Purpose: Almost all cervical cancers are caused by human papillomavirus (HPV) and patients with advanced stage are at high risk for relapse. Circulating HPV DNA (HPV ctDNA) may serve as a residual tumor marker at the end of chemoradiation or to predict relapse during the follow-up period. Experimental Design: We analyzed serum samples from 94 HPV16- or HPV18-related CCs from the BioRAIDs prospective cohort. Samples were collected before and after treatment and during an 18-month follow-up period. Using digital droplet PCR (ddPCR), we assessed the relevance of circulating HPV E7 gene as a marker for residual disease compared to HPV integration site and PIK3CA mutations. Finally, the prognostic impact of circulating HPV E7 gene was assessed with its prediction value of relapse. Results: HPV E7 gene was the most sensitive tumor marker, superior to both HPV integration sites and PIK3CA mutations in serum. Circulating HPV DNA (HPV ctDNA) was detected in 63% (59/94) of patients, before treatment. HPV ctDNA detection in serum sample was associated with high FIGO stage (P = 0.02) and para-aortic lymph node involvement (P = 0.01). The level of HPV ctDNA was positively correlated with HPV copy number in the tumor (R = 0.39, P < 0.001). Complete clearance of HPV ctDNA by the end of treatment was significantly associated with a longer PFS (P < 0.0001). Patients with persistent HPV ctDNA in serum relapsed with a median time of 10 months (range, 2–15) from HPV ctDNA detection. Conclusions: HPV ctDNA detection is a useful marker to predict relapse in cervical cancer. See related commentary by Wentzensen and Clarke, p. 5733

Background: Cervical cancer (CC) remains a leading cause of gynaecological cancer-related mortality with infection by human papilloma virus (HPV) being the most important risk factor. We analysed the association between different viral integration signatures, clinical parameters and outcome in pre-treated CCs. Methods: Different integration signatures were identified using HPV double capture followed by next-generation sequencing (NGS) in 272 CC patients from the BioRAIDs study [NCT02428842]. Correlations between HPV integration signatures and clinical, biological and molecular features were assessed. Results: Episomal HPV was much less frequent in CC as compared to anal carcinoma (p < 0.0001). We identified >300 different HPV-chromosomal junctions (inter- or intra-genic). The most frequent integration site in CC was in MACROD2 gene followed by MIPOL1/TTC6 and TP63. HPV integration signatures were not associated with histological subtype, FIGO staging, treatment or PFS. HPVs were more frequently episomal in PIK3CA mutated tumours (p = 0.023). Viral integration type was dependent on HPV genotype (p < 0.0001); HPV18 and HPV45 being always integrated. High HPV copy number was associated with longer PFS (p = 0.011). Conclusions: This is to our knowledge the first study assessing the prognostic value of HPV integration in a prospectively annotated CC cohort, which detects a hotspot of HPV integration at MACROD2; involved in impaired PARP1 activity and chromosome instability.

Chemoradiotherapy (CRT) is the standard of care for patients diagnosed with locally advanced cervical cancer (LACC), a human papillomavirus (HPV)-related cancer that relapses in 30%-60% of patients. This study aimed to (i) design HPV droplet digital PCR (ddPCR) assays for blood detection (including rare genotypes) and (ii) monitor blood HPV circulating tumor DNA (HPV ctDNA) levels during CRT in patients with LACC. We analyzed blood and tumor samples from 55 patients with HPV-positive LACC treated by CRT in a retrospective cohort (n = 41) and a prospective cohort (n = 14). HPV-ctDNA detection was carried out by genotype-specific ddPCR. HPV ctDNA was successfully detected in 69% of patients (n = 38/55) before CRT for LACC, including nine patients with a rare genotype. HPV-ctDNA level was correlated with HPV copy number in the tumor (r = 0.41, P < 0.001). HPV-ctDNA positivity for HPV18 (20%, n = 2/10) was significantly lower than for HPV16 (77%, n = 27/35) or other types (90%, n = 9/10, P = 0.002). HPV-ctDNA detection (positive versus negative) before CRT was associated with tumor stage (P = 0.037) and lymph node status (P = 0.02). Taking into account all samples from the end of CRT and during follow-up in the prospective cohort, positive HPV-ctDNA detection was associated with lower disease-free survival (DFS) (P = 0.048) and overall survival (OS) (P = 0.0013). This is one of the largest studies to report HPV-ctDNA detection before CRT and showed clearance of HPV ctDNA at the end of treatment in most patients. Residual HPV ctDNA at the end of CRT or during follow-up could help to identify patients more likely to experience subsequent relapse.

The role of human papillomavirus (HPV) in anal squamous cell carcinoma (ASCC) carcinogenesis has been clearly established, involving the expression of viral oncoproteins and optional viral DNA integration into the host genome. In this article, we describe the various mechanisms and sites of HPV DNA insertion and assess their prognostic and predictive value in a large series of patients with HPV-positive ASCC with long-term follow-up. We retrospectively analyzed 96 tumor samples from 93 HPV-positive ASCC patients using the Capture-HPV method followed by Next-Generation Sequencing, allowing determination of HPV genotype and identification of the mechanisms and sites of viral genome integration. We identified five different mechanistic signatures of HPV insertions. The distribution of HPV signatures differed from that previously described in HPV-positive cervical carcinoma (p < 0.001). In ASCC samples, the HPV genome more frequently remained in episomal form (45.2%). The most common signature of HPV insertion was MJ-SC (26.9%), i.e., HPV–chromosomal junctions scattered at different loci. Functionally, HPV integration signatures were not associated with survival or response to treatment, but were associated with viral load (p = 0.022) and PIK3CA mutation (p = 0.0069). High viral load was associated with longer survival in both univariate (p = 0.044) and multivariate (p = 0.011) analyses. Finally, HPV integration occurred on most human chromosomes, but intragenic integration into the NFIX gene was recurrently observed (n = 4/51 tumors). Overall, the distribution of mechanistic signatures of HPV insertions in ASCC was different from that observed in cervical carcinoma and was associated with viral load and PIK3CA mutation. We confirmed recurrent targeting of NFIX by HPV integration, suggesting a role for this gene in ASCC carcinogenesis.

Immune checkpoint inhibitors improve the treatment of many solid tumors and have shown encouraging results in advanced squamous cell carcinoma (SCC), yet only a minority of patients respond to immune checkpoint inhibitor monotherapy. We conducted the PEVOsq trial, an open-label, nonrandomized, multicenter, basket phase 2 trial to evaluate the combination of pembrolizumab and vorinostat in recurrent/metastatic SCC of various origins. The primary endpoint was the objective response rate (ORR) in each tumor cohort during treatment as per the investigators’ assessment. Secondary endpoints included safety and antitumor activity evaluation in terms of centrally confirmed ORR, progression-free survival, overall survival and duration of response. In the efficacy population (n = 107), the ORR was met in cervical (39%), anal (31%) and vulvar/vaginal (19%) cancer cohorts, but not in head and neck SCC (19%) or penile (18%) cancer cohorts (overall ORR = 26%). Median progression-free survival was 4.0 months (95% confidence interval: 2.6–4.3), and median overall survival was 11.1 months (95% confidence interval: 9.2–17.4). In the safety population, 101 (91%) of 111 patients developed at least one treatment-related adverse event, with 39% and 5.4% of patients experiencing at least one grade 3 and grade 4 treatment-related adverse event, respectively. Vorinostat-related toxicity prompted a dose reduction/interruption in 66% of patients. Whole-exome sequencing analyses revealed several potential predictive biomarkers of response to treatment. Further studies in a larger number of patients are required to validate these findings. ClinicalTrials.gov identifier: NCT04357873.