Note: Descriptions are shown in the official language in which they were submitted.
CA 02557134 2006-08-23
42137-0029
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
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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
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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
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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
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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)
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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
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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
T94350 22
L42611 Keratin 6 isoform K6e KRT6E Under-expressed
mRNA
M33653
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.
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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
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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
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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.
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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
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= ~
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.
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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
NME1
Fi ure4
EIFIAX Figure 5
CENPF Figure 6
AMPH Figure 7
RANBP1 Figure 8
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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
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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.
