CXCR6-positive circulating mucosal-associated invariant T cells can identify patients with non-small cell lung cancer responding to anti-PD-1 immunotherapy.

BACKGROUND: Mucosal-associated invariant T (MAIT) cells have been reported to regulate tumor immunity. However, the immune characteristics of MAIT cells in non-small cell lung cancer (NSCLC) and their correlation with the treatment efficacy of immune checkpoint inhibitors (ICIs) remain unclear. PATIENTS AND METHODS: In this study, we performed single-cell RNA sequencing (scRNA-seq), flow cytometry, and multiplex immunofluorescence assays to determine the proportion and characteristics of CD8+MAIT cells in patients with metastatic NSCLC who did and did not respond to anti-PD-1 therapy. Survival analyses were employed to determine the effects of MAIT proportion and C-X-C chemokine receptor 6 (CXCR6) expression on the prognosis of patients with advanced NSCLC. RESULTS: The proportion of activated and proliferating CD8+MAIT cells were significantly higher in responders-derived peripheral blood mononuclear cells (PBMCs) and lung tissues before anti-PD-1 therapy, with enhanced expression of cytotoxicity-related genes including CCL4, KLRG1, PRF1, NCR3, NKG7, GZMB, and KLRK1. The responders' peripheral and tumor-infiltrating CD8+MAIT cells showed an upregulated CXCR6 expression. Similarly, CXCR6+CD8+MAIT cells from responders showed higher expression of cytotoxicity-related genes, such as CST7, GNLY, KLRG1, NKG7, and PRF1. Patients with ≥15.1% CD8+MAIT cells to CD8+T cells ratio and ≥35.9% CXCR6+CD8+MAIT cells to CD8+MAIT cells ratio in peripheral blood showed better progression-free survival (PFS) after immunotherapy. The role of CD8+MAIT cells in lung cancer immunotherapy was potentially mediated by classical/non-classical monocytes through the CXCL16-CXCR6 axis. CONCLUSION: CD8+MAIT cells are a potential predictive biomarker for patients with NSCLC responding to anti-PD-1 therapy. The correlation between CD8+MAIT cells and immunotherapy sensitivity may be ascribed to high CXCR6 expression.

Procedures for application of the extended dosing after antitumor drug clinical trials. / æŠ—è‚¿ç˜¤è¯ç‰©ä¸´åºŠè¯•éªŒå—è¯•è€ å‡ºç»„åŽå»¶ä¼¸ç»™è¯ç”³è¯·ç¨‹åº.

New drug clinical trials have been considered as a positive way for treating cancer by cancer patients and doctors, and the extended dosing is a special way for patients' withdrawal from antitumor clinical trials to obtain investigational new drugs. However, neither the regulations of expanded dosing nor the detail documents for expanded dosing have been officially published in China. At present, expanded dosing of investigational drugs is still at the exploratory stage in various medical institutions, and a complete management system has not been established to meet patients' urgent needs for drug use. Based on the practical experience of extended dosing in Hunan Cancer Hospital, this paper preliminarily explored the application procedures and ethical review requirements of extended dosing for subjects in antitumor clinical trials. It is necessary to clarify the responsibilities of all patients in the procedure and establish a patient-medical institution-sponsor joint application system. In the process of ethical review, it is recommended that all parties fully consider the risks and benefits of extended dosing for patients, and then the ethics committee makes a comprehensive assessment to decide whether to approve extended dosing.

Benefit assessment of extended dosing in cancer patients after their withdrawal from clinical trials.

Background: Clinical trials have been widely recognized as an effective treatment approach by physicians and cancer patients alike. Physicians' evaluations suggest that many patients are likely to continue experiencing benefits from extended dosing of investigational new drugs even after withdrawing from clinical trials. Objective: Given the uncertainty surrounding the efficacy and safety of investigational new drugs, it is essential to continually assess the benefits of extended dosing for patients. Methods: The trial group for this study comprised patients who requested extended dosing after withdrawing from clinical trials at Hunan Cancer Hospital between 2016 and 2020. The control group consisted of patients who received conventional treatment and were enrolled in a 1:1 ratio. Follow-up assessments were conducted every 3 months for both groups, and included monitoring of patients' health status, survival time, disease control or remission, treatment modalities received, and medical costs. Results: A total of twenty-three patient pairs were successfully matched for this study. The Ethics Committee approved extended dosing for all patients in the trial group, with an average gap period of 16.48 days between their withdrawal from clinical trials and continuous access to the investigational drugs. The median overall survival for patients after withdrawal from clinical trials was 17.3 months in the extended dosing group and 12.9 months in the control group, with no significant difference observed between the two groups (p > 0.250). The median total cost of treatment after the previous clinical trial was 38,006.76 RMB, of which the median cost of therapeutic drugs for conventional treatment was 15,720 RMB, while extended dosing was provided free of charge. Conclusion: Extended dosing can indeed provide benefits, including survival benefits and economic benefits, to cancer patients after their withdrawal from clinical trials and will clinically present an additional treatment option for patients.

Establishment and Validation for Predicting the Lymph Node Metastasis in Early Gastric Adenocarcinoma.

Lymph node metastasis (LNM) is considered to be one of the important factors in determining the optimal treatment for early gastric cancer (EGC). This study aimed to develop and validate a nomogram to predict LNM in patients with EGC. A total of 842 cases from the Surveillance, Epidemiology, and End Results (SEER) database were divided into training and testing sets with a ratio of 6 : 4 for model development. Clinical data (494 patients) from the hospital were used for external validation. Univariate and multivariate logistic regression analyses were used to identify the predictors using the training set. Logistic regression, LASSO regression, ridge regression, and elastic-net regression methods were used to construct the model. The performance of the model was quantified by calculating the area under the receiver operating characteristic curve (AUC) with 95% confidence intervals (CIs). Results showed that T stage, tumor size, and tumor grade were independent predictors of LNM in EGC patients. The AUC of the logistic regression model was 0.766 (95% CI, 0.709-0.823), which was slightly higher than that of the other models. However, the AUC of the logistic regression model in external validation was 0.625 (95% CI, 0.537-0.678). A nomogram was drawn to predict LNM in EGC patients based on the logistic regression model. Further validation based on gender, age, and grade indicated that the logistic regression predictive model had good adaptability to the population with grade III tumors, with an AUC of 0.803 (95% CI, 0.606-0.999). Our nomogram showed a good predictive ability and may provide a tool for clinicians to predict LNM in EGC patients.

Effect of CDCA5 on Proliferation and Metastasis of Triple Negative Breast Cancer Cells under shRNA Interference Technology.

Objective: It was to explore the effect of cell division cycle associated 5 (CDCA5) under shRNA interference on proliferation and metastasis of triple negative breast cancer (TNBC) cells. Methods: MDA-ME-231 and BT549 cells were selected as the research objects. According to the different interference methods and CDCA5 interference sequences, they were divided into the interference group 1MDA-ME-231, the interference group 2MDA-ME-231, the interference group 1BT549, the interference group 2BT549 (using shRNA technology), the control group MDA-ME-231, and the control group BT549 (breast cancer cells under normal culture conditions). MCF10A cells were routinely cultured as the negative control group to analyze the effect of CDCA5 expression on the proliferation and migration of cancer cells. Results: The expression of CDCA5 protein in MDA-ME-231 and BT549 cells in control group was significantly higher than that in negative control group (P < 0.05). Compared with the control group, the inhibition rates of CDCA5 expression in 1MDA-ME-231, 2MDA-ME-231, 1BT549, and 2BT549 cells in the interference group were 39.01%, 42.98%, 49.57%, and 60.98%, respectively (P < 0.05). From 12 h, the proliferation level of TNBC cells at different culture time was lower than that of the control group (P < 0.05). Compared with the number of staining cells in the control group, the positive staining cells in 1MDA-ME-231 (61.42%), 2MDA-ME-231 (72.06%), 1BT549 (52.53%), and 2BT549 (59.65%) in the interference group were significantly decreased (P < 0.05). Conclusion: The results show that the expression of CDCA5 in TNBC is increased, which plays an important role in the proliferation and migration of cancer cells. shRNA interference technology can knock down the expression of CDCA5 and inhibit its "promoting cancer" effect.

Effect of fluconazole on the pharmacokinetics of fuzuloparib: an open-label, crossover study in Chinese healthy male volunteers.

PURPOSE: Fuzuloparib (AiRuiYiTM, formerly fluzoparib, SHR3162) is a new orally active poly adenosine diphosphate ribose polymerase (PARP) inhibitor. It has multiple pharmacological activities in breast, ovarian, and prostatic cancer. Fuzuloparib is mainly metabolized through the enzyme CYP3A4 may slow fuzuloparib metabolism and increase its concentrations in blood. We evaluated the pharmacokinetics and tolerability of fuzuloparib by fluconazole, which is a broad antifungal agent and a moderate inhibitor of CYP3A4. METHODS: In this study, the effects of CYP3A4 inhibition on the pharmacokinetics of fuzuloparib were assessed in a total of 20 healthy Chinese male subjects in an open-label, two-period, single-sequence, crossover study. RESULTS: Pharmacokinetic parameters, including the maximal plasma concentration (Cmax), the plasma concentration-time curve from time 0 to last measurable area under concentration (AUC0-t), and from time 0 to infinity (AUC0-∞), were increased by 32.4%, 104.5%, and 109.6%, with corresponding 90% confidence intervals of (23-43%), (93-116%), and (98-122%), respectively, when fluconazole was combined with fuzuloparib compared to fuzuloparib alone. There was also a slight increase in the incidence of treatment emergent adverse events, including hyperlipidemia and elevated aspartate transaminase. CONCLUSION: The fuzuloparib is 150 mg b.i.d in clinics use. Our results suggest that fuzuloparib could well be tolerated when administered as a single 20 mg oral dose alone or co-administered with 400 mg fluconazole in healthy male subjects. It is recommended to avoid using moderate CYP3A4 inhibitors together with fuzuloparib or instead of 50 mg when necessary.

A Large Real-World Study on the Effectiveness of the Combined Inhibition of EGFR and MET in EGFR-Mutant Non-Small-Cell Lung Cancer After Development of EGFR-TKI Resistance.

BACKGROUND: MET proto-oncogene amplification (amp) is an important mechanism underlying acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). However, the optimal treatment strategy after acquiring MET-amp-mediated EGFR-TKI resistance remains controversial. Our study compared three treatment strategies for patients with EGFR-mutant non-small-cell lung cancer (NSCLC) who were detected with MET-amp at EGFR-TKI progression using next-generation sequencing. METHODS: Of the 70 patients included in the study, 38 received EGFR-TKI + crizotinib, 10 received crizotinib monotherapy, and 22 received chemotherapy. Clinical outcomes and molecular profiles were analyzed. RESULTS: The objective response rate was 48.6% for EGFR-TKI + crizotinib group, 40.0% for crizotinib monotherapy group, and 18.2% for chemotherapy group. Patients who received EGFR-TKI + crizotinib had significantly longer progression-free survival than those who received crizotinib or chemotherapy (5.0 vs. 2.3 vs. 2.9 months, p = 0.010), but overall survival was comparable (10.0 vs. 4.1 vs. 8.5 months, p = 0.088). TP53 mutation (58.5%) and EGFR-amp (42.9%) were frequent concurrent mutations of the cohort. Progression-free survival was significantly longer for patients with either concurrent TP53 mutation (n = 17) (6.0 vs. 2.3 vs. 2.9 months, p = 0.009) or EGFR-amp (n = 13) (5.0 vs. 1.2 vs. 2.4 months, p = 0.016) in the EGFR-TKI + crizotinib group than the other two regimen. Potential acquired resistance mechanisms to EGFR-TKI + crizotinib included EGFR-T790M (n = 2), EGFR-L718Q (n = 1), EGFR-S645C (n = 1), MET-D1228H (n = 1), BRAF-V600E (n = 1), NRAS-Q61H (n = 1), KRAS-amp (n = 1), ERBB2-amp (n = 1), CDK4-amp (n = 1), and MYC-amp (n = 1). CONCLUSION: Our study provides real-world clinical evidence from a large cohort that simultaneous inhibition of EGFR and MET could be a more effective therapeutic strategy for patients with MET-amp acquired from EGFR-TKI therapy.

Association Between Sex and Immune-Related Adverse Events During Immune Checkpoint Inhibitor Therapy.

BACKGROUND: Accumulated evidence supports the existence of sex-associated differences in immune systems. Understanding the role of sex in immune-related adverse events (irAEs) is important for management of irAE in patients receiving immunotherapy. METHODS: We performed meta-analysis on published clinical study data and multivariable logistic regression on pharmacovigilance data and applied a propensity algorithm to The Cancer Genome Atlas omics data. We further validated our observations in 2 independent in-house cohorts of 179 and 767 cancer patients treated with immune checkpoint inhibitors. RESULTS: A meta-analysis using 13 clinical studies that reported on 1096 female patients (36.8%, 95% confidence interval [CI] = 35.0% to 38.5%) and 1886 male patients (63.2%, 95% CI = 61.5% to 65.0%) demonstrated no statistically significant irAE risk difference between the sexes (odds ratio [OR] = 1.19, 95% CI = 0.91 to 1.54, 2-sided P = .21). Multivariable logistic regression analysis of 12 225 patients from the Food and drug administration Adverse Event Reporting System (FAERS) and 10 979 patients from VigiBase showed no statistically significant difference in irAEs by sex. A propensity score algorithm used on multi-omics data for 6019 patients from The Cancer Genome Atlas found no statistically significant difference by sex for irAE-related factors or pathways. The retrospective analysis of 2 in-house patient cohorts validated these results (OR = 1.55, 95% CI = 0.98 to 2.47, false discovery rate = 0.13, for cohort 1; OR = 1.16, 95% CI = 0.86 to 1.57, false discovery rate = 0.39, for cohort 2). CONCLUSIONS: We observed minimal sex-associated differences in irAEs among cancer patients who received immune checkpoint inhibitor therapy. It may be unnecessary to consider sex effects for irAE management in clinical practice.

Evaluation of the pharmacokinetic effects of itraconazole on alflutinib (AST2818): an open-label, single-center, single-sequence, two-period randomized study in healthy volunteers.

Alflutinib (AST2818) is a newly developed third-generation EGFR tyrosine kinase inhibitor for the treatment of lung cancer patients with T790M-resistant mutations. It is metabolized mainly by the CYP3A4 enzyme. At the same time, it has the potential to induce CYP3A4. In this study, we aimed to estimate the effect of itraconazole (a strong inhibitor of CYP3A4) on the pharmacokinetics of alflutinib. For this aim, a single-center, open-label, single-sequence, two-period trial was designed. The pharmacokinetic parameters of AST2818 and its active metabolite AST5902 were established from blood concentration measurements, and adverse events (AEs) of two periods of treatment were documented. For AST2818, the Cmax, AUC0-t, and AUC0-∞ in period II (coadministration of itraconazole) increased by 6.5 ng/mL, 1263.0 h*ng/mL, and 1067.0 h*ng/mL, respectively. And the corresponding 90% CIs were 1.23 (1.14-1.32), 2.41 (2.29-2.54), and 2.22 (2.11-2.34), respectively. The Cmax, AUC0-t, and AUC0-∞ of AST5902 in period II decreased by 4.849 ng/mL, 415.60 h*ng/mL, and 391.4 h*ng/mL, respectively. Moreover, the corresponding 90% CIs were 0.09 (0.08-0.10), 0.18 (0.17-0.19), and 0.14 (0.13-0.15), respectively. Nonetheless, in period II, plasma concentrations of total active components (AST2818 and AST5902) changed marginally. The AUC0-∞ of total active components increased 60%, and the corresponding Cmax increased 8%. Possible treatment-related AEs assessed by investigators were fewer in period II (23.3% vs 36.7%). In conclusion, the total exposure of AST2818 and active metabolite AST5902 increased following the coadministration of itraconazole, but it was still safe and well-tolerated.

Pharmacokinetic and bioequivalence study of new S-1 capsule in Chinese cancer patients.

S-1 is a multicomponent capsule containing tegafur, gimeracil, and oteracil potassium that has shown anticancer activity against numerous tumor types. However, S-1 capsules from different manufacturing companies have shown variations in pharmacokinetics and safety. Therefore, this multicenter, single-dose, randomized-sequence, open-label, two-way, self-crossover study was conducted to evaluate the bioequivalence of a newly developed generic S-1 (New Times Pharmaceutical Co., Ltd., Shandong, China) and the original brand-name S-1 capsule (Taiho Pharmaceutical Co., Ltd., Japan). Furthermore, the safety profiles of both products were compared. A total of 70 patients with 18 types cancer including breast, lung, gastric, and colorectal recruited at 5 hospitals who were randomly and alternatively administered 50 mg of the reference and test S-1 with a 7-day interval. Plasma concentrations of tegafur, 5-chloro-2,4-dihydroxypyridine (CDHP), oteracil potassium, and 5-fluorouracil were detected using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pharmacokinetic parameters, including maximum drug concentration (Cmax), time to achieve Cmax (Tmax), half-life (t1/2, area under the concentration-time curve from 0-time t (AUC0-t), and AUC from 0-infinity (AUC0-∞) were determined using non-compartmental analysis with DAS2.0 software. Bioequivalence of the reference and test S-1 was evaluated according to 90% confidence intervals (CIs) for ratios of AUC and Cmax of S-1. Adverse events were evaluated by monitoring symptoms, physical and laboratory examinations, electrocardiogram, and subject interviews. No significant difference was observed in plasma concentrations and pharmacokinetic profiles of tegafur, CDHP, oteracil potassium, or 5-fluorouracil (p > 0.05) among cancer patients treated with the reference or test S-1 formulation. The 90% CIs of Cmax, AUC0-t, and AUC0-∞ ratios were within the 80%-125% limit. The generic S-1 caused eight mild adverse events including liver dysfunction, diarrhea, nausea, fatigue, abnormal blood electrolytes, hyperglycemia, and dermal toxicity. Similarly, 18 mild adverse events were observed including dysarteriotony, diarrhea, nausea, fatigue, fever, hematotoxicity, abnormal blood electrolytes, hyperglycemia, dermal toxicity, and joint pain. There were no differences in the adverse event incidence between the two formulations. In conclusion, the newly developed generic S-1 showed similar pharmacokinetics to those of an original brand-name S-1 in cancer patients, thereby indicating bioequivalence. Furthermore, both treatments were well tolerated, suggesting that the cost-effective generic S-1 should be considered as a feasible option when treating patients.

The novel TP53 3'-end methylation pattern associated with its expression would be a potential biomarker for breast cancer detection.

BACKGROUND: Deficiency or silence of TP53 is an early event in breast tumorigenesis. Aberrant methylation and mutation in regulatory regions were considered as crucial regulators of gene expression. METHODS: Using multiplex-PCR and next-generation sequencing technology, we analyzed TP53 mutation spectrum in its promoter region. Using PCR target sequence enrichment and next-generation bisulfite sequencing technology, we analyzed the methylation profile of the promoter and 3'-end regions of TP53 gene in paired breast tumor and normal tissues from 120 breast cancer patients. The expression of TP53 and the flanking gene ATP1B2 was explored with qPCR method in the same cohort. RESULTS: No promoter mutation of TP53 gene was found in the cohort of the 120 breast cancer patients. The 3'-end of TP53 gene was hyper-methylated (average 78.71%) compared with the promoter region (average less than 1%) in breast tumor tissues. TP53 was significantly lower expressed (P = 1.68E-15) and hyper-methylated in 3'-end (P = 1.82E-18) in tumor. Negative cis correlation was found between the TP53 expression and its 3'-end methylation (P = 9.02E-8, R = 0.337). TP53 expression was significantly associated with PR status (P = 0.0128), Ki67 level (P = 0.0091), and breast cancer subtypes (P = 0.0109). TP53 3'-end methylation and expression showed a good performance in discriminating breast cancer and normal tissues with an AUC of 0.930. CONCLUSIONS: The 3'-end methylation of TP53 might be a crucial regulator for its expression in breast cancer, suggesting that TP53 3'-end hyper-methylation associated with its lower expression could be a potential biomarker for breast cancer diagnosis.

Mast cells induce epithelial-to-mesenchymal transition and migration in non-small cell lung cancer through IL-8/Wnt/ß-catenin pathway.

[This retracts the article DOI: 10.7150/jca.29953.].

Mast cells induce epithelial-to-mesenchymal transition and migration in non-small cell lung cancer through IL-8/Wnt/ß-catenin pathway.

Background: In the various cancer, mast cells (MCs) infiltration is correlated with a worse prognosis. There is an increasing evidence that MCs and their mediators are participated in remodeling of the tumor microenvironment and facilitate tumor growth, epithelial-to-mesenchymal transition (EMT) and metastasis. Methods: The transwell was conducted to evaluate the correlations between MCs and non-small cell lung cancer (NSCLC) cells in vitro. The RNA interference of ß-catenin was performed to further explore the signaling pathway. Lung adenocarcinoma cell line A549 and human MC (HMC-1) were subcutaneously injected into BALB/c nude mice. The conventional experiment methods (such as quantitative RT-PCR Western Blot, Immunofluorescence, and ELISA) were used in the present study. Results: We found that high density of MCs in NSCLC correlates with worse prognosis. The NSCLC cells could release CCL5 and recruit MCs to the tumor microenvironment. Then, we explored that HMC-1 transplantation accelerated the growth of A549 cell in nude mice. Moreover, the MCs-derived factors were responsible for tumor growth. When NSCLC cells were activated, MCs produced various factors that induced EMT and migration. We also identified that CXCL8/interleukin (IL)-8 served as the major modulator containing in the activated MC conditioned medium. Furthermore, MCs and exogenous IL-8 promoted ß-catenin phosphorylation in NSCLC cells. Inhibiting the Wnt/ß-catenin pathway by RNA interference could revert EMT and migration of NSCLC. Conclusions: Our study suggests that MCs are recruited into NSCLC microenvironment and improve the EMT and migration of cancer cells, thereby accelerating the growth of NSCLC.

Comprehensive analysis of serum tumor markers and BRCA1/2 germline mutations in Chinese ovarian cancer patients.

BACKGROUND: The serum tumor markers has been widely used in ovarian cancer diagnosis. BRCA1/2 germline mutations are the most common predisposing factors for ovarian cancer development. This study aimed to comprehensively investigate serum tumor markers and BRCA1/2 germline mutations and analyze their associations with ovarian cancer. METHODS: Levels of 11 serum tumor markers were examined in ovarian cancer patients and controls with benign gynecologic diseases. By integrating multiplex PCR and next-generation sequencing technologies, BRCA1/2 germline mutations were analyzed and confirmed by Sanger sequencing. The discriminative models with serum tumor markers and BRCA1/2 mutation status were constructed for ovarian cancer detection and patient stratification. RESULTS: Among 11 markers, six of them were significantly elevated and only beta-human chorionic gonadotropin (ß-HCG) was significantly reduced in ovarian cancer patients. A total of 54 (23.3%) ovarian cancer patients were found to harbor BRCA1/2 deleterious mutations, and BRCA1/2 mutations were significantly associated with Hereditary Breast and Ovarian Cancer-related tumors and family history of cancer. Carbohydrate antigen 125 showed a good performance in ovarian cancer detection as a single marker (AUC = 0.799), while a panel of eight markers showed a good performance in BRCA1 mutation detection with an AUC value of 0.974. In addition, a panel of five serum tumor markers combined with BRCA1/2 mutation status showed a good performance in lymph node metastasis prediction (AUC = 0.843). CONCLUSIONS: We found the association between BRCA1/2 germline mutation status and serum tumor marker levels, and identified discriminative models that combined serum tumor markers with BRCA1/2 mutation status for ovarian cancer detection and patient stratification.

Crizotinib presented with promising efficacy but for concomitant mutation in next-generation sequencing-identified ROS1-rearranged non-small-cell lung cancer.

INTRODUCTION: Data of standard tyrosine kinase inhibitor (TKI) treatment outcome in next-generation sequencing (NGS)-identified ROS1-rearranged non-small-cell lung cancer (NSCLC) were rare. Thus, it is practical and necessary to evaluate the efficacy and influential factors of crizotinib in real-world practice. PATIENTS AND METHODS: A total of 1,466 NSCLC patients with positive targeted NGS test results from September 2015 to January 2018 were enrolled in this real-world retrospective study. Twenty-two patients had ROS1 rearrangement detected by NGS. The efficacy and safety of crizotinib were evaluated. Subgroups of concomitant mutations, brain metastasis, and fusion variants were also analyzed. RESULTS: Among all the patients, the occurrence rate of ROS1 rearrangement was 1.5% (22 of 1,466). Ten ROS1 fusion partners were detected, and the most common variant was CD74, which accounted for 50% (11 of 22). Five patients were found to carry dual ROS1 fusion partners, and 23% (5 of 22) of patients were detected with concomitant mutations, including TP53&PIK3CA&mTOR mutation, TP53&CDKN2A mutation, TP53&BRCA2 mutation, ALK missense mutation (p.R311H), and MET amplification. Among 22 patients with ROS1-rearranged NSCLC, 20 patients were diagnosed at stage IV, and 19 patients received crizotinib treatment. The average follow-up period was 16 months. The overall response rate (ORR) of crizotinib in unselected crizotinib-treated patients was 89%, and the median progression-free survival time (mPFS) was 13.6 months. It was shown that NSCLC patients with exclusive ROS1 rearrangement had a longer PFS than those carrying concomitant mutations (15.5 vs 8.5 months, P=0.0213). There were no newly occurring intolerant adverse events in this study. CONCLUSION: Crizotinib is highly effective in NGS-identified ROS1-rearranged advanced NSCLC in real-word clinical practice, and the data are consistent with previous clinical trials applying fluorescence in situ hybridization/real-time PCR for ROS1 companion diagnosis. Concomitant mutations may not be rare and may deteriorate the PFS of crizotinib in patients with ROS1-rearranged NSCLC.

Phase III Randomized Trial of Palonosetron and Dexamethasone With or Without Aprepitant to Prevent Nausea and Vomiting Induced by Full-dose Single-day Cisplatin-based Chemotherapy in Lung Cancer.

INTRODUCTION: This study aimed to determine the efficacy and safety of aprepitant, palonosetron, and dexamethasone to prevent chemotherapy-induced nausea and vomiting in patients with locally advanced or metastatic lung cancer receiving full-dose single-day cisplatin-based combination chemotherapy. MATERIALS AND METHODS: Patients diagnosed with locally advanced or metastatic lung cancer who received full dose single-day cisplatin-based chemotherapy were randomized (1:1) to aprepitant plus palonosetron and dexamethasone, or placebo plus palonosetron and dexamethasone. The primary endpoint was complete response of nausea and vomiting in the first cycle. The secondary endpoints were the proportion of patients with nausea and vomiting who received rescue antiemetic medication, the response of cross-over patients, and safety. RESULTS: A total of 244 patients were randomized. There was no difference between the 2 groups regarding personal characteristics. The administration of aprepitant significantly improved the complete response for vomiting in the overall period (92.6% vs. 79.93%; P < .01), but not a nausea-free response (75.4% vs. 71.3%; P > .05) in the first cycle. The percentage of patients who received rescue antiemetic medication was decreased for the aprepitant group (14.8% vs. 37.1%; P < .001). Patients who did not use aprepitant and suffered with nausea and vomiting in cycle 1 were crossed over to the aprepitant group (N = 32), and the rate of nausea and vomiting in cycle 2 was decreased to 37.5% (P < .05) and 25% (P < .05), respectively. There were no drug-related adverse effects. CONCLUSIONS: Aprepitant plus palonosetron and dexamethasone proved to be effective and well-tolerated in preventing chemotherapy-induced nausea and vomiting after administration of full-dose single-day cisplatin-based combination chemotherapy.

Integrated analysis of promoter methylation and expression of telomere related genes in breast cancer.

Telomeres at the ends of eukaryotic chromosomes play a critical role in tumorgenesis. Using microfluidic PCR and next-generation bisulfite sequencing technology, we investigated the promoter methylation of 29 telomere related genes in paired tumor and normal tissues from 184 breast cancer patients. The expression of significantly differentially methylated genes was quantified using qPCR method.We observed that the average methylation level of the 29 telomere related genes was significant higher in tumor than that in normal tissues (P = 4.30E-21). A total of 4 genes (RAD50, RTEL, TERC and TRF1) showed significant hyper-methylation in breast tumor tissues. RAD51D showed significant methylation difference among the four breast cancer subtypes. The methylation of TERC showed significant association with ER status of breast cancer. The expression profiles of the 4 hyper-methylated genes showed significantly reduced expression in tumor tissues. The integration analysis of methylation and expression of these 4 genes showed a good performance in breast cancer prediction (AUC = 0.947).Our results revealed the methylation pattern of telomere related genes in breast cancer and suggested a novel 4-gene panel might be a valuable biomarker for breast cancer diagnosis.

Integrated analysis of promoter mutation, methylation and expression of AKT1 gene in Chinese breast cancer patients.

BACKGROUND: As downstream mediators of PI3K /PTEN /AKT /mTORC1 pathway, the AKT isoforms play critical roles in tumorgenesis. Although the pleiotropic effects of AKT1 in breast cancer have been reported, the genetic and epigenetic characteristics of AKT1 promoter region in breast cancer remains to be identified. In this study we aimed to investigate the promoter mutation spectrum, methylation and gene expression pattern of AKT1 and their relationship with breast cancer. METHODS: By using PCR target sequence enrichment and next-generation sequencing technology, we sequenced AKT1 promoter region in pairs of breast tumor and normal tissues from 95 unselected Chinese breast cancer patients. The methylation of the promoter region and the expression profile of AKT1 in the same cohort were detected with bisulfite next-generation sequencing and qPCR, respectively. RESULTS: We identified 28 somatic mutations in 23 of the 95 (24.2%) breast cancer samples. And 19 of the 28 mutations were located in transcription factor (TF) binding sites. In the 23 patients with somatic mutations, no significant change of methylation or expression was found comparing with other patients. AKT1 promoter region was significantly hypo-methylated in tumor compared with matched normal tissue (P = 0.0014) in the 95 patients. The expression of AKT1 was significantly suppressed in tumor tissue (P = 0.0375). In clinicopathological factor analysis, AKT1 showed significant hypo-methylation (P = 0.0249) and suppressed expression (P = 0.0375) in HER2 negative subtype. And a trend of decrease in expression level (P = 0.0624) of AKT1 in the ER negative subtype was observed, which is significantly decreased in basal-like breast tumor (P = 0.0328). CONCLUSIONS: Hypo-methylation and suppressed expression of AKT1 was observed to be associated with breast cancer in our cohort. The methylation and expression of AKT1 were both significantly associated with HER2 status. The promoter mutation of AKT1 did not show significant association with its methylation and expression status. These results suggested that the promoter mutation, methylation and gene expression of AKT1 may play distinct roles in tumorgenesis of breast cancer and the integrated analysis of methylation and expression of AKT1 might serve as potential biomarkers for diagnosis and classification of breast cancer.

Integrated analysis of gene expression and methylation profiles of 48 candidate genes in breast cancer patients.

PURPOSE: Gene-specific methylation and expression have shown biological and clinical importance for breast cancer diagnosis and prognosis. Integrated analysis of gene methylation and gene expression may identify genes associated with biology mechanism and clinical outcome of breast cancer and aid in clinical management. METHODS: Using high-throughput microfluidic quantitative PCR, we analyzed the expression profiles of 48 candidate genes in 96 Chinese breast cancer patients and investigated their correlation with gene methylation and associations with breast cancer clinical parameters. RESULTS: Breast cancer-specific gene expression alternation was found in 25 genes with significant expression difference between paired tumor and normal tissues. A total of 9 genes (CCND2, EGFR, GSTP1, PGR, PTGS2, RECK, SOX17, TNFRSF10D, and WIF1) showed significant negative correlation between methylation and gene expression, which were validated in the TCGA database. Total 23 genes (ACADL, APC, BRCA2, CADM1, CAV1, CCND2, CST6, EGFR, ESR2, GSTP1, ICAM5, NPY, PGR, PTGS2, RECK, RUNX3, SFRP1, SOX17, SYK, TGFBR2, TNFRSF10D, WIF1, and WRN) annotated with potential TFBSs in the promoter regions showed negative correlation between methylation and expression. In logistics regression analysis, 31 of the 48 genes showed improved performance in disease prediction with combination of methylation and expression coefficient. CONCLUSIONS: Our results demonstrated the complex correlation and the possible regulatory mechanisms between DNA methylation and gene expression. Integration analysis of methylation and expression of candidate genes could improve performance in breast cancer prediction. These findings would contribute to molecular characterization and identification of biomarkers for potential clinical applications.

Identification and expression profiling analysis of grass carp Ctenopharyngodon idella LGP2 cDNA.

LGP2 (laboratory of genetics and physiology 2), a homologue of RIG-I (Retinoic acid inducible gene-I) and MDA5 (Melanoma differentiation associated gene 5) without the CARD (caspase activation and recruitment domain) required for signaling, plays a pivotal role in modulating signaling by RIG-I and MDA5 for interferon (IFN) synthesis. In this study, a novel LGP2 gene from grass carp Ctenopharyngodon idella (designated as CiLGP2) was isolated and characterized. The full-length cDNA of CiLGP2 was of 2920 bp with five instability motifs (ATTTA). The open reading frame was of 2043 bp and encoded a polypeptide of 680 amino acids, including five main overlapping structural domains: two DEXDc (DEAD/DEAH box helicase domain), one ResIII (conserved restriction domain of bacterial type III restriction enzyme), one HELICc (helicase superfamily c-terminal domain) and one RD (regulatory domain). There was one more alpha-helix in the RD, compared with that in human. The CiLGP2 mRNA was ubiquitous expression in the tested tissues, was high level in spleen, skin, heart and intestine tissues, and was up-regulated by grass carp reovirus (GCRV) injection by semi-quantitative RT-PCR (sqRT-PCR) assay. The CiLGP2 expression in spleen was significantly up-regulated at 12 h (14.5 folds, P < 0.05), reached the crest at 24 h (19.0 folds, P < 0.05), and then dropped a little at 48 h (10.4 folds) post-injection of GCRV and kept this level in the following test period (P < 0.05). In liver, the temporal expression of CiLGP2 mRNA was significantly increased at 24 h (3.8 times, P < 0.05), reached peak at 48 h (10.7 times, P < 0.05), and then decreased a little bit at 72 h (5.8 times, P < 0.05) and kept this high level by the end of the test (P < 0.05). These results collectively suggested that CiLGP2 was a novel member of RLR gene family, engaging in the early stage of antiviral innate immune defense in grass carp, and laid the foundation for the further mechanism research of LGP2 in fishes.