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PDF file - 2.35MB, Experimental set of 65 BLCs, Processing SNP-arrays, Inferring chromosome number, Detection of tumor ploidy, Breakpoints estimation, Validation of LSTs by Next Generation Sequencing and Sanger sequencing, Features of LSTs, Distribution of LSTs along the genome, LSTs in detection of BRCA1 tumors in Experimental and Validation sets, Somatic BRCA1 mutations detected in BLCs with high LST, BRCA2 mutations detected in BLCs with high LST, Triple negative cell lines, & Stability of LSTs in xenograft passages as compared to primary tumors.

PDF file - 1.7MB, Figure S1. Near-diploid (A) and near-tetraploid (B) profiles and patterns of alteration detected with the GAP method in the series of BLCs (Affymetrix SNP6.0). Figure S2. Four outlying patterns of alteration found in the series of BLCs. Figure S3. Deciphering the cutoff for the �small-scale� variation. Figure S4. LSTs detected in SNP array profile and validated by the NGS, Sanger sequencing and translocation specific PCR. Figure S5. Translocation specific PCR for validation of inter-chromosomal translocations detected by NGS and found within LSTs boundaries.

BRCA1 inactivation is a frequent event in basal-like breast carcinomas (BLC). However, BRCA1 can be inactivated by multiple mechanisms and determining its status is not a trivial issue. As an alternate approach, we profiled 65 BLC cases using single-nucleotide polymorphism arrays to define a signature of BRCA1-associated genomic instability. Large-scale state transitions (LST), defined as chromosomal break between adjacent regions of at least 10 Mb, were found to be a robust indicator of BRCA1 status in this setting. Two major ploidy-specific cutoffs in LST distributions were sufficient to distinguish highly rearranged BLCs with 85% of proven BRCA1-inactivated cases from less rearranged BLCs devoid of proven BRCA1-inactivated cases. The genomic signature we defined was validated in a second independent series of 55 primary BLC cases and 17 BLC-derived tumor cell lines. High numbers of LSTs resembling BRCA1-inactivated BLC were observed in 4 primary BLC cases and 2 BLC cell lines that harbored BRCA2 mutations. Overall, the genomic signature we defined predicted BRCA1/2 inactivation in BLCs with 100% sensitivity and 90% specificity (97% accuracy). This assay may ease the challenge of selecting patients for genetic testing or recruitment to clinical trials of novel emerging therapies that target DNA repair deficiencies in cancer. Cancer Res; 72(21); 5454–62. ©2012 AACR.

PDF file - 511K, Figure S6. Persistence and robustness of the discriminating �window� independently of the method of LSTs evalutation (supplementary to Figure 2B). Figure S7. Two tumors with similar total numbers of breakpoints. Figure S8. Distribution of LSTs along the genome, GC content and common fragile site for the chromosome bands with LSTs detected in more than 6 tumors. Figure S9. Somatic mutation in BRCA1 detected in two BLCs. Figure S10. LSTs in xenograft passages from GSE32530.

PDF file - 198K, Table S1. Number of chromosomes in the cell lines according to ATCC description, SKY images, and GAP estimation. Table S2. Tumor ploidy and percentage of genomic states in tumor genome. Table S3. Validation of LSTs by NGS, PCR and Sanger sequencing. Table S4. Final results obtained on the Experimental set of BLCs. Table S5. Final results obtained on the Validation set of BLCs. Table S6. LSTs fit validated rearrangements in triple-negative cell lines. Table S7. LSTs are conserved in xenografts.

Uveal melanoma arises from melanocytes of the uveal tract (iris, ciliary body and choroid) and represents the most common intraocular malignancy in adults. Some rare clinical situations (young age at diagnosis, bilateral or multifocal forms, association with cutaneous malignant melanoma and/or familial aggregations of melanomas) are suggestive of genetic susceptibility. The aim of this study was to evaluate the contribution of CDKN2A/P16INK4A, P14ARF and CDK4 gene germline mutations in a series of patients with uveal melanoma recruited in a single institution with a clinical presentation indicative of genetic predisposition. Molecular analyses were proposed to 36 patients and were performed in 25 cases. The contribution of BRCA1/2 gene germline mutations in patients with uveal melanoma and a personal and/or family history of breast/ovarian cancers was also evaluated. Molecular analysis of BRCA1/2 genes was proposed to 35 patients and was performed in 25 patients. No deleterious germline mutation was identified in either group of patients. These results indicate that the CDKN2A/P16INK4A, P14ARF, CDK4 genes are not responsible for the vast majority of genetic susceptibility to uveal melanoma. They also suggest that one case of uveal melanoma in a family with a history of breast cancer is not sufficient to justify BRCA1/2 genetic testing when the classical criteria for molecular analysis are not present. International studies are ongoing in melanoma-prone families in an attempt to identify uveal melanoma susceptibility loci and genes.

BRCA1 inactivation is a frequent event in basal-like breast carcinomas (BLC). However, BRCA1 can be inactivated by multiple mechanisms and determining its status is not a trivial issue. As an alternate approach, we profiled 65 BLC cases using single-nucleotide polymorphism arrays to define a signature of BRCA1-associated genomic instability. Large-scale state transitions (LST), defined as chromosomal break between adjacent regions of at least 10 Mb, were found to be a robust indicator of BRCA1 status in this setting. Two major ploidy-specific cutoffs in LST distributions were sufficient to distinguish highly rearranged BLCs with 85% of proven BRCA1-inactivated cases from less rearranged BLCs devoid of proven BRCA1-inactivated cases. The genomic signature we defined was validated in a second independent series of 55 primary BLC cases and 17 BLC-derived tumor cell lines. High numbers of LSTs resembling BRCA1-inactivated BLC were observed in 4 primary BLC cases and 2 BLC cell lines that harbored BRCA2 mutations. Overall, the genomic signature we defined predicted BRCA1/2 inactivation in BLCs with 100% sensitivity and 90% specificity (97% accuracy). This assay may ease the challenge of selecting patients for genetic testing or recruitment to clinical trials of novel emerging therapies that target DNA repair deficiencies in cancer. Cancer Res; 72(21); 5454–62. ©2012 AACR.

International audience; The detection of unknown mutations remains a serious challenge and, despite the expected benefits for the patient's health, a large number of genes are not screened on a routine basis. We present the diagnostic application of EMMA (Enhanced Mismatch Mutation Analysis®, Fluigent), a novel method based on heteroduplex analysis by capillary electrophoresis using innovative matrices. BRCA1 and BRCA2 were screened for point mutations and large rearrangements in 1,525 unrelated patients (372 for the validation step and 1,153 in routine diagnosis) using a single analytical condition. Seven working days were needed for complete BRCA1/2 screening in 30 patients by one technician (excluding DNA extraction and sequencing). A total of 137 mutations were found, including a BRCA2 duplication of exons 19 and 20, previously missed by Comprehensive BRACAnalysis®. The mutation detection rate was 11.9%, which is consistent with patient inclusions. This study therefore suggests that EMMA represents a valuable short-term and mid-term option for many diagnostic laboratories looking for an easy, reliable and affordable strategy, enabling fast and sensitive analysis for a large number of genes.