Senescent Tumor Cells Are Frequently Present at the Invasion Front: Implications for Improving Disease Control in Patients with Locally Advanced Prostate Cancer.

INTRODUCTION: Local tumor invasion is a critical factor for the outcome of men with prostate cancer. In particular, seminal vesicle invasion (SVI) has been reported to be associated with a more unfavorable prognosis. A better understanding of the functional state of invading prostate cancer cells is crucial to develop novel therapeutic strategies for patients with locally advanced disease. METHODS: The prognostic impact of local tumor progression was ascertained in over 1,000 men with prostate cancer. Prostate cancer specimens were stained by double-immunohistochemistry for the proliferation marker Ki-67 and the senescence marker p16INK4A. The migratory properties of senescent prostate cancer cells were analyzed in vitro using a wound healing assay and immunofluorescence microscopy for p16INK4A. RESULTS: We confirm the notion that patients with SVI have a more unfavorable prognosis than patients with extraprostatic extension alone. Surprisingly, we found that the tumor invasion front frequently harbors p16INK4A-positive and Ki-67-negative, i.e., senescent, tumor cells. While the intraprostatic tumor periphery was a hotspot for both proliferation and expression of p16INK4A, the area of SVI showed less proliferative activity but was at the same time a hotspot of cells with increased nuclear p16INK4A expression. Senescence was associated with an accelerated migration of prostate cancer cells in vitro. CONCLUSION: This proof-of-concept study shows that invading prostate cancer cells frequently show signs of cellular senescence. This finding may open new avenues for neoadjuvant and adjuvant treatment concepts in men with locally advanced prostate cancer.

Predictors for Survival of Patients with Squamous Cell Carcinoma of Unknown Primary in the Head and Neck Region.

BACKGROUND: Human papillomavirus (HPV) status is the most important predictor of survival in oropharyngeal squamous cell carcinoma (OPSCC). In patients with cervical lymph node metastases of squamous cell carcinoma of unknown origin (CUPHNSCC), much less is known. METHODS: We assessed a consecutive cohort of CUPHNSCC diagnosed from 2000-2018 for HPV DNA, mRNA, p16INK4a (p16) expression, and risk factors to identify prognostic classification markers. RESULTS: In 32/103 (31%) CUPHNSCC, p16 was overexpressed, and high-risk HPV DNA was detected in 18/32 (56.3%). This was mostly consistent with mRNA detection. In recursive partitioning analysis, CUPHNSCC patients were classified into three risk groups according to performance status (ECOG) and p16. Principal component analysis suggests a negative correlation of p16, HPV DNA, and gender in relation to ECOG, as well as a correlation between N stage, extranodal extension, and tobacco/alcohol consumption. CONCLUSIONS: Despite obvious differences, CUPHNSCC shares similarities in risk profile with OPSCC. However, the detection of p16 alone appears to be more suitable for the classification of CUPHNSCC than for OPSCC and, in combination with ECOG, allows stratification into three risk groups. In the future, additional factors besides p16 and ECOG may become important in larger studies or cases with special risk profiles.

Prognostic value of high-risk human papillomavirus DNA and p16INK4a immunohistochemistry in patients with anal cancer: An individual patient data meta-analysis.

BACKGROUND: High-risk human papillomavirus (hrHPV) types represent the aetiological agents in a major proportion of anal squamous cell carcinomas (ASCC). Several studies have suggested a prognostic relevance of HPV-related markers, particularly hrHPV DNA and p16INK4a (p16) protein expression, in patients with ASCC. However, broader evaluation of these prognostic marker candidates has been hampered by small cohort sizes and heterogeneous survival data among the individual studies. We conducted an individual patient data (IPD) meta-analysis to determine the prognostic value of hrHPV DNA and p16 in patients with ASCC while controlling for major clinical and tumour covariates. PATIENTS AND METHODS: A systematic literature search was conducted to identify all published studies analysing p16 alone or in combination with hrHPV DNA and reporting survival data in patients with ASCC. Clinical and tumour-related IPD were requested from authors of potentially eligible studies. Survival analyses were performed with a proportional hazard Cox model stratified by study and adjusted for relevant covariates. The study-specific hazard ratios (HRs) for the exposures were pooled using a random-effects model. Kaplan-Meier curves from different studies were pooled per exposure group and weighted by the study's total sample size. RESULTS: Seven studies providing IPD from 693 patients with ASCC could be included in the meta-analysis. Seventy-six percent of patients were p16+/hrHPV DNA+, whereas 11% were negative for both markers. A discordant marker status was observed in 13% of cases. Patients with p16+/hrHPV DNA+ ASCC showed significantly superior overall survival (OS) compared with patients with p16-/hrHPV DNA- tumours (pooled adjusted HR = 0.26 [95% confidence interval {CI}, 0.14-0.50]) with pooled three-year OS rates of 86% (95% CI, 82-90%) versus 39% (95% CI, 24-54%). Patients with discordant p16 and hrHPV DNA status showed intermediate three-year OS rates (75% [95% CI, 56-86%] for p16+/hrHPV DNA- and 55% [95% CI, 35-71%] for p16-/hrHPV DNA+ ASCC). CONCLUSION: This first IPD meta-analysis controlling for confounding variables shows that patients with p16+/hrHPV DNA+ ASCC have a significantly better survival than patients with p16-/hrHPV DNA- tumours.

Treatment resistance analysis reveals GLUT-1-mediated glucose uptake as a major target of synthetic rocaglates in cancer cells.

Rocaglates are natural compounds that have been extensively studied for their ability to inhibit translation initiation. Rocaglates represent promising drug candidates for tumor treatment due to their growth-inhibitory effects on neoplastic cells. In contrast to natural rocaglates, synthetic analogues of rocaglates have been less comprehensively characterized, but were also shown to have similar effects on the process of protein translation. Here, we demonstrate an enhanced growth-inhibitory effect of synthetic rocaglates when combined with glucose anti-metabolite 2-deoxy-D-glucose (2DG) in different cancer cell lines. Moreover, we unravel a new aspect in the mechanism of action of synthetic rocaglates involving reduction of glucose uptake mediated by downregulation or abrogation of glucose transporter GLUT-1 expression. Importantly, cells with genetically induced resistance to synthetic rocaglates showed substantially less pronounced treatment effect on glucose metabolism and did not demonstrate GLUT-1 downregulation, pointing at the crucial role of this mechanism for the anti-tumor activity of the synthetic rocaglates. Transcriptome profiling revealed glycolysis as one of the major pathways differentially regulated in sensitive and resistant cells. Analysis of synthetic rocaglate efficacy in a 3D tissue context with a co-culture of tumor and normal cells demonstrated a selective effect on tumor cells and substantiated the mechanistic observations obtained in cancer cell lines. Increased glucose uptake and metabolism is a universal feature across different tumor types. Therefore, targeting this feature by synthetic rocaglates could represent a promising direction for exploitation of rocaglates in novel anti-tumor therapies.

Deep Learning Predicts HPV Association in Oropharyngeal Squamous Cell Carcinomas and Identifies Patients with a Favorable Prognosis Using Regular H&E Stains.

PURPOSE: Human papillomavirus (HPV) in oropharyngeal squamous cell carcinoma (OPSCC) is tumorigenic and has been associated with a favorable prognosis compared with OPSCC caused by tobacco, alcohol, and other carcinogens. Meanwhile, machine learning has evolved as a powerful tool to predict molecular and cellular alterations of medical images of various sources. EXPERIMENTAL DESIGN: We generated a deep learning-based HPV prediction score (HPV-ps) on regular hematoxylin and eosin (H&E) stains and assessed its performance to predict HPV association using 273 patients from two different sites (OPSCC; Giessen, n = 163; Cologne, n = 110). Then, the prognostic relevance in a total of 594 patients (Giessen, Cologne, HNSCC TCGA) was evaluated. In addition, we investigated whether four board-certified pathologists could identify HPV association (n = 152) and compared the results to the classifier. RESULTS: Although pathologists were able to diagnose HPV association from H&E-stained slides (AUC = 0.74, median of four observers), the interrater reliability was minimal (Light Kappa = 0.37; P = 0.129), as compared with AUC = 0.8 using the HPV-ps within two independent cohorts (n = 273). The HPV-ps identified individuals with a favorable prognosis in a total of 594 patients from three cohorts (Giessen, OPSCC, HR = 0.55, P < 0.0001; Cologne, OPSCC, HR = 0.44, P = 0.0027; TCGA, non-OPSCC head and neck, HR = 0.69, P = 0.0073). Interestingly, the HPV-ps further stratified patients when combined with p16 status (Giessen, HR = 0.06, P < 0.0001; Cologne, HR = 0.3, P = 0.046). CONCLUSIONS: Detection of HPV association in OPSCC using deep learning with help of regular H&E stains may either be used as a single biomarker, or in combination with p16 status, to identify patients with OPSCC with a favorable prognosis, potentially outperforming combined HPV-DNA/p16 status as a biomarker for patient stratification.

Organotypic Co-Cultures as a Novel 3D Model for Head and Neck Squamous Cell Carcinoma.

Background: Head and neck squamous cell carcinomas (HNSCC) are phenotypically and molecularly heterogeneous and frequently develop therapy resistance. Reliable patient-derived 3D tumor models are urgently needed to further study the complex pathogenesis of these tumors and to overcome treatment failure. Methods: We developed a three-dimensional organotypic co-culture (3D-OTC) model for HNSCC that maintains the architecture and cell composition of the individual tumor. A dermal equivalent (DE), composed of healthy human-derived fibroblasts and viscose fibers, served as a scaffold for the patient sample. DEs were co-cultivated with 13 vital HNSCC explants (non-human papillomavirus (HPV) driven, n = 7; HPV-driven, n = 6). Fractionated irradiation was applied to 5 samples (non-HPV-driven, n = 2; HPV-driven n = 3). To evaluate expression of ki-67, cleaved caspase-3, pan-cytokeratin, p16INK4a, CD45, ∝smooth muscle actin and vimentin over time, immunohistochemistry and immunofluorescence staining were performed Patient checkup data were collected for up to 32 months after first diagnosis. Results: All non-HPV-driven 3D-OTCs encompassed proliferative cancer cells during cultivation for up to 21 days. Proliferation indices of primaries and 3D-OTCs were comparable and consistent over time. Overall, tumor explants displayed heterogeneous growth patterns (i.e., invasive, expansive, silent). Cancer-associated fibroblasts and leukocytes could be detected for up to 21 days. HPV DNA was detectable in both primary and 3D-OTCs (day 14) of HPV-driven tumors. However, p16INK4a expression levels were varying. Morphological alterations and radioresistant tumor cells were detected in 3D-OTC after fractionated irradiation in HPV-driven and non-driven samples. Conclusions: Our 3D-OTC model for HNSCC supports cancer cell survival and proliferation in their original microenvironment. The model enables investigation of invasive cancer growth and might, in the future, serve as a platform to perform sensitivity testing upon treatment to predict therapy response.

Rearranged ERG confers robustness to prostate cancer cells by subverting the function of p53.

OBJECTIVE: ERG rearrangements are frequent and early events in prostate cancer. The functional role of rearranged ERG, however, is still incompletely understood. ERG rearrangements are maintained during prostate cancer progression suggesting that they may confer a selective advantage. The molecular basis of this notion is the subject of this study. METHODS: A variety of immunological methods were used to characterize the effects of rearranged ERG on p53. Consequences of an overexpression of N-terminally deleted ERG on p53 function were interrogated by measuring apoptosis and cellular senescence in the presence or absence of exogenous DNA damage. Effects of N-terminally deleted ERG on the transactivation function of p53 were analyzed by qRT-PCR. RESULTS: We show that overexpression of ERG leads to an increased basal level of DNA damage and a stabilization of p53 that involves a sequestration of its E3 ubiquitin ligase, MDM2, into nucleoli. A higher p53 expression was also observed in vivo in an ERG-overexpressing prostatic intraepithelial neoplasia mouse model. The correlation between ERG and p53 expression was corroborated in 163 patients with prostate cancer. ERG overexpression was found to inhibit both apoptosis and cellular senescence induced by exogenous DNA damage. Mechanistically, this protective effect of ERG involved an abrogation of the DNA damage-induced expression of p53 target genes. CONCLUSIONS: By protecting tumor cells from the antiproliferative consequences of genotoxic stress, ERG may allow the survival and proliferation of genomically unstable tumor cells. Targeting ERG may therefore represent a promising strategy to suppress such adverse features during prostate cancer progression.

Evaluation of p16INK4a expression as a single marker to select patients with HPV-driven oropharyngeal cancers for treatment de-escalation.

BACKGROUND: A remarkably better prognosis is associated with oropharyngeal squamous cell carcinomas (OPSCC) driven by human papillomaviruses (HPV) compared with HPV-negative OPSCC. Consequently, de-escalation of standard treatment has been suggested. Due to modest specificity rates, debates are ongoing, whether p16INK4a, a surrogate marker for HPV-driven OPSCC, is sufficient to correctly identify those tumours and avoid substantial HPV misattribution and thus undertreatment of patients by de-escalation. Robust data estimating the proportion of potentially undertreated patients are missing. METHODS: We assessed a large-scale cohort of consecutively included OPSCC diagnosed between 2000 and 2017 for HPV-DNA, HPV genotypes, p16INK4a expression and multiple tumour- and patient-related risk factors, and investigated their impact on patients' survival in comprehensive uni- and multivariate analyses. RESULTS: Aetiological relevance of HPV (p16INK4a- and high-risk HPV-DNA-positivity) was detected in 27.1% (n = 192) of OPSCC, with HPV16 being the most abundant HPV type (94.6%). In 5.5% patients (n = 39), p16INK4a overexpression but no HPV-DNA was detected. Principal component and survival analyses revealed that 60.6% of these p16INK4a-positive OPSCC lacking HPV-DNA did not resemble HPV16-driven but HPV-negative OPSCC regarding risk-factor profile and overall survival. Notably, this group represented 10.6% of all p16INK4a-overexpressing OPSCC. CONCLUSIONS: p16INK4a as a single marker appears insufficient to indicate OPSCC patients suitable for treatment de-escalation.

Prognostic Significance of HPV DNA and p16INK4a in Anal Cancer: A Systematic Review and Meta-Analysis.

We conducted a systematic review and meta-analysis of observational studies evaluating survival in patients with anal cancer, according to human papillomavirus (HPV) DNA, p16INK4a, and combined HPV DNA/p16INK4a status. We systematically searched PubMed, EMBASE, and Cochrane Library databases to identify studies published in English until July 25, 2018, directly providing or allowing estimation of survival of patients with anal cancer according to the presence of HPV DNA and/or overexpression of p16INK4a We estimated pooled HRs and 95% confidence intervals (CI) for overall survival (OS) using a random-effects model. We included 16 studies, comprising 1,724 patients with anal cancer tested for HPV DNA (65% positive), and 567 patients tested for p16INK4a (87% positive). The pooled HR for OS was 0.54 (95% CI, 0.33-0.89) for HPV DNA positive versus negative, 0.37 (95% CI, 0.24-0.57) for p16INK4a positive versus negative, and 0.36 (95% CI, 0.22-0.58) for HPV DNA positive/p16INK4a positive versus HPV DNA positive/p16INK4a negative patients with anal cancer. Patients with HPV DNA or p16INK4a positive anal cancer have significantly better OS compared with HPV DNA or p16INK4a negative. This points to the possible value of HPV DNA and/or p16INK4a testing when planning the management and follow-up strategy for patients diagnosed with anal cancer.

Overexpression of p16INK4a Serves as Prognostic Marker in Squamous Cell Vulvar Cancer Patients Treated With Radiotherapy Irrespective of HPV-Status.

Purpose: We aimed to evaluate the impact of HPV-driven carcinogenesis on outcome in vulvar squamous cell carcinoma patients (VSCC) treated with radiotherapy. Methods and Materials: Analysis of clinical, pathological, and treatment data, HPV DNA-detection and -genotyping as well as p16INK4a immunohistochemistry were performed in 75 VSCC patients. Kaplan-Meier-method was used to estimate locoregional control (LC), Progression-free survival (PFS), and Overall Survival (OS). Univariate survival time comparisons were performed using the log-rank-test. Chi-square/Fisher exact test was used to assess correlations between HPV DNA and p16INK4a data, pathological, clinical, and treatment characteristics. Results: 23/75 (30.67%) of all women had locoregional relapse, 7/75 (9.3%) systemic recurrence, and 35/75 (46.67%) died after a median follow-up of 26.4 months. 21.3% of the tumors were HPV DNA-positive, mostly (93.75%) for the high-risk (HR) HPV type 16. 25.3% showed p16INK4a-overexpression. 17.3% showed concomitant HPV DNA- and p16INK4a-positivity (cHPPVC). Patients with p16INK4a-overexpression, irrespective of the HPV DNA status, showed significantly better PFS (5-year-PFS 69.3 vs. 39.2%, p = 0.045), LC (5-year-LC 86.7 vs. 56.7%, p = 0.033) and a strong trend for better OS (5-year-OS 75.6 vs. 43.9%, p = 0.077). Patients with cHPPVC showed a trend for better PFS (5-year-PFS 72.7 vs. 41.3%, p = 0.082) and OS (5-year-OS 81.1 vs. 45.7%, p = 0.084) but no significant benefit for LC. Conclusions: Patients with cHPPVC, indicating an etiological relevance of HPV in the respective tumors, showed a better, albeit not significant, prognosis. The sole detection of p16INK4a-overexpression is a prognostic factor for survival in vulvar cancer and indicates better prognosis after radiotherapy, independent of detection of HPV DNA. p16INK4a should be used as surrogate marker for HPV-driven carcinogenesis in vulvar cancer with caution.

Overcoming radioresistance in WiDr cells with heavy ion irradiation and radiosensitization by 2-deoxyglucose with photon irradiation.

BACKGROUND AND PURPOSE: Radiosensitizers and heavy ion irradiation could improve therapy for female patients with malignant tumors located in the pelvic region through dose reduction. Aim of the study was to investigate the radiosensitizing potential of 2-deoxy-d-glucose (2-DG) in combination with carbon ion-irradiation (12C) in representative cell lines of cancer in the female pelvic region. MATERIALS AND METHODS: The human cervix carcinoma cell line CaSki and the colorectal carcinoma cell line WiDr were used. 2-DG was employed in two different settings, pretreatment and treatment simultaneous to irradiation. Clonogenic survival, α and ß values for application of the linear quadratic model and relative biological effectiveness (RBE) were determined. ANOVA tests were used for statistical group comparison. Isobolograms were generated for curve comparisons. RESULTS: The comparison of monotherapy with 12C versus photons yielded RBE values of 2.4 for CaSki and 3.5 for WiDr along with a significant increase of α values in the 12C setting. 2-DG monotherapy reduced the colony formation of both cell lines. Radiosensitization was found in WiDr for the combination of photon irradiation with synchronous application of 2-DG. The same setup for 12C showed no radiosensitization, but rather an additive effect. In all settings with CaSki, the combination of irradiation and 2-DG exhibited additive properties. CONCLUSION: The combination of 2-DG and photon therapy, as well as irradiation with carbon ions can overcome radioresistance of tumor cells such as WiDr.

PGAM5 is a key driver of mitochondrial dysfunction in experimental lung fibrosis.

RATIONALE: Mitochondrial homeostasis has recently emerged as a focal point in the pathophysiology of idiopathic pulmonary fibrosis (IPF), but conflicting data have been reported regarding its regulation. We speculated that phosphoglycerate mutase family member 5 (PGAM5), a mitochondrial protein at the intersection of multiple cell death and mitochondrial turnover pathways, might be involved in the pathogenesis of IPF. METHODS: PGAM5-deficient mice and human pulmonary epithelial cells were analyzed comparatively with PGAM5-proficient controls in a bleomycin-based model of pulmonary fibrogenesis. Mitochondria were visualized by confocal and transmission electron microscopy. Mitochondrial homeostasis was assessed using JC1 (ΔΨ) and flow cytometry. RESULTS: PGAM5 plays an important role in pulmonary fibrogenesis. Pgam5-/- mice displayed significantly attenuated lung fibrosis compared to controls. Complementary, in vitro studies demonstrated that PGAM5 impaired mitochondrial integrity on a functional and structural level independently of mtROS-production. On a molecular level, reduced mitophagy caused by PGAM5 deficiency improved mitochondrial homeostasis. CONCLUSIONS: Our study identifies PGAM5 as an important regulator of mitochondrial homeostasis in pulmonary fibrosis. Our data further indicate PGAM5-mediated mitophagy itself as a pivotal gateway event in the mediation of self-sustaining mitochondrial damage and membrane depolarization. Our work hereby highlights the importance of mitochondrial dynamics and identifies a potential therapeutic target that warrants further studies. Toxic agents lead to mitochondrial damage resulting in depolarization of the mitochondrial membrane potential (ΔΨ) which is a gateway event for the initiation of PGAM5-mediated mitophagy. PGAM5-mediated mitophagy in turn leads to a self-perpetuating escalation of ΔΨ depolarization. Loss of the mitophagy-based damage-enhancing loop under PGAM5-deficient conditions breaks this vicious cycle, leading to improved mitochondrial homeostasis.

Increasing Incidence rates of Oropharyngeal Squamous Cell Carcinoma in Germany and Significance of Disease Burden Attributed to Human Papillomavirus.

Increasing incidences of head and neck cancers and rising proportions of these associated with human papillomavirus (HPV), especially in the oropharynx, have been reported in international studies. So far, the trends and contribution of HPV to the number of newly diagnosed cases of oropharyngeal squamous cell carcinomas (OPSCC) in Germany are uncertain. We investigated HPV association and incidence rates in a cohort of consecutively included patients with OPSCC in Giessen 2000-2017, and compared our results with regional (Giessen and the federal state of Hesse), national (Germany), and international (United States) databases. Regional data show a significant increase in the overall incidence rates of oropharyngeal cancers and in the incidence of HPV-associated cancers of the subsites tonsils and oropharynx, whereas other oropharyngeal subsites show no significant change. Analysis of national databases shows a significant incidence increase in Germany and in the United States. The rise in incidence is predominantly attributable to male patients in the US population, whereas in Germany rising OPSCC incidence is more associated with females. There is a significant elevation of OPSCC incidence rates in Germany, which corresponds to the recognized incidence increase of HPV-related oropharyngeal cancers based on experimental data from consecutively included patients of our cohort. Our investigation shows different patterns of this increase in Germany and in the United States, which demonstrates spatial heterogeneity and the need for population-based investigations regarding the role of HPV in oropharyngeal cancer.

Role of DNA methylation in HPV associated lesions.

Papillomavirus replication is tightly linked to squamous epithelial differentiation which in turn is governed to a large extent by epigenetic remodeling of genomes within the differentiating squamous epithelial cells. Over the past years it became evident that epigenetic and in particular differential methylation events substantially contribute to the regulation of the papillomavirus life cycle. Moreover, there is now good evidence that the initial trigger for HPV-mediated transformation of squamous epithelial cells is mediated by methylation of distinct CpG dinucleotides within E2-binding sites of the papillomavirus upstream regulatory region (URR). These findings have important implications for novel diagnostic markers but also for novel and indeed targeted therapy strategies for HPV linked neoplastic lesions.

APOBEC3A Expression in Penile Squamous Cell Carcinoma.

BACKGROUND: APOBECs (apolipoprotein B mRNA-editing catalytic polypeptides) are cytidine deaminases that have been implicated in the host defense against viruses by blocking viral replication. They have also been shown to play a role in genome hypermutation in several human cancers. An APOBEC3 hypermutation signature has been discovered in cervical cancer, which is intimately associated with infection by high-risk human papillomaviruses (HPVs). At the same time, HPV genomes themselves are subject to DNA editing by APOBECs. Similar to cervical cancer, a proportion of penile squamous cell carcinomas (SCCs) is etiologically driven by high-risk HPVs, but very little is known about the role of APOBECs in penile SCC development and progression. METHODS: A series of 34 penile SCCs was analyzed for the expression of APOBEC3A protein by immunohistochemistry. HPV genotyping was carried out using a bead-based multiplex hybridization assay preceded by BSGP5+6+ primer-based amplification. RESULTS: We found a frequent reduction of APOBEC3A protein expression in the invasive parts of the majority of HPV-negative penile SCCs. In contrast, the majority of HPV-positive penile SCCs retained APOBEC3A expression during malignant progression. CONCLUSION: Our results suggest that APOBEC3A expression is downregulated during progression towards invasiveness in HPV-negative penile SCC, but maintained in HPV-positive penile SCC. How high-risk HPV-infected tumor cells tolerate high APOBEC3A, which appears to exert tumor suppressive functions in HPV-negative penile SCCs, remains to be elucidated.

5-aza-2'-deoxycytidine (DAC) treatment downregulates the HPV E6 and E7 oncogene expression and blocks neoplastic growth of HPV-associated cancer cells.

High-risk human papillomaviruses (hr HPVs) may cause various human cancers and associated premalignant lesions. Transformation of the host cells is triggered by overexpression of the viral oncogenes E6 and E7 that deregulate the cell cycle and induce chromosomal instability. This process is accompanied by hypermethylation of distinct CpG sites resulting in silencing of tumor suppressor genes, inhibition of the viral E2 mediated control of E6 and E7 transcription as well as deregulated expression of host cell microRNAs. Therefore, we hypothesized that treatment with demethylating agents might restore those regulatory mechanisms. Here we show that treatment with 5-aza-2'-deoxycytidine (DAC) strongly decreases the expression of E6 and E7 in a panel of HPV-transformed cervical cancer and head and neck squamous cell carcinoma cell lines. Reduction of E6 and E7 further resulted in increased target protein levels including p53 and p21 reducing the proliferation rates and colony formation abilities of the treated cell lines. Moreover, DAC treatment led to enhanced expression of tumor the suppressive miRNA-375 that targets and degrades E6 and E7 transcripts. Therefore, we suggest that DAC treatment of HPV-associated cancers and respective precursor lesions may constitute a targeted approach to subvert HPV oncogene functions that deserves testing in clinical trials.

Prognostic Impact of AJCC/UICC 8th Edition New Staging Rules in Oropharyngeal Squamous Cell Carcinoma.

INTRODUCTION: The purpose of this study was to test whether the 8th edition of the AJCC/UICC TNM staging system (UICC) precisely differentiates between stages and reflects disease outcome in human papilloma virus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC). PATIENTS AND METHODS: OPSCC patients that were diagnosed between 2000 and 2016 were included in this analysis and HPV status was determined by combined DNA and p16 testing. Stratification was done according to 7th and 8th UICC staging rules. Incidence trends of HPV-associated tumorigenesis, 5-year overall survival (OS) according to tumor stages as well as the influence of therapy and prognostic factors toward the outcome were calculated using Kaplan-Meier method and Cox proportional-hazards model. RESULTS: A significant increase [2000; n = 8/39 (21%)-2015; n = 17/32 (53%); p = 0.002] in HPV-associated OPSCC was seen in the observation period. Together, 150/599 (25.0%) of the patients had HPV-driven OPSCC and 64.7% of curative treatments in all OPSCC patients included upfront surgery of the primary and the neck. 7th edition staging rules led to no discrimination in all respective four UICC stages in HPV OPSCC underlining the need for new staging rules. However, only discrimination between stages I vs. II and III vs. IV was significant in our patients with HPV-OPSCC (94.4 vs. 77.5%; p = 0.031 and 63.9 vs. 25.0%; p = 0.013), and stages II vs. III did not differ in OS rates (p = 0.257), when applying the new staging rules. For HPV-negative OPSCC, significant outcome differences were only seen between UICC stages III vs. IV (57.6 vs. 35.2%; p = 0.012). DISCUSSION: While the 7th edition of UICC shows invalid discrimination between stages, the 8th edition is more suitable for HPV-associated carcinoma. Due to lack of differentiation between stages II and III further adaption is essential.