Angelo Minucci

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.

A Case Report to Reflect on the Origins of MMRd Mesonephric-like Ovarian Adenocarcinoma: Can It Be Defined as a Mϋllerian Neoplasm?

Mesonephric-like adenocarcinoma (MLA) of ovaries is a new and rare neoplastic entity, recently classified by the World Health Organization. Its morphological and immunohistochemical profile is similar to primitive cervical mesonephric adenocarcinoma, but its origin has not been determined yet. Some authors believe that this neoplasm originates from Wolffian remnants in the ovarian hilum, while others suggest an origin from the Mϋllerian epithelium, followed by a mesonephric trans-differentiation. Starting from a recently diagnosed mismatch repair-deficient ovarian MLA, we try to further develop this line of research. A detailed molecular analysis of the studied tumor helps clarify our ideas. In fact, the typical KRAS mutation was not present. We found mutations in numerous other genes, which are rarely described in the literature or are already described in the endometrioid histotype. We reached some interesting conclusions, which, if supported by future studies, will clarify the true nature of these tumors, allowing for better stratification and a better therapeutic framework.

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.

Epithelial ovarian cancer and brain metastases: might the BRCA status, PARP inhibitor administration, and surgical treatment impact the survival?

To evaluate disease characteristics and survival according to This is a monocentric retrospective cohort of patients with ovarian cancer and brain metastases treated between 2000 and 2021. Data were collected by a retrospective review of medical records and analyzed according to: (1) Eighty-five patients with ovarian cancer and brain metastasis and known

Survival outcomes in patients with BRCA mutated, variant of unknown significance, and wild type ovarian cancer treated with PARP inhibitors

Correlation between Patients with ovarian cancer whose somatic Of 67 patients identified, 20 (29.9%), 24 (35.8%), and 23 (34.3%) had BRCA variant of unknown significance, mutated, and wild type, respectively. Patients received PARPi as maintenance treatment at the time of the first relapse after a complete response or partial response to platinum-based chemotherapy without differences in the previous platinum-free interval among the analyzed groups. The median progression-free survival of patients with Our study suggests that carriers of BRCA variant of unknown significance have survival outcomes comparable to patients with

Ovarian cancer onset across different BRCA mutation types: a view to a more tailored approach for BRCA mutated patients

To evaluate the role of different specific types of germline breast cancer susceptibility This was a multicenter, international, retrospective cohort of 474 patients diagnosed with recurrent or newly diagnosed high grade serous ovarian cancer, with known germline mutations in Excluding the 29 patients with a splicing mutation, 474 patients were enrolled: 309 (65.2%) with frameshift mutations, 102 (21.5%) with nonsense mutations, and 63 (13.3%) with missense mutations. The Different types of germline

BRCA testing delay during the COVID-19 pandemic: How to act?

Recently, our lab, part of a referral center in Italy, reported its experience regarding the execution of germline BRCA1/2 (gBRCA) testing during the first months of the coronavirus disease-2019 (COVID-19) pandemic, which highlights a substantial reduction (about 60%) compared with the first 2 months of the current year. This evidence appeared to be a lockdown effect due to extraordinary restriction measures to slow down the spread of SARS-CoV-2. In this study, we aimed to evaluate the overall effects of the ongoing pandemic on gBRCA testing in our institution and to understand how COVID-19 has influenced testing after the complete lockdown (March 8-May 5, 2020). Additionally, we compared this year's trend with trends of the last 3 years to better monitor gBRCA testing progress. This detailed analysis highlights two important findings: (1) gBRCA testing did not increase significantly after the lockdown period (May-October 2020) compared with the lockdown period (March-April 2020), emphasizing that even after the lockdown period testing remained low. (2) Comparing the total tests per year (January-October 2017, 2018, 2019, with 2020), the impact of COVID-19 on gBRCA testing is apparent, with similarities of trends registered in 2017. These evidences reveal a gBRCA testing delay for cancer patients and healthy patients at this moment, and the new era of gBRCA testing in the management of ovarian, breast, pancreas and prostate cancer patients has been seriously questioned due to the COVID-19 pandemic. As consequence, we underline that measures to guarantee oncogenetic testing (e.g., gBRCA testing) along with new diagnostic/clinic strategies are mandatory. For these reasons, several proposals are presented in this study.

BRCA testing in a genomic diagnostics referral center during the COVID-19 pandemic

The first person-to-person transmission of the 2019-novel coronavirus in Italy on 21 February 2020 led to an infection chain that represents one of the largest known COVID-19 outbreaks outside Asia. Hospitals have been forced to reorganized their units in response to prepare for an unforeseen healthcare emergency. In this context, our laboratory (Molecular and Genomic Diagnostics Unit, Fondazione Policlinico Universitario Agostino Gemelli IRCCS) re-modulated its priorities by temporarily interrupting most of the molecular tests guaranteeing only those considered "urgent" and not postponable. In particular, this paper details changes regarding the execution of germline BRCA (gBRCA) testing in our laboratory. A substantial reduction in gBRCA testing (about 60%) compared to the first 2 months of the current year was registered, but the requests have not been reset. The requesting physicians were mainly gynaecologists and oncologists. These evidences further emphasize the new era of gBRCA testing in the management of cancer patients and confirms definitively the integration of gBRCA testing/Next Generation Sequencing (NGS) into clinical oncology. Finally, a re-organization of gBRCA testing in our Unit, mainly related to delayed and reduced arrival of tests was necessary, ensuring, however, a high-quality standard and reliability, mandatory for gBRCA testing in a clinical setting.

Tumor BRCA testing in ovarian cancer and EQA scheme: our experience of a critical evaluation

AbstractNext generation sequencing (NGS) is a widespread molecular biology method integrated into clinical practice to detect genetic variants, for diagnostic and prognostic purposes. The scheduled external quality assessments (EQA) is integral part of clinical molecular laboratory quality assurance. The EQA provides an efficient system to compare analytic test performances among different laboratories, which is essential to evaluate consistency of molecular test. EQA failures demands targeted corrective action plans. In this context, the complexity of the NGS techniques requires careful and continuous quality control procedures. We report a tumor BRCA1/2 (tBRCA) testing benchmark discrepancy provided by the European Molecular Genetics Quality Network in our laboratory during a round of EQA for somatic mutation testing of BRCA genes in relation to ovarian cancer. The critical analysis emerging from the tBRCA EQA is presented. We underline that harmonization processes are still required for the EQA in the molecular biology field, especially if applied to the evaluation of methods characterized by high complexity.

The BRCA1 c.788G > T (NM_007294.4) variant in a high grade serous ovarian cancer (HGSOC) patient: foods for thought

In this report we described the case of a BRCA1/2 (BRCA) molecular testing performed on tumor sample in a High Grade Serous Ovarian Cancer (HGSOC) patient with two different Next Generation Tumor Sequencing (NGTS) pipelines. The two clinical reports leaded to apparently different BRCA status, providing important foods for thought. After NGTS, the gene sequencing information (i.e., reads) are aligned to the reference gene sequences obtained from public databases, in order to provide an uniform nomenclature for unambiguous variant designation. However, the criteria adopted for variant reporting in tissue test are not always univocal. Particularly, this is the case of rare and unclassified BRCA variants for which the molecular evaluation may be a relevant challenge. Here we described a BRCA1 unclassified variant that may be re-evaluated in the context of alternative BRCA1 transcripts due to its different biological effect. We underlined that an in-depth knowledge of BRCA testing is mandatory for its appropriate use.

Deep-Learning to Predict BRCA Mutation and Survival from Digital H&E Slides of Epithelial Ovarian Cancer

BRCA 1/2 genes mutation status can already determine the therapeutic algorithm of high grade serous ovarian cancer patients. Nevertheless, its assessment is not sufficient to identify all patients with genomic instability, since BRCA 1/2 mutations are only the most well-known mechanisms of homologous recombination deficiency (HR-d) pathway, and patients displaying HR-d behave similarly to BRCA mutated patients. HRd assessment can be challenging and is progressively overcoming BRCA testing not only for prognostic information but more importantly for drugs prescriptions. However, HR testing is not already integrated in clinical practice, it is quite expensive and it is not refundable in many countries. Selecting patients who are more likely to benefit from this assessment (BRCA 1/2 WT patients) at an early stage of the diagnostic process, would allow an optimization of genomic profiling resources. In this study, we sought to explore whether somatic BRCA1/2 genes status can be predicted using computational pathology from standard hematoxylin and eosin histology. In detail, we adopted a publicly available, deep-learning-based weakly supervised method that uses attention-based learning to automatically identify sub regions of high diagnostic value to accurately classify the whole slide (CLAM). The same model was also tested for progression free survival (PFS) prediction. The model was tested on a cohort of 664 (training set: n = 464, testing set: n = 132) ovarian cancer patients, of whom 233 (35.1%) had a somatic BRCA 1/2 mutation. An area under the curve of 0.7 and 0.55 was achieved in the training and testing set respectively. The model was then further refined by manually identifying areas of interest in half of the cases. 198 images were used for training (126/72) and 87 images for validation (55/32). The model reached a zero classification error on the training set, but the performance was 0.59 in terms of validation ROC AUC, with a 0.57 validation accuracy. Finally, when applied to predict PFS, the model achieved an AUC of 0.71, with a negative predictive value of 0.69, and a positive predictive value of 0.75. Based on these analyses, we have planned further steps of development such as proving a reference classification performance, exploring the hyperparameters space for training optimization, eventually tweaking the learning algorithms and the neural networks architecture for better suiting this specific task. These actions may allow the model to improve performances for all the considered outcomes.