Rare tumours: some recent data and ideas

Part two – endometriosis associated cancer

David G. Huntsman BC Cancer Agency Vancouver General Hospital University of British Columbia

Canada Research Chair in Molecular and Genomic Pathology Clear cell and endometrioid ovarian carcinoma represent around 20% of cases in North America Clinical disease heterogeneity Treated as one disease despite different clinical presentation and survival MP10

In retrospective cohorts expect up to a 20% misclassification in chart based pathology Impact of histotype changes • 300 cases centrally reviewed in 2002 • Reviewed again by same pathologist using 2014 WHO criteria : 54% concordance • New histotypes showed 98% concordance with second reviewer and stronger associations with outcome and biomarkers

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F Kommoss 2002 << F Kommoss 2014 = B Gilks 2014 Clear cell carcinoma of the ovary

• 2nd most common ovarian carcinoma subtype in NA (12%) and more frequent in Asia • Do not respond to standard ovarian chemotherapy • No other treatments available • May respond to radiotherapy • Molecular basis little understood • Weird cousins of renal CCC ARID1a mutations • Common in OCCC, Endometrioid of ovary and uterus and MSI positive gastric cancers • Found in cancer types without tp53 mutations • Occur in precancerous lesions but may not be initiating events • Not prognostic • Apart from association with PIK3Ca mutations no reproducible evidence that ARID1a mutant ovarian cancers are different from non mutant cases of the same type • Specific targeting of ARID1a mutant cancers has been challenging ARID1A Clear cell ovarian carcinoma

2010 NEJM Wiegand K, et al.; 2010 Science Jones S, et al. Genomic perspective Clear cell ovarian carcinoma

…and more recently highlighted… ERBB2 overexpressed and amplified Pro-oncogenic/transforming growth factor MET overexpressed and amplified Pro-oncogenic/transforming growth factor receptor

2010 GynOnc Anglesio M, et al. Activated pathways in OCCC

IL6  STAT3  HIF1A & HIF2A(EPAS1) (activation of hypoxia-related survival pathways)

Elevated levels: IL6 (Activated) STAT3 (Nuclear) HIF1A HIF2A (EPAS1)

Nuclear HIF1a in OCCC

Anglesio et al 2011. Clin Can Res Genomic disruptions Clear cell ovarian carcinoma

ERBB2 MET HNF1B

Anglesio 2011

Tan 2011 Endometrioid carcinomas

• Almost all are low grade yet some progress to higher grade cancers • Stage 1 low grade endometrioid carcinomas of ovary have a very good prognosis • Higher grade endometriod carcinomas and recurrent low grade need new treatment approaches • POLE mutations in 5% of cases, MSI in >20% • Beta catenin mutations in 50% of cases • Often present with synchronous uterine carcinoma Synchronous uterine and ovarian carcinomas • Up to 50% of low grade endometrioid carcinomas • Most are low grade and T1a • Due to excellent prognosis are considered to be separate primaries • Genomic and data molecular studies low resolution and interpreted as supporting separate primaries • Data to be shown non-validated comparisons of somatic mutations SEO_ SEO_ SEO_ SEO_ SEO_ SEO_ SEO_ SEO_ SEO_ TBG_ VAN_ TBG_ TBG_ VAN_ VAN_ VAN_ VAN_ VAN_ 22 54 15 31 08 04 07 33 40

Ovarian

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5 Endometriosis? 0 Endometrial

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In almost all cases the uterine and ovarian cancers share somatic mutations SEO_ SEO_ SEO_ SEO_ SEO_ SEO_ SEO_ SEO_ SEO_ VAN_ VAN_ VAN_ VAN_ VAN_ VAN_ VAN_ VAN_ VAN_ 22 27 29 60 14 43 56 58 65

Anglesio JNCI 2016 Copy number plots showing clonality Data and Conclusions • Clonal relationships between the endometrial and ovarian cancers seen in but one of 20 cases studies so far • Analysis of endometriosis and normal endometrium should inform whether these are metastatic cancers or whether a mutant field defect leads to both uterine cancer and through endometriosis • Are these true ? Endometriosis: the main risk factor for CCC and ENOC

• First described by Sampson in 1925. Pearce et al Lancet Oncology 2012

• 13,226 controls, 7,919 cases including 674 CCC and 1220 endometrioid Are these uterine cancers in the wrong place?

Gounaris et al, J Pathology 2011 Features found in OCCC can be found in adjacent endometriosis

MET (HGFR) amplification and overexpression in OCCC?

Regions of endometriosis that are synchronous to OCCC H&E IHC CISH Endometriosis

Adjacent atypical endometriosis

Fig 1 from Yamamoto et al, 2012. Mod Path

In second study by Yamamoto et al. MET overexpression and copy number changes were also correlated with atypical endometriosis that was synchronous with OCCC VOA1048 Adjacent Atypical Endometriosis vs. OCCC

21 DAH145 - VOA1048 (in some cases the adjacent atypical endometriosis is essentially cancer

X:153219079:HCFC1:coding:snvs:DAH145 X:153187162:ARHGAP4:coding:snvs:DAH145 X:117540879:WDR44:coding:snvs:DAH145 X:117043429:Y:RNA:coding:snvs:DAH145 X:37026831:FAM47C:coding:snvs:DAH145 X:8764386:FAM9A:coding:snvs:DAH145 22:39884587:MGAT3:coding:snvs:DAH145 22:37447918:7SK:coding:snvs:DAH145 22:24829598:ADORA2A:coding:snvs:DAH145 20:25263878:PYGB:coding:snvs:DAH145 19:814453:PTBP1:coding:snvs:DAH145 18:9859309:RAB31:coding:snvs:DAH145 17:73499325:KIAA0195:coding:snvs:DAH145 17:68129103:KCNJ16:coding:snvs:DAH145 17:42284886:UBTF:coding:snvs:DAH145 17:18226316:SHMT1:coding:snvs:DAH145 16:30980953:SETD1A:coding:snvs:DAH145 16:22825976:HS3ST2:coding:snvs:DAH145 16:4016933:ADCY9:coding:snvs:DAH145 14:92548659:ATXN3:coding:snvs:DAH145 13:73539509:PIBF1:coding:snvs:DAH145 13:23906156:SACS:coding:snvs:DAH145 12:102053560:MYBPC1:coding:snvs:DAH145 12:101682807:UTP20:coding:snvs:DAH145 12:57586646:LRP1:coding:snvs:DAH145 12:6078430:VWF:coding:snvs:DAH145 11:125853858:CDON:coding:snvs:DAH145 11:56143251:OR8U8:coding:snvs:DAH145 value 11:55135884:OR4A15:coding:snvs:DAH145 SOMATIC 11:45907403:CRY2:coding:snvs:DAH145 UNDETERMINED 10:104130515:GBF1:coding:snvs:DAH145 10:102566211:PAX2:coding:snvs:DAH145 WILDTYPE 10:95069866:MYOF:coding:snvs:DAH145 9:130270400:LRSAM1:coding:snvs:DAH145 9:2718192:KCNV2:coding:snvs:DAH145 8:113301714:CSMD3:coding:snvs:DAH145 7:101944369:AC005088.3−2:coding:snvs:DAH145 6:74073560:OOEP:coding:snvs:DAH145 6:7246723:RREB1:coding:snvs:DAH145 5:168180893:SLIT3:coding:snvs:DAH145 5:140615717:PCDHB18:coding:snvs:DAH145 5:127681270:FBN2:coding:snvs:DAH145 5:524228:SLC9A3:coding:snvs:DAH145 4:187629068:FAT1:coding:snvs:DAH145 3:184580707:VPS8:coding:snvs:DAH145 3:149700912:C1orf37:coding:snvs:DAH145 3:132172461:DNAJC13:coding:snvs:DAH145 2:219602546:TTLL4:coding:snvs:DAH145 2:211085473:ACADL:coding:snvs:DAH145 1:186324779:TPR:coding:snvs:DAH145 1:109197458:C1orf59:coding:snvs:DAH145 1:89523838:GBP1:coding:snvs:DAH145 1:46105922:GPBP1L1:coding:snvs:DAH145 1:29631897:PTPRU:coding:snvs:DAH145 1:6266355:C1orf188:coding:snvs:DAH145 20:46386033:SULF2:coding:snvs:DAH145 19:50840381:NAPSB:coding:snvs:DAH145 15:100890253:AC015723.8:coding:snvs:DAH145 1:22408228:CDC42:coding:snvs:DAH145 V V V V OA OA OA OA aty 10 Le 10 En 10 Le 10 p 48 ft 48 do 48 ft 48 ica .A Ov .A m .B Ov .T l e 15 ar 6 etr 6 ar nd y C ia y C om C l P C e C oly C trio p sis −a dja ce 22 nt Fig 3

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CCC (3a) AT-E-osis (3b) E-osis (3e) E-osis (3f)

Anglesio J Path 2015 DAH72 – VOA734

ARID1A

24 Conclusions • Adjacent atypical endometriosis can have a near complete complement of mutations -final transformation events may not be mutations • So far no explanation for why endometriosis can lead to two such distinct cancers • Are there more sensitive clonal marks for tracking relatedness • Is there a screening window ? • What about endometriosis not associated with cancer? Deep infiltrating endometriosis

• Will other clinically relevant forms of endometriosis have somatic mutations as have been seen in endometriosis associated with cancer

? Is endometriosis a partially competent neoplasm Deep Infiltrating Endometriosis “Case 2”

WT KRAS Double-mutation G12A positive

G12V

CASE2 Endometriosis (LCM)

CASE2 G12V CTRL NTC CTRL (LCM) Normal Tissue Endometriosis, CCOC and ENOCa

?

How do two such different cancers arise from the same precursor? How do different cancers arise from the same precursor? Do distinct mutations drive distinct oncogenic pathways

? ENOCA and CCC: commonly mutated Summary of specific genomic findings

• No single feature exclusive to endometrioid or CCC discovered • No feature seen exclusively in ARID1a wild type cancers seen • KMT2B (MLL4) the most commonly mutated “new” of interest Landscapes: Can the genomic landscape inform our understanding of the pathogenesis of these cancers • Higher level view of cancer genome enables identification of signatures that point to mutational process • ENOCa and CCC compared to GCT and HGSCa Copy number changes

GCT<

Signatures of mutational processes in human cancers: Alexandrov et al Nature 2014 ENOCA and CCC: genomic landscapes ENOCA and CCC: genomic landscapes

MMR APOBEC AGE How do different cancers arise from the same precursor?

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Although differences no mutation is exclusive to these cancer types some landscape features are enriched Cancer associated mutations may precede transformation process (Anglesio) Cysteine Biosynthetic Pathway

Higher in clear cell

CTH CBS Methionine Homocysteine Cystathionine Cysteine

Glutathione CTH is Highly Expressed in Clear Cell Ovarian Cancer

HGS ENOC CCOC CTH and CBS Expression in Cell Lines

ENOC CCC HGS

CaOV3 A2780 IGROV1 TOV112D 2008 JHOC5 JHOC7 JHOC9 OVISE OVMANA OVTOKO RMG2 TOV21G CaOV3 Hey Hey Kuramochi 3 OVCAR 4 OVCAR 5 OVCAR OVSAYO CTH

CBS

a-Tubulin

CBS CTH Homocysteine Cystathionine Cysteine The Origins of Endometriosis Associated Ovarian Cancer?

Cell of Origin for Endometrioid Ovarian Cancer?

Cell of Origin for Clear Cell Ovarian Cancer? CCOC and EndoCa and the ovary • Both cancers are associated with endometriosis • Although cancer associated mutations occur in endometriosis at other sites, transformation occurs almost exclusively within ovarian endometriomas • CCOC and EndoCa look similar to their endometrial counterparts and have similar mutations – do these cancers arise from different cells of origin? • The IL6 pathway is dominant in OCCC, whereas ARID1A/PIK3CA mutation occurs in approximately 50% of cases • Is OCCC more than on disease and if so what marks each type (proteomics screen) Thanks • My lab:, Niki Boyd ,Michelle Woo, Leah Prentice, Melissa McConechy, Winnie Yang, Sarah Mains-Bandiera, Clara Salamanca, Michael Anglesio, Alicia Tone, Hector Li Chang, Yemin Wang, Jay Chen, Tony Karnezis,, Madlen Maassen and Janine Senz • Sohrab Shah -- Bioinformatics: Jairhu Ding, Yikan Wang, Ali Bashashati, Gavin Ha, Andrew McPherson, Gavin Ha • GSC: Marco Marra, Martin Hirst, Gregg Morin • Collaborators: Stefan Kommoss, M Kobel, Blaise Clarke, J Brenton, AM Mes-Masson, D Bowtell, B Vanderhyden, A Okamoto and Sam Aparicio • OvCaRe BC: Blake Gilks, Dianne Miller, Ken Swenerton, Paul Hoskins, YZ Wang, Nelly Auersperg, Brad Nelson, Cal Roskelly, Tom Ehlen, Anna Tinker, Jessica McAlpine