Download journal club paper presentation - Yengage

Presenter:
Mrs. Anjali K.
Research Scholar
Yenepoya Research Centre
Yenepoya University
Guide:
Dr. Arun A. B.
Deputy Director
Yenepoya Research Centre
Yenepoya University
17.10.2015
Article
Changes in the abundance of oral
microbiota associated with oral cancer
Schmidt BL 1,2, Kuczynski J3, Bhattacharya A 4, Bing H4, Patricia MC1, Queiroz
ELS1, Nightingale K1, Ross KA 5, DeLacure DM6, Ratna Veeramachaneni4,
Olshen AB 4, AlbertsonDG 4
1 Bluestone Centre for Clinical Research, New York University College of Dentistry, New York, New York, United States of America
2 Department of Oral and Maxillofacial Surgery, New York University College of Dentistry, New York, New York, United States of America
3 Bioinformatics Department, Second Genome, San Bruno, California, United States of America, 4 Helen Diller Family Comprehensive
Cancer Center, University of California San Francisco, San Francisco, California, United States of America,
5 Department of Oral and Maxillofacial Pathology, Radiology and Medicine, New York University College of Dentistry, New York, New
York, United States of America
6 Departments of Otolaryngology and Plastic Surgery, New York University, New York, New York, United States of America
Journal: PLoS one, 2014; (9) e98741
Impact factor: 3.234
Aim of the study
To explore the oral microbiome for treatment and
monitoring of oral cancer initiation, progression
and recurrence.
Introduction
• Americans are diagnosed with oral cancer of
which 90% are squamous cell carcinoma (SCC)
• The incidence of oral cancer is increasing,
particularly among young people.
• The major risk factors are: Tobacco, alcohol
and human papillomavirus.
Introduction
• Changes in the microbial community - associated
with periodontal disease, oral –precancers and
cancers
• Analysis of relatively small number of known and
culturable oral bacterial species
• Culture independent methods, particularly
16Sribosomal RNA more comprehensive
Introduction
• Most commonly reported phyla: Firmicutes,
Proteobacteria, Bacteroidetes, Actinobacteria,
Fusobacterium
• Streptococcus is dominant genus in healthy oral
microbiome
• Prevotella, Veillonella, Neisseria, and
Haemophilus occur less frequently
Materials and Methods
• Study location:New York University College of
Dentistry
• Ethical clearance: Ethics Committee-New York
University College of Dentistry
• Study design: Prospective
Materials and Methods
• Sample collection:
Study 1: Samples from cancer and anatomically
matched contralateral clinically normal regions
of oral cavity obtained from five patients with oral
cancer.
Study 2: Cancer and pre-cancer patients, left and
right side of the lateral tongue and floor of the
mouth of healthy individuals.
Materials and Methods
 Samples collected from the cancer or pre-cancer
 Lesion dried by blotting with gauze and stroked
with an Isohelix SK-2 swab
 Swab held at an angle 20˚
 One side of the swab was stroked across the
lesion 10 times.
 Rotate swab at 180˚stroked 10 times
Materials and Methods
 Similarly swabs collected from anatomically
matched contralateral normal tissue and tissues
from healthy normal individuals
 Similarly samples collected using OralCDx
Brush.
Materials and Methods
 Collected Isohelix SK-2, OralCDx Brush swab
taken in microfuge tube, add Isohelix cell lysis,
DNA stabilization solution, proteinase K solution
 Samples stored at room temperature for nucleic
acid extraction.
Materials and Methods
DNA extraction
 Samples in nucleic acid extraction buffer were
vortexed, centrifuged and supernatant collected
 DNA extracted by using DNeasy blood and tissue kit
16S
rRNA
amplicon
preparation
and
454
pyrosequencing
• Study 1:
Primer set 515F (59-GTGCCAGCGCCGCGGTAA39) and 806R (59-GGACTACSGGGTATCTAAT-
39)
• Study 2:
515F and 806R fusion primers tailed with
sequences with illumina flow cell adapters,
indexing barcodes
Statistical analysis
• Binomial test to evaluate significance of finding
consistent changes in abundance
• Paired t-test to identify operational taxonomic
units (OTU)
• Welch’s t test to identify (OTU) significantly
increased or decreased across samples
Results
Study 1-Discovery of cohort
• Shows diversity of microbes
• Firmicutes, Bacteroidetes, Proteobacteria
Fusobacteria, Actinobacteria
• Proportion of Fusobacteria increases
• Proportion of Firmicutes,Actinobacteria
decreases
Results
Study-2 -Confirmation cohort
• Shows diversity of organisms
• Reduction in the abundance of Firmicutes,
Actinobacteria, in cancer and precancer patients
than the normal healthy individual
Table 1: Pathologic tumor stage according to the American Joint
Committee on Cancer guidelines (T = tumor size, N = regional lymph node
metastasis, M=distant metastasis)
Patien
t
Ag
e
Se
x
Site
Tumou pTNM1
r size
Immuno
compro
mised
smoker
alcohol
113
62
M
Right buccal
mucosa
2.5×3.
0
pT2N2bM
x
Yes
Previous
Current
116
84
F
Right retromolar
trigone
6.0
pT4aN0M
x
No
Previous
Previou
s
117
68
M
Left posterior
lateral tongue
1.5×2.
0
pT2NOMx
No
Previous
Current
136
68
M
Left lateral tongue
5.0×2.
0
pT2N2BM
x
No
Never
Current
142
64
M
Right dorsum of
tongue
0.3×1.
5
pT3N0M0
yes
current
current
Table 2: Pathologic tumor stage according to the American Joint Committee
on Cancer guidelines (T = tumor size, N = regional lymph node metastasis, M
= distant metastasis, NS= Non smoker)
Patien Age Se
t
x
Site
Tumou pTNM1
r size
smoker alcohol
104
55
F
Right lateral tongue
2.5×2.5 pT4N2M0
Current
108
39
F
Right lateral tongue corner 3.0
pT4N2bM0 Previous Nondrinker
114
48
M
Left post mandibular
gingiva
1.5
pT1N0M0
NS
current
114
48
M
Left post maxillery gingiva 2.5
pT2N0M0
NS
current
128
71
M
Left alveolar bridge
4.0×3.0 PT4bN0M0 Previous previous
143
61
M
Right floor of mouth
1.6×1.0 pT1N0M0
Current
144
70
M
Left floor of mouth
3.5×1.5 pT1N0M0
Previous current
146
78
M
Right retromolar triagone
1.5×1.5 pT1N2bMx Previous current
153
51
F
Left ventral tongue
8.0×8.0 pT2N1Mx
NS
current
156
69
F
Right mandibular gingiva
1.0×1.0 pT1N0M0
NS
current
current
current
Fig 1: Change in the relative abundance of phyla associated with
cancer compared to anatomically matched contra lateral clinically
normal samples in study 1.(a-e)
Fig 2: Change in relative abundance as difference between of phyla
associated with cancers compared to anatomically matched contra
lateral clinically normal samples
Fig 3: The relative distribution of phyla (percent of sequences) is shown
for each patient sample with clinically normal samples shown together on
top and cancer samples on the bottom.
Fig 4: Distribution of phyla in cancer, pre-cancer and healthy normal
samples in Study 2
Firmicutes
Actinobacteria
Fig 5: Change in relative abundance of phyla associated with cancers
compared to normal
Discussion
• changes in abundance of Actinobacteria and
Firmicutes.
• Confirmation Cohort (Study 2) further found
significant changes in the abundance of
microbiota
In cancer
• Phyla: Actinobacteria, genus Rothia
• Phyla: Firmicutes, genus Streptococcus
Discussion
• Significant increase in abundance of the
Fusobacteria genus in cancer
• No significant changes in abundance of
microbiota in smokers and non-smokers and
immunosuppressed individuals
• Decrease in prevalence of Streptococcus and
increased abundance of Fusobacterium genera
in pre-cancers could reflect the altered surface
properties of the cancer cells
Conclusion
Shifts in the abundance of Fusobacterium and
Streptococci
Enhanced pro inflammatory environment for
cancer cells
Increasing the complications
Microbiome from normal and cancer tissues can
be screened by non-invasive methods