To the Editor:
Treatment of advanced melanoma has been revolutionized by targeted therapy (TT) and immune checkpoint blockade (ICB), but not all patients respond; identification of predictive biomarkers remains a critical need. Our prior investigations demonstrated the circulating level of C-reactive protein (CRP) as an independent melanoma prognostic biomarker (Fang et al., 2016, Fang et al., 2017, Fang et al., 2015). Interleukin-6 (IL-6) is upstream of CRP, induces hepatocyte release of CRP, and is a pleiotropic inflammatory cytokine involved in progression of melanoma (Hoejberg et al., 2012). Higher circulating IL-6 levels are associated with poorer prognosis in multiple cancers (Guo et al., 2012). We therefore evaluated IL-6 as a prognostic and predictive biomarker of TT and ICB response in melanoma.
This study was conducted under protocols LAB00-063 and PA11-0957 approved by The University of Texas MD Anderson Cancer Center Institutional Review Board (IRB). All patients provided written, informed consent before participation. Demographic and clinical factors for study cohorts are summarized in Table 1. Analysis of the exploratory cohort found plasma IL-6 levels associated with age (correlation coefficient: 0.21), gender, disease stage, tumor thickness, ulceration, tumor burden, B-Raf mutation status, body mass index (BMI) and CRP (Supplementary Table S1). Elevated CRP (≥10 μg/ml) and elevated IL-6 (≥4 pg/ml), dichotomized by recursive partitioning, demonstrated association with poorer overall survival (OS) and melanoma specific survival (MSS) (Supplementary Table S2; Supplementary Fig. S1). Prior proposed cut-offs for IL-6 have varied from 2-20 pg/ml (Soubrane et al., 2005); our cut-off (≥4 pg/ml) is preliminary, requiring confirmation. Multivariable analysis identified IL-6 (≥4 pg/ml) as an independent predictor of poorer OS and MSS, while CRP was not (Supplementary Table S2). These results suggest that IL-6, upstream in the inflammatory cascade to CRP, could influence association of CRP with poorer melanoma patient outcome. Comparing change in IL-6 with change in disease status (responding disease/stable disease/progressive disease) using sequential samples identified an increase in IL-6 level with disease progression (Supplementary Table S3).
Table 1.
Patient Demographics and Clinical Parameters
| Patient Demographics and Clinical Parameters |
No. (%) | Median (Interquartile Range) |
|---|---|---|
| Exploratory Cohort | ||
| Age at blood draw (Years) | 462 | 58.6 (47.3 – 68.0) |
| Gender | 462 | |
| Female | 176 (38) | - |
| Male | 286 (62) | |
| Disease Stage at blood draw | 462 | |
| I | 152 (33) | |
| II | 34 (7) | - |
| III | 155 (34) | |
| IV | 121 (26) | |
| B-Raf Status | 251 | |
| Wild Type | 149 (59) | - |
| Mutant (V600E/K) | 102 (41) | |
| Not available | 211 | |
| Vital Status | 462 | |
| Alive | 390 (84) | - |
| Dead (Any cause death) | 72 (16) | |
| Dead (Melanoma specific death) | 48/72 (67) | |
| BMI (kg/m2) | 455 | 29.0 (25.4 – 32.6) |
| IL-6 (pg/ml) | 462 | 2.0 (1.2 – 3.7) |
| CRP (μg/ml) | 459 | 2.0 (0.6 – 4.9) |
| Validation Cohort (For ICB Response) | ||
| Age at blood draw (Years) | 62 | 60 (49 – 69) |
| Gender | 62 | |
| Female | 22 (35) | - |
| Male | 40 (65) | |
| Disease Stage at blood draw | 62 | |
| III | 28 (45) | - |
| IV | 34 (55) | |
Abbreviations: BMI - Body Mass Index; IL-6 - Interleukin-6; CRP - C-Reactive Protein.
We further evaluated potential associations between IL-6 and response to treatment in the exploratory cohort (Table 2). Treatment included TT (dabrafenib with or without trametinib) alone; ICB (ipilimumab, and/or nivolumab, and/or pembrolizumab) alone; or the combination of ICB plus TT (ICB+TT). Treatment response was evaluated using both Anytime-Treatment samples (Any-Tx, samples collected before, during or after treatment) and Pre-Treatment samples (Pre-Tx, samples collected ≤90 days prior to treatment). Among patients who received ICB alone, 3-8-fold higher IL-6 levels were observed in non-responders compared to responders (using Anytime-Tx samples, 10.68 ± 17.34 pg/ml vs. 3.20 ± 2.98 pg/ml, P = 0.001; using Pre-Tx samples, 19.97 ± 26.51 pg/ml vs. 2.39 ± 1.48 pg/ml, P = 0.01). Similarly, among patients who received ICB+TT, 3-8-fold higher levels of IL-6 were observed among non-responders compared to responders using Anytime-Tx or Pre-Tx samples, respectively. No significant correlation was observed between IL-6 levels and non-response among patients who received TT alone; however, the relatively small subset of TT-only patients raises the possibility of a Type II error in this group.
Table 2.
IL-6 Level as a Predictive Biomarker of Response to Immune Checkpoint Blockade (ICB)
| Sets | Treatment | Response Status |
IL-6 Level (pg/ml) | |||||
|---|---|---|---|---|---|---|---|---|
| Anytime-Treatment Sample, “Any-Tx” (before, during or after treatment samples - collected at any time point) |
Pre-Treatment Sample, “Pre-Tx” (before treatment samples ONLY – collected within 90 days prior to treatment) |
|||||||
| n | Mean ± SD | P Value | n | Mean ± SD | P Value | |||
| Exploratory Set | TT | Responders | 29 | 3.69 ± 3.65 | 0.40 | 5 | 2.54 ± 1.23 | 0.57 |
| Non-Responders | 11 | 11.08 ± 23.25 | 3 | 3.38 ± 4.22 | ||||
| ICB+TT | Responders | 110 | 3.09 ± 2.81 | <0.0001 | 18 | 2.30 ± 1.49 | 0.005 | |
| Non-Responders | 68 | 10.02 ± 16.19 | 12 | 19.27 ± 25.39 | ||||
| ICB | Responders | 80 | 3.20 ± 2.98 | <0.001 | 17 | 2.39 ± 1.48 | 0.01 | |
| Non-Responders | 58 | 10.68 ± 17.34 | 11 | 19.97 ± 26.51 | ||||
| ICB - Stage III | Responders | 31 | 3.15 ± 3.04 | 0.41 | 6 | 2.43 ± 2.0 | 0.39 | |
| Non-Responders | 19 | 4.08 ± 4.21 | 3 | 8.43 ± 9.42 | ||||
| ICB - Stage IV | Responders | 46 | 3.30 ± 3.05 | 0.0001 | 11 | 2.38 ± 1.23 | 0.03 | |
| Non-Responders | 35 | 15.16 ± 21.03 | 8 | 24.29 ± 30.00 | ||||
| Validation Set | ICB - Stage III | Responders | NA | 17 | 1.66 ± 0.97 | 0.65 | ||
| Non-Responders | 11 | 1.95 ± 2.21 | ||||||
| ICB - Stage IV | Responders | NA | 17 | 3.59 ± 4.05 | 0.03 | |||
| Non-Responders | 17 | 7.58 ± 8.60 | ||||||
| Combination of Both Sets | ICB - Stage IV | Responders | NA | 28 | 3.11 ± 3.26 | 0.002 | ||
| Non-Responders | 25 | 12.93 ± 19.37 | ||||||
Abbreviations: ICB - Immune Checkpoint Blockade; CR - Complete Response; PR - Partial Response; SD - Stable Disease; PD - Progressive Disease; Responders (CR+PR); Non-Responders (SD+PD).
To further explore the relationship between IL-6 and response to ICB with disease stage, subset analysis of stage III and IV patients was conducted. Elevated IL-6 was correlated with non-response to ICB in stage IV patients (stage IV Any-Tx non-responders vs. responders, 15.16 ± 21.03 pg/ml vs. 3.30 ± 3.05 pg/ml, P = 0.0001; stage IV Pre-Tx non-responders vs. responders, 24.29 ± 30.00 pg/ml vs. 2.38 ± 1.23 pg/ml, P = 0.03). In contrast, no association was observed between IL-6 and ICB response among stage III patients. These data suggested that elevated IL-6 levels could be a stratification marker for stage IV ICB treatment resistance.
To validate this finding, we evaluated Pre-Tx plasma IL-6 levels and response to ICB in an independent cohort (Table 2). Among stage IV patients in the validation cohort, Pre-Tx elevated IL-6 was again associated with ICB non-response (non-responders vs. responders, 7.58 ± 8.60 pg/ml vs. 3.59 ± 4.05 pg/ml, P = 0.03); no association was identified among stage III patients. Analysis of the combined cohorts yielded similar findings.
We evaluated potential confounders of tumor mutation status and tumor burden. While B-raf tumor mutation status was correlated with elevated IL-6 (Supplementary Table S1), we did not find B-raf status to be correlated with ICB response (data not shown). Tumor burden may predict response to immunotherapy (Kim et al., 2020); we found increased tumor burden associated with elevated IL-6 (Supplementary Table S1), and with Pre-Tx ICB non-response (Supplementary Table S4), thus suggesting that tumor burden could contribute to the IL-6 correlation with ICB non-response. Since elevated IL-6 could provide at least one mechanism explaining the association between tumor burden and ICB resistance, further investigation of these complex associations is indicated.
IL-6 levels have been reported higher in melanoma patients not responding to chemotherapy or interferon-alpha (Mouawad et al., 1996, Mouawad et al., 1999). In a report of melanoma patients treated with ipilimumab, IL-6 levels tended to be higher in those with treatment failure, but the association did not reach statistical significance (Bjoern et al., 2016). In a report of patients treated with ICB, elevated IL-6 was associated with poorer OS; treatment response was not evaluated (Laino et al., 2020). Importantly, our study is the first to our knowledge to specifically identify IL-6 level as a possible predictor of ICB response in melanoma patients.
IL-6-mediated immune suppression has been linked to myeloid-derived suppressor cells (Chen et al., 2014) and tumor-associated macrophages (TAMs) (Tsukamoto et al., 2018); these mechanisms could contribute to resistance to ICB. Our results indirectly suggest that targeting IL-6 either alone or in combination with ICB could be beneficial in advanced melanoma. Murine models suggest that combined IL-6 and Programmed Death-1 Ligand-1 (PDL-1) blockade can reduce melanoma metastasis (Tsukamoto et al., 2018). Interestingly, Tocilizumab, a humanized monoclonal antibody against IL-6 receptor, may improve immune-related adverse events without affecting therapeutic efficacy of ICB in melanoma (Hopkins et al., 2017) and lung adenocarcinoma (Horisberger et al., 2018). Thus, the results reported here in context with other evidence supports continued evaluation of the role of IL-6 as a predictive biomarker of ICB response and a potential therapeutic target.
Supplementary Material
Funding:
This work was supported by the NCI/NIH SPORE grant P50 CA093459 and the UTMDACC Support Grant P30 CA016672 (Clinical Trials Support Resource); philanthropic contributions to UTMDACC Moon Shots Program; UTMDACC Various Donors Melanoma and Skin Cancers Priority Program Fund; Miriam and Jim Mulva Research Fund; McCarthy Skin Cancer Research Fund and Marit Peterson Fund for Melanoma Research.
Footnotes
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Conflict of Interest: JW serves as a consultant/advisory board member for Roche/Genentech, Novartis, AstraZeneca, GlaxoSmithKline, Bristol-Myers Squibb, Merck, Biothera Pharmaceuticals and Ella Therapeutics. JW reports compensation for speaker’s bureau and honoraria from Imedex, Dava Oncology, Omniprex, Illumina, Gilead, PeerView, Physician Education Resource, MedImmune and Bristol-Myers Squibb. JW receives research support from GlaxoSmithKline, Roche/Genentech, Bristol-Myers Squibb, and Novartis. JLM is a consultant for Roche, BMS, Merck, and Novartis. JLM reports honoraria from BMS and Roche. JEG serves as a consultant and/or as an advisory board member for Merck, Syndax, Regeneron, Novartis, and Bristol Myers Squibb. MIR is a consultant for Amgen and a consultant and advisory board member for Merck. All other authors declare that they have no competing interests.
Data Availability:
All data relevant to the study are included in the article or uploaded as supplementary information.
REFERENCES
- Bjoern J, Juul Nitschke N, Zeeberg Iversen T, Schmidt H, Fode K, Svane IM. Immunological correlates of treatment and response in stage IV malignant melanoma patients treated with Ipilimumab. Oncoimmunology 2016;5(4):e1100788. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen MF, Kuan FC, Yen TC, Lu MS, Lin PY, Chung YH, et al. IL-6-stimulated CD11b+ CD14+ HLA-DR-myeloid-derived suppressor cells, are associated with progression and poor prognosis in squamous cell carcinoma of the esophagus. Oncotarget 2014;5(18):8716–28. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fang S, Sui D, Wang Y, Liu H, Chiang YJ, Ross MI, et al. Association of Vitamin D Levels With Outcome in Patients With Melanoma After Adjustment For C-Reactive Protein. J Clin Oncol 2016;34(15):1741–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fang S, Wang Y, Dang Y, Gagel A, Ross MI, Gershenwald JE, et al. Association between Body Mass Index, C-Reactive Protein Levels, and Melanoma Patient Outcomes. J Invest Dermatol 2017;137(8):1792–5. [DOI] [PubMed] [Google Scholar]
- Fang S, Wang Y, Sui D, Liu H, Ross MI, Gershenwald JE, et al. C-reactive protein as a marker of melanoma progression. J Clin Oncol 2015;33(12):1389–96. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Guo Y, Xu F, Lu T, Duan Z, Zhang Z. Interleukin-6 signaling pathway in targeted therapy for cancer. Cancer Treat Rev 2012;38(7):904–10. [DOI] [PubMed] [Google Scholar]
- Hoejberg L, Bastholt L, Schmidt H. Interleukin-6 and melanoma. Melanoma Res 2012;22(5):327–33. [DOI] [PubMed] [Google Scholar]
- Hopkins AM, Rowland A, Kichenadasse G, Wiese MD, Gurney H, McKinnon RA, et al. Predicting response and toxicity to immune checkpoint inhibitors using routinely available blood and clinical markers. Brit J Cancer 2017;117(7):913–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Horisberger A, La Rosa S, Zurcher JP, Zimmermann S, Spertini F, Coukos G, et al. A severe case of refractory esophageal stenosis induced by nivolumab and responding to tocilizumab therapy. J Immunother Cancer 2018;6(1):156. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kim SI, Cassella CR, Byrne KT. Tumor Burden and Immunotherapy: Impact on Immune Infiltration and Therapeutic Outcomes. Front Immunol 2020;11:629722. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laino AS, Woods D, Vassallo M, Qian X, Tang H, Wind-Rotolo M, et al. Serum interleukin-6 and C-reactive protein are associated with survival in melanoma patients receiving immune checkpoint inhibition. J Immunother Cancer 2020;8(1). [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mouawad R, Benhammouda A, Rixe O, Antoine EC, Borel C, Weil M, et al. Endogenous interleukin 6 levels in patients with metastatic malignant melanoma: correlation with tumor burden. Clin Cancer Res 1996;2(8):1405–9. [PubMed] [Google Scholar]
- Mouawad R, Khayat D, Merle S, Antoine EC, Gil-Delgado M, Soubrane C. Is there any relationship between interleukin-6/interleukin-6 receptor modulation and endogenous interleukin-6 release in metastatic malignant melanoma patients treated by biochemotherapy? Melanoma Res 1999;9(2):181–8. [DOI] [PubMed] [Google Scholar]
- Soubrane C, Rixe O, Meric JB, Khayat D, Mouawad R. Pretreatment serum interleukin-6 concentration as a prognostic factor of overall survival in metastatic malignant melanoma patients treated with biochemotherapy: a retrospective study. Melanoma Res 2005;15(3):199–204. [DOI] [PubMed] [Google Scholar]
- Tsukamoto H, Fujieda K, Senju S, Ikeda T, Oshiumi H, Nishimura Y. Immune-suppressive effects of interleukin-6 on T-cell-mediated anti-tumor immunity. Cancer Sci 2018;109(3):523–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
All data relevant to the study are included in the article or uploaded as supplementary information.