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Supplementary Figure describing TIMP-1 expression and its effect in clonogenic survival in-vitro.

Purpose:Combination PD-1 and CTLA-4 inhibitor therapy has dramatically improved the survival of patients with advanced melanoma but is also associated with significant immune-related toxicities. This study sought to identify circulating cytokine biomarkers of treatment response and immune-related toxicity.Experimental Design:The expression of 65 cytokines was profiled longitudinally in 98 patients with melanoma treated with PD-1 inhibitors, alone or in combination with anti-CTLA-4, and in an independent validation cohort of 49 patients treated with combination anti-PD-1 and anti-CTLA-4. Cytokine expression was correlated with RECIST response and immune-related toxicity, defined as toxicity that warranted permanent discontinuation of treatment and administration of high-dose steroids.Results:Eleven cytokines were significantly upregulated in patients with severe immune-related toxicities at baseline (PRE) and early during treatment (EDT). The expression of these 11 cytokines was integrated into a single toxicity score, the CYTOX (cytokine toxicity) score, and the predictive utility of this score was confirmed in the discovery and validation cohorts. The AUC for the CYTOX score in the validation cohort was 0.68 at PRE [95% confidence interval (CI), 0.51–0.84; P = 0.037] and 0.70 at EDT (95% CI, 0.55–0.85; P = 0.017) using ROC analysis.Conclusions:The CYTOX score is predictive of severe immune-related toxicity in patients with melanoma treated with combination anti-CTLA-4 and anti-PD-1 immunotherapy. This score, which includes proinflammatory cytokines such as IL1a, IL2, and IFNα2, may help in the early management of severe, potentially life-threatening immune-related toxicity.See related commentary by Johnson and Balko, p. 1452

Supplementary Methods including MRI imaging, immunofluorescence, Western blot, flow

Supplementary Table 1: Number of plasma samples included in the study Supplementary Table 2: Association of toxicity with treatment response Supplementary Table 3: Differentially expressed cytokines in patients with severe irAEs compared to those with no-severe irAEs at PRE in Cohort 2 Supplementary Table 4: Differentially expressed cytokines in patients with severe irAEs compared to those with no-severe irAEs at EDT in Cohort 2 Supplementary Table 5: Univariate analysis of cytokine expression and association with overall survival Supplementary Table 6: Univariate analysis of cytokine expression and association with RECIST response Supplementary Figure 1: Distribution of relative fluorescence intensity units of 65 circulating cytokines in plasma collected from 98 melanoma patients (cohorts 1 and 2) prior to therapy initiation. Supplementary Figure 2: Hierarchical clustering of cytokine expression profiles in cohorts 1 and 2.

Supplementary figure legends for Supplementary Figures 1 (Survival of PDAC cells in-vitro and in-vivo), 2 (ELISA, Western blot and Immunostaining), 3 (RTqPCR), 4 (Migration assay) and 5 (Flow cytometry).

Supplementary Figure showng Real time qPCR data on the expression of cytokines in-vitro.

Supplementary Figure describing the impact of TIMP-1 on immune cell populations in the in-vivo experiments.

Gemcitabine constitutes one of the backbones for chemotherapy treatment in pancreatic ductal adenocarcinoma (PDAC), but patients often respond poorly to this agent. Molecular markers downstream of gemcitabine treatment in preclinical models may provide an insight into resistance mechanisms. Using cytokine arrays, we identified potential secretory biomarkers of gemcitabine resistance (response) in the transgenic KRasG12D; Trp53R172H; Pdx-1 Cre (KPC) mouse model of PDAC. We verified the oncogenic role of the cytokine tissue inhibitor of matrix metalloproteinases 1 (TIMP1) in primary pancreatic tumors and metastases using both in vitro techniques and animal models. We identified potential pathways affected downstream of TIMP1 using the Illumina Human H12 array. Our findings were validated in both primary and metastatic models of pancreatic cancer. Gemcitabine increased inflammatory cytokines including TIMP1 in the KPC mouse model. TIMP1 was upregulated in patients with pancreatic intraepithelial neoplasias grade 3 and PDAC lesions relative to matched normal pancreatic tissue. In addition, TIMP1 played a role in tumor clonogenic survival and vascular density, while TIMP1 inhibition resensitized tumors to gemcitabine and radiotherapy. We observed a linear relationship between TIMP-1 expression, liver metastatic burden, and infiltration by CD11b+Gr1+ myeloid cells and CD4+CD25+FOXP3+ Tregs, whereas the presence of tumor cells was required for immune cell infiltration. Overall, our results identify TIMP1 upregulation as a resistance mechanism to gemcitabine and provide a rationale for combining chemo/radiotherapy with TIMP1 inhibitors in PDAC. Cancer Res; 77(21); 5952–62. ©2017 AACR.

Supplementary Figure describing the effect of TIMP-1 in PDAC cells in-vitro.

Supplementary Figure describing the impact of TIMP-1 on cell migration and invasion.