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

Desmoid fibromatosis Aim: 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 -catenin observed 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.

Sanger sequencing of CTNNB1 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 S45 T41 S45F

CTNNB1 mutation is not clearly associated with outcome WES of desmoids CTNNB1 mutation is not clearly associated with outcome CTNNB1 mutation and tumor location S45P T41A Extremity Abdominal wall Other Total S45F 19 (59%) 4 (12%) 9 (28%) 32 T41A 9 (19%) 8 (17%) 31 (65%) 48 None 3 (20%) 6 (40%) 15 S45P 1 (20%) 1 (20%) 3 (60%) 5 32 (32%) 19 (19%) 49 (49%) 100 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 Univariate Multivariate HR p Mutation (vs. T41A) None S45F 1.31 2.66 0.66 0.025 1.14 1.68 0.83 0.29 Extremity vs. Non-extremity 3.31 0.001 2.58 0.029 intraabdominal chest extremity other Extremity vs. abdominal wall p<0.05 Extremity vs. intraabdominal p<0.05

based on gene expression 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’

Whole exome sequencing of desmoid tumors 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 Illumina HiSeq flowcells 87% of the exomes were covered >88x Range 4-29 mutations per sample 29Mb sequenced per tumor <1 mutation/Mb

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

Whole exome sequencing of wild-type desmoid tumors WES of desmoids Whole exome sequencing of wild-type desmoid tumors Sample CTNNB1 mutation mutant allele frequency DES_1008 S45F 32% DES_884 T41A 36% DES_888 33% DES_931 DES_936 22% DES_938 46% DES_940 54% DES_926 38% DES_881 H36del 30% DES_1003 10% DES_956 21% DES_961 DES_890 None N/A DES_975 DES_955 DES_999 DES_1002 APC mutation I1918fs 61% K1462fs 70% known CTNNB1 mutation average 37% reads average 16% reads ‘wild-type’ tumors We were able to detect b-catenin mutations in 12/17 desmoid tumors, including 3 mutations not detected by clinical testing. We also found an additional 2 APC mutations (b-catenin pathway) in 2/17 tumors; mutually exclusive with b-cat mutations. Therefore, 14/17 tumors show b-catenin activation. RNA-seq confirms that b-catenin is highly expressed in both mutant and WT tumors, suggesting that they all rely on this pathway. Only 3 of nine ‘wild-type’ tumors without CTNNB1 or APC mutation.

Copy number alterations in desmoid fibromatosis 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.

Chromosome 6 loss in desmoid fibromatosis 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 Chromosome 6 CEP6 staining (red) 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

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.

BMI1 mutation in desmoid 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 Depth Forward Reverse Reference (Cytosine) 81014 (96.7%) 36784 44230 Alternate (Guanine) 2754 (3.28%) 1303 1451 Total 83898 38087 45681

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.

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).

Acknowledgements Funding: Samuel Singer, M.D., FACS Meera Hameed, M.D. 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. Gouri Nanjangud 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

Sequenced samples (n=110) WES of desmoids Clinical characteristics of patient cohort Characteristic Whole cohort (n=495) Sequenced samples (n=110) Age 15-25y.o. 26-45y.o. 46-65y.o. >65y.o. 95 (19%) 244 (49%) 110 (22%) 46 (9.3%) 17 (15%) 62 (56%) 24 (22%) 7 (6.4%) Location Abdominal wall Chest wall GI/Intraabdominal Extremity Other 88 (18%) 76 (15%) 100 (20%) 177 (46%) 54 (11%) 19 (17%) 12 (11%) 32 (29%) 34 (31%) 13 (12%) Primary 382 (77%) 85 (77%) Margin R0 R1 R2 Unknown 267 (54%) 173 (35%) 63 (13%) 2 (0.40%) 50 (45%) 52 (47%) 8 (7.2%) 0 (0%) Size <=5cm >5cm, <=10cm >10cm 148 (30%) 200 (40%) 125 (25%) 22 (4.5%) 20 (18%) 47 (43%) 42 (38%) 1 (0.91%) FAP 15 (3.0%) 2 (1.8%) 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).

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

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