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.

The increasing knowledge of the molecular pathogenesis of cancer and the rapid development of new molecular techniques are promoting the study of early molecular alterations involved in cancer development in body fluids. Specific genetic and epigenetic alterations could be found in plasma, serum, and urine cell-free DNA (cfDNA) and could potentially be used as diagnostic biomarkers for several types of cancers. This review focuses on the role of cfDNA in diagnosis: a PubMed search was performed by selecting papers according to journal impact factor and robustness of statistical analysis. A comprehensive evaluation of “liquid biopsy”, including cfDNA analysis, will be one of the critical challenges to better understand the early mechanisms of cancer development.

Breast and ovarian cancers are some of the most common tumors in females, and the genetic predisposition is emerging as one of the key risk factors in the development of these two malignancies. BRCA1 and BRCA2 are the best-known genes associated with hereditary breast and ovarian cancer. However, recent advances in molecular techniques, Next-Generation Sequencing in particular, have led to the identification of many new genes involved in the predisposition to breast and/or ovarian cancer, with different penetrance estimates. TP53, PTEN, STK11, and CDH1 have been identified as high penetrance genes for the risk of breast/ovarian cancers. Besides them, PALB2, BRIP1, ATM, CHEK2, BARD1, NBN, NF1, RAD51C, RAD51D and mismatch repair genes have been recognized as moderate and low penetrance genes, along with other genes encoding proteins involved in the same pathways, possibly associated with breast/ovarian cancer risk. In this review, we summarize the past and more recent findings in the field of cancer predisposition genes, with insights into the role of the encoded proteins and the associated genetic disorders. Furthermore, we discuss the possible clinical utility of genetic testing in terms of prevention protocols and therapeutic approaches. the recruitment of the recombinase RAD51 to the DNA double-strand breaks (DSBs) through the formation of a BRCA1-PALB2-BRCA2 complex. The BRCA2 protein contains a helical domain, three oligonucleotide binding domains, and a tower domain, which allow BRCA2 binding to both single-stranded DNA and double-stranded DNA [39,48,49].Germline PV/LPVs in the BRCA2 gene are associated with a 45-55% and 11-18% risk of developing BC and OC by the age of 70, respectively [20][21][22]. BRCA2 PV/LPVs have also been associated with an increased risk of BC in males, which is estimated at 6.8% by the age of 70 [43]. In addition, BRCA2 PV/LPVs have been associated with an increased risk of prostate cancer [50], pancreatic cancer [47,51], and uveal melanoma [52,53].According to the National Comprehensive Cancer Network (NCCN) guidelines, women with BRCA1/2 PV/LPVs should undergo a surveillance protocol, including clinical breast examination every 6-12 months and annual breast magnetic resonance imaging (MRI), starting at the age of 25, annual mammography with consideration of tomosynthesis, starting at the age of 30, and annual transvaginal ultrasound and serum CA-125 concentration, although of uncertain benefit, beginning at age 30-35 years [54]. Moreover, they should evaluate the opportunity of a bilateral risk-reducing mastectomy (RRM) and of a bilateral risk-reducing salpingo-oophorectomy (RRSO), typically at between 35 and 40 years and upon completion of childbearing [54]. Men with BRCA1/2 PV/LPVs should undergo clinical breast examination every 6-12 months, starting at the age of 35, and annual prostate cancer screening, starting at the age of 40 (in particular in BRCA2 PV/LPV carriers) [55]. In both sexes, screening for melanoma and pancreatic cancer should be evaluated on the basis of family...

HER2-positive advanced gastric cancer patients frequently develop resistance to trastuzumab through mechanisms still poorly understood. In breast cancer, other members of the HER-family are known to be involved in trastuzumab-resistance, as is overexpression of the scaffold protein IQGAP1. In the present work, we investigated acquired resistance to trastuzumab in gastric cancer experimental models. Trastuzumab-resistant (HR) subclones derived from 3 HER2-overexpressing gastric cancer cells were generated and characterized for alterations in HER2-signaling mechanisms by next-generation sequencing, immunohistochemical, western blot and qRT-PCR techniques, and molecular modeling analysis. All subclones showed a reduced growth rate with respect to parental cell lines but each had a different resistance mechanism. In NCI N87 HR cells, characterized by a marked increase in HER2-signaling pathways with respect to the parental cell line, trastuzumab sensitivity was restored when IQGAP1 expression was silenced. AKG HR subclone showed higher HER3 protein expression than the parental line. High nuclear HER4 levels were observed in KKP HR cells. In conclusion, our study revealed that high IQGAP1 expression leads to resistance to trastuzumab in gastric cancer. Furthermore, 2 new mutations of the HER2 gene that may be involved in acquired resistance were identified in AKG HR and KKP HR subclones.

CDH1 gene, encoding E-cadherin, is a tumor suppressor gene frequently altered in gastric cancers (GCs) of both diffuse (DGC) and intestinal (IGC) histotypes, albeit through different mechanisms. The study aimed to characterize CDH1 expression in sporadic IGC and to investigate whether microRNAs (miRs) are involved in its transcriptional control. We evaluated CDH1 expression by quantitative real-time PCR (RT-qPCR) in 33 IGC patients and found a significant downregulation in tumor tissues compared to normal counterparts (p-value = 0.025). Moreover, 14 miRs, predicted to be involved in CDH1 regulation in both a direct and indirect manner, were selected and analyzed by RT-qPCR in an independent case series of 17 IGCs and matched normal tissues. miR-101, miR-26b, and miR-200c emerged as significantly downregulated and were confirmed in the case series of 33 patients (p-value < 0.001). Finally, we evaluated EZH2 expression, a target of both miR-101 and miR-26b, which showed significant upregulation in IGCs (p-value = 0.005). A significant inverse correlation was observed between EZH2 overexpression and CDH1, miR-101, and miR-26b levels (p-value < 0.001). Our results reinforce the link between CDH1 and IGC, highlighting the role of miRs in its transcriptional control and improving our understanding of GC subtypes and biomarkers.

Purpose To describe a snapshot of international genetic testing practices, specifically regarding the use of multigene panels, for hereditary breast/ovarian cancers. We conducted a survey through the Evidence-Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium, covering questions about 16 non-BRCA1/2 genes. Methods Data were collected via in-person and paper/electronic surveys. ENIGMA members from around the world were invited to participate. Additional information was collected via country networks in the United Kingdom and in Italy. Results Responses from 61 cancer genetics practices across 20 countries showed that 16 genes were tested by > 50% of the centers, but only six (PALB2, TP53, PTEN, CHEK2, ATM, and BRIP1) were tested regularly. US centers tested the genes most often, whereas United Kingdom and Italian centers with no direct ENIGMA affiliation at the time of the survey were the least likely to regularly test them. Most centers tested the 16 genes through multigene panels; some centers tested TP53, PTEN, and other cancer syndrome–associated genes individually. Most centers reported (likely) pathogenic variants to patients and would test family members for such variants. Gene-specific guidelines for breast and ovarian cancer risk management were limited and differed among countries, especially with regard to starting age and type of imaging and risk-reducing surgery recommendations. Conclusion Currently, a small number of genes beyond BRCA1/2 are routinely analyzed worldwide, and management guidelines are limited and largely based on expert opinion. To attain clinical implementation of multigene panel testing through evidence-based management practices, it is paramount that clinicians (and patients) participate in international initiatives that share panel testing data, interpret sequence variants, and collect prospective data to underpin risk estimates and evaluate the outcome of risk intervention strategies.

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.

BackgroundCHEK2 is a multi-cancer susceptibility gene whose common germline mutations are known to contribute to the risk of developing breast and prostate cancer.Case presentationHere, 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.ConclusionsThis 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.