Purpose: Lung cancer is the worldwide leading cause of death from cancer. DNA methylation in gene promoter regions is a major mechanism of gene expression regulation that may promote tumorigenesis. However, whether clinically relevant subgroups based on DNA methylation patterns exist in lung cancer remains unclear.Experimental Design: Whole-genome DNA methylation analysis using 450K Illumina BeadArrays was performed on 12 normal lung tissues and 124 tumors, including 83 adenocarcinomas, 23 squamous cell carcinomas (SqCC), 1 adenosquamous cancer, 5 large cell carcinomas, 9 large cell neuroendocrine carcinomas (LCNEC), and 3 small-cell carcinomas (SCLC). Unsupervised bootstrap clustering was performed to identify DNA methylation subgroups, which were validated in 695 adenocarcinomas and 122 SqCCs. Subgroups were characterized by clinicopathologic factors, whole-exome sequencing data, and gene expression profiles.Results: Unsupervised analysis identified five DNA methylation subgroups (epitypes). One epitype was distinctly associated with neuroendocrine tumors (LCNEC and SCLC). For adenocarcinoma, remaining four epitypes were associated with unsupervised and supervised gene expression phenotypes, and differences in molecular features, including global hypomethylation, promoter hypermethylation, genomic instability, expression of proliferation-associated genes, and mutations in KRAS, TP53, KEAP1, SMARCA4, and STK11. Furthermore, these epitypes were associated with clinicopathologic features such as smoking history and patient outcome.Conclusions: Our findings highlight one neuroendocrine and four adenocarcinoma epitypes associated with molecular and clinicopathologic characteristics, including patient outcome. This study demonstrates the possibility to further subgroup lung cancer, and more specifically adenocarcinomas, based on epigenetic/molecular classification that could lead to more accurate tumor classification, prognostication, and tailored patient therapy. Clin Cancer Res; 20(23); 6127-40. Ó2014 AACR.

Refined classification of LCLC has implications for diagnosis, prognostics, and therapy decisions. Our molecular analyses support the WHO 2015 classification of LCLC and LCNEC tumors, which herein follow different tumorigenic paths and can accordingly be stratified into different transcriptional subgroups, thus linking diagnostic immunohistochemical staining-driven classification with the transcriptional landscape of lung cancer.

Large cell carcinoma with or without neuroendocrine features (LCNEC and LC, respectively) constitutes 3–9% of non-small cell lung cancer but is poorly characterized at the molecular level. Herein we analyzed 41 LC and 32 LCNEC (including 15 previously reported cases) tumors using massive parallel sequencing for mutations in 26 cancer-related genes and gene fusions in ALK, RET, and ROS1. LC patients were additionally subdivided into three immunohistochemistry groups based on positive expression of TTF-1/Napsin A (adenocarcinoma-like, n = 24; 59%), CK5/P40 (squamous-like, n = 5; 12%), or no marker expression (marker-negative, n = 12; 29%). Most common alterations were TP53 (83%), KRAS (22%), MET (12%) mutations in LCs, and TP53 (88%), STK11 (16%), and PTEN (13%) mutations in LCNECs. In general, LCs showed more oncogene mutations compared to LCNECs. Immunomarker stratification of LC revealed oncogene mutations in 63% of adenocarcinoma-like cases, but only in 17% of marker-negative cases. Moreover, marker-negative LCs were associated with inferior overall survival compared with adenocarcinoma-like tumors (p = 0.007). No ALK, RET or ROS1 fusions were detected in LCs or LCNECs. Together, our molecular analyses support that LC and LCNEC tumors follow different tumorigenic paths and that LC may be stratified into molecular subgroups with potential implications for diagnosis, prognostics, and therapy decisions.

The fourth edition of the WHO Classification of Lung Tumours leads to changes in histological type in 5% of resected NSCCs. The incorporation of IHC staining into NSCC diagnostics demands awareness that the choice of ancillary stains has an effect on diagnosis.

The complex interaction between cancer cells and the microenvironment plays an essential role in all stages of tumourigenesis. Despite the significance of this interplay, alterations in protein composition underlying tumour-stroma interactions are largely unknown. The aim of this study was to identify stromal proteins with clinical relevance in non-small cell lung cancer (NSCLC). A list encompassing 203 stromal candidate genes was compiled based on gene expression array data and available literature. The protein expression of these genes in human NSCLC was screened using the Human Protein Atlas. Twelve proteins were selected that showed a differential stromal staining pattern (BGN, CD99, DCN, EMILIN1, FBN1, PDGFRB, PDLIM5, POSTN, SPARC, TAGLN, TNC and VCAN). The corresponding antibodies were applied on tissue microarrays, including 190 NSCLC samples, and stromal staining was correlated with clinical parameters. Higher stromal expression of CD99 was associated with better prognosis in the univariate (p 5 0.037) and multivariate (p 5 0.039) analysis. The association was independent from the proportion of tumour stroma, the fraction of inflammatory cells and clinical and pathological parameters like stage, performance status and tumour histology. The prognostic impact of stromal CD99 protein expression was confirmed in an independent cohort of 240 NSCLC patients (p 5 0.008). Furthermore, double-staining confocal fluorescence microscopy showed that CD99 was expressed in stromal lymphocytes as well as in cancer-associated fibroblasts. Based on a comprehensive screening strategy the membrane protein CD99 was identified as a novel stromal factor with clinical relevance. The results support the concept that stromal properties have an important impact on tumour progression.Non-small cell lung cancer (NSCLC) represents the leading cause of cancer-related death world-wide. 1 Despite advances in NSCLC management, the effectiveness of available treatments is limited and the general prognosis remains poor. Although clinically important molecular biomarkers, like epidermal growth factor receptor mutations and ALK-EML4 translocations, have been identified in subgroups of patients, 2,3 the failure of long-lasting therapy responses and the absence of other valuable biological targets stress the need for new treatment strategies.NSCLC is a heterogeneous entity, comprising defined histological subtypes of adenocarcinoma, squamous cell carcinoma and large cell cancer, each with distinguishing morphological features. However, the stromal cell compartment in lung cancer is characterised by a common desmoplastic

CK5, TTF1, and napsin A are sensitive markers for squamous cell carcinoma and adenocarcinoma of the lung.

Precision medicine requires accurate multi-gene clinical diagnostics. We describe the implementation of an Illumina TruSight Tumor (TST) clinical NGS diagnostic framework and parallel validation of a NanoString RNA-based ALK, RET, and ROS1 gene fusion assay for combined analysis of treatment predictive alterations in non-small cell lung cancer (NSCLC) in a regional healthcare region of Sweden (Scandinavia). The TST panel was clinically validated in 81 tumors (99% hotspot mutation concordance), after which 533 consecutive NSCLCs were collected during one-year of routine clinical analysis in the healthcare region (˜90% advanced stage patients). The NanoString assay was evaluated in 169 of 533 cases. In the 533-sample cohort 79% had 1-2 variants, 12% >2 variants and 9% no detected variants. Ten gene fusions (five ALK, three RET, two ROS1) were detected in 135 successfully analyzed cases (80% analysis success rate). No ALK or ROS1 FISH fusion positive case was missed by the NanoString assay. Stratification of the 533-sample cohort based on actionable alterations in 11 oncogenes revealed that 66% of adenocarcinomas, 13% of squamous carcinoma (SqCC) and 56% of NSCLC not otherwise specified harbored ≥1 alteration. In adenocarcinoma, 10.6% of patients (50.3% if including KRAS) could potentially be eligible for emerging therapeutics, in addition to the 15.3% of patients eligible for standard EGFR or ALK inhibitors. For squamous carcinoma corresponding proportions were 4.4% (11.1% with KRAS) vs 2.2%. In conclusion, multiplexed NGS and gene fusion analyses are feasible in NSCLC for clinical diagnostics, identifying notable proportions of patients potentially eligible for emerging molecular therapeutics.

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