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 DI, cyclin D2, cyclin 0 3 and cyclin E. For this purpose, a series of I, I 7 I breast and 237 ovarian tumors tested for DNA amplification by Southern blotting and a subset of I32 breast and 22 ovarian cancers were analyzed for RNA expression levels by slot-blot and Northern blotting. In breast tumors, only cyclin DI 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 DI 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 DI activation determines the evolution of a particular subset of estrogen-responsive tumors. Data obtained in ovarian tumors contrasted with observations in breast cancer. Cyclin DI DNA amplification was much less frequent in ovarian than in breast tumors (3.3% vs. I 2.6%), whereas cyclin E amplification and overexpression were observed in a significant number of cases (I 2.5% and 18.0% respectively). Cyclin A, cyclin D2 and D3 rarely showed anoma- o 1996 Wiley-Liss, Inc.
The EWS/TEC gene fusion generated by the t(9;22) chromosomal translocation found in extraskeletal myxoid chondrosarcomas encodes a fusion protein containing the amino-terminal domain of the EWS protein fused to the whole coding sequence of the orphan nuclear receptor TEC. We have compared the DNA-binding and transcriptional activation properties of various TEC isoforms and the corresponding EWS/TEC fusion proteins. Band-shift experiments show that the fulllength TEC receptor can e ciently bind the NGFI-B Response Element (NBRE), whereas an isoform lacking the entire carboxyl-terminal domain of the receptor binds much less e ciently the NBRE. Addition of the aminoterminal domain of EWS to either isoforms does not alter signi®cantly their DNA-binding properties to the NBRE. Co-transfection experiments of COS cells and human chondrocytes indicate that whereas TEC moderately activates transcription from a NBRE-containing promoter, the corresponding EWS/TEC fusion protein is a highly potent transcriptional activator of the same promoter, being approximately 270-fold more active than the native receptor. EWS/TEC may thus exert its oncogenic potential in chrondrosarcomas by activating the transcription of target genes involved in cell proliferation.
Summary 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, 20q 13 correlated, as did MDM2 amplification, to aneuploidy. In parallel, we had also tested our tumour DNAs for amplification of CCNDJ, 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 CCNDI 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.
Chromosome 17q is frequently rearranged in breast cancer. Allelotyping studies have proposed the existence of at least four regions of allelic imbalance (AI). Here we present a study combining allelotyping using 19 CA repeat markers mapping in the 17q21 ± 25 region and molecular cytogenetics (CGH and FISH). Allelotyping was undertaken on 178 pairs of cognate tumor and normal DNA in order to determine the number of regions of AI and de®ne the shortest overlaps. AI ranged from 34 ± 54% of the informative cases according to the marker and, overall, 66% of the tumors presented AI at one of the markers tested. Analysis of the patterns of imbalances revealed at least ®ve common regions of imbalance respectively de®ned by markers: D17S855, which is intragenic of BRCA1 (SRO 1), D17S1607 (SRO 2), D17S1855 (SRO 3), between D17S789 and D17S785 (SRO 4) and D17S784 (SRO 5). In order to characterize the nature of the genetic events revealed by allelotyping we performed CGH analysis on a subset of 43 tumors presenting variable patterns of imbalance. CGH showed that AI at 17q could represent four di erent types of genetic events: loss of chromosome 17, gain of 17q, gain of 17q22 ± q24, loss of 17q11 ± q21 and/ or 17q25 ± qter. Some of these anomalies could occur concomitantly within the same tumor. Since 35% of the tumors analysed by CGH presented gains, these data indicated that AI at 17q were not solely indicative of losses of genetic material and could also represent DNA ampli®cation. Gains were most commonly observed in the 17q23 ± q24 regions. This suggested that AI in SRO 2 and SRO 3 corresponded to DNA ampli®cation. To assess this, we isolated BAC clones by PCR screening for markers D17S1607 and D17S1855 and used these in FISH experiments on six breast tumor cell lines and 14 breast cancer specimens. FISH results showed that both D17S1607 and D17S1855 were frequently involved in DNA ampli®cation (8 ± 30 copies). Altogether, our data show that allelotyping can be e ciently used in amplicon mapping. Clinico-pathological correlations indicated that imbalance at 17q preferentially occurred in high grade, PR-and ERBB2 ampli®ed tumors.
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