Methods of detecting colorectal cancer

Abstract

A method of detecting a predisposition to, or the incidence of colorectal cancer in a faecal sample comprises, in a first step (a), detecting the presence of blood in the faecal sample, wherein detection of the presence of blood is indicative of a predisposition to, or the incidence of colorectal cancer. The method additionally comprises, in second step (b), detecting an epi-genetic modification in the DNA contained within the faecal sample, wherein detection of the epigenetic modification is indicative of a predisposition to, or the incidence of colorectal cancer. Based upon a positive result obtained in either (a) or (b) or in both (a) and (b) a predisposition to, or the incidence of colorectal cancer is detected. Related methods and kits involve detecting an epigenetic modification in a number of specific genes.

1 . A method of detecting a predisposition to, or the incidence of, colorectal cancer in a fecal sample comprising:
 (a) detecting the presence of blood in the fecal sample, wherein detection of the presence of blood is indicative of a predisposition to, or the incidence of, colorectal cancer,   (b) detecting an epigenetic modification in the DNA contained within the fecal sample, wherein detection of the epigenetic modification is indicative of a predisposition to, or the incidence of, colorectal cancer; and   (c) based upon a positive result obtained in either (a) or (b), or in both (a) and (b), detecting a predisposition to, or the incidence of, colorectal cancer.   
     
     
         2 . The method of  claim 1 , wherein detecting the presence of blood in the fecal sample comprises detection of hemoglobin in the fecal sample. 
     
     
         3 . The method of  claim 2 , wherein hemoglobin in the fecal sample is detected through immunochemical means. 
     
     
         4 . The method of  claim 2 , wherein the result in step (a) is considered positive if the concentration of hemoglobin detected is:
 (a) at least between about 50 to 150 ng/ml;   (b) at least about 100 ng/ml;   (c) greater than a predetermined threshold level, which threshold level is lower than is typically employed as the threshold concentration of hemoglobin in hemoglobin detection tests, but for those samples in which a lower concentration of hemoglobin is detected, which would be considered a negative result in typically employed tests, step (b) is performed only on these samples and the detection of the epigenetic modification in step (b) is then used to confirm the positive result in step (a); or   (d) at least about 10 ng/ml and wherein step (b) is performed only in the event that the concentration of hemoglobin detected is between about 10 ng/ml and about 200 ng/ml and the detection of the epigenetic modification in step (b) is then used to confirm the positive result in step (a).   
     
     
         5 .- 7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein the epigenetic modification is detected in at least one gene:
 (a) selected from the group consisting of PHACTR3, NDRG4, FOXE1, GATA4, GPNMB, TFPI2, SOX17, SYNE1, LAMA, MMP2, OSMR, SFRP2 and CDO1, with detection of the epigenetic modification in at least one of the genes providing an indication of a predisposition to, or incidence of, colorectal cancer; or   (b) selected from the group consisting of PHACTR3, NDRG4 and FOXE1, with detection of the epigenetic modification in at least one of the genes providing an indication of a predisposition to, or incidence of, colorectal cancer.   
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 8  wherein the epigenetic modification is detected in a panel of at least two genes selected from the group consisting of PHACTR3, NDRG4 and FOXE1, with detection of the epigenetic modification in at least one of the genes providing an indication of a predisposition to, or incidence of, colorectal cancer. 
     
     
         11 . The method of  claim 10  wherein the panel of genes comprises:
 (a) PHACTR3, NDRG4 and FOXE1, 
 (b) NDRG4 and FOXE1, 
 (c) PHACTR3 and NDRG4, or 
 (d) PHACTR3 and FOXE1. 
 
     
     
         12 . The method of  claim 1 , wherein the epigenetic modification is methylation. 
     
     
         13 . The method of  claim 1 , which utilises primers selected from primers comprising the nucleotide sequences set forth in table 1, or which utilises primers selected from primers comprising the nucleotide sequences set forth as SEQ ID Nos: 1 and 2 (PHACTR3), 4 and 5 (FOXE1), and 7 and 8 (NDRG4) in order to detect methylation status in the DNA. 
     
     
         14 . The method of  claim 1 , which utilises probes selected from probes comprising the nucleotide sequences set forth in table 1, or which utilises probes selected from probes comprising the nucleotide sequences set forth as SEQ ID Nos: 3 (PHACTR3), 6 (FOXE1), and 9 (NDRG4) in order to detect methylation status in the DNA. 
     
     
         15 . The method of  claim 1 , wherein:
 (a) a single fecal sample is utilised as the source of the sample for (a) and (b); or   (b) the fecal sample is split to permit (a) and (b) to be carried out.   
     
     
         16 . (canceled) 
     
     
         17 . A method of sample processing, prior to carrying out the method of  claim 1 , comprising removing a portion of a collected fecal sample and adding the removed portion of the sample to a buffer which prevents denaturation or degradation of blood proteins found in the sample. 
     
     
         18 . The method of  claim 17 , which further comprises:
 (a) forwarding or otherwise delivering the remaining portion of the collected fecal sample to a laboratory for performing step (b) of the method of  claim 1  on the remaining portion of the collected fecal sample;   (b) forwarding or otherwise delivering the removed portion of the collected fecal sample to a laboratory for performing step (a) of the method of  claim 1  on the removed portion of the sample;   (c) performing step (b) of the method of  claim 1  on the remaining portion of the collected fecal sample; or   (d) performing step (a) of the method of  claim 1  on the removed portion of the sample.   
     
     
         19 .- 21 . (canceled) 
     
     
         22 . The method of  claim 1 , wherein the fecal sample utilised to perform step (b) weighs no more than approximately 4 grams. 
     
     
         23 . A method of detecting a predisposition to, or the incidence of, cancer in a sample comprising:
 (a) detecting an epigenetic modification in a panel of at least two genes selected from the group consisting of PHACTR3, NDRG4 and FOXE1, wherein detection of the epigenetic modification in at least one of the genes in the panel is indicative of a predisposition to, or the incidence of, cancer;   (b) detecting an epigenetic modification in at least one gene selected from LAMA1 and CDO1, wherein detection of the epigenetic modification in the at least one gene is indicative of a predisposition to, or the incidence of, cancer; or   (c) detecting an epigenetic modification in at least one gene selected from GPNMB and MMP2, wherein detection of the epigenetic modification in the at least one gene is indicative of a predisposition to, or the incidence of, cancer.   
     
     
         24 . A method for monitoring treatment of cancer with a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or HDAC inhibitor comprising:
 (a) detecting an epigenetic modification in a panel of at least two genes selected from the group consisting of PHACTR3, NDRG4 and FOXE1 in a sample, wherein detection of a reduction in the epigenetic modification in at least one of the genes in the panel as treatment progresses is indicative of successful treatment;   (b) detecting an epigenetic modification in at least one gene selected from LAMA1 and CDO1 in a sample, wherein detection of a reduction in the epigenetic modification in the at least one gene as treatment progresses is indicative of successful treatment; or   (c) detecting an epigenetic modification in at least one gene selected from GPNMB and MMP2 in a fecal sample, wherein detection of a reduction in the epigenetic modification in the at least one gene as treatment progresses is indicative of successful treatment.   
     
     
         25 . A method for predicting the likelihood of successful treatment of cancer with a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or HDAC inhibitor comprising:
 (a) detecting an epigenetic modification in a panel of at least two genes selected from the group consisting of PHACTR3, NDRG4 and FOXE1 in a sample, wherein detection of the epigenetic modification in at least one of the genes in the panel is indicative that the likelihood of successful treatment is higher than if the epigenetic modification is not detected;   (b) detecting an epigenetic modification in at least one gene selected from LAMA1 and CDO1 in a sample, wherein detection of the epigenetic modification in the at least one gene is indicative that the likelihood of successful treatment is higher than if the epigenetic modification is not detected; or   (c) detecting an epigenetic modification in at least one gene selected from GPNMB and MMP2 in a fecal sample, wherein detection of the epigenetic modification in the at least one gene is indicative that the likelihood of successful treatment is higher than if the epigenetic modification is not detected.   
     
     
         26 . A method for predicting the likelihood of resistance to treatment of cancer with a DNA demethylating agent and/or DNA methyltransferase inhibitor and/or HDAC inhibitor comprising:
 (a) detecting an epigenetic modification in a panel of at least two genes selected from the group consisting of PHACTR3, NDRG4 and FOXE1 in a sample, wherein detection of the epigenetic modification in at least one of the genes in the panel is indicative that the likelihood of resistance to treatment is lower than if the epigenetic modification is not detected;   (b) detecting an epigenetic modification in at least one gene selected from LAMA1 and CDO1 in a sample, wherein detection of the epigenetic modification in the at least one gene is indicative that the likelihood of resistance to treatment is lower than if the epigenetic modification is not detected; or   (c) detecting an epigenetic modification in at least one gene selected from GPNMB and MMP2 in a fecal sample, wherein detection of the epigenetic modification in the at least one gene is indicative that the likelihood of resistance to treatment is lower than if the epigenetic modification is not detected.   
     
     
         27 . A method of selecting a suitable treatment regimen for cancer comprising:
 (a) detecting an epigenetic modification in a panel of at least two genes selected from the group consisting of PHACTR3, NDRG4 and FOXE1 in a sample, wherein: (i) detection of the epigenetic modification in at least one of the genes in the panel results in selection of a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or a HDAC inhibitor for treatment; and (ii) if the epigenetic modification is not detected, a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or a HDAC inhibitor is not selected for treatment;   (b) detecting an epigenetic modification in at least one gene selected from LAMA1 and CDO1 in a sample, wherein: (i) detection of the epigenetic modification in the at least one gene results in selection of a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or a HDAC inhibitor for treatment; and (ii) if the epigenetic modification is not detected, a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or a HDAC inhibitor is not selected for treatment; or   (c) detecting an epigenetic modification in at least one gene selected from GPNMB and MMP2 in a fecal sample, wherein (i) detection of the epigenetic modification in the at least one gene results in selection of a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or a HDAC inhibitor for treatment and (ii) if the epigenetic modification is not detected, a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or a HDAC inhibitor is not selected for treatment.   
     
     
         28 . The method of  claim 23 , wherein the epigenetic modification is methylation and thus the method involves determining the methylation status of the panel of genes. 
     
     
         29 . The method of  claim 23 , which utilises primers selected from primers comprising the nucleotide sequences set forth as SEQ ID Nos: 1 and 2 (PHACTR3), 4 and 5 (FOXE1), and 7 and 8 (NDRG4). 
     
     
         30 . The method of  claim 23 , which utilises probes selected from probes comprising the nucleotide sequences set forth as SEQ ID Nos: 3 (PHACTR3), 6 (FOXE1), and 9 (NDRG4). 
     
     
         31 . The method of  claim 23 , wherein the sample is a fecal sample. 
     
     
         32 . The method of  claim 31 , wherein the fecal sample weighs no more than approximately 4 grams. 
     
     
         33 . A method of treating colorectal cancer in a subject comprising administration of a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or a HDAC inhibitor, wherein the subject has been selected for treatment on the basis of a method as claimed in  claim 23 . 
     
     
         34 .- 38 . (canceled) 
     
     
         39 . The method of  claim 23 , wherein the cancer is colorectal cancer. 
     
     
         40 . The method of  claim 24 , wherein the epigenetic modification is methylation and thus the method involves determining the methylation status of the at least one gene. 
     
     
         41 . The method of  claim 24 , which utilises primers selected from primers comprising the nucleotide sequences set forth as SEQ ID Nos: 28 and 29 (LAMA1) and 34 and 35 (CDO1). 
     
     
         42 . The method of  claim 24 , which utilises probes selected from probes comprising the nucleotide sequences set forth as SEQ ID Nos: 30 (LAMA1) and 36 (CDO1). 
     
     
         43 . The method of  claim 24 , wherein the sample is a fecal sample. 
     
     
         44 . The method of  claim 43 , wherein the fecal sample weighs no more than approximately 4 grams. 
     
     
         45 . A method of treating colorectal cancer in a subject comprising administration of a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or a HDAC inhibitor wherein the subject has been selected for treatment on the basis of a method as claimed in  claim 27 . 
     
     
         46 .- 50 . (canceled) 
     
     
         51 . The method of  claim 25 , wherein the epigenetic modification is methylation and thus the method involves determining the methylation status of the at least one gene. 
     
     
         52 . The method of  claim 25 , which utilises primers selected from primers comprising the nucleotide sequences set forth as SEQ ID Nos: 22 and 23 (GPNMB) and 31 and 32 (MMP2). 
     
     
         53 . The method of  claim 25 , which utilises probes selected from probes comprising the nucleotide sequences set forth as SEQ ID Nos: 24 (GPNMB) and 33 (MMP2). 
     
     
         54 . A method of treating colorectal cancer in a subject comprising administration of a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or a HDAC inhibitor wherein the subject has been selected for treatment on the basis of a method as claimed in  claim 25 . 
     
     
         55 . A kit for detecting a predisposition to, or the incidence of, colorectal cancer in a fecal sample comprising:
 (a) means for detecting the presence of blood in the fecal sample, wherein detection of the presence of blood is indicative of a predisposition to, or the incidence of, colorectal cancer, and   (b) means for detecting an epigenetic modification in the DNA contained within the fecal sample, wherein detection of the epigenetic modification is indicative of a predisposition to, or the incidence of, colorectal cancer.   
     
     
         56 . The kit of  claim 55  wherein the epigenetic modification is detected in at least one gene selected from the group consisting of PHACTR3, NDRG4, FOXE1, GATA4, GPNMB, TFPI2, SOX17, SYNE1, LAMA, MMP2, OSMR, SFRP2 and CDO1. 
     
     
         57 . The kit of  claim 55 , wherein the epigenetic modification is detected in at least one gene selected from the group consisting of PHACTR3, NDRG4 and FOXE1. 
     
     
         58 . The kit of  claim 57 , wherein the epigenetic modification is detected in a panel of at least two genes selected from the group consisting of PHACTR3, NDRG4 and FOXE1, with detection of the epigenetic modification in at least one of the genes providing an indication of a predisposition to, or incidence of, colorectal cancer. 
     
     
         59 . The kit of  claim 55 , wherein the means for detecting an epigenetic modification in the DNA contained within the fecal sample comprise primers and/or probes which permit the methylation status of the DNA to be determined directly. 
     
     
         60 . The kit of  claim 59  wherein the primers are selected from primers comprising the nucleotide sequences set forth as SEQ ID Nos: 1 and 2 (PHACTR3), 4 and 5 (FOXE1), and 7 and 8 (NDRG4). 
     
     
         61 . The kit of  claim 59 , wherein the probes are selected from probes comprising the nucleotide sequences set forth as SEQ ID Nos: 3 (PHACTR3), 6 (FOXE1), and 9 (NDRG4). 
     
     
         62 . The kit of  claim 55 , wherein the means for detecting the presence of blood in the fecal sample comprises means for detection of hemoglobin in the fecal sample. 
     
     
         63 . The kit of  claim 62  wherein hemoglobin in the fecal sample is detected through immunochemical means. 
     
     
         64 . A kit for any of:
 (a) detecting a predisposition to, or the incidence of, colorectal cancer in a sample;   (b) predicting the likelihood of successful treatment of colorectal cancer with a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or HDAC inhibitor;   (c) predicting the likelihood of resistance to treatment of colorectal cancer with a DNA demethylating agent and/or DNA methyltransferase inhibitor and/or HDAC inhibitor; or   (d) selecting a suitable treatment regimen for colorectal cancer,   wherein the kit comprises:
 (i) means for detecting an epigenetic modification in a panel of at least two genes selected from the group consisting of PHACTR3, NDRG4 and FOXE1; 
 (ii) means for detecting an epigenetic modification in at least one gene selected from LAMA1 and CDO1; 
 (iii) means for detecting an epigenetic modification in at least one gene selected from GPNMB and MMP2 and means for processing a fecal sample. 
   
     
     
         65 . The kit of  claim 64 (i), wherein the means for detecting an epigenetic modification in a panel of at least two genes selected from the group consisting of PHACTR3, NDRG4 and FOXE1 comprise primers and/or probes which permit the methylation status of the genes to be determined directly. 
     
     
         66 . The kit of  claim 65  wherein the primers are selected from primers comprising the nucleotide sequences set forth as SEQ ID Nos: 1 and 2 (PHACTR3), 4 and 5 (FOXE1), and 7 and 8 (NDRG4). 
     
     
         67 . The kit of  claim 65 , wherein the probes are selected from probes comprising the nucleotide sequences set forth as SEQ ID Nos: 3 (PHACTR3), 6 (FOXE1), and 9 (NDRG4). 
     
     
         68 . (canceled) 
     
     
         69 . The kit of  claim 55 , which further comprises means for processing a fecal sample. 
     
     
         70 . The kit of  claim 64 (i), wherein the means for detecting an epigenetic modification in at least one gene selected from LAMA1 and CDO1 comprise primers and/or probes which permit the methylation status of the DNA to be determined directly. 
     
     
         71 . The kit of  claim 70 , wherein the primers are selected from primers comprising the nucleotide sequences set forth as SEQ ID Nos: 28 and 29 (LAMA1) and 34 and 35 (CDO1). 
     
     
         72 . The kit of  claim 70 , wherein the probes are selected from probes comprising the nucleotide sequences set forth as SEQ ID Nos: 30 (LAMA1) and 36 (CDO1). 
     
     
         73 . (canceled) 
     
     
         74 . The kit of  claim 64 (iii), wherein the means for detecting an epigenetic modification in at least one gene selected from GPNMB and MMP2 comprise primers and/or probes which permit the methylation status of the DNA to be determined directly. 
     
     
         75 . The kit of  claim 74  wherein the primers are selected from primers comprising the nucleotide sequences set forth as SEQ ID Nos: 22 and 23 (GPNMB) and 31 and 32 (MMP2). 
     
     
         76 . The kit of  claim 74 , wherein the probes are selected from probes comprising the nucleotide sequences set forth as SEQ ID Nos: 24 (GPNMB) and 33 (MMP2). 
     
     
         77 . The kit of  claim 64 , wherein the means for processing a fecal sample comprises a sealable vessel for collection of a fecal sample. 
     
     
         78 . The kit of  claim 55 , comprising a container for a portion of the collected fecal sample suitably dimensioned to contain a fecal sample weighing no more than approximately 4 gams. 
     
     
         79 . The kit of  claim 78 , further comprising a collection device adapted to retrieve and/or deposit the fecal sample in the container. 
     
     
         80 . The kit of  claim 55 , which further comprises a reagent which selectively modifies unmethylated cytosine residues in the DNA contained in the sample to produce detectable modified residues but which does not modify methylated cytosine residues. 
     
     
         81 . A method of detecting a predisposition to, or the incidence of, colorectal cancer in a fecal sample comprising detecting an epigenetic modification in the DNA contained within the fecal sample, wherein detection of the epigenetic modification is indicative of a predisposition to, or the incidence of, colorectal cancer, characterised in that the fecal sample has previously been stored for at least about 6 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, or about 6 or more years and/or is less than about 4, less than about 3, less than about 2, or less than about 1 g in weight. 
     
     
         82 . The method of  claim 24 , wherein the cancer is colorectal cancer. 
     
     
         83 . The method of  claim 25 , wherein the cancer is colorectal cancer. 
     
     
         84 . The method of  claim 26 , wherein the cancer is colorectal cancer. 
     
     
         85 . The method of  claim 27 , wherein the cancer is colorectal cancer.