Real-world analysis of 2IR immune response score in urothelial carcinoma (UC).
Authors: Brown Jason Robert , Adeyelu Tolulope Tosin , Elliott Andrew , Basu Arnab , Bilen Mehmet Asim , Li Roger , Stewart Tyler F. , McKay Rana R. , Heath Elisabeth I. , Geynisman Daniel M. , Smith Norm , Barata Pedro C. 
Published: February 2025

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Background:
Immune checkpoint inhibition (ICI) is often used to treat UC, however many patients (pts) exhibit resistance. Using bulk RNA sequencing of pre-treatment tissue from ImVigor 210 and CheckMate 275 trials, an immune response score (2IR) was previously developed to predict ICI response by determining the comparative expression of genes involved in anti-tumorigenic adaptive immune response and pro-tumorigenic inflammation. Here, we evaluated the 2IR score as a prognostic or predictive biomarker in real-world pts with UC.
Methods:
Specimens from pts with UC (n = 6395) were profiled at Caris Life Sciences (Phoenix, AZ) using next generation sequencing (NGS) of DNA and RNA. 2IR was calculated by comparative RNA expression of 10 adaptive immune genes and 39 pro-tumorigenic genes. Tumors were classified as 2IR-Low (2IR ≤ -0.5), -Mid (-0.5 < 2IR < 0), and -High (2IR ≥ 0) as previously described. Spearman correlation analysis was utilized to compare 2IR with PD-L1 combined proportion score (CPS), tumor mutation burden (TMB), interferon score (IFN) and the tumor microenvironment (TME) cell fractions estimated using quanTIseq. Clinical outcomes included real-world overall survival (OS) from ICI start to last contact and time on treatment (ToT) with pembrolizumab, obtained via matched insurance claims data and calculated using Kaplan-Meier methods while Hazard ratio (HR) was calculated by Cox proportional model.
Results:
Of the 6395 UC samples, 9.5% (n = 611) were classified as 2IR-High, 42.7% (n=2730) 2IR-Mid, and 47.8% (n=3054) 2IR-Low. In pts with Upper Tract UC primary (UTUC, n = 1104), 2IR was comparatively lower than in pts with bladder cancer primary (BC, n = 4923) (median: -0.53 vs. -0.47, p < 0.001). 2IR shows a positive correlation with TMB (r = 0.23; p < 0.001), IFN score (r = 0.21; p < 0.001) and insignificant correlation with PD-L1 CPS (r = 0.09; p <0.001). 2IR-High positively correlated with mutations in
TP53
,
RB1
, and
ARID1A
(all p < 0.0001), amplification of
ERBB2
(p < 0.0001), and microsatellite-instability high status (p < 0.0001), but not with
FGFR3
mutations or fusions. 2IR positively correlated with immune cell fractions, such as CD4
+
T (r = 0.09), CD8
+
T (r = 0.22), and dendritic cell (r = 0.18), but negatively correlated with B cells (r = -0.06), M1 macrophages (r = -0.25), M2 macrophages (r = -0.08) and neutrophils (r = -0.12). Pts treated with 2IR-High tumors had longer ToT on pembrolizumab (HR = 0.65 CI 0.55 - 0.77, p <0.001) and OS from ICI start (HR = 0.51, CI 0.41 – 0.62, p < 0.001) compared to pts with 2IR-Low UC. This association held for both primary BC and UTUC.
Conclusions:
In a real-world cohort, we validate the 2IR score and suggest that it is prognostic for OS and predictive for pembrolizumab ToT, consistent with a positive correlation with known predictors of ICI response. Prospective studies are needed to further validate this biomarker for use in clinical practice, especially given the evolving UC treatment landscape.