Juraj Marcinek

Detection of JAK2 mutations in paraffin marrow biopsies by high resolution melting analysis: identification of L611S alone and in cis with V617F in polycythemia vera.

Abstract JAK2 mutations provide an objective major criterion for diagnosis of myeloproliferative neoplasms in the 2008 World Health Organization classification, and are particularly useful for cases of polycythemia vera (PV). High resolution melting analysis (HRM) using DNA from fresh samples for mutation detection in JAK2 exons 12 and 14 has been reported. Here, we describe two new HRM techniques that use both fresh and formalin-fixed paraffin-embedded marrow samples, allowing for both prospective and retrospective analyses. We used these novel assays to screen DNA isolated from paraffin-embedded marrow biopsies from 104 patients with PV for mutations. HRM mutation-positive samples were subsequently sequenced. Ninety-seven samples (93.3%) were positive solely for the JAK2 p.V617F. One harbored a 33 bp duplication in exon 12 along with an exon 14 p.V617F mutation, another had an exon 12 p.H538_K539delinsL, and a third carried a previously unreported mutation in PV, p.L611S, alone, and in cis with p.V617F. This indicates that these assays can detect known and novel JAK2 mutations in exons 12 and 14 using either fresh or paraffin-embedded archival materials.

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.

Tumefactive demyelination of the spinal cord: a case report.

Study design:Case report.Objectives:We report on a 52-year-old male patient with tumefactive demyelination of the spinal cord.Setting:University Hospital and Jessenius Faculty of Medicine, Comenius University, Martin, Slovakia.Background:In contrast to relatively frequent tumefactive fulminant lesions in the brain, cases affecting the spinal cord in isolation have been reported less frequently.Methods:Description of the case report.Results:Clinical, neuroradiological and necropsy findings are described in a 52-year-old man with tumefactive fulminant demyelination of the spinal cord. Progression of the demyelination process produced paraplegia, mild paresis of the right upper limb, neurogenic bladder and sensitive loss over 2 weeks. MRI scans revealed several ovoid lesions in cervical segments and tumefactive T2-hyperintense signals with oedema and post-contrast enhancement located in thoracic segments Th3 to Th6. Cerebrospinal fluid (CSF) examination displayed lymphomonocytic pleocytosis with normal proteinorhachia, positive CSF oligoclonal IgG bands (OCB) and elevated IgG index (1.55). Serum anti-AQP4-Ab was not tested. Stored frozen CSF samples were later repeatedly examined with negative findings of anti-AQP4-Ab. Treatment with high-dose methylprednisolon and plasma exchange had limited effect. Immunosuppressive medication was interrupted because of an acute urinary infection. The patient died suddenly because of pulmonary embolism as a secondary complication. Histopathology of the spinal cord confirmed active demyelination. We considered that tumefactive demyelination could be a variant of neuromyelitis optica.Conclusion:Our case could be anti-AQP4-Ab-negative longitudinally extensive transverse myelitis, a variant of neuromyelitis optica.

Relationship Between Potassium Ion Channels and Airways Defence Reflexes Influenced by Experimentally Induced Allergic Inflammation in Guinea Pigs

Relationship Between Potassium Ion Channels and Airways Defence Reflexes Influenced by Experimentally Induced Allergic Inflammation in Guinea Pigs Previously, we declared important role of ATP-sensitive (KATP) and calcium-sensitive (BKCa) potassium ion channels in cough and other defence reflexes of the airways coupled with reactivity of airways smooth muscle (ASM) and suggested their potential use as antitussives and antiasthmatic drugs. The aim of presented studies was prove whether openers of potassium ion channels, KATP - pinacidil and BKCa - NS 1619, inhibit the cough reflex and modulate the ASM reactivity in conditions of experimental allergic inflammation of the airways in guinea pigs and if their influence on airways defence reflexes is changed by developing airways inflammation. Presented studies were realized in 4 partial experimental procedures with unsensitized guinea pigs and animal on 7th, 14th and 21th day of sensitization. Allergic inflammation of airways was induced by repetitive exposure of guinea pigs to ovalbumine and the degree of allergic inflammation was determined by histological analysis of tracheal and pulmonary samples. The cough reflex was induced by 0, 3 M citric acid aerosol for 3 min interval in which total number of coughs was counted. ASM reactivity in vivo was expressed as values of specific airway resistance (R. V) calculated by Pennock. The cough response on the citric acid was significantly increased on 7th and 14th days of sensitization. The experiments showed persistent cough suppressive effect of pinacidil almost similar to codeine. The antitussive activity of NS 1619 remained only on 7th day of sensitization similar to its effect in group of unsensitized animals. Sensitization by ovalbumine gradually increased the values of R. V on bronchoprovoking agent citric acid. Pinacidil suppressed R. V values of both, unsensitized and sensitized animals, more significantly than salbutamol. In unsensitized guinea pigs, NS 1619 significantly reduced R. V values, but allergic inflammation attenuated its bronchodilatory activity on 7th and 14th days. Histological analysis of specimens showed increasing signs of allergic inflammation during sensitization procedure as well as significant proinflammatory effect of pinacidil and NS 1619. Introduction of non-selective KATP agonists in clinical practice is strongly limited due to proinflammatory effect, but KATP of ASM represents a rational therapeutic target for novel drugs - tissue selective agonists of KATP.

Differential mRNA expression of the main apoptotic proteins in normal and malignant cells and its relation to in vitro resistance

BackgroundApoptosis plays an important role in the development and homeostasis of multicellular organisms and its deregulation may result in many serious diseases, including cancer. Now it is clear that some oncogenic mutations disrupt apoptosis, leading to tumour initiation, progression or metastasis. Here, expression of apoptotic genes in context of drug resistance was investigated.MethodsWe examined total of 102 samples from leukemic patients (n = 60) and patients with solid tumours (n = 42). We used RT-PCR to determine the levels of mRNA expression and the in vitro chemoresistance of leukemic cells was evaluated using the MTT assay.ResultsWe found statistically significant increase in mRNA expression of all investigated proteins (p53, BAX, Bcl-2 and Bcl-XL) between the leukemia samples and leukocytes from healthy volunteers. We did not find any significant difference in mRNA levels among the solid tumour samples. Notably, we showed a significant positive correlation in both leukemic and solid tumour patient groups between p53 and BAX mRNA. We found that the highest values for the Bcl-2/BAX ratio were in solid tumours in comparison to leukemic cells or normal leukocytes. Moreover, we assessed the impact of p53 and BAX mRNA levels on the sensitivity of the leukemic cells to selected cytostatics.ConclusionsElevated levels of p53 and BAX mRNA may indicate cellular response to possible changes in genomic DNA integrity associated with malignant transformation. We suggest that the BAX gene is regulated by the p53 protein but the initiation of apoptosis through the transcription activation of BAX is blocked by the high levels of Bcl-2. Given that the apoptosis resistance mechanisms are different among oncological patients as well as stages of identical malignancy cases, personalized and specific combination therapy is proposed to be more effective in clinical application.

DNA methylation as mechanism of apoptotic resistance development in endometrial cancer patients

DNA methylation is a significant epigenetic modification which plays a key role in regulation of gene expression and influences functional changes in endometrial tissue. Aberrant DNA methylation changes result in deregulation of important apoptotic proteins during endometrial carcinogenesis and apoptosis resistance development. Evading apoptosis is still a major problem in the successful treatment of endometrial cancer patients. The aim of our study was to examine the promoter DNA methylation changes in 22 apoptosis-associated genes in endometrioid endometrial cancer patients, precancerous lesions and healthy tissue from various normal menstrual cycle phases using a unique pre-designed methylation platform. We observed as the first a significant difference in promoter DNA methylation status in genes: BCL2L11 (p < 0.001), CIDEB (p < 0.03) and GADD45A (p < 0.05) during endometrial carcinogenesis and BIK gene (p < 0.03) in different phases of normal menstrual cycle. The results of our study indicate that deregulation of mitochondrial apoptotic pathway can considerably contributes to the apoptosis resistance development and may be helpful in identifying of new potent biomarkers in endometrial cancer.