IntroductionExpression of PD-L1 on cancer cells is the only validated predictive factor for immunotherapy in NSCLC (Non-Small Cell Lung Cancer) patients. However, on this basis, it is difficult to predict the occurrence of resistance to immune checkpoint inhibitors (ICIs). MicroRNAs are widely studied as biomarkers of cancers. Our study was designed to determine whether microRNAs can be sensitive predictive factors in the qualification of NSCLC patients to first-line immunotherapy or chemoimmunotherapy.Material and methodsThe two-stage research on validation group (n=20) and study group (n=35) of patients with advanced NSCLC was conducted. Analysis of microRNAs expression by qPCR in plasma collected prior to the start of immunotherapy (pembrolizumab) or chemoimmunotherapy (combination of pembrolizumab with chemotherapy) was made. Broad-spectrum analysis of microRNAs expression was used in the studied group. Three microRNAs selected in that group as important for the effectiveness of ICIs were then examined in the validation group.ResultsIn the studied group, significantly higher expression of miRNA-126-3p, miR-144-3p and miR-146-5p was observed in patients with long PFS compared to those with short PFS. In the validation group, low miRNA-126 expression indicated lower median progression-free survival and overall survival (2.3 vs. 5.0 months and 5.2 vs 11.2, respectively). These patients had a significantly higher risk of progression (HR= 2.92, 95% CI: 1.01 to 8.40, p=0.04) and death (HR=3.64, 95% CI: 1.22 to 10.84, p=0.02).ConclusionOur study showed that the expression of miR-126 in blood plasma may be a predictive factor for the effectiveness of first-line immunotherapy or chemoimmunotherapy in advanced NSCLC patients.

A flail chest is one of the possible medical conditions suffered by individuals who were injured in traffic accidents, caused by multiple fractures of the ribs and sternum. Which often results in paradoxical chest movements. The consequence may be respiratory failure and need for long-term mechanical ventilation. Such treatment require Intensive Care Unit and may be associated with the possibility of numerous complications.Modified Nuss procedure was performed in 79-year-old man, a victim of a car crash to obtain stabilization of the flail chest. After compensation of paradoxical movements on the third day it was possible to end mechanical ventilation. A quick procedure dedicated to the congenital deformation of the chest made it possible to avoid long, expensive intensive therapy with possible respiratory complications.The NUSS procedure enables the effective and safe treatment of a flail chest in a selected group of patients.

Background: Lung cancer is the leading cause of cancer-related deaths. Early diagnosis may improve the prognosis. Methods: Using quantitative methylation-specific real-time PCR (qMSP-PCR), we assessed the methylation status of two genes (in two subsequent regions according to locations in their promoter sequences) related to carcinogenesis, DICER and DROSHA, in 101 plasma samples (obtained prior to the treatment) of lung cancer patients and 45 healthy volunteers. Results: The relative level of methylation of DROSHA was significantly lower (p = 0.012 for first and p < 0.00001 for the second region) and DICER significantly higher (p = 0.029 for the first region) in cancer patients. The relative level of methylation of DROSHA was significantly (p = 0.037) higher in patients with early-stage NSCLC (IA-IIIA) and could discriminate them from healthy people with a sensitivity of 71% and specificity of 76% (AUC = 0.696, 95% CI: 0.545–0.847, p = 0.011) for the first region and with a sensitivity of 60% and specificity of 85% (AUC = 0.795, 95% CI: 0.689–0.901, p < 0.0001) for the second region. Methylation analysis of the first region of the DICER enabled the distinction of NSCLC patients from healthy individuals with a sensitivity of 96% and specificity of 60% (AUC = 0.651, 95% CI: 0.517–0.785, p = 0.027). The limitations of the study include its small sample size, preliminary nature, being an observational type of study, and the lack of functional experiments allowing for the explanation of the biologic backgrounds of the observed associations. Conclusion: The obtained results indicate that the assessment of DICER and DROSHA methylation status can potentially be used as a biomarker for the early detection of lung cancer.

IntroductionExpression of PD-L1 protein on tumor cells, which is so far the only validated predictive factor for immunotherapy, is regulated by epigenetic and genetic factors. Among the most important ones that regulate gene expression are microRNAs.Materials and MethodsThe study included 60 patients with NSCLC who underwent first or second line immunotherapy with pembrolizumab or nivolumab. FFPE materials were collected before the start of immunotherapy. We examined relative expression of microRNAs (miR-141, miR-200a, miR-200b, miR-200c, miR-429, miR-508-3p, miR-1184, miR-1255a) and PD-L1 mRNA expression. Copy number variation (CNV) of PD-L1 gene by qPCR and FISH methods were assessed. Two single nucleotide polymorphisms (SNPs) in promoter region of PD-L1 gene (rs822335 and rs822336) were examined. Expression of PD-L1 protein on tumor cells was assessed by immunohistochemistry (IHC). The response rate to immunotherapy and progression free survival (PFS) measured in weeks and overall survival (OS) measured in months from the start of immunotherapy were evaluated.ResultsResponse to immunotherapy was observed in nine patients (15%, including one complete response), disease stabilization in 22 patients (36.7%), and progression in 29 patients (48.3%). Significantly higher (p=0.015) expression of miR-200b and significantly lower (p=0.043) expression of miR-429 were observed in responders compared to patients who did not respond to immunotherapy. The median PFS in the whole group of patients was 16 weeks, and the median OS was 10.5 month. In univariate analysis, the median PFS was significantly higher in patients with high miR-200b expression (HR=0.4253, 95%CI: 0.1737–1.0417, p=0.05) and high miR-508 expression (HR=0.4401, 95%CI: 0.1903–1.0178, p=0.05) and with low expression of miR-429 (HR=0.1288, 95%CI: 0.01727–0.9606, p=0.0456) compared to patients with low and high expression of these molecules, respectively. The median OS was higher in patients with low expression of miR-429 (HR=0,6288, 95%CI: 0,3053–1,2949, p=0.06) compared with patients with high expression of this microRNA. In multivariate analysis, we found that patients with PD-L1 expression on ≥1% of tumor cells compared to patients without PD-L1 expression on cancer cells had a significantly lower risk of progression (HR=0.3857, 95%CI: 0.1612–0.9226, p=0.0323) and death (HR=0.377, 95%CI: 0.1636–0.8688, p=0.022).ConclusionThe miR-200b and miR-429 molecules in tumor cells seem to have greatest impact on the effectiveness of immunotherapy in NSCLC patients.

Background: Lung resection changes intra-thoracic anatomy, which may affect electrocardiographic results. While postoperative cardiac arrhythmias have been recognized after lung resection, no study has documented changes in vectorcardiographic variables in patients undergoing this surgery. The purpose of this study was to analyse changes in spatial QRS-T angle (spQRS-T) and corrected QT interval (QTc) after lung resection. Methods: Adult patients undergoing elective lung resection under general anaesthesia were studied. The patients were allocated into four groups: those undergoing (1) left lobectomy (LL); (2) left pneumonectomy (LP); (3) right lobectomy (RL); and (4) right pneumonectomy (RP). The spQRS-T angle and QTc interval were measured one day before surgery (baseline) and 24, 48 and 72 h after surgery. Results: Seventy-one adult patients (47 men and 24 women) aged 47–80 (65 ± 7) years were studied. In the study group as a whole, lung resection was associated with significant increases in spQRS-T (p < 0.001) and QTc (p < 0.05 at 24 and 48 h and p < 0.01 at 72 h). The greatest changes were noted in patients undergoing LP. Postoperative atrial fibrillation (AF) was noted in 6.4% of patients studied, in whom the widest spQRS-T angle and the most prolonged QTc intervals were also noted. Conclusions: Lung resection widens the spQRS-T angle and prolongs the QTc interval, especially in patients undergoing LP. While postoperative AF was a relatively rare complication after lung resection in this study, it was associated with the widest spQRS-T angles and most prolonged QTc intervals.

INTRODUCTION:Air pollution is one of the most important issues of our times. Air quality assessment is based on the measurement of the concentration of substances formed during the combustion process and micro-particles suspended in the air in the form of an aerosol. Microscopic atmospheric particulate matters (PM) 2.5 and 10 are mixtures of organic and inorganic pollutants smaller than 2.5 and 10 µm, respectively. They are the main cause of negative phenomena in the earth's atmosphere of Earth and human health, especially on the respiratory and cardiovascular systems. Particulates have the ability to cause permanent mutations of tissue, leading to neoplasms and even premature deaths. Nitrogen dioxide (NO2) is one of the main pollutants which arises mainly during the burning of fossil fuels. Based on numerous scientific researches, it has been proved that long-term exposure to NO2 could increase morbidity of cancer due to inflammatory processes increasing abnormal mutations.MATERIAL AND METHODS:Data available in the Polish National Cancer Registry, Chief Inspectorate for Environmental Protection and Map of Health Needs in the Field of Oncology for Poland, WHO Air Quality Guidelines 2005 were analyzed. Air pollution was also evaluated: PM2.5, PM10, NO2, and compared with lung cancer morbidity.RESULTS AND CONCLUSIONS:Based on the available data and literature, it can be concluded that in 2009-2017, on average, each Pole smoked ten cigarettes a day +/- 2. Therefore, it can be estimated that after 60 years everyone had 30 package-years of smoking, leading to a high risk of lung cancer and other smoking related diseases. Additionally air quality in Poland is not satisfactory, exceeding the standards presented in the WHO Guidelines 2005. It can be assumed that this may translate into an additional, independent continuous increase in morbidity and mortality dependent on smoking.

OBJECTIVE: Lung cancer (LC) is diagnosed mostly in advanced, non-operable stage, with poor prognosis. The analysis of microRNAs may be a useful tool for early and non-invasive detection of cancer. Dicer and Drosha are enzymes with an essential role for microRNA biogenesis. The aim of our study was to analyze the expression of miRNA-27a-3p, miRNA-31. miRNA-182, miRNA-195 with the ability to reciprocal regulation of Dicer and Drosha expression in lung cancer patients. PATIENTS AND METHODS: The relative expression of microRNAs was detected by qP-CR in plasma of 160 LC patients. The U-Mann Whitney test was used to compare the relative expression between particular groups of lung cancer patients and healthy individuals. The diagnostic value of microRNAs examination was analyzed using a receiver operating curve. RESULTS: We demonstrated that the plasma levels of miRNA-27, miRNA-31 and miRNA-182 were significantly higher and miRNA-195 significantly lower in the whole group of LC patients and in patients with early stages of NSCLC, in comparison with healthy donors. ROC analysis showed that four studied microRNAs have a potential diagnostic value far early stages of NSCLC with AUC-0.95 for miRNA-27a (94% sensitivity and 81% specificity, p=0.0001). 0.71 for miRNA-31 (73% sensitivity and 61% specificity. p=0.001) 0.77 for miRNA-182 (70% sensitivity and 79% specificity. p=0.0001) and 0.82 for miRNA-195 (74% sensitivity and 80% specificity, p=0.0001). CONCLUSIONS: We have proved that the expression of miRNA-27a-3p, miRNA-31, miRNA-182, and miRNA-195 in patients with LC is different from the expression of these molecules in healthy people. The examination of these microRNAs in plasma could be used in non-invasive lung cancer diagnosis.

Abstract Background Topoisomerase 2‐alpha (TOP2A) is an enzyme that controls topologic changes in DNA during transcription and replication. ERCC1 is an enzyme that takes part in DNA repair processes. The purpose of this study was to assess the predictive role of particular single nucleotide polymorphisms (SNPs) in the promoter regions of TOP2A and ERCC1 genes in non‐small cell lung cancer patients (NSCLC) treated with chemotherapy. Materials and methods We enrolled 113 NSCLC patients treated in the first line with platinum‐based chemotherapy. Effectiveness was available for 71 patients. DNA was isolated from whole blood using the Qiamp DNA Blood Mini kit (Qiagen). We examined five SNPs: rs11615 (ERCC1), rs3212986 (ERCC1), rs13695 (TOP2A), rs34300454 (TOP2A), rs11540720 (TOP2A). Quantitative PCR using the TaqMan probe (ThermoFisher) was performed on a Eco Illumina Real‐Time PCR system device (Illumina Inc). Results Patients with the A/A genotype in rs11615 of the ERCC1 gene had significantly longer median progression free survival (PFS) (8.5 months; P = .0088). Patients with the C/C genotype in rs3212986 of the ERCC1 gene had longer median PFS (7 months; P = .05). Patients with the C/C genotype in rs34300454 of TOP2A gene had significantly higher median PFS (7.5 months; P = .0029). Carriers of the C/C genotype in rs34300454 of the TOP2A gene had significantly longer median OS (15.5 months; P = .0017). Patients with the A/A genotype in rs11615 of the ERCC1 gene had significantly higher risk of neutropenia (P = .0133). Conclusions Polymorphisms of the TOP2A and ERCC1 genes may be a predictive factor of toxicities and survival for chemotherapy in NSCLC patients.

The qualification of patients with non-small cell lung cancer (NSCLC) for anti-programmed cell death 1 (PD-1) or anti-programmed death ligand 1 (PD-L1) antibody therapy is based on an immunohistochemistry (IHC) assessment of PD-L1 expression. Immunological checkpoint inhibitors improve the overall survival of patients with expression of PD-L1; however certain PD-L1-negative patients may also benefit from immunotherapy. This indicates the requirement for novel predictive factors for the qualification of immunotherapy. It is also necessary to understand the mechanisms that effect the expression of PD-L1 in tumor cells. The expression of PD-L1 in 47 formalin-fixed, paraffin-embedded, NSCLC specimens was assessed using IHC and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression of 8 microRNAs (miRNAs, miRs) complementary to PD-L1-mRNA was also evaluated using RT-qPCR. A positive correlation was revealed between the expression level of PD-L1-mRNA and 2 miRs, miR-141 (R=0.533; P=0.0029) and miR-1184 (R=0.463; P=0.049). There was also a positive correlation between the percentage of PD-L1-positive tumor cells and the expression levels of miR-141 (R=0.441; P=0.0024), miR-200b (R=0.372; P=0.011) and miR-429 (R=0.430; P=0.0028), and between the percentage of the tumor area with immune cell infiltration and the expression levels of miR-141 (R=0.333; P=0.03) and miR-200b (R=0.312; P=0.046). Additionally, the percentage of tumor cells expressing PD-L1 positively correlated with miR-141 expression (R=0.407; P=0.0055). Correlations between the expression of the investigated miRs (particularly miR-141) and PD-L1 indicated that miRs may regulate PD-L1 expression at a post-transcriptional level.

Most drugs targeting PD-1 or PD-L1 are more effective when cancer cells of non-small cell lung cancer (NSCLC) patients express PD-L1 protein. The polymorphisms of PD-L1 gene and PD-L1 gene copy number could be responsible for PD-L1 mRNA and protein expression. We analyzed PD-L1 protein expression using two IHC assays, mRNA (PD-L1) expression by qRT-PCR, PD-L1 gene promoter region polymorphisms (rs822335 and rs822336) by qPCR and PD-L1 gene copy number by fluorescence in situ hybridization method. Patients with CC genotype in rs822335 had significantly (p = 0.043) higher percentage of tumor cells with PD-L1 expression (test with 22C3 antibody) than patients with CT or TT genotypes. PD-L1 gene copy number significantly positively correlated with percentage of tumor cells with PD-L1 expression detected in tests with 22C3 antibody (p = 0.005, R = +0.442) and with SP142 antibody (p = 0.021, R = +0.369). PD-L1 gene copy number did not correlate with PD-L1 mRNA expression. Patients with PD-L1 expression tested with 22C3 antibody had significantly higher expression of PD-L1 mRNA (p = 0.023), number of chromsosme 9 centromeres (p = 0.023) and PD-L1 gene copy number (p = 0.003) than patients without PD-L1 expression on tumor cells PD-L1 gene polymorphisms and PD-L1 gene copy number may be a predictor for PD-L1 protein expression on tumor cells.

Lung cancer (LC) is the leading cause of cancer mortality worldwide. The majority of LC patients will develop distant metastases at some point during their disease, and brain is among the most common sites of relapse. However, little is known about the mutational landscape of brain metastases (BM) and their potential inter-tumor heterogeneity. Better knowledge on this subject may pave the way to new therapeutic strategies. Here, we map the DNA copy number alterations (CNAs) by sequencing a cohort of primary LC samples and matched BM. The study group included 57 patients (21 females and 36 males, median age 61±8 years; 35 adenocarcinomas, 18 squamous-cell carcinomas, 2 large-cell carcinomas, 1 adenosquamous carcinoma and 1 small-cell lung cancer). From all patients pair-matched tissue samples from primary tumor and corresponding BM were collected, fixed in formalin and embedded in paraffin. All patients were therapy-naïve at the time of primary tumor collection. Genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Germany), followed by NGS library preparation using the NEBNext Ultra II DNA kit (NEB, USA). Samples were sequenced shallowly (average depth 26 Mreads) on the NextSeq 500 system (Illumina, USA). The R package QDNAseq was used to call and visualize DNA copy number levels. The P value of <0.05 (Wilcoxon paired test) was considered statistically significant. The median time between primary LC diagnosis and BM occurrence was 13 months range, 0 to 91 months), and synchronous BM were diagnosed in 12% of patients. Overall survival in the entire group was 22.5 months. The number of CNA was significantly higher in BM than in primary tumor, regardless of clinical/demographic data or type of aneuploidy (gains/losses). Primary tumors harbored significantly more gains and almost no losses. In both tumor sites, the most frequent gains affected 1q, 5p, 7p, 8q and 20q, whereas gains of 17q and 19q, and losses of 4p, 4q, 5q, 8p, 9p, 16q, 17p, 18q, 22q were identified only in BM. The fraction of the genome affected by mutational events in BM correlated positively with time to BM development. Three the top altered genes (IL7R, MLT11, SETDB1) were identical in both primary lesions and BM. Our results indicate that while primary LC lesions harbor frequent amplifications, the CNA landscape of BM is dominated by deletion events. Higher number of CNA harbored by late compared to synchronous BM suggests high levels of genomic instability.

TOP2A is an enzyme that control topologic changes in DNA during transcription and replication. ERCC1 is an enzyme takes part in DNA repair processes. Purpose of our studies was to assess predictive role of particular single nucleotide polymorphisms (SNPs) in promoter regions of TOP2A and ERCC1 genes in non-small cell lung cancer patients (NSCLC) treated with chemotherapy. We enrolled 116 NSCLC patients qualified to first line chemotherapy. Information on the chemotherapy regimens was available in 106 patients. All chemotherapy regimens were based on platinum compounds. 66 (62%) patients received additionally inhibitors of cell divisions (vinorelbine, taxanes). 40 (38%) patients were treated with nucleoside analogs or antimetabolites (gemcytabine, pemetrexed). DNA was isolated from whole blood with Qiamp DNA Blood Mini kit (Qiagen, Germany) according to the manufacture’s instruction. We examined five SNPs: rs11615 (ERCC1), rs3212986 (CD3EAP), rs13695 (TOP2A), rs34300454 (TOP2A), rs11540720 (TOP2A). Quantitative PCR using TaqMan probe (ThermoFisher, USA) was performed on Eco Illumina Real-Time PCR system device (Illumina Inc., USA). Statistical analysis were performed with MedCalc and Statistica 13.1 softwares. In whole group of patients, median of progression free survival (PFS) was 3 months. Patients with CC genotype in rs34300454 had significantly higher median PFS (8 months) compared to patients with CT genotype (4 months, p=0.0026; HR=0.36 with 95% CI: 0,19 to 0,7). We did not detect patients with TT genotype of this SNP. However, the differences in median PFS between patients with different genotypes of the TOP2A gene were significant only in the group receiving inhibitors of cell divisions (p=0.011). In second group of patients, we did not observed such significant relationship. Control of disease (response to chemotherapy or stable disease) were observed insignificantly more often in patients with AA genotype in rs11615 of ERCC1 gene than in patients with AG genotype of this SNP (X2=3.453, p=0.063). Polymorphism of TOP2A gene could influence PFS in NSCLC patients treated witch chemotherapy and CC genotype of this polymorphism may be a good predictive factor for chemotherapy regimens containing cell division inhibitors.

Drosha and Dicer are the enzymes necessary during the miRNA biogenesis. They have the same effect on all microRNAs. Many studies have focused on profiling the expression of selected miRNAs or whole miRNom's analysis in serum or plasma, as potential tool for early detection of diseases. We would like to check, whether the expression of Drosha and Dicer mRNA is detectable in plasma of NSCLC patients, and whether it can differentiate early and advanced stages of this disease. We enrolled 59 (43.1%) NSCLC patients in early (I-IIIA) stages and 78 (56.9%) in locally advanced or advanced (IIIB-IV) stages. We isolated total mRNA and reverse transcription PCR (RT-PCR) was performed. RT-PCR was made using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time PCR (qPCR) was performed for assessment of Drosha and Dicer mRNA expression on Eco Illumina Real-Time PCR system device (Illumina Inc.). We used TaqMan probe Hs00203008_m1 (Applied Biosystems) for Drosha and Hs00229023_ m1 (Applied Biosystems) for Dicer mRNA expression measurement. GAPDH was used a housekeeping gene. Statistical analysis were performed with use of Statistica 13.1 software (Tibco). Drosha mRNA expression was detectable in plasma of 57 (41.6%) patients. 17 (28.8%) patients with Drosha mRNA expression were in early stages and 40 (51.3%) patients were in stages IIIB or IV (χ2=6.98, p=0.008). Expression of Dicer mRNA was detectable in plasma of 71 (57.8%) patients. 15 (25.4%) of patients from this group were in early stages and 56 (71.8%) – in IIIB or IV stages (χ2=28.93, p<0.0001). Significant higher expression of Drosha mRNA was observed in group of patients with lymph node involvement compared with group of patients without lymph node metastases (p=0.001). Moreover, significantly higher expression of Dicer mRNA was observed in group of patients with distant metastases compared with group without metastases (p=0.0002). Furthermore, we found statistically nonsignificant (p=0.07) lower expression of Drosha mRNA in stages IIIB-IV compared with early stages. We did not find any differences between Drosha or Dicer mRNAs expression in patients stratified by age, tumor size or histopathological diagnosis (p<0.1). Plasma expression of Drosha and Dicer mRNAs is detected more often in advanced stages of NSCLC. Probably, different mRNAs from more damaged tumor cells in more advanced disease stages are present in higher expression in blood stream. However, this proves that free mRNAs of Drosha and Dicer are mainly produced by cancer cells in NSCLC patients. indirectly, it can be concluded that cancer cells have disturbed production of microRNAs. There are necessity to use more sensitive tools (i.e. Next Generation Sequencing method) to asses expression of Dicer and Drosha mRNAs in early stages of NSCLC.

Non-small-cell lung cancer (NSCLC) is characterized by aggressive clinical course including frequent occurrence of distant metastases. Central nervous system (CNS) metastases are diagnosed in 20%– 40% of NSCLC patients and they are considered as a pharmacological sanctuary lesions for most cytotoxic agents. Molecularly targeted therapies have shown relatively high activity in CNS metastases in patients harboring “drugable” abnormalities in EGFR or ALK genes. Especially promising activity of the secondgeneration ALK inhibitors (alectinib, ceritinib) and sequential or concurrent application of EGFR/ALK TKIs and WBRT/stereotactic radiotherapy has been postulated (4, 6, 8). To date the knowledge on the effectiveness of molecularly targeted therapies in NSCLC patients with CNS metastases is relatively scarce. The patients with untreated CNS metastases are excluded from recent clinical trials investigating new therapies in lung cancer (4, 8). Therefore, we retrospectively assessed the spectrum of “drugable” abnormalities in 10 genes, in CNS metastases of NSCLC (145 FFPE tissue samples—45 females and 100 males; median age 60 6 8.8 years; PS 5 0 or 1; all patients chemotherapy and TKI na€ıve), and determined the relationship between molecular status and clinical characteristics of our patients. The studied group was heterogeneous in terms of histopathology (80 adenocarcinoma, 29 squamous cell carcinoma, 22 large cell cancer and 14 nototherwise-specified NSCLC patients) and smoking status (73 current-smokers, 21 former smokers, 36 non-smokers). The molecular profile of selected mutations was assessed using different molecular methods. Mutations in EGFR (exons 18–21), KRAS (codons: 12; 13; 61), NRAS (codons: 12; 61), BRAF (codon: 600), PTEN (codon: 233) and AKT1 (codon: 17) genes were analyzed with commercially available kits certified for in vitro diagnostic (Entrogen, Woodland Hills, California, USA) or TaqMan probes for research use only (Applied Biosystem, Carlsbad, California, USA). To analysis mutation in, PIK3CA (codons: 542; 545; 1047), MEK1 (codons: 56; 57; 67), HER2 (exon 20) and DDR2 (codon: 768) genes we used originally designed methods which based on allele-specific PCR (ASP-PCR) and highresolution melting PCR (HRM-PCR). Moreover, direct sequencing and multi-temperature single strand conformation polymorphism (MSSCP) techniques were used to confirm results obtained by originally designed methods. ALK abnormal protein was determined using automated immunohistochemistry with Positive Rabbit Monoclonal Antibody D5F3 (Ventana, Tucson, Arizona, USA) according to manufacturer instructions (9). To confirm ALK gene rearrangement we used FISH technique with Vysis ALK Break Apart FISH Probe Kit (Abbot Molecular, Des Plaines, Illinois, USA). The criteria of FISH analysis were in accordance with FDA guidelines (10). In 30 patients, the material was simultaneously available from primary and metastatic NSCLC tumors. We identified at least one abnormality in 59 cases (41%): KRAS—21.4% (31/145), EGFR—6.2% (9/145), ALK—4.8% (7/145), DDR2—2.1% (3/145), PIK3CA—2.1% (3/145), NRAS— 1.4% (2/145) and HER2, AKT1, PTEN, MEK1 respectively in 0.7% of patients (1/145) (Figure 1A). Coexistence of two mutations (KRAS and DDR2) was found in one patient. Mutations were significantly more frequently observed in adenocarcinoma compared to other histologic types of NSCLC (P 5 0.001; v 5 15.9; Figure 1B,C) and in non-smokers compared to former/current smokers (P 5 0.034; v 5 11.39, Figure 1D,E). The mOS of patients with mutations was insignificantly longer than in patients with wild-type of analyzed genes (16 vs. 11.7 months; P 5 0.084; HR 5 1.36). Cox multivariate logistic regression demonstrated that the factors significantly prolonging patients’ survival were younger age (<60 years) and mutations’ presence (overall model: P 5 0.0459; v 5 6.161). In 30 matched primary NSCLC tumors, 23% had EGFR and 7% had KRAS mutations. Heterogeneity between primary tumor and CNS metastases concerned only KRAS mutations. In five cases KRAS gene mutations were identified in both specimens, in one case only in primary tumor and in one case only in CNS metastases. Most of our observations are in concordance with large epidemiological data from studies focusing on primary tumors of NSCLC. Barlesi et al indicated the presence of driver mutations in six examined genes in 50% out of 17 664 European and Caucasian patients. Prevalence of analyzed mutations was as follows: KRAS mutations in 29% (4894/17 001) of cases, EGFR mutations in 11% (1947/17 706) of cases, ALK rearrangement in 5% (388/8134) of cases, BRAF mutations in 2% (262/13 906) of cases, PIK3CA mutations in 2% (252/ 10 678) of cases and HER2 mutations in 1% of patients (98/11 723). The presence of a genetic alterations was significantly associated with longer duration of response to both firstand second-line of treatment, as well as with longer first-line PFS and with longer mOS. Cox multivariate analysis confirmed that the presence of ALK rearrangements and EGFR and HER2 genes mutations had a favorable effect on prognosis (1). Kris et al reported the driver mutations in 64% (466/733) of American patients with adenocarcinoma. The analysis included ten genes and spectrum of particular disorders was fallowing: KRAS mutations in 25% (182/733) of cases, EGFR activation mutations in 17% (122/733) of cases, ALK rearrangements in 8% (57/733) of cases, HER2 mutations in 3% (19/733) of cases, BRAF mutations in 2% (16/733) of cases; PIK3CA mutations in <1% (6/733) cases; MET amplification in <1% (5/733) of cases; NRAS mutations in <1% (5/733) of cases; MEK1 mutation in <1% (1/733) of cases. The authors have not identified a mutation in AKT1 gene. Moreover, they noted the co-existence of two or more mutations in 3% (24/733) of cases. PIK3CA gene mutations commonly overlapped with others genetic abnormalities. Kris et al observed significantly longer mOS in patients with molecular abnormalities who received targeted therapies

Examination of microRNAs expression in plasma could be useful in screening and in early detection of non-small cell lung cancer (NSCLC). One of the most important enzymes in miRNA biogenesis are Dicer and Drosha. Disrupted expression of miRNA with ability to reciprocal regulation of Dicer and Drosha could participate in cancer development. Plasma expression of miR-27-3p, miR-31, miR-182 and miR-195 were analysed in 138 Polish NSCLC patients (median age 65 years, 83 male and 55 female) and in 45 healthy people (median age 62 years, 28 male and 17 female). 57 (41.3%) NSCLC patient were in I-IIIA stage and 81 (58.7%) patients were in IIIB-IV stage. Relative expression of microRNAs between studied groups was compared using U Mann-Whitney test. For assessment of diagnostic accuracy (test sensitivity and specificity), the receiver operating curves (ROC) with area under curve (AUC) analysis were generated. We demonstrated that plasma levels of miR-27-3p, miR-31 and miR-182 were significantly higher (p<0.000001, p=0.00008, p=0.006 respectively) and miR-195 significantly lower (p=0.000002) in NSCLC patients in comparison with healthy donors. Moreover, patients with early stages (I-IIIA) of NSCLC showed significantly higher expression of miR-27a-3p (p<0.000001), miR-31 (p=0.0003) and miR-182 (p=0.000003) than healthy persons. Expression of miR-195 was significantly lower in patients with early stages (I-IIIA) of NSCLC than in healthy donors (p<0.000001). AUC for miR-27a was 0.95 (94% sensitivity and 81% specificity, p<0.00001), for miR-31 was 0.71 (73% sensitivity and 61% specificity, p=0.001), for miR-182 was 0.77 (70% sensitivity and 79% specificity, p<0.00001) and for miR-195 was 0.82 (74% sensitivity and 80% specificity, p=0.00001). Expression of miR-27a, miR-31, miR-182 and miR-195 could distinguish patients with NSCLC from healthy people. The examination of these microRNAs in plasma could be used in non-invasive lung cancer diagnosis. Deregulation of Dicer and Drosha expression by microRNAs could have oncogenic character.

Somatic mutations in NRAS, PTEN and AKT1 genes are rarely (~1%) reported in primary NSCLC, but their role in carcinogenesis have been proven. Therefore, we assessed the frequency of them in 145 FFPE tissue samples from CNS metastases of NSCLC using the real-time PCR technique. We identified four (two NRAS and single AKT1 and PTEN) mutations in CNS metastases of NSCLC. All mutations were observed in current male smokers (4% out of the male group; 4/100 and 4.25% out of smokers; 4/94). Three mutations have been detected in patients with SqCC (10.3% out of SqCC patients; 3/29), and only one mutation in the NRAS gene—in a patient with adenocarcinoma (1.25% out of AC patients; 1/80). The examined genes were mutually exclusive in terms of molecular background in KRAS; EGFR; DDR2; PIK3CA; HER2 and MEK1 genes that were evaluated in our previous studies. The OS of the patients who harbored NRAS, AKT1 and PTEN mutations was 10.1, 12.1, 7.3 and 4 months, respectively (vs 13.5 months of the studied group). Our results suggest that the presence of NRAS, PTEN and AKT1 gene mutations may have an influence on the occurrence of CNS metastases in patients with SqCC.

Anaplastic lymphoma kinase (ALK) gene rearrangement was reported in 3%-7% of primary non-small-cell lung cancer (NSCLC) and its presence is commonly associated with adenocarcinoma (AD) type and non-smoking history. ALK tyrosine kinase inhibitors (TKIs) such as crizotinib, alectinib and ceritinib showed efficiency in patients with primary NSCLC harboring ALK gene rearrangement. Moreover, response to ALK TKIs was observed in central nervous system (CNS) metastatic lesions of NSCLC. However, there are no reports concerning the frequency of ALK rearrangement in CNS metastases. We assessed the frequency of ALK abnormalities in 145 formalin fixed paraffin embedded (FFPE) tissue samples from CNS metastases of NSCLC using immunohistochemical (IHC) automated staining (BenchMark GX, Ventana, USA) and fluorescence in situ hybridization (FISH) technique (Abbot Molecular, USA). The studied group was heterogeneous in terms of histopathology and smoking status. ALK abnormalities were detected in 4.8% (7/145) of CNS metastases. ALK abnormalities were observed in six AD (7.5%; 6/80) and in single patients with adenosuqamous lung carcinoma. Analysis of clinical and demographic factors indicated that expression of abnormal ALK was significantly more frequently observed (P = 0.0002; χ2  = 16.783) in former-smokers. Comparison of IHC and FISH results showed some discrepancies, which were caused by unspecific staining of macrophages and glial/nerve cells, which constitute the background of CNS tissues. Their results indicate high frequency of ALK gene rearrangement in CNS metastatic sites of NSCLC that are in line with prior studies concerning evaluation of the presence of ALK abnormalities in such patients. However, they showed that assessment of ALK by IHC and FISH methods in CNS tissues require additional standardizations.

Purpose RT-PCR technique has showed a promising value as pre-screening method for detection of mRNA containing abnormal ALK sequences, but its sensitivity and specificity is still discussable. Previously, we determined the incidence of ALK rearrangement in CNS metastases of NSCLC using IHC and FISH methods. Materials We evaluated ALK gene rearrangement using two-step RT-PCR method with EML4-ALK Fusion Gene Detection Kit (Entrogen, USA). The studied group included 145 patients (45 females, 100 males) with CNS metastases of NSCLC and was heterogeneous in terms of histology and smoking status. Results 21% of CNS metastases of NSCLC (30/145) showed presence of mRNA containing abnormal ALK sequences. FISH and IHC tests confirmed the presence of ALK gene rearrangement and expression of ALK abnormal protein in seven patients with positive result of RT-PCR analysis (4.8% of all patients, 20% of RT-PCR positive patients). RT-PCR method compared to FISH analysis achieved 100% of sensitivity and only 82.7% of specificity. IHC method compared to FISH method indicated 100% of sensitivity and 97.8% of specificity. In comparison to IHC, RT-PCR showed identical sensitivity with high number of false positive results. Conclusion Utility of RT-PCR technique in screening of ALK abnormalities and in qualification patients for molecularly targeted therapies needs further validation.

Background: In non-small cell lung cancer (NSCLC) the phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) gene mutations have been reported in fewer than 5% of primary tumors. Materials and Methods: We assessed PIK3CA gene mutations in 145 tissue samples from central nervous system (CNS) metastases of NSCLC using three polymerase chain reaction (PCR) techniques: high resolution melting-PCR (HRM-PCR), allele-specific-quantitative PCR (ASP-qPCR) and TaqMan PCR. Results: HRM analysis allowed us to select three PIK3CA-positive specimens (2.1% of the studied group) and ASP-qPCR techniques identified them as one E542K and two H1047R substitutions, which were confirmed by TaqMan probes. The PIK3CA mutations were indicated only in males (3% of all males). One of the patients was reported to be a non-smoker with adenocarcinoma (AC; 2.5% of the AC group), however, the other two patients were smokers with squamous cell carcinoma (SCC; 3.4% of SCC group). Conclusion: This is the first report of the presence of PIK3CA gene mutation in CNS-metastatic lesions of NSCLC worldwide that could broaden therapeutic choices in such patients.

The mitogen-activated protein kinases 1 and 2 (MEK1, MEK2) are fundamental partners in the RAS-RAF-MEK-ERK pathway that is involved in regulation of cell proliferation, differentiation and survival. Downregulation of the MEK cascades has been implicated in acquiring of the malignant phenotype in various cancers. Somatic mutations in MEK1 gene (substitutions K57N, Q56P, D67N) were described in < 1 % of non-small cell lung cancer (NSCLC) and they were more commonly reported in adenocarcinoma patients with current or former smoking status.In the following study, we assessed the MEK1 gene mutations in 145 FFPE tissue samples from central nervous system (CNS) metastases of NSCLC using HRM-PCR and ASP-qPCR techniques. The studied group was heterogeneous in terms of histopathology and smoking status. The prevalence of the MEK1 gene mutation was correlated with the occurrence of mutations in KRAS, EGFR, DDR2, PIK3CA, NRAS, HER2, AKT1 and PTEN genes.Using HRM and ASP-qPCR methods we identified one (0.7 %; 1/145) MEK1 substitution (Q56P) in CNS metastases of NSCLC. The mutation was identified in a single, 50-year-old, current smoking men with adenocarcinoma (1.25 %; 1/80 of all adenocarcinomas).According to the current knowledge, the incidence of MEK1 gene mutation in CNS metastatic lesion of NSCLC is the first such report worldwide. The analysis of gene profile in cancer patients may extend the scope of molecularly targeted therapies used both in patients with primary and metastatic tumors of NSCLC.