Comparative analysis of the tumor microbiome, molecular profiles, and immune cell abundances by HPV status in mucosal head and neck cancers and their impact on survival.

Head and Neck Squamous Cell Carcinoma (HNSCC) comprises a diverse group of tumors with variable treatment response and prognosis. The tumor microenvironment (TME), which includes microbiome and immune cells, can impact outcomes. Here, we sought to relate the presence of specific microbes, gene expression, and tumor immune infiltration using tumor transcriptomics from The Cancer Genome Atlas (TCGA) and associate these with overall survival (OS). RNA sequencing (RNAseq) from HNSCC tumors in TCGA was processed through the exogenous sequences in tumors and immune cells (exotic) pipeline to identify and quantify low-abundance microbes. The detection of the Papillomaviridae family of viruses assessed HPV status. All statistical analyses were performed using R. A total of 499 RNAseq samples from TCGA were analyzed. HPV was detected in 111 samples (22%), most commonly Alphapapillomavirus 9 (90.1%). The presence of Alphapapillomavirus 9 was associated with improved OS [HR = 0.60 (95%CI: 0.40-0.89, p = .01)]. Among other microbes, Yersinia pseudotuberculosis was associated with the worst survival (HR = 3.88; p = .008), while Pseudomonas viridiflava had the best survival (HR = 0.05; p = .036). Microbial species found more abundant in HPV- tumors included several gram-negative anaerobes. HPV- tumors had a significantly higher abundance of M0 (p < .001) and M2 macrophages (p = .035), while HPV+ tumors had more T regulatory cells (p < .001) and CD8+ T-cells (p < .001). We identified microbes in HNSCC tumor samples significantly associated with survival. A greater abundance of certain anaerobic microbes was seen in HPV tumors and pro-tumorigenic macrophages. These findings suggest that TME can be used to predict patient outcomes and may help identify mechanisms of resistance to systemic therapies.

The intratumor microbiome varies by geographical location and anatomical site in head and neck squamous cell carcinoma.

Head and Neck Squamous Cell Carcinoma (HNSCC) is a highly heterogeneous cancer that is characterized by distinct phenotypes based on anatomical site and etiological agents. Recently, the intratumor microbiome has been implicated in cancer pathogenesis and progression. Although it is well established that the gut microbiome varies with geographical location and is highly influenced by factors such as diet, environment, and genetics, the intratumor microbiome is not very well characterized. In this review, we aim to characterize the HNSCC intratumor microbiome by geographical location and anatomical site. We conducted a review of primary literature from PubMed and assessed studies based on relevancy and recency. To the best of our knowledge, we are the first to comprehensively examine the tumor microenvironment of HNSCC with respect to these two primary factors on a large scale. Our results suggest that there are unique bacterial and fungal biomarkers for HNSCC for each of the following geographical locations: North America, Asia, Europe, Australia, and Africa. We also identified a panel of microbial biomarkers that are unique to two primary HNSCC anatomic sites, as well as microbial biomarkers associated with various etiological agents of HNSCC. Future study of these microbes may improve HNSCC diagnostic and therapeutic modalities by accounting for differences based on geographic regions and anatomical sites.

Prospective manipulation of the gut microbiome with microbial ecosystem therapeutic 4 (MET4) in HPV-related locoregionally-advanced oropharyngeal cancer squamous cell carcinoma (LA-OPSCC) undergoing primary chemoradiation: ROMA2 study.

BACKGROUND: Gut microbiome modulation to boost antitumor immune responses is under investigation. METHODS: ROMA-2 evaluated the microbial ecosystem therapeutic (MET)-4 oral consortia, a mixture of cultured human stool-derived immune-responsiveness associated bacteria, given with chemoradiation (CRT) in HPV-related oropharyngeal cancer patients. Co-primary endpoints were safety and changes in stool cumulative MET-4 taxa relative abundance (RA) by 16SRNA sequencing. Stools and plasma were collected pre/post-MET-4 intervention for microbiome and metabolome analysis. RESULTS: Twenty-nine patients received ≥1 dose of MET-4 and were evaluable for safety: drug-related adverse events (AEs) occurred in 13/29 patients: all grade 1-2 except one grade 3 (diarrhea). MET-4 was discontinued early in 7/29 patients due to CRT-induced toxicity, and in 1/29 due to MET-4 AEs. Twenty patients were evaluable for ecological endpoints: there was no increase in stool MET-4 RA post-intervention but trended to increase in stage III patients (p = 0.06). MET-4 RA was higher in stage III vs I-II patients at week 4 (p = 0.03) and 2-month follow-up (p = 0.01), which correlated with changes in plasma and stool targeted metabolomics. CONCLUSIONS: ROMA-2 did not meet its primary ecologic endpoint, as no engraftment was observed in the overall cohort. Exploratory findings of engraftment in stage III patients warrants further investigation of microbiome interventions in this subgroup.

Prevotella intermedia boosts OSCC progression through ISG15 upregulation: a new target for intervention.

PURPOSE: Periodontitis-associated bacteria, such as Porphyromonas gingivalis and Fusobacterium nucleatum, are closely linked to the risk of oral squamous cell carcinoma (OSCC). Emerging studies have indicated that another common periodontal pathogen, Prevotella intermedia (P. intermedia), is enriched in OSCC and could affect the occurrence and progression of OSCC. Our aim is to determine the effects of P. intermedia on the progression of OSCC and the role of antibiotics in reversing these effects. METHODS: In this study, a murine xenograft model of OSCC was established, and the mice were injected intratumorally with PBS (control group), P. intermedia (P.i group), or P. intermedia combined with an antibiotic cocktail administration (P.i + ABX group), respectively. The effects of P. intermedia and ABX administration on xenograft tumor growth, invasion, angiogenesis, and metastasis were investigated by tumor volume measurement and histopathological examination. Enzyme-linked immunosorbent assay (ELISA) was used to investigate the changes in serum cytokine levels. Immunohistochemistry (IHC) was adopted to analyze the alterations in the levels of inflammatory cytokines and infiltrated immune cells in OSCC tissues of xenograft tumors. Transcriptome sequencing and analysis were conducted to determine differential expression genes among various groups. RESULTS: Compared with the control treatment, P. intermedia treatment significantly promoted tumor growth, invasion, angiogenesis, and metastasis, markedly affected the levels of inflammatory cytokines, and markedly altered M2 macrophages and regulatory T cells (Tregs) infiltration in the tumor microenvironment. However, ABX administration clearly abolished these effects of P. intermedia. Transcriptome and immunohistochemical analyses revealed that P. intermedia infection increased the expression of interferon-stimulated gene 15 (ISG15). Correlation analysis indicated that the expression level of ISG15 was positively correlated with the Ki67 expression level, microvessel density, serum concentrations and tissue expression levels of inflammatory cytokines, and quantities of infiltrated M2 macrophages and Tregs. However, it is negatively correlated with the quantities of infiltrated CD4+ and CD8+ T cells. CONCLUSION: In conclusion, intratumoral P. intermedia infection aggravated OSCC progression, which may be achieved through upregulation of ISG15. This study sheds new light on the possible pathogenic mechanism of intratumoral P. intermedia in OSCC progression, which could be a prospective target for OSCC prevention and treatment.

Transcriptomic analysis of Porphyromonas gingivalis-infected head and neck cancer cells: Identification of PLAU as a candidate prognostic biomarker.

Periodontal disease is a risk factor for head and neck squamous cell carcinoma (HNSCC), and Porphyromonas gingivalis, a major periodontal pathogen, has been identified as a specific and potentially independent microbial factor that increases the risk of cancer mortality. Gene expression in HNSCC due to P. gingivalis infection and how changes in gene expression affect the prognosis of HNSCC patients are not clarified. When P. gingivalis was cultured with HNSCC cells, it efficiently adhered to these cells and enhanced their invasive ability. A transcriptome analysis of P. gingivalis -infected HNSCC cells showed that genes related to migration, including CCL20, CITED2, CTGF, C8orf44-SGK3, DUSP10, EGR3, FUZ, HBEGF, IL1B, IL24, JUN, PLAU, PTGS2, P2RY1, SEMA7A, SGK1 and SIX2, were highly up- or down-regulated. The expression of up-regulated genes was examined using the expression data of HNSCC patients obtained from The Cancer Genome Atlas (TCGA) database, and the expression of 5 genes, including PLAU, was found to be higher in cancer tissue than in solid normal tissue. An analysis of protein-protein interactions revealed that these 5 genes formed a dense network. A Cox regression analysis showed that high PLAU expression levels were associated with a poor prognosis in patients with TCGA-HNSCC. Furthermore, the prognostic impact correlated with tumour size and the presence or absence of lymph node metastasis. Collectively, these results suggest the potential of PLAU as a molecular prognostic marker in HNSCC patients. Further in vivo and in vitro studies are needed to verify the findings of this study.

Candida albicans induces upregulation of programmed death ligand 1 in oral squamous cell carcinoma.

BACKGROUND: The potential association between Candida albicans (C. albicans) infection and oral squamous cell carcinoma (OSCC) has been noticed for a long time. Programmed death ligand-1 (PD-L1) is a key molecule of tumor immune escape and tumor progression. This study aimed to explore whether C. albicans could influence PD-L1 expression in OSCC in vitro and in mouse model. METHODS: OSCC cell lines (Cal27 and HN6) were infected with C. albicans for 2 and 24 h, then PD-L1 expression was detected by quantitative real-time polymerase chain reaction (RT-qPCR), western blot (WB), and flow cytometry (FCM). To identify the underlying mechanisms, PD-L1 expression in OSCC cells treated with heat-inactivated C. albicans or with biofilm metabolites derived from C. albicans were explored respectively. Meanwhile, signaling pathways involved in PD-L1 regulation were explored by RT-qPCR, and the candidate genes were verified by WB. Moreover, an OSCC mouse model induced by 4-nitroquinoline-1 oxide was used to further explore the role of C. albicans infection in PD-L1 expression in vivo. RESULTS: C. albicans and heat-inactivated C. albicans upregulated the PD-L1 expression in Cal27 and HN6 cells. Various signaling pathways involved in PD-L1 regulation were influenced by C. albicans infection. Among them, TLR2/MyD88 and TLR2/NF-κB pathways might participate in this process. Furthermore, PD-L1 expression in oral mucosa epithelium was upregulated by C. albicans infection in both normal and OSCC mice. CONCLUSIONS: This study suggests that C. albicans could induce upregulation of PD-L1 in OSCC in vitro and in mouse model, which might due to the activation of TLR2/MyD88 and TLR2/NF-κB pathways.

Treponema denticola Promotes OSCC Development via the TGF-ß Signaling Pathway.

Numerous studies have demonstrated an association between periodontitis and oral squamous cell carcinoma (OSCC), and periodontal pathogens such as Treponema denticola are implicated in the pathogenesis of OSCC. Previous studies have mainly focused on T. denticola surface proteins-for example, chymotrypsin-like proteinase, which was detected in the majority of orodigestive tumor tissues.T. denticola may influence the development of OSCC. Nevertheless, the potential direct regulatory mechanism of T. denticola in OSCC is still unclear. Therefore, this study aimed to explore the direct effect of T. denticola on OSCC cell proliferation and elucidate potential mechanisms of T. denticola in contributing to cell proliferation. A series of in vitro experiments (e.g., CCK-8, EdU, flow cytometry) were performed to explore the effect of T. denticola on cell proliferation, cell cycle, and apoptosis. Mice experiments were performed to explore the effect of T. denticola on tumor growth. Whole mRNA transcriptome sequencing and quantitative real-time polymerase chain reaction were performed to explore the intracellular signaling pathway. Our study found that T. denticola could invade Cal-27 cells and directly promote cell proliferation, regulate the cell cycle, and inhibit apoptosis. T. denticola could also promote the growth of OSCC tumors in mice, and it upregulated Ki67 expression. Regarding the mechanism, T. denticola could promote the development of OSCC by activating the TGF-ß pathway. In conclusion, T. denticola could promote OSCC cell proliferation directly, and the mechanism was associated with intracellular TGF-ß pathway activation.

Oral microbiome associated with lymph node metastasis in oral squamous cell carcinoma.

Oral microbiota can alter cancer susceptibility and progression by modulating metabolism and inflammation. We assessed the association between the oral microbiome and lymph node (LN) metastasis in oral squamous cell carcinoma (OSCC). We collected a total of 54 saliva samples from patients with OSCC before surgery. LN metastasis was assessed based on postoperative pathological examination. We used QIIME2, linear discriminant analysis effect size (LEfSe), and PICRUSt2 methods to analyze microbial dysbiosis. A random forest classifier was used to assess whether the oral microbiome could predict LN metastasis. Among the 54 OSCC samples, 20 had LN metastasis, and 34 had no evidence of metastasis. There was a significant difference in ß-diversity between the metastasis and no metastasis groups. Through LEfSe analysis, the metastasis group was enriched in the genera Prevotella, Stomatobaculum, Bifidobacterium, Peptostreptococcaceae, Shuttleworthia and Finegoldia. Pathways related to signal peptidase II were predominant in the no metastasis group. The RF model showed a modestly high accuracy for predicting metastasis. Differences in microbial community composition and functions were observed in the oral microbiome of patients with OSCC with and without LN metastasis. However, the finding that specific taxa may be associated with LN metastasis should be verified in a further prospective study.

Fusobacterium nucleatum and oral cancer: a critical review.

There is a growing level of interest in the potential role inflammation has on the initiation and progression of malignancy. Notable examples include Helicobacter pylori-mediated inflammation in gastric cancer and more recently Fusobacterium nucleatum-mediated inflammation in colorectal cancer. Fusobacterium nucleatum is a Gram-negative anaerobic bacterium that was first isolated from the oral cavity and identified as a periodontal pathogen. Biofilms on oral squamous cell carcinomas are enriched with anaerobic periodontal pathogens, including F. nucleatum, which has prompted hypotheses that this bacterium could contribute to oral cancer development. Recent studies have demonstrated that F. nucleatum can promote cancer by several mechanisms; activation of cell proliferation, promotion of cellular invasion, induction of chronic inflammation and immune evasion. This review provides an update on the association between F. nucleatum and oral carcinogenesis, and provides insights into the possible mechanisms underlying it.

The effects of periodontitis associated microbiota on the development of oral squamous cell carcinoma.

Epidemiological data have shown that periodontal bacterial infection, periodontitis, and oral squamous cell carcinoma have close relationship on the disease progress and risk. However, the specific role of periodontal microbes and their mechanism in the development of oral squamous cell carcinoma is not yet clear. In our previous work, metagenomic Illumina Mi-seq analysis was used to identify tstructure and abundance of periodontital microbiome. Accoding to the results, we used Porphyromonas.spp. and Fusobacterium.spp. as the periodontitis positive microbiota; Neisseria.spp and Corynebacterium.spp as periodontitis negative microbiota (their average relative abundance were >5%). These representative strains of the above genus were used to infect OSCC cells to explore their effect on tumor cell biology behavior, and detect the expression level of the gene in related to inflammation, migration, invasion and cell cycle. We find that periodontitis positive correlated microbiota had a promoting effect on the development of oral squamous cell carcinoma in vitro by regulating mRNA and protein expression of IL-6, IL-8, MMP-9 and Cyclin-D1. Periodontitis negative correlated microbiota had suppression effect on the development of oral squamous cell carcinoma in vitro analysis.

The microbiome of HPV-positive tonsil squamous cell carcinoma and neck metastasis.

BACKGROUND: Oropharyngeal squamous cell carcinoma (OPSCC) has now surpassed cervical cancer as the most common site of HPV-related cancer in the United States. HPV-positive OPSCCs behave differently from HPV-negative tumors and often present with early lymph node involvement. The bacterial microbiome of HPV-associated OPSCC may contribute to carcinogenesis, and certain bacteria may influence the spread of cancer from the primary site to regional lymphatics. OBJECTIVE: To determine the bacterial microbiome in patients with HPV-associated, early tonsil SCC and compare them to benign tonsil specimens. METHOD: The microbiome of primary tumor specimens and lymph nodes was compared to benign tonsillectomy specimens with pan-pathogen microarray (PathoChip). RESULTS: A total of 114 patients were enrolled in the study. Patients with OPSCC had a microbiome that shifted towards more gram-negative. Numerous signatures of bacterial family and species were associated with the primary tumors and lymph nodes of cancer patients, including the urogenital pathogens Proteus mirabilis and Chlamydia trachomatis, Neisseria gonorrhoeae, Shigella dysenteriae, and Orientia tsutsugamushi. CONCLUSION: Our results suggest that detection of urogenital pathogens is associated with lymph node metastasis for patients with HPV-positive OPSCCs. Additional studies are necessary to determine the effects of the OPSCC microbiome on disease progression and clinical outcomes.

Fusobacterium nucleatum Promotes Cisplatin-Resistance and Migration of Oral Squamous Carcinoma Cells by Up-Regulating Wnt5a-Mediated NFATc3 Expression.

Bacterial infection contributes to tumor development and malignant progression. Fusobacterium nucleatum (F. nucleatum) is reported to promote oral squamous cell carcinoma. However, molecular bases of F. nucleatum regulating oral cancer cells have not been fully elucidated. We report here that F. nucleatum down-regulates p53 and E-cadherin via the Wnt/NFAT pathway to promote cisplatin-resistance and migration in oral squamous carcinoma cells. We pretreated Cal-27 and HSC-3 cells with F. nucleatum and the survival rates against cysplatin (Cis-diamminedichloroplatinum, CDDP) were significantly higher in treated cells. The expressions of migration and apoptosis-related proteins like E-cadherin and p53 were lower in western blot analysis. We observed that F. nucleatum was an activator of the Wnt/NFAT pathway. The expression levels of the Wnt pathway gene wnt5a and Nuclear factors of activated T cells 3 (NFATc3) were notably higher in treated cells. With the inhibition effect of NFAT-inhibitory peptide VIVIT, the expressions of E-cadherin and p53 in response to F. nucleatum infection were up-regulated reversely. We concluded that F. nucleatum might promote cisplatin-resistance and migration of oral squamous cell carcinoma cells through the Wnt/NFAT pathway.

Prognostic value of intratumoral Fusobacterium nucleatum and association with immune-related gene expression in oral squamous cell carcinoma patients.

Changes in the oral microbiome, particularly Fusobacterium nucleatum, are associated with oral squamous cell carcinoma (OSCC). F. nucleatum has been reported to modulate local immunity in cancers. We aimed to assess the association between intratumoral F. nucleatum and clinico-pathological features, relapse, and overall survival (OS) in two independent cohorts of patients with OSCC, and to explore the interplay with immune-related genes. We retrospectively analyzed tissue samples from a first cohort of 122 patients with head and neck squamous cell carcinoma, including 61 OSCC (cohort #1), and a second cohort of 90 additional OSCC (cohort #2). We then performed a sensitivity analysis on the merged cohort of OSCC patients (N = 151). F. nucleatum 16S rRNA gene sequences were quantified using real-time quantitative PCR. The presence of gram-negative bacteria and macrophages was confirmed by LPS and CD163 immunostainings, respectively. F. nucleatum positivity was associated with older age, less alcohol and combined alcohol plus tobacco consumption, and less frequent lymph node invasion. There was a trend for a lower recurrence rate in F. nucleatum-positive cases, with less metastatic relapses compared to F. nucleatum-negative tumors, and significantly longer OS, relapse-free and metastasis-free survival. F. nucleatum status was independently associated with OS in multivariate analysis. Immune-related gene and immunohistochemistry analyses showed that gram-negative bacteria load inversely correlated with M2 macrophages. F. nucleatum-associated OSCC has a specific immune microenvironment, is more frequent in older, non-drinking patients, and associated with a favorable prognosis.

Meta-omics analysis indicates the saliva microbiome and its proteins associated with the prognosis of oral cancer patients.

Saliva is a biofluid that maintains the health of oral tissues and the homeostasis of oral microbiota. Studies have demonstrated that Oral squamous cell carcinoma (OSCC) patients have different salivary microbiota than healthy individuals. However, the relationship between these microbial differences and clinicopathological outcomes is still far from conclusive. Herein, we investigate the capability of using metagenomic and metaproteomic saliva profiles to distinguish between Control (C), OSCC without active lesion (L0), and OSCC with active lesion (L1) patients. The results show that there are significantly distinct taxonomies and functional changes in L1 patients compared to C and L0 patients, suggesting compositional modulation of the oral microbiome, as the relative abundances of Centipeda, Veillonella, and Gemella suggested by metagenomics are correlated with tumor size, clinical stage, and active lesion. Metagenomics results also demonstrated that poor overall patient survival is associated with a higher relative abundance of Stenophotromonas, Staphylococcus, Centipeda, Selenomonas, Alloscordovia, and Acitenobacter. Finally, compositional and functional differences in the saliva content by metaproteomics analysis can distinguish healthy individuals from OSCC patients. In summary, our study suggests that oral microbiota and their protein abundance have potential diagnosis and prognosis value for oral cancer patients. Further studies are necessary to understand the role of uniquely detected metaproteins in the microbiota of healthy and OSCC patients as well as the crosstalk between saliva host proteins and the oral microbiome present in OSCC.

Oral microbiota and vitamin D impact on oropharyngeal squamous cell carcinogenesis: a narrative literature review.

An emerging body of research is revealing the microbiota pivotal involvement in determining the health or disease state of several human niches, and that of vitamin D also in extra-skeletal regions. Nevertheless, much of the oral microbiota and vitamin D reciprocal impact in oropharyngeal squamous cell carcinogenesis (OPSCC) is still mostly unknown. On this premise, starting from an in-depth scientific bibliographic analysis, this narrative literature review aims to show a detailed view of the state of the art on their contribution in the pathogenesis of this cancer type. Significant differences in the oral microbiota species quantity and quality have been detected in OPSCC-affected patients; in particular, mainly high-risk human papillomaviruses (HR-HPVs), Fusobacterium nucleatum, Porphyromonas gingivalis, Pseudomonas aeruginosa, and Candida spp. seem to be highly represented. Vitamin D prevents and fights infections promoted by the above identified pathogens, thus confirming its homeostatic function on the microbiota balance. However, its antimicrobial and antitumoral actions, well-described for the gut, have not been fully documented for the oropharynx yet. Deeper investigations of the mechanisms that link vitamin D levels, oral microbial diversity and inflammatory processes will lead to a better definition of OPSCC risk factors for the optimization of specific prevention and treatment strategies.

Dysbiosis of salivary microbiome and cytokines influence oral squamous cell carcinoma through inflammation.

Advanced combinatorial treatments of surgery, chemotherapy, and radiotherapy do not have any effect on the enhancement of a 5-year survival rate of oral squamous cell carcinoma (OSCC). The discovery of early diagnostic non-invasive biomarkers is required to improve the survival rate of OSCC patients. Recently, it has been reported that oral microbiome has a significant contribution to the development of OSCC. Oral microbiome induces inflammatory response through the production of cytokines and chemokines that enhances tumor cell proliferation and survival. The study aims to develop saliva-based oral microbiome and cytokine biomarker panel that screen OSCC patients based on the level of the microbiome and cytokine differences. We compared the oral microbiome signatures and cytokine level in the saliva of OSCC patients and healthy individuals by 16S rRNA gene sequencing targeting the V3/V4 region using the MiSeq platform and cytokine assay, respectively. The higher abundance of Prevotella melaninogenica, Fusobacterium sp., Veillonella parvula, Porphyromonas endodontalis, Prevotella pallens, Dialister, Streptococcus anginosus, Prevotella nigrescens, Campylobacter ureolyticus, Prevotella nanceiensis, Peptostreptococcus anaerobius and significant elevation of IL-8, IL-6, TNF-α, GM-CSF, and IFN-γ in the saliva of patients having OSCC. Oncobacteria such as S. anginosus, V. parvula, P. endodontalis, and P. anaerobius may contribute to the development of OSCC by increasing inflammation via increased expression of inflammatory cytokines such as IL-6, IL-8, TNF-α, IFN-γ, and GM-CSF. These oncobacteria and cytokines panels could potentially be used as a non-invasive biomarker in clinical practice for more efficient screening and early detection of OSCC patients.

Oral microbial dysbiosis and its performance in predicting oral cancer.

Dysbiosis of oral microbiome may dictate the progression of oral squamous cell carcinoma (OSCC). Yet, the composition of oral microbiome fluctuates by saliva and distinct sites of oral cavity and is affected by risky behaviors (smoking, drinking and betel quid chewing) and individuals' oral health condition. To characterize the disturbances in the oral microbial population mainly due to oral tumorigenicity, we profiled the bacteria within the surface of OSCC lesion and its contralateral normal tissue from discovery (n = 74) and validation (n = 42) cohorts of male patients with cancers of the buccal mucosa. Significant alterations in the bacterial diversity and relative abundance of specific oral microbiota (most profoundly, an enrichment for genus Fusobacterium and the loss of genus Streptococcus in the tumor sites) were identified. Functional prediction of oral microbiome shown that microbial genes related to the metabolism of terpenoids and polyketides were differentially enriched between the control and tumor groups, indicating a functional role of oral microbiome in formulating a tumor microenvironment via attenuated biosynthesis of secondary metabolites with anti-cancer effects. Furthermore, the vast majority of microbial signatures detected in the discovery cohort was generalized well to the independent validation cohort, and the clinical validity of these OSCC-associated microbes was observed and successfully replicated. Overall, our analyses reveal signatures (a profusion of Fusobacterium nucleatum CTI-2 and a decrease in Streptococcus pneumoniae) and functions (decreased production of tumor-suppressive metabolites) of oral microbiota related to oral cancer.

Preliminary insights into the impact of primary radiochemotherapy on the salivary microbiome in head and neck squamous cell carcinoma.

Squamous cell carcinoma is the most common type of throat cancer. Treatment options comprise surgery, radiotherapy, and/or chemo(immuno)therapy. The salivary microbiome is shaped by the disease, and likely by the treatment, resulting in side effects caused by chemoradiation that severely impair patients' well-being. High-throughput amplicon sequencing of the 16S rRNA gene provides an opportunity to investigate changes in the salivary microbiome in health and disease. In this preliminary study, we investigated alterations in the bacterial, fungal, and archaeal components of the salivary microbiome between healthy subjects and patients with head and neck squamous cell carcinoma before and close to the end point of chemoradiation ("after"). We enrolled 31 patients and 11 healthy controls, with 11 patients providing samples both before and after chemoradiation. Analysis revealed an effect on the bacterial and fungal microbiome, with a partial antagonistic reaction but no effects on the archaeal microbial community. Specifically, we observed an individual increase in Candida signatures following chemoradiation, whereas the overall diversity of the microbial and fungal signatures decreased significantly after therapy. Thus, our study indicates that the patient microbiome reacts individually to chemoradiation but has potential for future optimization of disease diagnostics and personalized treatments.

Breath methane to hydrogen ratio as a surrogate marker of intestinal dysbiosis in head and neck cancer.

Exhaled breath compounds can non-invasively detect head and neck squamous cell carcinoma (HNSCC). Here we investigated exhaled compounds related to intestinal bacterial carbohydrate fermentation. Fasting breath samples were collected into 3 litre FlexFoil PLUS bags from patients awaiting a biopsy procedure for suspected HNSCC. Samples were analysed using a Syft selected ion flow-tube mass spectrometer and a Quintron BreathTracker. Two tailed non-parametric significance testing was conducted with corrections for multiple imputations. 74 patients were diagnosed (histological) with HNSCC and 61 patients were benign (controls). The methane to hydrogen ratio was significantly different between cancer and non-cancer controls (p = 0.0440). This ratio increased with tumour stage with a significant difference between T1 and T4 tumours (p = 0.0259). Hydrogen levels were significantly higher in controls who were smokers (p = 0.0129), with no smoking dependent methane changes. There were no differences in short chain fatty acids between groups. Exhaled compounds of intestinal carbohydrate fermentation can detect HNSCC patients. These findings suggest a modified carbohydrate fermentation profile in HNSCC patients that is tumour stage and smoking status dependent.

Oral microbiome and onset of oral mucositis in patients with squamous cell carcinoma of the head and neck.

BACKGROUND: Oral mucositis (OM) is a debilitating sequela for patients treated for squamous cell carcinoma of the head and neck (HNSCC). This study investigated whether oral microbial features before treatment or during treatment are associated with the time to onset of severe OM in patients with HNSCC. METHODS: This was a cohort study of newly diagnosed patients with locoregional HNSCC who received chemotherapy with or without radiotherapy from April 2016 to September 2017. OM was based on the National Cancer Institute's Common Terminology Criteria for Adverse Events, version 4.0. The oral microbiome was characterized on the basis of the 16S ribosomal RNA V4 region with the Illumina platform. A mixture cure model was used to generate hazard ratios for the onset of severe OM. RESULTS: Eighty-six percent of the patients developed OM (n = 57 [33 nonsevere cases and 24 severe cases]) with a median time to onset of OM of 21 days. With adjustments for age, sex, and smoking status, genera abundance was associated with the hazard for the onset of severe OM as follows: 1) at the baseline (n = 66), Cardiobacterium (P = .03) and Granulicatella (P = .04); 2) immediately before the development of OM (n = 57), Prevotella (P = .03), Fusobacterium (P = .03), and Streptococcus (P = .01); and 3) immediately before the development of severe OM (n = 24), Megasphaera (P = .0001) and Cardiobacterium (P = .03). There were no differences in α-diversity between the baseline samples and Human Microbiome Project data. CONCLUSIONS: Changes in the abundance of genera over the course of treatment were associated with the onset of severe OM. The mechanism and therapeutic implications of these findings need to be investigated in future studies.