Jacek Kordiak

The positive correlation between gene expression of the two angiogenic factors: VEGF and BMP-2 in lung cancer patients

Lung cancer is a particular challenge in oncology. More than 1 million new cases occur worldwide every year and despite many clinical trials and modern diagnostic techniques, long-term survival rate has only marginally improved. The aim of the current research is to explore new molecular prognostic factors and identify new targets for anticancer therapy. Current evidence shows that angiogenesis is controlled by several angiogenic factors including VEGF and BMP-2. It has been also demonstrated that VEGF plays a key role in this process that is essential in carcinogenesis. Our study has shown that the expressions of the VEGF, BMP-2 and BMP-4 mRNAs were significantly higher (7.1-fold, 25.6-fold and 2.3-fold, respectively) in lung cancer samples than in adjacent normal lung tissues (real-time RT-PCR). Analysis based on the Pearsons correlation coefficient indicated the positive correlation between VEGF and BMP-2 gene expression, whereas no significant correlation between VEGF and BMP-4 gene expression was found. The mean+/-standard deviation serum level of VEGF was 423+/-136 pg/ml. Significant differences in the serum levels of VEGF between patients with T1 tumors and patients with T2, T3 or T4 tumors were observed. Patients with T2, T3 and T4 tumors, respectively, had 1.6-fold, 1.8-fold and 2.3-fold greater serum levels of VEGF than their peers with T1 tumors. In current study patients homozygous for the 936T allele of the +936C/T VEGF gene polymorphism had 12-fold lower VEGF gene expression and 1.3-fold lower VEGF serum level than patients homozygous for the 936C allele. In conclusion, our findings underline the importance of the two angiogenic factors namely VEGF and BMP-2 as well as +936C/T VEGF gene polymorphism in the evaluation of lung cancer patients.

CTLA-4 Expression and Polymorphisms in Lung Tissue of Patients with Diagnosed Non-Small-Cell Lung Cancer

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) is a potent immunoregulatory molecule that downregulates T-cell activation and thus influences the antitumor immune response. CTLA-4 polymorphisms are associated with various cancers, and CTLA-4 mRNA/protein increased expression is found in several tumor types. However, most of the studies are based on peripheral blood mononuclear cells, and much less is known about the relationship between CTLA-4 expression, especially gene expression, and its polymorphic variants in cancer tissue. In our study we assessed the distribution of CTLA-4 two polymorphisms (+49A/G and −318C/T), using TaqMan probes (rs231775 and rs5742909, resp.), and CTLA-4 gene expression in real-time PCR assay in non-small-cell lung cancer (NSCLC) tissue samples. The increased CTLA-4 expression was observed in the majority of NSCLC patients, and it was significantly correlated with TT genotype (−318C/T) and with tumor size (T2 versus T3 + T4). The presence of G allele and GG genotype in cancer tissue (+49A/G) was significantly associated with the increased NSCLC risk. Additionally, we compared genotype distributions in the corresponding tumor and blood samples and found statistically significant differences. The shift from one genotype in the blood to another in the tumor may confirm the complexity of gene functionality in cancer tissue.

Myocardial oxidative stress in patients with active infective endocarditis

BACKGROUND Infective endocarditis (IE) induces the rise of pro-inflammatory cytokines. Some of them can stimulate oxidants production in myocardium with subsequent peroxidative damage to various biomolecules. We compared indices of oxidative stress: H2O2, thiobarbituric acid-reactive substances (TBARs), thiols in myocardium specimens between patients with active IE and those with valvular heart disease (VHD) of rheumatic etiology who underwent surgical valve replacement. METHODS 17 left ventricle papillary muscle specimens and 28 specimens of auricle of the right heart were collected from 45IE patients, and 16 papillary muscle and 12 auricle specimens from 28 VHD patients, respectively. Patients groups had similar NYHA functional class and majority of echocardiographic indices of heart morphology. H2O2 and TBARs were determined fluorometrically in myocardium homogenates whereas thiols with photometric method. Between and within groups comparisons and mutual correlations between variables were analyzed. RESULTS H2O2 generation from all myocardium specimens and auricles was 2.14- and 2.59- times higher (p<0.001) in IE patients than in VHD group. Auricles had the highest H2O2 levels within IE group. TBARs were 10-times higher (p<0.05) in IE when compared to VHD group in auricles and papillary muscles. Thiols did not differ between groups. H2O2 positively correlated with TBARs and negatively with thiols in all IE myocardium specimens (r=0.31 and r=-0.46, p<0.05) and auricles (r=0.58 and r=-0.67, p<0.05), respectively. No such associations were noted in VHD specimens. CONCLUSIONS Active IE induces enhanced myocardial production of H2O2 and formation of TBARs which proves occurrence of oxidative stress in the heart.

Complete surgical resection of lung tumor decreases exhalation of mutated KRAS oncogene.

Exhaled breath condensate (EBC) contains extracellular DNA that may originate from pathological lesions of the respiratory tract and can be a genetic marker of pulmonary malignancy. We tested whether complete surgical excision of lung cancer will decrease exhalation of mutated KRAS oncogene. Fifty seven patients with clinical diagnosis of lung cancer and detectable KRAS mutations in pre-surgery EBC-DNA were qualified for surgical treatment. Point mutations at codon 12 of KRAS oncogene were detected using mutant-enriched PCR technique in DNA from pre-surgery blood, EBC collected before, 7 and 30 days after surgery and from specimens of resected tumor and normal pulmonary parenchyma. The ratio of mutated to wild type KRAS DNA (R mut/wild KRAS) was calculated for each specimen after electrophoresis and densitometry of the final amplification and digestion product. In 46 patients non-small cell lung cancer (NSCLC) and in 11 benign lesion (BL) were confirmed. All blood and tumor specimens were positive for KRAS mutations, while 41 specimens of normal pulmonary parenchyma were negative. In NSCLC patients pre-surgery EBC R mut/wild KRAS of 0.20 ± 0.03 decreased by 1.3- and 3.7-times (p < 0.001) at 7th and 30th day and 10 EBC specimens at day 30th became negative. The highest R mut/wild KRAS was found in NSCLC specimens - 1.36 ± 0.29 while the lowest in pulmonary parenchyma - 0.02 ± 0.03 (p < 0.001). R mut/wild KRAS in EBC did not correlate with the blood and cancer ratios. Determination of mutated KRAS oncogene in EBC can be potentially helpful in the follow-up of surgical treatment of pulmonary malignancy.

Decreased FAM107A Expression in Patients with Non-small Cell Lung Cancer

Lung cancer is the leading cause of cancer-related death in the world. Early detection, based on molecular markers, could decrease mortality from this disease. Tumor development is often associated with inactivation or loss of tumor suppressor genes (TSGs). The aim of the present study was to analyze the expression level of FAM107A gene, a TSG located in 3p21.1, in lung cancer tumors and in tumor adjacent normal lung samples. Promoter methylation status of FAM107A was evaluated as the potential mechanism of its epigenetic silencing. The relationship between gene mRNA expression and tumor staging, metastasis status, and non-small cell lung cancer (NSCLC) histopathological subtypes in 60 patients was analyzed. Total RNA was isolated from tissue samples and gene expression was assessed in qPCR assay. Gene promoter methylation status was evaluated in MSP reactions, using bisulfite converted DNA and two pairs of primers: methylated and unmethylated. We found that the expression of the gene was dramatically decreased in all NSCLC samples and was significantly lower than in tumor adjacent normal lung tissue. Promoter methylation of FAM107A gene was confirmed only in the minority of NSCLCs. The results highlight the importance of FAM107A in lung carcinogenesis, although indicate other than promoter hypermethylation mechanism of the gene decreased expression.

Elevated resting and agonist-induced whole blood chemiluminescence in patients with active infective endocarditis

Infective endocarditis (IE) and surgical procedures related to cardiac surgery are accompanied by inflammatory responses that may alter production of oxidants by phagocytes. This study evaluates luminol enhanced whole blood chemiluminescence (LBCL) as a measure of oxidative production by circulating phagocytes in 26 IE patients in comparison to 27 matched patients with acquired valvular heart disease and 25 healthy controls. Blood was collected the day before and 3, 7, 12 and 21 days after valve replacement surgery for LBCL measurement; resting (rCL) and agonist (fMLP)-stimulated total light emission (tCL). Preoperative rCL and tCL with values observed after 3, 7, 14, and 21 days from surgery were higher (P<0.01) in patients with IE than in healthy controls. Median preoperative rCL, and tCL was about 2.5-times higher (P<0.01) in IE group than in patients with valvular heart disease (4.3 vs. 1.7 U/10(4) phagocytes and 2473 vs. 782 Uxs/10(4) phagocytes). Three days after valve replacement, LBCL rose three times (P<0.01) in both operated groups. With patient recovery, LBCL decreased and no differences were noted between groups. Patients with IE had elevated LBCL reflecting increased oxidants release from circulating phagocytes that may predispose to the development of oxidative stress.

Expression analysis of three miRNAs, miR-26a, miR-29b and miR-519d, in relation to MMP-2 expression level in non-small cell lung cancer patients: a pilot study

Lung cancer is the most common cause of death in men and second only to breast cancer in women. MicroRNAs (miRNAs) are involved in tumorigenesis and function as oncogenes or tumor suppressor genes. Among other genes, miRNAs regulate matrix metalloproteinases (MMPs), the proteolytic enzymes playing a significant role in the degradation of extracellular matrix, enhancing tumor invasion and metastasis. The aim of the study was to evaluate the expression levels of selected miRNAs: miR-26a, miR-29b and miR-519d, and their target gene, matrix metalloproteinase-2 (MMP-2) in patients with non-small cell lung cancer (NSCLC). The results were correlated with tumor staging, NSCLC histopathological subtypes and patients’ demographical features to assess the possible diagnostic/prognostic value of the studied miRNAs and MMP-2. Total RNA was isolated from 38 NSCLC tissue samples, and the expression analysis was performed using TaqMan® probes in qPCR assay. The results indicated underexpression of selected miRNAs and overexpression of MMP-2. The decrease in miRNA-29b expression was statistically significant and differentiated NSCLC histopathological subtypes. Additionally, statistically significant negative correlation was found between MMP-2 expression and its regulatory miR-26a. There are very few studies reporting miRNA-MMPs analysis on mRNA level in lung cancer, and no similar reports are available from Polish population. The results of our pilot study indicated the diagnostic potential of miR-29b and MMP-2, an inverse association between miR-26a and MMP-2, and proved the role of MMP-2 and the studied miRNAs in lung carcinogenesis. Further studies are needed to verify their potential usefulness for the treatment of lung cancer.

RARβ Promoter Methylation as an Epigenetic Mechanism of Gene Silencing in Non-small Cell Lung Cancer.

The retinoid acid receptor-p (RARβ) gene is one of the tumor suppressor genes (TSGs), which is frequently deleted or epigenetically silenced at an early stage of tumor progression. In this study we investigated the promoter methylation and expression status of the RARβ gene in 60 surgically resected non-small cell lung cancer (NSCLC) tissue samples and 60 corresponding unchanged lung tissue samples, using methylation-specific PCR and real-time-polymerase chain reaction (qPCR) techniques. We correlated the results with the pathological features of tumors and clinical characteristics of patients. qPCR analysis detected a significantly lower RARβ expression in the patients with adenocarcinoma (AC) and large cell carcinoma (LCC) than in those with squamous cell carcinoma (SCC) (AC vs. SCC, p = 0.032; AC and LCC vs. SCC, p = 0.0 13). Additionally, significantly lower expression of the RARβ gene was revealed in the patients with non-squamous cell cancer with a history of smoking assessed as pack-years (PY < 40 vs. PY ≥ 40, p = 0.045). Regarding RARβ promoter methylation, we found significant differences in the methylation index in the SCC group when considering pTNM staging; with higher index values in T1a + T1b compared with T2a + T2b and T3 + T4 groups (p = 0.024). There was no correlation between the methylation status and expression level of the RARβ gene, which suggests that other molecular mechanisms influence the RARβ expression in NSCLC patients. In conclusion, different expression of the RARβ gene in SCC and NSCC makes the RARβ gene a valuable diagnostic marker for differentiating the NSCLC subtypes.

Acute mediastinitis: evaluation of clinical risk factors for death in surgically treated patients

Acute mediastinitis (AM) is the most lethal form of infection within the thorax. The authors of this study, using statistical tools, made an attempt to determine the most important clinical risk factors in retrospective material of patients treated surgically due to AM.

Intratumor heterogeneity and tissue distribution of KRAS mutation in non-small cell lung cancer: implications for detection of mutated KRAS oncogene in exhaled breath condensate

PurposeMutated KRAS oncogene in exhaled breath condensate (EBC) can be a genetic marker of non-small cell lung cancer (NSCLC). However, a possibility of inhomogeneous distribution in cancer tissue and intratumor heterogeneity of KRAS mutation may decrease its significance. We investigated a status of KRAS point mutation and its sequence at codon 12 in 51 NSCLC patients after tumor resection. The comparison of KRAS mutation status between EBC–DNA and cancer tissue was performed in 19 cases.MethodsFive cancer tissue samples from disparate tumor regions and one from normal lung were harvested at surgery. EBC was collected for DNA analysis the previous day. KRAS point mutations at codon 12 were detected using mutant-enriched PCR technique and pyrosequenced.ResultsForty-six cancers revealed concordance of KRAS mutation status: 27 contained mutated KRAS and 19 had only wild KRAS. Five NSCLCs revealed inhomogeneous distribution of KRAS mutation. Two different mutations were found in 14 NSCLCs and the most frequent one was G12D and G12V (n = 8). No mutated KRAS was found in normal lung. The concordance ratios of KRAS sequence in codon 12 between EBC–DNA and cancer were 18/19 for NSCLC patients and 11/12 for KRAS mutation positive NSCLC.ConclusionsIntratumor heterogeneity and inhomogeneous distribution of KRAS point mutation in codon 12 in cancer tissue can occur in NSCLCs. There was a high accordance between KRAS mutation status in EBC–DNA and cancer tissue in NSCLC patients what suggests usefulness of monitoring KRAS mutation in EBC–DNA as a biomarker of NSCLC.