1. INTERNATIONAL SARCOMA KINDRED STUDY: FIRST GENE SCREENS

2. ISKS cohort

3. ISKS hereditary cancer syndromes 658 ISKS families 109 Uninformative 104 No cancer in 1 o /2 o rels 337 No defined syndrome 108 Recognised syndrome 69 Clinically suspicious 268 Not suspicious 56 Chomp/L FS 21 BRCA 17 Lynch like 14 Other 16 ++ 42 + 11 +++

4. 658 ISKS families 109 Uninformative 104 No cancer in 1 o /2 o rels 337 No defined syndrome 108 Recognised syndrome 69 Clinically suspicious 268 Not suspicious 56 Chomp/L FS 21 BRCA 17 Lynch like 14 Other 16 ++ 42 + 11 +++ 19% of these families were referred to a familial cancer clinic ISKS hereditary cancer syndromes

5. Cancer genes: frequency and penetrance Common low penetrance Rare high penetrance

6. Common low penetrance Rare high penetrance Cancer genes: frequency and penetrance

7. QUAL>60 and total read depth ≥20 and ≥8 reads with variant allele and ≥20% reads had variant allele 17,001 variants No dbSNP ID or allele frequency<0.05, EVS EA population 26,365 variants Class I GenomeTrax disease mutant AND NOT benign by Condel Class II STOP, FS, essential splice, initiator codon variant Class III Missense, Condel 4 ISKS Filtering pipeline 582,235 variants non-synonymous, STOP, essential splice site, frameshifts 54,718 variants Strand bias (FS) -10 8,701 variants Occurs ≤3 times in cohort 1452 variants Rest

8. QUAL>60 and total read depth ≥20 and ≥8 reads with variant allele and ≥20% reads had variant allele 17,001 variants No dbSNP ID or allele frequency<0.05, EVS EA population 26,365 variants Class I GenomeTrax disease mutant AND NOT benign by Condel Class II STOP, FS, essential splice, initiator codon variant Class III Missense, Condel 4 582,235 variants non-synonymous, STOP, essential splice site, frameshifts 54,718 variants Strand bias (FS) -10 8,701 variants Occurs ≤3 times in cohort 1452 variants Rest ISKS Filtering pipeline CLASS I 86 variants in 85 probands CLASS II 41 variants in 38 probands CLASS III 299 variants in 225 probands No pathogenic variation 379 probands 116/681 (17%) ISKS probands carry 123 Class I or II variants 302/681 (44%) carry 426 class I-III variants

9. Mutation status and age of cancer onset HR 1.28, 95%CI 1.1-1.5, P<0.0001 HR 1.46, 95%CI 0.99-2.16, P=0.026HR 1.2, 95%CI 0.92-1.56, P=0.008 HR 1.32, 95%CI 1.1-1.6, P<0.0001 Hazard ratios: Mantel-Haenszel; Curve comparisons: Gehan-Breslow-Wilcoxon Median tumor latency Nil51y Class I40y Class II41.5y Class III42y All42y

10. QUAL>60 and total read depth ≥20 and ≥8 reads with variant allele and ≥20% reads had variant allele 17,001 variants No dbSNP ID or allele frequency<0.05, EVS EA population 26,365 variants Class I GenomeTrax disease mutant AND NOT benign by Condel Class II STOP, FS, essential splice, initiator codon variant Class III Missense, Condel 4 582,235 variants non-synonymous, STOP, essential splice site, frameshifts 54,718 variants Strand bias (FS) -10 8,701 variants Occurs ≤3 times in cohort 1452 variants Rest ISKS Filtering pipeline 90 (13%) individuals carry 2+ (214) variants 24 (4%) individuals carry 3+ (82) variants 90 (13%) individuals carry 2+ (214) variants 24 (4%) individuals carry 3+ (82) variants CLASS I 86 variants in 85 probands CLASS II 41 variants in 38 probands CLASS III 299 variants in 225 probands No pathogenic variation 379 probands

11. Polygenic contribution to age of cancer onset Curve comparisons: Logrank for trend; Gehan-Breslow-Wilcoxon for paired comparisons NumberMedian tumor latency Nil34751y 121044y* 26737y*** 3+2336y** TP531936y * P=0.008 **P=0.0001 ***P<0.0001 Trend: P<0.0001

12. Enrichment for deleterious variants in the ISKS cohort 950 case Australian control cohort – 55-80 years of age, selected on high/low bone density – Free of co-morbidity, including cancer Whole exome panel To correct for platform bias, compare ratios of deleterious to synonymous variation – Deleterious = FS, stop, ESS, initiator codon variant; or missense variant with Condel score > 0.7

13. Gene-specific mutation patterns in the ISKS cohort

14. * * * * CHEK2 TP53 CDKN2A ATM

15. (17) ATM (17) NBN (11) NBN (11) RAD50 (13) RAD50 (13) MRE11A (4) MRE11A (4) BRCA1 (2) BRCA1 (2) PALB2 (6) PALB2 (6) BRCA2 (18) BRCA2 (18) BRIP1 (12) BRIP1 (12) RAD51C (1) RAD51C (1) FANCI/D2 (5/2) FANCI/D2 (5/2) TP53 (19) TP53 (19) FANC A-C/E-G/L/M (62) FANC A-C/E-G/L/M (62) CHEK2 (5) CHEK2 (5) Homologous recombination/TP53 pathway n = 201 (Classes I-III) DNA damage and homologous recombination pathway ATR (14) ATR (14) TP53BP1 (6) TP53BP1 (6) FAM175A (4) FAM175A (4) WRAP53 (4) WRAP53 (4) CDKN2A (2) CDKN2A (2)

16. LDE225 PTCH1 ISKS therapeutic implications

17. ISKS Acknowledgements ISKS participating families International Steering Committee Isabelle Ray-Coquard Ajay Puri Study manager – Mandy Ballinger Data Manager – Eveline Niedermayr Research Assistant – Kim Riddell Thomas lab David Goode Tiffany Pang Arcadi Cipponi PeterMac FCC Gillian Mitchell Paul James Mary-Anne Young Alex Lewis Funding Rainbows for Kate Foundation NHMRC VCA PeterMac Stephen Wong Alex Dobrovic Genomics Core Richard Tothill Aga Borcz University of Queensland Matt Brown Paul Leo kConFab staff Heather Thorne

18. ATM (5) ATM (5) NBN (8) NBN (8) MRE11A (1) MRE11A (1) BRCA1 (1) BRCA1 (1) PALB2 (2) PALB2 (2) BRCA2 (3) BRCA2 (3) BRIP1 (3) BRIP1 (3) RAD51C (1) RAD51C (1) FANCI/D2 TP53 (19) TP53 (19) FANC A-C/E-G/L/M (12) FANC A-C/E-G/L/M (12) CHEK2 (5) CHEK2 (5) Homologous recombination/TP53 pathway n = 62 (Class I/II) Genetic pathways ATR (2) ATR (2) TP53BP1 (1) TP53BP1 (1) WRAP53 (-) WRAP53 (-) RAD50 (2) RAD50 (2)

19. 116/681 (17%) ISKS probands carry 123 Class I or II variants 302/681 (44%) carry 426 class I-III variants 90 individuals carry 2+ (214) variants 24 individuals carry 3+ (82) variants ISKS Genotypes

20. ISKS Phenotypes & FCC referral Clinically suspicious Chomp/LFS BRCA Lynch -like Other + ++ +++

21. ISKS Sarcoma subtypes

22. ISKS gene panel APCCDH1EXT1FANCMMLH3PMS2RETSQSTM1WRN ARID1ACDKN2AEXT2FHMRE11APOLHRMI1STK11WT1 ATMCHEK1FAM175AIDH1MSH2PPARGRMI2TAF15XPA ATRCHEK2FANCAIDH2MSH3PRKAR1ARPA3TGFBR2XPC AXIN1DDB2FANCBKIF1BMSH6PTCH1RPA70 TNFRSF11A XRCC2 AXIN2DICER1FANCCKITMUTYHPTENRPS19TOP1 BARD1DKC1FANCD2LIG1NBNPTPN11SDHATOP3A BLMDNA2FANCELIG4NEIL2RAD50SDHBTP53 BRCA1ERCC2FANCFMDM2NF1RAD51CSDHCTP53BP1 BRCA2ERCC3FANCGMEN1NF2RAD51L3SDHDTSC1 BRIP1ERCC4FANCIMETPALB2RB1SMARCA4TSC2 BUB1BERCC5FANCLMLH1PMS1RECQL4SMARCB1VHL Batch 1 – genes in black Batch 2 – genes in black & red Agilent Haloplex custom panel of 85-101 gene CDS capture 681 ISKS probands