High-resolution ROMA CGH and FISH analysis of aneuploid and diploid breast tumors.

Combining representational oligonucleotide microarray analysis (ROMA) of tumor DNA with fluorescence in situ hybridization (FISH) of individual tumor cells provides the opportunity to detect and validate a wide range of amplifications, deletions, and rearrangements directly in frozen tumor samples. We have used these combined techniques to examine 101 aneuploid and diploid breast tumors for which long-term follow-up and detailed clinical information were available. We have determined that ROMA provides accurate and sensitive detection of duplications, amplifications, and deletions and yields defined boundaries for these events with a resolution of <50 kbp in most cases. We find that diploid tumors exhibit fewer rearrangements on average than aneuploids, but rearrangements occur at the same locations in both types. Diploid tumors reflect at least three consistent patterns of rearrangement. The reproducibility and frequency of these events, especially in very early stage tumors, provide insight into the earliest chromosomal events in breast cancer. We have also identified correlations between certain sets of rearrangement events and clinically relevant parameters such as long-term survival. These correlations may enable novel prognostic indicators for breast and other cancers as more samples are analyzed.

Frequent co-amplification of two different regions on 17q in aneuploid breast carcinomas.

Chromosome 17q is highly susceptible to rearrangement mutations in breast cancer. c-erbB-2 at 17q11.2 approximately q21.1 is frequently amplified, as is a region at 17q22 approximately q24. As a step in the search for the target gene(s) of the 17q22-q24 amplification we determined whether the placental lactogen (PL) genes at 17q23 were amplified in 59 breast carcinomas. These genes were selected as their upregulation could theoretically be involved in breast cancer tumorigenesis. Amplification of the PL genes, and also of c-erbB-2, was detected using semi-quantitative PCR. The reliability of this method was confirmed since c-erbB-2 results obtained using PCR, Southern blotting and immunohistochemistry were in good agreement. The PL genes were amplified in 13 (22%) of the tumors. Furthermore, the PL and c-erbB-2 genes were frequently co-amplified although there is a non-amplified region between them. Expression of PL was investigated in 26 tumors and was detected in 16 of these cases including all 10 tumors with amplification of the PL genes. The tumors with PL gene amplification were all aneuploid. A trend was seen towards an increased incidence of lymph node involvement for tumors with amplification of the PL genes and for tumors with co-amplification of PL and c-erbB-2, which suggests a possible association with high malignancy.

Relationship between oncogene amplification, aneuploidy and altered expression of p53 in breast cancer.

Many studies have noted an association between amplification of single oncogenes and poor prognosis in breast cancer. We are investigating whether measurement of amplification in a larger number of proto-oncogenes increases the reliability of the prognostic information provided. As the first stage of this investigation, amplification (of c-erbB-2, cycD1, int-2, c-myc and MDM2), aneuploidy and altered expression of p53, which all indicate genetic instability, were studied in 117 primary breast adenocarcinomas. Amplification was correlated with aneuploidy (p=0.002) but not with altered expression of p53 even though the tumours with p53 overexpression were all aneuploid. Our results suggest that measurement of amplification is a potentially valuable prognostic factor.