A cryptic familial rearrangement of 11p15.5, involving both imprinting centers, in a family with a history of short stature.

Silver-Russell syndrome (SRS) is a heterogeneous disorder characterized by intrauterine and postnatal growth retardation, dysmorphic facial features and body asymmetry. Both hypomethylation of the telomeric imprinting control region 1 (ICR1) at 11p15.5 and maternal duplication of 11p15.5 have been implicated in the etiology of this disorder. Here we report the origin and segregation of the first reported between-arm intrachromosomal insertion of 11p15.5 that encompasses both ICR1 and ICR2 in a multigenerational family with a history of short stature. One (or any odd number) crossover within the centromeric segment during meiosis would produce recombinant chromosomes; one with a duplication of the inserted segment and the other a deletion. In this 4-generation family, there were six instances of transmission of the recombinant chromosome with duplication of the11p15.5 segment, which leads to a SRS phenotype when maternally inherited and a Beckwith-Wiedemann phenotype when paternally transmitted. The size of the duplicated region is ~1.9 Mb as determined by microarray analysis. This study provides further evidence that maternally inherited duplications of 11p15.5 result in a SRS phenotype that includes short stature and other variable features. The methylation status of the extra copy of the duplicated region of 11p15.5 ultimately predicts the resulting phenotype. Thus, the different phenotype based on parental mode of transmission is of importance in the genetic counseling of these patients.

Co-amplification of CCND1 and EMSY is associated with an adverse outcome in ER-positive tamoxifen-treated breast cancers.

Amplification of chromosome 11q13 is commonly seen in breast carcinomas and candidate genes from this region include CCND1 and EMSY. Here, we investigate the prognostic significance of CCND1 and EMSY amplification in a large series of breast carcinomas and in BRCA1 and BRCA2 mutation positive breast cancers. Amplification of CCND1 and EMSY was assessed by fluorescent in situ hybridization. Both CCND1 and EMSY amplifications were associated with a significantly worse outcome in ER-positive patients treated with tamoxifen only, in contrast to nonamplified tumors (P = 8.55 x 10(-4) and P = 8.35 x 10(-5), respectively). In multivariable Cox models, which included standard prognostic markers, co-amplification of CCND1 and EMSY was significantly more predictive of outcome than was amplification of either gene alone or neither gene amplified in ER-positive tamoxifen-treated patients (P = 5.47 x 10(-5)). EMSY gene amplification was a significantly less common event in BRCA2 mutation carriers as compared to BRCA1 mutation carriers (9 versus 24%, respectively). In contrast, CCND1 amplification occurred at a similar frequency in both BRCA1 and BRCA2 breast cancers (22 versus 18%, respectively). In summary, co-amplification of CCND1 and EMSY identified a poor prognostic subset of ER-positive tamoxifen-treated patients. In addition, EMSY amplification occurred at a lower frequency in BRCA2 mutation carriers providing evidence to support EMSY amplification as a somatic surrogate for BRCA2 loss in sporadic breast cancer.

Amplification of 11q13 in ovarian carcinoma.

Amplification at the 11q13 locus is commonly observed in breast, ovarian, head and neck, oral, and esophageal cancer. Studies of this region led to the identification of multiple amplicons containing several potential oncogenes including EMSY, PAK1, RSF1, and GAB2. Here, we investigate the amplification of the above four genes and their prognostic significance in histologically and clinically defined subsets of ovarian cancer. Amplification of all four genes was assessed by fluorescent in situ hybridization in tissue microarrays containing 538 clinically annotated ovarian carcinomas with 12 years of follow-up data. Overall, for the entire cohort, EMSY was amplified in 44 (16%) of 269 cases, PAK1 was amplified in 38 (15%) of 255 cases, RSF1 was amplified in 37 (12%) of 310 cases, and GAB2 was amplified in 41 (16%) of 255 cases. Amplification of EMSY, PAK1, RSF1, and GAB2 were all highly correlated with each other and with a serous histology. Univariate survival analysis showed that tumors with EMSY and RSF1 amplification were associated with a significantly worse outcome. A molecular inversion probe array was then used to study the 11q13 amplicon in 33 high grade serous carcinomas. The core of the amplicon mapped to a 6-Mb region encompassing EMSY, PAK1, RSF1, and GAB2. However, a second more telomeric amplicon was also observed for which no candidate genes have been identified. In summary, amplification of these four putative oncogenes from 11q13 in early ovarian cancer is associated with a serous histology and in the case of EMSY and RSF1 a poor outcome. These findings support the hypothesis that the11q13 amplicon in ovarian cancer is likely driven by a cassette of genes rather than by a single oncogene. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.

Distribution and significance of caveolin 2 expression in normal breast and invasive breast cancer: an immunofluorescence and immunohistochemical analysis.

BACKGROUND: The aims of this study were to define the distribution of caveolin 2 (CAV2) in frozen and formalin fixed, paraffin embedded (FFPE) normal breast samples and the significance of CAV2 expression in breast cancer. METHODS: Caveolin 2 distribution in frozen and paraffin-embedded whole tissue sections of normal breast was evaluated using immunohistochemistry and immunofluorescence, in conjunction with antibodies to define luminal epithelial cells (oestrogen receptor and cytokeratin 8/18) and myoepithelial/ basal cells (cytokeratins 14 and 5/6, p63 and smooth muscle actin). CAV2 expression was also immunohistochemically analysed in two independent cohorts of invasive breast carcinomas (n = 245 and n = 418). RESULTS: In normal breast, CAV2 was expressed in myoepithelial cells, endothelial cells, fibroblasts and adipocytes. Luminal epithelial cells showed no or only negligible staining. CAV2 expression was observed in 9.6% of all breast cancers and was strongly correlated with high histological grade, lack of oestrogen receptor, progesterone receptor and cyclin D1 expression, and positivity for epidermal growth factor receptor, basal markers, p53 expression, and high proliferation index. Furthermore, CAV2 expression was significantly associated with basal-like immunophenotype and proved to be a prognostic factor for breast cancer-specific survival on univariate analysis. CONCLUSION: Our results demonstrate that CAV2 is preferentially expressed in basal-like cancers and is associated with poor prognosis. Further in vitro studies are required to determine whether CAV2 has oncogenic properties or is only a surrogate marker of basal-like carcinomas.

Fluorescent in situ hybridization on tissue microarrays: challenges and solutions.

Tissue microarray (TMA) technology has provided a high throughput means of evaluating potential biomarkers and therapeutic targets in archival pathological specimens. TMAs facilitate the rapid assessment of molecular alterations in hundreds of different tumours on a single slide. Sections from TMAs can be used for any in situ tissue analysis, including fluorescent in situ hybridization (FISH). FISH is a molecular technique that detects numerical and structural abnormalities in both metaphase chromosomes and interphase nuclei. FISH is commonly used as a prognostic and diagnostic tool for the detection of translocations and for the assessment of gene deletion and amplification in tumours. Performing FISH on TMAs enables researchers to determine the clinical significance of specific genetic alterations in hundreds of highly characterized tumours. The use of FISH on archival paraffin embedded tissues is technically demanding and becomes even more challenging when applied to paraffin embedded TMAs. The problems encountered with FISH on TMAs, including probe preparation, hybridization, and potential applications of FISH, will be addressed in this review.

Amplification of EMSY, a novel oncogene on 11q13, in high grade ovarian surface epithelial carcinomas.

OBJECTIVES: Amplification of the 11q13 locus is commonly observed in a number of human cancers including both breast and ovarian cancer. Cyclin D1 and EMS1 have been implicated as candidate oncogenes involved in the emergence of amplification at this locus. Detailed analysis of the 11q13 amplicon in breast cancer led to the discovery of four regions of amplification suggesting the involvement of other genes. Here, we investigate the role of EMSY, a recently described BRCA2 interacting protein, as a key element of the 11q13 amplicon in ovarian cancer. EMSY maps to 11q13.5 and is amplified in 13% of breast and 17% of ovarian carcinomas. METHODS: EMSY amplification was assessed by fluorescent in-situ hybridization (FISH) in 674 ovarian cancers in a tissue microarray and correlated with histopathological subtype and tumor grade. A detailed map of the 11q13 amplicon in 51 cases of ovarian cancer was obtained using cDNA-array-based comparative genomic hybridization (aCGH). To further characterize the role of EMSY within this amplicon, we evaluated both the amplification profiles and RNA expression levels of EMSY and two other genes from the 11q13 amplicon in an additional series of 22 ovarian carcinomas. RESULTS: EMSY amplification was seen in 52/285 (18%) high grade papillary serous carcinomas, 4/27 (15%) high grade endometrioid carcinomas, 3/38 (8%) clear cell carcinomas, and 3/10 (30%) undifferentiated carcinomas. aCGH mapping of 11q13 in ovarian cancer showed that EMSY localized to the region with the highest frequency of copy number gain. Cyclin D1 and EMS1 showed a lower frequency of copy number gain. A highly significant correlation between EMSY gene amplification and RNA expression was also observed (P = 0.0001). This was a stronger correlation than for other genes at 11q13 including Cyclin D1 and PAK1. CONCLUSIONS: These findings support the role of EMSY as a key oncogene within the 11q13 amplicon in ovarian cancer.

NRG1 gene rearrangements in clinical breast cancer: identification of an adjacent novel amplicon associated with poor prognosis.

Rearrangements of the neuregulin (NRG1) gene have been implicated in breast carcinoma oncogenesis. To determine the frequency and clinical significance of NRG1 aberrations in clinical breast tumors, a breast cancer tissue microarray was screened for NRG1 aberrations by fluorescent in situ hybridization (FISH) using a two-color split-apart probe combination flanking the NRG1 gene. Rearrangements of NRG1 were identified in 17/382 cases by FISH, and bacterial artificial chromosome array comparative genomic hybridization was applied to five of these cases to further map the chromosome 8p abnormalities. In all five cases, there was a novel amplicon centromeric to NRG1 with a minimum common region of amplification encompassing two genes, SPFH2 and FLJ14299. Subsequent FISH analysis for the novel amplicon revealed that it was present in 63/262 cases. Abnormalities of NRG1 did not correlate with patient outcome, but the novel amplicon was associated with poor prognosis in univariate analysis, and in multivariate analysis was of prognostic significance independent of nodal status, tumor grade, estrogen receptor status, and human epidermal growth factor receptor (HER)2 overexpression. Of the two genes in the novel amplicon, expression of SPFH2 correlated most significantly with amplification. This amplicon may emerge as a result of breakpoints and chromosomal rearrangements within the NRG1 locus.

EMSY links the BRCA2 pathway to sporadic breast and ovarian cancer.

The BRCA2 gene is mutated in familial breast and ovarian cancer, and its product is implicated in DNA repair and transcriptional regulation. Here we identify a protein, EMSY, which binds BRCA2 within a region (exon 3) deleted in cancer. EMSY is capable of silencing the activation potential of BRCA2 exon 3, associates with chromatin regulators HP1beta and BS69, and localizes to sites of repair following DNA damage. EMSY maps to chromosome 11q13.5, a region known to be involved in breast and ovarian cancer. We show that the EMSY gene is amplified almost exclusively in sporadic breast cancer (13%) and higher-grade ovarian cancer (17%). In addition, EMSY amplification is associated with worse survival, particularly in node-negative breast cancer, suggesting that it may be of prognostic value. The remarkable clinical overlap between sporadic EMSY amplification and familial BRCA2 deletion implicates a BRCA2 pathway in sporadic breast and ovarian cancer.

CDH1 c-160a promotor polymorphism is not associated with risk of stomach cancer.

We have combined data from case control studies designed to test the hypothesis that the c-160a promotor polymorphism in the gene coding for the cell adhesion molecule E-cadherin (CDH1) is associated with stomach cancer. A total of 899 individuals (433 patients and 466 controls) were analyzed. The genotype frequencies did not differ significantly between cases and controls, and the genotype-specific risks were not significantly different from unity, with an odds ratio for heterozygotes compared with the common homozygote of 1.3 (95% CI 0.98-1.8) and 1.2 (0.68-2.0) for rare homozygotes compared with common homozygotes. We found no evidence for differences in risk for the intestinal- and diffuse-type histopathologic subgroups.