Cancer Microenvironment and Immunology Research Serum Immunoregulatory Proteins as Predictors of Overall Survival of Metastatic Melanoma Patients Treated with Ipilimumab Yoshinobu Koguchi1, Helena M. Hoen1, Shelly A. Bambina1, Michael D. Rynning2, Richard K. Fuerstenberg2, Brendan D. Curti1, Walter J. Urba1, Christina Milburn3, Frances Rena Bahjat3, Alan J. Korman3, and Keith S. Bahjat1 Abstract Treatment with ipilimumab improves overall survival (OS) [log10 CXCL11: HR, 1.88; 95% confidence interval (CI), 1.14– in patients with metastatic melanoma. Because ipilimumab 3.12; P ¼ 0.014; and log10 sMICA quadratic effect P ¼ 0.066; targets T lymphocytes and not the tumor itself, efficacy may sMICA ( 247 vs. 247): HR, 1.75; 95% CI, 1.02–3.01]. Mul- be uniquely sensitive to immunomodulatory factors present at tivariate analysis of an independent ipilimumab-treated cohort the time of treatment. We analyzed serum from patients with confirmed the association between log10 CXCL11 and OS (HR, metastatic melanoma (247 of 273, 90.4%) randomly assigned 3.18; 95% CI, 1.13–8.95; P ¼ 0.029), whereas sMICA was less to receive ipilimumab or gp100 peptide vaccine. We quantified strongly associated with OS [log10 sMICA quadratic effect P ¼ candidate biomarkers at baseline and assessed the association 0.16; sMICA (247 vs. 247): HR, 1.48; 95% CI, 0.67–3.27]. of each using multivariate analyses. Results were confirmed in High baseline CXCL11 and sMICA were associated with poor an independent cohort of similar patients (48 of 52, 92.3%) OS in patients with metastatic melanoma after ipilimumab treated with ipilimumab. After controlling for baseline covari- treatment but not vaccine treatment. Thus, pretreatment ates, elevated chemokine (C-X-C motif) ligand 11 (CXCL11) CXCL11 and sMICA may represent predictors of survival benefit and soluble MHC class I polypeptide–related chain A (sMICA) after ipilimumab treatment as well as therapeutic targets. Cancer were associated with poor OS in ipilimumab-treated patients Res; 75(23); 5084–92. Ó2015 AACR. Introduction received ipilimumab monotherapy achieved a 28.4% four-year OS rate (6). Despite the success of ipilimumab, the majority of the Ipilimumab is a human monoclonal antibody targeting CTLA- patients on this study died as a consequence of melanoma. With 4 (cytotoxic T lymphocyte antigen-4). CTLA-4 is expressed on an increasing number of treatment options available and activated T cells, has structural similarities to the costimulatory increased use of targeted therapies, predictive biomarkers that molecule CD28, and binds to the same ligands as CD28 albeit identify those patients most likely to benefit from a specific with higher affinity. Binding of CTLA-4 to CD80/CD86 inhibits T- treatment are needed (7, 8). cell activation by limiting IL2 production and expression of the Unlike traditional cancer therapies, immunotherapeutics act IL2 receptor (CD25; ref. 1). Ipilimumab prevents CTLA-4 from primarily upon cells of the immune system. The requirement for binding its ligands, thus promoting activation of effector T cells the immune system as a third-party mediator of the drug's activity via prolonged CD28 signaling (2). In addition, anti-CTLA-4 suggests the balance of positive and negative regulators of the antibodies can deplete intratumoral regulatory T cells, subverting immune response at the time of therapy may be a critical deter- yet another mechanism of immunosuppression (3). minant of efficacy for any immunotherapy. Cytokines, chemo- Ipilimumab improved overall survival (OS) in patients with kines, and soluble receptors regulate the survival, activity, and metastatic melanoma in a randomized, double-blinded phase III location of immune effector cells and thus represent potential clinical trial (4, 5). Patients with metastatic melanoma who players in determining drug efficacy. Of particular interest are soluble factors involved in the recruitment and regulation of effector T cells representing the most readily measurable clinical 1Earle A. Chiles Research Institute, Providence Cancer Center, Port- biomarkers. 2 3 land, Oregon. R&D Systems, Minneapolis, Minnesota. Bristol-Myers To identify candidate soluble factor(s) predictive of improved Squibb, Redwood City, California. survival following ipilimumab treatment, we analyzed pretreat- Note: Supplementary data for this article are available at Cancer Research ment sera from treatment (ipilimumab) and "active control" Online (http://cancerres.aacrjournals.org/). (gp100 vaccine) patients from the pivotal phase III clinical trial Corresponding Author: Keith S. Bahjat, Earle A. Chiles Research Institute, of ipilimumab (4) for a variety of factors and correlated their Providence Cancer Center, 4805 NE Glisan Street, 2N83, Portland, OR 97213. levels with OS. A hypothesis-guided panel of candidate biomar- Phone: 503-215-7229; Fax: 503-215-6841; E-mail: [email protected] kers was selected, including biomarkers previously reported to doi: 10.1158/0008-5472.CAN-15-2303 associate with response to ipilimumab. Each analyte was assessed Ó2015 American Association for Cancer Research. in univariate and multivariate models for its correlation with OS. 5084 Cancer Res; 75(23) December 1, 2015 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2015 American Association for Cancer Research. Immunoregulatory Proteins Predict Outcome of Ipilimumab Treatment Correlative biomarkers identified in the initial screen were further phase III trial, differences within treatment group were of primary validated by testing sera from an independent cohort of ipilimu- interest and thus tested in separate models. Survival was defined mab-treated patients at our institution. as time from beginning of ipilimumab treatment to date of death, censoring at date of last follow-up. To calculate median follow-up time, deaths were censored. Patients and Methods Univariate survival analysis was performed for each treatment Clinical trials subgroup using Cox proportional hazards regression. Effects of Detailed information regarding the phase III clinical trial of CXCL11, sMICA, sMICB, sCD25, VEGF, absolute lymphocyte ipilimumab (NCT00094653) was reported elsewhere (4). Briefly, counts (ALC), tumor burden, and effect of LDH [ vs. > upper patients with metastatic melanoma having failed at least one prior limit of normal (ULN)] were shown. Models of quadratic effects therapy that may have included IL2, dacarbazine, and/or temo- to examine possible nonlinear effects and models of linear effects zolomide were enrolled excluding those with ocular melanoma. of continuous variables were tested. When the quadratic effect was à þ All patients were HLA-A 0201 as the restricting element for the not significant or the linear effect was more strongly significant, gp100 peptides used. All ipilimumab-treated patients received main effects model results were reported. For sMICA and VEGF, ipilimumab alone at 3 mg/kg every 3 weeks for 4 treatments. In quadratic effect was significant in some models. To present an HR, the gp100 group, patients received two peptides (1 mg each), results are also reported for a categorized variable, with the cutoff injected subcutaneously as an emulsion with incomplete Freund's point determined as the quintile where a threshold effect was adjuvant (Montanide ISA-51). Peptide injections were given observed in the phase III trial. Kaplan–Meier plots used for immediately after 90-minute intravenous infusion of placebo. determining cutoff points are shown in Supplementary Fig. S1. Tumor burden was assessed by the treating physician as previ- In multivariate analyses, model results of the remaining variables ously described (4). (other than sMICA), however, are from the model containing the Serum samples were also obtained from patients treated on an continuous form of the variable with the quadratic effect. Con- expanded access program at the Earle A. Chiles Research Institute tinuous measures were approximately log normal and analyzed as (EACRI cohort). Detailed information regarding this Compas- log10-transformed. sionate Use Trial for Unresectable Melanoma with Ipilimumab is In multivariate analysis of OS, Cox proportional hazards available elsewhere (NCT00495066). All patients received ipili- regression was used to test effects of biomarker candidates on mumab alone (3 or 10 mg/kg every 3 weeks for 4 treatments) with survival after controlling for other biomarkers and baseline no exclusions for ocular primary melanomas or HLA type. patient characteristics. Only CXCL11 and sMICA were included, All patients provided written informed consent and all studies as they were significant in univariate survival models of the were carried out in accordance with the Declaration of Helsinki ipilimumab group but not the gp100 group. Covariates in models under good clinical practice and Institutional Review Board for both studies were age, gender, ECOG status, prior immuno- approval. therapy, LDH, and ALC. Tumor burden was also included in multivariate model for the phase III trial cohort, but not the EACRI Serum cytokine analysis cohort as these data were not captured. Serum was collected and stored at À80 C. Chemokine (C-C Analyses were performed using SAS 9.3 (SAS Institute Inc.). motif) ligand 2 (CCL2), CCL3, CCL4, CCL8, CCL18, CCL26, Forest plots were prepared using Forest Plot Viewer (9) and edited chemokine (C-X-C motif) ligand (CXCL9), CXCL10, CXCL11, using Adobe Illustrator. GraphPad Prism was used for depicting CXCL13, and VEGF were measured using a bead-based multi- some