Silencing Rac1 and Prex1 Inhibit Epithelial-Mesenchymal Transition in Human Gastric Cancer Cells Induced by Transforming Growth Factor-ß1.

BACKGROUND/AIMS: Transforming growth factor-beta can influence tumor cells, causing epithelial-mesenchymal transition and enhancing their invasion and metastasis ability. Rac1 protein could be used as an independent tumor diagnostic marker and survival predictor. Prex1 is closely related to cell metastasis. In this study, the impact of silencing Rac1 and Prex1 on transforming growth factor-beta 1-induced epithelial-mesenchymal transition and apoptosis of human gastric cancer cells MGC-803 and MKN45 was investigated. MATERIALS AND METHODS: MGC-803 and MKN45 cells received recombinant transforming growth factor-beta 1 (rTGF-ß1) treatments at various concentrations. Cell Counting Kit-8 kit was used to determine cell viability. Rac1 and Prex1 interference vectors were transfected into the rTGF-ß1-treated MGC-803 and MKN45 cells. Cell apoptosis and migration were detected by flow cytometry and scratch test, respectively. Western blot was used to detect the epithelial-mesenchymal transition-related markers E-cadherin, N-cadherin, vimentin, and PDLIM2 expression levels. RESULTS: The rTGF-ß1 (10 ng/mL) could promote MGC-803 and MKN45 cell viability. Silencing Rac1 and Prex1 could increase E-cadherin and PDLIM2 expression, decrease N-cadherin and vimentin expression, inhibit cell viability and migration, and promote apoptosis in rTGF-ß1-treated MGC-803 and MKN45 cells. CONCLUSIONS: Silencing Rac1 and Prex1 could inhibit epithelial-mesenchymal transition, reduce cell viability and migration, and promote apoptosis in human gastric cancer cells.

KMT2D promotes proliferation of gastric cancer cells: evidence from ctDNA sequencing.

BACKGROUND: ctDNA sequencing could be used for early cancer screening, prognosis prediction, and medication guidance. However, data of its application in gastric cancer are still lacking. In this study, using ctDNA sequencing, we aimed to screen the mutant genes closely associated with gastric cancer and to explore the impact of these genes on gastric cancer development. METHODS: ctDNA for high-throughput sequencing was obtained from gastric cancer patients, and the high-frequency mutant gene KMT2D was identified. Immunohistochemical examination was conducted to assess the expression of KMT2D in gastric cancer tissues. KMT2D knockdown was performed to establish the stably transfected gastric cancer cells. Then, CCK8, plate clone formation assay, and Transwell assay were conducted, and a subcutaneous tumor-bearing model was induced in nude mice to investigate the changes in cell proliferation and invasion capability. Transcriptome sequencing was also performed to investigate the differences in cellular gene expression. RESULTS: Detection of ctDNA found 113 gastric cancer-related mutations, 11 of which are the top 20 high-frequency mutations of gastric cancer recorded by COSMIC (Catalogue of Somatic Mutations in Cancer, COSMIC). They are TP53, ARID1A, CDH1, PIK3CA, KMT2C, KMT2D, APC, SPEN, CTNNB1, SETBP1, and KMT2A. The gene closely related to the clinical characteristics of the patient is KMT2D. The high-frequency mutant gene KMT2D was identified in gastric cancer tissues. The positive rate of KMT2D expression in cancer tissues was 74.3%, which was higher than that in para-carcinoma tissues (56.8%). The knockdown of KMT2D inhibited the proliferation, invasion, and tumor formation capacity of the gastric cancer cells, causing differences in the gene expression profiles, and the expression of different functional gene clusters was up- or downregulated. CONCLUSION: The findings of this study revealed that KMT2D could be an oncogene capable of promoting gastric cancer proliferation.

Comprehensive Analysis of CDC27 Related to Peritoneal Metastasis by Whole Exome Sequencing in Gastric Cancer.

INTRODUCTION: The peritoneum is the most common metastatic site of gastric cancer and is associated with a dismal prognosis. However, there is no reliable biomarker for predicting peritoneal metastasis (PM). MATERIALS AND METHODS: Whole-exome sequencing (WES) was performed on formalin-fixed, paraffin-embedded (FFPE) samples from 63 patients with stage I-III gastric cancer and circulating tumor DNA (ctDNA) samples from 10 patients with stage IV gastric cancer. Differentially expressed genes (DEGs) were identified between the PM and non-PM groups and analyzed by multiple bioinformatics analyses. Univariate and multivariate Cox regression analyses were used to identify the risk factors for PM and a risk score model was developed. RESULTS: The number of mutant genes and the tumor mutation burden (TMB) in the PM group were higher than those in the non-PM group (p < 0.05). There was a significant positive correlation between the number of mutant genes and the TMB (R2 = 0.9997). The risk of PM was significantly higher in the high TMB group than in the low TMB group (p = 0.045). Forty-nine DEGs were identified as associated with PM in gastric cancer. CDC27 mutations were associated with a higher risk for PM and poor survival. The CDC27 mutations were located in the Apc3 region, the TPR region, and the phosphorylation region, and new mutation sites were not included in the TCGA database. Multivariable Cox regression analysis demonstrated that pathological T stage, poor tumor differentiation, Borrmann type, and CDC27 mutations were independent predictive factors of PM. A risk score model was constructed that demonstrated good performance. CONCLUSION: Through WES, we identified 49 DEGs relevant to PM in gastric cancer. CDC27 mutations were independently associated with PM by statistical and bioinformatics analyses. A risk score model was built and was demonstrated to effectively discriminate gastric cancer patients with and without PM.

Detection of circulating tumor cell DNA for monitoring advanced gastric cancer.

INTRODUCTION: Circulating tumor DNA (ctDNA) for monitoring the effects of chemotherapy and predicting prognosis in advanced gastric cancer have not been thoroughly investigated. METHODS: We performed next-generation sequencing (NGS) of ctDNA from 23 gastric cancer patients. Then the genetic information and clinical information were statistically analyzed. RESULTS: In this study, the frequency of TP53 was significantly different between the effective and ineffective groups (P = 0.040), and the number of TP53 mutations was more frequent in the ineffective group. Missense mutation was a significant difference between the treatment effect groups (P = 0.026). The number of gene mutations and the change in copy number levels were related to therapeutic effect. Among the ineffective group, there was a significant difference in the number of gene mutations (P = 0.0006). We further divided the number of gene mutations into an increase group and a decrease group, and found that there was a significant difference between the effective and ineffective groups (P = 0.038). Finally, it was found that patients with high mutation abundance of gastric cancer had a shorter overall survival than patients with low mutation abundance (P<0.05). CONCLUSION: ctDNA can be used as an effective tool to monitor the efficacy of chemotherapy and predict prognosis in advanced gastric cancer.

A Comprehensive Survey of Genomic Alterations in Gastric Cancer Reveals Recurrent Neoantigens as Potential Therapeutic Targets.

Immunotherapy directed against cancer-specific neoantigens derived from non-silent mutants is a promising individualized strategy for cancer treatment. Neoantigens shared across patients could be used as a public resource for developing T cell-based therapy. To identify potential public neoantigens for therapy in gastric cancer (GC), 74 GC patients were enrolled in this study. Combined with the TCGA cohort and other published studies, whole exome sequencing data from 942 GC patients were used to detect somatic mutations and predict neoantigens shared by GC patients. The mutations pattern between our study and the TCGA cohort is comparable, and C > T is the most common substitution. The number of neoantigens was significantly higher in older patients (age ≥60) compared to younger patients (age <60), both in this study and the TCGA cohort. Recurrent neoantigens were found in eight genes (TP53, PIK3CA, PGM5, ERBB3, C6, TRIM49C, OR4C16, and KRAS) in this study. The neoantigen-associated mutations PIK3CA (p.H1047R) and TP53 (p.R175H) are common across several cancer types, indicating their potential usage. Overall, our study illustrates a comprehensive genomic landscape of GC and provides the recurrent neoantigens to facilitate further immunotherapy.

Molecular Profiles and Metastasis Markers in Chinese Patients with Gastric Carcinoma.

The goal of this work was to investigate the molecular profiles and metastasis markers in Chinese patients with gastric carcinoma (GC). In total, we performed whole exome sequencing (WES) on 74 GC patients with tumor and adjacent normal formalin-fixed, paraffin-embedded (FFPE) tissue samples. The mutation spectrum of these samples showed a high concordance with TCGA and other studies on GC. PTPRT is significantly associated with metastasis of GC, suggesting its predictive role in metastasis of GC. Patients carrying BRCA2 mutations tend not to metastasize, which may be related to their sensitivity to chemotherapy. Mutations in MACF1, CDC27, HMCN1, CDH1 and PDZD2 were moderately enriched in peritoneal metastasis (PM) samples. Furthermore, we found two genomic regions (1p36.21 and Xq26.3) were associated with PM of GC, and patients with amplification of 1p36.21 and Xq26.3 have a worse prognosis (P = 0.002, 0.01, respectively). Our analysis provides GC patients with potential markers for single and combination therapies.

Paclitaxel, 5-fluorouracil, and leucovorin combination chemotherapy as first-line treatment in patients with advanced gastric cancer.

The aim of this retrospective analysis was to evaluate the efficacy and toxicity of combination chemotherapy with paclitaxel, 5-fluorouracil, and leucovorin (TFL) as first-line treatment in patients with advanced gastric cancer (AGC). One hundred and thirteen patients were enrolled in the study who were confirmed to have AGC by histopathology. These patients were treated with TFL: paclitaxel at a dose of 135 mg/m as a 3-h intravenous infusion on day 1, LV 400 mg/m as an intravenous infusion over 2 h on day 1, followed by 5-fluorouracil 2400 mg/m as an infusion over a 46-h period on 3 consecutive days. Cycles were repeated every 2 weeks. A total of 113 patients were assessed for their response to therapy. A total of three patients achieved complete responses and 46 patients achieved partial responses, yielding an overall objective response rate of 43.4% [95% confidence interval (CI): 34.3-52.5%]. Fifty-four cases of stable disease and 10 cases of progressive disease were observed in the remaining patients. The median time to progression and overall survival were 5.2 months (95% CI: 4.7-5.8 months) and 14.1 months (95% CI: 12.5-15.8 months), respectively. Toxicities were tolerable and moderate. The most common grade 3-4 toxicities included leukopenia (16.8%), neutropenia (17.7%), anemia (8.0%), thrombocytopenia (5.3%), and fatigue (6.2%). Combination chemotherapy with TFL offers an active and safe therapeutic approach for patients with AGC.

Next-generation sequencing of circulating tumor DNA for detection of gene mutations in lung cancer: implications for precision treatment.

BACKGROUND: Lung cancer remains a major global health problem, which causes millions of deaths annually. Because the prognosis is mainly determined by the stage of lung cancer, precise early diagnosis is of great significance to improve the survival and prognosis. Circulating tumor DNA (ctDNA) has been recognized as a sensitive and specific biomarker for the detection of early- and late-stage lung cancer, and next-generation sequencing (NGS) of ctDNA has been accepted as a noninvasive tool for early identification and monitoring of cancer mutations. This study aimed to assess the value of NGS-based ctDNA analysis in detecting gene mutations in lung cancer patients. METHODS: A total of 101 subjects with pathological diagnosis of lung cancer were enrolled, and blood samples were collected. ctDNA samples were prepared and subjected to NGS assays. RESULTS: There were 31 cases harboring 40 gene mutations, and EGFR was the most frequently mutated gene (27.72%). In addition, there were seven cases with double mutations and one case with triple mutations, with EGFR p.T790M mutation exhibiting the highest frequency. CONCLUSION: Our findings demonstrate that NGS of ctDNA is effective in detecting gene mutations in lung cancer patients, and may be used as a liquid biopsy for lung cancer, which facilitates the development of precision treatment regimens for lung cancer.

Insight into resistance mechanism of anaplastic lymphoma kinase to alectinib and JH-VIII-157-02 caused by G1202R solvent front mutation.

BACKGROUND: Mutated anaplastic lymphoma kinase (ALK) drives the development of advanced non-small cell lung cancer (NSCLC). Most reported small-molecule inhibitors targeting the ALK domain do not display good inhibition of the G1202R solvent front mutation. The solvent front mutation was assumed to hinder drug binding. However, a different fact could be uncovered by the simulations reported in this study through a structural analog of alectinib (JH-VIII-157-02), which demonstrated potent effects against the G1202R mutation. METHODS: Molecular docking, conventional molecular dynamics (MD) simulations, free energy calculations, and umbrella sampling (US) simulations were carried out to make clear the principles of the binding preferences of alectinib and JH-VIII-157-02 toward ALKWT and the ALK G1202R (ALKG1202R) mutation. RESULTS: JH-VIII-157-02 has similar binding affinities to both ALKWT and ALKG1202R whereas it has has a much lower binding affinity for alectinib to ALKG1202R. Analysis of individual energy terms indicate the major variation involves the van der Waals and entropy terms. Structural analysis reveals that the conformational change of the ATP-binding glycine-rich loop was primarily responsible for the alectinib resistance, not JH-VIII-157-02. In addition, US simulations prove JH-VIII-157-02 has similar dissociative processes from both ALKWT and ALKG1202R, while alectinib is more easily dissociated from ALKG1202R than from ALKWT, thus indicating lesser residence time. CONCLUSION: Both the binding affinity and the drug residence time should be emphasized in rational drug design to overcome the G1202R solvent front mutation in ALK resistance.

Combination chemotherapy with paclitaxel and oxaliplatin as first-line treatment in patients with advanced gastric cancer.

PURPOSE: This study is a retrospective analysis evaluating the efficacy and toxicity of combination chemotherapy with Paclitaxel (PTX) and Oxaliplatin (OXA) as first-line treatment for patients with advanced gastric cancer (AGC). METHODS: One hundred and seven patients with locally advanced or metastatic gastric adenocarcinoma received intravenous infusions of PTX at 135 mg/m2 and OXA at 85 mg/m2 on day 1 every 14 days. RESULTS: Among 107 patients enrolled, 9 patients could not be evaluated for a response because of the absence of any measurable lesions. Assessment of the response of 98 patients was made. The overall objective response rate was 42.9% (95% CI 32.9-52.8%), with two complete responses and 40 partial responses. The disease control was 79.6% (95% CI 71.5-87.7%). With 29 months of the median time of follow-up, the median progression-free survival was 5.8 months (95% CI 4.30-7.30 months) and the median overall survival was 11.5 months (95% CI 9.08-13.9 months). The 1-year survival rate was 48.0%. The most common grades 3 and 4 toxicities included neutropenia (32.7%), leucopenia (17.8%), fatigue (5.61%), and anemia (4.67%). Peripheral neuropathy occurred in 23.4% patients and grade 2 or higher peripheral neuropathy occurred in 12.1% of the patients. CONCLUSIONS: Combination chemotherapy with PTX and OXA offers a new, effective and safe regimen for patients with advanced gastric cancer.

Paclitaxel, oxaliplatin, 5-fluorouracil and leucovorin combination chemotherapy in patients with recurrent or metastatic gastric cancer.

PURPOSE: This study evaluated the efficacy and toxicity of combination chemotherapy with paclitaxel, oxaliplatin, 5-fluorouracil and leucovorin (POFL) in patients with recurrent or metastatic gastric cancer. METHODS: One hundred and thirty-eight patients with histologically confirmed recurrent or metastatic gastric adenocarcinoma were treated with the POFL regimen: paclitaxel at a dose of 135 mg/m2 as a 3-hour intravenous infusion on day 1, oxaliplatin 85 mg/m2 and leucovorin 400 mg/m2 as an intravenous infusion over 2 hours on day 1, followed by 5-fluorouracil 2,400 mg/m2 as an infusion over a 46-hour period on 3 consecutive days, in a 2-week cycle. RESULTS: Twelve patients could not be evaluated for response because of the absence of any measurable lesions or early discontinuation of therapy, so responses were assessed in 126 patients. The overall objective response rate was 56.3% (95% CI, 47.5%-64.9%). The median time to progression was 6.7 months (95% CI, 5.8-7.6 months), and the median overall survival was 12.6 months (95% CI, 11.3-13.9 months). The most common grade 3 and 4 toxicities were neutropenia (50.7%), peripheral neurotoxicity (16.7%) and alopecia (27.5%). CONCLUSIONS: Combination chemotherapy with POFL offers a new, active and safe approach to the treatment of recurrent or metastatic gastric cancer.

Peritoneal recurrence in gastric cancer following curative resection can be predicted by postoperative but not preoperative biomarkers: a single-institution study of 320 cases.

To determine the risk factors for peritoneal recurrence in gastric cancer patients after curative resection, we included 320 patients with stage I-III primary gastric cancer between January 2008 and June 2012. Data on each patient's surgical and pathological information, preoperative and postoperative tumor markers were collected and analyzed retrospectively. The risk factors for peritoneal recurrence were investigated by univariate and multivariate analysis. In patients with peritoneal recurrence, advanced T or N stage, low differentiation, vascular/lymphatic invasion, perineural invasion, and elevated postoperative CEA/CA19-9 were more common than in patients without peritoneal recurrence. Patients with peritoneal recurrence showed a worse overall survival (OS) compared to those without peritoneal recurrence. In addition, patients with peritoneal recurrence within the first year had a worse OS compared to those with recurrence after 1 year. The univariate and multivariate analyses revealed that elevated number of metastatic lymph nodes and elevated postoperative CEA and CA19-9 were three independent risk factors for peritoneal recurrence in gastric cancer patients. For patients with N3 stage and high postoperative CEA and CA19-9, we found an initial steep slope within approximately 1 year and a subsequent gentle slope in the risk curve. Combined receiver operating characteristic curve analysis using the three independent risk factors for peritoneal recurrence yielded an area under the curve value of 0.73 with 73.7% sensitivity and 64.2% specificity. Therefore, the risk factors may be associated with peritoneal recurrence after curative resection in selected gastric cancer patients.

Paclitaxel combined with oxaliplatin as first-line chemotherapy for locally advanced or metastatic gastric cancer.

PURPOSE: This retrospective study was designed to evaluate the efficacy and safety of Paclitaxel (PTX) combined with Oxaliplatin (OXA) as first-line chemotherapy for locally advanced or metastatic gastric cancer (AGC). METHODS: Untreated patients with histologically confirmed AGC who received PTX at 135 mg/m(2) and OXA at 85 mg/m(2) every 2 weeks were studied. Antitumor activity was assessed by imaging and toxicities were evaluated. RESULTS: Thirty-nine (39) patients were enrolled. With 9.83 months median time of follow-up, 1 year OS rate was 42.0%. Complete response, partial response, stable disease and progressive disease was 2.6, 66.7, 17.9 and 12.0% respectively, the overall response rate was 69.2%. The mPFS was 8.5 months and the mOS 14.4 months. Grade 3/4 of toxicities included neutropenia (38.5%), febrile neutropenia (20.5%), vomiting (7.7%) and hypertransaminasemia (7.7%). Grade 2 peripheral neuropathy occurred in 33.3% patients. CONCLUSIONS: The combination of PTX combined with OXA is an active and safe regime for AGC and has a high overall response rate.

Combination Chemotherapy with S-1 and Oxaliplatin (SOX) as First-Line Treatment in Elderly Patients with Advanced Gastric Cancer.

This study is a retrospective analysis evaluating the efficacy and toxicity of combination chemotherapy with S-1 and oxaliplatin (SOX) as first-line treatment in elderly patients with advanced gastric cancer. One hundred and twenty-nine patients with recurrent or metastatic gastric adenocarcinoma were treated with SOX; S-1 (40-60 mg depending on patient's body surface area) was given orally, twice daily on days 1 to 14 followed by a 7-day rest period, 130 mg/m(2) oxaliplatin was given as an intravenous infusion over 2-hours on day one. The cycle was repeated every three weeks. All of the patients were older than 65 years. Among 129 patients enrolled, nine patients could not be evaluated for responses because of the absence of any measurable lesions or early discontinuation of therapy. Assessment of the response of 120 patients was made. The overall objective response rate was 54.2 % (95 %CI, 45.3-63.1 %), with three complete responses and 62 partial responses. The disease control rate was 80.8 % (95 %CI, 73.8-87.8 %). The median follow-up period was 23 months (range, 5-42 months). The median time to progression was 6.9 months (95 %CI, 5.5-8.3 months) and the median overall survival was 12.8 months (95 %CI, 11.4-14.2 months). The one-year survival rate was 57.5 % (95 %CI, 48.7-66.3 %). In 129 patients assessed safety, grade 3 and 4 toxicities included leucopenia (20.9 %), neutropenia (24.0 %), anemia (10.9 %), thrombocytopenia (10.1 %), anorexia (3.1 %), peripheral neurotoxicity (15.5 %), and fatigue (12.4 %). No treatment-related deaths occurred. Combination chemotherapy with SOX offers an effective, safe and well-tolerated regimen for elderly patients with advanced gastric cancer.