HPV-positive Tumors Research Articles

Transoral Laser or Robotic Surgery Outcomes for Oropharyngeal Carcinoma

Transoral robotic surgery (TORS) or transoral laser microsurgery (TLM) offer excellent oncological outcomes for oropharyngeal squamous cell carcinoma caused by human papillomavirus (HPV) infection. TORS may offer greater margin clearance around tumors than TLM. To determine whether the differing energy sources used and surgical technique of TORS or TLM is associated with postoperative early swallowing function, feeding tube use, and specific factors related to quality of life. This prespecified cohort study was performed within the Postoperative Adjuvant Treatment for HPV-Positive Tumours (PATHOS) randomized clinical trial at 40 centers in the UK, Germany, France, the US, and Australia between November 1, 2015, and August 31, 2023. PATHOS trial participants with HPV-positive oropharyngeal carcinoma of stages T1 to T3 and N0 to N2b M0 (TNM7) who underwent TLM or TORS were eligible. Of 989 consecutively recruited patients on the PATHOS trial, 508 were eligible for this substudy. The exposure of interest was TORS or TLM. Preplanned outcome measures included nasogastric tube insertion rates within 4 weeks after surgery, length of in-hospital stay following surgery, specific scales from the MD Anderson Dysphagia Inventory (MDADI), 35-item European Organization for Research and Treatment of Cancer Head and Neck Questionnaire (H&N35), and 30-item Quality of Life Questionnaire (QLQ C30), water swallow test results, and videofluoroscopy scores. Of the 508 patients included in the analysis (390 [76.8%] male; median age, 58.3 [IQR, 52.8-63.6] years), 195 had TLM and 313 had TORS. Nasogastric tube insertion rates were higher after TORS than TLM (85 of 189 [45.0%] vs 10 of 126 [7.9%]; adjusted odds ratio [OR], 4.41 [95% CI, 1.01-19.32]). Mean scores favored TLM with small effect sizes in all MDADI domains and the H&N35 swallowing item at 4 weeks after surgery; between-group difference for the MDADI composite score was -4.89 (95% CI, -8.27 to -1.50); for the MDADI physical functioning score, -6.37 (95% CI, -10.15 to -2.59); for the MDADI global score, -10.02 (95% CI, -16.50 to -3.54); and for H&N35 swallowing score, 7.24 (95% CI, 2.17-12.30). No other measures showed evidence of clinically meaningful differences. In this cohort study, functional outcomes were moderately less impaired 4 weeks following TLM compared with TORS. Once the longer-term outcomes for these patients are known, these findings could aid the design and use of future head and neck-specific surgical robots. ClinicalTrials.gov Identifier: NCT02215265.

Prognostic Significance of Human Papillomavirus Genotypes in Oropharyngeal Squamous Cell Carcinoma.

The prognostic significance of human papillomavirus (HPV) genotypes in oropharyngeal squamous cell carcinoma (OPSCC) has garnered considerable attention due to the increasing reliance on HPV status for clinical decision-making. This study aimed to compare the survival outcomes associated with different HPV genotypes in patients with OPSCC relative to HPV-negative tumors, providing insights into the potential implications for treatment de-intensification strategies. Patients diagnosed with invasive OPSCC were included from the National Cancer Database (NCDB). Patients were stratified based on HPV status and genotype, with HPV-negative tumors serving as the reference group. Multivariable Cox regression analysis was performed to assess the independent prognostic value of different HPV genotypes. Th majority of patient were classified as HPV-positive (N = 17,358, 70.0%), with HPV 16 being the most common genotype (N = 15410/17358, 88.8%) compared with other high-risk (N = 1217/17,358, 7.0%) and low-risk (N = 731/17,358, 4.2%) HPV genotypes. A significantly lower risk of death was measured for all HPV-positive compared with HPV-negative tumors (HPV 16: adjusted HR 0.51; 95% CI: 0.49-0.54; other high-risk HPV: adjusted HR 0.56; 95% CI: 0.49-0.63; low-risk HPV: adjusted HR 0.59; 95% CI: 0.50-0.68; p < 0.001). This study highlights the significant prognostic value of HPV genotypes in OPSCC, underscoring the superior survival outcomes of HPV-positive tumors across all genotypes compared with HPV-negative tumors. Detailed HPV subtype analysis can inform better treatment decisions and support de-intensification strategies for patients with low-risk genotypes. 3 Laryngoscope, 2024.

Deciphering the interplay of HPV infection, MHC-II expression, and CXCL13+ CD4+ T cell activation in oropharyngeal cancer: implications for immunotherapy

BackgroundHuman papillomavirus (HPV) infection has become an important etiological driver of oropharyngeal squamous cell carcinoma (OPSCC), leading to unique tumor characteristics. However, the interplay between HPV-associated tumor cells and tumor microenvironment (TME) remains an enigma.MethodsWe performed a single-cell RNA-sequencing (scRNA-seq) on HPV-positive (HPV+) and HPV-negative (HPV‒) OPSCC tumors, each for three samples, and one normal tonsil tissue. Ex vivo validation assays including immunofluorescence staining, cell line co-culture, and flow cytometry analysis were used to test specific subtypes of HPV+ tumor cells and their communications with T cells.ResultsThrough a comprehensive single-cell transcriptome analysis, we uncover the distinct transcriptional signatures between HPV+ and HPV‒ OPSCC. Specifically, HPV+ OPSCC tumor cells manifest an enhanced interferon response and elevated expression of the major histocompatibility complex II (MHC-II), potentially bolstering tumor recognition and immune response. Furthermore, we identify a CXCL13+CD4+ T cell subset that exhibits dual features of both follicular and pro-inflammatory helper T cells. Noteworthily, HPV+ OPSCC tumor cells embrace extensive intercellular communications with CXCL13+CD4+ T cells. Interaction with HPV+ OPSCC tumor cells amplifies CXCL13 and IFNγ release in CD4+T cells, fostering a pro-inflammatory TME. Additionally, HPV+ tumor cells expressing high MHC-II and CXCL13+CD4+ T cell prevalence are indicative of favorable overall survival rates in OPSCC patients.ConclusionsTogether, our study underscores a synergistic inflammatory immune response orchestrated by highly immunogenic tumor cells and CXCL13+CD4+ T cells in HPV+ OPSCC, offering useful insights into strategy development for patient stratification and effective immunotherapy in OPSCC.

HPV-18 E6 enhances the interaction between EMILIN2 and SNX27 to promote WNT signaling.

This study demonstrates that HPV E6 blocks EMILIN2 inhibition of WNT1 signaling, thereby enhancing cell proliferation in HPV-positive tumor cells. This involves a novel mechanism whereby the E6 PBM actually contributes toward enhancing the interaction between SNX27 and EMILIN2, suggesting that the mode of recognition of SNX27 by E6 and EMILIN2 is different. This is the first example of the E6 PBM altering a PDZ domain-containing protein to enhance potential substrate recognition.

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.

LRG1 and SDR16C5 protein expressions differ according to HPV status in oropharyngeal squamous cell carcinoma

The increasing incidence of oropharyngeal squamous cell carcinoma (OPSCC) is primarily due to human papillomavirus, and understanding the tumor biology caused by the virus is crucial. Our goal was to investigate the proteins present in the serum of patients with OPSCC, which were not previously studied in OPSCC tissue. We examined the difference in expression of these proteins between HPV-positive and -negative tumors and their correlation with clinicopathological parameters and patient survival. The study included 157 formalin-fixed, paraffin-embedded tissue samples and clinicopathological data. Based on the protein levels in the sera of OPSCC patients, we selected 12 proteins and studied their expression in HPV-negative and HPV-positive OPSCC cell lines. LRG1, SDR16C5, PIP4K2C and MVD proteins were selected for immunohistochemical analysis in HPV-positive and -negative OPSCC tissue samples. These protein´s expression levels were compared with clinicopathological parameters and patient survival to investigate their clinical relevance. LRG1 expression was strong in HPV-negative whereas SDR16C5 expression was strong in HPV-positive tumors. Correlation was observed between LRG1, SDR16C5, and PIP4K2C expression and patient survival. High expression of PIP4K2C was found to be an independent prognostic factor for overall survival and expression correlated with HPV-positive tumor status. The data suggest the possible role of LRG1, SDR16C5 and PIP4K2C in OPSCC biology.

Aspartate-β-hydroxylase and hypoxia marker expression in head and neck carcinomas: implications for HPV-associated tumors

BackgroundA proportion of head and neck carcinomas (HNSCCs) are induced by high-risk human papillomaviruses (HPVs) and are associated with better patient outcomes compared to patients with HNSCCs related to tobacco and alcohol abuse. In the microenvironment of solid tumors, including HNSCCs, oxygen levels are often reduced, and a hypoxic state is induced. This can lead to a poor treatment response and a worse patient prognosis. One of the hypoxia-responsive genes is aspartate-β-hydroxylase (ASPH), whose activity promotes the growth, invasiveness, and metastasis of many types of solid tumors.MethodsIn our study, HNSCC samples were analyzed for the expression of ASPH and selected endogenous hypoxia markers by real-time PCR and/or multiplex fluorescence immunohistochemistry.ResultsExcept for the EPAS1 gene, which had higher mRNA expression in the HPV-negative group of HNSCC (p < 0.05), we found no other differences in the expression of the tested genes that were related to HPV status. On the contrary, a statistically significantly higher number of cells producing ASPH (p < 0.0001), HIF1A (p < 0.0001), GLUT1 (p < 0.0001), and MMP13 (p < 0.05) proteins were detected in the HPV-positive tumor group than in the HPV-negative sample group. All the evaluated markers, except for MMP9/13, were more abundant in the tumor parenchyma than in the tumor stroma. The Cox proportional hazard models showed that increased numbers of cells with GLUT1 and HIF1A protein expression were positive prognostic markers for overall and disease-specific survival in patients independent of HPV tumor status.ConclusionThe study examined HNSCC samples and found that elevated ASPH and hypoxia marker proteins, typically associated with poor prognosis, may actually indicate active HPV infection, the strongest prognostic factor in HNSCC patients. In cases where HPV status is uncertain, increased expression of HIF1A and GLUT1 can serve as positive prognostic factors.

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...

Integrative analysis in head and neck cancer reveals distinct role of miRNome and methylome as tumour epigenetic drivers

Head and neck cancer is the sixth most common malignancy worldwide, with the relatively low 5-year survival rate, mainly because it is diagnosed at a late stage. Infection with HPV is a well known aetiology, which affects the nature of these cancers and patients’ survival. Besides, it is considered that the main driving force for this type of cancer could be epigenetics. In this study we aimed to find potential epigenetic biomarkers, by integrating miRNome, methylome, and transcriptome analyses. From the fresh head and neck cancer tissue samples, we chose a group for miRNome, methylome and transcriptome profiling, in comparison to adequate control samples. Bioinformatics analyses are performed in R v4.2.2. Count normalisation and group differential expression for mRNA and the previously obtained miRNA count data was performed with DESeq2 v1.36. Gene set enrichment analysis was performed and visualised using gProfiler2 v0.2.1 Identification of miRNA targets was performed by querying in miRTarBase using multiMiR v1.18.0. Annotation of CpG sites merging into islands was obtained from RnBeads.hg19 v1.28.0. package. For the integrative analysis we performed kmeans clustering using stats v4.2.2 package, using 8–12 clusters and nstart 100. We found that transcriptome analysis divides samples into cancers and controls clusters, with no relation to HPV status or cancer anatomical location. Differentially expressed genes (n = 2781) were predominantly associated with signalling pathways of tumour progression. We identified a cluster of genes under the control of the transcription factor E2F that are significantly underexpressed in cancer tissue, as well as T cell immunity genes and genes related to regulation of transcription. Among overexpressed genes in tumours we found those that belong to cell cycle regulation and vasculature. A small number of genes were found significantly differentially expressed in HPV-positive versus HPV-negative tumours (for example NEFH, ZFR2, TAF7L, ZNF541, and TYMS). In this comprehensive study on an overlapping set of samples where the integration of miRNome, methylome and transcriptome analysis were performed for head and neck cancer, we demonstrated that the majority of genes were associated exclusively with miRNome or methylome and, to a lesser extent, under the control of both epigenetic mechanisms.

Human Papillomavirus Is Rare and Does Not Correlate with p16INK4A Expression in Non-Small-Cell Lung Cancer in a Jordanian Subpopulation.

Background and Objectives: Human papillomavirus (HPV) was previously investigated in lung cancer with wide inter-geographic discrepancies. p16INK4a has been used as a surrogate for detecting high-risk HPV (HR-HPV) in some cancer types. This study assessed the evidence of HPV in non-small-cell lung cancer (NSCLC) among Jordanian patients, investigated the expression of p16INK4a, and evaluated its prognostic value and association with HPV status. Materials and Methods: The archived samples of 100 patients were used. HPV DNA detection was performed by real-time polymerase chain reaction (RT-PCR). p16INK4a expression was assessed by immunohistochemistry (IHC). The Eighth American Joint Committee on Cancer protocol (AJCC) of head and neck cancer criteria were applied to evaluate p16INK4a positivity considering a moderate/strong nuclear/cytoplasmic expression intensity with a distribution in ≥75% of cells as positive. Results: HPV DNA was detected in 5% of NSCLC cases. Three positive cases showed HR-HPV subtypes (16, 18, 52), and two cases showed the probable HR-HPV 26 subtype. p16INK4a expression was positive in 20 (20%) NSCLC cases. None of the HPV-positive tumors were positive for p16INK4a expression. A statistically significant association was identified between p16INK4a expression and the pathological stage (p = 0.029) but not with other variables. No survival impact of p16INK4a expression was detected in NSCLC cases as a group; however, it showed a statistically significant association with overall survival (OS) in squamous cell carcinoma (SqCC) cases (p = 0.033). Conclusions: This is the first study to assess HPV and p16INK4a expression in a Jordanian population. HPV positivity is rare in NSCLC among a Jordanian subpopulation. P16 INK4a reliability as a surrogate marker for HPV infection in lung cancer must be revisited.

Risk factors for head and neck squamous cell carcinoma in disease pathogenesis and prognosis: the importance of smoking cessation and the concept of harm reduction

Introduction. Cigarette smoking along with alcohol consumption, infection with human papilloma (HPV) and the Epstein–Barr viruses, is one of the main risk factors for head and neck squamous cell carcinoma. It can also contribute to virus-associated carcinogenesis and affect the clinical and prognostic features of the tumor. Continued smoking at diagnosis of head and neck squamous cell carcinoma is a negative prognostic factor and contributes to reduced patient survival rates. Aim. To analyze the smoking status and the effect of etiopathogenesis on molecular and clinical features in patients with oropharyngeal squamous cell carcinoma (OpSCC). Materials and methods. In patients with OPSCC, the smoking status and the level of motivation to its cessation were clarified. Immunohistochemical analysis of tumor sections from paraffin blocks from the ThermoScentific immunohis-tosteiner was performed using monoclonal antibodies to estrogen receptors α (ERα), programmed death-ligand 1 (pD-L1), Bcl-2, p53 and p16. The percentage of tumor-infiltrating lymphocytes (TILs) in the stromal component of the tumor (hematoxylin and eosin staining) was also analyzed. Results. Of 113 patients with OpSCC, 80 % of patients were current smokers, including 75 % of patients with Hpv-positive tumor status. men with OpSCC were statistically significantly more likely to smoke than women (p = 0.001), as were men with Hpv-positive tumor (p = 0.015). Smoking patients were characterized by a larger size of the primary tumor (T3–4), including those with Hpv (p = 0.015). positive Hpv status of the tumor is associated with autoimmune diseases (autoimmune thyroiditis, psoriasis) (p ˂ 0.05). A high level of TILs is characteristic of smokers, including those with p16+-status (p ˂0.05), but not for all patients with this status. The positive expression of ERα is associated with p16+-status, and the latter is associated with the absence of p53 expression (p ˂0.05). During conservative therapy, tumor regression is associated with the primary tumor size T1–2, p16 + status, female sex, age less than 55 years, TILs level ≥ 57.5 %, pD-L1 expression levels on tumor cells ≥2.2 %, Bcl-2 ≥5 % and ERα ≥65 % (p ˂ 0.05). At the same time, quality of good predictive model for tumor regression showed a TILs level of ≥57.5 % (area under curve, AuC) 0.796; sensitivity 78.6 %; specificity 75 %) and ERα (AuC 0.725; sensitivity 65 %; specificity 83 %). The level of motivation to quit smoking ranged from 1 to 7 points according the 10-point motivation scale in 89 % of respondents. Only 10 patients were ready to quit smoking immediately (10 points on the motivation scale). Conclusion. Molecular heterogeneity of OPSCC was revealed, including in the group of p16+-tumors. Correlation of clinical and molecular parameters with response to conservative therapy can be used in planning of primary therapy and earlier use of surgical treatment. The level of motivation to quit smoking in patients with squamous cell carcinoma of the head and neck may be increased by drawing attention to the problem at each doctor’s appointment as well as by informing about how to quit smoking and therapeutic approaches. In unmotivated patients, the concept of reducing the harm of smoking can be used involving the replacement of cigarettes with electronic tobacco heating systems, eliminating the carcinogenic effect of its combustion products.

Abstract CT226: Phase 2 study of oral CCR4 antagonist FLX475 (tivumecirnon) plus pembrolizumab in subjects with head and neck squamous cell carcinoma (HNSCC) previously treated with checkpoint inhibitor

Abstract BACKGROUND: FLX475 (tivumecirnon) is a selective CCR4 antagonist designed to block the recruitment of immunosuppressive regulatory T cells (Treg) into the tumor microenvironment. The FLX475-02 trial (NCT03674567) is a phase 1/2 study of FLX475 as monotherapy and in combination with pembrolizumab in subjects with advanced cancer. Early encouraging data on the biologic effects, safety and antitumor activity of FLX475 have previously been presented. We now present the results from the Phase 2 cohort of combination therapy in subjects with head and neck squamous cell carcinoma (HNSCC) previously treated with checkpoint inhibitor (CPI-experienced). METHODS: Subjects with CPI-experienced, recurrent or metastatic (R/M) HNSCC received FLX475 100 mg orally once daily with pembrolizumab (200 mg IV Q3 weeks). The primary study objectives were safety and tolerability, and antitumor activity. The primary efficacy endpoint was objective response rate (ORR), based on RECIST 1.1 criteria. Additional efficacy endpoints included progression-free survival (PFS). Safety was evaluated as per CTCAE v4.03. Data cutoff was 11DEC2023. RESULTS: For the 32 subjects with HNSCC evaluable for response, median follow-up was 14.8 months (range 2.2-40.1) and median lines of prior therapy were 3 (1-6). Median age of subjects was 59 yrs; 94% were male and 75% were Caucasian. Primary tumor location was oropharynx in 56%. As previously reported, the only adverse event clearly determined to be treatment related to FLX475 to date has been asymptomatic and reversible QT prolongation (managed by dose reduction). Among the subjects evaluable for response regardless of HPV or PD-L1 status (n=32), confirmed partial response (cPR) was observed in 5 subjects (ORR: 15.6%, 95% CI 6-32%). Among the subgroup of subjects whose tumors were HPV positive (n=18), 8 (44%) experienced reduction from baseline in target lesion measurements and cPR was observed in 4 (ORR: 22.2%, 95% CI 9-46%). Among the subgroup of subjects whose tumors expressed PD-L1 (combined positive score [CPS] ≥1%) (n=22), cPR was observed in 4 (ORR: 18.2%, 95% CI 7-39%). At data cutoff, the HPV-positive subgroup median PFS was 2.9 months (95% CI 2-10.3) and 3 subjects were still on treatment. CONCLUSIONS: FLX475, an oral CCR4 antagonist, has previously demonstrated clear monotherapy activity in EBV+ extranodal NK/T-cell lymphoma and promising combination activity with pembrolizumab in EBV+ gastric cancer and CPI-naïve non-small cell lung cancer. In this completed Phase 2 cohort of subjects with CPI-experienced R/M HNSCC, FLX475 in combination with pembrolizumab was shown to be well tolerated and has demonstrated encouraging clinical activity particularly in those with HPV-positive tumors, supporting the continued development of this combination therapy for CPI-experienced HNSCC. Citation Format: Jameel Muzaffar, Kedar Kirtane, Rebecca Redman, Muh-Hwa Yang, Tae Min Kim, Stephen Liu, Ryan Lynch, Julie Brahmer, Patricia LoRusso, Brian Henick, Sung-bae Kim, Yin-Hsun Feng, Michael Chisamore, Paul Kassner, Dirk Brockstedt, Nicole Nasrah, Rakesh Goyal, William Y. Ho, Victoria Villaflor. Phase 2 study of oral CCR4 antagonist FLX475 (tivumecirnon) plus pembrolizumab in subjects with head and neck squamous cell carcinoma (HNSCC) previously treated with checkpoint inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT226.

Human papillomavirus circulating tumor DNA: a diagnostic tool in squamous cell carcinoma of unknown primary-a pilot study.

Neck mass is the most common presentation of human papillomavirus-related (HPV-related) oropharyngeal squamous cell carcinoma (OPSCC). Recently, circulating tumor HPV-DNA (ctHPVDNA) assays have been developed to detect active OPSCC. This pilot study investigates the diagnostic accuracy of ctHPVDNA in establishing HPV status for known vs. unknown OPSCC presenting as a neck mass. A single-institution pilot study was conducted on all patients with OPSCC presenting as a neck mass between 2021 and 2022. The diagnostic accuracy of ctHPVDNA was compared to that of standard diagnostic procedures used to obtain HPV status according to the American Society of Clinical Oncology (ASCO) guideline for squamous cell carcinoma of unknown primary (SCCUP). Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of ctHPVDNA were calculated. A total of 27 patients were included; 70.4% were current or former smokers, 48.1% (N = 13) had identifiable primaries, and 51.9% (N = 14) had SCCUP. Four patients with known primaries required operative direct laryngoscopy with biopsy (DLB) to establish HPV status. Two patients with SCCUP underwent diagnostic transoral robotic surgery (TORS) to establish HPV status and localize the primary. Twelve patients underwent therapeutic TORS and neck dissection. The gold standard for HPV status was based on final histopathologic p16 or HPV in situ hybridization (ISH) staining during workup/treatment. ctHPVDNA had 95.8% sensitivity, 100% specificity, 100% PPV, and 75% NPV in predicting HPV-positive OPSCC in the whole sample. Binary logistic regression model using ctHPVDNA results to predict HPV-positive OPSCC was significant (-2 log likelihood = 5.55, χ2=8.70, p <.01, Nagelkerke's R squared = .67). Among patients with identifiable primaries, all patients had HPV-positive tumors on final pathology, and ctHPVDNA was positive in 100%. In the unknown primary patients, ctHPVDNA had 90.9% sensitivity, 100% specificity, 100% PPV, and 75% NPV. ctHPVDNA demonstrated good diagnostic accuracy for both known and unknown primaries. Incorporation of ctHPVDNA into the diagnostic algorithm for SCCUP may reduce the need for multiple procedures to establish HPV status.

Abstract 2955: HPV E6* and senescence-related gene expression in cervical cancer

Abstract Previous work in our lab found that increased expression of the E6* variant of the HPV E6 viral protein was significantly correlated with worse response to standard-of-care chemoradiation (CRT) and progression-free survival, independently of HPV genotype. In vitro validation showed that overexpression of E6* induces senescence in an HPV 16-positive cervical cancer cell line. Given these findings, we hypothesized that increased E6* expression mediates resistance to CRT by inducing a reversible state of tumor cell senescence. We created a senescence score using ten genes that have been consistently reported in the literature as associated with senescence. Using an institutional cohort of 68 patients with cervical cancer with prospectively collected clinical outcome data, we performed bulk RNA sequencing to assess senescence scores before CRT and determine whether there is an association between senescence scores and E6* expression. Using a second institutional cohort of 35 patients with cervical cancer treated with CRT with pre and posttreatment tumor biopsies, we performed bulk RNA sequencing to evaluate the same relationship and determine whether expression of senescence-related genes was affected by CRT. Nine cervical cancer cell lines stratified into low- and high-E6* expression groups will be assessed regarding their senescence score, beta-galactosidase staining, and SASP expression before and after CRT. In our first cohort, there was no correlation between E6*:E6 expression ratio and senescence score, regardless of HPV genotype. In our second cohort, CRT increased senescence score in CRT-treated samples (p &amp;lt; 0.01), but p16INK4a expression significantly decreased compared to pre-treatment samples (p &amp;lt; 0.01). Increased senescence score was not associated with cancer recurrence. There was no significant difference in senescence scores between different HPV genotypes before therapy. Tumors that were positive for HPV 16 or HPV 18 had significantly lower senescence scores compared to HPV negative tumors (p = 0.037 and p = 0.038, respectively) after CRT. Among HPV 16-positive tumors (n = 12), there was no correlation between E6*:E6 expression ratio and senescence score before or after CRT. We found no association between senescence score and E6* expression, and between senescence score and cancer recurrence after CRT in our dataset. Additionally, our results suggest that p16INK4a might not be an appropriate senescence marker in tumors that have intrinsically high p16INK4a expression, such as in cervical cancer, reinforcing the need for a cervical cancer-specific senescence signature. We are currently testing beta-galactosidase and SASP expression in vitro and in vivo before and after CRT. Citation Format: Louise Medina Bengtsson, Matthew Inkman, Naoshad Muhammad, Leahan Castillo, Naomi Msengi, Tyler McKinnish, Nishanth Gabriel, Jin Zhang, Julie Schwarz. HPV E6* and senescence-related gene expression in cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2955.

Abstract 2685: Activated phenotype of intraepithelial type 1 innate lymphoid cells is enriched in HPV-positive head and neck cancers

Abstract Innate lymphoid cells (ILCs) and natural killer (NK) cells are a heterogeneous family of innate immune cells that regulate inflammation, immune tolerance and tissue homeostasis in infection, chronic inflammation, metabolic diseases and cancer. The ILC family lacks the expression of antigen-specific receptors and demonstrate effector function by generating cytokines and secreted proteins. In tumor microenvironment, ILCs play a dual role including both tumor regression and tumor promotion, suggesting their diverse phenotypes and functional states. Among the ILC family, ILC1s and NK cells share some features, including the expression of the transcription factor T-bet and the ability to produce IFN-γ. In the present study, we analyzed single-cell RNA sequencing (scRNAseq) data to investigate the landscape and functional status of ILC families in patients with head and neck squamous cell carcinoma (HNSCC). The GSE164690 dataset, including pre-processed scRNAseq data and clinical data, was obtained from Gene Expression Omnibus. A total of 17 pairs of tumor-derived CD45-positive cells and peripheral blood lymphocytes, consisting of 6 HPV-positive HNSCCs and 11 HPV-negative HNSCCs, were analyzed using the Seurat v4 R package. After quality control, a total of 95,809 cells were obtained and clustered into 16 immune cell clusters. Next, a total of 7,278 NK cells were extracted and sub-clustered into 11 clusters. To characterize the 11 clusters, the abundances of NK subsets were estimated by CIBERSORTx. This approach identified eight NK cell clusters, two intraepithelial ILC1 (ieILC1) clusters, and one ieILC1-NK-intermediate (ieILC1-NK-int) cluster. Single-sample GSEA (ssGSEA) analysis was performed to further characterize each subset. ieILC1 clusters expressed tissue-resident markers ITGA1, CD69, and CXCR6, whereas ieILC1-NK-int cluster expressed ILC1 markers CCR7, IL7R, and SELL. Principle component analysis (PCA) based on ssGSEA indicated that ieILC1 clusters and ieILC1-NK-int showed the different PCA distribution from NK clusters. Inflammation and effector function-related pathways, including inflammatory response, interferon-gamma response, and interferon-alpha response, were enriched in ieILC1 clusters compared with the others. Among ieILC1 clusters, several pathways, including hypoxia, TNFA signaling via NFKB, allograft rejection, TGF beta signaling, were more enriched in ieILC1-1 than in ieILC1-2, suggesting that ieILC1-1 is the most immunologically active phenotype among NK subclusters. Of note, ieILC1-2 mainly comprised cells isolated from HPV-negative tumors, whereas ieILC1-1 mainly comprised cells isolated from HPV-positive tumors. In conclusion, we have demonstrated the diverse landscape of NK and ieILC1 cells in patients with HNSCC. Further characterization of the ILC family would lead to a novel therapeutic approach against HNSCC. Citation Format: Hideyuki Takahashi, Shota Ida, Hiroe Tada, Kazuaki Chikamatsu. Activated phenotype of intraepithelial type 1 innate lymphoid cells is enriched in HPV-positive head and neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2685.

Abstract 3401: Uncovering virus induced dysregulated processes in cancer and their impact on outcomes

Abstract The National Cancer Institute has designated several viruses such as EBV, HBV, HCV, HIV, HPV, and HTLV as agents that either cause cancer or increase the risk of developing cancer. However, extensive analyses of tumor genome and transcriptome datasets have identified numerous additional virus types, the functional implications of which remain largely unknown. Viral infection often dysregulates unique molecular processes within tumor cells and alters the composition of immune cells in the tumor microenvironment. For instance, HPV-positive tumors have been shown to exhibit a significantly elevated expression of the DNA editing enzyme APOBEC3B compared to HPV-negative tumors. Furthermore, HPV positive tumors display and increased number of C-to-T mutations induced by this protein. The genome and transcriptome of tumor tissue biopsies contain a wealth of information about these virus-associated signatures. However, advanced signal processing approaches are needed to extract these viral signatures from complex tumor datasets, and elucidate their specific roles in cancer. Here, we developed a quantitative approach to investigate the link between viral infection and dysregulation of a seven-membered A3 enzyme family (A3A/B/C/D/F/G/H). The primary role of these enzymes is to restrict viral infections by inducing C-to-U mutations in viral genomes. However, at least three members of this enzyme family (A3A/B/H) can also induce mutations in cellular DNA, and drive tumor formation and evolution. We used NMF (Nonnegative Matrix Factorization) to deconvolute the expression profiles of A3 enzymes in &amp;gt;10,000 bulk RNAseq datasets from diverse TCGA tumors. This analysis revealed the expression profiles of A3 enzymes within tumor and tumor microenvironment cells including various immune cells. Next, we conducted an association analysis to identify the link between the presence of viral infection and the proportion of each of these tumor and tumor microenvironment expression profiles. As expected, our analyses revealed strong associations between tumor-specific A3 expression profiles and infections by oncolytic viruses such as HPV. Notably, and somewhat unexpectedly, our analyses also revealed associations with viruses about which little is known in the field of cancer. For example, in certain tumors, we identified a robust association between bacteriophages and a tumor-specific A3 expression profile characterized by A3B upregulation. Taken together, our analyses provide evidence for the link between A3 dysregulation and infection by viruses including bacteriophages. Citation Format: Mohadeseh Soleimanpour moghadam, Azad Khosh, Diako Ebrahimi. Uncovering virus induced dysregulated processes in cancer and their impact on outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3401.

Clinical and genomic characterization of chemoradiation-resistant HPV-positive oropharyngeal squamous cell carcinoma.

Most patients with HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) have an excellent response to chemoradiation, and trials are now investigating de-escalated treatment. However, up to 25% of patients with HPV-positive OPSCC will experience recurrence, and up to 5% will even progress through primary treatment. Currently, there are no molecular markers to identify patients with poor prognosis who would be harmed by de-escalation. Herein we report the clinical and genomic characteristics of persistent HPV-positive OPSCC after definitive platinum-based chemoradiation therapy. Patients with HPV-positive OPSCC treated with curative intent platinum-based chemoradiation between 2007 and 2017 at two institutions and with a persistent locoregional disease were included. We evaluated clinical characteristics, including smoking status, age, stage, treatment, and overall survival. A subset of five patients had tissue available for targeted exome DNA sequencing and RNA sequencing. Genomic analysis was compared to a previously published cohort of 47 treatment-responsive HPV+ OPSCC tumors after batch correction. Mutational landscape, pathway activation, and OncoGPS tumor states were employed to characterize these tumors. Ten patients met the inclusion criteria. The tumor and nodal stages ranged from T1 to T4 and N1 to N2 by AJCC 8th edition staging. All patients were p16-positive by immunohistochemistry, and eight with available in situ hybridization were confirmed to be HPV-positive. The 1-year overall survival from the time of diagnosis was 57%, and the 2-year overall survival was 17%. TP53 mutations were present in three of five (60%) persistent tumors compared to 2% (one of 47) of treatment-responsive HPV-positive tumors (p = 0.008). Other genes with recurrent mutations in persistent HPV-positive OPSCC tumors were NF1, KMT2D, PIK3C2B, and TFGBR2. Compared to treatment-responsive HPV-positive tumors, persistent tumors demonstrated activation of DNA Repair and p53, EMT, MYC, SRC, and TGF-beta signaling pathways, with post-treatment samples demonstrating significant activation of the PI3K-EMT-Stem pathways compared to pretreatment samples. Chemoradiation-resistant HPV-positive OPSCC occurs infrequently but portends a poor prognosis. These tumors demonstrate higher rates of p53 mutation and activation of MYC, SRC, and TGF-beta pathways. A comparison of tumors before and after treatment demonstrates PI3K-EMT-Stem pathways post-treatment in HPV-positive tumors with persistent disease after platinum-based chemoradiation.

Our experience diagnosing 225 patients with cervical glandular lesions: current technologies, lessons learned, and areas for improvement

ObjectiveTo explore the relative sensitivity of different methods for detecting cervical glandular lesions.MethodsA total of 225 patients with cervical glandular lesions diagnosed from January 2018 to February 2023 were retrieved from the pathology database of Guangdong Maternal and Child Health Hospital, and their clinicopathological features were reviewed.ResultsFour human papillomavirus (HPV) genotypes: HPV18, 16, 45, and 52, dominated all glandular lesions, and accounting for 74.10% of HPV-positive tumors. Furthermore, 36.89% of abnormal squamous cells were diagnosed as abnormal based on cytological examinations leading to the detection of cervical glandular lesions; only 16.89% were diagnosed based on the initial detection of abnormal glandular cytology. The most common abnormal cervical screening result was ASC-US on cytology (14.22%), followed by HSIL (11.56%). Only few number of patients were diagnosed with or suspected of having cervical adenopathy via a Pap test (18.22%). Nearly one-third of cervical glandular lesions cases were not detected on the Pap test; but were diagnosed upon cervical biopsy or based on the histological examination of ECC, LEEP, or CKC specimens. The LEEP or CKC biopsy specimens had negative margins in 49 cases (40.83%), while the margins were positive in the other 71 cases (59.17%). Five cases (10.20%) with negative margins still had residual lesions following total hysterectomy, and 19 (26.76%) with positive margins had no residual lesions after total hysterectomy.ConclusionThe ability to detect cervical glandular lesions varies for routine HPV genotyping, Pap test, or biopsy/ECC, with different sensitivities and advantages and disadvantages for each method.

Impact of pretreatment body mass index on the survival of head and neck cancer patients.

Differences in pretreatment body mass index (BMI) have been associated with survival in squamous cell carcinoma of head and neck (SCCHN). We examined effects of BMI on survival in SCCHN patients after stratifying patients by tumor human papillomavirus (HPV) status and subsite. Totally 2204 SCCHN patients in a prospective study were included in this secondary analysis. Multivariable Cox models were used to evaluate associations between pretreatment BMI and overall survival, disease-specific survival, and disease-free survival. BMI was significantly higher among patients with HPV-positive tumors than HPV-negative tumors. BMI >25 kg/m2 was associated with improved survival, while BMI <18.5 kg/m2 was associated with reduced survival, particularly in patients with HPV-positive oropharyngeal cancer tumors. This exploratory analysis suggests that pretreatment BMI could be an independent prognostic factor of survival outcomes in SCCHN patients, particularly in patients with HPV-positive oropharyngeal cancer tumors. Further prospective investigations are warranted.

Abstract A003: HPV infection causes dependence on alternative DNA damage response pathways providing cancer specific targets for radiosensitization

Abstract Targeted radiosensitization is an attractive approach to improving the efficacy of radiotherapy in cancer treatment. However, drugs targeting ubiquitous DNA damage response (DDR) pathways are not cancer specific and can increase radiation related side effects. Thus, there is an unmet clinical need for tumor specific molecular targets. To address this need we sought to identify DNA damage signaling pathways that are unique to cancer cells. We hypothesized that by expressing its genome in host cells high-risk Human Papilloma Virus (HPV) can rewire DNA damage signaling making HPV-positive tumor cells dependent on alternative pathways to survive radiation, which can be exploited for targeted radiosensitization of HPV-induced cancers. Using a CRISPR/Cas9 screen in cells that selectively express HPV16 proteins E6 or E7 we uncovered genes that cells rely on to survive radiation-induced DNA damage in the context of E6 or E7 expression. We demonstrate that targeting these newly identified genes sensitizes cancer cells to radiation specifically in the presence of E6 or E7. We provide mechanistic insights showing that E6 and E7 alter the cellular epigenome by inhibiting histone lysine acetylation and tri-methylation, respectively. In this setting, a member of a centromeric protein complex identified in the screen is critical for mitotic progression and survival in E6-expressing cells following radiation exposure. Deletion of this factor resulted in delayed mitotic progression, chromosomal missegregation and rapid post-mitotic death in irradiated HPV-positive cells, a phenotype that was mitigated by ablation of E6 expression. Furthermore, we identified an E3 ubiquitin ligase that promotes survival in E7-expressing cells following radiation exposure by alleviating perturbations during DNA replication. Together, we have uncovered DDR pathways that are critical for cell survival after radiation exposure specifically in HPV-positive cells. Our findings suggest that expression of viral proteins E6 and E7 rewires DNA damage signaling networks causing dependence on alternative response pathways. We propose that these pathways can be targeted for tumor-specific radiosensitization of HPV-induced cancers. Citation Format: Michael Goldstein, Akhil Kotwal, Nishanth Gabriel, William Buchser, Monica Sentmanat, Stephanie Markovina, Julie Schwarz, Xia Cui. HPV infection causes dependence on alternative DNA damage response pathways providing cancer specific targets for radiosensitization [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A003.