Aimee M. Crago, Juliann Chmielecki, Mara Rosenberg, Rachael O’Connor,

1. Whole Exome Sequencing and INTEGRATED GENOMIC ANALYSIS OF ‘WILD-TYPE’ DESMOIDS IDENTIFIES POTENTIAL DRIVERS OF TUMOR INITIATION Aimee M. Crago, Juliann Chmielecki, Mara Rosenberg, Rachael O’Connor, Caitlin Byrne, Mono Pirun, Nicholas D. Socci, Gouri Nanjangud, Margaret Leversha, Meera Hameed, Matthew Meyerson and Samuel Singer Memorial Sloan Kettering Cancer Center Connective Tissue Oncology Society October 31, 2013

2. WES of desmoids Desmoid fibromatosis • Locally aggressive tumor without metastatic potential. • Historically treated with surgery though high rates of local recurrence were reported (25-50%). • Associated in 85% of patients with mutation in CTNNB1 gene. • Nuclear -cateninobserved in >70% of samples by IHC. • Component of Gardner’s syndrome in conjunction with familial adenomatosis polyposis and APC mutation. Aim: • Determine molecular events that modulate desmoid initiation in the absence of CTNNB1 mutation.

3. WES of desmoids Sanger sequencing of CTNNB1 • Frozen samples collected from 2002-2013 (n=110). • 77% primary tumors. • Median follow-up 43 months. • Pathology confirmed by cryomold. • Normal tissue macrodissected from specimen. • 17/110 (15%) tumors without classic exon 3 mutation . T41A mutant AG T41A S45 ‘wild-type’ S45F mutant CT TCT ACC T41 S45F T41 S45

4. WES of desmoids CTNNB1 mutation is not clearly associated with outcome CTNNB1 mutation and tumor location S45P T41A none S45F None vs. T41A – n.s. None vs. S45F – n.s. T41A vs. S45F – p<0.05 p=0.001 Multivariate analysis abdominal wall intraabdominal chest extremity other Extremity vs. abdominal wall p<0.05 Extremity vs. intraabdominal p<0.05

5. WES of desmoids • Unsupervised • clustering • based on gene expression • 47 desmoid tumors • Evaluated by U133A 2.0 microarrays • S45F – Red • T41A – Blue • ‘wild-type’ – Black • No S45P included 7/36 ‘wild-type’ 4/11 ‘wild-type’

6. WES of desmoids Whole exome sequencing of desmoid tumors • 17 tumor samples. • 9 with no mutation in CTNNB1 – ‘wild-type’ desmoids • 8 tumors with CTNNB1 mutation • Normal blood from the same patients. • Agilent SureSelect v2 Exome bait for capture • IlluminaHiSeqflowcells • 87% of the exomes were covered >88x Range 4-29 mutations per sample 29Mb sequenced per tumor <1 mutation/Mb

7. WES of desmoids Somatic mutation in desmoid tumors • 46 significant mutations • Called by MuTect • Significance by MutSig • CTNNB1 mutationin 12/17 tumors (71%) not just 8 as expected based on Sanger. • 2 tumors with APC mutation. * *

8. WES of desmoids Whole exome sequencing of wild-type desmoid tumors known CTNNB1 mutation average 37% reads average 16% reads ‘wild-type’ tumors Only 3 of nine ‘wild-type’ tumors without CTNNB1 or APC mutation.

9. WES of desmoids Copy number alterations in desmoid fibromatosis Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y CTNNB1 mutations APC mutation Wild-type APC CTNNB1 mutations APC mutation Wild-type • CapSeg algorithm from Broad Institute (McKenna et al., in preparation) • Few copy number events are observed in desmoid fibromatosis. • APC mutants with likely heterozygous deletion of the gene.

10. WES of desmoids Chromosome 6 loss in desmoid fibromatosis Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y CTNNB1 mutations APC mutation Wild-type CEP6 staining (red) Chromosome 6 24% nuclei with single chromosome 6 by FISH CTNNB1 mutations APC mutation Wild-type 56% nuclei with single chromosome 6 by FISH • - CapSeg algorithm from Broad

11. WES of desmoids Chromosome 6 deletions • Copy number events affecting chromosome 6 are uncommon in oncogenesis. • High incidence in HNPCC families without aberrations in the microsatellite instability pathway. • Identified in a subset of medulloblastoma patients with Wnt pathway activation • Potential tumor suppressors on chromosome 6 known to downregulate Wnt signaling: • QKI, DACT2, BMP6, LATS1 Clifford et al. (2006) Cell Cycle 5(22): 2666-70. Blӓker et al. (2008) Genes Chrom. Cancer 47(2): 159-64.

12. WES of desmoids BMI1 mutation in desmoid APC chr 6 del chr 6 del APC ? CTNNB1 mutations - BMI1 c.175C>G - 12/142 reads (7.9%) - Confirmed by orthogonal 454 sequencing

13. WES of desmoids Whole exome sequencing identifies BMI1 mutation in a ‘wild-type’ desmoid • Stem cell marker • Positively regulated by Wnt • Positive regulator of Wnt • BMI1 mutation a rare event – • Not detected by Sanger sequencing in any additional (n=96) desmoids Cho et al. (2013) J. Biol. Chem. 288(5): 3406-18.

14. WES of desmoids Conclusions • Patient outcomes and gene expression profiles associated with ‘wild-type’ desmoids do not differ significantly from those associated with CTNNB1 mutated tumors. • Next generation sequencing identifies CTNNB1 mutation in a significant number of ‘wild-type’ desmoids • ~95% of tumors can now be associated with mutated CTNNB1 or APC. • Genomic events that have potential to regulate Wnt signaling are identified in all desmoids without mutation in CTNNB1 or APC (e.g., chromosome 6 loss, BMI1 mutation).

15. WES of desmoids Acknowledgements Samuel Singer, M.D., FACS Meera Hameed, M.D. Matthew Meyerson, M.D., Ph.D. Juliann Chmielecki, Ph.D. Nicholas Socci, Ph.D. Li-Xuan Qin, Ph.D. Raya Khanin, Ph.D. Caitlyn Byrne, Ph.D. Margaret Leversha, Ph.D. GouriNanjangud Ph.D. Mara Rosenberg, Ph.D. Mono Pirun, Ph.D. Rachael O’Connor, B.S. Katherine Thorn, B.A. Funding: Kristin Ann Carr Foundation Cycle for Survival NCI SPORE in Soft Tissue Sarcoma American College of Surgeons Slim Initiative for Genomic Medicine

16. WES of desmoids Clinical characteristics of patient cohort • Frozen samples collected from 2002-2013 (n=110). • Clinical characteristics as compared to all surgically resected tumors between 1982 and 2011. • Median follow-up 43 months (range 0 -126 months).

17. WES of desmoids 34 y.o with 10cm rectus sheath tumor.

18. WES of desmoids 454 sequencing – signal to noise ratio BMI1 c.175C>G