Steinar Solberg

Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer

Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor prognosis. We sequenced 29 SCLC exomes, 2 genomes and 15 transcriptomes and found an extremely high mutation rate of 7.4 ± 1 protein-changing mutations per million base pairs. Therefore, we conducted integrated analyses of the various data sets to identify pathogenetically relevant mutated genes. In all cases, we found evidence for inactivation of TP53 and RB1 and identified recurrent mutations in the CREBBP, EP300 and MLL genes that encode histone modifiers. Furthermore, we observed mutations in PTEN, SLIT2 and EPHA7, as well as focal amplifications of the FGFR1 tyrosine kinase gene. Finally, we detected many of the alterations found in humans in SCLC tumors from Tp53 and Rb1 double knockout mice. Our study implicates histone modification as a major feature of SCLC, reveals potentially therapeutically tractable genomic alterations and provides a generalizable framework for the identification of biologically relevant genes in the context of high mutational background.

Risk Factors for Abdominal Aortic Aneurysms A 7-Year Prospective Study: The Tromsø Study, 1994–2001

Background— Abdominal aortic aneurysm is an asymptomatic condition with a high mortality rate related to rupture. Methods and Results— In a cohort of 2035 men and 2310 women in Tromsø, Norway, who were 25 to 82 years old in 1994, the authors identified risk factors for incident abdominal aortic aneurysm over the next 7 years. The impact of smoking was studied in particular. Ultrasound examination was performed initially in 1994/1995 and repeated in 2001. There were 119 incident cases of abdominal aortic aneurysms (an incidence of 0.4% per year). Male sex and increasing age were strong risk factors. In addition, the following variables were significantly associated with increased abdominal aortic aneurysm incidence: Smoking (OR=13.72, 95% CI 6.12 to 30.78, comparing current smokers of ≥20 cigarettes/d with never-smokers), hypertension (OR=1.54, 95% CI 1.03 to 2.30), hypercholesterolemia (OR=2.11, 95% CI 1.23 to 3.64, comparing subjects with serum total cholesterol ≥7.55 mmol/L with those with total cholesterol <5.85 mmol/L), and low high-density lipoprotein cholesterol (OR=3.25, 95% CI 1.68 to 6.27, comparing subjects with high-density lipoprotein cholesterol <1.25 mmol/L with those with high-density lipoprotein ≥1.83 mmol/L). In addition, use of statins was associated with increased risk of abdominal aortic aneurysm (OR=3.77, 95% CI 1.45 to 9.81), but this was probably a marker of high risk of cardiovascular diseases. Conclusions— The results demonstrate strong associations between traditional atherosclerosis risk factors and the risk of incident abdominal aortic aneurysms.

Intra- and interobserver variability in ultrasound measurements of abdominal aortic diameter. The Tromsø study

OBJECTIVES To assess the variability of ultrasonographic measurements at different levels of the abdominal aorta. DESIGN Reproducibility study as part of a population health screening for abdominal aortic aneurysm. MATERIALS AND METHODS In 1994/1995 a total of 6892 subjects underwent ultrasound examination of the abdominal aorta. Variability of measurements was assessed in the beginning and end of the survey period by inviting 112 randomly selected participants to a second ultrasound scan within 3 weeks of the first scan. The subjects were examined by an experienced radiologist and three sonographers who had been given a short course in ultrasonography. All examiners were blinded to each others results. RESULTS Variability was similar in the beginning and end of the survey period. Both the intra- and interobserver variability were less than 4 mm for all sonographers in measurements of maximal infrarenal aortic diameter, and variability was similar for measurements in the anterior-posterior and transverse plane. Variability was greater for measurements at the renal level than aortic bifurcation level. The radiologist had lower variability than the other sonographers. CONCLUSION Ultrasound measurements of the maximal diameter can be obtained with a high degree of accuracy. Inexperienced sonographers may achieve acceptable performance given appropriate training and surveillance.

Frequent mutations in chromatin-remodelling genes in pulmonary carcinoids

Pulmonary carcinoids are rare neuroendocrine tumors of the lung. The molecular alterations underlying the pathogenesis of these tumors have not been systematically studied so far. Here we perform gene copy number analysis (n=54), genome/exome (n=44) and transcriptome (n=69) sequencing of pulmonary carcinoids and observe frequent mutations in chromatin-remodeling genes. Covalent histone modifiers and subunits of the SWI/SNF complex are mutated in 40% and 22.2% of the cases respectively, with MEN1, PSIP1 and ARID1A being recurrently affected. In contrast to small-cell lung cancer and large-cell neuroendocrine tumors, TP53 and RB1 mutations are rare events, suggesting that pulmonary carcinoids are not early progenitor lesions of the highly aggressive lung neuroendocrine tumors but arise through independent cellular mechanisms. These data also suggest that inactivation of chromatin remodeling genes is sufficient to drive transformation in pulmonary carcinoids.

Liver transplantation for nonresectable liver metastases from colorectal cancer.

Objective:The objective of this pilot study was to investigate the potential for long-term overall survival (OS) after liver transplantation for colorectal liver metastases (CLMs). Background:Patients with nonresectable CLMs have poor prognosis, and few survive beyond 5 years. CLMs are currently considered an absolute contraindication for liver transplantation, although liver transplantation for primary and some secondary liver malignancies shows excellent outcome in selected patients. Before 1995, several liver transplantations for CLMs were performed, but outcome was poor (5-year survival rate: 18%) and liver transplantation for CLMs was abandoned. Since then, the survival rate after liver transplantation in general has improved by almost 30%. On the basis of this, a 5-year survival rate of about 50% after liver transplantation for CLMs could be anticipated. Methods:In a prospective pilot study, liver transplantation for nonresectable CLMs was performed (n = 21). Main inclusion criteria were liver-only CLMs, excised primary tumors, and at least 6 weeks of chemotherapy. Results:Kaplan-Meier estimates of the OS rate at 1, 3, and 5 years were 95%, 68%, and 60%, respectively. Metastatic recurrence of disease was common (mainly pulmonary). However, a significant proportion of the recurrences were accessible for surgery, and at follow-up (after median of 27 months; range, 8–60), 33% had no evidence of disease. Hepatic tumor load before liver transplantation, time from primary surgery to liver transplantation, and progressive disease on chemotherapy were identified as significant prognostic factors. Conclusions:OS exceeds by far reported outcome for chemotherapy, which is the only treatment option available for this patient group. Furthermore, OS is comparable with liver resection for resectable CLMs and survival after repeat liver transplantation for nonmalignant diseases. Selection strategies based on prognostic factors may further improve the outcome (ClinicalTrials.gov: NCT01311453).

Identification and Characterization of Cells with Cancer Stem Cell Properties in Human Primary Lung Cancer Cell Lines

Lung cancer (LC) with its different subtypes is generally known as a therapy resistant cancer with the highest morbidity rate worldwide. Therapy resistance of a tumor is thought to be related to cancer stem cells (CSCs) within the tumors. There have been indications that the lung cancer is propagated and maintained by a small population of CSCs. To study this question we established a panel of 15 primary lung cancer cell lines (PLCCLs) from 20 fresh primary tumors using a robust serum-free culture system. We subsequently focused on identification of lung CSCs by studying these cell lines derived from 4 representative lung cancer subtypes such as small cell lung cancer (SCLC), large cell carcinoma (LCC), squamous cell carcinoma (SCC) and adenocarcinoma (AC). We identified a small population of cells strongly positive for CD44 (CD44high) and a main population which was either weakly positive or negative for CD44 (CD44low/−). Co-expression of CD90 further narrowed down the putative stem cell population in PLCCLs from SCLC and LCC as spheroid-forming cells were mainly found within the CD44highCD90+ sub-population. Moreover, these CD44highCD90+ cells revealed mesenchymal morphology, increased expression of mesenchymal markers N-Cadherin and Vimentin, increased mRNA levels of the embryonic stem cell related genes Nanog and Oct4 and increased resistance to irradiation compared to other sub-populations studied, suggesting the CD44highCD90+ population a good candidate for the lung CSCs. Both CD44highCD90+ and CD44highCD90− cells in the PLCCL derived from SCC formed spheroids, whereas the CD44low/− cells were lacking this potential. These results indicate that CD44highCD90+ sub-population may represent CSCs in SCLC and LCC, whereas in SCC lung cancer subtype, CSC potentials were found within the CD44high sub-population.

Pan-cancer analysis of somatic copy-number alterations implicates IRS4 and IGF2 in enhancer hijacking

Extensive prior research focused on somatic copy-number alterations (SCNAs) affecting cancer genes, yet the extent to which recurrent SCNAs exert their influence through rearrangement of cis-regulatory elements (CREs) remains unclear. Here we present a framework for inferring cancer-related gene overexpression resulting from CRE reorganization (e.g., enhancer hijacking) by integrating SCNAs, gene expression data and information on topologically associating domains (TADs). Analysis of 7,416 cancer genomes uncovered several pan-cancer candidate genes, including IRS4, SMARCA1 and TERT. We demonstrate that IRS4 overexpression in lung cancer is associated with recurrent deletions in cis, and we present evidence supporting a tumor-promoting role. We additionally pursued cancer-type-specific analyses and uncovered IGF2 as a target for enhancer hijacking in colorectal cancer. Recurrent tandem duplications intersecting with a TAD boundary mediate de novo formation of a 3D contact domain comprising IGF2 and a lineage-specific super-enhancer, resulting in high-level gene activation. Our framework enables systematic inference of CRE rearrangements mediating dysregulation in cancer.

EGFR gene alterations in a Norwegian cohort of lung cancer patients selected for surgery.

Introduction:Lung cancer is the leading cause of cancer-related deaths worldwide. New therapies targeting the epidermal growth factor receptor (EGFR) tyrosine kinase are promising and show high response rates in the subset of patients with activating mutations in EGFR. The frequency of these mutations is largely unknown in unselected Caucasian patients. Methods:Mutation analysis of EGFR exons 18–21 was performed on 240 lung cancer samples using the TheraScreen EGFR mutation kit and denaturing high-performance liquid chromatography in addition to sequencing. Results:In a cohort of 240 Norwegian lung cancer patients selected for surgery, we identified 18 tumors with EGFR-activating mutations (7.5%, 14 women and 4 men), of which 14 were adenocarcinomas, 2 squamous cell carcinomas, and 2 bronchoalveolar carcinomas. Five of the mutations were found in patients with more than 20 pack-years of smoking history. Conclusion:The frequency of EGFR mutations is lower in our cohort than among Asian lung cancer patients and present in both men and women and smokers and never-smokers. However, the frequency is significantly higher among women and never-smokers and among patients with adenocarcinomas.

Strategies for clinical implementation of TNM-Immunoscore in resected nonsmall-cell lung cancer.

Immunoscore is a prognostic tool defined to quantify in situ immune cell infiltrates and appears highly promising as a supplement to the tumor-node-metastasis (TNM) classification of various tumors. In colorectal cancer, an international task force has initiated prospective multicenter studies aiming to implement TNM-Immunoscore (TNM-I) in a routine clinical setting. In breast cancer, recommendations for the evaluation of tumor-infiltrating lymphocytes (TILs) have been proposed by an international working group. Regardless of promising results, there are potential obstacles related to implementing TNM-I into the clinic. Diverse methods may be needed for different malignancies and even within each cancer entity. Nevertheless, a uniform approach across malignancies would be advantageous. In nonsmall-cell lung cancer (NSCLC), there are several previous reports indicating an apparent prognostic importance of TILs, but studies on TILs in a TNM-I setting are sparse and no general recommendations are made. However, recently published data is promising, evoking a realistic hope of a clinical useful NSCLC TNM-I. This review will focus on the TNM-I potential in NSCLC and propose strategies for clinical implementation of a TNM-I in resected NSCLC.

Identification of novel fusion genes in lung cancer using breakpoint assembly of transcriptome sequencing data

Genomic translocation events frequently underlie cancer development through generation of gene fusions with oncogenic properties. Identification of such fusion transcripts by transcriptome sequencing might help to discover new potential therapeutic targets. We developed TRUP (Tumor-specimen suited RNA-seq Unified Pipeline) (https://github.com/ruping/TRUP), a computational approach that combines split-read and read-pair analysis with de novo assembly for the identification of chimeric transcripts in cancer specimens. We apply TRUP to RNA-seq data of different tumor types, and find it to be more sensitive than alternative tools in detecting chimeric transcripts, such as secondary rearrangements in EML4-ALK-positive lung tumors, or recurrent inactivating rearrangements affecting RASSF8.