clonality, heterogeneity, and evolution of synchronous ... · and management. early evidence based...

Molecular and Cellular Pathobiology

Clonality, Heterogeneity, and Evolution ofSynchronous Bilateral Ovarian CancerXia Yin1,2, Ying Jing1, Mei-Chun Cai3, Pengfei Ma1, Yi Zhang1,2, Cong Xu1,2,Meiying Zhang1,2,Wen Di1,2, and Guanglei Zhuang1,2

Abstract

Synchronous bilateral ovarian cancer (SBOC) represents arelatively frequent occurrence and clinically relevant diagnosticdilemma. Delineation of its clonal architecture, genetic hetero-geneity, and evolutionary trajectories may have important impli-cations for prognosis and management of patients with SBOC.Here, we describe the results of next-generation whole-exome orwhole-genome sequencing of specimens from12 SBOC cases andreport that bilateral tumors from each individual display a com-parable number of genomic abnormalities and similar mutation-al signatures of single-nucleotide variations. Clonality indicesbased on tumor-specific alterations supported monoclonal ori-gins of SBOC. Each of the ovarian lesions was neverthelessoligoclonal, with inferred metastatic tumors harboring more

subclones than their primary counterparts. The phylogeneticstructure of SBOC indicated that most cancer cell disseminationoccurred early, when the primary carcinoma was still relativelysmall (<100million cells). Accordingly, themutation spectra andmutational signatures of somatic variants exhibited pronouncedspatiotemporal differences in each patient. Overall, these findingssuggest that SBOCs are clonally related and form through pelvicspread rather than independent multifocal oncogenesis. Meta-static dissemination is often an early event, with dynamic muta-tional processes leading to divergent evolution and intratumorand intertumor heterogeneity, ultimately contributing substan-tially to phenotypic plasticity and diverse clinical course in SBOC.Cancer Res; 77(23); 6551–61. �2017 AACR.

IntroductionMultiple synchronous gynecological lesions represent a rela-

tively frequent occurrence in female patients suffering fromovary-associated malignant diseases (1–4). Clinicians are often facedwith the diagnostic dilemma of whether the simultaneouslypresented tumors arise independently or result from metastaticdissemination (5–7). Such a distinction is especially importantyet challenging in the case of epithelial ovarian cancer (EOC),which contributes to the highest mortality among gynecologicalmalignancies (8–10). At the time of diagnosis, the majority ofwomen with EOC display multisite tumor spread and are empir-ically treated asmetastatic disease, although individual foci couldpossibly originate from independent ancestral clones (11–15).Of

particular perplexity, advanced EOC often affects both sides ofovaries, and synchronous bilateral ovarian cancers (SBOC) areeven observed in 20% to 25% of early-stage patients whoseneoplasm is still limited to the ovaries without pelvic extensionor widespread metastasis (16). However, currently there is noconsensus on the clonal architecture of SBOCs, the delineationof which may hold profound implications for patient prognosisand management. Early evidence based on X chromosomeinactivation, microsatellite alterations, cytogenetic analyses,loss of heterozygosity, and somatic mitochondrial DNA muta-tions has led to unclear conclusions (5, 16–20). In addition,these previous studies only provided incomplete molecularportraits of SBOCs and did not address the potential impactof genetic heterogeneity and evolutionary trajectories on shap-ing ovarian tumorigenesis.

Recent advancement of next-generation sequencing allows foraccurate diagnosis and comprehensive characterization of syn-chronous gynecological tumors. For example, massively parallelsequencing-based analyses reveal that synchronous endometrialand ovarian carcinomas are often clonally related and constitutemetastatic deposits disseminated from one site to the other,despite undergoing notable genetic divergence upon separation(21–23). Furthermore, genomic studies centered on understand-ing the phylogenetic relationships of primary gynecologicalcancer and concurrent abdominopelvic metastases depict theirclonal lineages and progression patterns, yielding new insightsinto the mechanisms of transcoelomic spread (24, 25). In prin-ciple, in-depth bioinformatics interrogation of high-resolutionsequencing data should not only enable definitive discriminationbetween two independent primary tumors andmetastatic disease,but also help infer the molecular origins and evolutionary courseof SBOCs.

1State Key Laboratory of Oncogenes and Related Genes, Department of Obstet-rics and Gynecology, Ren Ji Hospital, School of Medicine, Shanghai Jiao TongUniversity, Shanghai, China. 2Shanghai Key Laboratory of Gynecologic Oncol-ogy, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University,Shanghai, China. 3State Key Laboratory of Oncogenes and Related Genes,Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai JiaoTong University, Shanghai, China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

X. Yin, Y. Jing, and M.-C. Cai contributed equally to this article.

Corresponding Authors: Guanglei Zhuang, Shanghai Jiao Tong University, 800Dongchuan Road, Wenxuan Building, Room 405, Shanghai 200240, China.Phone: 86-21-34207995; Fax: 86-21-34207995; E-mail: [email protected];and Wen Di, No. 1630 Dongfang Road, Ren Ji Hospital, Shanghai 200127, China,E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-1461

�2017 American Association for Cancer Research.

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In the current study, we subjected a series of 12 SBOC cases towhole-exomeorwhole-genome sequencing (WGS),with the aimsto (i) clarify the clonal relatedness of synchronous ovarian tumorspresent in a given patient and (ii) determine the temporal dynam-ics and spatial heterogeneity underlying SBOC development. Ourdata unambiguously indicated that SBOCs in all individuals werederived froma commonancestry, irrespective of histological typesor clinical stages.We further demonstrated that the disseminationof unifocal bilateral tumors could occur early during the patho-genesis of ovarian cancer, resulting in disparate repertoires ofsomaticmutations and varied degree of intratumor heterogeneity.These findings lay the basis for prospective prognostic investiga-tions of unilateral versus bilateral EOCwithmatched clinicopath-ologic criteria and may potentially change current guidelines forthe prevention, early detection, precise diagnostication, and ratio-nal intervention of ovarian cancer.

Patients and MethodsPatient cohort

The study was conducted in accordance with ethical guidelinesof U.S. Common Rule and approved by the Ren Ji Hospital EthicsCommittee. Written informed consent was obtained from allpatients. Between 2008 and 2011, women diagnosed with EOCswere selected from a review of patients who had been treated andfollowed up in Ren Ji Hospital, Shanghai Jiao Tong University.Our cohort for retrospective analysis comprised a total of 121ovarian cancer patients. Clinical information, including age atdiagnosis, FIGO stage, pathological grade, CA-125 concentration,survival status and clinical intervention, was obtained frommedical records. The c2 test was used to evaluate the associationbetween ovarian cancer subtype and the clinicopathologic para-meters of the patients. P < 0.05 was considered statisticallysignificant. Biospecimens for sequencing were collected frompatients diagnosed with pathologically confirmed ovarian cancerduring initial debulking surgery in Ren Ji Hospital, who hadreceived no prior treatment for their disease. The sequencedcohort was selected based on the availability of paired ovariantumor tissues and archived blood cell controls, as well as theproportion of tumor cell nuclei (>50%) and necrosis (<30%;reviewed by a certified pathologist). Genomic DNA quantity andquality were determined to further exclude unqualified speci-mens. Eventually, 33 formalin-fixed and paraffin-embedded(FFPE) samples from 11 patients with SBOC (RJOC1-11) weresubjected to whole-exome sequencing (WES), concerning limitedDNA materials, and 5 fresh-frozen samples from RJOC12 withmetastatic disease were subjected to WGS.

Genomic DNA preparationTumor DNA was extracted from tissue shavings of fresh-frozen

specimens (RJOC12) or 10 sections with a thickness of 10 mmfrom FFPE tissues (RJOC1-11) using the QIAamp DNA FFPETissue Kit. Paired blood cell DNAwas extracted following instruc-tionsusing theQIAampDNABloodMiniKit.DNAwasquantifiedbyQubit (Life Technologies), andDNA integritywas examined byagarose gel electrophoresis.

Whole-exome sequencingA paired-end DNA library was constructed according to the

manufacturer's instructions (Agilent). Genomic DNA frompatients RJOC1 to 11 was sheared into 180 to 280 bp fragments

by Covaris S220 sonicator and purified using AMPure SPRI beadsfrom Agencourt. The DNA fragments were enriched by 6 cycles ofPCR using SureSelect Primer and SureSelect ILM Indexing PreCapture PCRReverse Primer. The size distributions of the librarieswere examined on an Agilent Bioanalyzer DNA 1000 chip. DNA(500 ng) was subjected to whole-exome capture, using the Agi-lent's SureSelect Human All Exon V5Kit. The capturedDNA–RNAhybrids were recovered using Dynabeads MyOne Streptavidin T1from Dynal. DNA was eluted from the beads and desalted usingQiagen MinElute PCR purification columns. The purified captureproducts were then amplified using the SureSelect ILM IndexingPost Capture Forward PCR Primer and PCR Primer Index 1through Index 16 (Agilent). DNA sequences (50 Mb) of334,378 exons from 20,965 genes were captured. DNA librarieswere sequenced on Illumina Hiseq 4000 sequencing platform(Illumina) according to the manufacturer's instructions forpaired-end 150 bp reads (Novogene). The targeted sequencingdepth was 200�.

Whole-genome sequencingOne microgram genomic DNA from each sample of patient

RJOC12 was used as input for the DNA library preparations.Sequencing library was prepared using Truseq Nano DNA HTSample Prep Kit (Illumina) following the manufacturer's recom-mendations. Briefly, genomic DNA sample was fragmented, end-polished, A-tailed, and ligated with the full-length adaptor forIllumina sequencing, followed by further PCR amplification. Theclustering of sampleswas performed on a cBot Cluster GenerationSystem using Hiseq PE Cluster Kit (Illumina) according to themanufacturer's instructions. After cluster generation, the DNAlibraries were sequenced on Illumina Hiseq 4000 sequencingplatform (Illumina) and 150 bp paired-end reads were generated(Novogene). The targeted sequencing depth was 60�.

Sequence alignment and variant callingClean reads in FastQ format generated by the Illumina platform

were aligned to UCSC hg19 human genome by Burrows-WheelerAligner to obtainmapping results in BAM format (26). SAMtools,Picard (http://broadinstitute.github.io/picard/), and GATK wereused to filter BAM files, local realignment, and base qualityrecalibration to generate final BAM files for computation of thesequence coverage and depth (27, 28). The somatic single-nucle-otide variations (SNV) were detected by MuTect with an addi-tional filter described below (29). The somatic indels detected byGATK Somatic Indel Detector. ANNOVAR was performed to doannotation for VCF (Variant Call Format; ref. 30). Variantsobtained from previous steps were compared against SNPs pres-ent in the dbSNP and 1000 Genomes databases (1000 GenomesProject Consortium) to discard known SNPs. The retained non-synonymous SNVs were submitted to PolyPhen and SortingIntolerant From Tolerant (SIFT) for functional prediction (31,32). Control-FREEC was utilized to detect somatic copy-numbervariations (33). For samples from patient RJOC12, which weresubjected to WGS, Breakdancer was implemented to identifypotential structural variants (34).

Filters for FFPE samplesAdditional filters were applied to exclude artifactual mutations

introduced by FFPE specimens. In brief, duplicates and softclipped reads removed data were analyzed in MuTect with theseparameters (align quality: 30; strand bias: 0.05; keep themutation

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sitewithhighest alignquality ifmore thanonemutation siteswereexamined within 11 bp; keep the mutation sites supported by atleast three different reads). Furthermore, we filtered out singlestrand bias based on a read pair orientation of larger than 20:1.

Identification of putative driver mutationsAll identified nonsynonymous mutations were compared with

potential driver genes in ovarian cancer or present in the COSMICcancer gene census. Putative driver mutations were determined ifthey satisfy one of the following criteria: (i) either the exactmutation, the same mutation site or at least three mutationslocated within 15 bp of the variant were found in COSMIC and(ii) if the candidate gene was marked as recessive in COSMIC andthe variant was predicted to be deleterious and had a SIFT score<0.05 or a PolyPhen score >0.995. The putative driver mutations(variant allele frequency, VAF, >0.1) were subjected to Sangersequencing for validation.

Mutational signature generationAll somatic SNVs were included to calculate relative weights

of mutational signatures in a given sample. The R package"deconstructSigs," based on the Wellcome Trust Sanger InstituteMutational Signature Framework, was used to statistically quan-tify the contribution of each signature for each tumor (35).

Bioinformatics analysisSciClone R package (https://github.com/genome/sciclone)

and PyClone (http://compbio.bccrc.ca/software/pyclone/) wereused to detect subclonality of a given sample (36, 37). Clonallyrelatedness was determined by clonality indices calculation,which was performed as described previously (21). Phylogenetictree was constructed through PHYLIP version 3.695. For eachvariant, the cancer cell fraction (CCF) was defined asVAF ¼ a�CCF

CT�aþCNð1�aÞ , where CT is the copy number of the tumor,

CN is the copy number of the matched normal sample, and a isthe tumor purity. Tumor purity and copy number were deter-mined by ABSOLUTE (38). VAF is defined with respect to thenumber of reads supporting the variant allele (xvar) and thenumber of reads supporting the reference allele (xref ):VAF ¼ xvar

xvarþxref. HLA typing was performed using Polysolver

(39). Nonsilent mutations were used to generate a list of peptidesranging 9 to 11 amino acids in length with the mutated residuesrepresented in each position. Prediction for binding affinity ofevery mutant peptide and its corresponding wild-type peptide tothe patient's germline HLA alleles was performed using theNetMHCpan (v3.0; ref. 40). Candidate neoantigens were identi-fied as those with a predicted mutant peptide binding affinity of<500 nmol/L.

Statistical analysisStatistical analysis was performed with GraphPad Prism soft-

ware. In all experiments, comparisons between two groups werebased on two-sided Student t test, and one-way analysis ofvariance (ANOVA) was used to test for differences among moregroups. P values of <0.05 were considered statistically significant.

ResultsClinical attributes and mutational profiles of SBOC

We set out to assess the prevalence and characteristics of SBOCby performing a retrospective analysis of 121 consecutive ovarian

cancer patients who underwent debulking surgery in our hospitalbetween 2008 and 2011 (Supplementary Table S1). A total of 46women (39%) with EOC had pathologically confirmed concur-rent tumors in both ovaries. At diagnosis, the median age ofpatients presenting SBOCs was similar to that of individuals withunilateral ovarian cancer (54 vs. 57). Most SBOCs (63%) werediagnosed as serous carcinomas, in comparison with 32% ofunilateral EOC cases (P ¼ 0.001, c2 test). Bilaterality was asso-ciated with EOC of higher histological grade (P < 0.001, c2 test)andmore advanced FIGO stage (P < 0.001, c2 test). The estimated5-year survival rate of SBOCs was significantly lower than that ofunilateral EOCs (30.7% vs. 65.4%, P ¼ 0.007, c2 test). Althoughthese data implied that SBOCsmight represent metastatic diseasewith a dismal prognosis, the exact molecular origin and relation-ship of the synchronous bilateral tumors remained largely elusive.

To resolve the clonal composition of SBOCs, we selectedarchived biospecimens from the 121-sample cohort formolecularanalysis, on the basis of availability of paired ovarian tumortissues and companion blood cell controls, as well as the pro-portion of tumor cell nuclei (>50%) and necrosis (<30%). Twen-ty-two tumors from paired ovaries of 11 EOC patients(Fig. 1A; Table 1), spanning various FIGO stages (from IB to IV)and different histological subtypes (Fig. 1B), passed sequencingquality control and were included in our study. Both representa-tive hematoxylin and eosin (H&E) staining and computed tomog-raphy (CT) images verified the presence of SBOC in each patient(Supplementary Fig. S1). Genomic DNA extracted from tumorsandmatched peripheral blood cells were subjected toWES with amedian depth of 212� (ranging from 99� to 393�). At least97.9%of targeted baseswere covered to a depth ofmore than 10�(Supplementary Table S2). We applied stringent filters to excludeenriched C>T sequence artifacts characteristic of DNA from FFPEtissues (41–43), and identified a median of 132 somatic exonicalterations per tumor (27–2,324), including 107 nonsynon-ymous mutations (17–1,537; Fig. 1C; Supplementary TableS3). A subset of somatic mutations with VAF of >0.1 wereanalyzed with Sanger sequencing, achieving a 90% validationrate (Supplementary Table S4). Consistent with previous reports(44), TP53 mutations were prevalent and detected in all EOCpatients (Fig. 1C). Analysis of genetic predisposition on the basisof 24 hereditary gynecologic cancer genes uncovered 13 single-nucleotide polymorphisms (SNP) possibly associated with ovar-ian cancer susceptibility in 9 patients (Supplementary Fig. S2;Supplementary Table S5). Notably, patient RJOC8 harboredgermline mutations in mismatch repair genes related to lynchsyndrome, including MSH2/6, and displayed a hypermutatorphenotype (Fig. 1C; Supplementary Fig. S2). Overall, the numberof genomic abnormalities, mutation spectra, and mutationalsignatures of SNVs were fairly consistent between two ovariantumors from each of the individuals (Fig. 1C–E). Additionally, weidentified putative driver genetic aberrations involving genesregulating multiple pivotal cancer-promoting pathways (Supple-mentary Table S6) and synchronous ovarian lesions from a givenpatient often shared mutations enriched in the same functionalcategories (Fig. 1D), further supportingmonoclonality of bilateraltumors within our SBOC cohort.

Establishing the clonal relationship of SBOCsRooted in the clonal evolution theory of tumorigenesis, SBOCs

are assumed to inherit an identical set of somatic variants if theyarise from a single ancestral progenitor cell; on the other hand,

Synchronous Bilateral Ovarian Cancers Are Clonally Related

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distinct genomic profiles of bilateral ovarian tumors would mostlikely indicate multifocal origins. Following this conceptualframework,weprobed the genetic aberrations of SBOCs identifiedby WES in order to yield important information about themolecular relationship of paired bilateral tumors in each patient.Our results revealed that the two synchronous tumors of a givencase contained considerably overlapping repertoires of somaticexonic mutations including SNVs and indels (Fig. 2A), though todiverse extents in different patients (11%–71%). Notably, clonal

architecture of SBOCs indicated that inmany cases, the number ofidentical shared variants was larger than or comparable with thatof private somatic events. Nonetheless, several patients such asRJOC3, RJOC8, and RJOC10 displayed relatively less similaritybetween bilateral ovarian carcinomas, raising the possibility thatshared mutations in these samples could had occurred by chance(Fig. 2B). To formally evaluate the phylogeny of SBOCs, we used aconservative analytic approach to estimate their clonality indicesbased on all SNVs and indels (21) and found that paired bilateral

Figure 1.

Clinical attributes and mutational profiles of SBOC. A, A schematic graph of anatomic sites from which tumor samples were obtained. B, Clinicopathologicalinformation of SBOC patients. FIGO, International Federation of Gynecology and Obstetrics stage; LNM, lymph node metastasis. C, Numbers of SNVs,small insertions and deletions (INDEL), and copy-number variations (CNV) detected by WES in each tumor sample. TP53 mutation status is indicated.D, Distributions of six substitution classes in all samples (top). Significantly mutated genes were classified according to the functional categories (bottom).E, Frequencies of the 96 trinucleotide mutation types in all tumors are displayed in a heat map.

Table 1. Clinicopathologic information of sequenced SBOCs

PatientID

Age(year) Gender Histology

FIGOstage

Tumorgrade

CA125(U/mL)

Lymph nodemetastasis Specimen sites

RJOC1 71 Female High-grade serous adenocarcinoma IIIC 3 152 Present Left and right fallopian tubeRJOC2 47 Female High-grade serous adenocarcinoma IIB 2 291.9 Absent Left and right ovaryRJOC3 48 Female High-grade serous adenocarcinoma IIIC 2–3 129 Absent Left and right ovaryRJOC4 60 Female High-grade serous adenocarcinoma IIIC 2–3 288.9 Present Left ovary and right adnexaRJOC5 62 Female High-grade serous adenocarcinoma IV 3 6,684 Present Left and right ovaryRJOC6 49 Female High-grade serous adenocarcinoma IIIC 2–3 3298 Present Left and right ovaryRJOC7 55 Female High-grade serous adenocarcinoma IIIC 2 9.46 Present Left and right ovaryRJOC8 58 Female High-grade serous adenocarcinoma IIIC 3 178 Present Left and right ovaryRJOC9 45 Female High-grade serous adenocarcinoma IV 2–3 374.8 Absent Left and right ovaryRJOC10 63 Female High-grade serous adenocarcinoma IIIC 2 1,443 Present Left and right ovaryRJOC11 70 Female Endometrioid adenocarcinoma IB 3 258 Absent Left and right ovaryRJOC12 37 Female High-grade serous adenocarcinoma IIIC 2 846.3 Present Left and right ovary, colon, stomach

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tumors from all 11 patients were clonally related with no excep-tion (Fig. 2C). Therefore, we concluded that SBOCs formedthrough metastatic spread rather than independent multifocaloncogenesis.

Monoclonal seeding and expansion of SBOCTo validate our findings in FFPE tissues (RJOC1-11) that are

prone to sequencing errors, we recruited a patient (RJOC12) withstage III serous ovarian cancer and sampled fresh-frozen bilateralovarian masses, in addition to two other anatomic sites (colonand stomach) with distant metastases. WGS achieved an averageof 50� coverage with 99% of targets sequenced at a depth of�10�, thus providingmore robust genetic data to better decipherthe metastatic routes and evolutionary dynamics of ovarian

malignancies. As with the WES-profiled subjects (RJOC1-11),RJOC12 exhibited generally similar mutation spectra and muta-tional signatures across all four specimens, although mutationburdens of the right ovarian tumor appeared to be relatively lower(Fig. 3A). We sought to infer the spatiotemporal origin of neo-plastic development by applying two complementary approacheson the basis of somatic mutations. First, SciClone (36) wasperformed to identify the number and genetic composition ofsubclones in each sample (Supplementary Fig. S3), followed bytwo-dimensional cluster correlation between tumor pairs of thefour samples. These analyses captured tumor-specific mutationsin cluster 2, which were observed in all samples but right ovarianlesion (Fig. 3B). Alternatively, a phylogenetic clustering method(45, 46) was used and revealed that the genome of right ovarian

Figure 2.

Establishing the clonal relationship of SBOCs. A, Venn diagrams present the total number of somatic exonic mutations unique to the left/right ovariantumor or shared between SBOCs. B, Heat maps show the presence (blue) or absence (gray) of somatic mutations in indicated tumor samples. C, Clonalityindices for the 11 cases of SBOC analyzed in our study, defined as the likelihood of two carcinomas sharing mutations not expected to have co-occurredby chance. Black dotted lines indicate the cutoff value to define clonal relatedness.






tumor was hierarchically most similar to germline DNA ofRJOC12, whereas sequenced samples from the other three ana-tomic sites located in parallel on the same branch (Fig. 3C). Takentogether, our results suggested that the right ovarian tumorrepresented the primary neoplasm, from which a subpopulationof cancer cells disseminated and implanted in the left ovary,colon, and stomach. Indeed, comparison of copy-number altera-tions or chromosomal structural variations harbored by foursamples confirmed this tumor topology (Fig. 3D; SupplementaryFig. S4). In addition, the strikingly recurrent patterns of somaticevents in distal sites and appreciably early dichotomy betweenprimary andmetastatic tumors supported amodel ofmonoclonalseeding and expansion during ovarian cancer progression ofRJOC12 (Fig. 3D). Interestingly, in line with the spatiotemporaltrajectories of malignant dissemination, CCF (the fraction oftumor cells harboring the SNVs) of somatic mutations in theright ovarian tumor was significantly lower than that in threepresumed metastatic lesions (Fig. 3E), which elicited our attemptto define the chronology of SBOC according to the CCF infor-mation. By comparing the relative CCF abundance of sharedsomatic alterations between bilateral ovarian tumors, we wereable to deduce the likely primary side of tumorigenesis with thesmaller median CCF value for each patient (Supplementary Fig.

S5). Future prospective clinical studies integrating bothmolecularand histopathological features to determine the metastatic direc-tionality of SBOC are warranted.

Spatiotemporal divergent evolution of SBOCTo further investigate the evolutionary process of SBOCs, we

performed phylogenetic reconstruction of the WES data for eachpatient (Fig. 4A). The first notable observation was that thephylogenetic structure varied considerably between differentSBOC cases, i.e., disparate timing of branched evolution in eachindividual resulting from tumor metastasis. By taking intoaccount the ovarian tumor volumes at surgery, we performed aquantitative analysis on the time point of metastatic spread,indicating that cancer dissemination to the contralateral ovarymostly occurred early when the primary carcinoma was stillrelatively small (<100 million cells; Fig. 4B). Accordingly, thespectra of point mutations in each patient displayed pronouncedtemporal difference between early (truncal) and late (branched)SNVs (Fig. 4A), with a statistically significant increase of C>Ttransitions and decrease of C>A or C>G transversions in latecompared with early mutations (Fig. 4C). We used deconstruct-Sigs framework (35) to extract known mutational signatures(47, 48) that might contribute to the specific mutation profiles

Figure 3.

Monoclonal seeding and expansion of SBOC.A, Numbers of somatic SNV, INDEL, and copy-number variation detected by whole-genome sequencing in each tumorsample of RJOC12 (left). Distributions of six substitution classes in all samples of RJOC12 (middle). Frequencies of the 96 trinucleotide mutation types in all tumors ofRJOC12 are displayed in a heat map (right). L, left ovary; R, right ovary; C, colon; S, stomach. B, Two-dimensional cluster correlation of SciClone analysisbetween tumor pairs of the four samples. C, Phylogenetic clustering of tumor and germline DNA sequences. D, Evolutionary history of RJOC12 is depicted.Representative H&E staining of all tumor samples is shown. Circos plots present inter- and intrachromosomal translocations, with events shared by alltumors indicated in red, events shared by metastatic lesions indicated in orange, and unique events indicated in black. The Circos plots depicting copy-number variations are shown at bottom. E, The heat map displays the CCF of all mutations in each sample of RJOC12.

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of early versus late SNVs (Fig. 4D; Supplementary Fig. S6). Early(truncal) SNVs exhibited an increased enrichment of signature 3(associated with failure of DNA double-strand break repair byhomologous recombination), signature 4 (attributable to tobaccomutagens), signature 13 (implicated with APOBEC activity), andsignature 24 (related to aflatoxin exposures). On the other hand,later (branched) SNVs were more consistent with mutationalprocesses characterized by signature 1 (correlated with age ofcancer diagnosis) and signature 6 (associatedwith defectiveDNA-mismatch repair). Additional investigations are required to fullyuncover the mechanistic underpinnings and biological signifi-cance of these observations, although the results pinpointeddifferential contributions of deficiency in different DNA repairmechanisms during ovarian cancer progression. Moreover, earlymutations included a large proportion of driver events, such asalterations in TP53, NOTCH1, and ARID1B; however, multipledriver mutations affecting cancer genes including EZH2 and

AXIN1 were acquired late (Fig. 4E). In addition, spatial hetero-geneity, as indicated by divergent mutation spectra and muta-tional signatures of late SNVs,was evident between geographicallyseparated bilateral tumors (Fig. 4A; Supplementary Fig. S7),suggesting that primary and metastatic cancers continued togenetically evolve upon disassociation. Together, our findingsimplied that dynamic mutational processes constantly shapedtumor genomes over time, conceivably leading to the substantialspatiotemporal divergent evolution of SBOC.

Intratumor and intertumor heterogeneity of SBOCPrevious studies have revealed profound intratumor genetic

heterogeneity of high-grade serous ovarian cancer, which mayhave predictive value for survival after chemotherapy treatment(49). To estimate the extent of intratumor heterogeneity inour SBOC cohort, we performed single-sample subclonality anal-ysis using SciClone (Supplementary Fig. S8) and PyClone

Figure 4.

Spatiotemporal divergent evolution of SBOC. A, Fraction of early (truncal) mutations and late (branched) mutations accounted for by each of the sixmutation types in bilateral ovarian lesions of RJOC1-11. Proportions of six substitution classes are indicated in pie charts. B, A schematic display of themutational timeline illustrating the time points of metastatic spread. One billion cells were equal to a tumor volume of �1 cm3 at surgery. Green arrowsindicate the inferred timing of tumor cell spread. C, Fraction of the early and late mutations in each of the substitution categories (unpaired Student t test).D, Plots of the inferred relative weights of SNVs attributed to each mutational signature for SNVs acquired on the early versus late stages of eachphylogenetic tree. Only signatures with a significant difference in the inferred relative weights between early and late SNVs are shown (unpaired Studentt test). E, Temporal distribution of driver mutated genes in 11 SBOC patients.






(Supplementary Fig. S9). Both methods consistently showed thatthemajority of sequenced samples were oligoclonal (Supplemen-tary Table S7) and the clonal structure was more complicated inmetastatic deposits relative to primary tumors (Fig. 5A). Conse-quently, most key putative driver mutations were subclonalevents in bilateral ovarian carcinomas, as evidenced by the CCFof below 1 (Fig. 5B). In addition, we evaluated the druggability ofsomatic aberrations by stratifying them based on the CancerDrivers Actionability Database (50). Although all 11 SBOC casescould potentially benefit from current or prospective anticancertargeted agents, the in silico prescription approach highlighted apatient-specific therapeutic landscape. More importantly, distincttailored drugs should be assigned to primary ormetastatic cancers(Fig. 5C).Of note, somatic alterations ofDNA repair genes such asBRCA1/2 often occurred only in one side of SBOC, in contrast tothe uniformly mutated germline cancer–predisposition genes(Supplementary Fig. S10), implying that currently approvedPARP inhibitors might display different efficacy against pairedtumors in some patients. Finally, bilateral tumors were predicted

to harbor distinct repertoires of neoantigens (Fig. 5D), suggestiveof heterogeneous tumor-infiltrating lymphocytes and potentiallydifferential clinical responses to immune checkpoint inhibitors.We concluded that the prominent intratumor and intertumorheterogeneity contributed to the genetic complexity and pheno-typic diversity of clonal related bilateral ovarian tumors, andmight have significant impact on the clinical course and thera-peutic interference of individuals who suffer from SBOC.

DiscussionIn this study, by performing next-generation genomic sequenc-

ing and detailed bioinformatics analysis, we have presented anexplicit view of the clonality, heterogeneity, and evolution ofSBOC. Our data support three major conclusions: first, bilateraltumors of SBOC most likely originate from a common ancestry,regardless of histotypes or pathological stages; second, ovariancancer cells may disseminate extremely early to give rise to theSBOC disease; third, each ovarian lesion comprises genetically

Figure 5.

Intratumor and intertumor heterogeneity of SBOC. A, Histogram and density (red line) of the number of SciClone inferred clonal clusters among 11 SBOCs(top). Density distribution of clonal clusters for primary (light blue) and metastatic (pink) tumors (bottom). B, The heat map displays the CCF of putativedriver mutations in bilateral ovarian lesions of RJOC1-11. C, Classification of nonsilent SNVs in genes with potential druggable implications according to theCancer Drivers Actionability Database. D, Binding affinity of the neoantigen was predicted across all 9-11 amino acids peptides generated from nonsilentmutations and the corresponding wild-type peptides using NetMHCpan algorithms. Mutated peptides with predicted binding affinity of <500 nmol/L wereplotted. Dark blue, neoantigens identified in both bilateral tumors; light blue, neoantigens identified only in the left ovarian tumor; yellow, neoantigensidentified only in the right ovarian tumor.

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heterogeneous composition and paired ovarian cancers displaynotable intertumor heterogeneity. These findings providenew insights into the phylogenetic principles of ovarian tumor-igenesis and malignant progression and may have considerableimplications for the prognosis and management of patientsbearing SBOC.

Previous studies, using a variety of experimental strategies,have attempted to evaluate the clonal relatedness of synchro-nous bilateral ovarian tumors and have reached inconsistentconclusions. For example, genetic markers based on the patternof X chromosome inactivation and microsatellite instability(MSI) revealed that SBOCs shared a unifocal origin (16).Similarly, cytogenetic analyses indicated that bilateral ovariancancer developed through metastatic spreading (19). In strikingcontrast, distinct somatic mitochondrial DNA variants weredetected between paired tumors in 24% (4 out of 17) of SBOCcases, indicative of different clonal populations of cancer cells(20). Additionally, evidence for monoclonality was found in 14of 16 SBOCs according to loss of heterozygosity (LOH) anal-ysis, whereas bilateral tumors in the remaining two casesshowed discordant LOH patterns, which was thought to bemost likely due to independent origins (18). Moreover, geneticcharacterization by combining LOH and MSI information intoa statistical algorithm classified 33% (5 out of 15) of SBOCs ashaving independently derived primary tumors (5). We specu-late that one plausible reason for these contradictory results liesin the insufficient resolution of limited numbers of molecularmarkers. Our study, on the other hand, exercised comprehen-sive genomic profiling of SBOCs for the first time (to ourknowledge) and conclusively demonstrated the uniformmonoclonality of bilateral ovarian lesions in most, if not all,cases. However, it is noteworthy that all the paired bilateraltumors have the same histologic appearance in our SBOCcohort. Therefore, it remains to be determined whether SBOCswith dissimilar histotypes are clonally related as well or rep-resent independent tumors.

It has been suggested that malignant dissemination can occurearly in the course of SBOC, based on the fact that some cases arediagnosed as stage IB diseasewith tumors affecting both ovaries inthe presence of seemingly intact capsules (16). Indeed, our SBOCcohort also included a stage IB patient and her bilateral ovariancarcinomas evolved as unifocal neoplasms through a yet-to-beidentifiedmechanism ofmetastatic spread.We further discoveredthat the extraorgan metastasis likewise represented a generallyearly event even in the advanced-stage SBOC, by quantitativelyinferring the time point of cancer cell dissemination to thecontralateral ovary. Taken together, these findings imply thatearly distal dissemination may be an intrinsic feature of ovariancancer, thereby causing delayed diagnosis and dismal prognosisin patients with EOC. This notion poses formidable challengesfor active surveillance, early detection, and optimal interventionof ovarian pathogenesis. It should be noted, though, that a"pseudo-metastatic" scenario has been proposed in the contextof gynecological cancers with a predilection for abdominopelvicimplantation (22), which advocates distinguishing between clin-ically indolent dissemination and widespread metastasis. There-fore, it is important to recognize the complexity underneath thesimple bilaterality regarding SBOC in order not to undertreat orovertreat patients.

Considering the early disassociation of bilateral ovariantumors, perhaps it was not surprising to learn that pronounced

spatiotemporal divergent evolution took place to shape SBOCtumorigenesis. As a consequence, notable differences in spatialand temporal acquisition of putative driver mutations, includ-ing those predicted targetable, were observed between pairedtumors. Importantly, we showed that the repertoire of neoanti-gens created by genetic mutations were heterogeneous inSBOCs, which might influence inflammatory microenviron-ment and tumor immunoreactivity. In addition, each tumorwas revealed to contain multiple subclones, and metastaticlesions seemed to bear a more complex subclonal structurerelative to primary tumors. Our findings of the prominentintratumor and intertumor heterogeneity were in accordancewith previous reports unveiling the extensive genomic diversityin high-grade serous ovarian cancer (49). Furthermore, dynam-ic mutational processes were pinpointed during SBOC progres-sion, and our data highlighted DNA repair deficiency as aprevalent mechanism involving the whole evolutionary historyof ovarian cancer. Future investigations are warranted to uncov-er the mechanistic underpinnings and therapeutic relevance ofthese observations.

In summary, we have presented a detailed delineation of themolecular origins and evolutionary trajectories of SBOCs, whichholds the promise to improve the diagnosis and treatment ofovarian cancer. Our results reveal the monoclonality, generallyearly metastatic dissemination and profound genetic heteroge-neity associated with SBOCs, and provide an impetus for furtherdissecting the underlying biological mechanisms. We envisionthat the sequencing-based approach described in this study,which is applicable to FFPE tissues, may be incorporated intoroutine histopathology to reliably identify the clonal relation-ships of synchronous gynecological lesions and other multiplemalignancies.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: W. Di, G. ZhuangDevelopment of methodology: W. DiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): X. Yin, Y. Jing, C. Xu, M. Zhang, W. DiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): X. Yin, Y. Jing, M.-C. Cai, P. Ma, M. Zhang, W. DiWriting, review, and/or revision of the manuscript: X. Yin, Y. Jing, M.-C. Cai,W. Di, G. ZhuangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): X. Yin, Y. Zhang, W. DiStudy supervision: Y. Jing, W. Di, G. Zhuang

AcknowledgmentsThe authors thank all lab members for helpful discussions and advice

regarding this work.

Grant SupportThis work was supported by the National Natural Science Foundation of

China (81472537 and 81672714 to G. Zhuang; 81502597 to Y. Jing; 81472426to W. Di), the Grants from the State Key Laboratory of Oncogenes and RelatedGenes (No. 91-15-12 to G. Zhuang; SB17-06 to M.-C. Cai), the grants fromShanghai Jiao Tong University School of Medicine (DLY201505 to W. Di;YG2016MS51 to X. Yin), Shanghai Municipal Education Commission-GaofengClinical Medicine Grant Support (20161313 to G. Zhuang), the ShanghaiInstitutions of Higher Learning (Eastern Scholar to G. Zhuang), ShanghaiRising-Star Program (16QA1403600 to G. Zhuang), Shanghai Municipal Com-mission of Health and Family Planning (2013ZYJB0202 and 15GWZK0701 to






W. Di), the grant from Shanghai Key Laboratory of Gynecologic Oncology(FKZL-2017-01 to Y. Jing), and the grant from Science and Technology Com-mission of Shanghai Municipality (16140904401 to X Yin).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received May 23, 2017; revised July 22, 2017; accepted September 25, 2017;published OnlineFirst September 28, 2017.

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2017;77:6551-6561. Published OnlineFirst September 28, 2017.Cancer Res Xia Yin, Ying Jing, Mei-Chun Cai, et al. Ovarian CancerClonality, Heterogeneity, and Evolution of Synchronous Bilateral

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