Zora Lasabova

RASSF1A and CDH1 hypermethylation as potential epimarkers in breast cancer

Breast cancer is the most common cancer in women worldwide, representing 28.2% of all female malignancies. In addition to genetic changes, epigenetic events, as aberrant DNA methylation and histone modification, are responsible for cancer development. Many tumour suppressor genes are inactivated by DNA hypermethylation, which could be utilized for identification of new epigenetic biomarkers. To investigate the relation between DNA methylation level and breast cancer progression, we analysed DNA methylation in RASSF1A and CDH1 promoters using quantitative multiplex methylation-specific PCR in paraffin-embedded tumour tissues and blood samples from 92 breast cancer patients and 50 controls, respectively. The associations between RASSF1A and CDH1 methylation levels and clinico-pathological parameters were tested by Kruskal-Wallis and van der Waerden ANOVA tests. Out of 92 breast cancer patients, 76 (82.6%) manifested various levels of RASSF1A (range from 1.20 to 92.63%) and 20 (21.7%) of CDH1 (range from 1.20 to 79.62%) methylation. However, no methylation was found in 50 controls. Increasing trends in RASSF1A methylation were observed in tumour size, lymph node status and TNM stage, but only CDH1 methylation levels showed statistically significant differences between the patient subgroups in lymph node status and IHC subtype. Overall, stable relatively high RASSF1A methylation could be utilised as universal tumour marker and the less frequent but highly methylated CDH1 promoter can serve for identification of potentially metastasising tumours.

Promoter hypermethylation of the tumor-suppressor genes RASSF1A, GSTP1 and CDH1 in endometrial cancer.

Endometrial cancer is a common gynecological malignancy with a good prognosis in early stages of the disease. The CpG island in the promoter region of tumor-suppressor genes are frequently methylated in various types of human cancers. In the present study, we examined the methylation status of the GSTP1, CDH1 and RASSF1A genes in endometrioid endometrial cancer (EEC), endometrial complex hyperplasia (EHP) and healthy endometrium with the aim to identify correlations between promoter hypermethylation, disease risk and clinicopathological parameters. A nested two-stage methylation-specific PCR (MSP) was performed to analyze the promoter CpG methylation status of GSTP1, CDH1 and RASSF1A genes in the population studied. A total of 92 subjects were initially included in the study of which 41 EEC, 19 EHP and 20 controls were processed for final analyses. A significant difference was found between the study groups and the presence of promoter CpG hypermethylation status in the GSTP1 (p<0.05) and RASSF1A (p<0.0001) genes. RASSF1A, GSTP1 and CDH1 gene promoter methylation was present in 85.4, 68.3 and 31.4% of EEC samples when compared to that in the controls with 30.0, 35.0 and 20.0%, respectively. CpG methylation of all three investigated tumor-suppressor genes was found in 12.2% of EEC patients, in 4.2% of EHP patients and in 3.7% of the controls, respectively. Positive findings for the promoter methylation of two investigated genes were found in 48.7% of EEC patients, 26.0% of EHP patients and in 18.5% of the controls. With regard to histopathological variables and CpG methylation, we found significant correlations between the RASSF1A and GSTP1 genes and higher tumor grade, deeper myometrial invasion and positive metastatic involvement of pelvic lymph nodes. No associations were noted between promoter hypermethylation of the CDH1 gene and biological features of the endometrial cancer cases. The results indicate that aberrant CpG methylation of the promoter region in the GSTP1 and RASSF1A tumor-suppressor genes is an important event in carcinogenesis of endometrial cancer and may have an impact on tumor aggressiveness. Finally, the present study suggests that epigenetic alterations may be of diagnostic value for the better clinical management of premalignant endometrial lesions.

Spectrum of mutations in gastrointestinal stromal tumor patients – a population‐based study from Slovakia

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of gastrointestinal tract and are characterized by presence of mutations in tyrosine kinases cKIT (KIT) and PDGFRα (PDGFRA). Mutations identified are highly heterogeneous, but some mutations are associated with specific clinical features of the tumor. Samples from 278 GIST patients collected during the period 2004–2011 were screened for mutations in exons 9, 11, 13, and 17 of KIT and 12, 14 and 18 of PDGFRA. Results of mutation screening were summarized and tested for possible association with clinical parameters of tumors. Mutations were identified in 83.81% of patients. Most frequent mutations were found in KIT exon 11 reaching frequency of 62.95%. Other exons contributed to the mutation pool with frequencies 8.27%, 7.55%, 2.52%, 1.44%, 1.08%, and 0.00%, in decreasing order KIT exon 9, PDGRFA exons 18 and 12, KIT exon 13, PDGFRA exon 14, and KIT exon 17. General linear model analysis showed no effect of any individual analyzed mutation on the phenotypic variables, but we confirmed association between mutations KIT exon 9 p. 503‐504_dup2, and PDGFRA exon 18 p. D842V and intestinal and gastric localization of tumors.

Whipple's disease-generalized stage.

Whipple’s disease is a chronic inflammatory systemic disorder in which all organs can be invaded by the rod-shaped bacterium Tropheryma whipplei. It is a rare disease and frequently misdiagnosed, though there is no valid estimate of its actual incidence and prevalence. Only about 1,000–1,500 cases have been reported. The clinical course of untreated Whipple’s disease can include three stages: (1) a non-specific prodromal stage which includes migratory polyarthralgias; (2) a classic abdominal manifestation which involves weight loss, weakness, chronic diarrhea, and abdominal pain; and (3) a generalized stage characterized by steatorhea, cachexia, lymphadenopathy, hyper-pigmentation, and cardiovascular, pulmonary, and neurological dysfunction. The authors describe a case of a 39-year-old male patient with about a year’s history of generalized adenopathy, inappetence, weight loss, progressive weakness, subfebrilities, and convulsive abdominal pain. Following primary exclusion of a tumor disease, a lymph node biopsy demonstrated a typical picture of a granulomatous inflammation—Whipple’s lymphadenitis with partial exemption of the Gram reaction, and stain features corresponding to T. whipplei, which is regarded as the etiological agent causing this disorder. Further tests confirmed the generalized form of the disorder, affecting the lymphatic tissues, gastrointestinal system, respiratory system, and nervous system, with sensory and motor polyneuropathy. HLA-B27 antigen, which is frequent among those with Whipple’s disease, was also present. Following treatment for three months with antibiotics a significant reduction of the changes typical of Whipple’s disease was found upon follow-up biopsy, hence we assume the applied therapy was successful. In our case study we emphasize the atypical course of the disease with dominant generalized lymphadenopathy and only mild gastrointestinal symptoms.

Antineoplastic effects of clove buds (Syzygium aromaticum L.) in the model of breast carcinoma

It is supposed that plant functional foods, rich in phytochemicals, may potentially have preventive effects in carcinogenesis. In this study, the anticancer effects of cloves in the in vivo and in vitro mammary carcinoma model were assessed. Dried flower buds of cloves (CLOs) were used at two concentrations of 0.1% and 1% through diet during 13 weeks after the application of chemocarcinogen. After autopsy, histopathological and immunohistochemical analyses of rat mammary carcinomas were performed. Moreover, in vitro evaluation using MCF‐7 cells was carried out. Dietary administered CLO caused the dose‐dependent decrease in tumour frequency by 47.5% and 58.5% when compared to control. Analysis of carcinoma cells in animals showed bcl‐2, Ki67, VEGFA, CD24 and CD44 expression decrease and Bax, caspase‐3 and ALDH1 expression increase after high‐dose CLO administration. MDA levels were substantially decreased in rat carcinomas in both CLO groups. The evaluation of histone modifications revealed increase in lysine trimethylations and acetylations (H4K20me3, H4K16ac) in carcinomas after CLO administration. TIMP3 promoter methylation levels of CpG3, CpG4, CpG5 islands were altered in treated cancer cells. An increase in total RASSF1A promoter methylation (three CpG sites) in CLO 1 group was found. In vitro studies showed antiproliferative and pro‐apoptotic effects of CLO extract in MCF‐7 cells (analyses of cytotoxicity, Brdu, cell cycle, annexin V/PI, caspase‐7, Bcl‐2 and mitochondrial membrane potential). This study showed a significant anticancer effect of clove buds in the mammary carcinoma model in vivo and in vitro.

A novel JAK2 exon 12 mutation identified in the retrospective analysis of paraffin-embedded tissues of polycythemia vera patients.

The aim of the study was to screen formalin-fixed, paraffin-embedded tissues of polycythemia vera patients for the presence of JAK2V617F and JAK2 exon 12 mutations. Of 64 cases, 60 were positive for JAK2V617F mutation using allele-specific polymerase chain reaction (PCR). Using modified allele-specific PCR, samples of 4 JAK2V617F-negative patients were analyzed for the presence of JAK2 exon 12 mutations. In one case, we found a PCR product matching with allele-specific primer, which was designed to detect the N542-E543del mutation. Surprisingly, in the result sequence we have detected another recently described mutation, R541-E543delinsK. In the other 3 JAK2V617F-negative patients, allele-specific PCR for the detection of JAK2 exon 12 mutations did not yield any product. However, in 1 case, the sequencing of JAK2 exon 12 PCR product revealed a novel mutation, H538-K539delinsF. We confirmed that paraffin-embedded tissues represent a valuable source of DNA, which can be used in diagnostics of both JAK2 exon 12 and exon 14 mutations and we described 1 novel JAK2 exon 12 mutation.

mRNA expression in cervical specimens for determination of severe dysplasia or worse in HPV-16/18-positive squamous lesions.

Objectives The objective of current study was to determine the p16INK4a mRNA level in cervical cells by relative quantification (RQ) and to test viral E6 expression in human papillomavirus (HPV) -16 or -18–positive specimens by widely used methods. We targeted the pivotal mRNA level associated with severe dysplasia or worse. Materials and Methods Cervical specimens were taken from 134 women with cervical disease and 132 women with normal cytologic results. The presence of HPV was analyzed by sequencing. The results of p16INK4a and E6 analyses were statistically processed in receiver operating characteristic curve analysis to predict severe dysplasia or worse. Results The HPV DNA was detected in 81.4% (109/134) of women with cervical disease and in 27.3% (36/132) of women with normal cytologic results. HPV-16 or -18 were present in 59.7% (80/134) of abnormal specimens. p16INK4a and E6 mRNA expression was increasing with severity of cervical dysplasia. p16INK4a mRNA expression was found 4.35-fold and 13.15-fold increased in high-grade squamous intraepithelial lesions and squamous cell carcinomas, respectively. E6 mRNA expression was significantly increased (p = .0038) in severe dysplasias or worse. The RQ method achieved better sensitivity (82.6%), and E6 mRNA got better specificity (80.6%) for the prediction of severe dysplasia or worse. Conclusions An increasing level of p16INK4a and E6 mRNA transcripts could mean the potential of cervical dysplasia progression to cancer, but further studies should be done to confirm this proposition. Nevertheless, we consider using both tests to improve the sensitivity and specificity for prediction of severe dysplasia or worse.

Active cigarette smoking and the risk of breast cancer at the level of N-acetyltransferase 2 (NAT2) gene polymorphisms

The aim of our study was to assess the correlation between the tobacco exposure and NAT2 gene (rs1041983 C/T, rs1801280 T/C, rs1799930 G/A) polymorphisms in association with breast cancer development. We wanted to determine the prognostic clinical importance of these polymorphisms in association with smoking and breast cancer. For the detection of possible association between smoking, NAT2 gene polymorphisms, and the risk of breast cancer, we designed a case-controlled study with 198 patients enrolled, 98 breast cancer patients and 100 healthy controls. Ten milliliters of peripheral blood from the cubital vein was withdrawn from every patient. The HRM (high resolution melting) analysis was used for the detection of three abovementioned NAT2 gene polymorphisms. When comparing a group of women smoking more than 5 cigarettes a day with the patients smoking fewer than 5 cigarettes a day, we found out that if women were the carriers of aberrant AA genotype for rs1799930, the first group of women had higher risk of breast carcinoma than the second group. If patients were the carriers of aberrant TT genotype for rs1041983, for rs1801280CC genotype, and rs1799930AA genotype and they smoked more than 5 cigarettes a day, they had higher risk of malignant breast disease than never-smoking women. Our results confirm the hypothesis that NAT2 gene polymorphisms (rs1041983 C/T, rs1801280 T/C, and rs1799930 G/A) in association with long-period active smoking could be the possible individual risk-predicting factors for breast cancer development in the population of Slovak women.

Genetic variations of the GP6 regulatory region in patients with sticky platelet syndrome and miscarriage.

Introduction: Thrombophilia increases the risk of venous thrombosis during pregnancy and may predispose to gestational vascular complications. Objective: The aim of this study is to evaluate the variability of GP6 regulatory regions in a group of patients with platelet hyperaggregability manifested as miscarriage compared with control subjects. Methods: We examined 27 female patients with platelet hyperaggregability and history of spontaneous abortion and 42 healthy women. Platelet hyperaggregability was established by light transmission aggregometry. We also assessed eight SNPs within the GP6 gene. Results: We found a higher occurrence of three SNPs in patients with platelet hyperaggregability and history of miscarriage (rs1671152, rs1654433, rs1671215). The haplotype analysis showed a significant higher occurrence of two haplotypes (ACGG, CCGT). Conclusions: Our results support the idea that genetic variability of GP6 regulatory regions can be associated with platelet hyperaggregability – a possible cause of miscarriage.

Fibrinogen Martin: A Novel Mutation in FGB (Gln180Stop) Causing Congenital Afibrinogenemia

We readwith great interest the topical article by deMoerloose et al1 in Seminars in Thrombosis and Hemostasis regarding congenital afibrinogenemia. Accordingly, we recently corresponded anovelmutation in an afibrinogenemicpatientwith a proposed new pathological molecular mechanism underlying afibrinogenemia.2Wehave named this novelmutation “fibrinogenMartin”, and nowwish to expand our intial report to also describefindingswith thehypofibrinogenemic kindreds of the index patient. Fibrinogen is a 340 kDa glycoprotein comprising pairs of three polypeptide chains termed Aα, Bβ, and γ. Fibrinogen has a trinodular structure with a central nodule (E-domain) that contains the N-terminus of each chain and two lateral globular domains (D-domains) that contain the C-terminus of Bβand γ-chains. The E-domain is linked to the two D-domains by a coiled-coil triple helix structure.3,4 The three genes encoding fibrinogen Bβ (FGB), Aα (FGA), and γ (FGG), ordered from centromere to telomere, are clustered in a region of 50 kb on human chromosome 4q28.1,4 Congenital afibrinogenemia is an autosomal recessive bleeding disorder, referring to the total absence of plasma fibrinogen asmeasured by an antigenic assay, and is caused by variations in these genes, and associated with homozygous or compound heterozygous mutations, while hypofibrinogenemia is usually linked with heterozygous mutations.5 Mutations of FGB are less common and of particular interest since the Bβ-chain is considered the rate-limiting factor in the hepatic production of the fibrinogen hexamer.6 The estimated prevalence of congenital afibrinogenemia is 1 in 1,000,000,1 and according to our present knowledge, in Slovakia is 1 in 5,000,000.7 We recently performed genetic analysis of FGA, FGB, and FGG as well as coagulation tests in a patient with congenital afibrinogenemia plus his immediate family (i.e., his parents and two older sisters). The patient with congenital