Molecular characterization of advanced muscle invasive bladder cancer (MIBC): Comparison of low-input targeted RNA sequencing (RNAseq) strategies.

Abstract

564 Background: 5-year survival for advanced MIBC is poor (~5%), but newer targeted agents (e.g., PD-1/PD-L1 or FGFR) have improved survival over traditional chemotherapy. To better select patients suited for targeted therapies, molecular characterization is more important than ever. The following study compares RNAseq using two low-RNA-input methods (SureSelect hybridization-capture and AmpliSeq PCR amplification, using 200 ng and 10 ng extracted RNA, respectively), as part of a retrospective study of advanced MIBC patients treated with anti-PD-1/PD-L1 therapy (Mayhew et al, 2019 ASCO). RNAseq results, including correlation between methods and molecular subtype agreement are presented herein. Methods: RNA extracts from 24 representative FFPE MIBC tumor samples were analyzed using SureSelect XT RNA (Agilent) and AmpliSeq transcriptome (Illumina) by NovaSeq6000 paired end sequencing. Samples were split between 4 molecular subtypes using the GeneCentric 60-gene MIBC nearest centroid subtyper (n = 6/subtype; luminal, luminal infiltrated, basal, basal infiltrated) (Mayhew et al, 2019 ASCO). Pearson correlation analysis was performed for all genes, as well as the 60 suptyper genes. Expression subtype calls were compared using RNAseq data obtained from both sequencing methods. Results: Of 58,000 genes queried (GrCH38.v22), 27,671 (55%) were found using both methods and 13,992 (28%) were not found using either method. 15,575 unique genes were found using SureSelect and 1,149 unique genes were found using AmpliSeq. The two methods were well correlated: median of 0.87 (0.73-0.89) overall and 0.80 (0.54-0.86) for the 60 subtyper genes. 22 of the 24 samples (92%) had congruent subtype calls using the 60-gene MIBC subtyper; one luminal-infiltrated called basal and one basal called basal-infiltrated. Conclusions: This study demonstrated comparable RNAseq analysis using two low-input FFPE-compatible methods. Using AmpliSeq, MIBC tumors were able to be molecularly characterized, including subtype analysis, with only 1/20th of the extracted RNA needed for SureSelect. Further validation of AmpliSeq methodology in MIBC and other tumor types is warranted.