JOURNAL CLUB PAPER PRESENTATION Presenter Mrs Anjali K

JOURNAL CLUB PAPER PRESENTATION 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 J 3, Bhattacharya A 4, Bing H 4, Patricia MC 1, Queiroz ELS 1, Nightingale K 1, Ross KA 5, De. Lacure DM 6, Ratna Veeramachaneni 4, Olshen AB 4, Albertson. DG 4 1 Bluestone Centre for Clinical Research, New York University College of Dentistry, New York, United States of America 2 Department of Oral and Maxillofacial Surgery, New York University College of Dentistry, 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, California, United States of America, 5 Department of Oral and Maxillofacial Pathology, Radiology and Medicine, New York University College of Dentistry, New York, United States of America 6 Departments of Otolaryngology and Plastic Surgery, New York University, New York, United States of America Journal: PLo. S one, 2014; (9) e 98741 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 16 Sribosomal 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 Ø Samples collected from the cancer or pre-cancer Ø Lesion dried by blotting with gauze and stroked with an Isohelix SK-2 swab Ø Swab held at an angle 20˚ Ø One side of the swab was stroked across the lesion 10 times. Ø Rotate swab at 180˚stroked 10 times

Materials and Methods Ø Similarly swabs collected from anatomically matched contralateral normal tissue and tissues from healthy normal individuals Ø Similarly samples collected using Oral. CDx Brush.

Materials and Methods Ø Collected Isohelix SK-2, Oral. CDx Brush swab taken in microfuge tube, add Isohelix cell lysis, DNA stabilization solution, proteinase K solution Ø Samples stored at room temperature for nucleic acid extraction.

Materials and Methods DNA extraction Ø Samples in nucleic acid extraction buffer were vortexed, centrifuged and supernatant collected Ø DNA extracted by using DNeasy blood and tissue kit

16 S r. RNA amplicon preparation and 454 pyrosequencing • Study 1: Primer set 515 F (59 -GTGCCAGCGCCGCGGTAA 39) and 806 R (59 -GGACTACSGGGTATCTAAT 39) • Study 2: 515 F and 806 R 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 Sex Site Tumo ur size 113 62 M Right buccal mucosa 116 84 F 117 68 136 142 p. TNM 1 Immuno compro mised smoker alcohol 2. 5× 3. 0 p. T 2 N 2 b. M x Yes Previous Current Right retromolar trigone 6. 0 p. T 4 a. N 0 M x No Previous Previou s M Left posterior lateral tongue 1. 5× 2. 0 p. T 2 NOMx No Previous Current 68 M Left lateral tongue 5. 0× 2. 0 p. T 2 N 2 BM x No Never Current 64 M Right dorsum of tongue 0. 3× 1. 5 p. T 3 N 0 M 0 yes 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 Sex Site t Tumou p. TNM 1 r size smoker alcohol 104 55 F Right lateral tongue 2. 5× 2. 5 p. T 4 N 2 M 0 Current 108 39 F Right lateral tongue corner 3. 0 p. T 4 N 2 b. M 0 Previous Nondrinker 114 48 M Left post mandibular gingiva 1. 5 p. T 1 N 0 M 0 NS current 114 48 M Left post maxillery gingiva 2. 5 p. T 2 N 0 M 0 NS current 128 71 M Left alveolar bridge 4. 0× 3. 0 PT 4 b. N 0 M 0 Previous previous 143 61 M Right floor of mouth 1. 6× 1. 0 p. T 1 N 0 M 0 Current 144 70 M Left floor of mouth 3. 5× 1. 5 p. T 1 N 0 M 0 Previous current 146 78 M Right retromolar triagone 1. 5× 1. 5 p. T 1 N 2 b. Mx Previous current 153 51 F Left ventral tongue 8. 0× 8. 0 p. T 2 N 1 Mx NS current 156 69 F Right mandibular gingiva 1. 0× 1. 0 p. T 1 N 0 M 0 NS 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 ØShifts in the abundance of Fusobacterium and Streptococci Enhanced pro inflammatory environment for cancer cells Increasing the complications ØMicrobiome from normal and cancer tissues can be screened by non-invasive methods

Thank You. .