Association of circulating tumor HPV16DNA levels and quantitative PET parameters in patients with HPV-positive head and neck squamous cell carcinoma.

Circulating tumor DNA (ctDNA), which circulates in the blood after being shed from cancer cells in the body, has recently gained attention as an excellent tumor marker. The purpose of this study was to evaluate whether ct human papillomavirus (HPV) 16 DNA (ctHPV16DNA) levels were associated with quantitative PET parameters in patients with HPV-positive head and neck (HN) squamous cell carcinoma (SCC). Fifty patients with oropharyngeal SCC (OPSCC) and 5 with SCC of unknown primary (SCCUP) before treatment were included. They all underwent blood sampling to test ctHPV16DNA levels and FDG PET-CT examinations. Quantitative PET parameters included SUVmax, metabolic tumor volume (MTV), MTV of whole-body lesions (wbMTV), and 56 texture features. ctHPV16DNA levels were compared to texture features of primary tumors in OPSCC patients (Group A) or the largest primary or metastatic lymph node lesions in OPSCC and SCCUP patients (Group B) and to other PET parameters. Spearman rank correlation test and multiple regression analysis were used to confirm the associations between ctHPV16DNA levels and PET parameters. ctHPV16DNA levels moderately correlated with wbMTV, but not with SUVmax or MTV in Groups A and B. ctHPV16DNA levels exhibited a weak negative correlation with low gray-level zone emphasis in Groups A and B. Multiple regression analysis revealed that wbMTV and high gray-level zone emphasis were the significant factors for ctHPV16DNA levels in Group B. These results were not observed in Group A. This study demonstrated that ctHPV16DNA levels correlated with the whole-body tumor burden and tumor heterogeneity visualized on FDG PET-CT in patients with HPV-positive HNSCC.

Circulating Plasma Exosomal PD-L1 Predicts Prognosis of Head and Neck Squamous Cell Carcinoma After Radiation Therapy.

Purpose: Radiation therapy is widely used to treat head and neck squamous cell carcinoma (HNSCC). This study evaluated the association between circulating plasma programmed death-ligand 1 (PD-L1) and the outcomes of patients with HNSCC after radiation therapy. Methods and Materials: In this retrospective observational study, plasma samples of 76 patients with HNSCC who underwent radiation therapy from June 2019 to August 2021 were analyzed. These plasma samples were obtained before radiation therapy. The median follow-up was 32.5 months. Total and exosomal PD-L1 was measured by enzyme-linked immunosorbent assay and retrospectively analyzed for association with overall survival (OS), progression-free survival (PFS), and local control (LC). Prognostic factors among patients' characteristics and circulating PD-L1 in plasma were evaluated by univariate (log-rank test) and multivariate (Cox proportional hazards model) analyses. Results: The median concentration of total PD-L1 in plasma was 115.1 pg/mL (95% CI, 114.7-137.9 pg/mL), and the median concentration of exosomal PD-L1 was 2.8 pg/mL (95% CI, 6.0-13.0 pg/mL). Univariate and multivariate analyses showed exosomal PD-L1 as a prognostic factor for PFS and LC. Patients with high exosomal PD-L1 in plasma had poor PFS and LC compared with those with low exosomal PD-L1, indicating that 1-year PFS was 79.2% versus 33.3% (P < .001) and 1-year LC was 87.3% versus 50.0% (P < .001) in patients with high and low exosomal PD-L1, respectively. However, exosomal PD-L1 in plasma had no significant effect on OS. Total PD-L1 in plasma did not correlate with PFS, LC, and OS. Conclusions: The pretreatment circulating exosomal PD-L1 in plasma of patients with HNSCC was a prognostic factor after radiation therapy.

Prognostic significance of nutritional indices in patients with head and neck squamous cell carcinoma treated with immune checkpoint inhibitors.

BACKGROUND: Although patients with head and neck squamous cell cancer (HNSCC) often show malnutrition, its effects on immune checkpoint inhibitor (ICI) treatment outcomes in these patients are unclear. OBJECTIVES: To investigate the prognostic influence of nutritional indices in patients with HNSCC treated with ICIs and determine the optimal indices. METHODS: This retrospective study included 106 patients with HNSCC treated with ICIs between 2017 and 2022. The prognostic influences of body mass index (BMI), geriatric nutritional risk index (GNRI), and prognostic nutritional index (PNI) on overall survival (OS) and progression-free survival (PFS) were analysed using the Kaplan-Meier method and Cox-regression models. RESULTS: The 1-year PFS rates in the groups with high and low BMI, GNRI, and PNI were, respectively, 24.2% and 28.4% (p = .731), 29.7% and 14.4% (p = .024), and 30.3% and 13.9% (p = .015). PNI was an independent prognostic factor for both PFS (hazard ratio (HR) = 1.89; 95% confidence interval (CI), 1.08-3.29) and OS (HR = 3.26; 95% CI, 1.66-6.40). CONCLUSIONS: PNI can predict ICI outcomes and should be assessed when ICI treatment is considered.

Microscopic Residual Disease After Nasal Squamous Cell Carcinoma Not Visualized on FDG PET-CT: A Case Report.

Nasal squamous cell carcinoma (SCC) is rare and aggressive. It often requires combination treatment. Precise post-treatment disease assessment is vital for determining the subsequent management and prognosis. We present the intriguing case of a 52-year-old man with T4bN0M0 stage IVB SCC. Post-treatment fluorodeoxyglucose (FDG) positron emission tomography-computed tomography (PET-CT) findings indicated a complete response; however, microscopic remnants of the cancer were detected during endoscopic sinus surgery. This report underscores the limitations of post-treatment assessment using FDG PET-CT and outpatient endoscopy alone due to these modalities' potential inability to detect microscopic residual disease. Endoscopic sinus surgery should be incorporated into routine post-treatment assessments of nasal SCC to improve disease detection and guide further treatment. Further large-scale studies are required to confirm these findings.

Clearance Profile of Circulating Tumor Human Papillomavirus DNA During Radiotherapy Predicts Clinical Outcomes in Human Papillomavirus-Related Oropharyngeal Cancer.

PURPOSE: This study aimed to examine whether circulating tumor human papillomavirus type 16 (HPV16) DNA (ctHPV16DNA) can help identify patients with locally advanced HPV16-related oropharyngeal squamous cell carcinoma who may benefit from deintensified treatment. MATERIALS AND METHODS: We serially collected blood samples before, during, and after treatment from 22 patients who received 70 Gy radiotherapy alone and longitudinally quantified ctHPV16DNA using droplet digital polymerase chain reaction. We correlated the clearance profile of ctHPV16DNA with clinical outcomes. RESULTS: The percentage of patients with detectable ctHPV16DNA decreased after every 10 Gy of radiotherapy. By contrast, the percentage of patients who later developed treatment failure among patients with detectable ctHPV16DNA gradually increased as radiotherapy proceeded, reaching 100% after 60 Gy of radiotherapy. We defined patients with and without detectable ctHPV16DNA after receiving 40 Gy as having slow and rapid clearance profiles, respectively. All 12 patients with a rapid clearance profile remained disease-free after radiotherapy. Of the 10 patients with a slow clearance profile, three had persistent or progressive disease at response evaluation after radiotherapy and one developed distant metastasis during follow-up (ie, four patients experienced treatment failure). The median follow-up for surviving patients was 38.6 months, and the 3-year failure-free survival rates of patients with rapid and slow clearance profiles were 100% and 58%, respectively (P = .02). Neither baseline ctHPV16DNA levels nor metabolic tumor volume was an independent predictor of the pattern of the clearance profile. CONCLUSION: In patients with HPV16-related oropharyngeal squamous cell carcinoma receiving radiotherapy, a slow ctHPV16DNA clearance profile could prelude unfavorable outcomes. Monitoring ctHPV16DNA is essential for determining the clearance profile, which might help optimize treatment intensity individually.

Performance of oral HPV DNA, oral HPV mRNA and circulating tumor HPV DNA in the detection of HPV-related oropharyngeal cancer and cancer of unknown primary.

A biomarker that is useful for the detection of human papillomavirus (HPV)-related oropharyngeal cancer (OPC) and cancer of unknown primary (CUP) is indispensable. We evaluated the diagnostic performance of HPV DNA and mRNA in oral gargle samples and circulating tumor HPV16 DNA (ctHPV16DNA) in blood samples. Oral HPV DNA and mRNA were analyzed using commercially available HPV assays of the GENOSEARCH HPV31 and Aptima, respectively. ctHPV16DNA was analyzed using in-house droplet digital polymerase chain reaction. Seventy-four patients with OPC and eight patients with CUP were included. The sensitivity and specificity of oral HPV DNA, oral HPV mRNA, and ctHPV16DNA were 82% (95% confidence interval [CI] = 66-92) and 100% (95% CI = 88-100), 85% (95% CI = 69-94) and 94% (95% CI = 73-100), and 93% (95% CI = 81-99) and 97% (95% CI = 84-100), respectively, for HPV16-related OPC, while those were 20% (95% CI = 1-72) and 100% (95% CI = 3-100), 0% (95% CI = 0-52) and 100% (95% CI = 3-100), and 100% (95% CI = 54-100) and 100% (95% CI = 16-100), respectively, for HPV16-related CUP. The sensitivity of ctHPV16DNA for HPV16-related OPC was higher than that of oral biomarkers, though the difference was not statistically significant. ctHPV16DNA remarkably correlated with the anatomic extent of disease, total metabolic tumor volume and HPV16 copy number per tumor genome in patients with HPV16-related OPC/CUP, whereas oral biomarkers did not. In conclusion, ctHPV16DNA is a potentially promising biomarker for HPV16-related OPC, while further studies are required for HPV16-related CUP.

Modified TPEx as First-line Treatment for Recurrent and/or Metastatic Head and Neck Cancer.

BACKGROUND/AIM: To retrospectively evaluate the efficacy and safety of modified TPEx (docetaxel 60 mg/m2 on day 1, cisplatin 60 mg/m2 on day 1, and weekly cetuximab 250 mg/m2 with loading dose of 400 mg/m2) followed by maintenance cetuximab as first-line treatment for inoperable recurrent and/or metastatic squamous cell carcinoma of the head and neck. PATIENTS AND METHODS: We analyzed 22 Japanese patients receiving modified TPEx every 21 days for four cycles with or without prophylactic granulocyte colony-stimulating factor (G-CSF). RESULTS: The best overall response rate was 55% [95% confidence interval (CI)=35-73]. The median progression-free survival and overall survival were 8.9 months (95%CI=3.9-10.2) and 14.3 months (95%CI=10.1-28.2), respectively. Without prophylactic G-CSF, Grade 3/4 neutropenia and febrile neutropenia was common (94% versus 20%; p=0.003 and 41% versus 0%; p=0.11, respectively). CONCLUSION: The modified TPEx is effective, while prophylactic G-CSF is essential.

Reciprocal activation of HEY1 and NOTCH4 under SOX2 control promotes EMT in head and neck squamous cell carcinoma.

Several comprehensive studies have demonstrated that the NOTCH pathway is altered in a bimodal manner in head and neck squamous cell carcinoma (HNSCC). In a previous study, it was found that the NOTCH4/HEY1 pathway was specifically upregulated in HNSCC and promoted epithelial­mesenchymal transition (EMT), and that HEY1 activation supported SOX2 expression. However, the interactions in this pathway have not yet been fully elucidated. The present study investigated the NOTCH4/HEY1/SOX2 axis in HNSCC using in vitro models and the Cancer Genome Atlas (TCGA) database. To explore the association, reporter and ChIP RT­qPCR assays using SOX2­overexpressing (SOX2­OE) cells were performed. The association between NOTCH4 and HEY1 was examined in the same manner using HEY1­overexpressing (HEY1­OE) cells. The results of the in vitro experiments indicated that HEY1 promoted EMT in the HNSCC cells. Furthermore, the overexpression of HEY1 also promoted sphere formation and increased murine xenograft tumorigenicity. Reporter assays and ChIP RT­qPCR experiments indicated that SOX2 regulated HEY1 expression via direct binding of the HEY1 promoter. HEY1 expression significantly correlated with SOX2 expression in primary lung SCC and other SCCs using the TCGA database. HEY1 also regulated NOTCH4 expression to create a positive reciprocal feedback loop. On the whole, the present study demonstrates that HEY1 expression in HNSCC is regulated via the promotion of SOX2 and promotes EMT. The NOTCH4/HEY1 pathway is specifically upregulated via a positive reciprocal feedback loop mediated by the HEY1­medaited regulation of NOTCH4 transcription, and SOX2 correlates with HEY1 expression in SCC from other primary sites.

Circulating tumor HPV DNA complements PET-CT in guiding management after radiotherapy in HPV-related squamous cell carcinoma of the head and neck.

Positron emission tomography and computed tomography (PET-CT) is widely used to assess the response to radiotherapy. However, the ability of PET-CT to predict treatment failure in human papillomavirus (HPV)-related squamous cell carcinoma of the head and neck (HNSCC) is unsatisfactory. We quantified circulating tumor HPV type16 DNA (ctHPV16DNA) using optimized droplet digital PCR in 35 patients with HPV16-related HNSCC, who received radiotherapy with or without chemotherapy, and prospectively correlated ctHPV16DNA and metabolic response with treatment failure. After a median follow-up of 21 months, ctHPV16DNA and PET-CT had similar negative predictive values (89.7% vs 84.0%), whereas the positive predictive value was much higher in ctHPV16DNA than in PET-CT (100% vs 50.0%). Notably, six patients who had detectable posttreatment ctHPV16DNA all had treatment failure irrespective of metabolic response, whereas none of five patients who had partial metabolic response without detectable posttreatment ctHPV16DNA had treatment failure. The risk of treatment failure was high in patients who had incomplete metabolic response with detectable posttreatment ctHPV16DNA (hazard ratio [HR], 138.8; 95% confidence interval [CI], 15.5-3366.4; P < .0001) and intermediate in patients who had discordant results between metabolic response and posttreatment ctHPV16DNA (HR, 4.7; 95% CI, 0.8-36.2, P = .09) as compared with patients who had complete metabolic response without detectable posttreatment ctHPV16DNA. One-year event-free survival rates of each risk group were 0%, 88% (95% CI, 46-98) and 95% (95% CI, 72-99), respectively (P < .0001). In conclusion, posttreatment ctHPV16DNA complements PET-CT and helps guide decisions managing patients with HPV16-related HNSCC after radiotherapy.

Radiation Therapy Alone for Human Papillomavirus-Related Squamous Cell Carcinoma of the Oropharynx: A Single-Arm, Phase 2 Study.

PURPOSE: Human papillomavirus (HPV)-related squamous cell carcinoma of the oropharynx (OPSCC) is extremely radiosensitive. Radiation therapy plus high-dose cisplatin remains the standard of care but causes long-term toxicity. Treatment deintensification approaches that reduce toxicity while maintaining survival are desirable for HPV-related OPSCC. METHODS AND MATERIALS: We conducted a single-arm, multicenter, phase 2 trial. Patients with newly diagnosed, biopsy-proven, American Joint Committee on Cancer (seventh edition) stage III or IV OPSCC positive for both p16 and HPV DNA were eligible. Patients with T4, N3, or T1N1 disease were excluded. Smoking history was not included in eligibility criteria. Patients received intensity modulated radiation therapy (IMRT) of 70 Gy in 35 fractions or 70.4 Gy in 32 fractions without chemotherapy. The primary endpoint was complete response or complete metabolic response 10 weeks after IMRT completion. RESULTS: Between September 13, 2013, and November 15, 2016, 39 patients were enrolled according to a 2-stage Simon design. Twenty-three patients (59%) had smoked for more than10 pack-years. Thirty-six patients (92%) had tumors genotyped as HPV16. Thirty-seven patients (95%) received full-dose radiation therapy and 35 (90%) had complete response or complete metabolic response. Median follow-up was 51 months (interquartile range, 41-63 months). One patient (3%) had regional recurrence and 3 (8%) had distant metastasis. One patient died of disease. The 2-year progression-free survival rate was 94% (95% CI, 81%-99%), and the 2-year overall survival rate was 100%. Common grade 3 adverse events during IMRT included mucositis in 10 patients (26%) and dysphagia in 7 patients (18%). No patients were dependent on a feeding tube at 1 month after IMRT completion. No grade 3 or 4 late adverse events were observed. CONCLUSIONS: IMRT alone is associated with excellent response as well as reduced toxicity and could be a treatment option for carefully selected patients with locally advanced "true" HPV-related OPSCC. Further studies are warranted.

Response Evaluation Criteria in Solid Tumors (RECIST) and PET Response Criteria in Solid Tumors (PERCIST) for response evaluation of the neck after chemoradiotherapy in head and neck squamous cell carcinoma.

BACKGROUND: An optimal approach to imaging assessment of neck after chemoradiotherapy must be established to avoid unnecessary neck dissection. METHODS: We retrospectively examined 101 patients and compared between Response evaluation criteria in solid tumors (RECIST), PET response criteria in solid tumors (PERCIST), and positron emission tomography/computed tomography (PET/CT) qualitative assessment. RESULTS: PERCIST was superior to RECIST in positive predictive value (PPV; 47% vs. 36%), with equivalent negative predictive value (NPV; 78%). Only 3 of 15 patients with incomplete responses on either RECIST or PERCIST alone had regional treatment failure, and the combination of RECIST and PERCIST improved PPV (55%) without reducing NPV. This combination yielded the highest hazard ratio of regional treatment failure. The combination of RECIST and PET/CT qualitative assessment also improved PPV (50%). In human papillomavirus (HPV)-related oropharyngeal cancer, NPV was 100% across the imaging assessments, while PPV was poor (14%-33%). CONCLUSIONS: Combining RECIST and PERCIST might optimize decision making in neck management after chemoradiotherapy. HPV status would affect the accuracy of response evaluation.

HPV E2, E4, E5 drive alternative carcinogenic pathways in HPV positive cancers.

The dominant paradigm for HPV carcinogenesis includes integration into the host genome followed by expression of E6 and E7 (E6/E7). We explored an alternative carcinogenic pathway characterized by episomal E2, E4, and E5 (E2/E4/E5) expression. Half of HPV positive cervical and pharyngeal cancers comprised a subtype with increase in expression of E2/E4/E5, as well as association with lack of integration into the host genome. Models of the E2/E4/E5 carcinogenesis show p53 dependent enhanced proliferation in vitro, as well as increased susceptibility to induction of cancer in vivo. Whole genomic expression analysis of the E2/E4/E5 pharyngeal cancer subtype is defined by activation of the fibroblast growth factor receptor (FGFR) pathway and this subtype is susceptible to combination FGFR and mTOR inhibition, with implications for targeted therapy.

Aberrant expression of CPSF1 promotes head and neck squamous cell carcinoma via regulating alternative splicing.

Alternative mRNA splicing increases protein diversity, and alternative splicing events (ASEs) drive oncogenesis in multiple tumor types. However, the driving alterations that underlie the broad dysregulation of ASEs are incompletely defined. Using head and neck squamous cell carcinoma (HNSCC) as a model, we hypothesized that the genomic alteration of genes associated with the spliceosome may broadly induce ASEs across a broad range of target genes, driving an oncogenic phenotype. We identified 319 spliceosome genes and employed a discovery pipeline to identify 13 candidate spliceosome genes altered in HNSCC using The Cancer Genome Atlas (TCGA) HNSCC data. Phenotypic screens identified amplified and overexpressed CPSF1 as a target gene alteration that was validated in proliferation, colony formation, and apoptosis assays in cell line and xenograft systems as well as in primary HNSCC. We employed knockdown and overexpression assays followed by identification of ASEs regulated by CPSF1 overexpression to identify changes in ASEs, and the expression of these ASEs was validated using RNA from cell line models. Alterations in expression of spliceosome genes, including CPSF1, may contribute to HNSCC by mediating aberrant ASE expression.

Cannabinoids Promote Progression of HPV-Positive Head and Neck Squamous Cell Carcinoma via p38 MAPK Activation.

PURPOSE: Human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) is associated with daily marijuana use and is also increasing in parallel with increased marijuana use in the United States. Our study is designed to define the interaction between cannabinoids and HPV-positive HNSCC. EXPERIMENTAL DESIGN: The expression of cannabinoid receptors CNR1 and CNR2 was analyzed using The Cancer Genome Atlas (TCGA) HNSCC data. We used agonists, antagonists, siRNAs, or shRNA-based models to explore the roles of CNR1 and CNR2 in HPV-positive HNSCC cell lines and animal models. Cannabinoid downstream pathways involved were determined by Western blotting and analyzed in a primary HPV HNSCC cohort with single-sample gene set enrichment analysis (ssGSEA) and the OncoGenome Positioning System (Onco-GPS). RESULTS: In TCGA cohort, the expression of CNR1 and CNR2 was elevated in HPV-positive HNSCC compared with HPV-negative HNSCC, and knockdown of CNR1/CNR2 expression inhibited proliferation in HPV-positive HNSCC cell lines. Specific CNR1 and CNR2 activation as well as nonselective cannabinoid receptor activation in cell lines and animal models promoted cell growth, migration, and inhibited apoptosis through p38 MAPK pathway activation. CNR1/CNR2 antagonists suppressed cell proliferation and migration and induced apoptosis. Using whole-genome expression analysis in a primary HPV HNSCC cohort, we identified specific p38 MAPK pathway activation signature in tumors from HPV HNSCC patients with objective measurement of concurrent cannabinoid exposure. CONCLUSIONS: Cannabinoids can promote progression of HPV-positive HNSCC through p38 MAPK pathway activation.

Rational genomic optimization of DNA detection for human papillomavirus type 16 in head and neck squamous cell carcinoma.

BACKGROUND: We aimed to use genomic data for optimizing polymerase chain reaction (PCR) primer/probe sets for detection of human papillomavirus (HPV)-16 in body fluids of patients with HPV-related head and neck squamous cell carcinoma (HPV-HNSCC). METHODS: We used genomic HPV-HNSCC sequencing data from a single institutional and a TCGA cohort. Optimized primer/probe sets were designed and tested for analytical performance in CaSki HPV-16 genome and confirmed in salivary rinse samples from patients with HPV-HNSCC. RESULTS: The highest read density was observed between E5 and L2 regions. The E1 region contained a region that was universally present. Among candidate PCR primer/probe sets created, six reliably detected 30 HPV-16 copy number. In a CLIA certified laboratory setting, the combination of two novel primer/probe with E7 sets improved performance in salivary rinse samples with a sensitivity of 96% and specificity of 100%. CONCLUSIONS: PCR-based detection of HPV-16 DNA in HPV-HNSCC can be improved using rational genomic design.

A novel splice variant of LOXL2 promotes progression of human papillomavirus-negative head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is one of the most frequently diagnosed cancers worldwide. LOXL2 demonstrates alternative splicing events in patients with human papillomavirus (HPV)-negative HNSCC. The current study explored the role of a dominant LOXL2 variant in HPV-negative HNSCC. METHODS: Expression of the LOXL2 variant was analyzed using The Cancer Genome Atlas cohorts and validated using quantitative reverse transcriptase-polymerase chain reaction in a separate primary tumor set. The authors defined the effect of LOXL2 splice variants in assays for cell proliferation using a cell viability assay and colony formation assay. Cell migration and invasion were examined using a cell scratch assay and transwell cell migration and invasion assay in LOXL2 splice variant gain and loss of expression cells. Western blot analysis and gene set enrichment analysis were used to explore the potential mechanism of the LOXL2 splice variant in HPV-negative HNSCC. RESULTS: Expression of a novel LOXL2 variant was found to be upregulated in The Cancer Genome Atlas HPV-negative HNSCC, and confirmed in the separate primary tumor validation set. Analyses of loss and gain of function demonstrated that this LOXL2 variant enhanced proliferation, migration, and invasion in HPV-negative HNSCC cells and activated the FAK/AKT pathway. A total of 837 upregulated and 820 downregulated genes and 526 upregulated and 124 downregulated pathways associated with LOXL2 variant expression were identified using gene set enrichment analysis, which helped in developing a better understanding of the networks activated by this LOXL2 variant in patients with HPV-negative HNSCC. CONCLUSIONS: The novel LOXL2 variant can promote the progression of HPV-negative HNSCC, in part through FAK/AKT pathway activation, which may provide a new potential therapeutic target among patients with HPV-negative HNSCC.

Publisher Correction: Chromatin dysregulation and DNA methylation at transcription start sites associated with transcriptional repression in cancers.

The original version of this Article contained an error in the author affiliations. Trey Ideker was incorrectly associated with 'Department of Medicine (Oncology), Stanford University School of Medicine, 875 Blake Wilbur Dr, Palo Alto, CA 94304, USA.' This has now been corrected in both the PDF and HTML versions of the Article.

Chromatin dysregulation and DNA methylation at transcription start sites associated with transcriptional repression in cancers.

Although promoter-associated CpG islands have been established as targets of DNA methylation changes in cancer, previous studies suggest that epigenetic dysregulation outside the promoter region may be more closely associated with transcriptional changes. Here we examine DNA methylation, chromatin marks, and transcriptional alterations to define the relationship between transcriptional modulation and spatial changes in chromatin structure. Using human papillomavirus-related oropharyngeal carcinoma as a model, we show aberrant enrichment of repressive H3K9me3 at the transcriptional start site (TSS) with methylation-associated, tumor-specific gene silencing. Further analysis identifies a hypermethylated subtype which shows a functional convergence on MYC targets and association with CREBBP/EP300 mutation. The tumor-specific shift to transcriptional repression associated with DNA methylation at TSSs was confirmed in multiple tumor types. Our data may show a common underlying epigenetic dysregulation in cancer associated with broad enrichment of repressive chromatin marks and aberrant DNA hypermethylation at TSSs in combination with MYC network activation.