Chemokine Analysis in Patients with Metastatic Uveal Melanoma Suggests a Role for CCL21 Signaling in Combined Epigenetic Therapy and Checkpoint Immunotherapy

Vasu R Sah, Henrik Jespersen, Joakim Karlsson, Lisa M Nilsson, Mattias Bergqvist, Iva Johansson, Ana Carneiro, Hildur Helgadottir, Max Levin, Gustav Ullenhag, Anders Ståhlberg, Roger Olofsson Bagge, Jonas A Nilsson, Lars Ny, Vasu R Sah, Henrik Jespersen, Joakim Karlsson, Lisa M Nilsson, Mattias Bergqvist, Iva Johansson, Ana Carneiro, Hildur Helgadottir, Max Levin, Gustav Ullenhag, Anders Ståhlberg, Roger Olofsson Bagge, Jonas A Nilsson, Lars Ny

Abstract

Purpose: Patients with metastatic uveal melanoma have limited therapeutic options and high mortality rate so new treatment options are needed.

Patients and methods: We previously reported that patients treated with the PD-1 inhibitor pembrolizumab and the histone deacetylase inhibitor entinostat in the PEMDAC trial, experienced clinical benefits if their tumor originated from iris or was wildtype for BAP1 tumor suppressor gene. Here we present the 2-year follow-up of the patients in the PEMDAC trial and identify additional factors that correlate with response or survival.

Results: Durable responses were observed in 4 patients, with additional 8 patients exhibiting a stable disease. The median overall survival was 13.7 months. Grade 3 adverse events were reported in 62% of the patients, but they were all manageable. No fatal toxicity was observed. Activity of thymidine kinase 1 in plasma was higher in patients with stable disease or who progressed on treatment, compared with those with partial response. Chemokines and cytokines were analyzed in plasma. Three chemokines were significantly different when comparing patients with and without response. One of the factors, CCL21, was higher in the plasma of responding patients before treatment initiation but decreased in the same patients upon treatment. In tumors, CCL21 was expressed in areas resembling tertiary lymphoid structures (TLS). High plasma levels of CCL21 and presence of TLS-like regions in the tumor correlated with longer survival.

Conclusions: This study provides insight into durable responses in the PEMDAC trial, and describes dynamic changes of chemokines and cytokines in the blood of these patients.

Significance: The most significant finding from the 2-year follow-up study of the PEMDAC trial was that high CCL21 levels in blood was associated with response and survival. CCL21 was also expressed in TLS-like regions and presence of these regions was associated with longer survival. These analyses of soluble and tumor markers can inform on predictive biomarkers needing validation and become hypothesis generating for experimental research.

© 2023 The Authors; Published by the American Association for Cancer Research.

Figures

FIGURE 1
FIGURE 1
A, Kaplan–Meier analysis showing PFS of all patients. B, Kaplan–Meier analysis showing OS of all patients except one. C, Swimmer plot showing time on treatment, time to best response, and duration of response in all patients who received at least one dose of study drug (n  =  29) are shown. D, Kaplan–Meier analysis comparing OS between patients with LDH baseline greater or lower than the upper limit of normal (ULN).
FIGURE 2
FIGURE 2
A and B, Kaplan–Meier analysis showing PFS and OS of patients with a wildtype BAP1 status and UV-damaged uveal melanoma genome. PFS (C) and OS (D) analyses comparing patients with GNAQ- or GNA11-mutated uveal melanoma. E, Volcano plot showing differentially expressed genes between short term (n = 16) and long term (alive patients, n = 4) from bulk RNA-seq. Genes with FDR-adjusted P values <0.05 were considered statistically significant. F, Individual box plots showing relevant gene signatures implicating long-term (alive patients) survival. Statistical tests were carried out using DESeq2 and FDR-adjusted P values were denoted with *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIGURE 3
FIGURE 3
A, Heatmap showing TK1 activity value (DuA) from PEMDAC patient plasma samples, grouped within response groups. Each square is a timepoint for each patient and shows TK1 levels from pretreatment to end of study, until otherwise stated. Total plasma samples analyzed for TK1 = 287. B, Longitudinal TK1 activity for individual patients, as shown in A. C and D, Kaplan–Meier analysis showing PFS and OS, respectively for pretreatment TK1 values using a threshold of 150 DuA (median TK1 for all samples = 113). Patients with nonavailability of pretreatment samples were excluded from the analysis. E, IHC of TK1 showing nuclear/cytoplasmic magenta staining in patient biopsies 2-027, 3-012, and 3-010. F, Comparison between pretreatment TK1 values for short- and long-term survivors. G, Correlation between pretreatment TK1 (DuA) and circulating tumor DNA (counts/mL) matched patient samples (n = 21). All statistical tests were unpaired two-tailed t tests, assuming equal variance, with *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIGURE 4
FIGURE 4
A, Heatmap of 71 chemokines and cytokines analyzed among all patients and their respective timepoints. Total plasma samples analyzed = 287. Each square represents a timepoint for each patient and shows response group-based levels from pretreatment until end of study, unless otherwise stated. B, Heatmap showing differential pretreatment values between PD and partial responders. Boxed chemokines are significant (Padjusted < 0.05) after FDR correction. C, Individual chemokine or cytokine values (pg/mL) compared among different response groups. Only significantly different chemokines from B are included. D, Fold change difference between pretreatment values and week 9 after start of treatment are shown for PD patients. Arrows indicate chemokines that are significant (Padjusted < 0.05) after FDR correction between patients that survived longer and those that survived shorter. E, Individual chemokine or cytokine values (pg/mL) compared among different response groups, only significant differences from D are included. Statistical tests in bar charts were unpaired two-tailed t tests (C), assuming unequal variance, or paired t tests (D) with *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIGURE 5
FIGURE 5
Kaplan–Meier analysis showing. PFS (A) and OS (B), respectively using pretreatment plasma CCL21 (pg/mL) measurements based on their median values. C, Correlation between CCL21 (pg/mL) and CD3+CCR7+CD45RA+ (% counts) matched patient samples (n = 23). D, Flow cytometry–based comparison between short- and long-term survivors for CD3+CCR7+CD45RA+ % (T naïve and T stem cell memory) analysis using pretreatment blood samples (n = 24). Statistical test was unpaired two-tailed t tests, assuming equal variance. E, Kaplan–Meier analysis showing OS using median CD3+CCR7+CD45RA+ % values.
FIGURE 6
FIGURE 6
IHC magenta showing low mag of CD20 varied staining of TLS-like (borderline-tertiary lymphoid structures) followed by high magnification images of CD20, CD3, CCL21 within serial sections for Pt 2-027 (A), 3-010 (B). Also see Supplementary Fig. S5. Low and high magnification images of CD20+ TLS-like staining for Pt. 2-026 (C) and no TLS sample Pt. 4-017 (D). Kaplan–Meier analysis showing PFS (E) and OS (F), respectively dividing patient population into two groups based on IHC, no-TLS (n = 4) and TLS-like (n = 18). Also see Supplementary Fig. S5. G, Heatmap showing top 10% genes with highest log2 fold change, among all positively and negatively significantly regulated genes comparing no-TLS and TLS-like groups using bulk RNA-seq (log2 Reads Per Kilobase per Million mapped reads [RPKM] normalized values). Arrows represent relevant TLS-based gene signatures. Genes with FDR-adjusted P values <0.05 were considered statistically significant. H, Individual box plots showing CCL19, CCL21, CCR7, and CD79A changes in expression between the groups. Statistical tests were carried out using DESeq2 and FDR-adjusted P values were denoted with *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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