Genomics of Ovarian Cancer Progression Reveals Diverse Metastatic Trajectories Including Intraepithelial Metastasis to the Fallopian Tube

Published OnlineFirst October 7, 2016; DOI: 10.1158/2159-8290.CD-16-0607

Research Brief

Mark A. Eckert1, Shawn Pan1, Kyle M. Hernandez2, Rachel M. Loth1, Jorge Andrade2, Samuel L. Volchenboum2,3, Pieter Faber4, Anthony Montag5, Ricardo Lastra5, Marcus E. Peter6, S. Diane Yamada1, and Ernst Lengyel1

abstract Accumulating evidence has supported the fallopian tube rather than the ovary as the origin for high-grade serous ovarian cancer (HGSOC). To understand the relationship between putative precursor lesions and metastatic tumors, we performed whole-exome sequencing on specimens from eight HGSOC patient progression series consisting of serous tubal intraepithelial carcinomas (STIC), invasive fallopian tube lesions, invasive ovarian lesions, and omental metastases. Integration of copy number and somatic mutations revealed patient-specific patterns with similar mutational signatures and copy-number variation profiles across all anatomic sites, suggesting that genomic instability is an early event in HGSOC. Phylogenetic analyses supported STIC as precursor lesions in half of our patient cohort, but also identified STIC as metastases in 2 patients.Ex vivo assays revealed that HGSOC spheroids can implant in the fallopian tube epithelium and mimic STIC lesions. That STIC may represent metastases calls into question the assumption that STIC are always indicative of primary fallopian tube cancers.

SIGNIFICANCE: We find that the putative precursor lesions for HGSOC, STIC, possess most of the genomic aberrations present in advanced cancers. In addition, a proportion of STIC represent intraepithelial metastases to the fallopian tube rather than the origin of HGSOC. Cancer Discov; 6(12); 1–10. ©2016 AACR.

See related commentary by Swisher et al., p. 1309.

INTRODUCTION For almost a century, it was believed that the surface epithe- High-grade serous cancer (HGSOC) encompasses several lium of the ovary gives rise to HGSOC. However, in 2001, a pathologic tumor entities, including ovarian, fallopian tube, Dutch research group described preneoplastic lesions in the and peritoneal cancers. The cell of origin of these cancers fallopian tubes of patients at high familial risk of HGSOC is currently unresolved, but their pattern of dissemination, (4). Careful sectioning of the fallopian tubes in patients with clinical behavior, and chemosensitivity are very similar (1–3). HGSOC has revealed serous tubal intraepithelial carcinomas

1Department of Obstetrics and Gynecology/Section of Gynecologic Oncol- Note: Supplementary data for this article are available at Cancer Discovery ogy, The University of Chicago, Chicago, Illinois. 2Center for Research Online (http://cancerdiscovery.aacrjournals.org/). 3 Informatics, The University of Chicago, Chicago, Illinois. Department Corresponding Author: Ernst Lengyel, The University of Chicago, 5841 4 of Pediatrics, The University of Chicago, Chicago, Illinois. University of South Maryland Avenue, MC 2050, Chicago, IL 60637. Phone: 773-702-6722; Chicago Genomics Facility, The University of Chicago, Chicago, Illinois. Fax: 773-702-5411; E-mail: [email protected] 5Department of Pathology, The University of Chicago, Chicago, Illinois. 6Department of Medicine, Northwestern University Feinberg School of doi: 10.1158/2159-8290.CD-16-0607 Medicine, Chicago, Illinois. ©2016 American Association for Cancer Research.

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Genomic Changes in STIC RESEARCH BRIEF

(STIC) with atypical histologic changes that resemble the tube, ovarian, and omental metastases (Fig. 1A). These four invasive serous component found in 50% of all patients with anatomic sites encompass the hypothetical progression HGSOC. Further molecular analysis of STIC found identical series; from in situ STIC precursors to locally invasive fallo- TP53 mutations in the STIC and corresponding HGSOC, as pian tube lesions, then to primary ovarian metastases, and well as a similar upregulation of cell cycle and DNA repair finally, to distant peritoneal omental metastases. All were proteins (5, 6). Because TP53 gene mutations represent one chemotherapy-naïve, with no germline BRCA1/2 mutations of the earliest genetic changes in HGSOC, and because they and no significant family history of ovarian or breast cancer are detected in all STIC, it was then considered evident that (clinic–pathologic features in Supplementary Table S1). For STIC may be the precursor lesions of HGSOC. This hypoth- each anatomic site, the tumor compartments were collected esis received additional support with the recent development using laser-capture microdissection (LCM; Fig. 1B). Microdis- of a genetic mouse model designed to determine if the fal- section was utilized to both eliminate stromal contamination lopian tube can give rise to HGSOC. In this model, the Pax8 and facilitate highly specific sequencing of smallin situ STIC promoter, specific to secretory fallopian tube epithelial cells lesions. Ovarian tumors were microdissected from the ovary (FTEC), was used to inactivate Brca1 (germline mutations associated with the STIC lesion in cases of bilateral ovarian in BRCA1/2 occur in about 13% of HGSOCs) and Pten and involvement. Because, by definition, there is limited mat- drive expression of mutant Trp53. These mice develop tumors erial in microdissected STIC (Fig. 1A), the DNA isolation mimicking human STIC lesions in the fallopian tube and method was optimized to include longer digestion times and have molecular alterations that recapitulate human HGSOC reduction of elution volumes. WES and data processing were (7). If one assumes that HGSOC advances along a linear performed to assess the spatiotemporal pattern of genomic progression series from in situ tumors to invasive tumors to alterations during HGSOC progression (Fig. 1C; Supplemen- metastasis, as is believed of colon cancer (8), these findings tary Fig. S1A). Despite low input amounts for some samples support the hypothesis that HGSOC originates in the fal- (50 ng), depth of coverage and on-target reads were similar lopian tube. across all anatomic sites surveyed (Supplementary Fig. S1B– Fully understanding the relationship of STIC and HGSOC S1D; Supplementary Table S2). requires a comprehensive understanding of the genomic An average of 1.0 single-nucleotide variants (SNV) per alterations underlying both STIC and HGSOC. Using next- megabase (Mb) were identified, corresponding to approxi- generation sequencing technologies, The Cancer Genome mately 50 de novo somatic mutations per sample, which Atlas (TCGA) and an Australian consortium have provided is comparable to rates identified in the TCGA analysis of a snapshot of the genomic changes and signaling pathways HGSOC (refs. 9, 15; Supplementary Table S3). The analy- characterizing invasive HGSOC at the ovary (2, 9). Compared sis of germline DNA did not detect BRCA1/2 mutations in with other carcinomas, HGSOC is uniquely characterized the patient cohort. The majority of mutated genes in our by recurrent copy-number variants (CNV), with TP53 the analysis had been identified in the TCGA analysis, confirm- most commonly mutated gene (10). In pancreatic and pros- ing that the patient cohort was representative of HGSOC. tate cancers, among others, multisite sequencing of primary Mutational burden was similar across all anatomic sites. tumors and metastases has revealed evidence of multistep The only exception was patient 539, who had a signifi- dissemination and unraveled the complex genomic trajec- cantly higher mutational burden in the ovarian and omental tories of metastasis (3, 11). Although several groups have metastasis (Fig. 1D). Overall, the pattern of mutations was performed sequencing of HGSOC and begun to understand enriched for C>T substitutions, a signature associated with its metastatic trajectory throughout the peritoneal cavity aging, mediated by spontaneous deamination of 5-methyl- (12–14), without integrative whole-exome sequencing (WES) cytosine (10). In contrast, C>A mutational rate, associated of STIC and metastatic lesions, the reconstruction of a com- with environmental carcinogenesis (15), was low (Fig. 1E). plete metastatic trajectory for HGSOC is not possible. The mutational signature was correlated with age at time We set out to characterize the dynamics of mutations of diagnosis and was patient-specific (Supplementary Fig. and copy-number variations during HGSOC dissemination, S1E and S1F). No significant differences were observed by using a systematic genomic characterization of a uniform anatomic site (Supplementary Fig. S1G). Mutational signa- group of patients with sporadic advanced HGSOC with STIC tures, including rates of indel detection, were similar to those and without BRCA1/2 germline mutational changes. We observed in the TCGA analysis of HGSOC (Supplementary hypothesized that sequencing both putative precursor lesions Fig. S1G). Patients with missense mutations in TP53 had evi- and metastatic HGSOC would elucidate early core events in dence of nuclear p53 stabilization as indicated by high pro- HGSOC and the genomic characters necessary to reconstruct tein expression in the tumor. One patient (patient 505) had its step-wise progression. a null splice site mutation and did not express p53 protein (Supplementary Fig. S1H). TP53 was the only gene mutated in every patient and ana- RESULTS tomic site (Fig. 2A; Supplementary Fig. S2A) and the only To begin to systematically understand the spatiotempo- gene identified as significantly mutated across more than 1 ral genomics of ovarian cancer progression, we identified patient by MutSig, an algorithm that corrects for high muta- a cohort of 8 patients who presented for primary tumor tional rates in late replicating and poorly transcribed genes debulking surgery with advanced, metastatic HGSOC. Germ (16). Common mutations in CSMD3 and other genes were line DNA was available for all patients, and the final pathol- not significant. Each patient had the sameTP53 mutation in ogy for these patients showed STIC and invasive fallopian all anatomic sites (Supplementary Table S4), whereas other

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A STIC FT Ov Om B Before LCM After LCM Captured tumor p53

C SNVs SNV and CNV classes 8 patients with HGSOC Whole-exome Tumor LCM Core genomic events STIC, FT, Ov, Om sequencing Metastatic phylogenies CNVs

D E SNVs by anatomic site SNVs by patient 40 2.0 2.0 30 1.5 1.5 20 1.0 1.0 SNVs/Mb SNVs/Mb 10 0.5 0.5 % of All SNVs & indels STIC FT Ov Om 0.0 0.0 0

FT Ov Om 442 451 505 530 539 559 563 754 STIC C>A C>G C>T T>A T>C T>G Indel Patient Mutation class

Figure 1. HGSOC mutational processes are established early and are patient-specific. A, Representative p53 IHC and hematoxylin and eosin images of the hypothetical progression series of HGSOC in a patient with STIC, and invasive lesions in the fallopian tube, ovary, and abdominal omentum. B, Laser capture microdissection of the tumor compartment from omentum. C, Workflow to elucidate the spatiotemporal pattern of genomic alterations in HGSOC to capture tumor phylogenies and core events. D, Frequencies of SNV by anatomic site and patient reveal that mutational burden is patient-specific rather than determined by anatomic tumor location. Average mutational burden across the patient cohort is approximately 1 somatic mutation per megabase (50 mutations total per tumor sample). E, Identification of an age-related mutational signature class characterized by high rates of >C T substitutions in all samples of the patient cohort reveals a comparable mutational process underlying disease progression in all patients examined. FT, invasive fallopian tube tumor; Ov, invasive ovarian tumor; Om, omental metastasis. mutations were limited to a subset of patients or anatomic mediating contact with extracellular matrices (ECM) or other sites (Supplementary Fig. S2A). Mutant allele frequencies cells may be an early event in HGSOC progression. (MAF) for TP53 were high at all sites (0.7188–1.000), sug- HGSOC has a disproportionately high number of CNVs gesting early LOH at the TP53 locus, highlighting the advan- compared with other cancers (10). The genomic location of tages of microdissection for obtaining high tumor purity CNVs in our data set was highly concordant with the TCGA (Supplementary Table S4). In general, an increase in MAF analysis of HGSOC (9), including frequent amplification across the progression from STIC to omental metastases was of chromosomes 1q, 3q, and 8q and deletion of 4p, 4q, and observed (Supplementary Fig. S2B). 8p (Fig. 3A; Supplementary Table S5). Across all anatomic To identify candidate driver molecular changes, mutations sites, patient-specific copy-number profiles were apparent, were assigned to three classes: (1) core mutations, present in including amplification of cytoband 8q24 in patient 754 all four anatomic sites; (2) shared mutations, present in two (Fig. 3B), but every patient had distinct amplifications and or three anatomic sites; and (3) private mutations unique to a deletions. Because the deletion of DNA damage repair genes single anatomic site. In all patients, a minority of SNVs were plays a role in the etiology of HGSOC (9, 17), we specifically “core” mutations, likely to participate in the early transform- investigated copy-number profiles for DNA damage repair ation of normal cells (Fig. 2B). Core and private mutations and response genes. Indeed, frequent deletion of genes impli- had identical mutational signatures (Supplementary Fig. cated in homologous recombination (BRCA2, FANCB), base S2C). A clear pattern emerged when we examined mutational excision repair (APEX2, NEIL3), nonhomologous end-joining class by anatomic site: The STIC and fallopian tube sites had (WRN), and DNA damage signaling (RAD23A) was observed a significantly higher proportion of “private” mutations. (Fig. 3C; Supplementary Fig. S3A). Although the extent and Gene ontology (GO) analysis of putative core mutations frequency of amplifications and deletions varied significantly across all patients (Fig. 2C) revealed enrichment of three across patients (Fig. 3D), we observed no significant dif- separate pathways involved in cell adhesion (Supplementary ferences among anatomic sites (Fig. 3E). We did not detect Fig. S2D), suggesting that mutation of genes involved in any metastasis-specific genes. Interestingly, the genomic

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Oncoprint of nonsynonymous mutations A Core Shared Private

Missense Indel 539 Nonsense OM OV FT STIC TTN ISL1 GLI1 VWF TP53 SI/2B PLD1 ETV1 NAI/3 SKA3 FUT7 TPTE CBLC AQP6 SETX ITGA8 DNA/1 KLF17 FBXL4 MAG/1 PATZ1 LOXL1 ARP/N RLBP1 SPG11 C3AR1 FRAS1 SVEP1 KRT37 LRFN5 DDX49 FADS6 SFRP5 CYTH4 CABP4 PXDNL DMXL1 MEF2A KCNS1 TRM49 PAPPA PRSS3 CETN2 NR1D2 ERAP2 STPG2 H2AFY ACAD8 SBNO1 ADCK3 SUSD5 L1CAM PTPRT DOCK1 DNAH3 PAQR3 PPFIA4 MYH7B WDR31 IGFLR1 NCAM1 CDHR2 CSMD3 MORC1 ZNF770 ZNF189 ZBTB33 MYOM2 VPS13A ALOX12 TUBB4A PARP10 PNPLA6 CRELD1 MAP4K1 OSBPL6 OR10A7 CRYBA1 LYSMD1 PCDHA1 PCDHA8 GABRB3 TMEM67 SFSWAP SLC19A2 COL17A1 COL21A1 L3MBTL2 TM4SF18 DNAJC10 K/AA1731 CREB3L3 TMEM186 RASGRP1 ARHGAP6 NEUROG1 SERPINB7 HNRNPUL2

Mutational class distribution Identification of core SNVs B C Core Shared Private 57 STIC 754 563 13 16 FT 71 559 14 539 28 12 142 13 Core SNVs/indels TP53 530

Patient 10 505 37 29 451 9 61 Ov 442 26 0 50 100 Om Percentage of SNVs/indels (%)

Figure 2. Core, recurrent SNVs in HGSOC are restricted to TP53 mutations. A, Oncoprint of all nonsynonymous mutations in patient 539 reveals mutation of TP53 and distribution of mutations into core, shared, and private classes. B, A minority of SNVs and insertions/deletions (indels) are present in all samples (“core”). The majority of mutations are shared between 2 and 3 anatomic sites (“shared”), or present in only one site (“private”). C, Euler dia- gram of all nonsynonymous SNVs and indels. Core SNVs/indels present in all anatomic sites are highlighted in red. FT, invasive fallopian tube tumor; Ov, invasive ovarian tumor; Om, omental metastasis. instability evident in omental and ovarian sites was already metastases to the fallopian tube (Fig. 4A; Supplementary present in the fallopian tube and STIC (Fig. 3E). Fig. S4; Supplementary Table S7). Histologically, the STIC Next, we utilized GISTIC, an algorithm to identify signifi- from these cases was indistinguishable from the other basal cant, recurrent amplifications and deletions (18), to detect STIC, although one of these cases presented with intralu- core CNVs collaborating with TP53 to drive HGSOC (Sup- minal HGSOC spheroids in the fallopian tube (Fig. 4B). plementary Fig. S3B and S3C). The Euler plot in Fig. 3F Intraluminal HGSOC spheroids were present in 3 of the 8 integrates significantly amplified or deleted genes at each patients in our cohort, including 1 patient in whom sphe- anatomic site, identifying several genomic events known roids adhered to the apical surface of the fallopian tube to be involved in HGSOC tumorigenesis, including CCNE1 epithelium (Fig. 4C). amplification, NF1 deletion, and other genes with previ- Although intraepithelial metastases of HGSOC to the fal- ously uncharacterized roles in HGSOC biology. These core lopian tube have not been described, they have been observed genomic events consisted of 93 amplified and 69 deleted in other tumor types, including endometrial and colon can- genes, which represented less than 5% of all significantly cers (12, 19, 20). Intraluminal HGSOC spheroids within amplified and deleted genes. Significantly amplified processes the fallopian tube are a common finding in patients with included genes involved in the regulation of transcription HGSOC (21). Because metastasis to the fallopian tube was and RNA metabolism, as well as several miRNAs (Supplemen- an unexpected finding, we sought to determine if HGSOC tary Fig. S3D; Supplementary Table S6). cells are able to implant into the fallopian tube epithelium. A To elucidate the metastatic trajectories of HGSOC in each model of intraepithelial metastasis was developed, in which patient, a phylogenetic clustering approach was used (11). full-thickness primary human fallopian tubes were co-cul- This analysis revealed three distinct classes of dissemination: tured with ovarian cancer spheroids (Fig. 4D; Supplementary (1) “basal STIC,” in which STIC represented the basal branch Fig. S5A). In this ex vivo model, the normal fallopian tube epi- (most similar to germline DNA of the patient) of a hierarchi- thelial morphology was preserved, including high expression cal, step-wise dissemination process; (2) “parallel” dissemina- of PAX8 and junctional expression of β-catenin and E-cadherin tion, in which no clear hierarchal dissemination pattern was (ref. 22; Supplementary Fig. S5B). Histologic examination of evident and all branches developed simultaneously from the fallopian tube–cancer cell explants revealed the presence of one precursor; and (3) “STIC metastases,” in which other implanted tumor cells that histologically resembled STIC and anatomic sites were hierarchically more basal than the STIC expressed the established molecular markers of STIC, includ- lesions, implying that in these patients, STIC represented ing p53, Ki67, and stathmin (Fig. 4E; Supplementary Fig. S5C;

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A B STIC FT Ov Om 100 1q 3q 6p 8q 12p 20p+q 442 451 505 530 539 559 563 754 75

50 1 25 2 0

25 3 50 % with gain or loss 4 75 5 100 4p+q 8p 15-19 p+q 22q 6 C 442 451 505 530 539 559 563 754 7

WRN 8 APEX2 XRCC6 FANCB 9 POLI LIG3 RAD23A POLD1 10 NEIL3 RFC1 LIG1 PNKP 11 XRCC1 POLN ERCC8 12 DDB2 RPA1 ALKBH3 RAD17 13 GTF2H2 RAD23B POLK 14 APTX BRCA2 15 RFC3 GTF2H1 CUL4A 16 FANCF PMS1 17 UNG ALKBH2 18 RFC2 NHEJ1 19 MSH2 MSH8 20 DCLRE1B FANCE 21 NEIL2 22 TOPBP1 RFC5 X XPC GTF2H3 MBD4 POLE MDC1 GTF2H4 MSH5-SAPCt LRIG3 SMUG2 POLH PCNA D CNVs by patient E CNVs by anatomic site F Identification of core CNVs

100 80 Amplified 263 STIC Amplified 726 Deleted 125 5 80 Deleted FT 60 30 0 303 368 70 250 224 8 0 93 60 1 Core SNVs 40 0 0 69 27 76 40 0 28 0 51 125 264 Ov

% of Genome % of Genome 20 2 20 22 30 313 Om 0 0

442 451 505 530 539 559 563 754 FT Ov Om STIC Patient

Figure 3. Genomic instability is a core feature of ovarian cancer that frequently involves DNA-damage repair genes. A, Frequency plot of CNVs across all patients and all four anatomic sites. Annotated arm level events were also identified as significant in the TCGA analysis of HGSOC. For all plots in the figure, amplifications are red and deletions are blue. B, Genomic aberration plot of CNVs across all anatomic sites and patients implies high degree of genomic instability in all anatomic sites. Chromosome numbers are indicated on margin. Amplifications are red; deletions are blue. C, CNV status of significantly altered DNA repair pathway genes (from REPAIRtoire) reveals common deletion of DNA damage response and repair genes known to be involved in HGSOC. Amplifications are red; deletions are blue.D and E, Extent of genomic instability is patient-specific (D) and does not vary by anatomic site (E). F, GISTIC identification and analysis of significantly altered genes identifies conserved core amplifications and deletions. Number of amplifications in red and deletions in blue. FT, invasive fallopian tube tumor; Ov, invasive ovarian tumor; Om, omental metastasis. refs. 5, 6, 12, 23). HT-29 colon adenocarcinoma cells were also FTEC–cancer cell adhesion and epithelial clearance. Human capable of implanting in the fallopian tube epithelium and FTECs (22) expressing PAX8 and preserving tight junctions could be distinguished by a lack of PAX8 immunoreactiv- (β-catenin expression; Supplementary Fig. S6A and S6B) were ity (Supplementary Fig. S5D). In addition, using a similar cocultured with HGSOC spheroids and adhesion measured. approach, we found that HGSOC spheroids were also capa- The cancer cells adhered to a monolayer of primary fal- ble of adhering to ex vivo omental explants (Supplementary lopian tube epithelium within 15 minutes in a β1-integrin– Fig. S5E), but did not adhere to endometrial explants after dependent manner (Fig. 4F and G; Supplementary Fig. S6C) extended culture (data not shown). and cleared the FTEC and fully integrated in the monolayer To mechanistically understand the interactions between within 12 hours (Fig. 4H). Combined, these in vitro and ex vivo FTECs and HGSOC cells, we established in vitro models of experiments suggest that ovarian cancer cells are capable of

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A Phylogeny Genomic events B gDNA 442 STIC C: TP53, EPHA8, NF1 C FT 1: WRN DEL 563 STIC DEL 1 Ov 2: CHD9, BRCA2 2 Om gDNA 539 STIC C: TP53, KLF17, CSMD3 C FT 1: MAP4K1 1 2 Ov 2: GLI1, PCDHA1 Om gDNA I 754 STIC C: TP53 C Ov 1: BACE2, CNOT1 Basal STIC 1 FT 2: SULT1A1 2 Om gDNA C 530 C STIC C: TP53 Ov 1: SCARF 1 2: 2 FT ARHGAP5 Om gDNA STIC C: TP53, CHD9, PCDH7 451 1 FT 1: MCM9 C Ov 2: TTN 2 II Om gDNA

Parallel STIC C: TP53, MUC5B 505 2 C Ov 1: DYSF, SMPDL3A FT 2: FBLN5, MPRIP 1° Ascites 1 Om D gDNA 559 Om C: TP53, ATM C Ov 1: KRT10, ZNF700 1 2: SLC3A2 2 STIC III FT gDNA 563 Om C: TP53, PCDHA4 C STIC 1: UGT2B17, VNN1 1 2: NBEAL1, MYO1E 2 FT TYK-nu

STIC metastases Ov 1° human FT

H&E p53 Ki67 E F G 1.5 HeyA8 + FTECs *** **

1.0 TYK-nu OvCa Ctrl IgG FTECs Phalloidin Merge Inset 0.5 H Cell adhesion (AU)

Inhibitor 0.0 - 1

β IgG AIIB2 IgG AIIB2 HeyA8 TYK-nu

Figure 4. Phylogenetic analyses of ovarian cancer progression reveal diverse metastatic processes and evidence of intraepithelial metastasis. A, Phylogenetic trees of each patient with key genomic events (SNVs, indels, CNVs) that characterize each branching event annotated [C (“core”), 1, or 2]. Deletions are in blue. B, Representative p53 staining of a “STIC Metastasis” in patient 563 (left), as well as intraluminal HGSOC spheroids within the same fallopian tube (right). C, p53 staining of intraluminal HGSOC spheroids adhering to the epithelium of the fallopian tube (patient 530). D, Human fallopian tube (FT) fimbriae and spheroids that were cocultured. E, Coculture of human fallopian tube explants with TYK-nu ovarian cancer spheroids mimic STIC with clearance of normal epithelium and implantation of tumor cells expressing Ki67 and nuclear p53. F and G, Adhesion of fluorescently labeled HGSOC cells (green) to primary FTEC monolayer (brightfield) after 15 minutes. Pretreatment withβ 1-integrin blocking antibody (AIIB2) attenuated adhesion of HeyA8 and TYK-nu cells to FTEC monolayer. **, P < 0.01; ***, P < 0.001. H, TYK-nu ovarian cancer spheroids (phalloidin only) clear a primary human fallopian tube epithelial monolayer (phalloidin and CellTracker Green) after 12 hours.

adhering to and integrating with fallopian tube epithelium, challenged by the intrinsically low sample amounts of STIC, we suggesting that a portion of putative precursor STIC are, show here that WES of low-input, in situ specimens is feasible instead, metastatic mimics. and can be integrated into established workflows. That this study was performed with archival paraffin blocks indicates that the sources of samples used for analysis of core genomic DISCUSSION events or phylogenetic reconstruction can be expanded to We set out to study the dynamics of genomic alterations paraffin blocks which are widely available. The laser-capture with the goal of understanding the relationships of STIC to the microdissection of the tumor compartment allowed the col- metastatic trajectory of HGSOC. Although we were initially lection of pure tumor samples, allowing us to make firm

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RESEARCH BRIEF Eckert et al. conclusions about genetic changes without the stromal con- represent the precursor of HGSOC. Endosalpingiosis to the tamination observed in other studies (3, 9). omentum occurs frequently (15% of women in an unselected We find that multisite sequencing is useful for identifying cohort; refs. 29, 30). Normal oviductal tissue displaced to the candidate driver genomic events, with putative core mutations omentum or peritoneum (endosalpingiosis) could undergo representing a minority of mutational events at any one site. transformation, thus mimicking an omental primary tumor In our study, the mutation rate and mutation signature, as or a primary peritoneal cancer with serous histology (30, 31). well as the CNV rates of STIC and HGSOC, were consistent Alternatively, it is possible either that the STIC metastases and unique to each patient. All samples had mutations at the originated from a precursor lesion within the fallopian tube TP53 locus, including all STIC lesions, demonstrating that or that a STIC precursor lesion colonized the peritoneum and the patients examined here are representative of HGSOC (5, later back-metastasized to the tube (11). 24). Every patient had very different genomic changes, consis- The data presented herein may lead us to develop our per- tent with the perception of HGSOC as a very heterogeneous ception of STIC further: STIC in HGSOC could be primary or disease that is not easily defined by a specific mutational metastatic. Previous studies observed precursor lesions exclu- change. There were no significant alterations between ana- sively in the fallopian tube of BRCA mutation carriers under- tomic sites within a single patient, suggesting that the biologi- going prophylactic salpingo-oophorectomy (32), whereas our cal processes underlying genomic instability, both CNVs and study focused on sporadic HGSOC. As clinical studies have SNVs, are established early during disease progression. The begun to investigate the utility and safety of salpingectomy extent and nature of mutations and CNVs are such that even in high-risk patients (33–35), it will be imperative to under- “basal STIC” possess the same genomic instability and all the stand if metastasis to the fallopian tube occurs in the context features of invasive HGSOC. During the progression to a dis- of germline BRCA mutations. Lastly, the frequent observation seminated HGSOC, the STIC have co-evolved with the tumor, of STIC detected after neoadjuvant treatment (occurring in thus increasing the extent of CNVs and SNVs. Common core 50% of all cases) could represent a late metastasis to a chemo events include mutation of TP53, frequent amplification of resistant niche (36). Therefore, a better understanding of the CCNE1, and deletion of DNA damage repair and signaling cross-talk between cell types unique to intraepithelial fal- genes. In addition, we found a previously unappreciated wide- lopian tube metastases could be both relevant clinically and spread (70%–100%) and early LOH at the TP53 locus. This important biologically. is in stark contrast to TP53 LOH rates in other cancers (15), indicating an exceptionally strong selective advantage for loss of the wild-type TP53 allele in HGSOC. METHODS Notably, we found evidence that a portion of STIC represent Surgical Treatment metastases to the fallopian tube epithelium, rather than All patients had newly diagnosed advanced, metastatic HGSOC the origin of the tumor. Another study had considered the and were undergoing primary debulking surgery by a gynecologic possibility of metastasis to the fallopian tube from HGSOC oncologist (S.D. Yamada and E. Lengyel) at the University of Chicago and uterine cancers (12). Targeted sequencing of multiple, (Supplementary Table S1). All tissues were collected with informed anatomically distinct STIC in patients with ovarian cancer consent under approved, University of Chicago Institutional Review has identified identicalTP53 mutations in these synchronous Board protocols and in accordance with the Declaration of Helsinki. in situ lesions, suggesting that they may represent clonal metastases within the fallopian tube (5). In the absence of LCM and DNA Extraction germline TP53 mutation, the acquisition of identical TP53 Sections (10 μm) of each tissue were cut onto PEN-Membrane mutations in two synchronous STIC is highly unlikely. Slides (Leica), deparaffinized with xylenes, rehydrated through Intraepithelial metastasis is thought to also occur in lung graded alcohols, and stained with hematoxylin. Tumor components from each tissue were microdissected with a Leica LMD 6500 (up to cancer, where cells metastasize to the bronchial epithelium 5 serial sections per sample). In cases of bilateral ovarian involvement, (25). Although we found that nongynecologic cancers may the tumor was microdissected from the ovary of the same laterality metastasize to the fallopian tube, these implants do not gain as the STIC lesion. DNA was extracted with a QIAamp DNA FFPE expression of FTEC markers and can be immunohistochemi- Tissue Kit (Qiagen), with proteinase K digestion extended to 12 cally differentiated from HGSOC. There is also evidence that hours and sequential elution of DNA in 20 μL volumes following the fallopian tube epithelium is a receptive site for metastasis 5-minute incubations. of nongynecologic cancers (19, 20). The peritoneal cavity, including the fallopian tubes and Sequencing and Alignment ovaries, is a continuous system bathed in peritoneal fluid, Libraries were prepared per standard protocols (Agilent Sure- which may facilitate complex patterns of dissemination. In Select Human All Exon v5) and sequenced on an Illumina HiSeq both cases of “STIC metastasis,” the omentum represented 2500 ultra high-throughput sequencing system (100 bp, paired-end). the most basal tumor site. The clinical presentation of pri- Sequence quality was assessed using FastQC v0.11.2. Adapters were mary peritoneal carcinoma, in which HGSOC is detected pri- then removed, and overlapping mates were merged using SeqPrep. Processed reads were then aligned to the human genome (hg19) marily in nonovarian anatomical sites (26), suggests that the using Novoalign v3.02.07 and filtered to remove low-quality align- omentum and peritoneal mesothelium represent a microen- ments and PCR duplicates with sambamba v0.5.4 (37). Alignment vironment that fosters the establishment and growth of early metrics were gathered using the CalculateHsMetrics tool from Picard metastases (27, 28). Based on expression of molecular mark- v1.129 and the Agilent SureSelect target files. Filtered alignments ers and the lack of putative precursor lesions (26), it would be were then refined as suggested by the Broad Institute’s “Best Prac- surprising for the peritoneal mesothelium of the omentum to tices” (38) using GATK v3.4 (39).

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SNV/Indel Analysis that was amplified or deleted by anatomic site or patient. Copy- Refined alignments were then used to detect somatic mutations number segments were grouped by tissue (e.g., all STIC samples were across the various combinations of normal (gDNA) and tumor sam- run together) and used as inputs for GISTIC v2.0.22 (18). ples within a patient. To reduce the number of false positives, three somatic mutation detection (SMD) tools were used: MuTect v1.1.4 Tumor Phylogenetics (40), VarScan2 v2.3.9 (41), and Virmid v1.2.0 (42). Important differ- Somatic mutations (both synonymous and nonsynonymous) were ences between these tools include (1) Virmid and VarScan2 can detect combined with high-confidence LOH loci calls >( 20× coverage, Var- LOH; (2) Virmid and VarScan2 can detect germline mutations; and Scan2) to generate a binary presence–absence matrix of genomic (3) only VarScan2 attempts to call indels. characters for each anatomic site within each patient. Inclusion of All detected somatic variants (including indels) were filtered to both LOH and SNV data lowers the likelihood of mutation reversion remove low-quality, low coverage, and ambiguous genotype calls confounding phylogenetic analyses. Distance matrices were calculated using in-house scripts (43). Filtered variants were then annotated using Jaccard–Tanimoto similarity coefficients with 100 bootstrap using Annovar (44) and further filtered to remove variants located in replicates and clustered using the unweighted pair group method with intergenic, upstream/downstream, intronic, UTR5/3, or noncoding arithmetic mean (UPGMA) method to generate dendrograms using regions. Next, results across the 3 SMD tools were combined and DendroUPGMA (48). Cophenetic correlation coefficients were greater merged at loci where the alleles were concordant or the mutation was than 0.98 for all trees (49). Resulting dendrograms were plotted with observed in more than one anatomic site of the same patient. For 1 the Interactive Tree of Life interface (50). patient, manual review of TP53 sequencing alignments with the Inte- grated Genome Viewer was used to confirm mutation ofTP53 at all Immunohistochemistry anatomic sites. To calculate mutational rates, per-locus coverage was Formalin-fixed, paraffin-embedded tissue sections (5μ m) were estimated using GATK’s DepthOfCoverage tool. Then, per-sample deparaffinized with xylene and rehydrated with a graded series of estimates of total target size were estimated by counting the number ethanol. Antigen retrieval was performed in 10 mmol/L sodium of loci with both normal and tumor sample coverage ≥8×. Next, citrate buffer (pH 6) with 0.05% Tween 20 at 100°C. Samples were using exonic and splicing somatic mutations, mutation rates were then incubated with 0.3% hydrogen peroxide for 20 minutes at room estimated for each tumor–normal combination. temperature (RT) and blocked with 2.5% normal horse serum (Vector To detect significant mutations, somatic variants detected by at Laboratories) for 1 hour at RT. Primary antibodies (Stathmin, 1:200, least 2 SMDs or by MuTect, as well as high-quality indels, were anno- Cell Signaling Technologies; Ki67, 1:200, Thermo Scientific; PAX8, tated by Oncotator (45) and converted to a single MAF file. The MAF 1:200, Cell Signaling Technologies; β-catenin, 1:200, BD Biosciences; file was then processed by MutSigCV v1.4 (16) using the default set- E-cadherin, 1:200, BD Biosciences; pan-p53, 1:200, Calbiochem) in tings and databases. Mutations detected as significant in only a single 1.25% normal horse serum/PBS were incubated overnight at 4°C patient were excluded. Mutational signatures were assessed by assign- and visualized using the R.T.U VectaStain Kit and DAB (Vector Lab- ing each SNV to one of seven classes as follows: T>C (A>G and T>C); oratories) and counterstained with hematoxylin. C>T (G>A and C>T); C>G (G>C and C>G); T>G (A>C and T>G); T>A (A>T and T>A); C>A (G>T and C>A); and indels (insertions and dele- Immunofluorescence tions; ref. 10). Mutational classes, based on their distribution between anatomic sites, were defined as follows: Core mutations were shared Cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% by all four anatomic sites within a patient; shared mutations were TritonX-100, and blocked with 10% goat serum. Primary antibodies present in two or three anatomic sites within a patient; and private (PAX8, 1:200, Cell Signaling Technologies; β-catenin, 1:200, BD Bio- mutations were detected in only a single anatomic site for a patient. sciences) were incubated overnight in 10% goat serum. Samples were To assess germline mutation status of BRCA1/2 and other ovarian probed with fluorescent secondary antibodies (Thermo Scientific) cancer susceptibility genes, Virmid and VarScan2 calls were utilized. and Hoechst (Invitrogen) for 1 hour before washing and mounting Germline variants were filtered to remove potential false positives with ProLong Gold. Images were collected on a Zeiss LSM 510 Meta following the suggestions presented by the VarScan2 developers (41). Confocal microscope and images processed with ImageJ.

CNV Analysis Cell Culture Refined alignments were scanned for CNVs using VarScan2 (41) HeyA8 (Gordon Mills, MD Anderson, Houston, TX; received June and custom scripts based on a previously published method (46). 2006), TYK-nu (Gottfried Koneczny, University of California, Los Due to the high overlap in segments between methods and less vari- Angeles; November 2014), and HT-29 (Marcus Peter, Northwestern ability in the in-house method, we utilized the method introduced by University, Chicago, IL; May 2009) cells were grown under recom- Lonigro and colleagues (46), which we term the “MI Method.” First, mended culture conditions and genotyped to confirm their authen- per-target (exon) coverage was estimated using GATK’s DepthOf- ticity (IDEXX Bioresearch short tandem repeat marker profiling Coverage tool (v3.4.0). Then, normalization was performed for each every 3 months; all cell lines last validated January–July 2016). All tumor–normal pair. Briefly, targets with coverage less than 10 in the cell lines were Mycoplasma-negative. Immortalized FT33-TAg FTEC matched normal sample were excluded. Then, per-target coverage cells were a gift from Dr. Ronnie Drapkin (University of Pennsylva- in the tumor sample was divided by the per-target coverage in the nia; December 2014) and were cultured in 2% Ultroser G (Crescent matched normal sample, resulting in coverage ratios for each target. Chemical) in DMEM/F12 50/50 (Corning; ref. 22). Coverage ratios were then globally normalized by dividing each of them by the ratio of human mappings between the two samples Ex Vivo Metastasis Assays

(tumor/normal) and log2 transforming. Finally, the overall median Normal fallopian tube, endometrial, and omental tissues were col- value was subtracted, resulting in a set of log2-transformed coverage lected from patients with benign gynecologic conditions at the time ratios with median zero for each tumor/normal matched pair. The of surgery. Fimbriae or 0.5 cm3 endometrial or omental tissues were normalized values were then segmented using the R/bioconductoR dissected and transferred into 500 μL of FTEC media, known to sup- package “copynumber” v1.6.0 (47). Segmented CNV calls were used port the proliferation and survival of primary FTECs (22). To each 4 to extract regions with log2R greater than 0.2 or less than 0.2, as well, 5 × 10 HGSOC cells (TYK-nu or HeyA8) were added. For some performed with TCGA data, to extract the percentage of the genome experiments, spheroids were labeled with CellTracker Green (Thermo

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RESEARCH BRIEF Eckert et al.

Scientific; 1:1,000 dilution). Following 72 hours of coculture, fim- Acknowledgments briae were fixed in 4% formaldehyde, dehydrated through graded We thank the Harris Family Foundation for their generous alcohols and xylenes, and embedded in paraffin blocks. Sections support. We thank Drs. H.A. Kenny, M. Curtis, K. Watters, and (5 μm) were cut for each tissue and processed for immunohistochem- A. Mukherjee from the University of Chicago ovarian cancer lab- istry (p53, Ki67, and stathmin). oratory for helpful discussions. We are very thankful to Gail Isenberg, University of Chicago, for carefully editing the manuscript. Isolation of Primary HGSOC Spheroids Ascites fluid from patients with HGSOC was passed through a Grant Support 40-μm nylon mesh and washed with PBS to remove single cells. Iso- This work was supported by a Marsha Rivkin Foundation lated spheroids were maintained in nonadherent plates (Corning). award (M.A. Eckert), National Cancer Institute grant CA111882 (E. Lengyel), a Harris Family Foundation award (S.D. Yamada), a Isolation of Primary FTECs University of Chicago Comprehensive Cancer Center Team Science Fallopian tubes were collected from patients with benign gyneco- award (E. Lengyel and S.L. Volchenboum), and University of Chicago logic conditions. FTECs were extracted and cultured as previously Cancer Center Support Grant P30CA014599. described (22). Received June 1, 2016; revised September 28, 2016; accepted October 3, 2016; published OnlineFirst October 7, 2016. In Vitro Fallopian Tube Adhesion Assay Plates (96-well) were coated with fibronectin (0.1μ g/mL). Immor- talized FTECs (FT33-TAg cells) were plated at 1 × 104 cells per well References and grown to confluency (48 hours). Ovarian cancer cells were labeled . 1 Lengyel E. Ovarian cancer development and metastasis. Am J Pathol 4 with CellTracker Green (1:1,000) and added to wells of plate (5 × 10 2010;177:1053–64. cells per well) and incubated for 15 minutes (37°C; 5% CO2). For 2. Norquist BM, Harrell MI, Brady MF, Walsh T, Lee MK, Gulsuner S, β1-inhibitor experiments, cells were treated in suspension with AIIB2 et al. Inherited mutations in women with ovarian carcinoma. JAMA blocking antibody or antibody control (5 μg/mL) for 30 minutes. Oncol 2015:1–9. Alternatively, 5 × 104 HGSOC cells (TYK-nu or HeyA8) were plated 3. Patch AM, Christie EL, Etemadmoghadam D, Garsed DW, George J, in a 96-well plate and allowed to form spheroids for 24 hours before Fereday S, et al. Whole-genome characterization of chemoresistant being labeled with CellTracker Green (1:1,000) and transferred to ovarian cancer. Nature 2015;521:489–94. FTEC plates as for single-cell suspensions. Plates were washed once 4. Piek JM, van Diest PJ, Zweemer RP, Jansen JW, Poort-Keesom RJ, with PBS, fixed in 4% paraformaldehyde, and imaged with a Zeiss Menko FH, et al. Dysplastic changes in prophylactically removed Axio Observer.A1 with AxioVision software (Rel 4.8). 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Perets R, Wyant GA, Muto KW, Bijron JG, Poole BB, Chin KT, et al. labeling with CellTracker Green (1:1,000). In parallel, 5 × 103 TYK-nu Transformation of the fallopian tube secretory epithelium leads to cells were plated in a nonadherent 96-well plate (Corning) to form high-grade serous ovarian cancer in Brca; Tp53; Pten models. Cancer spheroids for 72 hours. Spheroids were directly transferred from the Cell 2013;24:751–65. 96-well plate to the FTEC monolayer and incubated for 12 hours 8. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigen- before fixation and staining for actin (AlexaFluor 647 phalloidin; esis. Cell 1990;61:759–67. Thermo Scientific). Images were collected on a Zeiss LSM 510 Meta 9. The Cancer Genome Atlas N. Integrated genomic analyses of ovarian Confocal microscope and images processed with ImageJ. carcinoma. Nature 2011;474:609–15. 10. Ciriello G, Miller ML, Aksoy BA, Senbabaoglu Y, Schultz N, Sander C. Disclosure of Potential Conflicts of Interest Emerging landscape of oncogenic signatures across human cancers. Nat Genet 2013;45:1127–33. No potential conflicts of interest were disclosed. 11. Yachida S, Jones S, Bozic I, Antal T, Leary R, Fu B, et al. Distant metastasis occurs late during the genetic evolution of pancreatic cancer. Authors’ Contributions Nature 2010;467:1114–7. Conception and design: M.A. Eckert, S.L. Volchenboum, E. Lengyel 12. McDaniel AS, Stall JN, Hovelson DH, Cani AK, Liu CJ, Tomlins SA, Development of methodology: M.A. Eckert, J. Andrade, S.L. et al. Next-generation sequencing of tubal intraepithelial carcinomas. Volchenboum JAMA Oncol 2015;1:1128–32. Acquisition of data (provided animals, acquired and managed 13. Schwarz RF, Ng CK, Cooke SL, Newman S, Temple J, Piskorz AM, patients, provided facilities, etc.): M.A. Eckert, S. Pan, R.M. Loth, et al. Spatial and temporal heterogeneity in high-grade serous ovarian S.L. Volchenboum, P. Faber, A. Montag, S.D. Yamada, E. Lengyel cancer: A phylogenetic analysis. PLoS Med 2015;12:e1001789. Analysis and interpretation of data (e.g., statistical analysis, 14. McPherson A, Roth A, Laks E, Masud T, Bashashati A, Zhang AW, et al. Divergent modes of clonal spread and intraperitoneal mixing in biostatistics, computational analysis): M.A. Eckert, S. Pan, K.M. high-grade serous ovarian cancer. Nat Genet 2016;48:758–67. Hernandez, S.L. Volchenboum, M.E. Peter, E. Lengyel 15. Kandoth C, McLellan MD, Vandin F, Ye K, Niu B, Lu C, et al. Writing, review, and/or revision of the manuscript: M.A. Eckert, Mutational landscape and significance across 12 major cancer types. S.L. Volchenboum, R. Lastra, M.E. Peter, S.D. Yamada, E. Lengyel Nature 2013;502:333–9. Administrative, technical, or material support (i.e., reporting or 16. Lawrence MS, Stojanov P, Polak P, Kryukov GV, Cibulskis K, organizing data, constructing databases): E. Lengyel Sivachenko A, et al. Mutational heterogeneity in cancer and the Study supervision: M.A. Eckert, J. Andrade, E. Lengyel search for new cancer-associated genes. Nature 2013;499:214–8.

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December 2016 CANCER DISCOVERY | OF10

Genomics of Ovarian Cancer Progression Reveals Diverse Metastatic Trajectories Including Intraepithelial Metastasis to the Fallopian Tube

Mark A. Eckert, Shawn Pan, Kyle M. Hernandez, et al.

Cancer Discov Published OnlineFirst October 7, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/2159-8290.CD-16-0607

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