Mutational profile of hereditary breast and ovarian cancer - Establishing genetic testing guidelines in a developing country.

PURPOSE: Because many countries lack the capacity to follow the international guidelines for genetic testing, we suggest the specific approach for establishing local genetic testing guidelines that could be applied in developing countries. We focus on hereditary breast (BC) and ovarian cancer (OC) in Serbia. METHODS: From the cohort of 550 persons who were referred for genetic counseling at the Institute for Oncology and Radiology of Serbia, 392 were selected. Personal and family histories were collected and germline DNA was sequenced with NGS in a panel of 20 genes. RESULTS: Pathogenic (PV) and likely-pathogenic variants (LPV) were detected in 8 genes with the frequency of 23.7%. The most frequent were in BRCA1(7.6%), BRCA2(4.8%), PALB2(4.1%) and CHEK2(3.8%). They were also detected in ATM(1.8%), NBN(0.8%), TP53(0.5%) and RAD51C(0.3%). Whereas high carrier probability (CP), bilateral BC, BC and OC in the same patient and family history (FH) of BC/OC, were the strongest predictors for BRCA1/2 PV/LPV, lower CP values and early age of BC onset without FH were associated with higher frequency of PALB2 and CHEK2 PV/LPV. CONCLUSIONS: Population specific studies to identify specific mutational patterns and predictors of PV/LPV should be conducted in order to make scientifically sound and cost-effective guidelines for genetic testing in developing countries.

Expression of SIRT1, SIRT3 and SIRT6 Genes for Predicting Survival in Triple-Negative and Hormone Receptor-Positive Subtypes of Breast Cancer.

Triple-negative breast cancer (TNBC) is characterized by aggressive phenotype and a poorer prognosis compared to the estrogen and progesterone receptor positive, Her2 negative (ER + PR + Her2-) breast cancer. Increasing evidence suggests that sirtuins, a family of histone deacetylases, could have an important role in aggressiveness of TNBC's. The current study evaluated the potential clinical relevance of SIRT1, SIRT3 and SIRT6 gene expressions in two prognostically distinctive subtypes of breast cancer, the most aggressive TNBC and the least aggressive ER + PR + Her2- tumors. Total RNAs were isolated from 48 TNBC and 63 ER + PR + Her2- tumor samples. Relative gene expression was determined by SYBR Green RT-PCR and delta-delta Ct method, normalized to GAPDH. Mean gene expression of both SIRT1 and SIRT3 was significantly lower in the TNBC compared to ER + PR + Her2- tumors (p = 0.0001). Low SIRT1 and SIRT6 expressions associated with worse overall survival in ER + PR + Her2- patients (p = 0.039, p = 0.006, respectively), while TNBC patients with high SIRT1 tend to have a poor prognosis (p = 0.057). In contrast, high expression of SIRT3 in TNBC patients associated with higher histological grade (p = 0.027) and worse overall survival (p = 0.039). The Cox regression analysis revealed that low SIRT1 expression could be an independent prognostic marker of poor survival in ER + PR + Her2- breast cancers (HR = 11.765, 95% CI:1.234-100, p = 0.033). Observed differential expression of SIRT1, SIRT3 and SIRT6 genes in TNBC and ER + PR + Her2- subtypes, with opposite effects on patients' survival, suggests context-dependent mechanisms underlying aggressiveness of breast cancer. Further investigations are necessary to evaluate sirtuins as potential biomarkers and therapeutic targets in breast cancer.

Confirmation of damaging effect of MSH2 c.2634+1G>C mutation on splicing, its classification and implications for counseling.

INTRODUCTION: Lynch syndrome (LS) is predisposing mainly to colorectal and endometrial carcinomas, but also to urinary tract cancers. LS association with upper urinary tract carcinomas is known, but its association with bladder cancer is not so clear. Confirmation of pathogenicity of detected mutations in LS-associated genes is required for adequate counseling. MATERIAL AND METHODS: Tested young female has family history of two early colorectal and two bladder carcinomas. NGS sequencing revealed MSH2 splice site mutation c.2634+1G>C, which was confirmed by Sanger sequencing. MSH2 cDNA part containing potential splicing change was sequenced. in silico softwares were used to predict the effect of detected mutation on splicing and protein structure. ACMG Guidelines were used for mutation classification. RESULTS: in silico softwares predict damaging effect of detected mutation on splicing and loss of protein-binding domains. cDNA sequencing confirmed this mutation causes exon 15 excision. ACMG Guidelines classify this mutation as Pathogenic. DISCUSSION: MSH2 c.2634+1G>C mutation was not reported previously as LS associated. We confirmed its damaging effect on splicing. in silico tools predict consequent loss of protein domains implicating disrupted protein function. Our results suggest that this mutation should be classified as Pathogenic, and indicate inclusion of bladder cancer in LS cancer spectrum.

Association of uPA and PAI-1 tumor levels and 4G/5G variants of PAI-1 gene with disease outcome in luminal HER2-negative node-negative breast cancer patients treated with adjuvant endocrine therapy.

BACKGROUND: The aim of this study was to evaluate the prognostic potential of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) tumor tissue levels and examine the association between these biomarkers and classical prognostic factors in early node-negative luminal breast cancer patients. The clinical value of 4G/5G variants of PAI-1 gene was evaluated. PATIENTS AND METHODS: This study involved 81 node-negative, estrogen receptor-positive and/or progesterone receptor-positive and human epidermal growth factor receptor 2-negative operable breast cancer patients who underwent radical surgical resection and received adjuvant endocrine therapy. Determination of uPA and PAI-1 concentrations in the breast cancer tissue extracts was performed using FEMTELLE® uPA/PAI-1 ELISA. An insertion (5G)/deletion (4G) polymorphism at position - 675 of the PAI-1 gene was detected by PCR-RFLP analysis. RESULTS: Our research showed that patients with uPA tumor tissue levels higher than 3 ng/mg of protein had significantly reduced disease-free survival (DFS) and overall survival (OS) when compared to patients with uPA tumor tissue levels lower or equal to 3 ng/mg of protein. Patients with PAI-1 tumor tissue levels higher than 14 ng/mg of protein had significantly decreased OS in comparison with patients with PAI-1 tumor tissue levels lower or equal to 14 ng/mg of protein. ROC analysis confirmed the uPA and PAI-1 discriminative potential for the presence/absence of relevant events in these patients and resulted in higher cut-off values (5.65 ng/mg of protein for uPA and 27.10 ng/mg of protein for PAI-1) than standard reference cut-off values for both biomarkers. The prognostic importance of uPA and PAI-1 ROC cut-off values was confirmed by the impact of uPA higher than 5.65 ng/mg of protein and PAI-1 higher than 27.10 ng/mg of protein on poorer DFS, OS and event-free survival (EFS). We observed that patients with dominant allele in PAI-1 genotype (heterozygote and dominant homozygote, - 675 4G/5G and - 675 5G/5G) had significantly increased DFS, OS and EFS when compared with patients with recessive homozygote genotype (- 675 4G/4G). CONCLUSION: Our study indicates that uPA and PAI-1 tumor tissue levels and 4G/5G variants of PAI-1 gene might be of prognostic significance in early node-negative luminal HER2-negative breast cancer patients treated with adjuvant endocrine therapy.

Germline mutations in cancer susceptibility genes in high grade serous ovarian cancer in Serbia.

Clinical criteria for genetic testing of genes other than BRCA1/2 in epithelial ovarian cancer (EOC) still do not exist. We assessed the frequency and predictors of deleterious mutations in 19 cancer predisposition genes in high-grade serous ovarian cancer (HGSOC) in Serbia. Next-generation sequencing was used to identify germline mutations in the whole coding regions of a gene panel. Patients' characteristics and sequencing data were summarized with descriptive statistics and compared using chi-square test. Among 131 HGSOC patients, 23 had BRCA1 (17.6%) while 5 had BRCA2 (3.8%) mutation. In addition, 9 (6.9%) pathogenic mutations were detected in other genes including BRIP1 (n = 2;1.5%), CHEK2 (n = 2;1.5%), NBN (n = 3;2.3%) and RAD51C (n = 2;1.5%). Factors that predicted for BRCA1/2 mutations were: breast and ovarian cancers in the same patient (p = 0.031), young age of EOC (p = 0.029), menstrual status (p = 0.004) and family history of cancer (p < 0.0001). However, these factors did not predict for mutations in other cancer susceptibility genes. Applying established referral criteria for genetic testing in Serbia will help identify BRCA1/2 mutation carriers but will not help identify mutations in other cancer susceptibility genes. Until better predictors emerge we should be performing wider genetic testing of EOC in order to identify all mutation carriers.

Promoter hypermethylation of p16, BRCA1 and RASSF1A genes in triple-negative breast cancer patients from Serbia.

PURPOSE: In order to investigate if aberrant promoter methylation of p16, BRCA1 and RASSF1A genes contributes to biological behavior of triple-negative breast cancer (TNBC), marked as the most aggressive phenotype of breast cancer, we compared the hypermethylation pattern between TNBC and ER+PR+Her2- breast cancer. METHODS: 131 patients with histologically confirmed breast cancers were included - 61 TNBC and 70 ER+PR+Her2- cases. The patients were followed up for 1-87 months (median 78). DNA from tumor tissues was isolated by the salting out procedure. The methylation status was assessed by nested methylation-specific PCR after bisulfite modification of DNA. RESULTS: The frequency of p16 hypermethylated breast cancer cases was significantly higher in TNBC than in ER+PR+Her2- group (33; 54.1% vs. 20; 28.6%, p=0.00298). Co-methylated p16 and RASSF1A genes were more frequent in the TNBC than in ER+PR+Her2- group (20; 32.8% vs. 10; 14.3%, p=0.0225). The same result was observed when hypermethylated BRCA1 gene was added in the analysis: 12; 19.7% vs. 3; 4.3%, p=0.00791. Although there was significant difference in disease-free survival (DFS) and overall survival (OS) between TNBC and ER+PR+Her2- group, further analysis of co-methylation of p16 and RASSF1A (p16+RASSF1A+) showed that DFS was significantly shorter in the patients with both genes co-methylated in TNBC than in ER+PR+Her-2- group (8/20; 40% vs. 2/10; 20%, p=0.03272). CONCLUSIONS: The obtained data indicate that hypermethylated p16 and RASSF1A cell-cycle inhibitor genes might be considered as biomarkers for bad prognosis in breast cancer. Hypermethylation of these genes may influence the clinical disease course, distinguishing a particular group of TNBC patients with even more aggressive phenotype.

Polymorphisms in cancer susceptibility genes XRCC1, RAD51 and TP53 and the risk of breast cancer in Serbian women.

BACKGROUND: Thanks to immense improvements in technology over the past few decades, we have witnessed a major shift towards the idea that breast cancer results from a combined effect of multiple common alleles conferring low risk. This study investigates the role of 3 nonsynonymous SNPs in the DNA repair genes XRCC1 (R399Q), RAD51 (G135C) and TP53 (Arg72Pro) in breast cancer in Serbian women. PATIENTS AND METHODS: Cases of BRCA1/2-negative hereditary breast cancer (n = 52), sporadic breast cancer (n = 106) and age-matched cancer-free female controls (n = 104) were obtained from the Institute for Oncology and Radiology of Serbia's blood bank. Restriction fragment length polymorphism analysis was used for genotyping. Descriptive analyses included genotype and allelic frequencies; the odds ratio and 95% confidence interval were calculated as an estimate of the relative risk. RESULTS: A significant difference in QQ+RQ versus RR genotype distribution of XRCC1 was observed between hereditary breast cancer patients and cancer-free controls. The association was confirmed among young breast cancer patients from these high-risk families. The existence of 3 recessive alleles in the RAD51 and XRCC1 genotype combination showed an association with hereditary breast cancer. Odds ratio analysis indicated a strong protective role of the RAD51 GG + TP53 ArgArg + XRCC1 RR combined genotype against hereditary breast cancer negative for BRCA1/2 mutations. CONCLUSIONS: The XRCC1 R399Q polymorphism showed an association with increased breast cancer risk in Serbia, especially in the hereditary form of the disease and in young breast cancer patients. Dominant alleles of RAD51, TP53 and XRCC1 combined genotypes indicated a strong protective role against hereditary breast cancer.

RAD51C mutation screening in high-risk patients from Serbian hereditary breast/ovarian cancer families.

BACKGROUND: In 2010 an important finding was published showing that heterozygous mutations in RAD51C were highly penetrant and were able to confer an increased risk for breast and ovarian cancers. The role of possible third high penetrance breast cancer susceptibility gene was assigned to RAD51C. OBJECTIVE: Because of its rising importance in breast cancer development and the lack of information about RAD51C in Slavic populations, our goal was to identify potential population specific mutations in this gene in order to determine more detailed genetic screening strategy and breast cancer risk assessment. METHODS: The study included 55 females from Serbian hereditary breast/ovarian cancer families negative for sequence alterations and large genomic rearrangements in BRCA1/2 genes. Whole coding region and exon-intron boundaries of RAD51C were analyzed by dHPLC. All mutations were confirmed by Sanger sequencing. SIFT and Polyphen were used to predict possible impact of non-synonymous variants. RESULTS: We found 5 variants in RAD51C including two missense, one intronic, one in the 5'UTR and one variant in the promoter region of the gene. Three detected variants are common - c.1-118G>A (rs16943176, MAF = 0,203); c.1-26C>T (rs12946397, MAF = 0,207) and c.904+34T>C (rs28363318, MAF = 0,186). We detected two missense variants, c.790G>A (p.Gly264Ser) in exon 5 and c.859A>G (p.Thr287Ala) in exon 6. Both of them were previously shown to exhibit reduced protein function but their contribution to cancer risk is still unknown. CONCLUSIONS: Although the initial reports implied that RAD51C might be promising candidate for next high penetrance breast cancer susceptibility gene, lack of confirmation suggested that RAD51C mutations are not as common as expected. Our study did not reveal truncating mutations in RAD51C suggesting that other breast cancer susceptibility genes may account for the increased susceptibility in our cohort of high-risk BRCA1/2 negative families.

RAD51 135G>C and TP53 Arg72Pro polymorphisms and susceptibility to breast cancer in Serbian women.

Breast cancer is a complex disease with both genetic and environmental factors involved in its etiology. An important role of polymorphisms in genes involved in DNA repair has been reported related to breast cancer risk. We conducted a case-control study in order to investigate the association of RAD51 135G>C and TP53 Arg72Pro polymorphisms with breast cancer in Serbian women.48 BRCA negative women with breast cancer and family history of breast/ovarian cancer (hereditary group), 107 women with breast cancer but without family history of the disease (sporadic group) and 114 healthy women without a history of the disease (control group) were included. Restriction fragment length polymorphism was used for genotyping. Genotype and allelic frequencies, the odds ratio (OR) and the 95 % confidence interval (CI) were calculated as an estimate of relative risk. The Hardy-Weinberg equilibrium was tested using χ(2) test. Significance was considered for p < 0.05. RAD51 135G>C showed statistically significant association of CC genotype and increased breast cancer risk (OR 10.28, 95 % CI 1.12-94.5) in hereditary group of patients compared to the control group. Regarding the TP53 Arg72Pro, we showed statistical significance for ProPro + ProArg comparing to ArgArg (OR 2.34, 95 %, CI 1.17-4.70) in hereditary compared to sporadic group. RAD51 135G>C contributes to hereditary breast cancer in Serbian population, with CC genotype as a risk factor. We also found that carriers of Pro allele of TP53 codon 72 is related to hereditary cancer comparing to sporadic one, which indicates it as a potential risk factor for hereditary form of disease.

Serbian high-risk families: extensive results on BRCA mutation spectra and frequency.

Mutations in BRCA genes elevate risk for breast and ovarian cancer. These mutations are population specific. As there are no data on BRCA mutation screening on larger number of probands in Serbia to date, aim of this study was to determine types and frequencies of BRCA mutations in individuals from high-risk families from Serbia, as well as to determine which BRCA mutations may be considered as founder for Serbian population. We analyzed 94 probands and detected 9 frameshift mutations in 12 individuals, 1 benign BRCA2 nonsense mutation and numerous missense and synonymous mutations in both genes. Frequency of frameshift mutations is 12.77%. In addition to two novel mutations detected in our population we reported previously, we detected another novel mutation--c.7283delT in BRCA2 exon 14. None of the detected deleterious mutations may be considered as founder mutations for Serbian population, as each of them was found in no more than two high-risk families. This mutation diversity is most probably due to high migration rate in history of this part of Europe. Interpretation of genetic testing results with missense mutations of unknown clinical importance is very challenging and should be approached with caution, using all available data sources for results' interpretation.

Combined NGS approaches identify mutations in the intraflagellar transport gene IFT140 in skeletal ciliopathies with early progressive kidney Disease.

Ciliopathies are genetically heterogeneous disorders characterized by variable expressivity and overlaps between different disease entities. This is exemplified by the short rib-polydactyly syndromes, Jeune, Sensenbrenner, and Mainzer-Saldino chondrodysplasia syndromes. These three syndromes are frequently caused by mutations in intraflagellar transport (IFT) genes affecting the primary cilia, which play a crucial role in skeletal and chondral development. Here, we identified mutations in IFT140, an IFT complex A gene, in five Jeune asphyxiating thoracic dystrophy (JATD) and two Mainzer-Saldino syndrome (MSS) families, by screening a cohort of 66 JATD/MSS patients using whole exome sequencing and targeted resequencing of a customized ciliopathy gene panel. We also found an enrichment of rare IFT140 alleles in JATD compared with nonciliopathy diseases, implying putative modifier effects for certain alleles. IFT140 patients presented with mild chest narrowing, but all had end-stage renal failure under 13 years of age and retinal dystrophy when examined for ocular dysfunction. This is consistent with the severe cystic phenotype of Ift140 conditional knockout mice, and the higher level of Ift140 expression in kidney and retina compared with the skeleton at E15.5 in the mouse. IFT140 is therefore a major cause of cono-renal syndromes (JATD and MSS). The present study strengthens the rationale for IFT140 screening in skeletal ciliopathy spectrum patients that have kidney disease and/or retinal dystrophy.

Novel BRCA1/2 mutations in Serbian breast and breast-ovarian cancer patients with hereditary predisposition.

Mutations in breast cancer susceptibility (BRCA) genes lead to defects in DNA repair processes resulting in elevated genome instability and predisposing to breast and ovarian cancer. The study was designed to detect mutational spectra of BRCA1/2 genes in a Serbian population. Using automated DNA sequencing, we tested individuals for BRCA mutations, based on positive family history of either breast or ovarian cancer or both. Two novel mutations (c.4765_4784del in BRCA1 exon 15 and c.4367_4368dupTT in BRCA2 exon 11) were detected, in three probands from two different families. These mutations have not been reported previously in the BIC or LOVD databases. Protein products of these mutated alleles lack domains necessary for their DNA repair functions, an indicator that these are deleterious mutations. Neither mutation was found in any proband from 50 other families with hereditary predisposition, so the two mutations are likely family-specific rather than population-specific. Although BRCA1-associated tumors are typically negative for estrogen receptor (ER), progesterone receptor (PR), and ERBB2, the novel BRCA1 mutation identified in this study was detected in a proband with ER- and PR-positive breast cancer. Steroid receptor-positive BRCA-related breast cancer in this proband supports the idea of characteristic pathological features and older age of onset among BRCA1-mutated ER-positive breast cancers.

Gene hypermethylation in tumor tissue of advanced oral squamous cell carcinoma patients.

Oral squamous cell carcinoma (OSCC) is characterized by high mortality rate and rising incidence. The aim of this study was to examine the methylation of particular tumor suppressor genes promoters in OSCC and to correlate the methylation status with the tumor-host features and patients survival. The genes selected for our investigation are involved in key cellular processes of malignant transformation, including cell cycle control (p16), apoptosis (Death Associated Protein Kinase, DAPK), Wnt signaling (Adenomatous Polyposis Coli, APC), cell-cell adhesion (E-cadherin, E-cad), and DNA repair (O(6)-methylguanine-DNA methyltransferase, MGMT, Werner syndrome gene, WRN). In 77 patients with OSCC, hypermethylation was determined by nested methylation-specific PCR method. Methylation of p16 gene promoter was detected in 58.4% of samples, E-cad in 42.9%, DAPK in 36.8%, MGMT in 33.8%, WRN in 23.8%, and APC in 18.2% of OSCC samples. Patients with E-cad promoter methylation had significantly worse overall survival (p=0.039, log-rank test), while hypermethylation of other genes did not have prognostic significance. Stratified analysis by the lymph node involvement and tumor stage showed that E-cad promoter methylation is associated to poor survival in N positive (p=0.024), and stage III tumors (p=0.027), as an opposite to N0 and stage II tumors, where E-cad methylation did not have prognostic significance (p=0.849, p=0.794, respectively). Our findings indicate that multiple genes are frequently aberrantly methylated in OSCC, and that E-cad promoter methylation should be considered as a potential molecular marker for the poor survival in advanced OSCC.

cDNA microarrays: identification of gene signatures and their application in clinical practice.

Sequencing of human genome introduced the new postgenomic era in medicine. A novel microarray technology represents a novel genetic platform which is being widely exploited to bridge the gap between gene sequence and function. Basically, with this technique it is possible to simultaneously measure the activity of virtually all genes in the genome. The result of these experiments are spots whose brightness varies from gene to gene corresponding to the transcriptional activity of the examined genes. It makes it possible to create experiments looking for genes whose expression correlates with the presence of tumor, with progression from a benign to a malignant condition, or with progression from a low grade to high grade malignancy. Tumor types can be more specifically identified, and differences in gene expression can be used to detect tumors that already demonstrate gene expression changes associated with increased risk of metastasis. Gene expression profiles will also help clinicians make decisions about the optimal treatment of cancer patients.

p53 mutations and human papillomavirus infection in oral squamous cell carcinomas: correlation with overall survival.

PURPOSE: The study investigated the pattern of p53 gene mutations and human papillomavirus (HPV) infection concerning their relation to overall survival in patients with oral squamous cell carcinomas of the tongue and floor of the mouth. PATIENTS AND METHODS: The presence of HPV infection in 50 patients, and p53 gene mutations (42 patients from the same group) in the tumour specimens were analysed by polymerase chain reaction and single-stranded conformational polymorphism method. The follow-up period ranged from 12 to 48 (median 29) months. RESULTS: p53 mutations were identified in 11/42 tumours. HPV infection was detected in 32/50 cases, mostly HPV16 (10/32), HPV18 and HPV31 (6/32). A significantly higher incidence of HPV infection was found among smokers (p<0.05) and among patients with poor oral hygiene (p<0.01). The highest incidence of p53 mutations was detected in tumours of histological grade I and nuclear grade III. Patients with p53 mutation or with HPV infection had significantly shorter overall survival when compared with those that were without p53 mutations (p<0.01) or HPV infection (p<0.05). HPV-infected patients with p53 mutation had the worst prognosis when compared with patients with HPV infection only (p<0.01) or with patients negative for both HPV and p53 (p<0.01). CONCLUSION: The results stress once more the importance of HPV for the prognosis of survival of patients with squamous cell carcinoma of lower parts of the oral cavity. The presence of p53 mutations in HPV-infected tumours was associated with an even poorer prognosis for the patients.

[Recommendations for treatment of individuals at risk for hereditary breast and ovarian carcinoma]. / Preporuke za lecenje osoba sa rizikom za nasledni karcinom dojke i jajnika.

Germline mutations in BRCA 1 and BRCA 2 gene have been recognized as hereditary predisposition for breast and ovarian cancer for many years. The optimal clinical management of individuals at risk for hereditary breast and ovarian cancer are not completely defined. Current surveillance options are restricted in their effectiveness as well as limitations of the techniques. Surgical prevention interventions are effective, but may be complicated by physical and psychological morbidity. Genetic testing, which plays a role in defining risk, requires careful pre- and post-test counseling to discuss the limitations of testing itself and available management strategies.