A new method of double immunofluorescent staining for flow cytometry has been created to evaluate quantitative expression of mesenchymal protein vimentin only in epithelial cells of a solid tumor that is a mix of different origin cells. De novo vimentin expression is strongly associated with epithelial–mesenchymal transition and therefore is a metastatic potential marker of epithelial tumor cells. In comparison with semiquantitative available methods, the proposed one has several advantages, such as the accurate measurement of the marker’s expression, and minimization of spatial and temporal tumor heterogeneity. Clinical validation of the method has revealed inverse correlation between the quantitative index of epithelial–mesenchymal transition level and progression-free survival using Kaplan–Meier curves and the COX proportional hazards ratio in 32 ovarian cancer patients.

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Background: DNAseq, RNAseq and determination of microsatellite instability (MSI) are now routine analyses in precision medicine for management of cancer patients. Analyzing DNA/RNA requires reliable assays often performed in two separate runs. Faced with limited quantity of total nucleic acids (TNA) extracted from FFPE this can be challenging. Sequential approaches (i.e. DNAseq followed by RNAseq) increase the turnaround time and cost, delaying treatment. Here, we evaluated a novel NGS approach developed by Invitae (San Francisco, USA). Using TNA from FFPE tissue, we detected SNVs, indels, MSI and structural rearrangements in one single NGS run.Methods: 24 samples from patients with various cancers were included, previously characterized using conventional methods: DNAseq, RNAseq, PCR, IHC or FISH. The study was approved by ICL ethical and scientific committee-all patients gave their consent. FFPE samples were qualified, selected followed by tumor cell content evaluation by a pathologist. After microdissection, TNAs were extracted and libraries were prepared using a parallel VariantPlex (DNA) and FusionPlex (RNA) kit from Invitae. Kits were designed to detect known and novel fusions in gene targets commonly encountered in solid tumors, MSI, and variants using a panel of 156 cancer-relevant target genes (71 DNA, 136 RNA).Results: Among 9 previous SNVs/indels and 11 MSI samples identified by orthogonal methods, NGS approach confirmed all (100%). Among 12 fusions, 11 were detected by NGS (91.7%). Single ROS1 fusion scored low (1+) using IHC. Further analyses are conducted to determine whether the ROS1 fusion is false positive by IHC, or false negative by NGS.Conclusions: Novel, all-in-one NGS approach from Invitae shows promising results by comprehensively detecting SNVs, indels, MSI and structural variants. Analyses of DNA and RNA using the novel NGS strategy improves the overall turn-around-time and cost-effectiveness. This can potentially aid clinicians and patients to take timely decisions regarding treatment modalities.

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Expression of tumor-associated protein beta-III tubulin (TUBB3) was evaluated quantitatively by immunofluorescence analysis using flow cytometry in lung tissue of intact animals and on day 11 after the Lewis lung carcinoma transplantation as well in lung metastasis tissue and in visually “normal” surrounding tissue. It was shown that the tumor is characterized by a high TUBB3 expression, while also the expression of the marker was detected in both variants of visually normal lung tissue: it was lower than in tumor tissue but significantly higher than in lung tissue of intact animals. The detection of TUBB3 outside the tumor indicates that the tissue appeared to be normal has already involved in malignancy and this supports the hypothesis that tumor-associated protein TUBB3 can be used as a molecular marker of local tumor spread.

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In the study of 125 patients after radical surgery for non-small cell lung cancer (NSCLC), distinctive clinicopathological parameters of this disease were found. Thus, the majority of patients with NSCLC were smokers (73%), men had significantly higher rate of the disease than women (80% vs 20%). Patients of different sex varied by the tumor histological type: squamous cell carcinoma was characteristic of male patients (70%), while adenocarcinoma predominated in women (80%). Authors conclude that the described incidence patterns and the association of NSCLC with significant clinical features are comparable with the literature data, which indicates the absence of significant changes over the past 8 years. Survival rates of the patients after radical surgery for NSCLC also coincided with results of the previous studies. Overall survival rate varied by the stage of the disease, smoking status, and the tumor histological type: patients with earlier stage at diagnosis, never-smokers and patients with adenocarcinoma rather than squamous cell carcinoma were associated with a more favorable prognosis. The heterogeneity of the patients who survived 5 years after surgical treatment was shown: the group included patients not only with stage I of the disease, but also with more advanced II and even III stages of the disease. Finally, the absence of gender differences in the overall survival of the patients with NSCLC was demonstrated; this fact differs from the literature data on the better survival of female patients compared to men. The authors suggest that the result may indicate the emergence of factors in the Russian population that improve the course of disease in men or adversely affect the disease in women.