Characteristics of chest high-resolution computed tomography in patients with anti-aminoacyl-tRNA synthetase antibody-positive interstitial lung disease.

BACKGROUND AND AIM: Anti-aminoacyl-tRNA synthetase (ARS) antibodies form a condition called Antisynthetase syndrome (ASSD). While interstitial lung disease (ILD) is a particularly frequent manifestation of ASSD and is closely associated with morbidity and mortality, few studies have been conducted on its characteristics on high-resolution computed tomography (HRCT). In this study, we clarified the HRCT findings in patients with anti-ARS antibody-positive ILD (ARS-ILD).  Methods: The HRCT findings at the time of the ILD diagnosis in 24 ARS-ILD patients were retrospectively evaluated by 2 pulmonologists and one radiologist. We also assessed the clinical symptoms, physical examination findings, and laboratory data including the type of anti-ARS antibodies. For a further analysis, the data of patients were divided into two groups: the polymyositis (PM)/dermatomyositis (DM) group and the non-PM/DM group. RESULTS: The ratio of men to women was almost 1:1. The median age at the time of the diagnosis was 53 years old. Anti-glycyl (anti-EJ) and anti-histidyl (anti-Jo-1) antibodies were more common than others. An analysis of the HRCT patterns of 23 ARS-ILD patients showed that the most common pattern was the nonspecific interstitial pneumonia (NSIP) pattern. The second most common pattern was the usual interstitial pneumonia (UIP) pattern. Between the PM/DM and non-PM/DM groups, no clear trends were noted in the age, sex ratio, proportion of HRCT patterns, or type of anti-ARS antibodies. CONCLUSIONS: This retrospective study demonstrated that ARS-ILD patients, regardless of myositis symptoms, most often showed the NSIP pattern on HRCT, as previously reported. However, unlike previous reports, the UIP pattern on HRCT was not rare.

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.

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.

Immune checkpoint inhibitor-related pneumonitis with atypical radiologic features in a patient with anti-aminoacyl-tRNA synthetase antibody.

A man with non-small-cell lung cancer who was negative for anti-nuclear antibodies was admitted for dyspnea after immune checkpoint inhibitor (ICI) administration. Computed tomography (CT) showed complexed radiologic features, including subpleural and basal predominant reticular shadow with cystic structures and peribronchovascular consolidation. Although we treated him with high-dose steroid under a diagnosis of ICI-related pneumonitis, he developed acute exacerbation of pneumonitis with progressive fibrosis and volume loss. A re-evaluation identified anti-aminoacyl-tRNA synthetase antibody in the serum collected before ICI administration. This case highlights the importance of re-evaluating pre-existing autoimmune disorders in patients who develop ICI-related pneumonitis with atypical radiologic features.

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.

Retroperitoneal hemorrhage in a patient with coronavirus disease 2019 (COVID-19):A case report.

INTRODUCTION: Early prophylactic administration of anticoagulants is recommended in patients with coronavirus disease 2019 (COVID-19). A case of retroperitoneal hemorrhage during inpatient treatment for COVID-19 is reported. CASE PRESENTATION: A 69-year-old man was diagnosed with COVID-19 6 days after symptom onset. After admission for difficulty of breathing, he was treated with steroid pulse therapy, remdesivir, and heparin sodium. On day 16 after admission, his hemoglobin and blood pressure dropped. Computed tomography showed a left retroperitoneal hematoma and multiple areas of extravasation in bilateral iliopsoas muscles. Anticoagulation therapy was stopped, and blood transfusion therapy was chosen by considering poor general condition caused by severe pneumonia. On day 19, the hemoglobin and blood pressure improved, and blood transfusion was stopped. However, he died on day 25 due to pneumonia. CONCLUSION: When retroperitoneal hemorrhage occurs as a complication of COVID-19, appropriate treatment decision, transcatheter arterial embolization or conservative treatment, should be chosen based on patient's condition. J. Med. Invest. 69 : 148-151, February, 2022.

Novel prognostic value and potential utility of opioid receptor gene methylation in liquid biopsy for oral cavity cancer.

Opioids are a class of recreational drugs and prescription medications that bind to a group of G-protein-coupled receptors known as opioid receptors (ORs). ORs are involved in the development of many types of cancer; however, their role in head and neck squamous cell carcinoma (HNSCC) is complex and poorly understood. Here, we analyzed the methylation status of five OR genes in verification (301 HNSCC primary samples) and validation (five circulating tumor DNA [ctDNA] samples) studies using quantitative methylation-specific PCR (Q-MSP). OPRL1 and OPRM1 methylation levels were significantly higher in HNSCC tissues than in corresponding normal tissues from the same individuals (P = 0.001 and P < 0.001, respectively). In Kaplan-Meier estimate and multivariate Cox proportional hazard analyses, two genes (OPRL1 and OPRM1) were significantly associated with increased recurrence in the methylation group with oral cavity cancer. Furthermore, a validation study of ctDNA demonstrated that OPRL1 genes exhibited predictive performance as emerging biomarkers and were each capable of discriminating the plasma from tumor-free individuals. We characterized the relationship between OR gene methylation status and outcomes in oral cavity cancer. Our results highlight the potential utility of ctDNA methylation-based detection in the clinical management of oral cavity cancer.

Lenvatinib-induced Interstitial Pneumonia in a Patient with Hepatocellular Carcinoma.

Lenvatinib is a multi-targeted tyrosine kinase inhibitor available for the treatment of unresectable hepatocellular carcinoma (HCC). We herein report an 84-year-old-man with interstitial pneumonia caused by lenvatinib. Four months after the start of lenvatinib administration for HCC, chest computed tomography revealed bilateral ground-glass opacity. However, he continued to take lenvatinib for four more months until he complained of dyspnea on exertion. This is a case of lenvatinib-induced interstitial pneumonia that progressed relatively slowly with a long asymptomatic period despite the appearance of pneumonia on image findings.

DNA Methylation and HPV-Associated Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC), especially oropharyngeal squamous cell carcinoma (OPSCC), has recently been found to be significantly associated with human papillomavirus (HPV) infection. The incidence of OPSCC has been increasing and surpassed the number of cervical cancer cases in the United States. Although HPV-associated OPSCC has a relatively better prognosis than HPV-negative cancer, approximately 20% of HPV-associated HNSCC patients show a poor prognosis or therapeutic response, and the molecular mechanism behind this outcome in the intermediate-risk group is yet to be elucidated. These biological differences between HPV-associated HNSCC and HPV-negative HNSCC are partly explained by the differences in mutation patterns. However, recent reports have revealed that epigenetic dysregulation, such as dysregulated DNA methylation, is a strikingly common pathological feature of human malignancy. Notably, viral infections can induce aberrant DNA methylation, leading to carcinogenesis, and HPV-associated HNSCC cases tend to harbor a higher amount of aberrantly methylated DNA than HPV-negative HNSCC cases. Furthermore, recent comprehensive genome-wide DNA-methylation analyses with large cohorts have revealed that a sub-group of HPV-associated HNSCC correlates with increased DNA methylation. Accordingly, in this review, we provide an overview of the relationship between DNA methylation and HPV-associated HNSCC.

Telomere shortening in head and neck cancer: association between DNA demethylation and survival.

A growing body of evidence indicates that telomere dysfunction is a biological marker of progression in several types of cancer. However, the association between head and neck squamous cell carcinoma (HNSCC) and telomere length (TL) remains unknown. We measured the absolute TL levels in a well-characterised dataset of 211 tumoral vs normal tissues obtained from the same patients by quantitative polymerase chain reaction assay. Normalised TL levels were significantly lower in tumour samples than in normal tissue (P < 0.001) and there was a positive correlation between tumour tissue and normal mucosal tissue (R2 = 0.176, P < 0.001). We were able to distinguish two classes, one with a tumour/normal TL ratio ≤ 0.3 (38.4%), which showed clear telomere erosion, and the other with a tumour/normal TL ratio > 0.3 (61.6%), in which the TL was slightly shorter or longer than that in normal tissue. Notably, the tumour/normal TL ratio was correlated with the likelihood of disease recurrence (P = 0.002), the 5-hydroxymethylcytosine level (P = 0.043), and expression of the ten-eleven translocation (TET) gene (P = 0.043). Our findings show that TL shortening and subsequent low levels of 5-hydroxymethylcytosine and TET expression may contribute to development of HNSCC.

G Protein-Coupled Receptor Genes, PTGDR1, PTGDR2, and PTGIR, Are Candidate Epigenetic Biomarkers and Predictors for Treated Patients with HPV-Associated Oropharyngeal Cancer.

Differences in the biology of human papillomavirus (HPV)-associated oropharyngeal cancers (OPCs) and HPV-negative OPCs may have implications in patient management. Early detection is imperative to reduce HPV-associated OPC mortality. Circulating tumor DNA (ctDNA) can potentially serve as a biomarker for monitoring clinically relevant cancer-related genetic and epigenetic modifications. We analyzed the methylation status of 24 G protein-coupled receptor (GPCR) genes in verification (85 OPC primary samples) and validation (8 OPC ctDNA samples) studies using quantitative methylation-specific polymerase chain reaction (Q-MSP). The Q-MSP-based verification study with 85 OPC primary samples revealed the GPCR genes that were significantly associated with recurrence in high methylation groups (≥14 methylated genes) with OPC and HPV-associated OPC (p < 0.001). In the Kaplan-Meier estimate and multivariate Cox proportional hazard analyses, 13 GPCR genes were significantly related to increased recurrence in the methylation group. Furthermore, the validation study on ctDNA showed that three of these genes (Prostaglandin D2 receptor 1: PTGDR1, Prostaglandin D2 receptor 2: PTGDR2, and Prostaglandin I2 Receptor: PTGIR) had a prediction performance as emerging biomarkers. We characterized the relationship between the methylation status of GPCR genes and outcomes in HPV-associated OPC. Our results highlight the potential utility of ctDNA methylation-based detection for the clinical management of HPV-associated OPC.

Long interspersed nuclear element 1 hypomethylation has novel prognostic value and potential utility in liquid biopsy for oral cavity cancer.

BACKGROUND: New biomarkers are urgently needed to improve personalized treatment approaches for head and neck squamous cell carcinoma (HNSCC). Global DNA hypomethylation has wide-ranging functions in multistep carcinogenesis, and the hypomethylation of long interspersed nucleotide element-1 (LINE-1) is related to increased retrotransposon activity and induced genome instability. However, little information is available regarding LINE-1 hypomethylation and its prognostic implications in HNSCC. METHODS: In this study, we analyzed LINE-1 hypomethylation levels in a well-characterized dataset of 317 primary HNSCC tissues and 225 matched pairs of normal mucosa tissues, along with five oral cavity cancer (OCC) circulating tumor DNA (ctDNA) samples using quantitative real-time methylation and unmethylation PCR. The analysis was performed according to various clinical characteristics and prognostic implications. RESULTS: The results demonstrated that LINE-1 hypomethylation levels were significantly higher in the HNSCC tissues than in corresponding normal tissues from the same individuals (P < 0.001). Univariate analysis revealed that high levels of LINE-1 hypomethylation were correlated with poor disease-free survival (DFS; log-rank test, P = 0.038), whereas multivariate analysis demonstrated that they were significant independent prognostic factor for DFS (hazard ratio: 2.10, 95% confidence interval: 1.02-4.36; P = 0.045). Moreover, samples with high LINE-1 hypomethylation levels exhibited the greatest decrease in 5-hydroxymethylcytosine (5-hmC) levels and increase in tumor-suppressor gene methylation index (P = 0.006 and P < 0.001, respectively). Further, ctDNA studies also showed that LINE-1 hypomethylation had high predictive ability in OCC. CONCLUSIONS: LINE-1 hypomethylation is associated with a higher risk of early OCC relapse, and is hence, a potential predictive biomarker for OCC. Furthermore, 5-hmC levels also exhibited predictive potential in OCC, based on their inverse correlation with LINE-1 hypomethylation levels. LINE-1 hypomethylation analysis, therefore, has applications in determining patient prognosis and real-time surveillance of disease recurrence, and could serve as an alternative method for OCC screening. SUPPLEMENTARY INFORMATION: Supplementary information accompanies this paper at 10.1186/s40364-020-00235-y.

Identification of novel methylation markers in HPV-associated oropharyngeal cancer: genome-wide discovery, tissue verification and validation testing in ctDNA.

Human papilloma virus (HPV)-associated oropharyngeal cancer (OPC) is an independent tumour type with regard to cellular, biological, and clinical features. The use of non-invasive biomarkers such as circulating tumour DNA (ctDNA) may be relevant in early diagnosis and eventually improve the outcomes of patients with head and neck squamous cell carcinoma (HNSCC). Genome-wide discovery using RNA sequencing and reduced representation bisulfite sequencing yielded 21 candidates for methylation-targeted genes. A verification study (252 HNSCC patients) using quantitative methylation-specific PCR (Q-MSP) identified 10 genes (ATP2A1, CALML5, DNAJC5G, GNMT, GPT, LY6D, LYNX1, MAL, MGC16275, and MRGPRF) that showed a significant increase recurrence in methylation groups with OPC. Further study on ctDNA using Q-MSP in HPV-associated OPC showed that three genes (CALML5, DNAJC5G, and LY6D) had a high predictive ability as emerging biomarkers for a validation set, each capable of discriminating between the plasma of the patients from healthy individuals. Among the 42 ctDNA samples, methylated CALML5, DNAJC5G, and LY6D were observed in 31 (73.8%), 19 (45.2%), and 19 (45.2%) samples, respectively. Among pre-treatment ctDNA samples, methylated CALML5, DNAJC5G, and LY6D were observed in 8/8 (100%), 7/8 (87.5%), and 7/8 (87.5%) samples, respectively. Methylated CALML5, DNAJC5G, and LY6D were found in 2/8 (25.0%), 0/8 (0%), and 1/8 (12.5%) of the final samples in the series, respectively. Here, we present the relationship between the methylation status of three specific genes and cancer recurrence for risk classification of HPV-associated OPC cases. In conclusion, ctDNA analysis has the potential to aid in determining patient prognosis and real-time surveillance for disease recurrences and serves as an alternative method of screening for HPV-associated OPC.

Overexpression of Sal-like protein 4 in head and neck cancer: epigenetic effects and clinical correlations.

BACKGROUND: Sal-like protein 4 (SALL4), an embryonic stem cell factor, has been reported to play an essential role in embryogenesis and oncogenesis. As yet, however, the expression and role of this transcription factor in head and neck squamous cell carcinoma (HNSCC) has not been established. METHODS: We assessed SALL4 mRNA expression in a well-characterised dataset of 230 HNSCC samples (test cohort 110 cases and validation cohort 120 cases). We also transfected HNSCC cells (FaDu and UM-SCC-6) with SALL4 siRNA and assessed its effects on proliferation and expression of specific epigenetic factors in order to uncover the role of SALL4 in HNSCC. RESULTS: Overexpression of SALL4 was detected in tumour samples of both cohorts. HNSCC cells treated with SALL4 siRNA showed a reduction in growth and a decrease in DNA methyltransferase 3 alpha (DNMT3A) expression. In the patient cohorts, SALL4 overexpression was found to significantly correlate with disease recurrence (p < 0.001) and SALL4 methylation status (p = 0.002). We also found that DNMT3A was significantly upregulated upon SALL4 upregulation (p < 0.001). High expression levels of SALL4 correlated with decreases in disease-free survival (DFS) rates (log-rank test, p < 0.001). Multivariate analysis revealed that SALL4 expression served as an independent prognostic factor for DFS (hazard ratio: 2.566, 95% confidence interval: 1.598-4.121; p < 0.001). CONCLUSIONS: Our findings indicate that SALL4 upregulation correlates with HNSCC tumour aggressiveness and an adverse patient outcome. Our findings also indicate that DNMT3A may synergistically contribute to the regulatory effects of SALL4. Our findings provide insight into SALL4-mediated HNSCC development via epigenetic modulation.

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.

Neuropeptide receptor genes GHSR and NMUR1 are candidate epigenetic biomarkers and predictors for surgically treated patients with oropharyngeal cancer.

Pathological staging and histological grading systems are useful, but imperfect, predictors of recurrence in head and neck squamous cell carcinoma (HNSCC). Aberrant promoter methylation is the main type of epigenetic modification that plays a role in the inactivation of tumor suppressor genes. To identify new potential prognostic markers, we investigated the promoter methylation status of five neuropeptide receptor genes. The methylation status of the target genes was compared with clinical characteristics in 278 cases; 72 hypopharyngeal cancers, 54 laryngeal cancers, 75 oropharyngeal cancers, and 77 oral cavity cancers were studied. We found that the NTSR1, NTSR2, GHSR, MLNR, and NMUR1 promoters were methylated in 47.8%, 46.8%, 54.3%, 39.2%, and 43.5% of the samples, respectively. GHSR and NMUR1 promoter methylation independently predicted recurrence in HNSCC. In patients with oropharyngeal cancer (n = 75), GHSR and NMUR1 promoter methylation significantly correlates with survival in surgically treated patients. We classified our patients as having a low, intermediate, or high-risk of death based on three factors: HPV status, and GHSR and NMUR1 promoter methylation. The disease-free survival (DFS) rates were 87.1%, 42.7%, and 17.0%, respectively. Combined data analysis of the methylation status of ten-eleven translocation (TET) family genes indicated a trend toward greater methylation indices as the number of TET methylation events increased. In the current study, we presented the relationship between the methylation status of the GHSR and NMUR1 genes and recurrence in HNSCC, specifically in risk classification of oropharyngeal carcinomas cases with HPV status.

5-Hydroxymethylcytosine and ten-eleven translocation dioxygenases in head and neck carcinoma.

Ten-eleven translocation (TET) enzymes are implicated in DNA demethylation through dioxygenase activity, which converts 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC). However, the specific roles of TET enzymes and 5-hmC levels in head and neck squamous cell carcinoma (HNSCC) have not yet been evaluated. In this study, we analyzed 5-hmC levels and TET mRNA expression in a well-characterized dataset of 117 matched pairs of HNSCC tissues and normal tissues. 5-hmC levels and TET mRNA expression were examined via enzyme-linked immunosorbent assay and quantitative real-time PCR, respectively. 5-hmC levels were evaluated according to various clinical characteristics and prognostic implications. Notably, we found that 5-hmC levels were significantly correlated with tumor stage (P = 0.032) and recurrence (P = 0.018). Univariate analysis revealed that low levels of 5-hmC were correlated with poor disease-free survival (DFS; log-rank test, P = 0.038). The expression of TET family genes was not associated with outcomes. In multivariate analysis, low levels of 5-hmC were evaluated as a significant independent prognostic factor of DFS (hazard ratio: 2.352, 95% confidence interval: 1.136-4.896; P = 0.021). Taken together, our findings showed that reduction of TET family gene expression and subsequent low levels of 5-hmC may affect the development of HNSCC.

SALL2 Is a Novel Prognostic Methylation Marker in Patients with Oral Squamous Carcinomas: Associations with SALL1 and SALL3 Methylation Status.

Staging and pathological grading systems are convenient, but imperfect predictors of recurrence of head and neck squamous cell carcinoma. Therefore, to identify potential alternative prognostic markers, we investigated the methylation status of the promoter of Sal-like protein 2 (SALL2). SALL2 mRNA expression was absent in 8/9 (88.9%) University of Michigan squamous cell carcinoma cell lines, whereas two nonmalignant cell lines had stable expression. The normalized methylation value of SALL2 in cancer cell lines was significantly higher than in normal cell lines. SALL2 methylation found in 74 of 233 (31.8%) tumor specimens was correlated with the methylation status of both SALL1 and SALL3. SALL2 methylation was not associated with any difference in disease-free survival (DFS). Therefore, the presence of SALL2 methylation was statistically correlated with a decrease in DFS in patients with oral cancer (log-rank test, p = 0.032). Furthermore, it was associated with disease recurrence in 36.2% of oral cancer cases, with an odds ratio of 2.922 (95% confidence interval = 1.198-7.130; p = 0.018) by multivariate Cox proportional hazard regression analysis. This study suggests that cytosine-phosphate- guanosine (CpG) hypermethylation is a likely mechanism of SALL2 inactivation and supports the hypothesis that SALL2 could serve as an important clinical risk assessment.

Genes Located on 18q23 Are Epigenetic Markers and Have Prognostic Significance for Patients with Head and Neck Cancer.

Loss of heterozygosity (LOH) on chromosome 18q23 is associated with significantly decreased survival in head and neck cancer. In agreement with such tumor suppressive roles, the loss of function of genes located in this region can be achieved through LOH and promotor hypermethylation. In this study, the methylation status of promoters of 18q23 genes in 243 head and neck cancer patients was assessed by quantitative methylation-specific PCR. Promoter methylation was then compared to various clinical characteristics and patient survival. GALR1 and SALL3 promoter methylation correlated with reduced disease-free survival (log-rank test, p = 0.018 and p = 0.013, respectively). Furthermore, based on multivariate Cox proportional hazards analysis, these methylation events were associated with poor disease-free survival, with hazard ratios of 1.600 (95% confidence interval: CI, 1.027⁻2.493; p = 0.038) and 1.911 (95% CI, 1.155⁻3.162; p = 0.012), respectively. By comparison, GALR1 and SALL3 methylation were not prognostic for overall survival in The Cancer Genome Atlas (TCGA) cohort. Our findings suggest that the methylation status of 18q23 genes could serve as important biomarkers for the prediction of clinical outcomes in well-annotated head and neck squamous cell carcinoma cohorts. GALR1 and SALL3 methylation could thus help to facilitate risk stratification for individualized treatment.