128PMultiplex chromogenic Immunohistochemistry (IHC) for spatial analysis of checkpoint-positive tumour infiltrating lymphocytes (TILs)

Abstract Background Spatial localization of TILs has been shown to correlate with immunotherapy response. Chromogenic IHC (cIHC) enables assessment of lineage (eg, CD8) or immune checkpoint expression (eg, T-cell immunoglobulin and mucin-domain–containing-3 [TIM-3]). Codetection of lineage and checkpoint molecules may help to better inform responders to checkpoint inhibitors, but colocalization is challenging by cIHC. Fluorescence (fIHC) allows colocalization but not spatial assessment and is not an approved CDx platform. Consequently, CDxs have been limited to single-plex cIHC assays. We developed a multiplex cIHC platform that enables image analysis colocalization and spatial assessment of TILs. Methods Image analysis of cIHC via HALO software was performed to detect colocalized CD8 and TIM-3. We compared % TIM-3+/CD8+ by cIHC to TIL multiparameter flow cytometry (MFC) on 10 procured renal cell carcinoma (RCC) samples. Pathologist annotation and a trained random forest machine-learning tissue classifier were used to determine spatial localization (tumor vs peritumoral vs nontumor) of CD8+ and TIM-3+/CD8+ TIL subsets. We also performed a validated fIHC assay, including CD8 and TIM-3, on 5 additional procured RCC samples. Results High concordance (r = 0.92) was observed between % TIM3+/CD8+ TILs measured by cIHC and MFC. Image analysis showed that within the CD8+ population, median % TIM-3+/CD8+ TILs were primarily intratumoral (18.0%), with fewer in peritumoral (9.4%) and nontumoral (6.4%) regions (intratumoral vs peritumoral, and intratumoral vs nontumor, both P = 0.015). TIM-3/CD8 coexpression detection appeared similar between cIHC and fIHC. Conclusions We developed a multiplex cIHC method that enables simultaneous quantitation of TIL subpopulations, checkpoint expression, and spatial analysis. cIHC, fIHC and MFC performed similarly in coexpression detection, but only multiplex cIHC enabled spatial localization. Specifications of this assay show promise for development as a CDx. Additional data will demonstrate the capability to substitute TIM-3 with other markers, suggesting an assay template for other lineage/target combinations. Editorial acknowledgement Amrita Dervan, MSc, and Jay Rathi, MA, of Spark Medica Inc, funded by Bristol-Myers Squibb. Legal entity responsible for the study Bristol-Myers Squibb. Funding Bristol-Myers Squibb. Disclosure S. Ely: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. G. Lee: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. L. Menard: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. J. Yan: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. P. Fischer: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. B. Kakrecha: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. D. Locke: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. P. Patah: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. K. Urbanska: Full / Part-time employment: Bristol-Myers Squibb.