Genetics of Serrated Polyposis Syndrome

Assoc. Prof. Dan Buchanan

NHMRC R.D. Wright Biomedical Fellow

Head, Colorectal Oncogenomics Group

Department of Clinical Pathology

The University of Melbourne

Centre for Cancer Research

Victorian Comprehensive Cancer Centre

Genomic Medicine and Family Cancer Clinic,

Royal Melbourne Hospital

Disclosure

• In 2017 and 2018, I was a paid consultant on the Merck Sharp

and Dohme Tumour Agnostic dMMR Advisory Board for

Pembrolizumab

Serrated Polyposis Syndrome

Serrated Polyposis Syndrome (SPS) is a clinically defined

polyposis condition characterised by the occurrence of

multiple serrated polyps throughout the colon and the rectum

➢ Increased risk of CRC

SPS is currently defined by the WHO criteria in 2010:

1. 5 serrated polyps proximal to sigmoid colon, where 2 of these are 10mm or

2. 1 serrated polyp proximal to sigmoid colon in an individual who has FDR with SPS or

3. 20 serrated polyps of any size, but distributed throughout the colon

Distinguished from other polyposis syndromes by morphology of polyps – serrated

WHO define serrated polyp subtypes as: Hyperplastic polyps (HPs), sessile serrated polyps

(SSPs) with or without dysplasia and traditional serrated adenomas (TSAs)

• Any histologic subtype of serrated polyps included in the polyp count

• Serrated polyp count is cumulative

• Conventional adenomas co-occur

Evidence of a genetic basis for SPS

• Multiplicity of polyps and CRCs

• Young age at diagnosis

• Familial aggregation of SPS – RR for SPS in FDRs was 39 (95%CI 13 - 121) Boparai et al, 2010 (n=77)

• Northern European ethnicity (Yeoman et al, 2007; Buchanan et al 2010)

• Increased risk of CRC in FDRs

SIR for CRC in FDRs

➢ = 5.4 (95%CI = 3.7 - 7.8) Boparai et al, 2010 (n=77)

➢ = 5.2 (95%CI = 3.7 - 7.3) Win et al, 2012 (n=100)

➢ = 3.3 (95%CI = 2.2 - 4.8) Egoavil, Juarez et al 2017 (n=53)

➢ = 3.6 (95%CI = 3.0 - 4.3) Win, Greeves et al, unpublished (n=358)

SIR for Pancreatic cancer in FDRs

➢ = 2.5 (95%CI = 1.5 - 4.4) Win, Greeves et al, unpublished (n=358)

CRC and polyposis syndrome genes

Clinical multi-gene panel testing for CRC/polyposis genes has very low yield (RMH)

MMR, MUTYH, PTEN, BMPR1A, SMAD4, GREM1 (Clendenning et al 2013)

• Germline mutation carriers are rare in individuals with SPS

MUTYH

• Chow et al 2006 - >40 adenomas + >30 serrated polyps

• Boparai et al 2008 – 3% of biallelic MUTYH carriers met SPS criteria

• Buchanan and Young 2009 - <1% of 126 SPS had biallelic MUTYH mutations

NTHL1

• Grolleman, de Voer et al 2019 – Biallelic carriers with multiple serrated polyps

• 1/418 (0.2%) SPS had biallelic NTHL1 mutations - >20 adenomas + 30 serrated polyps

MMR

• GCPS - 6 x MLH1, MSH2 and MSH6 mutation carriers that meet SPS criteria 1 or 3

• Walsh et al 2009 – mixed families Lynch + SPS

PTEN

• Heald et al 2010 - ~24% of PTEN mutation carriers met SPS criteria

Macrocephaly/presence of mixed polyps types

MSH2 or MSH6 IHC loss and/or family cancer history

>20 adenomas

>20 adenomas

RNF43 as a susceptibility gene for SPS

Quintana et al 2018 identified one SPS patient with RNF43 c.394C>T p.Arg132*

The role of RNF43 in SPS and CRC

Aims

1) Determine the prevalence of and clinical and molecular phenotypes associated

with germline RNF43 mutations in a large cohort of individuals with SPS

2) Determine the contribution of germline RNF43 mutations to population-based CRC

from large case-control study

Study Participants

Aim 1: N= 418 individuals with SPS recruited to the Genetics of Colonic Polyposis Study based

on WHO criteria 1 and 3 for SPS, regardless of any family history of polyps or cancer

1. SPS probands n=67 + 8 FDRs with SPS

N=58 Whole exome sequencing + N=17 Whole genome sequencing

2. SPS probands n=351 – targeted sequencing of RNF43

Aim 2: CRC cases and controls recruited to the Colon Cancer Family Registry (CCFR)

1. N=1987 incident CRC-affected individuals unselected for family history of CRC

2. N=1207 cancer-unaffected population-based controls

Variant QC and filtering pipeline

Germline screen of coding regions of RNF43• HiPlex Targeted sequencing (www.HiPLEX.org)

• Highly multiplexed PCR-based targeted amplicon sequencing

Predicted pathogenic variants in RNF43

Marie Lorans, Khalid Mahmood, Mark Clendenning

Prevalence of germline RNF43 variants in SPS

GenegnomAD

(n=123,134)

SPS

WGS/WES

(n=67)

SPS

HiPlex

(n=351)

Total SPS

(n=418)OR (95%CI) P value

RNF43396

(0.32%)1 5

6

(1.44%)4.46 [2.0-9.94] 2.6E-03

Marie Lorans, Khalid Mahmood, Mark Clendenning

Serrated Polyposis Syndrome Total

Probands 418

Female 262 (62.7%)

Mean age at diagnosis (SD) yrs 44.3 (SD=15.3)

Range (yrs) 15-83

Mean polyp count (SD) 34.7 (SD=32.2)

Range 5-300

ID Age Dx

Serrated

polyp

count

CRCFDR with CRC or

SPS (age Dx)Method RNF43 Variant

gnomAD_

All

gnomAD_

NFECADD REVEL

ID181 27 40 Yes -- HiPlex c.394C>T p.Arg132* -- -- 37 --

ID039 43 21 -- Brother CRC (39) HiPlex c.376G>C p.Ala126Pro -- -- 29.5 0.478

ID054 55 23 -- Id. Twin CRC (52) HiPlex c.1478G>A p.Ser493Asn 1.65E-05 3.00E-05 24.1 0.132

ID197 60 50 -- -- HiPlex c.136G>A p.Ala46Thr -- -- 27.5 0.351

ID264 15 5--

Sister SPS (21) HiPlex c.340C>T p.Arg114Trp 1.70E-05 1.53E-05 32 0.267

ID007 57 34 Yes -- WES c.443C>G p.Ala148Gly 4.99E-05 6.03E-05 21 0.17

Results - RNF43 Predicted Pathogenic Variants

Marie Lorans, Khalid Mahmood, Mark Clendenning

Somatic hotpots

RNF43 loss of function variant carrier family

RNF43 c.394C>T, p.Arg132*

RNF43 missense carrier families

RNF43 c.443C>G, p.Ala148Gly

RNF43 c.376G>C, p.Ala126Pro

RNF43 c.340C>T, p.Arg114Trp

Histological and Molecular Characterisation of

RNF43 p.Arg132* serrated lesions

RNF43 c.394C>T, p.Arg132*

Histologic type Stage Location MMR status

1. Mucinous

adenocarcinomaIIIC Ascending Proficient

2. Adenocarcinoma IIA Transverse Proficient

1. SSA/P No dysplasia Ascending Proficient

2. SSA/P No dysplasia Ascending Proficient

3. SSA/P No dysplasia Ascending Proficient

4. SSA/P No dysplasia Transverse Proficient

5. SSA/P No dysplasia Transverse Proficient

6. SSA/P No dysplasia Transverse Proficient

7. SSA/P No dysplasia Descending Proficient

8. SSA/P No dysplasia Descending Proficient

9. SSA/P No dysplasia Descending Proficient Marie Lorans and Peter Georgeson

Loss of heterozygosity

➢ LOH across RNF43 - 2/2 CRCs

➢ LOH across RNF43 - 7/9 Serrated

polyps

Mutational profile of RNF43 carrier CRCs

RNF43 c.394C>T, p.Arg132*

Peter Georgeson and Khalid Mahmood

FFPE whole exome sequencing of CRCs

Histologic

type Stage Location MMR IHC Somatic SNV CIMP

1. Mucinous IIIC Ascending Proficient BRAFV600EHigh

2. Adeno-

carcinoma IIA Transverse Proficient BRAFV600EHigh

Sporadic - MMR-proficient CRC

Sporadic - MMR-proficient CRC

Lynch - MMR-deficient CRC

Lynch - MMR-deficient CRC

Biallelic MUTYH - MMR-prof CRC

Biallelic NTHL1 - MMR-prof CRC

CRC#1 – RNF43 p.Arg132*

CRC#2 – RNF43 p.Arg132*

TMB

3.4

2.3

3.4

2.4

65.1

57.6

4.8

6.5

RNF43 c.394C>T, p.Arg132*

Loss of RNF43 in Adenocarcinoma

Retention of RNF43

RNF43 protein expression

Loss of RNF43 in Adenocarcinoma

Retention of RNF43

Ryan Hutchinson and Julia Como

Aim 2: Germline RNF43 mutations in CRC

Australian cohort

Cases: Incident CRC dx

between 18-59 yrs

(independent of FHx)

Controls: 18-59 yo from

electoral role

Cases = 861

Controls = 247

Ontario cohort

Cases: Incident CRC dx

between 20-74 yrs

(weighted to FHx)

Controls: Random

population sampling

Cases = 633

Controls = 575

Seattle cohort

Cases: Incident CRC dx

between 18-74 yrs

(independent of FHx)

Controls: Unaffected

individuals from

Department of Licencing

Cases = 459

Controls = 385

Population-based CRC-affected probands from Colon Cancer Family Registry

Case-control

cohort

No. Gender <40yrs 40-49yrs 50-69yrs >= 70yrs unknown

CRC-affected

Cases

1953 51% male 160

(8.2%)

682

(34.9%)

944

(48.3%)

131

(6.7%)

36

(1.8%)

Controls 1207 53% male 76

(6.3%)

273

(22.6%)

662

(54.9%)

196

(16.2%)

0

Mark Clendenning and Khalid Mahmood

Predicted Pathogenic RNF43 variants in CRC

Case ID Variant gnomad_all gnomad_nfe CADD REVEL

4052 c.1020C>A, p.His340Gln - - 22.7 0.11

9940 c.697C>T, p.Gln233Ter - - 38 -

Control ID Variant gnomad_all gnomad_nfe CADD REVEL

1215 c.2309-2A>G - - 23.8 -

1107 c.1085C>T, p.Ser362Phe - - 21.4 0.05

9990 c.1082C>T, p.Pro361Leu - - 21.3 0.05

0265 c.86G>C, p.Gly29Ala 1.63E-05 3.58E-05 24.7 0.23

Cases

N=1953

Controls

N=1207OR (95%CI) P value

2 (0.1%) 4 (0.33%) 0.31 (0.06 - 1.68) 0.2

Cases

N=1953

gnomAD

N=123,134OR (95%CI) P value

2 (0.1%) 396 (0.32%) 0.32 (0.08 - 1.28) 0.1

HiPlex Analysis

1. Bioinformatic analysis (GATK and UNDR-ROVER) - high quality variants

2. Identified ultra-rare variants - variant allele frequency in ExAC <5.0E-05

3. In silico Variant Effect Prediction - CADD >20 or REVEL >0.5

Mark Clendenning and Khalid Mahmood

Whole Genome and Exome Sequencing in SPS families

Mark Clendenning, Khalid Mahmood, Sharelle Joseland

Selection criteria – familial, young onset, high polyp count

• 11 families with multiple SPS (n=24 SPS total)

• 64 singletons

Candidate SPS susceptibility gene

• Rare variants

• High impact variants

• Segregation with all SPS affected

• Candidate gene list

• Prevalence of predicted pathogenic variants in candidate genes in n=64 SPS singletons

Mark Clendenning, Khalid Mahmood, Sharelle Joseland

Variant filtering strategy

Candidate

gene

SPS FamiliesN=11 (24)

SPS

SingletonsN=64

High impact variant

Predicted pathogenic

Gene A 3 2

Gene B 3 3

Gene C 2 3

Gene D 2 27

Gene E 1 0

Summary

Genetics of SPS

• Low clinical utility of screening SPS for known CRC and polyposis genes

– Other syndromic features (>20 adenomatous polyps, dMMR, macrocephaly)

are red flags for clinical genetic testing in patients who meet SPS criteria

RNF43 in SPS and CRC

• Significant increased burden of predicted pathogenic variants in SPS in GCPS

• Accounts for 1-2% of SPS

• RNF43 c.394C>T, p.Arg132* carrier serrated lesions showed LOH

• Genomics – currently CRCs from RNF43 carriers indistinguishable from sporadic CRCs

• Limited role of germline RNF43 in CRC

WGS/WES studies

• Families with multiple SPS-affected members to identify SPS susceptibility genes

Future directions

• Expand on molecular profile of RNF43-associated lesions - IHC and mutation spectrum

• Validation of candidate SPS susceptibility genes

• Exploring the role of common low-penetrance variants in SPS

Acknowledgments - Genetics of Colonic Polyposis Study

Funding – NHMRC GNT1125268

Family Cancer Clinics, AustraliaNZL Familial Gastrointestinal Cancer Service

• Prof. Susan Parry

• Dr Julie Arnold

• Varnika Vijay

• Dr Sonja Woodall

Centre for Epidemiology & Biostatistics, UoM

• Prof. Mark Jenkins

• A/Prof Aung Win

Melbourne Bioinformatics, UoM

– A/Prof Bernie Pope

– A/Prof Danny Park

Ohio State UMC, USA

• Dr Kevin Sweet

Cancer Care Ontario, Canada

• Prof. Steve Gallinger

• Dr Melyssa Aronson

• Ms Kara Semotiuk

• Dr Spring Holter

Cassandra Nichols Lesley Andrews Tess Schenberg

Lyn Schofield Linda Warwick Lara Lipton

Nick Patcher Stephanie Badman Alexandra Lewis

Geoff Forbes Meera Warby Katrina Monohan

Nicola Seed Hilda High Alisha Harpur

Bhavya Vora Belinda Dopita Ellen Pieper

Helen Mountain Emilia Ip Cass Hoskins

Jo Burke Tanya Dwarte Joshua Schultz

Julia Mansour Annabelle NG Joanne Ibister

Briony Patterson Skye McKay Lucie Hallenstein

Sarah Leighton Stephanie Groube Linda Cicciarelli

Mike Field Lynne McKay Tiffany MacIntosh

Mathilda Wilding Yoland Antill Lucinda Salmon

Ashley Crook Kirsty West Ainsely Campbell

Stephanie White Kirsty Storey Eryn Dow

Kathy Tucker Catherine Beard Rhiana Spinoso

Rachel williams Maie Walsh Giulia Valente

Zia Risha Michael Bogwitz Megan Cotter

Allan Spigelman Emily Allen Jasmine Bojadzieva

Annabel Goodwin Jessica Taylor Stephanie Di Palma

Alexandra Groves Adrienne Sexton Shona O'Connell

April Morrow Rebecca Purvis Marion Harris

Louise Lynach Lindy Hodgkin Cassandra Hoskins

Carol Ann Verrenkamp Joshua Schultz Jan Wakeling

Margaret Gleeson Frida Djukiadmodjo Michael Gattas

Sian Greening Maira Kentwell Rachel Susman

Emma Healey Shelby Taylor Simon Troth

Genomic Medicine & Family Cancer Clinic,

Royal Melbourne Hospital

• Prof. Ingrid Winship

• Prof. Finlay Macrae

Colorectal Oncogenomics Group, UoM• Dr Mark Clendenning

• Sharelle Joseland

• Dr Khalid Mahmood

• Marie Lorans

• A/Prof Christophe Rosty

• Dr Ryan Hutchinson

• Julia Como

• Peter Georgeson

• Tessa Greeves

• Dr Eric Joo

• Dr Harindra Jayasekara

• Romy Walker

• Susan Preston

• Magda Kloc

• Chris Cotsopoulous

• Neil O’Callaghan

• Thomas Green

• A/Prof Bernie Pope

• Dr Abi Ragunathan

• Dr Simin Daneshvar

Acknowledgements:

Colorectal Oncogenomics Group

© Copyright The University of Melbourne 2011