Purpose: Human papillomavirus (HPV) is found in 90% of squamous cell carcinomas of the anal canal (SCCA). We investigated the clinical validity of HPV circulating tumor DNA (ctDNA) detection in patients enrolled in the Epitopes-HPV02 trial that demonstrated the efficacy of docetaxel, cisplatin, and 5-FU as first-line chemotherapy in advanced SCCA.Experimental Design: According to the protocol, serum samples were collected before chemotherapy and on completion of chemotherapy. HPV16 ctDNA was quantified by droplet digital PCR (ddPCR) and correlated with prospectively registered patient characteristics and outcomes. A landmark was set at the time of chemotherapy completion for postchemotherapy progression-free survival (PFS) analyses.Results: Among 57 patients with HPV16-related advanced SCCA, HPV ctDNA was detected in 91.1% (95% confidence interval, 81.1-96.2) of baseline samples. Baseline HPV ctDNA levels were not associated with any patient characteristics; baseline ctDNA level below the cutoff obtained by AUC (area under the curve) was associated with a longer PFS (HR ¼ 2.1; P ¼ 0.04). Among the 36 patients who completed 5 months of chemotherapy, residual HPV ctDNA was detected after chemotherapy in 38.9% of patients. Residual HPV ctDNA detected at chemotherapy completion was associated with shorter postchemotherapy PFS (median PFS 3.4 months vs. not reached; HR ¼ 5.5; P < 0.001) and a reduction of 1-year overall survival rate (OR ¼ 7.0; P ¼ 0.02).Conclusions: This prospective study in advanced SCCA demonstrated a significant prognostic impact of HPV ctDNA level before first-line chemotherapy and HPV ctDNA negativity after chemotherapy completion. With a limited cost and short turnaround, this assay is a promising tool to optimize the therapeutic management of SCCA.See related commentary by Morris, p. 2030

Human papillomavirus (HPV) 16 exhibits different variants that may differ in their carcinogenic risk. To identify some high-risk variants, we sequenced and compared HPV16 whole genomes obtained from a longitudinal cohort of 34 HPV16-infected women who had either spontaneously cleared their infection (clearance group or "C"), or developed cervical high-grade lesions following a viral persistence (group persistence or "P"). Phylogenetic analysis of paired samples obtained at the beginning (C0 or P0) and at the end (C2 or P2) of the follow-up (median intervals between C0-C2 and between P0-P2 were 16 and 36.5 months, respectively) revealed a low genetic variability within the host compared to the genetic interhost diversity. By comparing our HPV16 sequences to a reference sequence, we observed 301 different substitutions, more often transitions (60.9%) than transversions (39.1%), that occurred throughout the viral genome, but with a low frequency in E6 and E7 oncogenes (10 and 9 substitutions), suggesting a high conservation of these genes. Deletions and insertions were mostly observed in intergenic regions of the virus. The only significant substitution found between the subgroups C2 and P2 was observed in the L2 gene (L330F), with an unclear biological relevance.

Simple and standardized approaches for genome analysis of human papillomavirus (HPV) by next-generation sequencing are needed. The aim of the study was to develop a protocol for direct deep sequencing of high-risk (hr) HPV strains, based on the widely used commercial Hybrid Capture 2 (QIAGEN) test, without any additional probe design. This protocol was applied to 15 HPV-positive and two HPV-negative cervical samples or cell lines and validated at the genotype level by comparing the sequencing results to those obtained using a commercial genotyping kit. The performance of our protocol, presented in this proof-of-principle study, supports its use for accurate characterization of genetic variants of hrHPV.Human papillomaviruses (HPVs) are a leading cause of anogenital and oropharyngeal cancers worldwide [1,2]. HPVs are classified into genera, species, lineages, sublineages, and variants. Bzhalava et al. estimated that at least 400 different HPV types exist [3]. Among the various HPV types infecting the squamous epithelium of the lower anogenital tracts of both males and females, some are rarely, if ever, found in cervical cancer and are therefore called "low-risk HPVs' (lrHPVs), while others are considered high-risk (hr) types because they have been detected in cancers. Many studies have confirmed that 13 hrHPVs (

<div>AbstractPurpose:<p>Human papillomavirus (HPV) is found in 90% of squamous cell carcinomas of the anal canal (SCCA). We investigated the clinical validity of HPV circulating tumor DNA (ctDNA) detection in patients enrolled in the Epitopes-HPV02 trial that demonstrated the efficacy of docetaxel, cisplatin, and 5-FU as first-line chemotherapy in advanced SCCA.</p>Experimental Design:<p>According to the protocol, serum samples were collected before chemotherapy and on completion of chemotherapy. HPV16 ctDNA was quantified by droplet digital PCR (ddPCR) and correlated with prospectively registered patient characteristics and outcomes. A landmark was set at the time of chemotherapy completion for postchemotherapy progression-free survival (PFS) analyses.</p>Results:<p>Among 57 patients with HPV16-related advanced SCCA, HPV ctDNA was detected in 91.1% (95% confidence interval, 81.1–96.2) of baseline samples. Baseline HPV ctDNA levels were not associated with any patient characteristics; baseline ctDNA level below the cutoff obtained by AUC (area under the curve) was associated with a longer PFS (HR = 2.1; <i>P</i> = 0.04). Among the 36 patients who completed 5 months of chemotherapy, residual HPV ctDNA was detected after chemotherapy in 38.9% of patients. Residual HPV ctDNA detected at chemotherapy completion was associated with shorter postchemotherapy PFS (median PFS 3.4 months vs. not reached; HR = 5.5; <i>P</i> < 0.001) and a reduction of 1-year overall survival rate (OR = 7.0; <i>P</i> = 0.02).</p>Conclusions:<p>This prospective study in advanced SCCA demonstrated a significant prognostic impact of HPV ctDNA level before first-line chemotherapy and HPV ctDNA negativity after chemotherapy completion. With a limited cost and short turnaround, this assay is a promising tool to optimize the therapeutic management of SCCA.</p><p><i>See related commentary by Morris, p. 2030</i></p></div>