Correlation of the peripheral blood DNA methylation profile and immune-related adverse events in patients with solid cancer treated with immune checkpoint inhibitors

Abstract

Background/Introduction: Immune-related adverse events (irAEs) remain unpredictable complications of immune checkpoint inhibitor (ICI) therapy. Current biomarkers, such as inflammatory blood parameter lack specificity and clinical robustness. Because DNA methylation reflects stable immune programming and predisposition to autoimmune diseases, blood methylation profiling may offer a tumour-agnostic approach for irAE prediction.

Material & methods: We analysed baseline leukocyte DNA methylation in a prospectively enrolled real-world cohort of advanced pan-cancer patients treated with ICI in a palliative setting. Genome-wide methylation was profiled using Infinium MethylationEPIC v1.0 BeadChips. Differentially methylated positions (DMPs) were identified by rank-based comparison and tested for Gene Ontology enrichment. Blood-cell deconvolution using EpiDISH was applied to exclude compositional bias.

Results: Among 195 patients (median age 65 years; 34.9% female), 32.8% experienced at least one irAE, most frequently involving thyroid, skin, or lung. Despite globally concordant DNA methylation between groups (ρ = 0.9996), DMP analysis revealed subtle but distinct site-specific differences. A methylation-based classifier discriminated patients who developed irAEs with good accuracy (area under the curve [AUC] 0.831, 95% confidence interval [CI] 0.735–0.927), which remained robust in an internal validation subset (AUC 0.874, 95% CI 0.757–0.992). EpiDISH confirmed no significant variation in immune cell fractions between groups. Functional enrichment of top-ranked DMPs showed significant overrepresentation of neuronal development, cell-cell signalling, and epithelial morphogenesis pathways, networks linked to immune-synapse formation, cytokine signalling, and tissue repair responses.

Discussion: Baseline blood DNA methylation captures systemic immune-developmental states predisposing to irAEs. The enrichment of cell-cell signalling and epithelial morphogenesis pathways suggests an epigenetically encoded predisposition for dysregulated immune-tissue communication. Blood-based methylation profiling thus represents a promising, tumour-agnostic biomarker approach for individualised irAE risk prediction in cancer immunotherapy.

Keywords

biomarker / epigenetics / immunotherapy / methylation / precision medicine

References

[1] Celarain N, Tomas-Roig J. Aberrant DNA methylation profile exacerbates inflammation and neurodegeneration in multiple sclerosis patients. J Neuroinflammation. 2020; 17(1): 21.

[2] Ling C, Rönn T. Epigenetics in human obesity and type 2 diabetes. Cell Metab. 2019; 29: 1028-1044.

[3] Xu C, Söderhäll C, Bustamante M, et al. DNA methylation in childhood asthma: an epigenome-wide meta-analysis. Lancet Respir Med. 2018; 6(5): 379-388.

[4] Mazzone R, Zwergel C, Artico M, et al. The emerging role of epigenetics in human autoimmune disorders. Clin Epigenetics. 2019; 11(1): 34.

[5] Svendsen AJ, Mengel-From J, Junker P, et al. Differential DNA methylation patterns in whole blood from ACPA-positive patients with DMARD-naive rheumatoid arthritis at clinical disease onset. Front Immunol. 2025; 16: 1488161.

[6] Starzer A, et al. Tumor DNA methylation profiles correlate with response to anti-PD-1 immune checkpoint inhibitor monotherapy in sarcoma patients. J Immunother Cancer. 2021(3). PMID: 33762319

[7] Starzer AM, Heller G, Tomasich E, et al. DNA methylation profiles differ in responders versus non-responders to anti-PD-1 immune checkpoint inhibitors in patients with advanced and metastatic head and neck squamous cell carcinoma. J Immunother Cancer. 2022; 10(3). PMID: 38360861

[8] Ibrahim J, Peeters M, Van Camp G, Op de Beeck K. Methylation biomarkers for early cancer detection and diagnosis: current and future perspectives. Eur J Cancer. 2023; 178: 91-113.

[9] Bai R, Lv Z, Xu D, Cui J. Predictive biomarkers for cancer immunotherapy with immune checkpoint inhibitors. Biomark Res. 2020; 8: 34.

[10] Marsit CJ, Koestler DC, Christensen BC, Karagas MR, Houseman EA, Kelsey KT. DNA methylation array analysis identifies profiles of blood-derived DNA methylation associated with bladder cancer. J Clin Oncol. 2011; 29(9): 1133-1139.

[11] Chennamadhavuni A, Abushahin L, Jin N, Presley CJ, Manne A. Risk factors and biomarkers for immune-related adverse events: a practical guide to identifying high-risk patients and rechallenging immune checkpoint inhibitors. Front Immunol. 2022; 13: 779691.

[12] Suijkerbuijk KPM, van Eijs MJM, van Wijk F, Eggermont AMM. Clinical and translational attributes of immune-related adverse events. Nat Cancer. 2024(4): 557-571.

[13] Michailidou D, Khaki AR, Morelli MP, Diamantopoulos L, Singh N, Grivas P. Association of blood biomarkers and autoimmunity with immune related adverse events in patients with cancer treated with immune checkpoint inhibitors. Sci Rep. 2021; 11(1): 9029.

[14] Chu X, Zhao J, Zhou J, et al. Association of baseline peripheral-blood eosinophil count with immune checkpoint inhibitor-related pneumonitis and clinical outcomes in patients with non-small cell lung cancer receiving immune checkpoint inhibitors. Lung Cancer. 2020; 150: 76-82.

[15] Brilli L, Danielli R, Campanile M, et al. Baseline serum TSH levels predict the absence of thyroid dysfunction in cancer patients treated with immunotherapy. J Endocrinol Invest. 2021; 44(8): 1719-1726.

[16] Luongo C, Morra R, Gambale C, et al. Higher baseline TSH levels predict early hypothyroidism during cancer immunotherapy. J Endocrinol Invest. 2021; 44(9): 1927-1933.

[17] Yoon JH, Hong AR, Kim HK, Kang H. Characteristics of Immune-Related Thyroid Adverse Events in Patients Treated with PD-1/PD-L1 Inhibitors. Endocrinol Metab. 2021; 36(2): 413-423.

[18] Oyanagi J, Koh Y, Sato K, et al. Predictive value of serum protein levels in patients with advanced non-small cell lung cancer treated with nivolumab. Lung Cancer. 2019; 132: 107-113.

[19] Valpione S, Pasquali S, Campana LG, et al. Sex and interleukin-6 are prognostic factors for autoimmune toxicity following treatment with anti-CTLA4 blockade. J Transl Med. 2018; 16(1): 94.

[20] Hasan Ali O, Berner F, Bomze D, et al. Human leukocyte antigen variation is associated with adverse events of checkpoint inhibitors. Eur J Cancer. 2019; 107: 8-14.

[21] Chaput N, Lepage P, Coutzac C, et al. Baseline gut microbiota predicts clinical response and colitis in metastatic melanoma patients treated with ipilimumab. Ann Oncol. 2019; 30(12): 2012.

[22] Dubin K, Callahan MK, Ren B, et al. Intestinal microbiome analyses identify melanoma patients at risk for checkpoint-blockade-induced colitis. Nat Commun. 2016; 7: 10391.

[23] Shahabi V, Berman D, Chasalow SD, et al. Gene expression profiling of whole blood in ipilimumab-treated patients for identification of potential biomarkers of immune-related gastrointestinal adverse events. J Transl Med. 2013; 11: 75.

[24] Müller F, Scherer M, Assenov Y, et al. RnBeads 2.0: comprehensive analysis of DNA methylation data. Genome Biol. 2019; 20(1): 55.

[25] Zheng SC, Breeze CE, Beck S, Teschendorff AE. Identification of differentially methylated cell types in epigenome-wide association studies. Nat Methods. 2018; 15(12): 1059-1066.

[26] Bender R, Lange S. Adjusting for multiple testing–when and how? J Clin Epidemiol. 2001; 54(4): 343-349.

[27] Wang J, Ma Y, Lin H, Wang J, Cao B. Predictive biomarkers for immune-related adverse events in cancer patients treated with immune-checkpoint inhibitors. BMC Immunol. 2024; 25(1): 8.

[28] Zhao N, Yi Y, Cao W, Fu X, Mei N, Li C. Serum cytokine levels for predicting immune-related adverse events and the clinical response in lung cancer treated with immunotherapy. Front Oncol. 2022; 12: 923531.

[29] Calle-Fabregat C, Morante-Palacios O, Ballestar E. Understanding the relevance of DNA methylation changes in immune differentiation and disease. Genes. 2020; 11(1): 110. PMID: 31963661.

[30] Farina A, Villagrán-García M, Vogrig A, et al. Neurological adverse events of immune checkpoint inhibitors and the development of paraneoplastic neurological syndromes. Lancet Neurol. 2024; 23(1): 81-94.

[31] Luoma AM, Suo S, Williams HL, et al. Molecular pathways of colon inflammation induced by cancer immunotherapy. Cell. 2020; 182(3): 655-671. e22.

[32] Reschke R, Enk A, Hassel J. Prognostic biomarkers in evolving melanoma immunotherapy. Am J Clin Dermatol. 2025; 26(2): 213-223.

[33] Braun LM, Giesler S, Andrieux G, et al. Adiponectin reduces immune checkpoint inhibitor-induced inflammation without blocking anti-tumour immunity. Cancer Cell. 2025; 43(2): 269-291. e19.

[34] Berglund A, Mills M, Putney RM, Hamaidi I, Mulé J, Kim S. Methylation of immune synapse genes modulates tumour immunogenicity. J Clin Invest. 2020; 130(2): 974-980.

[35] Watson AS, Goutam S, Stukalin I, et al. Association of immune-related adverse events, hospitalization, and therapy resumption with survival among patients with metastatic melanoma receiving single-agent or combination immunotherapy. JAMA Netw Open. 2022; 5(12): e2245596.

[36] Cook S, Samuel V, Meyers DE, et al. Immune-related adverse events and survival among patients with metastatic NSCLC treated with immune checkpoint inhibitors. JAMA Netw Open. 2024; 7(1): e2352302.

[37] Kfoury M, Najean M, Lappara A, et al. Analysis of the association between prospectively collected immune-related adverse events and survival in patients with solid tumour treated with immune-checkpoint blockers, taking into account immortal-time bias. Cancer Treat Rev. 2022; 110: 102452.

[38] Wang S, Dougan S, Dougan M. Immune mechanisms of toxicity from checkpoint inhibitors. Trends Cancer. 2023; 9(7): 543-553.

[39] Vaziri H, Turshudzhyan A, Vecchio E. Immunotherapy-induced colitis: a comprehensive review of epidemiology, clinical presentation, diagnostic workup, and management plan. J Clin Gastroenterol. 2022; 56(7): 555-564.

[40] Sturm D, Capper D, Andreiuolo F, et al. Multiomic neuropathology improves diagnostic accuracy in pediatric neuro-oncology. Nat Med. 2023; 29(4): 917-926.

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2026 The Author(s). Clinical and Translational Discovery published by JohnWiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.