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Supplementary Figure from Tumor-Naïve Multimodal Profiling of Circulating Tumor DNA in Head and Neck Squamous Cell Carcinoma

Supplementary Data from HPV Sequencing Facilitates Ultrasensitive Detection of HPV Circulating Tumor DNA

Supplementary Figure from HPV Sequencing Facilitates Ultrasensitive Detection of HPV Circulating Tumor DNA

Supplementary Data from Tumor-Naïve Multimodal Profiling of Circulating Tumor DNA in Head and Neck Squamous Cell Carcinoma

Purpose:Circulating tumor DNA (ctDNA) enables personalized treatment strategies in oncology by providing a noninvasive source of clinical biomarkers. In patients with low ctDNA abundance, tumor-naïve methods are needed to facilitate clinical implementation. Here, using locoregionally confined head and neck squamous cell carcinoma (HNSCC) as an example, we demonstrate tumor-naïve detection of ctDNA by simultaneous profiling of mutations and methylation.Experimental Design:We conducted CAncer Personalized Profiling by deep Sequencing (CAPP-seq) and cell-free Methylated DNA ImmunoPrecipitation and high-throughput sequencing (cfMeDIP-seq) for detection of ctDNA-derived somatic mutations and aberrant methylation, respectively. We analyzed 77 plasma samples from 30 patients with stage I–IVA human papillomavirus–negative HNSCC as well as plasma samples from 20 risk-matched healthy controls. In addition, we analyzed leukocytes from patients and controls.Results:CAPP-seq identified mutations in 20 of 30 patients at frequencies similar to that of The Tumor Genome Atlas (TCGA). Differential methylation analysis of cfMeDIP-seq profiles identified 941 ctDNA-derived hypermethylated regions enriched for CpG islands and HNSCC-specific methylation patterns. Both methods demonstrated an association between ctDNA abundance and shorter fragment lengths. In addition, mutation- and methylation-based ctDNA abundance was highly correlated (r > 0.85). Patients with detectable pretreatment ctDNA by both methods demonstrated significantly worse overall survival (HR = 7.5; P = 0.025) independent of clinical stage, with lack of ctDNA clearance post-treatment strongly correlating with recurrence. We further leveraged cfMeDIP-seq profiles to validate a prognostic signature identified from TCGA samples.Conclusions:Tumor-naïve detection of ctDNA by multimodal profiling may facilitate biomarker discovery and clinical use in low ctDNA abundance applications.

Purpose:Human papillomavirus (HPV) DNA offers a convenient circulating tumor DNA (ctDNA) marker for HPV-associated malignancies, but current methods, such as digital PCR (dPCR), provide insufficient accuracy for clinical applications in patients with low disease burden. We asked whether a next-generation sequencing approach, HPV sequencing (HPV-seq), could provide quantitative and qualitative assessment of HPV ctDNA in low disease burden settings.Experimental Design:We conducted preclinical technical validation studies on HPV-seq and applied it retrospectively to a prospective multicenter cohort of patients with locally advanced cervix cancer (NCT02388698) and a cohort of patients with oropharynx cancer. HPV-seq results were compared with dPCR. The primary outcome was progression-free survival (PFS) according to end-of-treatment HPV ctDNA detectability.Results:HPV-seq achieved reproducible detection of HPV DNA at levels less than 0.6 copies in cell line data. HPV-seq and dPCR results for patients were highly correlated (R2 = 0.95, P = 1.9 × 10–29) with HPV-seq detecting ctDNA at levels down to 0.03 copies/mL plasma in dPCR-negative posttreatment samples. Detectable HPV ctDNA at end-of-treatment was associated with inferior PFS with 100% sensitivity and 67% specificity for recurrence. Accurate HPV genotyping was successful from 100% of pretreatment samples. HPV ctDNA fragment sizes were consistently shorter than non–cancer-derived cell-free DNA (cfDNA) fragments, and stereotyped cfDNA fragmentomic patterns were observed across HPV genomes.Conclusions:HPV-seq is a quantitative method for ctDNA detection that outperforms dPCR and reveals qualitative information about ctDNA. Our findings in this proof-of-principle study could have implications for treatment monitoring of disease burden in HPV-related cancers. Future prospective studies are needed to confirm that patients with undetectable HPV ctDNA following chemoradiotherapy have exceptionally high cure rates.

Background: Human papillomavirus (HPV)-associated cancers often present with locoregionally confined disease and are treated with curative intent. An emerging treatment paradigm includes radical therapy (i.e., chemoradiotherapy or surgery) followed by adjuvant treatment. Accurate detection of minimal residual disease (MRD) following radical therapy could enable personalized use of adjuvant treatment. The HPV genome offers a convenient circulating tumor (ct)DNA marker for HPV-associated cancers, but current methods such as digital (d)PCR provide insufficient accuracy for accurate MRD detection and for other clinical applications in patients with low disease burden. We asked whether a next-generation sequencing approach (HPV-seq) could provide quantitative and qualitative assessment of HPV ctDNA in low-disease-burden settings. Methods: We conducted preclinical technical validation studies on HPV-seq using cervix cancer cell lines. We then applied HPV-seq retrospectively to a prospective multicenter cohort study of locally advanced cervix cancer patients accrued from 2015 to 2016 (NCT02388698). Patients were treated with radical chemoradiotherapy with blood obtained at baseline, end of treatment, and 3 months post-treatment. Median follow-up was 27.5 months. In 38 plasma samples from 17 patients, HPV-seq results were compared with dPCR. The primary outcome was progression-free survival according to end-of-treatment HPV ctDNA detectability. Results: HPV-seq achieved reproducible detection of HPV DNA at levels Conclusions: HPV-seq is a quantitative method for ctDNA detection that outperforms dPCR. HPV-seq also reveals qualitative information about ctDNA fragments such as HPV genotype and ctDNA fragment length distribution. Our findings will have implications for MRD detection in HPV-related cancers. Future prospective studies are needed to confirm that patients with undetectable HPV ctDNA following chemoradiotherapy have exceptionally high cure rates. Citation Format: Eric Leung, Kathy Han, Jinfeng Zou, Zhen Zhao, Yangqiao Zheng, Ting Ting Wang, Lillian L. Siu, Trevor J. Pugh, Scott V. Bratman. HPV sequencing facilitates ultrasensitive detection of HPV circulating tumor DNA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A45.