Methods of screening for cell proliferation or neoplastic disorders
The invention relates to methods and compositions for identifying subjects having, or predisposed to having, a neoplastic or cell proliferation or neoplastic disorder. The methods are applicable to any type of tissue sample and can be conducted on otherwise normal tissue.
1. A method of determining predisposition of a subject to developing colorectal cancer comprising:
a) detecting at least one biomarker in undifferentiated cells and differentiated cells of a colorectal tissue sample from the subject, the at least one biomarker comprising Musashi 1 (Msi1);
b) calculating a ratio of undifferentiated to differentiated cells in the sample from the subject by identifying and quantitating via computerized analysis the at least one biomarker;
c) performing statistical analysis via a computerized algorithm to compare the ratio of undifferentiated to differentiated cells to a normal reference ratio;
d) determining a predisposition of the subject to develop colorectal cancer by detecting an increase in the ratio of undifferentiated to differentiated cells, as compared to the noimal reference ratio, the increase being indicative of a predisposition for developing colorectal cancer;
e) storing the comparison on a computer readable medium; and
f) isolating a cell displaying abnormal expression of a target gene that directly or indirectly results from loss of imprinting and analyzing methylation status of the target gene, wherein analyzing comprises a bisulfite treatment of the target gene.
2. The method of claim 1 , wherein the target gene is selected from H19 or IGF2.
3. The method of claim 1 , wherein the target gene is selected from the group consisting of Igf1R, IRS-1, IRS-2, PI3K, Akt, p70S6 kinase, FOXO, GSK3, MDM2, mTOR, Cyclin D1, c-Myc, Shc, Grb2, SOS, Ras, Raf, MEK, Erk, and MAPK gene.
4. The method of claim 1 , wherein the change in methylation is hypomethylation.
5. The method of claim 4 , wherein the method comprises analyzing the Biological sample for hypomethylation of both a DMR of the H19 gene and a DMR of the IGF2 gene.
6. The method of claim 2 , wherein the reference ratio is generated from tissue obtained from a subject comprising cells displaying normal imprinting of at least one of the H19 gene and the IGF2 gene.
7. The method of claim 1 , wherein the at least one biomarker further comprises Shh (Sonic hedgehog), Tcf4, Lef1, Twist, EphB2, EphB3, Hes1, Notch1, Hoxa9, Dkk1, Tle6, Tcf3, Bmi1, Kit, Cdx1, Hes5, Oct4, Ki-67, β-catenin, Noggin, BMP4, PTEN (phosphorylated PTEN), Akt (phosphorylated Akt), Villin, Aminopeptidase N (anpep), Sucrase isomaltase (SI), Ephrin-B1 (EfnB1), Cdx2, Crip, Apoa1, Aldh1b1, Calb3, Dgat1, Dgat2, Clu, Hephaestin, Gas1, Ihh (Indian hedgehog), Intrinsic factor B12 receptor, IFABP, and KLF4 or combination thereof.
8. The method of claim 1 , wherein determining the ratio of undifferentiated to differentiated cells in the sample comprises:
a) imaging the sample using immunohistochemical identification of the at least one biomarker;
b) imaging the sample using standard microscopy and distinguishing differentiated from undifferentiated cells using morphologic measurements;
c) imaging the sample using immunohistochemical identification of proliferation antigens and their distribution within colonic crypts;
d) imaging the sample using immunoflourescent identification of molecules specific to the at least one biomarker;
e) measuring RNA levels;
f) measuring gene expression;
g) whole genome expression analyses; or
h) allele specific expression.
9. The method of claim 1 , wherein the cells are epithelial cells.
10. The method of claim 9 , wherein the epithelial cells are obtained from a rectal Pap test.
11. The method of claim 9 , wherein the epithelial cells are obtained from intestinal tissue.
12. The method of claim 11 , wherein the intestinal tissue is obtained from the colon.
13. The method of claim 11 , wherein the epithelial cells are obtained from the lumen of the intestinal tissue.
14. The method of claim 13 , wherein the epithelial cells are obtained from the crypts of the lumen.
15. The method of claim 1 , wherein the subject is not previously known to have a colorectal neoplasm.
16. The method of claim 1 , further comprising correlating the ratio derived from the subject with the subject's family genetic history.
17. The method of claim 1 , wherein the subject is subjected to additional tests selected from the group consisting of chest X-rays, colorectal examinations, endoscopic examination, MRI, CAT scanning, gallium scanning, and barium imaging.
18. The method of claim 1 , wherein the subject is a human.