[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.

Population-based study of BRCA1/2 mutations: family history based criteria identify minority of mutation carriers.

The two major susceptibility genes, BRCA1 and BRCA2, are involved in hereditary breast and ovarian cancer syndrome. Early detection of mutation carriers has crucial clinical importance, as it allows identification of women who may benefit from intensive clinical follow-up or prophylactic surgery. Generally accepted inclusion criteria for BRCA1/2 mutation testing are based either upon family history of breast or ovarian cancer or young age at cancer diagnosis. In order to analyze the impact of BRCA1/2 mutations on breast cancer development in the Czech population and to confront the clinical and histopathological data of mutation carriers with current criteria for mutation testing we examined the frequency of mutations in unselected breast cancer cases. Mutational analysis of BRCA1/2 genes performed in 679 unselected female breast cancer patients included all recurrent deleterious alterations previously identified in the Prague area and truncating mutations in the whole exon 11 of BRCA1. Within analyzed gene sequences more than 80% of mutations were identified previously in high-risk patients. A total of 16 breast cancer patients (2.4%) carried a mutation. BRCA1 mutations were identified in 14 (2.1%) whereas BRCA2 in 2 (0.3%) women. Family history of ovarian cancer was a strong predictor of a BRCA1/2 mutation (OR = 8.3; p = 0.01), however, family history of breast cancer was not indicative of carrier status. A significant association between medullary breast cancer and mutation status was observed. Current criteria for BRCA1/2 mutation testing would distinguish only 6 out of 16 (37.5%) carriers identified in our study. Ten breast cancer patients with confirmed BRCA1/2 germ-line mutation exhibited no clinical characteristics that would predict their carrier status. Therefore, we believe that the testing for BRCA1/2 mutations in the Czech Republic may not be restricted only to high-risk patients. Our results indicate that analysis of locally prevalent BRCA1/2 mutations in all breast cancer patients might extend substantially the percentage of identified mutation carriers.

[Prerequisites for preimplantation genetic diagnosis (PGD in carriers of mutations responsible for hereditary cancers]. / Predpoklady pro preimplantacní genetickou diagnostiku (PGD) u nosicu mutací v nádorových predispozicních genech.

BACKGROUNDS: Carriers of hereditary mutations in cancer susceptibility genes represent a limited but high-risk population characterized by a high probability of cancer development, frequently with its manifestation in early age and with a 50% chance of pathogenic allele inheritance by offspring. In case of monogenic disorders, preimplantation genetic diagnosis (PGD) could be used for characterization of the DNA region affected by pathogenic mutation in the early stages of an embryo created by in vitro fertilization (IVF). Therefore, the transfer of unaffected embryos could be performed based on the results of PGD genotyping, enabling the development of offspring not carrying the pathogenic alteration. AIM: Here we present the consensus of the collaborative group of the Society for Medical Genetics, the Czech Society for Oncology and other professionals for use of PGD in the Czech Republic for carriers of mutations in cancer susceptibility genes. We address the conditions, prerequisites, and limits of practical application of this method. We also point out specific issues of ovarian hyperstimulation in carriers of mutations in BRCA1, BRCA2, and p53, anticipating the increased risk of hormonally dependent breast and ovarian cancers development. CONCLUSIONS: We assume that a narrow but non-negligible subgroup of cancer susceptibility gene mutation carriers may benefit from PGD.They are mainly individuals deciding to undergo IVF and PGD recruited from mutation carriers with extreme concerns about transmitting the mutation to their children. The PGD in these individuals should be managed by a closely cooperating multidisciplinary team of professionals responsible for indication of PGD, giving complete information regarding the IVF and PGD procedures including their limits, evaluating individual risks and performing instrumental and laboratory procedures with respect to up-to-date good laboratory and clinical practice.

[Inactivation of BRCA1, BRCA2 and p53 genes in sporadic ovarian cancer]. / Inaktivace genu BRCA1, BRCA2 a p53 u sporadického karcinomu vajecníku.

OBJECTIVE: To analyze loss of heterozygosity (LOH), loss of expression and somatic mutations of BRCA1, BRCA2 and p53 genes in sporadic epithelial ovarian cancer samples. DESIGN: Original paper. SETTING: Oncogynecologic center, Clinic of Obstetrics and gynecology, First Faculty of Medicine, Charles University in Prague and General Teaching Hospital, Prague. MATERIAL AND METHODS: We used genomic DNA and total RNA from peripheral blood and fresh frozen tumor as a template for LOH, loss-of-expression and mutation analyses. RESULTS: LOH in at least one region was found in 60% of tumors. Majority of these alterations occurred not solely, but in conjunction with other region deletions. CONCLUSION: Our study confirms high frequency of somatic alteration of BRCA1, BRCA2 and p53 genes in sporadic epithelial ovarian cancer.

Role of single nucleotide polymorphisms and haplotypes in BRCA1 in breast cancer: Czech case-control study.

We aimed at determining whether any association exists between six single nucleotide polymorphisms in breast cancer associated gene (BRCA1) and the risk of breast cancer. We constructed haplotypes and analyzed their importance as well. Clinico-pathological characteristics of breast cancer patients were included in the study to evaluate the prognostic impact of BRCA1 polymorphisms and haplotypes. Polymerase chain reaction-restriction fragment length polymorphism-based genotyping assays were used to determine the frequency of polymorphisms in codons 356, 871, 1038, 1183, 1436, and 1613 of BRCA1 in a group of 306 incident breast cancer patients and 313 unaffected controls of Czech origin. Statistical analyses revealed that the BRCA1 Arg356 allele may play a protective role in breast cancer (age-adjusted OR = 0.61, CI = 0.39-0.94, p = 0.026). We also observed a significant correlation between polymorphism Gln356Arg and stage (p = 0.026) in premenopausal cases suggesting that carriers of the wild Gln356Gln allele are at significantly higher risk of advanced disease. The most common haplotypes of BRCA1 did not play a significant role in breast cancer either as risk factors or as prognostic factors. The study on rare BRCA1 haplotypes however should be repeated using larger groups. In conclusion, the BRCA1-Gln356 allele presents risk factor in the onset and progression of breast cancer in Czech population and its use as a possible screening tool should be considered.

[Hereditary ovarian cancer]. / Hereditární karcinom ovaria.

OBJECTIVE: This article reviews the topic of hereditary ovarian cancer, describes persons at risk of hereditary disposition to cancer and gives instructions for genetic counselling and molecular analysis, including contacts to specialized centres in the Czech Republic. SUBJECT: Review. SETTING: Institute of Biochemistry and Experimental Oncology, Charles University in Prague. METHODS: Hereditary ovarian cancer occurs in three autosomal dominant syndromes: appropriate hereditary ovarian cancer (HOC), hereditary breast and ovarian cancer (HBOC) and hereditary non-poliposis colorectal cancer (HNPCC). Physician in practice or specialist at the clinic should focus interest on patients form families with frequent occurrence of breast and/or ovarian cancer, patients with early onset disease or tumour duplicity (breast and ovarian cancer). Hereditary disposition to ovarian (and breast) cancer could be assessed by molecular genetic analysis of two main susceptibility genes BRCA1 and BRCA2, or other genes in families with diverse tumours. Molecular genetic analysis should be in any cases indicated by experienced clinical genetic. In the Czech Republic, the consensus of genetic and clinical care of risk patients was published and specialized centres for families with hereditary predisposition were settled in Prague and Brno. CONCLUSION: Persons with hereditary susceptibility to cancer constitute noted group where painstaking dispensarisation and preventive care may prevent malignancy or detect it in the early stage.

Mutational analysis of the BRCA1 gene in 30 Czech ovarian cancer patients.

Ovarian cancer is one of the most severe of oncological diseases. Inherited mutations in cancer susceptibility genes play a causal role in 5-10% of newly diagnosed tumours. BRCA1 and BRCA2 gene alterations are found in the majority of these cases. The aim of this study was to analyse the BRCA1 gene in the ovarian cancer risk group to characterize the spectrum of its mutations in the Czech Republic. Five overlapping fragments amplified on both genomic DNA and cDNA were used to screen for the whole protein-coding sequence of the BRCA1 gene. These fragments were analysed by the protein truncation test (PTT) and direct sequencing. Three inactivating mutations were identified in the group of 30 Czech ovarian cancer patients: the 5382insC mutation in two unrelated patients and a deletion of exons 21 and 22 in another patient. In addition, we have found an alternatively spliced product lacking exon 5 in two other unrelated patients. The 5382insC is the most frequent alteration of the BRCA1 gene in Central and Eastern Europe. The deletion of exons 21 and 22 affects the BRCT functional domain of the BRCA1 protein. Although large genomic rearragements are known to be relatively frequent in Western European populations, no analyses have been performed in our region yet.

[Hereditary predisposition for the development of breast and ovarian carcinoma]. / Hereditární dispozice ke vzniku karcinomu prsu a ovaria.

Part of breast and ovarian cancer cases develops on the hereditary predisposition, i.e. mutation in one of predisposing genes. Although this proportion is relatively small, 5-10% of all breast and ovarian carcinomas, it represents a group with clearly defined etiologic factor. Predictive analysis of unaffected family members allows to identify individuals at high risk of cancer and to include them into the programme of primary and secondary cancer prevention. Following article presents basic review of the hereditary predisposition to breast and ovarian cancer focusing especially on BRCA1 and BRCA2 genes, which are responsible for almost three-quarters of those hereditary tumours.

Microsatellite markers in breast cancer studies.

Microsatellites are tandem repeats of simple polymorphic sequences randomly distributed in non-coding regions of DNA. They can be used in cancer genetics and indirect cancer diagnosis and can help unraveling the genetic basis of tumor formation and progression of cancer. Breast cancer is a complex disease in which numerous genetic alterations occur. The knowledge of specific genetic changes and their biological consequences is critical to an understanding of breast cancer tumorigenesis, screening and treatment of patients. Microsatellites can undergo two events during tumor progression. Loss of heterozygosity indicates absence of one allele in a given locus, which is associated with the loss of a corresponding genes. Microsatellite instability reflects replication errors induced by defective function of mismatch repair genes and is demonstrated with the appearance of novel, noninherited alleles in tumor cells and represents a specific pathway of tumor development. Both events serve as prognostic markers, which can be correlated with clinicopathological features and can help exploring breast cancer formation.

Detection of the most frequent mutations in BRCA1 gene on polyacrylamide gels containing Spreadex Polymer NAB.

Heterozygous carriers of germ-line mutations in the BRCA1 gene are at high risk for the development of breast and ovarian cancer. Inactivating mutations have been identified in the whole coding region of the gene, however, repeatedly occuring mutations can explain a large proportion of gene alterations detected in certain ethnic groups. In Czech patients, the 5382insC and 185delAG mutations may account for approximately 50% of all BRCA1 abnormalities (unpublished data). In the present study, a rapid and simple method to identify these short insertions and deletions that alter the size of the polymerase chain reaction (PCR) product is described. The analysis involves the separation of fragments amplified with primers that flank altered sites in the BRCA1 gene on non-denaturing polyacrylamide gels containing Spreadex Polymer NAB. The increased resolving power of Spreadex gels enables full separation of two DNA fragments that differ by 1-bp on gels that are 5 cm long. The method gave interpretable results with the genetic material obtained from all tested mutation carriers and control persons. Defective alleles were also detected in DNA samples from carriers of the 1135delA mutation in BRCA1 and the 4206ins4 mutation in BRCA2. These results suggest that electrophoresis on Spreadex gels can be used universally for detection of the most frequent frameshift mutations in BRCA genes. The method is suitable even for rapid screening of frequent germ-line mutations in BRCA genes in breast and ovarian cancer patients not selected for family history of cancer or age at diagnosis.

[Detection and occurrence of BRCA 1 gene mutation in patients with carcinoma of the breast and ovary]. / Detekce a výskyt mutací genu BRCA 1 u pacientek s karcinomem prsu a ovaria.

OBJECTIVE: The article presents a review of basic information on incidence and detection of BRCA 1 and BRCA 2 genes mutations. Results of investigation in a group of women with ovarian and breast cancer are presented. DESIGN: Retrospective clinical-laboratory study and review. SETTING: Department of Gynaecology and Obstetrics, 1st Medical Faculty of Charles University, Prague, Apolinárská 18, Czech Republic. MATERIAL AND METHODS: Investigated group consisted of 16 persons--12 patients with ovarian or breast cancer and 4 healthy relatives of a woman--breast cancer patient and a carrier of BRCA 1 gene mutation. Protein truncation test (PTT) was performed in order to detect BRCA 1 gene mutation. This test detects mutations leading to premature termination of protein synthesis. Truncated proteins are easily discriminated from full size. RESULTS: Three BRCA 1 gene alterations were identified in the investigated group of women suffering from ovarian or breast cancer. One asymptomatic person--carrier of BRCA 1 gene mutation--was identified in this study. She was daughter of a woman, a carrier of BRCA 1 gene mutation, with early onset of breast cancer and positive family history. CONCLUSIONS: BRCA 1 and BRCA 2 gene mutations are of particular importance in the increasing risk of ovarian cancer and early onset of breast cancer as well as some other malignancies. Genetic testing and counselling including investigation of some other genetic and environmental factors, related to cancer risk, may be of clinical significance in patients with increased risk of certain malignancies.

Mutations of the BRCA1 gene in hereditary breast and ovarian cancer in the Czech Republic.

OBJECTIVE: Mutations in the BRCA1 gene confer a high risk for the development of breast or ovarian cancer. The aim of this study was to establish the frequency and spectrum of BRCA1 mutations in Czech breast or ovarian cancer families. SUBJECTS AND METHODS: We have screened 28 patients with early-onset cancer and 54 patients from risk families for germ-line mutations in BRCA1. All coding exons were analyzed by the protein truncation test (PTT) and the reverse transcription polymerase chain reaction (RT-PCR). Detected variants were characterized by direct sequencing of PCR products. RESULTS: Five distinct deleterious mutations have been identified in six families. All the mutations led to the premature termination of translation. One mutation was detected in a group of 11 (9.1%) patients with early-onset breast cancer. Two mutations were observed in 32 (6.3%) families with a history of breast cancer only. Three mutations were found in 22 (13.6%) families with both breast and ovarian cancer. The 5382insC mutation was the only abnormality detected twice. In addition, two variant transcripts, the loss of exon 5 and in-frame 3-bp deletion at the beginning of exon 8, were identified only at the cDNA level. Their biological significance remains unknown. CONCLUSION: PTT analysis enables examination of long PCR products. The technique is useful for rapid detection of mutations in hereditary breast cancer.

[Apoptosis and its importance in the development and therapy of tumors (review)]. / Apoptóza a její význam z hlediska vzniku a lécby nádorových onemocnení (prehledový clánek).

This review focuses on apoptosis and its regulation as a tool of principal control mechanism of tissue homeostasis. Defects in regulation of apoptosis contribute to a various pathological processes, including tumor development, chronic inflammatory diseases, immunological disorders and many others. Apoptosis influences sensitivity to radiotherapy and chemotherapy of tumours. Microscopically the apoptosis is characterized by morphological changes, which result in the formation of apoptotic bodies. Apoptosis is an active process, which require synthesis and activation of a set of the specific cellular proteins. Among them, the key role belongs to the family of cystein proteases--caspases activated either through the death receptors or via activation steps starting with the release of a mitochondrial cytochrome c. Activation of caspases promotes the activation of downstream effectors leading to the cleavage of target cellular proteins and genomic DNA. The members of Bcl-2 family and p53 are- the others important proteins influencing the regulation of apoptosis. Enhancing of our knowledge about apoptosis and its mechanisms highly improves the rationale for diagnostics and therapy in oncology.

[Detection of tumor circulating cells in patients with ocular melanoma]. / Prukaz cirkulujících nádorových bunek u pacientu s ocním melanomem.

The authors investigated a group of 51 patients (29 men and 22 women) with intraocular melanoma: 41 patients with melanoma of the chorioid, 10 patients with melanoma of the ciliary body. They evaluated the clinical and pathological finding according to the TNM classification recommended by UICC (International Union Against Cancer). In all investigated patients they assessed circulating tumour cells (melanocytes) in the peripheral blood stream based on the detection of mRNA tyrosinase and marker MART 1. When evaluating the presence of markers according to the diagnosis irrespective of time, they found in patients in the clinical stage of T2 choroidal melanoma a 19% positivity of different markers and very rare a concurrent positivity of both markers. Patients in the clinical stage T3 had a 51% positivity of one marker and 34% concurrent positivity of both markers. In melanoma of the ciliary body evidence of individual markers was positive in 17% and only in 11% both markers were positive concurrently. On comparison of therapeutic procedures from the aspect of development in time in patients treated by brachytherapy only rare positivity was found at the time of administration the radioactive plaque, following an eight-month interval after brachytherapy the positivity of markers increased to 28%. On evaluation of markers of choroidal melanoma and ciliary body melanoma resolved by enucleation had their positivity at the time of operation was 36%, and during check-ups up to one year or longer it persisted at similar levels. Concurrent presence of both markers before this operation was rare, during postoperative check-up examinations it was within a range of 23 and 33%. The presence of both markers was repeatedly proved in five patients with chooidal melanoma after enucleation of the eye, in four of them in direct correlation with a metastatic process.

Expression of attractin and its differential enzyme activity in glioma cells.

Attractin/mahogany protein was previously shown to be involved in a number of physiological and pathological events, including immune system regulation, body weight control, pigmentation, myelinization, and tumor susceptibility. Human attractin has an enzymatic activity resembling dipeptidyl peptidase IV (DPP-IV). In the central nervous system, attractin has been detected in neurons but not in glial cells up to now. We show the expression of attractin mRNA and protein in glioma cell lines at different degree of transformation. In human U373 and U87 glioma cells (Grades III and IV), membrane-bound attractin displays hydrolytic activity amounting to 5 and 25% of total cellular DPP-IV-like enzyme activity, respectively. Such activity has not been observed in the rat C6 glioma cells (Grade I). Attractin presence in glioma, but not in normal glial cells, together with its differential enzymatic activity, suggests its role in growth properties of tumors of glial cell origin.

[Principles of gene therapy]. / Principy genové terapie.

Treatment of disease by gene transfer into the genome of target host cells has become possible. This article presents current clinical applications of gene therapy and describes possible corrections of genetic defects.

[Vectors used in gene therapy]. / Vektory pouzívané v genové terapii.

The ability to direct gene transfer to particular target cells is important to achieve a therapeutic effect. In this review we describe the development of methods for gene delivery to specific cell types.

Molecular aspects of malignant melanoma. Role of protooncogenes and oncosuppressor genes.

This review summarizes current knowledge of oncogenes and tumour-suppressor genes in development and progression of malignant melanoma.

Particulate protein-synthesis factors associated with translatable mRNA in mouse hybridoma cells.

Besides of mRNA, the postribosomal pellet of mouse hybridoma cells contains RNA species which become labeled more rapidly than rRNA. Their synthesis is inhibited by actinomycin D. Density-gradient centrifugation of the postribosomal pellet yielded fractions of approx. 55-60 and 90S, synthesizing after the addition of both ribosomal subunits and energy-sources light and heavy chains of immunoglobulin, as demonstrated by indirect immunoprecipitation. Analysis of translation products by electrophoresis indicated the presence of precursors of mRNAs for immunoglobulin chains in these particles. Postribosomal pellets thus apparently contain different particles composed of similar polypeptide chains and containing protein-synthesis factors associated with translatable mRNA.

The postribosomal particle of rabbit liver contains protein-synthesis factors and serum albumin mRNA.

As demonstrated by indirect immunoprecipitation and polyacrylamide gel electrophoresis, an 85S particle separated by sucrose density-gradient centrifugation from the postribosomal pellet of rabbit liver, is able to synthesize serum albumin if supplemented with both ribosomal subunits and sources of energy. It is retained on heparin bound to Sepharose 4B, contains translatable mRNA and apparently all protein factors required for translation. This particle may represent a highly organized protein synthesizing machinery, the combination of which with ribosomes results in formation of new protein molecules.