Frauke Leenders

Mapping the Hallmarks of Lung Adenocarcinoma with Massively Parallel Sequencing

Lung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for more than 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole-genome sequence analysis revealed frequent structural rearrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.

Frequent and Focal FGFR1 Amplification Associates with Therapeutically Tractable FGFR1 Dependency in Squamous Cell Lung Cancer

FGFR1 amplification provides a therapeutic target for squamous cell lung cancer, which is resistant to other targeted lung cancer drugs. A Smoking Gun for Lung Cancer Detectives and scientists alike need strong evidence to take their cases to the judge, who for scientists is often a patient with a deadly disease. Yet, new culprits are sometimes found that can break a case wide open. Lung cancer, which accounts for more than 10% of the global cancer burden, has a poor prognosis and inadequately responds to chemotherapy and radiotherapy. New targeted treatments for lung adenocarcinomas inhibit the oncogenic versions of signaling protein kinases that arise from mutations typically found in lung cancer patients who have never smoked. However, smokers frequently suffer from a different deviant, squamous cell lung cancers, for which there are no known molecular genetic targets for therapy. Now, Weiss et al. have fingered a new suspect in smoking-related lung cancer: amplification of the FGFR1 gene, which encodes the fibroblast growth factor receptor 1 tyrosine kinase (FGFR1). To identify therapeutically viable genetic alterations that may influence squamous cell lung cancer, Weiss et al. performed genomic profiles on a large set of lung cancer specimens. Squamous cell lung cancer samples showed FGFR1 amplification, which was not found in other lung cancer subtypes. The authors then determined that a molecule that broadly inhibits FGF receptor function could block tumor growth and cause cell death in the cancers that expressed high amounts of the FGFR1 gene product in a manner that was dependent on FGFR1 expression. Moreover, FGFR1 inhibition resulted in a considerable decrease in tumor size in a mouse model of FGFR1-amplified lung cancer. This culmination of evidence implies that inhibition of this receptor tyrosine kinase should be explored as a candidate therapy for corralling squamous cell lung cancer in smokers. Lung cancer remains one of the leading causes of cancer-related death in developed countries. Although lung adenocarcinomas with EGFR mutations or EML4-ALK fusions respond to treatment by epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) inhibition, respectively, squamous cell lung cancer currently lacks therapeutically exploitable genetic alterations. We conducted a systematic search in a set of 232 lung cancer specimens for genetic alterations that were therapeutically amenable and then performed high-resolution gene copy number analyses. We identified frequent and focal fibroblast growth factor receptor 1 (FGFR1) amplification in squamous cell lung cancer (n = 155), but not in other lung cancer subtypes, and, by fluorescence in situ hybridization, confirmed the presence of FGFR1 amplifications in an independent cohort of squamous cell lung cancer samples (22% of cases). Using cell-based screening with the FGFR inhibitor PD173074 in a large (n = 83) panel of lung cancer cell lines, we demonstrated that this compound inhibited growth and induced apoptosis specifically in those lung cancer cells carrying amplified FGFR1. We validated the FGFR1 dependence of FGFR1-amplified cell lines by FGFR1 knockdown and by ectopic expression of an FGFR1-resistant allele (FGFR1V561M), which rescued FGFR1-amplified cells from PD173074-mediated cytotoxicity. Finally, we showed that inhibition of FGFR1 with a small molecule led to significant tumor shrinkage in vivo. Thus, focal FGFR1 amplification is common in squamous cell lung cancer and associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in this cohort of patients.

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.

Mutations in the DDR2 Kinase Gene Identify a Novel Therapeutic Target in Squamous Cell Lung Cancer

UNLABELLED While genomically targeted therapies have improved outcomes for patients with lung adenocarcinoma, little is known about the genomic alterations which drive squamous cell lung cancer. Sanger sequencing of the tyrosine kinome identified mutations in the DDR2 kinase gene in 3.8% of squamous cell lung cancers and cell lines. Squamous lung cancer cell lines harboring DDR2 mutations were selectively killed by knock-down of DDR2 by RNAi or by treatment with the multi-targeted kinase inhibitor dasatinib. Tumors established from a DDR2 mutant cell line were sensitive to dasatinib in xenograft models. Expression of mutated DDR2 led to cellular transformation which was blocked by dasatinib. A squamous cell lung cancer patient with a response to dasatinib and erlotinib treatment harbored a DDR2 kinase domain mutation. These data suggest that gain-of-function mutations in DDR2 are important oncogenic events and are amenable to therapy with dasatinib. As dasatinib is already approved for use, these findings could be rapidly translated into clinical trials. SIGNIFICANCE DDR2 mutations are present in 4% of lung SCCs, and DDR2 mutations are associated with sensitivity to dasatinib. These findings provide a rationale for designing clinical trials with the FDA-approved drug dasatinib in patients with lung SCCs.

Comprehensive genomic profiles of small cell lung cancer

We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Δex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer.

Crizotinib Therapy for Advanced Lung Adenocarcinoma and a ROS1 Rearrangement: Results From the EUROS1 Cohort

PURPOSE Approximately 1% of lung adenocarcinomas are driven by oncogenic ROS1 rearrangement. Crizotinib is a potent inhibitor of both ROS1 and ALK kinase domains. PATIENTS AND METHODS In the absence of a prospective clinical trial in Europe, we conducted a retrospective study in centers that tested for ROS1 rearrangement. Eligible patients had stage IV lung adenocarcinoma, had ROS1 rearrangement according to fluorescent in situ hybridization, and had received crizotinib therapy through an individual off-label use. Best response was assessed locally using RECIST (version 1.1). All other data were analyzed centrally. RESULTS We identified 32 eligible patients. One patient was excluded because next-generation sequencing was negative for ROS1 fusion. Median age was 50.5 years, 64.5% of patients were women, and 67.7% were never-smokers. Thirty patients were evaluable for progression-free survival (PFS), and 29 patients were evaluable for best response. We observed four patients with disease progression, two patients with stable disease, and objective response in 24 patients, including five complete responses (overall response rate, 80%; disease control rate, 86.7%). Median PFS was 9.1 months, and the PFS rate at 12 months was 44%. No unexpected adverse effects were observed. Twenty-six patients received pemetrexed (either alone or in combination with platinum and either before or after crizotinib) and had a response rate of 57.7% and a median PFS of 7.2 months. CONCLUSION Crizotinib was highly active at treating lung cancer in patients with a ROS1 rearrangement, suggesting that patients with lung adenocarcinomas should be tested for ROS1. Prospective clinical trials with crizotinib and other ROS1 inhibitors are ongoing or planned.

Porcine 80-kDa Protein Reveals Intrinsic 17-Hydroxysteroid Dehydrogenase, Fatty Acyl-CoA-hydratase/Dehydrogenase, and Sterol Transfer Activities

Four types of 17β-hydroxysteroid dehydrogenases have been identified so far. The porcine peroxisomal 17β-hydroxysteroid dehydrogenase type IV catalyzes the oxidation of estradiol with high preference over the reduction of estrone. A 2.9-kilobase mRNA codes for an 80-kDa (737 amino acids) protein featuring domains which are not present in the other 17β-hydroxysteroid dehydrogenases. The 80-kDa protein is N terminally cleaved to a 32-kDa fragment with 17β-hydroxysteroid dehydrogenase activity. Here we show for the first time that both the 80-kDa and the N-terminal 32 kDa (amino acids 1-323) peptides are able to perform the dehydrogenase reaction not only with steroids at the C17 position but also with 3-hydroxyacyl-CoA. The central part of the 80-kDa protein (amino acids 324-596) catalyzes the 2-enoyl-acyl-CoA hydratase reaction with high efficiency. The C-terminal part of the 80-kDa protein (amino acids 597-737) is similar to sterol carrier protein 2 and facilitates the transfer of 7-dehydrocholesterol and phosphatidylcholine between membranes in vitro. The unique multidomain structure of the 80-kDa protein allows for the catalysis of several reactions so far thought to be performed by complexes of different enzymes.

YB-1 Provokes Breast Cancer through the Induction of Chromosomal Instability That Emerges from Mitotic Failure and Centrosome Amplification

YB-1 protein levels are elevated in most human breast cancers, and high YB-1 levels have been correlated with drug resistance and poor clinical outcome. YB-1 is a stress-responsive, cell cycle-regulated transcription factor with additional functions in RNA metabolism and translation. In this study, we show in a novel transgenic mouse model that human hemagglutinin-tagged YB-1 provokes remarkably diverse breast carcinomas through the induction of genetic instability that emerges from mitotic failure and centrosome amplification. The increase of centrosome numbers proceeds during breast cancer development and explanted tumor cell cultures show the phenotype of ongoing numerical chromosomal instability. These data illustrate a mechanism that might contribute to human breast cancer development.

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.

CD74-NRG1 fusions in lung adenocarcinoma

UNLABELLED We discovered a novel somatic gene fusion, CD74-NRG1, by transcriptome sequencing of 25 lung adenocarcinomas of never smokers. By screening 102 lung adenocarcinomas negative for known oncogenic alterations, we found four additional fusion-positive tumors, all of which were of the invasive mucinous subtype. Mechanistically, CD74-NRG1 leads to extracellular expression of the EGF-like domain of NRG1 III-β3, thereby providing the ligand for ERBB2-ERBB3 receptor complexes. Accordingly, ERBB2 and ERBB3 expression was high in the index case, and expression of phospho-ERBB3 was specifically found in tumors bearing the fusion (P < 0.0001). Ectopic expression of CD74-NRG1 in lung cancer cell lines expressing ERBB2 and ERBB3 activated ERBB3 and the PI3K-AKT pathway, and led to increased colony formation in soft agar. Thus, CD74-NRG1 gene fusions are activating genomic alterations in invasive mucinous adenocarcinomas and may offer a therapeutic opportunity for a lung tumor subtype with, so far, no effective treatment. SIGNIFICANCE CD74–NRG1 fusions may represent a therapeutic opportunity for invasive mucinous lung adenocarcinomas, a tumor with no effective treatment that frequently presents with multifocal unresectable disease.