Multiplex Quantitative Analysis of Tumor-Infiltrating Lymphocytes and Immunotherapy Outcome in Metastatic Melanoma

Abstract

Purpose: Because durable response to programmed cell death 1 (PD-1) inhibition is limited to a subset of melanoma patients, new predictive biomarkers could have clinical utility. We hypothesize that pretreatment tumor-infiltrating lymphocyte (TIL) profiles could be associated with response.

Experimental design: Pretreatment whole tissue sections from 94 melanoma patients treated with anti-PD-1 therapy were profiled by multiplex immunofluorescence to perform TIL quantification (CD4, CD8, CD20) and assess TIL activation (CD3, GZMB, Ki67). Two independent image analysis technologies were used: inForm (PerkinElmer) to determine cell counts, and AQUA to measure protein by quantitative immunofluorescence (QIF). TIL parameters by both methodologies were correlated with objective response or disease control rate (ORR/DCR) by RECIST 1.1 and survival outcome.

Results: Pretreatment lymphocytic infiltration, by cell counts or QIF, was significantly higher in complete or partial response than in stable or progressive disease, particularly for CD8 (P < 0.0001). Neither TIL activation nor dormancy was associated with outcome. CD8 associations with progression-free survival (HR > 3) were independently significant in multivariable analyses and accounted for similar CD3 associations in anti-PD-1-treated patients. CD8 was not associated with melanoma prognosis in the absence of immunotherapy. Predictive performance of CD8 cell count (and QIF) had an area under the ROC curve above 0.75 (ORR/DCR), which reached 0.83 for ipilimumab plus nivolumab.

Conclusions: Pretreatment lymphocytic infiltration is associated with anti-PD-1 response in metastatic melanoma. Quantitative TIL analysis has potential for application in digital precision immuno-oncology as an "indicative" companion diagnostic.

Figure 1.. RECIST categories of melanoma patients treated with anti-PD-1 therapy and TIL parameters by cell counts.

Representative multispectral immunofluorescence images of TIL quantification (CD4, CD8, CD20) (A) and the three major states of the tumor immune microenvironment (B) in melanoma: immune desert (CD3 low), TIL dormancy (CD3 high, Ki67 and GZMB low), and TIL activation (CD3 high, Ki67 and/or GZMB high) (magnification × 200; scale bar = 100 μm). TIL quantification (C) and TIL activation (D) parameters by cell counts per RECIST categories of best overall response. Data are presented as mean with standard deviation (error bars). Abbreviations: CR, complete response; HI, high; LO, low; PD, progressive disease; PR, partial response; RECIST, Response Evaluation Criteria in Solid Tumors; SD, stable disease; TIL, tumor-infiltrating lymphocyte.

Figure 2.. TIL activation parameters by cell counts and survival of melanoma patients treated with anti-PD-1 therapy.

Kaplan–Meier analysis of progression-free survival according to TIL activation (CD3, GZMB, Ki67) (A) parameters by cell counts, and the three states of the tumor immune microenvironment (B): immune desert (CD3 low), TIL dormancy (CD3 high, Ki67 and GZMB low), and TIL activation (CD3 high, Ki67 and/or GZMB high). The immune infiltration (CD3 high) category was stratified according to GZMB alone, Ki67 alone, or both GZMB and Ki67. Low and high statuses were objectively defined using thresholds determined by Joinpoint regression (see Methods). Abbreviations: ACT, TIL activation; DES, immune desert; DOR, TIL dormancy; HI, high; LO, low; NS, not significant; TIL, tumor-infiltrating lymphocyte.

Figure 3.. TIL quantification parameters by cell counts and survival of melanoma patients treated with anti-PD-1 therapy and untreated melanoma patients.

Kaplan–Meier analysis of progression-free survival of anti-PD-1 treated melanoma patients (A) and disease-specific survival of untreated melanoma patients (B) according to TIL quantification (CD4, CD8, CD20) parameters by cell counts. Low and high statuses were objectively defined using thresholds determined by Joinpoint regression (see Methods). Abbreviations: HI, high; LO, low; TIL, tumor-infiltrating lymphocyte.

Figure 4.. Receiver operating characteristic (ROC) curve analysis of CD8 by cell counts for the prediction of anti-PD-1 objective response rate or disease control rate in melanoma.

ROC curves constructed from logistic regression models for the prediction of anti-PD-1 response in terms of ORR (A) and DCR (B) for the total cohort, monotherapy (pembrolizumab or nivolumab), or dual therapy (ipilimumab plus nivolumab). AUC of 0.50 represents performance of random chance (line of identity, dotted); 1.00 represents perfect predictive performance. P values indicate probability that the AUC is significantly different from 0.50. Abbreviations: AUC, area under curve; CI, confidence interval; DCR, disease control rate; ORR, objective response rate.