Genetic Testing in Hereditary Breast and Ovarian Cancer Using Massive Parallel Sequencing

High throughput methods such as next generation sequencing are increasingly used in molecular diagnosis. The aim of this study was to develop a workflow for the detection ofBRCA1andBRCA2mutations using massive parallel sequencing in a 454 GS Junior bench top sequencer. Our approach was first validated in a panel of 23 patients containing 62 unique variants that had been previously Sanger sequenced. Subsequently, 101 patients with familial breast and ovarian cancer were studied.BRCA1andBRCA2exon enrichment has been performed by PCR amplification using the BRCA MASTR kit (Multiplicom). Bioinformatic analysis of reads is performed with the AVA software v2.7 (Roche). In total, all 62 variants were detected resulting in a sensitivity of 100%. 71 false positives were called resulting in a specificity of 97.35%. All of them correspond to deletions located in homopolymeric stretches. The analysis of the homopolymers stretches of 6 bp or longer using the BRCA HP kit (Multiplicom) increased the specificity of the detection ofBRCA1andBRCA2mutations to 99.99%. We show here that massive parallel pyrosequencing can be used as a diagnostic strategy to test forBRCA1andBRCA2mutations meeting very stringent sensitivity and specificity parameters replacing traditional Sanger sequencing with a lower cost.

Loss of Heterozygosity At the C Wild-Type Allele of rs1042522 in the TP53 Gene Frequently Occurs During Progression of Adult BCR-ABL1 Positive Acute Lymphoblastic Leukemia (ALL).

Abstract 2497 Introduction: The gene TP53 encoding the tumour suppressor protein p53 is among the most commonly mutated genes in human cancer. TP53 tumour-associated alterations often cause dramatic defects in p53 function and correlate with increased malignancy, dismal survival and resistance to treatment. In contrast, only a small fraction, if any, of the >200 single nucleotide polymorphisms (SNPs) of TP53 in human populations are expected to cause measurable perturbation of p53 function. Aim: Since the pattern, frequency and significance of TP53 aberrations and SNPs in adult BCR-ABL1 positive ALL have still to be investigated, in this study we used a massively parallel pyrosequencing technique to address these issues. Patients and methods: Forty-three adults with BCR-ABL1 positive ALL were analyzed (median age 63 years, range 18–84). Twenty-four cases (56%) were analyzed only at the time of diagnosis, four cases (9%) only at the time of relapse and fifteen cases (35%) at both time points. Massively parallel pyrosequencing in picoliter-sized wells was used to allow highly-sensitive deep-sequencing detecting molecular aberrations at a low burden rate. As part of the IRON (Interlaboratory RObustness of Next generation sequencing) II study network, we applied preconfigured plates including primers for TP53 (exons 4 to 11) and allowing the simultaneous screening of 11 patients, each being recognized by a unique molecular identifier. For each plate, after generating 88 amplicons, 8 per each patient, PCR products were purified using Agencourt AMPure XP beads and Biomek 3000 Laboratory Automation Workstation (Beckam Coulter) and quantified using the Quant-iT PicoGreen kit (Invitrogen). All amplicons were pooled in an equimolar ratio to generate one single library. Subsequent emulsion PCR and amplicon sequencing was performed according to the manufacturer's recommendations on the Genome Sequencer Junior Instrument (Roche Applied Science). Data were analyzed using the GS Amplicon Variant Analyzer software version 2.7 (Roche Applied Science). For the detection of variances, filters were set to display sequence variances occurring in more than 5% of bidirectional reads per amplicon in at least one sample. Results: On average, we generated 63,068 sequencing beads (key pass wells) per plate (range, 50,798-79,486) with a median read length range between 284 and 365 base pairs. The median number of generated reads per case was 5,413 (range, 687-9,604). The median number of reads per amplicon was as follows: exon 4: 275 (range, 0–888), exon 5: 222 (range, 0–1,013); exon 6: 316 (range, 84–854); exon 7: 317 (range, 5–720); exon 8: 313 (range, 0–784); exon 9: 215 (range, 0–785); exon 10: 328 (range, 0–826), exon 11: 447 (range, 0–1,511). Forward and reverse reads were homogeneously distributed allowing a sensitive detection of variances. In total, 8 single nucleotide variations were identified. All variances, except for one nucleotide substitution occurring at position 7576743 (GRCh37/hg19), were found to represent SNPs according to the NCBI dbSNP Build 137. They included: rs1042522 C/G (41/43, 95%) and rs1800370 A/G (1/43, 2%) in exon 4, rs1800372 A/G (2/43, 5%) in exon 6, rs1625895 A/G (42/43, 98%) in intron 6–7, rs12947788 A/G (3/43, 7%) and rs12951053 A/C (4/43, 9%) in intron 7–8 and rs1800899 C/T (1/43, 2%) in intron 9–10. Interestingly, in 2 cases (12%) loss of heterozygosity occurred at the relapse at the C wild-type allele of rs1042522 in leukemia cells. The same mechanism has been identified for one case at the wild-type allele of rs1625895 with the expansion of the variant form at relapse. Both rs1042522 and rs1625895 have been described to alter p53 functionality and increase susceptibility to cancers (Whibley et al.,2009). Although the role of rs12947788 and rs12951053 has not yet deeply investigated, in our study they were found in 3 cases that all relapsed. Conclusion: Comprehensive next generation deep-sequencing of TP53 by a screening assay set up within the IRON II study has demonstrated its ability to efficiently detect TP53 variant. The inactivation of the wild-type allelic forms of rs1042522 and rs1625895, altering the p53 functionality, may serve as an important background for leukemia progression in BCR-ABL1-positive ALL. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Luppi:CELGENE CORPORATION: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.