Tomasz Powrózek

Polymorphisms in TS, MTHFR and ERCC1 genes as predictive markers in first‑line platinum and pemetrexed therapy in NSCLC patients

AbstractPurpose We presented retrospective analysis of up to five polymorphisms in TS, MTHFR and ERCC1 genes as molecular predictive markers for homogeneous Caucasian, non-squamous NSCLC patients treated with pemetrexed and platinum front-line chemotherapy.MethodsThe following polymorphisms in DNA isolated from 115 patients were analyzed: various number of 28-bp tandem repeats in 5′-UTR region of TS gene, single nucleotide polymorphism (SNP) within the second tandem repeat of TS gene (G>C); 6-bp deletion in 3′-UTR region of the TS (1494del6); 677C>T SNP in MTHFR; 19007C>T SNP in ERCC1. Molecular examinations’ results were correlated with disease control rate, progression-free survival (PFS) and overall survival.ResultsPolymorphic tandem repeat sequence (2R, 3R) in the enhancer region of TS gene and G>C SNP within the second repeat of 3R allele seem to be important for the effectiveness of platinum and pemetrexed in first-line chemotherapy. The insignificant shortening of PFS in 3R/3R homozygotes as compared to 2R/2R and 2R/3R genotypes were observed, while it was significantly shorter in patients carrying synchronous 3R allele and G nucleotide. The combined analysis of TS VNTR and MTHFR 677C>T SNP revealed shortening of PFS in synchronous carriers of 3R allele in TS and two C alleles in MTHFR. The strongest factors increased the risk of progression were poor PS, weight loss, anemia and synchronous presence of 3R allele and G nucleotide in the second repeat of 3R allele in TS. Moreover, lack of application of second-line chemotherapy, weight loss and poor performance status and above-mentioned genotype of TS gene increased risk of early mortality.ConclusionThe examined polymorphisms should be accounted as molecular predictor factors for pemetrexed- and platinum-based front-line chemotherapy in non-squamous NSCLC patients.

EGFR gene mutations in patients with adenosquamous lung carcinoma.

Adenosquamous (ADSQ) carcinoma accounts for 1–4% of non‐small cell lung cancer (NSCLC). The origin of ADSQ carcinoma and its genetic background is not fully understood. Most studies concerning epidermal growth factor receptor (EGFR) mutation status are performed in adenocarcinoma, while there is limited information about the prevalence of this mutation in ADSQ‐bearing Caucasian patients and the efficacy of EGFR tyrosine kinase inhibitors.

Mechanisms of resistance to reversible inhibitors of EGFR tyrosine kinase in non-small cell lung cancer

Abnormalities of epidermal growth factor receptor (EGFR) in non-small-cell lung cancer (NSCLC) patients consist of EGFR overexpression and EGFR (HER1) gene mutations. Structural dysfunction of the tyrosine kinase domain of EGFR is associated with the clinical response to tyrosine kinase inhibitors (TKI) in patients with NSCLC. The most common EGFR gene mutations occur as either deletions in exon 19 or as substitution L858R in exon 21 and cause a clinically beneficial response to gefinitib or erlotinib treatment. Unfortunately, the majority of patients finally develop resistance to these drugs. Acquired resistance is linked to secondary mutations localised in the EGFR gene, mainly substitution T790M in exon 20. Through intense research a few different mechanisms of resistance to reversible tyrosine kinase inhibitors have been identified: amplification of MET or IGF-1R genes, abnormalities of PTEN and mTOR proteins as well as rare mutations in EGFR and HER2 genes. Extensively investigated new drugs could be of significant efficiency in NSCLC patients with secondary resistance to reversible EGFR TKI.

The diagnostic role of plasma circulating precursors of miRNA-944 and miRNA-3662 for non-small cell lung cancer detection

INTRODUCTION microRNA (miRNA) seem to be most attractive cancer markers due their crucial role in tumor development and possibility of their analysis using liquid biopsy. To date there is little known about role of miRNA precursors (pri-miRNA) in carcinogenesis and their utility as tumor markers. MATERIAL AND METHODS miRNA-944 and miRNA-3662 precursors as potential non-small cell lung cancer (NSCLC) markers were analyzed in plasma samples of 56 patients in an early stage of NSCLC and 100 healthy individuals. RESULTS Diagnostic test based on two studied markers for stage I-IIIA of the disease allowed to distinguish NSCLC from healthy individuals with 75.7% sensitivity and 82.3% specificity (AUC=0.898). pri-miRNA-944 distinguished SCC from AC with sensitivity of 78.6% and specificity of 91.7% (AUC=0.771), and pri-miRNA-3662 distinguished AC from SCC with 57.1% sensitivity and 90% specificity (AUC=0.845). CONCLUSION Circulating pri-miRNA-944 and 3662 can improve non-invasive NSCLC detection of operable stages of SCC and AC. miRNA precursors could be considered as novel potential lung cancer biomarkers.

Impact of I/D polymorphism of ACE gene on risk of development and course of chronic obstructive pulmonary disease

Introduction Chronic obstructive pulmonary disease (COPD) affects more than 10% of the worlds population over 40 years of age. The main exogenous risk factor is cigarette smoking; however, only 20% of smokers develop COPD, indicating that some other factors, e.g. genetic, may play an important role in the disease pathogenesis. Recent research indicates that ACE (angiotensin-converting enzyme) may be a susceptibility gene for asthma or COPD. The aim of our study was to determine the influence of I/D (insertion/deletion) polymorphism of the ACE gene (AluYa5, rs4646994) on the risk and course of COPD. Material and methods We investigated ACE I/D polymorphism in 206 COPD and 165 healthy Caucasian subjects. Results In the generalized linear model (GLZ) analysis of the influence of selected factors on presence of COPD we found a significant independent effect for male sex (repeatedly increases the risk of COPD, OR = 7.7, p = 0.049), as well as smoking or lower body mass index, but only in combination with older age (OR = 0.96, p = 0.003 and OR = 1.005, p = 0.04 respectively). Interestingly, analysis of factors which may influence the risk of a higher number of exacerbations demonstrated that occurrence of DD genotype, but only in men, is associated with a lower risk (OR = 0.7, p = 0.03) of this complication. Conclusions We suggest that ACE may not be a susceptibility gene for the origin of COPD but a disease-modifying gene. Since the impact of I/D polymorphism of the ACE gene on COPD risk is moderate or negligible, other molecular changes, that will help predict the development of this disease, should still be sought.

Prevalence of rare EGFR gene mutations in nonsmall-cell lung cancer: a multicenter study on 3856 Polish Caucasian patients

Department of Pneumonology, Oncology and Allergology, Medical University of Lublin, Poland GENIM Ltd. Institute of Genetics and Immunology, Lublin, Poland Department of Clinical Oncology, Medical University of Poznan, Poland Department of Pathology, Specialist Pulmonary Hospital of Sokolowski, Zakopane, Poland Regional Centre of Oncology M. Kopernik Hospital, Lodz, Poland Mazovian Centre for Treatment of Lung Diseases and Tuberculosis, Otwock, Poland Department of Neurosurgery and Pediatric Neurosurgery, Medical University of Lublin, Poland E.J. Zeyland Wielkopolska Center of Pulmonology and Thoracic Surgery, Poznan, Poland Regional Oncology Centre in Gdansk, Poland Department of Clinical Patomorphology, Medical University of Lublin, Poland

Comparison of the effectiveness of erlotinib, gefitinib, and afatinib for treatment of non‑small cell lung cancer in patients with common and rare EGFR gene mutations

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are routinely used to treat non-small cell lung cancer (NSCLC) in patients with common activating mutations of the EGFR gene. The aim of the study was to compare the efficacies of EGFR-TKIs in patients with common (exon 19 deletions and exon 21 p.Leu858Arg) and rare EGFR mutations. A retrospective analysis of 180 NSCLC patients with common (n=167) and rare (n=13) EGFR mutations treated with erlotinib (n=98), gefitinib (n=66) and afatinib (n=16) was performed. EGFR mutations were determined using RT-PCR and the EntroGen EGFR Mutations Analysis kit. Partial and complete response (PR and CR), progression-free survival (PFS), and overall survival (OS) were analyzed. Demographic and clinical factors had no impact on PFS or OS in patients treated with EGFR-TKIs. Erlotinib, gefitinib, and afatinib showed similar efficacies based on treatment response, median PFS, and OS. The type of EGFR mutation had no impact on median OS; however, median PFS was significantly longer in patients with the exon 19 deletion compared to patients with the exon 21 p.Leu858Arg substitution and rare EGFR gene mutations (P=0.013). Patients with common EGFR mutations showed significantly longer median PFS than those with rare EGFR mutations (10 vs. 5 months; P=0.009). Erlotinib, gefitinib, and afatinib show similar efficacies in NSCLC patients with both common and rare EGFR mutations. When undergoing EGFR-TKI treatment, patients with rare EGFR mutations showed similar OS but poorer PFS. Further investigation into the associations between particular rare EGFR mutations and EGFR-TKIs treatment outcomes is required.

Sensitive methods for the detection of an insertion in exon 20 of the HER2 gene in the metastasis of non‑small cell lung cancer to the central nervous system

The HER2 (ErbB2/neu) protein is a member of the HER (ErbB) receptor family (EGFR, HER2, HER3 and HER4) that expresses tyrosine kinase activity in the intracellular domain. EGFR and HER2 overexpression is observed in numerous types of cancer, nevertheless, the susceptibility of patients with non-small cell lung cancer (NSCLC) to therapy with EGFR and HER2 tyrosine kinase inhibitors (TKIs) depends on mutations present in the respective coding genes (driver mutations). In the present study, PCR and amplified DNA fragment length analysis (FLA) were used along with the multi-temperature single-strand conformation polymorphism (MSSCP) technique in order to identify the 12 base pair insertion in exon 20 of the HER2 gene in 143 patients with NSCLC metastasis to the central nervous system. The prevalence of the HER2 gene mutation was correlated with mutations in the EGFR and BRAF genes. The insertion in exon 20 of the HER2 gene was observed in a single 77-year-old, non-smoking male, with poorly-differentiated adenocarcinoma of the lung (1.5% of adenocarcinoma patients). No other genetic abnormalities were identified in this patient. In the therapy of NSCLC patients with HER2 gene mutations, drugs that inhibit the EGFR and HER2 receptors, for example afatinib, may be effective. The identification of other driving mutations in NSCLC cells appears to be key to the appropriate qualification of molecular targeted therapies.

Relationship between microRNA-146a expression and plasma renalase levels in hemodialyzed patients

Background microRNA (miRNA) belongs to the non-coding RNAs family responsible for the regulation of gene expression. Renalase is a protein composed of 342 amino acids, secreted by the kidneys and possibly plays an important role in the regulation of sympathetic tone and blood pressure. The aim of the present study was to investigate plasma renalase concentration, and explore the relationship between miRNA-146a-5p expression and plasma renalase levels in hemodialyzed patients. Methods The study population comprised 55 subjects who succumbed to various cardiac events, 27 women and 28 men, aged 65–70 years. The total RNA including miRNA fraction was isolated using QiagenmiRNEasy Serum/Plasma kit according to the manufacturer’s protocol. The isolated miRNAs were analyzed using a quantitative polymerase chain reaction (qRT-PCR) technique. The plasma renalase levels were measured using a commercial ELISA kit. Results In the group of patients with high levels of renalase, higher miRNA-146a expression was found, compared with those with low concentration of renalase. Patients with simultaneous low miRNA-146a expression and high level of renalase were confirmed to deliver a significantly longer survival time compared with other patients. Conclusions miRNA-146a and plasma renalase levels were estimated as independent prognostic factors of hemodialyzed patients’ survival time. Patients with low miRNA-146a expression demonstrated a significantly longer survival time in contrast to the patients with a high expression level of miRNA-146a. Moreover, a significantly longer survival time was found in patients with high renalase activity compared with patients with low activity of the enzyme.

Cytology Smears as Diagnostic Material for EGFR Gene Testing in Non-small Cell Lung Cancer:

Aims and Background Cytology smears can be effectively used for EGFR mutation testing in the qualification of NSCLC patients for EGFR tyrosine kinase inhibitor therapy. However, tissue specimens are preferred for EGFR mutation analysis. The aim of this study was to estimate the effectiveness of the real-time PCR method for EGFR testing in histology and cytology materials obtained simultaneously from NSCLC patients. Methods Fourteen adenocarcinoma patients with EGFR–mutation-positive primary tumor tissues were included in the study. Corresponding cytological smears of metastatic lymph nodes obtained by EBUS-TBNA were examined. EGFR Mutation Analysis Kit (EntroGen, USA) and real-time PCR (m2000rt system, Abbott, USA) were used for EGFR mutation analysis in both types of material. Results In primary tumor tissues, 12 deletions in exon 19 and 2 substitutions in exon 21 (L858R mutation) of the EGFR gene were found. Except for 1 deletion in exon 19, the same EGFR gene mutations were detected in all corresponding cytology samples. The percentage of tumor cells, DNA concentration, percentage of mutated DNA as well as ΔCt values were similar in cytology slides and histology material. In both types of materials, no significant correlations were found between the percentage of tumor cells and the percentage of mutated DNA nor between the DNA concentration and the percentage of mutated DNA. Conclusions We demonstrated the high effectiveness of a sensitive real-time PCR method in EGFR gene mutation detection in cytology smears.