|Home | About | Journals | Submit | Contact Us | Français|
Small cell lung cancer (SCLC) is one of the four major histologic types of lung cancer. The incidence of SCLC in developed countries has declined in recent years, presumably due to changes in cigarette composition. In the United States (US) SCLC is estimated to represent about 16% of new lung cancer diagnoses, which equates to about 35,000 new cases annually. In underdeveloped countries the percentage of SCLC cases may be higher. SCLC presents with a very large number of genetic alterations, including tumor suppressor genes, copy number gains and other somatic mutation in transcription factors, enzymes involved in chromatin modification, receptor tyrosine kinases and their downstream signaling components 1. SCLC has a high propensity for early spread and a high initial responsiveness to cytotoxic chemotherapy usually followed by rapid development of resistance. Thus, essentially all patients of any stage receive a doublet combination of etoposide with cisplatin or carboplatin. For the rare patient without nodal involvement, the chemotherapy may follow surgery and for the patient with nodal disease without distant metastases, a combination of chemotherapy with chest radiotherapy is usually given concurrently. Unfortunately, these therapies are short in duration and not curative in most instances with 5-year survival rates below 7%. No major treatment advances have occurred over the past 30 years2. Since the approval of topotecan in 1996, the US Food and Drug Administration (FDA) has not approved any new drugs for the treatment of SCLC patients 3. For these reasons SCLC was declared a “recalcitrant” cancer in the US. However, considerable therapeutic opportunities, including targeted therapies, exist because of recent developments in understanding the biology and molecular biology of SCLC in part due to the new model systems.
It is important to understand the specific cell type(s) present in the lung that are capable of transformation to SCLC, with special emphasis on their proliferation, differentiation, and migration control programs, so that the malignancy can be viewed within a framework of normal biological processes 6. Unlike the cells of origin for two other major lung cancer subtypes, adenocarcinoma (alveolar type 2 cells 7) and squamous cell carcinoma (presumed to be tracheo-bronchiolar basal cells 8), a rare sensory cell type termed pulmonary neuroendocrine cells are the predominant cell of origin for SCLC 9. Dr. Krasnow’s laboratory has demonstrated that the transformation processes for each of these cell types are similar in that characteristic oncogenes such as EGFR, KRAS, etc. (for lung adenocarcinoma 10) and tumor suppressors such as RB1, TP53, etc. (for SCLC 4) promote self-renewal in the corresponding normal cell types of origin 11 (MAK unpublished findings). These findings help explain why mutated versions of those genes are such powerful transformation inducers and why the mature cancer cells often phenotypically resemble their normal counterparts. Moreover, by assessing copy number neutral loss-of-heterozygosity mutations in human SCLC biopsies, Dr. Peifer’s laboratory has discovered that TP53 mutations likely occur earlier in SCLC tumorigenesis than mutations to RB1 (MP unpublished findings). This suggests that the fidelity of RB1’s programmed role(s) in pulmonary neuroendocrine cells is somewhat sensitive to the status of TP53.
An important distinguishing feature between SCLC and adenocarcinoma, on the other hand, appears to be the acquisition of additional mutations once transformation is achieved. Based on computational assessment of subclonal architecture in biopsied human SCLC versus adenocarcinoma samples by high-throughput sequencing, it appears that SCLC typically has significantly reduced genetic heterogeneity compared to adenocarcinoma 4. The basic biological and clinical implications of this finding are so far unclear.
While pulmonary neuroendocrine cells are agreed to be the predominant SCLC cell of origin9 (at least in mice), a question remains whether they are the only possible cell of origin, especially in light of additional oncogenes and tumor suppressors discovered recurrently mutated in human SCLC patients 4. In order to take an unbiased approach as to which genes might potentially induce SCLC transformation in different lung epithelial cell types, Dr. Berns’ laboratory has combined the canonical SCLC-relevant genetically engineered mouse model (GEMM), wherein both Rb1 and Trp53 are deleted from any cell type of interest 12, with transposon-mediated insertional mutagenesis to generate large numbers of epithelial clones with distinct mutations in individual animals 13. This system allows the experimenter to assess whether, in a background of compound Rb1/Tp53 deletions, randon additional mutations can transform previously refractory cell types in the lung to SCLC. These experiments verified the unrivaled vulnerability of pulmonary neuroendocrine cells to SCLC transformation, but also pointed to an, albeit less efficient, vulnerability of alveolar type 2 cells (AB unpublished findings). The relevant gene(s) that, when mutated in combination with Rb1 and Trp53 can induce alveolar type 2 cells to adopt a SCLC phenotype, remain unclear, but will be important targets for both basic biological and clinical investigations. Finally, because many orders of magnitude more alveolar type 2 cells are present in adult mouse and human lungs than pulmonary neuroendocrine cells 14, 15, these findings suggest that alveolar type 2 cells may in fact be a relevant target cell type for SCLC transformation in human patients.
In addition, the Berns laboratory has generated GEMMs designed to constitutively activate expression of candidate oncogenes, notably Mycl and Nfib, together with Rb1/Trp53 compound deletion in the mouse lung epithelium. Activated together, Mycl and Nfib accelerated SCLC development, including increasing both the growth rate of primary tumors and the appearance of metastases in the liver, bone, and kidney (AB unpublished findings). Interestingly, when studied in isolation, the two phenotypes were genetically separable: Mycl activation explained the primary tumor growth phenotype, and Nfib the metastatic progression. These results provide a rational framework to clinically intervene in two critical steps of SCLC tumorigenesis.
The growing understanding of the biologic and molecular characteristics of the SCLC cell of origin has therapeutic implications. Recently, the potential SCLC stem cell niche has emerged as a potential therapeutic target. Treatment strategies under investigation that may result in preferential targeting of SCLC stem cells include inhibition of developmental pathways such as WNT and Hedgehog, the transcription factors ASCL1 and NEUROD1, and the focal adhesion kinase (FAK) and PI3K/mTOR signaling pathways.
Aside from proliferation and transformation, the differentiation control program of pulmonary neuroendocrine and malignant SCLC cells, most notably the remarkable plasticity evident in response to commonly administered cancer therapies 16-18, requires increased understanding. By analyzing global gene expression patterns in a large panel of SCLC cell lines, as well as primary human SCLC samples, the laboratories of Drs. Massion and Quaranta have demonstrated that the samples can be subdivided into two general classes: those exhibiting a ’neuroendocrine’ expression pattern versus those exhibiting a ’mesenchymal-like’ pattern (PPD and VQ unpublished findings). Key genes underpinning the two states include transcription factors and cell adhesion proteins. Interestingly, both classes can coexist in a primary SCLC tumor biopsied from the same individual patient, and chemotherapy (e.g., topoisomerase inhibitor) can modulate representation of the two classes within the overall tumor. Previous studies have implicated the Ras/MAPK 19 and Notch 4 signaling pathways in controlling differentiation of pulmonary neuroendocrine and/or SCLC cells to alternative fates, and comprehensive integration of these results with those previous should shed new light on cellular heterogeneity in SCLC.
In addition to heterogeneity within and between individual SCLCs, phenotypic conversion to SCLC is a newly identified mechanism of drug resistance observed in lung adenocarcinomas progressing on EGFR tyrosine kinase inhibitor (TKI) treatment, occurring in roughly 15% of patients 16, 18. In most cases these adenocarcinoma-to-SCLC transformed tumors retain the original EGFR mutation, arguing against them being newly generated primary tumors posttherapy, but can continue to proliferate malignantly despite downregulation of EGFR protein. Moreover, in a recent study, 10/10 patients with adenocarcinoma-to-SCLC transformed tumors exhibited homozygous deletion of the RB1 gene, suggesting that RB1 deletion is required for phenotypic conversion 16. However, RB1 was also found deleted in 1/9 adenocarcinoma cases where TKI resistance was achieved independently of SCLC conversion 16, suggesting that RB1 deletion alone is insufficient explain the phenotypic switch.
Finally, the migration control program of normal pulmonary neuroendocrine cells and malignant SCLC cells is extremely relevant to both understanding and treating SCLC metastasis. Dr. Krasnow’s laboratory recently demonstrated a novel form of epithelial cell migration exhibited by normal pulmonary neuroendocrine cells during lung epithelial development, termed ’slithering,’ which is used to organize neuroendocrine cells into stereotyped clusters 20. The normal slithering program involves transient activation of an epithelial-to-mesenchymal transition (EMT) wherein recently specified pulmonary neuroendocrine cells migrate over and around other epithelial cells to find one another, without ever invading basally into the lung mesenchyme. Further investigation of the slithering program should hopefully reveal molecular dependencies that can be targeted in SCLC to attenuate or perhaps even prevent metastasis to extrapulmonary organs, which is the major cause of patient death 17.
Pulmonary carcinoids are low and intermediate grade neuroendocrine malignancies characterized by the expression of neuroendocrine differentiation markers and a low proliferation index. Pulmonary carcinoids (like carcinoids arising in other sites) are divided in typical (TC) and atypical carcinoids (AC) on the basis of histopathological objective criteria which are <2 mitosis/2mm2 and lack of necrosis for TC and 2-10/2mm2 mitoses and/or focal necrosis for the AC21. While the etiology is still not completely understood, carcinoids appear to arise from a different stem/progenitor cell than the high grade neuroendocrine lung tumors; SCLC and large cell neuroendocrine carcinoma (LCNEC). The first clue to them being different is a possible preneoplastic lesion “Diffuse Idioplastic Neuroendocrine Cell Hyperplasia” (DIPNECH) unique for pulmonary carcinoids and their occurrence in the setting of MEN1 disease (5% of carcinoids arise in MEN1 disease and less than 5% on the background of DIPNECH). However, 20 to 40% of carcinoids display somatic double allelic inactivation of MEN1 with mutation of one allele and allele loss (LOH) at the MEN1 gene (11q.13) location22. A more definitive clue was provided by the results of integrated genome analysis on 61 carcinoids (29 genomes, 5 exosomes, 69 RNA-Seq)23, which revealed a distinct mutational profile with 52% of the mutations affecting chromatin remodelling genes (MEN1, PSIP1, ARID1A), 34% belonging to the methylation complex and 25% to the SWI/SNF complex, in a mutually exclusive fashion. Overall, 73% of candidate drivers are found in pulmonary carcinoids. In contrast to high grade NE tumors, pulmonary carcinoids have a low rate of mutation (0.4 Muts/Mb), no significant focal copy number alteration, and TP53 or RB1 mutations/loss are very rare and never found together. Gene expressions profiling revealed wide differences. The pulmonary carcinoids appear to be etiologically independent of the high grade neuroendocrine tumors (SCLC / LCNEC) with no transitions or combinations occurring in patients. The molecular pathology and biology of carcinoids from other sites may be different.
LCNEC is a highly aggressive malignancy whose prognosis approaches that of SCLC24, 25. Based on similarity in expression profiling studies, a close biologic relationship between LCNEC and SCLC has been suggested 26. In the new WHO classification, LCNEC is categorized into the same category with SCLC under the “neuroendocrine carcinoma”, as LCNEC has many genetic features shared with SCLC despite some exceptions. Whether LCNEC should be therapeutically treated as SCLC or NSCLC remains controversial27, 28. Currently, there are only limited comprehensive molecular data on genomic alterations in LCNEC29. Such studies can be anticipated to yield insight into the biologic relationship between LCNEC and SCLC, which may inform clinical management of patients with these tumors. An analysis of a series of LCNEC utilizing custom targeted next-generation sequencing of 300 key cancer genes using the MSKIMPACT™ test, suggested that LCNEC comprises distinct molecular subsets: SCLC-like (characterized by RB1 + TP53 co-mutation and absent/rare NSCLC-type mutations) and NSCLC-like (characterized by the presence of NSCLC-type mutations, including KRAS, STK11, KEAP1, MAP2K1). The clinical relevance of these findings is under investigation.
SCLC sequencing efforts 30, 31 on 110 whole genomes found evidence for a nearly universal and bi-allelic loss of TP53 and RB14 (Figure 1). Rare tumors lacking RB1 mutations showed alternative mechanisms of RB1 activation by overexpression of Cyclin D1 due to chromothripsis events affecting chromosome 3 and 11. Alterations (point mutations, small deletions) of the PTEN gene, located at 10q23.3, are observed in 10-18% of SCLC tumors 32 and deregulation of MYC function has also been noted to be important in SCLC33. In addition, the analysis of somatic rearrangements showed that translocation within TP73 lead to the generation of an oncogenic version of the gene, TP73 Δex2 or TP73 Δex2-34. About 25% of human SCLC tumors showed inactivating mutations of NOTCH family genes suggesting that these genes are tumor suppressors in SCLC. This notion was further supported by the observation that activation of Notch signaling leads to significantly fewer tumors and a prolonged survival in SCLC transgenic mouse models 4.
In a large transcriptome analysis of 19 fresh frozen tumors and 23 cell lines, 60 fusion events were detected but none of them involved any targetable kinases. Only two genes were involved in recurrent fusion events: RLF and PTV1 - in accordance with the previously described low abundance of recurrent fusion events in SCLC 31. Combining these results with copy number analyses of these tumors showed that RLF- and PTV1-fusions most likely evolved as a byproduct of MYCL1 and MYC amplifications, respectively34.
The clinical characteristics and multigene mutation profiling of SCLC in never-smokers 35, 36 has been evaluated. The never-smokers with SCLC (50/391) had a better prognosis than smokers with SCLC in the Korean cohort. Although they found that the EGFR mutation rate was high in never-smokers, it is still unclear how effective EGFR TKIs are for EGFR-mutated SCLC. Other mutations found were TP53, RB1, PTEN, MET and SMAD4. Therefore, further global investigation of SCLC to determine differences in genetic or clinical characteristics between never-smokers and smokers is warranted.
A global CpG-site methylation analysis on 47 SCLC tumors (34 fresh frozen specimens, 6 patient derived xenografts (PDX), 7 cell lines) found that PDX samples better represent the methylome of primary tumors than cell lines. Furthermore, by using methylome and transcriptome analysis, distinct subtypes of SCLC can be defined that are indistinguishable by standard histological approaches 37. EZH2 showed increased overexpression in comparison to normal lung tissue and this increase was correlated with a higher methylation of the EZH2 promoter 38. Recent developments in drug discovery 39, 40 and pre-clinical data suggest that EZH2 is amenable to targeted therapy.
Proteomic analyses of SCLC cell lines and tumors led to the discovery that PARP1 is overexpressed and that PARP inhibition has activity in pre-clinical models and in a subset of SCLC patients 41, 42, with proteomic markers of DNA repair and PI3K pathway activation predictive for response of PARP inhibition in SCLC 43. Beyond PARP, proteomic analysis revealed other potential targets, such as EZH241 and Chk144.
Progress has been made in 1) the development and use of sophisticated SCLC GEMMs and PDXs, 2) the identification and propagation of circulating tumor cells (CTCs) from SCLC patients and 3) renewing interest in available SCLC cell lines 45.
Mouse models of cancer are initiated by oncogene and tumor suppressor mutations engineered into the mouse genome. These initiating mutations cause cancer in the mouse or, at minimum, predispose to the development of tumors46 (Figure 2). Additional acquired somatic genetic alterations have been described in GEMMs, suggesting that additional mutations might contribute to tumor formation or progression47, 48.
Comprehensive exome sequencing of primary and metastatic SCLC tumors from the Trp53; Rb1-mutant GEMM 12, 49 showed GEMM SCLC tumors harbor very few point mutations, most likely because the mice develop SCLC without exposure to cigarette smoke. The most frequently observed alterations in mouse SCLC were Mycl (encoding the L-myc oncogene) amplification, inactivating point mutations targeting the tumor suppressor Pten and DNA copy number loss of Chr19, which encodes the mouse Pten gene. Consistent with Pten loss acting as an important driver in SCLC, deletion of Pten in the Trp53, Rb1 model accelerated tumor progression50, 51. Genomic analysis of the triple mutant Trp53/Rb1/Pten tumors revealed persistence of the Mycl1 DNA amplification. This implies that MYCL1 is a key driver in SCLC. Analysis of the clonal evolution of tumors in the SCLC GEMM identified spread of multiple primary tumor subclones to regional thoracic lymph nodes. Considering the fecundity with which SCLC establishes multiple genetically-distinct metastatic subclones in regional lymph nodes, it is intriguing to wonder if these nodes might serve as a reservoir of disease after treatment, as in other cancer models 52.
Dr. MacPherson’s lab performed genomic characterization of human SCLC cell lines and tumor tissues to identify recurrent mutated potential driver genes. They showed the histone methylase MLL2/KMT2D as a putative tumor suppressor with frequent truncating mutations. Use of a conditional Mll2 knockout mouse suggested that Mll2 loss can cooperate with loss of Trp53 to promote cellular proliferation in mouse embryo fibroblasts, promote lung cancer and expand the tumor spectrum in the Trp53/Rb1 mouse model. The accelerated Trp53/Rb1/Pten triple mutant SCLC model49, 50 was used to investigate the role of Mycl as a driver of SCLC tumorigenesis. Conditional deletion of Mycl1 potently suppressed SCLC tumorigenesis, complementary to the Berns’ lab finding that overexpression of Mycl promoted SCLC53. These results confirm the role of MYCL1 as critical driver in SCLC, and implicate MLL2 loss and more broadly epigenetic dysregulation, as important events in a subset of human SCLC.
Human SCLC often exhibits a dramatic response to platinum-based chemotherapy, only to subsequently recur and become resistant to subsequent treatments 45. Preclinical models have largely failed to recapitulate this pattern of response to therapy. CTCs and CTC-derived xenografts (CDXs) may be a useful model for genetic characterization and preclinical studies assessing the development of resistance. Patients with SCLC exhibit a high burden of CTCs as previously described 54. Dr. Dive’s group demonstrated that CTCs from SCLC patients efficiently form tumors when implanted into immunocompromised mice. Importantly, the CDX tumors recapitulated the histological features of human SCLC, and mirrored the response to chemotherapy of the patient from which the cells were obtained. The genomic profile of expanded CDX tumors was also similar to the parental CTCs. It is enticing to envision using these models to uncover the genetic evolution of SCLC between initial therapy and the emergence of resistance 55. Additional samples are needed to validate this approach, but coupling preclinical therapeutic studies using CDXs with analysis of genetic progression is a potentially powerful approach to the identification of the mediators of chemotherapy resistance and to targeting these pathways with rationally designed drugs
The WNT56 and Hedgehog (Hh)57 signaling pathways are frequently disrupted in SCLC. SmoM2 is a mutant form of the smoothened receptor derived from human basal cell skin cancer that is constitutively active independent of the Hh ligand. Cells expressing this allele (SmoM2/+) display constitutive activation of the Hh signaling pathway. The Sage lab compared Rb1lox/lox/Trp53lox/lox/SmoM2/+ mice to Rb1lox/lox/Trp53lox/lox/Smolox/lox mice 57 and observed that the wild type Smo (SmoM2/+) had more, larger and earlier tumors. Inhibition of Smo through silencing or with LDE225 therapy, a smoothened inhibitor, inhibited colony growth and inhibition of chemo-resistant SCLC tumors was greater than inhibition of chemonaive SCLC tumors, indicating that Smo inhibitors may preferentially inhibit SCLC stem cells. The same group conducted studies indicating that the combination of E/P plus LDE225 produced superior in vivo growth inhibition compared to E/P alone or LDE225 alone. Thus, there is a definite role for Hedgehog signaling in SCLC. Based on these and other data, a randomized phase II trial in first-line SCLC therapy comparing etoposide/cisplatin (E/P) to etoposide/cisplatin plus vismodegib with vismodegib maintenance was designed. Unfortunately, there were no differences in efficacy with median PFS of 4.4-4.6 months in the two arms 58.
The WNT pathway may be involved in SLC as well as NSCLC pathogenesis. This pathway deserves further investigation in SCLC as a number of druggable targets exist in this signal pathway 56.
ASCL1, a transcription factor, regulates tumor initiating capacity in SCLC and is required during development of neural and neuroendocrine lineages9. Interestingly, the expression of ASCL1 and NEUROD1, another transcription factor, are mutually exclusive in SCLC cell lines. ASCL1 activates NOTCH signaling by direct regulation of DLL1 expression. NOTCH signaling represses ASCL1 expression via the transcriptional repressor HES1. ASCL1 but not NEUROD1 is required for neuroendocrine tumor formation in the Trp53/Rb1/p130 mouse model. ASCL1 directly regulates genes in SCLC tumor growth including MYC, NFIB, RET and genes in the NOTCH pathway 59 (and unpublished data JE Johnson).
NEUROD1 appears to play an important in some SCLC cell lines 60, 61. Studies have indicated that ERK 1/2 signaling is low in all SCLC cell lines and that blocking ERK activity has no effect on cell growth while activating ERK inhibits SCLC growth. NEUROD1 can inhibit ERK and stimulate metastases. SCLC cell lines such as NCI-H2171, NCI-H82, and NCI-H1962 that express NeuroD1 produce fast growing tumors in mouse xenografts. In cell lines with high NEUROD1 expression, knockdown of NEUROD1 inhibits formation of soft agar colonies and cell migration in vitro and metastases in vivo. NEUROD1 binds the TrkB promoter and knockdown of TrkB suppressed the growth and inhibition of TrkB by chemical means blocked SCLC proliferation in vitro and in vivo.
A key therapeutic goal is the induction of tumor cell death to achieve regression. The anti-apoptotic protein Bcl-2 is overexpressed in many cancers, including 40-60% of SCLCs 62-64. Treatment of SCLC PDX models with ABT-737, a Bcl-2/Bcl-xL inhibitor, resulted in reduced tumor growth, but the response was limited 65. Characterization of ABT-737 resistant tumor lead to the identification of PI3K/mTOR inhibitors as possible agents that could augment ABT-737 responses. The mTOR inhibitor rapamycin dramatically enhanced ABT-737 activity in the PDX models, possibly by inducing the pro-apoptotic proteins BAX and BAK1.
The Bcl-2/Bcl-xL/Bcl-w inhibitor ABT-263 also promotes apoptosis in SCLC cell lines, and a highthroughput drug screen showed that high expression of the pro-apoptotic regulator BIM and low expression of the anti-apoptotic MCL1 gene correlated strongly with sensitivity66, 67. However, a phase II trials of ABT-263 monotherapy revealed minimal clinical activity68. In SCLC cell lines, cells with high MCL-1 expression exhibited relative resistance to ABT-263 but inhibition of mTOR activity using AZD8055 suppressed MCL-1 protein levels and sensitized cells to ABT-263. This combination also suppressed growth of autochthonous Trp53/Rb1 double mutant GEMM SCLC tumors 67. Taken together, these studies67, 69 suggest that dual inhibition of Bcl-2/Bcl-xl and mTOR might be a rational therapeutic approach in SCLC.
eIF4A is sufficient and required for MYC driven T-ALL and lymphoma models 70. Silvestrol is a natural compound that blocks eIF4A RNA helicase and effectively inhibits T-ALL cells and many, but not all, SCLC cell lines. Silvestrol sensitive transcripts include MYC, NOTCH, MyB and others71.
High expression of Aurora kinase A or B imparts a poor prognosis in lung cancers 72, 73. This is in keeping with several trials showing that cell cycle gene signatures are also associated with prognosis. Aurora kinase tyrosine inhibitors may be directed at Aurora A, Aurora B or may inhibit all Aurora kinases at concentrations that can be achieved in humans. Aurora kinases inhibitors of all classes have been shown to inhibit the growth of several NSCLC cell lines in vitro and in preclinical mouse models. It is not clear whether any class of Aurora kinase inhibitor is preferred.
In SCLC cell lines, Sos et al showed that the specific Aurora A inhibitors MLN8237 and PHA680632 inhibited many, but not all, SCLC cell lines and that there was a significant association between MYC amplification and sensitivity to the TKI 74. The specific Aurora Kinase B inhibitor AZD1152 was studied on a panel of SCLC cell lines and there was a significant relationship between MYC and MYCL1 amplification and expression and drug sensitivity. There was also a significant relationship between a reported MYC gene signature and sensitivity75.
AZD1152 was studied in patients with hematologic malignancies and while there was some evidence of activity, hematologic toxicity was considerable. MLN8237 (alisertib) has been studied specifically in patients with SCLC, breast and ovarian cancers. In the phase II study in SCLC there was an objective response rate of 21% that included an OR of 19% in those with a sensitive relapse and 27% in those with a resistant relapse 76. The overall PFS was 2.6 months in the sensitive and 1.4 months in the resistant relapse. Millenium conducted trials of the combination of MLN8237 plus paclitaxel in patients with breast and ovarian cancers and found that the agents could be combined safely. Thus, they have instituted an ongoing randomized phase II trial comparing paclitaxel alone to the combination of paclitaxel plus MLN8237 in patients with SCLC who progress after initial etoposide/platinum therapy (NCT02038647).
MEDI0639 is a human monoclonal IgG1 antibody directed against Delta-like ligand 4 (DLL4) in the NOTCH developmental signaling pathway and believed to block angiogenesis by promoting formation of non-functional vasculature and inhibiting tumor initiating (stem) cells (TICs) 77. A phase Ib/II study of OMP-59R5, a fully human monoclonal IgG2 antibody targeting the Notch 2/3 receptors, in combination with etoposide and platinum in untreated extensive-stage SCLC showed promise with 13/16 (81.3%) attaining a partial response and 3 achieving stable disease 78. Demcizumab is another monoclonal antibody directed against DLL4 79 with ongoing trials in NSCLC (NCT02259582, NCT01189968).
The fibroblast growth factor receptor (FGFR) family represents promising targets for the development of targeted therapies in SCLC. Several studies have reported that the FGFR1 gene is amplified in 5-6% of SCLC patients 80. A study with 83 SCLC patients explored the correlations between FGFR1 and its ligands and the results from Dr. Hirsch’s group showed that a subset of SCLCs were potentially characterized by activated FGF/FGFR1 pathway, as is evidenced by positive FGFR1, FGF2, FGF9 protein and/or mRNA expression or gene copy number 81. Combined analysis of FGFR1 and ligand expression may allow selection of SCLC patients for FGFR1 inhibitor therapy. Dr. Hirsch’s group is studying the FGFR inhibitor ponatinib in a biomarker driven trial (NCT01935336). Another phase II trial to assess the efficacy and safety of lucitanib 82, an inhibitor of FGFR1-3, VEGFR1-3, and PDGFRα/β, in patients with advanced lung cancer is currently recruiting participants (NCT02109016).
Whole exon sequencing (n = 51) and copy number analysis (n =47) on Japanese SCLC patients 83 detected genetic alterations in the PI3K/AKT/mTOR pathway in 36% of the tumors. Importantly, the SCLC cells harboring active PIK3CA mutations were potentially targetable with currently available PI3K inhibitors. Therefore, a sequencing-based comprehensive analysis could stratify SCLC patients for potential therapeutic targets.
A possible approach to targeting cancer stem cells is through dual inhibition of PI3 kinase and mTOR using VS5584. In models of SCLC, breast, and ovarian cancer, VS5584 demonstrates preferential targeting of the stem niche, as defined by aldefluor, tumorsphere, and limiting dilution assays. Both PI3K and mTOR inhibition appear requisite for these effects. In the SCLC model, VS5584 also demonstrated marked reduction in tumor initiating capacity in contrast to platinum/etoposide, which had essentially no activity 84. VS5584 is currently in early phase clinical trials.
An activating M918T RET somatic mutation in a metastatic SCLC tumor specimen has been described 85. SCLC cell lines, which have the stable overexpression of both mutant M918T and wild-type RET, became sensitized to the RET TKIs, vandetanib and ponatinib. These results indicate that a subpopulation of SCLC patients may derive benefit from TKIs targeting RET.
A high-throughput cellular screen of a diverse chemical library discovered that SCLC is sensitive to THZ1, a covalent inhibitor of CDK7. Moreover, expression of super-enhancer-associated transcription factor genes, including MYC family proto-oncogenes and neuroendocrine lineage-specific factors, is highly vulnerability to THZ1 treatment 83, 86. Hence, the downregulation of these transcription factors may contribute to SCLC sensitivity to transcriptional inhibitors and THZ1 may represent a prototype drug for tailored SCLC therapy.
Cytokines and growth factor binding to their cognate receptors leads to activation of the Janus Kinase- Signal Transducer and Activator of Transcription (JAK-STAT) pathway. STATs result in cell proliferation and activation of both tumor and inflammatory cells 87, 88. STAT 3 is phosphorylated in NSCLC and pancreatic cancer and predicts for poor survival. There are a number of JAK inhibitors in development. Ruxolitinib (INCB1824), an inhibitor of JAK1/2, is FDA approved for myelofibrosis and tofacitinib 89, an inhibitor of JAK1/3, is approved in rheumatoid arthritis. Ruxolitinib has been evaluated in vitro in NSCLC cell lines and will inhibit STAT 3 activation 90. Randomized trials of ruxolitinib in addition to cisplatin/pemetrexed, docetaxel and erlotinib are in progress in NSCLC. In SCLC, evaluation of on line databases as well as tumors and cell lines demonstrated that 30-40% have copy number gain for JAK1/2 gene. In vitro and animal data indicates that targeting JAK2 with siRNA will inhibit the growth of SCLC. AZD1480 (which targets JAK1/2/3, FLT3 and Aurora kinase) has single agent activity as well as synergy with existing chemotherapy 91.
Phase II trials of amrubicin conducted in the US 92 and Asia 93 showed that amrubicin has activity in SCLC patients with had progressed after first line etoposide/platinum therapy. Activity was observed both in “sensitive” and “resistant” relapse. These studies were followed by a randomized phase III trial comparing amrubicin to topoteacan chemotherapy in patients who progressed on etop/platinum 94. Unfortunately, amrubicin, while suggestive of some clinical benefit, was not superior to topoteacan in this trial and thus has not been approved for use in SCLC except in Asia.
Since palifosfamide plus doxorubicin showed supportive results in soft tissue sarcoma (NCT00718484), a phase I trial with palifosfamide plus carboplatin and etoposide is in process (NCT01555710). Another global phase II trial with aldoxorubicin 95 in patients with relapsed and refractory SCLC is in process (NCT02200757). The Southwest Oncology Group pooled data from trials in second- and/or third-line ES-SCLC. Univariate and multivariate Cox regression models were fit to assess the relationship between baseline characteristics and PFS and OS. Of 329 patients, 151 were platinum sensitive and 178 refractory. In this analysis platinum sensitivity status was not associated with OS, however prognostic groups with differential OS outcomes (high, intermediate and poor risk) were identified. Elevated lactate dehydrogenase (LDH), weight loss, performance status and male sex were all associated with worse OS. The authors concluded that, platinum sensitivity status was no longer independently associated with OS and that validation of this model in an independent SCLC dataset is warranted 96.
Focal adhesion kinases (FAK) play a critical role in cancer initiation and proliferation as well as resistance to chemotherapy and radiation. Importantly, inhibition of FAK with the novel TKI VS6063 has demonstrated reduction in tumor initiating capacity (i.e. deplete cancer stem cells) in contrast to paclitaxel 97. VS6063 is currently in early phase clinical trials for SCLC, however, negative results were recently announced in a maintenance setting trial conducted in malignant pleural mesothelioma 98.
Chemokine receptor 4 (CXCR4) is a G protein-coupled chemokine receptor that is functionally expressed or overexpressed in a number of cancers 99, 100. CXCR4 plays a role in invasion, survival, angiogenesis and metastasis 101. Elevated CXCR4 expression is associated with inferior outcome in NSCLC 102. LY2510924 is a peptide antagonist of CXCR4 blocking the signal cascade of SDF-1, which has in vitro and in vivo activity in a number of cell lines and tumor models. A randomized phase 2 trial of this agent combined with carboplatin/etoposide vs. carboplatin/etoposide alone was conducted, however, it increased toxicity and failed to demonstrate any evidence of benefit in terms of either PFS or OS 103.
Poly ADP ribose polymerase (PARP) is critical for DNA damage repair 104. Given that all current treatment for SCLC relies on DNA damaging agents (e.g. chemotherapy and radiotherapy) and that resistance to these modalities is at least in part due to repair of DNA damage, inhibition of DNA damage repair is an extremely logical approach. Single agent activity of the PARP inhibitors olaparib, rucaparib, talazoparib and ABT-888 (veliparib) and synergy with platinum and etoposide was demonstrated in cell lines and animal models41, 43, 105. A randomized, Phase 2, double-blind Small cell lung cancer Trial of Olaparib as Maintenance Program (or “STOMP”) which compares PARP inhibition to placebo following first-line chemotherapy (Cancer Research UK, trial number CRUK/10/037) has recently closed to recruitment and results are awaited 106. A phase I trial combining veliparib with cisplatin and etoposide (ECOG-ACRIN E2518) 107 successfully completed and is now active as a randomized phase II trial. The newer PARP inhibitor talazoparib kills SCLC cells more efficiently than the older PARP inhibitor olaparib43 and had single agent activity in SCLC in a recently completed Phase 1 trial 42. High levels of PARP and other proteins involved in DNA damage repair, like FANCD2 and pCHK2, are strongly associated with the sensitivity of SCLC cells to talazoparib. High expression of a “DNA repair protein score” is also associated with greater response to talazoparib, while higher expression of a “PI3K score” is more resistant to the drug 108.
Antibody drug conjugates (ADCs) have been some of the most active agents developed in oncology, combining the specificity of antibodies with the cytotoxicity of traditional chemotherapeutic agents 109. An ADC, lorvotuzamab mertansine, targeting the neural cell adhesion molecule CD56 found on over 90% of SCLC had excellent activity in vitro, in preclinical models, as well as single agent activity 110. However, further development was abandoned due to unacceptable toxicity when combined with carboplatin/etoposide 111.
SC16LD6.5 (rovalpituzumab tesirine) is an ADC that targets SCLC and LCNEC tumors by way of binding the atypical Notch ligand delta-like ligand 3 (DLL3) on the cell surface and then delivering the DNA damaging agent D6.5, pyrrolobenzodiazepine dimer toxin. Data from a tissue microarray shows that the majority of SCLCs express DLL3 by IHC analysis and, using an H score, high expression (H-score >120) was observed in 60%; moderate expression (H-score 60-120) in 22% and low expression (H-score <60) in 18%. Similar rates (61%, 14% and 25%) were found in patients on the phase 1 trial (NCT01901653). The ADC inhibits the growth of SCLC PDX models in relation to the level of expression of DLL3 and the growth inhibition was far superior to the combination of etoposide/cisplatin112. Overall response rate in the Phase 1 was 20%, with 70% receiving clinical benefit. For those with an H-score of 180 or above, the ORR was 39% and clinical benefit was 75%113.
Targeting moieties (including antibodies and small peptides) can be conjugated to radioisotopes. Radioisotopes that have the potential for use as cancer therapeutics include alpha emitting agents, which have high energy but short path lengths, and beta emitters, with lower energy, but longer path lengths. The latter agents uniquely possess the possibility of overcoming tumor heterogeneity by “crossfire effect”, i.e. the particle travels a distance of > 1 cell and therefore has the ability to damage cells that may not express the target. In addition, gamma emitting radioconjugates (which can be a separate agent or the same drug, depending upon the radioisotope) can be used for imaging and therefore allow for real time assessment of the presence of the target within a tumor site. Somatostatin receptors, specifically SSTR2, are frequently expressed in neuroendocrine cancers, including SCLC. A preliminary trial demonstrated the feasibility of targeting SSTR2 with a rhenium 188 labeled SSTR2 peptide fragment 114.
Based on the sequencing the entire coding region and the intron–exon boundaries of MAX in lung cancer cell lines, EZH2 and BRG1 can be biomarkers to predict sensitivity to an EZH2 specific inhibitor, GSK126, plus etoposide 115, 116.
Targeted therapy trials in SCLC that did not statistically reach their endpoints include antiangiogenic agents like thalidomide, bevacizumab and sorafenib 117-121. A phase II study of cediranib (single agent) failed to demonstrate any objective response in recurrent or refractory SCLC 122. Similarly vandetanib in maintenance setting 123 and aflibercept (VEGF-trap) in a phase II clinical trial testing aflibercept with or without topotecan did not show any significant clinical benefit 124. Sunitinib as a single agent was tested in a single arm Phase II trial (EORTC-08061) in patients with chemo-naive extensive small cell lung cancer or who had a “chemosensitive” relapse. The trial was stopped early due to poor accrual 125. However, sunitinib maintenance met its primary endpoint of prolonged PFS in a randomized Phase II CALGB trial126.
Clinical trials of signaling pathway inhibitors for c-kit and PDGFR (imatinib in all comers or in KIT positive patients), MET/HGF or IGF1R (AMG 102 or AMG 479, respectively, in combination with platinum-based chemotherapy) and AKT/mTOR (everolimus, temsirolimus etc.) have had disappointing results despite pre-clinical evidence of activity 127-133.
AZD1775 is a small molecule wee-1 kinase inhibitor believed to abrogate the G2/M checkpoint, leading to mitotic catastrophe and cell death in the presence of chemotherapy induced DNA damage in TP53 mutant SCLC tumors 134.
Prophylactic cranial irradiation (PCI) is known to reduce the risk of brain metastases and improves survival, however higher dose PCI was not found to be better. Neurocognitive changes mainly occur after high doses and in elderly patients, but are also observed without PCI. In a phase 3 randomized controlled trial 498 patients were assigned (1:1) to receive either thoracic radiotherapy (30 Gy in ten fractions) or no thoracic radiotherapy. All underwent PCI. Overall survival at 1 year was not significantly different between the groups, 33% (95% CI 27–39) for the thoracic radiotherapy group versus 28% (95% CI 22–34) for the control group (hazard ratio [HR] 0.84, 95% CI 0.69–1.01; p=0.066). However, in a secondary analysis, 2-year overall survival was 13% (95% CI 9–19) versus 3% (95% CI 2–8; p=0.004). Progression was less likely in the thoracic radiotherapy group than in the control group (HR 0.73, 95% CI 0.61–0.87; p=0.001). At 6 months, PFS was 24% (95% CI 19–30) versus 7% (95% CI 4–11; p=0.001). The authors concluded that thoracic radiotherapy in addition to PCI should be considered for all patients with ES-SCLC who respond to chemotherapy 135.
The development of immunotherapies for SCLC is not a novel endeavor 136-140, but recent successes in this field for other cancer types suggest that these approaches may provide a degree of prolonged clinical benefit that has not been observed in SCLC patients with traditional treatments 141 (Figure 4).
SCLC patients with paraneoplastic syndromes develop T-cell responses 142, 143, appear to live longer that than SCLC patients without such syndromes144 and long-term survivors of SCLC maintained a high ratio of effector T-cells to regulatory T-cells 145. One would expect SCLC tumors to be sensitive to the activation of T-cell checkpoints due to the high mutation burden in these tumors 30, 31, even if immunosuppression mechanisms may limit the efficacy of T-cell clones 146, 147. A significant increase in survival in patients with a high number of lymphocytes in their blood has been reported148. In addition, neural-specific antibodies can be used as diagnostic and prognostic biomarkers for SCLC patients in large cohorts 149. Importantly, there is a strong correlation between auto-immune phenotypes or paraneoplastic syndromes and exceptional longevity in SCLC patients, suggesting that activation of the immune system is beneficial to these patients in the long term.
Manipulation of T-cell immune checkpoints, particularly CTLA-4, PD-1, and PD-L1, 150 in SCLC to enhance the anti-cancer effects of T-cells has been or is being evaluated. In a phase II clinical trial in ES-SCLC patients ipilimumab, a fully human IgG1 cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) monoclonal antibody, in combination with standard chemotherapy, improved the immune-related progression-free survival 151 and a phase III trial is in progress (NCT01450761). In the phase I/II CheckMate-032 study, SCLC patients with progressive disease after >1 prior line of therapy received nivolumab (a fully human IgG4 programmed death 1 (PD-1) inhibitor antibody) + ipilimumab or nivolumab monotherapy. The combination or monotherapy showed activity and durable responses with tolerable toxicity. Overall response was reported in 7/40 pts (18%) with nivolumab alone and in 8/46 pts (17%) in the combination therapy 152. In the phase IB KEYNOTE-028 study SCLC patients with PD-L1 positive tumor who failed or were ineligible for standard therapy were treated with pembrolizumab (humanized monoclonal IgG4 PD-1 inhibitor antibody); the overall response was reported to be 35% and safety profile consistent with other PDL-1 studies 153. Another two studies are exploring the efficacy of combination of pembrolizumab with chemotherapy/radiotherapy for extensive SCLC (NCT02359019, NCT02402920). Additionally, blocking PD-L1 signaling by infiltrating macrophages may be sufficient to generate a positive response from T-cells and improve the survival of SCLC patients 154. A number of clinical trials including or focusing on SCLC patients with PD-1, PD-L1 or CTLA-4 inhibitors are ongoing (e.g., NCT01693562, -02261220, -02046733, 02538666).
Gangliosides are cell surface oligoglycosylceramides that contain a sialic acid linked onto the sugar chain and are predominantly found in the nervous system and one such antigen, Fucosyl-GM1 (Fuc-GM1), is selectively expressed at high levels on the majority of SCLC tumors155. In the last 15 years, vaccines have been developed against Fuc-GM1156, 157, and these have demonstrated safety and immunogenicity in pilot trials enrolling patients with SCLC who have completed first-line chemotherapy158. A phase I/II clinical trial using a high-affinity antibody against this antigen is ongoing (BMS-986012, NCT02247349).
Chimeric antigen receptor (CAR) T-cells 159, 160 have a possible use in SCLC and potential targets of CAR T-cells in SCLC can be identified from auto-antibodies found in patients. In particular, NCAM/CD56 is an attractive target with high expression on the surface of SCLC cells. Pre-clinical data indicate potent anti-tumor effects of CD56 CAR T-cells in SCLC. The expression of CD56 on other cells, including neural tissue and natural killer (NK) cells, might limit the use of this particular antigen, but this strategy may be developed with other SCLC-specific cell surface markers and in combination with other immunotherapies.
CD47 is a cell-surface molecule that acts as a “marker of self” and prevents cells of the innate immune system from attacking hematologic malignancies and certain types of solid tumors 161-163. CD47 normally promotes immune evasion by signaling through SIRPα, an inhibitory receptor on macrophages and other myeloid cells 164. CD47 levels are high on the surface of SCLC cells and pre-clinical data from human cell lines and xenografts suggest that blocking CD47 strongly promotes the phagocytosis of SCLC cells by macrophages and inhibits tumor growth by T cellmediated processes165. It is possible that the anti-cancer effects of anti-CD47 strategies could be enhanced with the concomitant activation of T-cell checkpoints.
The Recalcitrant Cancer Research Act of 2012 (H.R. 733) that was signed into a bill in early 2013 stipulates the National Cancer Institute (NCI) to “develop scientific frameworks” in SCLC research as this cancer fulfils the criteria of a recalcitrant disease.
The available resources to conduct broad scale SCLC research are suboptimal. New initiatives are needed that would include changes in standard of care and standardization of tissue collection protocols to gain access to specimens that reflect the dynamic biology of the disease. The National Cancer Institute has conducted a preliminary high throughput drug screen on a panel of 63 SCLC cell lines using 103 approved oncology drugs and 420 investigational agents. mRNA and miRNA gene expression profiles were determined for all cell lines and correlations between mRNA/miRNA expression patterns and drug sensitivity were robust and provide another approach to begin to understand the molecular determinants of therapy response and resistance in SCLC. Additionally this approach may potentially identify new drugs and drug combinations with improved efficacy in this recalcitrant disease.
Despite the paucity of therapeutic advances in SCLC, considerable progress in understanding the biology, molecular biology, model systems and potential therapeutic targets has been made. Studies of early lung and neuroendrocrine cell development models have provided insights into the cell of origin for SCLC. New GEMMs have illustrated the universal importance of TP53 and RB1 gene mutations in the pathogenesis and the potential role of additional genetic changes as well as changes in transcription factor expression. PDXs and CDXs provide new means for preclinical testing of new therapies. Molecular studies have identified the high mutation burden found in SCLC and have identified differences between SCLC, carcinoids and large cell neuroendocrine tumors. Potential therapeutic targets including EZH2, PARP, CDK1, MCL1, BCL2, BIM, SHH (Sonic Hedgehog), WNT, NOTCH1, Aurora Kinase, FGFR, PIK3CA, RET, THZ1, JAK-STAT, FAK, CXCR4, PD-L1, Fuc-GM1, CD56 and CD47. Ongoing and future clinical trials have to show which of these candidates can be translated into an effective targeted therapy. Thus, the future of improving outcomes for SCLC patients appears promising but there are still a number of unanswered questions which need to be addressed in the future and these are outlined below.
Most recently the US National Cancer Institute released a Request for Application (RFA) (PAR-16-049, PAR-16-050, PAR-16-051) for grants specifically focusing on SCLC, and hopefully through these grants many of the above questions will be addressed.
Paul A. Bunn, Jr, MD has received consulting fees from AstraZeneca, BMS, Clovis, Daiichi, Genentech, Merck, Novartis, Sanofi, Lilly, Merck-Serono, and Pfizer. His institution has received grants from NCI and Eisai.
John Minna, MD reports grants from National Cancer Institute, grants from Cancer Prevention and Research Institute of Texas (CPRIT), personal fees from National Institutes of Health, personal fees from Genentech, other from University of Texas Southwestern Medical Center, during the conduct of the study; grants from National Cancer Institute, grants from CPRIT (State of Texas), personal fees from National Institutes of Health, personal fees from Genentech, other from University of Texas Southwestern Medical Center, outside the submitted work;.
Alexander Augustyn PhD has nothing to disclose.
Adi Gazdar MD has nothing to disclose.
Youcef Ouadah BS has nothing to disclose
Mark A. Krasnow, MD, PhD has nothing to disclose
Anton Berns, PhD has nothing to disclose.
Elisabeth Brambilla, MD has nothing to disclose
Natasha Rekhtman, MD, PhD has nothing to disclose.
Pierre P. Massion, MD has nothing to disclose.
Matthew Niederst, PhD reports personal fees from Boehringer Ingelheim, during the conduct of the study; personal fees from Boerhinger Ingelheim, outside the submitted work;.
Martin Peifer, MD reports personal fees and other from NEO New Oncology AG, outside the submitted work;.
Jun Yokota, MD has nothing to disclose.
Ramaswamy Govindan, MD reports personal fees from Celgene, Bayer, Roche, Clovis, Boehringer Ingelheim, Helsinn, Genentech, AbbVie, GSK, Novartis, Genecentric, Merck, and Pfizer, outside the submitted work;
John Poirier, PhD has nothing to disclose.
Lauren A. Byers, MD serves on consulting/advisory boards for BioMarin, AstraZeneca, and AbbVie
Murry W. Wynes, PhD has nothing to disclose.
David McFadden, MD, PhD has nothing to disclose.
David MacPherson, PhD has nothing to disclose.
Christine L. Hann, MD, PhD has nothing to disclose.
Anna F. Farago, MD, PhD reports personal fees from Intervention Insights, outside the submitted work;
Caroline Dive, PhD reports personal fees from Intervention Insights, outside the submitted work;
Beverly A. Teicher, PhD has nothing to disclose.
Craig Peacock, PhD reports grants from Flight Attendant Medical Research Institute (FAMRI), non-financial support from Novartis, during the conduct of the study;.
Jane E. Johnson, PhD has nothing to disclose.
Melanie H. Cobb, PhD has nothing to disclose.
Hans-Guido Wendel, MD has nothing to disclose.
David Spigel, MD is a non-compensated advisor to BMS, Genentech/Roche, Novartis, and Pfizer.
He also serves on the Data Safefy Monitoring Board for Merck.
Julien Sage, PhD has nothing to disclose.
Ping Yang, MD, PhD has nothing to disclose.
M. Catherine Pietanza, MD reports personal fees from Genentech, CelGene Corp, Abbvie, and Clovis Oncology, grants and personal fees from Novartis and Bristol Myers Squibb, grants from Stemcentrx, Inc and OncoMed Pharmaceuticals, Inc, outside the submitted work;.
Lee M. Krug, MD reports grants from Bristol-Myers Squibb, during the conduct of the study; other from Bristol-Myers Squibb, outside the submitted work;.
John Heymach, MD, PhD reports grants from AstraZeneca, GlaxoSmithKline, and Bayer, during the conduct of the study; grants from Genentech, other from AstraZeneca, other from Novartis, other from GlaxoSmithKline, other from Lilly, other from Boerhinger Ingelheim, other from Synta, other from Exelixis, outside the submitted work;
Peter Ujhazy, MD, PhD has nothing to disclose.
Caicun Zhou, MD, PhD has nothing to disclose.
Koichi Goto, MD reports grants from MSD K.K., grants and personal fees from Astra Zeneka K.K, grants and personal fees from Taiho Pharmaceutical Co., Ltd., grants and personal fees from Chugai Pharmaceutical Co., Ltd., grants and personal fees from Nippon Boehringer Ingelheim CO., Ltd, grants and personal fees from Ono Pharmaceutical Co., Ltd., grants from Quintiles Inc., grants from GlaxoSmithKline K.K, grants from OxOnc, grants and personal fees from Pfizer Inc., grants and personal fees from Kyowa Hakko Kirin Co., Ltd, grants and personal fees from Eli Lilly Japan K.K, personal fees from Yakult Honsha Co., Ltd., grants from Sumitomo Dainippon Pharma Co., Ltd., grants from Takeda Pharmaceutical Co., Ltd., grants and personal fees from Novartis Pharma K.K., grants and personal fees from DAIICHI SANKYO Co., Ltd., grants from Astellas Pharma Inc., grants from Eisai Co., Ltd., grants from Amgen Astellas BioPharma K.K., personal fees from Bristol-Myers Squibb Co., outside the submitted work;.
Afshin Dowlati, MD has nothing to disclose.
Camilla Laulund Christensen, PhD has nothing to disclose.
Keunchil Park, MD, PhD reports other from Astellas, grants and other from Astra Zeneca, other from Boehringer Ingelheim, other from Clovis, other from Eli Lilly, other from Hanmi, other from KHK, other from ONO, other from Novartis, other from Roche, outside the submitted work;.
Lawrence H. Einhorn, MD has nothing to disclose.
Martin J. Edelman, MD reports personal fees from Andarix Pharmaceuticals, during the conduct of the study; grants from BMS, grants from Oncomed, grants from Genentech, grants from Novartis, grants from Peregrine, grants from Heat Biologics, grants from Adaptimmune, grants from Endocyte, outside the submitted work;.
Giuseppe Giaccone, MD, PhD has nothing to disclose.
David E. Gerber, MD has nothing to disclose.
Ravi Salgia, MD, PhD has nothing to disclose.
Taofeek Owonikoko, MD, PhD has nothing to disclose.
Shakun Malik, MD has nothing to disclose.
Niki Karachaliou, MD has nothing to disclose.
David R. Gandara, MD has nothing to disclose.
Ben J. Slotman, MD, PhD reports grants and personal fees from Varian medical systems, grants and personal fees from BrainLAB AG, outside the submitted work;
Fiona Blackhall, MD, PhD has nothing to disclose.
Glenwood Goss, MD, FCPSA, FRCPC has nothing to disclose.
Roman Thomas, MD reports grants from German Cancer Aid, grants from BMBF e:Med, grants from Deutsche Forschungsgemeinschaft, during the conduct of the study; other from New Oncology AG, personal fees from Sanofi-Aventis, grants and personal fees from Merck, personal fees from Roche, personal fees from Lilly, personal fees from Boehringer Ingelheim, grants and personal fees from AstraZeneca, personal fees from Atlas-Bioloabs, personal fees from Daiichi-Sankyo, personal fees from MSD, personal fees from Puma, grants from EOS, outside the submitted work;.
Charles M. Rudin, MD, PhD reports consulting fees from Abbvie, Boehringer Ingelheim, Celgene, GSK, and Merck, and a grant from Biomarin, outside the submitted work.
Fred. R. Hirsch, MD, PhD is the Chief Executive Director of IASLC
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.