Abstract 3375: Molecular subtyping of circulating tumor cells in patients with small cell lung cancer
Authors: Kopparapu Prasad R. , Duplessis Melinda , Lo Edward , Yan Yingjun , Iams Wade T. , Nordberg Josh , Ramirez Arturo , George Tad , Lovly Christine 
Published: June 2022

Abstract
Background: Recently, a new molecular classification of small cell lung cancer (SCLC), defined by expression of four key transcription regulators - ASCL1, NEUROD1, YAP1 and POU2F3 - has been proposed. These molecular subtypes may confer differential biology and therapeutic vulnerabilities, however, studies to date have been constrained by the paucity of available longitudinal tumor biopsy samples obtained from patients with SCLC. Herein, we utilized circulating tumor cells (CTCs) to characterize subtype marker heterogeneity at the time of diagnosis and evaluate the dynamic nature of marker expression during treatment in patients with SCLC.
Methods: Informed consent was obtained from patients with SCLC using an IRB approved protocol (IRB#030763). We have collected blood samples from 32 patients, including treatment naïve samples from the entire cohort as well as on-treatment samples (median of 4 collections per patient). We utilized the RareCyte rare-cell analysis platform to isolate and quantify CTCs, which were defined as CK/EpCAM positive and CD45 negative. Each sample was also evaluated for expression of neuroendocrine markers (ASCL1, NEUROD1) and non-neuroendocrine markers (YAP1, POU2F3). Each set of neuroendocrine and non-neuroendocrine markers were co-stained along with the CTC markers. Mean fluorescence intensity of each marker was extracted using a python program.
Results: To date, we have analyzed 22/32 (69%) treatment naïve samples, including n=6 patients with early stage disease and n=16 patients with advanced/metastatic disease. We detected CTCs from 16/22 (73%) patients, with quantities ranging from 1 - 3406 (median = 66) per 7.5ml of blood. CTCs were positive for the neuroendocrine markers ASCL1 in 16/16 (100%) and NEUROD1 in 10/16 (62%); all NEUROD1 positive CTCs were also positive for ASCL1. The non-neuroendocrine markers YAP1 and POU2F3 were detected in 6/16 (37%) and 11/16 (69%) samples, respectively, including YAP1/POU2F3 double positives in 6/16 (37%). Analysis of on-treatment samples is ongoing.
Conclusion: We have developed a protocol for monitoring expression of subtype markers on CTCs isolated from patients with SCLC. We found CTCs expressing both neuroendocrine and non-neuroendocrine markers at the time of diagnosis. Ongoing work will attempt to delineate the dynamic nature of subtype marker expression during therapy, to correlate with clinical outcomes. These studies have the potential to offer critical insights regarding the heterogeneity and evolution of SCLC, a tumor type in which novel disease insights and innovative treatment strategies are urgently needed to improve patient survival.
Citation Format: Prasad R. Kopparapu, Melinda Duplessis, Edward Lo, Yingjun Yan, Wade T. Iams, Josh Nordberg, Arturo Ramirez, Tad George, Christine Lovly. Molecular subtyping of circulating tumor cells in patients with small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3375.