Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
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