Rare tumours: some recent data and ideas Part one – Stromal tumours David G. Huntsman BC Cancer Agency Vancouver General Hospital University of British Columbia Canada Research Chair in Molecular and Genomic Pathology
COI – disclaimer • Relationships with commercial entities – Contextual Genomics – founder and CMO – Research support received from - Sanofi, Pfizer, Takeda, Novartis, GSK • COI-Mitigating strategies – Generic drug names only used – No products or other work from Contextual Genomics to be discussed
Cell context and mutation: The evil twins of cancer Partners in crime from the origin of cancers through to the development of acquired resistance to targeted therapy
Outline • GCT – time for a rethink • SCCOHT – new treatment approaches imminent
FOXL2 mutation 4 granulosa cell tumors of the ovary V V O O A A 4 1 9 1 9 V V O O A A 1 2 5 9 9 2 Shah et al (2009) NEJM
FOXL2 IHC and mutational analysis as a standard diagnostic (Kommoss et al Mod Path2013) Confirmation of FOXL2 aGCT specific c.402C>G mutation by Sanger Sequencing TaqMan based digital mutation assay for FOXL2 aGCT specific c.402C>G mutation
Cross-cancer mutation summary for FOXL2 (147 studies ) None of the cancers included in c-bioportal had the C134W mutation as none were GCT *No mutations at C134W Missense Mutations Truncating Mutations Question : what does a cancer specific mutation mean to patients 7
Melissa McConechy Winnie Yang Blake Gilks Aline Talhouk Anniina Färkkilä Mikko Anttonen Markku Heikinheimo Leila Unkila-Kallio Hugo Horlings Hannah van Meurs Maaike Bleeker Stefan Kommoss Sarah Brucker
3 European Centers (n =336) Finland, The Netherlands & Germany McConechy, Färkkilä, Horlings et al JNCI 2016
• Patient’s disease relapsed after 2.6 years and died of disease 3 years from Diagnosis • She had been misdiagnosed as GCT – personal opportunity cost
Overall survival of women with molecularly define AGCT is not distinct from population based controls
Why is the recurrent FOXL2 mutation so specific for adult type GCT In adults expression is restricted to specialized stroma of ovary, uterus and fallopian tube Image from protein atlas
FOXL2 in Ovarian and Mullerian Stroma Ovary Fallopian tube Endometrium Endocervix
The FOXL2 mutation is diagnostic for aGCT and its oncogenicity linked to granulosa cell pathophysiology Follicle Corpus luteum Ovulation AGCT Granulosa cell Luteinized granulosa cell FOXL2-mutant granulosa cell Theca cell Oocyte
Other targets What else is going on in GCT genomes? • TERT promoter mutations in 1/3 • Smattering of other cancer mutations including targetable PIK3CA pathway mutations • LOH of chromosome 22
Molecularly defined AGCT The power of a correct diagnosis • Prior studies overestimated death from disease due to contaminating misdiagnosed cases. • Missed diagnosed cases dominate early relapses and likely past trials studied in clinical • Usually an indolent disease or managed well by primary and secondary surgeries • Treatment needed for inoperable cases of molecularly defined AGCT • Medium time to relapse >7 years therefore current follow-up strategy likely useless – needs testing • Cell free DNA could be used as an adjunct to monitor patients in trials • If no means of targeting FOXL2 derived then analysis for other targetable mutations through inclusion in a basket type trial may be feasible
Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) • Described by Scully 1979 • A rare disease in young women • Sometimes familial • Undifferentiated small cells • Elevated serum calcium level (60%) • Very lethal 35% 2yr survival
Small Cell Carcinoma of the Ovary, Hypercalcemic type (SCCOHT) • Highly aggressive tumor with 2- • Rare and rapid growth, but poorly year survival less than 35% differentiated tumor with small rounded cells and variable numbers of larger cells, 60% with hypercalcemia • Sporadic and familial • Mean age at diagnosis: 24 yrs (range 14 months – 43 yrs.) • Cell of origin is unclear • Surgical debulking followed by aggressive chemotherapy/radiotherapy combination Estel et al. Arch Gynecol Obstet 2011
SMARCA4 inactivating mutation- the only recurrent somatic mutation in SCCOHT SMARCA4 Germline 1 QLQ HSA BRK DEXDc SNF2_N HELICc Bromo 1,647 a.a. Somatic Nonsense mutation Missense mutation Splice site mutation Kupryjanczyk et al. Polish J Patho l 2013. Ramos, Karnezis et al. Nature Genetics 2014. Witkowski et al. Nature Genetics 2014. Jelinic et al. Nature Genetics 2014 Ramos et al. Rare diseases 2014 Karnezis et al J Path 2016
• • David Huntsman Jeff Trent • • Anthony Karnezis Holly Yin • Shary Chen • Will Hendricks • Winnie Yang • Jessica Lang • Sarah Maines-Bandiera • Pilar Ramos • Christine Chow • David Craig • Clara Salamanca • Alex Sekulic • BC cancer agency • Ashley Wang • • Marcel Bally Patrick Pirrotte • Michelle Woo • Nancy Dos Santos • Jeff Kiefer • Michael Anglesio • Nicole Wretham • • Dawn Cochrane Dana Masin • • Hong Yan Friedrich Kommoss • Ada Leung • Bernard Weissman • Gregg Morin • Krystal Orlando • Shane Colborne • Shujie Song
Re-expression of SMARCA4 induces neuronal differentiation in SCCOHT cells GFP SMARCA4 MAP2 BIN67 SCCOHT1 100 % cell confluence pLDpuro-GFP 80 pLDpuro-SMARCA4 60 40 20 0 0 24 48 72 96 120 144 168 Time (hours)
Cross-cancer mutation summary for SMARCA4 (147 studies ) Missense Mutations Truncating Mutations In-frame Mutations 24
SCCOHT can be familial: associated with germline SMARCA4 mutations in 40% of cases Witkowski, Nature Genetics 2014 and Gyn Onc 2016
SWI/SNF chromatin remodeling complex SMARCA4/2 SMARCA4/2 SMARCA4 - Complex composition: Mutually exclusive ATPases: SMARCA4/A2 Core subunits: BAF155, BAF170 and SNF5 Accessory subunits: i.e. ARID1A/B, BAF180, etc - Open chromatin structure for transcriptional regulation of gene expression - Regulate many biologic pathways, i.e. cell cycle control, cell death, cell differentiation - Frequently mutated in cancer - SMARCA4-deficient lung cancer cells depend on SMARCA2 activity for survival Wilson BG and Roberts CW. Nature Reviews Cancer 2011
SWI/SNF ATP-Dependent Chromatin Remodeling Complex SCCO-012 SCCO-002 SCCO-014 SCCO-015 SCCOHT1 PDX-040 PDX-065 BIN67 ACTB BCL11B DPF3 SMARCA2 PHF10 SMARCD2 ARID1A DPF2 SMARCA4 BCL11A BAF47/INI1 BRD9 BRD7 ACTL6A ARID1A SMARCB1 ARID1B PBRM1 SMARCC1 BCL7B DPF1 SMARCD1 SMARCE1 ACTL6B SS18 SMARCC2 SS18L1 ARID2 SMARCD3 Log 2 � ra o� 3� 2� 1� 0� -1� -2� -3� Figure modified from Riccio, Nat Neurosci 2010
Dual loss of SMARCA4/A2 in SCCOHT SCCOHT1 SVOG3e BIN67 B KGN SMARCA4 SMARCA2 Vinculin 4 Relative mRNA level SMARCA2 3 SMARCA4 2 1 0 This cancer is defined by two features that would be lethal Karnezis, Wang, Ramos et al. J Path 2015 In most other cell types
Model of SCCOHT Pathogenesis Why is it so rare? synthetic BRM+ lethality Normal cell ovarian cells ubiquitous SMARCA4 SCCOHT mutation Immature BRM- teratoma cell rare
Working model and Hypothesis Neuronal differentiation EZH2 suppression? Progenitor cells Stalled differentiation X oncogenic transformation • Histone methylatransferase activity (EZH2) • Primarily trimethylates histone H3K27 • Silence gene • Maintain stem cells • Upregulated by SMARCB1 depletion • Required for SMARCB1-induced tumorigenesis
EZH2 is highly expressed in SCCOHT Strong diffused staining 19/24 (79%) 12 Normal ovary 10 SCCOHT Relative gene expression 8 6 4 Variable staining 2 5/24 (21%) 0 EZH2 EZH1 EED SUZ12
EZH2 is downregulated in SMARCA4- reexpressing SCCOHT cells SCCOHT1 BIN67 SMARCA4 - + - + SMARCA4 EZH2 Vinculin H3K27Me3 Total H3
Depletion of EZH2 suppresses the growth of SCCOHT cells 140 BIN67 120 shEZH2_1 shEZH2_2 shctrl 100 shCtrl % Growth shEZH2_1 80 EZH2 shEZH2_2 60 H3K27Me3 40 H3 20 0 BIN67 COV434 SCCOHT1 ES-2 SCCOHT cells
EZH2 inhibitors suppress SCCOHT cell growth in vitro A B BIN67 SCCOHT1 BIN67 SCCOHT1 GSK126 0 1 5 0 0.1 1 μM EPZ-6438 0 0.1 0.5 0 0.1 0.5 μM H3K27Me3 H3K27Me3 H3 H3 GSK126 EPZ-6438 C D 100 100 BIN67 SCCOHT1 COV434 % Survival % Survival G401 BIN67 ES-2 50 SCCOHT1 RMG1 50 COV434 OVCAR-8 G401 OVISE ES-2 OVTOKO RMG1 OVCAR-8 0 0 -2 -1 0 1 -2 -1 0 1 Log(conc) Log(conc)
EPZ-6438 induces neuronal differentiation in SCCOHT cells MAP2 DAPI Merged DMSO EPZ-6438 day 7 EPZ-6438 day 12 BIN67 cells
EPZ-6438 suppressed SCCOHT xenograft tumor growth in mouse A B BIN67 xenograft model BIN67 xenograft model 1200 120 vechicle 100mg/Kg EPZ-6438 1000 100 200mg/Kg EPZ-6438 QD 800 80 Tumor volume BID Percentage Survival 60 600 40 400 vechicle 20 200 100mg/Kg EPZ-6438 200mg/Kg EPZ-6438 0 0 0 10 20 30 40 50 13 18 23 28 33 38 43 48 Days after cell inoculation Days after cell inoculation EPZ-6438 Control day 28 1 2 3 1 2 3 H3K27Me3 H3
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