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Erin L Stewart



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    MA27 - Novel Drugs and PDX Models (ID 931)

    • Event: WCLC 2018
    • Type: Mini Oral Abstract Session
    • Track: Targeted Therapy
    • Presentations: 2
    • Now Available
    • Moderators:
    • Coordinates: 9/26/2018, 13:30 - 15:00, Room 206 BD
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      MA27.01 - Establishment of PDX From Tumors Characterized by EGFR Mutations or ALK Fusion Genes from Resections, Biopsies and Pleural Fluids (Now Available) (ID 12144)

      13:30 - 13:35  |  Author(s): Erin L Stewart

      • Abstract
      • Presentation
      • Slides

      Background

      Patient-derived xenograft (PDX) models allow for cancer tissue expansion, providing an effective method to evaluate tumor biology and mechanisms of response or resistance. Our study aims to establish models in patients enriched for lung adenocarcinoma (LUAD) with EGFR mutations or ALK fusion genes which respond initially to oral targeted therapy, but typically develop resistance and disease relapse within 2 years. The PDXs will be evaluated for their potential to model therapy outcomes, to determine resistance mechanisms and to evaluate novel therapy strategies to overcome resistance.

      a9ded1e5ce5d75814730bb4caaf49419 Method

      From August 2015 to January 2018, we collected 109 samples from patients with EGFR- or ALK-driven LUAD and from never-smoker LUAD patients with unknown mutation status. Five samples with low tissue viability (i.e. necrotic) or very low tumor content (<100 malignant cells) were excluded. Adequate samples were implanted into the subcutaneous tissue of NOD-SCID mice. At this time, 16 samples have reached the study endpoint (tumor growth ≥1.5cm3) and 60 showed no tumor-growth following implantation (median follow-up: 8m). Results are currently pending for 18 models.

      4c3880bb027f159e801041b1021e88e8 Result

      Samples were collected from surgical resections (31, 36%), CT-guided biopsies (12, 14%), EBUS (19, 22%) and pleural fluid effusions (24, 28%). Most patients were female (51/86, 59%), never smokers (62/85, 73%), and had stage III or IV cancer (55/79, 70%). Mutations in EGFR and ALK were found in 55/81 (68%) and 12/84 (14%) primary cancers, respectively. Early-passage xenograft engraftment (XG) was observed in only 16 (19%) PDXs, including 9/55 (16%) EGFR- and 1/12 (8%) ALK-mutant cancers. The phenotype and molecular changes (EGFR and ALK) were consistent within the PDX model and its corresponding patient sample. Samples collected from surgical-resection specimens showed a trend towards higher engraftment rates (p=0.084). Conversely, the presence of EGFR or ALK mutations showed a trend towards non-engraftment (noXG, p=0.075). Patient smoking status and tumor stage did not influence engraftment rate. To identify reasons for no tumor-growth, we conducted histological analysis in the subcutaneous fat-pads (nodes in the implant sites) of 28 noXG mice. Interestingly, we identified small non-palpable foci of carcinoma in 8 animals (4 EGFR+ and 2 ALK+).

      8eea62084ca7e541d918e823422bd82e Conclusion

      Environmental or molecular factors may impair engraftment rates of EGFR+ and ALK+ LUAD samples in PDX models. Nevertheless, these models recapitulate the primary disease and could be useful for population-based drug-screening studies.

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      MA27.03 - Multi-Omic Characterization of TKI-Treated Drug-Tolerant Cell Population in an EGFR-Mutated NSCLC Primary-Derived Xenograft (Now Available) (ID 13370)

      13:40 - 13:45  |  Presenting Author(s): Erin L Stewart

      • Abstract
      • Presentation
      • Slides

      Background

      Sixty to eighty percent of advanced stage lung adenocarcinoma patients with epidermal growth factor receptor (EGFR) mutated tumors respond to first generation EGFR tyrosine kinase inhibitors (TKIs). However, cure is not yet achievable with any EGFR TKI monotherapy, as patients eventually progress due to acquired resistance. In vitro evidence suggests that minor populations of epigenetically modified drug tolerant cells (DTCs) may be important for tumor cells surviving TKI. We hypothesize that molecularly characterizing DTCs in vivo and comparing them to the untreated tumor in a patient-derived xenograft (PDX) model may delineate mechanisms of tolerance that closely mimic those occurring in patients.

      a9ded1e5ce5d75814730bb4caaf49419 Method

      DTCs were produced via chronic exposure to erlotinib in a lung adenocarcinoma PDX harbouring an exon 19 deletion. Histological, genomic, transcriptomic (including single-cell RNA-seq), and epigenetic characterizations were performed on DTCs and compared to untreated baseline (BL) tumors.

      4c3880bb027f159e801041b1021e88e8 Result

      Compared to BL, DTCs exhibit decreased levels of proliferation (Ki67 by immunohistochemistry (IHC) and increased expression of senescence/quiescence (p21) and anti-apoptosis (BCL-XL) immunohistochemistry (IHC) markers, while maintaining EGFR pathway signaling (pEGFR, pAKT, pERK, pS6 IHC). Whole exome-sequencing provides evidence that DTCs likely do not represent mutationally distinct subclones from the bulk tumor. Instead, DTCs exhibit a number of differentially expressed genes compared to BL tumors that are involved in cell cycle arrest, senescence/quiescence, differentiation, vesicles, and inflammation. Genes with epigenetic differences (chromatin openness and/or promoter methylation) are involved in similar cellular processes. A minor (<2%) subpopulation of transcriptomically-defined DTC-like cells in the BL tumors are very similar to the DTCs, supporting the hypothesis that DTCs may exist prior to treatment. A number of transcription regulators are found to have differential gene expression and epigenetic regulation as well as DNA-binding motifs found in regions of chromatin uniquely open in DTCs or baseline tumors. These transcription regulators are involved in cell maintenance, proliferation, and differentiation, and may play key roles in promoting DTC phenotype.

      8eea62084ca7e541d918e823422bd82e Conclusion

      In this specific EGFR mutant PDX model sensitive to first generation TKIs, DTC-like cells are found in the BL untreated tumors, and its resultant phenotype after exposure to TKI appears to be involved in cell cycle, differentiation, senescence/quiescence, proliferation and maintenance. PDX models may provide insights into therapeutic strategies to target DTCs, and further improve the survival of EGFR-mutated NSCLC patients.

      6f8b794f3246b0c1e1780bb4d4d5dc53

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    MTE01 - Preclinical Models of Lung Cancer (Ticketed Session) (ID 811)

    • Event: WCLC 2018
    • Type: Meet the Expert Session
    • Track: Biology
    • Presentations: 1
    • Now Available
    • Moderators:
    • Coordinates: 9/24/2018, 07:00 - 08:00, Room 206 F
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      MTE01.02 - Lung Patient Derived Xenograft and Organoid (Now Available) (ID 11547)

      07:30 - 08:00  |  Author(s): Erin L Stewart

      • Abstract
      • Presentation
      • Slides

      Abstract

      Establishment of preclinical lung cancer models that closely match patient tumor biology is imperative for developing therapeutic strategies with the most translational relevance. Non small cell lung cancer (NSCLC) cell lines grown under 2D conditions or as cell line-derived xenografts (CDXs) are the most widely used models. They have been complemented with murine models engineered to develop lung cancer after introduction of specific genetic alterations (GEMMs). Lung cancer cell lines are readily amenable to mechanistic studies and economical high-throughput drug screening. However, for many NSCLC cell lines, the spectrum of mutations and copy number alterations have drifted considerably relative to patient tumors 1. This finding, in conjunction with long-term adaption to heterologous in vitro growth conditions, raise concerns about the extent to which cell line biology and potential drug responses may have deviated from clinical tumors. GEMMs are powerful tools for studying specific oncogenic mechanisms in isolation in vivo, but these models lack the intratumoral heterogeneity of patient tumors, which is thought to play a major role in the development of drug resistance. Furthermore, ideally, GEMMs should be constructed using the appropriate cell of origin context, which is challenging, as there are differences in the compositions of human and murine airways, and the cellular origins for most forms of lung cancer have not been established.

      NSCLC patient-derived xenografts (PDXs) overcome some of the limitations of these other models. They show much less genetic drift than cell lines, and their mRNA expression and the phospho-tyrosyl proteome more closely match patient tumors 1, 2. We have established a large collection of NSCLC PDXs from surgically resected tumors and endobronchial ultrasound-guided (EBUS) and CT-guided biopsies. Tumor specimens were initially implanted in the subcutaneous flanks of NSG mice (NOD SCID gamma, non-obese diabetic severe combined immunodeficiency, gamma). The PDX tumors have been viably cryopreserved and can be serially passaged in NOD SCID mice. Most of our collection comprises the major histologic subtypes of NSCLC [52 adenocarcinomas (LUAD) and 62 squamous cell carcinomas (LUSC)]. They, along with the primary patient tumors, are being molecularly profiled at multiple levels so that they can be optimally used for personalized medicine studies and novel integrated approaches to understand NSCLC pathogenesis, prognosis, and treatment. These levels include copy number variations, exome mutations, DNA methylation, mRNA and miRNA expression, and proteomics. In general, the PDX models recapitulate the mutation spectrum, copy number variations, and gene expression of matched patient histologies. They also recapitulate sensitivity and resistance to known targeted therapeutics (e.g. EGFR inhibitors), and thus, can be used to dissect mechanisms underlying differential drug responses. Such studies are ongoing, including investigation of potentially new biomarker-targeted therapeutic combinations. We have also found that not all patient tumor fragments engraft successfully, and that successful engraftment correlates with poor prognosis of the patient 3. We are using this relationship to discover a new molecular fingerprint to predict clinical outcome, as well as understand the bases that distinguish less and more aggressive tumor behavior.

      In parallel, we have developed methods to grow organoids from primary patient tumors and PDX models in 3D culture using Matrigel (PDO and XDO, respectively). For LUAD, both the PDO and XDO success rate of establishing bona fide organoid models is ~20%. Our stringent criteria include a minimum capacity of 10 passages and a split ratio of at least 1:3. LUSC has been more difficult to establish as organoid models, with a success rate of 17%, and only from PDXs, so far. Using these methods, we have established 4 models of each histology, which we have confirmed form tumors when transplanted into mice. Molecular profiling indicates that the organoids maintain the same mutation spectrum and copy number variations of their parental tumor tissue. These models offer distinct advantages over PDXs and cell lines. As compared to standard 2D cultures, they recapitulate the appropriate tissue histology, and thus, possibly clinically relevant growth control mechanisms, even while growing ex vivo. This notion is further supported by the ex vivo conditions supporting gene expression patterns, which allows the organoids to be segregated into their respective tumor histologies when using signatures derived from patient or PDX material. Given the low cost, rapid growth rates, and ease of in vitro manipulation, these models are ideally suited for rapid discovery and testing of new therapeutic strategies that can be matched to specific patient molecular profiles.

      In summary, generation of molecularly profiled PDX and organoid models offer great opportunity for translational and personalized medicine in NSCLC.

      1. Gao, H. et al. High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response. Nat. Med. 21, 1318-1325 (2015).

      2. Wang, D. et al. Molecular heterogeneity of non-small cell lung carcinoma patient-derived xenografts closely reflect their primary tumors. Int. J. Cancer 140, 662-673 (2017).

      3. John, T. et al. The ability to form primary tumor xenografts is predictive of increased risk of disease recurrence in early-stage non-small cell lung cancer. Clin. Cancer Res. 17, 134-141 (2011).

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