HPV-positive Cancer Research Articles

Germline variants of homology-directed repair or mismatch repair genes in cervical cancer.

While cervical cancer is associated with a persistent human papillomavirus (HPV) infection, the progression to cancer is influenced by genomic risk factors that have remained largely obscure. Pathogenic variants in genes of the homology-directed repair (HDR) or mismatch repair (MMR) are known to predispose to diverse tumour entities including breast and ovarian cancer (HDR) or colon and endometrial cancer (MMR). We here investigate the spectrum of HDR and MMR germline variants in cervical cancer, with particular focus on the HPV status and histological subgroups. We performed targeted next-generation sequencing for 5 MMR genes and 12 HDR genes on 728 German patients with cervical dysplasia or invasive cancer. In total, 4% of our patients carried a pathogenic germline variant, based on ClinVar classifications and additional ESM1b and AlphaMissense predictions. These included 15 patients with truncating variants in HDR genes (BARD1, BRCA1, BRCA2, BRIP1, FANCM, RAD51D and SLX4). MMR-related gene variants were less prevalent and mainly of the missense type. While MMR-related gene variants tended to associate with adenocarcinomas, HDR gene variants were commonly observed in squamous cancers. While one patient with HPV-negative cancer carried a pathogenic MMR gene variant (in MSH6), the HDR germline variants were found in patients with HPV-positive cancers and tended to associate with HPV18. Taken together, our study supports a potentially risk-modifying role of MMR and HDR germline variants in cervical cancer but no association with HPV-negative status. These variants may be exploitable in future therapeutic managements.

Research progress on human papillomavirus-negative cervical cancer: A review.

Cervical cancer is the fourth most common cancer in women worldwide. The vast majority of cervical cancers are associated with human papillomavirus (HPV) infection, but a small proportion of cervical cancers occur independently of HPV infection, with different subtypes having varying rates of occurrence. Despite the presence of false negatives in current testing, improving the accuracy of detection is crucial for studying the pathogenesis of HPV-negative cervical cancer and improving the prognosis of these patients. Existing research suggests that HPV-negative cervical cancer has a different pathogenesis from HPV-positive cervical cancer, although the exact mechanism is not yet clear. It is currently believed to be associated with the immune microenvironment, certain tumor gene mutations, and some long noncoding RNAs. This article provides an overview of the latest research progress on HPV-negative cervical cancer, including possible reasons, pathogenesis, pathological features, and clinical characteristics, aiming to provide new insights for diagnosis, treatment, and prognosis improvement.

74 (PB062): The transcriptional regulation of TBX3 by the c-Myc/E6/E7 axis and targeting this axis for the treatment of HPV-positive cervical cancer

74 (PB062): The transcriptional regulation of TBX3 by the c-Myc/E6/E7 axis and targeting this axis for the treatment of HPV-positive cervical cancer

Human papillomavirus 16 replication converts SAMHD1 into a homologous recombination factor and promotes its recruitment to replicating viral DNA.

Human papillomaviruses (HPVs) are causative agents in around 5% of all human cancers. The development of anti-viral therapeutics depends upon an increased understanding of the viral life cycle. Here, we demonstrate that HPV16 replication converts sterile alpha motif and histidine-aspartic domain HD-containing protein 1 (SAMHD1) into a homologous recombination (HR) factor via phosphorylation. This phosphorylation promotes recruitment of SAMHD1 to viral DNA to assist with replication. A SAMHD1 mutant that mimics phosphorylation is hyper-recruited to viral DNA and attenuates viral replication. Expression of this mutant in HPV16-immortalized cells attenuates the growth of these cells, but not cells immortalized by the viral oncogenes E6/E7 alone. Finally, we demonstrate that the phosphatase inhibitor endothall promotes hyper-recruitment of endogenous SAMHD1 to HPV16 replicating DNA and can attenuate the growth of both HPV16-immortalized human foreskin keratinocytes (HFKs) and HPV16-positive head and neck cancer cell lines. We propose that phosphatase inhibitors represent a novel tool for combating HPV infections and disease.

932P Accuracy and prognostic implications of extranodal extension on radiologic imaging in HPV-positive oropharyngeal cancer (HNCIG-ENE): A multinational, real-world study

932P Accuracy and prognostic implications of extranodal extension on radiologic imaging in HPV-positive oropharyngeal cancer (HNCIG-ENE): A multinational, real-world study

Overall Survival, Treatment Duration, and Rechallenge Outcomes With ICI Therapy for Recurrent or Metastatic HNSCC

Immune checkpoint inhibition (ICI) is a frontline treatment for recurrent or metastatic head and neck squamous cell carcinoma (R/M HNSCC), but questions remain surrounding optimal duration of therapy, benefits and risks of ICI rechallenge, and efficacy in first vs subsequent lines of therapy. To estimate survival in US patients receiving ICI-based treatment for R/M HNSCC, compare outcomes associated with treatment discontinuation vs continuation at 1 or 2 years, and assess outcomes after immunotherapy rechallenge. This retrospective, population-based cohort study included adult patients in the Flatiron Health nationwide oncology database treated with immunotherapy for R/M HNSCC from 2015 to 2023. Data cutoff was August 31, 2023; data analysis was conducted from December 2023 to February 2024. Treatment continuation vs discontinuation at 1 and 2 years; rechallenge with ICI after at least a 60-day period off ICI therapy without intervening systemic treatment (immediate rechallenge), or with intervening systemic treatment (delayed rechallenge). Overall survival (OS) from ICI initiation was analyzed using the Kaplan-Meier method. Cox multivariable regression was used to examine associations of key variables (line of therapy, human papillomavirus [HPV] status, Eastern Cooperative Oncology Group [ECOG] performance status) with survival. The cohort included 4549 patients with R/M HNSCC who received ICI-containing therapy (median [IQR] age, 66 [59-72] years; 3551 [78.1%] male; 56 [1.2%] Asian, 260 [5.7%] Black or African American, 3020 [66.4%] White, 1213 [26.7%] other or unknown race; 3226 [70.9%] ECOG performance status 0 or 1). There were 3000 patients (65.9%) who received ICI in frontline and 1207 (26.5%) in second line; 3478 patients (76.5%) received ICI monotherapy. Median (IQR) OS was 10.9 (4.1-29.1) months and was longer in patients who received ICI in frontline therapy (12.2 [4.8-32.0] vs 8.7 [3.2-22.4] months), had HPV-positive cancer (16.6 [6.5-43.9] vs 8.8 [3.5-24.0] months), and had ECOG performance status 0 or 1 (13.5 [5.2-33.9] vs 5.5 [2.0-13.7] months). There were no survival differences on adjusted analysis between patients who stopped vs those who continued ICI at 1 or 2 years. Median (IQR) OS after ICI rechallenge was 15.7 (13.7-21.9) months in the immediate rechallenge group and 9.9 (3.7-18.1) months in the delayed rechallenge group. In this large cohort study of patients with R/M HNSCC receiving ICI-based therapy, survival estimates closely mirrored clinical trial results, both in frontline and later-line settings. Discontinuation of ICI in long-term responders at 1 or 2 years may be a reasonable strategy that does not appear to compromise survival. ICI rechallenge was associated with clinical benefit in a subset of patients.

Hypofractionated radiation therapy alone for human papillomavirus-related oropharyngeal cancer.

To report a single-institutional experience with hypofractionated radiation therapy alone for human papillomavirus (HPV)-positive oropharyngeal cancer. A total of 101 consecutive patients were treated by radiation therapy alone using a regimen of 66 Gy in 30 fractions (60 patients) or 70 Gy in 33 fractions (41 patients) for newly diagnosed p16-positive squamous cell carcinoma of the oropharynx. Sixty-seven patients (67%) were never smokers. The 3-year actuarial rates of overall survival, local-regional control, and progression-free survival were 94%, 93%, and 89%, respectively. Among never-smokers, the 3-year rates of overall survival and local-regional control were 98% and 100%, respectively. The grade 3+ acute toxicity rate was 21%, with the most commonly observed side effects related to mucositis. Hypofractionated radiation alone resulted in excellent outcomes for patients with HPV-positive oropharyngeal cancer. A prospective clinical trial investigating this modality in the setting of de-escalation is currently underway.

Differential expression of host oncogenes in human papillomavirus-associated nasopharyngeal and cervical epithelial cancers.

Human papillomavirus (HPV)-related cervical and nasopharyngeal cancers differ in molecular mechanisms underlying the oncogenic processes. The disparity may be attributed to differential expression of oncoproteins. The current study investigated the host oncogenes expression pattern in HPV-associated cervical and nasopharyngeal cancer. Formalin-fixed paraffin-embedded tissues originating from the nasopharyngeal and cervical regions were screened using Hematoxylin and Eosin staining. Genomic DNA and total RNA were extracted from confirmed cancer biopsies and non-cancer tissues (NC). HPV was detected by PCR using MY09/GP5+/6+ primers. Protein expression levels of AKT, IQGAP1, and MMP16 in HPV-infected cancers and controls were determined by immunohistochemistry. RT-qPCR was used to profile mRNAs of the oncogenes. AKT and IQGAP1 proteins were highly expressed in the epithelial cancers compared with the non-cancer tissues (p < 0.05). IQGAP1 and MMP16 mRNAs level was significantly higher in the cancers than in the NC (p < 0.05), but not AKT mRNA levels. MMP16 protein was ubiquitously expressed in all tissues. AKT mRNA level was significantly elevated in CC compared with NPC (p < 0.001). However, the difference in AKT, IQGAP1 and MMP16 proteins level between CC and NPC was not significant (p > 0.05). The oncoproteins expression level between the HPV-positive and HPV-negative cancer biopsies showed no significant difference (p < 0.05). Current study reports AKT but not IQGAP1 and MMP16 mRNAs differentially expression in cervical and nasopharyngeal cancers, independent of HPV infection status.

Gd-GQDs as nanotheranostic platform for the treatment of HPV-positive oropharyngeal cancer.

In this study, we developed new gadolinium-graphene quantum dot nanoparticles (Gd-GQDs) as a theranostic platform for magnetic resonance imaging and improved the efficiency of radiotherapy in HPV-positive oropharyngeal cancer. Based on cell toxicity results, Gd-GQD NPs were nontoxic for both cancer and normal cell lines up to 25µg/ml. These NPs enhance the cytotoxic effect of radiation only on cancer cells but not on normal cells. The flow cytometry analysis indicated that cell death mainly occurred in the late phase of apoptosis. The immunocytochemical analysis was used to evaluate apoptosis pathway proteins. The Bcl-2 and p53 protein levels did not differ statistically significantly between radiation alone group and those that received irradiation in combination with NPs. In contrast, the combination group exhibited a significant increase in Bax protein expression, suggesting that cells could undergo apoptosis independent of the p53 pathway. Magnetic resonance (MR) imaging showed that Gd-GQD NPs, when used at low concentrations, enhanced T1-weighted signal intensity resulting from T1 shortening effects. At higher concentrations, the T2 shortening effect became predominant and was able to decrease the signal intensity. Gd-GQD appears to offer a novel approach for enhancing the effectiveness of radiation treatment and facilitating MR imaging for monitoring HPV-positive tumors.

Structure and transcription of integrated HPV DNA in vulvar carcinomas

HPV infections are associated with a fraction of vulvar cancers. Through hybridization capture and DNA sequencing, HPV DNA was detected in five of thirteen vulvar cancers. HPV16 DNA was integrated into human DNA in three of the five. The insertions were in introns of human NCKAP1, C5orf67, and LRP1B. Integrations in NCKAP1 and C5orf67 were flanked by short direct repeats in the human DNA, consistent with HPV DNA insertions at sites of abortive, staggered, endonucleolytic incisions. The insertion in C5orf67 was present as a 36 kbp, human-HPV-hetero-catemeric DNA as either an extrachromosomal circle or a tandem repeat within the human genome. The human circularization/repeat junction was defined at single nucleotide resolution. The integrated viral DNA segments all retained an intact upstream regulatory region and the adjacent viral E6 and E7 oncogenes. RNA sequencing revealed that the only HPV genes consistently transcribed from the integrated viral DNAs were E7 and E6*I. The other two HPV DNA+ tumors had coinfections, but no evidence for integration. HPV-positive and HPV-negative vulvar cancers exhibited contrasting human, global gene expression patterns partially overlapping with previously observed differences between HPV-positive and HPV-negative cervical and oropharyngeal cancers. A substantial fraction of the differentially expressed genes involved immune system function. Thus, transcription and HPV DNA integration in vulvar cancers resemble those in other HPV-positive cancers. This study emphasizes the power of hybridization capture coupled with DNA and RNA sequencing to identify a broad spectrum of HPV types, determine human genome integration status of viral DNAs, and elucidate their structures.

Interplay of miR-542, miR-126, miR-143 and miR-26b with PI3K-Akt is a Diagnostic Signal and Putative Regulatory Target in HPV-Positive Cervical Cancer

Interplay of miR-542, miR-126, miR-143 and miR-26b with PI3K-Akt is a Diagnostic Signal and Putative Regulatory Target in HPV-Positive Cervical Cancer

Development and validation of an assay to quantify plasma cell-free human papillomavirus DNA for 13 high-risk types that cause 98% of HPV-positive cancers.

6065 Background: Plasma cell-free HPV DNA (cfHPV) may be a valuable tool for identification of patients with local-regionally advanced HPV-positive cancers at risk for recurrence after chemoradiation. Technical validation is required for use as an integral biomarker in a prospective clinical trial. Methods: Cell-Free 13 (i.e., CF13) is a digital-droplet PCR assay for quantification of 13 high-risk HPV types and control ERV3 in plasma that considered both variant sequences into primer/probe design and a distance matrix in phylogenetic analysis in multiplex combinations. Oligonucleotides/plasmids encoding type-specific E6/E7 regions were used as positive controls. Limit of blank, detection, quantification (LoB, LoD, LoQ), linear range, inter- and intra-assay coefficients of variation (CoV), and type-specific cross-reactivity were determined as were sensitivity and specificity for an HPV-positive diagnosis in a cohort of 278 oropharyngeal/unknown primary/oral cavity cancers tested for HPV by E6/E7 mRNA qRT-PCR or p16 IHC and HPV in situ hybridization. Results: Under identical assay conditions, the LoB, LoD and linear range were &lt;1, 5 and 5 to 200,000 virus copies without HPV type-specific cross-reactivity. Multiplexing had no effect on LoD or linearity. LoQ was 16 copies/ml plasma for all 13 HPV types. For 80 and 10,000 copies per ml, inter-assay CoV ranged from ~15-28% and ~3.2-4.6% and intra-assay CoV ranged from ~16-38% and 0.2-3.3%, respectively. cfDNA purification method (manual v automated; extraction efficiency 142% and 91% for 16 and 10,000 HPV16 copies/ml), input plasma volume (1, 2, or 4 ml), total background cfDNA (&lt;1800ng) or genomic DNA (&lt;700ng) did not affect quantification. For a diagnosis of HPV-positive cancer, sensitivity and specificity were 91.1% (214/235) and 97.7% (42/43), respectively. cfHPV was associated with gender, race, T stage, N stage, and primary treatment modality. When compared to below the median (≤230 copies/ml), cfHPV above the median was associated with worse progression-free survival (HR=2.26, 95% CI 1.23-4.17, p=0.009) in univariate analysis. However, this was no longer significant after adjustment for age, T stage, and M stage (HRadj=1.78, 95% CI 0.95-3.35, p=0.07). Conclusions: CF13 has high sensitivity, accuracy, precision, linearity, HPV type-specificity, and robustness, offering a rigorously validated assay applicable to ~98% of patients with HPV-positive cancer. CF13 had excellent clinical sensitivity and specificity for a diagnosis of HPV-positive OPC.

Results of a randomized, double-blind, placebo-controlled, phase 2 study (OpcemISA) of the combination of ISA101b and cemiplimab versus cemiplimab for recurrent/metastatic (R/M) HPV16-positive oropharyngeal cancer (OPC).

6003 Background: ISA101b (peltopepimut-S) is a therapeutic vaccine targeting the HPV16 E6/E7 oncoproteins. The synthetic long peptides of ISA101b induce specific expansion of both CD4+ T-helper cells and CD8+ cytotoxic T-cells against E6/7 oncogenes1. Combination of cemiplimab, an anti-PD-1 antibody with ISA101b elicits a synergistic anti-tumor effect2. Methods: First and second line anti-PD-1 naïve patients with confirmed HPV16+ R/M OPC were randomized to treatment with either ISA101b (subcutaneously 100µg/peptide on days 1, 29, and 50) or placebo, with cemiplimab (intravenously 350mg q/21 days) for up to 24 months or until disease progression or treatment withdrawal. The primary efficacy endpoint was ORR after ≥6 months of follow-up by independent review as per RECIST1.1. Data cut-off for this analysis was 5 July 2023. The primary safety endpoint was frequency and severity of AEs. Secondary endpoints included PFS and OS. For the latter 12-month survival data are shown. Combined Positive Score (CPS) analyses were planned subgroup analyses. A p-value &lt;0.1 was defined as statistically significant. Predefined analysis sets are the full analysis set (FAS), i.e. all patients who received ≥1 dose of study drug, and the per protocol set (PPS) which includes patients with centrally confirmed HPV16-positivity who received all 3 doses of ISA101b/placebo, had at least 1 post-baseline tumor assessment and no major protocol deviations. Results: A total of 198 patients (mean age 62.8 ±9.6 years) received ≥1 dose of study drug: 173 (87.4%) male, and 25 (12.6%) female; 110 (55.6%) were treated in first, and 74 (37.4%) in second line. Baseline characteristics were well balanced. In the ISA101b arm, ORR was 25.3% compared to 22.9% in the control arm (NS, Table). SAEs occurred in 33.0% of patients in the ISA101b arm vs 31.6% in the control arm. Patients with a CPS ≥20 treated with cemiplimab and 3 doses of ISA101b had a significantly better ORR and OS compared to patients in the control arm (Table; mOS (95% CI) not reached (28.1, -) vs 23.3 (11.9, 30.1) months, P = 0.0232 (PPS)). Patients with CPS &lt;20 had on average a shorter OS in the ISA101b arm. Conclusions: Whereas there was no advantage of the addition of ISA101b to cemiplimab regarding ORR on the overall population, in patients with CPS ≥20 ISA101b significantly improved the ORR. Median OS was better in patients with CPS ≥20 who completed a full course of ISA101b. In contrast, patients with lower CPS did not benefit. Toxicity was comparable between the 2 arms. Clinical trial information: NCT03669718 . [Table: see text]

HB-200 arenavirus-based immunotherapy plus pembrolizumab as first-line treatment of patients with recurrent/metastatic HPV16-positive head and neck cancer: Updated results.

6005 Background: Treatment options are limited for patients with recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) with no approved treatment specifically targeting human papillomavirus (HPV) 16-positive tumors. In the first-line (1L) R/M setting, only a minority of patients (~20%) respond to pembrolizumab monotherapy, including those with high programmed death ligand 1 (PD-L1) expression in the tumor. HB-200 comprises an alternating sequence of two replicating attenuated arenavirus vectors derived from LCMV (HB-201) and Pichinde virus (HB-202), respectively. HB-200 vectors express a non-oncogenic HPV16 E7E6 fusion protein and induce E6 and E7-specific CD8 T-cell responses. Previously, we reported promising preliminary clinical activity of HB-200 in combination with pembrolizumab as 1L treatment for patients with HPV16+ PD-L1+ R/M HNSCC. Here, we report updated results with a focus on PD-L1 status. Methods: In this non-randomized Phase 2 part of the study, participants with HPV16+ PD-L1 combined positive score (CPS) ≥1 R/M HNSCC were treated with HB-200 intravenously (every 3 weeks [Q3W] then Q6W) in combination with pembrolizumab (200 mg Q3W or 400 mg Q6W). Safety, T cell response, and preliminary antitumor activity were assessed. Results: As of 12 January 2024, 42 patients with HPV16+ PD-L1+ R/M HNSCC (41 of oropharynx as primary cancer site) were treated with HB-200 plus pembrolizumab in the 1L setting. Median follow-up time was 5.6 months. Characteristics of this cohort included: median age 66 years (range 50-77), 41 (98%) male, 37 (88%) White, 14 (33%) with ≥ 10 pack/year smoking history, 40 (95%) check point inhibitor (CPI) naïve (2 received CPI in the adjuvant setting), and 21 (50%) with PD-L1 CPS ≥ 20. HB-200 + pembrolizumab were generally well tolerated. Grade ≥3 treatment-related adverse events (TRAEs) were reported in 6 (14%) patients, serious TRAEs in 3 (7%) patients, and TRAEs leading to treatment discontinuation in 2 (5%) patients. No treatment-related death were reported. Among 35 evaluable patients (those with ≥ 1 tumor response assessments), the overall response rate (ORR) was 43% (3 complete response [CR], 9 partial response [PR], 3 unconfirmed PR) and the disease control rate (DCR) was 71%. Notably, among patients with PD-L1 CPS ≥20 (N = 17), ORR was 59% (3 CR, 6 PR, 1 unconfirmed PR) and DCR was 88%. Conclusions: Updated data of HB-200 arenavirus-based immunotherapy plus pembrolizumab continued to demonstrate a favorable safety profile and promising clinical activity as 1L treatment in patients with HPV16+ PD-L1+ R/M HNSCC and confirms previously reported data. The results suggest that the subgroup of patients with PD-L1 CPS ≥20 may benefit more from this treatment, which warrants further development in a randomized pivotal study. Clinical trial# NCT04180215. Clinical trial information: NCT04180215 .

Single-cell analysis reveals cellular and molecular factors counteracting HPV-positive oropharyngeal cancer immunotherapy outcomes

BackgroundOropharyngeal squamous cell carcinoma (OPSCC) induced by human papillomavirus (HPV-positive) is associated with better clinical outcomes than HPV-negative OPSCC. However, the clinical benefits of immunotherapy in patients with HPV-positive OPSCC...

Biomarker-driven radiation therapy dose reduction after transoral robotic surgery for the treatment of HPV-positive oropharyngeal cancer.

TPS6119 Background: HPV-related oropharyngeal squamous cell carcinoma (OPSCC) is a highly curable cancer with a considerable burden of treatment-related toxicities. Transoral surgery (TOS) is a treatment option for patients with early-stage disease; however, adjuvant radiotherapy (RT) is often recommended and can add to these late toxicities. Circulating tumor HPV DNA (ctHPVDNA) is a sensitive marker of tumor recurrence, and undetectable post-operative ctHPVDNA is associated with improved recurrence-free survival. We hypothesize that patients with undetectable post-TOS ctHPVDNA without high risk pathologic factors can safely undergo de-escalation of adjuvant RT to 36 Gy, and this reduction will improve long-term swallowing function. Methods: We opened a multi-institutional phase II study (NCT05387915) including patients with pT1-2, N0-1, M0 HPV-related OPSCC who had TOS resection of their primary site and at least ipsilateral neck dissection. Pertinent inclusion criteria include a &lt;10 pack-year smoking history, or a &lt;30 pack-year smoking history if &gt;10 years since last tobacco consumption, and pathologic findings of one or more intermediate risk factors: close margin (1 – 4 mm), perineural invasion, 2 – 4 positive lymph nodes or a single lymph node &gt;3 cm. Patients with high risk factors (positive margin, extranodal extension, 5 or more positive lymph nodes) are excluded. Patients must have a detectable pre-operative ctHPVDNA and a post-operative ctHPVDNA drawn 7-14 days after TOS; if negative, patients receive 36 Gy of adjuvant RT in 15 fractions of 2.4 Gy delivered daily; if positive, they receive care per the discretion of the treating radiation oncologist. If the final surgical margins at the site of the primary tumor are &gt;2mm, patients are eligible for omission of the primary tumor post-operative bed from the radiation volume. Our primary endpoint is 1-year swallowing function as measured by the composite MD Anderson Dysphagia Index (MDADI) score. Secondary endpoints include overall/progression-free survival and other quality of life metrics. The study is powered to detect an 8.5-point improvement in MDADI composite score at one year over the historical control from ECOG 3311 Arm B. With an alpha of 0.05 and a power of 0.90 we require 27 evaluable patients. Accounting for dropout and patients lost to follow-up, we aim to enroll 35 total patients. Since accrual began in June 2022, we have enrolled 18 patients as of January 2024. We recently opened the study at a second institution and are in the process of opening at a third institution. Clinical trial information: NCT05387915 .

Molecular characteristics of non-human papillomavirus driven cervical adenocarcinoma and adenosquamous carcinoma.

e17532 Background: Cervical cancer is the fourth most common malignancy in women worldwide. Although infection from human papillomavirus (HPV) has been the major cause of cervical cancer, about 3-8% of cervical cancer cases are HPV-negative. Clinical data showed that patients with HPV-negative cervical cancer had significantly lower DFS than those with HPV-positive cervical cancer. The detection of HPV DNA tends to be lower in adenosquamous carcinoma (AC/ASC) than in squamous cell carcinoma. Because the mechanism of HPV-negative cervical cancer development is unclear, this study aims to find the pattern of differential gene expression in HPV-negative AC/ASC and verify the underlying potential mechanism. Methods: In this study, we aimed to exam the differential gene expression profiling by exome-capture RNA sequencing on formalin-fixed, paraffin-embedded tumor tissue of prospectively enrolled cervical AC/ASC patients (HPV-negative: HPV-positive: 1:1) and validate the selected biomarkers on the remaining (unmatched; n = 110) prospective cohort and the 274 retrospective cohort patients. Subsequently, dysregulated differentially expressed genes specifically existed in HPV-negative cervical AC/ASC tissues and HPV-negative cell lines were validated by Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical staining (IHC staining). Results: Exome-capture RNA sequencing on HPV-negative and age/grade matched HPV-positive AC/ASC (10:6) for discovery initially. We found differentially activated WNT pathway from HPV positive/negative AC/ASC in the prospective cohort. In the preliminary results of 28 prospective cohort patients, SOX17 and MMP7 highly expressed genes in HPV-negative (n = 13) then the HPV-positive (n = 11) cervical AC/ASC by IHC staining (p = 0.1029 and p = 0.0306, respectively). Moreover, plasma MMP7 levels were upregulated in HPV-negative AC/ASC (n = 10) compared with HPV-positive AC/ASC (n = 17) (p = 0.0086) by ELISA analysis in the prospective cohort. Conclusions: Further studies and more analyses will be continued to investigate the effects of changes in the expression of SOX17 and MMP7 related mechanisms, and targeting WNT/SOX17/MMP7 pathways specifically associated with HPV-negative AC/ASC to provide new insights into the early screening and effective treatment of HPV-negative AC/ASC.

EVOLVE-HNSCC: A global phase 3 study of volrustomig as sequential therapy for unresected locally advanced head and neck squamous cell carcinoma (LA-HNSCC) following definitive concurrent chemoradiotherapy (cCRT).

TPS6120 Background: Currently there are no approved maintenance therapies for patients (pts) with unresected LA-HNSCC following definitive cCRT. The PD-1 inhibitors pembrolizumab and nivolumab are licensed for treatment of pts with recurrent or metastatic HNSCC who have progressed on or after a platinum-based therapy. Dual inhibition of CTLA-4 and PD-L1 is approved in solid tumors including renal cell carcinoma (RCC), NSCLC, and melanoma, and has shown a numerical trend towards improved survival in first-line pts with recurrent/metastatic HNSCC whose tumors express PD-L1. Volrustomig (MEDI5752) is a monovalent, PD-1/CTLA-4 bispecific, humanized IgG1 monoclonal antibody engineered to specifically inhibit PD-1, with increased CTLA-4 blockade on PD-1+ activated T cells compared to PD-1– resting peripheral T cells. In a first-in-human phase 1/2 study (NCT03530397), volrustomig monotherapy showed promising efficacy with acceptable tolerability in advanced clear cell RCC and in combination with chemotherapy in advanced NSCLC. The phase 3, randomized, open-label, multicenter, eVOLVE-HNSCC study will evaluate the efficacy and safety of sequential therapy with volrustomig compared with observation in pts with unresected LA-HNSCC who have not progressed after receiving definitive cCRT (NCT06129864). Methods: Key eligibility criteria include histologically or cytologically confirmed, unresected LA-HNSCC with no evidence of metastatic disease, i.e. AJCC 8th edition (TNM staging system) stage IVA/B cancers of the hypopharynx, oral cavity, larynx or HPV-negative oropharynx, or stage III HPV-positive oropharynx cancer; age ≥18 years; WHO/ECOG performance score of 0 or 1; and adequate organ and bone marrow function. Key exclusion criteria include requiring further treatment with curative intent after definitive cCRT, resected LA-HNSCC, recurrent/metastatic disease, and &gt;1 primary tumor. Following initial screening and definitive cCRT (cisplatin or carboplatin + paclitaxel or carboplatin + 5-FU, plus concomitant radiotherapy), approximately 1145 pts whose disease has not progressed within 12 weeks of the last dose of cCRT will be randomized 1:1 to Arm A or B. Arm A will receive volrustomig intravenously every 3 weeks for a maximum of 12 months or 18 cycles, or until RECIST v1.1-defined radiological progressive disease (PD) or unacceptable toxicity. Arm B will undergo observation for a maximum of 12 months or until PD. The primary endpoint is PFS in pts with PD-L1-expressing tumors. Secondary endpoints include PFS in all randomized pts, OS in pts with PD-L1-expressing tumors and in all randomized pts, PFS2, safety, patient-reported outcomes, pharmacokinetics, and immunogenicity. Exploratory biomarker analyses will also be conducted. Enrollment began in December 2023. Clinical trial information: NCT06129864 .

De-escalation treatment of patients with HPV-positive oropharyngeal squamous cell carcinoma.

e18052 Background: According to Globocan in 2020 were identified 98 412 new cases of oropharyngeal squamous cell carcinoma in the world. It was proved that the human papillomavirus (HPV) is a favorable prognostic factor. Patients with HPV-positive oropharynx cancer have higher indicators of progression-free survival (PFS) and overall survival (OS). As induction chemotherapy, the only TPF regimen (cisplatin/ docetaxel/ 5-fluorouracil) has been approved which has high toxic profile. Published results of randomized trials demonstrate the possibility of de-escalation CRT without reduction OS and PFS, but there is no data about safety of de-escalated IC regimen. Given the high toxicity of the standard regimen, as well as the presence of a favorable prognostic factor (HPV infection), since 2021 has started a prospective trial, investigating the possibility of use the dual-component IC regimen according to the TP scheme (cisplatin/ docetaxel). Objective: Improve the results of treatment of patients with locally advanced HPV- positive squamous cell carcinoma of the oropharynx by optimizing the scheme IC. Methods: In the prospective trial included 68 patients with p16-positive oropharyngeal squamous cell carcinoma (T3-4N0-1M0, or T1-4N2-3M0), who were divided into 2 equal groups. The research group received 3 cycles of IC to the de-escalated TP regimen and the control group according to the standard three-component TPF regimen. After completion IC patients of both groups had a standard CRT 70 Gy with carboplatin AUC 2.0 weekly. The primary endpoints were the assessment of objective response rate after the IC and toxicity. The Secondary endpoints: 1-year OS and PFS. Results: Complete response (CR) was achieved in 3 patients (8,8%), a partial response (PR) in 22 (64,7%), stable disease (SD) in 8 (23,5%) patients in the research arm. In the control arm: CR 4 patients (11,8%), PR 24 (70,6%), SD 6 (17,6%) patients. Both IC regimens demonstrated high efficacy rates, and there was no statistically significant ORR in the group (p=0,2133).Toxic manifestations were often observed in the arm of standard IC. The higher incidence of mucositis in the control group was statistically significant (p=0,025). The effect of IC regimen on subsequent CRT was also assessed. In the research arm, the time before the start of CRT was statistically significantly less than in the control arm (30 days vs 63 days, p=0,0035.). And the period of development mucositis of CRT in the TP group was longer than in the TPF group (34G vs 32G, p=0,0298). 1 year - PFS 94%, 1 year - OS 100% in the research group and 92% and 100% in the control arm. There was no statistically significant 1 year – PFS and 1 year - OS in the group (р=0,6547). Conclusions: The research scheme of IC demonstrated high results of an objective response, 1-year OS and 1 year - PFS, comparable to the standard TPF scheme, against the background of better tolerance.

Time dependency for HPV ctDNA detection as a prognostic biomarker for anal cancer.

3513 Background: Prior HPV infection is associated with &gt; 90% of anal cancers, a malignancy with rising incidence in the United States. Standard treatment for localized anal cancer is concurrent chemoradiation (cRT). While detection of circulating tumor DNA (ctDNA) within weeks after surgery is linked to recurrence in other solid tumors, the optimal time point for ctDNA detection as a prognostic biomarker following cRT for HPV-associated anogenital cancers is not well characterized. We utilized a novel, highly sensitive HPV ctDNA assay to evaluate clinical outcomes according to HPV ctDNA status among patients with localized anal cancer treated with cRT. Methods: ctDNA was isolated from patients with stages I-III anal cancer treated at our institution with cRT prior to treatment, at the end of treatment (week 5), and at months 3, 6, 9, and 12 after treatment under an IRB-approved protocol. A droplet digital HPV ctDNA PCR assay evaluating HPV E6 and E7 oncogenes for 13 oncogenic HPV types was utilized for quantification of HPV ctDNA. A limit of quantification at 16 HPV copies/mL plasma was set for “HPV ctDNA detection.” Median recurrence-free survival (RFS) according to HPV ctDNA status was estimated via Kaplan-Meier and compared using a log-rank test. Associations between selected clinical factors and recurrence were evaluated with a Chi-square test, with a one-sided p &lt; .05 considered significant. Results: Following cRT, HPV ctDNA was detected in 9/41 (22%), 4/30 (13%), 2/20 (10%), and 0/12 (0%) patients at week 5 and months 3, 6, and 12, respectively. Detection of HPV ctDNA 3 months after cRT was associated with clinical recurrence (100% versus 8%; odds ratio 88, 95% CI 4-2000; p = .006) and inferior RFS (5.9 months vs not reached (NR); hazard ratio (HR) 24, 95% CI 1.2-475; p &lt; .001) relative to HPV ctDNA-negative status. Differences in RFS according to HPV ctDNA status were not observed at week 5/end of treatment (median RFS NR for both; HR 2.4, 95% CI .5-10; p = .15). At month 3, sensitivity and specificity for recurrence according to HPV ctDNA detection were 57% and 100%, respectively, with a PPV and NPV of 100% and 89%, respectively. Baseline clinical stage, T stage, N stage, age, and gender were not associated with clinical recurrence after cRT (p &gt; .25 for all). Conclusions: With a novel, highly sensitive assay, detection of HPV ctDNA at 3 months after cRT was associated with RFS. Further, an HPV ctDNA-positive status outperformed baseline clinical features for prognosticating anal cancer recurrence after cRT in this retrospective series. Future clinical trials should incorporate the 3-month post-treatment time point for identification of patients with HPV-positive anal cancer at elevated risk for recurrence according to HPV ctDNA status. A clinical trial evaluating anti-PD-L1 + anti-TIGIT immunotherapy for patients with HPV-associated cancers with detected HPV ctDNA after cRT is forthcoming at our institution.