International Society for Gastrointestinal Hereditary

Tumours (InSiGHT):

Edited highlights

Düsseldorf, 2009

InSiGHT’s history• In 1985 polyposis experts (Dukes, Bussey, Morton et al … ) met at Leeds

Castle, Kent, UK. In 1989 the Leeds Castle Polyposis Group (LCPG) was formally established.

• The International Collaborative Group on Hereditary Non Polyposis Colorectal Cancer (ICG-HNPCC) was conceived in Jerusalem, in 1989.

• In 1990 the first formal meeting of the ICG-HNPCC was organized in Amsterdam.

• In 1997 the first joint meeting of LCPG and ICG-HNPCC took place in Noordwijk.

• At the meeting in Venice in 2001 it was agreed that the ICG-HNPCC should formally merge with the LCPG to form a new society.

• InSiGHT’s journal is Familial Cancer

• www.insight-group.org

• Members include physicians, geneticists, surgeons, pathologists, scientists, counsellors etc

• It co-ordinates Registries on an International basis

Pre-meeting

• UVs• Feedback from Sian Tavtigian post-Lyon Feb 09• Adding phenotype to InSiGHT mutation database• Functional assays• Worldwide collaborative project on UVs

– International interpretation panel – Feb 2013 InSiGHT meeting in Melbourne will be linked

to HVP– Nature Genetics paper of all 10,000 MMR mutations

Phenotype

• No phenotype• FHx +/-• AC +/-• Summary FH based on Barnetson & Chao• Pedigree (for segregation)

Algorithms• Family mutation probability: Wijnen• Individual mutation probability: Barnetson R et al NEJM 2006; 354:2751-63.• [See also the PREMM1,2,6 model]

• Developed from a population <55y and validated on a population <45y. ?performance >55

• https://hnpccpredict.hgu.mrc.ac.uk• Pr/(1-Pr) = 1.39 x 0.89Age x 2.57Gender x 4.45Location x 9.53Sync/Met x

46.26CRCFH(<50) x 7.04CRCFH(=>50) x 59.36ECFH• Stage 1 and Stage 2

* Age is expressed in years at time of diagnosis * Gender is 1 = male, 0 = female * Location is 1 = proximal, 0 = distal tumours (Proximal colon = any of the following sites Splenic flexure, Transverse colon, Ascending colon/hepatic flexure, Caecum,

Appendix) * Synchronous and/or metachronous tumours are: Sync/MetTumour = 1 and no Sync/MetTumour = 0; * There are three possible colorectal cancer family history categories CRCFH(<50) and CRCFH(>=50): 1 and 0 if the youngest affected first degree relative was aged <50yrs 0 and 1 if the youngest affected first degree relative was aged >=50yrs 0 and 0 if there were no affected first degree relatives; * Family history of endometrial cancer: ECFH = 1 if there are any first degree relatives with endometrial cancer ECFH = 0 if none.

InSiGHT summary FH phenotype dataset

• As these phenotype parameters are used to assess mutation probability, they can be used to inform the Bayesian probability of risk for any unclassified variant. Hence:

• Stage 1

– Age, gender, location, sync/met, FH, ECFH

• Stage 2– IHC– MSI

• Stage 3– Additional family data: e.g. # affecteds– Co-segregation

UVs likelihood ratios

• Five classes

• 5: >0.99• 4: 0.95-0.99• 3: 0.05-0.95• 2: 0.001-0.05• 1: <0.001

• Need to quantitate “natural variation” in results

• HGVS @ UNESCO HQ, Paris, Jan [May] 2010

Functional assays

• (IHC)

• Robert Hofstra, Leiden• Couch FJ Hum Mutation 29:1314-26

• Assymetrical F-tagged PCR construct with an engineered mismatch

• In vitro expression of MSH2 etc• Add to –MSH2 brew• Relative heights of cut/uncut peaks gives measure of in vitro

activity

• Quick, cheap, simple, easy …

PMS2 & BRAF status

• Abnormal PMS2 expression alone in tumours• Strong indicator of PMS2 mutations• MLH1 + PMS2 absent … but weak MLH1 on re-testing !• iQC & EQA

• Others find (uninterpretable) complex combinations of IHC abnormality

• Tumours with BRAF V600E in PMS2 families• Probably sporadic• Due to ascertainment bias consequent upon clinical referral

criteria• PMS2 carriers ascertained secondary to homoz children have v

little if any FH

• Houlston, Dunlop, Tomlinson, Gallinger, Peters et al

• 10 SNPs = 6% of FCRC risk• Nature Genetics 40:1426-35

• [100 SNPs = 80%]• [172 SNPs = 100%]

• Issues:• Population heterogeneity• Rare private variants• Incomplete SNP capture• Copy Number Variants need studying• Gene-Environment and GxG interactions• Deep re-sequencing needed, to find private variants

% of FCRC risk

SNPs (n)

Genome-Wide Association Studies

Genome-Wide Association Studies

• LS Modifiers (Houlston)

• CycD1 G242A MSH2, but not MLH1• MTHFR

– C677T & age of onset – iffy

– A1298C – iffy

– but compound hets 15 y younger

• IGF1(dinuc) <18rpts = younger onset & age of onset α 1/rpt

• RNASEL R462Q• HFE (?)• … 4 others

Main meeting

PMS2-PMS2CL hybrids, Wimmer

• PMS2-PMS2CL hybrid alleles• Inv dup 7p22.1• φ = exons 9, 11-15• Non-homol recomb• Allele drop out

• Hybrid 1-12,φ13-15• PCR by exon specific SNPs - 13: 2253T/C; 14: 2324A/G; 15+

*92dupA

• 98/300 alleles are hybrid

MSI tumour immunology• Tumour infiltrating lymphocytes . in tumours with MSI• Novel antigens in MSI tumours due to coding microsatellite

(MS) mutations

MSI tumour immunology

• T cell Immune responses• FrameShiftPolypeptide-specific T cells are present but tumours grow• FSP-specific T cells are present in LS patients, without tumours …• Patients are probably being immunised by tumours which never

present clinically

• Humoural IR• Highest after resection or adv tumours• Low level ab in healthy LS pts

• Immune evasion:• HLA Class I expression is lost because of beta2M/HLA A2 – coding

MS muts• Known for a long time• New finding:• HLA Class II is lost through CIITA 1962_1963insC , RFX5 56delC,

RFXANK, RFXAP muts

MLH1 epimutations, Megan Hitchins

• MLH1 epimutation segregates in a 3 gen fam• Soma-wide, allele specific, mosaic• T allele methylated• Loss of T allele in mRNA• + loss of C allele in tumour• 3 affecteds and 5 unaffecteds had soma methylation (14-49%)• AD transmission• Father’s sperm demethylated• Son has soma methylation• So, now reversible non-Mendelian & Mendelian MLH1 epimutation• ?distinct mechanisms

EPCAM/TACSTD1

• Different deletions, vary in size, cause same effect

• Methylation of MSH2 promotor• Common Dutch founder EPCAM c.859-1462_*1999del• 5 patients

– 1 AC+– 4 FH– CRCa x31, DUCa x1, SBCa x2, BRCa x1, PRCa x3, Leuk x2, ENCa x0

• Homoz mutn of EPCAM causes congenital tufting enteropathy• ?hemizygosity contributes to cancer

EPCAM MSH2

AluJo

CAPP2, Burn

• LS pts treated with

• Aspirin 600 mg/d• Resistant starch 30 g/d• Both• Neither (placebo)• for• 29 months (7-74 months)

• No apparent initial effect on tumour incidence, but delayed action, and significant effect now being seen (as in other aspirin studies)

• Fodde: aspirin attenuation of betaCATenin/TCF4 pathway, ^stability of betaCAT

• Ca stem cells produce IL4, but aspirin inhibits IL4

Novel CRCa genes, Katachalam

• 32 YOCC with MSS tumours• aCGH• Novel CNVs in 5• Dup PTPRJ/DEP-1 11p11.2• 110-160 Kb, contains 1-2 genes• Candidate human TSG & mouse susceptibility locus• So, 3 lines of evidence suggesting significance

• 1500 Fam CRCa + controls tested for the CNV• 2 dups and 4 dels• Various sizes• ? Why both del and dup associated with same phenotype

MMR mut prob model, Kastrinos

• MMRpredict• MMRpro

• PREMM1,2,6

• http://www.dfci.org/premm

MSH6 risks, Jenkins

• 70y 80y HR• CRCa M 22% 44% 7.6• F 10% 20%

• ENCa 26% 44% 26

• Others* M 3% 6% 0.8• F 11% 22% 6

• *Gastric, Small bowel, Kidney, Ureter, Ovary, Brain

• Differences between 70 and 80 are due to large rise in population risks at this age

MSH2 extracolonic risks (Standardised Incidence Ratios), Engel (German Registry)

• M F SIR• Bladder x75y 10.1 15.4 7.6• 5.8% 4.8%

• Breast 1.84 (1.4 – 2.4)• 15.1%

• Ovary 13.4 MSH2/6 highest• 8.0% MLH1 lower

• MLH1 MSH2 MSH6• Gastric, SB High High Low• Bladder Low High Low• Ovary (low) High High

HFE and CRCa risk, Scott

• HFE C282Y and H63D

• x3 CRCa risk

• H63D HR 2.93 (1.3 – 6.4) p 0.007

• If H63D homoz then ~6y earlier onset

• Shiu et al Int J Ca 125:78-83

• However:

• HFE is within HLA on chr 6, so is this an association with HLA and not HFE per se …

• Another group reported an association with C282Y but not H63D, so could well be either a ‘spoof’ association or very population specific, implying HLA not HFE.

LS Modifiers, Wijnen

• 14 SNPs associated with CRCa• Are they associated with LS risks?• On testing Leiden’s mega set of LS families:

• All M F• 8q23.3 (x2) x3 rs16982766 C allele• (related to MYC)

• 11q23.1 x3 rs3802842

• 4q13.1 x2 rs4355419

• >3 risk alleles increase risks further• ?warrants more intensive surveillance

Prostate Ca risk in LS, Pål Møller

• MSH2, MLH1, MSH6

• Obs 9 cases of PRCa, expected 1.52, p <0.01

• MSH2 MLH1 MSH6 PMS2• 6 0 2 1

• 7/8 tumours abnormal IHC• Age 60.4, cf. 66.6, p <0.01

• 5/8 had high Gleason scores (8-10), v signif.

IHC in LS, Klarskov & Frayling

• Interobserver variability– Variability

– Tyros and experts no different

• UK NEQAS ICC– Technical scheme

– ~50% of 55-74 labs score <12

MutYH

• Elke Holinski-Feder• MutYH mutations now observed in a wide variety of phenotypes: FAP, AFAP,

ATFAP

• Christina Thirlwell, London• E466X diagnosed 10y earlier than Caucasians with 165/382• Later stage Ca• ?more adenomas• Café-au-lait spots ?!

• Sampson• CRCa risk in carriers• SIR 2.12 (1.3-3.3)

MutYH

• Elke Holinski-Feder• MutYH mutations now observed in a wide variety of phenotypes: FAP,

AFAP, ATFAP

• Christina Thirlwell, London• E466X diagnosed 10y earlier than Caucasians with 165/382• Later stage Ca, ?more adenomas, Café-au-lait spots ?!

• Sampson• CRCa risk in carriers SIR 2.12 (1.3-3.3)

Hereditary Pancreatic Ca, Bartsch

• Testing

• BRCA2 if 2 FDR with PACa, or any 3 rels with PACa

• p16 if malignant melanoma in F/SDR of PACa case

• PRSS1, MMR, LKB1, ATM, APC as per FH