.5318 0.1935 −0.1193
    CD1116pax 0 −0.2072 −0.0543 −0.2818 0.2982 1.4885 0.4107
    CD1120pax 0 −0.8872 0.0357 −0.5868 0.2032 1.1885 −0.2443
    CD1198pax 0 0.5253 0.5317 0.7908 0.7509 0.7915 0.9786
    PB1179pax 0 1.0003 1.0617 0.8808 0.9909 2.4315 1.2286
    PB1277pax 0 0.8803 1.4817 0.4258 0.9859 1.5765 0.8286
    PB1301pax 0 −1.7247 −0.9183 −0.9092 −0.5091 −0.6985 −1.2864
    PB1315pax 0 −1.5572 0.2257 −0.6218 −0.3118 −0.2015 0.9007
    PB1345pax 0 0.1953 0.6617 0.7508 1.0459 0.3615 2.0236
    PB1518pax 0 1.2653 1.6967 0.2058 0.8359 1.3565 1.3036
    PB1520pax 0 1.0953 2.0267 0.9708 0.0109 1.6565 2.2236
    PB1574pax 0 1.0753 1.5467 0.8008 0.6109 1.0465 1.1836
    PB1783pax 0 1.5978 1.7007 1.1982 1.2632 2.1135 1.5907
    PB1799pax 0 0.6978 1.0157 0.5282 1.0632 2.0785 0.5957
    PB1811pax 0 0.8628 1.4057 0.8232 1.2632 1.3685 0.9507
    PB1830pax 0 0.1428 0.6807 0.2532 0.1182 0.8985 2.3407
    PB1833pax 0 0.2028 1.1407 0.4782 0.5532 0.9385 1.5457
    PB1843pax 0 0.5553 0.4717 0.0708 0.5909 −0.5285 1.1486
    PB1851pax 0 0.4428 0.1007 0.2632 0.3232 2.7035 1.6007
    PB1919pax 0 1.4703 2.1067 0.8108 1.7959 2.1365 2.5086
    PB1922pax 0 0.3103 1.1867 0.4258 1.3759 2.1215 0.9986
    PB1924pax 0 0.2428 0.5357 0.1632 0.3632 1.1535 1.2357
    PB1937pax 0 1.3128 2.1057 1.0982 2.9132 2.4835 2.2007
    PB1964pax 0 0.4828 2.6207 0.7382 1.5332 2.4035 2.7507
    PB2027pax 0 −0.1422 0.3857 0.1582 0.3182 1.1835 1.5807
    PB2029pax 0 0.1953 0.5917 0.0708 −0.0391 1.8615 0.7336
    PB2073pax 0 0.4478 0.9057 0.4382 1.6632 1.5085 0.9257
    PB2086pax 0 0.1153 0.1817 0.3108 0.8409 0.3215 0.5386
    PB2099pax 0 −0.4622 −0.5993 −0.1568 0.0732 −0.1265 0.1057
    PB2100pax 0 1.1628 1.1057 0.6532 1.5482 2.1385 1.1507
    PB2132pax 0 0.5503 1.1517 0.3558 0.6109 2.1315 1.4536
    PB2168pax 0 1.3278 1.5357 0.8482 1.1682 2.3435 1.3307
    PB2192pax 0 0.3153 0.8967 0.3308 −0.0291 1.2515 1.9286
    PB2196pax 0 0.8328 1.6107 0.7932 1.4432 2.0535 1.2107
    PB2200pax 0 1.1028 1.4807 0.3732 0.4432 0.9635 0.5757
    PB2213pax 0 2.1753 2.2717 1.1658 0.9009 3.1465 1.9736
    PB2224pax 0 −0.2772 0.6207 0.0482 0.3582 2.1235 1.2607
    PB2228pax 0 1.7703 2.1017 1.3658 1.6859 3.6965 2.7186
    PB2229pax 0 −0.2047 −0.3183 −0.0542 −0.9241 0.5615 −0.1764
    PB2277pax 0 1.3578 0.8507 0.6332 0.3582 1.0785 1.7457
    PB2297pax 0 0.4178 1.1507 0.1282 0.0332 0.9235 −0.3343
    PB2312pax 0 1.3628 2.0057 1.4132 0.9632 2.0885 2.0357
    PB2398pax 0 −0.5822 0.1007 −0.2868 −0.7518 −0.1865 1.3307
    PB2409pax 0 0.1153 0.6817 0.3008 0.3359 1.0115 1.5036
    PB2414pax 0 2.7128 2.3707 1.2132 2.1832 3.6985 1.9707
    PB2467pax 0 0.7478 1.1457 0.5482 0.6382 2.2385 1.3607
    PB2473pax 0 0.3828 0.8857 0.4432 1.0932 0.9185 1.8407
    PB2512pax 0 0.7003 1.7267 0.8458 0.4709 1.8115 2.0136
    PB2568pax 0 −0.4772 0.0157 −0.3718 0.4132 1.2435 1.0857
    PB2571pax 0 0.3003 1.0017 0.5558 0.2609 1.8915 1.9336
    PB2603pax 0 1.0128 1.4757 1.1082 1.1632 2.3335 0.7457
    PB2624pax 0 0.4403 0.5167 0.7208 1.3259 1.3865 1.2286
    PB2824pax 0 0.5178 0.9907 0.4582 1.0132 1.8135 1.3207
    PB2880pax 0 1.0403 1.0617 0.6208 −0.2491 1.8615 1.5586
    PB3088pax 0 1.6503 1.7517 1.3208 1.1359 3.4165 2.3786
    RC0882pax 0 0.2728 1.4507 0.6582 0.7682 2.0985 1.8007
    RC0888pax 0 −0.7872 −0.3193 −0.2118 0.2932 0.7835 0.2957
    RC0968pax 0 −0.8522 −0.6493 0.0182 1.4382 0.7835 2.1457
    RC2114pax 0 −0.4722 0.7557 0.0282 0.4132 0.1335 1.9357
    RC2238pax 0 0.9528 1.8557 0.5982 1.5182 2.4435 2.8857
    RC2681pax 0 0.3278 1.1957 0.4582 0.5532 0.7435 0.9507
    RC2703pax 0 1.6403 1.8317 0.7658 1.1959 1.1615 1.1436
    RC2749pax 0 0.3978 1.1407 0.3632 0.4682 1.6635 1.5157
    RC2750pax 0 −1.7872 −1.2393 −1.0418 −0.3118 −0.1115 −0.6943
    RC2756pax 0 1.3803 0.9167 0.5508 1.0009 1.6415 1.9636
    RC2771pax 0 −0.9547 0.3567 −0.6792 −0.3341 1.1965 1.4536
    RC2790pax 0 0.8953 0.9817 0.4808 0.7259 1.0815 1.3536
    RC2792pax 0 1.2903 0.8667 0.8058 1.4559 1.9415 1.5836
    RC2808pax 0 0.7953 1.9117 0.7558 1.0009 1.1715 1.4036
    RC2822pax 0 0.7728 1.2607 0.3282 0.5932 2.5785 0.5807
    RC2834pax 0 −0.6247 0.2917 −0.5742 0.9309 2.0765 0.9236
    RC2871pax 0 0.9028 1.4107 1.1032 1.5332 2.6485 2.6307
    RC2879pax 0 −0.2047 0.3867 −0.0342 0.8509 0.6315 0.5086
    RC2892pax 0 −0.0022 0.5557 −0.0968 0.7032 2.0385 1.1857
    RC2895pax 0 1.7178 1.9857 1.2632 1.3682 1.4435 2.0557
    RC2921pax 0 1.3153 1.2967 1.0058 1.3709 2.0315 2.0886
    RC2958pax 0 1.0553 1.1017 0.4958 0.1409 0.4965 1.4836
    RC3022pax 0 0.3028 0.3007 0.3582 1.0432 1.7635 0.4707
    RC3112pax 0 1.2553 1.4667 0.5908 0.5759 2.5515 1.3286
    RC3146pax 0 −0.1572 −0.2793 −0.4918 0.1432 1.0235 0.8307
    RC3184pax 0 2.1353 2.5967 1.0758 1.3559 2.2865 2.2336
    RC3232pax 0 −0.3747 0.5667 −0.5942 0.5309 −0.4385 2.3786
    RC3324pax 0 0.2128 0.8557 0.0082 0.3282 1.3985 −0.0693
    RC3327pax 0 0.2003 −0.1333 −0.0292 −0.2491 1.2315 1.8086
    RC3355pax 0 0.0328 0.5657 −0.2668 −0.0418 1.2935 0.4857
    RC3380pax 0 −0.4372 0.4907 −0.3518 −0.0318 0.5235 0.0757
    RC3413pax 0 0.6028 1.1907 0.4532 0.0182 0.8435 2.3907
    RC3421pax 0 −0.0047 0.2917 −0.0142 0.1409 −0.0735 −0.0964
    RC3468pax 0 −0.1022 −0.0143 −0.0618 −0.3368 1.1485 0.2907
    RC3498pax 0 0.0353 −0.2633 −0.1892 0.2909 1.2415 0.2486
    CC0003pax 1 −1.4122 −0.3593 −0.9318 −0.6468 0.8435 −0.8893
    CD0157pax 1 1.5153 1.5517 1.1308 1.3209 3.0715 2.8186
    CD0164pax 1 −0.0247 0.4667 0.6758 1.0059 2.5315 1.2886
    CD0256pax 1 −0.6322 0.0807 0.0032 0.0382 0.8935 1.1557
    CD0322pax 1 −1.1572 −0.4693 −0.8368 −0.2818 0.5435 1.0507
    CD0356pax 1 −0.6772 −1.1393 −0.7718 −0.8668 −0.2215 −1.1643
    CD0371pax 1 0.1028 0.9607 −0.1918 −0.3018 0.8385 −0.2843
    CD0629pax 1 −0.0772 0.5407 0.5382 1.3882 0.2885 1.2507
    CD1050pax 1 0.8153 0.1417 0.2208 0.7559 1.3815 1.0836
    DC0001pax 1 0.1353 −0.4183 0.1208 −0.0941 0.3065 0.0936
    DC0002pax 1 0.9878 0.2857 0.3632 −0.1718 1.7035 0.9057
    DS0003pax 1 −1.8347 −0.7183 −1.5192 −1.5591 −0.3485 0.6886
    FC0005pax 1 0.0928 0.1507 0.2682 0.1732 0.4235 1.8157
    FC0011pax 1 −0.4572 −0.0793 −0.1118 −0.2318 0.8635 0.2707
    FC0012pax 1 −2.5847 −0.4033 −1.3792 −1.2541 −0.2885 −0.3614
    JGA0001pax 1 −2.3247 −1.6483 −1.5592 −0.7241 −1.2535 −1.1814
    JGA0008pax 1 −0.6772 1.0357 0.5732 0.0932 0.8485 0.7057
    JH0002pax 1 0.5453 0.9667 0.5608 0.4459 1.4065 0.7586
    JH0003pax 1 0.2853 0.1667 −0.2292 −0.3941 −0.1385 1.0886
    JH0004pax 1 −0.1747 −0.1733 −0.3592 −0.5591 1.1515 0.6686
    JH0005pax 1 0.0928 1.2207 0.0682 0.1482 1.5735 1.1757
    JH0006pax 1 −0.6397 0.0667 −0.2692 −0.2741 0.8715 −0.3114
    JH0007pax 1 −2.6372 −2.2043 −1.6168 −0.5018 −0.0865 −1.2993
    JH0008pax 1 0.5453 2.4167 0.2208 0.8859 2.1315 0.6336
    JH0009pax 1 −0.8522 −0.7793 −0.0968 −0.6668 −0.1815 0.4507
    JH0010pax 1 −0.3847 0.6517 0.2208 0.3059 0.6365 −0.2664
    JH0012pax 1 −0.1072 0.3857 0.2882 0.0432 0.6685 0.9807
    JH0013pax 1 −0.3022 0.2007 −0.3118 −0.0568 2.0485 0.8357
    JH0014pax 1 1.1828 1.2107 0.3182 −0.0418 2.0485 2.5207
    JH0016pax 1 0.5053 0.9167 0.6658 0.2659 0.4715 0.8636
    JH0018pax 1 −1.1972 −0.1493 −0.4268 −0.4318 −0.1465 0.3207
    JH0019pax 1 0.1153 0.6267 0.0558 −0.2091 0.1565 0.1936
    JH0020pax 1 0.2728 2.0207 0.7332 −0.1518 0.7585 1.3107
    JH0021pax 1 −0.8297 0.7867 −0.3992 −0.8541 0.2615 0.0586
    JH0023pax 1 −0.2272 1.3307 0.5182 1.6882 1.2985 1.9507
    JH0024pax 1 1.0653 2.5467 1.2508 1.4159 3.3465 2.9786
    JH0025pax 1 −0.7847 −0.3833 −0.4292 −0.8891 −0.2435 0.5136
    JH0026pax 1 −1.1522 0.5857 −0.0418 −0.3468 1.1035 1.1507
    JH0027pax 1 −3.3947 −2.5983 −2.3842 −2.0791 −2.3435 −1.8814
    JH0028pax 1 0.3253 1.0967 0.9908 0.7459 2.5415 1.4586
    JH0029pax 1 −1.3522 −0.6093 −0.5918 −0.7818 −0.5615 0.5557
    JH0031pax 1 −0.7297 0.2817 −0.2092 −0.2791 0.5615 0.5136
    JH0032pax 1 1.5478 1.7557 0.4532 0.3732 1.7785 1.8307
    JH0033pax 1 −0.8797 1.0817 −0.4242 −0.6141 1.3865 −0.1914
    JH0034pax 1 −0.5397 1.0667 0.4908 0.3909 1.5465 1.5186
    JH0035pax 1 −0.3222 1.3857 0.2782 −0.3268 1.9385 0.8207
    JH0036pax 1 0.8128 1.0107 0.4032 0.2182 1.9235 1.1757
    JH0038pax 1 0.3103 1.5367 0.2258 0.4109 2.1215 0.8386
    JH0039pax 1 0.5278 0.6207 0.5032 1.1232 3.1135 0.0857
    JH0040pax 1 0.4353 0.8117 0.5508 0.3259 1.2465 0.8636
    JH0041pax 1 0.7303 1.7117 0.4508 0.2759 1.9565 1.0436
    JH0042pax 1 −2.2922 −0.6293 −1.2268 −0.7568 −1.0515 −1.7443
    JH0043pax 1 −0.8022 −0.1993 −0.2918 −0.9318 −0.3015 −0.2393
    JH0046pax 1 −0.0447 0.0567 0.6408 −0.2541 0.8965 0.0336
    JH0047pax 1 0.1728 1.9907 0.6082 1.2782 1.6085 2.0757
    JH0051pax 1 −0.9647 −0.1533 −0.3892 −1.0491 −0.2135 −0.4064
    JH0052pax 1 −0.6597 0.3067 −0.4442 −0.9141 −0.4585 −0.7914
    JH0053pax 1 −0.9522 −0.1693 −0.5918 −0.0318 −0.2515 0.1107
    JH0057pax 1 −0.1872 1.9757 0.1432 0.6682 1.4335 1.2057
    JH0059pax 1 −0.7897 0.0717 −0.3442 −0.3241 0.3215 0.1136
    JH0060pax 1 −1.0122 0.1207 −0.2818 0.0482 0.3385 0.3557
    JH0061pax 1 0.7803 2.5567 0.5558 1.0309 3.2565 0.3136
    JH0063pax 1 −0.2197 1.0467 0.2458 −0.4141 0.8115 2.0486
    JH0065pax 1 −2.7497 −1.4333 −2.0542 −2.8691 −1.5035 −0.4814
    JH0066pax 1 −1.1972 −0.0343 −0.8418 −1.6318 −1.0715 −0.4643
    JH0068pax 1 0.3653 0.8867 0.7108 0.4809 1.7165 2.1986
    JH0069pax 1 −0.6447 0.3967 −0.0892 −0.1791 −0.0735 −0.4014
    JH0071pax 1 −2.3272 −2.1943 −1.6968 −1.0318 −1.2715 −0.3743
    JH0072pax 1 −0.1197 −0.7533 −0.0892 −0.6741 1.1515 0.0586
    JH0077pax 1 −0.3022 0.2957 0.0882 −0.0368 0.4435 1.6307
    JH0078pax 1 1.2953 1.2767 1.2058 0.2609 1.2165 1.2786
    JH0080pax 1 −2.1122 −1.1393 −1.0418 −0.7568 −1.5315 0.3507
    JH0082pax 1 −0.6722 −0.5393 −0.0518 −0.2368 −0.3065 1.0857
    JH0083pax 1 −1.3997 −0.0133 −0.3542 −1.2991 0.3115 0.7236
    JH0086pax 1 0.2553 0.1667 0.0158 −0.7941 0.4015 1.1086
    JH0092pax 1 −0.2647 1.6167 0.2158 0.2559 0.1565 1.3386
    MH0001pax 1 1.1653 1.7717 1.0908 1.1159 2.8015 1.9086
    MH0009pax 1 −0.4972 −0.0193 −0.7268 −0.5768 −0.3015 −0.3493
    MH0012pax 1 −0.1347 1.0017 0.3658 0.8559 0.9765 0.9186
    MH0014pax 1 0.8278 1.4457 0.8482 0.5432 3.0535 1.8007
    MH0016pax 1 −1.1672 −0.8793 −0.9168 −1.2168 −0.4165 −0.7243
    MH0017pax 1 0.3403 1.4267 0.5658 −0.0241 1.7565 2.0536
    MH0018pax 1 1.2128 0.7657 0.3232 0.3682 2.1235 0.7057
    MH0021pax 1 0.8103 0.2517 −0.3192 0.0709 1.6865 1.7086
    MH0022pax 1 0.6903 1.3667 0.6308 0.5459 1.2515 1.4236
    MH0024pax 1 0.2228 0.4757 −0.0568 −0.1768 0.0635 0.9057
    MH0026pax 1 −1.2897 0.0417 −0.4392 0.1059 −0.3935 −0.1914
    MH0028pax 1 −0.0272 0.3557 −0.1068 −0.8368 −0.0765 −0.0493
    MH0029pax 1 −0.0697 0.0167 0.0658 −0.6641 1.0215 0.3536
    MH0035pax 1 0.6603 2.0217 0.9458 0.7759 1.0965 1.4036
    MH0037pax 1 −0.1697 1.0717 −0.0342 0.1309 1.1665 1.6186
    MH0038pax 1 1.7453 1.5967 1.3108 1.4609 2.2365 2.3736
    MH0039pax 1 −1.1922 −0.3093 −0.1218 −0.5718 −0.0965 0.5557
    MH0042pax 1 −0.1522 0.0657 0.2332 −0.1318 −0.0815 0.9357
    MH0050pax 1 −1.4222 0.5507 −0.6818 −0.3468 0.8885 1.6307
    MH0051pax 1 −1.2197 −1.0683 −0.7092 −1.6041 −2.1735 −0.1814
    MIP0004pax 1 −2.1722 −2.1793 −1.4118 −1.1218 −0.9865 −2.2593
    MP0013Apax 1 0.4028 1.2307 0.0782 0.2282 1.4535 0.1557
    MP0014Bpax 1 −0.4347 0.6117 −0.5542 −0.0441 1.2765 −0.1964
    MP0018Apax 1 −1.0022 −0.4193 −0.9668 −1.5668 −0.3165 −1.4293
    MP0019Bpax 1 −0.6222 −0.2993 −0.3768 −0.6268 −0.3465 0.8757
    MP0024pax 1 −1.2597 −0.2333 −0.4542 0.1409 1.3465 0.7386
    NK2001pax 1 −0.8347 −0.1233 −0.6992 −1.5741 −0.0785 1.1036
    NK2002pax 1 0.0203 −0.0183 0.1908 −0.4741 0.8115 0.9786
    NK2003pax 1 −0.7372 0.0407 −0.3218 −0.2868 1.2085 −0.1993
    NK2004pax 1 −0.9022 0.3107 −0.5118 −0.9568 0.5285 −0.3843
    PB1829pax 1 0.3378 1.5057 0.0682 0.0782 1.5485 1.7407
    PB1842pax 1 1.0953 1.3467 0.8708 0.2009 2.3265 0.9536
    PB1872pax 1 0.3928 0.6507 0.5682 −0.5068 1.2035 0.1807
    PB2857pax 1 −0.9422 0.5057 −0.3968 0.2982 −0.2165 0.8657
    RC2919pax 1 2.0678 2.0357 1.6382 3.0282 2.8985 3.9757
    RC3062pax 1 0.1453 0.5917 0.1408 0.3759 0.1365 0.3386
    RC3277pax 1 −0.1122 0.1707 −0.0568 −0.0168 0.7285 0.2457
    RC3297pax 1 0.2078 0.9457 0.4032 0.5332 2.7135 0.9807
    RC3445pax 1 −0.8497 −0.1233 −0.3442 −0.7391 0.6765 0.3086
    RC3467pax 1 2.0928 2.9557 1.5782 1.4332 2.8485 2.8457
  • Surprisingly, analysis of the data showed that RNA encoded by ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 is present on average at a significantly higher level (p-value less than 0.05) in blood of subjects having colorectal cancer relative to subjects having no colorectal pathology (Table 10). The ranges of fold-change in the levels of RNA encoded by these genes normalized to levels of RNA encoded by IL2RB in blood of the training set subjects having colorectal cancer relative to the training set subjects not having any colorectal pathology are shown in Table 10.
  • TABLE 10
    Sample training set ranges of fold-change in levels of RNA encoded by
    ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 normalized to
    levels of RNA encoded by IL2RB in blood of subjects having colorectal
    cancer relative to subjects not having any colorectal pathology.
    Gene
    ANXA3 CLEC4D LMNB1 PRRG4 TNFAIP6 VNN1
    Average normalized RNA level in −0.30 0.42 −0.05 −0.12 0.79 0.66
    subjects having colorectal cancer
    (ΔCt)
    Average normalized RNA level in 0.46 0.99 0.39 0.65 1.42 1.25
    subjects not having any colorectal
    pathology (ΔCt)
    Average RNA level fold-change 1.69 1.48 1.55 1.35 1.55 1.35
    p-value for average RNA level fold- 5.5E−09 5.5E−06 6.4E−07 2.5E−13 6.0E−06 2.7E−06
    change
    Maximum observed RNA level 14.43 12.01 6.83 11.46 13.58 11.36
    directional fold-change
  • As can be seen in Table 10, a test subject having a blood level of RNA encoded by ANXA3, normalized to a level of RNA encoded by IL2RB, which is 1.7 to 14.4 fold higher than the average level of RNA encoded by this gene in blood of subjects not having any colorectal pathology is more likely to have colorectal cancer than to not have any colorectal pathology.
  • As can be seen in Table 10, a test subject having a blood level of RNA encoded by CLEC4D, normalized to a level of RNA encoded by IL2RB, which is 1.5 to 12.0 fold higher than the average level of RNA encoded by this gene in blood of subjects not having any colorectal pathology is more likely to have colorectal cancer than to not have any colorectal pathology.
  • As can be seen in Table 10, a test subject having a blood level of RNA encoded by LMNB1, normalized to a level of RNA encoded by IL2RB, which is 1.5 to 6.8 fold higher than the average level of RNA encoded by this gene in blood of subjects not having any colorectal pathology is more likely to have colorectal cancer than to not have any colorectal pathology.
  • As can be seen in Table 10, a test subject having a blood level of RNA encoded by PRRG4, normalized to a level of RNA encoded by IL2RB, which is 1.3 to 11.5 fold higher than the average level of RNA encoded by this gene in blood of subjects not having any colorectal pathology is more likely to have colorectal cancer than to not have any colorectal pathology.
  • As can be seen in Table 10, a test subject having a blood level of RNA encoded by TNFAIP6, normalized to a level of RNA encoded by IL2RB, which is 1.5 to 13.6 fold higher than the average level of RNA encoded by this gene in blood of subjects not having any colorectal pathology is more likely to have colorectal cancer than to not have any colorectal pathology.
  • As can be seen in Table 10, a test subject having a blood level of RNA encoded by VNN1, normalized to a level of RNA encoded by IL2RB, which is 1.3 to 11.4 fold higher than the average level of RNA encoded by this gene in blood of subjects not having any colorectal pathology is more likely to have colorectal cancer than to not have any colorectal pathology.
  • Generation of Logistic Regression Models for Determining the Probability that a Test Subject has Colorectal Cancer Versus not Having any Colorectal Pathology Via Measurement of Levels of RNA Encoded by ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 Normalized to Levels of RNA Encoded by IL2RB:
  • Linear regression analysis of levels of RNA encoded by ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 normalized to IL2RB surprisingly showed that logistic regression models could be generated, based on blood expression levels normalized to IL2RB for all 63 possible combinations of one or more of these genes, for discriminating, with a ROC AUC of at least 0.67, between subjects having colorectal cancer and subjects not having any colorectal pathology. Examples of these logistic regression models are shown in Table 11. A logistic regression model based on ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 (Table 11, Model #128) was surprisingly found to enable discrimination between subjects having colorectal cancer and subjects not having any colorectal pathology with a ROC AUC of 0.80.
  • By way of example, Model #128 of Table 11 corresponds to:

  • P={1+ê−[(−0.196)+(−1.042)(L ANXA3)+(0.393)(L CLEC4D)+(1.272)(L LMNB1)+(−1.837)(L PRRG4)+(0.289)(L TNFAIP6)+(−0.153)(L VNN1)]}̂−1,
      • where P is the probability that a test subject has colorectal cancer as opposed to not having any colorectal pathology, where LANXA3 is a ratio of a level of RNA encoded by ANXA3 to a level of RNA encoded by IL2RB in blood of the test subject, LCLEC4D is a ratio of a level of RNA encoded by CLEC4D to a level of RNA encoded by IL2RB in blood of the test subject, LLMNB1 is a ratio of a level of RNA encoded by LMNB1 to a level of RNA encoded by IL2RB in blood of the test subject, LPRRG4 is a ratio of a level of RNA encoded by PRRG4 to a level of RNA encoded by IL2RB in blood of the test subject, LTNFAIP6 is a ratio of a level of RNA encoded by TNFAIP6 to a level of RNA encoded by IL2RB in blood of the test subject, and LVNN1 is a ratio of a level of RNA encoded by VNN1 to a level of RNA encoded by IL2RB in blood of the test subject.
  • Further by way of example, Model #157 of Table 11 corresponds to:

  • P={1+ê−[0.288+(−1.392)(L PRRG4)]}̂−1,
      • where P is the probability that a test subject has colorectal cancer as opposed to not having any colorectal pathology, and LPRRG4 is a ratio of a level of RNA encoded by PRRG4 to a level of RNA encoded by IL2RB in blood of the test subject.
  • TABLE 11
    Logistic regression models based on blood expression levels for any possible combination of one or
    more of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, normalized to IL2RB expression levels for
    determining the probability that a test subject has colorectal cancer as opposed to not having colorectal cancer.
    ROC AUC values for the models are shown for the sample training set used to generate the models, as well as for
    an independent blind sample test set used to test the models. The models, listed in order of decreasing ROC AUC
    value for the training set, are based on expression levels determined via quantitative reverse transcriptase-PCR
    analysis using IL2RB as duplex partner for normalization. The form of these models is: P = {1 + e{circumflex over ( )}−[K0 + K1L1 +
    K2L2 + K3L3 . . . + KnLn]}{circumflex over ( )}−1, where P is the probability that a test subject has colorectal cancer as opposed to not
    having any colorectal pathology; K0 is a constant; K1 is a coefficient specific to a first gene; L1 is a ratio of a level
    of RNA encoded by the first gene in blood to a level of RNA encoded by IL2RB in blood; K2 is a coefficient
    specific to a second gene; L2 is a ratio of a level of RNA encoded by the second gene in blood to a level of RNA
    encoded by IL2RB in blood; K3 is a coefficient specific to a third gene; L3 is a ratio of a level of RNA encoded by
    the third gene in blood to a level of RNA encoded by IL2RB in blood; Kn is a coefficient specific to an nth gene;
    and Ln is a ratio of a level of RNA encoded by the nth gene in blood to a level of RNA encoded by IL2RB in
    blood. No regression coefficients are specified for genes which are not included in the gene combination (indicated
    by “—”) on which a given logistic regression model is based.
    No. of
    Logistic genes ROC AUC Gene-specific regression coefficient
    Regression in Training Constant (Kn)
    Model # Model Set Test Set (K0) ANXA3 CLEC4D LMNB1 PRRG4 TNFAIP6 VNN1
    128 6 0.80 0.78 −0.196 −1.042 0.393 1.272 −1.837   0.289 −0.153
    129 5 0.80 0.79 −0.298 −1.058 0.366 1.187 −1.854   0.285
    130 5 0.80 0.79 0.034 −0.945 0.456 1.300 −1.715 −0.146
    131 5 0.79 0.78 −0.070 −0.937 1.469 −1.774   0.336 −0.115
    132 4 0.79 0.79 −0.065 −0.961 0.428 1.221 −1.733
    133 4 0.79 0.78 −0.154 −0.955 1.394 −1.790   0.330
    134 5 0.79 0.78 −0.305 −0.630 0.575 −1.632   0.311 −0.049
    135 4 0.79 0.78 0.229 −0.799 1.537 −1.620 −0.097
    136 4 0.79 0.79 −0.337 −0.645 0.562 −1.642   0.309
    137 4 0.79 0.78 −0.058 −0.516 0.645 −1.493 −0.041
    138 5 0.79 0.76 0.239 0.173 0.355 −1.782   0.144 −0.210
    139 3 0.79 0.78 0.153 −0.817 1.474 −1.637
    140 3 0.79 0.79 −0.085 −0.528 0.634 −1.503
    141 4 0.79 0.76 0.128 0.280 −1.707   0.174 −0.162
    142 4 0.79 0.76 0.337 0.216 0.413 −1.721 −0.203
    143 3 0.79 0.77 0.229 0.355 −1.615 −0.144
    144 4 0.79 0.76 0.279 0.492 −1.757   0.172 −0.189
    145 4 0.78 0.76 0.109 0.128 0.225 −1.802   0.134
    146 3 0.78 0.76 0.053 0.209 −1.747   0.157
    147 3 0.78 0.76 0.416 0.607 −1.673 −0.173
    148 3 0.78 0.76 0.206 0.169 0.285 −1.745
    149 3 0.78 0.76 0.150 0.339 −1.781   0.156
    150 2 0.78 0.77 0.153 0.285 −1.659
    151 2 0.78 0.76 0.284 0.457 −1.703
    152 3 0.78 0.76 0.123 −1.591   0.257 −0.078
    153 3 0.78 0.77 −0.102 −0.352 −1.463   0.390
    154 4 0.78 0.77 −0.130 −0.368 −1.474   0.388   0.037
    155 2 0.78 0.76 0.082 −1.630   0.237
    156 3 0.77 0.77 0.216 −0.179 −1.276   0.061
    157 1 0.77 0.76 0.288 −1.392
    158 2 0.77 0.77 0.267 −0.150 −1.256
    159 2 0.77 0.76 0.296 −1.384 −0.011
    160 5 0.73 0.75 0.162 −0.907 0.153 0.321 −0.073 −0.270
    161 4 0.73 0.75 0.108 −0.930 0.130 0.296 −0.273
    162 3 0.73 0.76 0.086 −0.818 0.185 −0.243
    163 4 0.73 0.76 0.125 −0.793 0.206 −0.055 −0.238
    164 4 0.72 0.75 0.208 −0.868 0.406 −0.046 −0.254
    165 3 0.72 0.75 0.167 −0.888 0.380 −0.258
    166 2 0.72 0.75 0.171 −0.698 −0.204
    167 3 0.72 0.75 0.179 −0.691 −0.009 −0.203
    168 3 0.72 0.76 −0.021 −0.870 0.134 −0.077
    169 4 0.72 0.76 −0.012 −0.929 0.103 0.155 −0.087
    170 2 0.72 0.77 −0.080 −0.907 0.102
    171 1 0.72 0.76 −0.014 −0.827
    172 2 0.72 0.76 0.031 −0.793 −0.043
    173 3 0.72 0.76 −0.079 −0.957 0.074 0.122
    174 2 0.72 0.76 −0.039 −0.932 0.178
    175 3 0.72 0.76 0.026 −0.902 0.221 −0.068
    176 3 0.71 0.72 0.514 −0.502   −0.191 −0.309
    177 4 0.71 0.72 0.519 −0.024   −0.482   −0.186 −0.306
    178 3 0.70 0.72 0.398 −0.097   −0.604   −0.319
    179 2 0.70 0.73 0.361 −0.706   −0.333
    180 2 0.70 0.70 0.727 −0.345 −0.451
    181 3 0.70 0.71 0.701 −0.192   −0.256 −0.383
    182 2 0.70 0.73 0.306 −0.769   −0.227
    183 3 0.69 0.73 0.331 −0.085   −0.690   −0.208
    184 1 0.69 0.73 0.102 −1.041  
    185 2 0.69 0.70 0.589 −0.367   −0.433
    186 2 0.69 0.73 0.186 −0.170   −0.838  
    187 1 0.68 0.69 0.552 −0.671
    188 2 0.68 0.72 0.548 −0.379   −0.331
    189 1 0.67 0.70 0.549 −0.576
    190 1 0.67 0.71 0.371 −0.649  
  • Blind Sample Test Set:
  • Quantitative reverse transcriptase-PCR analysis of expression of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 in an independent test set of blood samples from 165 subjects having colorectal cancer and 171 subjects not having any colorectal pathology was performed as described above for the training set. The normalized RNA levels measured are shown in Table 12.
  • TABLE 12
    Sample test set levels of RNA encoded by ANXA3, CLEC4D, LMNB1, PRRG4,
    TNFAIP6 and VNN1 in blood of subjects having colorectal cancer (Group 1) and
    subjects not having any colorectal pathology (Group 0), normalized to levels of RNA
    encoded by IL2RB. Levels shown correspond to ΔCt.
    Gene
    Sample ID Group ANXA3 CLEC4D LMNB1 PRRG4 TNFAIP6 VNN1
    CD0036pax 0 −0.0922 −0.0443 −0.1868 −1.0168 0.5035 −0.0543
    CD0053pax 0 1.1828 2.1407 1.5032 1.3032 2.9135 1.5907
    CD0092pax 0 1.2028 0.9057 0.8232 0.6032 1.6285 0.2907
    CD0108pax 0 −0.0522 −0.1893 0.2582 −0.1318 0.3385 0.2757
    CD0122pax 0 0.5628 1.2857 0.5582 0.6382 2.0485 1.9357
    CD0148pax 0 0.6778 2.0057 0.6732 1.4432 2.0335 1.5607
    CD0192pax 0 0.6878 0.8157 0.7682 1.5882 0.7085 0.8907
    CD0204pax 0 0.1978 0.6907 0.0782 −0.1418 1.0185 0.9557
    CD0214pax 0 0.4478 0.1557 1.1682 1.1932 0.6235 0.6157
    CD0237pax 0 0.3828 0.8707 −0.0168 0.0582 1.1685 0.8007
    CD0238pax 0 0.2428 0.8957 0.3982 1.0232 1.5385 0.9657
    CD0242pax 0 0.9528 1.3957 0.5082 −0.3818 0.9685 1.0307
    CD0244pax 0 0.4928 0.3507 0.4182 0.8982 1.4535 −0.0493
    CD0277pax 0 0.1453 1.7617 0.2358 0.4909 1.8265 0.1486
    CD0282pax 0 −0.1572 0.2207 −0.2118 0.0632 0.7585 −1.2243
    CD0295pax 0 0.1578 0.7957 0.5482 0.5332 1.1735 0.1907
    CD0354pax 0 0.5178 0.9557 0.2432 1.0732 1.6235 1.0307
    CD0367pax 0 0.7778 0.6857 0.5232 0.3432 1.8935 0.8457
    CD0369pax 0 1.0328 2.1057 0.9982 1.9532 2.1885 1.7607
    CD0398pax 0 2.1128 2.2157 1.6682 2.0432 3.7635 3.1407
    CD0409pax 0 −0.2272 0.7757 0.0282 0.7082 0.5735 1.2357
    CD0419pax 0 0.2503 0.9617 0.5758 0.3759 2.1815 −0.3114
    CD0432pax 0 0.3628 0.5607 0.2532 0.0282 2.0485 0.4407
    CD0437pax 0 −0.8872 −0.1793 −0.6918 −0.7768 0.4635 −0.1593
    CD0472pax 0 −2.1022 −0.4543 −1.0718 −0.6968 0.0235 −0.4243
    CD0482pax 0 3.2778 3.4857 2.2282 2.2532 4.6085 3.4507
    CD0484pax 0 0.2778 1.1907 0.3932 1.3182 1.7635 0.4107
    CD0507pax 0 1.4228 1.0757 1.0982 0.4282 2.4985 1.5357
    CD0547pax 0 −1.6622 −0.1543 −0.5068 0.0632 0.1035 −0.3643
    CD0571pax 0 0.9378 1.2407 1.0382 1.8282 2.3535 0.5757
    CD0580pax 0 1.2978 1.6557 0.5582 0.4382 2.3585 1.2807
    CD0583pax 0 −0.4472 −0.9493 −0.4968 −0.5218 0.7935 −0.6093
    CD0603pax 0 0.6928 0.5507 0.1582 −0.0818 1.4135 1.2457
    CD0604pax 0 0.0028 0.6557 −0.1218 −0.1268 1.6635 −0.1443
    CD0619pax 0 −0.3072 0.2557 0.1532 0.5182 0.3285 0.8407
    CD0637pax 0 0.3428 0.0357 −0.0618 0.3132 0.8935 0.6657
    CD0667pax 0 −0.5672 −0.4993 −0.5168 −0.1568 0.0235 −0.2643
    CD0670pax 0 3.0478 2.9307 1.7582 1.5882 3.3985 2.6957
    CD0676pax 0 −0.7022 −0.6343 −0.5518 −1.0468 0.2485 0.0357
    CD0687pax 0 1.4528 2.5807 1.6032 1.9382 1.9035 2.4357
    CD0715pax 0 −2.1372 −1.5893 −1.0468 −0.7768 −0.3665 0.4757
    CD0721pax 0 0.1778 1.1807 0.0382 0.0582 1.1935 0.7607
    CD0726pax 0 −0.2472 −0.2193 0.0432 −0.0318 0.8985 −1.1393
    CD0743pax 0 0.4178 −0.4243 0.2032 0.2782 1.6735 −0.0243
    CD0786pax 0 0.6678 1.0807 0.6132 0.9682 1.8735 1.0607
    CD0800pax 0 2.6478 2.7757 1.9282 2.4682 3.8585 3.0107
    CD0829pax 0 −0.0722 0.4757 0.2082 −0.3318 0.5785 0.7957
    CD0833pax 0 0.1378 −0.3693 0.0982 0.5032 0.8185 −0.2293
    CD0840pax 0 −0.5072 0.4157 −0.2018 −0.5618 0.6035 0.1307
    CD0843pax 0 0.2853 −0.1433 −0.2192 −0.5291 0.3415 0.2136
    CD0937pax 0 1.2178 0.8507 0.9632 1.4432 2.6285 1.8457
    CD1001pax 0 0.7553 0.8367 0.2758 −0.3541 2.7865 0.9686
    CD1032pax 0 −3.0422 −2.3193 −0.7918 −0.7218 −1.1065 0.0557
    CD1068pax 0 −0.1722 −0.0343 0.1382 0.5532 2.0935 −0.2243
    CD1134pax 0 0.4253 1.2867 0.2608 0.4409 1.3565 1.9236
    CD1269pax 0 0.0378 1.3507 0.1732 −0.2218 1.1735 1.1007
    CD1270pax 0 0.8328 0.8557 0.6482 0.3532 1.4535 1.4157
    CD1271pax 0 1.6778 2.4257 1.2282 0.6282 0.9885 1.8207
    CD1278pax 0 0.2578 1.0757 0.5232 0.1582 1.2685 1.3507
    CD1285pax 0 0.6903 0.3917 0.8458 0.3609 2.7765 0.8986
    CD1313pax 0 1.7278 1.3757 0.8182 1.0282 2.8285 0.6057
    CD1320pax 0 −0.8972 0.2757 −0.6068 −0.0668 0.1335 0.7907
    CD1329pax 0 0.1903 −0.0983 0.2308 −0.3091 1.1115 0.8936
    CD1349pax 0 −0.1622 0.5757 −0.3668 −0.9268 0.3935 0.5807
    CD1401pax 0 −0.0397 −0.2933 −0.2492 0.2409 0.2715 0.2036
    CD1428pax 0 2.0228 2.1257 1.1632 2.0382 2.5385 1.4807
    CD1438pax 0 −0.1572 0.3757 −0.1418 0.3782 0.6935 −0.1493
    CD1441pax 0 −0.6972 0.3307 0.0582 0.1132 0.7235 −0.3693
    CD1458pax 0 0.2328 0.2407 0.9082 0.7332 1.8935 0.5707
    CD1487pax 0 0.2378 1.9757 0.9582 1.6032 1.9785 0.5407
    CD1559pax 0 −0.0022 0.8557 0.1632 0.3532 1.8685 1.0657
    CD1561pax 0 1.1728 0.7557 0.6132 0.5432 1.8635 1.9307
    CD1567pax 0 0.6928 1.4807 0.4982 1.2232 1.6035 1.2807
    CD1627pax 0 −1.0072 −0.6693 −0.1868 0.2632 −0.4965 −0.2743
    CD1708pax 0 1.3903 1.8417 1.1458 0.6909 2.4365 1.8636
    CD1719pax 0 −0.4022 0.0407 −0.3068 −0.7568 0.1285 0.4657
    CD1728pax 0 1.8578 2.0957 1.2732 2.0332 1.9635 3.0907
    CD1741pax 0 −0.4922 −0.5493 −0.1868 −0.4618 1.0035 0.3257
    PB0662pax 0 0.0003 0.3867 0.6908 0.6809 1.2815 −0.1464
    PB0701pax 0 0.8803 1.5017 1.2058 2.1159 1.6765 3.1036
    PB0790pax 0 0.4253 0.2867 0.5158 0.5459 0.1665 −0.2414
    PB1222pax 0 −0.0272 0.4407 0.2932 −0.6868 −0.0465 0.8957
    PB1260pax 0 −1.3447 −1.3983 −0.3742 −1.2441 −0.6585 −0.5814
    PB1275pax 0 −0.7447 0.3767 −0.3092 0.2359 −0.1835 2.1786
    PB1324pax 0 −0.1597 0.5667 0.4508 1.1109 1.7015 1.8886
    PB1336pax 0 0.4853 0.6817 0.2158 −0.5991 1.1365 1.5136
    PB1446pax 0 −0.9897 −0.0133 −0.1992 0.0159 0.7765 1.0086
    PB1514pax 0 −1.2222 −0.2093 −0.3418 −0.4868 0.8185 −0.0293
    PB1540pax 0 −0.7672 −0.2493 −0.2668 −0.0068 0.5185 0.8157
    PB1700pax 0 0.5403 0.7517 0.5258 0.8759 1.3265 0.8836
    PB1763pax 0 2.0953 2.5067 1.7958 2.6409 3.0115 1.9736
    PB1785pax 0 0.9903 1.6717 0.6008 1.4309 1.3415 0.9486
    PB1871pax 0 0.4853 0.3717 0.2008 0.3059 1.3365 1.4286
    PB1918pax 0 2.6678 2.3457 1.4632 1.7882 3.8835 2.6107
    PB1944pax 0 0.0028 −0.4843 0.4432 0.7482 0.5435 1.3507
    PB1952pax 0 1.9703 1.9167 0.5508 1.8859 2.9865 2.0386
    PB1973pax 0 2.7753 2.8667 1.9308 3.1009 3.7065 3.7736
    PB1984pax 0 −2.2397 −1.7683 −1.8492 −1.7341 −1.4835 −1.3114
    PB2005pax 0 1.4803 1.5917 0.8408 1.1159 3.3915 1.8386
    PB2015pax 0 2.4703 1.9567 2.0908 3.2409 4.2865 2.5086
    PB2024pax 0 0.8053 1.3967 0.8908 1.1359 2.6515 2.2286
    PB2041pax 0 −0.5647 0.7517 −0.0692 0.1909 1.7565 0.1836
    PB2062pax 0 2.4703 2.4517 1.7908 2.2809 3.3065 2.6786
    PB2084pax 0 1.3553 1.0517 0.6808 1.5959 2.3965 1.4886
    PB2130pax 0 0.4353 0.6717 0.0408 −0.1091 0.6315 0.6436
    PB2179pax 0 0.6453 1.5017 0.5358 0.3559 0.7115 0.8686
    PB2184pax 0 −0.8047 −0.1983 −0.3292 0.0859 0.2715 1.4886
    PB2258pax 0 −0.0397 −0.9183 −0.5042 −0.4191 0.8415 −1.1164
    PB2272pax 0 2.2653 2.2717 1.6408 1.7259 4.0015 1.8136
    PB2342pax 0 2.9678 2.7557 1.1682 1.6732 2.4335 2.7607
    PB2464pax 0 1.0003 1.6717 0.8458 0.4759 1.4465 3.2686
    PB2516pax 0 0.1603 0.8067 0.4858 0.2109 0.6065 1.9036
    PB2564pax 0 0.1253 1.3217 0.0258 0.3009 0.5815 0.7836
    PB2634pax 0 0.0153 1.1567 0.8958 0.7009 2.5465 0.5586
    PB2682pax 0 −1.2297 −0.1383 −0.5242 −1.4441 0.0415 −0.0114
    PB2709pax 0 1.2978 1.7457 1.3832 1.9982 2.2535 1.3107
    PB2711pax 0 −0.8797 −1.1033 −0.3892 −0.8391 0.5215 0.4736
    PB2757pax 0 0.5353 1.6117 0.8508 0.6659 1.5865 1.9836
    PB2809pax 0 −0.1722 0.9957 0.0032 −0.1768 0.6335 0.9057
    PB2842pax 0 −0.5547 0.3867 −0.3492 0.0209 −0.2585 0.3486
    PB2875pax 0 −0.0497 1.2967 0.3958 0.3959 1.2365 1.1736
    PB2889pax 0 0.6453 1.9717 1.0808 1.4209 1.4015 1.5286
    PB2909pax 0 −0.1372 −1.0543 −0.4368 −0.7268 0.2735 −0.3993
    PB2924pax 0 0.7803 1.5167 1.1858 1.2159 1.3115 1.2786
    PB2927pax 0 2.1453 0.8717 0.7158 0.1309 1.5815 2.1286
    PB2931pax 0 0.2203 −0.3283 −0.8242 −0.8241 1.8315 −0.3664
    PB2951pax 0 −0.0897 −1.6683 −1.0792 −0.2891 −0.3035 −0.9164
    PB2974pax 0 −0.2547 0.8417 0.1958 0.2259 1.0515 2.2086
    PB2978pax 0 3.1253 3.2667 2.2458 2.4059 3.7165 2.9886
    PB2988pax 0 −1.5847 −1.3333 −0.7892 −1.1941 −0.2435 −0.3764
    PB3014pax 0 1.3953 1.9017 1.2458 0.7959 2.3615 2.5236
    PB3021pax 0 0.3053 2.1667 0.4108 0.9459 0.9015 0.7786
    PB3032pax 0 −0.1547 0.4867 0.6658 0.0409 1.5065 1.1036
    PB3163pax 0 0.3003 1.4117 0.5908 1.1459 2.4115 1.7336
    PB3193pax 0 −0.0047 0.3717 0.0058 0.4559 1.1065 0.7036
    PB3200pax 0 0.2653 1.0967 0.8608 1.0859 1.4265 1.5786
    PB3226pax 0 0.3203 0.9467 0.4658 0.6709 1.9165 1.1536
    PB3227pax 0 −0.0147 0.7317 0.4658 0.6559 2.0115 1.3936
    PB3361pax 0 −0.8197 −0.1133 −0.5592 −0.9891 0.8715 −0.6564
    PB3439pax 0 −1.2272 −1.8643 −1.1768 −1.2568 −0.9015 −1.7543
    PB3445pax 0 1.6203 2.4017 1.5208 2.2859 2.7265 2.2836
    PB3481pax 0 −0.0397 0.9167 0.3308 1.1359 0.5015 1.3636
    PB3513pax 0 −0.0347 −0.2133 −0.5142 −0.6891 0.8765 −0.9214
    PB3524pax 0