Abstract 3638: Quantitative assessment of tumor-infiltrating lymphocytes and immunotherapy outcome in metastatic melanoma
Authors: Wong Pok Fai , Smithy James W. , Blenman Kim R. , Kluger Harriet M. , Rimm David L. 
Published: August 2018

Background: Durable responses to immune checkpoint blockade against programmed cell death 1 (PD-1) are limited to a subset of metastatic melanoma patients, so there is a need for predictive biomarkers. Tumor-infiltrating lymphocytes (TILs) are the major cellular target of anti-PD-1 therapy, so we hypothesized that pretreatment TIL profiles would be associated with response.

Methods: Pretreatment whole-tissue sections from 94 melanoma patients treated with anti-PD-1 therapy (pembrolizumab, nivolumab, or ipilimumab plus nivolumab) from 2011-17 were collected from Yale Pathology archives, and stained to visualize nuclei (DAPI) and melanoma cells (S100 & HMB45) in combination with two multiplex immunofluorescence panels to: (1) perform TIL quantitation of helper T cells by CD4 (SP35, Spring), cytotoxic T cells by CD8 (C8/144B, Dako), and B cells by CD20 (L26, Dako); and (2) assess TIL activation by identifying T cells by CD3 (SP7, Novus), cytolytic activity by GZMB (4E6, Abcam), and proliferation by Ki67 (MIB-1, Dako). Cell phenotyping and counting were performed using inForm software (PerkinElmer) and protein expression was measured by the AQUA method of quantitative immunofluorescence (QIF). Response Evaluation Criteria In Solid Tumors (RECIST) 1.1 were used to classify best overall response as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD). Objective response rate (ORR; CR/PR), disease control rate (DCR; CR/PR/SD), and progression-free survival (PFS) were correlated with TIL parameters measured by both methodologies.

Results: Pretreatment lymphocytic infiltration, by cell counts or QIF, was significantly higher in CR/PR than in SD/PD, particularly for CD3 and CD8 (P &lt; 0.0001). Neither TIL activation (CD3 high, Ki67 and/or GZMB high) nor TIL dormancy (CD3 high, Ki67 and GZMB low) was significantly associated with outcome. Multivariable analyses revealed significant CD8 associations (HR &gt; 3) with PFS independent of age, sex, mutation, stage, treatment, and prior immune checkpoint blockade, which accounted for similar CD3 PFS associations as expected. The favorable predictive performance of CD8 cell count (and QIF) had an area under the receiver operating characteristic (ROC) curve of 0.75 for ORR and 0.78 for DCR, which reached 0.83 (ORR or DCR) for dual therapy (ipilimumab plus nivolumab). Interestingly, in contrast to previous hypothetical classifications, there were a number of responders (CR/PR) in the CD3 or CD8 low (immune desert) category.

Conclusions: Pretreatment lymphocytic infiltration, by cell counts or QIF, is associated with anti-PD-1 response in metastatic melanoma. Multiplex analysis of the tumor microenvironment has the potential to be used as a companion diagnostic test for precision immunotherapy.

Citation Format: Pok Fai Wong, James W. Smithy, Kim R. Blenman, Harriet M. Kluger, David L. Rimm. Quantitative assessment of tumor-infiltrating lymphocytes and immunotherapy outcome in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3638.