Impact of the time interval between primary or interval surgery and adjuvant chemotherapy in ovarian cancer patients.

Introduction: Primary debulking surgery (PDS), interval debulking surgery (IDS), and platinum-based chemotherapy are the current standard treatments for advanced ovarian cancer (OC). The time to initiation of adjuvant chemotherapy (TTC) could influence patient outcomes. Methods: We conducted a multicenter retrospective cohort study of advanced (International Federation of Gynecology and Obstetrics (FIGO) stage III or IV) OC treated between 2014 and 2018 to assess progression-free survival (PFS) and overall survival (OS) in relation to TTC. All patients underwent a germline multigene panel for BRCA1/2 evaluation. Results: Among the 83 patients who underwent PDS, a TTC ≥ 60 days was associated with a shorter PFS (hazard ratio (HR) 2.02, 95% confidence interval (CI) 1.04-3.93, p = 0.038), although this association lost statistical significance when adjusting for residual disease (HR 1.52, 95% CI 0.75-3.06, p = 0.244, for TTC and HR 2.73, 95% CI 1.50-4.96, p = 0.001, for residual disease). Among 52 IDS patients, we found no evidence of an association between TTC and clinical outcomes. Ascites, type of chemotherapy, or germline BRCA1/2 mutational status did not influence TTC and were not associated with clinical outcomes in PDS or IDS patients. Discussion: In conclusion, longer TTC seems to negatively affect prognosis in patients undergoing PDS, especially those with residual disease.

A Novel FLCN Variant in a Suspected Birt-Hogg-Dubè Syndrome Patient.

Subjects with pathogenic (PV) and likely pathogenic (LPV) FLCN variants have an increased risk of manifesting benign and malignant disorders that are related to Birt-Hogg-Dubé syndrome (BHDS): an autosomal dominantly inherited disorder whose severity can vary significantly. Renal cell carcinoma (RCC) development in BHD (Birt-Hogg-Dubé) patients has a very high incidence; thus, identifying this rare syndrome at early stages and preventing metastatic spread is crucial. Over the last decade, the advancement of Next Generation Sequencing (NGS) and the implementation of multigene panels for hereditary cancer syndromes (HCS) have led to a subsequent focus on additional genes and variants, including those of uncertain significance (VUS). Here, we describe a novel FLCN variant observed in a subject manifesting disorders that were suspected to be related to BHDS and with a family history of multiple cancers.

Ovarian cancer pathology characteristics as predictors of variant pathogenicity in BRCA1 and BRCA2.

BACKGROUND: The distribution of ovarian tumour characteristics differs between germline BRCA1 and BRCA2 pathogenic variant carriers and non-carriers. In this study, we assessed the utility of ovarian tumour characteristics as predictors of BRCA1 and BRCA2 variant pathogenicity, for application using the American College of Medical Genetics and the Association for Molecular Pathology (ACMG/AMP) variant classification system. METHODS: Data for 10,373 ovarian cancer cases, including carriers and non-carriers of BRCA1 or BRCA2 pathogenic variants, were collected from unpublished international cohorts and consortia and published studies. Likelihood ratios (LR) were calculated for the association of ovarian cancer histology and other characteristics, with BRCA1 and BRCA2 variant pathogenicity. Estimates were aligned to ACMG/AMP code strengths (supporting, moderate, strong). RESULTS: No histological subtype provided informative ACMG/AMP evidence in favour of BRCA1 and BRCA2 variant pathogenicity. Evidence against variant pathogenicity was estimated for the mucinous and clear cell histologies (supporting) and borderline cases (moderate). Refined associations are provided according to tumour grade, invasion and age at diagnosis. CONCLUSIONS: We provide detailed estimates for predicting BRCA1 and BRCA2 variant pathogenicity based on ovarian tumour characteristics. This evidence can be combined with other variant information under the ACMG/AMP classification system, to improve classification and carrier clinical management.

Prevalence of a BRCA2 Pathogenic Variant in Hereditary-Breast-and-Ovarian-Cancer-Syndrome Families with Increased Risk of Pancreatic Cancer in a Restricted Italian Area.

PVs and LPVs in BRCA1/2 genes are correlated to a high risk of developing breast cancer and/or ovarian cancer (Hereditary Breast and Ovarian Cancer syndrome, HBOC); additionally, in recent years, an increasing number of BRCA 1/2 variants have been identified and associated with pancreatic cancer. Epidemiologic studies have highlighted that inherited factors are involved in 10% to 20% of PCs, mainly through deleterious variants of BRCA2. The frequency of BRCA1/2 germline alterations fluctuates quite a lot among different ethnic groups, and the estimated rate of PVs/LPVs variants in Italian HBOC families is not very accurate, according to different reports. The aim of our study is to describe the prevalence of a BRCA2 PV observed in a selected cohort of HBOC patients and their relatives, whose common origin is the eastern coast of Emilia Romagna, a region of Italy. This study provides insight into the frequency of the variant detected in this area and provides evidence of an increased risk of pancreatic and breast cancer, useful for genetic counseling and surveillance programs.

Clinical Impact of Next-Generation Sequencing Multi-Gene Panel Highlighting the Landscape of Germline Alterations in Ovarian Cancer Patients.

BRCA1 and BRCA2 are the most frequently mutated genes in ovarian cancer (OC) crucial both for the identification of cancer predisposition and therapeutic choices. However, germline variants in other genes could be involved in OC susceptibility. We characterized OC patients to detect mutations in genes other than BRCA1/2 that could be associated with a high risk of developing OC and permit patients to enter the most appropriate treatment and surveillance program. Next-generation sequencing analysis with a 94-gene panel was performed on germline DNA of 219 OC patients. We identified 34 pathogenic/likely pathogenic variants in BRCA1/2 and 38 in other 21 genes. The patients with pathogenic/likely pathogenic variants in the non-BRCA1/2 genes mainly developed OC alone compared to the other groups that also developed breast cancer or other tumors (p = 0.001). Clinical correlation analysis showed that the low-risk patients were significantly associated with platinum sensitivity (p < 0.001). Regarding PARP inhibitors (PARPi) response, the patients with pathogenic mutations in the non-BRCA1/2 genes had worse PFS and OS. Moreover, a statistically significantly worse PFS was found for every increase of one thousand platelets before PARPi treatment. To conclude, knowledge about molecular alterations in genes beyond BRCA1/2 in OC could allow for more personalized diagnostic, predictive, prognostic, and therapeutic strategies for OC patients.

Case Report: A BRCA2 Mutation Identified Through Next-Generation Sequencing in a Birt-Hogg-Dubè Syndrome Family.

Birt-Hogg-Dubé syndrome (BHDS) is a rare autosomal dominant inherited disorder caused by a mutation in folliculin (FLCN) gene transmitted via germline autosomal dominant pattern. Patients with this syndrome have an increased susceptibility to renal cell carcinoma, lung cysts, spontaneous pneumothorax, and benign skin hamartomas, and its diagnosis is not easy and consequently underestimated. Several mutations have been identified in FLCN gene, among which the majority of alterations are frameshift (insertion/deletion), nonsense, or splice-site mutations that generally produce unfunctional truncated FLCN proteins. Our aim is to present a case of a BHDS family whose proband is a 56-year-old patient who has been experiencing multiple disorders, has an FLCN genetic mutation, and has also been identified to have a pathogenic variant in BRCA2 gene. Our further purpose is to emphasize the importance of the next-generation sequencing (NGS) approach to identify potential multiple germline mutations in complex and rare oncologic disorders, allowing strict and more targeted cancer screening programs.

Comprehensive analysis of DNA damage repair genes reveals pathogenic variants beyond BRCA and suggests the need for extensive genetic testing in pancreatic cancer.

BACKGROUND: Pancreatic cancer (PC) is a major cause of cancer death. In an effort to improve treatment strategies and outcomes, DNA damage repair (DDR) pathways have been introduced as a new target in PC and in other cancers, through the exploitation of synthetic lethality. Furthermore, genes involved in DDR are among the major determinants of cancer susceptibility. In addition to the well-known BRCA1 and BRCA2 genes, a plethora of other targets in the same pathways are now emerging. METHODS: We analyzed samples from 60 patients, affected by PC and already tested for BRCA, using a panel with 24 other cancer susceptibility genes. RESULTS: We detected 8 pathogenic or likely pathogenic mutations (13.3% of samples analyzed), 4 of which were found in non-BRCA genes (2 in ATM, 1 each in PALB2 and RAD50). Furthermore, 4 pathogenic or likely pathogenic mutations were found in patients without a personal or familial history of cancer. CONCLUSIONS: Our results suggest that genetic testing with a comprehensive gene panel should be perfomed in all patients with PC, in order to allow screening for PC and other gene-related cancers in all at risk family members and to assess patients' eligibility for emerging therapeutic options.

Male Breast Cancer: Results of the Application of Multigene Panel Testing to an Italian Cohort of Patients.

Male breast cancer (MBC) is a rare tumor, accounting for less than 1% of all breast cancers. In MBC, genetic predisposition plays an important role; however, only a few studies have investigated in depth the role of genes other than BRCA1 and BRCA2. We performed a Next-Generation Sequencing (NGS) analysis with a panel of 94 cancer predisposition genes on germline DNA from an Italian case series of 70 patients with MBC. Moreover, we searched for large deletions/duplications of BRCA1/2 genes through the Multiplex Ligation-dependent Probe Amplification (MLPA) technique. Through the combination of NGS and MLPA, we identified three pathogenic variants in the BRCA1 gene and six in the BRCA2 gene. Besides these alterations, we found six additional pathogenic/likely-pathogenic variants in PALB2, CHEK2, ATM, RAD51C, BAP1 and EGFR genes. From our study, BRCA1 and BRCA2 emerge as the main genes associated with MBC risk, but also other genes seem to be associated with the disease. Indeed, some of these genes have already been implicated in female breast cancer predisposition, but others are known to be involved in other types of cancer. Consequently, our results suggest that novel genes could be involved in MBC susceptibility, shedding new light on their role in cancer development.

Insights into Genetic Susceptibility to Melanoma by Gene Panel Testing: Potential Pathogenic Variants in ACD, ATM, BAP1, and POT1.

The contribution of recently established or candidate susceptibility genes to melanoma missing heritability has yet to be determined. Multigene panel testing could increase diagnostic yield and better define the role of candidate genes. We characterized 273 CDKN2A/ARF and CDK4-negative probands through a custom-designed targeted gene panel that included CDKN2A/ARF, CDK4, ACD, BAP1, MITF, POT1, TERF2IP, ATM, and PALB2. Co-segregation, loss of heterozygosity (LOH)/protein expression analysis, and splicing characterization were performed to improve variant classification. We identified 16 (5.9%) pathogenic and likely pathogenic variants in established high/medium penetrance cutaneous melanoma susceptibility genes (BAP1, POT1, ACD, MITF, and TERF2IP), including two novel variants in BAP1 and 4 in POT1. We also found four deleterious and five likely deleterious variants in ATM (3.3%). Thus, including potentially deleterious variants in ATM increased the diagnostic yield to about 9%. Inclusion of rare variants of uncertain significance would increase the overall detection yield to 14%. At least 10% of melanoma missing heritability may be explained through panel testing in our population. To our knowledge, this is the highest frequency of putative ATM deleterious variants reported in melanoma families, suggesting a possible role in melanoma susceptibility, which needs further investigation.

Identification of a novel large EPCAM-MSH2 duplication, concurrently with LOHs in chromosome 20 and X, in a family with Lynch syndrome.

BACKGROUND: Lynch syndrome (LS) is associated with germline mutations in one of the mismatch repair genes or EPCAM. The majority of the causative alterations are point mutations. Large genomic rearrangements represent only 5-20%. Hypothetically, the allelic imbalance, like the loss of heterozygosity, may be another high penetrance risk factor. CASE PRESENTATION: We describe the case of a patient who developed 5 tumors during her lifetime and with a family history characterized by a high frequency of tumors associated with LS. The proband was tested for mutations and copy number alterations with a panel of hereditary cancer genes and by SNP array. She showed a 187 Kb duplication including EPCAM and the first 7 exons of MSH2, plus two loss of heterozygosity (LOHs) in chromosome 20 and one in chromosome X which include many tumor suppressor genes. CONCLUSION: We found a novel large EPCAM-MSH2 duplication associated with LS and the presence of LOHs in regions containing numerous tumor suppressors, raising the hypothesis that these alterations could contribute to cancer susceptibility. Our results underline the importance to deepen the knowledge of molecular mechanisms in order to determine the role in cancer predisposition of novel genetic alterations.

Multigene Panel Testing Increases the Number of Loci Associated with Gastric Cancer Predisposition.

The main gene involved in gastric cancer (GC) predisposition is CDH1, the pathogenic variants of which are associated with diffuse-type gastric cancer (DGC) and lobular breast cancer (LBC). CDH1 only explains a fraction (10-50%) of patients suspected of DGC/LBC genetic predisposition. To identify novel susceptibility genes, thus improving the management of families at risk, we performed a multigene panel testing on selected patients. We searched for germline pathogenic variants in 94 cancer-related genes in 96 GC or LBC Italian patients with early-onset and/or family history of GC. We found CDH1 pathogenic variants in 10.4% of patients. In 11.5% of cases, we identified loss-of-function variants in BRCA1, BRCA2, PALB2, and ATM breast/ovarian cancer susceptibility genes, as well as in MSH2, PMS2, BMPR1A, PRF1, and BLM genes. In 78.1% of patients, we did not find any variants with clear-cut clinical significance; however, 37.3% of these cases harbored rare missense variants predicted to be damaging by bioinformatics tools. Multigene panel testing decreased the number of patients that would have otherwise remained genetically unexplained. Besides CDH1, our results demonstrated that GC pathogenic variants are distributed across a number of susceptibility genes and reinforced the emerging link between gastric and breast cancer predisposition.

Multiple-gene panel analysis in a case series of 255 women with hereditary breast and ovarian cancer.

As new genes predisposing to breast (BC) and ovarian cancer (OC) are constantly emerging, the use of panels of genes analyzed by Next-Generation Sequencing (NGS) is increasing in clinical diagnostics. The identification of a large number of new germline mutations allows for deeper knowledge of cancer predisposition, although raising many questions about patient management.BC and OC patients recruited by our counseling service between 2012-2015 were included in this study. DNA was extracted from peripheral blood and a panel of 94 genes involved in hereditary tumors was analyzed by NGS. Patient clinical features of BC and OC and cancer family history were collected and compared to the patient genetic profile.A total of 255 women were analyzed, 57 of whom had a pathogenic mutation in BRCA1/2 genes, and 17 carried pathogenic mutations in other genes, such as PALB2, ATM, BRIP1, RAD51D, MSH6, PPM1D, RECQL4, ERCC3, TSC2, SLX4 and other Fanconi anemia genes.Patients with a pathogenic mutation in genes other than BRCA1 and BRCA2 showed no significant difference from the BRCA1/2-mutated carriers with respect to age at diagnosis and clinical features, suggesting that mutations in other genes could pose a high risk of cancer development.These patients had a much higher percentage of bilateral breast cancer (BBC) and a lower rate of OC than BRCA-mutated patients and patients with no pathogenic mutations: as a consequence, the surveillance protocol should be customized to the patient genetic characteristics.

BRCA1 p.His1673del is a pathogenic mutation associated with a predominant ovarian cancer phenotype.

We have investigated the clinical significance of the BRCA1 variant p.His1673del in 14 families from the Emilia-Romagna region of Italy, including 20 breast and 23 ovarian cancer cases; four families displayed site-specific ovarian cancer.The variant, absent in human variation databases, has been reported three times in BRCA1 specific databases; all probands shared the same rare haplotype at the BRCA1 locus, consistent with a common ancestor.The multifactorial likelihood method by Goldgar, used to estimate the probability of the variant being causative, gave a ratio of 2,263,474:1 in favor of causality. Moreover, in silico modeling suggested that His1673-lacking BRCA1 protein may have a decreased ability to bind BARD1 and other related proteins. All six ovarian carcinomas and two out of four breast carcinomas available showed a loss of the BRCA1 wild-type allele, which in three out of four ovarian carcinomas analyzed by FISH was associated with duplication of the chromosome 17 containing the variant. Although the pathogenicity of the allele is strongly supported by the multifactorial ratio,we cannot exclude that p.His1673del is not itself deleterious, but is linked to another undetected mutation on the same ancestral allele.

Multiple primary tumors in a family with Li-Fraumeni syndrome with a TP53 germline mutation identified by next-generation sequencing.

Li-Fraumeni syndrome (LFS) is an autosomal dominant disorder occurring at a young age that predisposes individuals to multiple forms of cancer and to a heterogeneous spectrum of malignancies. We describe the clinical history of a patient who had 5 primary malignant cancers and a familiar history consistent with LFS. We analyzed the genomic DNA of the proband and her relatives by next-generation sequencing (NGS) technology using an enrichment protocol for the simultaneous sequencing of 94 genes involved in hereditary cancers. Genetic analysis of the proband revealed a TP53 germline mutation in exon 5 determining a nucleotide alteration at codon 175 (R175H), a hot spot mutation site related to LFS and a reported pathogenic mutation. The proband daughter's and brother's DNA did not carry the TP53 mutation but they had some rare variants in common with the proband, in addition to other variants with a still unclear role. In conclusion, we identified a TP53 mutation in a patient with multiple primary tumors and a family history characterized by a severe susceptibility to cancer. The genetic analysis by targeted NGS led to the identification of the genetic background and to the exclusion of a cancer risk for the family members. Targeted NGS represents an efficient approach for the identification of mutations in families with a heterogeneous phenotype.

First evidence of a large CHEK2 duplication involved in cancer predisposition in an Italian family with hereditary breast cancer.

BACKGROUND: CHEK2 is a multi-cancer susceptibility gene whose common germline mutations are known to contribute to the risk of developing breast and prostate cancer. CASE PRESENTATION: Here, we describe an Italian family with a high number of cases of breast cancer and other types of tumour subjected to the MLPA test to verify the presence of BRCA1, BRCA2 and CHEK2 deletions and duplications. We identified a new 23-kb duplication in the CHEK2 gene extending from intron 5 to 13 that was associated with breast cancer in the family. The presence and localisation of the alteration was confirmed by a second analysis by Next-Generation Sequencing. CONCLUSIONS: This finding suggests that CHEK2 mutations are heterogeneous and that techniques other than sequencing, such as MLPA, are needed to identify CHEK2 mutations. It also indicates that CHEK2 rare variants, such as duplications, can confer a high susceptibility to cancer development and should thus be studied in depth as most of our knowledge of CHEK2 concerns common mutations.

Disease family history and modification of breast cancer risk in common BRCA2 variants.

A number of BRCA1 and BRCA2 polymorphisms have been extensively studied in order to test their association with breast cancer risk. Subsequently, discordant results were reported. In the present study, the genotypes of one BRCA1 (Q356R) and three BRCA2 (203G>A, N372H, IVS21-66T>C) common variants were evaluated in a series of 252 breast cancer patients, 155 age-matched controls and analysed in relation to family history (low- or high-risk) and BRCA1/2 mutation status. A complete analysis of the BRCA1/2 coding regions was performed on the 217 women from high-risk families and 44 BRCA1/2 mutation carriers were identifed. According to a dominant inheritance model, the BRCA2 IVS21-66T>C variant showed a 1.79-fold (95% CI, 1.16-2.78; P=0.009) increased breast cancer risk for the overall series. The BRCA2 N372H polymorphism was associated with a 2.29-fold (95% CI, 1.16-4.49; P=0.016) increased risk in the subgroup of high-risk families with no BRCA1/2 mutations. Conversely, the BRCA1 Q356R and BRCA2 203G>A polymorphisms did not show any significant associations with breast cancer risk. In conclusion, the analysis of some BRCA2 variants could help to identify women at a higher risk of developing breast cancer who could be candidates for chemoprevention protocols.

Results of a population-based screening for hereditary breast cancer in a region of North-Central Italy: contribution of BRCA1/2 germ-line mutations.

BRCA1/2 mutation status is of paramount importance to identify families at risk of Hereditary Breast and Ovarian Cancer (HBOC). Most HBOC and BRCA1/2 mutation studies have focused on highly selected sub-populations, and few data are available for large population cohorts. For this reason, as part of a regional cancer prevention strategy in North-Central Italy, we set up a population-based screening programme to identify all resident HBOC families, and to determine their BRCA1/2 mutation status. To date, 44 different BRCA1/2 variants have been identified in 55 HBOC families. Of the seven newly reported mutations, only BRCA1 Q284X is clearly deleterious. The analysis of clinical disease characteristics in relation to age of disease onset and family history showed a difference between BRCA1/2 wild type and mutation carrier families. Interestingly, BRCA1/2 mutations were significantly more common in women who developed breast cancer