Droplet digital PCR as a novel dia-gnostic tool.

BACKGROUND: Nowadays, modern treatment methods for cancer patients are based on targeting specific molecules involved in cellular signaling system associated with tumor initiation and progression. The success of such approach depends on a correctly chosen dia-gnostic test with high sensitivity that identifies the occurrence and level of bio-markers in patients to select those who will respond and benefit from the treatment. The development of new technologies and the upgrades of the known ones contribute to the innovations in molecular characterization of cancer, which allows the detection of patients mutational status with high sensitivity and specificity. PURPOSE: Here, we discuss the utilization of the third-generation type of polymerase chain reaction (PCR), droplet digital PCR (ddPCR), in the molecular dia-gnostics of oncology diseases. According to the studies reported in our review, ddPCR represents a promising tool in genetic profiling of cancer patients. Therefore, the optimization and precise validation may enable gradual implementation of ddPCR into clinical practice in the field of oncology.

Beta-adrenergic receptors gene polymorphisms are associated with cardiac contractility and blood pressure variability.

Beta-adrenergic receptors (beta-ARs) play a pivotal role in the cardiovascular regulation. In the human heart beta1- and beta2-ARs dominate in atria as well as in ventricle influencing heart rate and myocardial contractility. Some single nucleotide polymorphisms (SNPs) of beta-ARs might influence cardiovascular function. However, the influence of beta-AR genes SNPs on hemodynamic parameters at rest and their reactivity under stress is still not well known. We aimed to explore the associations between four selected beta-ARs gene polymorphisms and selected cardiovascular measures in eighty-seven young healthy subjects. While in beta1-AR polymorphism rs1801252 no significant association was observed, second beta1-AR polymorphism rs1801253 was associated with decreased cardiac output and cardiac index during all phases and with decreased flow time corrected and ejection time index at rest and during mental arithmetics. Polymorphism rs1042713 in beta2-AR was associated with alterations in blood pressure variability at rest and during head-up-tilt, while rs1042714 was associated predominantly with decreased parameters of cardiac contractility at rest and during mental arithmetics. We conclude that complex analysis of various cardiovascular characteristics related to the strength of cardiac contraction and blood pressure variability can reveal subtle differences in cardiovascular sympathetic nervous control associated with beta-ARs polymorphisms.

Quantitative analysis of 2-hydroxyglutarate as a controversial oncometabolite in malignant gliomas.

There is a great effort to connect the accumulation of 2-hydroxyglutarate (2-HG) oncometabolite with cellular onco-epigenetic status and subsequently predict the prognoses of glioma patients. In this observational study, the concentrations of D- and L- 2-HG were determined in 57 tumor tissue samples of glioma patients (n=57) WHO grade I through IV (astrocytoma, oligodendroglioma, secondary glioblastoma, and glioblastoma multiforme) in vitro. Also, genetic mutation status on isocitrate dehydrogenase 1 and 2 (IDH 1/2) was determined from these samples. The objective of this study was to confirm or to reject the hypothesis of the direct correlation of 2-HG concentration in tumor tissue and the results from IDH 1/IDH 2 point mutation analyses. The concentrations of 2-HG were quantified using high sensitive HPLC and Q-TOF HRMS spectrometer setup. Concurrently, the genetic mutation analyses of both IDH 1 (cytosolic) and IDH 2 (mitochondrial) were performed by the isolation of tumor tissue DNA, PCR amplification, and subsequent Sanger forward sequencing. Our results indicate that there is no definite correlation between the two as we identified cases of glioma tumors with significantly increased concentration of one or both L- and D- 2-HG but no IDH 1/2 mutations (44% 2-HG positive cases).

Detection of driver mutations in FFPE samples from patients with verified malignant melanoma.

Malignant melanoma is an oncological disease characterized by etiologic heterogeneity and it has increasing incidence and mortality in the Slovak Republic. While it is treated surgically in combination with chemotherapy, targeted therapy, and immunotherapy, malignant melanomas can ulcerate and are susceptible to infections. These are highly aggressive cancers with metastasis, and recent studies have shown the presence of mutations in RAC1, PPP6C and STK19 genes in melanoma patients. Mutations in these genes are driver mutations; important in oncogenesis and providing selective advantage to tumor cells. The aim of our study is to establish a method to detect driver mutations in formalin-fixed, paraffin embedded (FFPE) tissue DNA. We applied Sanger sequencing to detect driver somatic mutations in RAC1, PPP6C, STK19 and BRAF genes in patients with malignant melanoma. Confirmation of BRAF V600E mutation was obtained by allele-specific PCR. The BRAF V600E mutation was present in 15 of 113 patients (13.2%) and the driver mutation in 7 of 113 patients (6.2 %). Our results demonstrate that Sanger sequencing analysis detects mutations in FFPE clinical samples. The identification of these somatic driver mutations in samples with verified malignant melanomas enabled development of a molecular classification of melanomas, and our study provides evidence of diversity of novel driver mutations implicated in malignant melanoma pathogenesis. These findings could have very important implications for targeted therapy.

Epigenetics: an alternative pathway in GISTs tumorigenesis.

Many diseases have different pathological backgrounds responsible for abnormal cell behavior and exhibiting altered function and signal transduction. This is especially true for tumors and although changes affecting DNA sequence, irreversible mutations and chromosomal aberrations in gastrointestinal stromal tumors (GISTs) have been widely studied, the importance of reversible epigenetic changes increasingly recognized in many cancers has received insufficient attention in these tumors. Epigenetic mechanisms are part of normal development and gene expression under normal conditions, but malfunction of these processes leads to malignant transformation by disturbing both intra- and intercellular communication. GISTs are a specific group of gastrointestinal tract tumors resistant to conventional chemotherapy and radiotherapy. Although they account for only 1% to 2% of tumors, they are among the most widespread gastrointestinal mesenchymal tumors. DNA hyper/hypomethylation overexpression/underexpression of miRNAs or abnormal histone modification may provide an alternative to the genetic modifications responsible for GIST pathology, response to treatment, prognosis and overall survival. This review summarizes the known epigenetic mechanisms involved in GIST pathogenesis; including onset, progression, and GISTs resistance. Reversible epigenetic changes are a novel and appropriate approach to halt the spread of metastases and the emergence of resistance in GIST treatment, and these changes depend on the type of epigenetic alternation, including inhibitors of histone acetyltranferase and deacetylase and DNA methyltransferases.

Impact of early aggressive nutrition on retinal development in premature infants.

The normal retinal development is interrupted by preterm birth and a retinopathy of prematurity (ROP) may develop as its consequence. ROP is characterized by aberrant vessel formation in the retina as a response to multiple risk factors influencing the process of retinal angiogenesis. Insulin-like growth factor I (IGF-1) and vascular endothelial growth factor (VEGF) play an important role in the process of normal retinal vascularization. Insufficient nutrition during the first 4 postnatal weeks results in low serum levels of IGF-1, which is essential for correct retinal vessels formation, ensuring survival of the newly formed endothelial cells. Low IGF-1 level results in stop of angiogenesis in the retina, leaving it avascular and prompting the onset of ROP. Keeping the newborns in a positive energetic balance by providing enough nutrients and energy has a beneficial impact on their growth, neurodevelopment and decreased incidence of ROP. The best way to achieve this is the early parenteral nutrition with the high content of nutrients combined with early enteral feeding by the own mother´s breast milk. Multiple studies confirmed the safety and efficacy of early aggressive nutrition but information about its long-term effects on the metabolism, growth and development is still needed.

Gastrointestinal stromal tumor after tyrosine kinase inhibition therapy: a review of biopsies of 34 patients with clinically suspected relapse and/or progression of the tumor.

Implementation of combined surgical and targeted therapy strategies using tyrosine kinase inhibitors improved the prognosis of patients with aggressive GISTs. The therapeutic answer may be individually different, some patients do not respond properly, or even progress in spite of the therapy. This together with intratumoral heterogeneity and possible development of secondary phenotypical and genetical changes represents a challenge for pathologists examining a biopsy of relapsed tumors and/or their metastases. For this study biopsy files of the national Slovak GIST registry were reviewed to identify patients examined bioptically both prior the therapy and during the TKI treatment due to suspected tumor relapse and/or progression. All the GIST biopsies were analyzed using a standardized algorithm of histological, immunohistochemical and molecular analyses of exon 7, 9, 11, 13 of c-KIT and exons 12, 14, and 18 of PDGFRA genes, with the aim to identify posttherapeutical changes of these parameters. From 34 patients fulfilling the criteria of selection, all were histologically examined during their clinically suspicious first GIST relaps, eight during the 2nd, three during 3rd and one during 4th and 5th relapse resp. All but one posttherapeutical biopsies showed "viable" GIST tissue and so 44 relapses of 33 patients could be evaluated in comparison with identical parameters of diagnostic biopsies. Distinguishing three major histological types (spindle-, epitheloid-cell and mixed cell type), a change of the GIST type was identified in 1/3 of 1st relapse and » of all relapse biopsies. Evaluation of three phenotypical GIST parameters CD117, CD34 and DOG-1, showed that phenotype alteration was always represented by a single change. The most common was either a gain or loss of CD34 positivity appearing in 1/3 of 1st relapse biopsies, while a loss of CD117 positivity was identified in one patient´s biopsy only. Altogether, the phenotypical changes were in » of all relapses. A changed mutational profile was recognized in 38,2% first relaps biopsies and in 33% of all relapses, the change was mostly isolated (in 10/45 relapses) and less often (in 4/45 relapses) it represented a gain of a new mutation in association with persisting original one. In conclusion, the biopsies of patients showing relapse and/or progression on TKI treatment show predominance of viable GIST cells with limited or even absent signs of scaring, as well as relatively low incidence of morphological, pheno- and genotypical changes.

[Genetic aspects of pelvic floor defects and stress urinary incontinence in women]. / Genetické aspekty defektov panvového dna a stresovej mocovej inkontinencie u zien.

OBJECTIVE: An overwiev of the genetic aspects of pelvic floor defects and stress urinary incontinence in women. DESIGN: A review article. SETTING: Department of Gynecology and Obstetrics, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Slovak Republic. METHODS: An analysis of the literature using database search engines PubMed, BLAST and Ensembl in field of POP and SUI. RESULTS: Pelvic organ prolapse (POP) and stress urinary incontinence (SUI) affect a large number of women and often requires surgical correction. Moreover, these diseases are not only individual health but also social and economic burden on patients and society. It is possible to prevent or at least timely intervene these pathologies by screening of risk patients. The incidence of these disorders leads to find possible genetic factors. In ethiopathogenetic studies of pelvic floor defects and stress urinary incontinence it is looking for genes associated with these diseases and optimization of molecular biology methods is necessary to determine the possible gene mutations or polymorphism site. Selection of relevant genetic factors tend to clarify the pathomechanism of SUI and POP that can greatly affect their current diagnostics and therapeutics. KEYWORDS: prolapse, stress urinary incontinence, polymorphism, gene expression.

[Correlation between the incidence of PIK3CA mutations in breast cancer and histopathological characteristics of the tumor]. / Korelácia výskytu PIK3CA mutácií v karcinóme prsníka s histologickými vlastnostami nádoru.

OBJECTIVE: To determine the presence of mutations in exon 9 (encoding the helical domain) and exon 20 (encoding the kinase domain) of phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) gene in DNA obtained from paraffin embedded tissue from patients with carcinoma of the mammary gland and to correlate results with clinicopathological characteristics of cancer. DESIGN: Prospective clinical study. SETTING: Department of Molecular Biology, Department of Obstetrics and Gynecology, Jessenius Faculty of Medicine, Commenius University, Martin, Slovak Republic. METHODS: In set of 95 tissue samples from patients with breast cancer, mutations in exon 9 and 20 were analysed by sequencing. We also observed the associations between mutations and histopathological characteristics of tumor. RESULTS: Overall, mutations were present in 25.3% (24/95) of PIK3CA gene, of this 14.7% (14/95) of mutations were located in exon 9 and 10.5% (10/95) of mutations were in exon 20. We detected three "hotspot" mutations, two were located in exon 9 (E542K, E545K) and the third mutation was found in exon 20 (H1047R). Mutations in exon 9 showed significant correlation with lower grade(p = 0.0074) and pN status without metastases(p = 0.0415). Mutations in exon 20 were associated with higher age of patient (p = 0.0249). The E545K mutation correlated with lower grade (p = 0.0013) and pN status (p = 0.0232) particularly; the H1047R mutation was significantly more frequent in lobular type of breast cancer (p = 0.0354). CONCLUSION: The PI3K signaling pathway plays a critical oncogenic role in the development of human breast cancer and the prevalence of its deregulation advocates its potential as a feasible therapeutic target. In our study we demonstrate a significant correlation between the presence of PIK3CA mutations and some clinicopathological characteristics of tumour. We have shown that the mutations in exon 9 of PIK3CA were associated with favourable prognostic factors. KEYWORDS: "hotspot" mutation, PIK3CA, PI3K pathway, breast cancer.

Rasopathies - dysmorphic syndromes with short stature and risk of malignancy.

OBJECTIVES: The term ´Rasopathies´ represents a group of five neurodevelopmental syndromes (Noonan, LEOPARD, Costello, Cardio-facio-cutaneous, and Neurofibromatose-Noonan syndrome) caused by germline mutation in genes encoding proteins involved in RAS/MAPK (rat sarcoma/mitogen-activated protein kinase) signaling pathway. The RAS/MAPK signaling pathway participates in regulation of cell determination, proliferation, differentiation, migration, and senescence and dysregulation of this pathway can lead to the risk of tumorigenesis. In this review, we aim to summarize the current clinical and molecular genetic knowledge on Rasopathies with special attention for the risk of cancer. We propose also clinical and therapeutic approach for patients with malignancy. METHODS: We are reviewing the clinical and molecular basis of Rasopathies based on recent studies, clinical examination, and molecular diagnostics (mutation analysis of causal genes for Rasopathies) in Slovak pediatric patients. RESULTS: Some clinical features, such as short stature, a specific facial dysmorphology and cardiac abnormalities are common to all of Rasopathy syndromes. However, there are unique signs by which the syndromes can differ from each other, especially multiple lentigo in LEOPARD syndrome, increased risk of malignancy in Costello syndrome, dry hyperkeratotic skin in patients with cardio-facio-cutaneous syndrome, and neurofibromas and cafe-au-lait spots in neurofibromatosis-Noonan syndrome. CONCLUSION: Despite the overlapping clinical features, Rasopathy syndromes exhibit unique fenotypical features and the precise molecular diagnostics may lead to confirmation of each syndrome. The molecular diagnostics may allow the detection of pathogenic mutation associated with tumorigenesis.

[Rapid detection of the most common chromosomal aneuploidies in the second-trimester amniotic fluid using QF-PCR]. / Rýchla detekcia najbeznejsích chromozómových aneuploídií z plodovej vody v druhom trimestri tehotnosti vyuzitím QF-PCR.

OBJECTIVE: To introduce QF-PCR method for detection of the most common chromosomal (trisomy 21, 18 and 13) and gonosomal aneuploidies at our department in the second-trimester amniotic fluid. To test the hypothesis of chromosomal aneuploidies detection using STR markers of Aneufast® kit via analysing free fetal DNA (ffDNA) isolated from plasma of pregnant women with confirmed trisomy 21 in fetus. DESIGN: A prospective clinical study. SETTING: Department of Obstetrics and Gynecology, Jessenius Faculty of Medicine and University Hospital in Martin, Slovak Republic. METHODS: The samples of amniotic fluid were obtained from 67 women (twin pregnancy in 3 cases) in the 2nd trimester (15th to 22nd gestational week (g.w.)). Samples were examined using multiplex QF-PCR via Aneufast kit. In the case of positivity for trisomy 21, they were re-examined using Devyser Resolution 21 kit. All samples were parallelly evaluated by cytogenetic karyotyping. We also analyzed ffDNA from the plasma of 3 high-risk women using Aneufast kit. The plasma samples were obtained in the 2nd trimester(17th to 21st g.w.). Qiaamp DSP Virus kit was used for ffDNA isolation. Trisomy 21 of 3 fetuses was confirmed by karyotyping after 2nd trimester amniocentesis. RESULTS: In the cohort of 70 samples, 7 pathological results (six trisomies 21 and one trisomy 18) were obtained. There was 100% concordance with cytogenetic karyotype in all samples examined by QF-PCR. The amplification of tracked chromosome 21 fragments was not evaluable in the case of ffDNA analysis. CONCLUSION: QF-PCR was approved as reliable, rapid, quite simple and financially bearable method of prenatal diagnostics. Despite the fact of good availability and work implementation of Aneufast® kit, results of ffDNA analysis are insufficient. We did not obtain interpretable results after ffDNA analysis from maternal plasma in trisomy 21 fetuses.

[The p16INK4A mRNA transcripts estimation in cervical smear with different degrees of cervical dysplasia]. / Stanovenie expresie p16INK4A mRNA transkriptu v steroch z krcka maternice s rôznymi stupnami cervikálnej dysplázie.

OBJECTIVE: To determine the presence of HPV infection and expression level of p16INK4A mRNA transcripts in cervical smears as adjunct biomarker in detection of cervical intraepithelial neoplasia or cancer. DESIGN: Prospective pilot clinical study assessing clinical utility and validity of ddCt method for qPCR mRNA expression of p16ink4a in comparison to immunohistochemistry. SETTING: Department of Molecular Biology, Department of Obstetrics and Gynecology, Jessenius Medical Faculty, Commenius University, Martin, Slovak Republic. METHODS: Cervical smears (OC) from patients with different cervical lesions (L-SIL, H-SIL, SCA; n=45) and from healthy controls (n=45) were tested for the presence of HPV infection and p16INK4A mRNA transcripts using relative quantification (RQ). Results were compared to H&E and IHC histological findings from biopsies (conization, hysterectomy). RESULTS: HPV 16 was the most frequent finding (53.3%) in the group of subjects with cervical dysplasia. The p16INK4A mRNA expression analysis revealed the slightly reduced expression in L-SIL group, 4-fold increased expression in H-SIL and 10-fold increase in women with SCA when compared to controls. The p16INK4A mRNA expression in OC was present in 30% of L-SIL, 75% of H-SIL and 85.7% of SCA samples, respectively. The test overall sensitivity was 81.48% (95% CI: 61.92-93.7) and specificity 60% (95% CI: 26.24- 87.84) with PPV of 84.62% and NPV of 54.55%. The likelihood ratio (LR) in case of test positivity was 2.04 and for negativity 0.31. The diagnostic accuracy of p16INK4A expression by RQ method in OC smears for prediction of p16 positivity in cervical dysplasia was 66.7% for the L-SIL lesions, 59.5% for H-SIL lesions, and 100% for SCA (r=0.9897, p<0.0913) when compared to IHC p16 positive findings in surgically treated samples. CONCLUSION: The relative quantification is able to determine the level of p16INK4A mRNA transcripts in cervical smear cells with active carcinogenesis nearly at the same level as IHC staining. The advance of biopsy sparing over IHC is qualifying this diagnostic approach for useful candidate in selective management of women with cervical dysplasia looking for cervix preservation or avoiding the unnecessary overtreatment.

[Methylation of selected tumor-supressor genes in benign and malignant ovarian tumors]. / Metylácia vybraných tumor-supresorických génov v benígnych a malígnych ovariálnych nádoroch.

OBJECTIVE: To evaluate the usefullness of examination of methylation status of selected tumor-supressor genes in early diagnosis of ovarian cancer. DESIGN: Prospective clinical study. SETTING: Department of Gynecology and Obstetrics, Department of Molecular Biology, Jessenius Medical Faculty, Commenius University, Martin, Slovak Republic. METHODS: In this study we analyzed hypermethylation of 5 genes RASSF1A, GSTP, E-cadherin, p16 and APC in ovarian tumor samples from 34 patients - 13 patients with epithelial ovarian cancer, 2 patients with border-line ovarian tumors, 12 patients with benign lesions of ovaries and 7 patients with healthy ovarian tissue. The methylation status of promoter region of tumor-supressor genes was determined by Methylation Specific Polymerase Chain Reaction (MSP) using a nested two-step approach with bisulfite modified DNA template and specific primers. RESULTS: Gene methylation analysis revealed hypermethylation of gene RASSF1A (46%) and GSTP (8%) only in malignant ovarian tissue samples. Ecad, p16 and APC genes were methylated both in maignant and benign tissue samples. Methylation positivity in observed genes was present independently to all clinical stages of ovarian cancer and to tumor grades. However, there was observed a trend of increased number and selective involvement of methylated genes with increasing disease stages. Furthermore, there was no association between positive methylation status and histological subtypes of ovarian carcinomas. CONCLUSION: RASSF1A and GSTP promoter methylation positivity is associated with ovarian cancer. The revealed gene-selective methylation positivity and the increased number of methylated genes with advancing disease stages could be considered as a useful molecular marker for early detection of ovarian cancer. However, there is need to find diagnostic approach of specifically and frequently methylated genes to determining a methylation phenotype for early detection of ovarian malignancies.

[Our experience with detection of JAK2 mutations in paraffin-embedded trephine bone marrow biopsies of patients with chronic myeloproliferative disorders]. / Nase skúsenosti s vysetrovaním JAK2 mutácií pacientov s myeloproliferatívnymi ochoreniami z trepanobioptického materiálu kostnej drene.

Polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) characterized by JAK2 mutation. The exon 14 V617F mutation is present in almost all patients with PV and in approx. 60% of patients with ET and PMF. The importance of JAK2V617F in the differential diagnostic considerations is still unclear and here the BM morphology examination still represents an important diagnostic tool. In the WHO classification of Ph1-negative MPNs, the identification of JAK2 mutations represents a major diagnostic criterion of these diseases. Therefore we decided to implement the examination of JAK2V617F mutation in formalin-fixed paraffin-embedded biopsy specimens of patients with Ph1-negative MPN using allele-specific PCR. In addition, in all JAK2 V617F negative patients with PV we sequenced the whole JAK2 exon 12. Until now we examined up to 200 patients with clinically confirmed MPN and our results in all three categories PV, ET and PMF are in agreement with earlier published data. Paraffin embedded tissues represent a valuable source of DNA which can be used in the diagnostics of both JAK2 exon 12 and exon 14 mutations. It is of particular importance if the fresh material is not available and there is a clinical and/or research utility for the performance of PCR on archival bone marrow samples with PV, ET or PMF suspicion.

One-year follow-up of kidney transplant recipients with increased expression of mRNA for granzyme B in urinary cells.

BACKGROUND: Measurement of mRNA encoding cytotoxic proteins in urinary cells is recognized as a potential noninvasive means to diagnose acute rejection in kidney allograft recipients. We sought to evaluate kidney graft function after 1 year follow-up without therapeutic intervention among patients with increased urinary expression of mRNA for granzyme B, albeit with stable graft function at the time of measurements. PATIENTS AND METHODS: The 29 randomly selected patients were at a median of 39 months (range, 10-156) after transplantation with stable graft function over the previous 3 months. The reference housekeeping gene GAPDH was used for expression measurement in a TaqMan Gene Expression Assay with the target granzyme B gene. Delta delta ct relative gene expression analysis compared results with reference samples from 10 healthy individuals. Kidney graft function was reassessed after 1 year follow-up; immunosuppression was not changed during this period. RESULTS: mRNA granzyme B expression was significantly higher among the group of randomly assessed out-clinic patients with stable graft function than among healthy volunteers (mean ± standard error of the mean, 6.18 ± 1.27; P < .01). Despite no therapeutic intervention, no significant changes were observed in delta glomerular filtration rate or quantitative proteinuria between groups with mRNA expression > 5× versus <2× higher than the healthy controls at 1 year of follow-up. CONCLUSION: Increased mRNA expression for granzyme B in urinary cells over the medium to long term among kidney transplant recipients did not predict changes in renal allograft function after 1 year follow-up.

[Detection of DNA hypermethylation as a potential biomarker for prostate cancer]. / Detekcia hypermetylácie DNA ako potenciálny biomarker pre karcinóm prostaty.

Prostate cancer is one of the most common malignant diseases in men above the age of 50. A genetic predisposition and/or acquired genetic and epigenetic changes together with lifestyle contribute to the development of the disease. The most studied epigenetic modification in prostate cancer is the methylation of the cytosine located within the dinucleotide CpG of promoter regions of different genes by methylation specific PCR. The evidence of gene silencing by DNA methylation in genes like GSTP1, APC or RASF1 is a common and relatively specific event in prostate cancer. DNA methylation testing can be performed on tissue samples or urine, ejaculate or serum. Translational research is searching for new biomarkers for early detection and prognosis of prostate cancer, but because of large methodological differences in applied techniques and patient cohorts, the investigations have yielded promising, but also some controversial results. More prospective randomized trials and standardized methods are needed to assess the true value of methylation for the diagnosis and prognosis of prostate cancer.

Hypermethylation of the GSTP1 promoter region in breast cancer is associated with prognostic clinicopathological parameters.

UNLABELLED: Breast cancer is one of the most common cancer affecting women and the recent research is focused on identifying new genetic and epigenetic prognostic and predictive factors. Glutathione S-transferase P1 (GSTP1) is abiotransformation enzyme expressed in normal breast epithelial cells which can be epigenetically inactivated in breast cancer. We have shown, that application of nested two-stage methylation-specific PCR (MSP) is asuitable method for analysis of epigenetically silenced GSTP1 in formalin-fixed paraffin-embedded (FFPE) tissues from breast cancer patients. Of 45 breast tumors, 11 (24, 4%) were found to have methylated GSTP 1promoter region. We were able to demonstrate the correlation between the hypermethylation of the GSTP1 promoter region and histological grade of the tumor (p KEYWORDS: breast cancer, prognostic factors, hypermethylation, GSTP1, methylation-specific PCR.

HER-2 [Ile655Val] polymorphism in association with breast cancer risk: a population-based case-control study in Slovakia.

Breast cancer belongs to the most frequent types of cancer affecting women and it occurs at any age. Around 1600-1800 women are getting ill annually in the Slovak republic. One of the most important factors in connection with cancer genesis refers to changes in specific genes. HER-2 proto-oncogene belongs to low penetrating genes, which increase susceptibility to breast cancer genesis. Clinical studies demonstrated an association between polymorphism at codon 655 of this gene and increased risk for breast cancer development. The aim of this case-control based prospective study was to determine the distribution of HER-2 genotype and its association with risk factors of breast cancer in the population of women in Slovak republic. HER-2 genotypes were determined with PCR-RFLP method. The DNA was isolated from white blood cell nuclei. The frequency of Val allele in the cancer group was 29.79% and was higher than in the control group 15.84% (p<0.05). The presence of the heterozygote (Ile/Val) genotype was identified in 46.81% of patients in the case group and in 28.33% in healthy individuals, and the homozygote (Val/Val) genotype in 6.38% and 1.67, respectively (p<0.01). The risk of breast cancer development for carriers of one valine (Val) allele in genotype was two-times lower (OR=2.47) than for carriers of two Val alleles (OR=5.73) (p<0.05). Risk of cancer genesis for Val allele carriers was higher in multiparas (OR=2.90), among women with positive family history of breast cancer (OR=5.0), BMI>24 (kg/m2), and late menopause (OR=1.5). Contraceptives in anamnesis contrariwise showed tend to decrease the risk in Val allele carriers (OR=0.3). In conclusion, this study revealed relatively high frequency of the Val allele among the women population of the Slovak republic. Ile655Val polymorphism of HER-2 gene was associated with a statistically significantly increased risk of breast cancer all above in homozygotes for Val allele.