mp3 file (6.8 MB). In the inaugural edition of the Cancer Discovery podcast, Executive Editor Mark Landis talks with Matthew Meyerson about his paper, which describes the identification of the DDR2 kinase as a therapeutic target in squamous cell lung cancer.

Supplementary Table 1 from Mutations in the DDR2 Kinase Gene Identify a Novel Therapeutic Target in Squamous Cell Lung Cancer

Supplementary Figure Legends 1-7 from Mutations in the DDR2 Kinase Gene Identify a Novel Therapeutic Target in Squamous Cell Lung Cancer

In humans, studies based on Developmental Origins of Health and Disease (DOHaD) concept and targeting short half-lived chemicals, including many endocrine disruptors, generally assessed exposures from spot biospecimens. Effects of early-life exposure to atmospheric pollutants were reported, based on outdoor air pollution levels. For both exposure families, exposure misclassification is expected from these designs: for non-persistent chemicals, because a spot biospecimen is unlikely to capture exposure over windows longer than a few days; for air pollutants, because indoor levels are ignored. We developed a couple-child cohort relying on deep phenotyping and extended personal exposure assessment aiming to better characterize the effects of components of the exposome, including air pollutants and non-persistent endocrine disruptors, on child health and development. Pregnant women were included in SEPAGES couple-child cohort (Grenoble area) from 2014 to 2017. Maternal and children exposure to air pollutants was repeatedly assessed by personal monitors. DNA, RNA, serum, plasma, placenta, cord blood, meconium, child and mother stools, living cells, milk, hair and repeated urine samples were collected. A total of 484 pregnant women were recruited, with excellent compliance to the repeated urine sampling protocol (median, 43 urine samples per woman during pregnancy). The main health outcomes are child respiratory health using early objective measures, growth and neurodevelopment. Compared to former studies, the accuracy of assessment of non-persistent exposures is expected to be strongly improved in this new type of birth cohort tailored for the exposome concept, with deep phenotyping and extended exposure characterization. By targeting weaknesses in exposure assessment of the current approaches of cohorts on effects of early life environmental exposures with strong temporal variations, and relying on a rich biobank to provide insight on the underlying biological pathways whereby exposures affect health, this design is expected to provide deeper understanding of the interplay between the Exposome and child development and health.

Pulmonary grade-1 typical (TC) and grade-2 atypical (AC) carcinoids share molecular characteristics with grade-3 large-cell neuroendocrine carcinoma (LCNEC) despite the distinct clinical behaviors. Most carcinoids can be surgically resected, however, limited treatment options exist for metastatic disease, present in 10-23% of TC and 40-50% of AC. Comprehensive genomic studies could help identify better therapeutic opportunities, novel diagnostic markers, and provide insight on the mechanisms responsible for the increased aggressiveness of AC versus TC. Such studies are rare due to the limited availability of suitable material. We have established a multi-center collaboration that has given us access to a unique collection of samples. We have already characterized 40 TC and 60 LCNEC genomes/exomes, and 61 TC, 8 AC and 69 LCNEC trancriptomes (published data). In the present study, we have performed whole-exome and transcriptome sequencing on 20 AC patients. Methylation data from 850K Illumina arrays were also generated for these samples, and for a subset of 20 TC and 20 LCNEC previously mentioned. When comparing the mutational data on AC with that of TC and LCNEC, we have found that similar to TC, AC harbor recurrent alterations in chromatin remodeling genes (such as MEN1 and ARID1A). They also carry alterations in genes involved in other cancer-related pathways (based on STRING), such as cell motility and cell death explaining their more aggressive phenotype. Integrative clustering analysis (MOFA and iCLUSTER) based on expression and methylation data tends to classify carcinoids into four groups: groups 1 and 2 are mostly composed of females with TC, and differ by their age composition and smoking status (Fisher9s exact test p=0.008 and 0.03, respectively). Groups 3 and 4 are mostly composed of males with AC (Fisher9s exact test for tumor type p=8x10-5). When including the LCNEC data, the samples from group 3 cluster with LCNEC, suggesting that AC can display a variety of expression and methylation patterns that may be linked to aggressiveness. This result was supported by the better survival of groups 1 and 2 compared to groups 3 and 4 (log-rank p=0.02), for which survival was similar to that of patients with LCNEC. Here, we present for the first time: (i) a multi-omics study on AC; (ii) the methylome characterization of TC, AC, and LCNEC; and (iii) the results of a comparative analysis of TC, AC, and LCNEC based on their molecular characteristics. We have identified the genes and pathways that might explain the progression from low-grade TC to intermediate-grade AC. Our expression and methylation data also supports the existence of a “super-AC” group, which clusters with LCNEC. Finally, we have identified a panel of molecular alterations that may help pathologist distinguishing between these three entities. NL and NA contributed equally. LFC and MF jointly supervised this work. Citation Format: Noemie Leblay, Nicolas Alcala, David Hervas Marin, Tiffany M. Delhomme, Theo Giffon, Akram Ghantous, Amelie Chabrier, Cyrille Cuenin, Janine Altmueller, Geoffroy Durand, Catherine Voegele, Philippe Lorimier, Anne-Claire Toffart, Jules Derks, Odd Terje Brustugun, Joachim H. Clement, Joerg Saenger, John K. Field, Alex Soltermann, Gavin M. Wright, Luca Roz, Lucia Anna Muscarella, Paolo Graziano, Zdenko Herceg, Ernst-Jan Speel, Peter Nuernberg, James McKay, Nicolas Girard, Sylvie Lantuejoul, Juan Sandoval, Elisabeth Brambilla, Matthieu Foll, Lynnette Fernandez-Cuesta. Multi-omics comparative analyses of pulmonary typical carcinoids, atypical carcinoids, and large-cell neuroendocrine carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5358.

Collected specimens for research purposes may or may not be made available depending on their scarcity and/or on the project needs. Their protection against degradation or in the event of an incident is pivotal. Duplication and storage on a different site is the best way to assure their sustainability. The conservation of samples at room temperature (RT) by duplication can facilitate their protection. We describe a security system for the collection of non-small cell lung cancers (NSCLC) stored in the biobank of the Nice Hospital Center, France, by duplication and conservation of lyophilized (dried), encapsulated DNA kept at RT. Therefore, three frozen tissue collections from non-smoking, early stage and sarcomatoid carcinoma NSCLC patients were selected for this study. DNA was extracted, lyophilized and encapsulated at RT under anoxic conditions using the DNAshell technology. In total, 1974 samples from 987 patients were encapsulated. Six and two capsules from each sample were stored in the biobanks of the Nice and Grenoble (France) Hospitals, respectively. In conclusion, DNA maintained at RT allows for the conservation, duplication and durability of collections of interest stored in biobanks. This is a low-cost and safe technology that requires a limited amount of space and has a low environmental impact.

Introduction: Individuals are repeatedly exposed to many chemicals with short half-lives, which presents a challenge for exposure assessment using biomarkers. We recently propose a within-subject pooling as an effective strategy to reduce exposure misclassification without increasing assay costs. Here, we empirically validate this promising approach. Methods: In 8 pregnant women we collected all micturitions over one week at each trimester of pregnancy, resulting in 1624 urine samples (on average 203 samples per woman over 3 weeks). Aliquots were pooled to have within-subject daily, weekly and 3-week pools. Urinary concentrations of creatinine, 6 phenols and 4 parabens were measured in all first-week individual assays in 2 women (about 57 spot samples per woman) and all the pooling assays in all 8 women (56 daily, 24 weekly and 8 3-week pools). Spearman correlations, regressions and method of moments for the hybrid design were utilized to estimate the sources of variability and evaluate the underlying pooling assumptions. Results: Biomarkers concentrations were detected in more than 70% of samples, except for benzophenone-3 (37%). Biomarkers were more likely to be quantified in the pooled compared to the individual samples. Correlation between concentrations in pools and the corresponding means of individual samples ranged from 0.80 to 1.00 for 7 compounds. Creatinine-adjusted regressions showed accurate biomarker concentration estimation in pooled samples, except for benzophenone-3, triclosan and 2,4-dichlorophenol, compounds with lower correlations (r = 0.16, 0.66 and 0.76 respectively). For those, given the biomarker concentration in pooled samples, the corresponding daily exposure average was undervalued. Conclusion: Within-subject pooling of biological samples allows estimating average biomarker levels over a week. This approach may overcome constraints of high temporal variability and short half-lives at much lower cost.

Roman Thomas and colleagues report exome sequencing of 29 small-cell lung cancers (SCLCs), 2 SCLC genomes and transcriptomes of 15 SCLCs. They identify recurrent mutations in the CREBBP, EP300 and MLL genes encoding histone modifiers. They identify mutations in SLIT2 and EPHA7, which have a role in axon guidance and cell migration, and focal amplifications of FGFR1. Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor prognosis1,2,3. 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 mice4. 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.

Abstract Although genomically targeted therapies have improved outcomes for patients with lung adenocarcinoma, little is known about the genomic alterations that drive squamous cell cancer (SCC) of the lung. Sanger sequencing of the tyrosine kinome identified mutations in the DDR2 kinase gene in 3.8% of lung SCCs and cell lines. Lung SCC cell lines harboring DDR2 mutations were selectively killed by knockdown of DDR2 by RNA interference or by treatment with the multitargeted 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 that was blocked by dasatinib. A patient with lung SCC that responded 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. Because dasatinib is already approved for use, these findings could be used to rapidly generate 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. Cancer Discovery; 1(1); 78–89. ©2011 AACR. Read the Commentary on this article by Ohashi and Pao, p. 23 This article is highlighted in the In This Issue feature, p. 4

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 (FGFR1(V561M)), 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.

The conclusions of a French National Cancer Institute task force on the constitution of a prospective tumour cohort provide a number of recommendations and identify domains for further investigation. A prospective program is mandatory to reach the medical and scientific objectives made accessible by present and incoming technologies. Retrospective collections may he used for test sets, pilot studies or development of new techniques or biomarkers. Approaches based on prospective collections are being implemented or discussed in several countries. The costs of sampling are high since high clinical and biological standards are required, The costs of analysis are difficult to predict and will depend upon the approach selected (targeted versus globa). The main bottlenecks for exploitation are clinical annotations and bioinformatics for data integration. A prospective collection should rely on clearly defined biological and medical objectives, providing a basis for protocol design and patient's inclusion criteria. International cooperation or selection of << niche projects >> will have to be considered. Economical and ethical consideration will have to be taken into account to obtain the participation of all actors as well as of patients. In the choice of tumour sites to he considered for banking, the input of cooperative bioclinical groups will be important, A strategy of call for applications may be used to identify participating centers, Identified centers may cooperate with each other on a principle of mutual sharing of biospecimen in the context of contract between national and regional coordination centers.

Un groupe de reflexion nomme par l’Institut national du cancer (INCa) sur la constitution d’une cohorte prospective de tumeur a formule un nombre limite de recommandations et a surtout identifie les domaines a preciser avant toute decision d’action. Un programme cohorte prospective est seul a meme de repondre aux enjeux medicaux, scientifiques et technologiques actuels, les cohortes retrospectives venant en amont ou en complement. Des programmes « cohorte prospective » sont identifies en Europe, Amerique du Nord et Asie, au stade de discussions ou mises en route. Le cout de collection des echantillons est eleve si l’on se fixe des criteres de qualite biologiques et cliniques exigeants. Le cout d’analyse et d’interpretation est impossible a fixer : 3 a 5 k€ par echantillon avec une approche ciblee et les moyens actuels, de l’ordre de 20 k€ si l’on envisage l’approche globale du NCI americain. Les facteurs limitants risquent de rester la mobilisation des acteurs cliniques, l’acquisition des annotations cliniques et le developpement bio-informatique pour l’integration des resultats plus que les plateformes d’analyse. Il faut preciser les objectifs du programme car les protocoles d’inclusion et de suivi des patients en dependent. Il faut soit envisager une cooperation internationale, soit se placer dans des creneaux technologiques tres specifiques. Il faut inclure dans la mise au point d’un programme la participation de groupes de bioethique et de valorisation economique. Le transfert des techniques vers la pratique doit etre envisage. Le choix des sites tumoraux ne peut se faire sur une seule base d’incidence, mais il devra etre fait en prenant en compte l’apport eventuel de groupes cooperateurs. Le choix des centres participant doit se faire sous la forme d’un contrat avec partage de materiel. Une procedure d’appel a expression d’interet peut aider a selectionner et/ou a regrouper les initiatives.

PURPOSE:Telomerase, a ribonucleoprotein complex whose activity is related to the expression of its catalytic subunit human telomerase reverse transcriptase (hTERT), restores telomere length in tumor cells and enables immortality after p53/Rb inactivation has been achieved. To determine the timing of hTERT derepression during bronchial carcinogenesis and its relationship with telomere shortening and the p53/Rb pathway alterations, we did an immunohistochemical and in situ hybridization study in preinvasive and invasive bronchial lesions. EXPERIMENTAL DESIGN:hTERT, P53, P16, cyclin D1, Bax-to-Bcl2 ratio, and Ki67 immunostainings were done in 106 preneoplastic lesions and in paired lung carcinoma and normal bronchial mucosae. Concomitantly, hTERT mRNA levels and qualitative telomere shortening were assessed by in situ hybridization and fluorescence in situ hybridization, respectively, in a subset of preneoplastic and neoplastic lesions. RESULTS:Telomerase was increasingly expressed from normal epithelium to squamous metaplasia, dysplasia, and carcinoma in situ, and decreased in invasive carcinoma (P < 0.0001), with a direct correlation between protein and mRNA levels of expression (P < 0.0001). hTERT expression was directly correlated with P53, Ki67, and Bcl2-to-Bax ratio, suggesting a coupling between telomerase reactivation, proliferation, and resistance to apoptosis. Telomere signals significantly decreased as early as squamous metaplasia and progressively increased over the spectrum of preneoplastic lesions. CONCLUSIONS:Telomere shortening represents an early genetic abnormality in bronchial carcinogenesis, preceding telomerase expression and p53/Rb inactivation, which predominate in high-grade preinvasive lesions.

Introduction TUNEL, or terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, is acknowledged as a method of choice in the rapid identification and quantification of the apoptotic cell fraction in cultured-cell preparations. Indeed, despite the now well-known apoptotic cell morphotype accurately described by Kerr and Wyllie (1), there are at least two reasons for preferring the labeling-assisted identification: (i) The process is rapid: 2 to 5 minutes for chromatin condensation and cell fragmentation, 3 hours for apoptotic body phagocytosis (2, 3); (ii) The “picture” of the apoptotic cell has broadened and gained certain nuances: the apoptotic nucleus may condense without splitting (pycnotic nuclei; 4, 5) and, in the early stages of apoptosis, the nucleus may be slightly enlarged while DNA is starting to fragment (6). Moreover, nuclear morphology is strongly dependent on sample fixation, with consequent effects on the identification and interpretation of the morphological aspects of apoptotic cells. The accessibility to DNA breaks for enzymatic reactions is reduced by the nuclear protein environment (7) and impaired by cell fixation (8) currently performed with bridging aldehydes, paraformaldehyde (8), or formaldehyde (9), and sometimes followed by an ethanol postfixation (10). Thus, chemical pretreatments of the biological samples have been devised to improve TUNEL sensitivity. These involve the incubation of cells with a detergent, most frequently Triton® X-100 (6, 11), which, in our experience, exhibited limited efficiency. Proteolytic enzymes, mainly proteinase K, have been used for tissue sections (7, 8, 12) but not yet applied to cultured cells. Since one of the effects of the widely used microwave antigen-retrieval method is peptidebond cleavage (13), we assumed that this approach, together with proteinases, might prove efficient for TUNEL when applied to cultured cells. Two recent articles (14, 15) reported a similar attempt on formaldehyde-fixed, paraffin-embedded, microwavepretreated tissue sections. As a result of these tests we propose an optimized TUNEL protocol for cell cultures (16). ADRIEN NEGOESCU1*, PHILIPPE LORIMIER1, FRANCOISE LABAT-MOLEUR2, LAURENT AZOTI1, CATHERINE ROBERT1, CHRISTIANE GUILLERMET2, CHRISTIAN BRAMBILLA1, and ELISABETH BRAMBILLA1,2