HPV-negative Cell Research Articles

Comparison of a modified staging system with 8th edition AJCC criteria in a North American cohort of pT2/pT3 HPV-negative penile squamous cell carcinoma

The staging for pT2/pT3 penile squamous cell carcinoma (pSCC) has undergone major changes. Some authors proposed criteria wherein the distinction between pT2/pT3 was made using the same histopathological variables that are currently utilized to differentiate pT1a/pT1b. In this single-institution, North American study, we focused on (HPV-negative) pT2/3 pSCCs (i.e., tumors invading corpus spongiosum/corpus cavernosum), and compared the prognostic ability of the following systems: (i) AJCC (8th edition) criteria; (ii) modified staging criteria proposed by Sali et al. (Am J Surg Pathol. 2020; 44:1112–7). In the proposed system, pT2 tumors were defined as those devoid of lymphovascular invasion (LVI) or perineural invasion (PNI), and were not poorly differentiated; whereas pT3 showed one or more of the following: LVI, PNI, and/or grade 3. 48 pT2/pT3 cases were included (AJCC, pT2: 27 and pT3: 21; Proposed, pT2: 22 and pT3: 26). The disease-free survival (DFS) and progression-free survival (PFS) did not differ between pT2 and pT3, following the current AJCC definitions (p = 0.19 and p = 0.10, respectively). When the pT2/3 stages were reconstructed using the modified criteria, however, a statistically significant difference was present in both DFS and PFS between pT2 and pT3 (p = 0.004 and p = 0.003, respectively). The proposed staging system has the potential to improve the prognostication of pT2/pT3 tumors in pSCC. Each of these histopathologic variables has been shown to have a significant association with outcomes in pSCC, which is an advantage. Further studies are needed to demonstrate the utility of this modified staging system in patient populations from other geographic regions.

Abstract LB156: Targeting epigenetic activity of beta-catenin/CBP impedes oral cancer progression

Abstract Head and neck cancer is a complex malignancy with its major anatomical subsite, cancer of the oral cavity, ranking among the most deadly and disfiguring cancers. Oral cancer presents primarily as HPV-negative oral squamous cell carcinoma (OSCC) whose etiology is associated with tobacco and alcohol use and for which there are limited treatments. Whereas immunotherapies, such as anti-PD-1 antibodies, have become a promising therapeutic option, only ~20% of head neck cancer patients benefit from anti-PD-1 immunotherapy. We and others have shown that non-mutational epigenetic reprogramming mediated by the β-catenin/CBP complex promotes aggressive cell phenotypes in OSCC by establishing open chromatin at the transcription start sites of genes associated with cancer stemness. Accordingly, pharmacological inhibition of this complex dramatically inhibits OSCC evolution in a syngeneic mouse model of OSCC. Here, we evaluated whether the inhibition of β-catenin/CBP epigenetic activity would enhance the effects of anti-PD-1 immunotherapy. We adapted a syngeneic mouse model of tobacco-associated oral carcinogenesis that utilizes a mouse cell line, 4MOSC1, derived from 4-nitroquinoline-1 oxide- (4NQO)-induced tongue tumors of C57BL/6J mice. After tumors developed for 7 days, mice were blindly separated into treatment cohorts (n=13, each) to receive vehicle control (PBS), monotherapy (either 10 mg/kg anti-PD-1 EOD IP, or 100 mg/kg E7386, a Wnt signaling pathway modulator, QD oral gavage) or combination therapy (anti-PD-1 + E7386). Tumor isografts were harvested 11 days post initiation of treatments and processed for histopathology, immunohistochemistry, and immunofluorescence analyses. Measurements of tumor volumes showed that combination therapy resulted in a statistically significant reduction by ~80% compared to control mice and that it surpassed tumor decrease by either monotherapy. These results were confirmed by histopathology analyses. Evaluation of tumor tissues by immunofluorescence revealed statistically significant decreases in Cbp, H3K4me3, Keratin 14, and Podoplanin expression in response to E7386 alone and in combination with anti PD-1. Collectively our results suggest that a combination therapy comprising anti PD-1 treatment with E7386-targeting β-catenin/CBP epigenetic signaling is likely to impede OSCC evolution to advanced disease. Supported by Eisai, Co Ltd Research Award (MAK), NIH 5R01 DE030350 (MAK, SM, XV) and 5R01 DE031413 (MVB). Citation Format: Emily Fisher, Anthony Spinella, Junji Matsui, Kenichi Nomoto, Xaralabos Varelas, Manish Bais, Maria Kukuruzinska. Targeting epigenetic activity of beta-catenin/CBP impedes oral cancer progression [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 LB156.

Relating pre-treatment non-Gaussian intravoxel incoherent motion diffusion-weighted imaging to human papillomavirus status and response in oropharyngeal carcinoma

Background and purposeDiffusion-weighted imaging (DWI) is a promising technique for response assessment in head-and-neck cancer. Recently, we optimized Non-Gaussian Intravoxel Incoherent Motion Imaging (NG-IVIM), an extension of the conventional apparent diffusion coefficient (ADC) model, for the head and neck. In the current study, we describe the first application in a group of patients with human papillomavirus (HPV)-positive and HPV-negative oropharyngeal squamous cell carcinoma. The aim of this study was to relate ADC and NG-IVIM DWI parameters to HPV status and clinical treatment response. Materials and methodsThirty-six patients (18 HPV-positive, 18 HPV-negative) were prospectively included. Presence of progressive disease was scored within one year. The mean pre-treatment ADC and NG-IVIM parameters in the gross tumor volume were compared between HPV-positive and HPV-negative patients. In HPV-negative patients, ADC and NG-IVIM parameters were compared between patients with and without progressive disease. ResultsADC, the NG-IVIM diffusion coefficient D, and perfusion fraction f were significantly higher, while pseudo-diffusion coefficient D* and kurtosis K were significantly lower in the HPV-negative compared to HPV-positive patients. In the HPV-negative group, a significantly lower D was found for patients with progressive disease compared to complete responders. No relation with ADC was observed. ConclusionThe results of our single-center study suggest that ADC is related to HPV status, but not an independent response predictor. The NG-IVIM parameter D, however, was independently associated to response in the HPV-negative group. Noteworthy in the opposite direction as previously thought based on ADC.

Treatment Delay and HPV Status on OPSCC With Upfront Surgery: Analysis of National Cancer Database.

Evaluate the effect of treatment delay on survival in human papillomavirus (HPV)-positive and HPV-negative oropharyngeal squamous cell carcinoma (OPSCC) patients undergoing primary surgical resection. Retrospective cohort study using the 2010-2017 National Cancer Database. Multicenter database study. Patients >18 years old with OPSCC and known HPV status, treated surgically with or without postoperative radiation/chemotherapy were included. Two cohorts based on HPV status were grouped by time to treatment initiation (TD-TI, ≤30, 31-60, ≥61 days) and surgery to radiotherapy (TS-RT, ≤42, 43-66, ≥67 days). Univariate, Kaplan-Meier, and multivariate analyses assessed correlations between demographic and clinical factors with overall survival in treatment delay groups. Included were 1643 HPV-positive OPSCC patients and 391 HPV-negative OPSCC patients. No associations between survival and gender, age, race, insurance, or radiotherapy length were observed. Regardless of HPV status, larger tumor size (>2 cm) and lymphovascular invasion predicted worse survival. HPV negative patients with >4 lymph nodes involved had 2.5×greater mortality risk (P = .039). Robotic surgery was associated with improved survival only in HPV positive patients (hazard ratio [HR]: 0.41, P < .001). In HPV positive patients, higher TD-TI related to lower mean survival, although this was not significant on multivariate analysis. HPV negative patients with >42 days of TS-RT had decreased survival (43-66 days, HR 1.63, P = .049; ≥67 days, HR 2.10, P = .032). Longer TS-RT was associated with lower overall survival in HPV negative patients. Treatment delay was not associated with survival in HPV positive OPSCC according to multivariate analysis. These findings enhance knowledge about treatment delay effects in OPSCC, aiding providers in decisions and patient communication.

Plumbagin as a preferential lead molecule to combat EGFR-driven matrix abundance and migration of cervical carcinoma cells.

The handshake between the complex networks of matrix components in the tumor micro-environment (TME) is considered as a crucial event in the progression of several cancers including cervical carcinoma (CC). A number of studies report a connection between epidermal growth factor (EGF) and matrix component production. Studies demonstrate that the mechano-transduction trigger by collagen, influences the tumor cells to undergo epithelial-mesenchymal transition (EMT) and block the entryof drugs. We hypothesize that the intervention to prevent EGF triggered deposition of matrix components could sensitize several therapies for CC cells. We utilized morphological assessment, MTT assay, mitored tracking, acridine orange (AO)/ ethidium bromide (EtBr) staining and bromodeoxyuridine (BrdU) assay to measure the cell viability, mitochondrial activity, cellular apoptosis, and DNA synthesis. Clonogenic assay and scratch healing assay were executed to address the stemness and migratory potential. Detection of glycosaminoglycan's (GAGs), collagen, matrix metalloproteinase (MMP)-2/9 secretion and calcium (Ca2+) ions were performed to assess the production of matrix components. Finally, the interaction between EGFR and plumbagin was evaluated by employing molecular dynamics (MD) simulation. Pre-treating the cells with plumbagin inhibited the EGF-induced EMT along with reduction in cell proliferation, migration, clonogenesis and depletion of matrix components. The actions of EGF and plumbagin were more pronounced in HPV-positive CC cells than HPV-negative CC cells. This study identified that increased matrix production triggered by EGF-rich milieu is inhibited by plumbagin in human papilloma viral (HPV) 68 positive ME180, HPV 16 positive SiHa and HPV-negative C33A cell lines. Delivery of plumbagin directly to TME would effectively accelerate the clearance of CC cells, reduce metastasis and matrix abundance by employing targeted delivery to minimize the undesired effects of plumbagin.

Abstract 4603: Single-cell and spatial dissection of the effect of combined PD-L1 and TGFb blockade by bintrafusp alfa from the ICING window-of-opportunity trial in resectable locally advanced HNSCC patients

Abstract Despite high tumor mutational burden, immune checkpoint blockade has shown limited efficacy in head and neck squamous cell carcinoma (HNSCC), highlighting the need for additional therapeutic targets. TGFb signaling has been associated with an immunosuppressive tumor microenvironment as well as a lack of response to anti-PD(L)1 therapy, which led to the development of combined therapeutic approaches. ICING is a prospective (NCT04428047) open label, multicenter, phase II, window-of-opportunity preoperative, single-agent trial. The primary objective was to evaluate the efficacy and biological activity of pre-operative combined PD-L1 and TGFb blockade by bintrafusp alfa in patients with untreated resectable stage III/IV locally advanced HNSCC and its effect on the tumor microenvironment (TME). Tumor samples were harvested at baseline prior to immunotherapy and at surgery after two doses (1200mg; D1 and D15) for pre- and post-treatment comparison. Treatment efficacy was assessed using the pathological response (PathR) with a 10% threshold for response as primary endpoint. Changes in the TME were determined by single-cell RNA sequencing (scRNAseq) and multiplexed imaging. Seven patients were included and six received the full treatment scheme (5 oral cavity and 2 oropharyngeal, all HPV-negative; median age = 61y, range 34y-74y). Four patients had a PathR exceeding 10%, including one with a major PathR above 50%. The TME pre-treatment of this major responder did not differ from the other samples, but this sample was set apart by the presence of proliferative, p16+, HPV-negative tumor cells with mildly higher genomic alterations whilst still classified as low tumor mutational burden. Regardless of treatment response, we report several changes in post-treatment samples: (i) the remodeling of the stromal architecture concomitant with a phenotypic shift in cancer-associated fibroblasts (CAF) with a reduction in activated LRRC15+ CAF and an increase in CD10+IL24+ CAF; (ii) a reduction of TGFb-induced genes in both stromal and cancer cells; (iii) a reduction in both conventional and regulatory CD4+ T cells; (iv) an increase in CD8+ T cell infiltration in previously excluded tumors; (v) an interferon gamma-mediated activation of monocytes, macrophages and dendritic cells expressing CXCL9 and CXCL10. Collectively, our study deciphers the effects of bintrafusp alfa both molecularly and spatially at unprecedented granularity, which is of major importance for the many combined PD(L)1 and TGFb blockade therapeutic approaches currently under clinical development. Citation Format: Caroline Hoffmann, Jan-Timon Werle, Jocelyn Gal, Philemon Sirven, Jerzy Klijanienko, Jeremy Mesple, Benjamin Demaille, Wail Zeitouni, Olivier Choussy, Gregoire Marret, Nathalie Badois, Antoine Dubray-Vautrin, Gilles Dolivet, Lionnel Geoffrois, Maud Kamal, Ivan Bieche, Laure Monard, Clotilde Simon, Christophe Le Tourneau, Helene Salmon. Single-cell and spatial dissection of the effect of combined PD-L1 and TGFb blockade by bintrafusp alfa from the ICING window-of-opportunity trial in resectable locally advanced HNSCC patients [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 4603.

Abstract 1729: SUV420H1 depletion reveals therapeutic potential in HPV-negative head and neck squamous cell carcinoma

Abstract Human Papilloma Virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) poses a significant clinical challenge with limited treatment options. SUV420H1, which encodes for a protein lysine methyltransferase (PMT) that methylates H4K20 (H4K20me3), is recurrently amplified in ~5% of HPV-negative HNSCC tumors and is the second most frequently amplified PMT in this disease, suggesting an oncogenic function in this subset of HPV-negative HNSCC patients. Gene set enrichment analysis using the TCGA database of HPV-negative HNSCC tumors revealed that SUV420H1 overexpressing tumors showed enrichment in the mitotic spindle related pathways, and repression of immune-related pathways. To assess whether SUV420H1 depletion affects the proliferation of HPV-negative HSNCC cells, CCK8 and colony formation assays were pursued after siRNA-mediated knockdown of SUV420H1 in five SUV420H1-overexpressing HPV-negative HNSCC cell lines and revealed a significant reduction in cell proliferation and colony forming capacity. SUV420H1 CRISPR knockout (KO) HPV-negative HNSCC human and mouse cell lines were generated. Proliferation and colony formation assays with the SUV420H1 KO cell lines are ongoing to validate the aforementioned phenotypes. To identify oncogenic mechanism of SUV420H1, genome-wide mapping of H4K20me3 after siRNA-mediated depletion or enzymatic inhibition of SUV420H1 (A-196) was pursued using CUT&amp;RUN assays combined with RNA-seq in a SUV420H1 amplified cell line (SCC-151), and analysis is ongoing. Preliminary RNA-seq analysis revealed upregulation of a number of type I IFN response genes after siRNA-mediated knockdown of SUV420H1. In vivo mouse experiments using SUV420H1 KO mouse cell lines are planned in a syngeneic HPV-negative HNSCC flank mouse model (MOC1) with or without anti-PD-1 treatment with the goal to assess whether SUV420H1 depletion affects tumor growth and/or sensitizes cancer cells to anti-PD-1 therapy. These data provide preliminary support for the function of SUV420H1 as an oncogene in HPV-negative HNSCC. Citation Format: Arfa Moshiri, Sohyoung Kim, Marie Luff, Vassiliki Saloura. SUV420H1 depletion reveals therapeutic potential in HPV-negative head and neck squamous cell carcinoma [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 1729.

Abstract 1734: Dissecting the bifaceted function of SMYD3 as a transcriptional activator and repressor in HPV-negative HNSCC

Abstract Background: Approximately 75% of Human-papilloma-virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) tumors carry genetic and expression alterations in the family of protein methyltransferases and demethylases, underscoring the importance of these enzymes in the pathogenesis of HNSCC. SMYD3 a lysine methyltransferase that is overexpressed in HPV-negative HNSCC. We recently reported that nuclear SMYD3 plays dual role in immune-related gene regulation through H3K4me3 or H4K20me3, leading to gene activation or silencing, respectively in HPV-negative HNSCC. While SMYD3 has been reported to write and bind to H3K4me3 activating target gene expression, its function in gene silencing is understudied. This study aims to determine the mechanism(s) through which SMYD3 functions both as a transcriptional activator and a repressor within the same cell context, specifically in HPV-negative HNSCC. Methods: immunoprecipitation (IP) of SMYD3 was performed from nuclear extracts isolated from HN-6 cells to identify candidate interacting partners of SMYD3. The nuclear immunoprecipitates were sent for MS analysis to identify candidate nuclear interacting partners of SMYD3. CUT&amp;RUN assays for selected candidate interacting proteins and SMYD3 will be performed in control HN-6 cells versus SMYD3 KO cells to assess genomic co-occupancy with SMYD3. Further, CUT&amp;RUN assays for repressive marks H4K20me3, H3K27me3 and H3K9me3 have been performed in HN-6 and SMYD3 KO cells to assess co-occupancy with SMYD3 as well as its effect of SMYD3 on the genome-wide mapping of these marks. Finally, CUT&amp;RUN assays for SMYD3 followed by re-CUT&amp;RUN of repressive histone marks are ongoing to identify co-occupied loci. Results: Preliminary analysis of our nuclear SMYD3 IP results has identified a chromatin modifier with known repressive function. Co-immunoprecipitation experiments to validate this interaction are planned. CUT&amp;RUN assays for repressive histone marks in HN-6 versus SMYD3 KO cells have revealed impact on H4K20me3 and H3K27me3. Conclusions: Our preliminary results suggest that SMYD3 may exert its silencing function by regulating the genome-wide deposition of H4K20me3.Our nuclear SMYD3 IP analysis has identified candidate interacting partners that may assist SMYD3 in promoting the deposition of these repressive marks. CUT&amp;RUN assays for SMYD3 followed by re-CUT&amp;RUN for H4K20me3 and H3K27me3 bound chromatin may provide further insight about whether SMYD3 co-occupies specific genomic regions together with these repressive marks. This project is expected to provide mechanistic insights into the bifaceted functions of SMYD3 within the same cell context using HPV-negative HNSCC cell lines as an experimental model system.. Citation Format: Jawad Akhtar, Sohyoung Kim, Vassiliki Saloura. Dissecting the bifaceted function of SMYD3 as a transcriptional activator and repressor in HPV-negative HNSCC [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 1734.

Combined IL6 and CCR2 blockade potentiates antitumor activity of NK cells in HPV-negative head and neck cancer

BackgroundWhile T cell-activating immunotherapies against recurrent head and neck squamous cell carcinoma (HNSCC) have shown impressive results in clinical trials, they are often ineffective in the majority of patients. NK cells are potential targets for immunotherapeutic intervention; however, the setback in monalizumab-based therapy in HNSCC highlights the need for an alternative treatment to enhance their antitumor activity.MethodsSingle-cell RNA sequencing (scRNA-seq) and TCGA HNSCC datasets were used to identify key molecular alterations in NK cells. Representative HPV-positive ( +) and HPV-negative ( −) HNSCC cell lines and orthotopic mouse models were used to validate the bioinformatic findings. Changes in immune cells were examined by flow cytometry and immunofluorescence.ResultsThrough integration of scRNA-seq data with TCGA data, we found that the impact of IL6/IL6R and CCL2/CCR2 signaling pathways on evasion of immune attack by NK cells is more pronounced in the HPV − HNSCC cohort compared to the HPV + HNSCC cohort. In orthotopic mouse models, blocking IL6 with a neutralizing antibody suppressed HPV − but not HPV + tumors, which was accompanied by increased tumor infiltration and proliferation of CD161+ NK cells. Notably, combining the CCR2 chemokine receptor antagonist RS504393 with IL6 blockade resulted in a more pronounced antitumor effect that was associated with more activated intratumoral NK cells in HPV − HNSCC compared to either agent alone.ConclusionsThese findings demonstrate that dual blockade of IL6 and CCR2 pathways effectively enhances the antitumor activity of NK cells in HPV-negative HNSCC, providing a novel strategy for treating this type of cancer.

256 comparison of clinicopathological and prognostic features of HPV positive and HPV negative head and neck squamous cell carcinoma (HNSCC)

256 comparison of clinicopathological and prognostic features of HPV positive and HPV negative head and neck squamous cell carcinoma (HNSCC)

Human papillomavirus-16 E6 activates the pentose phosphate pathway to promote cervical cancer cell proliferation by inhibiting G6PD lactylation

High-risk human papillomaviruses (HPVs) are the causative agents of cervical cancer. Here, we report that HPV16 E6E7 promotes cervical cancer cell proliferation by activating the pentose phosphate pathway (PPP). We found that HPV16 E6 activates the PPP primarily by increasing glucose-6-phosphate dehydrogenase (G6PD) enzyme activity. Mechanistically, HPV16 E6 promoted G6PD dimer formation by inhibiting its lactylation. Importantly, we suggest that G6PD K45 was lactylated during G6PD-mediated antioxidant stress. In primary human keratinocytes and an HPV-negative cervical cancer C33A cells line ectopically expressing HPV16 E6, the transduction of G6PD K45A (unable to be lactylated) increased GSH and NADPH levels and, correspondingly, decreasing ROS levels. Conversely, the re-expression of G6PD K45T (mimicking constitutive lactylation) in HPV16-positive SiHa cells line inhibited cell proliferation. In vivo, the inhibition of G6PD enzyme activity with 6-aminonicotinamide (6-An) or the re-expression of G6PD K45T inhibited tumor proliferation. In conclusion, we have revealed a novel mechanism of HPV oncoprotein-mediated malignant transformation. These findings might provide effective strategies for treating cervical and HPV-associated cancers.

INHBA is Enriched in HPV-negative Oropharyngeal Squamous Cell Carcinoma and Promotes Cancer Progression.

Patients with HPV-negative OPSCC have a poorer prognosis due to distinct molecular pathways. This study reveals significant transcriptomic differences between HPV-negative and HPV-positive OPSCC, identifying INHBA as a key upregulated gene in HPV-negative OPSCC's oncogenic pathways. INHBA is crucial in promoting EMT, cell proliferation, and an immunosuppressive tumor environment, suggesting its potential as a therapeutic target for HPV-negative OPSCC.

575 - Differences in mitochondrial gene expression between white and african americans with HPV-negative oropharyngeal squamous cell carcinoma

575 - Differences in mitochondrial gene expression between white and african americans with HPV-negative oropharyngeal squamous cell carcinoma

Human papillomavirus-associated head and neck squamous cell carcinoma cells rely on glycolysis and display reduced oxidative phosphorylation.

Head and neck squamous cell carcinoma (HNSCC) constitutes a heterogeneous group of cancers. Human papilloma virus (HPV) is associated with a subtype of HNSCC with a better response to treatment and more favorable prognosis. Mitochondrial function and metabolism vary depending on cancer type and can be related to tumor aggressiveness. This study aims to characterize the metabolism of HPV-positive and HPV-negative HNSCC cell lines. Oxidative phosphorylation (OXPHOS) and glycolysis were assessed in intact cells, in four HNSCC cell lines using Seahorse XF Analyzer. OXPHOS was further studied in permeabilized cells using high-resolution respirometry in an Oroboros O2K. Metabolomic analysis was performed using mass spectroscopy. The HPV-negative cell lines were found to display a higher OXPHOS capacity and were also able to upregulate glycolysis when needed. The HPV-positive cell line had a higher basal glycolytic rate but lower spare OXPHOS capacity. These cells were also unable to increase respiration in response to succinate, unlike the HPV-negative cells. In the metabolomic analysis, the HPV-positive cells showed a higher kynurenine/tryptophan ratio. HPV-positive HNSCC preferred glycolysis to compensate for lower OXPHOS reserves, while the HPV-negative HNSCC displayed a more versatile metabolism, which might be related to increased tumor aggressiveness. The higher kynurenine/tryptophan ratio of HPV-positive HNSCC might be related to increased indoleamine 2,3-dioxygenase activity due to the carcinoma's viral origin. This study highlights important metabolic differences between HPV-positive and HPV-negative cancers and suggests that future metabolic targets for cancer treatment should be individualized based on specific tumor metabolism.

Human papillomavirus E7 protein induces homologous recombination defects and PARPi sensitivity

PurposeCervical cancer is a common gynecological malignancy, pathologically associated with persistent infection of high-risk types of human papillomavirus (HPV). Previous studies revealed that HPV-positive cervical cancer displays genomic instability; however, the underlying mechanism is not fully understood.MethodsTo investigate if DNA damage responses are aggravated in precancerous lesions of HPV-positive cervical epithelium, cervical tissues were biopsied and cryosectioned, and subjected to immunofluorescent staining. Cloned HA-tagged E6 and E7 genes of HPV16 subtype were transfected into HEK293T or C33A cells, and indirect immunofluorescent staining was applied to reveal the competency of double strand break (DSB) repair. To test the synthetic lethality of E7-indued HRD and PARP inhibitor (PARPi), we expressed E7 in C33A cells in the presence or absence of olaparib, and evaluated cell viability by colony formation.ResultsIn precancerous lesions, endogenous DNA lesions were elevated along with the severity of CIN grade. Expressing high-risk viral factor (E7) in HPV-negative cervical cells did not impair checkpoint activation upon genotoxic insults, but affected the potential of DSB repair, leading to homologous recombination deficiency (HRD). Based on this HPV-induced genomic instability, the viability of E7-expressing cells was reduced upon exposure to PARPi in comparison with control cells.ConclusionIn aggregate, our findings demonstrate that HPV-E7 is a potential driver for genome instability and provides a new angle to understand its role in cancer development. The viral HRD could be employed to target HPV-positive cervical cancer via synthetic lethality.

Preclinical Evaluation of the ATR Inhibitor BAY 1895344 as a Radiosensitizer for Head and Neck Squamous Cell Carcinoma

Despite aggressive multimodal treatment that typically includes definitive or adjuvant radiation therapy (RT), locoregional recurrence rates approach 50% for patients with locally advanced human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC). Thus, more effective therapeutics are needed to improve patient outcomes. We evaluated the radiosensitizing effects of ataxia telangiectasia and RAD3-related (ATR) inhibitor (ATRi) BAY 1895344 in preclinical models of HNSCC. Murine and human HPV-negative HNSCC cells (MOC2, MOC1, JHU-012) were treated with vehicle or ATRi with or without 4 Gy. Checkpoint kinase 1 phosphorylation and DNA damage (γH2AX) were evaluated by Western blot, and ATRi half-maximal inhibitory concentration was determined by MTT assay for HNSCC cells and immortalized murine oral keratinocytes. In vitro radiosensitization was tested by clonogenic assay. Cell cycle distribution and mitotic catastrophe were evaluated by flow cytometry. Mitotic aberrations were quantified by fluorescent microscopy. Tumor growth delay and survival were assessed in mice bearing MOC2 or JHU-012 transplant tumors treated with vehicle, ATRi, RT (10 Gy × 1 or 8 Gy × 3), or combined ATRi + RT. ATRi caused dose-dependent reduction in checkpoint kinase 1 phosphorylation at 1 hour post-RT (4 Gy) and dose-dependent increase in γH2AX at 18 hours post-RT. Addition of RT to ATRi led to decreased BAY 1895344 half-maximal inhibitory concentration in HNSCC cell lines but not in normal tissue surrogate immortalized murine oral keratinocytes. Clonogenic assays demonstrated radiosensitization in the HNSCC cell lines. ATRi abrogated the RT-induced G2/M checkpoint, leading to mitosis with unrepaired DNA damage and increased mitotic aberrations (multinucleated cells, micronuclei, nuclear buds, nucleoplasmic bridges). ATRi and RT significantly delayed tumor growth in MOC2 and JHU-012 in vivo models, with improved overall survival in the MOC2 model. These findings demonstrated that BAY 1895344 increased in vitro and in vivo radiosensitivity in HPV-negative HNSCC preclinical models, suggesting therapeutic potential warranting evaluation in clinical trials for patients with locally advanced or recurrent HPV-negative HNSCC.

HPV-Negative and HPV-Positive Oral Cancer Cells Stimulate the Polarization of Neutrophils towards Different Functional Phenotypes In Vitro.

High-risk human papillomavirus (HPV) infection is one of the leading causes of oropharyngeal squamous cell carcinoma (OPSCC), while the correlation between HPV and oral squamous cell carcinoma (OSCC) remains controversial. The inflammatory infiltrate involved in these epithelial neoplasms differs based on their association with HPV. HPV- tumors show higher tumor-associated neutrophil (TAN) infiltration. It is believed that TANs can play a dual role in cancer by exerting either anti-tumorigenic or pro-tumorigenic effects. However, the impact of HPV status on neutrophil polarization remains unknown. Therefore, this study aimed to investigate the effect of OSCC cells, both HPV- and HPV16+, on the functional phenotype of neutrophils. Peripheral blood neutrophils were stimulated with supernatants from OSCC cell lines and non-tumorigenic HaCaT keratinocytes transduced with HPV16 E6/E7 oncogenes. Subsequently, cytokine production, cell viability, metabolism, expression of degranulation markers, and PD-L1 expression were evaluated. Our findings demonstrate that in contrast to UPCI:SCC154 (HPV+ OSCC) cells, the SCC-9 (HPV- OSCC) cell line induced a highly activated functional state in neutrophils, which is potentially associated with a pro-tumorigenic effect. The HaCaT 16-E7 supernatant only stimulated the activation of some neutrophil functions. Understanding the complex interplay between neutrophils and their microenvironment has the potential to identify TANs as viable therapeutic targets.

Analysis of cells of epithelial, connective tissue and immune differentiation in HPV-positive-, HPV-negative oropharyngeal carcinoma and normal oropharyngeal tissue by immunofluorescence multiplex image cytometry: a preliminary report

BackgroundEpithelial, connective tissue and immune cells contribute in various ways to the pathophysiology of HPV positive (HPV+) and HPV negative (HPV-) oropharyngeal squamous cell carcinoma (OPSCC). We aimed to investigate the abundance of these cell lineages and their coexpression patterns in patients with HPV + and HPV- OPSCC.MethodsWe used a 4-channel immunofluorescence-microscopy technique for the simultaneous detection of three direct-conjugated antibodies (pancytokeratin, vimentin and CD45/CD18) and DAPI (4’,6-Diamidin-2-phenylindole) in formalin fixed paraffin-embedded tissue samples (FFPE) of patients with HPV + and HPV- OPSCC, and of control patients. Image acquisition and analysis were performed with TissueFAXS and StrataQuest (TissueGnostics, Vienna, Austria), respectively, in tumor cell clusters/stroma in OPSCC specimens and epithelial layer/lamina propria in control specimens. Cell populations were created based on antibodies’ coexpression patterns. Isotype and positive controls were examined for plausibility.ResultsThe proportion of cells of epithelial differentiation in tumor cell clusters was higher in HPV + OPSCC (55%) than in HPV- OPSCC samples (44%). The proportion of connective tissue cells in tumor cell cluster was lower in HPV + OPSCC patients (18%) than in HPV- OPSCC patients (26%). The proportion of immune cells in tumor cell clusters was higher in HPV + OPSCC patients (25%) than in HPV- OPSCC patients (18%). The percentage of anaplastic, potentially de-differentiated cells, was 2% in control patients, and it was higher in HPV- OPSCC (21%) than in HPV + OPSCC samples (6%).ConclusionsThis study provided the first quantitative data for the abundance of cells of epithelial, connective tissue and immune differentiation, in patients with OPSCC and control patients. The abundance of these different crucial cell populations was consistently originating from the same tissue sample. De-differentiation of tumor cells was higher in HPV- OPSCC than in HPV + OPSCC. In tumor cells clusters, the antitumoral host immune response was higher in HPV + OPSCC than in HPV- OPSCC, whereas the fibroblast response was higher in HPV- OPSCC than in HPV + OPSCC. This study contributed to the understanding of histopathologic differences between HPV + OPSCC and HPV- OPSCC patients.

A novel tailed primer nucleic acid test for detection of HPV 16, 18 and 45 DNA at the point of care

Cervical cancer is a leading cause of death for women in low-resource settings despite being preventable through human papillomavirus (HPV) vaccination, early detection, and treatment of precancerous lesions. The World Health Organization recommends high-risk HPV (hrHPV) as the preferred cervical cancer screening strategy, which is difficult to implement in low-resource settings due to high costs, reliance on centralized laboratory infrastructure, and long sample-to-answer times. To help meet the need for rapid, low-cost, and decentralized cervical cancer screening, we developed tailed primer isothermal amplification and lateral flow detection assays for HPV16, HPV18, and HPV45 DNA. We translated these assays into a self-contained cartridge to achieve multiplexed detection of three hrHPV genotypes in a disposable cartridge. The developed test achieves clinically relevant limits of detection of 50–500 copies per reaction with extracted genomic DNA from HPV-positive cells. Finally, we performed sample-to-answer testing with direct lysates of HPV-negative and HPV-positive cell lines and demonstrated consistent detection of HPV16, HPV18, and HPV45 with 5000–50,000 cells/mL in < 35 min. With additional optimization to improve cartridge reliability, incorporation of additional hrHPV types, and validation with clinical samples, the assay could serve as a point-of-care HPV DNA test that improves access to cervical cancer screening in low-resource settings.

A comparative analysis of phyto-components on EGFR binding, viability, and migration in HPV positive ME180 and HPV negative C33A cervical cancer cells

A comparative analysis of phyto-components on EGFR binding, viability, and migration in HPV positive ME180 and HPV negative C33A cervical cancer cells