Pan-cancer assessment of antineoplastic therapy-induced interstitial lung disease in patients receiving subsequent therapy immediately following immune checkpoint blockade therapy.

BACKGROUND: Drug-induced interstitial lung disease (DIILD) is a serious adverse event potentially induced by any antineoplastic agent. Whether cancer patients are predisposed to a higher risk of DIILD after receiving immune checkpoint inhibitors (ICIs) is unknown. METHODS: This study retrospectively assessed the cumulative incidence of DIILD in consecutive cancer patients who received post-ICI antineoplastic treatment within 6 months from the final dose of ICIs. There was also a separate control cohort of 55 ICI-naïve patients with non-small cell lung cancer (NSCLC) who received docetaxel. RESULTS: Of 552 patients who received ICIs, 186 met the inclusion criteria. The cohort predominantly comprised patients with cancer of the lung, kidney/urinary tract, or gastrointestinal tract. The cumulative incidence of DIILD in the entire cohort at 3 and 6 months was 4.9% (95% confidence interval [CI] 2.4%-8.7%) and 7.2% (95% CI 4.0%-11.5%), respectively. There were significant differences according to cancer type (Gray's test, P = .04), with the highest cumulative incidence of DIILD in patients with lung cancer being 9.8% (95% CI 4.3%-18.0%) at 3 months and 14.2% (95% CI 7.3%-23.3%) at 6 months. DIILD was caused by docetaxel in six of these 11 lung cancer patients (54.5%). After matching, the cumulative incidence of docetaxel-induced ILD in patients with NSCLC in the post-ICI setting was higher than that in the ICI-naïve setting: 13.0% (95% CI 3.3%-29.7%) vs 4.3% (95% CI 0.3%-18.2%) at 3 months; and 21.7% (95% CI 7.9%-39.9%) vs 4.3% (95% CI 0.3%-18.2%) at 6 months. However, these were not significant differences (hazard ratio, 5.37; 95% CI 0.64-45.33; Fine-Gray P = .12). CONCLUSIONS: Patients with lung cancer were at high risk of developing DIILD in subsequent regimens after ICI treatment. Whether NSCLC patients are predisposed to additional risk of docetaxel-induced ILD by prior ICIs warrants further study.

Identifying Active Progeny Virus Particles in Formalin-Fixed, Paraffin-Embedded Sections Using Correlative Light and Scanning Electron Microscopy.

Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) tissue blocks is routinely used to identify virus-infected cells. However, detecting virus particles in FFPE sections using light microscopy is difficult because of the light diffraction resolution limitations of an optical microscope. In this study, light microscopy and field emission scanning electron microscopy were performed to observe 3-dimensional virus particles in FFPE sections in a nondestructive manner using NanoSuit or osmium conductive treatment methods. The virus particles in FFPE sections were immunostained with specific antibodies against the surface antigens of the viral particles and stained with 3,3'-diaminobenzidine. A metal solution (0.2% gold chloride or 2% osmium tetroxide) was applied to enhance the 3,3'-diaminobenzidine-stained area. This procedure is nondestructive for FFPE sections and is a simpler method than transmission electron microscopy. To validate the applicability of this technique, we performed 3-dimensional imaging of the virus particles of different sizes, such as human papillomavirus, cytomegalovirus, and varicella-zoster virus. Furthermore, ultrathin sections from the FFPE sections that were observed to harbor viral particles using field emission scanning electron microscopy were prepared and assessed using transmission electron microscopy. In the correlative areas, transmission electron microscopy confirmed the presence of large numbers of virus particles. These results indicated that the combination of marking viral particles with 3,3'-diaminobenzidine/metal staining and conductive treatment can identify active progeny virus particles in FFPE sections using scanning electron microscopy. This easy correlative imaging of field emission scanning electron microscopy of the identical area of FFPE in light microscopy may help elucidate new pathological mechanisms of virus-related diseases.

Tumorigenic activation around HPV integrated sites in head and neck squamous cell carcinoma.

Human papillomavirus (HPV) is causally involved in the development of head and neck squamous cell carcinoma (HNSCC). The integration of HPV drives tumorigenesis through expression of oncogenic viral genes as well as genomic alterations in surrounding regions. To elucidate involvement of epigenetic dysregulation in tumorigenesis, we here performed integrated analyses of the epigenome, transcriptome and interactome using ChIP-seq, RNA-seq and Hi-C and 4C-seq for HPV(+) HNSCCs. We analyzed clinical HNSCC using The Cancer Genome Atlas data and found that genes neighboring HPV integration sites were significantly upregulated and were correlated with oncogenic phenotypes in HPV(+) HNSCCs. While we found four HPV integration sites in HPV(+) HNSCC cell line UPCI-SCC-090 through target enrichment sequencing, 4C-seq revealed 0.5 to 40 Mb of HPV-interacting regions (HPVIRs) where host genomic regions interacted with integrated HPV genomes. While 9% of the HPVIRs were amplified and activated epigenetically forming super-enhancers, the remaining non-amplified regions were found to show a significant increase in H3K27ac levels and an upregulation of genes associated with GO terms, for example, Signaling by WNT and Cell Cycle. Among those genes, ITPR3 was significantly upregulated, involving UPCI-SCC-090-specific super-enhancer formation around the ITPR3 promoter and in the 80-kb-downstream region. The knockdown of ITPR3 by siRNA or CRISPR deletions of the distant enhancer region led to a significant suppression of cell proliferation. The epigenetic activation of HPVIRs was also confirmed in other cell lines, UM-SCC-47 and UM-SCC-104. These data indicate that epigenetic activation in HPVIRs contributes, at least partially, to genesis of HPV(+) HNSCC.

Identification and characterization of primary cilia-positive salivary gland tumours exhibiting basaloid/myoepithelial differentiation.

Primary cilia (PC) are non-motile, antenna-like structures on the cell surface. Many types of neoplasms exhibit PC loss, whereas in some neoplasms PC are retained and involved in tumourigenesis. To elucidate the PC status and characteristics of major salivary gland tumours (SGTs), we examined 100 major SGTs encompassing eight histopathological types by immunohistochemical analysis. PC were present in all (100%) of the pleomorphic adenomas (PAs), basal cell adenomas (BCAs), adenoid cystic carcinomas (AdCCs), and basal cell adenocarcinomas (BCAcs) examined, but absent in all (0%) of the Warthin tumours, salivary duct carcinomas, mucoepidermoid carcinomas, and acinic cell carcinomas examined. PC were also detected by electron-microscopic analysis using the NanoSuit method. It is worthy of note that the former category and latter category of tumours contained and did not contain a basaloid/myoepithelial differentiation component, respectively. The four types of PC-positive SGTs showed longer PC than normal and exhibited a characteristic distribution pattern of the PC in the ductal and basaloid/neoplastic myoepithelial components. Two PC-positive carcinomas (AdCC and BCAc) still possessed PC in their recurrent/metastatic sites. Interestingly, activation of the Hedgehog signalling pathway, shown by predominantly nuclear GLI1 expression, was significantly more frequently observed in PC-positive SGTs. Finally, we identified tau tubulin kinase 2 (TTBK2) as being possibly involved in the production of PC in SGTs. Taken together, our findings indicate that SGTs that exhibit basaloid/myoepithelial differentiation (PA, BCA, AdCC, and BCAc) are ciliated, and their PC exhibit tumour-specific characteristics, are involved in activation of the Hedgehog pathway, and are associated with TTBK2 upregulation, providing a significant and important link between SGT tumourigenesis and PC. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Stratification of HPV-associated and HPV-negative oropharyngeal squamous cell carcinomas based on DNA methylation epigenotypes.

While the incidence of oropharyngeal squamous cell carcinoma (OPSCC) has been increasing in these two decades, primarily due to human papillomavirus (HPV), stratification of OPSCC into molecular subgroups showing different clinicopathological features has not been fully investigated. We performed DNA methylome analysis using Infinium 450k for 170 OPSCC cases, including 89 cases in our cohort and 81 cases reported by The Cancer Genome Atlas, together with targeted exon sequencing analysis. We stratified OPSCC by hierarchical clustering analysis using methylome data. Methylation levels of classifier markers were validated quantitatively using pyrosequencing, and area under the curve (AUC) values of receiver operating characteristics (ROC) curves were calculated. OPSCC was stratified into four epigenotypes: HPV(+) high-methylation (OP1), HPV(+) intermediate-methylation (OP2), HPV(-) intermediate-methylation (OP3) and HPV(-) low-methylation (OP4). Ten methylation marker genes were generated: five to classify HPV(+) cases into OP1 and OP2, and five to classify HPV(-) cases into OP3 and OP4. AUC values of ROC curves were 0.969 and 0.952 for the two marker panels, respectively. While significantly higher TP53 mutation and CCND1 copy number gains were observed in HPV(-) than in HPV(+) groups (p < 0.01), no significant difference of genomic aberrations was observed between OP1 and OP2, or OP3 and OP4. The four epigenotypes showed significantly different prognosis (p = 0.0006), distinguishing the most favorable OPSCC subgroup (OP1) among generally favorable HPV(+) cases, and the most unfavorable OPSCC subgroup (OP3) among generally unfavorable HPV(-) cases. HPV(+) and HPV(-) OPSCC are further divided into distinct DNA methylation epigenotypes, showing significantly different prognosis.

Establishment of epigenetic markers to predict irradiation efficacy against oropharyngeal cancer.

Irradiation, or chemoradiotherapy, is a curative treatment for oropharyngeal squamous cell carcinoma (OPSCC). Its invasiveness, however, can often negate its efficacy. Therefore, developing methods to predict which patients would benefit from irradiation is urgent. Promoter DNA hypermethylation was recently reported to correlate with favorable OPSCC prognosis. It is still unclear, however, whether there is an association between promoter DNA methylation and response to irradiation. In this study, we analyzed DNA methylation in the specimens from 40 OPSCC patients who had undergone irradiation, using the Infinium assay. Our results showed significant correlation between high levels of promoter DNA methylation and better response to treatment (P < 0.01). We used the 10 most differentially-methylated genes between responders and non-responders to develop a panel of predictive markers for efficacy. Our panel had high sensitivity, specificity and accuracy (92%, 93% and 93%, respectively). We conducted pyrosequencing to quantitatively validate the methylation levels of 8 of the 10 marker genes (ROBO1, ULK4P3, MYOD1, LBX1, CACNA1A, IRX4, DPYSL3 and ELAVL2) obtained by Infinium. The validation by pyrosequencing showed that these 8 genes had a high prediction performance for the training set of 40 specimens and for a validation set of 35 OPSCC specimens, showing 96% sensitivity, 89% specificity and 94% accuracy. Methylation of these markers correlated significantly with better progression-free and overall survival rates, regardless of human papillomavirus status. These results indicate that increased DNA methylation is associated with better responses to irradiation therapy and that DNA methylation can help establish efficacy prediction markers in OPSCC.

Prostanoid receptor genes confer poor prognosis in head and neck squamous cell carcinoma via epigenetic inactivation.

BACKGROUND: Chronic inflammation is a risk factor for head and neck squamous cell carcinoma (HNSCC) and other diseases. Prostanoid receptors are clearly involved in the development of many types of cancer. However, their role is not simple and is poorly understood in HNSCC. METHODS: Methylation profiles of prostanoid receptor family genes were generated for tumour samples obtained from 274 patients with HNSCC, including 69 hypopharynx, 51 larynx, 79 oral cavity, and 75 oropharynx tumour samples, by quantitative methylation-specific PCR. Promoter methylation was then evaluated with respect to various clinical characteristics and patient survival. RESULTS: The mean number of methylated genes per sample was 2.05 ± 2.59 (range 0 to 9). Promoters of PTGDR1, PTGDR2, PTGER1, PTGER2, PTGER3, PTGER4, PTGFR, PTGIR, and TBXA2R were methylated in 43.8%, 18.2%, 25.5%, 17.5%, 41.2%, 8.0%, 19.3%, 20.4%, and 11.3% of the samples, respectively. Methylation indices for prostanoid receptor family genes tended to be higher as the number of TET methylation events increased. Patients with 5-9 methylated genes had a significantly lower survival rate than that of patients with 0-4 methylated genes (log-rank test, P= 0.007). In multivariate analyses, PTGDR1 methylation was most highly correlated with recurrence in patients with hypopharyngeal cancer (P = 0.014). A similar correlation was observed for PTGER4 in patients with laryngeal cancer (P = 0.046). Methylation of the PTGIR and TBXA2R promoters was positively correlated with recurrence in oropharyngeal cancer (P = 0.028 and P = 0.006, respectively). Moreover, Patients with 5-9 methylated genes were extremely lower of 5hmC levels (P = 0.035) and was correlated with increasing expression of DNMT3A and DNMT3B (P < 0.05 and P < 0.05, respectively). CONCLUSION: We characterised the relationship between the methylation status of prostanoid receptor genes and recurrence in HNSCC. These results provide new perspectives for the development of molecular targeted treatment approaches.

Management of elderly patients with head and neck carcinoma: analysis of outcomes for radical versus palliative treatment.

BACKGROUND: There are no universally accepted treatment recommendations for elderly patients with head and neck carcinomas. This study investigated whether radical treatment in elderly patients resulted in better survival compared with palliative treatment. METHODS: We retrospectively reviewed the medical records of 724 patients aged > 60 years who underwent treatment for primary head and neck carcinomas at Hamamatsu University Hospital. We evaluated the impact of the following: age, sex, the clinical stage, smoking history, alcohol use history, primary tumor site, performance status, and Osaka Head and Neck Comorbidity Index score on overall survival using a Cox proportional hazards model. RESULTS: The 5-year overall survival rate was significantly greater for the 646 patients initially treated with radical (curative) therapy than for the 78 patients treated with palliative therapy (p < 0.01). Patients who received palliative treatment in all age groups were more likely to die than were those in the radical treatment group, after controlling for age, sex, and clinical stage of the cancer. Information on the survival status of patients was obtained after a mean follow-up period of 46 months (range 6-205 months). CONCLUSIONS: In the absence of contraindications associated with comorbidities, radical treatment protocols should be recommended for elderly patients with head and neck carcinomas because they confer better survival.

Relevance of Level IIb Neck Dissection in Patients with Head and Neck Squamous Cell Carcinomas.

BACKGROUND: Cervical nodal metastasis is the most important prognostic factor in patients with head and neck cancers. Unfortunately, nodal dissection at level IIb carries a risk of damage to the spinal accessory nerve. We aimed to determine the prevalence of level IIb metastasis and the relevance of nodal dissection at level IIb in patients with head and neck squamous cell carcinomas. METHODS: During neck dissection, level IIb lymph nodes obtained from 181 patients with head and neck squamous cell carcinomas were removed, processed, and histopathologically examined. All specimens were divided into two groups according to the side (affected and unaffected sides). The number of dissected lymph nodes and prevalence of level IIb metastasis in each group were then determined and compared according to the preoperative clinical N stage (cN0 and cN+). RESULTS: The study included 158 men and 23 women with a median age of 65 years (range, 17-89 years). The prevalence of pathologically confirmed level IIb metastasis was 0% for clinically node-negative (cN0) necks on the unaffected side and 10.34% for clinically node-positive necks (cN+), with an overall prevalence of 2.4%. There was a significant association between clinically determined and pathologically confirmed node negativity at level IIb. CONCLUSION: Our findings suggest that level IIb neck dissection in patients with head and neck squamous cell carcinomas may be required only if preoperative examination reveals multilevel or level IIa metastasis or suspicious level IIb metastasis.

Risk and prognostic factors for multiple primary carcinomas in patients with head and neck cancer.

BACKGROUND: Unexpected multiple primary carcinomas (MPCs) that develop in patients with head and neck carcinomas complicate approaches to their management. We therefore investigated the clinical factors associated with survival outcomes after the treatment of MPCs. METHODS: We performed a retrospective review of records of 1104 patients who underwent treatment for primary head and neck carcinoma at Hamamatsu University Hospital. We evaluated clinical staging, age, sex, smoking, alcohol consumption, the primary tumor site (particularly the involvement of the mucosal epithelial lining of the aerodigestive tract), and overall survival (OS) as determined by Kaplan-Meier analysis. Information on patients' survival status was obtained after a mean follow-up period of 43.8 months (range, 1-144 months). RESULTS: Among 566 patients with mucosa-associated carcinoma arising in the epithelial lining, the 5- and 10-year OS rates (68.49% and 58.96%, respectively) were significantly shorter than those of patients with mucosa non-associated carcinoma (74.22%, and 66.76%, respectively) (log-rank P = 0.0219). Older age (P = 0.016) and male sex (P < 0.001) were likely independent risk factors for developing MPCs; smoking (P < 0.001) and alcohol consumption (P < 0.001) were also significant risk factors. CONCLUSION: Mucosa-associated carcinomas arising in the epithelial lining of the aerodigestive tract in the head and neck are a significant risk factor for developing MPC and are a poor prognostic factor. Careful follow-up and more frequent examinations of the aerodigestive tracts of these patients are recommended.

Aberrant Epigenetic Regulation in Head and Neck Cancer Due to Distinct EZH2 Overexpression and DNA Hypermethylation.

Enhancer of Zeste homologue 2 (EZH2) overexpression is associated with tumor proliferation, metastasis, and poor prognosis. Targeting and inhibition of EZH2 is a potentially effective therapeutic strategy for head and neck squamous cell carcinoma (HNSCC). We analyzed EZH2 mRNA expression in a well-characterized dataset of 230 (110 original and 120 validation cohorts) human head and neck cancer samples. This study aimed to investigate the effects of inhibiting EZH2, either via RNA interference or via pharmacotherapy, on HNSCC growth. EZH2 upregulation was significantly correlated with recurrence (p < 0.001) and the methylation index of tumor suppressor genes (p < 0.05). DNMT3A was significantly upregulated upon EZH2 upregulation (p = 0.043). Univariate analysis revealed that EZH2 upregulation was associated with poor disease-free survival (log-rank test, p < 0.001). In multivariate analysis, EZH2 upregulation was evaluated as a significant independent prognostic factor of disease-free survival (hazard ratio: 2.085, 95% confidence interval: 1.390⁻3.127; p < 0.001). Cells treated with RNA interference and DZNep, an EZH2 inhibitor, showed the most dramatic changes in expression, accompanied with a reduction in the growth and survival of FaDu cells. These findings suggest that EZH2 upregulation is correlated with tumor aggressiveness and adverse patient outcomes in HNSCC. Evaluation of EZH2 expression might help predict the prognosis of HNSCC patients.

Site-specific methylation patterns of the GAL and GALR1/2 genes in head and neck cancer: Potential utility as biomarkers for prognosis.

The aim of this study was to evaluate the prognostic value of the promoter methylation status of galanin (GAL) and galanin receptor 1/2 (GALR1/2) by assessing their association with disease-free survival and known prognostic factors in head and neck cancer. We generated methylation profiles of GAL and GALR1/2 in tumor samples obtained from 202 patients with head and neck squamous cell carcinoma (HNSCC); these included 43 hypopharynx, 42 larynx, 59 oral cavity, and 58 oropharynx tumor samples. CpG island hypermethylation status of the three genes was analyzed using quantitative methylation-specific PCR (Q-MSP). In order to determine the prognostic value of the methylation status of these genes, the associations between methylation index and various clinical characteristics, especially tumor site, were assessed for tumors from patients with HNSCC. The methylation index was positively correlated with female gender (P = 0.008) and disease recurrence (P = 0.01) in oral cancer and human papillomavirus (HPV)-positive (P = 0.004) status and disease recurrence (P = 0.005) in oropharyngeal cancer. Among patients with oral and oropharyngeal cancer, promoter hypermethylation of GAL, GALR1, or GALR2 was statistically correlated with a decrease in disease-free survival (log-rank test, P = 0.036 and P = 0.042, respectively). Furthermore, methylation of GAL, GALR1, or GALR2 exhibited the highest association with poor survival (log-rank test, P = 0.018) in patients with HPV-negative oropharyngeal cancers. As such, GAL and GALR1/2 methylation status may serve as an important site-specific biomarker for prediction of clinical outcome in patients with HNSCC. © 2016 Wiley Periodicals, Inc.

Evaluation of epigenetic inactivation of vascular endothelial growth factor receptors in head and neck squamous cell carcinoma.

The aim of this study was to determine the methylation status of the genes encoding the vascular endothelial growth factor receptors and to evaluate the usefulness of VEGFR methylation as a prognostic indicator in head and neck squamous cell carcinoma. VEGFR messenger RNA expression and promoter methylation were examined in a panel of cell lines via quantitative reverse transcription and methylation-specific polymerase chain reaction, respectively. Promoter methylation was compared with clinical characteristics in 128 head and neck squamous cell carcinoma samples. The normalized methylation values for the VEGFR1, VEGFR2 and VEGFR3 promoters tended to be higher in the tumour cell lines than in normal tonsil samples, whereas amounts of VEGFR1, VEGFR2 and VEGFR3 messenger RNA were significantly higher. Methylation of the VEGFR1 promoter (p = 0.003; 66/128 head and neck squamous cell carcinoma samples, 52%) and VEGFR3 promoter (p = 0.043; 53/128 head and neck squamous cell carcinoma samples, 41%) significantly correlated with recurrence, whereas methylation of the VEGFR2 promoter significantly correlated with lymph node metastasis (p = 0.046; 47/128 head and neck squamous cell carcinoma samples, 37%). Concurrent methylation of the VEGFR1 and VEGFR3 promoters significantly correlated with reduced disease-free survival (log-rank test, p = 0.009). In a multivariate logistic regression analysis, methylation of the VEGFR1, VEGFR3 and both the VEGFR1 and VEGFR3 promoters independently predicted recurrence (odds ratios and 95% confidence intervals: 3.19, 1.51-6.75 (p = 0.002); 2.24, 1.06-4.76 (p = 0.035); and 2.56, 1.09-6.05 (p = 0.032), respectively). Methylation of the VEGFR promoters predicts poor prognosis in head and neck squamous cell carcinoma patients.

Novel anti-tumor mechanism of galanin receptor type 2 in head and neck squamous cell carcinoma cells.

Galanin and its receptors, GALR1 and GALR2, are known tumor suppressors and potential therapeutic targets in head and neck squamous cell carcinoma (HNSCC). Previously, we demonstrated that, in GALR1-expressing HNSCC cells, the addition of galanin suppressed tumor proliferation via upregulation of ERK1/2 and cyclin-dependent kinase inhibitors, whereas, in GALR2-expressing cells, the addition of galanin not only suppressed proliferation, but also induced apoptosis. In this study, we first transduced HEp-2 and KB cell lines using a recombinant adeno-associated virus (rAAV)-green fluorescent protein (GFP) vector and confirmed a high GFP expression rate (>90%) in both cell lines at the standard vector dose. Next, we demonstrated that GALR2 expression in the presence of galanin suppressed cell viability to 40-60% after 72 h in both cell lines. Additionally, the annexin V-positive rate and sub-G0/G1 phase population were significantly elevated in HEp-2 cells (mock vs GALR2: 12.3 vs 25.0% (P < 0.01) and 9.1 vs 32.0% (P < 0.05), respectively) after 48 h. These changes were also observed in KB cells, although to a lesser extent. Furthermore, in HEp-2 cells, GALR2-mediated apoptosis was caspase-independent, involving downregulation of ERK1/2, followed by induction of the pro-apoptotic Bcl-2 protein, Bim. These results illustrate that transient GALR2 expression in the presence of galanin induces apoptosis via diverse pathways and serves as a platform for suicide gene therapy against HNSCC.

Tumor suppressor activity and inactivation of galanin receptor type 2 by aberrant promoter methylation in head and neck cancer.

BACKGROUND: There is accumulating evidence that galanin receptors (GALRs) may be tumor suppressors in head and neck squamous cell carcinoma (HNSCC). Promoter methylation status and gene expression were assessed in a large panel of head and neck primary tumors, based on the hypothesis that cytosine-guanine dinucleotide (CpG) hypermethylation might silence the galanin receptor 2 (GALR2) gene. METHODS: GALR2 expression was examined in a panel of cell lines by using quantitative reverse transcription polymerase chain reaction (RT-PCR). The methylation status of the GALR2 promoter was studied using quantitative methylation-specific PCR (Q-MSP). UM-SCC-1 was stably transfected to express GALR2. RESULTS: GALR2 expression was suppressed in UM-SCC cell lines, whereas nonmalignant cell lines exhibited stable expression. GALR2 methylation found in 31 of 100 (31.0%) tumor specimens was significantly correlated with the methylation status of both GALR1 and Galanin. The observed GALR2 promoter hypermethylation was statistically correlated with a decrease in disease-free survival (log-rank test, P=.045). A multivariate logistic-regression analysis revealed a high odds ratio for recurring methylation of GALR2 and the gene pair GALR2 and Galanin, 8.95 (95% confidence interval, 2.29-35.03; P=.024) and 9.05 (95% confidence interval, 1.76-46.50; P=.008), respectively. In addition, exogenous expression of GALR2 suppressed cell proliferation in UM-SCC-1 cells with hypermethylated Galanin and GALR2-proficient cell lines. CONCLUSIONS: Frequent promoter hypermethylation in association with prognosis, and growth suppression after re-expression, supports the hypothesis that GALR2 may act to suppress tumor activity. GALR2 is a potentially significant therapeutic target and prognostic factor for this cancer type.

Enhancer infestation drives tumorigenic activation of inactive B compartment in Epstein-Barr virus-positive nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated malignant epithelial tumor endemic to Southern China and Southeast Asia. While previous studies have revealed a low frequency of gene mutations in NPC, its epigenomic aberrations are not fully elucidated apart from DNA hypermethylation. Epigenomic rewiring and enhancer dysregulation, such as enhancer hijacking due to genomic structural changes or extrachromosomal DNA, drive cancer progression. METHODS: We conducted Hi-C, 4C-seq, ChIP-seq, and RNA-seq analyses to comprehensively elucidate the epigenome and interactome of NPC using C666-1 EBV(+)-NPC cell lines, NP69T immortalized nasopharyngeal epithelial cells, clinical NPC biopsy samples, and in vitro EBV infection in HK1 and NPC-TW01 EBV(-) cell lines. FINDINGS: In C666-1, the EBV genome significantly interacted with inactive B compartments of host cells; the significant association of EBV-interacting regions (EBVIRs) with B compartment was confirmed using clinical NPC and in vitro EBV infection model. EBVIRs in C666-1 showed significantly higher levels of active histone modifications compared with NP69T. Aberrant activation of EBVIRs after EBV infection was validated using in vitro EBV infection models. Within the EBVIR-overlapping topologically associating domains, 14 H3K4me3(+) genes were significantly upregulated in C666-1. Target genes of EBVIRs including PLA2G4A, PTGS2 and CITED2, interacted with the enhancers activated in EBVIRs and were highly expressed in NPC, and their knockdown significantly reduced cell proliferation. INTERPRETATION: The EBV genome contributes to NPC tumorigenesis through "enhancer infestation" by interacting with the inactive B compartments of the host genome and aberrantly activating enhancers. FUNDING: The funds are listed in the Acknowledgements section.

Analysis of Speech Fundamental Frequencies for Different Tasks in Japanese.

PURPOSE: Speech fundamental frequency (SFF) assessment is essential for all dysphonia patients to effectively evaluate the therapeutic effects of voice therapy, especially in patients with disturbances in their voice pitch due to mutational dysphonia, Reinke's edema, or as side effects of hormone therapy. A standard method of SFF measurement remains unknown. Speech tasks such as sustained vowel phonation, counting, reading passage, and spontaneous speech have generally been used for SFF measurements. Ideally, spontaneous speech best reflects SFF; however, this task has not yet been clearly defined and is limited with regard to its adaptation to a clinical setting. A reliable task for SFF measurement in Japanese, which corresponds to a speech task that most closely reflects the value that would be observed with typical spontaneous speech, has not been investigated. This study aimed to identify a reliable speech task by measuring the SFF values elicited by different widely used speech tasks in Japanese, and assess its reliability and coefficient of determination (R2). METHODS: Sixty healthy volunteers (30 men and 30 women; aged 19-30 years; mean age 22.5 years) were enrolled. All experimental procedures were performed in Japanese. The SFF values for the speech tasks were determined through the voice samples recorded using a Pulse Code Modulation (PCM) recorder. Each task, except spontaneous speech, was repeated five times, and the average fundamental frequency in each task was determined as the SFF. To assess the reliability of the SFF values across daily variations within individual speakers, the SFF measurements were repeated on two different days, separated by at least 1 week. RESULTS: The SFF values of sustained /a/ phonation, sustained vowel-average, counting, reading passage, and spontaneous speech had excellent reliability, in terms of their reproduction based on intraclass correlation. Significantly high SFF values were observed, in decreasing order, for sustained vowels-average, counting, reading passage, and spontaneous speech in both males and females. The highest R2 for spontaneous speech was that of reading passage in both males (R2 = 0.771) and females (R2 = 0.806) (P < 0.01). CONCLUSION: When spontaneous speech was presented as a task most reflective of daily conversation, reading passage was determined to be the reliable task to assess the therapeutic effect of voice therapy in Japanese.

Tumor-derived exosomes elicit cancer-associated fibroblasts shaping inflammatory tumor microenvironment in head and neck squamous cell carcinoma.

OBJECTIVES: Exosome-mediated reciprocal crosstalk between tumor and stromal cells plays a crucial role in tumor development and progression. This study investigated whether exosomes released from head and neck squamous cell carcinoma (HNSCC) tumor cells can convert normal fibroblasts into cancer-associated fibroblasts (CAF)-like cells and further analyzed the functional characterization of fibroblasts educated by tumor-derived exosomes. MATERIALS AND METHODS: Exosomes secreted from HNSCC cell lines were isolated and normal fibroblasts were established from normal oropharyngeal mucosa. The effects of the exosomes on fibroblasts were examined by proliferation and migration assays, and exosome-educated fibroblasts were analyzed for the expression of eight genes (IL1B, IL6, CXCL8, TGFB1, ACTA2, FAP, CD274, and PDCD1LG2) by RT-qPCR. Moreover, T cells or CD14-positive cells were co-cultured with culture supernatants from exosome-educated fibroblasts. T-cell proliferation and macrophage polarization were examined using flow cytometry. Then, RNA sequencing (RNA-seq) of exosome-educated fibroblasts and the corresponding control fibroblasts was performed. RESULTS: Tumor-derived exosomes enhanced fibroblast proliferation and migration. Moreover, gene expression analysis revealed upregulation of the gene expression of proinflammatory cytokines and immunoregulatory genes, and activated fibroblast marker genes. The culture supernatants of tumor-derived exosome-educated fibroblasts suppressed T cell proliferation and the induction of protumoral macrophages compared with those of control fibroblasts. Next, comprehensive RNA-seq analysis data revealed the activation of 11 signaling pathways, including IL-6- and IL-17-related signaling. CONCLUSION: These results indicate that HNSCC tumor cells induce and/or differentiate into CAFs through exosome-based cell-to-cell communication to create an inflammatory tumor microenvironment.

Repression of DERL3 via DNA methylation by Epstein-Barr virus latent membrane protein 1 in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is Epstein-Barr virus (EBV)-associated invasive malignancy. Increasing evidence indicates that epigenetic abnormalities, including DNA methylation, play important roles in the development of NPC. In particular, the EBV principal oncogene, latent membrane protein 1 (LMP1), is considered a key factor in inducing aberrant DNA methylation of several tumour suppressor genes in NPC, although the mechanism remains unclear. Herein, we comprehensively analysed the methylome data of Infinium BeadArray from 51 NPC and 52 normal nasopharyngeal tissues to identify LMP1-inducible methylation genes. Using hierarchical clustering analysis, we classified NPC into the high-methylation, low-methylation, and normal-like subgroups. We defined high-methylation genes as those that were methylated in the high-methylation subgroup only and common methylation genes as those that were methylated in both high- and low-methylation subgroups. Subsequently, we identified 715 LMP1-inducible methylation genes by observing the methylome data of the nasopharyngeal epithelial cell line with or without LMP1 expression. Because high-methylation genes were enriched with LMP1-inducible methylation genes, we extracted 95 high-methylation genes that overlapped with the LMP1-inducible methylation genes. Among them, we identified DERL3 as the most significantly methylated gene affected by LMP1 expression. DERL3 knockdown in cell lines resulted in significantly increased cell proliferation, migration, and invasion. Lower DERL3 expression was more frequently detected in the advanced T-stage NPC than in early T-stage NPC. These results indicate that DERL3 repression by DNA methylation contributes to NPC tumour progression.