Jiri Drabek

CC chemokine receptor (CCR)2 polymorphism in Czech patients with myocardial infarction.

Examining an association between myocardial infarction (MI) and the Val/Ile polymorphism in the gene for CC chemokine receptor (CCR)2 at the position 64 (CCR2-V64I), 122 MI Czech patients and 277 unrelated control (C) subjects were genotyped by PCR-SSP. The frequency of the VI genotype of CCR2-V64I was increased in MI patients in comparison with the control population (P=0.03). Further analysis revealed that relationship between the VI genotype and MI is specific only for females and, strikingly, this genotype was associated to an early MI onset (before or at the age of 50 years). Females with the VI genotype were seven times more prone to suffer from MI before 50 years than those with the VV genotype (P<0.01). If the VI genotype of the CCR2-V64I is indeed a risk factor for an earlier MI onset in females must be checked by independent studies in other centres and/or populations.

A comparison of Direct sequencing, Pyrosequencing, High resolution melting analysis, TheraScreen DxS, and the K-ras StripAssay for detecting KRAS mutations in non small cell lung carcinomas

BackgroundIt is mandatory to confirm the absence of mutations in the KRAS gene before treating metastatic colorectal cancers with epidermal growth factor receptor inhibitors, and similar regulations are being considered for non-small cell lung carcinomas (NSCLC) and other tumor types. Routine diagnosis of KRAS mutations in NSCLC is challenging because of compromised quantity and quality of biological material. Although there are several methods available for detecting mutations in KRAS, there is little comparative data regarding their analytical performance, economic merits, and workflow parameters.MethodsWe compared the specificity, sensitivity, cost, and working time of five methods using 131 frozen NSCLC tissue samples. We extracted genomic DNA from the samples and compared the performance of Sanger cycle sequencing, Pyrosequencing, High-resolution melting analysis (HRM), and the Conformité Européenne (CE)-marked TheraScreen DxS and K-ras StripAssay kits.Results and conclusionsOur results demonstrate that TheraScreen DxS and the StripAssay, in that order, were most effective at diagnosing mutations in KRAS. However, there were still unsatisfactory disagreements between them for 6.1% of all samples tested. Despite this, our findings are likely to assist molecular biologists in making rational decisions when selecting a reliable, efficient, and cost-effective method for detecting KRAS mutations in heterogeneous clinical tumor samples.

Expression of the chemokine PARC mRNA in bronchoalveolar cells of patients with sarcoidosis

BACKGROUND Chemotactic cytokines (chemokines) have been recently implicated in the pathogenesis of interstitial lung diseases. A novel chemokine called pulmonary and activation-regulated chemokine (PARC/CCL18), which attracts lymphocytes in vitro, has been detected in the human lung. We have, therefore, investigated PARC mRNA expression in bronchoalveolar lavage fluid (BALF) cells of patients with pulmonary sarcoidosis-a disease characterised by a lymphocytic infiltrate. Further, because several immunomodulators are used in the treatment of sarcoidosis, we have determined the effects of selected drugs on PARC mRNA expression in vitro. SUBJECTS AND METHODS BALF cells were obtained by standard bronchoalveolar lavage (BAL) from 30 patients with pulmonary sarcoidosis (S) and 16 control subjects (C). BALF cells from seven subjects were cultured in the presence of dexamethasone (Dx), cyclosporin A (CyA) and pentoxifylline (Px). PARC mRNA expression was semiquantitated by reverse-transcription polymerase chain reaction (RT-PCR) using normalisation to the expression of the beta-actin gene. RESULTS PARC mRNA transcripts were detected in 87% of all investigated BALF samples. The expression (ODR PARC/beta-actin; median, the first to the third quartile range) was similar in both groups tested (S, 0.60 (0.50-0.95); C, 0.59 (0.36-0.93); S vs. C: P>0.05). PARC mRNA expression was not associated with the number of lymphocytes in bronchoalveolar space. PARC mRNA expression was significantly suppressed by Dx (P=0.02); CyA and Px showed a moderate inhibitory effect which did not attain significance. CONCLUSION mRNA for the chemokine PARC is expressed in the lower respiratory tract in both healthy subjects and patients with pulmonary sarcoidosis. Out of the three immunomodulatory drugs tested, Dx downregulates PARC mRNA expression in BALF cells in vitro.

CC chemokine receptor 5 (CCR5) mRNA expression in pulmonary sarcoidosis

The CC chemokine receptor 5 (CCR5) binds the chemokine ligands RANTES (CCL5) and MIP-1alpha (CCL3), which have been implicated in the development of alveolitis in sarcoidosis. We have, therefore, investigated CCR5 mRNA expression in bronchoalveolar lavage fluid (BALF) cells from patients with sarcoidosis. Further, we explored whether there was any association between CCR5 mRNA expression and the presence of the CCR5Delta32 DNA polymorphism. Semiquantitative RT-PCR was used to determine CCR5 mRNA expression from BALF cells from 16 control subjects (C) and 39 patients with sarcoidosis (S). The data on the CCR5Delta32 polymorphism, determined by PCR-SSP, were available for 37 patients. CCR5 mRNA expression was significantly upregulated in sarcoidosis (median+/-SEM, C, 0.00+/-0.07; S, 0.12+/-0.07; P<0.05). When patients were evaluated according to their CCR5Delta32 genotype, an interesting trend emerged with Delta32 positive patients (wt, mt) expressing less mRNA than the patients with both wild-type alleles (wt, wt): 0.00+/-0.09, and 0.26+/-0.09, respectively; P>0.05). In conclusion, upregulation of CCR5 mRNA in BALF of patients with sarcoidosis is consistent with its chemokine ligands RANTES and MIP-1alpha playing a pivotal role in inflammatory cell recruitment to disease sites. Though the data from this pilot study had no clinical correlations we suggest that further studies are warranted on the role of this Th1 subset marker in the pathogenesis of sarcoidosis.

Non-small cell lung cancer - genetic predictors

BACKGROUND Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer that is the leading cause of cancer-related mortality worldwide. Several predictive markers have been found in NSCLC patients to date but only a few are currently used for tailored therapy. METHODS AND RESULTS PubMed and Web of Science online databases were used to search review and original articles on the most important predictive markers in NSCLC. CONCLUSION EGFR activating mutations (exons 18 to 21) and EML4-ALK rearrangement are clinically important markers able to select NSCLC patients which benefit from EGFR or ALK tyrosine kinase inhibitors (gefitinib, erlotinib, crizotinib). Other markers, such as KRAS mutation, EGFR T790M mutation and C-MET amplification, are responsible for resistance to these inhibitors. Overcoming of this resistance as well as discovery of new potential markers and inhibitors is the main goal of ongoing research and clinical trials in NSCLC.

Isocitrate Dehydrogenase 1 and 2 Mutations in Gliomas

Over the past few years, new biomarkers have allowed a deeper insight into gliomagenesis and facilitated the identification of possible targets for glioma therapy. Isocitrate dehydrogenase (IDH) 1 and IDH2 mutations have been shown to be promising biomarkers for monitoring disease prognosis and predicting the response to treatment. This review summarizes recent findings in this field. Web of Science, Science Direct, and PubMed online databases were used to search for publications investigating the role of IDH in glioma. References were identified by searching for the keywords “IDH1 or IDH2 and glioma and diagnostic or predictive or prognostic” in papers published from January, 2008, to April, 2014. Only papers in English were reviewed. Publications available only as an abstract were not included. IDH1/2 mutations are tightly associated with grade II and III gliomas and secondary glioblastomas, with better prognosis and production of a recently described oncometabolite, 2‐hydroxyglutarate (2HG). Although the contradictory positive effect of IDH mutation on prognosis and negative role of 2HG in tumor transformation remain unresolved, the future direction of personalized treatment strategies targeted to glioma development is likely to focus on IDH1/2 mutations.

Glioblastoma multiforme in patients with history of extracranial cancer: Case series

OBJECTIVES Significant progress in treatment strategies improves the expectations of patients with extracranial cancers. Metastases are the primary consideration in patients with cancer history. In the case of neurologic disorders, the patient should undergo brain MRI. A rationale is presented for surgery, whole-brain or stereotactic radiotherapy, or chemotherapy. Recently, we have encountered misdiagnosed primary malignant brain tumours in patients with oncologic history who had been admitted for surgery for brain metastases. The aim of our study is to evaluate the incidence of concurrent cancers, to assess the relationship between previous cancer staging and primary brain tumour evaluation as well as to determine treatment efficiency. METHODS From January 2007 to December 2011, we prospectively followed up patients with concurrent history of both extracranial cancer and subsequent glioblastoma multiforme. Information was collected on the clinical condition, imaging, history of extracranial cancer, previous and present surgical and oncologic procedures, and GBM histologic, cytogenetic, and molecular genetic investigations. RESULTS Five patients were recruited: three females and two males. The average patient age at the time of GBM diagnosis was 65.6 years. Three patients had a history of breast carcinoma, one of renal carcinoma and one of colorectal carcinoma. Following the diagnosis of carcinoma, three patients received chemotherapy and radiotherapy, one patient had radiotherapy alone, and one had no adjuvant therapy. In all the cases, surgery revealed primary GBM, with a standard occurrence of genetic abnormalities (Table 1). The average time from the diagnosis of extracranial cancer to that of GBM was 4 years. Four patients underwent chemoradiotherapy and one had palliative radiotherapy. Two patients completed oncotherapy and their OS was 27 months and 19 months, respectively. One patient had post-surgical progression of hemiparesis. One patient had pulmonary embolism during oncotherapy and one had paraplegia caused by a pathological fracture of vertebras T5 due to breast carcinoma metastases. The OS was 11.8 months (range 3-27 months). All the patients succumbed to GBM progression.

Significant phenotype variability of congenital central hypoventilation syndrome in a family with polyalanine expansion mutation of the PHOX2B gene

BACKGROUND Congenital central hypoventilation syndrome (CCHS) is a rare genetic disorder resulting from mutations in the PHOX2B gene located on chromosome 4p12.3, characterized by hypoventilation secondary to missing responses to both hypercapnia and hypoxia. CASE REPORT Proband. A girl, hospitalised 5 times for respiratory failure from 6 weeks old, presented at 4 years of age severe cyanosis related to pneumonia. Tracheostomy was done, and she was discharged home using a portable positive pressure ventilator during sleep. Probands father: The father was retrospectively found out to suffer from severe headache and excessive daytime sleepiness. Molecular genetic evaluation of PHOX2B gene was performed and casual polyalanine repeat expansion mutation c.741_755dup15 in exon 3 was found both in proband and her father in heterozygous form. The probands grandmother died of respiratory failure after administration of benzodiazepine at the age of fifty years. Considering the grandmothers history, she is highly suspected of having had CCHS as well. CONCLUSION Repeated respiratory failure of girl was explained by PHOX2B mutation and Ondina curse. Proband´s father has incompletely penetrated PHOX2B heterozygous mutation as well and proband´s grandmother died probably from the consequences of drug interaction with PHOX2B mutated background as well. Both daughter and father currently require overnight mechanical ventilatory support. Although most PHOX2B mutations occur de novo, our case is a rare three generation family affected by autosomal dominant inheritance with incomplete penetrance manifested as the late-form of CCHS and proven PHOX2B mutation in two generations.

Whole genome amplification induced bias in the detection of KRAS-mutated cell populations during colorectal carcinoma tissue testing

Whole genome amplification replicates the entire DNA content of a sample and can thus help to circumvent material limitations when insufficient DNA is available for planned genetic analyses. However, there are conflicting data in the literature whether whole genome amplification introduces bias or reflects precisely the spectrum of starting DNA.

Isocitrate Dehydrogenase Mutations are Better Prognostic Marker than O6-methylguanine-DNA Methyltransferase Promoter Methylation in Glioblastomas – a Retrospective, Single-centre Molecular Genetics Study of Gliomas

BACKGROUND Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are a promising prognostic biomarker of gliomas. The purpose of our study was to examine the clinical prognostic properties of IDH1/2 mutations in a glioma patient cohort from the Czech Republic using an improved platform for simple and reliable IDH genotyping. MATERIAL AND METHODS We retrospectively analyzed a group of 145 glioma patients by testing for the three most frequent IDH mutations, IDH1 R132H, IDH1 R132C, and IDH2 R172K, through the competitive amplification of differentially melting amplicons (CADMA) polymerase chain reaction (PCR). O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation, copy number of EGFR, p53, RB1, MDM2, CDKN2A genes, and deletions in 1p, 19q and 10p chromosomal regions were also analyzed and correlated with clinical characteristics. RESULTS Of 145 gliomas, 36 harbored IDH1 R132H mutation and 1 IDH1 R132C mutation. We did not detect any IDH2 R172K mutation. IDH1 mutations were positively associated with MGMT methylation (OR 3.08, 95% CI 1.387-7.282; p = 0.007), 1p/19q co-loss (OR 8.85, 95% CI 2.367-42.786; p = 0.002) and negatively associated with epidermal growth factor receptor amplification (OR 0.12, 95% CI 0.019-0.437; p = 0.006) and 10p loss (OR 0.09, 95% CI 0.005-0.436; p = 0.019). The overall survival of IDH-mutant was 25 months, but only 9 months in IDH-wild type gliomas (p = 0.035); at the same time, survival associated with methylated vs. unmethylated MGMT promoter did not significantly differ (p = 0.166). CONCLUSION Despite IDH1 mutations being closely associated with MGMT methylation in glioma patients, IDH1 mutations in glioblastoma patients are stronger marker of overall survival than MGMT methylation and should be the marker of choice, especially when using genotyping by CADMA PCR.Key words: isocitrate dehydrogenase - polymerase chain reaction - glioma - glioblastoma.