Classification of germline variants identified in cancer predisposition genetic testing - consensus of the CZECANCA consortium.

BACKGROUND: Hereditary cancer syndromes are an important subset of malignant cancers caused by pathogenic variants in one of many known cancer predisposition genes. Diagnosis of cancer predisposition is based on genetic testing using next-generation sequencing. This allows many genes to be analysed at once, increasing the number of variants identified. The correct classification of the variants found is essential for the clinical interpretation of genetic test results. PURPOSE: The aim of this study is to summarise the rules for classifying identified variants within individual laboratories and to present the process for creating a common classification. In the Czech Republic, the sharing of identified genetic variants and the development of their consensus classification among national laboratory diagnostic communities is carried out within the Czech Cancer Panel for Clinical Application (CZECANCA) consortium of scientific and diagnostic oncogenetic laboratories. Consensus for variant classification follows a defined protocol. Sharing the results and consensus classification accelerates and refines the release of genetic test results, harmonises results between laboratories and thus contributes to improving the care of individuals at high risk of cancer and their relatives.

Latest findings on the placenta from the point of view of immunology, tolerance and mesenchymal stem cells.

OBJECTIVE: Overview of current placental findings from the point of view of immunology, tolerance and mesenchymal stem cells. TYPE OF STUDY: Review. SETTING: Medicínské centrum Praha. CONCLUSION: The placenta is an important organ that connects mother and developing fetus during pregnancy. For the uncomplicated course of pregnancy and fetal development the placental function is crucial. The placenta provides not only the replacement of breathing gases, nutrients and waste materials, but also creates an immunological interface between the mother and the fetus. Maternal tolerance towards the fetus carrying paternal antigens is induced at the fetomaternal interface due to the mutual molecular interactions. Immune tolerance at the interface between placenta and decidua is ensured mainly due to the expression of HLA-C, HLA-E, HLA-F, and HLA-G on trophoblasts and their interactions with receptors expressed on uterine NK cells. Regulatory T cells and DC-10 cells also play an important role at the fetomaternal interface on the mothers side of placenta. However, some fetal cells, such as Hofbauer cells or granulocytic myeloid-derived suppressor cells are also partially involved in inducement of maternal tolerance towards the fetus. Recently, considerable attention is also paid to mesenchymal stem cells derived from both placental and umbilical tissues. These mesenchymal stem cells play an important role in inducement of immune tolerance and exhibit better immunomodulatory properties than mesenchymal stem cells isolated from adult human tissues.

[Recommended Extension of Indication Criteria for Genetic Testing of BRCA1 and BRCA2 Mutations in Hereditary Breast and Ovarian Cancer Syndrome]. / Doporucení rozsírení indikacních kriterií ke genetickému testování mutací v genech BRCA1 a BRCA2 u hereditárního syndromu nádoru prsu a ovarií.

Genetic testing for hereditary breast and ovarian cancer syndrome is indicated by a genetic counselor on the basis of personal and family history evaluation, with regards to consensual criteria, reflecting the current knowledge. The latest recommendation accepted by Czech Oncology Society and Society of Medical Genetics was published in the supplement 22 to the Journal of Clinical Oncology in 2009. Since the availability of PARP inhibitors for treatment of ovarian cancer in BRCA1/ 2 mutation carriers, an update of these guidelines is urgently needed. Another reason is a higher incidence of other malignancies in high-risk families, such as prostate or pancreatic cancer. The goal is to refine the detection of mutations in selected families, to improve preventive care and collect data necessary for targeted cancer treatment.

[Retrospective NGS Study in High-risk Hereditary Cancer Patients at Masaryk Memorial Cancer Institute]. / Retrospektivní NGS studie u vysoce rizikových pa-cientu s hereditární predispozicí k nádorovému onemocnení v Masarykove onkologickém ústavu.

BACKGROUND: Currently, more than 200 hereditary cancer syndromes have been described, yet, in most countries genetic testing is restricted to a narrow spectrum of genes within a limited group of people tested. METHODS: For this retrospective study we used the TruSight cancer panel (Illumina)--NGS panel targeting 94 cancer predisposition genes in order to analyze 50 high-risk cancer patients with significant personal and family history of cancer who did not carry mutations in BRCA1, BRCA2, MLH1, MSH2, MSH6, TP53 or APC genes. All pathogenic and potentially pathogenic mutations detected by NGS technology have been confirmed by Sanger sequencing. RESULTS: There were several deleterious (frame-shift/nonsense) mutations detected in ATM, BAP1, FANCC, FANCI, PMS2, SBDS, ERCC2, RECQL4 genes. Various pathogenic or potentially pathogenic (missense, predicted splice site, in-frame insertion/deletion) mutations were detected in ATM, BRIP1, CDH1, CHEK2, ERCC2, ERCC3, ERCC4, FANCA, MC1R, MEN1, MRE11A, MUTYH, PALB2, RAD51C, RET, SDHB, STK11. These mutations affect highly conserved protein domains and affect their function as proved by the available functional assays. They were confirmed to be pathogenic as an "Parent No2 " in serious recessive diseases such as Ataxia telangiectasia or Fanconi anemia. The clinical significance of the majority of detected missense variants still remains to be identified. CONCLUSION: Moderate or low penetrance variants are of limited clinical importance. Panel genetic testing in high-risk individuals with cancer provides important information concerning the cause of the investigated cancer, and may assist in the risk assesment and optimal management of the cancer, as well as in further preventive care.

[Fanconi Anemia, Complementation Group D1 Caused by Biallelic Mutations of BRCA2 Gene--Case Report]. / Fanconiho anémie, komplementacní skupina D1 v dusledku bialelické mutace genu BRCA2--kazuistika.

Fanconi anemia is a rare autosomal recessive disorder, clinically and genetically heterogeneous, characterized by typical clinical features, such as short stature, microcephaly, skeletal abnormalities, abnormal skin pigmentations, developmental delay and congenital heart, kidney anomalies etc. Pancytopenia leading to bone marrow failure occurs in the first decade. Patients with Fanconi anemia have a high risk of hematologic malignancies and solid tumors. The diagnosis of Fanconi anemia is based on cytogenetic testing for increased rates of spontaneous chromosomal breakage and increased sensitivity to diepoxybutane or mitomycin C. Fanconi anemia is a heterogeneous disorder, at least 15 complementation groups are described, and 15 genes in which mutations are responsible for all of the 15 Fanconi anemia complementation groups have been identified. Unlike other Fanconi anemia complementation groups, for complementation group D1 (FANCD1), the bone marrow failure is not a typical feature, but early-onset leukemia and specific solid tumors, most often medulloblastoma and Wilms tumor, are typical for this complementation group.

[Breast cancer in monozygotic twins]. / Karcinom prsu u monozygotních dvojcat.

SUMMARY: Breast cancer is a multifactorial disease. Twin studies comparing the disease concordance rate in identical twin pairs serve to differentiate the influence of genetic and environmental factors in the disease development. AIM OF THE STUDY: To assess breast cancer risk for an identical twin sister of a patient with breast cancer. PATIENTS AND METHODS: Five monozygotic twin families were examined during 2005- 2011 in which at least one of the monozygotic sisters developed breast cancer.In 4 breast cancer women from 4 families, molecular genetic analysis of BRCA1 and BRCA2 genes was performed. RESULTS: The median followup period was 12.6 years (7 to 24 years). No pair of monozygotic sisters was concordant for breast cancer. Familial breast/ ovarian cancer syndrome due to BRCA1 gene mutation was confirmed in one pair. These twins were phenotypically discordant, the first one developing breast cancer at the age of 54 years, her co sister suffering from ovarian cancer at the age of 43 years. In the other 4 nonBRCA families, breast cancer was diagnosed at the age of 38- 50 years (median 44 years) in one of the sisters; the other twins remain healthy through the followup period. CONCLUSION: We did not observe concordance for breast cancer in 5 pairs of monozygotic twins. Based on results of published studies, the life  time breast cancer risk for a healthy identical twin of a breast cancer nonBRCA woman is around 20- 30 %. Other nonhereditary risk factors must exist to explain the discordant phenotype. This highlights that environmental factors play an important role in breast cancer development. In case of BRCA associated breast cancer, breast cancer risk for the healthy co twin is the same as that for other BRCA mutation carriers, i.e. 45- 85%.

[Hereditary diffuse gastric cancer]. / Hereditární difuzní karcinom zaludku.

Hereditary diffuse gastric cancer is an autosomal dominant syndrome with a high lifetime risk of diffuse gastric cancer and also a high risk of lobular breast carcinoma. Hereditary diffuse gastric cancer (HDGC) is characterized by late presentation and a poor prognosis. The average age of onset of HDGC is 38 years, with a range of 14-69 years. The estimated lifetime risk of developing gastric cancer by age 80 is 67% for men and 83% for women. Many families with HDGC have germline mutations in the E-cadherin (CDH1) gene. We describe indication for genetic testing of germline mutations in CDH1 gene, possibilities of predictive testing, preventive care, prophylactic gastrectomy and preimplantation diagnosis.

[Li-Fraumeni syndrome - a proposal of complex prevention care for carriers of TP53 mutation with total-body MRI]. / Li-Fraumeni syndrom - návrh komplexní preventivní péce o nosice TP53 mutace s pouzitím celotelové magnetické rezonance.

Li-Fraumeni syndrome (LFS) is one of the most serious hereditary cancer syndromes with high risk of malignancy already in childhood. Adrenocortical carcinoma, brain tumor, leukemia, sarcoma are the most frequent malignancies in children. Early breast cancer, brain tumor, sarcoma, skin cancer, gastrointestinal, lung, gynecological, hematological and other malignancies can be seen in adults. Predictive testing in families with detected LFS and TP53 mutation is offered from the age of 18 for various reasons. One of the most important reasons is a very limited effectivity of prevention especially in children, also the possible risk of psychological harm to the child and his family caused by the diagnosis of this syndrome. Progress in diagnostic methods, especially total body MRI, enables to propose preventive care for early cancer diagnoses for children and adults. Biochemical tests, ultrasound, MRI may improve survival of these high risk individuals and support the possibility of predictive testing in children.

[The clinical importance of a genetic analysis of moderate-risk cancer susceptibility genes in breast and other cancer patients from the Czech Republic]. / Klinický význam analýz genu stredního rizika pro hodnocení rizika vzniku karcinomu prsu a dalsích nádoru v Ceské republice.

BACKGROUND: Analysis of the major breast cancer (BC) predisposition genes BRCA1 and BRCA2 enables identification of high-risk individuals. Specialized programs enrolling the carriers of BRCA1/2 mutations facilitate an improvement in prevention and early diagnostics in asymptomatic individuals and rationalize the selection of individualized treatment in case of a BC onset. However, the carriers of mutations in the major predisposition genes represent only approximately 25% of cases among high-risk BC patients. Numerous candidate predisposing genes for breast and other cancers have recently been identified. The risk of cancer development associated with alterations in these genes is lower, and there is a considerable population variability in different regions worldwide. AIM: We have performed mutation analyses of moderate-risk cancer susceptibility genes to evaluate their clinical importance for genetic counseling in high-risk patients suffering from breast and other cancers in the Czech population. RESULTS: Czech oncological patients were analysed for mutation in ATM, CHEK2, NBS1 (NBN) and PALB2 genes. The majority of analyzed individuals represent the population of high-risk BRCA1/2-negative BC patients. CONCLUSIONS: Based on results of this study, we recommend an analysis of recurrent truncating mutations in the CHEK2 gene (the c.1100delC mutation and a large deletion affecting exons 9-10) in BRCA1/2-negative patients from high-risk BC families. A clinical assessment of missense variants in CHEK2 is not suitable. A routine mutation analysis of the ATM and NBS1 (NBN) genes is not recommended in BC patients due to the low frequency of alterations in these genes in the Czech Republic. An identification of truncating mutations in the PALB2 gene is important in BRCA1/2-negative BC patients from families with a strong history of BC (HBC families). The frequency of PALB2 mutations may be comparable to the frequency of mutations in the BRCA2 gene in Czech HBC families.

[Evaluation of variants of unknown significance in the BRCA2 gene]. / Hodnocení variant nejasného významu v genu BRCA2.

BACKGROUND: Endogenous processes and exogenous agents cause constant DNA damage. DNA double-strand breaks are among the most serious types of damage. They are mainly repaired by homologous recombination, where the BRCA2 protein plays a dominant role. Heterozygous germline BRCA2 mutations predispose to breast, ovarian, pancreatic and other types of cancer. The presence of a pathogenic mutation in patients or their family members warrants close surveillance and prophylactic surgery. Apart from clearly pathogenic mutations, variants leading only to a single amino acid substitution are often identified. Since the influence of these variants on cancer risk is unknown, they represent a major clinical problem. AIMS: The aim of this paper is to summarize the current possibilities of predicting pathogenicity of BRCA2 variants. In some cases, genetic methods are able to classify variants with high probability; however, their use is often limited by low frequency of the variants or inaccessibility of samples for mRNA isolation or DNA from family members. Alternatively, functional assays performed in various cellular models may be employed. Multiple functional tests and cellular models are presented and characterized, including their advantages and limitations. A new model of human syngeneic cell lines developed by the authors is presented, in which one BRCA2 allele is deleted and the variant is introduced into the other allele by homologous recombination. This model has the potential to evaluate function of variants without some of the unwanted effects of the other models. Currently, this model is being applied to variants identified in patients with hereditary cancer predisposition in the Masaryk Memorial Cancer Institute. CONCLUSION: Functional assays in cellular models including a new model of syngeneic cell lines described by the authors have a great potential in evaluating clinical importance of unclassified variants in the BRCA2 gene, especially in cases where genetic tests are not applicable.

Genetic testing and prevention of hereditary cancer at the MMCI--over 10 years of experience.

Hereditary cancer syndromes are frequently seen in young cancer patients and patients with a positive family history. Genetic testing is important for the identification of high-risk individuals, and for the early introduction of specialized preventive care or prophylactic surgeries. High-risk tumour suppressor genes (BRCA1 and BRCA2) and DNA repair genes (MLH1, MSH2 and MSH6) are responsible for a substantial part of hereditary breast, ovarian and colorectal cancer. Other hereditary cancers are seen less frequently, but genetic testing has increased for many other site-specific cancers and complex syndromes. Genetic centres and molecular genetic laboratories are located mostly within university or regional hospitals. Some genetic centres are private. It is highly recommended (Czech Society for Medical Genetics) that all laboratories are accredited according to ISO 15,189 and that genetic testing of hereditary cancer syndromes is indicated by medical geneticists. The indication criteria and prevention strategies were published in Supplement 22 of Clinical Oncology 2009 (in Czech). Preventive care for high-risk individuals is organized by thirteen Oncology Centres, which provide most of the oncology care in the Czech Republic. Genetic testing and preventive care for high-risk individuals and mutation carriers is covered by health insurance. The molecular genetic laboratory at the MMCI provides molecular genetic testing of BRCA1, BRCA2, CHEK2 for hereditary breast/ovarian cancer, MLH1, MSH2, MSH6 for Lynch syndrome,TP53 for Li-Fraumeni syndrome, CDKN2A for familial malignant melanoma syndrome and CDH1 gene for hereditary diffuse gastric cancer. Other syndromes are tested in specialized laboratories elsewhere.The use of genetic testing is increasing because of more frequent referrals from oncologists and other specialists and the increasing variety of genes tested. However, in some patients the testing is not recommended and other family members are dying because of the late diagnosis of hereditary syndrome. Greater awareness of the importance of genetic testing in oncology is needed.

[Limitations of genetic testing in oncology]. / Limitace genetického testování v onkologii.

Genetic testing of cancer syndromes is based on the existing knowledge of monogenic causes of oncologic diseases. In cases of high-risk genes, the findings concerning the carrier status of pathogenic mutation can be of clinical use in the prediction of risks and for preventive care. In non-carriers in families with mutation in the high-risk gene, the risk of cancer diseases may not be the same as the population risk and some preventive follow-up is recommended. The clinical use of genes with mild or moderate risk of cancer is problematic and could lead to distorted conclusions about the actual cause of the familial form of the disease. Predictive testing in genes with moderate risk of cancer (2-3 times) is not offered, or the non-carriers are followed in the same way as carriers. The use of genes with mild risk is not recommended in clinical practice. Molecular genetic testing also has its limitations and its quality depends on the methods and technology used and the existing knowledge of the significance of mutations. In some variants it is not clear yet whether they are just insignificant polymorphisms or pathogenic mutations. The interpretation of test results in the context of the whole family history is important.

[Disease-causing mutations versus neutral polymorphism: use of bioinformatics and DNA diagnosis]. / Onemocnení zpusobující mutace versus neutrální polymorfizmus: vyuzití bioinformatiky v DNA diagnostice.

Molecular genetic diagnostics is available for increasing number of genetically determined diseases. A wide spectrum of mutations can be detected by laboratory methods. A mutation can be defined as a change in a specific DNA sequence when compared with the reference sequence published in the gene database. However, in some cases it is difficult to distinguish if the detected sequence variant is a causal mutation or a neutral (polymorphic) variation without any effect on phenotype. The interpretation of rare sequence variants of unknown significance detected in disease-causing genes becomes an increasingly important problem. Further analysis on DNA and on protein levels with the use of bioinformatics are needed to reveal the effect of rare sequence variants. Inherited complex disorders, for example rare hereditary forms of cancer diseases, represent a challenge to molecular geneticists. The identification of exact causal mutation directly responsible for the development of the disease and for the assessment of disease risk resulting from this genetic variation has further implications. Predictive genetic diagnostics allows identify relatives at high risk of genetically determined disease and use of targeted preventive and therapeutic approaches. In severe cases it allows also prenatal or pre-implantation diagnostics.

Novel germline BRCA1 and BRCA2 mutations in breast and breast/ovarian cancer families from the Czech Republic.

Germline mutations in breast cancer susceptibility genes, BRCA1 and BRCA2, are responsible for a substantial proportion of high-risk breast and breast/ovarian cancer families. To characterize the spectrum of BRCA1 and BRCA2 mutations, we screened Czech families with breast/ovarian cancer using the non-radioactive protein truncation test, heteroduplex analysis and direct sequencing. In a group of 100 high-risk breast and breast/ovarian cancer families, four novel frame shift mutations were identified in BRCA1 and BRCA2 genes. In BRCA1, two novel frame shift mutations were identified as 3761-3762delGA and 2616-2617ins10; in BRCA2, two novel frame shift mutations were identified as 5073-5074delCT and 6866delC. Furthermore, a novel missense substitution M18K in BRCA1 gene in a breast/ovarian cancer family was identified which lies adjacent just upstream of the most highly conserved C3HC4 RING zinc finger motif. To examine the tertiary structure of the RING zinc finger domain and possible effects of M18K substitution on its stability, we used threading techniques according to the crystal structure of RAG1 dimerization domain of the DNA-binding protein.

A high occurrence of BRCA1 and BRCA2 mutations among Czech hereditary breast and breast-ovarian cancer families.

BACKGROUND: About 5-10% of breast and ovarian cancer can be of hereditary origin. Germline mutations in BRCA1 or BRCA2 and probably other yet unknown genes may cause predisposition to these cancers. METHODS AND RESULTS: Molecular genetic testing of BRCA1 and BRCA2 genes in 21 high-risk breast and breast/ovarian cancer families was performed in order to find the types and the frequency of mutations in the South Moravian region of the Czech Republic. A germline mutation was found in 12 of 21 tested families (57%), 9 mutations in BRCA1 gene and 3 mutations in BRCA2 gene. In 4 unrelated families the same germline mutation in the BRCA1 gene (5382insC) was identified. In 12 families diagnosed with breast cancer only syndrome 3 families harbouring BRCA1 mutations and 3 families harbouring BRCA2 mutations were found. In 9 families with breast-ovarian cancer syndrome 6 families carrying BRCA1 mutations were detected. CONCLUSION: Molecular genetic testing of BRCA1 and BRCA2 genes in high-risk women with breast/ovarian cancer is effective in determining genetic predisposition to cancer. Spectrum of mutations found in both genes is variable and further investigation is needed for estimation of more frequent or "founder" mutations. The genetic counselling and preventive clinical follow-up of gene carriers has to be part of the genetic program.

Mutation analysis of the BRCA1 and BRCA2 genes in the Belgian patient population and identification of a Belgian founder mutation BRCA1 IVS5 + 3A > G.

Since the identification of the BRCA1 and BRCA2 genes, several hundred different germline mutations in both genes have been reported. Recurrent mutations are rare and mainly due to founder effects. As the mutational spectrum of the BRCA1 and BRCA2 genes in the Belgian patient population is largely unknown, we initiated mutation analysis for the complete coding sequence of both genes in Belgian families with multiple breast and/or ovarian cancer patients and in "sporadic" patients with early onset disease. We completed the analysis in 49 families and in 19 "sporadic" female patients with early onset breast and/or ovarian cancer. In 15 families we identified a mutation (12 mutations in BRCA1 and 3 mutations in BRCA2). In 5 apparently unrelated families the same splice site mutation was identified (BRCA1 IVS5 + 3A > G). Haplotype analysis revealed a common haplotype immediately flanking the mutation in all families suggesting that disease alleles are identical by descent. In none of the 19 sporadic patients was a mutation found.

Mutation analysis of the BRCA1 and BRCA2 genes results in the identification of novel and recurrent mutations in 6/16 flemish families with breast and/or ovarian cancer but not in 12 sporadic patients with early-onset disease. Mutations in brief no. 224. Online.

Since the identification of the BRCA1 and BRCA2 genes (MIM#s 113705 and 600185), more than hundred different mutations throughout both genes have been reported. Recurrent mutations are rare and mainly due to founder effects. We analyzed 12 sporadic female patients with breast cancer before age 35, as well as 16 unrelated families, presenting with either (i) at least 3 first degree relatives with breast and/or ovarian cancer diagnosed at any age, or (ii) at least 2 first and/or second degree relatives with breast and/or ovarian cancer before age 45 years. We performed a protein truncation test for BRCA1 exon 11 and BRCA2 exons 10 and 11 and heteroduplex analysis for all the remaining exons of BRCA1 and 2. Presence of genomic deletions encompassing exons 13 or 22 of BRCA1, known to be Dutch founder mutations, was investigated by PCR. In 6/16 (37.5%) unrelated families the causal mutation in either the BRCA1 or BRCA2 gene was identified. Four different mutations were found in the BRCA1 gene: IVS5+3A>G (intron 5), 1191delC (exon 11), R1443X (exon 13), IVS22+5G>A (intron 22) and two in the BRCA2 gene: 6503delTT (exon 11), 6831delTG (exon 11). 1191delC (BRCA1) and 6831delTG (BRCA2) are novel mutations. IVS5+3A>G in exon 5 of BRCA1 published by Peelen et al. (1997) as a novel Belgian mutation, was identified in one additional family, not fulfilling our inclusion criteria. In the group of 12 sporadic female patients no mutations were found.