Luciano Giacò

Detection of Clinically Significant BRCA Large Genomic Rearrangements in FFPE Ovarian Cancer Samples: A Comparative NGS Study

Background: Copy number variations (CNVs), also referred to as large genomic rearrangements (LGRs), represent a crucial component of BRCA1/2 (BRCA) testing. Next-generation sequencing (NGS) has become an established approach for detecting LGRs by combining sequencing data with dedicated bioinformatics pipelines. However, CNV detection in formalin-fixed paraffin-embedded (FFPE) samples remains technically challenging, and there is the need to implement a robust and optimized analysis strategy for routine clinical practice. Methods: This study evaluated 40 FFPE ovarian cancer (OC) samples from patients undergoing BRCA testing. The performance of the amplicon-based NGS Diatech Myriapod® NGS BRCA1/2 panel (Diatech Pharmacogenetics, Jesi, Italy) was assessed for its ability to detect BRCA CNVs and results were compared to two hybrid capture-based reference assays. Results: Among the 40 analyzed samples (17 CNV-positive and 23 CNV-negative for BRCA genes), the Diatech pipeline showed a good concordance with the reference method—all CNVs were correctly identified in 16 cases with good enough sequencing quality. Only one result was inconclusive due to low sequencing quality. Conclusions: These findings support the clinical utility of NGS-based CNV analysis in FFPE samples when combined with appropriate bioinformatics tools. Integrating visual inspection of CNV plots with automated CNV calling improves the reliability of CNV detection and enhances the interpretation of results from tumor tissue. Accurate CNV detection directly from tumor tissue may reduce the need for additional germline testing, thus shortening turnaround times. Nevertheless, blood-based testing remains mandatory to determine whether detected BRCA CNVs are of hereditary or somatic origin, particularly in cases with a strong clinical suspicion of inherited predisposition due to young age and a personal and/or family history of OC.

POLE mutations in endometrial carcinoma: Clinical and genomic landscape from a large prospective single‐center cohort

AbstractBackgroundTo date, 11 DNA polymerase epsilon (POLE) pathogenic variants have been declared “hotspot” mutations. Patients with endometrial cancer (EC) characterized by POLE hotspot mutations (POLEmut) have exceptional survival outcomes. Whereas international guidelines encourage deescalation of adjuvant treatment in early‐stage POLEmut EC, data regarding safety in POLEmut patients with unfavorable characteristics are still under investigation. On the other hand, the spread of comprehensive genome profiling programs has underscored the need to interpret POLE variants not considered to be hotspots.MethodsThis study provides a comprehensive analysis of 596 sequenced patients with EC. The genomic landscape of POLEmut EC was compared with cases harboring nonhotspot POLE mutations within the exonuclease domain. Additionally, the genomic characteristics of multiple classifiers, as well as those exhibiting unfavorable histopathological and clinical features, were examined.ResultsNo significant genomic differences were observed among patients with POLEmut EC when comparing multiple classifiers to not‐multiple classifiers or those with unfavorable clinical features. However, the tumor mutational burden differed in both comparisons, whereas the percentage of C>G mutations only differed in the comparison based on clinical features. Specific POLE mutations, even if not considered to be hotspots, have genomic features comparable to POLEmut.ConclusionsThe present findings confirm the absence of significant genomic differences among POLEmut patients regardless of multiple‐classifier status or association with high‐risk clinical features. Prognostic data will be essential to elucidate the clinical significance of POLE mutations not classified as hotspots that exhibit genomic characteristics similar to those in POLEmut patients.

Germline reflex BRCA1/2 testing following tumor-only comprehensive genomic profiling: why, when, and how

Abstract The majority of tumor comprehensive genomic profiling (CGP) currently does not include a matched normal control. The use of a tumor-only CGP approach needs the development of a strategy to refine germline pathogenic/likely pathogenic variants (gP/LPVs) calls, so as to limit the performance of unnecessary germline reflex tests and instead successfully identify patients who are carriers of likely gP/LPVs. Guidelines have been developed for the identification of gP/LPVs in BRCA1/2 genes on the basis of tumor-only CGP results and for the evaluation of the appropriateness of performing germline reflex BRCA1/2 testing. In this study, an algorithm to assist decision-making for germline reflex testing of BRCA1/2 variants following tumor-only CGP is proposed.