Immune Gene Expression Profiles in High-Grade Urothelial Carcinoma of the Bladder: A NanoString Study - Beyond the Abstract

Recent advancements in immunology have led to immunotherapy taking center stage in the treatment of urothelial carcinoma (UC). In particular, immune checkpoint inhibitors (ICIs), such as programmed cell death protein (PD-1) and programmed death-ligand 1 (PD-L1) inhibitors, have emerged as therapeutic approaches for advanced and metastatic UC. However, only a subset of patients benefit from these inhibitors. To identify patients most likely to benefit from anti-PD-1/PD-L1 inhibitors, assessment of tumor and/or immune cell PD-L1 expression by immunohistochemistry (IHC) is typically used as a complementary diagnostic, with better overall response rates noted in PD-L1-positive/high disease. However, we now know that the response to anti-PD1/PD-L1 therapy cannot be solely predicted by PD-L1 IHC status. Evidence suggests that response to these inhibitors may be associated with the type and degree of immune infiltration in the tumor microenvironment. In this regard, targeted gene expression panels have the ability to comprehensively assess the tumor microenvironment and immune milieu of UC. As a first step to understanding discrepancies in therapeutic response to ICIs, we sought to evaluate the transcriptome of PD-L1 IHC high and PD-L1 IHC low high-grade muscle-invasive urothelial carcinomas of the bladder (HGUC).

In this pilot study, we first determined the degree of concordance between PD-L1 IHC assay (VENTANA SP263) and messenger RNA levels profiled using the NanoString nCounter® Human V.1.1 PanCancer Panel. We found that detection of PD-L1 RNA in 40 HGUC cystectomy FFPE clinical samples using NanoString-based profiling strongly correlated with PD-L1 protein expression (Pearson's correlation coefficient r=0.720, p<0.001). The sensitivity, specificity, positive and negative predictive values of PD-L1 RNA for protein expression were 85%, 96%, 92%, and 93%, respectively. Our findings are in line with several other studies reporting a strong correlation between PD-L1 RNA and protein expression.

Secondly, our NanoString immune transcriptome analysis of HGUC identified a 16 immune gene expression signature associated with PD-L1 protein expression. Pathway analysis determined enrichment of these 16 differentially expressed genes in the production of interleukin-10 (e.g. CD46, NOD2) previously shown to induce PD-L1 expression, lymphocyte chemotaxis (e.g. CXCL14, CXCL16), leucocyte activation (eg, CD46, NOD2), suppression of T cell activation (e.g. FCER1G, PDCD1LG2) and regulation of ERK1/ERK2 cascade (e.g. CXCL16, CCL3) known to be involved in PD-L1 upregulation. The findings further indicate a cross-talk between PD-L1 in the adaptive immunosuppressive pathway and the innate immune response genes C1QA, CD46 (complement system), and NOD2 (NOD2 signaling). Lastly, we noted an association between expression of PD-L1 and the proinflammatory cytokine OSM which has been shown to stimulate several signaling pathways (Janus kinases/signal transducer and activator of transcription proteins, MAPK, and PI3 kinase), some of which are involved in the upregulation of PD-L1 expression. Our findings also imply cross-talk between PD-L1 expression and downregulation of SMAD3 (a key component of TGFβ signaling) in inhibition of antitumor immunity. This is consistent with previous reports that SMAD3-deficient mice have an increase in constitutively activated T cells and expression of cytokines such as interleukin-2, interleukin-13, interleukin-15, and IFNγ.

There are limitations to this study, which include its retrospective design, data from a single institution, inclusion of only muscle-invasive HGUC, and small cohort size. Nonetheless, these initial findings are encouraging and warrant further exploration in large ICI treated cohorts to assess how these gene expression profiles correlate with therapeutic outcomes.

Written by: Ekaterina Olkhov-Mitsel, PhD, Laboratory Medicine and Molecular Diagnostics, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada., and Michelle R. Downes, MD, Pathology, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada

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