Targeted Sequencing in Gastric Cancer: Association with Tumor Molecular Characteristics and FLOT Therapy Effectiveness.

Heterogeneity of gastric cancer (GC) is the main trigger of the disease's relapse. The aim of this study was to investigate the connections between targeted genes, cancer clinical features, and the effectiveness of FLOT chemotherapy. Twenty-one patients with gastric cancers (GCs) were included in this study. Tumor-targeted sequencing was conducted, and real-time PCR was used to assess the expression of molecular markers in tumors. Seven patients with stabilization had mutations that were related to their response to therapy and were relevant to the tumor phenotype. Two patients had two mutations. The number of patients with TP53 mutations increased in HER2-positive tumor status. PD-L1-positive cancers had mutations in KRAS, TP53, PIK3CA, PTEN, and ERBB, which resulted in an increase in PD-1 expression. TP53 mutation and PTEN mutation are associated with changes in factors associated with neoangiogenesis. In concusion, patients who did not have aggressive growth markers that were verified by molecular features had the best response to treatment, including complete morphologic regression.

Safety and Efficacy of Neoadjuvant Chemoimmunotherapy in Gastric Cancer Patients with a PD-L1 Positive Status: A Case Report.

INTRODUCTION: The landscape of gastric cancer treatment has changed owing to the widespread use of immune checkpoint inhibitors. Autophagy, involved in regulating the immune system, is a potential trigger of immunity in tumors. This study aims to find molecular-based evidence for the effectiveness of FLOT chemotherapy with immune checkpoint inhibitors in gastric cancer patients. MATERIALS AND METHODS: Three patients with advanced gastric cancer received FLOT neoadjuvant chemotherapy with immunotherapy and surgery. IHC was used to determine the PD-L1 status. Real-time PCR was used to analyze expression patterns of transcriptional growth factors, AKT/mTOR signaling components, PD-1, PD-L1, PD-L2 and LC3B. The LC3B content was measured via Western blotting analysis. RESULTS: The combination of FLOT neoadjuvant chemotherapy and immunotherapy was found to be efficient in patients with a PD-L1-positive status. Gastric tumors with a PD-L1-positive status exhibited autophagy activation and decreased PD-1 expression. CONCLUSIONS: FLOT chemotherapy combined with immune checkpoint inhibitors showed high efficacy in gastric cancer patients with a positive PD-L1 status. Autophagy was involved in activating the tumor immunity. Further research is needed to clarify the mechanism of effective anticancer treatment.

LC3B, mTOR, AMPK Are Molecular Targets for Neoadjuvant Chemotherapy in Gastric Cancers.

Autophagy plays a dual role in oncogenesis processes. On one hand, autophagy enhances the cell resistance to oncogenic factors, and on the other hand, it participates in the tumor progression. The aim of the study was to find the associations between the effectiveness of the FLOT regimen in resectable gastric cancers (GCs) with the key autophagy-related proteins. Materials and Methods: The study included 34 patients with morphologically verified gastric cancer. All patients had FLOT neoadjunvant chemotherapy (NACT) (fluorouracil, leucovorin, oxaliplatin, and docetaxel) followed by gastrectomy. The studied tissue material was the non-transformed and tumor tissues obtained during diagnostic video gastroscopy in patients before the start of the combined treatment and after surgical treatment, frozen after collection. The LC3B, mTOR, and AMPK expression was determined by real-time PCR. The content of the LC3B protein was determined by Western blotting analysis. Results: The mRNA level and the content of the LC3B protein were associated with the tumor stage and the presence of signet ring cells. The AMPK mRNA level was increased in patients with the T4N0-2M0 stage by 37.7 and 7.33 times, which was consequently compared with patients with the T2N0M0 and T3N0-1M0 stages. The opposite changes in the mTOR and AMPK in the GCs before anti-cancer therapy were noted. The tumor size and regional lymph node affections were associated with a decrease in the mTOR mRNA level. A decrease in the mTOR expression was accompanied by an increase in the AMPK expression in the GCs. The mTOR expression was reduced in patients with a cancer spreading; in contrast, AMPK grew with the tumor size. There was an increase in the LC3B expression, which can probably determine the response to therapy. An increase in LC3B mRNA before the start of treatment and the protein content in cancers after NACT with a decrease in therapy effectiveness was recorded. There was an increase in the protein level in patients with partial regression and stabilization by 3.65 and 5.78 times, respectively, when compared with patients with complete tumor regression was noted. Conclusions: The anticancer effectiveness in GCS is down to the LC3B, mTOR, and AMPK expression. These were found to be entire molecular targets affecting the cancer progression and metastasis as well as the NACT effectiveness.

Functional activity of natural killer cells in biological fluids in patients with colorectal and ovarian cancers.

AIM OF THE STUDY: To compare the functional activity of natural killer cells depending on the presence of a malignant process and its dissemination. MATERIAL AND METHODS: The study included 20 patients with Stage IIIB, C (FIGO, 2009) ovarian cancer, 10 patients with benign ovarian tumours (BOT), and 20 patients with colorectal cancer (T2-4N0-2M0). The control group consisted of 9 healthy donors. To evaluate the number and functional activity of NK cells, multicolour flow cytometry was performed. RESULTS: In cancer patients, the relative number of activated NK cells secreting granzyme B (GB) (CD56+CD107a+GB+PF-) was significantly decreased, and the proportion of degranulated NK cells (CD56+CD107a+GB-PF-) was significantly increased, compared to those observed in healthy donors. The total number of NK cells in peripheral blood was low in ovarian cancer patients (p < 0.05). The proportion of activated peripheral blood NK cells containing cytolytic granules GB and perforin (PF) in colorectal cancer patients increased with tumour growth. However, lymph node metastasis did not affect the content and activation of NK cells. Comparative analysis of NK-cell populations in patients with benign and malignant ovarian tumours revealed that the level of CD56+ cells was significantly higher in ascites than in peripheral blood. However, CD56+CD107a+ activated cells and CD56+CD107a+GB+PF+ cells were found more frequently in ascites of BOT patients than in ovarian cancer patients. The degranulated population of NK cells (CD56+CD107a+GB-PF-) was mainly observed in the peripheral blood of ovarian cancer patients.

The Composition of Small Extracellular Vesicles (sEVs) in the Blood Plasma of Colorectal Cancer Patients Reflects the Presence of Metabolic Syndrome and Correlates with Angiogenesis and the Effectiveness of Thermoradiation Therapy.

The majority of colorectal cancer patients (CRCPs) develop tumors on the background of "metabolically healthy obesity" or metabolic syndrome. The aim of the work was to study the levels of matrix metalloproteinases (MMPs) and heat shock proteins (HSPs) on the surface of blood plasma CD9-positive and FABP4-positive small extracellular vesicles (sEVs) from CRCPs depending on metabolic status and tumor angiogenesis, as well as to evaluate the sEVs markers as predictors of the effectiveness of thermoradiotherapy. In CRCPs, compared with patients with colorectal polyps (CPPs), the proportion of triple positive EVs and EVs with the MMP9+MMP2-TIMP1+ phenotype increased significantly among FABP4-positive EVs (adipocyte-derived EVs), which in general may indicate the overexpression of MMP9 and TIMP1 by adipocytes or adipose tissue macrophages in CRCPs. The results obtained have prospects for use as markers to clarify cancer risk in CPPs. One can assume that for CRCPs with metabolic syndrome or metabolically healthy obesity, it is the FABP4+MMP9+MMP2-TIMP1- population of circulating sEVs that is the most optimal biomarker reflecting tumor angiogenesis. Determining this population in the blood will be useful in monitoring patients after treatment for the early detection of tumor progression. CD9+MMP9+MMP2-TIMP1- and MMP9+MMP2-TIMP1+ subpopulations of circulating sEVs are the most promising predictors of the efficacy of thermoradiation therapy because their levels at baseline differ significantly in CRCPs with different tumor responses.

Nanoparticles in Gastric Cancer Management.

Gastric cancer is the second most common cause of cancer-related deaths in the world. The surgical management of the tumor is the best therapeutic option for gastric cancer patients. A combination of a heterogeneous distribution of genetic and environmental factors appears to be required to explain patients' poor prognosis. A search for targeted and molecular-based approaches is affected by the optimal gastric cancer drug management. The modern multidisciplinary approach to treating the pathology used worldwide prolongs the overall patient survival and decreases the rate of recurrence. An understanding of the mechanisms that underlie therapies will provide new insights into gastric cancer treatment. The improvement in medicine will probably be associated with a study of tumor biology, followed by a personalized and molecular-based approach development in anticancer drugs administration. The modern perspective in gastric cancer detection and treatment is the application of nanoparticles. Nanoparticles affecting the intensity of biological processes in cancer cells can be used to treat cancers to increase the effectiveness of anti-tumor therapy. Their cytotoxicity involves a wide range of pathological events. Their targets are the extracellular matrix degradation, epithelial-mesenchymal transition, tumor angiogenesis, tumor microenvironment modulation. These are accompanied by lipid peroxidation, apoptosis, and autophagic flux. Preliminary studies on the efficacy of the use of nanoparticles in cultured gastric cancers open new opportunities for anti-tumor treatment to overcome the toxicity of therapeutic agents and decrease the rate of resistance to anticancer drugs and therapies.

Exosomal Protease Cargo as Prognostic Biomarker in Colorectal Cancer.

OBJECTIVE: The aim of the study was to develop a model for predicting cancer risk in colorectal polyps' patients (CPPs), as well as to reveal additional prognosis factors for Stage III colorectal cancer based on differences in subpopulations of tetraspanins, tetraspanin-associated and tetraspanin-non-associated proteases in blood plasma exosomes of CPPs and colorectal cancer patients (CRCPs). METHODS: The subpopulations of CD151- and Tspan8-positive exosomes, the subpopulations of metalloproteinase at the surface of СD9-positive exosomes and the level of 20S proteasomes in plasma exosomes in 15 CPPs (tubulovillous adenomas) and 60 CRCPs were evaluated using flow cytometry and Western blotting. Logistic regression analysis was performed to predict cancer risk of CPPs. RESULTS: The levels of 20S proteasomes in exosomes, MMP9+, MMP9+/MMP2+/EMMPRIN+ in CD9-positive blood plasma exosomes are associated with the risk of malignant transformation of colorectal tubulovillous adenomas.  In patients with Stage III CRC, the levels of 20S proteasomes (less than 2 units) and MMP9+ subpopulations (more than 61%) in plasma exosomes are unfavorable prognostic factors for overall survival. The levels of 20S proteasomes and ADAM10+/ADAM17- subpopulations in CD9-positive blood plasma exosomes are the most significant values for predicting relapse-free survival. CONCLUSION: Protease cargo in CD9-positive blood plasma exosomes is prognostic biomarker for colorectal polyps and colorectal cancer.

Molecular Mechanism of Resistance to Chemotherapy in Gastric Cancers, the Role of Autophagy.

Gastric cancer (GC) is biologically and genetically heterogeneous with complex carcinogenesis at the molecular level. Despite the application of multiple approaches in the GC treatment, its 5-year survival is poor. A major limitation of anti-cancer drugs application is intrinsic or acquired resistance, especially to chemotherapeutical agents. It is known that the effectiveness of chemotherapy remains debatable and varies according to the molecular type of GC. Chemotherapy has an established role in the management of GC. Perioperative chemotherapy or postoperative chemotherapy is applied for localized ones. Most of the advanced GC patients have a poor response to treatment and unfavorable outcomes with standard therapies. Resistance substantially limits the depth and duration of clinical responses to targeted anticancer therapies. Through the use of complementary experimental approaches, investigators have revealed that cancer cells can achieve resistance through adaptation or selection driven by specific genetic, epigenetic, or microenvironmental alterations. Ultimately, these diverse alterations often lead to the activation of MAPK, AKT/mTOR, and Wnt/ß-catenin signaling pathways that, when co-opted, enable cancer cells to survive drug treatments. We have summarized the mechanisms of resistance development to cisplatin, 5-fluorouracil, and multidrug resistance in the GC management. The complexity of molecular targets and components of signaling cascades altered in the resistance development results in the absence of significant benefits in GC treatment, and its efficacy remains low. The universal process responsible for the failure in the multimodal approach in GC treatment is autophagy. Its dual role in oncogenesis is the most unexplored issue. We have discussed the possible mechanism of autophagy regulation upon the action of endogenous factors and drugs. The experimental data obtained in the cultured GC cells need further verification. To overcome the cancer resistance and to prevent autophagy as the main reason of ineffective treatment, it is suggested the concept of the direct influence of autophagy molecular markers followed by the standard chemotherapy. Dozen of studies have focused on finding the rationale for the benefits of such complex therapy. The perspectives in the molecular-based management of GC are associated with the development of molecular markers predicting the protective autophagy initiation and search for novel targets of effective anticancer therapy.

Relation between Tetraspanin- Associated and Tetraspanin- Non- Associated Exosomal Proteases and Metabolic Syndrome in Colorectal Cancer Patients

Purpose: Exosomal proteases are important in regulation of molecular signaling from growth factor receptors and adhesion molecules and also the regulation of cell motility and protein folding. The aim of this study was to evaluate the level of ADAM10, ADAM17 and 20S proteasomes in exosomes isolated from colorectal cancer patients (CRCPs) in relation with clinical and histopathological parameters. Methods: Blood plasma exosomes of 60 CRCPs at stage T2-4N0-2M0-1 and 10 control subjects (CSs) with colorectal polyps were isolated using ultrafiltration in combination with ultracentrifugation. The level of tetraspanin-associated (ADAM20 and ADAM17) and tetraspanin-non-associated (20S proteasome) proteases were evaluated by flow cytometry and western blot analysis. Results: The ADAM10-/ ADAM17- population predominated in plasma exosomes of CRCPs and the level of ADAM10+ exosomes was significantly higher in exosomes of CSs compared with CRCPs. No difference was found between subpopulations of ADAM10/ADAM17 exosomes and level of exosomal 20S proteasomes in terms of sex, age and tumor grade. Simultaneous decrease of ADAM10+/ADAM17-subpopulation of exosomes and level of exosomal 20S proteasomes in patients with metastatic CRC was observed compared with patients with non-metastatic CRC. The level of ADAM17+ exosomes significantly reduced in exosomes of CRCPs with metabolic syndrome compared to CRCPs without metabolic syndrome( 3.97±0.71 (%) vs. 13.04±1.34 (%), respectively (p<0.05). A decrease in the 20S proteasomes level in plasma exosomes was revealed in CRCPs with metabolic syndrome compared with CRCPs without metabolic disorders ( 1.90±0.25 (r.u.) vs. 2.92±0.42 (r.u.) respectively( (p<0.05). Conclusion: According to findings of this study, it seems that exosomal proteases can be promising molecular predictors of hematogenous metastasis in patients with non-metastatic CRC. Further studies on subpopulation composition of exosomes CRCPs are need for elucidating the role of tetraspanin-associated and tetraspanin-non-associated exosomal proteases in CRC development and progression.

The role of metabolic syndrome variant in the malignant tumors progression.

Metabolic syndrome (MS) is one of the leading risk factors for the development of some common cancers (endometrial cancer, postmenopausal breast cancer, colorectal cancer). Currently, a drug-induced metabolic syndrome related with androgen deprivation therapy in patients with prostate cancer represents a serious medical problem. Not only MS, or its individual components, but MS variants with different levels of leptin, adiponectin, visfatin, resistin are associated with tumor invasion, metastasis and survival rates in patients with MS-associated malignancies.

Molecular targets for the therapy of cancer associated with metabolic syndrome (transcription and growth factors).

Metabolic syndrome (MS) is one of the leading risk factors for the development of cardiovascular diseases, type II diabetes mellitus and reproductive system diseases. Currently, not only cardiovascular disease and reproductive history risks related with MS are frequently discussed, but it has been also shown that MS is associated with increased risk of some common cancers (endometrial cancer, postmenopausal breast cancer, colorectal cancer, biliary tract cancers and liver cancer for men). Further studies are required to understand the mechanisms of the involvement of MS components in the pathogenesis of malignant neoplasms. Changes in the expression of transcription and growth factors in the peripheral tissues as well as in cancer tissues of patients with MS were revealed. Transcription factors (AMP-activated protein kinase-1, STAT3, sterol regulatory element-binding protein-1 and peroxisome proliferator-activated receptor-γ), leptin and adiponectin receptors seem to be the most promising molecular targets for the therapy of cancers associated with MS.

Association of IGFBP-6 Expression with Metabolic Syndrome and Adiponectin and IGF-IR Receptor Levels in Colorectal Cancer.

PURPOSE: To assess IGFBP-6 expression in relation with the presence of the metabolic syndrome, adiponectin receptors (AdipoR1 and AdipoR2) and IGF-IR levels in colorectal adenocarcinoma cases. MATERIALS AND METHODS: IGFBP-6 mRNA and protein levels were analyzed using real-time quantitative PCR and Western blotting in 46 patients. ELISA and ow cytometry were used for evaluation of AdipoR1, AdipoR2 and IGF-IR. RESULTS: The results showed that IGFBP-6 mRNA expression and the IGFBP-6 content were higher in tumor tissue samples of colorectal cancer patients with and without the metabolic syndrome. In addition, IGFBP-6 mRNA expression was associated with tumor invasion (tumor size) and the IGFBP-6 protein level was associated with nodal status. Positive correlations and positive nonlinear relations were found between the IGFBP-6 level and the AdipoR1 and AdipoR2 contents in colorectal cancer patients. CONCLUSIONS: The IGFBP-6 mRNA level and protein level were found to be associated with presence of the metabolic syndrome. Positive correlations indicated probable cross-talk between the IGF-IR-mediated and adiponectin-mediated signaling pathways in colorectal carcinomas. IGFBP-6 may be considered as a potential biomarker associated with lymphogenous metastasis and the metabolic syndrome in colorectal cancer.