Likun Hou

High Discrepancy of Driver Mutations in Patients with NSCLC and Synchronous Multiple Lung Ground-Glass Nodules

Background: The aim of this study was to investigate the discordance rates of eight known driver mutations among multiple matched intrapulmonary ground-glass nodules (GGNs) in non–small-cell lung cancer (NSCLC) patients. Methods: Tumors from 35 patients with multiple lesions resected, including confirmed NSCLC and at least one GGN, were analyzed for mutations in EGFR, KRAS, HER2, BRAF, and PIK3CA together with fusions in ALK, ROS1, and RET. Results: From 35 patients, a total of 72 lesions (60 were GGNs) were analyzed. These included nine adenocarcinoma in situ, nine minimal invasive adenocarcinoma, and 54 invasive adenocarcinoma. Among them, 33 tumor lesions (45.8 %) were found harboring EGFR mutations: 13 tumors with exon 19 deletion, 18 with L858R on exon 21, and two with both exon 19 del and L858R mutation. There were 5 tumors (6.9 %) harboring EML4-ALK fusion, four HER2 mutations (5.6%), three KRAS mutations (4.2%), one ROS1 fusion and one BRAF mutation. When we used the matched tumors to determine the intertumor discrepancy, only six out of 30 patients harbored identical mutations. The discordance rate of driver mutations was 80% (24 of 30) in those patients harboring at least one of the detected driver mutations. The median disease-free survival was 41.2 months (95% confidence interval: 35.8–52.6 months) and the median overall survival was “still not reached” in this cohort. Conclusions: We found a high discrepancy of driver mutations among NSCLC patients with GGNs and a favorable prognosis after multiple lesions resection, which support surgical resection in this situation as a reasonable approach.

Epithelial phenotype as a predictive marker for response to EGFR-TKIs in non-small cell lung cancer patients with wild-type EGFR.

Epithelial‐to‐mesenchymal transition (EMT) has profound impacts on cancer progression and also on drug resistance, including epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‐TKIs). Nowadays, there is still no predictive biomarker identified for the use of EGFR‐TKIs in non‐small cell lung cancer (NSCLC) patients with wild‐type EGFR. To clarify the role of EMT phenotype as a predictive marker for EGFR‐TKI, we performed a retrospective study in 202 stage IV or recurrent NSCLC patients receiving gefitinib or erlotinib therapy from June 2008 to September 2012 in our institute. Clinical data and EGFR mutational status were collected, while epithelial, epithelial to mesenchymal, not specified or mesenchymal phenotype were classified according to EMT markers such as E‐cadherin, fibronectin, N‐cadherin and vimentin by immunohistochemistry. Epithelial phenotype was more frequently found in patients with EGFR mutation (p = 0.044). Epithelial phenotype was associated with a significantly higher objective response rate (23.5 vs. 11.1 vs. 0.0 vs. 2.4%, p = 0.011), longer progression‐free survival (4.4 vs. 1.9 vs. 1.7 vs. 1.0 months, p < 0.001) and longer overall survival (11.5 vs. 8.9 vs. 4.5 vs. 4.9 months, p < 0.001) compared to epithelial to mesenchymal, not specified and mesenchymal phenotype in the wild‐type EGFR subgroup. In the subgroup with EGFR mutation, the trend remained but without a statistically significant difference. In conclusion, epithelial phenotype was more likely expressed in patients with EGFR mutation and was associated with a better outcome in advanced NSCLC patients with wild‐type EGFR, which indicates that the EMT phenotype might be a potential marker to guide EGFR‐TKI therapy in this population.

EML4-ALK Fusion Detected by RT-PCR Confers Similar Response to Crizotinib as Detected by FISH in Patients with Advanced Non-Small-Cell Lung Cancer.

Introduction: Reverse transcriptase polymerase chain reaction (RT-PCR) assay has been proved to have high sensitivity and specificity to detect anaplastic lymphoma kinase (ALK) rearrangements. The aim of this study was to investigate the response to crizotinib in patients of advanced non–small-cell lung cancer (NSCLC) with ALK rearrangements detected by RT-PCR. Methods: Only patients with advanced NSCLC who had their ALK rearrangement status detected by RT-PCR were included in this analysis. The utility of RT-PCR and fluorescence in situ hybridization (FISH) assay were compared in patients who were treated with crizotinib based on their positive ALK rearrangements. Results: One thousand ten patients were included in this study. Among them, 104 patients were ALK RT-PCR positive and 53 of them received crizotinib treatment. Among 255 tumors simultaneously analyzed by FISH and RT-PCR, the latter successfully detected all the 25 tumors with arrangements, including two cases that were missed by FISH. The overall response rate and median progression-free survival of the 53 patients with ALK rearrangements who received crizotinib treatment were 60.4% (95% confidence interval [CI], 47.2–73.6) and 8.4 months (95% CI, 6.75–10.05), respectively, which were similar to the 21 patients detected by FISH with overall response rate of 57.1% (95% CI, 33.3–76.2; p = 0.799) and median progression-free survival of 7.4 months (95% CI, 4.43–10.38; p = 0.833) after crizotinib treatment. Interestingly, there were two patients responded to crizotinib had their ALK rearrangement detected by RT-PCR but not FISH. Conclusions: RT-PCR should be considered as an alternative/supplemental approach to detect ALK fusion oncogene in NSCLC patients who might benefit from crizotinib treatment.

Long non-coding RNA UCA1 is a predictive biomarker of cancer

Human urothelial carcinoma associated 1 (UCA1) is a long noncoding RNA that is putatively oncogenic in solid tumors. This meta-analysis investigated an association between UCA1 levels and survival times of cancer patients. The primary endpoints were overall survival (OS) and progression-free survival (PFS). A comprehensive, computerized literature search was conducted of the databases PubMed, EMBASE, Chinese National Knowledge Infrastructure (CNKI), and Wanfang. The strength of association between UCA1 and cancer prognosis was assessed by computing the hazard ratio (HR) with its corresponding 95% confidence interval (CI). Twelve studies comprising 954 cancer patients met the criteria for this meta-analysis. Overall, a significant negative association was found between UCA1 levels and OS time (HR1.81, 95% CI1.52−2.17), including the following cancers analyzed independently: colorectal (HR2.61, 95% CI1.56−4.37), non-small cell lung (HR1.49, 95% CI1.16−1.90), gastric (HR2.19, 95% CI1.36−3.51), and ovarian (HR1.89, 95% CI1.14−3.12). There was also a significant negative association between UCA1 levels and PFS time (HR2.59, 95% CI1.61−4.16). In conclusion, this meta-analysis indicated that higher levels of UCA1 correlate with shorter PFS and OS times in cancers.

Association of microRNA-33a Molecular Signature with Non-Small Cell Lung Cancer Diagnosis and Prognosis after Chemotherapy

Objective This study aims to explore the expression pattern and prognostic significance of miR-33a in non-small cell lung cancer (NSCLC) treated with adjuvant chemotherapy. Methods MiR-33aexpression in NSCLC was analyzed in silico using the GEO database and was subsequently confirmed by quantitative RT-PCR in 147 NSCLC biopsies. Among these, 32 of these biopsies were paired with adjacent non-neoplastic tissues. The survival analysis of NSCLC by Kaplan-Meier estimates was stratified based on miR-33a expression. In addition, multivariate survival analysis in corresponding groups of NSCLC patients was conducted by Cox proportional hazards regression model. Results The in silico analysis of miR-33a expression in NSCLC resulted to its down-regulation in different tumor types. The expression level of miR-33a was lower in each grade of NSCLC tumor biopsies than in normal lung tissues. Univariate and multivariate survival analysis further established that low miR-33a expression was an important risk factor for overall survival and disease free survival in NSCLC patients. Conclusion Our study implied that miR-33a expression levels may have an essential role in NSCLC progression, and could act as a specific and sensitive biomarker for NSCLC patients who have undergone adjuvant chemotherapy.

LAG-3 Protein Expression in Non–Small Cell Lung Cancer and Its Relationship with PD-1/PD-L1 and Tumor-Infiltrating Lymphocytes

Introduction: Immunotherapy targeting the programmed death 1 (PD‐1)/programmed death ligand 1 (PD‐L1) checkpoint has shown promising efficacy in patients with NSCLC. Lymphocyte activating 3 gene (LAG‐3) is another important checkpoint, and its role in NSCLC is still not clear. In this study we investigated lymphocyte activing 3 (LAG‐3) protein expression; its correlation with PD‐1, PD‐L1, and tumor‐infiltrating lymphocytes (TILs); and its association with survival in NSCLC. Methods: The expression of LAG‐3 (EPR4392 [Abcam, Cambridge, MA]) protein was assessed in 55 NSCLC cell lines by immunohistochemistry. LAG‐3, PD‐1 (NAT 105 [Cell Marque, Rocklin, CA]), and PD‐L1 (22C3 [Dako, Carpenteria, CA]) protein expression was evaluated by immunohistochemistry, and TIL abundance was scored in 139 surgically resected specimens from patients with NSCLC. We also verified results in samples from 62 patients with untreated NSCLC and detected a correlation between LAG‐3 expression and EGFR and KRAS mutation and echinoderm microtubule associated protein like 4 gene (EML4)–anaplastic lymphoma receptor tyrosine kinase gene (ALK) rearrangement. Results: LAG‐3 was not expressed on any of the 55 NSCLC cell lines. However, LAG‐3 was expressed on the TILs in 36 patients with NSCLC (25.9%). Sixty patient samples (43.2%) were positive for PD‐1 on the TILs, and 25 (18.0%) were positive for PD‐L1 on tumor cells. Neither LAG‐3 nor PD‐1 was expressed on the tumor cells. LAG‐3 was overexpressed on the TILs in nonadenocarcinoma compared with in adenocarcinoma (p = 0.031). LAG‐3 expression on TILs was significantly correlated with that of PD‐1 on TILs (p < 0.001) and PD‐L1 on tumor cells (p = 0.041) but not with TIL percentage (p = 0.244). With the logistic regression model, the ORs for LAG‐3 were 0.320 (95% confidence interval [CI]: 0.110–0.929) and 4.364 (95% CI: 1.898–10.031) when nonadenocarcinoma was compared with adenocarcinoma and TILs that were negative for PD‐1 were compared with those positive for PD‐1. Recurrence‐free survival was significantly different in patients whose TILs were LAG‐3–negative as opposed to LAG‐3–positive (1.91 years [95% CI: 0.76–3.06] versus 0.87 years [95% CI: 0.27–1.47] [p = 0.025]). Likewise, LAG‐3 status of TILs (negative versus positive) did significantly affect overall survival (OS) (3.04 years [95% CI: 2.76–3.32] versus 1.08 years [95% CI: 0.42–1.74] [p = 0.039]). Using Kaplan‐Meier analysis, we found that patients with both PD‐L1–negative tumor cells and LAG‐3–negative TILs have longer recurrence‐free survival than patients who are either PD‐L1– or LAG‐3–positive or both PD‐L1– and LAG‐3–positive (2.09 years [95% CI: 0.90–3.28] versus 1.42 years [95% CI: 0.46–2.34] versus 0.67 years [95% CI: 0.00–1.45] [p = 0.007]). In the verification stage, high expression of LAG‐3 was also significantly correlated with higher expression of PD‐1 on TILs (p = 0.016) and PD‐L1 on tumor cells (p = 0.014). There was no correlation between LAG‐3 expression and EGFR (p = 0.325) and KRAS mutation (p = 1.000) and ALK fusion (p = 0.562). Conclusions: LAG‐3 is expressed on TILs in tumor tissues of some patients with NSCLC. Its expression was higher in nonadenocarcinoma and correlated with PD‐1/PD‐L1 expression. LAG‐3 positivity or both LAG‐3 and PD‐L1 positivity was correlated with early postoperative recurrence. LAG‐3 was related to poor prognosis.

Incidence and physiological mechanism of carboplatin-induced electrolyte abnormality among patients with non-small cell lung cancer

To clarify the association between carboplatin and electrolyte abnormality, a pooled-analysis was performed with the adverse event reports of non-small cell lung cancer patients. A total of 19901 adverse events were retrieved from the FDA Adverse Event Reporting System (FAERS). Pooled reporting odds ratios (RORs) and 95% CIs suggested that carboplatin was significantly associated with hyponatremia (pooled ROR = 1.57, 95% CI 1.182.09, P = 1.99 × 10-3) and hypokalemia (pooled ROR = 2.37, 95% CI 1.803.10, P = 5.24 × 10-10) as compared to other therapies. In addition, we found that dehydration was frequently concurrent with carboplatin therapy (pooled ROR = 2.01, 95% CI 1.522.66, P = 8.37 × 10-7), which may prompt excessive water ingestion and decrease serum electrolyte concentrations. This information has not been mentioned in the FDA-approved drug label and could help explain the physiological mechanism of carboplatin-induced electrolyte abnormality. In conclusion, the above results will facilitate clinical management and prompt intervention of life-threatening electrolyte imbalance in the course of cancer treatment.

Feasibility of cytological specimens for ALK fusion detection in patients with advanced NSCLC using the method of RT-PCR.

OBJECTIVES Histological tissues are preferred for anaplastic lymphoma kinase (ALK) fusion detection in non-small cell lung cancer (NSCLC). The aim of this study was to evaluate the feasibility of cytological sample as an alternative specimen for ALK fusion testing in patients with advanced NSCLC. MATERIALS AND METHODS Advanced NSCLC patients with cytological specimens or tumor tissue who had their ALK fusion status detected by the method of reverse transcriptase polymerase chain reaction (RT-PCR) in Shanghai Pulmonary Hospital, Tongji University were included into this study. The efficacy was evaluated in those with ALK fusion positive and received the therapy of crizotinib. RESULTS 1274 patients were included in this study. Among them, 108 patients were ALK RT-PCR positive and 69 of them received crizotinib treatment. Among 1002 patients with cytological specimens, the average concentration of RNA extracted from cytological specimens was 60.99 ng/μl (95% confidence interval [CI], 55.56-66.60) and the incidence rate of ALK fusion was 8.3% (83/1002), which were similar to 63.16 ng/μl (95% CI, 51.88-76.34) (p=0.727) and 9.2% (25/272, p=0.624) in 272 patients with tumor tissue. Also, there were no statistically significant differences regarding to the objective response rate (ORR) (62.0% vs. 42.1%, p=0.177) and the median progression free survival (mPFS) [8.6 months (95% CI 7.30-9.84) vs. 7.0 months (95% CI 4.54-9.47), p=0.736] in patients of cytological group and tissue group after the treatment of crizotinib. CONCLUSION Cytological specimens showed a high feasibility to detect ALK fusion status, which could be regarded as alternative samples for ALK fusion detection by the method of RT-PCR in patients with advanced NSCLC.

Distinct clinicopathologic features, genomic characteristics and survival of central and peripheral pulmonary large cell neuroendocrine carcinoma: From different origin cells?

BACKGROUND Pulmonary large cell neuroendocrine carcinoma (LCNEC) represents a rare entity in lung cancer with dismal prognosis. In the present study, we investigated whether there are significant differences between central and peripheral tumors of LCNEC, in terms of clinicopathologic features, genomic profiles, and survival. METHODS AND MATERIALS A total of 126 cases of LCNEC were included. The tumors with invasion of the segmental and/or lobar bronchus were classified as central LCNEC and those without as peripheral LCNEC. EGFR/BRAF/Kras mutations and ALK/ROS1 translocations were detected. Overall survival (OS) was evaluated by the Kaplan-Meier plots. RESULTS The majority of LCNEC proved to be of the peripheral type (64.3%, 81/126). Central tumors were associated with smoking habit (p = 0.047), higher TNM-stage (p = 0.014) and larger tumor size (p < 0.001). Expression of neuroendocrine markers (CD56, CGA, and SYN) was not significantly different by tumor location but central tumors had higher serum levels of NSE (p = 0.004). Peripheral tumors had a higher incidence of EGFR mutations (18.8% vs. 0%, p = 0.023). ROS1 translocation was detected in 1 patient with peripheral LCNEC. RB1 protein was more frequently expressed in peripheral tumor than central tumor. The median OS was 3.71 years in the entire cohort. Peripheral tumors had better survival compared with central tumors (median OS: 4.04 vs. 1.51 years, p < 0.001). Multivariate analyses demonstrated tumor location (hazard ratio [HR], 6.07, 95% confidence interval [CI], 1.57-23.44, p = 0.009), resection status (HR, 6.58, 95% CI, 1.92-22.51, p = 0.003) and EGFR mutational status (HR, 0.18, 95% CI, 0.04-0.75, p = 0.018) were independent prognostic factors for OS. CONCLUSION Primary tumor location of LCNEC, divided into central and peripheral type, has distinct clinicopathologic features, genomic characteristics and survival. These differences are likely due to differences in the origin cells and pathogenesis of these tumors.

Heterogeneity of PD-L1 Expression Among the Different Histological Components and Metastatic Lymph Nodes in Patients With Resected Lung Adenosquamous Carcinoma.

&NA; In this study, we investigated heterogeneity of programmed death‐ligand 1 (PD‐L1) expression and accordance of metastatic nodes. PD‐L1 expression in 72 resected lung adenosquamous carcinoma and paired metastatic nodes was assessed. We found that PD‐L1 expression was inconsistent in different histologic components within the tumor, and consistent in paired (primary and lymph node) histologic components. Introduction: Programmed death‐ligand 1 (PD‐L1) expression using immunohistochemistry is approved by the US Food and Drug Administration to guide treatment with anti‐programmed death‐1/PD‐L1 monoantibodies. However, intratumoral heterogeneity of PD‐L1 expression and the accordance between primary and metastatic lesions remains unresolved. Materials and Methods: PD‐L1 expression was evaluated in tumor cells and tumor‐infiltrating lymphocytes (TILs) of surgically resected lung adenosquamous carcinoma. Discrepancy of PD‐L1 expression and cluster of differentiation 8‐positive TILs of different histologic components was investigated. PD‐L1 expression was also compared between primary tumor and nodal metastases. Results: The study included a total of 72 lung adenosquamous carcinomas. Fifteen patients (20.8%) and 25 patients (34.7%) were positive for PD‐L1 expression in adenomatous and squamous components respectively, with a cutoff value of 5%. We found that 57.8% of cases showed consistent PD‐L1 expression in tumor cells in the different histological components at a cutoff of 1%, and 48.1% of cases were likewise consistent at a cutoff of 5%. In paired squamous components of metastatic nodes and primary lesions, 90% and 80% of cases of PD‐L1 expression were consistent, at cutoffs of 1% and 5%, respectively. For the adenomatous component of tumor/metastasis pairs, 77.8% of cases at the 1% cutoff and 74.1% of cases at the 5% cutoff were consistent, partially attributed to the difference of predominant histologic patterns. Conclusion: PD‐L1 expression showed discrepancy in different histological components within a tumor and consistency in paired histological type between tumor and metastases. Different pathological features might contribute to the heterogeneous PD‐L1 expression in patients with non–small‐cell lung cancer.