CA2557134A1

CA2557134A1 - Molecular method for diagnosis of colon cancer

Info

Publication number: CA2557134A1
Application number: CA 2557134
Authority: CA
Inventors: Nabil Belacel; Miroslava Cuperlovic-Culf; Rodney Ouellette
Original assignee: National Research Council of Canada
Current assignee: National Research Council of Canada
Priority date: 2006-08-23
Filing date: 2006-08-23
Publication date: 2008-02-23

Abstract

Methods for diagnosing or detecting cancerous colon tissue. A panel of 21 specific marker genes are provided. The overexpression of some of these marker genes compared to their expression in normal colon tissue and the underexpression of the rest of these marker genes are indicative of cancerous colon tissue. By using these 21 marker genes as a diagnostic tool, smaller tissue samples, such as those obtained by core needle biopsies and from patient stool samples, can be used.

Description

MOLECULAR METHOD FOR DIAGNOSIS OF COLON CANCER

Field of the Invention [00001] The present invention relates to diagnosis methods and, more particularly, to diagnosis methods for detecting colon cancer.

Background to the Invention [00002] With 19,200 new cases in Canada in 2004, colon cancer is one of the three most prevalent cancers in Canada for both men and women (Canadian Cancer Statistiscs, 2004). Invasive biopsy procedures require long hospitalizations and may have numerous possible side effects. Other alternative diagnostic procedures, such as digital rectal examination, fecal occult blood procedure, double-contrast barium enema, flexible sigmoidoscopy, and total colonoscopy are mostly invasive. The fecal occult blood test, while non-invasive, requires confirmation by way of additional invasive procedures. Unfortunately, such invasive procedures can possibly lead to side effects and/or long hospitalizations.

[00003] There is therefore a need for a non-invasive and accurate testing procedure for detecting colon cancer. Ideally, such a test should be able to detect cancerous colon cells even from small sample sizes.

[00004] There is therefore a need for a more accurate diagnostic method that does not require an invasive biopsy to detect or diagnose colon cancer.
Ideally, such a method should be usable even with very small sample sizes and may be combined with other, pathologist-based diagnosis methods.

Summary of the Invention [00005] The present invention provides methods for diagnosing or detecting cancerous colon tissue. Colon tissue samples are acquired and are tested for the expression of specific marker genes. A panel of 21 specific marker genes are provided. The overexpression of some of these marker genes compared to their expression in normal colon tissue and the underexpression of the rest of these marker genes compared to normal colon tissue are indicative of cancerous colon tissue. By using these 21 marker genes as a diagnostic tool, small tissue samples, such as those obtained by core needle biopsies and from stool samples can be used.

[00006] In a first aspect, the present invention provides a method for determining if colon cells are cancerous, the method comprising:
a) obtaining said colon cells;
b) determining if at least one specific gene is overexpressed or underexpressed in said colon cells compared to an expression of said at least one specific gene in normal colon cells;
c) determining that said colon cells are cancerous based on whether said at least one gene is overexpressed or underexpressed in said colon cells.

[00007] In another aspect, the present invention provides a use of at least one marker gene for identifying cancerous colon tissue, an overexpression or underexpression of said at least one marker gene in colon tissue compared to an expression of said at least one marker gene in normal colon tissue being indicative of cancerous colon tissue.

[00008] Yet another aspect of the invention provides a method of diagnosing colon cancer, the method comprising:
a) obtaining colon tissue to be diagnosed;
b) determining if specific marker genes are overexpressed or underexpressed in said colon tissue to be diagnosed compared to non-cancerous colon tissue;
c) determining if said colon tissue to be diagnosed is cancerous based on an underexpression or overexpression of said specific marker genes.

Brief Description of the Drawings [00009] A better understanding of the invention will be obtained by considering the detailed description below, with reference to the following drawings in which:

[00010] Figure 1 is an expression plot for the 21 genes which is the subject of the present invention;

[00011] Figures 2-20 illustrate box plots of the expression of the 21 genes in both cancerous and non-cancerous tissue; and [00012] Figure 21 is a table which, taken in conjunction with a table in the description, denotes which sample sets were used in which experiments for the box plotted results in Figures 2-20.

Detailed Description [00013] The present invention relates to the use of a panel of 21 specific marker genes to diagnose or detect cancerous colon tissue. The panel of 21 marker genes is listed in Table 1 below. Experiments have shown that this panel of marker genes give high accuracy in colon cancer diagnosis due to the expression levels of the marker genes in cancer tissue relative to their expression levels in normal tissue.

[00014] The panel of 21 marker genes is given in Table 1. The marker genes were determined from two different microarray data sets. The first 14 genes were found to give 100% of correct classification for the data set described by Notterman DA, et al. ((2001) Transcriptional Gene Expression Profiles of Colorectal Adenoma, Adenocarcinoma and Normal Tissue Examined by Oligonucleotide Arrays. Cancer Res. 61:3124-3130). The rest of the genes in the panel were selected from the data set published by Alon, U. et al. ((1999) Broad Patterns of Gene Expression Revealed by Clustering Analysis of Tumour and Normal Colon Tissue Probed by Oligonucleotide Arrays. Proc. Natl. Acad. Sci. 96: 6745-6750).

[00015] The data set from Alon, et al. consisted of 40 tumour and 22 normal samples for a total of 66 samples. Samples were obtained from colon adenocarcinoma specimens snap-frozen in liquid nitrogen within 20 min of removal/collection from patients. From some of these patients paired normal colon tissue also was obtained. The microarrays were hybridized using Affymetrix Hum600 array using standard protocol. The 2,000 highest intensity genes were selected and published on the web at http://microarray.princeton.edu/oncology/. From this subset were selected seven diagnostic genes that give 100 % of correct classification (the last 6 genes in Table 1). The dataset from Alon et al. is limited in size and therefore biomarker selection was performed on another data set also found in the Notterman et al. paper. In this data set, samples of colon adenocarcinoma and paired normal tissue from the same patient were obtained from the Cooperative Human Tissue Network. The tissue was snap-frozen in liquid nitrogen within 20-30 min of harvesting and stored thereafter at -80 C. mRNA was extracted from the bulk tissue samples and hybridized to the array using standard procedure (see Notterman et al., 2001). This data set was also cited by Rhodes et al. in 2004 (see Rhodes, D.R.
et al. (2004) Large-scale Meta-Analysis of Cancer Microarray Data Identifies Common Transcriptional Profiles of Neoplastic Transformation and Progression. Proc.
Natl.
Acad. Sci. 101: 9309). The adenocarcinoma samples were specifically re-reviewed by a pathologist at the institution where the samples were obtained using paraffin-embedded tissue that was adjacent or in close proximity to the frozen sample from which the RNA was extracted. The publicly available data set consists of 18 adenocarcinoma and 18 normal samples. The set consists of -6600 genes. The 14 genes that give 100% accurate diagnosis of adenocarcinomas and normal colon tissue were selected using another method.

Table 1- Panel of 21 genes found to give high accuracy in colon cancer diagnosis and their expression level in cancer relative to normal tissue.

Over or Under-GeneBank Gene Name Symbol expressed in cancer Accession tissue relative to Number normal tissue Pyrroline-5-carboxylate PYCR1 Overexpressed M77836 reductase 1 General transcription X63468 factor IIE, polypeptide 1, GTF2E1 Over-expressed alpha 56kDa Transcribed locus, NME1 H20426 strongly similar to Over-expressed NP 937818.1 nucleoside-diphosphate kinase 1 isoform a [Homo sapiens]

Eukaryotic translation EIF1AX Over-expressed L18960 initiation factor 1A, X-linked U30872 Centromere protein F, CENPF Over-expressed 350/400ka (mitosin) Amphiphysin (Stiff-Man AMPH Over-expressed syndrome with breast X81438 cancer 128kDa autoantigen) H67367 RAN binding protein 1 RANBP1 Over-expressed D13645 KIAA0020 KIAA0020 Over-expressed Membrane cofactor R33367 protein (CD46, MCP Over-expressed trophoblast-lymphocyte cross-reactive antigen) similar to Homo sapiens similar to T94834 acidic (leucine-rich) ANP32B Over-expressed nuclear phosphoprotein 32 family, member B.
L20852 Solute carrier family 20 (phosphate transporter), SLC20A2 Under-expressed member 2 R39540 TU3A protein TU3A Under-expressed H86039 Adenylate kinase 1 AK1 Under-expressed T78477 Zinc finger protein 297 ZNF297 Under-expressed T84082 ER Lumen Protein KDELR1 Under-expressed Retaining Receptor 1 X05610 Human mRNA for type IV COL4A2 Under-expressed collagen alpha (2) chain R39130 S27965 Hypothetical LOC51035 Under-expressed protein Src homology 2 domain T49397 contating transforming SHC Under-expressed protein 1 Peripheral myelin protein PMP22 Under-expressed L42611 Keratin 6 isoform K6e KRT6E Under-expressed mRNA

Collagen, type XIII, Under-expressed alpha 1 COL13A1 [00016] The genes listed above were derived using a microarray gene expression experiment, the gene expression plot being provided as Figure 1 for the 21 genes. For this expression plot, the samples are normal and cancerous tissues.

In the plot, positive expression levels are shown in red while negative expression levels are shown in green. All experiments were normalized (scaled) to have a mean of zero and a standard deviation of one.

[00017] By following the procedure noted above, the expression of the above genes can be determined from sample tissue obtained from a patient. By determining the expression of the above noted genes in the sample tissue, the presence or absence of cancerous colon tissue may be determined.

[00018] It should be noted that the procedure for determining the expression of genes in tissue is well-known in the art. Furthermore, procedures for the extraction and collection of tissue, in this case colon tissue, are also well-known. As noted above, colon tissue samples may be obtained from patient stool samples or core needle biopsies. These tissue samples may then be tested for the expression of the above genes and then compared to the expression of the above genes in tissue samples known to be non-cancerous. If the first 10 genes listed above are overexpressed in the patient sample tissue relative to their expression levels in normal tissue, and if the next 11 genes listed above are underexpressed in the patient sample tissue relative to their expression levels in normal tissue, then this would indicate the presence of cancerous colon tissue in the patient sample tissue.

[00019] It should be noted that expression analysis can be carried out using any method for measuring gene expression. Such methods as microarrays, diagnostic panel mini-chip, PCR, real-time PCR, and other similar methods may be used.
Similarly, methods for measuring protein expression may also be used.

[00020] As noted above, the cancerous colon cells can be obtained from a patient using minimally invasive core needle biopsy or from techniques such as from a patient's stool samples. Normal or non-cancerous colon cells against which the cancerous cells can be compared can also be obtained from the patient or from other patients. Experiments have shown that the diagnosis can be possible from just a small number of cancer cells.

[00021] Referring to Figures 2 - 20, boxplots of test results for the above noted genes are illustrated. The boxplots illustrate that, for each particular gene, that gene is either underexpressed or overexpressed in cancerous tissue relative to normal tissue. The tissue samples which were used for the experiments were those used and referred to in the following publications as set out in the table below :

Sample Publication Sample Sample type Set subset A Notterman DA, Alon U, Sierk AJ, 1 Normal tissue Levine AJ. Transcriptional gene expression profiles of colorectal adenoma, adenocarcinoma, and normal tissue examined by 2 Adenocarcionoma oligonucleotide arrays. Cancer tissue Res. 2001 Apr 1;61(7):3124-30 B Zou TT, Selaru FM, Xu Y, 1 normal colonic Shustova V, Yin J, Mori Y, epithelium Shibata D, Sato F, Wang S, Olaru A, Deacu E, Liu TC, Abraham JM, Meltzer SJ. Application of cDNA
microarrays to generate a molecular taxonomy capable of 2 colon carcinoma distinguishing between colon cancer and normal colon.
Oncogene. 2002 Jul 18;21(31):4855-62.

C Notterman DA, Alon U, Sierk AJ, 1 Duke Stage A
Levine AJ. Transcriptional gene expression profiles of colorectal 2 Duke Stage B
adenoma, adenocarcinoma, and normal tissue examined by 3 Duke Stage C
oligonucleotide arrays. Cancer Res. 2001 Apr 1;61(7):3124-30 4 Duke Stage D

D Notterman DA, Alon U, Sierk AJ, 1 Stage A(1) Levine AJ. Transcriptional gene expression profiles of colorectal 2 Stage B(7) adenoma, adenocarcinoma, and normal tissue examined by oligonucleotide arrays. Cancer 3 Stage C(5) Res. 2001 Apr 1;61(7):3124-30.
4 Stage D(5) E Notterman DA, Alon U, Sierk AJ, 1 p53 mutation negative Levine AJ. Transcriptional gene expression profiles of colorectal adenoma, adenocarcinoma, and normal tissue examined by 2 p53 mutation positive oligonucleotide arrays. Cancer Res. 2001 Apr 1;61(7):3124-30.

F Shyamsundar R, Kim YH, Higgins 1 Multitissue JP, Montgomery K, Jorden M, Sethuraman A, van de Rijn M, Botstein D, Brown PO, Pollack JR.
A DNA microarray survey of gene expression in normal human 2 Colon Normal tissues. Genome Biol.
2005;6(3):R22. Epub 2005 Feb 14 G Notterman DA, Alon U, Sierk AJ, 1 Female Levine AJ. Transcriptional gene expression profiles of colorectal adenoma, adenocarcinoma, and normal tissue examined by 2 Male oligonucleotide arrays. Cancer Res. 2001 Apr 1;61(7):3124-30.

H Ramaswamy S, Tamayo P, Rifkin 1 Cancer progression R, Mukherjee S, Yeang CH, normal Angelo M, Ladd C, Reich M, Latulippe E, Mesirov JP, Poggio T, Gerald W, Loda M, Lander ES, Golub TR. Multiclass cancer 2 cancer progression diagnosis using tumor gene primary expression signatures. Proc Natl AcadSci U S A. 2001 Dec 18;98 Su AI, Welsh JB, Sapinoso LM, 1 multi-tissue cancer Kern SG, Dimitrov P, Lapp H, Schultz PG, Powell SM, Moskaluk CA, Frierson HF Jr, Hampton GM.
Molecular classification of human carcinomas by use of gene 2 colorectal expression signatures. Cancer Res. adenocarcinoma 2001 Oct 15;61(20):7388-93.

J Ramaswamy S, Tamayo P, Rifkin 1 R, Mukherjee S, Yeang CH, Multi-tissue cancer Angelo M, Ladd C, Reich M, Latulippe E, Mesirov JP, Poggio T, Gerald W, Loda M, Lander ES, Golub TR. Multiclass cancer 2 colorectal diagnosis using tumor gene adenocarcinoma expression signatures. Proc Natl Acad Sci U S A. 2001 Dec 18;98 K 1 primary Ramaswamy S, Tamayo P, Rifkin R, Mukherj ee S, Yeang CH, Angelo M, Ladd C, Reich M, Latulippe E, Mesirov JP, Poggio T, Gerald W, Loda M, Lander ES, Golub TR. Multiclass cancer 2 metastatic diagnosis using tumor gene expression signatures. Proc Natl AcadSci U S A. 2001 Dec 18;98 = ~

L Ramaswamy S, Tamayo P, Rifkin 1 R, Mukherj ee S, Yeang CH, Angelo M, Ladd C, Reich M, Primary Latulippe E, Mesirov JP, Poggio T, Gerald W, Loda M, Lander ES, Golub TR. Multiclass cancer 2 metastatic diagnosis using tumor gene expression signatures. Proc Natl AcadSci U S A. 2001 Dec 18;98 M Alon U, Barkai N, Notterman DA, 1 Gish K, Ybarra S, Mack D, Levine normal colon AJ. Broad patterns of gene expression revealed by clustering analysis of tumor and normal colon tissues probed by oligonucleotide 2 arrays. Proc Natl Acad Sci U S A. colon adenocarcinoma 1999 Jun 8;96 N Ramaswamy S, Tamayo P, Rifkin 1 R, Mukherjee S, Yeang CH, multi-tissue normal Angelo M, Ladd C, Reich M, Latulippe E, Mesirov JP, Poggio T, Gerald W, Loda M, Lander ES, Golub TR. Multiclass cancer 2 diagnosis using tumor gene expression signatures. Proc Natl Colon normal Acad Sci U S A. 2001 Dec 18;98 [00022] For the experiments for which the results are in the boxplots of Figures 2-20, the genes tested and the sample sets used are as noted in Figure 21. The second row in the table of Figure 21 notes the symbol of the gene being tested while the first column denotes the experiment number. The intersection between the gene symbol and the experiment number shows the sample set used for that experiment.
The experiment number corresponds to the bottom row of the box plot for that gene.

As an example, for the gene denoted by symbol AK1, the boxplot of which is in Figure 13, experiment 1 used sample set A noted above. Since sample set A has two sample subsets, then there are two sub-columns for the first column in the box plot of Fig 13. The first sub-column shows the expression level for the gene AK1 in normal tissue (as noted in the table above) while the second sub-column for this experiment is the expression level for the gene AK1 in adenocarcionoma tissue (again as noted above for sample set A).

[00023] As another example, experiment 7 for the gene PYCR1 used the sample set C with four subsample sets (see Fig 2) which tested the expression level of PYCR1 in tissues at various Duke stages.

[00024] The correspondence between the test results in the figures and the genes being tested are as follows :

Gene Figure containing Symbol box plot results PYCR1 Figure 2 GTF2E1 Figure 3 Fi ure4 EIFIAX Figure 5 CENPF Figure 6 AMPH Figure 7 RANBP1 Figure 8 KIAA0020 Figure 9 MCP Figure 10 SLC20A2 Figure 11 TU3A Figure 12 AK1 Figure 13 ZNF297 Figure 14 COL4A2 Figure 15 LOC51035 Figure 16 SHC Figure 17 PMP22 Figure 18 KRT6E Figure 19 COL13A1 Figure 20 [00025] It should be noted that the underexpression or the overexpression of the above noted genes in cancerous tissue relative to their expression in normal tissue is readily evident in the box plots. Specifically, the experiments which used the samples sets A, B, M, and N compare the expression levels of specific genes in both cancerous and non-cancerous tissue in a side-by-side manner. For the genes which were not tested for sample sets A, B, M, and N, their expression levels for sample set F (normal tissue) may be compared with their expression levels for sample sets H and I (cancerous tissue). For the genes for which sample set E
was used, the presence of p53 mutation indicates cancerous tissue, sample subset 2 for this sample set being cancerous tissue.

[00026] While it is preferable that the complete panel of 21 marker genes be used in the diagnosis of possible colon cancer, using a subset of the 21 marker genes will also yield useful results. Using a panel of anywhere from 1 to 21 marker genes out of the 21 marker genes on suspect colon tissue will still provide a useful indication as to whether cancerous colon tissue may be present or whether further and more involved tests are required.

[00027] A person understanding this invention may now conceive of alternative structures and embodiments or variations of the above all of which are intended to fall within the scope of the invention as defined in the claims that follow.

Claims

1. A method for determining if colon cells are cancerous, the method comprising:
a) obtaining said colon cells;
b) determining if at least one specific gene is overexpressed or underexpressed in said colon cells compared to an expression of said at least one specific gene in normal colon cells;
c) determining that said colon cells are cancerous based on whether said at least one gene is overexpressed or underexpressed in said colon cells.

2. A method according to claim 1 wherein said colon cells are obtained by a core needle biopsy.

3. A method according to claim 1 wherein step b) comprises determining if a plurality of specific genes selected from a specific panel of marker genes are overexpressed in said colon cells.

4. A method according to claim 3 wherein step c) comprises determining that said colon cells are cancerous if said plurality of specific genes selected from said specific panel of marker genes are overexpressed in said colon cells.

5. A method according to claim 1 wherein step b) comprises determining if a plurality of specific genes selected from a selected panel of marker genes are underexpressed in said colon cells.

6. A method according to claim 5 wherein step c) comprises determining that said colon cells are cancerous if said plurality of specific genes selected from said specific panel of marker genes are underexpressed in said colon cells.

7. A method according to claim 1 wherein said at least one gene is selected from a group comprising :

8. A method according to claim 3 wherein said specific panel of marker genes comprises:

9. A method according to claim 5 wherein said specific panel of marker genes comprises:

10. Use of at least one marker gene for identifying cancerous colon tissue, an overexpression or underexpression of said at least one marker gene in colon tissue compared to an expression of said at least one marker gene in normal colon tissue being indicative of cancerous colon tissue.

11. A use according to claim 10 wherein an overexpression of said at least one marker gene is indicative of a presence of cancerous colon tissue, said at least one marker gene being selected from a group comprising:

12. A use according to claim 10 wherein an underexpression of said at least one marker gene is indicative of a presence of cancerous colon tissue, said at least one marker gene being selected from a group comprising:

13. A method of diagnosing colon cancer, the method comprising:
a) obtaining colon tissue to be diagnosed;
b) determining if specific marker genes are overexpressed or underexpressed in said colon tissue to be diagnosed compared to non-cancerous colon tissue;
c) determining if said colon tissue to be diagnosed is cancerous based on an underexpression or overexpression of said specific marker genes.

14. A method according to claim 13 wherein said colon tissue is obtained by a core needle biopsy.

15. A method according to claim 13 wherein said specific marker genes are selected from a group comprising:

16. A method according to claim 15 wherein step b) comprises determining if a subset of said marker genes are overexpressed in said colon tissue to be diagnosed, the subset comprising:

17. A method according to claim 15 wherein step b) comprises determining if a subset of said marker genes are underexpressed in said colon tissue, the subset comprising:

18. A method according to claim 1 wherein said colon cells are obtained from at least one stool sample.

19. A method according to claim 13 wherein said colon tissue are obtained from at least one stool sample.