1. Gastroenterological Society of Queensland Young Investigator Awards The Traditional Serrated Adenoma A Clinicopathological and Molecular Analysis Presented by Mark Bettington 24 May 2014

2. Background Colorectal carcinoma (CRC) is a heterogeneous disease Most CRC (70-80%) arise from conventional adenomas A subset (20-30%) arise from serrated polyps and are characterised by MAP kinase pathway activation (via BRAF or KRAS mutation) and widespread promoter methylation (CIMP) Cancers derived from the serrated pathway can be either microsatellite unstable (MSI), due to MLH1 methylation, or less frequently, microsatellite stable (MSS) Sessile serrated adenomas (SSA) are perhaps the best known and understood precursor of the serrated pathway In contrast much less is known about the TSA and how it progresses to carcinoma Current guidelines recommend a three year surveillance interval for TSAs © QIMR Berghofer Medical Research Institute | 2

3. Aims 1.To describe the clinicopathological features of ordinary and advanced TSAs 2.To describe the molecular features of ordinary and advanced TSAs 1.To use immunohistochemistry to better define the pathways by which TSAs progress to carcinoma © QIMR Berghofer Medical Research Institute | 3

4. Methods 200 cases were collected from the files of Envoi Specialist Pathologists Performed a molecular analysis (BRAF, KRAS, CIMP and IHC) on all polyps Further assessed for the presence of a precursor polyp and for any histologically advanced areas (overt dysplasia or invasive carcinoma) © QIMR Berghofer Medical Research Institute | 4

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8. Aim 1 – Clinicopathological analysis Ordinary TSAs 48.1% associated with a precursor SSA or MVHP Mean size 13.8mm (median 12mm) 162 cases (81%) 51.2% female Mean age 63.6 years 67.9% distal Advanced TSAs 15.8% associated with a precursor SSA or MVHP Mean size 24.7mm (median 20mm) 38 cases (19%) 44.7% female Mean age 64.5 years 81.6% distal © QIMR Berghofer Medical Research Institute | 8 TSAs have frequent origin in a SSA or MVHP (42% overall)

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11. Aim 2 - Molecular analysis Ordinary TSAs Mutation and CIMP status - BRAF mutation in 112 (69.1%) - KRAS mutation in 33 (20.4%) -Wild type in 17 (10.5%) -CIMP+ 46.3% Advanced TSAs Mutation and CIMP status -BRAF mutation in 22 (57.9%) -KRAS mutation in 10 (26.3%) -Wild type in 6 (15.8%) -CIMP+ 44.7% Two thirds of TSAs are BRAF mutant / BRAF mutation correlates with CIMP CIMP+ by mutation status

12. Each polyp was stained for MLH1, β-catenin, p53 and p16 Each of these proteins are critical components of independent cancer pathways known to be involved in colorectal carcinoma progression © QIMR Berghofer Medical Research Institute | 12 Aim 3 – Pathways to carcinoma

13. © QIMR Berghofer Medical Research Institute | 13 MLH1 expression is retained in 99.5% of TSAs indicating a MICROSATELLITE STABLE PHENOTYPE Aim 3 – Pathways to carcinoma

14. © QIMR Berghofer Medical Research Institute | 14 Nuclear β-catenin (advanced TSAs) Aim 3 – Pathways to carcinoma Nuclear β-catenin (ordinary TSAs) Wnt pathway signaling becomes activated in a subset of advanced TSAs p=<0.0001 (ordinary vs advanced)

15. © QIMR Berghofer Medical Research Institute | 15 Aim 3 – Pathways to carcinoma P53 is mutated in the majority of advanced TSAs Nuclear p53 (advanced TSAs) p=<0.0001 (ordinary vs advanced) Nuclear p53 (ordinary TSAs)

16. © QIMR Berghofer Medical Research Institute | 16 Aim 3 – Pathways to carcinoma P16 is silenced in carcinomas arising from BRAF mutant TSAs P16 staining in TSAs with dysplasiaP16 staining in TSAs with CRC

17. © QIMR Berghofer Medical Research Institute | 17 KRAS pathway Normal mucosa TSA TSAD KRAS mutant MSS CRC BRAF pathway Normal mucosa MVHP / SSA TSA arising in MVHP/SSA TSAD BRAF mutant MSS CRC BRAF / CIMP KRAS P53 mutation, Wnt pathway activation Proposed Progression of TSAs to Carcinoma P16 silencing

18. Summary 1.BRAF mutant TSAs are more common than KRAS mutant TSAs 2.BRAF mutant TSAs arise from SSAs/MVHPs and are CIMP-H 3.Advanced TSAs are overwhelmingly microsatellite stable and show p53 mutation, Wnt pathway activation and p16 silencing © QIMR Berghofer Medical Research Institute | 18

19. Conclusions The TSA is an important precursor of the aggressive BRAF mutant, microsatellite stable subtype of CRC Close follow up is recommended if advanced features are identified by histology © QIMR Berghofer Medical Research Institute | 19

20. Acknowledgments The Conjoint Gastroenterology Laboratory Prof Barbara Leggett A/Prof Vicki Whitehall Sally-Ann Pearson Diane McKeone Troy Dumenil Catherine Bond Ron Buttenshaw Winnie Fernando Envoi Specialist Pathologists A/Prof Neal Walker Dr Ian Brown A/Prof Christophe Rosty A/Prof Andrew Clouston