The oropharyngeal microbiome and metabolome as potential novel biomarker for disease progression or treatment response in head and neck squamous cell carcinoma (HNSCC); a pilot study.

The human microbiota is defined as the collective of microorganisms that reside
in the human body or its surfaces. The *microbiome* is reserved for the genomes
of this collective group of microorganisms. Microbial imbalance (dysbiosis) on
or inside the body may affect oncogenesis, tumor progression and response to
cancer therapy1,2. First of all, risk factors for dysbiosis, such as
antibiotics, smoking, diet and alcohol consumption, may also promote
carcinogenesis directly. Secondly, an altered gut microbiota may affect the
incidence and progression of both locoregional carcinogenesis and
extra-intestinal cancers1. Of interest, microbes present at mucosal sites may
exert different immune-modulatory effects. Segment-ed filamentous bacteria,
which can breach the gut mucus layer and attach to intestinal epi-thelial cells
(IECs) are a potent inducer of T helper 17 cells and for instance Helicobacter
pylori may lead to an increase in loco-regional Tregs3,4.

During cancer therapy, radiation or drug regimens may exert toxic effects on
bacteria, there-by promoting dysbiosis. On the other hand, (gut) microbiota may
influence the therapeutic efficacy and side effects of cancer drugs via
pharmacodynamics and immunological mecha-nisms1,2. Indeed, recent work has
shown that gut microbiota interfere with tumor responses to chemotherapy and
aPD1 or aCTLA4 immunotherapy3,5,6.

*Omic* technologies aim to comprehensively study the molecules that make up a
cell, tissue or an organism. The primary aim of this holistic approach is the
global detection of molecules in a biological sample, in a non-targeted and
unbiased manner. Among all the *omic* tech-nologies, metabolomics is the
closest link to the phenotypes of biological systems. The analysis of the
metabolome of a cell, a tissue, a bio-fluid or an organism has the aim to
iden-tify alterations in biochemical networks. Oral diseases are known to be
closely associated with oral biofilm metabolism. The collection of saliva
specimen is inexpensive and non-invasive, presents high potential of biomarkers
discovery, as well as longitudinal disease monitoring7-9. Despite these
opportunities, today, only few reports addressed the metabolic composition and
changes of salivary samples. However, a deeper understanding of perturba-tions
of the salivary metabolome in association with the microbiome composition is
essential for clarifying complex pathogenic mechanisms, such as cancer or
dysbiosis related disorders. At first, the detection of metabolic alterations
will allow for a more comprehensive un-derstanding of disease progression, the
possible evaluation of therapeutic outcomes, and simultaneously shed light on
cancer metabolism, the involvement of the oral microbiome as well as their
interplay. On the long term, a more complete understanding of these
alterations, could very well contribute to the development of personalized
diagnosis, boost the improve-ment of safety and efficacy of treatment as well
as promote personalized therapy.

In head and neck squamous cell carcinoma (HNSCC), the oral cavity harbors more
than 700 bacterial species and is one of the most densely microbial populated
areas of the human body. The oral cavity is the largest core of commonly shared
microbes among unrelated indi-vidual and provides an ideal source for biomarker
discoveries due to low inter- and intra- bio-logical variations. The oral
cavity includes several distinct habitats as the tooth sur-face/gingival
crevice, tongue, tonsils and oropharynx, each host a distinct microbiota10.
Dif-ferences in bacterial- and viral composition have been described for oral
cancers in patient series11, potentially serving as biomarker for oral cancer.
The microbiome, its metabolome, and its potential effect on cancer treatment in
HNSCC were never studied before. The ac-cessibility of the oral region allows
spatial and temporal analyses of the connection between the micro- and
metabolome in a non-invasive manner.

As previous research suggests a potential key role for microbiome and
metabolome analyses in tumor response to treatment, the results of this study
may have implications for the devel-opment of novel biomarkers for the
prediction of treatment response and clinical outcome in head and neck cancer.

Primary objectives:
• To define any alterations in the oral and gut microbiome and metabolome in
patients with HNSCC compared to healthy controls;
• To investigate the dynamics oral and gut microbiome and metabolome in HNSCC
before, during and after different treatment regimens.

Secondary Objective(s):
To investigate the correlation of the microbiome and metabolome with
demographic, clinical, pathological and imaging data as well as genomic date
(if available).

This is a prospective, bicenter (NKI-AVL and LUMC), observational cohort study
in which 60 patients with locally HNSCC will be included. In addition, 20
healthy volunteers will serve as a control population. There will be no
randomization or blinding.

When swabs are taken during a routine physical investigations or routine
general anesthesia (in case of surgery), this will hardly cause any extra
burden to the patient as these procedures were already planned as part of the
treatment. The risk of swabs and fecal sampling is negligible. Patients will
collect stool at home using a stool collection kit and hand in the sample
during their routine hospital visits. This procedure is considered simple and
non-invasive.
After surgery, tissue is standard fixed in formaldehyde for pathology. A
section will be nitrogen frozen for metabolome analyses by tissue mass
spectrometry and the pathology section and metabolome section will be matched
later. This does not provide any additional burden to the patient and will
include only additional handling of the surgical specimen.
The participation of a patient in this trial will not affect the treatment that
the patient will receive and will not directly have a benefit for individual
patients. The data that will be acquired in this study will be of benefit to
the general cancer patient population as they can help to answer complicated
research questions in the field of chemical immunology and oncology.