Molecular Karyotyping of Breast Cancer by DNA Fingerprinting

Institution: The Burnham Institute for Medical Research
Investigator(s): Sergei Malkhosyan, Ph.D. -
Award Cycle: 1997 (Cycle III) Grant #: 3IB-0130 Award: $290,550
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (1997)
The initiation and progression of human breast tumors involves a wide spectrum of known, suspected, and undiscovered genetic alterations. Thus, it is believed that tumor cells are genetically different from the surrounding normal breast cells. One potential approach to detect, analyze, and enumerate these genetic differences in breast cancer is to use a technique of DNA fingerprinting. We plan to use this technique to provide a comprehensive view of the role of known genetic aberrations in breast tumorigenesis and to facilitate the identification, mapping and eventual isolation of novel cancer genes. Our specific detection method is termed ‘Arbitrarily Primed PCR’ (AP-PCR). This is an unbiased method, which generates a profile of quantitative differences between the tumor and corresponding (from the same patient) normal tissues. We call it quantitative allelotype or ‘amplotype’. This ‘amplotype’ reflects tumor-specific changes of different DNA segments randomly distributed throughout the genome, and the chromosomal origin of the fingerprint bands can be easily determined. We have previously used this method to analyze colon cancer, where progression is closely associated with specific genetic defects.

Our experimental approach features three specific aims. First, about 100 human breast tumors with corresponding normal tissues will be analyzed by AP-PCR fingerprinting, which is anticipated to yield a ‘fingerprint’ of about 500 different DNA pieces (bands). Each difference between the matched tumor and normal sample will be linked to a chromosomal site and will be scored either as an allelic (genetic) loss, allelic gain, or structural change. Secondly, the extent (genetic damage index) and spectrum of genetic alterations will be determined for each tumor. Thus, we will compare breast tumors from different patients. Our goal is to associate these genetic aberrations with specific biological and clinical parameters. Finally, a sub-chromosomal map of recurrent genetic aberrations will be determined. These maps will then be compared with known genetic regions for breast cancer development. Our goal is to identify novel genetic aberrations for further study. Our experimental approach directly studies samples from breast cancer patients. It should provide a comprehensive view of the role of known genetic aberrations in breast tumorigenesis and facilitate the identification, mapping and eventual isolation of novel cancer genes.


Final Report (1999)
This project aimed to apply technique of Arbitrarily Primed PCR (AP-PCR) for the analysis of genetic alterations occurring in breast cancer. AP-PCR is an unbiased DNA fingerprinting method which generates a profile of quantitative differences between the fingerprints of tumor and corresponding normal tissues (from the same patient). We call it quantitative allelotype or "amplotype." This amplotype reflects quantitative tumor-specific changes of different DNA segments randomly distributed throughout the genome. Because the chromosomal origin of the fingerprint bands can easily be determined, amplotyping therefore represents a molecular karyotyping.

Our strategy was, first, to analyze breast tumors with corresponding normal tissues by AP-PCR fingerprinting with a set of arbitrary primers. The differences between the normal and tumor tissue fingerprints were interpreted as allelic losses, allelic gains, or structural changes. The subchromosomal origin of AP-PCR fingerprint bands which detect frequent genetic alterations was determined. Secondly, we mapped the recurrent genetic alterations by allelotyping with microsatellite repeat markers. Finally, we correlated these genetic alterations with specific biological and clinical tumor parameters.

We carried out all planned experiments and completed the project. AP-PCR fingerprinting identified six major genetic alterations which occur in breast tumors with a frequency from 40% to 50%: gains of chromosomes 4 and 8 and losses of chromosomes 2, 4, 6, and X. The common region of gain on chromosome 8 coincided with the position of the c-myc gene, and thus confirmed a previously known amplification of the c-myc gene in breast tumors. The other genetic alterations were localized to the chromosomal areas which had not been previously described. Thus, these genetic alterations unmask the presence of a novel oncogene on chromosome 4 and novel tumor suppressor genes on chromosomes 2, 4, 6, and X. In more detailed experiments, we found significant correlation between losses of chromosomes 2q and Xq and high-grade tumors, and between losses of chromosome Xq and the presence of axillary lymph node metastases.

Our plan is to clone the putative tumor suppressor gene on the Xq chromosome. This gene seems to be the most interesting target for future experiments, since it may be responsible for breast cancer progression and could be used as a prognostic marker for the development of metastases.

Frequent loss Xq25 on the inactive X chromosome in primary breast carcinomas is associated with tumor grade and axillary lymph node metastasis.
Periodical:Genes Chromosomes Cancer
Index Medicus:
Authors: Piao Z, Malkhosyan SR.
Yr: 2002 Vol: 33 Nbr: 3 Abs: Pg:262-9