Small cell lung cancer (SCLC) is an aggressive malignancy characterized by rapid onset of platinum-resistance. However, mechanisms underlying platinum-resistance remain obscure due to scarcity of tissue samples from relapsed patients. We generated circulating tumor cell (CTC)-derived xenograft (CDX) models from SCLC patients that recapitulate patient tumor genomics and response to platinum chemotherapy. Little is known about whether intratumoral heterogeneity (ITH) exists in SCLC and how it may contribute to clinical outcomes and development of treatment resistance. To investigate this, we performed baseline single-cell RNAseq analyses of platinum-sensitive and -resistant CDX models, as well as longitudinal single-cell RNAseq analyses of CDX models and patient CTCs over the course of therapy. Within each CDX model, we observe not only increased ITH with resistance (variance-based metric, P=0.018), but distinct cellular populations with unique gene signatures associated with resistance (e.g. EMT, DNA damage repair, MYC activation, etc.). To confirm this relationship between ITH and resistance, platinum-sensitive CDX models were subjected to extended treatment with DNA damage response targeted therapies until relapse occurred. Single-cell RNAseq confirmed that, as predicted, untreated tumors were molecularly homogeneous, while relapse was associated with increased ITH and multiple, concurrent mechanisms of resistance, including TGF beta signaling and G2/M checkpoints. Unexpectedly, we found variations in the mechanisms of resistance within replicate treatment-relapsed mice, suggesting that resistance even to molecularly targeted therapies does not follow a predictable, reproducible pathway. For example, onset of resistance to a PARP inhibitor resulted in upregulation of NOTCH signaling in one tumor, but not others. Similarly, longitudinal single-cell profiling of CTCs directly from patient blood before, during, and after platinum-relapse confirmed increased ITH post-relapse accompanying unique mechanisms of resistance within specific cell populations (e.g., MYC activation, EMT, and TNFα signaling). We independently found ITH of protein expression (e.g., SLFN11, EZH2, EMT) in CTCs isolated from patient blood, signifying a method for measuring ITH clinically. These data suggest that treatment resistance in SCLC entails a fluid process of shifting expression profiles to generate an increasingly heterogeneous tumor with multiple, disparate mechanisms of resistance. Clinically, these findings imply that drug development efforts in this disease should focus on combination or maintenance therapies for treatment-naïve SCLC tumors to maximize depth of initial responses and delay the onset of resistance defined by ITH.

Citation Format: C. Allison Stewart, Carl M. Gay, Yuanxin Xi, Junya Fujimoto, Neda Kalhor, Patrice M. Hartsfield, Hai Tran, Luisa Fernandez, David Lu, Yipeng Wang, Ryan Dittamore, Jianjun Zhang, Stephen G. Swisher, Jack A. Roth, Trudy G. Oliver, John V. Heymach, Ignacio I. Wistuba, Bonnie S. Glisson, Paul Robson, Jing Wang, Lauren A. Byers. Single-cell analyses reveal increasing intratumoral heterogeneity as an essential component of treatment resistance in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2899.

©2019 American Association for Cancer Research.
2019
American Association for Cancer Research.