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Dr.
Michael Wigler, Dr.
Robert Lucito and their colleagues have
developed a reproducible, high resolution technique for detecting
changes in gene copy number that are associated with the initiation
of breast, ovarian, and pancreatic cancer, or with the progression
of these cancers from a non-invasive to an invasive or metastatic
state. The technique, called Representational Oligonucleotide Microarray
Analysis, or ROMA, combines DNA microarray technology with a method
Dr. Wigler previously invented to simplify the search for differences
between two sets of DNA (Representational Difference Analysis). By
using ROMA, the entire genomes of both normal cells and cancer cells
taken from the same patient can be scanned, and the differences between
the two can be detected. Typical data from this type of analysis
can be viewed at http://roma.cshl.org.
For the ROMA technique, sufficient material can be obtained with
as few as 1,000 to 10,000 cells from tumor tissue, followed by isolation
of the normal and cancer cell DNAs. In the current format, a resolution
of 30kb throughout the entire genome is achieved; higher density
arrays are being developed that will allow even better resolution.
RDA itself has already proven useful in the
discovery of novel tumor suppressors and oncogenes, which are the
subject of ongoing studies. One of the first genes identified by
RDA was PTEN, a tyrosine phosphatase gene. Follow-up studies indicate
that the key effect of PTEN is through regulation of PI3 kinase signaling.
Recently, in collaboration with Masaaki Hamaguchi, a novel gene,
DBC2, was found by RDA to be mutated in breast cancers. Expression
of wild type DBC2 inhibits cell growth in cells that do not normally
express it, but expression of the mutant form does not. This work
confirms preliminary findings that DBC2 is a tumor suppressor gene.
Finally, in collaboration with Scott Powers and his group at Tularik
Inc., KCNK9 has been identified as a gene frequently amplified in
breast cancers, enhancing the oncogenicity of host cells. KCNK9 is
unusual among oncogenes in that it appears to encode an ion channel.
Using ROMA, it is possible to identify chromosomal
amplifications and deletions that are frequently associated with
cancer. Genes that are amplified or deleted in some cancers may be
modified by mutation in others. Analysis of genes identified by ROMA
will include screening within the candidate genes for additional
common lesions to fully assess the contribution of the gene to cancer.
Novel genes identified through this technique are prime targets for
improved methods to diagnose and treat cancer. Furthermore, by generating
a cancer gene profile for each cancer examined, and following the
progression of the cancer and response to treatment, important clinical
correlations should be possible.
A project of this scale requires an enormous
amount of computational support. Tools for analysis of genomic sequences,
design and evaluation of oligonucleotide probes and data interpretation
have been developed in parallel with the ROMA techniques. The analysis
and cataloging of genomic array information, and correlation of these
results with clinical data, will continue to provide analytical challenges.
KEY PUBLICATIONS
RDA: Lisitsyn,
N.A., N.M. Lisitsina, G. Dalbagni, P. Barker, C.A. Sanchez, J. Gnarra,
W.M. Linehan, B.J. Reid, and M.H. Wigler. 1995. Comparative genomic
analysis of tumors: Detection of DNA losses and amplification. Proc.
Natl. Acad. Sci. 92: 151–155. abstract
PTEN: Li, J., C. Yen,
D. Liaw, K. Podsypanina, S. Bose, S.I. Wang, J. Puc, C. Miliaresis,
L. Rodgers, R. McCombie, S.H. Bigner, B.C. Giovanella, M. Ittmann,
B. Tycko, H. Hibshoosh, M.H. Wigler, and R. Parsons. 1997. PTEN,
a putative protein tyrosine phosphatase gene mutated in human brain,
breast, and prostate cancer. Science 275: 1943–1947. abstract
DBC2. Hamaguchi, M.,
J.L. Meth, C.von Klitzing, W. Wei, D. Esposito, L. Rodgers, T. Walsh,
P. Welcsh, M.C. King, and M.H. Wigler. 2002. DBC2, a candidate for
a tumor suppressor gene involved in breast cancer. Proc. Natl. Acad.Sci.
99: 13647-13652. abstract
KCNK9. Mu D, Chen L,
Zhang X, See LH, Koch CM, Yen C, Tong JJ, Spiegel L, Nguyen KC, Servoss
A, Peng Y, Pei L, Marks JR, Lowe S, Hoey T, Jan LY, McCombie WR,
Wigler MH, Powers S. (2003) Genomic amplification and oncogenic properties
of the KCNK9 potassium channel gene. Cancer Cell 3(3):297-302. abstract
ROMA. Lucito, R, West,
J., Reiner, A., Alexander, J., Esposito, D., Mishra, B., Powers,
S., Norton, L, and Wigler, M. (2000). Detecting Gene Copy Number
Fluctuations in Tumor Cells by Microarray Analysis of Genomic Representations.
Genome Research 10: 1726-1736. abstract |
