Fig. S1. E7 sequence alignments of HPV 16 variants and HPV18 variants for ddPCR assay design Fig. S2. ddPCR dual detection assay for HPV 16 and HPV18 with genomic DNA from cervical cancer cell lines Fig. S3. Dilution linearity and quantification range with HPV DNA spiked plasma samples Fig. S4. Cervical cancer patient blood HPV genotyping for 14 HPV types Table S1. Record on healthy blood donors Table S2. HPV variants covered by the HPV16 and HPV18 specific detection assays Table S3. Sequences of ddPCR primers and probes used for HPV16 and HPV18 ccfDNA detection Table S4. Interference study with plasma containing potential interferents and spiked HPV16 DNA
ARTICLE ABSTRACTPurpose: Circulating cell-free (ccf) human papillomavirus (HPV) DNA may serve as a unique tumor marker for HPV-associated malignancies, including cervical cancer. We developed a method to genotype and quantify circulating HPV DNA in patients with HPV16- or HPV18-positive metastatic cervical cancer for potential disease monitoring and treatment-related decision making.Experimental Design: In this retrospective study, HPV ccfDNA was measured in serum samples from 19 metastatic cervical cancer patients by duplex digital droplet PCR (ddPCR). Nine patients had received tumor-infiltrating lymphocyte (TIL) immunotherapy. ccfDNA data were aligned with the tumor HPV genotype, drug treatment, and clinical outcome.Results: In blinded tests, HPV ccfDNA was detected in 19 of 19 (100%) patients with HPV-positive metastatic cervical cancer but not in any of the 45 healthy blood donors. The HPV genotype harbored in the patients' tumors was correctly identified in 87 of 87 (100%) sequential patient serum samples from 9 patients who received TIL immunotherapy. In three patients who experienced objective cancer regression after TIL treatment, a transient HPV ccfDNA peak was detected 2–3 days after TIL infusion. Furthermore, persistent clearance of HPV ccfDNA was only observed in two patients who experienced complete response (CR) after TIL immunotherapy.Conclusions: HPV ccfDNA represents a promising tumor marker for noninvasive HPV genotyping and may be used in selecting patients for HPV type–specific T-cell-based immunotherapies. It may also have value in detecting antitumor activity of therapeutic agents and in the long-term follow-up of cervical cancer patients in remission. Clin Cancer Res; 23(22); 6856–62. ©2017 AACR.