Whole-exome sequencing reveals an association of rs112065068 in TGOLN2 gene with distant metastasis of non-small cell lung cancer.

Early prediction and prevention of recurring illness is critical for improving the survival rates of patients with non-small cell lung cancer (NSCLC). Previously, we demonstrated that the presence of premalignant epithelial changes in the small bronchi distant to the primary tumor is associated with NSCLC progression: isolated basal cell hyperplasia (iBCH) indicates a high risk of distant metastasis, BCH combined with squamous metaplasia (BCHSM) - a high risk of locoregional recurrence. Here, we aimed to identify germline single nucleotide variants (SNVs) and insertions and deletions (InDels) associated with distant metastasis and locoregional recurrence in cases with iBCH and BCHSM using whole-exome sequencing of 172 NSCLC patients. The rs112065068 of the TGOLN2 gene was identified only in iBCH patients and was associated with a high risk of distant metastasis (P < .001) and worse metastasis-free survival (HR = 4.19 (95 %CI 1.97-8.93); P < .001). This variant was validated in a group of 109 NSCLC patients using real-time PCR and Sanger sequencing analyses. To our knowledge, this study is the first to identify a germline variant associated with NSCLC distant metastasis.

Integrin-associated transcriptional characteristics of circulating tumor cells in breast cancer patients.

Background: Integrins enable cell communication with the basal membrane and extracellular matrix, activating signaling pathways and facilitating intracellular changes. Integrins in circulating tumor cells (CTCs) play a significant role in apoptosis evasion and anchor-independent survival. However, the link between CTCs expressing different integrin subunits, their transcriptional profile and, therefore, their functional activity with respect to metastatic potential remains unclear. Methods: Single-cell RNA sequencing of CD45-negative cell fraction of breast cancer patients was performed. All CTCs were divided into nine groups according to their integrin profile. Results: СTCs without the gene expression of integrins or with the expression of non-complementary α and ß subunits that cannot form heterodimers prevailed. Only about 15% of CTCs expressed integrin subunits which can form heterodimers. The transcriptional profile of CTCs appeared to be associated with the spectrum of expressed integrins. The lowest potential activity was observed in CTCs without integrin expression, while the highest frequency of expression of tumor progression-related genes, namely genes of stemness, epithelial-mesenchymal transition (EMT), invasion, proinflammatory chemokines and cytokines as well as laminin subunits, were observed in CTCs co-expressing ITGA6 and ITGB4. Validation on the protein level revealed that the median of integrin ß4+ CTCs was higher in patients with more aggressive molecular subtypes as well as in metastatic breast cancer patients. One can expect that CTCs with ITGA6 and ITGB4 expression will have pronounced metastatic potencies manifesting in expression of EMT and stemness-related genes, as well as potential ability to produce chemokine/proinflammatory cytokines and laminins.

Complex Analysis of Single-Cell RNA Sequencing Data.

Single-cell RNA sequencing (scRNA-seq) is a revolutionary tool for studying the physiology of normal and pathologically altered tissues. This approach provides information about molecular features (gene expression, mutations, chromatin accessibility, etc.) of cells, opens up the possibility to analyze the trajectories/phylogeny of cell differentiation and cell-cell interactions, and helps in discovery of new cell types and previously unexplored processes. From a clinical point of view, scRNA-seq facilitates deeper and more detailed analysis of molecular mechanisms of diseases and serves as a basis for the development of new preventive, diagnostic, and therapeutic strategies. The review describes different approaches to the analysis of scRNA-seq data, discusses the advantages and disadvantages of bioinformatics tools, provides recommendations and examples of their successful use, and suggests potential directions for improvement. We also emphasize the need for creating new protocols, including multiomics ones, for the preparation of DNA/RNA libraries of single cells with the purpose of more complete understanding of individual cells.

Metastasis prevention: How to catch metastatic seeds.

Despite considerable advances in the evolution of anticancer therapies, metastasis still remains the main cause of cancer mortality. Therefore, current strategies for cancer cure should be redirected towards prevention of metastasis. Targeting metastatic pathways represents a promising therapeutic opportunity aimed at obstructing tumor cell dissemination and metastatic colonization. In this review, we focus on preclinical studies and clinical trials over the last five years that showed high efficacy in suppressing metastasis through targeting lymph node dissemination, tumor cell extravasation, reactive oxygen species, pre-metastatic niche, exosome machinery, and dormancy.

Spatial Profile of Tumor Microenvironment in PD-L1-Negative and PD-L1-Positive Triple-Negative Breast Cancer.

The problem of finding more precise stratification criteria for identifying the cohort of patients who would obtain the maximum benefit from immunotherapy is acute in modern times. In our study were enrolled 18 triple-negative breast cancer patients. The Ventana SP142 test was used for PD-L1 detection. Spatial transcriptomic analysis by 10x Genomics was used to compare PD-L1-positive and PD-L1-negative tumors. The seven-color multiplex immunofluorescence (by Akoya) was used for the detection of the type of cells that carried the PD1 receptor and the PD-L1 ligand. Using pathway analysis, we showed that PD-L1-positive tumors demonstrate signatures of a cell response to cytokines, among others, and PD-L1-negative tumors demonstrate signatures of antigen presentation. PD-L1-positive and PD-L1-negative tumors have different tumor microenvironment (TME) compositions according to CIBERSORT analysis. Multiplex immunohistochemistry (IHC) confirmed the prevalence of PD1-negative M2 macrophages and PD1-negative T lymphocytes in PD-L1-positive tumors. PD-L1-positive tumors are not characterized by direct contact between cells carrying the PD1 receptor and the PD-L1 ligand. So, the absence of specific immune reactions against the tumor, predominance of pro-tumor microenvironment, and rare contact between PDL1 and PD1-positive cells may be the potential reasons for the lack of an immune checkpoint inhibitor (ICI) effect in triple-negative breast cancer patients.

Heterogeneity of Circulating Epithelial Cells in Breast Cancer at Single-Cell Resolution: Identifying Tumor and Hybrid Cells.

Circulating tumor cells and hybrid cells formed by the fusion of tumor cells with normal cells are leading players in metastasis and have prognostic relevance. This study applies single-cell RNA sequencing to profile CD45-negative and CD45-positive circulating epithelial cells (CECs) in nonmetastatic breast cancer patients. CECs are represented by transcriptionally-distinct populations that include both aneuploid and diploid cells. CD45- CECs are predominantly aneuploid, but one population contained more diploid than aneuploid cells. CD45+ CECs mostly diploid: only two populations have aneuploid cells. Diploid CD45+ CECs annotated as different immune cells, surprisingly harbored many copy number aberrations, and positively correlated to tumor grade. It is noteworthy that cancer-associated signaling pathways areabundant only in one aneuploid CD45- CEC population, which may represent an aggressive subset of circulating tumor cells. Thus, CD45- and CD45+ CECs are highly heterogeneous in breast cancer patients and include aneuploid cells, which are most likely circulating tumor and hybrid cells, respectively, and diploid cells. DNA ploidy analysis can be an effective instrument for identifying tumor and hybrid cells among CECs. Further follow-up study is needed to determine which subsets of circulating tumor and hybrid cells contribute to breast cancer metastasis.

Metastasis Prevention: Focus on Metastatic Circulating Tumor Cells.

Metastasis is the main cause of cancer death. Metastatic foci are derived from tumor cells that detach from the primary tumor and then enter the circulation. Circulating tumor cells (CTCs) are generally associated with a high probability of distant metastasis and a negative prognosis. Most CTCs die in the bloodstream, and only a few cells form metastases. Such metastatic CTCs have a stem-like and hybrid epithelial-mesenchymal phenotype, can avoid immune surveillance, and show increased therapy resistance. Targeting metastatic CTCs and their progenitors in primary tumors and their descendants, particularly disseminated tumor cells, represents an attractive strategy for metastasis prevention. However, current therapeutic strategies mainly target the primary tumor and only indirectly affect metastasis-initiating cells. Here, we consider potential methods for preventing metastasis based on targeting molecular and cellular features of metastatic CTCs, including CTC clusters. Also, we emphasize current knowledge gaps in CTC biology that should be addressed to develop highly effective therapeutics and strategies for metastasis suppression.

Prognosis of Different Types of Non-Small Cell Lung Cancer Progression: Current State and Perspectives.

Despite advances in diagnostics and therapy of non-small cell lung cancer (NSCLC), the problem of prognosis and prevention of tumor progression is still highly important. Even if NSCLC is diagnosed in the early stages, almost a quarter of patients develop relapse; most of them die from recurrent disease. A large number of different markers have been proposed to predict the risk of NSCLC progression; however, none of them are used in clinical practice. It is obvious that this situation is related to the economic and methodological complexity of the proposed markers and/or their insufficient efficiency due to a lack of effective study models and tumor heterogeneity. Another reason may be that potential markers are developed for NSCLC progression in general, which is represented by at least four pathogenetically-distinct processes: synchronous lymph node metastasis, local, regional, and distant recurrence. In this review, we summarize data from published literature on clinicopathological, genetic, and molecular factors associated with different types of NSCLC progression and emphasize challenges and approaches to developing prognostic factors. In conclusion, we highlight the importance of further studies to reveal molecular mechanisms of NSCLC progression and the need for differential analysis of markers of local, regional, and distant recurrences for disease prognosis.