Umberto Malapelle

In-house homologous recombination deficiency testing in ovarian cancer: a multi-institutional Italian pilot study

Aims Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPIs) represent a standard of care for the clinical management of high-grade serous ovarian cancer (HGSOC). The recognition of homologous recombination deficiency (HRD) has emerged as a predictive biomarker of response for first-line PARPIs treatment in patients with HGOSC. On the other hand, this test is extremely complex and therefore it is often externalised. Regrettably, the reliability of outsourced HRD testing can be troubled by inconclusive results and high rejection rates. In this methodological study, we assessed the technical feasibility, interassay and interlaboratory reproducibility of in-house HRD testing using three different commercially available next-generation sequencing assays. Methods A total of n=20 epithelial ovarian cancer samples previously analysed with MyChoice CDx were subjected to HRD retesting using three different platforms in three different major pathology laboratories, that is, SOPHiA DDM HRD Solution, HRD focus and Oncomine homologous recombination repair pathway predesigned panel. Concordance was calculated by Cohen’s (dual) and Fleiss (triple) κ coefficients. Results In-house BRCA1/2 molecular testing yielded a concordance rate >90.0% among all participating centres. HRD scores were successfully calculated by each institution with a concordance rate of 76.5%. Concerning the external gold standard test, the overall percentage of agreement ranged from 80.0% to 90.0% with a positive percentage agreement ranging from 75.0% to 80.0% and a negative percentage agreement ranging from 80.0% to 100%. Conclusions In-house testing for HRD can be reliably performed with commercially available next-generation sequencing assays.

Reliability and reproducibility among different platforms for tumour BRCA testing in ovarian cancer: a study of the Italian NGS Network

Introduction BRCA tumour testing is a crucial tool for personalised therapy of patients with ovarian cancer. Since different next-generation sequencing (NGS) platforms and BRCA panels are available, the NGS Italian Network proposed to assess the robustness of different technologies. Methods Six centres, using four different technologies, provided raw data of 284 cases, including 75 cases with pathogenic/likely pathogenic variants, for a revision blindly performed by an external bioinformatic platform. Results The third-party revision assessed that all the 284 raw data reached good quality parameters. The variant calling analysis confirmed all the 75 pathogenic/likely pathogenic variants, including challenging variants, achieving a concordance rate of 100% regardless of the panel, instrument and bioinformatic pipeline adopted. No additional variants were identified in the reanalysis of a subset of 41 cases. Conclusions BRCA tumour testing performed with different technologies in different centres, may achieve the realibility and reproducibility required for clinical diagnostic procedures.

BRCA1/2 NGS Somatic Testing in Clinical Practice: A Short Report

High-grade serous ovarian carcinoma (HGSOC) is the most common subtype of all ovarian carcinomas. HGSOC harboring BRCA1/2 germline or somatic mutations are sensitive to the poly (adenosine diphosphate-ribose) polymerase inhibitors (PARPi). Therefore, detecting these mutations is crucial to identifying patients for PARPi-targeted treatment. In the clinical setting, next generation sequencing (NGS) has proven to be a reliable diagnostic approach BRCA1/2 molecular evaluation. Here, we review the results of our BRCA1/2 NGS analysis obtained in a year and a half of diagnostic routine practice. BRCA1/2 molecular NGS records of HGSOC patients were retrieved from our institutional archive covering the period from January 2020 to September 2021. NGS analysis was performed on the Ion S5™ System (Thermo Fisher Scientific, Waltham, MA, USA) with the Oncomine™ BRCA Research Assay panel (Thermo Fisher Scientific). Variants were classified as pathogenic or likely pathogenic according to the guidelines of the American College of Medical Genetics and Genomics by using the inspection of Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) and ClinVar (NCBI) databases. Sixty-five HGSOC patient samples were successfully analyzed. Overall, 11 (16.9%) out of 65 cases harbored a pathogenic alteration in BRCA1/2, in particular, six BRCA1 and five BRCA2 pathogenic variations. This study confirms the efficiency and high sensitivity of NGS analysis in detecting BRCA1/2 germline or somatic variations in patients with HGSOC.

Leveraging Real-World Data From a Clinicogenomic Database Addresses the Treatment Gap in Patients With High-Grade Serous Ovarian Cancer

PURPOSE This study used a deidentified nationwide (US-based) high-grade serous ovarian cancer (HGSOC) clinicogenomic database (CGDB) to enrich our understanding of HGSOC's genomic heterogeneity and assess the utility of comprehensive genomic profiling (CGP) in clinical settings. PATIENTS AND METHODS We conducted a retrospective observational analysis on 856 patients with HGSOC profiled with CGP genomic tests, retrieved from CGDB from January 2011 to September 2023. RESULTS In addition to BRCA1 (11.7%) and BRCA2 (6.5%) variants, CGP revealed further potentially actionable alterations (amplifications and/or mutations) in CCNE1 (16%), FGFR1/2/3/4 (6.5%), PIK3CA (3.9%), TP53 Y220C (3.7%), ERBB2 (3.5%), CDK12 (2.3%), ARID1A (2.2%), KRAS (2.1%), and BRAF (1%) genes. Then, 439 patients were selected, presenting both CGP test performed on specimen collected at the time of surgery and initiation of first-line therapy within ±8 months from surgery, and categorized into no (NS, n = 74), interval (IS, n = 157), and upfront surgery (n = 208) groups, each comparable by clinical features. The CGP revealed BRCA mutations, at similar frequency in the three groups, in 54/439 patients (12.3%). Patients with pathogenic BRCA mutations had better event-free survival (EFS) compared with those with BRCA wt . Loss-of-heterozygosity (LOH) ≥16 (LOH-positive patients) was found in 142/433 (32.8%) patients, with different prevalence across treatment groups (12.8% NS; 9% IS; 8.8% upfront surgery). Patients treated with poly (ADP-ribose) polymerase inhibitors (PARPi) had improved EFS (hazard ratio for other drugs v PARPi 1.77 [95% CI, 1.21 to 2.58]). Interestingly, in 206 BRCA wt and LOH-negative patients, not eligible for PARPi, CGP detected potentially targetable alterations in 99 of them (48%). CONCLUSION Overall, our study provides evidence that CGP significantly improves the identification of molecular targets in HGSOC, supporting its importance in the clinical practice to provide patients with more therapeutic options.