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D. Carney

Moderator of

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    MS 07 - SCLC Biology & Models (ID 25)

    • Event: WCLC 2015
    • Type: Mini Symposium
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 6
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      Introduction (ID 2072)

      • Abstract

      Abstract not provided

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      MS07.01 - PDX Models (ID 1872)

      C.M. Rudin

      • Abstract
      • Presentation
      • Slides

      Abstract not provided

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      MS07.02 - GEM Models (p53/Rb) (ID 1873)

      K. Sutherland

      • Abstract
      • Presentation
      • Slides

      Abstract:
      Small cell lung cancer (SCLC) is an aggressive neuroendocrine (NE) tumour associated with poor 5-year survival rates. Given the difficulties associated with obtaining human material, genetically engineered mouse models (GEMMs) for SCLC have emerged as powerful pre-clinical tools for translational research. Inactivation of the tumour suppressor genes TRP53 and RB1 is almost universally found in human SCLC. Based on this observation, the Berns Laboratory generated a mouse model of sporadic SCLC whereby p53 and Rb1 loss was restricted to lung epithelial cells by intra-tracheal instillation of an Adeno-Cre virus (Cre expression is under the control of a ubiquitous CMV promoter). These mice develop NE lung tumours with striking morphological and genomic similarities to SCLC observed in human patients[1]. This model allows us to address questions that would not be possible using patient samples or cancer cell lines alone. In my presentation, I will provide an overview on the GEMMs for SCLC currently available. I will also touch upon the emergence of new gene editing technologies, such as CRISPR-Cas9, and how these techniques can be used to further manipulate current models to address clinically relevant questions. Lung cancers exhibit a high level of intra-tumoral heterogeneity. The histopathology of individual tumour subtypes, suggests that these tumours have distinct cells-of-origin, but this has not been formally shown. I will present the work we carried out to address the cellular origins of lung cancer, with a focus on the research we performed using the GEMM of SCLC (p53[f/f];Rb1[f/f]). Briefly, we generated a series of recombinant adenoviruses that target Cre-recombinase expression selectively in Club (Ad5-CC10-Cre), alveolar type 2 (Ad5-SPC-Cre) and neuroendocrine (Ad5-CGRP-Cre) cells[2]. To address the cellular origins of SCLC, we infected p53[f/f];Rb1[f/f] mice with our cell type-restricted Adeno-Cre viruses, listed above. Results from these studies show that inactivation of p53 and Rb1 can efficiently transform neuroendocrine (CGRP-positive) and to a lesser extent, alveolar type 2 (SPC-positive) cells leading to SCLC. In contrast, CC10-expressing cells were largely resistant to transformation. The results clearly indicate that neuroendocrine cells serve as the predominant cell-of-origin of SCLC. Interestingly genome-sequencing studies have revealed genetic aberrations that overlap with squamous cell carcinomas in a subset of SCLCs. Does this reflect a common cellular origin? I will present some recent data we have generated to address this question. References 1. Meuwissen R, Linn SC, Linnoila RI, Zevenhoven J, Mooi WJ and Berns A. Induction of small cell lung cancer by somatic inactivation of both Trp53 and Rb1 in a conditional mouse model. Cancer Cell 2003 vol. 4(3) pp. 181-189. 2. Sutherland KD, Proost N, Brouns I, Adriaesen D, Song J-Y and Berns A. Cell of origin in small cell lung cancer: inactivation of Trp53 and Rb1 in distinct cell types of the adult mouse lung. Cancer Cell 2011 vol. 19(6) pp. 754-764.

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      MS07.03 - Pre-Clinical Mouse Models of SCLC to Identify and Validate New Therapeutic Targets (ID 1874)

      J. Lim, N. Jahchan, D. Yang, J. George, M. Peifer, R.K. Thomas, J. Sage

      • Abstract
      • Presentation
      • Slides

      Abstract:
      Small cell lung cancer (SCLC) is a neuroendocrine subtype of lung cancer characterized by a fast growth rate, extensive dissemination, and rapid resistance to chemotherapy. Survival rates are dismal and have not significantly improved in the past few decades. The group of Roman Thomas and Martin Peifer sequenced the genomes of over 100 human SCLC, which demonstrates universal inactivation of p53 and RB and identified inactivating mutations in NOTCH family genes in ~25% of tumors. Accordingly, we found that activation of Notch signaling in a pre-clinical SCLC mouse model dramatically reduces the number of tumors and extends the survival of the mutant mice. In addition to suppressing proliferation, active Notch inhibits neuroendocrine gene expression in SCLC cells. Thus, Notch plays a key tumor suppressive role in SCLC and strategies to re-activate Notch in SCLC tumors may be beneficial to patients (George, Lim, et al., in press). At the histological level, SCLC tumor cells are often viewed as homogeneous. These studies and previous studies (e.g. Calbo et al., Cancer Cell, 2011 – Berns lab) have identified several levels of intra-tumor heterogeneity in SCLC, which may contribute significantly to SCLC aggressive nature and resistance to therapy. We will also discuss the existence and the role of several subpopulations of SCLC tumor cells involved in the long-term propagation of this cancer type, the rapid acquisition of chemoresistance, and metastasis. A better understanding of the molecular underpinnings of these cellular heterogeneity may help identify novel therapeutic targets in SCLC.

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      MS07.04 - From GEMs to ROCKs - An Assessment of In Vitro Models for the Study of SCLC (ID 1875)

      A.F. Gazdar

      • Abstract
      • Presentation

      Abstract:
      Because SCLC tumors are seldom resected, in vitro models to study this “recalcitrant disease” are of crucial importance. The major strengths and limitations of the three basic preclinical model systems are summarized in Table 1. Table 1: Strengths and Limitations of Preclinical model systems for the study of SCLC

      Preclinical Model Strengths Limitations
      Tumor Cell lines (TCLs) Spheroidal growth, cytological appearances and neuroendocrine (NE) cell properties. May represent oligoclonal selection. Lacks stroma and vasculature.
      Patient-derived xenografts (PDXs) Histology and gene expression profile of tumors closely resemble human counterpart. Stroma and vasculature are of host mouse origin. Lacks intact immune system. Metastatic spread limited. Possible contamination with murine xenotropic virus.
      Genetically Engineered Mouse Models (GEMMs) Reproduces pathology of NE carcinomas and similar metastatic pattern. Only model for studying multistage pathogenesis Long latent time. Precise histology mixture variable.
      Tumor Cell Lines SCLC lines have been established since the early 1970s. A large series of cell lines was established by Drs. Gazdar, Desmond Carney and John Minna.[1]Most lines retained the cytological and NE cell features of SCLC tumors. We have confirmed that vast majority of the NCI series of lines have retained these features even after 4 decades in culture. Some of the lines, especially those established after prior therapy and which had amplification of a MYC family gene, had atypical morphology and lacked some of the NE cell program. These were termed variant SCLC cell lines.[2]They remain the major resource for most of the biology studies performed in SCLC.[3] Constitutional sources of DNA are available for some of the lines. A major shortcoming is lack of cell lines established from the putative precursor cell, the NE cells of the respiratory epithelium. While most TCLs grow as two dimensional adherent monolayers, SCLC cultures naturally grow as three dimensional floating aggregates or spheroids. Several recent reports have suggested that three dimensional in vitro growth more closely resembles the natural growth characteristics of patient tumors, and may be more representative of drug response.[4] While they are an estimated 150 SCLC TCLs established worldwide, recent reports have been scarce. Two recent developments offered innovative new approaches to the establishment of SCLC lines. The finding that the circulating tumor cell burden in SCLC cases were extremely high and could be used to establish PDXs[5]was promising and also suggested that the circulating cells could be used to establish new SCLC TCLs. Recently a method for the propagation of epithelial cells of non-malignant and malignant origin, termed “Conditionally Reprogrammed Cells” (CRC) was described. CRC cells have properties of epithelial stem cells.[6]This method was widely utilized to generate many new putative lung cancer TCLs, mainly of NSCLC origin. Our extensive characterization (led by Boning Gao and John Minna) of CRC cells from NSCLC specimens indicated robust growth of epithelial cells apparently free of fibroblast contamination. However, characterization of the cells indicated that they mostly had properties of stem cells derived from non-malignant cells, and were diploid and lacked mutations present in the corresponding tumors. These results suggest, at least for lung cancer specimens, that the CRC method preferentially grows the non malignant epithelial stem cell component present in all lung cancer resections. Patient Derived Xenografts (PDXs) PDX tumors are generated by direct transfer of human tumor fragments or cell isolates from patient tumors to immune-deficient mice (or other rodent species). At least during early serial passage, PDXs retain the genetic and morphological characteristics of the original human tumor, including histological features, gene expression profiles, copy number variations and chromosomal stability of PDX tumors.[7] Thus, PDXs have been proposed as an advanced preclinical tool for therapy testing in a number of tumor types including lung cancers.[8] Most PDXs are inoculated subcutaneously. Orthotopic models for SCLC may increase metastatic potential and relevance for chemotherapy evaluation.[9] Intracranial heterotransplantation of SCLC into the brain provides a model to study intracranial and leptomeningeal meatastases.[10] The mouse genome contains over 500,000 copies of integrated strains of mouse leukemia virus virus. Some strains are xenotropic and grow efficiently in human cells. Serial transplantation of PDXs, especially SCLC, is associated with a high frequency of xenotropic virus contamination,[11]which poses potential health risks and may influence genetic analyses. Genetically engineered mouse models (GEMMs) Berns developed the double knockout model (lacking p53 and Rb1 that closely recapitulated the histology and metastatic pattern of SCLC, but had a relatively long latent period.[12]Several triple knockout variants of the basic model have been developed, specifically to reduce the long latent period. However, these variations often have more complex histologies, reflecting the spectrum of high grade NE carcinoma of the lung. The resultant histological phenotypes were influenced by multiple factors. The lengthy latent time permitted observations of the preneoplastic and premalignant stages of SCLC development, which are seldom observed in human tumors because of the explosive growth of SCLC once it becomes invasive. The long latent period is caused by the development of secondary genetic changes required for tumor formation such as alterations of the PTEN and NFIB genes.[13]A recent review[12]concluded that GEMM models studied are representative for the entire spectrum of human high-grade NE carcinomas and are also useful for the study of multistage pathogenesis and the metastatic properties of these tumors. Summary The major In vitro models for SCLC each have their individual strengths and weaknesses. Each has to be carefully evaluated for its suitability for the proposed experimental approach. Despite their limitations, In vitro models remain the single most important source of knowledge about the non-clinical aspects of SCLC and will likely remain so into the foreseeable future. 1. Phelps RM, Johnson BE, Ihde DC, et al. NCI-Navy Medical Oncology Branch cell line data base. J Cell Biochem 1996;Suppl. 24:32-91. 2. Gazdar AF, Carney DN, Nau MM, et al. Characterization of variant subclasses of cell lines derived from small cell lung cancer having distinctive biochemical, morphological, and growth properties. Cancer Res 1985;45:2924-2930. 3. Gazdar AF, Girard L, Lockwood WW, et al. Lung cancer cell lines as tools for biomedical discovery and research. Journal of the National Cancer Institute 2010;102:1310-1321. 4. Breslin S, O'Driscoll L. Three-dimensional cell culture: the missing link in drug discovery. Drug Discov Today 2013;18:240-249. 5. Hodgkinson CL, Morrow CJ, Li Y, et al. Tumorigenicity and genetic profiling of circulating tumor cells in small-cell lung cancer. Nat Med 2014;20:897-903. 6. Liu X, Ory V, Chapman S, et al. ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells. The American journal of pathology 2012;180:599-607. 7. Rosfjord E, Lucas J, Li G, et al. Advances in patient-derived tumor xenografts: from target identification to predicting clinical response rates in oncology. Biochem Pharmacol 2014;91:135-143. 8. Moro M, Bertolini G, Tortoreto M, et al. Patient-derived xenografts of non small cell lung cancer: resurgence of an old model for investigation of modern concepts of tailored therapy and cancer stem cells. J Biomed Biotechnol 2012;2012:568567. 9. Isobe T, Onn A, Morgensztern D, et al. Evaluation of novel orthotopic nude mouse models for human small-cell lung cancer. J Thorac Oncol 2013;8:140-146. 10. Gazdar AF, Carney DN, Sims HL, et al. Heterotransplantation of small-cell carcinoma of the lung into nude mice: comparison of intracranial and subcutaneous routes. Int J Cancer 1981;28:777-783. 11. Zhang YA, Maitra A, Hsieh JT, et al. Frequent detection of infectious xenotropic murine leukemia virus (XMLV) in human cultures established from mouse xenografts. Cancer Biol Ther 2011;12:617-628. 12. Gazdar AF, Savage TK, Johnson JE, et al. The comparative pathology of genetically engineered mouse models for neuroendocrine carcinomas of the lung. J Thorac Oncol 2015;10:553-564. 13. McFadden DG, Papagiannakopoulos T, Taylor-Weiner A, et al. Genetic and clonal dissection of murine small cell lung carcinoma progression by genome sequencing. Cell 2014;156:1298-1311.

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      MS07.05 - Circulating Tumour Cells (ID 1876)

      F. Blackhall, C. Dive

      • Abstract
      • Presentation
      • Slides

      Abstract:
      Circulating Tumour Cells Dr Fiona Blackhall and Professor Caroline Dive Progress in understanding the molecular biology of small cell lung cancer has undoubtedly been hampered by lack of tissue resources suitable for comprehensive systems biology analysis. Tissue quantities sufficient for molecular analysis are more commonly from surgical resections and open biopsies from patients with very limited stage disease and therefore not representative of the majority of SCLC patients. Serial biopsies are even rarer to obtain. As an alternative to tumour tissue, circulating tumour cells (CTCs) are highly prevalent and abundant in patients with SCLC. These surrogate biomarkers, increasingly referred to as ‘virtual’ or ‘liquid’ biopsies, may be more relevant to understanding the biology of this disease that is hallmarked by early and widespread haematogenous dissemination. In our own series (Hou et al. JCO 2012) blood samples from 97 treatment naive patients, 31 with limited stage (LS) and 66 with extensive stage (ES), were assessed for CTCs using the EpCam-based immunomagnetic detection method, CellSearch. CTCs were detectable in the majority (85%) of patients and abundant. The mean ± standard deviation for CTC number(#) in a 7.5ml blood sample was 1,589 ± 5,565 and median CTC# was 24 (range 0 – 44, 896). CTC# was significantly associated (higher) with ES, lactate dehydrogenase, presence of liver metastases and number of sites of metastases. In multivariate analysis, adjusting for these clinical associations, pretreatment CTC# and change in CTC# after one cycle of chemotherapy were independent prognostic factors. A statistically derived cut off of 50 CTCs demonstrated most significant discrimination in survival estimation. The overall survival was 5.4 months for patients with ≥ 50 CTCs/7.5 mL of blood compared with 11.5 months (P < .0001) for patients with less than 50 CTCs/7.5 mL of blood before chemotherapy (hazard ratio = 2.45; 95% CI, 1.39 to 4.30; P =0 .002). In addition to prognostic information CTCs are pharmacodynamic and amenable to biomarker assay development (protein expression, omic profiling, FISH etc). CTCs ex vivo are also tumourigenic. We have established a series of CTC derived xenografts (CDX) in immune compromised (IC) mice (Hodgkinson et al. Nat Med 2014). Of 6 initial patients whose CTCs were implanted in IC mice, 4 gave rise to tumours in less than 5 months. Implantation and CDX tumour formation was associated with higher CTC# (>400 CTCs / 7.5mls of blood). The immunohistochemical characteristics of the CDX tumours were consistent with SCLC morphology and neuroendocrine marker expression. Whole genome sequencing demonstrated that the tumours had mutations (e.g. TP53 and RB1) and copy number variation (e.g. loss of 3p and 13q) commonly observed in SCLC. Furthermore, the same genetic abnormalities as the CDX were present in single cells CTCs isolated from the corresponding patient. On exposure of the CDX to platinum and etoposide chemotherapy a remarkable correlation was observed for the tumour responses compared to the patients’ tumour responses and survival. For example the most chemoresistant CDX was established from CTCs of a patient who survived for only 0.9 months and who had chemorefractory disease, whereas the most chemosensitive CDX was obtained from a patient who responded to platinum/etoposide chemotherapy and who survived for 9.7 months. A CDX of intermediate chemosensitivity was derived from a patient who survived for 3.5 months. Once the CDX tumours are established they can be harvested for passage, frozen and resurrected. Ongoing work aims to establish serial CDX models from patients who have progressed after initial treatment for study of biology, particularly that of acquired chemoresistance, and for preclinical testing of novel therapeutics in treatment naïve and previously treated SCLC. There is also possibility to incorporate serial CTC analysis and CDX model generation into clinical trials as ‘co-clinical trials’ with interrogation of pharmacodynamic and putative predictive biomarkers in addition to discovering mechanisms of resistance to novel therapeutics. CTC analysis and CDX model generation are technically challenging and resource intensive, but essential tools to further develop if we are to end the impasse on a targeted therapy breakthrough for this disease. References Hou JM, Krebs MG, Lancashire L, Sloane R, Backen A, Swain RK, Priest LJ, Greystoke A, Zhou C, Morris K, Ward T, Blackhall FH, Dive C. Clinical significance and molecular characteristics of circulating tumor cells and circulating tumor microemboli in patients with small-cell lung cancer. J Clin Oncol. 2012 Feb 10;30(5):525-32. Hodgkinson CL, Morrow CJ, Li Y, Metcalf RL, Rothwell DG, Trapani F, Polanski R, Burt DJ, Simpson KL, Morris K, Pepper SD, Nonaka D, Greystoke A, Kelly P, Bola B, Krebs MG, Antonello J, Ayub M, Faulkner S, Priest L, Carter L, Tate C, Miller CJ, Blackhall F, Brady G, Dive C. Tumorigenicity and genetic profiling of circulating tumor cells in small-cell lung cancer. Nat Med. 2014 Aug;20(8):897-903.

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    YIS - Young Investigator Session incl. Q & A with Longstanding IASLC Members (ID 238)

    • Event: WCLC 2015
    • Type: Young Investigator Session
    • Track: Other
    • Presentations: 1
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      YIS.07 - Q & A with Longstanding IASLC Members (ID 3517)

      D. Carney

      • Abstract
      • Slides

      Abstract not provided

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