Mapping of DNA amplifications at 15 chromosomal localizations in 1875 breast tumors: definition of phenotypic groups.

DNA amplification is frequent in breast cancer and has been associated with specific clinicopathological parameters and/or worsened course of the disease. In the present work, we were interested in further defining the association linking the occurrence of DNA amplification to the emergence of specific breast tumor phenotype. To this aim, we studied by Southern blotting a total of 1875 breast tumor DNAs with 26 probes mapping at 15 distinct chromosomal localizations. Of the 26 loci tested, 11 loci showed elevated levels of amplification, 9 loci showed occasional and/or low level of DNA copy number increase, and 6 loci showed very rare or no variation. This allowed us to define six amplified domains mapping at 8p12, 8q24, 11q13, 12q13, 17q12, and 20q13.2, respectively. Over 60% of the tumors analyzed presented at least one amplification at one of these localizations. Amplifications often covered large regions of DNA and bore complex patterns involving coamplification of several colocalized markers. Statistical analysis revealed correlations associating DNA amplification with breast tumor phenotype, as well as sets of preferential coamplifications. Based on these correlations, we defined three subsets of breast cancer according to their patterns of DNA amplification. The first subset (group A) was organized around the amplifications at 11q13 and/or 8p12 and was predominantly composed of estrogen receptor-positive tumors and presented a large proportion of lobular cancers. The second subset (group B) was organized around the amplifications of ERBB2 and/or MYC. These tumors were mostly estrogen receptor-negative and of the ductal invasive type. The third subset (group C) corresponded to tumors in which no amplification was detected in the present screen. Tumors in this group were largely diploid and of low histopathological grading.

DNA amplifications at 20q13 and MDM2 define distinct subsets of evolved breast and ovarian tumours.

DNA amplification seems to be particularly frequent in human breast tumours and has been associated with cancer evolution and aggressiveness. Recent data indicate that new events should be added to the list, such as the amplifications at chromosome 20q13 or the MDM2 gene. The present work aimed at determining the incidence and clinicopathological signification of these amplifications in a large series of breast and ovarian tumours. We tested 1371 breast and 179 ovarian tumours by Southern blotting and observed amplification of 20q13 in 5.4% breast and 2.8% ovarian carcinomas, whereas MDM2 was found amplified in 5.3% and 3.8% of breast and ovarian tumours respectively. MDM2 RNA expression levels were analysed in a subset of 57 breast tumours and overexpression was observed in 4/57 (7%) of the tumours. Elevated expression levels coincided with amplification of the gene. In breast cancer, 20q13 and MDM2 amplifications seem to define subsets of aggressive tumours. Indeed, 20q13 was correlated to axillary nodal involvement and occurred preferentially in younger patients (< 50 years). Furthermore, 20q13 correlated, as did MDM2 amplification, to aneuploidy. In parallel, we had also tested our tumour DNAs for amplification of CCND1, ERBB-2 and MYC, which made it possible to test for correlations with 20q13 or MDM2 amplifications. Whereas 20q13 showed a very strong correlation to CCND1 amplification, that of MDM2 was prevalent in MYC-amplified tumours. Interestingly, 20q13 and MDM2 amplifications showed some degree of correlation to each other, which may possibly be owing to the fact that both events occurred preferentially in aneuploid tumours. In ovarian cancer, no statistically significant correlation was observed. However, 20q13 amplification occurred preferentially in stage 3 tumours and MDM2 was correlated to ERBB-2 amplification. This may suggest that in ovarian tumours also, 20q13 and MDM2 amplifications occur in late or aggressive cancers.

Cyclin gene amplification and overexpression in breast and ovarian cancers: evidence for the selection of cyclin D1 in breast and cyclin E in ovarian tumors.

Evidence of the involvement of cyclin genes in genetic alterations in human cancer is growing. In the present study, we investigated the amplification, in human breast and ovarian cancer, of 5 cyclin genes; cyclin A, cyclin D1, cyclin D2, cyclin D3 and cyclin E. For this purpose, a series of 1,171 breast and 237 ovarian tumors tested for DNA amplification by Southern blotting and a subset of 132 breast and 22 ovarian cancers were analyzed for RNA expression levels by slot-blot and Northern blotting. In breast tumors, only cyclin D1 was found to be activated in a sizeable fraction of the tumors (amplification 12.6%, overexpression 19%). Cyclin A, D2, D3, and E genes never, or only on rare occasions, showed increased DNA copy numbers and were never found overexpressed at the RNA level. Amplification of cyclin D1 correlated with ER+ breast cancer and the presence of lymph-node metastasis. Interestingly, we were also able to determine an association with invasive lobular carcinoma. Our data suggest that cyclin D1 activation determines the evolution of a particular subset of estrogen-responsive tumors. Data obtained in ovarian tumors contrasted with observations in breast cancer. Cyclin D1 DNA amplification was much less frequent in ovarian than in breast tumors (3.3% vs. 12.6%), whereas cyclin E amplification and overexpression were observed in a significant number of cases (12.5% and 18.0% respectively). Cyclin A, cyclin D2 and D3 rarely showed anomalies at the DNA level and were never overexpressed. No clear correlation could be observed between amplification of the cyclin E gene and tumor type, stage or grade in ovarian cancer. Data presented here suggest distinct pathways of cyclin activation in human breast and ovarian cancer.

Concurrent abnormal expression of erbB-2, myc and ras genes is associated with poor outcome of ovarian cancer patients.

Epithelial ovarian cancer probably occurs due to activation of several different combinations of genes, which produce cancers that vary biologically and clinically. We tested this hypothesis in 100 consecutive ovarian carcinomas by molecular biology techniques at the DNA and protein levels in three genes (erbB-2, myc, ras), which are frequently altered in this tumor system. Abnormally high expression of erbB-2 gene encoded p185 protein was observed in 31% of the samples, while erbB-2 gene amplification was detected by Southern analysis in 8%. ErbB-2 abnormal gene expression did not significantly affect the clinical outcome of patients, conferring a marginal worsening of survival. In 25 out of 96 (26%) tumor samples there was myc amplification. Higher levels of the ras-encoded p21 protein than in normal ovaries and benign ovarian tumors were found in 45% of the samples. Simultaneous overexpression of p185 and p21 was associated with shorter disease free (p = 0.02) and overall survival (p = 0.04) at significance levels notably higher than those observed for these oncoproteins singly. In addition, survival of patients with myc amplification and high p185/p21 coexpression was significantly worse (p < 0.05) than that of patients with normal levels. Our data suggest that concurrent abnormal gene expression may act synergistically to endow ovarian tumor cells with a highly aggressive phenotype. Evaluation of these genes may be helpful in the biological characterization of ovarian cancer and in defining individual patient prognosis.

Quantification of ERBB2 protein expression in breast cancer: three levels of expression defined by their clinico-pathological correlations.

Activation of the ERBB2 oncogene seems to be an early event in breast cancer progression and prevalent in in situ carcinomas. However, its prognosis value, albeit recognized for node-positive patients, remains controversial for those without apparent nodal involvement. One possible reason for this problem is likely to be the difficulty of defining threshold levels for ERBB2 protein overexpression. ERBB2 protein expression was therefore analyzed in primary invasive breast tumors. Quantification of the gene product by a commercial ELISA test was compared to results obtained by immunohistochemistry and western blotting, as well as to gene amplification status determined by Southern blotting. Correlations between results obtained by the different techniques were highly significant (P value < 10(-6)). Nevertheless, ELISA permitted us to determine three levels of protein expression corresponding to distinct tumor subsets. 1) Tumors with p185/ERBB2 expression levels exceeding 10 U/microgram exhibited in most cases amplification of the gene (83% of cases), DNA aneuploidy (81%) and absence of estrogen receptor (ER) (44%). Such high levels of protein expression were exclusively observed in invasive ductal carcinomas and were prevalent in those showing a significant in situ component. 2) "Intermediate" levels of expression (3-10 U/micrograms) were rarely observed in tumors exhibiting gene amplification (9%), but were preferentially found in cancers of more favorable prognosis (only 49% were aneuploid and 9% estrogen receptor negative). 3) Levels of p185/ERBB2 below 3 U/micrograms were detected in benign mastopathies and, thus, carcinomas presenting such levels were scored ERBB2 negative. Interestingly, invasive lobular carcinomas were rarely ERBB2 positive, and if so, only at intermediate levels. Moreover, our data show a complex interrelationship between p185/ERBB2 expression and ER levels. Indeed, tumors with more than 10 U/micrograms of p185 were prevalently ER, whereas those with p185 ranging from 3 to 10 U presented elevated levels of ER.

Patterns of allele losses suggest the existence of five distinct regions of LOH on chromosome 17 in breast cancer.

Chromosome 17 is a frequent target during breast-cancer formation and progression. It has been shown to be affected by allele losses at multiple sites, as well as by DNA amplification. Our aim was to delineate a map of the genetic alterations on chromosome 17 in a given set of breast tumors. To this end we analyzed 151 pairs of tumor and cognate lymphocyte DNAs by Southern blotting with 5 RFLP or VNTR probes and by PCR at 8 CA repeat polymorphic loci for LOHs. Moreover, we studied DNA amplification of the evi2, erbB2, thraI, gcsf and rara genes. Data presented here point strongly to the existence of 5 distinct regions of allele losses on chromosome 17:2 on 17p, 3 on 17q. Of the 2 regions on 17p, one involves tp53 while the second is located more distally toward the telomere. LOH was found in 45.9% and 58.8% respectively. The 3 regions on 17q are located: (i) on the proximal portion of the long arm band q21, corresponding to the brcaI region; (ii) in a central region defined by the marker D17S74; (iii) on the distal part of 17q (band q25) characterized by losses of the marker D17S24. Each of these regions presented respectively allele losses in 47.5%, 33.3% and 40.8% of the informative tumors. Whereas some tumors presented patterns of LOH consistent with the loss of a complete chromosomal arm or of large portions of the chromosome, a high proportion of the analyzed tumors showed interstitial losses. Amplifications were found in 15% of the tumors and were centered around erbB2.(ABSTRACT TRUNCATED AT 250 WORDS)

FGFRI and PLAT genes and DNA amplification at 8p12 in breast and ovarian cancers.

Several chromosomal regions are found to be consistently amplified in human breast cancers. For two of these regions, 8p12 and 10q26, we previously reported the amplification of genes encoding FGF receptors, FGFRI/FLG and FGFR2/BEK, in about 12% of breast tumors. The PLAT gene, encoding the tissue-type plasminogen activator, is also located close to or within the 8p12 region. In the present study, we show that both FGFRI and PLAT can be amplified in breast as well as ovarian carcinomas. FGFRI amplification was detected in 14.5% of breast and 7.8% of ovarian tumors, whereas PLAT was found to be amplified in 15.6% and 19.4% of the tumors, respectively. Each gene could be amplified independently of the other. These data raised the question of which gene is selected for amplification at 8p12. In most cases, the levels of expression of FGFRI and PLAT in breast tumors were comparable to their level of expression in normal mammary tissue. However, FGFRI was expressed above the normal level in a certain number of cases. This gene could be a good candidate as "driver" of the 8p12 amplification, but it cannot account for all complex molecular events taking place in this region.

An attempt to define sets of cooperating genetic alterations in human breast cancer.

The extent and the variation of losses of genetic material were examined in a series of 191 human breast cancers by means of a set of 18 polymorphic DNA probes, specific of 7 chromosomal arms (1p, 1q, 3p, 11p, 13q, 17p and 18q) known to be frequently affected by allele losses. Frequencies of losses of heterozygosity ranged from a low of 3.5% (chromosome 13q) to a high of 27% (chromosome 3p). The number of sites involved in breast cancer added to the frequent occurrence of concomitant losses at several chromosomal arms within the same tumor suggest cooperative effects of these LOHs. We were therefore interested in assessing the existence of preferential associations between sets of LOHs in our panel of tumors. Statistically significant associations were found between LOHs at chromosomes 1p and 17p, and between LOHs at chromosomes 11p and 17p. Furthermore, since all the tumors presently studied had previously been analyzed for proto-oncogene amplification at 5 distinct chromosomal sites, we tested for associations between LOH and DNA amplification. Such associations were indeed observed as exemplified by the correlations observed between the LOH at 11p and amplification of the erbB2 gene and LOH at 17p and the amplification of the flg gene. The only correlation with clinico-pathological parameters that could be observed linked the occurrence of LOHs on 11p with recurrent breast cancer (p = 0.015). Sets of several LOHs or LOHs and gene amplifications could not be significantly related to any marker of tumor aggressiveness.