June Koo Lee

The transcriptional landscape and mutational profile of lung adenocarcinoma

All cancers harbor molecular alterations in their genomes. The transcriptional consequences of these somatic mutations have not yet been comprehensively explored in lung cancer. Here we present the first large scale RNA sequencing study of lung adenocarcinoma, demonstrating its power to identify somatic point mutations as well as transcriptional variants such as gene fusions, alternative splicing events, and expression outliers. Our results reveal the genetic basis of 200 lung adenocarcinomas in Koreans including deep characterization of 87 surgical specimens by transcriptome sequencing. We identified driver somatic mutations in cancer genes including EGFR, KRAS, NRAS, BRAF, PIK3CA, MET, and CTNNB1. Candidates for novel driver mutations were also identified in genes newly implicated in lung adenocarcinoma such as LMTK2, ARID1A, NOTCH2, and SMARCA4. We found 45 fusion genes, eight of which were chimeric tyrosine kinases involving ALK, RET, ROS1, FGFR2, AXL, and PDGFRA. Among 17 recurrent alternative splicing events, we identified exon 14 skipping in the proto-oncogene MET as highly likely to be a cancer driver. The number of somatic mutations and expression outliers varied markedly between individual cancers and was strongly correlated with smoking history of patients. We identified genomic blocks within which gene expression levels were consistently increased or decreased that could be explained by copy number alterations in samples. We also found an association between lymph node metastasis and somatic mutations in TP53. These findings broaden our understanding of lung adenocarcinoma and may also lead to new diagnostic and therapeutic approaches.

Immunohistochemical screening for anaplastic lymphoma kinase (ALK) rearrangement in advanced non-small cell lung cancer patients

Fluorescence in situ hybridization (FISH) is currently used to detect non-small cell lung cancer (NSCLC) patients with anaplastic lymphoma kinase (ALK) gene rearrangement, who are candidates for ALK inhibitor therapy. However, FISH may not be a practical method for screening for ALK-positive patients in a large population due to its cost and difficulty in interpretation. We investigated the role of immunohistochemistry (IHC) to screen for ALK rearrangement in advanced NSCLC. We identified 1,166 stage IIIB or IV NSCLC patients with non-squamous histology from the Seoul National University Hospital NSCLC database. To enrich ALK-positive cases, we selected 262 patients who were either EGFR wild-type or non-responders to previous EGFR tyrosine kinase inhibitors (TKI). ALK IHC and ALK FISH were performed on formalin-fixed, paraffin-embedded tissue. ALK protein was expressed in 28 (10.7%) tumors in 262 patients. ALK FISH was positive in 25 (9.5%) cases. All patients with IHC score of 3 (n=9) were FISH-positive and all patients with score of 0 (n=234) were FISH-negative. Among patients with IHC scores of 1 and 2, five (83.3%, 5/6) and eleven (84.6%, 11/13) were FISH-positive, respectively. The sensitivity and specificity of ALK IHC with intensity score of 1 or more were 100% and 98.7%, respectively. IHC can be a useful test for screening ALK FISH-positive cases in advanced NSCLC. FISH testing should be considered for advanced NSCLC patients with tumors showing mild to moderate staining for ALK by IHC to confirm ALK translocation.

Epidermal growth factor receptor tyrosine kinase inhibitors vs conventional chemotherapy in non-small cell lung cancer harboring wild-type epidermal growth factor receptor: a meta- analysis

IMPORTANCE Current guidelines recommend both epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) and cytotoxic chemotherapy drugs as standard treatment options for patients with wild-type (WT) EGFR who were previously treated for non-small cell lung cancer (NSCLC). However, it is not clear that EGFR TKIs are as efficacious as chemotherapy in patients with WT EGFR. OBJECTIVE To determine the association between first-generation EGFR TKI vs chemotherapy and survival in advanced NSCLC patients with WT EGFR. DATA SOURCES PubMed, EMBASE, Cochrane database, and meeting abstracts of the American Society of Clinical Oncology and European Society of Medical Oncology through December 2013. STUDY SELECTION Eligible studies were randomized controlled trials comparing EGFR TKI with conventional chemotherapy in patients with advanced NSCLC. Out of 1947 retrieved articles, 11 trials incorporating 1605 patients with WT EGFR were included. DATA EXTRACTION AND SYNTHESIS Two reviewers extracted trial characteristics and outcomes. The risk of bias was evaluated using the Cochrane tool. All measures were pooled using random-effects models and 95% CIs were calculated. MAIN OUTCOMES AND MEASURES The primary outcome was progression-free survival (PFS), measured as hazard ratios (HRs). The secondary outcomes were objective response rate and overall survival, expressed as relative risks and HRs, respectively. RESULTS Among patients with WT EGFR tumors, chemotherapy was associated with improvement of PFS, compared with TKI (HR for TKI, 1.41; 95% CI, 1.10-1.81). No statistically significant subgroup difference was identified in terms of line of treatment (first-line vs second- or later-line), experimental drug, dominant ethnicity, or EGFR mutation analysis method. Trials using more sensitive platforms than direct sequencing were associated with a significant PFS benefit with chemotherapy (HR for TKI, 1.84; 95% CI, 1.35-2.52). The association of chemotherapy with improvement in PFS was also significant in second- or later-line trials (HR, 1.34; 95% CI, 1.09-1.65). The objective response rate was higher with chemotherapy (92/549, 16.8%, vs 39/540, 7.2%, for TKI; relative risk for TKI, 1.11; 95% CI, 1.02-1.21); however, no statistically significant difference was observed with respect to overall survival (HR for TKI, 1.08; 95% CI, 0.96-1.22). CONCLUSIONS AND RELEVANCE Among patients with advanced NSCLC harboring WT EGFR, conventional chemotherapy, compared with first-generation EGFR TKI, was associated with improvement in PFS but not overall survival.

Primary resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in patients with non-small-cell lung cancer harboring TKI-sensitive EGFR mutations: an exploratory study

BACKGROUND The mechanism of primary resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small-cell lung cancer (NSCLC) has not been clearly understood. PATIENTS AND METHODS Eleven patients exhibiting primary resistance (disease progression <3 months) were identified among 197 consecutive NSCLC patients with TKI-sensitive EGFR mutations who received EGFR TKIs at Seoul National University Hospital. Treatment-naïve tumors were examined for concurrent genetic alterations using fluorescence in situ hybridization and targeted deep sequencing of cancer-related genes. Deletion polymorphism of Bcl-2-interacting mediator of cell death (BIM) gene was examined to validate its predictive role for TKI outcome. RESULTS The median progression-free survival (PFS) for patients receiving EGFR TKIs was 11.9 months, and the response rate 78.8%. Among the 11 patients exhibiting primary resistance, a de novo T790M mutation was identified in one patient, and two exhibited mesenchymal-epithelial transition amplification and anaplastic lymphoma kinase fusion. Targeted deep sequencing identified no recurrent, coexistent drivers of NSCLC. Survival analysis revealed that patients with recurrent disease after surgery had a longer PFS than those with initial stage IV disease. However, BIM deletion polymorphism, line of treatment, EGFR genotype, and smoking were not predictive of PFS for EGFR TKIs. CONCLUSIONS We identified coexistent genetic alterations of cancer-related genes that could explain primary resistance in a small proportion of patients. Our result suggests that the mechanism of primary resistance might be heterogeneous.

Actin remodelling factors control ciliogenesis by regulating YAP/TAZ activity and vesicle trafficking

Primary cilia exert a profound impact on cell signalling and cell cycle progression. Recently, actin cytoskeleton destabilization has been recognized as a dominant inducer of ciliogenesis, but the exact mechanisms regulating ciliogenesis remain poorly understood. Here we show that the actin cytoskeleton remodelling controls ciliogenesis by regulating transcriptional coactivator YAP/TAZ as well as ciliary vesicle trafficking. Cytoplasmic retention of YAP/TAZ correlates with active ciliogenesis either in spatially confined cells or in cells treated with an actin filament destabilizer. Moreover, knockdown of YAP/TAZ is sufficient to induce ciliogenesis, whereas YAP/TAZ hyperactivation suppresses serum starvation-mediated ciliogenesis. We also identify actin remodelling factors LIMK2 and TESK1 as key players in the ciliogenesis control network in which YAP/TAZ and directional vesicle trafficking are integral components. Our work provides new insights for understanding the link between actin dynamics and ciliogenesis.

Differential sensitivities to tyrosine kinase inhibitors in NSCLC harboring EGFR mutation and ALK translocation.

Epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) translocations in non-small cell lung cancer (NSCLC) are mutually exclusive. However, several exceptional cases harboring both genetic alterations have been reported. In this study, a total of 444 patients with lung adenocarcinoma were examined for their EGFR and ALK status at Seoul National University Hospital between July 2008 and September 2011. EGFR mutations and ALK translocations were detected in 228 (51.4%) and 34 (7.7%) patients, respectively. Four patients (0.9%) had both genetic alterations and three underwent curative surgery. One patient who received both EGFR tyrosine kinase and ALK inhibitors, separately showed an objective response to the ALK inhibitor alone. Considering our and previous studies, patients harboring both EGFR mutation and ALK translocation showed differential sensitivities to both targeted therapies, suggesting a variable dependence on EGFR and ALK oncogenes.

Comparative analyses of overall survival in patients with anaplastic lymphoma kinase-positive and matched wild-type advanced nonsmall cell lung cancer†‡

The purpose of this study was to investigate the overall survival (OS) of patients with advanced ALK‐positive nonsmall cell lung cancer (NSCLC) who were managed in the pre‐ALK inhibitor era and to compare their survival with that of a matched case cohort of ALK wild‐type (WT) patients.

Clonal History and Genetic Predictors of Transformation Into Small-Cell Carcinomas From Lung Adenocarcinomas.

Purpose Histologic transformation of EGFR mutant lung adenocarcinoma (LADC) into small-cell lung cancer (SCLC) has been described as one of the major resistant mechanisms for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). However, the molecular pathogenesis is still unclear. Methods We investigated 21 patients with advanced EGFR-mutant LADCs that were transformed into EGFR TKI-resistant SCLCs. Among them, whole genome sequencing was applied for nine tumors acquired at various time points from four patients to reconstruct their clonal evolutionary history and to detect genetic predictors for small-cell transformation. The findings were validated by immunohistochemistry in 210 lung cancer tissues. Results We identified that EGFR TKI-resistant LADCs and SCLCs share a common clonal origin and undergo branched evolutionary trajectories. The clonal divergence of SCLC ancestors from the LADC cells occurred before the first EGFR TKI treatments, and the complete inactivation of both RB1 and TP53 were observed from the early LADC stages in sequenced tumors. We extended the findings by immunohistochemistry in the early-stage LADC tissues of 75 patients treated with EGFR TKIs; inactivation of both Rb and p53 was strikingly more frequent in the small-cell-transformed group than in the nontransformed group (82% v 3%; odds ratio, 131; 95% CI, 19.9 to 859). Among patients registered in a predefined cohort (n = 65), an EGFR mutant LADC that harbored completely inactivated Rb and p53 had a 43× greater risk of small-cell transformation (relative risk, 42.8; 95% CI, 5.88 to 311). Branch-specific mutational signature analysis revealed that apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC)-induced hypermutation was frequent in the branches toward small-cell transformation. Conclusion EGFR TKI-resistant SCLCs are branched out early from the LADC clones that harbor completely inactivated RB1 and TP53. The evaluation of RB1 and TP53 status in EGFR TKI-treated LADCs is informative in predicting small-cell transformation.

Actin remodeling confers BRAF inhibitor resistance to melanoma cells through YAP/TAZ activation

The activation of transcriptional coactivators YAP and its paralog TAZ has been shown to promote resistance to anti‐cancer therapies. YAP/TAZ activity is tightly coupled to actin cytoskeleton architecture. However, the influence of actin remodeling on cancer drug resistance remains largely unexplored. Here, we report a pivotal role of actin remodeling in YAP/TAZ‐dependent BRAF inhibitor resistance in BRAF V600E mutant melanoma cells. Melanoma cells resistant to the BRAF inhibitor PLX4032 exhibit an increase in actin stress fiber formation, which appears to promote the nuclear accumulation of YAP/TAZ. Knockdown of YAP/TAZ reduces the viability of resistant melanoma cells, whereas overexpression of constitutively active YAP induces resistance. Moreover, inhibition of actin polymerization and actomyosin tension in melanoma cells suppresses both YAP/TAZ activation and PLX4032 resistance. Our siRNA library screening identifies actin dynamics regulator TESK1 as a novel vulnerable point of the YAP/TAZ‐dependent resistance pathway. These results suggest that inhibition of actin remodeling is a potential strategy to suppress resistance in BRAF inhibitor therapies.

Metabolic and metastatic characteristics of ALK-rearranged lung adenocarcinoma on FDG PET/CT

INTRODUCTION ALK rearrangement in lung cancer has been identified as a novel molecular target in lung adenocarcinoma. In this study, we evaluated metabolic and metastatic features of lung adenocarcinoma by using FDG PET/CT, with regard to specific genotypes of ALK and EGFR mutation. METHODS Patients with lung adenocarcinoma initially diagnosed and examined with FDG PET/CT and molecular genotyping with biopsy specimen, from September 2009 to September 2011, were selected retrospectively. ALK fluorescence in situ hybridization and EGFR mutations were tested. Maximum standardized uptake value (SUVmax) and metastatic characteristics on FDG PET/CT were analyzed with regard to ALK and EGFR status. RESULTS Of the 331 lung adenocarcinoma patients, 18 were ALK positive (ALK(+)), 156 were EGFR mutation positive (EGFR(+)), and 157 were wild type (WT) for both ALK and EGFR mutation. The ALK(+) tumor showed significantly higher SUVmax and more common metastasis to lymph nodes and distant organs than those of other genotypes in overall patients (P<0.01). In a subgroup analysis of advanced stage (stage IIIb and IV), ALK(+) lung cancer showed significantly higher SUVmax (P<0.05) than EGFR(+) tumors. In another subgroup analysis of size matched groups, ALK(+) tumors showed significant difference in SUVmax, lymph node and distant metastasis (P<0.01), particularly in the moderate-sized tumors (1.5-3cm). CONCLUSION ALK-rearranged lung adenocarcinoma represents higher glucose metabolism and more rapid metastasis to lymph nodes or distant sites compared with those with EGFR mutation and wild type, which suggests more aggressive features of ALK rearrangement.