Drug-Resistant Tuberculosis on the Balkan Peninsula: Determination of Drug Resistance Mechanisms with Xpert MTB/XDR and Whole-Genome Sequencing Analysis.

The new molecular assay Xpert MTB/XDR (Cepheid, Sunnyvale, CA, USA) was launched in 2021 to detect Mycobacterium tuberculosis (MT) complex with mutations conferring resistance to isoniazid (INH), ethionamide (ETH), fluoroquinolone (FQ), and second-line injectable drugs (SLIDs). The aim of our study was to evaluate the performance of the Xpert MTB/XDR rapid molecular assay on rifampicin-resistant, multidrug-resistant, and pre-extensively resistant tuberculosis (TB) isolates in a clinical laboratory in the Balkan Peninsula compared to a phenotypic drug susceptibility test (pDST). Xpert MTB/XDR was used to test positive Bactec MGIT 960 (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) cultures or DNA isolates. In the case of discrepant results between Xpert MTB/XDR and pDST, the usefulness of whole-genome sequencing (WGS) was emphasized. In our study, 80 MT isolates from different Balkan countries were selectively chosen from the National Mycobacterial Strain Collection in Golnik, Slovenia. Isolates were tested with the Xpert MTB/XDR assay, conventional pDST, and WGS. Xpert MTB/XDR showed high sensitivities of 91.9%, 100%, and 100% for detecting INH, FQ, and SLID resistance, respectively, compared to pDST. In contrast, low sensitivity (51.9%) for ETH resistance was achieved because isolates harbored widespread mutations across the ethA gene. The specificity of Xpert MTB/XDR was 100% for all drugs except for INH (66.7%). Further investigation with WGS revealed -57c→t mutations in the oxyR-ahpC region marked with uncertain significance, which caused the low specificity for detecting INH resistance with the new assay. Xpert MTB/XDR can be used in clinical laboratories for the rapid detection of INH, FQ, and SLID resistance. Moreover, it can be used to rule in resistance to ETH. Additional use of WGS is recommended in cases of discrepant results between pDST and Xpert MTB/XDR. Future improvements of Xpert MTB/XDR with the inclusion of additional genes may increase the usefulness of the assay. IMPORTANCE 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. According to the results of our study with Xpert MTB/XDR, sensitivities for the detection of SLID, FQ, and INH resistance were sufficient (>90%) for the assay to be implemented into diagnostic algorithms. In our study, WGS revealed lesser-known mutations in genes conferring INH and ETH resistance, and their impact on resistance is still unknown. Mutations in the ethA gene causing resistance to ETH were scattered along structural gene without high-confidence markers for resistance. Therefore, resistance to ETH should be reported based on a combination of methods. Because the Xpert MTB/XDR assay was found to have good performance, we propose that it should be the method of choice for confirming resistance to INH, FQ, and SLID and conditionally for resistance to ETH.

Next-generation sequencing to characterise pyrazinamide resistance in Mycobacterium tuberculosis isolates from two Balkan countries.

OBJECTIVES: Next-generation sequencing (NGS) can provide a comprehensive analysis of the genetic alterations most commonly linked with pyrazinamide (PZA) resistance. However, there are no studies reporting the molecular background of PZA resistance in Mycobacterium tuberculosis (TB) isolates from the Balkan Peninsula. We aimed to examine the feasibility of full-length analysis of a gene linked with PZA resistance (pncA) using Ion Torrent technology compared with phenotypic BACTEC MGIT 960 drug susceptibility testing (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 an AmpliSeq™ Kit for Chef DL8 and Ion AmpliSeq TB Research Panel. RESULTS: Of the 61 TB isolates, 56 were phenotypically resistant to any antibiotic. Among them, 38/56 isolates (67.9%) were phenotypically resistant to pyrazinamide, and pncA mutations were detected in 34/38 cases (89.5%). A mutation in the pncA promoter region was the most prevalent genetic alteration, detected in eight TB isolates. Comparison of NGS data with conventional BACTEC MGIT 960 DST revealed very strong agreement (91.8%) between the two methods in identifying PZA resistance, with high sensitivity (89.5%) and specificity (95.7%) for NGS. CONCLUSION: Detection of PZA resistance using NGS appears to be a valuable tool for surveillance of TB drug resistance in the Balkan Peninsula, with great potential to provide useful information at least 1 weak earlier than is possible with phenotypic DST.

Nationwide analysis of Mycobacterium chimaera and Mycobacterium intracellulare isolates: Frequency, clinical importance, and molecular and phenotypic resistance profiles.

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.

High BMI1 mRNA expression in peripheral whole blood is associated with favorable prognosis in advanced non-small cell lung cancer patients.

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<0.001). Similarly, we observed decreased BMI1 mRNA expression in primary tumors compared to normal lungs from operable NSCLC patients (P=0.001). We found high BMI1 mRNA expression in blood was associated with longer progression-free survival (PFS) (P=0.049) and overall survival (OS) (P=0.012) in advanced NSCLC patients treated with first-line platinum-based chemotherapy. However, no association between the BMI1 mRNA level and response to chemotherapy was found (P=0.21). Multivariate Cox proportional hazards regression analysis showed elevated BMI1 mRNA level in whole blood was an independent prognostic factor for longer PFS (P=0.012) and OS (P<0.001). In conclusion, BMI1 mRNA expression in whole blood might represent a new biomarker for the diagnosis and prognosis of NSCLC.

The prognostic value of whole blood SOX2, NANOG and OCT4 mRNA expression in advanced small-cell lung cancer.

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.

BMI1, ALDH1A1, and CD133 Transcripts Connect Epithelial-Mesenchymal Transition to Cancer Stem Cells in Lung Carcinoma.

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 between BMI1 and ALDH1A1 and between BMI1 and CD133 mRNA expressions were identified. No correlations between TWIST1 and CSC markers were observed. BMI1 mRNA expression in tumors positively correlated with BMI1 mRNA 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.

Prognostic value of cytokeratin-7 mRNA expression in peripheral whole blood of advanced lung adenocarcinoma patients.

BACKGROUND: The rarity of circulating tumour cells (CTCs) in peripheral blood requires the application of sensitive techniques for their detection. The aim of our study was to (i) first determine the sensitivity of cytokeratin-7 (KRT7) mRNA expression levels for the molecular detection of CTCs using spiked-in lung adenocarcinoma (AC)-derived A549 cells and (ii) evaluate the impact of KRT7 mRNA expression in peripheral whole blood on the response to treatment and prognosis of patients with advanced lung AC who were treated with first-line platinum-based chemotherapy. METHODS: A549 cells were micro-manipulated before being spiked into whole blood samples obtained from healthy donors. Additionally, whole blood samples from 65 lung AC patients were collected in PAXgene blood tubes before the start of first-line platinum-based chemotherapy. KRT7 mRNA expression was measured using RT-qPCR. RESULTS: Through the spike-in experiment we found that it is feasible to detect a single A549 tumour cell in 2.5 ml whole blood and that the KRT7 mRNA levels were linearly correlated with the number of spiked-in tumour cells with a high reproducibility. In lung AC patients, no significant differences in response rate to chemotherapy, progression-free survival or overall survival and KRT7 mRNA levels were found. CONCLUSIONS: Our data show that KRT7 mRNA expression measured by RT-qPCR serves as a sensitive approach for the molecular detection of KRT7-positive CTC-resembling A549 cells in peripheral whole blood. The KRT7 mRNA levels measured were not significantly associated with the response to chemotherapy or the survival of patients with advanced lung AC. Additional studies are required to establish the possible clinical significance of KRT7 mRNA expression in whole blood after chemotherapy.

Efficacy of tyrosine kinase inhibitors in routine clinical practice: epidermal growth factor mutations and their implications.

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.

let-7b and miR-126 are down-regulated in tumor tissue and correlate with microvessel density and survival outcomes in non--small--cell lung cancer.

Angiogenesis is a critical event in the development, progression, and spread of various human cancers, including lung cancer. Molecular mechanisms that underlie the complex regulation of angiogenic processes are poorly understood. However, an increasing body of evidence indicates miRNAs as important regulators of tumor angiogenesis. Forceps biopsies were collected from tumor tissue, surrounding tissue, and non-tumor tissue from 50 NSCLC patients. Lung tissue samples from individuals with no clinical evidence of a cancerous disease served as controls. Immunohistochemical staining for Factor VIII was used to evaluate microvessel density (MVD). TaqMan® primer-probe sets were used in quantitative real-time RT-PCR reactions to determine expression levels of let-7b, miR-126, miR-9, and miR-19a. We demonstrated significantly higher MVD and decreased expression levels of let-7b and miR-126 in tumor tissue and surrounding tissue in comparison to corresponding non-tumor tissue or lung tissue from the control group. In addition, no differences in MVD and expression levels of both miRNAs between tumor tissue and surrounding tissue from NSCLC patients were observed. Low expression of both miRNAs correlated with high MVD and worse progression-free survival and overall survival. These observations strongly suggest similar molecular alternations within tumor tissue and surrounding tissue that comprise a specific microenvironment. Low expression of let-7b and miR-126 seems to have a possible anti-angiogenic role in lung tumor tissue and significantly correlates with worse survival outcomes for lung cancer patients. Moreover, the regulation of let-7b and miR-126 expression could have therapeutic potential because it could reduce tumor angiogenesis and therefore suppress tumor growth in lung cancer patients.

Impact of ERCC1 expression on treatment outcome in small-cell lung cancer patients treated with platinum-based chemotherapy.

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.

Posttranslational modification of 6-phosphofructo-1-kinase as an important feature of cancer metabolism.

BACKGROUND: Human cancers consume larger amounts of glucose compared to normal tissues with most being converted and excreted as lactate despite abundant oxygen availability (Warburg effect). The underlying higher rate of glycolysis is therefore at the root of tumor formation and growth. Normal control of glycolytic allosteric enzymes appears impaired in tumors; however, the phenomenon has not been fully resolved. METHODOLOGY/PRINCIPAL FINDINGS: In the present paper, we show evidence that the native 85-kDa 6-phosphofructo-1-kinase (PFK1), a key regulatory enzyme of glycolysis that is normally under the control of feedback inhibition, undergoes posttranslational modification. After proteolytic cleavage of the C-terminal portion of the enzyme, an active, shorter 47-kDa fragment was formed that was insensitive to citrate and ATP inhibition. In tumorigenic cell lines, only the short fragments but not the native 85-kDa PFK1 were detected by immunoblotting. Similar fragments were detected also in a tumor tissue that developed in mice after the subcutaneous infection with tumorigenic B16-F10 cells. Based on limited proteolytic digestion of the rabbit muscle PFK-M, an active citrate inhibition-resistant shorter form was obtained, indicating that a single posttranslational modification step was possible. The exact molecular masses of the active shorter PFK1 fragments were determined by inserting the truncated genes constructed from human muscle PFK1 cDNA into a pfk null E. coli strain. Two E. coli transformants encoding for the modified PFK1s of 45,551 Da and 47,835 Da grew in glucose medium. The insertion of modified truncated human pfkM genes also stimulated glucose consumption and lactate excretion in stable transfectants of non-tumorigenic human HEK cell, suggesting the important role of shorter PFK1 fragments in enhancing glycolytic flux. CONCLUSIONS/SIGNIFICANCE: Posttranslational modification of PFK1 enzyme might be the pivotal factor of deregulated glycolytic flux in tumors that in combination with altered signaling mechanisms essentially supports fast proliferation of cancer cells.

Predictive value of multidrug resistance proteins, topoisomerases II and ERCC1 in small cell lung cancer: a systematic review.

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.