LncRNA H19 downregulation confers erlotinib resistance through upregulation of PKM2 and phosphorylation of AKT in EGFR-mutant lung cancers.

First-generation EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib have significant activity in NSCLC patients with activating EGFR mutations. However, EGFR-TKI resistance inevitably occurs after approximately 12 months of treatment. Acquired mechanisms of resistance, other than secondary mutations in EGFR (T790 M) which account for 50-60%, are less well understood. Here, we identified lncRNA H19 as a significantly downregulated lncRNA in vitro models and clinical specimens with acquired EGFR-TKI resistance, H19 knockdown or overexpression conferred resistance or sensitivity, respectively, both in vitro and in vivo models. H19 downregulation contributed to erlotinib resistance through interaction and upregulation of PKM2, which enhanced the phosphorylation of AKT. AKT inhibitors restored the sensitivity of erlotinib-resistant cells to erlotinib. In EGFR-mutant patients treated with EGFR-TKIs, low H19 levels were associated with a shorter progression-free survival (PFS) (P = 0.021). These findings revealed a novel mechanism of low-level H19 in the regulation of erlotinib resistance in EGFR-mutant lung cancers. Combination of AKT inhibitors and EGFR-TKIs could be a rational therapeutic approach for some subgroups of EGFR-mutant lung cancer patients.

Expressions of CLDN1 and insulin-like growth factor 2 are associated with poor prognosis in stage N2 non-small cell lung cancer.

BACKGROUND: Patients with single station mediastinal lymph node (N2) non-small cell lung cancer (NSCLC) have a better prognosis than those with multilevel N2. The molecular factors which are involved in disease progression remain largely unknown. The purpose of this study was to investigate gene expression differences between single station and multilevel N2 NSCLC and to identify the crucial molecular factors which are associated with progress and prognosis of stage N2 NSCLC. METHODS: Gene expression analysis was performed using Agilent 4×44K Whole Human Genome Oligo Microarray on 10 freshfrozen lymph node tissue samples from single station N2 and paired multilevel N2 NSCLC patients. Real-time reverse transcription (RT)-PCR was used to validate the differential expression of 14 genes selected by cDNA microarray of which four were confirmed. Immunohistochemical staining for these validated genes was performed on formalin-fixed, paraffinembedded tissue samples from 130 cases of stage N2 NSCLC arranged in a high-density tissue microarray. RESULTS: We identified a 14 gene expression signature by comparative analysis of gene expression. Expression of these genes strongly differed between single station and multilevel N2 NSCLC. Four genes (ADAM28, MUC4, CLDN1, and IGF2) correlated with the results of microarray and real-time RT-PCR analysis for the gene-expression data in samples from 56 NSCLC patients. Immunohistochemical staining for these genes in samples from 130 cases of stage N2 NSCLC demonstrated the expression of IGF2 and CLDN1 was negatively correlated with overall survival of stage N2 NSCLC. CONCLUSIONS: Our results suggest that the expression of CLDN1 and IGF2 indicate a poor prognosis in stage N2 NSCLC. Further, CLDN1 and IGF2 may provide potential targeting opportunities in future therapies.

Combined Erlotinib and Cetuximab overcome the acquired resistance to epidermal growth factor receptors tyrosine kinase inhibitor in non-small-cell lung cancer.

PURPOSE: Non-small-cell lung cancer (NSCLC) cells with somatic mutations in epidermal growth factor receptors (EGFR) are initially susceptible to tyrosine kinase inhibitor (TKI); however, eventually resistance to TKI is developed in these cells, which leads to the failure of treatment. The most common mechanism of this acquired drug resistance is development of a secondary T790M mutation in EGFR. In this study, we investigated the effects of the combination of Erlotinib and Cetuximab on T790M and L858R mutation lung cancer cells lines (H1975), in the primary NSCLC cells with the T790M mutation and TKI-resistant EGFR mutations human tumor xenograft model (H1975). METHODS: The effects of these two agents on cell proliferation, apoptosis, and EGFR-dependent signaling were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V staining, and Western blotting. Sensitivity of EGFR inhibitors was detected in the primary tumor cell suspension and human tumor xenograft model (H1975). RESULTS: Compared with single-agent treatment, the combination of Cetuximab and Erlotinib increased apoptosis of EGFR TKI-resistant NSCLC cells (H1975), resulting in more pronounced growth inhibition on cell proliferation and significant inhibition of EGFR-dependent signaling. CONCLUSIONS: These data suggest that treatment with a combination of Erlotinib and Cetuximab overcomes T790M-mediated drug resistance.

Value of the metastatic lymph node ratio for predicting the prognosis of non-small-cell lung cancer patients.

BACKGROUND: The aim of this study was to investigate the relation between the metastatic lymph node ratio (LNR) and the prognosis of non-small-cell lung cancer (NSCLC). METHODS: A total of 301 patients with N1 or N2 NSCLC who underwent complete pulmonary resection were analyzed retrospectively. The correlations between the LNR and clinical and pathologic data were analyzed using χ(2) test analysis. The prognostic value of the LNR was calculated by univariate Kaplan-Meier survival analysis and multivariate Cox proportional hazard model analysis. The risk groups were classified by a combination of the LNR and pN stage. RESULTS: The LNR was correlated with age, smoking status, pathologic type, subcarinal lymph node, clinical staging, N stage (P < 0.05), and the number of positive lymph nodes and positive lymph node stations (P < 0.0001). In the univariate analysis, the LNR played an important role in predicting overall survival (OS) (P < 0.0001) and disease-free survival (P < 0.0001) by Kaplan-Meier survival analysis. In the multivariate analysis, high LNR (>18%) was an independent poor prognostic factor for OS [hazard ratio (HR) 2.5034, 95% confidence interval (CI) 1.6096-3.8933, P < 0.0001] and DFS (HR 1.9023, 95% CI 1.2465-2.9031, P = 0.0031). Stratification into high-, medium-, and low-risk groups-based on high-risk factors (LNR > 18%, N2) intermediate-risk factors (LNR > 18%, N1 or LNR < 18%, N2), and low-risk factors (LNR < 18%, N1)-could efficiently predicted outcomes (P < 0.0001) of patients with lymph node-positive NSCLC. CONCLUSIONS: The combination of the LNR and pN status provides a valuable help with prognosis. However, these results must be evaluated further in a large prospective randomized clinical trial.

[Diagnostic value of (18)F-FDG PET/CT imaging plus serum tumor marker assays for pulmonary lesions and clinical significance of SUVmax].

OBJECTIVE: To evaluate the diagnostic value of (18)F-FDG positron emission tomography/computed tomography (PET/CT) plus serum tumor marker assay in lung cancer and explore the correlation between standard uptake value (SUVmax) with clinicopathologic factors in lung cancer. METHODS: A total of 177 cases of lung cancer diagnosed by radiography or computed tomography (CT) were recruited.(18)F-FDG PET/CT imaging and detection of three lung cancer related serum markers (carcinoembryonic antigen, CYFRA21-1 and neuron specific enolase) were performed within one week in all cases. The sensitivity, specificity and accuracy of those approaches were calculated through comparing the results with pathologic examinations. Also the associations between SUVmax and clinicopathologic features were analyzed. RESULTS: Among them, 145 patients were detected to have lung cancer by pathologic diagnosis while the other 32 patients had benign lung diseases. The sensitivity, specificity, accuracy of (18)F-FDG PET/CT imaging, serum tumor markers and their combination in assessing lung cancers were 89.7%, 78.1%, 87.6%; 89.7%, 78.1%, 87.6% and 96.6%, 56.3%, 89.3% respectively. The combination of (18)F-FDG PET/CT and serum tumor markers in lung lesions showed significantly higher sensitivity than serum tumor markers and (18)F-FDG PET/CT alone (P = 0.000, P = 0.002). Its accuracy was also significantly higher than those of tumor markers (P < 0.05). Compared with (18)F-FDG PET/CT alone, the accuracy was higher in combination group. But the difference showed no statistical significance (P > 0.05). SUVmax was significantly associated with tumor staging, tumor size and pathologic type. CONCLUSION: The combination of (18)F-FDG PET/CT and tumor markers may improve the positive diagnostic rate of lung cancer. And SUVmax can help to evaluate tumor staging and determine pathological types.

[Epithelial growth factor receptor mutation status to the effective of survival in non-small cell lung cancer after surgery].

OBJECTIVES: To investigate the relationship between the epithelial growth factor receptor (EGFR) mutation status and clinicopathological factors, and to analyze the mutation on the effect in non-small cell lung cancer (NSCLC) after surgery. METHODS: The NSCLC patients who were resected and detected EGFR gene from March 2009 to March 2011 were retrospectively reviewed. The relationship between EGFR mutation status and clinicopathological factors, tumor markers, prognostic was analyzed. RESULTS: The mutation and the wild group had 169 and 214 patients respectively. EGFR mutation in female, non-smoking, adenocarcinoma and less than 60 years old accounted for 63.91%, 61.54%, 88.76% and 62.13% with statistical significance compared with male (χ(2) = 53.490, P = 0.000), smoking (χ(2) = 48.568, P = 0.000), non-adenocarcinoma (χ(2) = 105.560, P = 0.000) and more than 60 years old (χ(2) = 6.057, P = 0.017). Disease free survival (DFS) of the wild group was better than mutation group (χ(2) = 11.329, P = 0.001). In addition, there were some relations between mutation status and excision repair cross complementing (ERCC1) protein, carcinoembryonic antigen (CEA), squamous cell carcinoma (SCC) and Cyfra21-1. ERCC1(+) (χ(2) = 6.739, P = 0.012), SCC(χ(2) = 16.839, P = 0.000) and Cyfra21-1(χ(2) = 6.638, P = 0.013) more than normal value was common in wild group. Increased CEA was common in mutation group (χ(2) = 5.436, P = 0.023). CONCLUSIONS: EGFR mutation is commonly found in female, non-smoking, adenocarcinoma and less than 60 years old NSCLC patients. The wild group obtains better DFS than mutation group. Tumor markers may predict the mutation status, which need further research.

[Value of metastatic lymph node ratio in predicting the prognosis of non-small cell lung cancer patients].

OBJECTIVE: To investigate the relationship between the metastatic lymph node ratio (LNR) and the prognosis of non-small cell lung cancer (NSCLC). METHODS: A total of 301 patients with N1 and N2 NSCLC undergoing curative pulmonectomy were analyzed retrospectively. There were 103 females and 198 males with a median age of 59 years (range: 31 - 78). The correlations between LNR and clinicopathological data were analyzed by χ(2) test. The effects of LNR on overall survival (OS) and disease free survival (DFS) of patients were analyzed by the methods of univariate Kaplan-Meier and multivariate Cox proportional hazard model. The risk groups were classified by LNR on the basis of N staging. RESULTS: LNR correlated with age, smoking status, pathological type, clinical stage and N stage (P < 0.05). And it also correlated with positive lymph nodes, resected lymph nodes and the number of positive lymph node station (P < 0.001). Kaplan-Meier survival analysis revealed that LNR influenced significantly the lengths of OS (P < 0.001) and DFS (P < 0.001). Cox proportional hazard model showed a high LNR was an independent poor prognostic factor for OS (HR = 2.507, 95%CI 1.612 - 3.900, P < 0.001) and DFS (HR = 1.872, 95%CI 1.182 - 2.964, P = 0.008); and at the same N stage, the low-LNR group was better in OS and DFS than the high-LNR group. After stratification into high-, medium- and low-risk groups, the high- (LNR: > 18%, N-status: N2), intermediate- (LNR: > 18%, N-status: N1; LNR: < 18%, N-status: N2) and low-risk factors (LNR: < 18%, N-status: N1) could efficiently predict the outcomes. The 5-year survival rate (32.8% vs 20.7% vs 6.9%), median survival time (MST) (57 vs 30 vs 16 months), 5-year disease-free survival rate (28.1% vs 16.3% vs 5.5%) and disease-free survival time (38 vs 19 vs 10 months) decreased progressively with the rising risk groups (P < 0.001). CONCLUSION: LNR may be used to accurately predict the prognosis, guide the treatment of NSCLC and improve its staging.

[Clinical and prognostic analysis of skip N2 metastases in stage IIIA-N2 non-small cell lung cancer].

OBJECTIVE: To investigate the clinicopathologic factors and the distribution pattern of N2 lymph nodes, to analyze the relationship between the survival rate and skip metastasis of non-small cell lung cancer (NSCLC) patients. METHODS: The clinical data of 478 patients with a pN2 stage who underwent resection for non-small cell lung cancer from January 2000 to December 2004 was retrospectively analyzed. Skip group and non-skip group were defined. Characteristics of tumors, ganglionar involvement and survival were analyzed in both groups. RESULTS: The incidence rate of skip N2 metastasis in stage IIIA-N2 NSCLC patients was 40.6%, which was correlated to sex, smoking and the type of histology (P < 0.05). Squamous carcinoma was the main type of skip group (chi² = 7.602, P = 0.022). The frequency of skip metastasis was higher in patients with a primary tumor in the upper lobe (57%) compared to the lower lobe (43%) (chi² = 5.097, P = 0.024). Superior nodes were more frequently involved by skip group (chi² = 7.046, P = 0.030). Moreover, the relationship between the primary tumor location and N2 positive lymph nodes were described as follows: right upper lobe cancer displayed skip-N2 nodal metastasis mostly in the 2nd, 3rd and 4th station, right middle and lower lobe mostly in the 7th station, left upper lobe mostly in the 5th and 6th station (71.7%), and left lower lobe mostly in the 7th and 9th station. The 5-year survival rate of pN2 patients with skip metastasis was 22.1% compared to 13.6% in patients with involvement of N1 and N2 nodes (P = 0.001). Survival analysis showed that skip N2 metastasis was an independent risk factor of stage IIIA NSCLC. CONCLUSIONS: The frequency of skip metastasis was higher in patients with a primary tumor in the upper lobe and in the superior nodes. Skip metastasis is an independent prognostic factor of survival. The presence of skip metastasis seems to be a unique subgroup of pN2 disease in NSCLC.

[A clinical and prognostic retrospective analysis of IIIA-N2 non-small cell lung cancer].

OBJECTIVES: To analyze the clinical conditions of postoperative patients with IIIA-N2 non-small cell lung cancer (NSCLC) and the prognostic factors related with survival of NSCLC, and to investigate the influence of operation and therapy on prognosis. METHODS: Clinical data of 657 inpatient cases with IIIA-N2 NSCLC admitted from January 2000 to December 2005 was retrospectively reviewed. The Kaplan-Meier method was used for survival analysis. The Log-rank law was applied to analyze the relationship between the variables and the prognosis in monovariate analysis, while Cox proportional hazard regression model was used to make multivariate analysis. RESULTS: The 1-, 3-and 5-year accumulative survival rates of the operative patience were 64.4%, 26.0% and 17.9%, respectively. The median survival time was 18 months. In monovariate analysis, the main unfavorable factors that affect life span involve were the diameter of tumor, T stage, skip metastasis of N2 lymph node, the number of metastatic lymph nodes, the metastasis of subcarinal lymph nodes, adjuvant chemotherapy, the cycle of adjuvant chemotherapy, postoperative radiotherapy, and the modality of therapy (the effect of naive surgery was disappointed, while the prognosis of the patients with adjuvant chemoradiotherapy was better than those with chemotherapy alone). A multivariate analysis using Cox regression identified 5 factors of prognosis: the diameter of tumor (P = 0.001), the metastasis of subcarinal lymph nodes (P = 0.019), the number of metastatic lymph nodes (P = 0.006), the cycle of adjuvant chemotherapy (P = 0.007), postoperative radiotherapy (P = 0.055), and adjuvant chemoradiotherapy (P = 0.026). CONCLUSIONS: The 5-year survival rate of the patients with IIIA-N2 Non-small cell lung cancer is poor. Tumor size, the metastasis of subcarinal lymph nodes, the number of metastatic LNs, the cycle of adjuvant chemotherapy, and postoperative radiotherapy have an effect on the prognosis. The prognosis of postoperative patients with single-level N2 and multi-level N2 disease is similar, and the key point of survival is the number of nodes involved. The therapeutic effect of patience given adjuvant chemoradiotherapy is superior to those treated with adjuvant chemotherapy.