Integrative analysis reveals associations between oral microbiota dysbiosis and host genetic and epigenetic aberrations in oral cavity squamous cell carcinoma.

Dysbiosis of the human oral microbiota has been reported to be associated with oral cavity squamous cell carcinoma (OSCC) while the host-microbiota interactions with respect to the potential impact of pathogenic bacteria on host genomic and epigenomic abnormalities remain poorly studied. In this study, the mucosal bacterial community, host genome-wide transcriptome and DNA CpG methylation were simultaneously profiled in tumors and their adjacent normal tissues of OSCC patients. Significant enrichment in the relative abundance of seven bacteria species (Fusobacterium nucleatum, Treponema medium, Peptostreptococcus stomatis, Gemella morbillorum, Catonella morbi, Peptoanaerobacter yurli and Peptococcus simiae) were observed in OSCC tumor microenvironment. These tumor-enriched bacteria formed 254 positive correlations with 206 up-regulated host genes, mainly involving signaling pathways related to cell adhesion, migration and proliferation. Integrative analysis of bacteria-transcriptome and bacteria-methylation correlations identified at least 20 dysregulated host genes with inverted CpG methylation in their promoter regions associated with enrichment of bacterial pathogens, implying a potential of pathogenic bacteria to regulate gene expression, in part, through epigenetic alterations. An in vitro model further confirmed that Fusobacterium nucleatum might contribute to cellular invasion via crosstalk with E-cadherin/ß-catenin signaling, TNFα/NF-κB pathway and extracellular matrix remodeling by up-regulating SNAI2 gene, a key transcription factor of epithelial-mesenchymal transition (EMT). Our work using multi-omics approaches explored complex host-microbiota interactions and provided important insights into genetic and functional basis in OSCC tumorigenesis, which may serve as a precursor for hypothesis-driven study to better understand the causational relationship of pathogenic bacteria in this deadly cancer.

Combining Transoral Nasopharyngeal Brush and Plasma Epstein-Barr Virus DNA in Detecting Locally Recurrent Nasopharyngeal Carcinoma.

OBJECTIVE: To evaluate the sensitivities and specificities of Epstein-Barr virus (EBV) DNA in the detection of locally recurrent or persistent nasopharyngeal carcinoma (NPC) through nasopharyngeal (NP) brush biopsy and plasma, respectively, and whether a combination of both would be superior to the individual tests. STUDY DESIGN: A case-control study was conducted from September 2016 to June 2022. SETTING: A multicentre study at 3 tertiary referral centers in Hong Kong was conducted by the Department of Otorhinolaryngology, Head and Neck Surgery, The Chinese University of Hong Kong. METHODS: Twenty-seven patients with biopsy-confirmed locally recurrent NPC were recruited as study subjects. Magnetic resonance imaging was performed to rule out regional recurrence. The control group consisted of 58 patients with a prior history of NPC who were now disease-free based on endoscopic and imaging findings. Patients underwent both the transoral NP brush (NP Screen®) and blood for plasma Epstein-Barr DNA levels. RESULTS: The sensitivity and specificity of the combined modalities were 84.62% and 85.19%, respectively. The positive predictive value was 73.33% and the negative predictive value was 92.0%. CONCLUSION: The combination of NP brush biopsy and plasma EBV DNA is potentially an additional surveillance modality in detecting the local recurrence of NPC. Further study with a larger sample size would be required to validate the cutoff values.

Convergent dysbiosis of upper aerodigestive microbiota between patients with esophageal and oral cavity squamous cell carcinoma.

The bidirectional association between primary esophageal squamous cell carcinoma (ESCC) and oral cavity squamous cell carcinoma (OSCC) suggests common risk factors and oncogenic molecular processes but it is unclear whether these two cancers display similar patterns of dysbiosis in their upper aerodigestive microbiota (UADM). We conducted a case-control study to characterize the microbial communities in esophageal lavage samples from 49 ESCC patients and oral rinse samples from 91 OSCC patients using 16S rRNA V3-V4 amplicon sequencing. Compared with their respective non-SCC controls from the same anatomical sites, 32 and 45 discriminative bacterial genera were detected in ESCC and OSCC patients, respectively. Interestingly, 20 of them were commonly enriched or depleted in both types of cancer, suggesting a convergent niche adaptation of upper aerodigestive SCC-associated bacteria that may play important roles in the pathogenesis of malignancies. Notably, Fusobacterium, Selenomonas, Peptoanaerobacter and Peptostreptococcus were enriched in both ESCC and OSCC, whereas Streptococcus and Granulicatelia were commonly depleted. We further identified Fusobacterium nucleatum as the most abundant species enriched in the upper aerodigestive SCC microenvironment, and the higher relative abundances of Selenomonas danae and Treponema maroon were positively correlated with smoking. In addition, predicted functional analysis revealed several depleted (eg, lipoic acid and pyruvate metabolism) and enriched (eg, RNA polymerase and nucleotide excision repair) pathways common to both cancers. Our findings reveal a convergent dysbiosis in the UADM between patients with ESCC and OSCC, suggesting a shared niche adaptation of host-microbiota interactions in the pathogenesis of upper aerodigestive tract malignancies.

Characterization of oral microbiota in HPV and non-HPV head and neck squamous cell carcinoma and its association with patient outcomes.

OBJECTIVE: To investigate the interplay among the oral microbiota, HPV infection, traditional risk factors and patient outcomes in head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: A multi-center study of HNSCC patients with paired tumor and control tissues. We characterized the oral microbiota and HPV infection of tissues in 166 Chinese adults by sequencing the bacterial 16S rRNA V3-V4 and HPV L1 regions, respectively, and examined the associations among the oral microbiota, HPV and clinical features. RESULTS: A total of 15.7% of the surveyed HNSCC patients were positive for HPV DNA, with infection rates varying from 66.7% in oropharyngeal SCC to 10.4% in oral cavity SCC (OSCC). No HPV infection was detected in the surveyed hypopharyngeal SCC. HPV16 was largely the predominant type. HPV infection in non-OSCC, especially oropharyngeal SCC, was associated with advanced N stage and superior survival outcomes. Oral microbiota dysbiosis was observed in HNSCC tumors, with differentially abundant taxa mainly associated with HNSCC subtype, T stage, survival/relapse, HPV infection, and smoking. Notably, the enrichment of Fusobacterium in tumor tissues of OSCC patients was associated with no smoking, early T stage, early N stage, and better 3-year disease-specific survival. CONCLUSION: Our findings underscore the involvement of oral microbiota dysbiosis in OSCC pathogenesis, Fusobacterium is involved with improved OSCC patient outcomes, especially in patients lacking traditional risk factors. Understanding the complex interactions among the oral microbiota, HPV infection and other risk factors for HNSCC will provide important insights into the pathogenesis of HNSCC.

Restoration of the Oral Microbiota After Surgery for Head and Neck Squamous Cell Carcinoma Is Associated With Patient Outcomes.

OBJECTIVE: To evaluate the dynamics of the oral microbiome and associated patient outcomes following treatment of head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: This was a prospective cohort study at a tertiary academic center in Hong Kong SAR of patients with head and neck squamous cell carcinoma evaluating the oral microbiome in pre- and postsurgery oral rinses (at 1, 3, and 6 months) with 16S rRNA gene V3-V4 amplicon sequencing. RESULTS: In total, 76 HNSCC patients were evaluated. There was a significantly depressed alpha diversities of oral microbial communities observed in HNSCC oral rinse samples within the first 6 months post-surgery when compared to presurgery or healthy controls. Distant clustering between pre- and postsurgery was also observed (p < 0.022). Following treatment, eight oral bacterial genera showed a trend towards the restoration in the relative abundances that approximate healthy persons. In evaluating patient outcomes, the decreased relative abundance of three periodontal bacteria (Capnocytophaga, Prevotella 7, and Leptotrichia) and the increased relative abundance of two commensal bacteria (Streptococcus and Rothia) at 6 months postsurgery compared to presurgery showed a better 3-year disease-specific survival (a cutoff of Kaplan-Meier survival curve test p < 0.3 at 36 months). In particular, the postsurgery restoration of Prevotella 7 was statistically significant in the surveyed patients (survival rate of 84% vs. 56% at 36 months, p = 0.0065). CONCLUSIONS: Oral microbiome dysbiosis associated with HNSCC is dynamic. These dynamics of the oral microbiome postsurgery are also associated with patient treatment and outcomes and may serve as potential biomarkers for patient management in HNSCC.

Droplet digital PCR of tumor suppressor gene methylation in serial oral rinses of patients with head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) currently lacks sensitive approaches to detect cancer-related traits in body fluid. METHODS: Methylation of tumor suppressor genes (TSGs) (PAX5, EDNRB, and DCC) were measured in the oral rinses from 50 HNSCC and 58 control subjects using droplet digital PCR (ddPCR). Diagnostic accuracies in detecting HNSCC and the detection rate of recurrence in the post-treatment monitoring were analyzed. RESULTS: ddPCR TSG methylation detection in oral rinses for diagnosis of HNSCC had an AUC of 0.892 for PAX5, 0.753 for EDNRB, and 0.729 for DCC. Significant drop of TSG methylation was observed after completion of surgery (p < 0.01). 76.9% of the relapse cases had a pre-emptive rebound of methylation above presurgery levels in at least one of the tested markers before confirmed recurrence. CONCLUSIONS: Utilizing ddPCR for TSG methylation detection in oral rinses shows potential for detection and monitoring of HNSCC.

The Intersection between Oral Microbiota, Host Gene Methylation and Patient Outcomes in Head and Neck Squamous Cell Carcinoma.

The role of oral microbiota in head and neck squamous cell carcinoma (HNSCC) is poorly understood. Here we sought to evaluate the association of the bacterial microbiome with host gene methylation and patient outcomes, and to explore its potential as a biomarker for early detection or intervention. Here we performed 16S rRNA gene amplicon sequencing in sixty-eight HNSCC patients across both tissue and oral rinse samples to identify oral bacteria with differential abundance between HNSCC and controls. A subset of thirty-one pairs of HNSCC tumor tissues and the adjacent normal tissues were characterized for host gene methylation profile using bisulfite capture sequencing. We observed significant enrichments of Fusobacterium and Peptostreptococcus in HNSCC tumor tissues when compared to the adjacent normal tissues, and in HNSCC oral rinses when compared to healthy subjects, while ten other bacterial genera were largely depleted. These HNSCC-related bacteria were discriminative for HNSCC and controls with area under the receiver operating curves (AUCs) of 0.84 and 0.86 in tissue and oral rinse samples, respectively. Moreover, Fusobacterium nucleatum abundance in HNSCC cases was strongly associated with non-smokers, lower tumor stage, lower rate of recurrence, and improved disease-specific survival. An integrative analysis identified that enrichment of F. nucleatum was associated with host gene promoter methylation, including hypermethylation of tumor suppressor genes LXN and SMARCA2, for which gene expressions were downregulated in the HNSCC cohort from The Cancer Genome Atlas. In conclusion, we identified a taxonomically defined microbial consortium associated with HNSCC that may have clinical potential regarding biomarkers for early detection or intervention. Host-microbe interactions between F. nucleatum enrichment and clinical outcomes or host gene methylation imply a potential role of F. nucleatum as a pro-inflammatory driver in initiating HNSCC without traditional risk factors, which warrants further investigation for the underlying mechanisms.

Prospective clinical trial to evaluate safety and feasibility of using a single port flexible robotic system for transoral head and neck surgery.

INTRODUCTION: The aim of this study was to determine the clinical safety and feasibility of a novel single-port flexible robot for Transoral Robotic Surgery (TORS). MATERIALS AND METHODS: This was a prospective phase II / IDEAL stage 2 clinical trial of both benign and malignant lesions of the head and neck. The primary endpoint included conversion rates and perioperative complications within 30 days following surgery. The study was registered on www.ClinicalTrials.gov (NCT03010813). The Fisher's exact test and Mann-Whitney U test were used to compare categorical, and non-parametric data for the trial. A p value <0.05 was considered to be statistically significant. Statistical analysis was performed with SPSS 20.0 (IBM Corp., Armonk, New York) RESULTS: Twenty-one patients safely underwent TORS with the da Vinci SP (Intuitive Surgical Inc., Sunnyvale, CA) demonstrating the feasibility of access to the nasopharynx, oropharynx, larynx and hypopharynx. There were no conversions of the robotic surgical system. There were no serious adverse events or adverse events related to the use of the robot at 30-day follow-up for all patients. CONCLUSIONS: In a prospective Phase II clinical trial, a novel single-port flexible robotic system appears safe and feasible to use for transoral endoscopic head and neck surgery to access the nasopharynx, oropharynx, larynx and hypopharynx.

Comparison of Opioid Utilization Patterns After Major Head and Neck Procedures Between Hong Kong and the United States.

Importance: The current opioid abuse epidemic in the United States requires evaluation of prescribing practices within all medical specialties. This examination includes a review of postoperative pain management for patients undergoing major head and neck procedures. Objective: To report differences in postoperative pain regimens between an international and domestic head and neck surgical program. Design, Setting, and Participants: Pain management patterns after head and neck surgery in the programs at Chinese University of Hong Kong (CUHK) and Oregon Health and Science University (OHSU) were compared with a focus on opioids, nonsteroidal anti-inflammatory drugs (NSAIDs), acetaminophen or paracetamol, and anxiolytics. Cases from July 1, 2013, through August 31, 2017, were reviewed. Standing medication orders the day before surgery (PRE1), postoperative day 6 (POD6), and postoperative day 14 (POD14) were compared between institutions. Exposures: Head and neck surgery. Results: A total of 253 cases from CUHK and 567 cases from OHSU were analyzed (mean [SD] age, 59.4 [14.3] and 60.1 [16.4] years, respectively). Patients from OHSU had a significantly higher frequency of opioid orders on PRE1 (15.3% vs 1.6%; odds ratio [OR], 11.3; 95% CI, 4.09-31.10), POD6 (86.8% vs 0.4%; OR, 1653.12; 95% CI, 228.51-11 959.01), and POD14 (71.4% vs 0.8%; OR, 313.75; 95% CI, 77.12-1276.52). There were no significant differences in acetaminophen or paracetamol, NSAID, or anxiolytic orders between institutions. Institution was the most significant indicator for the presence of opioid orders on POD6 (OR, 4271.10; 95% CI, 380.04-47 999.70) and POD14 (OR, 330.35; 95% CI, 79.67-1369.82). In addition to treating institution, multivariate analysis showed that PRE1 opioid orders indicated a significant increase in likelihood of opioid orders on POD6 (OR, 4.77; 95% CI, 1.23-18.57) but not POD14. POD6 anxiolytic orders remained a significant indicator of opioid orders for POD6 (95% CI, 1.49-113.10) and POD14 (95% CI, 1.17-5.03), respectively. Conclusions and Relevance: A significantly lower frequency of postoperative opioid orders was observed from CUHK compared with OHSU across similar major head and neck procedures. This contrast encourages a careful examination of (1) cultural and patient expectations of pain control, (2) the metrics by which control is assessed, (3) industry and economic drivers of opioid use, and (4) alternatives to opioid pain regimens. A thoughtful shift in postoperative pain protocols that deemphasizes opioid use may be an opportunity to counter the epidemic of opioid abuse in the United States.

Case Report: Exome sequencing reveals recurrent RETSAT mutations and a loss-of-function POLDIP2 mutation in a rare undifferentiated tongue sarcoma.

Soft tissue sarcoma of the tongue represents a very rare head and neck cancer with connective tissue features, and the genetics underlying this rare cancer are largely unknown. There are less than 20 cases reported in the literature thus far. Here, we reported the first whole-exome characterization (>×200 depth) of an undifferentiated sarcoma of the tongue in a 31-year-old male. Even with a very good sequencing depth, only 19 nonsynonymous mutations were found, indicating a relatively low mutation rate of this rare cancer (lower than that of human papillomavirus (HPV)-positive head and neck cancer). Yet, among the few genes that are somatically mutated in this HPV-negative undifferentiated tongue sarcoma, a noticeable deleterious frameshift mutation (with a very high allele frequency of >93%) of a gene for DNA replication and repair, namely POLDIP2 (DNA polymerase delta interacting protein 2), and two recurrent mutations of the adipogenesis and adipocyte differentiation gene RETSAT (retinol saturase), were identified. Thus, somatic events likely affecting adipogenesis and differentiation, as well as potential stem mutations to POLDIP2, may be implicated in the formation of this rare cancer. This identified somatic whole-exome sequencing profile appears to be distinct from that of other reported adult sarcomas from The Cancer Genome Atlas, suggesting a potential unique genetic profile for this rare sarcoma of the tongue. Interestingly, this low somatic mutation rate is unexpectedly found to be accompanied by multiple tumor protein p53 and NOTCH1 germline mutations of the patient's blood DNA. This may explain the very early age of onset of head and neck cancer, with likely hereditary predisposition. Our findings are, to our knowledge, the first to reveal a unique genetic profile of this very rare undifferentiated sarcoma of the tongue.