Single Cell RNA-Sequencing Highlights Underlying Cellular And Transcriptomic Differences In Non-Muscle Invasive Bladder Cancers Of Identical Stage And Grade
Benjamin T. Ristau, MD1, Dylan Baker, PhD2, Paul Robson, PhD2.
1University of Connecticut Health Center, Farmington, CT, USA, 2The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA.
BACKGROUND: Clinical decisions in patients with non-muscle invasive bladder cancer (NMIBC) are based primarily on tumor grade and stage. Despite creation of prognostic risk scores using clinical parameters, recurrence (15-61% for Ta and T1) and progression (up to 54% for CIS) remain variable. We hypothesize that underlying molecular and cellular heterogeneity within stage exists and may explain variation in clinical outcome. METHODS: : Bladder tumors were obtained from patients via transurethral resection of bladder tumor during routine clinical care. Tissues were obtained fresh or cryopreserved. Samples were dissociated, live cells sorted by FACS, and single cell suspensions used for single cell RNA sequencing (scRNA-seq) library preparation utilizing 10x Chromium Gene Expression Kit. Sequencing was done on Illumina Hiseq and Novaseq platforms and subsequent analysis performed with Scanpy (Python) and consensus non-negative matrix factorization (NMF). Novel molecular signatures derived from scRNA-seq data were mapped to progression data from the UROMOL project, a European multi-center study on NMIBC.
RESULTS: : scRNA-seq was performed on tumors from 16 patients with NMIBC. We observed differences in cellular and transcriptomic composition between tumors that would traditionally have been characterized by the same stage and grade (Figure 1). Molecular signatures derived from NMF identified a higher degree of urothelial heterogeneity than previously appreciated in prior studies using bulk tissue. Novel molecular signatures identified by scRNA-seq correlate with progression free survival when mapped onto bulk RNA seq datasets (r = -0.718, p<0.001). CONCLUSIONS:NMIBC tumors of the same stage and grade harbor underlying differences in molecular and cellular composition. Molecular signatures derived from scRNA-seq data correlate with risk of clinical progression. Underlying heterogeneity within NMIBC stage may explain observed variability in clinical outcomes and holds promise for the development personalized treatment approaches.
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