The Oral Microbiome as a Prognostic Tool in Oral Malignant and Premalignant Lesions and in Medication Related Osteonecrosis of the Jaw

Study Overview
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Brief Summary
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Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the head and neck, and its incidence has increased in recent years. Extensive surgery with neck dissection and chemo/radio/ targeted therapy is the current treatment for OSCC, and despite great progress in chemotherapy, radiotherapy, and targeted therapy in the last three decades, the prognosis of OSCC is still poor due to aggressive local invasion and metastasis, which lead to recurrence. Postoperative tumor recurrence confers a poor prognosis in OSCC and a poor quality of life. The 5-year survival rate is over 90% in OSCC patients without recurrence and 30% in patients with recurrence, with a median survival of 76.8 months in patients without recurrence and 42.5 months in patients with recurrence . Therefore, it is important to identify biomarkers that may predict the postoperative recurrence of OSCC. Also, some of the OSCC are preceded by precursor lesions. In the oral cavity the most common lesions recognized as potentially malignant are leukoplakia and erythroplakia, but it is also apparent that as many as 50% of OSCC arise from apparently clinically normal mucosa. The prognostic significance of an individual lesion is difficult to determine. At present therefore, the gold standard for the assessment of oral potentially malignant lesions is microscopic evaluation of haematoxylin and eosin stained sections for the presence of architectural and cytological changes, which are generally referred to as oral epithelial dysplasia (OED). The human microbiome is defined as the collective genomes of the microbes (composed of bacteria, bacteriophages, fungi, protozoa and viruses) that live inside and on the human body, and there are approximately 10 microbes and 100 microbial genes for each human cell and gene respectively. With the advent of next generation sequencing technology, the Human Microbiome Project delineated the composition of healthy microbial communities associated to different body sites in healthy individuals, including the oral cavity [Human microbiome consortium]. As opposed to a normal (healthy) microbiome, a disrupted microbiome or dysbiosis represents the lack of equilibrium, and is hypothetically related to disease. Interestingly, the healthy oral microbiome shows relative intraindividual stability over time, suggesting that differences in microbiome profiles may serve as useful tools for the identification of disease states. The working hypothesis is that in OSCC patients, the oral microbiome is altered in comparison to healthy individuals and certain microbial signatures are characteristic of healthy versus disease. In addition, in precursor conditions, i.e., oral epithelial dysplasia (OED), a partial alteration in the composition of the microbiome may predict the progression to malignancy.Also, during treatment, it could be that specific microbial signatures are associated with incomplete eradication, tendency to local recurrence or metastatic potential.Correlations to local recurrence (LR), distant metastases (DM) or disease free survival (DFS) adjusted to clinicopathologic correlations will be sought. In this study, buccal mucosa samples will be collected from patients with OSCC, OED and from healthy individuals , after signing for informed consent, according to Helsinki protocol. Routine pathologic diagnosis will be performed by expert Pathology physicians in our center. Data will be correlated to demographic and clinical data obtained from medical records. This will be carried out in line with institutional ethical guidelines.

Official Title
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The Oral Microbiome as a Prognostic Tool in Oral Malignant and Premalignant Lesions and in Medication Related Osteonecrosis of the Jaw

Conditions
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Oral Squamous Cell Carcinoma, Medication Related Osteonecrosis of the Jaw, Oral Epithelial Dysplasia

Intervention / Treatment
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* Diagnostic Test: Buccal mucosa samples for Extraction of BACTERIAL DNA


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Patients biopsied with OSCC Patients biopsied with oral premalignant lesions Patients biopsied with osteonecrosis of the jaw healthy age matched patients that visit the oral and maxillofacial clinic for other purposes such as extraction, reactive lesions Exclusion Criteria: Patients receiving antimicrobial treatment in the previous 3 months Patients receiving radiotherapy Patients receiving chemo/immunotherapy

Ages Eligible for Study
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Minimum Age: 20 Years
Maximum Age: 90 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Oral Squamous Cell Carcinoma<br>Buccal mucosa samples for Extraction of BACTERIAL DNA | Diagnostic Test: Buccal mucosa samples for Extraction of BACTERIAL DNA<br>* Sampling of the buccal mucosa after informed consent and prior to routine treatment. Similar sampling on follow up after 3,6,9,12 months. Extracted BACTERIAL DNA (and not human DNA) from clinical samples will be sequenced and analyzed for the 16s rRNA (BACTERIAL ribosome).<br>|
| Oral Epithelial Dysplasia<br>Buccal mucosa samples for Extraction of BACTERIAL DNA | Diagnostic Test: Buccal mucosa samples for Extraction of BACTERIAL DNA<br>* Sampling of the buccal mucosa after informed consent and prior to routine treatment. Similar sampling on follow up after 3,6,9,12 months. Extracted BACTERIAL DNA (and not human DNA) from clinical samples will be sequenced and analyzed for the 16s rRNA (BACTERIAL ribosome).<br>|
| Control: Healthy age matched patients<br>Buccal mucosa samples for Extraction of BACTERIAL DNA | Diagnostic Test: Buccal mucosa samples for Extraction of BACTERIAL DNA<br>* Sampling of the buccal mucosa after informed consent and prior to routine treatment. Similar sampling on follow up after 3,6,9,12 months. Extracted BACTERIAL DNA (and not human DNA) from clinical samples will be sequenced and analyzed for the 16s rRNA (BACTERIAL ribosome).<br>|
| Osteonecrosis of the Jaw<br>Buccal mucosa samples for Extraction of BACTERIAL DNA | Diagnostic Test: Buccal mucosa samples for Extraction of BACTERIAL DNA<br>* Sampling of the buccal mucosa after informed consent and prior to routine treatment. Similar sampling on follow up after 3,6,9,12 months. Extracted BACTERIAL DNA (and not human DNA) from clinical samples will be sequenced and analyzed for the 16s rRNA (BACTERIAL ribosome).<br>|
| Underlying disease, no necrosis of the jaw<br>Buccal mucosa samples for Extraction of BACTERIAL DNA | Diagnostic Test: Buccal mucosa samples for Extraction of BACTERIAL DNA<br>* Sampling of the buccal mucosa after informed consent and prior to routine treatment. Similar sampling on follow up after 3,6,9,12 months. Extracted BACTERIAL DNA (and not human DNA) from clinical samples will be sequenced and analyzed for the 16s rRNA (BACTERIAL ribosome).<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Microbiome characterization day 1 of the study | Extracted BACTERIAL DNA sequenced by amplification of the V4 region of 16s rRNA (BACTERIAL ribosome). The samples will be obtained by buccal mucosa swabs from all subjects at the clinic visit (day 1). FLOQ swabs (Copan) will be rubbed on the inner cheek mucosa that looks macroscopically normal. The swab will be immediately placed in a 15ml polypropylene tube containing 1 ml of Molecular grade PBS. Samples will be kept at 4oC until frozen at -80oC. Each sample will be assigned a protocol serial number, de-identified from the subject. | Day 1 of each participant | 
| Microbiome characterization month 3 of the study | 1Extracted BACTERIAL DNA sequenced by amplification of the V4 region of 16s rRNA (BACTERIAL ribosome). The samples will be obtained by buccal mucosa swabs from all subjects at the clinic visit (month 3). FLOQ swabs (Copan) will be rubbed on the inner cheek mucosa that looks macroscopically normal. The swab will be immediately placed in a 15ml polypropylene tube containing 1 ml of Molecular grade PBS. Samples will be kept at 4oC until frozen at -80oC. Each sample will be assigned a protocol serial number, de-identified from the subject. | Month 3 of each participant | 
| Microbiome characterization month 6 of the study | Extracted BACTERIAL DNA sequenced by amplification of the V4 region of 16s rRNA (BACTERIAL ribosome). The samples will be obtained by buccal mucosa swabs from all subjects at the clinic visit (month 6). FLOQ swabs (Copan) will be rubbed on the inner cheek mucosa that looks macroscopically normal. The swab will be immediately placed in a 15ml polypropylene tube containing 1 ml of Molecular grade PBS. Samples will be kept at 4oC until frozen at -80oC. Each sample will be assigned a protocol serial number, de-identified from the subject. | Month 6 of each participant | 
| Microbiome characterization month 9 of the study | Extracted BACTERIAL DNA sequenced by amplification of the V4 region of 16s rRNA (BACTERIAL ribosome). The samples will be obtained by buccal mucosa swabs from all subjects at the clinic visit (month 9). FLOQ swabs (Copan) will be rubbed on the inner cheek mucosa that looks macroscopically normal. The swab will be immediately placed in a 15ml polypropylene tube containing 1 ml of Molecular grade PBS. Samples will be kept at 4oC until frozen at -80oC. Each sample will be assigned a protocol serial number, de-identified from the subject. | Month 9 of each participant | 
| Microbiome characterization month 12 of the study | 3. Extracted BACTERIAL DNA sequenced by amplification of the V4 region of 16s rRNA (BACTERIAL ribosome). The samples will be obtained by buccal mucosa swabs from all subjects at the clinic visit (month 12). FLOQ swabs (Copan) will be rubbed on the inner cheek mucosa that looks macroscopically normal. The swab will be immediately placed in a 15ml polypropylene tube containing 1 ml of Molecular grade PBS. Samples will be kept at 4oC until frozen at -80oC. Each sample will be assigned a protocol serial number, de-identified from the subject. | Month 12 of each participant |