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The introduction of immune checkpoint inhibitors has transformed the treatment landscape of metastatic non-small cell lung cancer. However, challenges remain to increase the fraction of patients achieving durable clinical responses to these drugs and to help monitor the treatment effect. In this phase II trial, we investigated the toxicity, systemic responses and circulating tumour DNA responses in patients (n = 21) with advanced non-small-cell lung cancer treated with atezolizumab and stereotactic body radiotherapy in the second or later line. We found the combined treatment to be safe with grade 3 toxicity reported in three patients. As the best overall response, four patients had a partial response, eight had stable disease and five had progressive disease. Median overall survival time was still not reached after a median follow-up of 26.5 months and 10/15 patients with programmed death-ligand 1 negative tumours were alive >18 months after the start of the study treatment. ctDNA was detectable at baseline in 11 patients. A rapid decline in ctDNA to

Among early‐stage non‐small‐cell lung cancer (NSCLC) patients, cg05293407TRIM27 was significantly and exclusively associated with survival of lung squamous cell carcinoma patients, who had higher smoking intensity compared to lung adenocarcinoma patients. Generally, the significant association between cg05293407TRIM27 and survival only remained in NSCLC patients having medium‐to‐high pack‐year of smoking. The cg05293407TRIM27‐smoking synergistic interaction might account for histologically heterogeneous effects of TRIM27 DNA methylation on NSCLC survival., Tripartite motif containing 27 (TRIM27) is highly expressed in lung cancer, including non‐small‐cell lung cancer (NSCLC). Here, we profiled DNA methylation of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) tumours from 613 early‐stage NSCLC patients and evaluated associations between CpG methylation of TRIM27 and overall survival. Significant CpG probes were confirmed in 617 samples from The Cancer Genome Atlas. The methylation of the CpG probe cg05293407TRIM27 was significantly associated with overall survival in patients with LUSC (HR = 1.65, 95% CI: 1.30–2.09, P = 4.52 × 10−5), but not in patients with LUAD (HR = 1.08, 95% CI: 0.87–1.33, P = 0.493). As incidence of LUSC is associated with higher smoking intensity compared to LUAD, we investigated whether smoking intensity impacted on the prognostic effect of cg05293407TRIM27 methylation in NSCLC. LUSC patients had a higher average pack‐year of smoking (37.49LUAD vs 54.79LUSC, P = 1.03 × 10−19) and included a higher proportion of current smokers than LUAD patients (28.24%LUAD vs 34.09%LUSC, P = 0.037). cg05293407TRIM27 was significantly associated with overall survival only in NSCLC patients with medium–high pack‐year of smoking (HR = 1.58, 95% CI: 1.26–1.96, P = 5.25 × 10−5). We conclude that cg05293407TRIM27 methylation is a potential predictor of LUSC prognosis, and smoking intensity may impact on its prognostic value across the various types of NSCLC.

Background Genetic alterations are common in non-small cell lung cancer (NSCLC), and DNA mutations and translocations are targets for therapy. Copy number aberrations occur frequently in NSCLC tumors and may influence gene expression and further alter signaling pathways. In this study we aimed to characterize the genomic architecture of NSCLC tumors and to identify genomic differences between tumors stratified by histology and mutation status. Furthermore, we sought to integrate DNA copy number data with mRNA expression to find genes with expression putatively regulated by copy number aberrations and the oncogenic pathways associated with these affected genes. Methods Copy number data were obtained from 190 resected early-stage NSCLC tumors and gene expression data were available from 113 of the adenocarcinomas. Clinical and histopathological data were known, and EGFR-, KRAS- and TP53 mutation status was determined. Allele-specific copy number profiles were calculated using ASCAT, and regional copy number aberration were subsequently obtained and analyzed jointly with the gene expression data. Results The NSCLC tumors tissue displayed overall complex DNA copy number profiles with numerous recurrent aberrations. Despite histological differences, tissue samples from squamous cell carcinomas and adenocarcinomas had remarkably similar copy number patterns. The TP53-mutated lung adenocarcinomas displayed a highly aberrant genome, with significantly altered copy number profiles including gains, losses and focal complex events. The EGFR-mutant lung adenocarcinomas had specific arm-wise aberrations particularly at chromosome7p and 9q. A large number of genes displayed correlation between copy number and expression level, and the PI(3)K-mTOR pathway was highly enriched for such genes. Conclusions The genomic architecture in NSCLC tumors is complex, and particularly TP53-mutated lung adenocarcinomas displayed highly aberrant copy number profiles. We suggest to always include TP53-mutation status when studying copy number aberrations in NSCLC tumors. Copy number may further impact gene expression and alter cellular signaling pathways.

Background Malignant pleural mesothelioma (MPM) is a rare and aggressive tumour. For patients with inoperable disease, few treatment options are available after first line chemotherapy. The combination of ipilimumab and nivolumab has recently shown increased survival compared to standard chemotherapy, but most patients do not respond and improvements are called for. Telomerase is expressed in mesothelioma cells, but only sparsely in normal tissues and is therefore an attractive target for therapeutic vaccination. Vaccination against telomerase is tolerable and has shown to induce immune responses associated with increased survival in other cancer types. There is a well-founded scientific rationale for the combination of a telomerase vaccine and checkpoint inhibition to improve treatment response in MPM patients. Methods NIPU is a randomized, multi-centre, open-label, phase II study comparing the efficacy and safety of nivolumab and ipilimumab with or without telomerase vaccine in patients with inoperable malignant pleural mesothelioma after first-line platinum-based chemotherapy. Participants (n = 118) are randomized 1:1 into two treatment arms. All participants receive treatment with nivolumab (240 mg every 2 weeks) and ipilimumab (1 mg/kg every 6 weeks) until disease progression, unacceptable toxicity or for a maximum of 2 years. Patients randomised to the experimental arm receive 8 intradermal injections of UV1 vaccine during the first three months of treatment. Tumour tissue, blood, urine, faeces and imaging will be collected for biomarker analyses and exploration of mechanisms for response and resistance to therapy. Discussion Checkpoint inhibition is used for treatment of mesothelioma, but many patients still do not respond. Increasing therapy response to immunotherapy is an important goal. Possible approaches include combination with chemotherapy, radiotherapy, targeted therapy and other immunotherapeutic agents. Predictive biomarkers are necessary to ensure optimal treatment for each patient and to prevent unnecessary side effects. This trial seeks to improve treatment response by combining checkpoint inhibition with a telomerase vaccine and also to explore mechanisms for treatment response and resistance. Knowledge gained in the NIPU study may be transferred to the first line setting and to other cancers with limited benefit from immunotherapy. Trial registration: ClinicalTrials.gov: NCT04300244, registered March 8th, 2020, https://clinicaltrials.gov/ct2/show/NCT04300244?term=NIPU&draw=2&rank=1.

Background DNA methylation and gene expression are promising biomarkers of various cancers, including non-small cell lung cancer (NSCLC). Besides the main effects of biomarkers, the progression of complex diseases is also influenced by gene-gene (G×G) interactions. Research Question Would screening the functional capacity of biomarkers on the basis of main effects or interactions, using multiomics data, improve the accuracy of cancer prognosis? Study Design and Methods Biomarker screening and model validation were used to construct and validate a prognostic prediction model. NSCLC prognosis-associated biomarkers were identified on the basis of either their main effects or interactions with two types of omics data. A prognostic score incorporating epigenetic and transcriptional biomarkers, as well as clinical information, was independently validated. Results Twenty-six pairs of biomarkers with G×G interactions and two biomarkers with main effects were significantly associated with NSCLC survival. Compared with a model using clinical information only, the accuracy of the epigenetic and transcriptional biomarker-based prognostic model, measured by area under the receiver operating characteristic curve (AUC), increased by 35.38% (95% CI, 27.09%-42.17%; P = 5.10 × 10–17) and 34.85% (95% CI, 26.33%-41.87%; P = 2.52 × 10–18) for 3- and 5-year survival, respectively, which exhibited a superior predictive ability for NSCLC survival (AUC3 year, 0.88 [95% CI, 0.83-0.93]; and AUC5 year, 0.89 [95% CI, 0.83-0.93]) in an independent Cancer Genome Atlas population. G×G interactions contributed a 65.2% and 91.3% increase in prediction accuracy for 3- and 5-year survival, respectively. Interpretation The integration of epigenetic and transcriptional biomarkers with main effects and G×G interactions significantly improves the accuracy of prognostic prediction of early-stage NSCLC survival.

DNA methylation changes during aging, but it remains unclear whether the effect of DNA methylation on lung cancer survival varies with age. Such an effect could decrease prediction accuracy and treatment efficacy. We performed a methylation-age interaction analysis using 1,230 early-stage lung adenocarcinoma patients from five cohorts. A Cox proportional hazards model was used to investigate lung adenocarcinoma and squamous cell carcinoma patients for methylation-age interactions, which were further confirmed in a validation phase. We identified one adenocarcinoma-specific CpG probe, cg14326354PRODH, with effects significantly modified by age (HRinteraction = 0.989; 95% CI: 0.986-0.994; P = 9.18×10-7). The effect of low methylation was reversed for young and elderly patients categorized by the boundary of 95% CI standard (HRyoung = 2.44; 95% CI: 1.26-4.72; P = 8.34×10-3; HRelderly = 0.58; 95% CI: 0.42-0.82; P = 1.67×10-3). Moreover, there was an antagonistic interaction between low cg14326354PRODH methylation and elderly age (HRinteraction = 0.21; 95% CI: 0.11-0.40; P = 2.20×10-6). In summary, low methylation of cg14326354PRODH might benefit survival of elderly lung adenocarcinoma patients, providing new insight to age-specific prediction and potential drug targeting.

Background: KDM lysine demethylase family members are related to lung cancer clinical outcomes and are potential biomarkers for chemotherapeutics. However, little is known about epigenetic alterations in KDM genes and their roles in lung cancer survival. Methods: Tumor tissue samples of 1230 early-stage non-small cell lung cancer (NSCLC) patients were collected from the five independent cohorts. The 393 methylation sites in KDM genes were extracted from epigenome-wide datasets and analyzed by weighted random forest (Ranger) in discovery phase and validation dataset, respectively. The variable importance scores (VIS) for the sites in top 5% of both discovery and validation sets were carried forward for Cox regression to further evaluate the association with patient's overall survival. TCGA transcriptomic data were used to evaluate the correlation with the corresponding DNA methylation. Results: DNA methylation at sites cg11637544 in KDM2A and cg26662347 in KDM1A were in the top 5% of VIS in both discovery phase and validation for squamous cell carcinomas (SCC), which were also significantly associated with SCC survival (HRcg11637544 = 1.32, 95% CI, 1.16-1.50, P = 1.1 x 10(-4); HRcg26662347 = 1.88, 95% CI, 1.37-2.60, P = 3.7 x 10(-3)), and correlated with corresponding gene expression (cg11637544 for KDM2A, P = 1.3 x 10(-10); cg26662347 for KDM1A P = 1.5 x 10(-5)). In addition, by using flexible criteria for Ranger analysis followed by survival classification tree analysis, we identified four clusters for adenocarcinomas and five clusters for squamous cell carcinomas which showed a considerable difference of clinical outcomes with statistical significance. Conclusions: These findings highlight the association between somatic DNA methylation in KDM genes and early-stage NSCLC patient survival, which may reveal potential epigenetic therapeutic targets.

Smoking cessation prolongs survival and decreases mortality of patients with non-small-cell lung cancer (NSCLC). In addition, epigenetic alterations of some genes are associated with survival. However, potential interactions between smoking cessation and epigenetics have not been assessed. Here, we conducted an epigenome-wide interaction analysis between DNA methylation and smoking cessation on NSCLC survival. We used a two-stage study design to identify DNA methylation–smoking cessation interactions that affect overall survival for early-stage NSCLC. The discovery phase contained NSCLC patients from Harvard, Spain, Norway, and Sweden. A histology-stratified Cox proportional hazards model adjusted for age, sex, clinical stage, and study center was used to test DNA methylation–smoking cessation interaction terms. Interactions with false discovery rate-q ≤ 0.05 were further confirmed in a validation phase using The Cancer Genome Atlas database. Histology-specific interactions were identified by stratification analysis in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) patients. We identified one CpG probe (cg02268510 SIPA 1L3 ) that significantly and exclusively modified the effect of smoking cessation on survival in LUAD patients [hazard ratio (HR) interaction = 1.12; 95% confidence interval (CI): 1.07–1.16; P = 4.30 × 10 –7 ]. Further, the effect of smoking cessation on early-stage LUAD survival varied across patients with different methylation levels of cg02268510 SIPA 1L3 . Smoking cessation only benefited LUAD patients with low methylation (HR = 0.53; 95% CI: 0.34–0.82; P = 4.61 × 10 –3 ) rather than medium or high methylation (HR = 1.21; 95% CI: 0.86–1.70; P = 0.266) of cg02268510 SIPA 1L3 . Moreover, there was an antagonistic interaction between elevated methylation of cg02268510 SIPA 1L3 and smoking cessation (HR interaction = 2.1835; 95% CI: 1.27–3.74; P = 4.46 × 10 −3 ). In summary, smoking cessation benefited survival of LUAD patients with low methylation at cg02268510 SIPA 1L3 . The results have implications for not only smoking cessation after diagnosis, but also possible methylation-specific drug targeting.

Hypoxia occurs frequently in human cancers and promotes stabilization and activation of hypoxia inducible factor (HIF). HIF-1α is specific for the hypoxia response, and its degradation mediated by three enzymes EGLN1, EGLN2 and EGLN3. Although EGLNs expression has been found to be related to prognosis of many cancers, few studies examined DNA methylation in EGLNs and its relationship to prognosis of early-stage non-small cell lung cancer (NSCLC). We analyzed EGLNs DNA methylation data from tumor tissue samples of 1,230 early-stage NSCLC patients, as well as gene expression data from The Cancer Genome Atlas. The sliding windows sequential forward feature selection method and weighted random forest were used to screen out the candidate CpG probes in lung adenocarcinomas (LUAD) and lung squamous cell carcinomas patients, respectively, in both discovery and validation phases. Then Cox regression was performed to evaluate the association between DNA methylation and overall survival. Among the 34 CpG probes in EGLNs, DNA methylation at cg25923056(EGLN2) was identified to be significantly associated with LUAD survival (HR = 1.02, 95% CI: 1.01–1.03, P = 9.90 × 10(–5)), and correlated with EGLN2 expression (r = – 0.36, P = 1.52 × 10(–11)). Meanwhile, EGLN2 expression was negatively correlated with HIF1A expression in tumor tissues (r = – 0.30, P = 4.78 × 10(–8)) and significantly (P = 0.037) interacted with HIF1A expression on overall survival. Therefore, DNA methylation of EGLN2- HIF1A is a potential marker for LUAD prognosis and these genes are potential treatment targets for further development of HIF-1α inhibitors in lung cancer therapy.

Limited studies have focused on developing prognostic models with trans-omics biomarkers for early-stage lung adenocarcinoma (LUAD). We performed integrative analysis of clinical information, DNA methylation, and gene expression data using 825 early-stage LUAD patients from 5 cohorts. Ranger algorithm was used to screen prognosis-associated biomarkers, which were confirmed with a validation phase. Clinical and biomarker information was fused using an iCluster plus algorithm, which significantly distinguished patients into high- and low-mortality risk groups (Pdiscovery = 0.01 and Pvalidation = 2.71×10-3). Further, potential functional DNA methylation–gene expression–overall survival pathways were evaluated by causal mediation analysis. The effect of DNA methylation level on LUAD survival was significantly mediated through gene expression level. By adding DNA methylation and gene expression biomarkers to a model of only clinical data, the AUCs of the trans-omics model improved by 18.3% (to 87.2%) and 16.4% (to 85.3%) in discovery and validation phases, respectively. Further, concordance index of the nomogram was 0.81 and 0.77 in discovery and validation phases, respectively. Based on systematic review of published literatures, our model was superior to all existing models for early-stage LUAD. In summary, our trans-omics model may help physicians accurately identify patients with high mortality risk.

Various feature selection algorithms have been proposed to identify cancer prognostic biomarkers. In recent years, however, their reproducibility is criticized. The performance of feature selection algorithms is shown to be affected by the datasets, underlying networks and evaluation metrics. One of the causes is the curse of dimensionality, which makes it hard to select the features that generalize well on independent data. Even the integration of biological networks does not mitigate this issue because the networks are large and many of their components are not relevant for the phenotype of interest. With the availability of multi-omics data, integrative approaches are being developed to build more robust predictive models. In this scenario, the higher data dimensions create greater challenges.We proposed a phenotype relevant network-based feature selection (PRNFS) framework and demonstrated its advantages in lung cancer prognosis prediction. We constructed cancer prognosis relevant networks based on epithelial mesenchymal transition (EMT) and integrated them with different types of omics data for feature selection. With less than 2.5% of the total dimensionality, we obtained EMT prognostic signatures that achieved remarkable prediction performance (average AUC values >0.8), very significant sample stratifications, and meaningful biological interpretations. In addition to finding EMT signatures from different omics data levels, we combined these single-omics signatures into multi-omics signatures, which improved sample stratifications significantly. Both single- and multi-omics EMT signatures were tested on independent multi-omics lung cancer datasets and significant sample stratifications were obtained.

// Ann Rita Halvorsen 1 , Maria Bjaanaes 1, 2 , Marissa LeBlanc 3 , Are M. Holm 4 , Nils Bolstad 5 , Luis Rubio 6 , Juan Carlos Penalver 7 , Jose Cervera 8 , Julia Cruz Mojarrieta 9 , Jose Antonio Lopez-Guerrero 6 , Odd Terje Brustugun 1, 2 , Aslaug Helland 1, 2 1 Department of Cancer Genetics, Institute for Cancer Research, OUS Radiumhospitalet, Oslo, Norway 2 Department of Oncology, OUS Radiumhospitalet, Oslo, Norway 3 Oslo Centre for Biostatistics and Epidemiology, Research Support Services, Oslo University Hospital, Oslo, Norway 4 Department of Respiratory Medicine, OUS Rikshospitalet, Oslo, Norway 5 Department of Medical Biochemistry, OUS Radiumhospitalet, Oslo, Norway 6 Laboratory of Molecular Biology, Fundacion Instituto Valenciano de Oncologia, Valencia, Spain 7 Department of Thoracic Surgery, Fundacion Instituto Valenciano de Oncologia, Valencia, Spain 8 Department of Radiology, Fundacion Instituto Valenciano de Oncologia, Valencia, Spain 9 Department of Pathology, Fundacion Instituto Valenciano de Oncologia, Valencia, Spain Correspondence to: Aslaug Helland, email: ahelland@medisin.uio.no Keywords: circulating microRNAs, biomarker, lung cancer, early detection, COPD Received: February 15, 2016 Accepted: April 16, 2016 Published: May 14, 2016 ABSTRACT Introduction: Circulating microRNAs are promising biomarkers for diagnosis, predication and prognostication of diseases. Lung cancer is the cancer disease accountable for most cancer deaths, largely due to being diagnosed at late stages. Therefore, diagnosing lung cancer patients at an early stage is crucial for improving the outcome. The purpose of this study was to identify circulating microRNAs for detection of early stage lung cancer, capable of discriminating lung cancer patients from those with chronic obstructive pulmonary disease (COPD) and healthy volunteers. Results: We identified 7 microRNAs separating lung cancer patients from controls. By using RT-qPCR, we validated 6 microRNAs (miR-429, miR-205, miR-200b, miR-203, miR-125b and miR-34b) with a significantly higher abundance in serum from NSCLC patients. Furthermore, the 6 miRNAs were validated in a different dataset, revealing an area under the receiver operating characteristic curve of 0.89 for stage I-IV and 0.88 for stage I/II. Materials and Methods: We profiled the expression of 754 unique microRNAs by TaqMan Low Density Arrays, and analyzed serum from 38 patients with NSCLC, 16 patients suffering from COPD and 16 healthy volunteers from Norway, to explore their potential as diagnostic biomarkers. For validation, we analyzed serum collected from high-risk individuals enrolled in the Valencia branch of the International Early Lung Cancer Action Program screening trial (n=107) in addition to 51 lung cancer patients. Conclusion: Considering the accessibility and stability of circulating miRNAs, these 6 microRNAs are promising biomarkers as a supplement in future screening studies.

9007 Background: Concurrent chemoradiotherapy is the standard treatment of LD SCLC. Some patients are cured, but most relapse and better treatment is needed. 45 Gy in 30 fractions BID is the most recommended TRT-schedule. Studies suggest that a higher TRT-dose might prolong survival, but hitherto, this has not been confirmed in randomized trials. We aimed to investigate whether high-dose BID TRT of 60 Gy in 40 fractions was feasible, tolerated, and improved survival. Methods: Patients > 18 years, performance status (PS) 0-2 and confirmed LD SCLC were to receive 4 courses of platinum/etoposide and were randomized to BID TRT of 60 or 45 Gy. Responders were offered prophylactic cranial irradiation of 25-30 Gy. Primary endpoint was 2-year survival; secondary endpoints were toxicity, progression free survival (PFS), and overall survival (OS). To demonstrate a 25% improvement of 2-year survival from 53% to 66% with a one-sided α = .10 and β = .80, 75 patients were required on each arm. Results: Between 2014-2018, 176 patients were enrolled at 22 Scandinavian hospitals. 160 completed TRT per protocol and were eligible for the present analyses (60 Gy: n = 84, 45 Gy: n = 76). Median age was 65, 58% women, 90% PS 0-1. There were no significant differences in grade 3–4 esophagitis (60 Gy: 19%, 45 Gy: 18%, p = .92) or grade 3–4 pneumonitis (60 Gy: 4%, 45 Gy: 0%, p = .10). There was a trend towards more neutropenic infections on the 45 Gy arm (60 Gy: 21%, 45 Gy: 36%, p = .05). There were no significant differences in other grade 3-4 toxicity. Three patients died during the study treatment period (60 Gy: one neutropenic infection and one aortic dissection; 45 Gy: one thrombocytopenic bleeding). There were no statistically significant differences in response rates (60 Gy: 88% [95% CI 81-95], 45 Gy: 85% [95% CI 76-93], p = .52) or median PFS (60 Gy: 20 months [95% CI 11-29], 45 Gy: 14 months [95% CI 10-19], p = .31). Significantly more patients on the 60 Gy arm were alive after 2 years (60 Gy: 73% [95% CI 63-83], 45 Gy: 46% [95% CI 36-60], p = .001), and they had a significantly longer median overall survival (60 Gy: 42 months [95% CI 32-51], 45 Gy: 23 months [95% CI 17-28], HR .63 [95% CI .41-.96], p = .031). Conclusions: LD SCLC patients who received BID TRT of 60 Gy had a statistically significant and numerically substantial benefit in terms of 2-year survival (primary endpoint) and median overall survival compared with those who received BID TRT of 45 Gy. The higher TRT dose did not cause more toxicity than the standard dose. Clinical trial information: NCT02041845.

B-cell translocation gene 2 (BTG2) is a tumour suppressor protein known to be downregulated in several types of cancer. In this study, we investigated a potential role for BTG2 in early-stage non-small cell lung cancer (NSCLC) survival. We analysed BTG2 methylation data from 1230 early-stage NSCLC patients from five international cohorts, as well as gene expression data from 3038 lung cancer cases from multiple cohorts. Three CpG probes (cg01798157, cg06373167, cg23371584) that detected BTG2 hypermethylation in tumour tissues were associated with lower overall survival. The prognostic model based on methylation could distinguish patient survival in the four cohorts [hazard ratio (HR) range, 1.51-2.21] and the independent validation set (HR = 1.85). In the expression analysis, BTG2 expression was positively correlated with survival in each cohort (HR range, 0.28-0.68), which we confirmed with meta-analysis (HR = 0.61, 95% CI 0.54-0.68). The three CpG probes were all negatively correlated with BTG2 expression. Importantly, an integrative model of BTG2 methylation, expression and clinical information showed better predictive ability in the training set and validation set. In conclusion, the methylation and integrated prognostic signatures based on BTG2 are stable and reliable biomarkers for early-stage NSCLC. They may have new applications for appropriate clinical adjuvant trials and personalized treatments in the future.

BACKGROUND: Previous studies support a tumor-suppressor role for LRRC3B across various types of cancers. We aim to investigate the association between DNA methylation of LRRC3B and overall survival for early-stage non-small cell lung cancer patients. METHODS: This study included 1,230 early-stage non-small cell lung cancer patients. DNA was extracted from lung tumor tissues and DNA methylation was measured using Illumina Infinium HumanMethylation450 BeadChips. The association between DNA methylation and overall survival was first tested using Cox regression on a discovery cohort and then validated in an independent cohort. Next, the association between DNA methylation and gene expression was investigated in two independent cohorts. Finally, the association between gene expression and overall survival was investigated in three independent groups of patients. RESULTS: Three novel DNA methylation sites in LRRC3B were significantly associated with overall survival in two groups of patients. Patients with hypermethylation in the DNA methylation sites had significantly longer survival than the others in both the discovery cohort (HR = 0.62; P = 2.02×10(−05)) and validation cohort (HR = 0.55; P = 4.44×10(−04)). The three DNA methylation sites were significantly associated with LRRC3B expression, which was also associated with overall survival. CONCLUSION: Using clinical data from a large population, we illustrated the association between DNA methylation of LRRC3B and overall survival of early-stage non-small cell lung cancer. IMPACT: We provide evidence of plausibility for building biomarkers on DNA methylation of LRRC3B for overall survival of early-stage non-small cell lung cancer, thus filling a gap between previous in vitro studies and clinical applications.

Background: Previous studies support a tumor-suppressor role for LRRC3B across various types of cancers. We aimed to investigate the association between DNA methylation of LRRC3B and overall survival (OS) for patients with early-stage non–small cell lung cancer (NSCLC). Methods: This study included 1,230 patients with early-stage NSCLC. DNA was extracted from lung tumor tissues and DNA methylation was measured using Illumina Infinium HumanMethylation450 BeadChips. The association between DNA methylation and OS was first tested using Cox regression on a discovery cohort and then validated in an independent cohort. Next, the association between DNA methylation and gene expression was investigated in two independent cohorts. Finally, the association between gene expression and OS was investigated in three independent groups of patients. Results: Three novel DNA methylation sites in LRRC3B were significantly associated with OS in two groups of patients. Patients with hypermethylation in the DNA methylation sites had significantly longer survival than the others in both the discovery cohort (HR, 0.62; P = 2.02 × 10−05) and validation cohort (HR, 0.55; P = 4.44 × 10−04). The three DNA methylation sites were significantly associated with LRRC3B expression, which was also associated with OS. Conclusions: Using clinical data from a large population, we illustrated the association between DNA methylation of LRRC3B and OS of early-stage NSCLC. Impact: We provide evidence of plausibility for building biomarkers on DNA methylation of LRRC3B for OS of early-stage NSCLC, thus filling a gap between previous in vitro studies and clinical applications.

Background Extensive research has increased our understanding of the molecular alterations needed for non-small cell lung cancer (NSCLC) development. Deregulation of a pathway including MYCN, HMGA2 and CDKN2A, with the participation of DICER1, is of importance in several solid tumours, and may also be of significance in the pathogenesis of NSCLC. Methods Gene expression of MYCN, HMGA2, CDKN2A and DICER1 were investigated with RT-qPCR in surgically resected NSCLC tumour tissue from 175 patients. Expression of the let-7 microRNA family was performed in 78 adenocarcinomas and 16 matching normal lung tissue samples using microarrays. The protein levels of HMGA2 were determined by immunohistochemistry in 156 tumour samples and the protein expression was correlated with gene expression. Associations between clinical data, including time to recurrence, and expression of mRNA, protein and microRNAs were analysed. Results Compared to adenocarcinomas, squamous cell carcinomas had a median 5-fold increase in mRNA expression of HMGA2 (p = 0.003). A positive correlation (r = 0.513, p

Background: DNA methylation alterations are early events in tumorigenesis and important in the regulation of gene expression in cancer cells. Lung cancer patients have in general a poor prognosis, and a deeper insight into the epigenetic landscape in lung adenocarcinoma tumors and its prognostic implications is needed. Results: We determined whole‐genome DNA methylation profiles of 164 fresh frozen lung adenocarcinoma samples and 19 samples of matched normal lung tissue using the Illumina Infinium 450K array. A large number of differentially methylated CpGs in lung adenocarcinoma tissue were identified, and specific methylation profiles were observed in tumors with mutations in the EGFR‐, KRAS‐ or TP53 genes and according to the patients' smoking status. The methylation levels were correlated with gene expression and both positive and negative correlations were seen. Methylation profiles of the tumor samples identified subtypes of tumors with distinct prognosis, including one subtype enriched for TP53 mutant tumors. A prognostic index based on the methylation levels of 33 CpGs was established, and was significantly associated with prognosis in the univariate analysis using an independent cohort of lung adenocarcinoma patients from The Cancer Genome Atlas project. CpGs in the HOX B and HOX C gene clusters were represented in the prognostic signature. Conclusions: Methylation differences mirror biologically important features in the etiology of lung adenocarcinomas and influence prognosis., Highlights Extensive DNA methylation changes linked to immune signaling.DNA methylation in EGFR‐mutated tumors associated with oncogenic pathways.DNA methylation with both positive and negative correlation with mRNA expression.DNA methylation signature with prognostic information.

The findings of mutations and the development of targeted therapies have improved lung cancer management. Still, the prognosis remains poor, and we need to know more about the genetic and epigenetic alterations in lung cancer. MicroRNAs are involved in crucial biological processes like carcinogenesis by regulating gene expression at the post-transcriptional level. In this project, we have studied the microRNA expression of lung adenocarcinomas and corresponding normal lung tissue and correlated the expression with clinical data and EGFR- and KRAS-mutational status. Agilent microarrays have been used, examining microRNA expression in 154 surgically resected lung adenocarcinomas and 20 corresponding normal lung tissue samples. Findings were confirmed by RT-qPCR in the same cohort and in an independent cohort of 103 lung cancer patients. EGFR and KRAS mutation analyses were also performed. 129 microRNAs were significantly differentially expressed in lung adenocarcinomas compared with normal lung tissue, and 17 microRNAs were differentially expressed between EGFR-mutated and EGFR wildtype tumors. We identified microRNAs associated with time to progression. We have identified several aberrantly expressed microRNAs that discriminate lung adenocarcinomas from normal lung tissue, and hence may be potential biomarkers for early detection. We have found microRNAs that are differentially expressed between EGFR-mutated and EGFR wildtype lung adenocarcinomas, suggesting that microRNAs can be used as molecular biomarkers in classification. We hypothesize that microRNA expression can be used as biomarkers for clinical course.

Introduction: Lung cancer is the most common cause of cancer deaths worldwide. The findings of new mutations and the development of targeted therapies have improved lung cancer management. Still, the prognosis remains poor and we need to know more about the genetic and epigenetic alterations in the tumors to better understand the biology of lung cancer. MicroRNAs are small non-coding RNAs that are involved in crucial biological processes in carcinogenesis by regulating gene expression at the post transcriptional level. In this project we have studied the microRNA expression patterns of lung adenocarcinomas and correspondent tumor-adjacent normal lung tissue and correlated the expression patterns with clinical data and mutational status. Methods: We have examined microRNA expression pattern in tumor tissues from 154 surgically resected lung adenocarcinomas and from 20 tumor-adjacent normal lung tissue samples. The expression of 1205 human microRNAs was conducted using the 60K microRNA microarray from Agilent technology. EGFR and KRAS mutation analyses were also performed. The analysis of differentially expressed microRNAs between groups of samples was done using significance analyses of microarrays (SAM) in the J-express software. We also performed survival analysis using univariate- and multivariate Cox regression analysis. The results are about to be validated by qRT-PCR. Results: Preliminary results show 129 differentially expressed microRNAs in tumor compared to the tumor-adjacent normal lung tissue. EGFR and KRAS mutations were found in 22/152 (14.5%) and 47/137 (34.3%) samples respectively. We have detected 17 microRNAs that are differentially expressed in EGFR mutated tumors compared to EGFR wildtype tumors. Two microRNAs were identified to have a strong association with time to progression in both univariate- and multivariate Cox regression analysis. Discussion: The microRNAs are thought to play an essential role in the development and progression of human malignancies, including lung cancer. We have identified several aberrantly expressed microRNAs that can discriminate lung adenocarcinoma tumor tissue from tumor-adjacent normal lung tissue samples. This can lead to the identification of biomarkers for early detection. 17 microRNAs were differentially expressed between EGFR mutated- and EGFR wt lung adenocarcinomas suggesting that microRNAs can be used as molecular biomarkers for lung cancer classification. We have also identified microRNAs that can be used as prognostic biomarkers. We are now confirming our results with qRT-PCR. We hypothesize that microRNA can be used as biomarkers for classification and clinical course. Citation Format: Maria Bjaanæs, Rita Halvorsen, Steinar Solberg, Lars Jørgensen, Odd-Terje Brustugun, Åslaug Helland. Differentially expressed microRNAs in adenocarcinomas of the lung and tumor-adjacent normal lung tissue. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1962. doi:10.1158/1538-7445.AM2013-1962

Non-small cell lung cancer is cancer killer #1, and consists of a variety of histological subtypes of which adenocarcinoma, squamous cell carcinoma and large cell carcinoma are most frequent. Genetic aberrations are regarded both prognostic and predicitive in NSCLC, and are found with various frequency and different significance in the subtypes. Mutation in the KRAS gene is a well-known factor in NSCLC, but the prognostic impact is still debated, and the role as target of therapy is under investigation. We intended to study the frequency and prognostic importance of KRAS mutations in a large number of early stage lung cancers of all major histological subtypes. Material and methods Tumor tissue was obtained from 321 consecutively operated lung cancer patients admitted to Oslo University Hospital-Rikshospitalet in the period 2006-2012. Tissue was taken from the excised tumours, snap frozen in liquid nitrogen in the operation room, and stored at -80oC until DNA isolation. The tumour cell content in the specimens was found to be more than 70% in most samples. DNA was isolated according to standard procedures, and mutation analysis was done with a TaqMan-based wobble-enhanced amplification refractory mutation system, for detecting the seven most commonly reported mutations of clinical importance in the KRAS gene; g.34G>C (p.G12R), g.34G>A (p.G12S), g.34G>T (p.G12C), g.35G>A (p.G12D), g.35G>C (p.G12A), g.35G>T (p.G12V), and g.38G>A (p.G13D). After thorough follow-up (median 34.6 months, range 6.3-80.6), relapse-free survival was calculated with the Kaplan-Meier method using Chi-Square for significance assessment. Results Tissues from 154 females and 167 males were analyzed; 212 (66%) adenocarcinomas, 89 (28%) squamous cell carcinomas and 20 (6%) large cell carcinomas. 182 (57%) were in stage I, 88 (27%) II and 51 (16%) stage III. 24 (7,5%) were never-smokers. Overall, 89 patients (27.7%) harbored a KRAS mutation, 78 (36.8%) of adenocarcinomas, 5 (5.6%) of squamous cell carcinomas and 6 (30%) of large cell carcinomas. The most frequent mutation subtype was G12C (51.7%). In the total group there was a significant relapse-free survival difference (p=0.021) in favor of the wild-type. In the adenocarcinoma subgroup, however, there was no survival difference (p=0.316), whereas there was a major difference in the non-adenocarcinoma group (p Conclusions KRAS mutations confer a survival disadvantage in non-small cell lung cancers. This effect is only seen in non-adenocarcinoma subtypes, which might have implications in therapy strategies. Citation Format: Odd Terje Brustugun, Marius Lund-Iversen, Maria M. Bjaanæs, Ann Rita Halvorsen, Martina L. Skrede, Lars H. Jørgensen, Elin H. Kure, Åslaug Helland. KRAS mutation status is a strong prognostic factor in some but not all subtypes of non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2398. doi:10.1158/1538-7445.AM2013-2398