While PD-1/L1 axis-targeted therapies have shown promising clinical responses, their use in combination therapies is associated with both benefits and safety concerns. Response rates for single-agent anti-PD-1 therapies are significantly higher in biomarker positive patients, therefore there is a need to utilize predictive diagnostics to enhance benefit-risk profiles and guide treatment decisions. To address this, we developed a novel quantitative multiplexed immunohistochemistry assay that provides objective quantitation of PD-L1 positive cells, but more importantly assesses interactions with immune cells (PD-1+ or CD8+) in formalin-fixed paraffin embedded (FFPE) clinical specimens. The clinical validity of this assay was verified in a small series of melanoma patients treated with anti-PD1 targeted therapies/agents.

FFPE melanoma tissues from patients who received anti-PD-1 therapy were fluorescently stained with a combination of anti-PD-1, PD-L1, and S100 antibodies plus DAPI or a combination of anti-CD8, PD-L1, and S100 antibodies plus DAPI. Each slide was then imaged on Vectra platform and the frequencies of biomarker positive cells (PD-L1, PD-1, and CD8) and their interaction scores were objectively evaluated using proprietary Automated Quantitative Analysis (AQUA®) algorithms. Analytical sensitivity, precision, and accuracy were established using standardized PD-L1 and PD-1 tissue control arrays composed of cell lines and lymphoid organs, while range of biomarker expression was verified on archived melanoma clinical specimens (n = 30), including samples taken from melanoma patients prior to anti-PD-1 therapies (n = 21).

Frequencies of PD-L1 positive cells could be accurately quantified within 1% to 100% range in predefined control cell line mixtures. PD-L1 and PD-1 measurements were highly reproducible (R2 = 0.98 and 0.97, respectively). A broad range of PD-L1 and PD-1 expression and interaction scores were observed in archival clinical specimens (n = 53). In a cohort of 21 advanced melanoma patients treated with nivolumab (n = 5) or pembrolizumab (n = 16), the PD-1/L1 interaction score was found to reliably distinguish responders from non-responders (p = 0.03) while PD-L1 alone (p = 0.15) or CD-8 alone (p = 0.23) did not. Additionally, patients exhibiting higher PD-1/L1 interaction scores had superior response rates (78% vs. 17%, p = 0.03). Responders experienced significantly longer median progression-free survival (177 vs. 85 days, p = 0.014), and fewer deaths (22% vs 58%) compared with patients having lower PD-1/L1 interaction scores.

In terms of diagnostic utility, the PD-1/L1 multiplex test showed superior predictive power (78% Positive Predictive Value, 83% Negative Predictive Value) compared with PD-L1 expression alone. Additional studies are underway to fully establish diagnostic utility and aid in treatment guidance.

Citation Format: Jennifer Bordeaux, Douglas Johnson, Jeffrey Sosman, Ju Young Kim, Christine Vaupel, Bashar Dabbas, Justin Cates, Jeff Hall, Jelveh Lameh, Shabnam Tangri, Naveen Dakappagari. Novel quantitative multiplexed PD-1/PD-L1 immunohistochemistry test provides superior prediction of treatment response in melanoma patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 853.