Phase III study of selpercatinib versus chemotherapy ± pembrolizumab in untreated RET positive non-small-cell lung cancer.

Selpercatinib, a novel, highly selective and potent, inhibitor of RET, demonstrated clinically meaningful antitumor activity with manageable toxicity in heavily pretreated and treatment-naive RET fusion-positive non-small-cell lung cancer patients in a Phase I/II clinical trial. LIBRETTO-431 (NCT04194944) is a randomized, global, multicenter, open-label, Phase III trial, evaluating selpercatinib versus carboplatin or cisplatin and pemetrexed chemotherapy with or without pembrolizumab in treatment-naive patients with locally advanced/metastatic RET fusion-positive nonsquamous non-small-cell lung cancer. The primary end point is progression-free survival by independent review. Key secondary end points include overall survival, response rate, duration of response and progression-free survival. Clinical trial registration: NCT04194944 (ClinicalTrials.gov).

Hydrogen sulfide protects against acetaminophen-induced acute liver injury by inhibiting apoptosis via the JNK/MAPK signaling pathway.

Acetaminophen (APAP) is a widely used over-the-counter analgesic and antipyretic. It can cause hepatotoxicity. Recent studies demonstrated that hydrogen sulfide (H2 S) exhibits cell protection in several cell types. This study was designed to investigate whether H 2 S ameliorated APAP-induced acute liver injury and to elucidate its mechanisms. In this study, we analyzed the detailed biological and molecular processes of APAP-induced hepatotoxicity using a bioinformatics analysis, which showed that apoptosis and the c-Jun N-terminal kinase (JNK)/mitogen-activated protein kinase pathway were confirmed to play critical roles in these processes. We further investigated the protective effects of H 2 S on APAP-induced hepatotoxicity. In vivo, we observed that the exogenous supplement of H 2 S ameliorated APAP-induced liver injury. Cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE) systems were the endogenous pathway of H 2 S. The expression of CBS/CSE was decreased in APAP-treated mice, while H 2 S could significantly restore it. In addition, APAP-induced JNK activation was inhibited by H 2 S in vivo. In vitro, H 2 S abolished the active effects of APAP on caspase3, Bax, and Bcl-2 expressions as well as JNK phosphorylation in hepatocytes. It was found through flow cytometry that the amount of APAP-induced apoptotic hepatocytes was decreased in the presence of H 2 S. In conclusion, our results suggested that H 2 S attenuated APAP-induced apoptosis in hepatocytes through JNK/MAPK siganaling pathway.

SPOP promotes FADD degradation and inhibits NF-κB activity in non-small cell lung cancer.

FAS-associated protein with death domain (FADD) is the pivotal adaptor protein, which transmits apoptotic signals mediated by the death receptors. Here we report that high FADD protein level predicts poor prognosis of non-small cell lung cancer (NSCLC) patients and its protein level is mainly regulated by the 26S proteasome. We also found that ubiquitin ligase SPOP (speckle-type POZ protein) binds to FADD and mediates its degradation, which can be blocked by MG132 treatment. Notably, SPOP inhibits NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activity and its target genes expression via FADD. These results reveal the function of SPOP-FADDNFκB axis in NSCLC cells, which is associated with prognosis of NSCLC patients.

Targeted therapies for patients with advanced NSCLC harboring wild-type EGFR: what's new and what's enough.

Historically, non-small cell lung cancer (NSCLC) is divided into squamous and nonsquamous subtypes based on histologic features. With a growing number of oncogenic drivers being identified in squamous and nonsquamous NSCLC, this malignancy has been recently divided into several distinct subtypes according to the specific molecular alterations. This new paradigm has substantially highlighted the treatment of advanced NSCLC, shifting it from standard chemotherapy according to specific histologic subtypes to targeted therapy according to specific oncogenic drivers. The application of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) in NSCLC patients harboring activating EGFR mutations has been a representative model of precise medicine in the treatment of NSCLC. As the role of EGFR-TKIs in routine management of patients with advanced NSCLC has been well established, this review provides an overview of alternative targeted therapy in the treatment of NSCLC, including EGFR-TKIs for patients with wild-type EGFR NSCLC, as well as other targeted agents either clinical available or in early- to late-stage development.

[Outcomes of chemotherapy in patients with EGFR mutation-negative non-small cell lung cancer].

OBJECTIVE: To evaluate the efficacy of chemotherapy for patients with EGFR (exon 19 and 21) mutation-negative non-small cell lung cancer (NSCLC). METHODS: One hundred and forty NSCLC patients with negative EGFR mutation (90 cases) or EGFR mutation (50 cases) underwent gemcitabine or vinorelbine plus cisplatin or carboplatin chemotherapy. RESULTS: In the EGFR mutation-negative patients, there were PR in 26 cases, SD in 48 cases, PD in 16 cases, the disease control rate was 82.2%. In the patients with EGFR mutation, there were PR in 14 cases, SD in 23 cases, PD in 13 cases, the disease control rate was 74.0%. The difference of disease control rates in the two groups was not significant (P = 0.250). The progression free survival (PFS) of EGFR mutation-negative patients was 4.2 months (95%CI 3.8-4.6) vs. 4.0 months (95%CI 3.6-4.4) in patients with EGFR mutation, with a significant difference (P = 0.021). The overall survival (OS) of EGFR mutation-negative patients was 9.2 months (95%CI 8.4-10.0) vs. 7.8 months (95%CI, 6.9-8.7) of patients with EGFR mutation (P = 0.028). CONCLUSIONS: Chemotherapy can prolong the PFS and OS of EGFR mutation-negative patients. However, only the extension of OS has practical significance.

[Effect of Wnt signaling suppression on gefitinib in non small cell lung cancer cell lines].

OBJECTIVE: To explore the effect of Wnt signaling suppression on proliferation of non small cell lung cancer to gefitinib, and its related mechanisms. METHODS: PC9 and PC9/AB2 cells of both gefitinib sensitive and resistant were treated with different concentrations of gefitinib, and the proliferation index was measured using CCK8 kit. The members of Wnt signaling pathway were detected by Western blot. Dual luciferase reportor gene assay (TOP Flash) was used to document the transcriptional level of ß-catenin. ß-catenin siRNA was transfected into PC9/AB2 cells to suppress the Wnt signaling transcription, followed by treatment with different concentrations of gefitinib. Western blot was then used to detect the expression of EGFR and its downstream signaling after inhibit the expression of ß-catenin. RESULTS: Treating with different concentrations of gefitinib, the resistance of PC9/AB2 cells to gefitinib was significantly increased (P < 0.05). The members of Wnt signaling expressed at higher level in PC9/AB2 cells than in PC9 cells (t = 24.590, P = 0.000). TOP Flash examination showed that the endogenous transcriptional activity of Wnt signaling was higher in PC9/AB2 cell than that in PC9 cell (t = 4.983, P = 0.008). Compared with the negative control group, apoptotic rate and sensitivity to gefitinib significantly increased in interfered group (P < 0.05). The expression of p-ERK1/2 significantly decreased after Wnt signaling suppression, although other proteins showed no significant alterations. CONCLUSION: Suppressing the activity of Wnt signaling can partly reverse the celluar resistance to gefitinib in non small cell lung cancer.

DNMT3A governs tyrosine kinase inhibitors responses through IAPs and in a cell senescence-dependent manner in non-small cell lung cancer.

Patients with non-small cell lung cancer (NSCLC) treated with tyrosine kinase inhibitors (TKIs) inevitably exhibit drug resistance, which diminishes therapeutic effects. Nonetheless, the molecular mechanisms of TKI resistance in NSCLC remain obscure. In this study, data from clinical and TCGA databases revealed an increase in DNMT3A expression, which was correlated with a poor prognosis. Using NSCLC organoid models, we observed that high DNMT3A levels reduced TKI susceptibility of NSCLC cells via upregulating inhibitor of apoptosis proteins (IAPs). Simultaneously, the DNMT3Ahigh subset, which escaped apoptosis, underwent an early senescent-like state in a CDKN1A-dependent manner. Furthermore, the cellular senescence induced by TKIs was observed to be reversible, whereas DNMT3Ahigh cells reacquired their proliferative characteristics in the absence of TKIs, resulting in subsequent tumour recurrence and growth. Notably, the blockade of DNMT3A/IAPs signals enhanced the efficacy of TKIs in DNMT3Ahigh tumour-bearing mice, which represented a promising strategy for the effective treatment of NSCLC.

Real-world evidence of osimertinib in Chinese patients with EGFR T790M-positive non-small cell lung cancer: a subgroup analysis from ASTRIS study.

PURPOSE: ASTRIS study aimed the largest to evaluate the effectiveness and safety of second- or higher-line osimertinib in patients with advanced/metastatic epidermal growth factor receptor (EGFR) T790M mutation-positive non-small cell lung cancer (NSCLC) in the real-world setting. Here we report the results of Chinese patients in ASTRIS study. METHODS: Adults with EGFR T790M-positive advanced NSCLC pretreated with EGFR-tyrosine kinase inhibitor (EGFR-TKI), having a WHO performance status score of 0-2 and asymptomatic, stable central nervous system (CNS) metastases were included. All patients received once-daily osimertinib 80 mg orally. The outcomes included investigator-assessed clinical response, progression-free survival (PFS), time-to-treatment discontinuation (TTD), and safety. RESULTS: A total of 1350 patients were included. Response rate was 55.7% (95% confidence interval [CI] 0.53-0.58). The median PFS and the median TTD were 11.7 months (95% CI 11.1-12.5) and 13.9 months (95% CI 13.1-15.2), respectively. Overall, 389 patients (28.8%) had at least one protocol-specified adverse event (AE); AEs of interstitial lung diseases/pneumonitis-like events and QT prolongation were reported in 3 (0.2%) and 59 (4.4%) patients, respectively. CONCLUSION: Osimertinib was effective in Chinese patients with T790M-positive NSCLC who had progressed after first- or second-generation EGFR-TKI in real-word setting and the results were consistent with ASTRIS study overall population and AURA studies. No new safety signals or events were identified. CLINICAL TRIAL NUMBER: NCT02474355.

[Clinical characteristics and diagnosis of pulmonary mucosa-associated lymphoid tissue-derived (MALT) lymphoma: a retrospective analysis of 29 cases].

OBJECTIVE: To study the clinical manifestations and radiological characteristics, diagnostic methods and outcomes of pulmonary mucosa-associated lymphoid tissue-derived(MALT) lymphoma. METHODS: A retrospective review of clinical, radiological and follow-up data of 29 pulmonary MALT lymphoma cases at Shanghai Pulmonary Hospital affiliated to Tong Ji University from January 2002 to June 2010 was performed. RESULTS: Among these patients, there were 19(65.5%) males and 10 (34.5%) females aged from 27 to 73 (median 53) years old. Common clinical manifestations were cough (51.7%), fever (20.7%), apnea (17.2%), chest pain (17.2%), fatigue (13.8%) and weight loss (13.8%), while 9(31.0%) cases had no symptoms at diagnosis. The characteristics of the chest CT showed that 22 (75.9%) of the cases had patch infiltration or consolidation of the lung, 7(24.1%) of the cases had mass, and 15 (51.7%) unilateral and 14(48.3%) bilateral lesions. Their diagnosis duration varied between 0.5 and 96 months. 18(62.1%) cases were confirmed by surgery (15 open lung and 7 video-assisted thoracic surgery, VAST), 4 (13.8%) by percutaneous lung biopsy, 5 (17.2%) by bronchoscopic biopsy, and 2 (6.9%) by peripheral lymph node biopsy. The treatment methods included surgery, combined chemotherapy, radiotherapy and Chinese herbal medicine. The 1- and 3-year-survival rates were 92.3% and 87.4%, respectively. CONCLUSIONS: Pulmonary MALT lymphoma is atypical in clinical manifestations and radiological characteristics, and easy to be misdiagnosed. Local diseases are mainly treated by operation while extensive diseases receive combined chemotherapy. A proper diagnosis is mainly based on pathological biopsy. Patients with MALT lymphoma have a favorable outcome. Poor prognosis may be connected with poor performance status and long diagnosis duration.

[Correlation analysis between cytokines levels in serum and bronchoalveolar lavage fluid, blood T cell subsets and pneumoconiosis severity].

OBJECTIVE: To analyze the correlation between the pneumoconiosis severity and the cytokines levels in serum and bronchoalveolar lavage fluid (BALF) or blood T cell subsets. METHODS: The subjects were divided into five groups: control group (6 cases), group exposed to dusts (6 cases) and 3 pneumoconiosis groups (36 in stage I, 12 in stage II and 10 in stage III). ELISA was used to detect IL-6, sIL-2R and TNF-α levels in serum and BALF. The subsets of blood T cells were classified by flow cytometer. RESULTS: As compared with control group and group exposed to dusts, the levels of serum IL-6 and sIL-2R in patients with II or III stages significantly increased, which were positively correlated with pneumoconiosis stages (r(1) = 0.74, r(2) = 0.81, P < 0.05). The level of serum TNF-α significantly decreased in patients with III stages, as compared with control group and group exposed to dusts. There was a negative correlation between serum TNF-α level and pneumoconiosis severity (r = -0.58, P < 0.05). There was a positive correlation between the levels of IL-6, sIL-2R and TNF-α in BALF and the levels of IL-6, sIL-2R and TNF-α in serum (r(1) = 0.77, r(2) = 0.96 and r(3) = 0.88, P < 0.05). The proportion of CD(4)(+)T cells and the ratio of CD(4)(+)/CD(8)(+) decreased dramatically in patients with II and III stages. But there was no correlation between these values and disease severity. CONCLUSION: The immune function in Th cell was inhibited. The levels of IL-6, sIL-2R and TNF-α in serum and BALF were associated with the severity of pneumoconiosis.

Individualized Chemotherapy in Advanced NSCLC Patients Based on mRNA Levels of BRCA1 and RRM1.

OBJECTIVE: Experimental evidence suggests that the overexpression of breast cancer-specific tumor suppressor protein 1 (BRCA1) gene enhances sensitivity to docetaxel and resistance to cisplatin and ribonucleotide reductase M1 (RRM1) gene overexpression enhances resistance to gemcitabine. To further examine the effect of BRCA1 and RRM1 mRNA levels on outcome in advanced non-small cell lung cancer (NSCLC), we performed this non-randomized phase II clinical trial which tested the hypothesis that customized therapy would confer improved outcome over non-customized therapy. METHODS: RNA was isolated from fresh tumor tissue. Patients received chemotherapy regimen based on their BRCA1 and RRM1 mRNA levels: both low-cisplatin plus gemcitabine (GP); both high-vinorelbine plus cisplatin (NP); BRCA1 low and RRM1 high-cisplatin plus docetaxel (TP); BRCA1 high and RRM1 low-vinorelbine plus gemcitabine (GN). RESULTS: From Dec 2005 to Nov 2008, 94 metastatic and locally advanced NSCLC patients from our institute were enrolled in this study. The median age was 58 years old. Among them, 21 patients received GP, 30 patients received TP and 43 patients received NP chemotherapy. GP group had a higher response rate, and longer median time to progression (TTP) and median overall survival (OS) time than the other 2 groups. The response rates in the GP, TP and NP groups were 42.9%, 36.7% and 27.9%, respectively (P=0.568). The median TTP was 5.6, 5.0, 4.8 months (P=0.975), respectively, and the median OS time was 12.5, 11.0, 9.7 months (P=0.808), respectively. CONCLUSION: Chemotherapy customized according to BRCA1 and RRM1 expression levels is associated with higher response rate and longer TTP and OS time in the GP group. This suggests that BRCA1 and RRM1 mRNA levels could be used as biomarkers in individual therapy in NSCLC.

Auricular Acupressure for Hemodialysis Patients with Insomnia: A Multicenter Double-Blind Randomized Sham-Controlled Trial.

Background and objectives: The effect of auricular acupressure (AA) for maintenance hemodialysis (MHD) patients with insomnia has been controversial. This study assessed the efficacy and safety of AA for MHD patients with chronic insomnia. Design, setting, participants, and measurements: This was a multicenter, double-blind (participant and assessor), randomized sham-controlled trial. A total of 133 subjects were randomized to receive AA on active points (AA group, n = 64) or on sham auricular acupressure (SAA) points (SAA group, n = 69) for 8 weeks and followed up for 12 weeks. AA was provided by assigned qualified nurses who were not involved in assessment. The primary outcome was the clinical response rate, which was defined as the percentage of participants who reached a reduction of Pittsburgh Sleep Quality Index (PSQI) global score ≥3 in each group. Secondary outcomes included changes in PSQI scores over time, PSQI scores and hypnotics use at each visit, and changes in the weekly dose of hypnotics for drug-dependent subjects. Results: At week 8, the AA group yielded a higher clinical response rate than the SAA group (AA: 55% vs. SAA: 36%, odds ratio: 1.5, 95% confidence interval: 1.0-2.2, p = 0.033). Both groups showed a reduction in PSQI global scores during treatment and follow-up, compared with the baseline, respectively. A significant change of PSQI global score was observed over time (F = 28.387, p < 0.001). PSQI global score of the AA group was relatively lower than that of the SAA group at each visit (p < 0.05 at week 16 and 20). For those depending on hypnotics, AA reduced their consumption of hypnotics. The intervention was safe, and its adherence was satisfactory. Conclusion: AA could serve as a complementary or alternative therapy for MHD patients with insomnia by improving their sleep quality and reducing their use of hypnotics. Clinical trial registration: Clinicaltrials.gov, Identifier: NCT03015766.

Effect of 5/6 nephrectomized rat serum on epithelial-to-mesenchymal transition in vitro.

OBJECTIVE: To investigate whether the 5/6 nephrectomized (5/6Nx) rats' 12-week serum could lead to tubular epithelial-to-mesenchymal transition (EMT) and its molecular mechanism, so as to probe the potential stimulation from circulation in chronic progressive kidney disease. METHODS: A total of 24 Sprague Dawley (SD) rats were randomly divided into two groups: sham operation group (sham group) and 5/6Nx group. Rats were killed 12 weeks after surgery to obtain 5/6Nx rats' 12-week serum. Then we detected the expression of E-cadherin in renal tubular epithelial cells of the remaining kidney and we investigated whether the 12th week serum of 5/6Nx rats could cause HK-2 (human kidney proximal tubular cell line) cells to transdifferentiate into fibroblasts. RESULTS: Our data confirmed that E-cadherin expression decreased significantly in the remaining kidney at 12 weeks, and the 5/6Nx rats' 12-week serum could suppress E-cadherin protein and mRNA expression (p < 0.05). We also found that the 5/6Nx rats' 12-week serum could upregulate ZEB1, ß-catenin, and wnt3 protein expression (p < 0.05). CONCLUSIONS: Our results demonstrated that the 5/6Nx rats' 12-week serum could suppress the expression of E-cadherin in HK-2 cells. It was partially through modulating the increase of ZEB1. The loss of E-cadherin could lead ß-catenin to localize to the cytoplasm and nucleus, and feed into the Wnt signaling pathway. It means that the pathogenic serum in chronic kidney disease (CKD) plays an important role in the loss of renal function and turns to be a new avenue of research with potential clinical implications.

Differences in Immunological Landscape between EGFR-Mutated and Wild-Type Lung Adenocarcinoma.

Recent clinical trials of lung adenocarcinoma with immune checkpoint inhibitors revealed that lung adenocarcinoma patients with EGFR mutations have a poor response to immunotherapy. However, the mechanisms have not been addressed. We performed immunohistochemistry analyses of resected lung adenocarcinoma tissues with and without EGFR mutations to investigate and compare the characteristics of the tumor microenvironment (TME). We retrospectively enrolled a total of 323 lung adenocarcinoma patients (164 had EGFR mutations), and their corresponding tissue samples were analyzed by the EGFR mutation test and immunohistochemistry. We selected the markers of the immune checkpoint molecule (PD1, PD-L1, and LAG-3) and immune cell (CD3, CD4, CD8, and Foxp3) as markers of the tumor microenvironment. Our results revealed that patients had a distinct tumor microenvironment between EGFR-mutant and wild-type lung adenocarcinomas; the expression of CD3, CD4, PD-L1, and Foxp3 in EGFR-mutant tumors was significantly higher than that in wild-type tumors, while the expression of LAG3 and PD-1 showed a positive correlation with EGFR-wild-type tumors. In survival analysis, EGFR-wild-type patients had longer disease-free survival (DFS) than EGFR-mutant patients (P = 0.0065). Our research demonstrates significant differences in tumor microenvironment composition between EGFR-mutant and wild-type patients. Our findings provide novel evidence that contributes to understanding the mechanism underlying the poor efficacy of immune checkpoint inhibitors.

A Novel Combined Conjugate Therapeutic Cancer Vaccine, Recombinant EGF-CRM197, in Patients With Advanced Solid Tumors: A Phase I Clinical Study.

INTRODUCTION: The therapeutic cancer vaccine recombinant Epidermal Growth Factor (EGF)-CRM197 is a novel combined conjugate EGF with CRM197 as a carrier protein. Immunization with the EGF-CRM197 vaccine can induce high levels of neutralizing anti-EGF antibodies that inhibit EGF/EGFR signaling and thereby suppress growth of tumors that rely on this signaling pathway. Herein, we characterize the humoral immune responses elicited by the recombinant EGF-CRM197 vaccine in patients with advanced solid tumors in a phase I clinical trial and assess the safety, tolerability, and immunogenicity of this vaccine (CTR20190473). METHODS: A total of 16 subjects were enrolled in this study. Under 6 + 3 design, patients in each dosing cohort were administrated subcutaneously at a dosage of 0.4 mg, 0.8 mg, and 1.6 mg, respectively. The patients received vaccinations for immune induction (once a week for 4 consecutive weeks) and booster vaccinations (once every 4 weeks). Safety evaluation was performed 1 week after the immune induction. Booster vaccination was given until the occurrence of disease progression, intolerance, withdrawal of informed consent by the patient, or negative result of anti-EGF test after two booster vaccinations. RESULTS: Vaccination with EGF-CRM197 is safe and well-tolerated in patients with advanced solid tumors. Adverse reactions at the injection site were the most common adverse events (AEs) in recipients. No severe adverse reactions post vaccination were observed in the present study. Vaccinated patients developed a robust neutralizing antibody response triggered by EGF-CRM197 that significantly reduced the levels of EGF in serum. For lung cancer patients who were super good antibody responders (sGAR) to EGF-CRM197, the median progress-free survival (PFS) was 4.83 months, significantly longer than that of the good antibody responder (GAR) patients with lung cancer whose median PFS was 2.10 months (P=0.0018). The median overall survival (OS) of GAR lung cancer patients was 10.67 months while the OS) for sGAR lung cancer patients was not reached until analysis was performed. The median follow-up of the sGAR lung cancer patients was 14.6 months. CONCLUSION: Our study demonstrates that the recombinant EGF-CRM197 therapeutic cancer vaccine can induce a good immune response in patients with advanced solid tumors and is safe and well tolerated, which ensures further clinical development of the vaccine for extending the survival time of EGF-CRM197 sensitive patients with advanced solid tumors. CLINICAL TRIAL REGISTRATION: http://www.chinadrugtrials.org.cn, identifier CTR20190473, EGF-CRM197.

[Efficacy and safety of Erlotinib in the treatment for advanced non-small cell lung cancer in Chinese patients].

OBJECTIVE: To observe the efficacy and the adverse effects of erlotinib in the treatment for advanced non-small cell lung cancer (NSCLC) in Chinese patients. METHODS: From November 2005 to March 2009, a total of 519 patients with unresectable, local advanced, relapsed or metastatic NSCLC were enrolled in the trial. All the patients were treated with erlotinib 150 mg/day until disease progression or intolerable toxicity or for other reasons. The response rate, time to disease progression, overall survival and toxicity were analyzed. RESULTS: Of these 519 patients, 1 case had complete response, 127 cases had partial response and 263 cases had stable disease, resulting in an overall response rate (CR + PR) of 26.7%, disease stable rate of 54.9% and disease control rate (CR + PR + SD) of 81.6%. The median time to progression was 6.44 months and median overall survival was 15.37 months. The major erlotinib treatment-related adverse events (AE) were mild (CTC AE 1/2), only 3 cases had severe adverse effect, 1 case had interstitial lung disease and died of respiratory failure. CONCLUSION: The study presents excellent response rates, time to progression and overall survival of erlotinib treatment for advanced NSCLC in Chinese patients, and its adverse events are tolerable.

[Drug resistance mechanism of non small cell lung cancer PC9/AB2 cell line with acquired drug resistance to gefitinib].

OBJECTIVE: To study the drug resistance mechanism of non-small cell lung cancer (NSCLC) cell line PC9/AB2 with acquired drug resistance to gefitinib. METHODS: The human lung adenocarcinoma cell line PC9 was cultured in vitro, and was induced by MNNG to obtain the cell line PC9/AB2 with acquired drug resistance to gefitinib. The sensitivity of the cell line PC9 and PC9/AB2 to gefitinib was determined by MTT assay. The effects of gefitinib on cell apoptosis of the 2 cell lines were determined by flow cytometry. The genomic DNA of the 2 cell lines were extracted, and then the exons 19-21 of EGFR gene were amplified by PCR and sequenced. The protein expression of c-MET and integrin beta1 in the 2 cell lines was determined by Western blot method. The adhesion ability and migration ability of the 2 cell lines were determined by adhesion test and scratch assay. RESULTS: (1) The data form MTT and apoptosis detection showed that the IC50 of PC9/AB2 cells was (24.2+/-5.5) micromol/L, 576 times higher than PC9 cells [IC50 (0.04+/-0.01) micromol/L]. Given the same concentration of gefitinib, the apoptosis rate of PC9 cells was 38.48%, while that of PC9/AB2 cells was 2.2%. (2) The results of gene sequencing showed that there was a deletion of 15 bp in both exon 19 of the 2 cell lines, while no T790M mutation occurred. (3) The results from Western blot showed that there was no significant difference in protein expression of c-MET between the 2 cell lines, while the protein expression of integrin beta1 in PC9/AB2 cells was significantly higher than that of the PC9 cells. (4) The result from adhesion test and scratch assay showed that the adhesion ability and migration ability of the PC9/AB2 cells was significantly higher that those of PC9 cells. CONCLUSION: The high expression of integrin beta1 may be associated with acquired drug resistance of NSCLC cell line PC9/AB2 to gefitinib.

Clinicopathological features and epidermal growth factor receptor mutations associated with epithelial-mesenchymal transition in non-small cell lung cancer.

BACKGROUND AND OBJECTIVE: Small molecular inhibitors of the epidermal growth factor receptor (EGFR) have been extensively studied in non-small cell lung cancer (NSCLC) patients. The discovery of molecular biomarkers that identify the subgroups of NSCLC patients benefiting from EGFR tyrosine kinase inhibitor (TKI) has become an important area of investigation. Recent studies have suggested that epithelial-mesenchymal transition (EMT) in tumours decreases the cellular requirements for EGFR signalling pathway, and this may provide a molecular signature to define those NSCLC patients most likely to respond to treatment with targeted EGFR TKI. This research explored the clinicopathological features and EGFR mutations associated with EMT in NSCLC. METHODS: The EMT status in surgically resected specimens from 62 patients with NSCLC was tested by immunohistochemical staining. The frequency of tumour epithelial phenotype was calculated and the strength of the association with clinicopathological features and EGFR genotype was determined by logistic regression. RESULTS: The overall frequency of the epithelial phenotype was 35.48% (22 of 62). Based on univariate analyses, the frequency of the epithelial phenotype (E-cadherin-positive) was greater for EGFR mutants versus wild types (77.78% vs 18.18%; P < 0.0001) and women versus men (54.55% vs 25%; P = 0.02). Multivariate logistic analysis showed that only the EGFR genotype (odds ratio, 0.063; 95% CI: 0.013-0.3; P = 0.0005) was significantly associated with the epithelial phenotype. CONCLUSION: In patients with NSCLC, there is a higher frequency of epithelial markers in patients with EGFR mutation.

Comparison among different presentations of venous thromboembolism because of lung cancer.

INTRODUCTION: Venous thromboembolism (VTE) because of lung cancer has been sufficiently studied, nevertheless, little is known regarding the discrepancy of clinical characteristics and predictive factors among different presentations of VTE because of lung cancer. OBJECTIVES: This study was designed to investigate the distinction of clinical characteristics and predictive factors among different presentations of VTE because of lung cancer. METHODS: All patients concomitant lung cancer and VTE were stratified into three groups: pulmonary embolism (PE) group in which patients had sole PE, deep vein thrombosis (DVT) group with sole DVT and concomitance group with both PE and DVT. RESULTS: Concomitance of PE and DVT (28.2 days) mostly occurred at the early stage after the diagnosis of lung cancer, by contrast with DVT (63.6 days) which did at the latest stage, whereas PE (36.7 days) generally developed intermediately in between (P = .02). In a Kaplan-Meier analysis, the cumulative survival rate of DVT group was higher than that of concomitance group, whereas the rate of PE group lied in between. (P = .002) The strongest correlated factors with the development of DVT, PE and concomitance were adenocarcinoma (HR 3.27, P = .003), chemotherapy (HR 2.62, P = .005) and D-Dimer (HR 3.88, P < .001), respectively. The strongest correlated factors with the mortality of DVT, PE and concomitance were comorbidity (HR 2.32, P = .003), metastasis (HR 3.12, P < .001), and metastasis (HR 4.29, P < .001), respectively. CONCLUSION: Concomitance of DVT and PE represents the severest state of lung cancer, the earliest occurrence of VTE, and the worst survival rate, whereas DVT stands for the mildest condition of lung cancer and stablest pattern of VTE.

[Sensitivity of two cell lines with acquired resistance to gefitinib to several chemotherapeutic drugs].

OBJECTIVE: To explore the sensitivity of tumor cell lines with acquired resistance to gefitinib to several chemotherapeutic drugs and provide preclinical basis of available chemotherapy regimens after failure of molecular targeted therapy. METHODS: Human lung adenocarcinoma cell lines PC9 and PC9/G with acquired resistance to gefitinib were cultured in vitro. The sensitivity to chemotherapeutic drugs and inhibition rate of cell proliferation was determined by MTT assay. Effects of drugs on apoptosis and expression of P-170 were determined by flow cytometry. Difference of gene expression profile between PC9 and PC9/G cells was analyzed by DNA microarray. Western blot was used to test the expression of Akt, phospho-Akt and integrin beta1. RESULTS: The resistance index of PC9/G cells to cisplatin was about 5.4-fold compared with that of PC9 cells. LY294002 may significantly elevate the sensitivity of PC9/G cells to cisplatin (P < 0.05). PC9/G cells were more sensitive to docetaxel than PC9 cells. No significant difference of sensitivity to pemetrexed was found between these two cell lines. Expression level of P-170 in PC9/G cells was lower than that in PC9 cells. In PC9/G cells, the expression of integrin beta1 and DNA healing gene was high and expression of gene during mitosis was low. The level of expression of Akt, phospho-Akt and integrin beta1 in PC9/G cells was higher than that in PC9 cells. CONCLUSION: In PC9/G cells, a cell line with acquired resistance to gefitinib, over-expression of PI3K, integrin and DNA restoration gene and continuous activation of PI3K is found to be correlated with resistance to cisplatin. Docetaxel or pemetrexed is a more reasonable choice than cisplatin for treatment of NSCLC patients who failed to respond to EGFR-TKI.