gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of <em>BRCA1</em>, <em>BRCA2</em>, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Centre Georges-François Leclerc

Expression of BIRC5 (survivin), a member of the inhibitor of apoptosis protein (IAP) family, is elevated in fetal tissues and in various human cancers. Mechanisms up-regulating BIRC5 in cancer are poorly understood. Here, we show that overexpression of BIRC5 induces a high proliferation level in MCF-7 breast tumor cells. In a population of 191 breast carcinomas, BIRC5 expression is not affected by BIRC5 promoter polymorphism at -31, or BIRC5 gene copy number. However, a significant correlation was found between expression of demethylase (dMTase) and expression of BIRC5. In addition, among 13 chromosomal regions tested for allelic loss [loss of heterozygosity (LOH)], two regions close to D3S1478 and D6S264 were related to BIRC5 expression. In tumors with LOH at D3S1478 and/or D6S264, BIRC5 expression was significantly increased. These regions have been suggested to harbor tumor suppressor genes and/or common fragile sites that may play a role in increasing genetic instability. These results suggest that genes located near D3S1478 and D6S264 might work by inhibiting, directly or indirectly, BIRC5 expression and thus their loss leads to its up-regulation. In addition, BIRC5 expression may induce breast tumor proliferation by promoting genetic instability. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.

The expression of BIRC5 is correlated with loss of specific chromosomal regions in breast carcinomas.

Survivin, an anti-apoptotic protein, was described as strongly expressed in human cancers including breast cancer. However, little is known about the association between Survivin variants (Survivin-2B, Survivin-ΔEx3, Survivin-3B, and Survivin-2α) and the other apoptotic-related genes. In this study, we analyzed the apoptosis gene signature of Survivin and its variant expression in breast cancer. Human Apoptosis Gene Arrays were used to screen genes that could be associated with Survivin variants. Expression of the five transcripts was measured by RT-PCR in 135 breast carcinomas and Cox survival analysis was analyzed according to the patient outcome. Significant associations between Survivin transcripts and apoptotic genes were found. Interestingly, Survivin-3B variant showed major inverse correlations with pro-apoptotic genes. In addition, in vitro results indicated that overexpression of Survivin-3B strongly inhibits 5-fluorouracil/epirubicin/cyclophosphamide-induced apoptosis in breast tumor cell lines. In breast carcinomas, uni- and multivariate analysis showed patients with high level of Survivin-3B expression had a shorter overall (P=0.030 and P=0.042 respectively), and disease-free (P=0.024 and P=0.009) survival. Our data suggest that Survivin-3B contributes to cell survival through the anti-apoptotic pathway and that its expression level could be an important factor in determining therapeutic strategies for breast carcinoma.

Apoptosis gene signature of Survivin and its splice variant expression in breast carcinoma.

The presence of a TP53 gene mutation can influence tumour response to some treatments, especially in breast cancer. In this study, we analysed p53 mRNA expression, LOH at 17p13 and TP53 mutations from exons 2 to 11 in 206 patients with breast carcinoma and correlated the results with disease-free and overall survival. The observed mutations were classified according to their type and location in the three protein domains (transactivation domain, DNA binding domain, oligomerization domain) and correlated with disease-free and overall survival. In our population, neither p53 mRNA expression nor LOH correlated with outcome. Concerning TP53 mutations, 27% of tumours were mutated (53/197) and the presence of a mutation in the TP53 gene was associated with worse overall survival (p = 0.0026) but not with disease-free survival (p = 0.0697), with median survival of 80 months and 78 months, respectively. When alterations were segregated into mutation categories and locations, and related to survival, tumours harbouring mutations other than missense mutations in the DNA binding domain of P53 had the same survival profiles as wild-type tumours. Concerning missense mutations in the DNA binding domain, median disease-free and overall survival was 23 months and 35 months, respectively (p = 0.0021 and p<0.0001, respectively), compared with 78 and 80 months in mutated tumours overall. This work shows that disease-free and overall survival in patients with a frameshift mutation of TP53 or missense mutation in the oligomerization domain are the same as those in wild-type TP53 patients.

Only missense mutations affecting the DNA binding domain of p53 influence outcomes in patients with breast carcinoma.

The development of targeted therapies in cancer has accelerated the development of molecular diagnosis. This new cancer discipline is booming, with an increasing number of gene alterations to analyze in a growing number of patients. To deal with this fast-developing activity, current analysis techniques (Sanger sequencing, allelic discrimination and high resolution melting) take more and more time. In recent years, next generation sequencing (NGS) technologies have appeared and given new perspectives in oncology. In this study, we analyzed FFPE lung and colon carcinomas using the Truseq Cancer Panel, which analyzes the mutation hotspots of 48 genes. We also tested the use of whole-genome amplification before NGS analysis. NGS results were compared with the data obtained from routine diagnosis. All of the alterations routinely observed were identified by NGS. Moreover, NGS revealed mutations in the KRAS and EGFR genes in patients diagnosed as wild-type by routine techniques. NGS also identified concomitant mutations in EGFR and KRAS or BRAF mutations, and a 15-nt deletion in exon 19 of EGFR in colon carcinomas. The study of the other genes sequenced in the Panel revealed 14 genes altered by 27 different mutations and three SNP with a possible role in cancer susceptibility or in the response to treatment. In conclusion, this study showed that NGS analysis could be used for the analysis of gDNA extracted from FFPE tissues. However, given the high sensitivity of this technology, high-throughput clinical trials are needed to confirm its reliability for the molecular diagnosis of cancer.

Sandy Chevrier

Department of Biology and Pathology of Tumors,
Unit of Molecular Biology,
Platform of Immunomonitoring and Genetics,
Department of Biology and Pathology of Tumors, Unit of Molecular Biology, Platform of Immunomonitoring and Genetics

Centre Georges-François Leclerc

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair