Fecal sample processing and analysis comprising detection of blood

Abstract

A method of processing a fecal sample from a human subject comprising removing a portion of a collected fecal sample and adding the removed portion of the sample to a buffer that prevents denaturation or degradation of blood proteins found in the sample, and detecting the presence of human blood in the removed portion of the fecal sample. The method further comprises stabilizing the remaining portion of the fecal sample.

I claim: 
     
         1 . A method of detecting a predisposition to, or the incidence of, colorectal cancer in a faecal sample comprising: (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, (b) detecting an epigenetic 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 and 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 faecal sample comprises detection of haemoglobin in the faecal sample. 
     
     
         3 . The method of  claim 2  wherein haemoglobin in the faecal sample is detected through immunochemical means. 
     
     
         4 . The method of  claim 2  or  3  wherein the result in step (a) is considered positive if the concentration of hemoglobin detected is at least between about 50 to 150 ng/ml. 
     
     
         5 . The method of  claim 2  or  3  wherein the result in step (a) is considered positive if the concentration of hemoglobin detected is at least about 100 ng/ml. 
     
     
         6 . The method of  claim 2  or  3  wherein the result in step (a) is considered positive if the concentration of hemoglobin detected is 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). 
     
     
         7 . The method of  claim 2  or  3  wherein the result in step (a) is considered positive if the concentration of hemoglobin detected is 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). 
     
     
         8 . The method of any preceding claim wherein the epigenetic modification is detected in at least one gene selected from 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. 
     
     
         9 . The method of any preceding claim wherein the epigenetic modification is detected in at least one gene selected from 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. 
     
     
         10 . The method of  claim 9  wherein the epigenetic modification is detected in a panel of at least two genes selected from 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, consists essentially of or consists of PHACTR3, NDRG4 and FOXE1, NDRG4 and FOXE1, PHACTR3 and NDRG4, or PHACTR3 and FOXE1. 
     
     
         12 . The method of any preceding claim wherein the epigenetic modification is methylation. 
     
     
         13 . The method of any preceding claim which utilises primers selected from primers comprising the nucleotide sequences set forth in table 1, in particular 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 any preceding claim which utilises probes selected from probes comprising the nucleotide sequences set forth in table 1, in particular 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 any preceding claim wherein a single faecal sample is utilised as the source of the sample for (a) and (b). 
     
     
         16 . The method of  claim 15  wherein the faecal sample is split to permit (a) and (b) to be carried out. 
     
     
         17 . A method of sample processing, prior to carrying out a method of any of  claims 1  to  16 , comprising removing a portion of a collected faecal 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 forwarding or otherwise delivering the remaining portion of the collected faecal sample to a laboratory for performing step (b) of the method of  claim 1 , optionally as further defined in any one of  claims 8  to  14 , on the remaining portion of the collected faecal sample. 
     
     
         19 . The method of  claim 17  or  18  which further comprises forwarding or otherwise delivering the removed portion of the collected faecal sample to a laboratory for performing step (a) of the method of  claim 1 , optionally as further defined in any of  claims 2  to  7 , on the removed portion of the sample. 
     
     
         20 . The method of any of  claims 17  to  19  which further comprises performing step (b) of the method of  claim 1 , optionally as further defined in any one of  claims 8  to  14 , on the remaining portion of the collected faecal sample. 
     
     
         21 . The method of any of  claims 17  to  20  which further comprises performing step (a) of the method of  claim 1 , optionally as further defined in any of  claim 2  or  7 , on the removed portion of the sample. 
     
     
         22 . The method of any preceding claim wherein the faecal 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, colorectal cancer in a sample comprising detecting an epigenetic modification in a panel of at least two genes selected from 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, colorectal cancer. 
     
     
         24 . A method for monitoring treatment of colorectal cancer with a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or HDAC inhibitor comprising detecting an epigenetic modification in a panel of at least two genes selected from 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. 
     
     
         25 . A method for predicting the likelihood of successful treatment of colorectal cancer with a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or HDAC inhibitor comprising detecting an epigenetic modification in a panel of at least two genes selected from 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. 
     
     
         26 . A method for predicting the likelihood of resistance to treatment of colorectal cancer with a DNA demethylating agent and/or DNA methyltransferase inhibitor and/or HDAC inhibitor comprising detecting an epigenetic modification in a panel of at least two genes selected from 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. 
     
     
         27 . A method of selecting a suitable treatment regimen for colorectal cancer comprising detecting an epigenetic modification in a panel of at least two genes selected from PHACTR3, NDRG4 and FOXE1 in a sample, wherein 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 wherein 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 any of  claims 23  to  27  wherein the epigenetic modification is methylation and thus the method involves determining the methylation status of the panel of genes. 
     
     
         29 . The method of any of  claims 23  to  28  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 any of  claims 23  to  29  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 any of  claims 23  to  30  wherein the sample is a faecal sample. 
     
     
         32 . The method of  claim 31  wherein the faecal 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 any of  claims 23  to  32 . 
     
     
         34 . A method of detecting a predisposition to, or the incidence of, cancer in a sample comprising 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. 
     
     
         35 . A method for monitoring treatment of cancer with a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or HDAC inhibitor comprising 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. 
     
     
         36 . 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 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. 
     
     
         37 . 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 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. 
     
     
         38 . A method of selecting a suitable treatment regimen for cancer comprising 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 results in selection of a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or a HDAC inhibitor for treatment and wherein 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. 
     
     
         39 . The method of any of  claims 34  to  38  wherein the cancer is colorectal cancer. 
     
     
         40 . The method of any of  claims 34  to  39  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 any of  claims 34  to  40  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 any of  claims 34  to  41  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 any of  claims 34  to  42  wherein the sample is a faecal sample. 
     
     
         44 . The method of  claim 43  wherein the faecal 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 any of  claims 34  to  44 . 
     
     
         46 . A method of detecting a predisposition to, or the incidence of, colorectal cancer in a faecal sample comprising 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, colorectal cancer. 
     
     
         47 . A method for monitoring treatment of colorectal cancer with a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or HDAC inhibitor comprising detecting an epigenetic modification in at least one gene selected from GPNMB and MMP2 in a faecal sample, wherein detection of a reduction in the epigenetic modification in the at least one gene as treatment progresses is indicative of successful treatment. 
     
     
         48 . A method for predicting the likelihood of successful treatment of colorectal cancer with a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or HDAC inhibitor comprising detecting an epigenetic modification in at least one gene selected from GPNMB and MMP2 in a faecal 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. 
     
     
         49 . A method for predicting the likelihood of resistance to treatment of colorectal cancer with a DNA demethylating agent and/or DNA methyltransferase inhibitor and/or HDAC inhibitor comprising detecting an epigenetic modification in at least one gene selected from GPNMB and MMP2 in a faecal 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. 
     
     
         50 . A method of selecting a suitable treatment regimen for colorectal cancer comprising detecting an epigenetic modification in at least one gene selected from GPNMB and MMP2 in a faecal sample, wherein 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 wherein 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. 
     
     
         51 . The method of any of  claims 46  to  50  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 any of  claims 46  to  51  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 any of  claims 46  to  52  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 any of  claims 46  to  53 . 
     
     
         55 . A kit for detecting a predisposition to, or the incidence of, colorectal cancer in a faecal sample comprising:
 (a) means for 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, and   (b) means for detecting an epigenetic 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.   
     
     
         56 . The kit of  claim 55  wherein the epigenetic modification is detected in at least one gene selected from PHACTR3, NDRG4, FOXE1, GATA4, GPNMB, TFPI2, SOX17, SYNE1, LAMA, MMP2, OSMR, SFRP2 and CDO1. 
     
     
         57 . The kit of  claim 55  or  56  wherein the epigenetic modification is detected in at least one gene selected from 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 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 any one of  claims 55  to  58  wherein the means for detecting an epigenetic modification in the DNA contained within the faecal 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  or  60  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 any of  claims 55  to  61  wherein the means for detecting the presence of blood in the faecal sample comprises means for detection of haemoglobin in the faecal sample. 
     
     
         63 . The kit of  claim 62  wherein haemoglobin in the faecal 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 comprising means for detecting an epigenetic modification in a panel of at least two genes selected from PHACTR3, NDRG4 and FOXE1.   
     
     
         65 . The kit of  claim 64  wherein the means for detecting an epigenetic modification in a panel of at least two genes selected from 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  or  66  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 . 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 comprising means for detecting an epigenetic modification in at least one gene selected from LAMA1 and CDOL   
     
     
         69 . The kit of any of  claims 55  to  68  which further comprises means for processing a faecal sample. 
     
     
         70 . The kit of  claim 68  or  69  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 any of  claims 68  to  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 any of  claims 68  to  71  wherein the probes are selected from probes comprising the nucleotide sequences set forth as SEQ ID Nos: 30 (LAMA1) and 36 (CDO1). 
     
     
         73 . 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 comprising means for detecting an epigenetic modification in at least one gene selected from GPNMB and MMP2 and means for processing a faecal sample.   
     
     
         74 . The kit of  claim 73  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 73  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 73  or  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 any of  claims 69  to  76  wherein the means for processing a faecal sample comprises a sealable vessel for collection of a faecal sample. 
     
     
         78 . The kit of any of  claims 55  to  77  comprising a container for a portion of the collected faecal sample suitably dimensioned to contain a faecal sample weighing no more than approximately 4 grams. 
     
     
         79 . The kit of  claim 78  further comprising a collection device adapted to retrieve and/or deposit the faecal sample in the container. 
     
     
         80 . The kit of any of  claims 55  to  79  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 faecal sample comprising detecting an epigenetic 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, characterised in that the faecal sample has previously been stored for at least approximately 6 months, 1, 2, 3, 4, 5, 6 or more years and/or is less than approximately 4, 3, 2, or 1 g in weight.