Association of Oral Microbiome with Incident Risk of Head and Neck Squamous Cell Cancer

Group 7: Joy Eskandar, Haowei Ni, Vaibhav Menon

Objective

To examine relationships between the oral microbiome and head and neck squamous cell cancer (HNSCC)

Why is this relevant?

• More than half a million new head and neck cancer (oral cavity, larynx, pharynx) cases and 380000 deaths worldwide each year
• HNSCC make up 85% of these cases
• Leads to physical disfigurement, decreased quality of life, and a 40% 5 year mortality rate.

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• There is a need to make new and effective approaches for prevention, in addition to smoking, alcohol, and HPV control
• The human mouth has diverse community of about 700 different bacterial species--oral microbiome
• The bacteria have many functions including maintaining oral health

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Methods

• Bacteria profiles were examined in these studies in relation to oral cavity cancers
• Prospective study in 2 US cohorts to determine if oral microbiome was associated with risk of HNSCC
• Directly assessed the oral microbiota by sequencing the 16S rRNA gene in oral samples from 129 HNSCC cases and 254 controls.

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• Compared patient cases and controls for overall microbiota composition and abundance of specific bacterial species.
• Examined the associations of oral microbiome with subsequent HNSCC differed in relation to tobacco use, cancer sites (oral cavity, larynx, pharynx), or by time from microbiome measurement to cancer diagnosis.

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American Cancer Society Cancer Prevention Study II Nutrition Cohort (CPS-II)

• This cohort had 184000 participants, ages 50 to 74 yo, from 21 US states, who completed a diet and lifestyle questionnaire. (1992)
• Follow- up questionnaires have been sent to cohort members every other year to get updated exposure info and incident cancer cases(1997)
• In 2001-2002 oral wash samples were collected from 70004 cohort members
• The analysis includes HNSCC diagnosed between oral wash collection and 2009

Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO)

• This cohort was a large population-based randomized trial that looked at the effects of screening on cancer related mortality in men and women (55 to 74) from 1993-2001
• They were followed for cancer incidence
• Randomized to either screening or control
• Oral wash samples collected in control (52000)
• Incident cancers were determined in follow-up
• This analysis includes cases of HNSCC diagnosed between oral wash collection to 2010

Nested Case-Control Study Selection

• Incident cases (129) of HNSCC were found during a 3.9 yr follow up
• 2 controls were matched to each patient case (by cohort, sex, age, date of oral wash)
• All participants selected for study had pre-diagnostic oral wash samples and no prior medical history of cancer

Measurement

How to get demographic and risk factor information?

Questionnaires!

Oral wash samples and DNA

How to characterize oral microbiota?

1 Extract using the PowerSoil DNA Isolation Kit (MO BIO) to bacterial genomic DNA

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2 Amplify the 16s rRNA gene

Universal Primer 347F 5′-GGAGGCAGCAGTRRGGAAT′-

3′ and 803R 5′-CTACCRGGGTATCTAATCC-3′

3 Amplified Result

How to do Upstream Sequence Analysis of microbiome data?

1 QIIME:

Bioinformatic pipeline for performing microbiome from raw sequencing data

Demultiplexing, quality filtering, OTU picking, taxonomic assignment, diversity analysis and visualization

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2 Cluster the sequence into operational taxonomic units (OTU)

3 Human Oral Microbiome DataBase

98% or greater identity

383 wash samples → 3217274 high-quality 16s rRNA gene sequencing reads.

How to ensure the quality?

Blind positive quality control Assess the temporal stability of microbiome

Statistical Analysis

1 Permutational multivariate analysis of variance to compare composition

2 adjust oral sample collection, ever-smokers, ever-drinkers

3 Differential gene expression analysis based on the negative binomial distribution to compare taxa (DESeq) to compare bacterial taxa

Filter and exclude low count taxa

5 phyla, 11 classes, 17 orders, 28 families,

50 genera and 167 species.

p < 0.05

P < 0.001

Results

Healthy Oral Microbiome Players

• Kingella, corynebacteria, Neisseria, Abiotrophia, capnocytophaga, streptococcus sanguinis all associated with lower risk of larynx cancer
• Actinomyces oris and veillonella denticariosi associated with reduced risk of oral cavity cancer
• However, another unnamed actinomyces associated with increased risk.

Lifestyle and Oral Microbiome

• Association of characteristic microbiome content and species richness more pronounced in tobacco smokers (current and former) than non-smokers
• However, not a statistically significant difference in populations based on smoker status

Figure 2a

Decrease in microbiome population

“Good” bacterial populations in these cancer patients show sweeping decrease

Fig 2b.

Discussion- Key Takeaways

• Corynebacteria and Kingella are associated with reduced risk of HNSCC
• Consistent across both cohorts
• Especially pronounced among tobacco smokers
• Healthy oral microbiome has a role in HNSCC prevention
• Floss and brush your teeth!

Oral Bacterial Mechanisms and Cancer

• Corynebacterium + Kingella- involved in biodegradation and metabolic pathways that can break down toxic compounds found in cigarette smoke
• Recall pronounced effect of characteristic microbiome in smokers
• Suggests that within oral cavity of smokers, these bacteria may play role in preventing development of cancer despite high-carcinogen environment
• Alcohol consumption– commensal Bacteria metabolize ethanol to carcinogen acetaldehyde
• While this was noted by organizers of PLCO cohort, studying this link not within paper scope
• HPV associated with 22-fold increase in oropharyngeal cancer, but presence of A. oris and V. denticariosi can mitigate this cancer risk in HPV noncarriers–some kind of protective mechanism

Study Limitations

• No causal link established–these are just associations based off the 16S rRNA sequence data
• Oral wash samples used; other oral sites–tongue, mucous, plaque– not tested.
• Not longitudinal-samples taken at single time point in each patient
• Change of microbiome community over time could be useful to study against disease progression in cancer patients
• Almost every patient studied–case + control, across both cohorts – was white.
• Mean age: 71 in CPS II cohort and 62.3 in PLCO cohort