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Background HPV-associated malignancies are a major health concern, with >630,000 cases occurring annually worldwide. Immune checkpoint blockade (ICB) with a dual inhibitor of programmed cell death ligand 1 (PD-L1) and transforming growth factor β (TGFβ), induced clinical responses in 30.5% of patients with HPV-associated malignancies who were ICB-naïve and 10% who were ICB-resistant. 1 2 Circulating tumor DNA (ctDNA) is a promising, non-invasive tool for monitoring tumor dynamics and potentially predicting therapeutic outcomes; however, few studies have evaluated ctDNA kinetics in patients with HPV-associated malignancies treated with immunotherapy.3 We evaluated HPV16/18-ctDNA as a predictive biomarker of clinical response and survival in patients with HPV-associated malignancies treated with dual blockade of PD-L1 and TGF-β.Methods Peripheral blood was collected before and after 1 and 3 cycles of administration of a dual inhibitor of PD-L1 and TGF-β, bintrafusp alfa, in 51 patients with HPV16/18-associated cancers ( NCT03427411). HPV status was confirmed through tumor-based commercial assays. Baseline levels and changes in plasma HPV16/18-ctDNA were evaluated by digital droplet PCR with primers for HPV16-E6 and HPV18-E6, and associations with tumor burden, peripheral immune parameters, and clinical outcomes were investigated.Results HPV16/18-ctDNA was detectable in 91.1% of patients, with detection rates of 87.1% in HPV16+ and 100% in HPV18+ cases. Baseline HPV16/18-ctDNA levels were positively correlated with tumor size (p=0.006) and elevated in ICB-resistant versus ICB-naïve patients (p=0.0002). Decreases in HPV16/18-ctDNA levels after 3 cycles of therapy associated with clinical response (p=0.0006), with decreases noted in 80% of patients developing partial or complete responses versus 26% of patients without objective responses. In contrast, greater increases in HPV16/18-ctDNA levels were associated with disease progression. Lower baseline and on-treatment HPV16/18-ctDNA levels, and changes in levels were also associated with prolonged overall survival (OS). A combined predictive model integrating baseline levels of HPV16/18-ctDNA with circulating interleukin 8 predicted OS of ≥180 days with 86% accuracy, where patients with a predicted response probability >0.5 had a longer median OS than patients with a poor predicted response (971 vs 121 days, p<0.0001).Conclusions This study revealed that baseline levels and longitudinal changes in HPV16/18-ctDNA correlate with clinical responses in patients with HPV-associated malignancies treated with dual blockade of PD-L1 and TGF-β. These data support the continued investigation of easily accessible peripheral biomarkers in patients with advanced cancer, and suggest that evaluation of multiple circulating biomarkers may be beneficial in predicting clinical response to maximize patient benefit.References Strauss J, Heery CR, Schlom J, Madan RA, Cao L, Kang Z, Lamping E, Marte JL, Donahue RN, Grenga I, Cordes L, Christensen O, Mahnke L, Helwig C, Gulley JL. Clin Cancer Res. 2018;24:1287-1295.Strauss J, Gatti-Mays ME, Cho BC, Hill A, Salas S, McClay E, Redman JM, Abdul Sater H, Donahue RN, Jochems C, Lamping E, Burmeister A, Marte JL, Cordes LM, Bilusic M, Karzai F., Ojalvo LS, Jehl G, Rolfe PA, Hinrichs C, Madan RA, Schlom J, Gulley JL. Bintrafusp alfa, a bifunctional fusion protein targeting TGF-β and PD-L1, in patients with human papillomavirus-associated malignancies. J Immunother Cancer. 2020;8:e001395.Goswami M, Schlom J, Donahue RN. Peripheral surrogates of tumor burden to guide chemotherapeutic and immunotherapeutic strategies for HPV-associated malignancies. Oncotarget. 2023;14:758-774.Ethics Approval All subjects gave written informed consent. Study protocol ( NCT03427411) was approved by the NIH’s IRB, and conducted in accordance with institutional and federal guidelines.