An optimizedBRCA1/2next-generation sequencing for different clinical sample types

Abstract

ObjectiveA simultaneous detection of germline and somatic mutations in ovarian cancer (OC) using tumor materials is considered to be cost-effective for BRCA1/2 testing. However, there are limited studies of the analytical performances according to various sample types. The aim of this study is to propose a strategy for routine BRCA1/2 next-generation sequencing (NGS) screening based on analytical performance according to different sample types.MethodsWe compared BRCA1/2 NGS screening assay using buffy coat, fresh-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) from 130 samples.ResultsThe rate of repeated tests in a total of buffy coat, FF and FFPE was 0%, 8%, and 34%, respectively. The accuracy of BRCA1/2 NGS testing was 100.0%, 99.9% and 99.9% in buffy coat, FFPE and FF, respectively. However, due to the presence of variant allele frequency (VAF) shifted heterozygous variants, tumor materials (FFPE and FF) showed lower sensitivity (95.5%–99.0%) than buffy coat (100%). Furthermore, FFPE showed 51.4% of the positive predictive value (PPV) on account of sequence artifacts. When performed in the post-filtration process, PPV was increased by approximately 20% in FFPE. Buffy coat showed 100% of sensitivity, specificity and accuracy in BRCA1/2 NGS test.ConclusionsOn the comparison of the analytical performance according to different sample types, the buffy coat was not affected by sequencing artifacts and VAF shifted variants. Therefore, the blood test should be given priority in detecting germline BRCA1/2 mutation, and tumor materials could be suitable to detect somatic mutations in OC patients without identifying germline BRCA1/2 mutation.

Cost-Effectiveness Analysis of Germline and Somatic BRCA Testing in Patients With Advanced Ovarian Cancer

“…The amplification of DNA extracted from FFPE results in sequence artifacts, e.g., DNA nucleotide substitutions of C with T and G with A, due to the deamination of cytosine to uracil [ 33 – 35 ]. Compared with BRCA testing by NGS using buffy coat samples, which yielded no false-positive results, BRCA testing by NGS using FFPE samples was associated with a higher rate of false-positive results, mainly due to C-to-T and G-to-A transitions [ 36 ]. Moreover, NGS using FFPE and fresh frozen tumor samples resulted in a disproportionate variant allele frequency (VAF) when compared with NGS using matched buffy coat samples; thus, the analytical performance of NGS using tumor tissues can be affected by sequencing artifacts and VAF-shifted variants [ 36 ].…”

“…Compared with BRCA testing by NGS using buffy coat samples, which yielded no false-positive results, BRCA testing by NGS using FFPE samples was associated with a higher rate of false-positive results, mainly due to C-to-T and G-to-A transitions [ 36 ]. Moreover, NGS using FFPE and fresh frozen tumor samples resulted in a disproportionate variant allele frequency (VAF) when compared with NGS using matched buffy coat samples; thus, the analytical performance of NGS using tumor tissues can be affected by sequencing artifacts and VAF-shifted variants [ 36 ]. In previous studies, tumor BRCA testing in ovarian cancer was unsuccessful in 1%–3% of cases [ 27 , 30 , 31 ].…”

Characterization of BRCA Deficiency in Ovarian Cancer

“…However, cancer tissue testing poses some critical issues, such as: differences between the types of samples, including formalin-fixed and paraffin-embedded (FFPE) tissues and snap-frozen (SF) tissues, the choice between primary tumor and relapse, the assessment of large rearrangements and the predictive value of specific variants for drug response [17][18][19][20]. Furthermore, a non-negligible fraction of ovarian tumors (11-16%) present BRCA deficiency due to epigenetic inactivation of BRCA1, not identifiable with routine somatic tests, and some tumors may present homologous recombination deficiency (HRD) due to alterations in other genes of the pathway [5,[21][22][23].…”

BRCA1 ve BRCA2 Mutasyonlarının Tespitine Yönelik Yeni Nesil Dizileme Temelli Kit Geliştirilmesi ve Rutinde Kullanılan Yöntemler ile Valide Edilmesi

“…Bununla birlikte, numune hazırlama tekniklerindeki standardizasyon eksiklikleri, farklı platformlar ve farklı veri analiz yöntemleriyle elde edilen varyant sınıflandırmaları arasındaki farklılıklar, NGS platformunun klinik uygulamada kullanımına ilişkin zorluklar arasında yer almaktadır 16,17…”