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Supp Tables S1-S6 (including variant classification, bioinformatics predictions versus experimental data, primer sequences, summary of biological, clinical, tumoral, familial and multifactorial data, and description of statistical analysis approaches)

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.

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.

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.

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.

// Gemma Bruera 1, 2, * , Francesco Pepe 3, * , Umberto Malapelle 3 , Pasquale Pisapia 3 , Antonella Dal Mas 4 , Daniela Di Giacomo 1, 2 , Giuseppe Calvisi 4 , Giancarlo Troncone 3 and Enrico Ricevuto 1, 2 1 Oncology Territorial Care, S. Salvatore Hospital, Oncology Network ASL1 Abruzzo, University of L'Aquila, L'Aquila, Italy 2 Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila, L’Aquila, Italy 3 Department of Public Health, University Federico II, Napoli, Italy 4 Pathology, S. Salvatore Hospital, Oncology Network ASL1 Abruzzo, L'Aquila, Italy * These authors equally contributed to this work Correspondence to: Enrico Ricevuto, email: enrico.ricevuto@univaq.it Keywords: FIr-B/FOx intensive first line triplet chemotherapy plus bevacizumab; metastatic colorectal cancer; next generation sequencing; RAS/BRAF mutations; 50 genes panel Received: October 06, 2017 Accepted: April 05, 2018 Published: May 29, 2018 ABSTRACT 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 ( KRAS 2-4 ), NRAS 2-4 , BRAF 15 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, KRAS 2-4 / NRAS 2-4 / BRAF 15 wild-type (wt) were 14 (22.2%), mutant (mut) 49 (77.8%): KRAS 2-4 42 (66.7%); NRAS 2-4 11 (16.4%); BRAF 15 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/ 1000×cov/>5% AF, 8 (12.7%) >500×cov/>5% AF, 1 (1.6%) >1000×cov/ 500×cov/>5% AF, 4 (6%) >1000×cov/ 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 KRAS 2-4 / NRAS 2-4 / BRAF 15 , 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 KRAS 2-4 / NRAS 2-4 / BRAF 15 mutations by NGS, often multiple and affecting few tumoral clones; 22% were triple wt. Clinical outcome is not significantly affected by KRAS 2-4 / NRAS 2-4 / BRAF 15 genotype, trendy different in triple wt, compared with KRAS 2-4 / NRAS 2-4 / BRAF 15 ≥2 mut.

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.

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 2 on days 1,2, 8, 9, 15, 16,22, 23), irinotecan plus BEV (160 mg/m2 and 5 mg/kg, respectively, on days 1,15); and oxaliplatin (80 mg/m2, 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.

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 2, 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.

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.

e14596 Background: Prognosis of MCRC patients (pts) treated with bevacizumab (BEV) and chemotherapy (CT) is not significantly different according to KRAS genotype. Specificmutations confer different aggressiveness. Prognostic relevance of the prevalent c.35 G > A KRAS mutation was retrospectively evaluated in pts treated with first line FIr-B/FOx, and post-progression. Methods: KRAS codon 12/13 and BRAF mutations were screened by SNaPshot and/or sequencing. FIr-B/FOx: weekly 2 days/12h-timed-flat-infusion/5-fluorouracil 900 mg/m2, weekly alternating irinotecan 160 mg/m2/BEV 5 mg/kg, or oxaliplatin 80 mg/m2. Second-line treatment was selected according to fitness, KRAS genotype, previous efficacy and safety. Efficacy was evaluated and compared according to treatment or KRAS genotype, using log-rank. Results: At 21.5 months, 59 pts were evaluated. 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%). ORR, PFS, OS were, respectively: c.35 G > A, 71%, 9 months, 14 months; other 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/BRAFwild-type (p = 0.03), other pts (p = 0.002), other mutants (p = 0.05), other codon 12 (p = 0.03) mutant pts; PFS was not significantly different. Post-progression, at 14 months follow-up, PFS and OS were significantly worse in c.35 G > A compared to wild-type and/or other mutant pts. ORR of second line treatments was 38%, metastasectomies 12.5%, PFS 10 months, OS 14 months. PFS and OS were significantly favourable in pts treated with triplet CT plus targeted agent compared to triplet, respectively: PFS 13 months versus 8 months; OS not reached versus 11 months. Conclusions: KRAS c.35 G > A mutation may significantly affect worse OS of MCRC pts. It does not significantly affect worse PFS of intensive first line FIr-B/FOx, while after progression significantly affect worse efficacy of second line treatments. Re-challenge of intensive regimens may affect significantly favourable PFS and OS.

e14010 Background: Bevacizumab (BEV) plus triplet chemotherapy can increase efficacy of first-line treatment of MCRC, particularly if integrated with secondary liver surgery in liver-limited (L-L) patients (pts). Clinical outcome of FIr-B/FOx regimen was evaluated according to KRAS genotype in L-L and other MCRC pts. Methods: Tumoral and metastatic samples were screened for KRAS codon 12 and 13, and BRAF mutations by SNaPshot and/or direct sequencing. MCRC pts were classified as L-L and other or multiple metastatic (O/MM). Activity and efficacy were evaluated and compared using log-rank test. Results: Fifty-nine pts 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, respectively: overall in 25 L-L compared to 32 O/MM evaluable pts, 17 and 12 months, 47 and 21 months, significantly different; in KRAS wild-type, 12 L-L compared to 18 O/MM, 21 and 12 months, 47 and 28 months, significantly different; in KRAS mutant, 13 L-L compared to 14 O/MMS, 11 months equivalently, 39 and 19 months, not significantly different. Conclusions: First line FIr-B/FOx regimen can increase activity and efficacy of KRAS wild-type and mutant MCRC pts; integration with secondary liver surgery significantly discriminates increased clinical outcome in KRAS wild-type L-L compared to O/MM pts while not in KRAS mutant pts.

521 Background: Bevacizumab (BEV) addition to doublet chemotherapy significantly increases efficacy without differentially affecting prognosis in KRAS wild-type (wt) and mutant (m) MCRC. Present study evaluates clinical outcome of BEV added to triplet chemotherapy, FIr-B/FOx (Bruera G et al, BMC Cancer 2010, 10:567), according to KRAS genotype. Methods: MCRC patients (pts) were treated with first line FIr-B/FOx regimen including BEV (5 mg/kg, days 1,15) and triplet chemotherapy, weekly alternating irinotecan (160 mg/m2 days 1,15) or oxaliplatin (80 mg/m2, days 8, 22) associated to fluorouracil (900 mg/m2, days 1-2, 8-9, 15-16, 22-23); every 4 weeks. Tumoral samples were simultaneously screened for KRAS codon 12 and 13, and BRAF c.1799 T > A mutations by SNaPshot multiplex assay (Di Fiore F, Br J Cancer 2007;96(8):1166-1169) and/or direct sequencing. Activity and efficacy were evaluated and compared using log-rank test. Results: Forty-five MCRC pts were evaluated: 25 KRAS wt (56%), 20 KRAS m (44%). After 30 months median follow-up, objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) were, respectively: KRAS wt 88% (C.I. ± 14), 14 months, 38 months; KRAS m 80% (C.I. ± 19), 12 months, 21 months. PFS and OS were not significantly different. Among KRAS m pts, ORR, PFS and OS at 19 months median follow-up in c.35 G > A (13 pts) were 69% (C.I. ± 26), 9 months, 11 months, respectively. OS of c.35 G > A KRAS m was significantly worse compared to KRAS wt (p = 0.003) and to other than c.35 G > A mutant pts (p = 0.022). Conclusions: First line FIr-B/FOx intensive regimen adding BEV to triplet chemotherapy can further increase activity and efficacy without significantly affecting different clinical outcome in KRAS wt and m MCRC pts. A significant interaction between the most prevalent c.35 G > A KRAS mutation and worse prognosis was observed, compared to KRAS wt pts and to other than c.35 G > A KRAS exon 2 mutations, depending from different biological aggressiveness and sensitivity to BEV addition to triplet chemotherapy.

Introduction: Bevacizumab (BEV) addition to doublet chemotherapy significantly increases efficacy without differentially affecting prognosis in KRAS wild-type (wt) and mutant (m) MCRC. Present study evaluates clinical outcome of BEV added to triplet chemotherapy, FIr-B/FOx (Bruera G et al, BMC Cancer 2010, 10:567), according to KRAS genotype. Methods: MCRCpatients (pts) were treated with first line FIr/FOx regimen including BEV and triplet chemotherapy (weekly alternating irinotecan or oxaliplatin associated to fluorouracil). Treatment schedule: weekly 12h-timed-flat-infusion/5-fluorouracil 900 mg/m2, days 1–2, 8–9, 15–16, 22–23; irinotecan 160 mg/m2 plus BEV 5 mg/kg, days 1,15; oxaliplatin, 80 mg/m2, days 8, 22; every 4 weeks. Tumoral samples were simultaneously screened for KRAS codon 12 and 13, and BRAF c.1799 T > A mutations by SNaPshot multiplex assay (Di Fiore F, Br J Cancer 2007;96(8):1166–1169) and/or direct sequencing. Activity and efficacy were evaluated and compared using log-rank test. Results: Forty-five MCRC pts were evaluated: 25 KRAS wt (56%), 20 KRAS m (44%). After 30 months median follow-up, objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) were, respectively: KRAS wt 88% (C.I. × 14), 14 months, 38 months; KRAS m 80% (C.I. × 19), 12 months, 21 months. PFS and OS were not significantly different. Among KRAS m pts, ORR, PFS and OS at 19 months median follow-up in c.35 G > A (13 pts) were 69% (C.I. × 26), 9 months, 11 months, respectively. OS of c.35 G > A KRAS m was significantly worse compared to KRAS wt (p = 0.003) and to other than c.35 G > A mutant pts (p = 0.022). Conclusions: First line FIr-B/FOx intensive regimen adding BEV to triplet chemotherapy can further increase activity and efficacy without significantly affecting different clinical outcome in KRAS wt and m MCRC pts. For the first time in MCRC pts treated with BEV-containing chemotherapy, present study shows a significant interaction between the most prevalent c.35 G > A KRAS mutation and worse prognosis compared to KRAS wt pts and to other than c.35 G > A KRAS exon 2 mutations, depending from different biological aggressiveness and sensitivity to BEV addition to triplet chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C5.