[Malignant Melanoma - from Classical Histology towards Molecular Genetic Testing]. / Maligní melanom - od klasické histologie k molekulárne genetickému testování.

BACKGROUND: Malignant melanoma is - in comparison with other skin tumors - a relatively rare malignant neoplasm with highly aggressive biologic behavior and variable prognosis. Recent data in pathology and molecular diagnostics indicate that malignant melanoma is in fact not a single entity but a group of different neoplasms with variable etiopathogenesis, biologic behavior and prognosis. New therapeutic options using targeted treatment blocking MAPK signaling pathway require testing of BRAF gene mutation status. This helps to select patients with highest probability of benefit from this treatment. AIM: This article summarizes information on the correlation of morphological findings with genetic changes, discusses the representation of individual genetic types in various morphological subgroups and deals with the newly proposed genetic classification of melanoma and the current possibilities, pitfalls and challenges in BRAF testing of malignant melanoma. It also describes the current testing situation in the Czech Republic - the methods used, the representation of BRAF mutations in the tested population and the future of testing. It also shows the limitations of the BRAF and MEK targeted treatment concept resulting from the heterogeneity of the tumor population. Mechanisms of acquired resistance to MAPK pathway inhibitors, possibilities of their detection, and issues of combination of targeted therapy and immunotherapy are discussed.Key words: malignant melanoma - BRAF - mutation - molecular targeted therapy - tumor microenvironment - tumor heterogeneity This work was supported by projects PROGRES Q40/11, BBMRICZ LM2015089, SVV 260398 and GACR 17-10331S. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 28. 3. 2017Accepted: 16. 5. 2017.

Prognostic and predictive molecular biological markers in prostate cancer - significance of expression of genes PCA3 and TMPRSS2.

UNLABELLED: Prostate specific antigen and digital rectal examination have low specificity for detecting prostate cancer and they poorly predict the presence of aggressive disease. We present recent findings on PCA3 and TMPRSS:ERG fusion and assessed the relationship between PSA, urine PCA3 and TMPRSS2:ERG and corelation with pathological findings. We tested the PCA3 score in two groups. The first comprised 96 men treated in urology out-patient units with suspicion of prostate cancer, who had elevated PSA and/or positive DRE. The second group comprised 28 patients, who were treated by radiation for localised prostate cancer, and whose PCA3 was regularly monitored. A further cohort comprised patients with already-diagnosed tumors, who had undergone radical prostatectomy. With these, using histopathological samples, we examined samples of the TMPRSS2:ERG fusion gene and compared the results with Gleason score values and level of PSA. We also examined the TMPRSS2:ERG gene in patients who had positive biopsy. Part of the genetical analysis was also an examination of the MSMB gene.The sensitivity of PCA3 testing was 66.7% and the specificity 78.5%. TMPRSS2:ERG gene was correllated with the Gleason score. Neither the TMPRSS2:ERG (p=0.13) nor the MSMB (p=0.556) genotype had an influence on the value of the Gleason score. However a difference was found between the homozygote and wild type (WT) in the TMPRSS2 gene.FISH analysis of TMPRSS/ERG gene fusion was evaluated as positive in 8 (36.8%) of the biopsically verified tumors and in 20 (37.3%) of the evaluated patients after RAPE of parafin slicing.We did not confirm a corellation between fusion and Gleason score (p=0.29).PCA3, with its higher sensitivity in comparison with PSA, is more useful for eventual screening examination. Identification of further molecular markers such as TMPRSS2, may be very promising ways to determine further prognosis of patients with prostate cancer. KEYWORDS: prostate cancer, PSA, PCA3.

[Screening of celiac disease in patients with osteoporosis and osteopenia]. / Screening celiakie a pacientu s osteoporózou a osteopenií.

The celiac disease is traditionally viewed as the children's disease with a typical form accompanied mainly by intestinal symptoms and malabsorption. This opinion is still generally accepted by the medical community. Findings based on the area-wide screening show that the prevalence has risen from the original 1 : 1 000-1 500 to 1 : 70-550. The average prevalence in the western countries is nearly 1 : 100. The prevalence of the celiac disease in the Czech republic is estimated to be approximately 1 : 200-250. It means that the number of people in the Czech republic who are likely to be affected is about 40,000-50,000 people. Currently only 10-15% of the total number of the ill people are diagnosed and monitored. Adult patients represent the main diagnostic problem because their clinical pictures are individual and the main symptoms are atypical (nonenteral). These are anaemia (mainly sideropnic), early/premature osteoporosis, herpetiformic (Duhring) dermatitis, polyneurititis, ataxia, depression, behavioural disorders, menstrual cycle disorders and infertility. Therefore our attention is currently focused on the screening of these groups of subjects. The purpose of our study was to check the frequency of the celiac disease with patients with diagnosed osteoporosis and osteopenia. In our study we have confirmed the assumption that the prevalence ofthe celiac disease in the group of subjects was 1 : 50, which means that 2.2% of patients with osteoporosis and osteopenia are affected by celiac sprue and therefore screening examination of these patients with the subsequent causal treatment (gluten-free diet) is recommended.

Absence of spinocerebellar ataxia type 3/Machado-Joseph disease within ataxic patients in the Czech population.

Although spinocerebellar ataxia type 3 (SCA3)/Machado-Joseph disease is the most common type of SCA worldwide, we did not identify any cases of the disease amongst SCA patients in the Czech population. It has been proposed that the prevalence of large normal alleles correlates with the frequency of various types of SCA. We have therefore attempted to resolve the absence of SCA3 in our population by investigating, within 204 normal chromosomes, the frequency and nature of CAG repeats as well as two intragenic polymorphisms. We found that large normal alleles with more than 33 CAG repeats were observed at a frequency of only 0.49%. Whereas most of the expanded alleles worldwide have the CA haplotype, this was the least common (5.4%) variant observed in our study, although it was associated with a larger mean CAG repeat length (26.9). We postulate that the absence of SCA3 in the Czech population might be explained by the lack of large normal alleles and consequently a relatively small reservoir for aberrant CAG expansions at the SCA3 locus.

Fluorescent multiplex PCR--fast method for autosomal dominant spinocerebellar ataxias screening.

Expansion of CAG trinucleotide repeats has been shown to cause a number of autosomal dominant spinocerebellar ataxias such as SCA1, SCA2, SCA3/MJD, SCA6 and SCA7. These disorders are characterized by a wide inter- and intrafamiliar variation in clinical features. The same mutation can result in different phenotypes and the very similar phenotypes can be caused by different mutations. Therefore it is necessary to investigate more SCA genes (according to prevalence) to identify the causal elongation. We developed a fast and efficient screening method based on touchdown multiplex PCR with fluorescent labelled primers for the most common types of SCAs (SCA 1, 2, 3 and 7). It has been reliable in 113 probands tested. Fragment analysis was performed by using 6% denaturing polyacrylamide gel and employing the automated DNA sequencer. This method considerably shortens the process of molecular genetic screening of SCAs and might be used as a tip for designing other SCA screening sets.

[DNA diagnosis of the fragile X chromosome syndrome--FRAXA using PCR]. / DNA diagnostika syndromu fragilního X chromozomu--FRAXA pomocí PCR.

BACKGROUND: Fragile X syndrome is gonosomal recessive mental retardation with the frequency 1:1000 in male population. Fragile X syndrome is caused by amplification of CGG repeat in 1. exon of FMT-1 gene. The aim of this study was to set up and validate a rapid and efficient PCR diagnosis to select FRAXA negative patients in population of mental retarded patients. METHODS AND RESULTS: In the set up phase of the method, 196 patients were diagnosed. We were using modified radioactive PCR of CGG. Obtained PCR fragments were separated on 6% denaturing PAGE. Results were correlated with Southern blot analysis using pE5.1 probe. STR-PCR was verified on a large set of patients and shows validity and efficiency of results in the case of pre- and full mutations in male hemizygous patients too. For estimation of carriers with pre- and full mutation by females modified diagnostic approach was developed. There was no difference found between results from PCR and Southern blot analysis. CONCLUSIONS: The PCR method is convenient not only for selection of FRAXA negative patients, but for diagnosis of full mutation and premutation of affected probands.

[Use of CA repeat polymorphism in direct and indirect diagnosis of Duchenne and Becker muscular dystrophy]. / Vyuzitie polymorfizmov CA opakovaní v priamej a nepriamej diagnostike Duchenneovej a Beckerovej muskulárnej dystrofie.

BACKGROUND: DNA analysis makes it possible to confirm the clinical diagnosis of the majority of cases DMD/BMD and to detect at the same time carries and the prenatal diagnosis for relatives at risk. The objective of the present work was to improve the haplotype analysis and to identify the most frequent deletions in carries. METHODS AND RESULTS: The method is based on the initial amplification of several DNA polymorphisms of CA repetitions and subsequent identification of alleles in the denaturation sequencing polyacrylamide gel using radioactive detection system. The system of CA polymorphisms provides information in the great majority of families, it detects the recombination in the DMD gene, which reduces to a minimum the risk of a diagnostic error and provides valuable information on the carriership of deletion. CONCLUSIONS: The introduction of haplotype analysis of CA repetitions is beyond doubt an asset to the prenatal diagnosis of DMD/BMD and assessment of carriership of this serious hereditary disease. The variability of the length of alleles of these markers improves analysis, prenatal diagnosis and makes it possible to rule out or identify deletion in cca 40% carriers.