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 realtime PCR apparatus (Roche Diagnostics).Results: KRAS mutations were detected in 190 patients (38%), NRAS mutations in 20 patients (4%), and BRAF mutations in 24 patients (4.8%). 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.

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.

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.

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.

Barrett’s esophagus (BE) is a premalignant adaptation to chronic gastroesophageal reflux disease (GERD) and the principal precursor lesion of esophageal adenocarcinoma (EAC). Carbon monoxide (CO), an endogenous gaseous mediator generated by heme oxygenases, regulates cellular homeostasis, stress responses, and survival pathways; however, its role in BE progression and EAC remains poorly understood. We investigated the biological and transcriptional effects of increased CO bioavailability in cellular models representing distinct stages of disease progression. Non-transformed esophageal squamous epithelial cells (NES-G2T), BE cells (BAR-T), and EAC-derived OE19 and OE33 cells were exposed to the CO donor CORM-A1 or its CO-depleted structural control (iCORM-A1). Cellular metabolic activity was assessed by the MTT assay, apoptosis by Annexin V/PI flow cytometry, and transcriptomic alterations by RNA sequencing in BAR-T, OE19, and OE33 cells following CORM-A1 treatment. Transient CO exposure was well tolerated by NES-G2T cells, with no significant effects on metabolic activity or apoptosis. While BAR-T cells showed alterations in MTT-derived metabolic activity, they exhibited only limited apoptotic changes following CORM-A1 exposure, indicating that the reduced metabolic activity was not accompanied by significant apoptotic cell death. In contrast, CORM-A1 induced a reduction in metabolic activity and increased apoptotic cell death in EAC-derived OE19 and OE33 cells. At 500 μM, CORM-A1 reduced metabolic activity of both EAC cell lines below 75% of the untreated control and exerted a significantly greater reduction in metabolic activity than iCORM-A1, suggesting a contribution of CO release to the observed response. Flow cytometric analyses confirmed a noticeable apoptotic response to CORM-A1 in both EAC cell lines. RNA sequencing revealed marked, highly cell type-dependent transcriptional responses to CO exposure. Non-neoplastic BAR-T cells exhibited the most pronounced transcriptomic remodeling, predominantly affecting pathways related to epithelial differentiation, tissue homeostasis, and extracellular matrix organization. In contrast, OE33 and OE19 cells displayed more restricted transcriptional responses that preferentially involved growth factor signaling, cellular adaptation, inflammatory regulation, and tumor-microenvironment interactions. Collectively, these findings suggest that responsiveness to CO is shaped by the degree of neoplastic transformation, with BE mounting a broad adaptive transcriptional response, showing limited drug-induced apoptosis. However, malignant cells exhibit greater vulnerability to CORM-A1-induced programmed cell death despite undergoing more limited transcriptomic remodeling. These data identify CORM-A1-responsive pathways and CO signaling as potential therapeutic targets in EAC.