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The goal of the CLOVER study was to perform a pairwise comparison of four tests based on the same patient population with non-small cell lung cancer (NSCLC): three validated PDL1 immunohistochemistry (IHC) assays (Ventana SP142, Ventana SP263, Dako 22C3) and one PCR test. Four hundred seventy-three NSCLC samples were obtained from a biobank and were stained using PDL1 IHC assays. Four trained pathologists independently evaluated the percentage of tumor cells (TC) and immune cells (IC) that stained positive at any intensity. PDL1 transcripts were quantified in 437 patients by a standard Taqman RT-PCR assay using SDHA as a reference gene. A concordance analysis was performed to assess (1) the correlation of TC and IC between different assays and (2) the predictive properties of one test for another. “High” RNA expression was detected in 187 of 437 (43%) patients. The percentage of PDL1-positive cells (≥1%) was higher among the IC than the TC in all IHC three assays. The Pearson correlation coefficients (PCC) for TC were 0.71, 0.87, and 0.75 between 22C3/SP142, 22C3/SP263, and SP263/SP142, respectively. The PCC for IC were 0.45, 0.61, and 0.68 for the same pairs. A low correlation was observed between the PCR test and each of the three IHC assays; however, if a patient tested low/negative by PCR, then they were likely to test negative by any single IHC test with a high probability (92–99%). Among patients who tested positive by PCR, only 9–45% tested positive by IHC assays. There was excellent positive and negative agreement (>91%) between 22C3 and SP263 staining using the recommended individual cutoffs for first-line treatment. PCR RNA expression analysis is not equivalent to IHC. However, this method may have some potential for the identification of PDL1-negative tumors. 22C3 could be considered as a substitute for SP263 in first-line treatment.

Distribution of actionable mutations in melanoma may show considerable geographic variations. Given the importance of genetic testing for the proper use of targeted drugs, we carried out the first-in-Russia nationwide molecular epidemiological study for melanoma. Sanger sequencing analysis for BRAF (exon 15) and NRAS (exons 2-4) genes was carried out for patients with the stage IIIB or IV disease from 46 cancer centers located throughout the country. BRAF mutations were identified in 625/1034 (60.4%) melanoma samples. BRAF c.1799T>A (p.V600E) substitution was the most prevalent, being detected in 561/1034 (54.3%) tumors. Non-V600E mutations constituted about 10% of activating BRAF genetic lesions (64/625, 10.2%), with a clear prevalence of c.1798_1799GT>AA (p.V600K) variant (52 tumors) and noticeable occurrence of c.1798_1799GT>AG (p.V600R) allele (five tumors). BRAF V600E mutations were associated with younger patient age and localization of melanoma on sun-protected areas of the skin, whereas BRAF V600K substitutions were characteristic of elderly patients and occurred more often at the chronically sun-exposed regions of the body. Activating NRAS mutations were detected in 86/601 (14.3%) of samples analyzed, with 79 events affecting codon 61 and seven mutations detected in codons 12 or 13. Six types of distinct NRAS codon 61 substitutions were identified, with c.181C>A (p.Q61K), c.182A>G (p.Q61R), and c.182A>T (p.Q61L) being frequent and c.181_182CA>TT (p.Q61L), c.183A>T (p.Q61H), and c.183A>C (p.Q61H) being rare. An age-related increase in the frequency of NRAS mutations was observed. Multiplicity and clinical distribution of BRAF and NRAS mutations have to be taken into account while considering molecular testing for melanoma patients.

SURF-6 is an evolutionarily conserved nucleolar protein that is important for cell viability; however, its function in mammals still remains uncertain. The aim of this study is to generate monoclonal antibodies to human SURF-6 protein suitable for fundamental and biomedical research. The full-size human SURF-6 was expressed as a recombinant GST-fusion protein and used as an antigen to generate monoclonal antibodies, S79 and S148, specific for SURF-6. The monoclonal antibody produced by hybridoma clone S79 specifically recognizes endogenous SURF-6 by Western and immunofluorescence analyses in various cultured human cells, and by immunohistochemistry in paraffin-embedded sections of human breast cancer samples. Moreover, S79 immunoprecipitates protein complexes containing SURF-6 from HeLa cells extracts. The antibody S79 recognizes SURF-6 only in human cells; however, the antibody produced by hybridoma clone S148 can detect SURF-6 of human and mouse origin. Monoclonal antibodies to the nucleolar protein SURF-6 described in this work can be a useful tool for studies of ribosome biogenesis in normal and cancer cells.