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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.

IntroductionDetection 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 monoclonal antibodies. However, data about prevalence of mutations in these genes, in different localizations of CRC tumors, is limited.Material and methodsWe 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 embeded (FFPE) tissues using Qiagen QIAmp DNA FFPE-kit. Analysis of mutations was carried out using KRAS/BRAF, NRAS and BRAF Mutation Analysis Kit for Real-Time PCR (EntroGen) using the Cobas 480 real-time PCR apparatus (Roche Diagnostics)ResultsKRAS mutations have been detected in 190 patients (38%), NRAS mutations in 20 patients (4%), whereas BRAF mutations in 24 patients (4,8%). There were no associations between age of CRC patients and frequency of KRAS, NRAS and BRAF genes mutations. These mutations were significantly more often diagnosed in women (55.5%) than in man (41%, p<0.005). Tumors of rectum and sigmoideum were the most often observed in both groups of CRC patients – with and without KRAS, NRAS and BRAF genes mutations. However, transverse colon, ascending colon and cecum cancers were the most often affected by mutationsConclusionsOur 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.

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.