Platform session presentation at the 2016 American Society of Human Genetics Conference in Vancouver by Dr. David Goode, Peter MacCallum Cancer Centre, Melbourne, Australia.

1. The connection between germline
risk variants and somatic mutation
patterns in sarcoma
Dr. David Goode
Bioinformatics and Cancer Genomics
Peter MacCallum Cancer Centre
Melbourne, Australia
Oct 19, 2016

2. Sarcomas tend to occur earlier in life
“Sarcomas are rare cancers that develop in the muscle, bone, nerves,
cartilage, tendons, blood vessels and the fatty and fibrous tissues”
- Sarcoma UK (sarcoma.org.uk)
Soft-tissue sarcomas All Cancers
Cancer Research UK (www.cancerresearchuk.org/cancer-info/cancerstats/incidence/age/#Cancer)

3. Investigating inherited sarcoma risk
 Sequenced exons of 72 genes with known or suspected roles in
cancer
 Germline (blood) samples from 1162 probands from International
Sarcoma Kindred Study (ISKS)
 Genetic variants inherited from parents (99%)
 Agilent Haloplex capture
Functional Class # of Genes Functional Class # of Genes
Tumour Suppressors 40 DNA Repair 39
Cell Cycle 28 Response to DNA damage 22
Recombination 22 Ser/Thr Kinases 11
Ballinger et al, The Lancet Oncology, 2016 17(9):1261-71.

4. Identifying putatively pathogenic variants
 Class 5
 Established association with cancer (HGMDPro)
 Functional evidence of pathogenicity
 Class 4
 Very likely protein damaging
 Frameshift, essential splice site or nonsense
 Class 3
 In silico indication of pathogenicity
 Predicted protein-damaging (Condel)
 Rare (<0.5%) in Exome Aggregation Consortium (ExAC)
Ballinger et al, The Lancet Oncology, 2016 17(9):1261-71.

5. DNA repair genes enriched in inherited
deleterious genetic variants in sarcoma patients
Sarcoma cases carry more damaging germline
SNVs and indels than controls
Age at first diagnosis associated
with # of deleterious variants
Ballinger et al, The Lancet Oncology, 2016 17(9):1261-71.
TP53, ATM, BRCA2, ERCC2 & ATR are top hit genes

6. How do inherited protein-damaging variants in DNA
damage response and repair genes lead to sarcoma?
 Sequence matched tumours from probands with variants in
 5 top hit genes
 Other DNA repair/DNA damage response genes
 Somatic mutations in same 72 genes targeted in the ISKS study
 Agilent SureSelect
 Varscan2
 DNA from FFPE blocks from 36 patients
 Average coverage per sample of ~1200X

7. From inherited deleterious variant to tumour (1)
Inherited
germline
variant Somatic
mutation?TumourGermline
Blood FFPE

8. Samples with second hits in same gene
Sample Gene(s) Germline Variant Somatic Mutation
Age at first
diagnosis
Sarcoma
Morphology
Clinical
Syndrome
12-1215 TP53 p.Arg156His p.Cys135Trp 45
Chondrosarcoma,
NOS
Chompret
12-1147 APC
p.His492ThrfsTer6 (C4
frameshift)
p.Ser1501LeufsTer6
(frameshift)
31
Aggressive
fibromatosis
FAP
11-654 PTCH1
p.Thr410MetfsTer15
(C4 frameshift)
p.Asn872Ile
+RB1 stop
+BRIP1 splice
[subclonal]
47
Leiomyosarcoma
(Kidney)
Gorlin
11-611
NBN
ATM
p.Leu238Ser (C3)
p.Gln466Ter
(C5 stop)
p.Arg215Trp (NBN)
+ TP53 frameshift
[subclonal]
61 Myxoid liposarcoma NS
NS = No familial cancer Syndrome

9. From inherited deleterious variant to tumour (2)
Inherited
germline
variant Somatic
deletionTumourGermline
Blood FFPE
Loss of
Heterozygosity
(LOH)

10. Samples with LOH at damaging variant
Sample Gene(s) Germline Variant Somatic Mutation
Age at
first
diagnosis
Sarcoma
Morphology
Clinical
Syndrome
11-807 TP53
c.559+1N>T
(C4 splice variant)
c.559+1N>T
(homozgyous)
25
Chondrosarcoma
(Testis)
Li-Fraumeni
11-920
(F)
BRCA2 p.Gly1529Arg (C5)
Gly1529Arg
(Homozygous) 35
Dedifferentiated
liposarcoma
NS
12-1108
ERCC4
FANCL
p.Ala13Thr (C3)
p.Thr372AsnfsTer13(C
4)
p.Ala13Thr
(homozygous)
79
Leiomyosarcoma
(Breast, Colon)
NS
12-1161
(F)
TP53
BRCA2
TP53 (Arg158His; C5)
& BRCA2
(Lys2950Asn; C3;
rs28897754)
TP53 & BRCA2
(homozygous)
NF2
(Arg291ValfsTer5)
26
Fibromyxosarcoma
(Breast)
Chompret

11. From inherited deleterious variant to tumour (3)
Inherited
germline
variant
Driver
mutation
TumourGermline
Blood FFPE
Somatic
mutations in
other genes

12. Samples with somatic mutations in TP53
NS = No familial cancer Syndrome
Sample Germline Variant Somatic Mutation
Age at
first
diagnosis
Sarcoma
Morphology
Clinical
Syndrome
11-561
(F)
BRCA2 (C5;
p.Asp596His)
TP53 (p.Leu238Ser) 64
Osteosarcoma
(Breast)
NS
10-335
ATR (C4;
p.Val316IlefsTer2)
TP53 (p.Arg248Gly) 28 Sarcoma NOS Uninformative
10-74
NF1 (C3;
p.Ser1838Cys)
TP53 (p.Tyr234His) 55 (68)
Liposarcoma
(secondary)
NS
10—91
(F)
4 C3 SNVs (FANCF,
BRCA2, MLH3, CDH1)
TP53
(Leu201SerfsTer43)
13 Ewings Chompret

13. # of somatic mutations by # of damaging germline variants
11-654
10-388
11-611
12-1147
12-1259
12-1131
11-720
372
12-1108
11-728
12-1161
10-163
10-79
11_900
10--91
13-1285
11-920
10-335
12-1215
11-559
11-561
12-1227
10-74
10--97
334
11-588
11-807
11-1040
11-1089
11-594
#ofsomaticmutations
0
50
100
150
200
4 Damaging Variants
2 Damaging Variants
1 Damaging Variants
P (2+ Variants) = 0.29

14. # of somatic mutations by # of damaging germline variants
11-654
10-388
11-611
12-1147
12-1259
12-1131
11-720
372
12-1108
11-728
12-1161
10-163
10-79
11_900
10--91
13-1285
11-920
10-335
12-1215
11-559
11-561
12-1227
10-74
10--97
334
11-588
11-807
11-1040
11-1089
11-594
#ofsomaticmutations
0
50
100
150
200
BRCA1/2
TP53
ATR
APC
ATM
P (ATM & APC) = 0.02
APC & ATM
carriers

15. Summary
 Potentially informative second hits in 12/36 samples to date
 Direct & indirect effects
 False positives or less obvious effects in remaining 23 samples?
 Point mutation burden higher in APC & ATM carriers
 Investigating association of germline variants with
 overall chromosomal instability
 somatic mutation signatures
 Sequencing additional patients

16. Acknowledgements
 PMC Molecular Genomics Core
 Gisela Mir Anau
 Sreeja Gadipally
 PMC Bioinformatics Core
 Jason Li
 Richard Lupat
 Peter Mac Pathology
 Familial Cancer Centre
 International Sarcoma Kindred Study (ISKS)
 David Thomas (Kinghorn Cancer Centre, Sydney)
 Mandy Ballinger (Kinghorn Cancer Centre, Sydney)
 Emma Galligan (Peter MacCallum Cancer Centre)
 The Sewell Family