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AIM: Most patients tested by a 44-gene panel for hereditary cancer (HerediGene), even though they had a strong family history, have a negative result or a finding in a low-risk gene. This creates a question about the risk for these individuals to develop breast cancer. The Polygenic Risk Score is a tool used to identify and calculate the lifetime risk of developing breast cancer and thus we are investigating whether it can be used in our cohort of patients to identify this risk. PATIENTS AND METHODS: Among 111 patients analyzed with a 44-gene hereditary cancer panel, we were able to produce the Polygenic Risk Score for 105 of them. All the patients were diagnosed with breast cancer, and they all had a strong family history. We analyzed the ability of the PRS to identify the risk of the patients in combination with the findings of the 44-gene panel. RESULTS: Overall, 74% of patients with a family history had a negative PRS (with a cut-off of 20%). Among the BRCA positive patients all of them had a positive PRS and among all the high-risk gene positive cases, 70% had a positive PRS. On the other hand, among the low-risk genes and the negative cases 18.5% had a positive PRS. There is a positive correlation between the findings from the NGS panel analysis and the PRS. CONCLUSION: Our analysis shows that PRS is correlated to the findings of the 44-gene NGS panel having the majority of the PRS positive patients carry high risk mutations. In addition to this, in 18.5% of the patients with low-risk findings or negative result from the 44-gene panel, the PRS was positive which can explain the outcome of the patient since breast cancer had developed. This is an indication that the combination of PRS with the findings from the 44-gene panel can identify individuals with a higher risk of developing breast cancer. Citation Format: Nikolaos Tsoulos, Konstantinos Agiannitopoulos, Helen Goga, Theodoros Troupis, Christos Markopoulos. Polygenic Risk Score in a cohort of 105 Breast Cancer patients previously tested with a multi gene panel for hereditary cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-12-07.

Background: Breast cancer is the most frequently diagnosed cancer in women and about 10% of breast cancer cases are hereditary. BRCA1 and BRCA2 are the genes most frequently associated with Hereditary Breast Cancer, although there are numerous other genes, such as PALB2, CHEK2 and ATM, that require to be considered as well. Germline Copy Number Variation (CNV) is one mutation type that is an important contributor to hereditary breast cancer. Nowadays, next-generation sequencing (NGS) technologies has contributed to multi-gene panel analysis used in clinical practice. Methods: In total, 1418 individuals were tested for breast cancer predisposition, using a solution-based capture approach. Targeted NGS was performed with a panel of 36 genes. The capture-based approach allowed for computational analysis of CNVs from NGS data. Results: We investigate the performance of the CNV module of the commercial software suite SeqPilot (JSI Medical Systems) and the non-commercial tool panelcn.MOPS. Both algorithms are specifically developed for CNV analysis of sequencing data reporting 99-100% sensitivity and up to 100% specificity for the prediction of CNVs up to the level of a single gene exon. All CNVs detected with these algorithms were then verified experimentally using the MLPA technique as an orthogonal assay. At least one pathogenic/likely pathogenic variant was identified in 289 samples (20.4%). CNVs accounted for 10.4% (30/289), referring to the deletion of one or more exons of a gene. Interestingly, 50% of deletions were single exon and approximately 36% of CNVs were detected in genes other than BRCA1/2. In specific, of the 30 CNVs detected, 60% occurred in BRCA1, 3.3% in BRCA2, 20% in CHEK2, 6.7% in FANCA, 6.7% in PMS2, and 3.3% in ATM. The majority of CNVs in BRCA1 were deletions of exons 19, 22, and 22-23 whereas deletions of exons 9-10 were the most common deletions in CHEK2. Detailed information of all CNVs detected is provided in Table 1. Conclusions: Our results suggest that CNV analysis should not be restricted to BRCA1/2 due to the significant proportion of CNVs (36%) in additional breast cancer predisposition genes. Furthermore, in silico CNV detection tools provide a cost-effective and feasible methodology for the identification of CNVs from NGS experiments. This outlines the clinical utility of comprehensive genetic testing that includes full sequencing and CNV analysis in hereditary breast cancer facilitating personalized management decisions for patients. Table 1.Pathogenic Copy Number Variations (CNVs) identified in this studyGeneHGVS nomenclatureOther nomenclature# detectedATMNM_000051:c.(-30+1_-29-1)_(331+1_332-1)deldeletion of exons 2-41BRCA1NM_007294:c.(5467+1_5468-1)-(*1_?)deldeletion of exon 237BRCA1NM_007294:c.(5406+1_5407-1)_(*1_?)deldeletion of exons 23-245BRCA1NM_007294:c.(5193+1_5194-1)-(5277+1_5278-1)deldeletion of exon 196BRCA2NM_000059:c.(6841+1_6842-1)_(7007+1_7008-1)deldeletion of exons 12-131CHEK2NM_007194:c.(908+1_909-1)_(1095+1_1096-1)deldeletion of exons 9-104CHEK2NM_007194:c.(792+1_793-1)_(846+1_847-1)deletion of exon 72FANCANM_000135:c.(1626+1_1627-1)_ (2852+1_2853-1)deldeletion of exons 18-291FANCANM_000135:c.(893+1_894-1)_(1359+1_1360-1)deldeletion of exons 11-141PMS2NM_000535: c.(903+1_904-1)_(988+1_989-1)deldeletion of exon 91PMS2NM_000535:c.(705+1_706-1)_(2006+1_2007-1)deldeletion of exons 7-111 Citation Format: Nikolaos Tsoulos, Konstantinos Agiannitopoulos, Georgia Pepe, Eirini Papadopoulou, Georgios N Tsaousis, Despina Apostolopoulou, Angeliki Meintani, Vassileios Venizelos, Christos Markopoulos, Rodoniki Iosifidou, Sofia Karageorgopoulou, Christos Christodoulou, Ioannis Natsiopoulos, Konstantinos Papazisis, Maria Vasilaki-Antonatou, Eleftherios Kabletsas, Amanta Psyrri, Stylianos Giassas, Dimitrios Ziogas, Efthalia Lalla, Anna Koumarianou, Christos Papadimitriou, Vahit Ozmen, Sualp Tansan, Kerim Kaban, Tahsin Ozatlı, Dan Tudor Eniu, Angelica Chiorean, Alexandru Blidaru, George Nasioulas. Different CNVs account for 10.4% of pathogenic variants in 1418 patients referred for hereditary breast cancer testing [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-09-10.

Male breast cancer (MBC) is a very rare disorder affecting approximately 1 in 833 men. Genetic predisposition is one of the most important risk factors of MBC with BRCA2 being the most commonly mutated gene in males diagnosed with breast cancer. However, a large part of MBC heritability is still unexplained. This study sought to add to the data already available on the genetics of MBC.Our study initially involved comprehensive analysis of BRCA1 and BRCA2, followed by analysis of 43 genes implicated in cancer predisposition in a series of 100 Greek patients diagnosed with MBC between 1995-2015.Pathogenic variants were identified in 13 patients, with BRCA2 being the most commonly affected gene, followed by BRCA1, RAD50, RAD51B, and MSH3.In agreement with previous reports, BRCA2 is the most important genetic factor of MBC predisposition, while the remaining known cancer predisposition genes are each very rarely involved, rendering conclusions as to their cumulative effect difficult to draw.

Objective Several microRNA (miRNA) polymorphisms have been associated with susceptibility to specific health disorders, including cardiovascular diseases. The aim of the present study was to investigate whether four well-studied miRNA polymorphisms in non-Caucasian populations, namely miR146a G>C (rs2910164), miR149 C>T (rs2292832), miR196a2 C>T (rs11614913) and miR499 A>G (rs3746444), contribute to the risk for the development of premature Coronary Artery Disease (CAD) in the Greek population. Methods We used a case-control study to examine these associations in 400 individuals: 200 CAD patients [including a subgroup of myocardial infraction (MI) patients] and 200 healthy controls, all of Greek origin. MiRNA polymorphisms were genotyped using three different assays: Polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP), High resolution Melting (HRM) and Sanger sequencing. Results Two of these polymorphisms, miR196a2 C>T (rs11614913) and miR499 A>G (rs3746444) were found to be strongly associated with increased risk for CAD (p=0.0388 and p=0.0013, respectively) and for MI (p=0.0281 and p=0.0273, respectively). Furthermore, miR146C-miR149C-miR196T-miR499G allele combination appeared to be significantly related to CAD (p=0.0185) and MI (p=0.0337) prevalence. Conclusions Our results suggest that at least two of the studied polymorphisms, miR196a2 C>T (rs11614913) and miR499 A>G (rs3746444), as well as the miR146C-miR149C-miR196T-miR499G allele combination could represent useful biomarkers of CAD and/or MI susceptibility in the Greek population. These special genetic characteristics, in combination with environmental factors and personal habits, might contribute to CAD and/or MI prevalence.

Background Classification of splicing variants (SVs) in genes associated with hereditary cancer is often challenging. The aim of this study was to investigate the occurrence of SVs in hereditary cancer genes and the clinical utility of RNA analysis. Material and methods 1518 individuals were tested for cancer predisposition, using a Next Generation Sequencing (NGS) panel of 36 genes. Splicing variant analysis was performed using RT-PCR and Sanger Sequencing. Results In total, 34 different SVs were identified, 53% of which were classified as pathogenic or likely pathogenic. The remaining 16 variants were initially classified as Variant of Uncertain Significance (VUS). RNA analysis was performed for 3 novel variants. Conclusion The RNA analysis assisted in the reclassification of 20% of splicing variants from VUS to pathogenic. RNA analysis is essential in the case of uncharacterized splicing variants, for proper classification and personalized management of these patients.

Background/Aim: The use of multi-gene panels for germline testing in breast cancer enables the estimation of cancer risk and guides risk-reducing management options. The aim of this study was to present data that demonstrate the different levels of actionability for multi-gene panels used in genetic testing of breast cancer patients and their family members. Materials and Methods: We performed an analysis in our clinical database to identify breast cancer patients undergoing genetic testing. We reviewed positive results in respect of risk estimation and management, cascade family testing, secondary findings and information for treatment decision-making. Results: A total of 415 positive test reports were identified with 57.1%, 18.1%, 10.8% and 13.5% of individuals having pathogenic/likely pathogenic variants in high, moderate, low and with insufficient evidence for breast cancer risk genes, respectively. Six point seven percent of individuals were double heterozygotes. Conclusion: Germline findings in 92% of individuals are linked to evidence-based treatment information and risk estimates for predisposition to breast and/or other cancer types. The use of germline findings for treatment decision making expands the indication of genetic testing to include individuals that could benefit from targeted treatments.

Next-generation sequencing (NGS) technology is used to evaluate hereditary cancer risks of patients worldwide; however, information concerning the germline multigene mutational spectrum among patients with breast cancer (BC) with consanguineous marriage (CM) is limited. Therefore, this prospective study aimed to determine the molecular characteristics of patients with BC who were tested with multigene hereditary cancer predisposition NGS panel and to show the effect of CM on cancer-related genes. Patients with BC with or without CM and family history (FH) of BC treated in our breast center were selected according to The National Comprehensive Cancer Network (NCCN) criteria for hereditary BC. In these patients, the analysis of a panel of 33 genes involved in hereditary cancer predisposition was performed after genetic counseling by using NGS. The pathogenic variant (PV) and the variant of uncertain significance (VUS) were found to be 15.8 and 47.4%, respectively. PVs were identified in 10/33 genes in 34 patients; 38.2% in BRCA1/2 genes; 6, 24, and 14% in other high, moderate and low-risk genes, respectively. The CM rate was 17.7% among the 215 patients with BC. The PV rate was 13.2% in patients with CM and 16.4% in patients without CM (P=0.80). When PV and VUS were evaluated together, the PV+VUS ratio was significantly higher in patients with CM and FH of BC than patients without CM and FH of BC (88.2 vs. 63.3%, P=0.045). Analysis of multigene panel provided 9.76% additional PVs in moderate/low-risk genes. The PV rate was similar in patients with BC with or without CM. A high PV+VUS ratio in patients with CM and FH of BC suggests that genes whose importance are unknown are likely to be pathogenic genes later.

Background Carriers with pathogenic variants in MSH2 have increased risk to develop colorectal, endometrium, ovarian, and other types of cancer. The PALB2 is associated with breast, ovarian, pancreatic, and prostate cancer. We describe the case of a 42‐year‐old female diagnosed with endometrial cancer at the age of 42 years with a strong family history of colorectal cancer, which was referred to our private diagnostic laboratory for genetic testing. Methods In this study, we performed next‐generation sequencing (NGS) using an amplicon based 26 genes panel. The presence of multi‐exonic copy number variations (CNVs) was investigated by computational analysis and Multiplex Ligation‐dependent Probe Amplification (MLPA). Results A gross deletion of the genomic region encompassing exons 11–16 of the MSH2 and the loss‐of‐function variant c.757_758delCT, p.(Leu253Ilefs*3) in the PALB2 were identified in the proband. Conclusions Multigene analysis using NGS technology allows the identification of pathogenic variants in genes that would normally not be tested based on the patient diagnosis. In our case these results explained not only the personal and/or family history of cancer but also allowed the surveillance for prevention of other cancer types. Moreover, the detection of large genomic rearrangements should be routinely included in hereditary cancer testing., We describe the case of a 42‐year‐old female diagnosed with endometrial cancer with a strong family history of colorectal cancer, which was referred to our private diagnostic laboratory for genetic testing. By next‐generation sequencing (NGS) and Multiplex Ligation‐dependent Probe Amplification (MLPA) analysis we identified a gross deletion of the genomic region encompassing exons 11–16 of the MSH2 gene and the loss‐of‐function variant c.757_758delCT, p.(Leu253Ilefs*3) in the PALB2 gene in our proband.

Circulating cell-free DNA (cf-DNA) is present in human biological fluids, mainly in plasma and serum, originating from cell death, a process that massively takes place during acute myocardial infarction (AMI). In the present study, cf-DNA was assessed by different quantification techniques, in order to determine its levels in patients admitted with AMI. A total of 130 subjects were included in the study: 80 ST elevation myocardial infarction (STEMI) patients and 50 healthy controls. Cf-DNA extracted from plasma was analyzed by: a) Qubit 3.0 with single (ss) and double (ds) stranded DNA assay kits, b) NanoDrop and c) quantitative PCR (qPCR). Cf-DNA levels were recorded elevated in AMI patients compared to those of healthy individuals. Specifically, Qubit 3.0 ss-DNA kit provided the highest cf-DNA concentration values for all the samples analyzed in comparison with ds-DNA assay kit and NanoDrop, approaching the values obtained by qPCR. Cf-DNA augments in massive cell death settings, including AMI, proposing that the quantification of its levels by novel methodologies could contribute to patient diagnosis and clinical management.

Background CHEK2 is involved in the DNA damage repair response Fanconi anemia (FA)-BRCA pathway. An increased risk for breast and other cancers has been documented in individuals who carry a single pathogenic CHEK2 variant. As for other genes involved in cancer predisposition, different types of pathogenic variants have been observed, including single nucleotide variations, short insertions/deletions, large genomic rearrangements and splicing variants. Splicing variants occurring in the splicing acceptor or donor site result in alternative mature mRNA produced and can cause intron retention, exon skipping, or creation of alternative 3′ and 5′ splice site. Thus, the pathogenicity of this type of alterations should always be explored experimentally and their effect in the mRNA and consequently the protein produced, should be defined. The aim of this study was the delineation of the effect of a splicing variant in the CHEK2 gene. Case presentation A healthy 28-year-old woman with a family history of breast and ovarian cancer was referred for genetic testing. The variant c.793-1G > A (rs730881687) was identified by Next Generation Sequencing (NGS) using a solution-based capture method, targeting 33 cancer predisposition genes (SeqCap EZ Probe library, Roche NimbleGen). Experimental analysis in patient-derived leukocytes using RT-PCR of mRNA followed by cDNA sequencing revealed the deletion of one base from the alternative transcript created (r.793del). This resulted in a frameshift leading to premature termination codon within exon 7 (p.(Asp265Thrfs*10)). Conclusions This finding suggests that the CHEK2 splicing variant c.793-1G > A is a deleterious variant. Our case shows that RNA analysis is a valuable tool for uncharacterized splice site variants in individuals referred for testing and facilitates their personalized management.

Gastrointestinal malignancies are among the leading causes of cancer-related deaths worldwide. Like all human malignancies they are characterized by accumulation of mutations which lead to inactivation of tumor suppressor genes or activation of oncogenes. Advances in Molecular Biology techniques have allowed for more accurate analysis of tumors' genetic profiling using new breakthrough technologies such as next generation sequencing (NGS), leading to the development of targeted therapeutical approaches based upon biomarker-selection. During the last 10 years tremendous advances in the development of targeted therapies for patients with advanced cancer have been made, thus various targeted agents, associated with predictive biomarkers, have been developed or are in development for the treatment of patients with gastrointestinal cancer patients. This review summarizes the advances in the field of molecular biomarkers in tumors of the gastrointestinal tract, with focus on the available NGS platforms that enable comprehensive tumor molecular profile analysis.

Background The CYP19 gene encodes aromatase, a key cytochrome P450 enzyme that converts androgens to estrogens in the ovarian tissues of premenopausal women. A polymorphic variation C/T in the CYP19 gene, rs10046, results in modified estrogen levels through increasing aromatase activity. In the present study, we investigated the possible association between the rs10046 polymorphism of the CYP19 gene and the risk of recurrent spontaneous abortions (RSA) in the Caucasian Greek population. Methods In the prospective case-control study 120 patients and 100 healthy controls were studied. Women who had at least three unexplained spontaneous abortions before 20 weeks of gestation were included in the patient group, while the control group consisted of women with at least two successful pregnancies and without history of abortions. The PCR-RFLP method was used in order to genotype the subjects. Results The frequencies of CC, CT, TT genotypes of the rs10046 variant were 0.275, 0.500, 0.225, respectively, in the patient group and 0.210, 0.480, 0.310, respectively, in the control group. The allele frequencies were 0.525 and 0.475 for C and T, respectively, in the patient group and 0.450 and 0.550 for C and T, respectively, in the control group. Statistical analysis of these results indicated that there are no significant differences in genotype ( P = 0.2897) or in allele frequencies [ P = 0.1261, OR (95%) = 1.351(0.9269–1.969)] between RSA patient and the control groups. Conclusion The rs10046 polymorphism of CYP19 gene seems that is not an important contributing factor in the aetiology of RSA in the Greek Caucasian population.

It has been reported that certain patients with non-small-cell lung cancer (NSCLC) that harbor activating somatic mutations within the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene may be effectively treated using targeted therapy. The use of EGFR inhibitors in patient therapy has been demonstrated to improve response and survival rates; therefore, it was suggested that clinical screening for EGFR mutations should be performed for all patients. Numerous clinicopathological factors have been associated with EGFR and Kirsten-rat sarcoma oncogene homolog (KRAS) mutational status including gender, smoking history and histology. In addition, it was reported that EGFR mutation frequency in NSCLC patients was ethnicity-dependent, with an incidence rate of ~30% in Asian populations and ~15% in Caucasian populations. However, limited data has been reported on intra-ethnic differences throughout Europe. The present study aimed to investigate the frequency and spectrum of EGFR mutations in 1,472 Greek NSCLC patients. In addition, KRAS mutation analysis was performed in patients with known smoking history in order to determine the correlation of type and mutation frequency with smoking. High-resolution melting curve (HRM) analysis followed by Sanger sequencing was used to identify mutations in exons 18-21 of the EGFR gene and in exon 2 of the KRAS gene. A sensitive next-generation sequencing (NGS) technology was also employed to classify samples with equivocal results. The use of sensitive mutation detection techniques in a large study population of Greek NSCLC patients in routine diagnostic practice revealed an overall EGFR mutation frequency of 15.83%. This mutation frequency was comparable to that previously reported in other European populations. Of note, there was a 99.8% concordance between the HRM method and Sanger sequencing. NGS was found to be the most sensitive method. In addition, female non-smokers demonstrated a high prevalence of EGFR mutations. Furthermore, KRAS mutation analysis in patients with a known smoking history revealed no difference in mutation frequency according to smoking status; however, a different mutation spectrum was observed.

Background/aim Certain microRNAs (miRs) present in human plasma are candidate biomarkers for cardiovascular diseases, including acute myocardial infarction (AMI). We examined the expression of two cardiac-specific miRs (miR-208b and miR-499) in a Greek pathological population. Materials and methods Plasma samples from AMI patients and healthy subjects (controls) were analyzed using TaqMan® MicroRNA assays. Results The concentration of both miRs was significantly elevated in AMI patients compared to healthy controls. Moreover, receiver-operating characteristic (ROC) curve analysis showed that miR-208b and miR-499 displayed similar properties with the established AMI biomarker cardiac troponin T (cTnT). Conclusion We showed, for the first time, that these miRs could be used as AMI biomarkers in our population as well. Our data are in agreement with those of studies based on different population groups and further strengthen the observation that plasma levels of circulating miR-208b and miR-499 could serve as potential AMI biomarkers.

Background Androgens are known to influence the risk of developing cardiovascular diseases. This study aims at investigating the possible association between G1733A polymorphism in the coding region of androgen receptor (AR) gene and premature coronary artery disease (CAD). Methods A total of 460 Greek subjects were investigated for the G1733A polymorphism. The patient group consisted of 250 CAD individuals, aged less than 58 years, while 210 healthy individuals served as controls. Genotyping was performed using the PCR-RFLP method. Results Significant differences in genotype distribution (P = 0.0067) and allele frequencies (P = 0.0060) have been observed between patients and controls in the women's subgroup. Conversely, the genotype/allele frequencies were similar between patients and controls in the subgroup of men. Conclusion We may conclude that the G1733A polymorphism of AR gene could be a useful genetic marker for the assessment of a woman's risk for CAD in our Caucasian Greek population.

To investigate whether the missense rs605059 polymorphism of HSD17B1 gene, which is expressed mainly in the placenta, is associated with recurrent spontaneous abortions (RSA).This study group consisted of 138 women with three or more unexplained spontaneous abortions, before the 20th week of gestation, with the same partner, while 140 healthy women served as controls. To genotype the individuals, we used the polymerase chain reaction-restriction fragment length polymorphism method.The genotyping of the rs605059 polymorphism revealed the frequencies 0.22, 0.45 and 0.33, for AA, GA and GG genotypes, respectively, for the patient group and 0.37, 0.41 and 0.22, respectively, for the control group. The A allele frequencies were 0.44 and 0.57 for the patient and control group, respectively, and the G allele frequencies were 0.56 and 0.43 for the patient and control group, respectively. Statistical analysis of the results indicated the existence of significant differences in genotype and allele frequencies between the two groups.The rs605059 polymorphism of the HSD17B1 gene is associated with increased risk of RSA in our Caucasian Greek population. Thus it could be used as a prognostic genetic marker for RSA.

Background The application of the Next Generation Sequencing (NGS) technology has facilitated multigene panel testing for hereditary breast cancer (BC) in clinical practice. We performed a retrospective analysis of individuals referred for testing in our lab aiming to investigate the contribution of included genes and evaluate current genetic testing guidelines in BC. Methods In total, 1141 BC patients and 184 unaffected individuals with family history (FH) of BC were referred from physicians for testing using a multigene panel. Genomic DNA was enriched for targeted regions of 33 genes and sequencing was carried out using the Illumina NGS technology. The presence of large genomic rearrangements (LGRs) was investigated computationally and by Multiplex Ligation-dependent Probe Amplification (MLPA). Results A pathogenic variant (PV) was identified in 22% (291/1325) of analyzed individuals and in specific in 23.2% of BC patients and 14.1% of unaffected individuals (P = 0.006). Among individuals with PVs, 49.1% were located in the BRCA1/2 genes whereas 8.6%, 22.7% and 19.6% occurred in other high, moderate and low-risk genes respectively. Notably, 21 of the 291 positive individuals (7.2%) carried clinically significant variants in two different genes and 6.5% had a LGR. A retrospective analysis of positive individuals showed that 88.3% of BC patients met the NCCN criteria for further genetic risk evaluation compared to 80.8% of unaffected individuals with FH of BC (P = 0.269). In BRCA-positive cases, NCCN criteria were met in 92.3% of the referrals compared to 81.8% in individuals positive for other genes (P = 0.008). Conclusions Extended multigene panel testing in hereditary BC facilitates the detection of nearly twice as many individuals that could benefit from personalized management. In our cohort, the currently used selection criteria for HBOC failed to identify only 12.7% of individuals positive for pathogenic variants, suggesting strong selection strategies from physicians. However, our results indicate that selection criteria perform better for the identification of BRCA-positive BC patients and should be revised to facilitate towards the inclusion of BC patients with PVs in other genes. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

AIM: The aim of this study was to further delineate the extent and nature of mutations in the BRCA1 and BRCA2 genes, responsible for hereditary breast and ovarian cancer in Greek families. MATERAILS & METHODS: Genomic DNA was isolated from whole peripheral blood of patients referred to our center for mutation analysis of the BRCA1 and BRCA2 genes. Patients were included on the basis of affected family members, types of cancer present in the family and age at diagnosis of breast cancer in the proband. Families were subdivided into high, medium and low risk depending on the number of affected family members, types of cancer diagnosed in the family and age at diagnosis of affected family members. In total, 675 families have been analyzed by our group in the past 4 years. Mutation analysis in all cases included sequencing of the coding region and the splice sites of the two genes. In addition, MLPA analysis was carried in 585 of the patients. RESULTS: In total, a pathogenic mutation has been identified in 12% of the 675 patients analyzed. Of the 78 mutations identified in total, 13 (17%) were large genomic rearrangements. These were deletions of exons 8, 20, 23, 23-24 and the entire BRCA1 gene, in addition to a duplication of exons 3-8 of the BRCA1 gene. As far as BRCA2 is involved deletions of exons 3, 15 and the entire BRCA2 gene were detected. All deletions were confirmed by use of other MLPA probe sets and/or relative quantitation by Real Time PCR. Of the rearrangements identified, two, namely deletions of exon 20 and exons 23-24 of the BRCA1 gene were identified in more than one unrelated families. In addition, the recurrent mutations 5382insC and G1738R, which have been previously identified as founder mutations in the Greek population, were identified in multiple unrelated analyzed families. CONCLUSIONS: Our results indicate that different large genomic rearrangements account for an important proportion (17%) of the mutations in the BRCA1 and BRCA2 genes, in Greek families at risk of carrying a germline mutation as judged by family / personal history. The use of the available technologies for the identification of such mutational events is therefore necessary when carrying out complete analysis of the genes in high risk families of Greek background. Citation Format: Angela Apessos, Eirini Papadopoulou, Vassiliki Metaxa-Mariatou, Konstantinos Agiannitopoulos, Christos Markopoulos, Vasileios Venizelos, Grigorios Xepapadakis, Maria Vasilaki-Antonatou, Antonios Keramopoulos, Nikolaos Bredakis, Aristeidis Tsiftsoglou, Georgios Kesisis, Stylianos Kakolyris, Nikolaos Touroutoglou, Ioannis Natsiopoulos, Konstantinos Papazisis, Georgios Nasioulas. Different genomic rearrangements account for 17% of BRCA1/2 mutations in Greece [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-03-08.

Germline mutations in the BRCA1 and BRCA2 genes are associated with hereditary predisposition to breast and ovarian cancer. Sensitive and accurate detection of BRCA1 and BRCA2 mutations is crucial for personalized clinical management of individuals affected by breast or ovarian cancer, and for the identification of at-risk healthy relatives. We performed molecular analysis of the BRCA1 and BRCA2 genes in 898 Greek families, using Sanger sequencing or Next Generation Sequencing for the detection of small insertion/deletion frameshift, nonsynonymous, truncating and splice-site alterations and MLPA for the detection of large genomic rearrangements. In total, a pathogenic mutation was identified in 12.9% of 898 families analyzed. Of the 116 mutations identified in total 9% were novel and 14.7% were large genomic rearrangements. Our results indicate that different types of mutational events in the BRCA1 and BRCA2 genes are responsible for the hereditary component of breast/ovarian cancer in the Greek population. Therefore the methodology used in the analysis of Greek patients must be able to detect both point and small frameshift mutations in addition to large genomic rearrangements across the entire coding region of the two genes.

BACKGOUND: Hereditary cancer predisposition syndromes are believed to be responsible for approximately 5-10% of all diagnosed cancer cases. In the past, single genes analysis of certain high risk genes was used for the determination of the genetic cause of cancer heritability in certain families. The selection of genes was mainly based on the family history of the individuals analyzed and included only highly associated genes (e.g. the BRCA1 and BRCA2 genes for families with breast cancer history. Nowadays though, the application of Next Generation Sequencing (NGS) technology has facilitated multigene panel analysis and is widely used in clinical practice, for the identification of individuals with cancer predisposition gene mutations. AIM: The aim of this study was to investigate the extent and nature of mutations in 36 genes implicated in hereditary cancer predisposition in individuals referred for testing in our lab. MATERIALS & METHODS: In total, 1197 individuals were referred for testing in our lab in the past four years from Greece, Romania and Turkey. The analysis of genes involved in hereditary cancer predisposition was performed using two NGS approaches. The first 451 individuals were analyzed using an amplicon based sequencing method (26 gene panel), while the following 746 individuals were analyzed using a capture based method (33 gene panel). Genomic DNA was enriched for targeted regions of 36 genes involved in hereditary predisposition to cancer included in both versions of the panel (APC, BMPR1A, BRCA1, BRCA2, CDH1, CDK4, CDKN2A, EPCAM, MEN1, MLH1, MSH2, MSH6, MUTYH, PALB2, PMS2, PTEN, RET, SMAD4, STK11, TP53, VHL, ATM, BRIP1, CHEK2, NBN, RAD51C, RAD51D, BARD1, BLM, CHEK1, ABRAXAS1 (FAM175A), MRE11 (MRE11A), NF1, RAD50, RAD51B, XRCC2). Sequencing was carried out using the Illumina NGS technology. Reads were aligned to the reference sequence (GRCh37), and sequence changes were identified and interpreted in the context of a single clinically relevant transcript. The presence of large genomic rearrangements was investigated by computational analysis of NGS results and the use of MLPA for 13 genes. All clinically significant observations were confirmed by orthogonal technologies. RESULTS: In total, a pathogenic mutation was identified in 259 of the 1197 individuals (21.6%) analyzed while a VUS was identified in 35.7% of the cases. Clinically significant mutations were identified in 29 of the genes analyzed. Concerning the mutation distribution among individuals with positive findings, 44.7% of them were located in BRCA1/2 genes whereas 20.9%, 19.9%, and 14.5% in high, moderate and low risk genes respectively. In addition to BRCA1 and BRCA2 genes other highly mutated genes were CHEK2 (10.6%), PALB2 (7.1%), MUTYH (7.1%) and ATM (4.3%). Of note is that 25 of the 259 positive individuals (9.7%) carried clinically significant mutations in two different genes and 5.8% had a large genomic rearrangement (LGR). CONCLUSIONS: Our results support the clinical significance of analysis of a panel of genes involved in hereditary cancer predisposition. In our cohort, analysis of this panel allowed for the identification of 8.3% additional pathogenic variants in moderate/low risk genes, enabling personalized management of these individuals. Citation Format: Tsoulos N, Tsaousis GN, Papadopoulou E, Agiannitopoulos K, Pepe G, Kambouri S, Apessos A, Diamantopoulos N, Floros T, Iosifidou R, Katopodi O, Koumarianou A, Markopoulos C, Papazisis K, Venizelos V, Xanthakis I, Xepapadakis G, Banu E, Eniu DT, Negru S, Stanculeanu DL, Ungureanu A, Ozmen V, Tansan S, Tekinel M, Yalcin S, Nasioulas G. Analysis of hereditary cancer syndromes by using a panel of genes: Novel and multiple pathogenic mutations [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-03-07.

The CYP17 gene encodes the enzyme cytochrome P450c17α, which functions at key steps in the synthesis process of human sex steroid hormones. A T/C polymorphism in the 5′ promoter region of the CYP17 gene has been described previously. Serum levels of androgens and estrogens have been shown to be elevated in individuals who carry the C substitution (Α2 allele). We hypothesized that variability in genes that control the sex hormone (estrogens, testosterone) biosynthesis might affect the pregnancy outcome. In the present study, we investigated the possible association between the T/C polymorphism of the promoter of CYP17 gene and the risk of recurrent spontaneous abortions in the Greek population. In the prospective case-control study, 148 patients and 134 healthy controls were studied. Women who had at least three unexplained spontaneous abortions before 20 weeks of gestation were included in the patient group. The PCR–RFLP method was used to genotype the subjects. The frequencies of A1A1, A1A2, A2A2 genotypes were 0.34, 0.52, 0.14, respectively, in the patient group and 0.32, 0.47, 0.21, respectively, in the control group. The allele frequencies were 0.595 and 0.405 for A1 and A2, in the patient group and 0.555 and 0.445 for A1 and A2, respectively, in the control group. The data between the two groups were analyzed by χ2 test. Our results showed that there are no significant differences in genotype (P = 0.3883) or in allele frequencies (P = 0.3800) between the patient and the control group. The T/C promoter polymorphism of the CYP17 gene is not associated with the risk for recurrent spontaneous abortions in our Caucasian population.

INTRODUCTION Hereditary breast cancer is estimated to account for approximately 10% of all breast cancer cases. In addition, an estimated 15-20% of those affected by breast cancer have a positive family history. Despite the fact that BRCA1 and BRCA2 are the two most significant genes in hereditary breast cancer predisposition, twenty years of analysis has highlighted the fact, that mutations in these two highly penetrant genes, are only present in approximately 20% of high risk families. Other genes, mutations in which are associated with high risk of breast cancer, were identified because of the strong association with familial cancer syndromes, in which breast cancer is one of the defining components. Technological advances in molecular biology and especially DNA sequencing, commonly designated as “Next Generation Sequencing – NGS” have aided in the concentrated efforts to identify new genes responsible for the missing heritability, allowing the application of this knowledge in the diagnostic setting. AIM The aim of this study was to investigate the extent and nature of mutations in 26 genes implicated in hereditary cancer predisposition in families of Romanian descent. MATERIALS & METHODS In total, 297 Romanian families have been analyzed by our group in the past three years. Genomic DNA was enriched for targeted regions of 26 genes involved in hereditary predisposition to cancer (ATM, BARD1, BLM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM (only intron 8, exon 9 and 3'UTR), FAM175A, MEN1, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53, XRCC2). Sequencing was carried out using the Illumina NGS technology. Reads were aligned to the reference sequence (GRCh38), and sequence changes were identified and interpreted in the context of a single clinically relevant transcript. The presence of large genomic rearrangements was investigated by use of MLPA. All clinically significant observations were confirmed by orthogonal technologies. RESULTS In total, a pathogenic mutation was identified in 79 of the 297 families (26.6%) analyzed. Clinically significant mutations were identified in 17 of the genes included in the panel. The most commonly mutated genes in the Romanian population were BRCA1 and BRCA2, accounting for 50% of the mutations identified, followed by PALB2 (12%), CHEK2 (9.4%) and ATM, NBN and RAD50 which accounted for 3.5% of the mutations each. Of note is that 7 of the 79 affected families (8.8%) carried clinically significant mutations in two different genes. CONCLUSIONS Our results support the clinical significance of analysis of a panel of genes involved in hereditary cancer predisposition. In this series of patients, analysis of this panel allowed for the identification of 14% additional pathogenic variants. This is especially true in those cases where more than one pathogenic variant was identified. Citation Format: Tsoulos N, Apessos A, Agiannitopoulos K, Pepe G, Tsaousis G, Kambouri S, Eniu DT, Ungureanu A, Banu E, Ciule L, Blidaru A, Chiorean A, Stanculeanu DL, Mateescu D, Nasioulas G. Analysis of hereditary cancer syndromes by use of a panel of genes: More answers than questions [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-03-03.

It has been reported that certain patients with non-small-cell lung cancer (NSCLC) that harbor activating somatic mutations within the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene may be effectively treated using targeted therapy. The use of EGFR inhibitors in patient therapy has been demonstrated to improve response and survival rates; therefore, it was suggested that clinical screening for EGFR mutations should be performed for all patients. Numerous clinicopathological factors have been associated with EGFR and Kirsten-rat sarcoma oncogene homolog (KRAS) mutational status including gender, smoking history and histology. In addition, it was reported that EGFR mutation frequency in NSCLC patients was ethnicity-dependent, with an incidence rate of ~30% in Asian populations and ~15% in Caucasian populations. However, limited data has been reported on intra-ethnic differences throughout Europe. The present study aimed to investigate the frequency and spectrum of EGFR mutations in 1,472 Greek NSCLC patients. In addition, KRAS mutation analysis was performed in patients with known smoking history in order to determine the correlation of type and mutation frequency with smoking. High-resolution melting curve (HRM) analysis followed by Sanger sequencing was used to identify mutations in exons 18–21 of the EGFR gene and in exon 2 of the KRAS gene. A sensitive next-generation sequencing (NGS) technology was also employed to classify samples with equivocal results. The use of sensitive mutation detection techniques in a large study population of Greek NSCLC patients in routine diagnostic practice revealed an overall EGFR mutation frequency of 15.83%. This mutation frequency was comparable to that previously reported in other European populations. Of note, there was a 99.8% concordance between the HRM method and Sanger sequencing. NGS was found to be the most sensitive method. In addition, female non-smokers demonstrated a high prevalence of EGFR mutations. Furthermore, KRAS mutation analysis in patients with a known smoking history revealed no difference in mutation frequency according to smoking status; however, a different mutation spectrum was observed.

Sex hormones are well known to increase the risk of coronary artery disease (CAD). The CYP17 gene encodes the enzyme cytochrome P450c17, which functions at key steps during the process of human sex steroid hormone synthesis. A T/C polymorphism in the 5 promoter region of the CYP17 gene influences its expression and the resulting serum levels of androgens and estrogens. The aim of this casecontrol study was to investigate the role of a T/C CYP17 polymorphism in premature CAD and the occurrence of myocardial infarction (MI) in the Caucasian Greek population.Our study group consisted of 230 CAD patients, aged less than 58 years, while 200 healthy individuals served as controls. The genotyping of the T/C CYP17 polymorphism was carried out using the PCRRFLP method.The frequencies of TT, TC, and CC genotypes were 0.38, 0.42, and 0.20, respectively, in the patient group, and 0.35, 0.44, and 0.21, respectively, in the control group. Allele frequencies for the patient group were 0.58 and 0.42 for T and C, respectively, and 0.57 and 0.43, respectively, for the control group. Statistical analysis revealed no significant differences between patients and controls in genotype frequencies (p=0.8746) or allele frequencies (p=0.6783).These findings do not support the hypothesis that the genetic variation T/C of the promoter of the CYP17 gene is an important contributing factor in the aetiology of premature CAD or occurrence of MI in the Caucasian Greek population.

HSD3B1 gene encodes the 3β-hydroxysteroid deydrogenases/isomerase (3β-HSD) enzyme, which plays a crucial role in the biosynthesis of all hormonal steroids. The aim of this study was to examine the potential impact of a T → C substitution at codon Leu(338) of HSD3B1 gene on pregnancy outcome.In this prospective case-control study, 162 patients and 139 healthy controls were investigated for the possible association between the HSD3B1 T/C polymorphism and the risk of recurrent spontaneous abortions (RSA). The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used in order to genotype the subjects.The frequencies of TT, TC, and CC genotypes were 0.20, 0.51, and 0.29, respectively, in the patient group and 0.20, 0.45, and 0.35, respectively, in the control group. The allele frequencies were 0.456 and 0.428 for T allele for the patient group and control group, respectively and 0.543 and 0.572 for C allele for the patient and control group, respectively. The data between the two groups were analyzed by chi-square test or Fisher's exact test. Our results showed that there are no significant differences in genotype (P = 0.56) or in allele frequencies (P = 0.51) between the patient and the control group.The HSD3B1 T/C polymorphism cannot be used as genetic marker for the risk for RSA in our Caucasian population.

e22178 Background: NSCLC patients harboring activating somatic mutations within the tyrosine kinase (TK) domain of the epidermal growth factor receptor (EGFR) significantly benefit from EGFR target...

e12536 Background: Approximately 5-10% of breast cancer cases are hereditary. Germline mutations in the BRCA1 and BRCA2genes are responsible for 80% of inherited breast cancer cases. Carriers of su...