Abstract 3473: Use of DNA methylation from tumor and plasma to identify four major small cell lung cancer subtypes with distinct biology and therapeutic vulnerabilities
Authors: Heeke Simon , Gay Carl M. , Estecio Marcos R. , Stewart Allison , Tran Hai , Zhang Bingnan , Tang Ximing , Raso Gabriela , Concannon Kyle , De Sousa Luana Guimaraes , Lewis Whitney E. , Nilsson Monique , Xi Yuanxin , Diao Lixia , Wang Qi , Zhang Jianjun , Wang Jing , Wistuba Ignacio I. , Byers Lauren A. , Heymach John V. 
Published: June 2022

Abstract
Small cell lung cancer (SCLC) is a highly aggressive cancer with limited treatment options and generally poor prognosis. Treatment of SCLC has not considerably changed over the last decades with therapeutic options focusing on unselected populations. Although in the past SCLC was thought to be a relatively homogenous malignancy, recent reports from our group and others identified four major distinct subgroups of SCLC, each with different therapeutic vulnerabilities. Three of the subtypes are defined by the expression of a specific transcription factor, ASCL1 (SCLC-A), NEUROD1 (SCLC-N) and POU2F3 (SCLC-P) while the fourth subtype is defined by an inflamed phenotype (SCLC-I). While our initial subtyping of SCLC is based on a gene expression signature comprised of ~1300 genes, which makes routine implementation challenging, we hypothesized that DNA methylation as a proxy to gene expression might be a more suitable approach for biomarker development in SCLC. We assembled a cohort of 105 SCLC formalin-fixed paraffin embedded (FFPE) samples (82/105 Stage &gt; IIIb) and performed matched RNA-Sequencing (RNAseq) and methylation profiling using reduced-representation bisulfite sequencing (RRBS). To validate our findings and expand our analysis across different sample types, we profiled a panel of 59 fully characterized SCLC cell lines as well as 68 patient-derived xenograft models. We found that methylation levels differ markedly between the four subtypes, with the SCLC-N presenting with a hypermethylated phenotype and the SCLC-P with a hypomethylated phenotype across the genome, highlighting the profound differences in the underlying epigenetic regulation among the SCLC subtypes and supporting DNA methylation analysis as a potential readout for identifying SCLC subtypes. Furthermore, in order to subtype the clinical SCLC samples, we developed a predictive model using an extreme gradient boost model using RNA expression and DNA methylation, respectively, to allow the classification with 94.5% accuracy in the tissue testing cohort. Using a cohort of matched plasma samples, we further demonstrated that the DNA methylation differences were indeed preserved in cell-free DNA (cfDNA) allowing subtype classification with an accuracy of 87.5%. These data indicate that DNA methylation can be used for reliable subtyping of SCLC in tissue and in liquid biopsy samples. In summary, using a large cohort of predominantly extensive stage SCLC clinical samples, we were able to identify profound differences in DNA methylation that can be exploited as a novel biomarker for the classification of SCLC into four distinct subtypes with both tissue biopsy and non-invasive using plasma. Considering the previously shown therapeutic vulnerabilities of the four subtypes, these findings will enable the rapid initiation of personalized clinical trials in SCLC.
Citation Format: Simon Heeke, Carl M. Gay, Marcos R. Estecio, Allison Stewart, Hai Tran, Bingnan Zhang, Ximing Tang, Gabriela Raso, Kyle Concannon, Luana Guimaraes De Sousa, Whitney E. Lewis, Monique Nilsson, Yuanxin Xi, Lixia Diao, Qi Wang, Jianjun Zhang, Jing Wang, Ignacio I. Wistuba, Lauren A. Byers, John V. Heymach. Use of DNA methylation from tumor and plasma to identify four major small cell lung cancer subtypes with distinct biology and therapeutic vulnerabilities [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 3473.