Patent Application
20080050723
A better understanding of the invention will be obtained by considering the detailed description below, with reference to the following drawings in which:
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.
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 D A, 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).
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. NatI. 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.
The genes listed above were derived using a microarray gene expression experiment, the gene expression plot being provided as
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.
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.
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.
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.
Referring to
For the experiments for which the results are in the boxplots of
As another example, experiment 7 for the gene PYCR1 used the sample set C with four subsample sets (see
The correspondence between the test results in the figures and the genes being tested are as follows:
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.
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.
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.
