A CD274, PDCD1LG2, CD8A, and IRF1 multiplex in a closed system RT-qPCR panel and immunotherapy outcome in metastatic melanoma

Background: In melanoma, there is no companion diagnostic test to predict response to programmed cell death 1 (PD-1) axis immune checkpoint inhibitor therapy (ICI). In the adjuvant setting, only 1 in 5 patients may benefit from ICI, so biomarker is needed to select those that may or may not benefit. Candidate techniques for the assessment of predictive markers include immunohistochemistry (IHC), multiplex fluorescence, genome sequencing, and RNA expression profiles. Here we test a new 4-gene research use only (RUO)* prototype mRNA expression profile on the GeneXpert closed system using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) for association with clinical benefit after treatment with ICI in metastatic melanoma patients. Methods: Pretreatment formalin-fixed paraffin-embedded (FFPE) tissue sections from melanoma patients treated with anti-PD-1 therapy (pembrolizumab, nivolumab, or ipilimumab plus nivolumab) between 2011-17 were selected from the Yale Pathology archives. FFPE sections were macrodissected to enrich for tumor for quantitative assessment of CD274 (PD-L1), PDCD1LG2 (PD-L2), CD8A, and IRF1 by RT-qPCR multiplex mRNA panel. Multiplex panel transcript levels were correlated with clinical benefit (CR/PR/SD); disease outcomes (progression-free survival, PFS and overall survival, OS); and protein levels assessed by quantitative immunofluorescence (QIF). Median values for each marker were used to define high versus low mRNA or protein expression groups. This study was approved by Yale Human Investigation IRB protocol ID 9505008219. Results: Inter-transcript regression was observed among all four markers with R2 ranging from 0.20 to 0.51. Transcript levels were significantly higher in CR/PR/SD than in PD for CD8A (p = 0.0001) and IRF1 (p = 0.0019). PFS was strongly associated with high CD274 (p = 0.0046), PDCD1LG2 (p = 0.0039), CD8A (p = 0.0002), and IRF1 (p = 0.0030) mRNA expression. Similar associations were observed for OS with high CD274 (p = 0.0004), CD8A (p = 0.0030), and IRF1 (p = 0.0096) mRNA expression. Multivariate analyses revealed significant associations with OS independent of age, sex, stage, mutation, treatment, and prior ICI for CD274 (HR = 0.30), CD8A (HR = 0.40) and IRF1 mRNA (HR = 0.36). Similar PFS association with CD8A (HR = 0.39) and IRF1 (HR = 0.48) parameters were observed by multivariate analyses. Nonlinear exponential relationship was observed between transcript and protein levels for CD8A (R2 = 0.66) and IRF1 (R2 = 0.40). Conclusions: Although tested in only a single melanoma cohort, CD274, CD8A and IRF1 mRNA levels show promising associations with outcome. The turnaround time of the test (2h) and easy standardization of the platform makes this an attractive approach for further study in the search for predictive biomarkers for ICI. *for Research Use Only - not approved or reviewed by any regulatory body Citation Format: Swati Gupta, Leena McCann, Yvonne G.Y. Chan, Edwin W. Lai, Pok Fai Wong, James W. Smithy, Jodi Weidler, Brian Rhees, Michael Bates, Harriet M. Kluger, David L. Rimm. A CD274, PDCD1LG2, CD8A, and IRF1 multiplex in a closed system RT-qPCR panel and immunotherapy outcome in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3142.