Abstract PO-059: Investigating gene expression profiles associated with clinical radiation resistance in KEAP1/NFE2L2 wildtype lung cancer
Authors: Binkley Michael S. , Jeon Young-Jun , Nesselbush Monica , Moding Everett J. , Nabet Barzin , Almanza Diego , Yoo Christopher , Kurtz David M. , Owen Susie Grant , Backhus Leah M. , Berry Mark F. , Shrager Joseph B. , Ramchandran Kavitha J. , Padda Sukhmani K. , Das Millie , Neal Joel W. , Wakelee Heather A. , Alizadeh Ash A. , Loo Billy W. , Diehn Maximilian 
Published: April 2021

Abstract
Background: We previously reported that approximately half of local recurrences (LR) after radiotherapy for localized NSCLC harbor mutations in KEAP1 or NFE2L2. Here we sought to explore factors associated with LR after radiotherapy in KEAP1/NFE2L2 wildtype NSCLC. Methods: We identified consecutive patients with stage IA1-IIIC NSCLC treated at our institution with chemoradiotherapy (CRT) or stereotactic ablative radiotherapy (SABR) from 2009-2018 and who had genotyping performed on tumor tissue with full coverage of common recurrent lung cancer driver genes including TP53, KRAS, KEAP1, and NFE2L2. We defined LR as tumor regrowth within the prescription dose radiotherapy volume. Our primary objective was to identify factors associated with LR in KEAP1/NFE2L2WT tumors. We performed RNA-seq on a subset of cases with available tissue using the SMARTer Stranded total RNA-seq Kit v2 (Takara Bio USA, Inc.). Statistical analysis was performed using R (version 3.6) with differential gene expression performed using ‘DESeq2’. All P-values were two-sided and considered significant at P&lt;0.05 with adjustment for multiple hypothesis testing when appropriate. Results: We identified 139 consecutive patients with localized NSCLC who received tumor genotyping and were treated with CRT for stage IIB-IIIC NSCLC (n=58) or SABR for stage I-IIB NSCLC (n=81). 26 (18.7%) of these patients harbored KEAP1/NFE2L2WT tumors. Clinical factors such as tumor volume (P=0.18), histology (P=0.87), and radiation dose (P=0.3) were not associated with LR in this subset. Similarly, somatic mutation analysis did not reveal association of any recurrent driver mutations with LR in these KEAP1/NFE2L2WT tumors, including in TP53 (n=19; P=0.73) or KRAS (n=9; P=0.98). Tissue was available for RNA-seq analysis of 38 KEAP1/NFE2L2WT tumors, of which the majority were adenocarcinomas (n=25, 65.8%) and approximately half each received CRT (n=20, 52.6%) and SABR (n=18, 47.4%). Gene set enrichment analysis revealed a trend for association of LR with expression of hypoxia genes (P=0.07, Q=0.28). Similarly, a previously reported 10-gene radiation sensitivity index (RSI) was not associated with LR (P=0.34). Individual gene analysis identified KRT14 as being significantly less expressed in cases with LR (adjusted P=2.2e-9). In a validation cohort of 24 stage I-IIA KEAP1/NFE2L2WT patients from the TCGA who were treated with radiotherapy, those who died had lower expression of KRT14 than those who did not (P=0.0003). Conclusions: In summary, we identify low expression of KRT14, a squamous cell carcinoma differentiation gene, as a potential biomarker for increased risk of LR after definitive radiotherapy of KEAP1/NFE2L2WT NSCLC. Validation in larger cohorts and biological characterization will be required to determine if this biomarker could be useful for guiding precision radiotherapy approaches.
Citation Format: Michael S. Binkley, Young-Jun Jeon, Monica Nesselbush, Everett J. Moding, Barzin Nabet, Diego Almanza, Christopher Yoo, David M. Kurtz, Susie Grant Owen, Leah M. Backhus, Mark F. Berry, Joseph B. Shrager, Kavitha J. Ramchandran, Sukhmani K. Padda, Millie Das, Joel W. Neal, Heather A. Wakelee, Ash A. Alizadeh, Billy W. Loo, Maximilian Diehn. Investigating gene expression profiles associated with clinical radiation resistance in KEAP1/NFE2L2 wildtype lung cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-059.