Molecular Determinants of Sotorasib Clinical Efficacy in KRAS G12C–Mutated Non–Small-Cell Lung Cancer

This study describes transcriptional subtypes of non-squamous non–small cell lung cancer (NSCLC). Baseline tissue samples (fresh/archival) were analyzed using a whole-transcriptome RNA assay (Tempus RS.v2; Tempus Labs, Inc., Chicago, IL, USA). Objectives included expression characterization of the KRAS G12C subject population with respect to genetic mutation (ATM, SMARCA4, KEAP1, STK11) and transcriptional signatures: KPCL (Skoulidis, Cancer Discov (2015) 5 (8): 860–877) & NRF2 activation (Singh, Clin Cancer Res (2021) 27 (3): 877–888). This data set consists of 234 NSCLC gene expression profiles of baseline tissue samples (fresh/archival), from patients with KRAS G12C‒mutated locally advanced or metastatic NSCLC after progression on prior therapies, who were randomized to the single-arm phase 2 CodeBreaK 100 study and the global randomized phase 3 CodeBreaK 200 study. Tumor specimens were prepared following standard pathology practices. Total nucleic acid (TNA) was extracted from tissue specimens using a Chemagic 360 or similar instrumentation, and the recovered TNA was quantified and qualified, and may have been concentrated if necessary. Extracted TNA that was intended for processing by Tempus|RS was treated with Invitrogen TURBO DNase to degrade DNA. The minimum amount of input total RNA required to perform the test was 50 ng. RNA was fragmented using heat and magnesium, with variable parameters, to yield similar sized fragments from RNA inputs with different starting size distributions. Strand-specific library preparation was performed using the KAPA RNA HyperPrep Kit for Illumina with KAPA single indexed 6 base pair adapters. The libraries were amplified with high fidelity, low-bias PCR using primers complementary to adapter sequences. After library preparation and amplification, targets were captured by hybridization, clean-up of captured targets was performed, and unbound fragments were washed away. The amplified target-captured libraries were sequenced with a 2x76 read length to an average of 50 million total reads on an Illumina HiSeq4000 System using patterned flowcells. Transcript level pseudo-alignment and quantification to the Ensembl GRCh37 Release 97 (July 2019) reference was performed using Kallisto (version 0.44). Raw counts or transcripts per million (TPM) were calculated for 180,253 transcripts in the Ensembl reference. 134,160 transcripts were covered by the Tempus|RS panel.