Role of Breast Cancer Risk Estimation Models to Identify Women Eligible for Genetic Testing and Risk-Reducing Surgery.

Hereditary breast and ovarian cancer (HBOC) syndrome is responsible for approximately 10% of breast cancers (BCs). The HBOC gene panel includes both high-risk genes, i.e., a four times higher risk of BC (BRCA1, BRCA2, PALB2, CDH1, PTEN, STK11 and TP53), and moderate-risk genes, i.e., a two to four times higher risk of BC (BARD1, CHEK2, RAD51C, RAD51D and ATM). Pathogenic germline variants (PGVs) in HBOC genes confer an absolute risk of BC that changes according to the gene considered. We illustrate and compare different BC risk estimation models, also describing their limitations. These models allow us to identify women eligible for genetic testing and possibly to offer surgical strategies for primary prevention, i.e., risk-reducing mastectomies and salpingo-oophorectomies.

Role of the Molecular Tumor Board for the Personalized Treatment of Patients with Metastatic Breast Cancer: A Focus on the State of the Art in Italy.

Molecular tumor boards (MTBs) are multidisciplinary groups that combine molecular and clinical data from cancer patients in order to formulate treatment recommendations for precision medicine. To date, there is insufficient data to support the use of singleplex or next-generation sequencing (NGS) technologies to select first-line therapy for patients with metastatic breast cancer (MBC), but considering the high number of level II alterations, according to the ESMO scale for clinical actionability of molecular targets (ESCAT), it is suggested to include patients in molecular screening programs in order to be able to offer targeted therapies for specific genomic alterations. This article aims at reviewing the most recent literature related to the most used methodologies/approaches for molecular diagnostics and variants' classification, summarizing the internationally published molecular screening studies in support of MTB activity and, in the end, discussing MTBs' current position and role in Italy, the number of which is increasing, also thanks to the thrust of institutions.

Validation of AmpliSeq NGS Panel for BRCA1 and BRCA2 Variant Detection in Canine Formalin-Fixed Paraffin-Embedded Mammary Tumors.

Mammary carcinomas are the most common neoplasms observed in women and in female dogs. Canine mammary tumors show epidemiological, clinical, genetic, and prognostic characteristics comparable to human breast cancers. The recent introduction of next generation sequencing (NGS) technologies has greatly improved research and diagnostics for humans, while these new tools still need to be implemented in animal models. In this study we developed and validated an AmpliSeq Panel assay for the identification of BRCA variants in twenty-two different dogs. The amplicon mean coverage was 5499× and uniformity was higher than 98% in all samples. The results of germline single nucleotide variants (SNVs) and insertions/deletions (INDELs) were fully concordant regardless of the types of samples considered (blood, fresh and FFPE tissues). Moreover, despite the high DNA degradation observed in older FFPE blocks (>5 years), the assay allowed full coverage of all amplicons for downstream analyses. We consider the NGS panel developed in this study as a useful tool for expanding information on BRCA genes in the veterinary field and for human health from a comparative oncology perspective.

Intensive multidisciplinary treatment strategies and patient resilience to challenge long-term survival in metastatic colorectal cancer: a case report in real life and clinical practice.

In fit metastatic colorectal cancer (MCRC), multidisciplinary treatment strategy integrating intensive FIr-B/FOx triplet chemotherapy associated to bevacizumab and secondary metastasectomies significantly improved clinical outcomes up to progression-free survival (PFS) 17 months and overall survival (OS) 44 months. A non-elderly woman affected by rectal cancer, lymph nodes involvement, synchronous unresectable liver metastases, was treated with first-line FIr-B/FOx integrated with two-stage liver resections, short course radiotherapy, anterior rectal resection, with a PFS 9 months and progression-free interval (PFI) 4 months off-treatment. After progression characterized by single liver and lymph node inferior mesenteric axis metastases, FIr-B/FOx was re-introduced, liver and lymph node resections were performed, with a PFS 8 months and PFI 3 months. FIr-B/FOx was further proposed due to bilateral lung, and liver metastases with stable disease, PFS 8 months. Patient experienced a limiting toxicity syndrome multiple sites (LTS-ms) with G3 diarrhea, G2 asthenia, nausea, requiring irinotecan reduction and 5-fluorouracil discontinuation, and subsequent oxaliplatin discontinuation, due to infusional hypersensitivity reaction. Overall, integrated first-line medical and surgical treatment strategies gained PFS 26 months. Further lines II-V of treatment obtained a combined PFS 28 months: modulated aflibercept/irinotecan, PFS 8 months; panitumumab, PFS 8 months, proposed due to KRAS/NRAS/BRAF wild-type and EGFR c.2156 G>C (p.G719A) mutation, achieving biomarkers reduction, lung, liver, lymph nodes partial responses; regorafenib, PFS 8 months; trifluridine-tipiracil, PFS 4 months and induced an LTS-ms, with febrile G4 leucopenia, G3 neutropenia, thrombocytopenia, asthenia, G2 anemia, diarrhea, hypotension. After 2 months of palliative care, patient died, at OS 58 months, gained by intensive medical/surgical treatments coupled with patient's resilience. To date, selection of tailored medical treatments, according to clinical (age, performance and comorbidity status) and molecular (RAS/BRAF and pharmacogenomic analyses) evaluations, careful monitoring of individual toxicity syndromes, potential integration of metastasectomies, and furthermore individual resilience as patient life priority need to challenge MCRC long-term survival.

Genome Sequences of Three SARS-CoV-2 P.1 Strains Identified from Patients Returning from Brazil to Italy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. We report the complete sequences of three SARS-CoV-2 P.1 strains obtained from nasopharyngeal swab specimens from three patients returning from Brazil to Italy.

Calibration of Pathogenicity Due to Variant-Induced Leaky Splicing Defects by Using BRCA2 Exon 3 as a Model System.

BRCA2 is a clinically actionable gene implicated in breast and ovarian cancer predisposition that has become a high priority target for improving the classification of variants of unknown significance (VUS). Among all BRCA2 VUS, those causing partial/leaky splicing defects are the most challenging to classify because the minimal level of full-length (FL) transcripts required for normal function remains to be established. Here, we explored BRCA2 exon 3 (BRCA2e3) as a model for calibrating variant-induced spliceogenicity and estimating thresholds for BRCA2 haploinsufficiency. In silico predictions, minigene splicing assays, patients' RNA analyses, a mouse embryonic stem cell (mESC) complementation assay and retrieval of patient-related information were combined to determine the minimal requirement of FL BRCA2 transcripts. Of 100 BRCA2e3 variants tested in the minigene assay, 64 were found to be spliceogenic, causing mild to severe RNA defects. Splicing defects were also confirmed in patients' RNA when available. Analysis of a neutral leaky variant (c.231T>G) showed that a reduction of approximately 60% of FL BRCA2 transcripts from a mutant allele does not cause any increase in cancer risk. Moreover, data obtained from mESCs suggest that variants causing a decline in FL BRCA2 with approximately 30% of wild-type are not pathogenic, given that mESCs are fully viable and resistant to DNA-damaging agents in those conditions. In contrast, mESCs producing lower relative amounts of FL BRCA2 exhibited either null or hypomorphic phenotypes. Overall, our findings are likely to have broader implications on the interpretation of BRCA2 variants affecting the splicing pattern of other essential exons. SIGNIFICANCE: These findings demonstrate that BRCA2 tumor suppressor function tolerates substantial reduction in full-length transcripts, helping to determine the pathogenicity of BRCA2 leaky splicing variants, some of which may not increase cancer risk.

KRAS, NRAS and BRAF mutations detected by next generation sequencing, and differential clinical outcome in metastatic colorectal cancer (MCRC) patients treated with first line FIr-B/FOx adding bevacizumab (BEV) to triplet chemotherapy.

BACKGROUND: First line triplet chemotherapy/BEV significantly improved clinical outcome of MCRC. KRAS/NRAS/BRAF mutations were evaluated by next generation sequencing (NGS) in MCRC patients treated with first line FIr-B/FOx. METHODS: KRAS exons 2-4 (KRAS2-4 ), NRAS2-4 , BRAF15 were evaluated in 67 tumours by ION Torrent platform. Mutation detection criteria: >500×sequence coverage (cov); >1% mutant allelic fraction (AF). Clinical outcomes were compared by log-rank. RESULTS: In 63 samples, KRAS2-4 /NRAS2-4 /BRAF15 wild-type (wt) were 14 (22.2%), mutant (mut) 49 (77.8%): KRAS2-4 42 (66.7%); NRAS2-4 11 (16.4%); BRAF15 5 (7.5%). Sixty mutations were detected, range 1-3 mut: 43 (71.7%) >1000×cov/>5% AF; 9 (15%) >500×cov/>5% AF; 8 (13.3%) >1000×cov/<5% AF. Mut distribution in KRAS2-4 /NRAS2-4 /BRAF15 : 40 (63.5%) >1000×cov/>5% AF, 8 (12.7%) >500×cov/>5% AF, 1 (1.6%) >1000×cov/<5% AF; BRAF15 1 (1.5%) >500×cov/>5% AF, 4 (6%) >1000×cov/<5% AF. Prevalence of ≥2 mut samples: KRAS2-4 /NRAS2-4 /BRAF15 8 (12.7%); KRAS2-4 7 (11.1%); NRAS2-4 5 (7.5%). BRAF15 mutant were all ≥2 mut (7.5%), atypical and associated to KRAS and/or NRAS mut: c.1405 G>A; c.1406 G>C; c.1756 G>A, 2 samples; c.1796 C>T. At 21 months (m) follow-up, clinical outcome wt compared to mut was not significantly different: in KRAS2-4 /NRAS2-4 /BRAF15 , progression-free survival (PFS) 18/12 m, overall survival (OS) 28/22 m; 1/≥2 mutations, PFS 14/11, OS 37/22. PFS was trendy worse in RAS/BRAF wt vs ≥2 mut genes (P 0.059). CONCLUSIONS: Most MCRC harboured KRAS2-4 /NRAS2-4 /BRAF15 mutations by NGS, often multiple and affecting few tumoral clones; 22% were triple wt. Clinical outcome is not significantly affected by KRAS2-4 /NRAS2-4 /BRAF15 genotype, trendy different in triple wt, compared with KRAS2-4 /NRAS2-4 /BRAF15 ≥2 mut.

Functional analysis of a large set of BRCA2 exon 7 variants highlights the predictive value of hexamer scores in detecting alterations of exonic splicing regulatory elements.

Exonic variants can alter pre-mRNA splicing either by changing splice sites or by modifying splicing regulatory elements. Often these effects are difficult to predict and are only detected by performing RNA analyses. Here, we analyzed, in a minigene assay, 26 variants identified in the exon 7 of BRCA2, a cancer predisposition gene. Our results revealed eight new exon skipping mutations in this exon: one directly altering the 5' splice site and seven affecting potential regulatory elements. This brings the number of splicing regulatory mutations detected in BRCA2 exon 7 to a total of 11, a remarkably high number considering the total number of variants reported in this exon (n = 36), all tested in our minigene assay. We then exploited this large set of splicing data to test the predictive value of splicing regulator hexamers' scores recently established by Ke et al. (). Comparisons of hexamer-based predictions with our experimental data revealed high sensitivity in detecting variants that increased exon skipping, an important feature for prescreening variants before RNA analysis. In conclusion, hexamer scores represent a promising tool for predicting the biological consequences of exonic variants and may have important applications for the interpretation of variants detected by high-throughput sequencing.

Worse prognosis of KRAS c.35 G > A mutant metastatic colorectal cancer (MCRC) patients treated with intensive triplet chemotherapy plus bevacizumab (FIr-B/FOx).

BACKGROUND: Prognosis of KRAS wild-type and mutant metastatic colorectal cancer (MCRC) patients (pts) treated with bevacizumab (BEV)-containing chemotherapy is not significantly different. Since specific KRAS mutations confer different aggressive behaviors, the prognostic role of prevalent KRAS mutations was retrospectively evaluated in MCRC pts treated with first line FIr-B/FOx, associating BEV to triplet chemotherapy. METHODS: Tumor samples were screened for KRAS codon 12, 13 and BRAF V600E mutations by SNaPshot and/or direct sequencing. MCRC pts <75-years-old were consecutively treated with FIr-B/FOx: weekly 12 hour-timed-flat-infusion/5-fluorouracil (900 mg/m(2) on days 1,2, 8, 9, 15, 16,22, 23), irinotecan plus BEV (160 mg/m(2) and 5 mg/kg, respectively, on days 1,15); and oxaliplatin (80 mg/m(2), on days 8,22). Pts were classified as liver-limited (L-L) and other/multiple metastatic (O/MM). Progression-free survival (PFS) and overall survival (OS) were compared using the log-rank test. RESULTS: Fifty-nine pts were evaluated at a median follow-up of 21.5 months. KRAS mutant pts: c.35 G > A, 15 (25.4%); c.35 G > T, 7 (11.8%); c.38 G > A, 3 (5%); other, 3 (5%). KRAS wild-type, 31 pts (52.7%). The objective response rate (ORR), PFS and OS were, respectively: c.35 G > A mutant, 71%, 9 months, 14 months; other than c.35 G > A mutants, 61%, 12 months, 39 months. OS was significantly worse in c.35 G > A pts compared to KRAS wild-type (P = 0.002), KRAS/BRAF wild-type (P = 0.03), other MCRC patients (P = 0.002), other than c.35 G > A (P = 0.05), other codon 12 (P = 0.03) mutant pts. OS was not significantly different compared to c.35 G > T KRAS mutant (P = 0.142). CONCLUSIONS: KRAS c.35 G > A mutant status may be significantly associated with a worse prognosis of MCRC pts treated with first line FIr-B/FOx intensive regimen compared to KRAS/BRAF wild type and other than c.35 G > A mutant pts.

Prognostic value of KRAS genotype in metastatic colorectal cancer (MCRC) patients treated with intensive triplet chemotherapy plus bevacizumab (FIr-B/FOx) according to extension of metastatic disease.

BACKGROUND: Bevacizumab (BEV) plus triplet chemotherapy can increase efficacy of first-line treatment of metastatic colorectal cancer (MCRC), particularly integrated with secondary liver surgery in liver-limited (L-L) patients. The prognostic value of the KRAS genotype in L-L and other or multiple metastatic (O/MM) MCRC patients treated with the FIr-B/FOx regimen was retrospectively evaluated. METHODS: Tumoral and metastatic samples were screened for KRAS codon 12 and 13 and BRAF mutations by SNaPshot and/or direct sequencing. Fit MCRC patients <75 years were consecutively treated with FIr-B/FOx regimen: weekly 12-h timed flat-infusion/5-fluorouracil (TFI 5-FU) 900 mg/m2, days 1, 2, 8, 9, 15, 16, 22 and 23; irinotecan (CPT-11) 160 mg/m2 plus BEV 5 mg/kg, days 1, 15; oxaliplatin (OXP) 80 mg/m2, days 8, 22; every 4 weeks. MCRC patients were classified as L-L and O/MM. Activity and efficacy were evaluated and compared using log-rank test. RESULTS: In all, 59 patients were evaluated: 31 KRAS wild-type (53%), 28 KRAS mutant (47%). At 21.5 months median follow-up, objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) were, respectively: KRAS wild-type 90%, 14 months, 38 months; KRAS mutant 67%, 11 months, 20 months. PFS and OS were not significantly different. PFS and OS were significantly different in L-L compared to O/MM evaluable patients. In KRAS wild-type patients, clinical outcome of 12 L-L compared to 18 O/MM was significantly different: PFS 21 versus 12 months and OS 47 versus 28 months, respectively. In KRAS mutant patients, the clinical outcome of 13 L-L compared to 14 O/MM was not significantly different: PFS 11 months equivalently and OS 39 versus 19 months, respectively. CONCLUSIONS: The KRAS genotype wild-type and mutant does not significantly affect different clinical outcomes for MCRC patients treated with the first-line FIr-B/FOx intensive regimen. KRAS wild-type patients with L-L disease may achieve a significantly prolonged clinical outcome due to integration with secondary liver surgery, with respect to KRAS mutant patients.

Multiple sequence variants of BRCA2 exon 7 alter splicing regulation.

BACKGROUND: Exonic variants of unknown biological significance (VUS) identified in patients can affect mRNA splicing, either by changing 5' or 3' splice sites or by modifying splicing regulatory elements. Bioinformatic predictions of these elements are still inaccurate and only few such elements have been functionally mapped in BRCA2. We studied the effect on splicing of eight exon 7 VUS, selected from the French UMD-BRCA2 mutation database. METHODS: We performed splicing minigene assays and analyses of patient RNA. We also developed a pyrosequencing-based quantitative assay, to measure, in patient RNA, the relative contribution of each allele to the production of exon 7-containing transcripts. Moreover, an exonic splicing enhancer (ESE)-dependent minigene assay was used to evaluate the splicing regulatory properties of wild-type and mutant segments. RESULTS: Six out of the eight variants induced splicing defects. In the minigene assay, c.517G>T and c.631G>A altered the natural splice sites, c.572A>G created a new 5' splice site, and c.520C>T, c.587G>A and c.617C>G induced exon 7 skipping (66%, 25% and 46%, respectively). Pyrosequencing of patient RNA confirmed these levels of exon skipping for c.520C>T and c.617C>G. Results from the ESE-dependent minigene assay indicated that c.520C>T and c.587G>A disturb splicing regulatory elements. CONCLUSIONS: BRCA2 exon 7 splicing is regulated by multiple exonic elements and is sensitive to disease-associated sequence variations. Measurements of allelic imbalance in patient-derived RNA and/or quantitative analyses using minigene assays provide valuable estimates of the extent of partial splicing defects. Assessment of pathogenicity of variants with partial splicing effect awaits additional evidence and especially the completion of segregation analyses.