Analysis of hereditary cancer syndromes by using a panel of genes: novel and multiple pathogenic mutations.

BACKGROUND: Hereditary cancer predisposition syndromes are responsible for approximately 5-10% of all diagnosed cancer cases. In the past, single-gene analysis of specific high risk genes was used for the determination of the genetic cause of cancer heritability in certain families. 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 predisposing gene variants. The purpose of this study was to investigate the extent and nature of variants in genes implicated in hereditary cancer predisposition in individuals referred for testing in our laboratory. METHODS: In total, 1197 individuals from Greece, Romania and Turkey were referred to our laboratory for genetic testing in the past 4 years. The majority of referrals included individuals with personal of family history of breast and/or ovarian cancer. The analysis of genes involved in hereditary cancer predisposition was performed using a NGS approach. Genomic DNA was enriched for targeted regions of 36 genes and sequencing was carried out using the Illumina NGS technology. The presence of large genomic rearrangements (LGRs) was investigated by computational analysis and Multiplex Ligation-dependent Probe Amplification (MLPA). RESULTS: A pathogenic variant was identified in 264 of 1197 individuals (22.1%) analyzed while a variant of uncertain significance (VUS) was identified in 34.8% of cases. Clinically significant variants were identified in 29 of the 36 genes analyzed. Concerning the mutation distribution among individuals with positive findings, 43.6% were located in the BRCA1/2 genes whereas 21.6, 19.9, and 15.0% in other high, moderate and low risk genes respectively. Notably, 25 of the 264 positive individuals (9.5%) carried clinically significant variants in two different genes and 6.1% had a LGR. CONCLUSIONS: In our cohort, analysis of all the genes in the panel allowed the identification of 4.3 and 8.1% additional pathogenic variants in other high or moderate/low risk genes, respectively, enabling personalized management decisions for these individuals and supporting the clinical significance of multigene panel analysis in hereditary cancer predisposition.

Genetic Predisposition to Male Breast Cancer: A Case Series.

BACKGROUND/AIM: 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. MATERIALS AND METHODS: 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. RESULTS: Pathogenic variants were identified in 13 patients, with BRCA2 being the most commonly affected gene, followed by BRCA1, RAD50, RAD51B, and MSH3. CONCLUSION: 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.

Culture of primary epithelial adenoma cells from familial adenomatous polyposis patients.

BACKGROUND: Colorectal tumors arise from unregulated cell proliferation of the intestinal epithelium through a multistep process the first step usually being premalignant adenomas. Familial adenomatous polyposis patients carry a germ line mutation in the APC gene leading to the development of thousands of polyps, which, if left untreated, lead to cancer. The goal of this study was the establishment of conditions for the culture of epithelial cells composing an adenomatic structure. MATERIALS AND METHODS: All colorectal specimens were obtained from FAP patients within 1-2 hours of surgery. Cells were cultured by standard procedures. Characterization was carried out by immunostaining with pancytokeratin, vimentin and desmoplakin antibodies. RESULTS: A culture protocol that gave rise to epithelial cell growth with high efficiency and efficacy was established. Successful subculturing of the cell sheets took place only when dispase prepared in Ca2+ and Mg2+ free medium, was used to digest polyp tissue taken from FAP patients. By using immunostaining these cells were characterized as epithelial. CONCLUSION: The protocol we developed here provides a means of preparing cell cultures from human colorectal adenomas, which aid in the research of the transition from adenoma to carcinoma.

Inherited cancer predisposition syndromes in Greece.

Hereditary cancer syndromes comprise approximately 5-10% of diagnosed carcinomas. They are caused by mutations in specific genes. Carriers of mutations in these genes are at an increased risk of developing cancer at a young age. When there is a suspicion of a hereditary cancer predisposition syndrome a detailed family tree of the patient requesting screening is constructed. DNA is isolated from all available members of the family. Mutation detection is carried out on DNA from an affected family member. If a mutation is found the remaining family is screened. The genetic basis of a large number of inherited cancer predisposition syndromes is known. In this paper the focus is on mutations in genes responsible for colorectal cancer, meaning adenomatous polyposis coli (APC), which is involved in familial adenomatous polyposis and homo sapiens mutL homolog 1 (hMLH1) and homo sapiens mutS homolog 2 (hMSH2), involved in hereditary non-polyposis colorectal cancer. In addition, the genes responsible for inherited breast and/or ovarian cancer, breast cancer genes 1 and 2 (BRCA1 and BRCA2), and the rearranged during transfection protooncogene RET which is responsible for multiple endocrine neoplasia type 2 are discussed. In all cases emphasis is given to the data available on the Greek population.

Different intrafamilial clinical presentation of FMF mutation carriers.

Familial Mediterranean fever (FMF) is a heterogeneous disorder; at present, it is diagnosed using only genetic methods. In the current study, we performed molecular analysis in two families presenting with FMF. In the first family, we report two brothers with a common genotype (M694V/V726A) but with different clinical presentation. In the second family, we identified the M694V and K695R mutations in a presymptomatic carrier.

Comprehensive BRCA mutation analysis in the Greek population. Experience from a single clinical diagnostic center.

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.

Heterogeneous molecular mechanisms underlie attenuated familial adenomatous polyposis.

PURPOSE: Familial adenomatous polyposis is a phenotypically heterogeneous disease predisposing to colorectal cancer. It is dominantly transmitted, when associated with the APC gene, and recessively inherited, when associated with MUTYH gene. We searched for APC and MUTYH germline alterations in Italian and Greek patients with attenuated polyposis, a phenotypic variant whose genetic cause remains unknown in many cases. METHODS: We studied 26 unrelated patients (and 16 relatives) with multiple colorectal adenomas (3-100, by endoscopic analysis) that had screened APC mutation-negative by protein truncation test. We searched for APC rearrangements by multiplex ligation-dependent probe amplification and for MUTYH mutations by sequencing. We performed a screening of five MUTYH recurrent pathogenic mutations in 501 Italian and 144 Greek controls. RESULTS: One patient proved to carry an APC whole-gene deletion; 4 of 25 (16%) patients showed biallelic and 3 of 25 (12%) monoallelic MUTYH mutations. In the three heterozygous subjects no pathogenetic variants were found in OGG1, MTH1, APE1, MSH2, and MSH6 genes. Frequency assessment of MUTYH mutations in healthy subjects showed that only Y165C and G382D reach a subpolymorphic frequency. CONCLUSION: Attenuated polyposis patients without "conventional" APC mutations are genetically heterogeneous, and the phenotype is not directly related to the germline defect. Therefore, the families' appropriate management requires an accurate genetic and clinical investigation.

Molecular predictive markers in tumors of the gastrointestinal tract.

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.

Rare mutations predisposing to familial adenomatous polyposis in Greek FAP patients.

BACKGROUND: Familial Adenomatous Polyposis (FAP) is caused by germline mutations in the APC (Adenomatous Polyposis Coli) gene. The vast majority of APC mutations are point mutations or small insertions/deletions which lead to truncated protein products. Splicing mutations or gross genomic rearrangements are less common inactivating events of the APC gene. METHODS: In the current study genomic DNA or RNA from ten unrelated FAP suspected patients was examined for germline mutations in the APC gene. Family history and phenotype were used in order to select the patients. Methods used for testing were dHPLC (denaturing High Performance Liquid Chromatography), sequencing, MLPA (Multiplex Ligation - dependent Probe Amplification), Karyotyping, FISH (Fluorescence In Situ Hybridization) and RT-PCR (Reverse Transcription - Polymerase Chain Reaction). RESULTS: A 250 Kbp deletion in the APC gene starting from intron 5 and extending beyond exon 15 was identified in one patient. A substitution of the +5 conserved nucleotide at the splice donor site of intron 9 in the APC gene was shown to produce frameshift and inefficient exon skipping in a second patient. Four frameshift mutations (1577insT, 1973delAG, 3180delAAAA, 3212delA) and a nonsense mutation (C1690T) were identified in the rest of the patients. CONCLUSION: Screening for APC mutations in FAP patients should include testing for splicing defects and gross genomic alterations.

Determination of EGFR and KRAS mutational status in Greek non-small-cell lung cancer patients.

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.

Characterization of a novel large deletion and single point mutations in the BRCA1 gene in a Greek cohort of families with suspected hereditary breast cancer.

BACKGROUND: Germline mutations in BRCA1 and BRCA2 predispose to breast and ovarian cancer. A multitude of mutations have been described and are found to be scattered throughout these two large genes. We describe analysis of BRCA1 in 25 individuals from 18 families from a Greek cohort. METHODS: The approach used is based on dHPLC mutation screening of the BRCA1 gene, followed by sequencing of fragments suspected to carry a mutation including intron--exon boundaries. In patients with a strong family history but for whom no mutations were detected, analysis was extended to exons 10 and 11 of the BRCA2 gene, followed by MLPA analysis for screening for large genomic rearrangements. RESULTS: A pathogenic mutation in BRCA1 was identified in 5/18 (27.7 %) families, where four distinct mutations have been observed. Single base putative pathogenic mutations were identified by dHPLC and confirmed by sequence analysis in 4 families: 5382insC (in two families), G1738R, and 5586G > A (in one family each). In addition, 18 unclassified variants and silent polymorphisms were detected including a novel silent polymorphism in exon 11 of the BRCA1 gene. Finally, MLPA revealed deletion of exon 20 of the BRCA1 gene in one family, a deletion that encompasses 3.2 kb of the gene starting 21 bases into exon 20 and extending 3.2 kb into intron 20 and leads to skipping of the entire exon 20. The 3' breakpoint lies within an AluSp repeat but there are no recognizable repeat motifs at the 5' breakpoint implicating a mechanism different to Alu-mediated recombination, responsible for the majority of rearrangements in the BRCA1 gene. CONCLUSIONS: We conclude that a combination of techniques capable of detecting both single base mutations and small insertions/deletions and large genomic rearrangements is necessary in order to accurately analyze the BRCA1 gene in patients at high risk of carrying a germline mutation as determined by their family history. Furthermore, our results suggest that in those families with strong evidence of linkage to the BRCA1 locus in whom no point mutation has been identified re-examination should be carried out searching specifically for genomic rearrangements.

Evaluation of dHPLC in mutation screening of the APC gene in a Greek FAP cohort.

BACKGROUND: Germline mutations in the APC gene predispose to colorectal adenomas leading to cancer in over 80% of patients. A multitude of mutations, dispersed throughout the gene, have been described. We wanted to evaluate the usefulness of denaturing high performance liquid chromatography (dHPLC) for mutation screening. MATERIALS AND METHODS: Ten amplicons containing 14 mutations in the APC, previously identified by sequencing in 22 FAP patients, were analysed by dHPLC. dHPLC was also used to screen members of a family for a mutation identified in the proband. RESULTS: We analysed 10 amplicons under a total of 59 temperatures. Successful results were obtained from 51 out of 59 tested temperatures (86.4%). In all cases a different heteroduplex-homoduplex pattern was obtained from mutant DNA in at least two of the temperatures. All 14 mutations identified by sequencing were also detected using dHPLC. Sequence analysis of a large family confirmed the dHPLC results. CONCLUSION: Using dHPLC we detected all mutations previously identified by sequencing.

KRAS, NRAS and BRAF mutations in Greek and Romanian patients with colorectal cancer: a cohort study.

OBJECTIVES: Treatment decision-making in colorectal cancer is often guided by tumour tissue molecular analysis. The aim of this study was the development and validation of a high-resolution melting (HRM) method for the detection of KRAS, NRAS and BRAF mutations in Greek and Romanian patients with colorectal cancer and determination of the frequency of these mutations in the respective populations. SETTING: Diagnostic molecular laboratory located in Athens, Greece. PARTICIPANTS: 2425 patients with colorectal cancer participated in the study. PRIMARY AND SECONDARY OUTCOME MEASURES: 2071 patients with colorectal cancer (1699 of Greek and 372 of Romanian origin) were analysed for KRAS exon 2 mutations. In addition, 354 tumours from consecutive patients (196 Greek and 161 Romanian) were subjected to full KRAS (exons 2, 3 and 4), NRAS (exons 2, 3 and 4) and BRAF (exon 15) analysis. KRAS, NRAS and BRAF mutation detection was performed by a newly designed HRM analysis protocol, followed by Sanger sequencing. RESULTS: KRAS exon 2 mutations (codons 12/13) were detected in 702 of the 1699 Greek patients with colorectal carcinoma analysed (41.3%) and in 39.2% (146/372) of the Romanian patients. Among the 354 patients who were subjected to full KRAS, NRAS and BRAF analysis, 40.96% had KRAS exon 2 mutations (codons 12/13). Among the KRAS exon 2 wild-type patients 15.31% harboured additional RAS mutations and 12.44% BRAF mutations. The newly designed HRM method used showed a higher sensitivity compared with the sequencing method. CONCLUSIONS: The HRM method developed was shown to be a reliable method for KRAS, NRAS and BRAF mutation detection. Furthermore, no difference in the mutation frequency of KRAS, NRAS and BRAF was observed between Greek and Romanian patients with colorectal cancer.

Elucidation of the transmission of a novel mutation in BRCA1 (1125delCT) in a family with multiple cases of breast cancer.

In a family with multiple members affected by breast cancer we identified the novel mutation 1125delCT (exon 11) in BRCA1. Three out of three offsprings have the novel mutation while the mother affected by breast cancer does not carry the mutation. Linkage analysis revealed the transmission of the healthy haplotype from the mother to the three offsprings while the children inherited the mutated haplotype from the father. Our data document in an unquestionable way where the mutated haplotype was inherited from. In some families, although the transmission pathway seems obvious, the molecular analysis yields surprising results.

Prenatal diagnosis of common aneuploidies using quantitative fluorescent PCR.

OBJECTIVE: Quantitative fluorescence-polymerase chain reaction (QF-PCR) has recently been used for the detection of common chromosomal aneuploidies in prenatal diagnosis. Here we describe our experience in prenatal diagnosis of 1100 samples. METHODS: Extraction of DNA was performed from amniotic fluid, chorionic villus samples (CVS), fetal blood and fetal tissue samples, using a simple, rapid protocol. Fluorescent multiplex PCR products of single tandem repeats (STRs) located on chromosomes 13, 18, 21, X and Y were then analyzed on an automated laser fluorescent sequencer. All samples were analyzed with at least two polymorphic markers for chromosomes 13, 18 and 21 and one for the X chromosome. The amelogenin locus was used for sexing. Analysis was performed twice on affected samples. When miscellaneous results were obtained extra markers were used. RESULTS: We evaluated the usefulness of different markers in the Greek population. In a total of 1100 samples, 25 chromosome aberrations were identified, including trisomy 13, 18 and 21, XYY, triploidies 69,XXX and 69,XXY and one Turner mosaic. All results but three were consistent with conventional cytogenetic analysis. One mosaic was missed. Most bloodstained samples were successfully analyzed. CONCLUSION: Successful analysis of a large number of prenatal samples proves QF-PCR to be an efficient adjunct in routine prenatal diagnosis.

Preimplantation genetic diagnosis for single gene disorders: experience with five single gene disorders.

We report our experience of 14 preimplantation genetic diagnosis (PGD) cycles in eight couples carrying five different single gene disorders, during the last 18 months. Diagnoses were performed for myotonic dystrophy (DM), cystic fibrosis (CF) [Delta F508 and exon 4 (621+1 G>T)], fragile X and CF simultaneously, and two disorders for which PGD had not been previously attempted, namely neurofibromatosis type 2 (NF2) and Crouzon syndrome. Diagnoses for single gene disorders were carried out on ideally two blastomeres biopsied from Day 3 embryos. A highly polymorphic marker was included in each diagnosis to control against contamination. For the dominant disorders, where possible, linked polymorphisms provided an additional means of determining the genotype of the embryo hence reducing the risk of misdiagnosis due to allele dropout (ADO). Multiplex fluorescent polymerase chain reaction (F-PCR) was used in all cases, followed by fragment analysis and/or single-stranded conformation polymorphism (SSCP) for genotyping. Embryo transfer was performed in 13 cycles resulting in one biochemical pregnancy for CF, three normal deliveries (a twin and a singleton) and one early miscarriage for DM and a singleton for Crouzon syndrome. In each case the untransferred embryos were used to confirm the diagnoses performed on the biopsied cells. The results were concordant in all cases. The inclusion of a polymorphic marker allowed the detection of extraneous DNA contamination in two cells from one case. Knowing the genotype of the contaminating DNA allowed its origin to be traced. All five pregnancies were obtained from embryos in which two blastomeres were biopsied for the diagnosis. Our data demonstrate the successful strategy of using multiplex PCR to simultaneously amplify the mutation site and a polymorphic locus, fluorescent PCR technology to achieve greater sensitivity, and two-cell biopsy to increase the efficiency and success of diagnoses.