Extraction-Free Comprehensive Transcriptome Sequencing of Carcinoma In-situ is Highly Feasible and Insightful in Non-Muscle Invasive Bladder Cancer

Abstract

Most carcinoma in-situ (CIS) molecular investigations to date have studied CIS in the context of concurrent papillary (Pap) tumors (conPap). A critical gap remains in our understanding of unique CIS biology. We investigated the feasibility of obtaining high-quality transcriptome data from CIS specimens utilizing the novel extraction-free EdgeSeq Precision Immuno-Oncology Panel (PIP) platform. Formalin-fixed paraffin embedded CIS, CIS with conPap, and pure Pap tumors were identified. Tumor cells were macro-dissected from two unstained 5-micron thick slides and analyzed on the PIP panel comprised of 1,410 immunomodulatory genes. Principle components analyses (PCA) were performed to identify distinct cohorts. The Bioconductor DEseq2 package was used to identify differential gene expression between CIS +/- conPap and pure Pap tumors. Functional and pathway analyses were performed using Ingenuity Pathway Analysis software. Secondary analyses assessed any confounding effects of relevant patient clinical features. 135 specimens were identified including: 51 CIS (32 CIS, 19 CIS + concurrent high grade (HG) Ta-T2), 55 HG Pap (21 HGTa, 21 HGT1, 12 HGT2), and 29 low grade (LG) Pap (28 LGTa, 1 LGT1) tumors. 85 patients (63.4%) were BCG-naïve. PIP data from 117 (87.3%) specimens, including 43 of 51 (84.3%) CIS specimens passed all QC measures and were included in analyses. PCA distinguished CIS from pure Pap specimens. Compared to HG Pap, CIS +/- conPap specimens demonstrated increased expression of EMT pathway genes including SNAI1/2, ZEB1, CDH1, GATA3, TWIST2, EPCAM (p<0.001, FDR<0.005). In evaluable patients who received subsequent BCG therapy (n=26), increased expression of antigen presentation and B-cell function genes (CCL19, CCL22, LTB, CD52, CCL17, FCMR, MS4A1) were associated with a recurrence-free survival of 12 months or more (p < 0.001, FDR<0.1). Comprehensive transcriptome profiling of archived CIS specimens is highly feasible including pure CIS samples. CIS tumors and tumors from durable responders to BCG therapy are characterized by unique gene expression signatures. Validation of these findings in prospective clinical trials is ongoing.