Physiological healing of chronic gastric ulcer is not impaired by the hydrogen sulphide (H2S)-releasing derivative of acetylsalicylic acid (ATB-340): functional and proteomic approaches.

Gastric ulcers affect approx. 10% of population. Non-steroidal anti-inflammatory drugs (NSAIDs), including acetylsalicylic acid (ASA) predispose to or impair the physiologically complex healing of pre-existing ulcers. Since H2S is an endogenous cytoprotective molecule, we hypothesized that new H2S-releasing ASA-derivative (ATB-340) could overcome pathological impact of NSAIDs on GI regeneration.Clinically translational gastric ulcers were induced in Wistar rats using state-of-the-art microsurgical model employing serosal application of acetic acid. This was followed by 9 days long i.g. daily treatment with vehicle, ATB-340 (6-24 mg/kg) or equimolar ASA doses (4-14 mg/kg). Ulcer area was assessed macro- and microscopically. Prostaglandin (PG)E2  levels, indicating pharmacological activity of NSAIDs and 8-hydroxyguanozine content, reflecting nucleic acids oxidation in serum/gastric mucosa, were determined by ELISA. Qualitative and/or quantitative pathway-specific alterations at the ulcer margin were evaluated using real-time PCR and mass spectrometry-based proteomics.ASA, unlike ATB-340, dose-dependently delayed/impaired gastric tissue recovery, deregulating 310 proteins at the ulcer margin, including Ras signalling, wound healing or apoptosis regulators. ATB-340 maintained NSAIDs-specific cyclooxygenase-inhibiting capacity on systemic and GI level but in time-dependent manner. High dose of ATB-340 (24 mg/kg daily), but not ASA, decreased nucleic acids oxidation and upregulated anti-oxidative/anti-inflammatory heme oxygenase-1, 24-dehydrocholesterol reductase or suppressor of cytokine signalling (SOCS3) at the ulcer margin.Thus, ASA impairs the physiological healing of pre-existing gastric ulcers, inducing the extensive molecularly functional and proteomic alterations at the wound margin. H2S-releasing ATB-340 maintains the target activity of NSAIDs with limited impact on gastric PGE2 signalling and physiological GI regeneration, enhancing anti-inflammatory and anti-oxidative response, and providing the pharmacological advantage.

Correlation between KRAS, NRAS and BRAF mutations and tumor localizations in patients with primary and metastatic colorectal cancer.

Introduction: Detection of abnormalities in the KRAS, NRAS and BRAF genes is extremely important for proper qualification of colorectal cancer (CRC) patients for therapy with anti-EGFR (epidermal growth factor receptor) monoclonal antibodies. However, data about prevalence of mutations in these genes, in different localizations of CRC tumors, are limited. Material and methods: We examined the frequency of mutations in the KRAS, NRAS and BRAF genes in 500 Caucasian CRC patients (200 women and 300 men, median age 66 years). DNA was isolated from formalin-fixed, paraffin-embedded (FFPE) tissues using a Qiagen QIAamp DNA FFPE-kit. Analysis of mutations was carried out using the KRAS/BRAF, NRAS and BRAF Mutation Analysis Kit for Real-Time PCR (EntroGen) with the Cobas 480 real-time PCR apparatus (Roche Diagnostics). Results: KRAS mutations were detected in 190 (38%) patients, NRAS mutations in 20 (4%) patients, and BRAF mutations in 24 (4.8%) patients. There were no associations between age of CRC patients and frequency of KRAS, NRAS and BRAF gene mutations. These mutations were significantly more often diagnosed in women (55.5%) than in men (41%, p < 0.005). Tumors of the rectum and sigmoideum were the most often observed in both groups of CRC patients - with and without KRAS, NRAS and BRAF gene mutations. However, transverse colon, ascending colon and cecum cancers were the most often affected by mutations. Conclusions: Our study showed that the occurrence of mutations in the KRAS, NRAS and BRAF genes is not accidental and depends on the location of CRC tumors.

Carbon monoxide (CO)/heme oxygenase (HO)-1 in gastrointestinal tumors pathophysiology and pharmacology - possible anti- and pro-cancer activities.

Gastrointestinal (GI) tract cancers pose a significant pharmacological challenge for researchers in terms of the discovery of molecular agents and the development of targeted therapies. Although many ongoing clinical trials have brought new perspectives, there is still a lack of successful long-term treatment. Several novel pharmacological and molecular agents are being studied in the prevention and treatment of GI cancers. On the other hand, pharmacological tools designed to release an endogenous gaseous mediator, carbon monoxide (CO), were shown to prevent the gastric mucosa against various types of injuries and exert therapeutic properties in the treatment of GI pathologies. In this review, we summarized the current evidence on the role of CO and heme oxygenase 1 (HO-1) as a CO producing enzyme in the pathophysiology of GI tumors. We focused on a beneficial role of HO-1 and CO in biological systems and common pathological conditions. We further discussed the complex and ambiguous function of the HO-1/CO pathway in cancer cells with a special emphasis on molecular and cellular pro-cancerous and anti-cancer mechanisms. We also focused on the role that HO-1/CO plays in GI cancers, especially within upper parts such as esophagus or stomach.

Barrett's Metaplasia Progression towards Esophageal Adenocarcinoma: An Attempt to Select a Panel of Molecular Sensors and to Reflect Clinical Alterations by Experimental Models.

The molecular processes that predispose the development of Barrett's esophagus (BE) towards esophageal adenocarcinoma (EAC) induced by gastrointestinal reflux disease (GERD) are still under investigation. In this study, based on a scientific literature screening and an analysis of clinical datasets, we selected a panel of 20 genes covering BE- and EAC-specific molecular markers (FZD5, IFNGR1, IL1A, IL1B, IL1R1, IL1RN, KRT4, KRT8, KRT15, KRT18, NFKBIL1, PTGS1, PTGS2, SOCS3, SOX4, SOX9, SOX15, TIMP1, TMEM2, TNFRSF10B). Furthermore, we aimed to reflect these alterations within an experimental and translational in vitro model of BE to EAC progression. We performed a comparison between expression profiles in GSE clinical databases with an in vitro model of GERD involving a BE cell line (BAR-T) and EAC cell lines (OE33 and OE19). Molecular responses of cells treated with acidified bile mixture (BM) at concentration of 100 and 250 µM for 30 min per day were evaluated. We also determined a basal mRNA expression within untreated, wild type cell lines on subsequent stages of BE and EAC development. We observed that an appropriately optimized in vitro model based on the combination of BAR-T, OE33 and OE19 cell lines reflects in 65% and more the clinical molecular alterations observed during BE and EAC development. We also confirmed previous observations that exposure to BM (GERD in vitro) activated carcinogenesis in non-dysplastic cells, inducing molecular alternations in the advanced stages of BE. We conclude that it is possible to induce, to a high extent, the molecular profile observed clinically within appropriately and carefully optimized experimental models, triggering EAC development. This experimental scheme and molecular marker panel might be implemented in further research, e.g., aiming to develop and evaluate novel compounds and prodrugs targeting GERD as well as BE and EAC prevention and treatment.

Impact of copy number variant and single nucleotide polymorphism of the programmed death-ligand 1 gene, programmed death-ligand 1 protein expression and therapy regimens on overall survival in a large group of Caucasian patients with non-small cell lung carcinoma.

Anti-programmed death-1 or anti-programmed death-ligand 1 (PD-L1) blockade may be ineffective in some patients with non-small cell lung cancer (NSCLC) with high percentage of tumor cells with PD-L1 expression. In addition, immunotherapy may provide great benefits in patients without PD-L1 expression. The present study assessed PD-L1 protein expression by immunohistochemistry, copy number variation (CNV) of PD-L1 and two single nucleotide polymorphisms (SNPs), rs822335 and rs822336, in the promoter of PD-L1 by quantitative PCR in 673 patients with NSCLC. Overall survival time of patients with NSCLC depending on the assessed predictive factors (PD-L1 CNV or SNP) and the treatment methods (immunotherapy in first/second line of treatment or chemotherapy) was analyzed. The present study revealed significantly higher PD-L1 copies number in patients with ≥10% and ≥50% of tumor cells with PD-L1 expression compared to patients with lower percentage of PD-L1-positive tumor cells (P=0.02 and P=0.0002, respectively). There was a significant positive correlation (R=0.2; P=0.01) between number of PD-L1 copies and percentage of tumor cells with PD-L1 protein expression. Percentage of tumor cells with PD-L1 expression was lower in patients with TT genotype of the rs822335 polymorphism compared to those with CC genotype (P=0.03). The present study observed significantly higher risk of death in patients treated with chemotherapy compared to those treated with immunotherapy (P<0.0001; hazard ratio=2.4768; 95% confidence interval, 2.0120-3.0490). The present study demonstrated a close relationship between PD-L1 copies number, genotype of rs822335 PD-L1 polymorphism and PD-L1 protein expression on tumor cells. However, the impact of CNV and SNPs of PD-L1 on overall survival of patients with NSCLC requires further investigation.

An Episode of Pseudothrombocytopenia during Pembrolizumab Therapy in NSCLC Patient.

Immunotherapy with immune checkpoint inhibitors (ICI) is a new option of treatment in a growing range of neoplasms. In addition to an antitumor effect, ICI are associated with autoimmune reactions resulting in a wide spectrum of toxicities that have not been seen in patients receiving chemotherapy. In this article, we present a case of a patient with advanced lung adenocarcinoma who developed an EDTA-dependent pseudothrombocytopenia (PTCP) during pembrolizumab therapy. To the best of our knowledge, this is the first reported case of EDTA-dependent PTCP occurring during immunotherapy treatment of nonsmall lung cell cancer with ICI. The phenomenon of EDTA-dependent PTCP may prompt clinical decisions, as unnecessary transfusions or even exclusion from pembrolizumab therapy. Therefore, it is important to be aware of PTCP as a possible side effect of this therapy.

Tissue MicroRNA Expression as a Predictor of Response to Immunotherapy in NSCLC Patients.

INTRODUCTION: Expression 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 METHODS: The 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. RESULTS: Response 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). CONCLUSION: The miR-200b and miR-429 molecules in tumor cells seem to have greatest impact on the effectiveness of immunotherapy in NSCLC patients.

PD-L1 gene copy number and promoter polymorphisms regulate PD-L1 expression in tumor cells of non-small cell lung cancer patients.

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.

Discrepancies between ALK protein disruption and occurrence of ALK gene rearrangement in Polish NSCLC patients.

BACKGROUND: Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations or anaplastic lymphoma kinase (ALK) rearrangement are predisposed to molecularly targeted therapies. Proper diagnostic is crucial for quick and correct patients qualification to optimal treatment method. Genetic tests to detect predictive factors could be performed sequentially. After excluding EGFR mutations, abnormal ALK protein expression should be tested using immunohistochemistry (IHC) method. In patients with disrupted ALK expression, the rearrangement of the ALK gene should be confirmed by FISH method. Despite few years of experience in analysis of these predictive factors, there are still problems in interpretation of diagnostic tests results. Especially, some recommendations for ALK IHC diagnosis are not precise. METHODS: Mutations in EGFR gene were examined using real-time PCR technique in 1,108 formalin-fixed paraffin-embedded (FFPE) tissues, 398 FFPE cell-blocks and 470 cytological specimens of NSCLC. The disrupted ALK protein expression was analysed in 1,100 samples including 782 histological and 306 cytological (cell-blocks) samples using IHC. Twelve materials (1.1%) were non-diagnostic in IHC. ALK gene rearrangement using FISH method was analysed in IHC positive cases. RESULTS: The frequency of EGFR mutations was 8.6%. EGFR mutations occurred significantly more often in females (P=0.00001, χ2=62.732) and in adenocarcinoma cases (P=0.0002, χ2=14.222). The exon 19 deletions (49%) and exon 21 Leu858Arg substitution (38%) were the most common, rare EGFR mutations occurred in 13% of patients. Any expression of abnormal ALK protein was detected in 202 cases (18.57%). ALK gene rearrangement was confirmed in 49 cases (4.5%). ALK gene rearrangement is significantly more common in female than in male (P=0.0105, χ2=6.541). In patients with ALK gene rearrangement, the median percentage of nuclei with ALK rearrangement was only 25.5%. The polysomy (≥4 gene copy number per nuclei) of ALK gene was observed in 39 cases (21.4% of patients with diagnostic result of FISH examination). Median number of ALK gene copy per nuclei was 2.9±0.77. Significant positive correlation between percentage of cells with abnormal ALK expression in IHC test and percentage of nuclei with ALK rearrangement in FISH method was detected (R=0.617, P<0.00001). Significant negative correlation between the number of copies of ALK gene and the percentage of cells with expression of abnormal ALK was observed (R=-0.2004, P<0.05). ALK gene rearrangement was significantly more frequently observed in the material with coarse-grained cytoplasmic and membranous IHC staining than in materials with light cytoplasmic stippling. The occurrence of cytoplasmic stippling correlated with the increase of ALK gene copy number. CONCLUSIONS: We indicated that diagnosis of ALK disruption in NSCLC patients should be notably careful using IHC and FISH methods. Recommendations for ALK diagnosis should include the way of interpretation of cases with low percentage of cells with abnormal ALK protein expression in IHC test, character of IHC reaction, and cases with ALK gene polysomy in FISH method.