Published OnlineFirst November 8, 2018; DOI: 10.1158/1078-0432.CCR-18-2795 Translational Cancer Mechanisms and Therapy Clinical Cancer Research Circulating Cytokines Predict Immune-Related Toxicity in Melanoma Patients Receiving Anti-PD-1–Based Immunotherapy Su Y. Lim1,2, Jenny H. Lee1,2, Tuba N. Gide2,3, Alexander M. Menzies2,3,4, Alexander Guminski2,3,4, Matteo S. Carlino2,3,5, Edmond J. Breen6, Jean Y.H. Yang7,8, Shila Ghazanfar7,8, Richard F. Kefford1,2,5, Richard A. Scolyer2,3,9, Georgina V. Long2,3,4, and Helen Rizos1,2 Abstract Purpose: Combination PD-1 and CTLA-4 inhibitor therapy line (PRE) and early during treatment (EDT). The expres- has dramatically improved the survival of patients with sion of these 11 cytokines was integrated into a single advanced melanoma but is also associated with significant toxicity score, the CYTOX (cytokine toxicity) score, and the immune-related toxicities. This study sought to identify cir- predictive utility of this score was confirmed in the dis- culating cytokine biomarkers of treatment response and covery and validation cohorts. The AUC for the CYTOX immune-related toxicity. score in the validation cohort was 0.68 at PRE [95% Experimental Design: The expression of 65 cytokines was confidence interval (CI), 0.51–0.84; P ¼ 0.037] and profiled longitudinally in 98 patients with melanoma treated 0.70 at EDT (95% CI, 0.55–0.85; P ¼ 0.017) using ROC with PD-1 inhibitors, alone or in combination with anti- analysis. CTLA-4, and in an independent validation cohort of 49 Conclusions: The CYTOX score is predictive of severe patients treated with combination anti-PD-1 and anti-CTLA- immune-related toxicity in patients with melanoma 4. Cytokine expression was correlated with RECIST response treated with combination anti-CTLA-4 and anti-PD-1 and immune-related toxicity, defined as toxicity that war- immunotherapy. This score, which includes proinflam- ranted permanent discontinuation of treatment and admin- matory cytokines such as IL1a, IL2, and IFNa2, may istration of high-dose steroids. help in the early management of severe, potentially life- Results: Eleven cytokines were significantly upregulated threatening immune-related toxicity. in patients with severe immune-related toxicities at base- See related commentary by Johnson and Balko Introduction patients with melanoma (3–5), and have now entered adjuvant therapy. The last 5 years have seen remarkable improvements in the Circulating biomarkers of response to anti-PD-1–based therapy survival of patients with advanced melanoma due to the use of have been widely investigated. For instance, circulating tumor immune checkpoint therapies that promote T-cell–mediated þ À DNA, frequency of activated monocytes (CD14 CD16 HLA- antitumor activity (1, 2). Immune checkpoint inhibitors targeting DRhi), and neutrophil counts have each been shown to predict the cytotoxic T-lymphocyte antigen-4 (CTLA-4; ipilimumab) and progression-free and overall survival in patients treated with programmed death-1 (PD-1; nivolumab and pembrolizumab) immune checkpoint inhibitors (6–8). In contrast, there have been receptors produce durable responses and prolong the survival of few studies exploring biomarkers that predict risk of immune- related adverse events (irAE; reviewed in ref. 9), despite grade 3/4 1Faculty of Medicine and Health Sciences, Macquarie University, Sydney, irAEs occurring in over 50% of patients treated with combination Australia. 2Melanoma Institute Australia, The University of Sydney, Sydney, immune checkpoint inhibitors (10, 11). Early recognition and Australia. 3Sydney Medical School, The University of Sydney, Sydney, Australia. intervention is critical for severe irAEs, and many patients require 4Royal North Shore and Mater Hospitals, Sydney, Australia. 5Westmead treatment interruption, discontinuation, and/or immunosup- Hospital, Sydney, Australia. 6Australian Proteome Analysis Facility, Macquarie fl 7 pressive agents such as high-dose steroids, in iximab, or myco- University, Sydney, Australia. School of Mathematics and Statistics, The phenolate (12). University of Sydney, Sydney, Australia. 8Charles Perkins Centre, The University of Sydney, Sydney, Australia. 9Royal Prince Alfred Hospital, Sydney, Australia. Cytokines are important regulators of host immune activity; they promote the recruitment of immune cells into the tumor Note: Supplementary data for this article are available at Clinical Cancer microenvironment (13–17) and induce the expression of Research Online (http://clincancerres.aacrjournals.org/). immune checkpoint receptors, including TIM-3 and PD-1 (18). S.Y. Lim and J.H. Lee contributed equally to this article. Consequently, these soluble mediators may serve as predictive Corresponding Author: Helen Rizos, Macquarie University, Talavera Road, biomarkers of immunotherapy response and/or immune-medi- Sydney, New South Wales 2109, Australia. Phone: 612-9850-2762; Fax: 612- ated toxicity. For instance, baseline serum levels of TGFb and IL10 9845-9102; E-mail: [email protected] correlated with clinical outcome, and elevated baseline IL17 levels doi: 10.1158/1078-0432.CCR-18-2795 predicted grade 3 colitis in patients with melanoma treated with Ó2018 American Association for Cancer Research. neoadjuvant ipilimumab (n ¼ 35; ref. 19). These findings are yet www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst November 8, 2018; DOI: 10.1158/1078-0432.CCR-18-2795 Lim et al. and OpACIN-neo clinical trials (NCT02374242, NCT01295827, Translational Relevance NCT02083484, NCT02089685, NCT02905266, NCT02599402, The identification of blood-based biomarkers that can pre- NCT02714218, and NCT02977052; refs. 20, 21). dict immunotherapy toxicity and response remains a crucial but unmet need. This study explored the utility of circulating Disease characteristics and clinical assessment plasma cytokines in predicting immune-related toxicity and Patient demographics and clinicopathologic features includ- treatment response in patients with melanoma treated with ing sex, age, baseline lactate dehydrogenase (LDH) levels, and anti-PD-1–based immunotherapy, alone or in combination American Joint Committee on Cancer (8th edition) M stage were with anti-CTLA-4. Plasma cytokine levels were generally collected. Baseline disease tumor burden was determined by the unchanged with immunotherapy, indicating that analysis of sum of the product of bidimensional diameters (SPOD) for either baseline (PRE) or early during treatment (EDT) plasma every metastasis 5mminlongaxis(15 mm short axis for may prove useful as a stable biomarker of toxicity. We defined lymph nodes). A SPOD of 1,000 mm2 was considered low- a toxicity score (CYTOX) that includes the expression of 11 disease burden and >1,000 mm2 was considered high-disease circulating cytokines, which was significantly higher in burden (6). Investigator-determined objective response was patients with severe immune-related toxicity, at PRE and EDT. assessed radiologically with CT and MRI of the brain at 2–3 We validated the predictive utility of the CYTOX score in an monthly intervals using the RECIST 1.1 criteria (22). Patients independent cohort of patients treated with combination PD- with a complete response (CR) or partial response as best 1 and CTLA-4 inhibitors. These findings have important clin- response were classified as responders, and those with stable ical implications in the prediction and early management of disease or progressive disease were classified as nonresponders. severe, potentially life-threatening immune-related toxicity. Follow-up duration was calculated from the date of first dose of systemic therapy to the following three dates: date of death, loss to follow-up, or May 31, 2018. Toxicity data were categorized according to the National Cancer to be validated, however, and the role of circulating cytokines in Institute Common Terminology Criteria for Adverse Events ver- fi predicting immune-related toxicity remains unexplored. sion 4.0. Severe irAEs were de ned as requiring dose interruption In this study, we analyzed the expression of 65 cytokines in and the use of high-dose steroids (prednisolone dose 50 mg/ longitudinal plasma samples collected prior to therapy and dur- day equivalent), or other immunomodulatory drugs such as fl ing treatment from patients with melanoma treated with PD-1 in iximab and mycophenolate. We included only severe irAEs inhibitors (nivolumab or pembrolizumab), alone or in combi- that developed within 6 months of starting treatment, as delayed fi nation with the CTLA-4 inhibitor ipilimumab. Cytokine expres- toxicity may be dif cult to predict using pretreatment and early sion was correlated to the development of immune-related tox- during treatment cytokine levels. Treatment of irAEs was in line icity. Although cytokine levels displayed large interpatient vari- with standard guidelines and at the treating clinician's discretion. ation, there was limited intrapatient variability over the course of Endocrinopathies requiring hormonal replacement including treatment. Importantly, elevated levels of 11 circulating cytokines uncomplicated hypophysitis were not considered severe toxici- at baseline (PRE) and early during treatment (EDT) were signif- ties. Patients who did not have severe toxicity according to the icantly associated with the development of high-grade immune- above description or no toxicity were categorized as "no severe related toxicity. The integration