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The microbiota and radiotherapy for head and neck cancer: What should clinical oncologists know?

Open AccessPublished:July 31, 2022DOI:https://doi.org/10.1016/j.ctrv.2022.102442

      Highlights

      • Radiotherapy is a linchpin in head and neck squamous cell carcinoma treatment.
      • The microbiota may modulate radiotherapy outcomes.
      • The oral microbiota may contribute to head and neck cancer development.
      • The microbiota may contribute to radiotherapy toxicity and efficacy.

      Abstract

      Radiotherapy is a linchpin in head and neck squamous cell carcinoma (HN-SCC) treatment. Modulating tumour and/or normal tissue biology offers opportunities to further develop HN-SCC radiotherapy. The microbiota, which can exhibit homeostatic properties and be a modulator of immunity, has recently received considerable interest from the Oncology community. Microbiota research in head and neck oncology has also flourished. However, available data are difficult to interpret for clinical and radiation oncologists.
      In this review, we focus on how microbiota research can contribute to the improvement of radiotherapy for HN-SCC, focusing on how current and future research can be translated back to the clinic. We include in-depth discussions about the microbiota, its multiple habitats and relevance to human physiology, mechanistic interactions with HN-SCC, available evidence on microbiota and HNC oncogenesis, efficacy and toxicity of treatment. We discuss clinically-relevant areas such as the role of the microbiota as a predictive and prognostic biomarker, as well as the potential of leveraging the microbiota and its interactions with immunity to improve treatment results. Importantly, we draw parallels with other cancers where research is more mature. We map out future directions of research and explain clinical implications in detail.

      Keywords

      Introduction

      Head and neck squamous cell carcinoma (HN-SCC) is the 6th most common cancer worldwide []. Tobacco and alcohol are the main risk factors for HN-SCC, though infection by human papilloma virus (HPV) is a major contributor. Five-year survival for non-metastatic advanced (stage III-IV) HN-SCC range 27.8–67 %, although there are significant differences between subtypes [,
      • Bird T.
      • De Felice F.
      • Michaelidou A.
      • Thavaraj S.
      • Jeannon J.-P.
      • Lyons A.
      • et al.
      Outcomes of intensity-modulated radiotherapy as primary treatment for oropharyngeal squamous cell carcinoma – a European single institution analysis.
      ].
      Radiotherapy is a linchpin in HN-SCC treatment and is the main treatment modality in curative organ-preserving strategies. Treatment outcomes for advanced HN-SCC have improved substantially since the introduction of concurrent radiochemotherapy. Sophisticated radiotherapy techniques such as intensity-modulated radiotherapy (IMRT) and image-guided radiotherapy also contribute to improved outcomes.
      Modulating tumour and/or normal tissue biology offers opportunities to further develop HN-SCC radiotherapy. Since the success of immune checkpoint inhibitors (ICI) in palliative systemic therapy for HN-SCC and the promise of their combination with radiotherapy, interactions between the tumour microenvironment and its immune landscape have become a hot topic of research [
      • Ferris R.L.
      Immunology and Immunotherapy of Head and Neck Cancer.
      ]. In addition, an in-depth understanding of the biology of HN-SCC would allow for better patient stratification and treatment personalisation. Of note, a study of the top 10 research priorities for patients, carers and clinicians, identified uncertainties in best overall treatment combination or regimen selection as the number 1 priority, and precision medicine strategies to help clinicians personalise treatment as number 3 [
      • Lechelt L.A.
      • Rieger J.M.
      • Cowan K.
      • Debenham B.J.
      • Krewski B.
      • Nayar S.
      • et al.
      Top 10 research priorities in head and neck cancer: Results of an Alberta priority setting partnership of patients, caregivers, family members, and clinicians.
      ].
      The microbiota, the communities of microorganisms found in and on all multicellular organisms such as humans, can exhibit homeostatic properties and modulate immunity. Its importance in cancer treatment is increasingly recognised. In this review, we will focus on how microbiota research can contribute to the improvement of radiotherapy for HN-SCC, focusing on how current and future research can be translated back to the clinic.

      Specific anatomical/tumoural habitats determine different microorganism communities

      The human organism harbours a varied and complex microbiota which includes representatives of bacteria, archaea, fungi, viruses and protozoa. Bacteria, the most researched element of the microbiota, slightly outnumber host cells in the human body by 1–1.3:1 [
      • Sender R.
      • Fuchs S.
      • Milo R.
      Revised Estimates for the Number of Human and Bacteria Cells in the Body.
      ]. This review will focus primarily on bacteria and their functions.
      Microbial communities differ between anatomical habitats in the body in terms of the number of different kinds of microbes (known as microbial richness), the proportions of those kinds of microbes (structure), and the total number of microbes (load). Each body site has its own characteristic microbiota but subjects vary greatly in the composition of their bacterial communities at individual sites [
      • Segata N.
      • Haake S.
      • Mannon P.
      • Lemon K.P.
      • Waldron L.
      • Gevers D.
      • et al.
      Composition of the adult digestive tract bacterial microbiome based on seven mouth surfaces, tonsils, throat and stool samples.
      ]. Table 1 shows the classification of bacteria in the upper aerodigestive tract ranked by subsite using the most recent Human Microbiome Project data [
      • Lloyd-Price J.
      • Mahurkar A.
      • Rahnavard G.
      • Crabtree J.
      • Orvis J.
      • Hall A.B.
      • et al.
      Strains, functions and dynamics in the expanded Human Microbiome Project.
      ]. There is, however, some degree of redundancy in that although the bacterial species community composition of individuals may be different, the functions of those communities may be more similar. The diversity of genetic potential among the strains of a bacterial species is so great that many functions have not yet been determined. Functional similarities are determined on the basis of core housekeeping genes. Because they are well-characterised and common, they may be over-estimated. Bacterial behaviour is further influenced by the growth conditions with very different properties being expressed in free-floating cells, for example in saliva, and those growing as biofilms, which are aggregations of microbial communities encased in a polymeric matrix adhering to surfaces including mucosae and tumours, one example of which is dental plaque [
      • Dejea C.M.
      • Wick E.C.
      • Hechenbleikner E.M.
      • White J.R.
      • Mark Welch J.L.
      • Rossetti B.J.
      • et al.
      Microbiota organization is a distinct feature of proximal colorectal cancers.
      ]. In addition, recent data suggest that host genetic makeup could influence host and bacterial functional expression [
      • DeStefano Shields C.E.
      • et al.
      Bacterial-Driven Inflammation and Mutant BRAF Expression Combine to Promote Murine Colon Tumorigenesis That Is Sensitive to Immune Checkpoint Therapy.
      ].
      Table 1Most abundant bacteria in the upper aerodigestive tract, taxonomy, Gram stain and respiration data.
      SiteAbundance (ranked)PhylumClassOrderFamilyGenusSpeciesGram stainAtmospheric requirement
      Anterior nares1ActinobacteriaActinomycetiaPropionibacterialesPropionibacteriaceaePropionibacteriumPropionibacterium acnesGram positiveFacultative anaerobe
      2ActinobacteriaActinomycetiaMycobacterialesCorynebacteriaceaeCorynebacteriumCorynebacterium accolensGram positiveObligate aerobe
      3FirmicutesBacilliLactobacillalesCarnobacteriaceaeDolosigranulumDolosigranulum pigrumGram positiveFacultative anaerobe
      4FirmicutesBacilliBacillalesStaphylococcaceaeStaphylococcusStaphylococcus epidermidisGram positiveFacultative anaerobe
      5FirmicutesBacilliBacillalesStaphylococcaceaeStaphylococcusStaphylococcus aureusGram positiveFacultative anaerobe
      Buccal mucosa1ProteobacteriaGammaproteobacteriaPasteurellalesPasteurellaceaeHaemophilusHaemophilus parainfluenzaeGram negativeFacultative anaerobe
      2FirmicutesBacilliLactobacillalesStreptococcaceaeStreptococcusStreptococcus mitisGram positiveFacultative anaerobe
      3FirmicutesBacilliBacillalesStaphylococcaceaeGemellaGemella haemolysansGram positiveFacultative anaerobe
      4ActinobacteriaActinomycetiaMicrococcalesMicrococcaceaeRothiaRothia mucilaginosaGram positiveObligate aerobe
      5ActinobacteriaActinomycetiaMicrococcalesMicrococcaceaeRothiaRothia dentocariosaGram positiveObligate aerobe
      6FirmicutesBacilliLactobacillalesStreptococcaceaeStreptococcusStreptococcus sanguinisGram positiveFacultative anaerobe
      7BacteroidetesBacteroidiaBacteroidalesPorphyromonadaceaePorphyromonasPorphyromonas sp oral taxon 279Gram negativeObligate anaerobe
      8FirmicutesClostridiaClostridialesVeillonellaceaeVeillonellaVeillonella parvulaGram negativeObligate anaerobe
      9FirmicutesBacilliLactobacillalesCarnobacteriaceaeGranulicatellaGranulicatella elegansGram positiveFacultative anaerobe
      10FirmicutesBacilliLactobacillalesStreptococcaceaeStreptococcusStreptococcus vestibularisGram positiveFacultative anaerobe
      11ProteobacteriaGammaproteobacteriaPasteurellalesPasteurellaceaeHaemophilusHaemophilus haemolyticusGram negativeFacultative anaerobe
      12FirmicutesBacilliLactobacillalesStreptococcaceaeStreptococcusStreptococcus salivariusGram positiveFacultative anaerobe
      13ActinobacteriaActinomycetiaActinomycetalesActinomycetaceaeActinomycesActinomyces orisGram positiveFacultative anaerobe
      14FirmicutesBacilliLactobacillalesStreptococcaceaeStreptococcusStreptococcus parasanguinisGram positiveFacultative anaerobe
      15FirmicutesBacilliLactobacillalesStreptococcaceaeStreptococcusStreptococcus infantisGram positiveFacultative anaerobe
      16ProteobacteriaBetaproteobacteriaBurkholderialesBurkholderiaceaeLautropiaLautropia mirabilisGram negativeFacultative anaerobe
      17FirmicutesNegativicutesVellionellalesVeillonellaceaeVeillonellaVeillonella sp oral taxon 780Gram positiveObligate anaerobe
      18ProteobacteriaBetaproteobacteriaNeisserialesNeisseriaceaeNeisseriaNeisseria flavescensGram negativeObligate aerobe
      19BacteroidetesBacteroidiaBacteroidalesPrevotellaceaePrevotellaPrevotella melaninogenicaGram negativeObligate anaerobe
      20ActinobacteriaActinomycetiaMycobacterialesCorynebacteriaceaeCorynebacteriumCorynebacterium matruchotiiGram positiveObligate aerobe
      Supragingival plaque1ActinobacteriaActinomycetiaMycobacterialesCorynebacteriaceaeCorynebacteriumCorynebacterium matruchotiiGram positiveObligate aerobe
      2ActinobacteriaActinomycetiaMicrococcalesMicrococcaceaeRothiaRothia dentocariosaGram positiveObligate aerobe
      3ProteobacteriaGammaproteobacteriaPasteurellalesPasteurellaceaeHaemophilusHaemophilus parainfluenzaeGram negativeFacultative anaerobe
      4FirmicutesBacilliLactobacillalesStreptococcaceaeStreptococcusStreptococcus sanguinisGram positiveFacultative anaerobe
      5ActinobacteriaActinomycetiaMicrococcalesMicrococcaceaeRothiaRothia aeriaGram positiveObligate aerobe
      6ProteobacteriaBetaproteobacteriaBurkholderialesBurkholderiaceaeLautropiaLautropia mirabilisGram negativeFacultative anaerobe
      7ActinobacteriaActinomycetiaActinomycetalesActinomycetaceaeActinobaculumActinobaculum sp oral taxon 183Gram positiveFacultative anaerobe
      8ActinobacteriaActinomycetiaActinomycetalesActinomycetaceaeActinomycesActinomyces massiliensisGram positiveFacultative anaerobe
      9ActinobacteriaActinomycetiaMycobacterialesCorynebacteriaceaeCorynebacteriumCorynebacterium durumGram positiveObligate aerobe
      10FirmicutesClostridiaClostridialesVeillonellaceaeVeillonellaVeillonella parvulaGram negativeObligate anaerobe
      11BacteroidetesFlavobacteriaFlavobacterialesFlavobacteriaceaeCapnocytophagaCapnocytophaga gingivalisGram negativeFacultative anaerobe
      12ProteobacteriaBetaproteobacteriaNeisserialesNeisseriaceaeNeisseriaNeisseria elongataGram negativeObligate aerobe
      13ActinobacteriaActinomycetiaActinomycetalesActinomycetaceaeActinomycesActinomyces orisGram positiveFacultative anaerobe
      14BacteroidetesFlavobacteriaFlavobacterialesFlavobacteriaceaeCapnocytophagaCapnocytophaga sp oral taxon 329Gram negativeFacultative anaerobe
      15ActinobacteriaActinomycetiaActinomycetalesActinomycetaceaeActinomycesActinomyces sp oral taxon 448Gram positiveFacultative anaerobe
      16ActinobacteriaActinomycetiaActinomycetalesActinomycetaceaeActinomycesActinomyces naeslundiiGram positiveFacultative anaerobe
      17FusobacteriaFusobacteriiaFusobacterialesFusobacteriaceaeFusobacteriumFusobacterium nucleatumGram negativeObligate anaerobe
      18ProteobacteriaGammaproteobacteriaCardiobacterialesCardiobacteriaceaeCardiobacteriumCardiobacterium hominisGram negativeFacultative anaerobe
      19ActinobacteriaActinomycetiaActinomycetalesActinomycetaceaeActinomycesActinomyces sp oral taxon 170Gram positiveFacultative anaerobe
      20ProteobacteriaBetaproteobacteriaNeisserialesNeisseriaceaeKingellaKingella oralisGram negativeFacultative anaerobe
      Tongue dorsum1ProteobacteriaGammaproteobacteriaPasteurellalesPasteurellaceaeHaemophilusHaemophilus parainfluenzaeGram negativeFacultative anaerobe
      2ActinobacteriaActinomycetiaMicrococcalesMicrococcaceaeRothiaRothia mucilaginosaGram positiveObligate aerobe
      3FirmicutesBacilliLactobacillalesStreptococcaceaeStreptococcusStreptococcus parasanguinisGram positiveFacultative anaerobe
      4ActinobacteriaActinomycetiaActinomycetalesActinomycetaceaeActinomycesActinomyces sp ICM47Gram positiveFacultative anaerobe
      5BacteroidetesBacteroidiaBacteroidalesPrevotellaceaePrevotellaPrevotella melaninogenicaGram negativeObligate anaerobe
      6FirmicutesBacilliLactobacillalesStreptococcaceaeStreptococcusStreptococcus salivariusGram positiveFacultative anaerobe
      7ActinobacteriaActinomycetiaActinomycetalesActinomycetaceaeActinomycesActinomyces graevenitziiGram positiveFacultative anaerobe
      8BacteroidetesBacteroidiaBacteroidalesPorphyromonadaceaePorphyromonasPorphyromonas sp oral taxon 279Gram negativeObligate anaerobe
      9FusobacteriaFusobacteriiaFusobacterialesFusobacteriaceaeFusobacteriumFusobacterium periodonticumGram negativeObligate anaerobe
      10BacteroidetesBacteroidiaBacteroidalesPrevotellaceaePrevotellaPrevotella histicolaGram negativeObligate anaerobe
      11ProteobacteriaBetaproteobacteriaNeisserialesNeisseriaceaeNeisseriaNeisseria flavescensGram negativeObligate aerobe
      12FirmicutesBacilliLactobacillalesStreptococcaceaeStreptococcusStreptococcus infantisGram positiveFacultative anaerobe
      13BacteroidetesBacteroidiaBacteroidalesPrevotellaceaePrevotellaPrevotella nanceiensisGram negativeAnaerobe
      14ProteobacteriaBetaproteobacteriaNeisserialesNeisseriaceaeNeisseriaNeisseria subflavaGram negativeAerobe
      15FirmicutesClostridiaClostridialesVeillonellaceaeVeillonellaVeillonella atypicaGram negativeAnaerobe
      16ActinobacteriaActinomycetiaActinomycetalesActinomycetaceaeActinomycesActinomyces sp oral taxon 172Gram positiveAnaerobe
      17FirmicutesClostridiaClostridialesVeillonellaceaeVeillonellaVeillonella disparGram negativeAnaerobe
      18ProteobacteriaEpsilonproteobacteriaCampylobacteralesCampylobacteraceaeCampylobacterCampylobacter concisusGram negativeAnaerobe
      19BacteroidetesBacteroidiaBacteroidalesPrevotellaceaePrevotellaPrevotella pallensGram negativeAnaerobe
      20FirmicutesBacilliBacillalesStaphylococcaceaeGemellaGemella sanguinisGram positiveFacultative anaerobe
      It is thus not surprising that the microbiota differs between cancers according to anatomical location. Nejman and colleagues conducted a study comparing human samples of breast, melanoma, lung, pancreas, ovary, bone and central nervous system tumours (n = 1010). They found tumour-specific microbiome signatures with most of the intratumoural bacteria located intracellularly in both tumour and immune cells [

      Nejman D. et al. The human tumor microbiome is composed of tumor type–specific intracellular bacteria. Science (1979) 2020; 368: 973 LP – 980.

      ]. Although the authors employed strategies to mitigate counting contaminants as relevant bacteria, they were only partially successful; for example, Rhodospirillum, a genus found in marine environments and soil, but not in the human microbiome, was strongly associated to cigarette smoking, suggesting contamination during methodological procedures. The issue of contamination was addressed by Poore and colleagues using data from the Cancer Genome Atlas (TCGA), where whole genomes and transcriptomes of 20,000 primary cancer and matched normal samples spanning 33 cancer types were sequenced, including any microbial DNA present in tumour samples. Focusing on treatment-naïve tumours (n = 18,116), the authors used machine learning and implemented an in-silico decontamination strategy identifying putative contaminants in sample batches by sequencing centre, removing taxa found to be contaminants, and manually reviewing the literature to re-include pathobiont or mixed-evidence genera. This strategy allowed for the creation of three distinct datasets with increasing stringency of contamination control, which in turn decreased discrimination of tumours in different sites, but increased discrimination in the same site of tumour versus normal tissue [
      • Poore G.D.
      • Kopylova E.
      • Zhu Q.
      • Carpenter C.
      • Fraraccio S.
      • Wandro S.
      • et al.
      Microbiome analyses of blood and tissues suggest cancer diagnostic approach.
      ]. No studies specific to HN-SCC exist, but these two examples highlight why any studies should be interpreted with care given the complex analytic pipelines employed.

      The microbiota and HN-SCC: Cause or effect?

      The oral cavity and connected habitats contain over 700 different bacterial species and any one individual will have around 350 in their mouth at any one time [
      • Mager D.L.
      • et al.
      The salivary microbiota as a diagnostic indicator of oral cancer: A descriptive, non-randomized study of cancer-free and oral squamous cell carcinoma subjects.
      ]. There are several microhabitats within upper aerodigestive tract mucosae each with their own characteristic bacterial composition. Environmental factors that affect community composition include temperature, pH, redox potential, oxygen concentration and these are further modulated by the dynamic aspects of saliva flow, local and systemic immunity [
      • Jenkinson H.F.
      • Lamont R.J.
      Oral microbial communities in sickness and in health.
      ]. In addition, variations in oral hygiene practice contribute to the amount and composition of biofilms on the teeth and other surfaces. Complex inter-relationships between bacteria also shape communities. For example, Streptococcus mitis, a prevalent micro-organism in the oral cavity, supports growth of Haemophilus parainfluenzae by providing nicotinamide adenine dinucleotide, which is necessary for the latter’s survival. However, when a ratio of 1:1 is reached, S. mitis inhibits H. parainfluenzae growth through production of hydrogen peroxide and therefore maintaining a balance between the species [
      • Perera D.
      • McLean A.
      • Morillo-López V.
      • Cloutier-Leblanc K.
      • Almeida E.
      • Cabana K.
      • et al.
      Mechanisms underlying interactions between two abundant oral commensal bacteria.
      ].
      Oral bacteria are associated with the two commonest human bacterial diseases: dental caries (tooth decay) and periodontal (gum) disease [
      • Wade W.G.
      The oral microbiome in health and disease.
      ]. The primary risk factor for caries is excessive and over-frequent consumption of fermentable carbohydrates [
      • Selwitz R.H.
      • Ismail A.I.
      • Pitts N.B.
      Dental caries.
      ]. Many oral bacteria produce acid from carbohydrate, which can degrade saliva's buffering capacity and reduce local pH which demineralises the teeth, causing cavity formation. The low pH selects for aciduric bacteria which can tolerate acidic conditions. Poor oral hygiene leads to plaque accumulation and an increase in the proportions of anaerobic and Gram-negative bacteria which inflame the gingivae (gums) causes gingivitis. Gingivitis affects around 90 % of individuals to some degree and is reversible via improved oral hygiene at affected sites resolving the inflammation [
      • Pihlstrom B.L.
      • Michalowicz B.S.
      • Johnson N.W.
      Periodontal diseases.
      ]. However, around 15 % of individuals develop a more serious form of gum disease termed periodontitis where the gingival tissues become detached from the teeth with the formation of pockets which become heavily colonised by anaerobic bacteria. The resulting chronic infection causes resorption of the bone supporting the teeth as well as loss of the ligament, often leading to tooth mobility. If left untreated, teeth can be lost. As some bacterial species, such as Porphyromonas gingivalis, are associated with late stages of periodontitis, they have been described as periodontal pathogens even if they are not pathogens in the traditional medical sense [
      • Socransky S.S.
      • Haffajee A.D.
      • Cugini M.A.
      • Smith C.
      • Kent R.L.
      Microbial complexes in subgingival plaque.
      ]. Bacteria associated with oral diseases are members of the normal microbiota although their numbers may fluctuate depending on the disease environment. For example, Porphyromonas gingivalis requires haemin for growth and their numbers are raised in periodontitis, where the gums bleed [
      • Mayrand D.
      • Holt S.C.
      Biology of asaccharolytic black-pigmented Bacteroides species.
      ]. A better description for bacterial species associated with oral disease might be pathobiont, defined as a symbiont which can become harmful under specific circumstances in susceptible individuals [
      • Loos B.G.
      • van Dyke T.E.
      The role of inflammation and genetics in periodontal disease.
      ].
      Poor oral hygiene and associated oral diseases are associated with HN-SCC, suggesting that specific microbiota profiles may associate with carcinogenesis. The International HN-SCC Epidemiology (INHANCE) consortium explored associations between clinical indicators of oral hygiene and HN-SCC in 8,925 HN-SCC cases and 12,527 controls and found significant inverse correlation between HN-SCC and either < 5 missing teeth, annual dental visit, daily tooth brushing or no gum disease. Importantly, these associations persisted when the cohort was stratified by smoking, alcohol consumption, localisation of gum disease, tooth brushing, and number of missing teeth [
      • Hashim D.
      • Sartori S.
      • Brennan P.
      • Curado M.P.
      • Wünsch-Filho V.
      • Divaris K.
      • et al.
      The role of oral hygiene in head and neck cancer: results from International Head and Neck Cancer Epidemiology (INHANCE) consortium.
      ]. Interestingly, although the association with oral cavity cancer was more marked, significance was also observed in other sublocations (including oropharynx, hypopharynx, other pharynx and larynx), similar to what was observed in 886 patients prior to radiotherapy [
      • Patel V.
      • Patel D.
      • Browning T.
      • Patel S.
      • McGurk M.
      • Sassoon I.
      • et al.
      Presenting pre-radiotherapy dental status of head and neck cancer patients in the novel radiation era.
      ].
      Several clinical studies (Table 2) have attempted to characterise associations between the microbiome and HN-SCC. However, methodological disparity and study-specific issues have hindered extrapolation of conclusions, reflected in often contradictory results and limited clinical applicability. Common limitations include: (1) the microbiome is often measured in oral wash/salivary samples, so salivary bacteria (which primarily represent tongue and palate communities) were measured rather than more biologically-relevant tumour-associated bacteria; (2) several HN-SCC sites (e.g. oral cavity, oropharynx, nasal cavity) with different biologies are often lumped together; (3) microbial DNA degradation and/or contamination due to non-ideal sample storage/processing; (4) limited sample sizes leading to over-interpretation of results. These studies should thus be considered as hypothesis-generating rather than definitive. If taken as such, some conclusions of interest can be drawn: tumours are often characterised by higher counts of fusobacteria, dominated by common oral bacteria such as streptococci, and tumour microbiome functionality is often characterised by increased pro-inflammatory and/or decreased xenobiotic degradation pathways.
      Table 2Clinical studies investigating associations between the microbiome and HN-SCC.
      StudyNCancer siteDesignSample typeTechniques usedMain reported findingsNotes
      Bahig et al., 2021
      • Bahig H.
      • Fuller C.D.
      • Mitra A.
      • Yoshida-Court K.
      • Solley T.
      • Ping Ng S.
      • et al.
      Longitudinal characterization of the tumoral microbiome during radiotherapy in HPV-associated oropharynx cancer.
      n = 9Oropharyngeal cancer (HPV + )CohortMucosal and tumoural swabs16S rRNA gene-based community profilingTumours had higher abundance of Veillonella and Leptotrichia compared to buccal mucosa.Also evaluated response to radiotherapy (see main text)
      Oliva et al., 2021
      • Oliva M.
      • Schneeberger P.H.H.
      • Rey V.
      • Cho M.
      • Taylor R.
      • Hansen A.R.
      • et al.
      Transitions in oral and gut microbiome of HPV+ oropharyngeal squamous cell carcinoma following definitive chemoradiotherapy (ROMA LA-OPSCC study).
      n = 22 casesOropharyngeal cancer (HPV + )CohortSaliva and oropharyngeal swabs, and stoolShotgun metagenomics (swabs and stools)Stage III (compared to stage I and II) patients had higher abundance of: (a) in oropharyngeal swabs: Fusobacterium nucleatum, Gemella morbillorum, Gemella haemolysans, Leptotrichia hofstadii, Selenomonas sputigena and Selenomonas infelix.; and (b) in stools: Actinobacteria and Proteobacteria.Also evaluated response to radiotherapy (see main text)
      Zhang et al., 2020

      Zhang L, Liu Y, Zheng HJ. & Zhang CP. The Oral Microbiota May Have Influence on Oral Cancer. Frontiers in Cellular and Infection Microbiology vol. 9 476 Preprint at 2020.

      n = 50 casesOral SCC and matched samples of oral tissueCohortTumour and tissue swab/brushing16S rRNA gene-based community profilingHN-SCC associated with higher bacterial diversity.

      Higher relative abundance in tumour → Bacterial genera: Aggregibacter, Alloprevotella, Campylobacter, Capnocytophaga, Catonella, Filifactor, Fusobacterium, Parvimonas, Peptococcus, Peptostreptoccus, Porphyromonas, Treponema. Pathways: Genes associated with pro-inflammatory bacterial components (e.g. lipopolysaccharide biosynthesis), with metabolism of cofactors and vitamins, genes related to cell motility.

      Higher relative abundance in normal tissue → Bacterial genera: Actinomyces, Corynebacterium, Granulicatella, Oribacterium, Rothia, Streptococcus, Tannerella, Veillonella. Pathways: carbohydrate metabolism and phosphotransferase system transport.
      Sharma et al, 2020

      Kumar-Sharma A, Debusk WT, Stepanov I, Gomez A. & Khariwala SS. Oral microbiome profiling in smokers with and without head and neck cancer reveals variations between health and disease. Cancer Prevention Research canprevres.0459.2019 (2020) doi:10.1158/1940-6207.CAPR-19-0459.

      n = 27 cases and n = 24 controlsOral cavity (n = 6), oropharynx (n = 12), larynx (n = 6), hypopharynx (n = 2), N/A (n = 1)Case-controlOral brushings from buccal mucosa16S rRNA gene-based community profilingHN-SCC associated with higher relative abundance of Stenotrophomonas, Comamonadaceae, and predicted bacterial pathways mainly involved in xenobiotic and amine degradationComparison of smokers with and without HN-SCC
      Chen et al, 2020

      Chen Z. et al. The Intersection between Oral Microbiota, Host Gene Methylation and Patient Outcomes in Head and Neck Squamous Cell Carcinoma. Cancers vol. 12 Preprint at https://doi.org/10.3390/cancers12113425 (2020).

      n = 68 cases and n = 68 controlsOral cavity (n = 48), larynx (n = 13), hypopharynx (n = 3), nasal cavity (n = 2), oropharynx (n = 1), paranasal sinus (n = 1)Case-controlTissue and oral rinse samples16S rRNA gene-based community profilingHN-SCC associated with higher abundance of Fusobacterium and PeptostreptococcusHigher abundance of Fusobacterium and Peptostreptococcus also in tumour compared to adjacent normal tissue. Association of F. nucleatum with better prognosis and hypermethylation of tumour suppressor genes (LXN and SMARCA2).
      Takahashi et al, 2019
      • Takahashi Y.
      • Park J.
      • Hosomi K.
      • Yamada T.
      • Kobayashi A.
      • Yamaguchi Y.
      • et al.
      Analysis of oral microbiota in Japanese oral cancer patients using 16S rRNA sequencing.
      n = 60 cases and n = 80 controlsOral cavityCase-controlSalivary samples16S rRNA gene-based community profilingHN-SCC associated with higher α-diversity, higher abundance of Peptostreptococcus, Fusobacterium, Alloprevotella, and Capnocytophaga; and lower abundance of Rothia and Haemophilus.Negative correlation between Rothia and T stage; multivariate analysis showed Chao1 (bacterial richness index) and sex as significant variables.
      Hayes et al., 2018
      • Hayes R.B.
      • Ahn J.
      • Fan X.
      • Peters B.A.
      • Ma Y.
      • Yang L.
      • et al.
      Association of Oral Microbiome With Risk for Incident Head and Neck Squamous Cell Cancer.
      n = 129 cases and n = 254 controlsOral cavity (n = 19), pharynx (n = 12), larynx (n = 27)Case-controlOral wash16S rRNA gene-based community profilingCorynebacterium and Kingella associated with decreased risk for HN-SCCStronger association in smokers or ex-smokers
      Perera et al 2018
      • Perera M.
      • Al-hebshi N.N.
      • Perera I.
      • Ipe D.
      • Ulett G.C.
      • Speicher D.J.
      • et al.
      Inflammatory Bacteriome and Oral Squamous Cell Carcinoma.
      N = 25 cases (OSCC), n = 27 controls (oral fibroepithelial polyp)Oral cavityCase-controlBiopsies16S rRNA gene-based community profilingHN-SCC associated with lower diversity, higher abundance of Capnocytophaga, Pseudomonas, and Atopobium; and lower abundance of Lautropia, Staphylococcus, and Propionibacterium.

      HN-SCC also associated with higher abundance of genes responsible for lipopolysaccharide biosynthesis and peptidases.
      At the species level, Campylobacter concisus, Prevotella salivae, Prevotella loeschii, and Fusobacterium oral taxon 204 were enriched in OSCC, while Streptococcus mitis, Streptococcus oral taxon 070, Lautropia mirabilis, and Rothia dentocariosa among others were more abundant in FEP.
      Yang et al, 2018
      • Yang C.-Y.
      • Yeh Y.-M.
      • Yu H.-Y.
      • Chin C.-Y.
      • Hsu C.-W.
      • Liu H.
      • et al.
      Oral Microbiota Community Dynamics Associated With Oral Squamous Cell Carcinoma Staging.
      n = 197 cases (41 stage I; 66 stage II/III; 90 stage IV) and n = 51 controlsOral cavityCohortOral rinse16S rRNA gene-based community profilingAbundance of Fusobacteria increased with OSCC stage. HN-SCC associated with higher abundance of Fusobacterium periodonticum, Parvimonas micra, Streptococcus constellatus, Haemophilus influenza, and Filifactor alocis and these bacteria increased in abundance with stage. HN-SCC associated with lower abundance of Streptococcus mitis, Haemophilus parainfluenzae, and Porphyromonas pasteri.

      Pathway analysis showed an association between late stage HN-SCC and higher abundance of genes involved in carbohydrate-related metabolism, such as methane metabolism, and energy-metabolism-related parameters, such as oxidative phosphorylation and carbon fixation in photosynthetic organisms; and with lower abundance of genes involved in amino acid biosynthesis.
      Zhu et al, 2017
      • Zhu X.-X.
      • Yang X.-J.
      • Chao Y.-L.
      • Zheng H.-M.
      • Sheng H.-F.
      • Liu H.-Y.
      • et al.
      The Potential Effect of Oral Microbiota in the Prediction of Mucositis During Radiotherapy for Nasopharyngeal Carcinoma.
      41 cases and 49 controlsNasopharyngeal cancerCase-controlOropharyngeal swabs16S rRNA gene-based community profilingΑ-diversity was lower in cases. Avibacterium, Pediococcus, Pseudomonas, Oscillibacter, and unclassified Firmicutes and Clostridiaceae were more abundant, and Fusobacteria, Porphyromonas, Treponema, Peptococcus, Tannerella and Corynebacterium less abdundant in NPC patients compared to controls.
      Wolf et al, 2017
      • Wolf A.
      • Moissl-Eichinger C.
      • Perras A.
      • Koskinen K.
      • Tomazic P.V.
      • Thurnher D.
      The salivary microbiome as an indicator of carcinogenesis in patients with oropharyngeal squamous cell carcinoma: A pilot study.
      and Kumpitsch et al 2020
      • Kumpitsch C.
      • Moissl-Eichinger C.
      • Pock J.
      • Thurnher D.
      • Wolf A.
      Preliminary insights into the impact of primary radiochemotherapy on the salivary microbiome in head and neck squamous cell carcinoma.
      Wolf et al: n = 11 cases and n = 11 controls

      Kumptisch et al: n = 31 cases (including 11 from Wolf et al) and n = 11 controls
      Oral cavity and oropharynxCase-controlSaliva16S rRNA gene-based community profilingWolf et al: Prevotella, Haemophilus, Neisseria, Streptococcus and Veilonella more abundant in controls; Actinomyces, Schwartzia, Treponema and Selenomonas more abundant in cases.



      Kumptisch et al: Veillonella and Bifidobacteriaceae more abundant in cases, and Pasteurellaceae, Eubacterium more abundant in controls.
      Sugar metabolism (including biofilm matrix formation) and stress response (spore formation) pathways more abundant in cases; lipid metabolism and defence mechanisms more abundant in controls (Wolf et al).
      Zhao et al, 2017
      • Zhao H.
      • et al.
      Variations in oral microbiota associated with oral cancer.
      n = 40 cases and n = 40 controlsOral cavityCase-controlTumour or normal tissue swabs16S rRNA gene-based community profilingHN-SCC associated with higher α-diversity; and higher abundance of Mycoplasma, Treponema, Campylobacter, Eikenella, Centipeda, Lachnospiraceae_G_7, Alloprevotella, Fusobacterium, Selenomonas, Dialister, Peptostreptococcus, Filifactor, Peptococcus, Catonella, Parvimonas, Capnocytophaga, and Peptostreptococcaceae_XI_G_7; and lower abundance of Megasphaera, Stomatobaculum, Granulicatella, Lautropia, Veillonella, Streptococcus, Scardovia, Rothia, and Actinomyces. Pathways related to genetic information processing associated with HN-SCC.Network analysis showed highly connected bacterial cluster of Fusobacterium that was heavily involved in the bacterial ecology structure of cancer samples compared with the controls, which separated cancer and control samples with good accuracy.
      Lee et al, 2017
      • Lee W.-H.
      • Chen H.-M.
      • Yang S.-F.
      • Liang C.
      • Peng C.-Y.
      • Lin F.-M.
      • et al.
      Bacterial alterations in salivary microbiota and their association in oral cancer.
      n = 127 normal controls, n = 124 epithelial cancer precursor lesions; n = 125 cases (cancer confirmed)Oral cavityCohortSaliva16S rRNA gene-based community profilingHN-SCC associated with higher abundance of Bacillus, Enterococcus, Parvimonas, Peptostreptococcus and Slackia.Epithelial precursor lesions included dysplasia, hyperplasia and hyperkeratosis.
      Al-Hebshi et al., 2017
      • Al-hebshi N.N.
      • Nasher A.T.
      • Maryoud M.Y.
      • Homeida H.E.
      • Chen T.
      • Idris A.M.
      • et al.
      Inflammatory bacteriome featuring Fusobacterium nucleatum and Pseudomonas aeruginosa identified in association with oral squamous cell carcinoma.
      n = 20 cases and n = 20 controlsOral cavityCase-controlBiopsies (cases) and swabs (controls)16S rRNA gene-based community profilingHN-SCC associated with higher abundance of Fusobacterium nucleatum polymorphum, Pseudomonas aeruginosa and Campylobacter spp.; and with lower abundance of Streptococcus mitis, Rothia mucilaginosa and Haemophilus parainfluenzae.

      Functional analysis (imputed metagenomics) showed an association between HN-SCC and higher abundance of genes involved in bacterial mobility, flagellar assembly, bacterial chemotaxis and lipopolysaccharide synthesis; and a lower abundance of genes related to DNA repair, purine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, ribosome biogenesis and glycolysis/gluconeogenesis.
      Case samples obtained from leftover DNA extracts obtained from biopsies from a previous study, collected between 2009 and 2011. Control samples obtained freshly by authors (swabs).
      Guerrero-Preston et al., 2016
      • Guerrero-Preston R.
      • Godoy-Vitorino F.
      • Jedlicka A.
      • Rodríguez-Hilario A.
      • González H.
      • Bondy J.
      • et al.
      16S rRNA amplicon sequencing identifies microbiota associated with oral cancer, Human Papilloma Virus infection and surgical treatment.
      n = 17 cases and n = 25 controlsOral (n = 6), oropharynx (n = 11)Cohort studySaliva16S rRNA gene-based community profilingHN-SCC associated with lower richness and diversity, higher abundance of Lactobacillus, Streptococcus, Staphylococcus, Parvimonas; lower counts of Neisseria, Aggregibacter, Haemophilus and GemellaceaeHPV-positive oropharyngeal tumours had higher abundance of Lactobacillus and Weeksellaceae; HPV negative had higher abundance of Eikenella, Neisseria, and Leptotrichia
      Schmidt et al., 2014
      • Schmidt B.L.
      • Kuczynski J.
      • Bhattacharya A.
      • Huey B.
      • Corby P.M.
      • Queiroz E.L.S.
      • et al.
      Changes in Abundance of Oral Microbiota Associated with Oral Cancer.
      n = 5 (discovery cohort); n = 10 (confirmation cohort)Oral cavityCohort studyTumour and adjacent normal mucosa swabs16S rRNA gene-based community profilingReduction in relative abundance of Firmicutes and Actinobacteria (phyla), and Streptococcus, Rothia; increase in Fusobacterium (genera) in tumour samples (note: results from several analyses including pooling of the 2 cohorts)
      Pushalkar et al, 2012
      • Pushalkar S.
      • Ji X.
      • Li Y.
      • Estilo C.
      • Yegnanarayana R.
      • Singh B.
      • et al.
      Comparison of oral microbiota in tumor and non-tumor tissues of patients with oral squamous cell carcinoma.
      n = 10 casesOral SCCCohortTumour and adjacent normal mucosa samples16S rRNA gene-based community profilingHN-SCC associated with higher abundance of Streptococcus sp. oral taxon 058, Peptostreptococcus stomatis, Streptococcus salivarius, Streptococcus gordonii, Gemella haemolysans, Gemella morbillorum, Johnsonella ignava and Streptococcus parasanguinis I; and lower abundance of Granulicatella adiacens.
      Hooper et al, 2007
      • Hooper S.J.
      • Crean S.-J.
      • Fardy M.J.
      • Lewis M.A.O.
      • Spratt D.A.
      • Wade W.G.
      • et al.
      A molecular analysis of the bacteria present within oral squamous cell carcinoma.
      n = 10 casesOral SCC and matched samples of oral tissueCohortTumour specimensFISH (one patient only) and 16S rRNA gene-based community profilingNo statistically significant differences between tumours and normal tissueMajority of species observed within tumours were saccharolytic and aciduric.
      Sasaki et al, 2005
      • Sasaki M.
      • Yamaura C.
      • Ohara-Nemoto Y.
      • Tajika S.
      • Kodama Y.
      • Ohya T.
      • et al.
      Streptococcus anginosus infection in oral cancer and its infection route.
      n = 49 casesOral SCC (n = 42: 21 gingiva, 15 tongue, 2 floor of mouth, 4 buccal mucosa), oral lymphoma (n = 2), rhabdomyosarcoma (n = 2), leukoplakia (n = 3)CohortTumour specimens, dental plaque and mixed saliva samplesPolymerase chain reaction (PCR) assayStreptococcus anginosus found in 19 (45.2 %) of oral SCC but not in other groups.This study was conducted specifically to assess the frequency of S. anginosus prevalence in OSCC.

      S. anginosus not detected in salivary samples, only in tumour and dental plaque.
      Mager et al, 2005
      • Mager D.L.
      • et al.
      The salivary microbiota as a diagnostic indicator of oral cancer: A descriptive, non-randomized study of cancer-free and oral squamous cell carcinoma subjects.
      n = 45 cases and n = 229 controlsOral cavityCase-controlSalivaCheckerboard DNA-DNA hybridization and whole genomic probe detectionHN-SCC associated with higher abundance of Capnocytophaga gingivalis, Prevotella melaninogenica and Streptococcus mitis
      Nagy et al, 1998
      • Nagy K.N.
      • Sonkodi I.
      • Szöke I.
      • Nagy E.
      • Newman H.N.
      The microflora associated with human oral carcinomas.
      n = 21 casesOral SCCCohortBiofilm samples (swabs) from central surface of tumoursCultureHN-SCC associated with higher number of colony forming units of Veillonella, Fusobacterium, Prevotella, Porphyromonas, Actinomyces, Clostridium, Haemophilus, Enterobacteriaceae, Streptococcus, Candida albicansComparison between tumour and normal tissue in cases only. Candida albicans was found at eight of the 21 tumour sites, but never at control sites.
      Mechanistic studies have provided interesting perspectives about upper aerodigestive tract physiology, tumour initiation and progression which are of interest to the radiation oncology community. In health, the oral ecosystem remains in balance through xenobiotic clearance, immune regulation and homeostasis. Inter-bacterial communication regulates population density sensing and growth rate. A healthy microbiota also limits the growth of potentially harmful micro-organisms residing in the pharyngo-laryngeal mucosa such as Streptococcus pyogenes and Haemophilus influenza [
      • Dewhirst F.E.
      • Chen T.
      • Izard J.
      • Paster B.J.
      • Tanner A.C.R.
      • Yu W.-H.
      • et al.
      The human oral microbiome.
      ]. In addition, some microorganisms modulate the effect of potential carcinogens. Ubiquitous oral species such as Candida spp. (a fungal genus) and Neisseria possess the alcohol dehydrogenase enzyme, which converts dietary ethanol into the carcinogenic acetaldehyde. Individuals with poor oral hygiene have higher acetaldehyde concentrations than healthy subjects [
      • Meurman J.H.
      • Uittamo J.
      Oral micro-organisms in the etiology of cancer.
      ]. Also, germ-free animal models have lower oral acetaldehyde concentrations [
      • Takahashi N.
      Oral Microbiome Metabolism.
      ]. Acetaldehyde induces oxidative stress, a common hallmark of cancer, an important aspect when employing oxidative cytotoxic therapies such as radiotherapy [
      • Roman J.
      • et al.
      Differential role of ethanol and acetaldehyde in the induction of oxidative stress in HEP G2 cells: effect on transcription factors AP-1 and NF-kappaB.
      ]. Acetaldehyde in turn can be oxidated by aldehyde dehydrogenase 1 (ALDH1), a component of alcohol metabolism, with ALDH1 + HN-SCC displaying resistance to radiotherapy [
      • Chen Y.-C.
      • Chen Y.-W.
      • Hsu H.-S.
      • Tseng L.-M.
      • Huang P.-I.
      • Lu K.-H.
      • et al.
      Aldehyde dehydrogenase 1 is a putative marker for cancer stem cells in head and neck squamous cancer.
      ]. It should be noted however that acetaldehyde produced by bacteria can be several orders of magnitude smaller than dietary acetaldehyde [
      • Miyake T.
      • Shibamoto T.
      Quantitative analysis of acetaldehyde in foods and beverages.
      ]. Smoking also significantly affects the oral microbiome. In a study involving 1204 individuals, depletion of bacterial genera related to carbohydrate, energy and xenobiotic metabolism was detected in current smokers, although the literature is highly contradictory and effects are not fully established [
      • Wu J.
      • Peters B.A.
      • Dominianni C.
      • Zhang Y.
      • Pei Z.
      • Yang L.
      • et al.
      Cigarette smoking and the oral microbiome in a large study of American adults.
      ].
      Bacteria can promote cancer proliferation, invasion, metastasis, angiogenesis, inhibit apoptosis and anti-tumour immunity. All of these are mechanisms of resistance to radiotherapy, chemo- and immunotherapy [
      • Longo D.L.
      • Citrin D.E.
      Recent Developments in Radiotherapy.
      ]. For example, Fusobacterium nucleatum, an anaerobic oral commensal, promotes cancer progression by inhibiting Natural Killer (NK) and T cell-mediated tumour killing through interactions between the F. nucleatum Fap2 outer membrane autotransporter and TIGIT, an inhibitory receptor present in T and NK cells. In addition, an unknown fusobacterial ligand interacts with CEACAM1, a member of the carcinoembryonic antigen-related cell adhesion molecules activating inhibitory or exhaustion pathways leading to T cell dysfunction [
      • Gur C.
      • Ibrahim Y.
      • Isaacson B.
      • Yamin R.
      • Abed J.
      • Gamliel M.
      • et al.
      Binding of the Fap2 protein of fusobacterium nucleatum to human inhibitory receptor TIGIT protects tumors from immune cell attack.
      ,
      • Gur C.
      • Maalouf N.
      • Shhadeh A.
      • Berhani O.
      • Singer B.B.
      • Bachrach G.
      • et al.
      Fusobacterium nucleatum supresses anti-tumor immunity by activating CEACAM1.
      ]. Evidence that F. nucleatum holds a role specifically in HN-SCC initiation and progression is accruing, although is not as yet conclusive. F. nucleatum promotes cancer aggressiveness and invasiveness in oral SCC (OSCC) cell lines, an effect which is not entirely cell contact-dependent, i.e., it is in part mediated by signalling molecules, particularly the LPS/TLR4 pathway [
      • Harrandah A.M.
      • Chukkapalli S.S.
      • Bhattacharyya I.
      • Progulske-Fox A.
      • Chan E.K.L.
      Fusobacteria modulate oral carcinogenesis and promote cancer progression.
      ]. Porphyromonas gingivalis is another oral anaerobe known to promote epithelial to mesenchymal transition in oral cancer (thus supporting cancer migration and invasion) and also immunosuppression through increased myeloid-derived suppressor cell activity in mouse models [
      • Wen L.
      • Mu W.
      • Lu H.
      • Wang X.
      • Fang J.
      • Jia Y.
      • et al.
      Porphyromonas gingivalis Promotes Oral Squamous Cell Carcinoma Progression in an Immune Microenvironment.
      ]. Combined, P. gingivalis and F. nucleatum promote tumour progression in mice [
      • Harrandah A.M.
      • Chukkapalli S.S.
      • Bhattacharyya I.
      • Progulske-Fox A.
      • Chan E.K.L.
      Fusobacteria modulate oral carcinogenesis and promote cancer progression.
      ]. It should however be noted that mice may not provide a good model for bacteria-mediated tumour initiation, illustrated by difficulties in achieving stable colonisation of mouse gastrointestinal tract by fusobacteria harvested from human colon cancers, with low in-vivo inflammation compared to higher cytokine production in human colon cancer cell lines [
      • Queen J.
      • Domingue J.C.
      • White J.R.
      • Stevens C.
      • Udayasuryan B.
      • Nguyen T.T.D.
      • et al.
      Comparative Analysis of Colon Cancer-Derived Fusobacterium nucleatum Subspecies: Inflammation and Colon Tumorigenesis in Murine Models.
      ]. In addition, F. nucleatum is now recognized to be comprised of four distinct subspecies and to be highly genetically variable. How this complexity bears on oral tumorigenesis remains to be investigated.
      Overall, careful interpretation of these data is necessary. We all harbour most of the bacteria discussed above in our mouths and most of us will not go on to develop oral cancer. The possibility that bacteria remain mostly confined to the role of a passenger rather than a driver in cancer initiation remains plausible, but the alternative where they play a pathological role is increasingly supported by an accruing body of evidence.

      Past, present and future of the microbiota in radiotherapy for HN-SCC

      Research into the influence of the commensal microbiota on cancer management is still in its infancy. Furthermore, the microbiota of the upper aerodigestive tract is difficult to research and more attention has been devoted to gut microbial communities. Nevertheless, many findings are of interest to head and neck oncologists and it is important to be aware of how recent discoveries may contribute to paradigm changes in the medium to long term.
      The oncology community became widely aware of the importance of the microbiota after several studies showed that specific gut bacterial community profiles are associated with response to systemic therapies. Animal models treated with antibiotics to eliminate gut bacteria showed reduced production of anti-tumoural oxygen species and hampered T helper responses when exposed to chemotherapy, with consequential sub-optimal treatment response [
      • Viaud S.
      • et al.
      The Intestinal Microbiota Modulates the Anticancer Immune Effects of Cyclophosphamide.
      ,
      • Iida N.
      • Dzutsev A.
      • Stewart C.A.
      • Smith L.
      • Bouladoux N.
      • Weingarten R.A.
      • et al.
      Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment.
      ]. However, the real explosion of interest came from studies showing convincing evidence that the composition of the gut microbiome affects the outcomes of ICI, which are drugs inhibiting proteins preventing anti-tumoural immune responses [

      Gopalakrishnan V, Helmink BA, Spencer CN, Reuben A. & Wargo JA. The Influence of the Gut Microbiome on Cancer, Immunity, and Cancer Immunotherapy. Cancer Cell Preprint at https://doi.org/10.1016/j.ccell.2018.03.015 (2018).

      ]. Nivolumab and pembrolizumab, which are two ICI, are successful in treating HN-SCC in palliative contexts and are now standard of care in the UK after positive results of the Checkmate-141 (nivolumab), Keynote-040 and Keynote-048 (pembrolizumab) trials, where they were compared to systemic cytotoxic chemotherapy regimens [
      • Burtness B.
      • Harrington K.J.
      • Greil R.
      • Soulières D.
      • Tahara M.
      • de Castro G.
      • et al.
      Pembrolizumab alone or with chemotherapy versus cetuximab with chemotherapy for recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-048): a randomised, open-label, phase 3 study.
      ,
      • Cohen E.E.W.
      • Soulières D.
      • Le Tourneau C.
      • Dinis J.
      • Licitra L.
      • Ahn M.-J.
      • et al.
      Pembrolizumab versus methotrexate, docetaxel, or cetuximab for recurrent or metastatic head-and-neck squamous cell carcinoma (KEYNOTE-040): a randomised, open-label, phase 3 study.
      ,
      • Ferris R.L.
      • Blumenschein G.
      • Fayette J.
      • Guigay J.
      • Colevas A.D.
      • Licitra L.
      • et al.
      Nivolumab for Recurrent Squamous-Cell Carcinoma of the Head and Neck.
      ]. It is now clear that microbiota-dependent immune regulation is very important for both innate and adaptive immune tumour surveillance, as well as for the success of treatment with these agents, clinically reflected by the observation that antibiotic treatment negatively affects their efficacy [
      • Zhou C.-B.
      • Zhou Y.-L.
      • Fang J.-Y.
      Gut Microbiota in Cancer Immune Response and Immunotherapy.
      ,
      • Pinato D.J.
      • Howlett S.
      • Ottaviani D.
      • Urus H.
      • Patel A.
      • Mineo T.
      • et al.
      Association of Prior Antibiotic Treatment With Survival and Response to Immune Checkpoint Inhibitor Therapy in Patients With Cancer.
      ]. Overall, microbial regulation of systemic therapies relies on five fundamental mechanisms, summarised by the acronym TIMER: (1) Translocation (bacteria crossing intestinal/mucosal barriers to enter secondary lymphoid organs), (2) Immunomodulation, (3) Metabolism (interference of bacterial products in drug pathways), (4) Enzymatic degradation (where bacterial enzymes degrade or metabolise chemotherapeutics) and (5) Reduced diversity (where therapy-induced changes in bacterial communities lead to toxicity) [
      • Alexander J.L.
      • Wilson I.D.
      • Teare J.
      • Marchesi J.R.
      • Nicholson J.K.
      • Kinross J.M.
      Gut microbiota modulation of chemotherapy efficacy and toxicity. Nature Reviews Gastroenterology &Amp.
      ].
      The field of microbiota research in radiation oncology is by comparison under-researched. However, radiation oncologists should be aware that microbes may be key players in radiotherapy responses both in tumours and normal tissues through direct and non-direct (e.g., immune-mediated) mechanisms. Again, the gut microbiota has been prioritised in research efforts for the reasons explained above. There is an evident cross-talk between the microbiota and the host in the setting of the changing environment caused by radiotherapy on either healthy tissues or tumours. For example, associations between gut microbiota, their products and radiation enteropathy have been researched and accruing evidence points towards a significant contribution of bacteria producing short chain fatty acids (SCFA) in protection against radiation-induced gastrointestinal toxicity, with potential mechanisms such as direct effects on enterocytes (which fuel on bacterially-produced SCFA) or modulation of mucosal Treg responses [

      Reis Ferreira M. et al. Microbiota and radiotherapy-induced gastrointestinal side-effects (MARS) study: a large pilot study of the microbiome in acute and late radiation enteropathy. Clinical Cancer Research clincanres.0960.2019 (2019) doi:10.1158/1078-0432.CCR-19-0960.

      ,
      • Ferreira M.R.
      • Muls A.
      • Dearnaley D.P.
      • Andreyev H.J.N.
      Microbiota and radiation-induced bowel toxicity: lessons from inflammatory bowel disease for the radiation oncologist.
      ,
      • Guo H.
      • Chou W.-C.
      • Lai Y.
      • Liang K.
      • Tam J.W.
      • Brickey W.J.
      • et al.
      Multi-omics analyses of radiation survivors identify radioprotective microbes and metabolites.
      ]. Bacterial products can also mediate responses to radiation, as illustrated by interactions between muramyl dipeptide, a peptidoglycan motif common to all bacteria which interacts with the NOD2 receptor in host enterocytes, leading to mitophagy and a decrease in intra-cellular reactive oxygen species in response to radiation, i.e., counteracting the latter’s effect [
      • Levy A.
      • Stedman A.
      • Deutsch E.
      • Donnadieu F.
      • Virgin H.W.
      • Sansonetti P.J.
      • et al.
      Innate immune receptor NOD2 mediates LGR5(+) intestinal stem cell protection against ROS cytotoxicity via mitophagy stimulation.
      ]. However, some conflicting evidence exists. For example, colon tumour response to radiotherapy is enhanced by vancomycin in mice, which in turn decreases levels of butyrate in tumours. In mouse models, administration of Kineothrix alysoides, a bacterium of the Lachnospiraceae family, abrogates this enhanced response through secretion of butyrate (a SCFA), which inhibits STING-activated type I IFN expression in dendritic cells through blockade of TBK1 and IRF3 phosphorylation, with a consequent decrease in cytotoxic T cell-mediated antitumour immunity [
      • Yang K.
      • Hou Y.
      • Zhang Y.
      • Liang H.
      • Sharma A.
      • Zheng W.
      • et al.
      Suppression of local type I interferon by gut microbiota–derived butyrate impairs antitumor effects of ionizing radiation.
      ]. These observations would suggest that butyrate promotes radioresistance. On the other hand, butyrate suppresses the proliferation of colon cancer organoids, and enhances tumour response to radiation in glucose-poor but not in glucose-rich media (i.e., through the Warburg effect); this effect is not observed in normal intestinal tissue organoids, suggesting that butyrate is a radiosensitizer [
      • Park M.
      • Kwon J.
      • Shin H.
      • Moon S.
      • Kim S.
      • Shin U.i.
      • et al.
      Butyrate enhances the efficacy of radiotherapy via FOXO3A in colorectal cancer patient–derived organoids.
      ]. Although this contradictory evidence is difficult to interpret at first glance, the fact remains that most studies indicate a radioprotective role for SCFA. Interestingly, F. nucleatum is a SCFA producer, which may have a role in radiation responses in HN-SCC [
      • Dahlstrand Rudin A.
      • et al.
      Short chain fatty acids released by Fusobacterium nucleatum are neutrophil chemoattractants acting via free fatty acid receptor 2 (FFAR2).
      ]. Overall, these contradictions stress the importance of waiting for accumulation of evidence, particularly from longitudinal human studies, otherwise there is a high risk of data overinterpretation.

      Impact of radiotherapy on the microbiota

      Radiation and other cytostatic or cytotoxic therapies have an impact on rapidly dividing cells such as bacteria, as do changes in the mucosal microenvironment. As such, radiotherapy may affect the microbiota.
      Two nearly identical studies by the same group with 8 and 3 patients claim that the microbiome of subgingival plaque (curetted from bucco-gingival surfaces of the maxillary first molar and analysed with sequencing of the 16S rRNA gene (16Sseq)) becomes less rich and diverse during radiotherapy, with reduced counts of Streptococcus, while Actinomyces increases [
      • Gao L.i.
      • Hu Y.
      • Wang Y.
      • Jiang W.
      • He Z.
      • Zhu C.
      • et al.
      Exploring the variation of oral microbiota in supragingival plaque during and after head-and-neck radiotherapy using pyrosequencing.
      ,
      • Hu Y.-J.
      • Shao Z.-Y.
      • Wang Q.
      • Jiang Y.-T.
      • Ma R.
      • Tang Z.-S.
      • et al.
      Exploring the dynamic core microbiome of plaque microbiota during head-and-neck radiotherapy using pyrosequencing.
      ]. Leung and colleagues used direct microscopy and culture to evaluate the subgingival plaque of 18 patients previously irradiated for nasopharyngeal carcinoma and detected a microbiota similar to the one observed in gingivitis [
      • Leung W.K.
      • Jin L.J.
      • Samaranayake L.P.
      • Chlu G.K.C.
      Subgingival microbiota of shallow periodontal pockets in individuals after head and neck irradiation.
      ]. Another study enrolled 31 OSCC patients and 11 controls; salivary samples were taken before and, in 11 patients, also after radiotherapy. Haemophilus, Veillonella, and Granulicatella increased post-treatment, whereas Lactobacillus, Scardovia, Acinetobacter, and Enterococcus decreased.[
      • Kumpitsch C.
      • Moissl-Eichinger C.
      • Pock J.
      • Thurnher D.
      • Wolf A.
      Preliminary insights into the impact of primary radiochemotherapy on the salivary microbiome in head and neck squamous cell carcinoma.
      ] Interestingly, this study provides a very different microbiological picture compared to its pilot predecessor study in humans from the same authors in associations between HN-SCC and the microbiome (Table 2, asterisk footnote).[
      • Wolf A.
      • Moissl-Eichinger C.
      • Perras A.
      • Koskinen K.
      • Tomazic P.V.
      • Thurnher D.
      The salivary microbiome as an indicator of carcinogenesis in patients with oropharyngeal squamous cell carcinoma: A pilot study.
      ] Also, Enterococcus and Acinetobacter are not members of the oral microbiota and could be a result of contamination or opportunistic colonisation as a result of impaired local immunity. A study involving 19 patients with disparate HN-SCC diagnoses (including oropharynx, pinna, parotid, etc) showed that salivary bacterial communities, analysed with 16Sseq, were dominated by Streptococcus, Prevotella, Fusobacterium and Granulicatella, and remained stable during radiotherapy [
      • Vesty A.
      • et al.
      Oral microbial influences on oral mucositis during radiotherapy treatment of head and neck cancer.
      ]. Oliva and colleagues conducted a study examining the impact of radiochemotherapy in 22 patients with HPV-related oropharyngeal cancer and observed a shift in microbial communities when comparing pre to post-radiotherapy oropharyngeal swab samples, but no differences in stool communities [
      • Oliva M.
      • Schneeberger P.H.H.
      • Rey V.
      • Cho M.
      • Taylor R.
      • Hansen A.R.
      • et al.
      Transitions in oral and gut microbiome of HPV+ oropharyngeal squamous cell carcinoma following definitive chemoradiotherapy (ROMA LA-OPSCC study).
      ]. Importantly, shotgun metagenomics were used in this study, which should be highlighted because the relatively low quantities of bacterial DNA present in swabs make this technique challenging. No comparison between shotgun metagenomics and 16Sseq was carried out for swab samples, which would have been informative. Schuurhuis et al followed a cohort of 26 and 27 oral or oropharyngeal cancer patients treated with radiotherapy and radiochemotherapy respectively and found that, when compared to patients treated with surgery alone, patients treated with radiotherapy had increased abundance of enteric rods, staphylococci and Candida one year after treatment [
      • Schuurhuis J.M.
      • Stokman M.A.
      • Witjes M.J.H.
      • Langendijk J.A.
      • van Winkelhoff A.J.
      • Vissink A.
      • et al.
      Head and neck intensity modulated radiation therapy leads to an increase of opportunistic oral pathogens.
      ]. Finally, another small study reported a shift in the composition of the bacterial community, with Actinomyces and Leptotrichia declining over the course of radiation, while Veillonella and Atopobium increased [
      • Bahig H.
      • Fuller C.D.
      • Mitra A.
      • Yoshida-Court K.
      • Solley T.
      • Ping Ng S.
      • et al.
      Longitudinal characterization of the tumoral microbiome during radiotherapy in HPV-associated oropharynx cancer.
      ].
      Overall, these studies provide inconsistent evidence of some impact of radiotherapy on the microbiota of the upper aerodigestive tract, but they are limited by small numbers of patients and the varying experimental design, leading to interpretational difficulties and questionable clinical significance. Furthermore, functional and/or mechanistic studies are lacking. However, there appears to be a shift by the bacterial community to a pro-cariogenic/pro-inflammatory-one after radiotherapy in all studies, although whether this is clinically relevant is unclear.

      Toxicity studies

      Radiotherapy causes acute and late reactions (Fig. 1) which have been reviewed elsewhere [
      • Alterio D.
      • Marvaso G.
      • Ferrari A.
      • Volpe S.
      • Orecchia R.
      • Jereczek-Fossa B.A.
      Modern radiotherapy for head and neck cancer.
      ]. Pre-radiotherapy dysbiosis of the oral microbiota and/or overgrowth of pathogens associating with subclinical inflammatory processes may facilitate radiation toxicity. It is therefore unsurprising that the role of the microbiota as contributor or biomarker for radiation-induced toxicity has been explored in HN-SCC.
      Figure thumbnail gr1
      Fig. 1Summary of mechanisms of radiation-induced acute oral mucositis by stage. Green boxes indicate processes where the oral microbiota has been causally implicated in non-radiotherapy-related research.
      Vesty and colleagues reported associations between acute oral mucositis and the microbiome in 19 patients treated with HN-SCC radiotherapy and found that Capnocytophaga leadbetteri, Neisseria mucosa, Olsenella uli, Parvimonas micra and Tannerella forsythia were more abundant in the saliva of patients with oral mucositis grade 2 +. Moreover, a comparison of buccal mucosa swabs at sites of grade 2 + oral mucositis showed increased abundance of genera Fusobacterium and Sneathia compared to sites where mucositis was graded ≤ 1. In addition, pre-radiotherapy low abundance of Streptococcus, Staphylococcus and Lactobacillus, and high abundance of Fusobacterium, Haemophilus, Tannerella, Porphyromonas and Eikenella were predictive of grade 2 + mucositis [
      • Vesty A.
      • et al.
      Oral microbial influences on oral mucositis during radiotherapy treatment of head and neck cancer.
      ]. Many of these microbes are members of the normal microbiota or health-associated, so these findings could be due to chance in a context of multiple comparisons. Another study investigated bacterial populations of buccal mucosal swabs of 66 patients with oral cancer treated with radiotherapy, chemotherapy, or radiochemotherapy, and reported positive associations between shorter times to onset of oral mucositis and (i) Cardiobacterium and Granulicatella at baseline, (ii) Prevotella, Fusobacterium, and Streptococcus immediately before the onset of mucositis, and (iii) Megasphaera and Cardiobacterium before the onset of grade 3 + mucositis [
      • Reyes‐Gibby C.C.
      • Wang J.
      • Zhang L.
      • Peterson C.B.
      • Do K.-A.
      • Jenq R.R.
      • et al.
      Oral microbiome and onset of oral mucositis in patients with squamous cell carcinoma of the head and neck.
      ]. In two distinct but very similar studies from the same group, 41 and 19 patients with nasopharyngeal cancer receiving definitive radiotherapy were sampled with oropharyngeal swabs at 8 timepoints from baseline to the end of radiotherapy. In one study, Phenylobacterium, Acinetobacter, Burkholderia, Sphingomonas, Azospirillum, Rhizobium, Hydrogenophaga, Paracoccus and Nocardioides (all of which are potential contaminants) positively, and Leptotrichia and Peptostreptococcus negatively associated with mucositis [
      • Zhu X.-X.
      • Yang X.-J.
      • Chao Y.-L.
      • Zheng H.-M.
      • Sheng H.-F.
      • Liu H.-Y.
      • et al.
      The Potential Effect of Oral Microbiota in the Prediction of Mucositis During Radiotherapy for Nasopharyngeal Carcinoma.
      ]. In the other, abundance of Fusobacterium, Treponema, Porphyromonas, and Prevotella peaked at the same time as mucositis, although statistical associations between microbes and mucositis are not reported [
      • Hou J.
      • Zheng HuiMin
      • Li P.
      • Liu HaiYue
      • Zhou HongWei
      • Yang XiaoJun
      Distinct shifts in the oral microbiota are associated with the progression and aggravation of mucositis during radiotherapy.
      ]. Vidal-Casariego and colleagues found that bacterial (but not yeast) colonisation pre-radiotherapy, evaluated by culturing buccal smears, was associated with severe mucositis [
      • Vidal-Casariego A.
      • et al.
      Nutritional, microbiological, and therapeutic factors related to mucositis in head and neck cancer patients: A cohort study.
      ]. A translational study reported the impact of HN-SCC radiotherapy in terms of diversity and structure (elevation of Streptococcaceae and Lactobacillaceae) in 44 patients, but did not identify relationships with mucositis. However, oral microbiota transplantation (OMT) from healthy mouse donors improved oral mucositis after irradiation. OMT led to a restructured oral microbiota, higher proliferation of the oral epithelium, and suppression of IL6, TNFα and TGFβ expression [
      • Xiao H.
      • Fan Y.
      • Li Y.
      • Dong J.
      • Zhang S.
      • Wang B.
      • et al.
      Oral microbiota transplantation fights against head and neck radiotherapy-induced oral mucositis in mice.
      ].
      Oral candidiasis is a common complication of oral mucositis. A study of 60 patients with suspected candidiasis reported a 77 % incidence of detection of yeasts, with the most commonly detected species including Candida albicans (48 %), Candida tropicalis (28 %), and Candida parapsilosis (13 %) [
      • Bulacio L.
      • Paz M.
      • Ramadán S.
      • Ramos L.
      • Pairoba C.
      • Sortino M.
      • et al.
      Oral infections caused by yeasts in patients with head and neck cancer undergoing radiotherapy. Identification of the yeasts and evaluation of their antifungal susceptibility.
      ]. Using culture methods, a recent study in 86 patients with HN-SCC undergoing definitive or postoperative radiotherapy where the field included the oral cavity and/or oropharynx also detected a 54 % incidence of oral candidiasis, with the accuracy of clinical diagnosis unaided by microbiological sampling ranging 52–60 % [
      • Chitapanarux I.
      • Wongsrita S.
      • Sripan P.
      • Kongsupapsiri P.
      • Phakoetsuk P.
      • Chachvarat S.
      • et al.
      An underestimated pitfall of oral candidiasis in head and neck cancer patients undergoing radiotherapy: an observation study.
      ]. Although it is tempting to assume that candidiasis is underdiagnosed by about 40 %, it is important to note that Candida is a common commensal of the oral cavity, so detection does not necessarily represent disease. Also, symptoms of candidiasis and oral mucositis are nearly indistinguishable. Candidiasis is not an exclusive problem of the acute setting. Radiotherapy for HN-SCC very often leads to late xerostomia and two separate cohort studies in patients with unspecified HN-SCC examined the impact on oral microbiota with culture of saliva (n = 18 patients) or sub/supragingival biofilm (n = 28 patients and matched controls without HN-SCC or previous irradiation). In both cases associations between xerostomia and increased counts of C. albicans were detected [
      • Arrifin A.
      • Heidari E.
      • Burke M.
      • Fenlon M.R.
      • Banerjee A.
      The Effect of Radiotherapy for Treatment of Head and Neck Cancer on Oral Flora and Saliva.
      ,
      • Gaetti-Jardim E.
      • Jardim E.C.G.
      • Schweitzer C.M.
      • da Silva J.C.L.
      • Oliveira M.M.
      • Masocatto D.C.
      • et al.
      Supragingival and subgingival microbiota from patients with poor oral hygiene submitted to radiotherapy for head and neck cancer treatment.
      ]. Another study cultured the microbiota of tongue, buccal mucosa, vestibulum, supragingival plaque and subgingival region in 13 patients with HN-SCC with xerostomia post-radiotherapy and compared them to matched controls, reporting an increase in positive cultures of C. albicans (54 vs 15 %), Enterococcus (38 % vs 0 %), and lactobacilli (92 % vs 70 %) [
      • Almståhl A.
      • Wikström M.
      • Fagerberg-Mohlin B.
      Microflora in oral ecosystems in subjects with radiation-induced hyposalivation.
      ]. These results stress the importance of oral hygiene counselling for HN-SCC patients previously treated with radiotherapy [
      • Vila T.
      • Rizk A.M.
      • Sultan A.S.
      • Jabra-Rizk M.A.
      • Hogan D.A.
      The power of saliva: Antimicrobial and beyond.
      ]. Importantly, Candida may contribute to malignant transformation in the upper aerodigestive tract, so infection may facilitate recurrence [
      • Ramirez-Garcia A.
      • et al.
      Candida albicans and cancer: Can this yeast induce cancer development or progression?.
      ].
      Bacteriotherapy to prevent mucositis has been attempted. Two randomized controlled trials (RCT) assessed lozenges of Lactobacillus brevis, which produces arginine deaminase and competes for arginine with eukaryotic nitric oxide synthase, a pro-inflammatory enzyme, as a preventative measure for oral mucositis. Although an initial double-blind RCT showed promising results with reduction of grade 3 + oral mucositis rates, the second trial (closed early at 75/106 planned patients due to drug supply issues) did not show any benefits [
      • De sanctis Vitaliana
      • Belgioia Liliana
      • Cante Domenico
      • La porta M.R.
      • Caspiani Orietta
      • Guarnaccia Roberta
      • et al.
      Lactobacillus brevis CD2 for Prevention of Oral Mucositis in Patients With Head and Neck Tumors: A Multicentric Randomized Study.
      ,
      • Sharma A.
      • Rath G.K.
      • Chaudhary S.P.
      • Thakar A.
      • Mohanti B.K.
      • Bahadur S.
      Lactobacillus brevis CD2 lozenges reduce radiation- and chemotherapy-induced mucositis in patients with head and neck cancer: A randomized double-blind placebo-controlled study.
      ]. Possible reasons for these incongruent results include better radiotherapy techniques (IMRT vs 2DCRT) in the second trial, and also because in the latter the drug was compared to standard-of-care rather than placebo/no treatment.
      Studies into other late side-effects of radiotherapy are scarce. A study of the bacteriology of the sinonasal compartment in patients with chronic rhinosinusitis after radiotherapy completion included 22 patients treated for sinonasal cancers of diverse histologies and used culture methods, with 16Sseq used in 54 % of patients. The most commonly identified bacteria were Staphylococcus aureus and Pseudomonas aeruginosa, closely resembling non-radiation-induced chronic sinusitis [
      • Stoddard T.J.
      • Varadarajan V.V.
      • Dziegielewski P.T.
      • Boyce B.J.
      • Justice J.M.
      Detection of Microbiota in Post Radiation Sinusitis.
      ]. Another study reported a higher prevalence of cultured gram-positive cocci and lower incidence of gram-negative bacilli in 30 patients treated for nasopharyngeal carcinoma with post-radiotherapy sinusitis lasting ≥ 6 months compared with 30 patients with non-radiation induced sinusitis [
      • Deng Z.-Y.
      • Tang A.-Z.
      Bacteriology of postradiotherapy chronic rhinosinusitis in nasopharyngeal carcinoma patients and chronic rhinosinusitis.
      ]. Only few historic studies have examined the microbiota in osteoradionecrosis (ORN). A study in 31 patients with ORN identified Actinomyces in 64.5 % [
      • Hansen T.
      • Kunkel M.
      • Kirkpatrick C.J.
      • Weber A.
      Actinomyces in infected osteoradionecrosis—underestimated?.
      ]. Prior to this, a study in 50 patients with ORN showed an Actinomyces isolation rate of 12 % and, where detected, ORN took longer to resolve (29.7 vs 13.4 months) [
      • Curi M.M.
      • Dib L.L.
      • Kowalski L.P.
      • Landman G.
      • Mangini C.
      Opportunistic actinomycosis in osteoradionecrosis of the jaws in patients affected by head and neck cancer: incidence and clinical significance.
      ]. A study evaluated 14 patients with mandibular ORN with exposed bone using electron microscopy and identified mostly rods, but also spirochetes and cocci in 64 % patients [
      • Støre G.
      • Olsen I.
      Scanning and transmission electron microscopy demonstrates bacteria in osteoradionecrosis.
      ]. A small study in 6 patients with mandibular ORN detected mostly bacterial commensals [
      • Aas J.A.
      • Reime L.
      • Pedersen K.
      • Eribe E.R.K.
      • Abesha-Belay E.
      • Støre G.
      • et al.
      Osteoradionecrosis contains a wide variety of cultivable and non-cultivable bacteria.
      ].
      Overall, available data remain clinically questionable, especially because reproducible evidence needs to be accrued and mechanisms elucidated. Nevertheless, some conclusions can be drawn: oral hygiene is crucial in decreasing side-effects, oral fungal disease needs to be addressed promptly, and superimposed infection on a background of ORN should always be suspected and treated.

      Efficacy studies

      Studies on the impact of microbiota in radiotherapy efficacy are rare, particularly in HN-SCC [
      • Liu J.
      • Liu C.
      • Yue J.
      Radiotherapy and the gut microbiome: facts and fiction.
      ]. This is intriguing, especially because a relevant biomarker of good prognosis in HN-SCC, HPV, is a microbe, albeit non-bacterial. However, it has, to date, not shown to be reliable as a predictive biomarker for treatment personalisation, although ongoing trials such as PATHOS (NCT02215265) may change this picture.
      The role of the bacterial microbiota in treatment efficacy is a hot topic in systemic therapies, which identically to radiotherapy are dependent on immunity for tumour response [
      • Longo D.L.
      • Citrin D.E.
      Recent Developments in Radiotherapy.
      ]. An interesting example was reported by Rui and colleagues, who collected oral rinse samples from 44 patients with OSCC prior to induction chemotherapy (TPF regimen prior to surgery and adjuvant radiotherapy) and found higher abundances of F. nucleatum and Mycoplasma in patients who did not respond. Microbiome pathways related to platinum resistance and chemical carcinogenesis were also more abundant in the non-responder group [
      • Rui M.
      • Zhang X.
      • Huang J.
      • Wei D.
      • Li Z.
      • Shao Z.
      • et al.
      The baseline oral microbiota predicts the response of locally advanced oral squamous cell carcinoma patients to induction chemotherapy: A prospective longitudinal study.
      ]. No such studies exist in radiotherapy for HN-SCC, but Nenclares and colleagues retrospectively assessed whether treatment with antibiotics during radiotherapy for locally-advanced HN-SCC affected survival outcomes in 272 patients. Most patients had stage IVA (69 %) and received induction chemotherapy followed by radiochemotherapy (85 %). No inter-group differences were found in performance status. Antibiotics prescribed were mostly penicillin and derivatives (68.6 %). At a median follow-up of 54 months, patients in the antibiotic group had lower overall, progression-free and disease-specific survival, which worsened with higher number of courses of antibiotics prescribed. The impact on survival was independent of type and timing of antibiotic prescription. Interestingly, antibiotic use had an impact on locoregional rather than distant metastatic recurrence, suggesting a preponderant local effect [
      • Nenclares P.
      • Bhide S.A.
      • Sandoval-Insausti H.
      • Pialat P.
      • Gunn L.
      • Melcher A.
      • et al.
      Impact of antibiotic use during curative treatment of locally advanced head and neck cancers with chemotherapy and radiotherapy.
      ]. Overall, this interesting study suggests that gross changes in the microbiota impact on curative HN-SCC radiotherapy efficacy and mirrors other studies showing an impact of antibiotics on systemic cancer therapies [
      • Pinato D.J.
      • Howlett S.
      • Ottaviani D.
      • Urus H.
      • Patel A.
      • Mineo T.
      • et al.
      Association of Prior Antibiotic Treatment With Survival and Response to Immune Checkpoint Inhibitor Therapy in Patients With Cancer.
      ,
      • Shaikh F.Y.
      • Gills J.J.
      • Sears C.L.
      Impact of the microbiome on checkpoint inhibitor treatment in patients with non-small cell lung cancer and melanoma.
      ]. However, one should interpret this data with caution. Antibiotic use may reflect an overall compromised immunity, including against cancer. Also, antibiotics have an impact beyond bacterial killing, particularly through microbiome-independent effects such as direct inhibition of cellular respiratory activity (including immune cells), as eukaryotic mitochondrial functionality is affected, thereby potentially selecting cancer cells, which have a competitive advantage caused via the Warburg effect [
      • Singh R.
      • Sripada L.
      • Singh R.
      Side effects of antibiotics during bacterial infection: Mitochondria, the main target in host cell.
      ,
      • Yang J.H.
      • et al.
      Antibiotic-Induced Changes to the Host Metabolic Environment Inhibit Drug Efficacy and Alter Immune Function.
      ]. Finally, it is difficult to dissociate whether the impact was on radiotherapy, chemotherapy, or both, particularly since most patients received significant courses of systemic treatment.
      As expected, mechanistic studies specific to how the microbiota affects radiotherapy efficacy in HN-SCC are still unavailable. However, a study in mice models demonstrating the importance of the buccal/oral microbiome in radioresistance is of interest. Dong and colleagues showed that F. nucleatum migrates from the oral cavity to the intestine, where it harbours within colorectal cancers and promotes radioresistance. Given the accumulated evidence pointing towards a role of F. nucleatum in modulating inflammation and driving HN-SCC, these results may offer new avenues of research [
      • Dong J.
      • et al.
      Oral microbiota affects the efficacy and prognosis of radiotherapy for colorectal cancer in mouse models.
      ].
      Overall, the evidence is promising but still immature, so clinical studies investigating specifically whether microbes, and particularly bacteria, impact on radiotherapy efficacy are sorely needed. Such studies may provide head and neck oncologists with the next HPV or pathway for radiation response modification.

      Conclusion: The time is now for microbiota research in head and neck radiation oncology

      The microbiota can impact cancer treatment through direct and indirect mechanisms. Radiation oncologists should be aware that the microbiota is likely to become as impactful in radiotherapy as it has in systemic therapies over the last decade. Studies conducted so far indicate a bidirectional effect between radiotherapy and microbiota. On one side, radiotherapy-induced tissue oxidation and inflammation may disrupt the microbiota, triggering a pro-inflammatory environment. On the other side, a disrupted microbiota can decrease radiotherapy effectiveness.
      It is now possible to conduct microbiome clinical studies with deep characterisation of microbial populations at competitive costs. High-quality longitudinal studies with carefully selected controls are necessary to establish the role of bacteria, fungi and viruses in the radiotherapy pathway and should be complemented by mechanistic studies. In the meantime, radiation oncologists should be vigilant for Candida infections, which should be promptly diagnosed and treated, and avoid prescribing broad-spectrum antibiotics in the absence of solid clinical reasons.

      CRediT authorship contribution statement

      Miguel Reis Ferreira: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Visualization, Writing – original draft, Writing – review & editing. Anna Pasto: Visualization, Writing – review & editing. Tony Ng: Writing – review & editing. Vinod Patel: Writing – review & editing. Teresa Guerrero Urbano: Writing – review & editing. Cynthia Sears: Writing – review & editing. William G. Wade: Conceptualization, Methodology, Writing – original draft, Writing – review & editing.

      Declaration of Competing Interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      Acknowledgements

      This work was supported by the Radiation Research Unit at the Cancer Research UK City of London Centre Award [C7893/A28990] and by the Guy’s and St Thomas’ Charity. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health and Social Care.

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