Article Figures & Data
Figures
- Figure 1.
Melphalan and CTLA-4 blockade in a model of melanoma. A, B16 cells were treated in vitro with melphalan or vehicle control at 50 μmol/L. After 24 hours, the expression of MHC class I, MHC class II, and PD-L1 was assessed by flow cytometry. This is a representative figure from the experiment performed 3 times. Fold change was calculated based upon mean fluorescence intensity change from untreated to treated. B, Melphalan synergizes with CTLA-4 blockade. Mice with palpable allografted B16 tumors were treated with single intratumoral dose of melphalan, alone or followed by CTLA-4 blockade, 100 μg IP, every 3 days for 4 doses. Control mice received intratumoral injection of vehicle control and IP injection of isotype antibody. Shown are pooled data of 3 separate experiments (3–5 mice per treatment group per experiment, 4 experiments). C and D, Combination therapy enhances the inflammatory environment of the tumor. C, CD4 effector and CD8+ cells per gram of tumor in B16 tumors from the four treatment groups, P < 0.05 for combination versus control treatment. D, CD4 and FoxP3 expression on cells from tumors from the four treatment groups. Experiments were performed 3 times; shown is representation of one experiment. E, IFNγR−/− mice had an improved median survival for melphalan plus CTLA-4 blockade over control- or melphalan-treated mice [combination (n = 9), median survival 22 days; control (n = 5), median survival 10 days; melphalan (n = 6), 12 days, P < 0.05). However, no combination mice were cured of tumors, suggesting the importance of IFNγ on the host.
- Figure 2.
Clinical benefit from combination limb infusion and CTLA-4 blockade. A, Schema of the phase II clinical trial. B, Swimmer plot of all patients, showing responses in the melphalan-treated limb. C, PFS for patients, including the 2 patients who died prior to the evaluation point. D, Example of durable response both in and outside of the limb of the infusion. This patient had an unknown primary melanoma and presented with multiple subcutaneous nodules of the limb, with groin adenopathy, biopsy-proven lung metastasis, and thoracic adenopathy. Three years after limb infusion and systemic ipilimumab, the patient remains free of disease (bottom plot).
- Figure 3.
Nanostring analysis of tumors after ILI and after combination limb infusion and ipilimumab. Gene expression was compared from available pretreatment biopsies (n = 11) to post-ILI (n = 4) and post-ipilimumab (IPI; n = 14) biopsies. Tumor biopsies were taken just prior to ILI, after ILI (7–15 days), and 3 weeks after the last dose of ipilimumab. The gene expression pattern after ILI favored upregulation of costimulatory ligands, and innate immune function, whereas after combination treatment, there was increased expression of cytotoxic function, particularly granzymes, IFNγ, perforin, and ICOS.
- Figure 4.
Analysis of cytokines and immune cell phenotypes in the phase II trial. A, Increase in serum cytokines after combination therapy. The cytokines indicated were quantitated by Meso Scale Discovery assay in serum taken pretreatment (pre), after ILI, and after the initial ipilimumab (IPI) treatment. *, P < 0.05; ***, P < 0.001 compared with pretreatment or as indicated by brackets. Note the increase in cytotoxic cytokines after combination treatment consistent with nanostring data. B–D, Changes in T cells in blood and tumor after ILI and ipilimumab treatments, assessed by flow cytometry. B, Increase in a percentage of CD4+ Ki67+ and CD8+ Ki67+ T cells, of total CD45+ cells, in peripheral blood mononuclear cells after ILI and after the first dose of ipilimumab. C, Increase in a percentage of CD4+ ICOS+ cells after ILI and after the each of the first (of 4) ipilimumab doses. D, Analysis of tumor samples demonstrating an increase in the ratio of CD8+ T cells to CD4+FOXP3+ T cells (left) and the percentage of CD4+ICOS+ T cells (right). The bars shown mean values and SD. E and F, Immune infiltration in tumors after ILI and ipilimumab treatment. E, Representative multiplex IHC at 20x showing CD4 (green), CD8 (red), CD68 (yellow), and PD-L1 (white) on samples of a tumor before treatment (top) and after ILI and ipilimumab (bottom). F, Quantitation of the IHC results from all available tumors. Note the increase in CD4+, CD8+, and CD68+ cells. Left, box-and-whisker plot showing the median, interquartile range, and range (excluding outliers) for cells positive for CD4, CD8, and CD68. Right, mean percentage of positive cells, with dark green indicating the PD-L1–positive subset. G, H&E and IHC of a single tumor, demonstrating the pretreatment melanoma tumor cells with a paucity of immune cells but with MHC Class II expression (top), and an increase in both immune cells and MHC after ILI and IPI (bottom).
- Figure 5.
TCRβ seqencing. A, TCR fraction in PBMCs and in tumor at baseline (Pre-TX), after limb infusion (post-ILI), and after the fourth dose of ipilimumab (Post-IPI). Note that there is not a difference in the starting T cells between patients that derived benefit (PFS at 1 year versus not). Although there was not an overall change in T cells in the periphery, there T cells in the tumors of patients with PFS at 1 year were somewhat increased, although the change was not significant. B, Box-and-whisker plots of the clonality of the TCR fraction. The clonality did not change in the PBMC; however, there was a significant increase in clonality, or a less even distribution of T cell clones, in the tumor versus the PBMC (P = 0.015, by Wiloxon signed-rank test), after combination treatment.
Tables
- Table 1.
Characteristics of the 26 patients in the phase II trial
Characteristic Number (%) Gender Female 10 (38) Male 16 (62) Stage IIIB 11 (42) IIIC 12 (46) IV 3 (12) Mutation status BRAF V600E 4 (15) NRAS 9 (35) WT 9 (35) Unknown 4 (15) Melanoma subtype Acral 3 (12) Cutaneous 19 (73) Unknown primary 4 (15) High tumor burden (>50 lesions or one >3 cm) Yes 7 (27) No 19 (73) - Table 2.
Number of patients affected by adverse events
Any Grade 3 or 4 Treatment-related adverse event 10 (38%) Any adverse event 13 (50%) Diarrhea 16 (62%) 3 (12%) Colitis 3 (12%) 3 (12%) Fatigue 18 (70%) 1 (4%) Pruritus 17 (26%) 2 (8%) Rash 18 (70%) 1 (4%) Nausea 8 (31%) 0 (0%) Anorexia 11 (42%) 2 (8%) Headache 10 (38%) 0 (0%) Arthralgia 4 (15%) 1 (4%) Myalgia 9 (35%) 1 (4%) Pneumonitis 1 (4%) 0 (0%) Elevated liver function test resulta 16 (62%) 1 (4%) Hypothyroidism 5 (19%) 0 (0%) Hypoadrenal 7 (27%) 2 (8%) Increased lipase 3 (0%) 0 (0%) ↵aIncludes any increase in aspartate aminotransferase (AST) or alanine aminotransferase (ALT).
- Table 3.
Genes upregulated in tumors after treatment relative to pretreatment
Gene Fold change P After limb infusion TREM1 12.49 0.023 CCL3L1 10.59 0.009 CCL3 10.36 0.013 TNFRSF10C 9.27 0.004 CXCL3 8.9 0.042 TREM2 8.09 0.003 CXCR2 7.72 0.030 CCL18 7.59 0.046 MARCO 7.43 0.002 CCL23 7.31 0.016 CXCL2 7.17 0.029 S100A12 7.05 0.047 SLC11A1 7.04 0.027 CXCL1 6.96 0.039 S100A8 6.9 0.038 LILRA5 5.7 0.025 C9 5.28 0.006 CCL4 5.03 0.027 PLAUR 5.03 0.041 IRGM 4.84 0.006 CCRL2 4.84 0.017 IL21R 4.82 0.016 IL3RA 4.79 0.001 CD70 4.77 0.010 ITGAX 4.76 0.032 After limb infusion and ipilimumab CHIT1 17.27 0.001 CCL18 8.09 0.001 GZMA 7.35 0.001 CXCR6 7.23 0.000 LY9 6.8 0.002 KLRC2 6.68 0.000 GZMH 6.39 0.001 GZMK 6.34 0.003 CCL26 6.25 0.004 CCL3 6.1 0.010 TREM2 6.01 0.003 CCL3L1 5.95 0.009 PDCD1 5.73 0.011 SH2D1A 5.4 0.005 KLRK1 5.39 0.001 CD3G 5.27 0.006 CD2 5.26 0.009 NCR1 4.97 0.001 IFNG 4.9 0.011 KLRG1 4.89 0.005 IL18RAP 4.88 0.006 CXCR3 4.88 0.019 CCL5 4.87 0.003 CD27 4.82 0.015 ICOS 4.81 0.010 NOTE: Shown are the 25 genes with the greatest significant fold change.
Supplementary Data
- Supplemental Data - Supplementary Figure 1. Gemcitabine and CTLA-4 blockade synergize to create an inflammatory microenvironment and anti-tumor response. Mice with palpable tumors were treated with low dose gemcitabine followed by systemic CTLA-4 blockade. Survival was improved (A) and tumors with combination treatment had a significant influx in CD4 and CD8 Tcells (B). Elispot (C) demonstrated improved enhanced IFNγ in response to SPAS-1 peptide (SNC9H8). Supplemental Figure S2. Lack of significant change of TIM3 or LAG3 over time in peripheral blood mononuclear cells (PBMC) over time. Supplementary Table S1. Toxicities in the limb according to Wieberdink criteria Supplemental Table 2. Nanostring fold change results of all genes
Volume 6, Issue 2
