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BackgroundIt is estimated that more than 20% of ovarian cancer cases are associated with a genetic predisposition that is only partially explained by germline mutations in theBRCA1andBRCA2genes. Recently, several pieces of evidence showed that mutations in three genes involved in the homologous recombination DNA repair pathway, i.e.,BRIP1,RAD51C, andRAD51D, are associated with a high risk of ovarian cancer. To more precisely estimate the ovarian cancer risk attributed toBRIP1,RAD51C, andRAD51Dmutations, we performed a meta-analysis based on a comparison of a total of ~ 29,400 ovarian cancer patients from 63 studies and a total of ~ 116,000 controls from the gnomAD database.ResultsThe analysis allowed precise estimation of ovarian cancer risks attributed to mutations inBRIP1,RAD51C, andRAD51D, confirming that all three genes are ovarian cancer high-risk genes (odds ratio (OR) = 4.94, 95%CIs:4.07–6.00,p p p ConclusionsThe meta-analysis provides evidence supporting the pathogenicity ofBRIP1,RAD51C, andRAD51Dmutations in relation to ovarian cancer. The level of ovarian cancer risk conferred by these mutations is relatively high, indicating that afterBRCA1andBRCA2, theBRIP1,RAD51C, andRAD51Dgenes are the most important ovarian cancer risk genes, cumulatively contributing to ~ 2% of ovarian cancer cases. The inclusion of the genes into routine diagnostic tests may influence both the prevention and the potential treatment of ovarian cancer.

// Magdalena Ratajska 1 , Magdalena Koczkowska 1 , Monika Żuk 1, 2 , Adam Gorczynski 3 , Alina Kuźniacka 1, 2 , Maciej Stukan 4 , Wojciech Biernat 3 , Janusz Limon 1, 2 and Bartosz Wasąg 1, 2 1 Department of Biology and Medical Genetics, Medical University of Gdansk, Gdansk, Poland 2 Laboratory of Clinical Genetics, University Clinical Centre, Gdansk, Poland 3 Department of Pathology, Medical University of Gdansk, Gdansk, Poland 4 Department of Gynecologic Oncology, Gdynia Oncology Center, Gdynia, Poland Correspondence to: Bartosz Wasąg, email: bwasag@gumed.edu.pl Keywords: ovarian cancer; ctDNA; BRCA1/2; PARP1 inhibitor; next-generation sequencing Received: May 26, 2017 Accepted: July 31, 2017 Published: September 08, 2017 ABSTRACT Approximately 25% of patients with ovarian cancer harbor a pathogenic BRCA1/2 mutation that has been associated with favorable responses for targeted therapy with poly (ADP-ribose) polymerase 1 (PARP1) inhibitors compared to wild-type individuals. The overall frequency of germline and somatic BRCA1/2 alterations is estimated at 13-15% and 3-10%, respectively. A high incidence of BRCA1/2 somatic variants significantly increases the number of patients eligible for treatment with PARP1 inhibitors. Here, we assessed circulating tumor DNA (ctDNA) from 121 patients with ovarian cancer for BRCA1/2 mutational analysis by next generation sequencing. A total number of patients carrying the pathogenic BRCA1/2 variants was 30/121 (24.8%), including 22 and 7 individuals with exclusively germline or somatic mutations, respectively and one patient with variants of both origin. Among this cohort, more than one known pathogenic BRCA1 and/or BRCA2 alterations were identified in 7/30 individuals. The most recurrent mutations were detected in the BRCA1 gene: c.5266dupC (p.Gln1756Profs * 74) with the frequency of ~18%, followed by c.3756_3759del (p.Ser1253Argfs * 10) and c.181T>G (p.Cys61Gly). In seven (5.8%) patients, coincidence of two or more BRCA1/2 pathogenic mutations have been identified. Our results clearly demonstrate that the detection of both germline and somatic BRCA1/2 mutations in ctDNA from ovarian cancer patients is feasible and may be a valuable complementary tool for identification of somatic alterations when the standard diagnostic procedures are insufficient. Finally, ctDNA can potentially allow to monitor the efficacy of PARP1 inhibitors and to detect a secondary reversion BRCA1/2 mutations.

// Ewa Szutowicz-Zielinska 1,* , Krzysztof Konopa 1,* , Anna Kowalczyk 1 , Malgorzata Suszko-Kazarnowicz 2 , Renata Duchnowska 3 , Aleksandra Szczesna 4 , Magdalena Ratajska 5 , Aleksander Sowa 6 , Janusz Limon 5 , Wojciech Biernat 7 , Tomasz Burzykowski 8 , Jacek Jassem 1 and Rafal Dziadziuszko 1 1 Department of Oncology and Radiotherapy, Medical University of Gdansk, Poland 2 Department of Oncology, The Centre for Pulmonary Diseases Olsztyn, Poland 3 Department of Oncology, Military Institute of Medicine, Warsaw, Poland 4 Department of Lung Diseases, Mazovian Centre for Treatment of Lung Diseases and Tuberculosis, Otwock, Poland 5 Department of Biology and Genetics, Medical University of Gdansk, Poland 6 Roche, Poland 7 Department of Pathomorphology, Medical University of Gdansk, Poland 8 Interuniversity Institute of Biostatistics and Statistical Bioinformatics, Hasselt University, Diepenbeek, Belgium * Ewa Szutowicz-Zielinska and Krzysztof Konopa contributed equally to this study Correspondence to: Ewa Szutowicz-Zielinska, email: // Keywords : non-small cell lung cancer, epidermal growth factor receptor, gene copy number, erlotinib Received : July 15, 2016 Accepted : October 12, 2016 Published : December 04, 2016 Abstract Background: First-line treatment with epidermal growth factor receptor (EGFR) inhibitors in NSCLC is effective in patients with activating EGFR mutations. The activity of erlotinib in patients harboring high EGFR gene copy number has been considered debatable. Patients and Methods: A multicenter, open-label, single-arm phase II clinical trial was performed to test the efficacy of erlotinib in the first-line treatment of NSCLC patients harboring high EGFR gene copy number defined as ≥4 copies in ≥40% of cells. Findings: Between December 2007 and April 2011, tumor samples from 149 subjects were screened for EGFR gene copy number by fluorescence in-situ hybridization (FISH), Out of 49 patients with positive EGFR FISH test, 45 were treated with erlotinib. Median PFS in the intent-to-treat population was 3.3 months (95%CI: 1.8–3.9 months), and median overall survival was 7.9 months (95% CI: 5.1–12.6 months). Toxicity profile of erlotinib was consistent with its known safety profile. The trial was stopped prematurely at 63% of originally planned sample size due to accumulating evidence that EGFR gene copy number should not be used to select NSCLC patients to first-line therapy with EGFR TKI. Data on erlotinib efficacy according to EGFR , KRAS and BRAF mutations are additionally presented. Interpretation: This trial argues against using high gene copy number for selection of NSCLC patients to first-line therapy with EGFR TKIs. The study adds to the discussion on efficacy of other targeted agents in patients with target gene amplified tumors.

Somatic mosaicism for DNA copy-number alterations (SMC-CNAs) is defined as gain or loss of chromosomal segments in somatic cells within a single organism. As cells harboring SMC-CNAs can undergo clonal expansion, it has been proposed that SMC-CNAs may contribute to the predisposition of these cells to genetic disease including cancer. Herein, the gross genomic alterations (>500 kbp) were characterized in uninvolved mammary glandular tissue from 59 breast cancer patients and matched samples of primary tumors and lymph node metastases. Array-based comparative genomic hybridization showed 10% (6/59) of patients harbored one to 359 large SMC-CNAs (mean: 1,328 kbp; median: 961 kbp) in a substantial portion of glandular tissue cells, distal from the primary tumor site. SMC-CNAs were partially recurrent in tumors, albeit with considerable contribution of stochastic SMC-CNAs indicating genomic destabilization. Targeted resequencing of 301 known predisposition and somatic driver loci revealed mutations and rare variants in genes related to maintenance of genomic integrity: BRCA1 (p.Gln1756Profs*74, p.Arg504Cys), BRCA2 (p.Asn3124Ile), NCOR1 (p.Pro1570Glnfs*45), PALB2 (p.Ser500Pro), and TP53 (p.Arg306*). Co-occurrence of gross SMC-CNAs along with point mutations or rare variants in genes responsible for safeguarding genomic integrity highlights the temporal and spatial neoplastic potential of uninvolved glandular tissue in breast cancer patients.

The breast cancer susceptibility gene BARD1 (BRCA1-associated RING domain protein, MIM# 601593) acts with BRCA1 in DNA double-strand break (DSB) repair and also in apoptosis initiation. We screened 109 BRCA1/2 negative high-risk breast and/or ovarian cancer patients from North-Eastern Poland for BARD1 germline mutations using a combination of denaturing high-performance liquid chromatography and direct sequencing. We identified 16 different BARD1 sequence variants, five of which are novel. Three of them were suspected to be pathogenic, including a protein truncating nonsense mutation (c.1690C>T, p.Gln564X), a splice mutation (c.1315-2A>G) resulting in exon 5 skipping, and a silent change (c.1977A>G) which alters several exonic splicing enhancer motifs in exon 10 and results in a transcript lacking exons 2–9. Our findings suggest that BARD1 mutations may be regarded as cancer risk alleles and warrant further investigation to determine their actual contribution to non-BRCA1/2 breast and ovarian cancer families.

Cornelia de Lange syndrome (CdLS) is a rare multisystem congenital anomaly disorder characterized by growth and developmental delay, distinctive facial dysmorphism, limb malformations and multiple organ defects. Approximately 60–65% of the CdLS subjects have mutation in one of three cohesin proteins, a main regulator of cohesin-associated protein, NIPBL, and two components of the cohesin ring structure SMC1A and SMC3. A prominent role for cohesin is to control chromosome segregation during cell divisions. We have performed MLPA analysis in a group of 11 children with the CdLS but without identifiable point mutations in the NIPBL and SMC1A genes. In a single patient, we identified a large deletion encompassing exons 35 to 47 of the NIPBL gene. Our finding was validated by aCGH and further characterized by long-range PCR and DNA sequencing of the breakpoint junction.

The overall prevalence of germline BRCA1/2 mutations is estimated between 11% and 15% of all ovarian cancers. Individuals with germline BRCA1/2 alterations treated with the PARP1 inhibitors (iPARP1) tend to respond better than patients with wild‐type BRCA1/2. Additionally, also somatic BRCA1/2 alterations induce the sensitivity to iPARP1. Therefore, the detection of both germline and somatic BRCA1/2 mutations is required for effective iPARP1 treatment. The aim of this study was to identify the frequency and spectrum of germline and somatic BRCA1/2 alterations in a group of Polish patients with ovarian serous carcinoma. In total, 100 formalin‐fixed paraffin‐embedded (FFPE) ovarian serous carcinoma tissues were enrolled to the study. Mutational analysis of BRCA1/2 genes was performed by using next‐generation sequencing. The presence of pathogenic variants was confirmed by Sanger sequencing. In addition, to confirm the germline or somatic status of the mutation, the nonneoplastic tissue was analyzed by bidirectional Sanger sequencing. In total, 27 (28% of patient samples) mutations (20 in BRCA1 and 7 in BRCA2) were identified. For 22 of 27 patients, nonneoplastic cells were available and sequencing revealed the somatic character of two BRCA1 (2/16; 12.5%) and two BRCA2 (2/6; 33%) mutations. Notably, we identified six novel frameshift or nonsense BRCA1/2 mutations. The heterogeneity of the detected mutations confirms the necessity of simultaneous analysis of BRCA1/2 genes in all patients diagnosed with serous ovarian carcinoma. Moreover, the use of tumor tissue for mutational analysis allowed the detection of both somatic and germline BRCA1/2 mutations.

The aim of the present study was to investigate novel molecular markers that could improve the diagnosis of ovarian cancer patients or be of predictive value. The sequence of the sodium‑hydrogen antiporter 3 regulator 1 (SLC9A3R1) gene that codes for the PDZ2 motif of the Na+/H+ exchanger regulatory factor 1 (NHERF1) protein was analyzed. Changes in migration and cell transformation, and alterations of growth factor signaling pathways have been described in cells lacking endogenous NHERF1 or expressing an isoform lacking the function of the PDZ2 domain. Exons 2 and 3, together with flanking intronic sequences of the SLC9A3R1 gene, were amplified and bi‑directionally sequenced in 31 primary tumor samples from epithelial ovarian cancer patients. In total, 3 different previously undescribed mutations were detected in 8 out of 31 serous adenocarcinoma tumor samples (25.8%). Bioinformatics analysis predicted a significant effect in the splicing process as a result of the mutations that could disrupt the NHERF1 PDZ2 domain. Point mutations in consensus splicing recognition are a major cause of the splicing defects that are found in several diseases, including cancer. It has previously been shown that a lack of exon 2 and disruption of the PDZ2 domain contribute to cell transformation and leads to modifications in the physiological regulation of the confor­mational state of NHERF1. Further studies in bigger groups of ovarian cancer patients will determine the importance of this mutation in disease progression and patient survival. Fil: Kreimann, Erica Lorena. Comisión Nacional de Energía Atómica. Gerencia de Area de Aplicaciones de la Tecnología Nuclear. Gerencia de Radiobiología (Centro Atómico Constituyentes); Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ratajska, Magdalena. Medical University of Gdansk; Polonia Fil: Kuzniacka, Alina. Medical University of Gdansk; Polonia Fil: Demacopulo, Brenda. Comisión Nacional de Energía Atómica. Gerencia de Area de Aplicaciones de la Tecnología Nuclear. Gerencia de Radiobiología (Centro Atómico Constituyentes); Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Stukan, Maciej. Gdynia Oncology Centre; Polonia Fil: Limon, Janusz. Medical University of Gdansk; Polonia

Introduction The aim of this study was to evaluate a patient presenting dysmorphic features and multiorgan malformations suggestive of a severe Cornelia de Lange syndrome (CdLS) phenotype. Initial studies included classical karyotype and nipped-B homolog (NIPBL) mutational screening, which showed a translocation t(3;5)(p13;q11) and a novel sequence variant in the NIPBL gene. Further evaluation of the findings allowed the determination of their pathogenic significance and hence molecular confirmation of the diagnosis of CdLS.

Sixty-four Polish families with a history of breast and/or ovarian cancer were screened for mutations in the BRCA1/2 genes using a combination of denaturing high performance liquid chromatography (DHPLC) and sequencing. Two thirds (43/64; 67%) of the families were found to carry deleterious mutations, of which the most frequent were BRCA1 5382insC (n=22/43; 51%) and Cys61Gly (n=9/43; 20%). Two other recurrent mutations were BRCA1 185delAG (n=3) and 3819del5 (n=4), together accounting for 16% of the 43 mutation-positive cases. We also found three novel mutations (BRCA1 2991del5, BRCA2 6238ins2del21 and 8876delC) which combined with findings from our earlier study of 60 Northern Polish families. Moreover, screening of 43 BRCA1/2 negative families for the presence of large rearrangements by multiplex ligation-dependent probe amplification (MLPA) resulted in the finding of two additional BRCA1 mutations: a deletion of exons 1A, 1B and 2, and a deletion of exons 17-19, both present in single families. We conclude that the Polish population has a diverse mutation spectrum influenced by strong founder effects. However, families with strong breast/ovarian cancer history who are negative for these common mutations should be offered a complete BRCA gene screening, including MLPA analysis.

Background. We estimated the prevalence of BRCA1/2 germline mutations in consecutive ovarian cancers and correlated the mutation status with clinicopathological features. Methods. 151 consecutive primary ovarian cancer patients were screened for BRCA1/2 germline mutations. Results. We identified BRCA1/2 germline mutations in 21 (13.9%) patients. Seventeen (81%) of carriers have BRCA1 and four (19%) have BRCA2 mutation. BRCA1/2 carriers have a distinctly longer overall survival than sporadic cases (log-rank, p =0.014). Conclusions. The relatively high proportion of BRCA1/2 carriers among unselected ovarian cancer patients indicates the necessity of searching for recurrent BRCA mutations in each case of ovarian carcinoma. This routine screen should be widened to include denaturing high performance liquid chromatography (DHPLC) analysis of both exons 11 of BRCA1 and BRCA2 genes in women with positive family history.

Whereas HER2 amplification is a well-known phenomenon in breast tumours, its frequency and clinical importance in ovarian cancer have not been established. The aim of the study was to compare the frequency of HER2 amplification in hereditary (BRCA-positive) and sporadic (BRCA-negative) ovarian tumours and to estimate the association of this gene alteration on clinical outcome in ovarian cancer patients. We analysed HER2 amplification in 53 ovarian tumours: 20 from mutation carriers (18 in BRCA1 and 2 in BRCA2 gene) and 33 from non-carriers. Fluorescence in situ hybridization for HER2 was performed on 'touch' slides from frozen tumour samples or formalin-fixed, paraffin-embedded tissue. Our results indicate that high amplification (HER2: centromere ratio>5) is an infrequent phenomenon in ovarian tumours (6/53 cases). It occurs in both hereditary (4/20) and sporadic (2/33) tumours and no difference in the frequency of HER2 amplification exists between these groups. There is no significant difference in the clinical outcome of patients with HER2 amplified and non-amplified tumours (p = 0.3). Our results suggest a different biological role of HER2 amplification in ovarian and breast cancer.

BARD1 has tumor suppressor functions by binding to BRCA1 and to p53 via RING finger and ankyrin repeats, respectively. The BARD1-BRCA1 dimer has E3 ligase activity, and BARD1-p53 interaction induces apoptosis. The C-terminus of BARD1 is binding to many proteins that are targets of the BRCA1-BARD1 ubiquitin ligase, and expression of N-terminally truncated RING-less BARD1 isoforms was correlated with poor prognosis in various cancers, consistent with their oncogenic functions by antagonistic binding to target proteins. Expression and interaction assays showed that the BARD1 BRCT domains were critically involved in telomere maintenance and interacted with the telomere binding protein TRF2. Overexpression of BARD1 or BRCA1 lead to TRF2 degradation, suggesting that TRF2 presents a novel target of the BARD1-BRCA1 E3 ubiquitination ligase. Repression of BARD1 or BRCA1 by siRNA leads to genetic instability, as shown before, and telomere alterations. We observed distinct telomeric alterations in blood cells of patients with a BARD1 germline mutation that causes a translation stop and translation of a truncated protein lacking the BRCT domains. Cells of carriers of a BRCA1 mutation that disrupts the BARD1-BRCA1 interaction show similar telomere alterations. These in vitro and in vivo observations suggest that BARD1 and BRCA1 play distinct roles in maintaining telomere integrity in a dosage dependent manner. BARD1δ, an isoform lacking RING and ankyrin motifs, but retaining the BRCT domains, is abundantly expressed in all cancers investigated so far. Overexpression of BARD1δ in vitro blocked cell cycle progression and induced chromosomal and telomere abnormalities, suggesting that BARD1δ also affected telomere integrity. We conclude that the correct dose of BARD1 and/ or its BRCT domains is critically important for maintaining telomere integrity. Expression of BARD1δ in cancer, or reduced expression of BARD1 in mutation carriers causes chromosomal instability and promotes carcinogenesis and tumor progression. Citation Format: Irmgard Irminger-Finger, Maxim Pilyugin, Magdalena Ratajska. The BARD1 BRCT domains are essential for maintenance of telomere integrity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2393. doi:10.1158/1538-7445.AM2014-2393

It is estimated that about 5–10% of ovarian and 2–5% of all breast cancer patients are carriers of a germline BRCA1 or BRCA2 gene mutation. Most families with detected BRCA1 or BRCA2 gene mutation are qualified for molecular testing on the basis of family history of breast or ovarian cancers. The purpose of our study was to establish the frequency of positive family history of cancer in a series of Polish consecutive breast and ovarian cancer patients in two groups, with and without the BRCA1 gene mutations. We analysed the prevalence of four of the most common BRCA1 mutations: 5382insC (c.5266dupC), 300T>G (p.181T>G), 185delAG (c.68_69delAG) and 3819del5 (c.3700_3704del5). The patient group consisted of 1,845 consecutive female breast and 363 ovarian cancer cases. 19 out of 37 (51%) of BRCA1-positive ovarian cancer patients and 21 out of 55 (39%) BRCA1-positive breast cancer had negative family history of breast and/or ovarian cancer among first- and second-degree relatives. In ovarian cancer patients, negative family history was more frequent in those with 300T>G BRCA1 gene mutation than in 5382insC carriers. This finding indicates the necessity of searching for 300T>G mutation in families with a single diagnosis of ovarian cancer in family. The high frequency of mutations detected in breast cancer patients lacking obvious family history shows that breast cancer patients should be qualified for genetic testing on the basis of wide clinical and pathological criteria.

BARD1 was identified as a protein interacting with BRCA1 - the heterodimer formed by BRCA1 and BARD1 acts in DNA repair, RNA processing, transcription and cell cycle regulation. BARD1 has also BRCA1-independent functions like mediating p53-dependent apoptosis. Additionally, BARD1 mRNA isoforms were found to be highly expressed in most human gynecological cancers. We report 17 different BARD1 variants, four of which were suspected to be pathogenic, including a novel substitution (c.1361C>T) leading to amino acid change in highly conserved ankirin domain motif, a splice mutation (c.1315-2A/G) resulting in exon 5 skipping and a silent change (c.1977A/G) which alters several ESE motifs in exon 10, and results in a transcript lacking exons 2-9. Finally we identified two unrelated patients carrying truncating nonsense mutation in exon 8 (c.1690C>T). Our findings suggest that BARD1 mutations may be regarded as cancer risk alleles and warrant further investigation to determine their actual contribution to non-BRCA1/2 breast and ovarian cancer families.