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The Xpert MTB/XDR was tested on drug-resistant Mycobacterium tuberculosis complex isolates from the Balkan Peninsula. Positive Bactec MGIT 960 cultures or DNA isolates were tested as starting material.

Objectives Next-generation sequencing (NGS) provide a comprehensive analysis of the genetic alterations that are most commonly linked with pyrazinamide (PZA) resistance. There are no studies reporting molecular background of PZA resistance in TB isolates from Balkan Peninsula. We aimed to examine the feasibility of full-length analysis of a gene linked with PZA resistance, pncA, using Ion Torrent technology in comparison to phenotypic BACTEC MGIT 960 DST in clinical TB isolates from two countries of the Balkan Peninsula. Methods Between 1996 and 2017, we retrospectively selected 61 TB isolates. To identify gene variants related to drug resistance in genomic DNA extracted from TB isolates, AmpliSeq libraries were generated automatically using the AmpliSeq™ Kit for Chef DL8 and the Ion AmpliSeq TB Research Panel. Results Of all 61 TB isolates included, 56 TB were phenotypically resistant to any antibiotic. Among them, 38/56 (67.9%) TB isolates were phenotypically resistant to pyrazinamide and pncA mutations were detected in 33/38 cases (86.8%). A mutation in the pncA promoter region was the most prevalent genetic alteration, detected in eight TB isolates. Comparison of NGS to conventional BACTEC MGIT 960 DST revealed very strong agreement (90.2%) between the two methods in identifying PZA resistance, with high sensitivity (89.5%) and specificity (95.7%) for NGS. Conclusions Detection of PZA resistance using NGS seems to be a valuable tool for surveillance of TB drug resistance also in the Balkan Peninsula, with great potential to provide useful information at least one weak earlier than is possible with phenotypic DST.

// Ana Koren 1 , Matija Rijavec 1 , Eva Sodja 1 , Izidor Kern 1 , Aleksander Sadikov 2 , Viljem Kovac 3 , Peter Korosec 1 , Tanja Cufer 1 1 University Clinic Golnik, Golnik, Slovenia 2 University of Ljubljana, Faculty of Computer and Information Science, Ljubljana, Slovenia 3 Institute of Oncology Ljubljana, Ljubljana, Slovenia Correspondence to: Ana Koren, email: ana.koren@klinika-golnik.si Keywords: non-small cell lung cancer, peripheral whole blood, BMI1 , mRNA expression, prognosis Received: August 26, 2016 Accepted: February 07, 2017 Published: March 06, 2017 ABSTRACT Polycomb group member protein BMI1 is involved in maintaining cell identity, proliferation, differentiation and human oncogenesis. In the present study, we determined BMI1 mRNA expression in whole blood and evaluated the impact of the expression level on the treatment response and survival of 96 advanced NSCLC patients treated with first-line platinum-based chemotherapy. We also determined BMI1 mRNA expression in primary tumors from 22 operable NSCLC patients treated with radical surgery. We found that compared with control subjects, BMI1 mRNA expression in whole blood of advanced NSCLC patients was decreased ( P

The burden of tuberculosis (TB) in Slovenia is low due to good ongoing preventive measures. However, analysis of data obtained from the Registry for Tuberculosis and National Reference Laboratory for Mycobacteria at University Clinic of Respiratory and Allergic Diseases Golnik from January 2000 to December 2017 revealed that the number of nontuberculous mycobacterial (NTM) isolates is increasing. A group of slowly growing NTM and a common cause of NTM disease among humans is the Mycobacterium avium complex, the taxonomy of which is rapidly changing. M. intracellulare and M. chimaera are part of the Mycobacterium avium complex, and together represent 19.6% of all isolated NTM species in Slovenia. Due to the high genotypic and phenotypic similarity between M. intracellulare and M. chimaera species, both species are difficult to differentiate. A method that can be used to successfully distinguish between M. intracellulare and M. chimaera is the molecular assay GenoType NTM-DR. Mutations in the rrl and rrs genes that are associated with macrolide and aminoglycoside resistance, respectively, can also be detected with this method. Overall, 222 clinical isolates were tested, and the nationwide study showed that 44.6% of the previously identified M. intracellulare species were actually M. chimaera. Further, this study showed that none of the tested M. intracellulare and M. chimaera isolates harboured mutations in the rrl and rrs genes. The genotyping result that no isolates were resistant to macrolides or aminoglycosides was also confirmed phenotypically with the broth microdilution method. Among isolates from the Slovenian Mycobacterial Isolates Collection all these tested strains (n = 222) were sensitive to macrolides and aminoglycosides.

Background The data on expression and clinical impact of cancer stem cell markers SOX2, NANOG and OCT4 in lung cancer is still lacking. The aim of our study was to compare SOX2, NANOG and OCT4 mRNA expression levels in whole blood between advanced small-cell lung cancer (SCLC) patients and healthy controls, and to correlate mRNA expression with progression-free survival (PFS) after first-line chemotherapy and overall survival (OS) in advanced SCLC patients. Patients and methods 50 advanced SCLC patients treated with standard chemotherapy and followed at University Clinic Golnik, Slovenia, between 2009 and 2013 were prospectively included. SOX2, NANOG and OCT4 mRNA expression levels were determined using TaqMan qPCR in whole blood collected prior to chemotherapy. Whole blood of 34 matched healthy individuals with no cancerous disease was also tested. Results SOX2 mRNA expression was significantly higher in whole blood of SCLC patients compared to healthy controls (p = 0.006). Significant correlation between SOX2 mRNA expression levels and the number of distant metastatic sites was established (p = 0.027). In survival analysis, patients with high SOX2 expression had shorter OS (p = 0.017) and PFS (p = 0.046). In multivariate Cox analysis, an independent value of high SOX2 expression for shorter OS (p = 0.002), but not PFS was confirmed. No significant differences were observed for NANOG or OCT4 expression levels when comparing SCLC patients and healthy controls neither when analysing survival outcomes in SCLC patients. Conclusions SOX2 mRNA expression in whole blood might be a promising non-invasive marker for molecular screening of SCLC and important prognostic marker in advanced chemotherapy-treated SCLC patients, altogether indicating important role of cancer stem-like cell (CSC) regulators in cancer spread. Further evaluation of SOX2 as a possible screening/prognostic marker and a therapeutic target of SCLC is warranted.

Background: Activating mutations in the epidermal growth factor (EGFR) gene confer sensitivity to the tyrosine kinase inhibitors (TKIs) in patients with advanced non-small cell lung cancer (NSCLC). TKI treatment efficacy and EGFR mutation implications were evaluated in clinically selected advanced NSCLC patients treated with TKIs in routine clinical practice. Materials and Methods: A retrospective chart review for clinicopathological characteristics and mutation status (EGFR, KRAS) analysis of 40 consecutive patients treated with TKIs between 2005 and 2010 was performed. Statistical Analysis Used: PFS and OS were estimated by the Kaplan-Meier method, the log-rank test was used to test for differences. The strength of the associations between the EGFR mutation status and clinicopathological characteristics were tested with the Mann-Whitney U-test or the Kruskal-Wallis H-test. Results: The prevalence of EGFR mutations was 45% with a predominance of deletion mutations in exon 19 (55.5%). Significant correlations between gender, histology, and EGFR mutations were observed. Median progression-free survival (mPFS) for the entire group of patients was 8.7 months and median overall survival (mOS) was not yet reached. Patients with EGFR mutant tumors derived significantly higher benefit from TKI therapy compared to patients with mutation-negative disease; with mPFS of 22.0 vs. 3.2 months (HR: 3.9, 95% CI 1.56-9.89) and with a trend towards better OS (probability of survival at 12 months 82.0 vs. 63.0%, P = 0.080). Conclusion: We demonstrated that screening for EGFR mutations is reliable in a routine clinical setting and might allow for a better selection of NSCLC patients for anti-EGFR TKI therapy.

Epithelial-mesenchymal transition (EMT) is the underlying mechanism of tumor invasion and metastasis. Evidences from lung cancer cellular models show EMT can trigger conversion to a cancer stem cell (CSC) phenotype. In this study, we assessed mRNA expression levels of EMT-inducing transcription factors (BMI1,TWIST1), CSC (CD133,ALDH1A1), and epithelial (EpCAM) markers in primary tumor and whole blood samples obtained from 57 patients with operable non-small-cell lung cancer (NSCLC) as well as in circulating tumor cells (CTCs) of 13 patients with metastatic disease; then possible associations between marker expressions were evaluated. In primary tumors as well as in whole blood, correlations betweenBMI1andALDH1A1and betweenBMI1andCD133mRNA expressions were identified. No correlations betweenTWIST1and CSC markers were observed.BMI1mRNA expression in tumors positively correlated withBMI1mRNA expression in blood. The immunohistochemical analysis confirmed coexpression of BMI1 and CSC markers in tumors. Gene expression profiling in CTCs revealed upregulated expression of EMT/CSC markers in CTCs. Our results suggest CSCs are present in both, tumor tissue and blood of NSCLC patients, whereas Bmi1 may play an important role in initiation and maintenance of CSCs and might be involved in the blood-borne dissemination of NSCLC.

In small-cell lung cancer (SCLC), resistance to cancer drugs presents a major problem, limiting the effectiveness of chemotherapy. A better understanding of the molecular biology is essential to improve currently available cytotoxic therapy. Herein, a systematic review of studies evaluating the predictive value of multidrug resistance-associated proteins (MDR1, MRP1, MRP2 and MVP), topoisomerase II and ERCC1 for chemotherapy outcomes is presented. The role of MDR1, MRP1 and MRP2 as predictive markers in SCLC has not yet been elucidated. The majority of studies reported an association between protein or gene expression and response to chemotherapy; however, the evidence is limited to univariate analyses performed in the frame of small retrospective trials. In addition, the largest trial did not confirm an independent predictive value for response rates or survival. Genetic variability may be overseen as a more promising marker. Available data on the predictive value of topoisomerase II are scarce and in contrast to the general idea that higher protein or gene expression correlate with greater chemo-sensitivity. The data on a possible predictive value of ERCC1 are also quite limited; in two retrospective studies, ERCC1 turned out to be a significant predictive marker for survival, but only for limited disease patients. In conclusion, a continuous research, with standardized and validated methodology of markers' determination, should be aspired at all times; a better understanding of the biology of SCLC is of utmost importance to enable personalized therapy and to improve survival rates in this, so far, poorly controlled disease.

Introduction The excision repair cross-complementing 1 (ERCC1) protein is an extensively investigated molecular marker because it may decrease sensitivity to platinum-based chemotherapy. Low ERCC1 expression has already been correlated with better treatment efficacy in non-small-cell lung cancer patients treated with platinum-based chemotherapy. However, the data on a prognostic and/or predictive value of ERCC1 in small-cell lung cancer (SCLC) are still very limited. Methods This retrospective pilot study evaluated the impact of ERCC1 expression levels on response to first-line platinum-based chemotherapy with or without radiotherapy and survival outcomes of 77 SCLC patients. ERCC1 protein expression was determined immunohistochemically in primary tumour tissue. Results ERCC1 protein expression was positive in 40/77 (51.9%) of our patients. No significant association was found between ERCC1 protein expression and response rate to first-line platinum-based chemotherapy, progression-free survival (PFS), or overall survival (OS), either in the overall population or in patients stratified by disease stage. Conclusions In our limited group of 77 SCLC patients, ERCC1 protein expression was not found to correlate with either response rate to platinum-based chemotherapy or survival outcomes. Multi-centric prospective trials using a validated method of ERCC1 determination are mandatory in order to obtain a definitive answer on the predictive value of ERCC1 in SCLC.