Patient-derived ovarian tumor xenografts recapitulate human clinicopathology and genetic alterations

Therapeutics, Targets, and Chemical Biology

Patient-Derived Ovarian Tumor Xenografts RecapitulateHuman Clinicopathology and Genetic Alterations

Francesca Ricci1, Francesca Bizzaro1, Marta Cesca1, Federica Guffanti1, Monica Ganzinelli1,Alessandra Decio1, Carmen Ghilardi1, Patrizia Perego2, Robert Fruscio3, Alessandro Buda3,Rodolfo Milani3, Paola Ostano4, Giovanna Chiorino4, Maria Rosa Bani1, Giovanna Damia1, andRaffaella Giavazzi1

AbstractEpithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. On the basis of its histopa-

thology and molecular-genomic changes, ovarian cancer has been divided into subtypes, each with distinctbiology and outcome. The aim of this study was to develop a panel of patient-derived EOC xenografts thatrecapitulate the molecular and biologic heterogeneity of human ovarian cancer. Thirty-four EOC xenograftswere successfully established, either subcutaneously or intraperitoneally, in nude mice. The xenografts werehistologically similar to the corresponding patient tumor and comprised all the major ovarian cancersubtypes. After orthotopic transplantation in the bursa of the mouse ovary, they disseminate into the organsof the peritoneal cavity and produce ascites, typical of ovarian cancer. Gene expression analysis and mutationstatus indicated a high degree of similarity with the original patient and discriminate different subsets ofxenografts. They were very responsive, responsive, and resistant to cisplatin, resembling the clinical situationin ovarian cancer. This panel of patient-derived EOC xenografts that recapitulate the recently type I and typeII classification serves to study the biology of ovarian cancer, identify tumor-specific molecular markers, anddevelop novel treatment modalities. Cancer Res; 74(23); 6980–90. �2014 AACR.

IntroductionEpithelial ovarian cancer (EOC) accounts for 90% of ovarian

cancer and is the most lethal gynecologic cancer in westerncountries accounting for more than 13,000 deaths/years (1).Even with optimal treatment, consisting of surgical cytoreduc-tion (debulking) followed by platinum- and taxane-basedchemotherapy, the 5-year survival for women with advancedstage disease is only 46% at best (2).

On the basis of histology and IHC analysis, five main sub-types of EOC can be recognized: high-grade serous carcinoma(70%), endometrioid carcinoma (10%), clear cell carcinoma(10%), mucinous carcinoma (3%), and low-grade serous car-cinoma (<5%). The different histologic subtypes of EOC are

unique entities as indicated by differences in epidemiologicand genetic risk factors, and each has its own distinct biologicbehavior (precursion lesions, pattern of tumor spread,response to chemotherapy and prognosis; refs. 3, 4). In recentyears, the remarkable progress in understanding themolecularand cellular biology of ovarian cancer has brought to classifyEOC in two main categories based on the pattern of tumorprogression and molecular genetic alterations (3, 5–7). Type IEOCs include low-grade serous, low-grade endometrioid,mucinous, and a subset of clear cell carcinomas; they aregenetically stable and relatively indolent. Most of the type IIare high-grade serous and endometrioid carcinomas with anaggressive clinical course, genetically unstable, and frequentlymutated in TP53 (8, 9).

The scarcity of in vivo preclinical models that closely repro-duce the complexity and heterogeneity of ovarian cancer limitsthe development of new therapeutic strategies. Preclinicalmodels of ovarian cancer rely on in vitro stabilized cancer celllines, on tumor xenografts obtained from in vitro cell lines and,to a lesser extent, on patient-derived tumors (10, 11). Cancercell lines are reproducible, easy to use, and useful for studyingspecific mechanisms, but their resemblance to the originaltumor and thus their therapeutic predictive value is verylimited (12). Two studies describing ovarian cancer patient-derived xenografts have been recently published (13, 14).However, in those studies little characterization was carriedout in relation to the recently proposed origin and pathogen-esis of ovarian cancer (i.e., type I or type II), that is now basis fornovel target therapy. Recently, genetically engineered mouse

1Department of Oncology, IRCCS-Istituto di Ricerche FarmacologicheMario Negri, Milan, Italy. 2Department of Pathology, San Gerardo Hospital,Monza, Italy. 3Obstetrics and Gynecology Clinic, San Gerardo Hospital,Monza, Italy. 4Cancer Genomics Laboratory, Fondazione Edo ed ElvoTempia Valenta, Biella, Italy.

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

F. Ricci and F. Bizzaro contributed equally to this article.

Corresponding Authors:Giovanna Damia and Raffaella Giavazzi, Depart-ment of Oncology, IRCCS-Mario Negri Institute for PharmacologicalResearch, Via La Masa 19, 20156 Milan, Italy. Phone: 39-02-39014234;Fax: 39-02-3546277; E-mail:[email protected];[email protected]

doi: 10.1158/0008-5472.CAN-14-0274

�2014 American Association for Cancer Research.

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(GEM) models of ovarian cancer have been obtained moreclosely resembling the origin and initiation of human ovariancancer; however, we are still a longway fromchemotherapeutictrials (8, 15, 16).New preclinical ovarian cancer models are needed to foster

and if possible to tumor-tailor drug development. Preclinicalmodels based on xenografts obtained by engraftment ofpatient-derived tumor samples directly into animals rely ontheir limited dissimilarity from the patient's tumor (17–19).Because ovarian tumors are known for their pathologic andbiologic heterogeneity, with considerable differences in histol-ogy, genetics, and sensitivity to chemotherapy, the ideal pre-clinical model of ovarian cancer should consist of tumorxenografts that recapitulate this heterogeneity and preservethe characteristics of the original tumor. We report the estab-lishment of transplantable patient-derived ovarian tumorgrafts (EOC xenografts) that retain the original patients'molecular and biologic features. Our investigation supportsthe use of this platform to develop novel treatment opportu-nities for ovarian cancer.

Materials and MethodsSpecimen collection and clinical dataOne hundred thirty-eight clinical specimens (primary ovar-

ian tumors, metastasis, ascitic fluid) were obtained frompatients undergoing surgery for ovarian tumor by laparotomyor paracentesis at the San Gerardo Hospital in Monza, Italy.Tumor specimens were engrafted in nude mice within 24hours, as described below. The study protocol for tissuecollection and clinical information was approved by the Insti-tutional Review Board and patients provided written informedconsent authorizing the collection and use of the tissue forstudy purposes. Detailed information is reported in the Sup-plementary Data section.

AnimalsFemale NCr-nu/numice obtained fromHarlan Laboratories

were used when 6 to 8 weeks old. Mice were maintained underspecific pathogen-free conditions, housed in isolated ventedcages, and handled using aseptic procedures. Proceduresinvolving animals and their care were conducted in conformitywith institutional guidelines at the IRCCS-Istituto di RicercheFarmacologiche Mario Negri, in compliance with national andinternational laws and policies and in line with Guidelines forthe Welfare and Use of Animals in Cancer Research (20).

Ovarian carcinoma xenograftsRoutinely solid specimens from tumor masses (ovary and

omentum) were engrafted subcutaneously (s.c.), whereas asci-tes were transplanted intraperitoneally (i.p.) as tumor suspen-sion (Table 1). The ability of EOC xenografts to disseminate andmetastasize was tested from intraperitoneal and intrabursaltransplantations as detailed below.Subcutis models. Primary tumors and metastases were

dissected free of necrotic tissue repeatedly rinsed in HBSS and2 to 4 mm of tissue was implanted subcutaneously in the flankof nudemice (11). Tumor growth wasmeasured with a Vernier

caliper, and weight (mg ¼mm3) calculated as follows: [length(mm) � width2 (mm2)]/2.

Intraperitoneal models. Ascites was centrifuged, washedrepeatedly, resuspended in HBSS, and implanted intraperito-neally in nudemice at a dose of 10 to 20� 106 cells. Criteria forgrowing tumors were abdominal distension and palpabletumor masses in the peritoneal cavity (11). Mice were killedwhen they presented signs of discomfort (survival), ascites washarvested, and the volume recorded.

Intrabursal transplantation. Ovarian cancer cells fromsolid tumor enzymatic digestion or ascites (1 � 106 cell sus-pension) were injected orthotopically under the bursa (i.b.)of the mouse ovary, as previously reported (21) and detailedin Supplementary Data. At necropsy, the ovary image wasacquired with a macrodigital imaging system (MacroPATH;Milestone S.r.l); the two diameters were determined, the meancalculated and taken as measure of tumor mass. Ascites washarvested and the volume recorded.

For mice transplanted intraperitoneally or intrabursally, acomplete necropsy was done on each mouse by two indepen-dent scientists. Tumor dissemination in representative organsof the peritoneal cavity (liver, diaphragm, omentum, pancreas,uterus/ovary, and enlarged lymph nodes) was rated using anarbitrary score for gross tumor dissemination: 0 ¼ not infil-trated; 1 ¼ small masses; 2 ¼ evident masses; 3 ¼ completelyinvaded, as previously described (22).

EOC xenograft samples were snap frozen for genomicanalysis, fixed in 10% formalin and embedded in paraffin(FFPE), or frozen in optimal cutting compound (OCT) forhistologic and IHC analysis. Established EOC xenografts weretransplanted serially in nude mice for further studies (i.e.,therapy) and cryopreserved frozen in DMSO at differentpassages.

Hystopathological analysisThe morphology of patient's tumor tissues was compared

with their corresponding xenografts using paraffin-embeddedsections and standard protocols (23), as detailed in Supple-mentary Data.

Molecular analysisMutational analysis. ARID1A (exons 1 to 20),BRAF (exons

11 and 15), CTNNB1 (exon 3), KRAS (exon 2), PI3KCA (exons 10and 21), PPP2R1A (exons 5 and 6), and TP53 (exons 5 to 9) weresequenced to assess their mutational status. Genomic DNAwas obtained from EOC xenografts (N ¼ 34) and patienttumors (N ¼ 23) and analyzed as described in SupplementaryData and Supplementary Table S1.

Gene copy number. The c-Met, c-Myc, PI3Ka, PTEN,FGFR1, ERBB2, RB1, and NF1 gene copy number was assessedusing the TaqMan Copy Number Assay (Applied Biosystems)using the ABI 7900, Applied Biosystems. RNAse P copy numberwas used as reference gene.

Genome-wide gene expression. EOC xenografts collectedfrom subcutis, abdominal masses, and ascites of miceengrafted with tumors at different passages (from 1 up to13) and from patient specimens, underwent one-color micro-array-based gene expression profiling. To assess the amount of

Patient-Derived Ovarian Carcinoma Xenografts

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human- andmouse-derived cells in the xenograft tumors, totalRNAwas evaluated by species-specific qPCR assays for b-actin,as described in Supplementary Data. Only samples with ahumanRNA content>75%underwent gene expression analysis

with SurePrint G3 Human GE V2 8� 60 K microarrays (50,599Biological Features/array; Agilent Technologies), as describedin Supplementary Data. Microarray data analysis of ninepatient specimens and 62 xenograft samples (representing

Table 1. Characteristics of the ovarian tumors from which EOC xenografts were derived

Patients EOC xenografts

Original diagnosis Sourced

IDa Hystotype Grade Stage Patient treatmentbPatientresponsec Origin Ov Om A

MNHOC8 Serous G3 IV CBDCA Y P XMNHOC8Y Serous G3 IV CBDCA N R1 XMNHOC10 Serous G3 IIIC CDDP N P XMNHOC18 Serous G3 IV EPIþCTX (neoadjuvant) N P XMNHOC22 Serous G3 III DDP N R2 XMNHOC76 Serous G3 IIIC PAC N RNA XMNHOC84 Serous G3 IIIC CDDP/PTX NA RNA XMNHOC94/2 Serous G2 IA NA NA R3 XMNHOC106 Serous G3 IIIC PAC N R3 XMNHOC107 Serous G3 IIIC PAC PS RNA XMNHOC111/2 Serous G3 IIIC CDDP/PTX N R4 XMNHOC125 Serous G3 IV CBDCA PS P XMNHOC229 Serous G3 IIIC CBDCA/PTX N P XMNHOC239 Serous G2 IV CBDCA/PTX Y R5 XMNHOC241 Serous G2 IC CBDCA/PTX Y P XMNHOC244 Serous G2 IV CBDCA/PTX Y P XMNHOC124 Serous/endometrioid G2 IIIC CBDCA/PTX N P XMNHOC212 Serous/endometrioid G2 IIIC CBDCA/PTX Y P XMNHOC232 Serous/endometrioid G2 IIB CBDCA/PTX/Beva Y P XMNHOC78 Endometrioid G2 IIIC CP Y RNA XMNHOC109 Endometrioid G2 IC CBDCA PS R6 XMNHOC154 Endometrioid G2 IIC CBDCA/PTX Y R7 XMNHOC218 Endometrioid G3 IIIC CBDCA/PTX Y P XMNHOC230 Endometrioid G3 IIB CBDCA/PTX Y RNA XMNHOC79 EndometrioidþClear cell G3 IIIC CDDP PS RNA XMNHOC119 Clear Cell G3 IC CBDCA Y P XMNHOC142 Clear Cell G3 IIIC CBDCA/PTX NA P XMNHOC164 Mucinous G2 IV CBDCA/PTX (neoadjuvant) N P XMNHOC182 Mucinous G1 IC no treatment — P XMNHOC135 Mixed Mullerian tumor G3 IIIB TIP Y P XMNHOC195 Mixed Mullerian tumor G3 IIIB TIP N P XMNHOC88 Undifferentiated G3 IIIC CDDP Y R8 XMNHOC213 Undifferentiated G3 IIIC CBDCA/PTX Y P XMNHOC9 Not classified NA IIIC CBDCA/PEC PS P X

Abbreviations: P, primary tumor; R, relapse. Treatment at relapse: 1CTX;CDDP;EPI; 2DDP; 3PAC; 4DDP/PTX; 5Caelyx;CBDCA/PTX;6CBDCA; 7CBDCA/PTX; CBDCA/Caelyx; CBDCA/Topotecan; CBDCA/PTX;CBDCA/Gemcitabine; 8CBDCA; NAnot available, pluri-treated but specific treatment not known. Ov, ovary; Om, omentum; A, ascites.aThirty-four ovarian cancers were established as xenografts in nude mice.bPatient treatment (first-line therapy):CBDCA (carboplatin); EPI (epirubicin);CDDP (cisplatin);CTX (cyclophosphamide); PAC (cisplatin-adriamicin-cyclophosphamide); PTX (paclitaxel); Beva (bevacizumab); CP (CDDP-cyclophosphamide); TIP (paclitaxel-ifosfamide-cisplatin); PEC (cisplatin-epirubicin-cyclophosphamide); NA (not available).cPatient response (adiujavant or neoadjuvant therapy): Y, sensitive tumor (relapsingafter 12months); PS, partially sensitive (relapsing in6–12 months); N, resistant tumors (relapsing in 0–6 months).dSource of the xenografts.

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Cancer Res; 74(23) December 1, 2014 Cancer Research6982




29 EOC xenograft models) was done with Bioconductor (24),using R statistical language, with MeV version 4.8 (25) and thefunctional annotation tool available in the DAVID bioinfor-matic resource (26), as detailed in Supplementary Data.Microarray data are MIAME compliant and have been

deposited into the NCBI (National Center for BiotechnicalInformation) database Gene Expression Omnibus (GEO acces-sion no.GSE56920).

Drugs and treatmentsPaclitaxel (PTX, Indena S.p.A.) was dissolved in 50% Cre-

mophorEL (Sigma-Aldrich) and 50% ethanol and further dilut-ed with saline before use. Cisplatin (CDDP, Sigma-Aldrich) wasdissolved in 0.9% NaCl. They were administered at theiroptimal dose and schedule as detailed in the Results.For subcutaneous tumors, mice were randomized to treat-

ment at approximately 150 mg of tumor weight (8–10 miceper group). Treatment efficacy was expressed as best tumorgrowth inhibition [%T/C ¼ (median weight of treatedtumors/median weight of control tumors) � 100]. Animalswere euthanized when primary tumor volume exceeded 15%of body weight.For intraperitoneal tumors, mice (8 to 10 per group) were

randomized to treatment at an advanced stage (i.e., 25% ofexpectedmedian survival time,MST), regularlymonitored, andkilled at the first signs of discomfort (the day of death beingconsidered the limit of survival). The increment of life span(ILS) was calculated as [(median survival day of treatedgroup � median survival day of control group)/median sur-vival day of control group] � 100.Drug activity was interpreted as follows: subcutaneous

tumors were considered resistant with T/C �50%, responsivewith 10%<T/C<50%, and very responsive with T/C�10%;intraperitoneal tumors were considered resistant withILS�40%, responsive with 40%<ILS<100%, and very responsivewith ILS � 100%, according to published criteria (27, 28).

ResultsGenerationofEOCxenografts frompatientswithovariancancerWe collected 138 tumor samples from patients with ovarian

cancer and xenotrasplanted them in nude mice; 34 EOCxenografts could be established (25% tumor take) and suc-cessfully maintained through multiple rounds of serial trans-plantation. Approximately another 10% of the specimensengrafted in the mice receded and could not be transplantedfurther. Twenty-two EOC xenografts were established subcu-taneously and twelve were obtained by transplanting ascitesinto the peritoneal cavity of themice (Table 1 and Fig. 1). Table1 and Supplementary Table S2 summarize the clinicopatho-logical data of patients' tumors fromwhich the EOC xenograftswere derived. Seventeen EOC xenografts came from chemo-therapy-na€�ve tumors, 17 from patients treated with a chemo-therapy, two of them (MNHOC18 and MNHOC164) derivedfrom patients who underwent neoadjuvant treatment. Tumorgrade and tumor stage did not seem to predict engraftment innude mice (Supplementary Table S3A); when only the serous

histotype was considered, a correlation between residualtumor and tumor engraftment was found (P ¼ 0.024; Supple-mentary Table S3B).

Figure 1 depicts the biologic behavior of the EOC xenograftsestablished in nude mice as s.c (Fig. 1A) or ascites (Fig. 1B–D).The growth rate of the EOC xenografts differed as suggested bythe time to reach 1 g (1–15 months for subcutaneouslytransplanted xenografts; Fig. 1A), by the MST (1–4 monthsfor intraperitoneally transplanted xenografts; Fig. 1B), and bythe production of ascites and the level of tumor disseminationin the organs of the peritoneal cavity of the mice (Fig. 1Cand D).

Morphologic and pathologic similarity of EOCxenografts to the original patient's tumor

To rule out any phenotypic drift that xenografted tumorsmight have acquired, the histology of tumors grown in micewas compared with the corresponding original patient tumor.In all the cases, the morphology and tissue architecture weresimilarly preserved (14 representative matched cases arereported in Fig. 2 and Supplementary Fig. S1).

The established xenografts (N ¼ 34) were histologicallysimilar to the patient tumors from which they were derivedand 16 were classified as serous, three as mixed serous-endo-metrioid, five as endometrioid, one as mixed endometrioid-clear cell, two as clear cell, two as mucinous, two as mixedMullerian tumors (carcinosarcoma), two as undifferentiated,and one as nonclassified.

All tumorgrafts retained positivity for a number of anti-bodies generally used for the diagnosis of ovarian cancer,including cytokeratin pool and CA-125 (Supplementary Fig.S2). After multiple passages in mice, the positivity for somemarkers decreased, but the tissue architecture of the tumor oforigin was maintained (Supplementary Fig. S3).

EOC xenografts reproduce the dissemination pattern ofhuman ovarian cancer

Ovarian carcinoma spreads into the peritoneal cavity witha clinical feature of disseminated carcinomatosis. The abilityto recapitulate the dissemination pattern of human EOC wasinvestigated in a subset of EOC xenografts transplanted inthe bursa of the mouse ovary (Fig. 1E–G). The growth rate inthe ovary seemed not to be influenced by the histotype (Fig.1E). A diffuse tumor dissemination in the peritoneal cavitysimilar to that of the EOC xenograft engrafted intraperito-neally and involving liver, pancreas, ovary-uterus (contro-lateral), lymph nodes, diaphragm, and omentum wasobserved (Fig. 1F and G). The growth rate in the bursa andtheir dissemination were not necessarily associated, withMNHOC10, MNHOC107, and MNHOC78 showing the great-est dissemination potential. Ascites did not always form,despite the ability of ovarian cancer cells to disseminatethrough the peritoneal cavity (11/17 EOC xenografts trans-planted intrabursally formed ascites; Fig. 1F). The ability toform abdominal effusion seemed to depend on the primarytumor source, not on the route of implantation, as almost allthe xenografts from patients' ascites gave rise to ascites inmice (except MNHOC8 and MNHOC84 when transplanted






Figure 1. Biologic behavior of established EOC xenografts. A, subcutaneous growth. EOC xenografts were engrafted as tumor fragments in the subcutisof nude mice. Growth rate was expressed as time to reach 1 gr. B–D, intraperitoneal tumor growth and dissemination. EOC xenografts were engraftedas tumor cell suspension in the peritoneal cavity of nude mice. Mice presenting abdominal distension and/or palpable tumor masses in theperitoneal cavity were killed when showing signs of discomfort. Tumor growth was expressed as MST (B); tumor burden was expressed as volume ofascites (C) and dissemination to the peritoneal organs (D). E–G, intrabursal tumor growth and dissemination. EOC xenografts were engrafted astumor cell suspension in the bursa of the mouse ovary (1 � 106 cell suspension). Tumor burden is expressed as ovarian tumor mass (E), volume ofascites (F), and dissemination to the peritoneal organs (G). (Continued on the following page.)

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intrabursally), while most of those from solid tumors didnot.

EOC xenografts retain the molecular features of humanovarian cancerMolecular characterization was undertaken in the original

patient tumor and corresponding xenograft. We investigatedthe mutational status of genes involved in the pathogenesis ofovarian carcinoma. The detailed mutational spectrum is sum-

marized in Supplementary Table S4. Nonsynonymous TP53mutations were found in 76% of the EOC xenografts. Interest-ingly, clear cell, mucinous, and low-grade serous/endometrioidcarcinomas harbored wt TP53, whereas the majority of high-grade serous carcinoma harbored a mutated TP53, in line withclinical data (29). No mutations were found in ARID1A, BRAF,CTNNB1, and PPP2R1A genes. MNHOC142 xenograft harboreda mutation in the catalytic subunit of PI3Ka and MNHOC84xenograft and its corresponding patient tumor had amutationaffecting KRAS (G12A). The EOC xenografts and the corre-sponding patient tumors whose DNAs were available generallydisplayed the same mutational status (18/23 ¼ 78%). Excep-tions were one case showing a different TP53 missense muta-tion and four were the mutations that could not be detected inthe patient tumor (Supplementary Table S4).

We then checked the copy number of different genes (cMet,cMyc, PI3Ka, PTEN, FGFR1, ERBB2, NF1, and RB1). Despite asimilar gene copy number distribution (Supplementary TableS5), EOCxenografts tended to harbor higher gene copynumbersthan patient tumors, suggesting greater tumor genomic insta-bility upon in vivo selection; matching patient tumor andxenograft gene copy number is shown in Supplementary Fig. S4.

We carried out genome-wide gene expression analysis ofEOC xenografts and patient tumors to evaluate their tran-scriptomic profiling. Unsupervised hierarchical clusteringrevealed a high correlation of global gene expression amongthe EOC regardless of their origin (patient or xenograft); EOCclustered far apart from other cancer types, tumor xenografts,and cell lines of different origin (Fig. 3A), obtained from publicrepositories and hybridized on the same Agilent platform. Thehigh degree of similarity between EOC xenografts and patienttumors was confirmed by the Pearson correlation coefficientranging from 0.84 to 0.99 (Supplementary Fig. S5). Two-classpaired comparison between patient tumors and their pairedEOC xenografts (9 cases) revealed 1,042 differentially expressedtranscriptswith log fold change greater than 1 or lower than�1(P value <0.01). Interestingly, the main biologic processesrepresented in this dataset belonged to the immune response(Supplementary Table S6). Clustering based on these tran-scripts (Fig. 3B) showed a clear distinction between the patienttumors and the EOC xenografts, with most genes being down-regulated in the latter. Unsupervised hierarchical clustering ofthe EOC xenografts (based on the expression of all probes; Fig.4) revealed a high reproducibility of global gene expressionprofiles among xenografts harvested from different in vivopassages or different site of implantation (i.e., subcutis ororthotopic-abdominal masses and ascitic effusion) of thesame patient lesion. Interestingly, 85% (17 out of 20) of thehigh-grade/high-stage serous and endometrioid carcinomasEOC models clustered together, likewise the majority of clearcells, mucinous, low-grade/low-stage serous, and endome-trioid ovarian cancer xenografts (83%; 5 out of 6; Fig. 4).

MNHOC124Serous

MNHOC154Endometrioid

MNHOC164Mucinous

MNHOC195Mixed mullerian tumor

MNHOC213Undifferentiated

EOC-XENO PATIENT

MNHOC119Clear cell

Figure 2. Representative histologic characteristics of the original patienttumors and corresponding EOC xenografts. Sections from the patienttumor and the corresponding xenograft are shown (hematoxylin andeosin). The EOC xenograft identification number and the original clinicaldiagnosis are indicated.

(Continued.) Tumor histotype are depicted as follows: serous ( ), serous/endometrioid ( ), endometrioid ( ), clear cell ( ), mucinous ( ),mixed Mullerian tumors ( ), undifferentiated ( ), and not classified ( ). Each experimental group consisted of 5 to 8 mice. Data in A–G areexpressed as median with the upper value limit. Histograms in D and G are the sum of the mean score of each organ evaluated as described in Materialsand Methods. Each organ is depicted as follows: ovary/uterus ( ), liver ( ), diaphragm ( ), pancreas ( ), omentum ( ), lymph nodes ( ).






EOC xenografts and response to platinum-based therapyEOC xenografts were pharmacologically characterized for

their response to CDDP and PTX at optimal dose schedules.CDDP antitumor activity was evaluated, not carboplatin, asprevious data from our laboratory have shown similar antitu-mor activity of the two drugs in xenograft models. A heatmaprepresentation of EOC xenograft response to therapy isreported in Table 2. The spectrum of response to both drugswas wide, ranging from very good activity, in which tumorregressions could be observed, to no activity. The patient'sresponse to a platinum-based therapy for 11 cases could becompared with the response of the corresponding xenograft(Table 3). Only two cases showed a completely differentresponse to a platinum-based therapy: the MNHOC124 xeno-graft was very responsive, while the patient's clinical responsewas stable disease, and theMNHOC119 xenograft was resistantto CDDP, while the patient achieved a complete response. In allthe other cases, activity was similar with three cases thatcompletely matched in patient and the corresponding xeno-graft (MNHOC8Y, MNHOC10, and MNHOC230).

DiscussionCollections of patient-derived tumor xenografts have been

established for different tumor types (for review see ref. 19),including breast (30, 31), colorectal (32, 33), lung (34), pancre-atic cancers (35, 36), and glioblastoma (37, 38). While we werepreparing this manuscript, two papers were published ontumor grafts obtained from ovarian tumors, one focusing ona small series of high-grade serous type (13), whereas the otherpresenting a large tumor bank of ovarian cancer of differenthistotype (14).

The present work shows that (i) our panel of 34 EOCxenografts comprises all the main subtypes of ovarian carci-noma; (ii) the EOC xenografts in general maintain the keyfeatures of the original tumor, including histopathology, genecopy number, and mutational spectrum; (iii) the EOC xeno-grafts reproduce the dissemination into the peritoneal cavity ofmice typical of ovarian carcinoma; (iv) comprehensivegenome-wide gene expression analysis confirms high-degreeof similarity among the xenografts and between the xeno-grafts and patient EOCs, and distinguishes different subsets of

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Prostate cancer (54)

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Bladder cancer (3) E-GEOD-45184

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MNHOC212ptMNHOC164ptMNHOC218ptMNHOC154ptMNHOC182ptMNHOC135ptMNHOC124ptMNHOC230ptMNHOC18_sc1MNHOC84_A3MNHOC84_A4MNHOC84_sc1MNHOC84_sc2MNHOC94/2_sc1MNHOC182_sc2MNHOH182_sc1MNHOC182_sc3MNHOC109_sc1MNHOC119_am1MNHOC119_am3MNHOC119_am2MNHOC142_am1MNHOC22_A3MNHOC22_am1MNHOC22_A4MNHOC22_am2MNHOC111/2_A1MNHOC111/2_A2MNHOC88_sc1MNHOC76_am1MNHOC76_am2MNHOC76_A3MNHOC76_A4MNHOC8_am1MNHOC8_A2MNHOC8_A3MNHOC8Y_am1MNHOC8Y_A3MNHOC8Y_A4MNHOC8Y_am2MNHOC125_sc1MNHOC125_sc2MNHOC107_sc1MNHOC106_sc1MNHOC106_sc2MNOC244ptMNHOC244_sc1MNHOC154_sc1MNHOC78_am1MNHOC78_am2MNHOC78_A4MNHOC78_A5MNHOC78_am3MNHOC239_sc1MNHOC230_sc1MNHOC9_sc1MNHOC212_sc1MNHOC10_A4MNHOC10_A5MNHOC10_am1MNHOC10_am2MNHOC10_am3MNHOC135_sc1MNHOC164_am1MNHOC164_am2MNHOC164_am3MNHOC79_am1MNHOC79_A2MNHOC79_A3MNHOC218_sc1MNHOC124_sc1

B–0.18

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Figure 3. Global gene expression of EOC xenografts and patient tumors. Gene expression for 29 EOC xenograft models (62 xenograft samples) and9 corresponding patient specimens was generated using SurePrint G3 Human GE V2 8 � 60 K microarrays (50,599 probes/array; Agilent Technologies).A, unsupervised hierarchical clustering of EOC together with tumors of different origin. Clustering using Pearson correlation and completelinkage was based on 33,536 common probes across Agilent SurePrint G3 8 � 60 K v1 and v2 platforms. To complement the analysis, external geneexpression datasets were retrieved from ArrayExpress (European Bioinformatic Institute – EBI) or GEO (NCBI database) and from experiments inG.C. laboratory). B, clustering and heatmap of EOC. Two-class paired comparison analysis based on 50,599 probes indicated 1,042 differentiallyexpressed transcripts between EOC xenografts and their corresponding patient tumors (9 cases). Clustering using Pearson correlation and averagelinkage was based on these transcripts. Data were divided by the mean of log Intensity values from patient tumors. A, ascites; am, abdominal masses;sc, subcutis of individual mice (1, 2, n).

Ricci et al.





EOCxenografts; (v) our EOCxenograft panel consists of tumorswith different sensitivity to chemotherapy from very respon-sive, responsive, to resistant tumors, well reproducing theresponse to therapy in ovarian patients.The EOC xenografts were obtained by transplanting subcu-

taneously or intraperitoneally in nude mice tumor samplesfreshly obtained after cytoreductive surgery for abdominalmasses or paracentesis of ascites. In these experimental con-ditions, we obtained 25% xenografts, regardless of the trans-plantation route, a tumor take in line with earlier studiesestablishing patient-derived EOC xenografts in athymic nudemice (11, 39, 40). A better take could probably be obtainedby transplanting ovarian tumors in more immunodeficientmice (SCID and NOD-SCID-IL2gR,) as recently reported(13, 14). Interestingly, we found the engraftment being corre-lated with residual tumor in high-serous ovarian carcinomas,further supporting residual tumor as a poor prognostic factorin this disease.The EOC xenografts showed consistency with the tumors

they derived from, on the histopathological and molecularlevels. The histotype and tissue architecture were fairly wellmaintained and, importantly, the xeno-panel reproduced theplethora of human ovarian carcinomas with all the differenthistotypes: serous, endometrioid, clear cell, mucinous, carci-nosarcoma, and undifferentiated, similarly to what was recent-ly reported by Weroha and colleagues (14).Our genome-wide studies indicate a high degree of similarity

among the ovarian cancers that clustered together far apartfrom clinical tumors of different types, xenograft models, andcell types. The correlation coefficients (0.84–0.99) confirmedthe high concordance of the clinical samples with the EOCxenografts. In similar studies, the correlation coefficient

between the patient tumors and their matching xenograftswas above 0.88 for pancreatic cancers (41), from 0.78 to 0.95 forNSCLC (42), and lower for melanomas (43). The genes differ-entially expressed in the patient's tumors and the xenograftsbelong to the human immune system and were mainly down-regulated in the xenografts. This is in accordancewith previousreports (41, 42) and suggests a loss of human-infiltratingimmune cells and a tumor stroma of murine origin. Althoughgene expression analysis on EOC xenografts and primarypatient tumor was available for only nine cases, our findingsare in agreement with previous studies showing molecularfidelity of tumor xenograft to its primary patient tumor(14, 30, 31). The overall gene expression profile of the individualEOC xenografts was conserved upon passaging and was notaltered in relation to the site of tumor growth, so it appears thatthe process of engraftment and expansion does not largelychange the molecular features of the cancer.

The general preservation of most of the patient tumormutations in the EOC xenografts suggests that they are a validmodel for functional and therapeutic studies. However, thealtered mutations and higher gene copy number in four of the34 xenografts call for caution in interpreting the results in thosemodels. An enriched pattern of mutations in xenografts com-paredwith primary tumors has been described (44, 45), and canbe explained by the xenotransplantation selecting cells with adistinct subset of the primary tumor mutation repertoire or,alternatively, by tumor genomic progression during xenograftestablishment.

Themajority of our EOCxenografts derived from stage III/IVand grade 3 ovarian tumors and diffuse dissemination into theperitoneal cavity was observed when transplanting EOC xeno-grafts intraperitoneally and intrabursally, with tumor masses

HGS MUC CC MUC HGE/CC

HGS HGS HGS HGE HGS HGS

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TYPE IITYPE I TYPE II TYPE I

KRAS mut

TP53 mut TP53 wt

PIK3CA mut

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Figure 4. Transcriptomic profiling of EOC xenografts. Unsupervised hierarchical clustering (based on 50,599 probes; Agilent SurePrint G3 Human GE V28 � 60 K microarrays) of 62 EOC xenograft replicates from different locations (A, ascites; am, abdominal masses; sc, subcutis) of individual mice (1, 2, n),representing 29 EOC xenograft models. Gray circles depict mutations of the indicated genes (Supplementary Table S4). HGS, high-grade serous;LGS, low-grade serous; MUC, mucinous; LGE, low-grade endometrioid; CC, clear cells; TMM, mixed Mullerian tumor; HGE, high-grade endometrioid.






growing and invading the visceral organs. In some cases, thiswas accompanied with the production of ascites. Our preclin-ical models mirror the clinical setting, where one third ofwomen with ovarian cancer develop ascites during the courseof their disease (46) and this seems not to be related to anyspecific histologic subtype.

The response to therapy is instrumental to validate theclinical predictive value of patient-derived xenografts. In 11cases, we could compare the xenograft response with theclinical response to a CDDP-based therapy: it was completelydifferent in two cases, while comparable in the remaining.More than in other tumors, ovarian cancer patient's outcomedepends on other factors, such as the disease disseminationand residual tumor (RT) after surgery (47). Our xeno-trialswereclearly not influenced by these factors as most of them weredone in very different experimental conditions (i.e., "neoadju-vant like" setting with a limited tumor burden). For example,the MNHOC124 xenograft responded very well; in contrastpatient MNHOC124, albeit achieving stable disease (SD) afteradjuvant therapy, had a RT >10 mm that probably influencedher prognosis, as her overall survival was 9 months. Anothercase is MNHOC119, a clear cell carcinoma with a RT ¼ NED(nonevidence of disease) that likely determined the patient'sgood response; however a poor prognosis with low responseto chemotherapy has been reported in advanced ovarian clearcell carcinomas (4), as in our corresponding xenograft. Re-sponse to platinum in ovarian cancer xenografts (eight cases)correlated well with patient's clinical response in Weroha andcolleagues study (14) and concordance was also reported byTopp and colleagues in their series of 12 high-grade serousovarian patient-derived xenografts (13). Our panel of EOCxenografts fulfills all the different responses to platinum-basedtherapy observed in the clinic and this strengthens the pos-sibility of this EOC xeno-bank being instrumental in under-standing the mechanism of the resistance to CDDP and intesting novel therapeutic strategies to overcome it.

A dualistic model of ovarian carcinoma pathogenesis hasbeen proposed that classifies them as type I and type II. Type IEOCs are low-grade, relatively indolent, and genetically stable;type II tumors include high-grade serous carcinoma, which isa highly aggressive cancer, genetically unstable, and frequentlymutated in TP53 (7). The EOC xenografts we established are ofdifferent histologic types and span from grade 1 to 3; theintegration of the clinical and preclinical data would allow toclassify these xenografts as type I and type II on the basis oftheir pathologic and molecular characteristics (Fig. 4). Thisclassification might help to a better understanding of ovariancancers and enable us to tackle specific questions such astailored therapy of high-grade serous ovarian carcinoma orisolation of tumor-initiating cells from type I and type IItumors (23).

The data reported reinforce the idea that EOC patient-derived xenografts largely retain the phenotypic and geno-mic characteristics of their original tumor. Our preclinicalplatform, along with the other two series of patient-derivedovarian cancer xenografts recently obtained (13, 14), offersan instructive framework for molecular target discovery/validation studies, for the identification of biomarker of

Table 2. Heatmap of EOC xenograft responseto chemotherapy

Response to

ID # CDDP PTX

MNHOC8 a

MNHOC8Y a

MNHOC10 a

MNHOC18 b

MNHOC22 a

MNHOC76 a

MNHOC84 b

MNHOC94/2 b

MNHOC106 b

MNHOC107 b

MNHOC111/2 a

MNHOC125 b

MNHOC239 b

MNHOC124 b

MNHOC212 b

MNHOC78 b

MNHOC109 b

MNHOC154 b

MNHOC218 b

MNHOC230 b

MNHOC79 a

MNHOC119 b

MNHOC142 a

MNHOC164 b

MNHOC182 b

MNHOC135 b

MNHOC88 b

MNHOC9 b

NOTE: Mice bearing EOC xenografts were randomized asdescribed in Materials and Methods. CDDP was given i.v.4 mg/kg Q4 � 3 or 5 mg/kg Q7 � 3. PTX was given i.v. 20mg/kgQ4� 3 orQ7� 3. All these schedules are reported to beeffective in different experimental models (22, 48).aIntraperitoneally transplanted xenografts- drug activity wasexpressed as ILS% as detailed in Material and Methods.bSubcutaneously transplanted xenografts-drug activity isexpressed as T/C% as detailed in Material and Methods,

very responsiver, responsive, resistant.

Ricci et al.





platinum resistance and for testing new investigationaltherapeutic agents.

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

Authors' ContributionsConception and design: F. Ricci, F. Bizzaro, R. Fruscio, M.R. Bani, G. Damia,R. GiavazziDevelopment of methodology: F. Ricci, F. Bizzaro, M. Ganzinelli, A. Decio,C. Ghilardi, M.R. Bani, G. DamiaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F. Ricci, F. Bizzaro, M. Cesca, F. Guffanti,M. Ganzinelli, A. Decio, C. Ghilardi, R. Fruscio, A. Buda, R. Milani, G. Chiorino,R. GiavazziAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F. Ricci, F. Bizzaro, M. Cesca, F. Guffanti, P. Ostano,G. Chiorino, M.R. Bani, G. DamiaWriting, review, and/or revision of the manuscript: F. Ricci, F. Bizzaro,M. Cesca, R. Fruscio, R. Milani, G. Chiorino, M.R. Bani, G. Damia, R. GiavazziAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): F. Ricci, G. DamiaStudy supervision: F. Ricci, G. Damia, R. GiavazziOther (histopathological contribution): P. Perego

AcknowledgmentsThe authors thank the Nerina and Mattioli Foundation, "Pandora," the

ovarian cancer tumor tissue collection IRCCS-Istituto di Ricerche Farm-acologiche Mario Negri of the Department of Oncology, Prof. C. Mangionifor his long-standing support and his special devotion to patients, MartaAlberti, Clinical Research Center for Rare Diseases "Aldo e Cele Dacco forassistance in running the sequencing reactions," IRCCS-Istituto di RicercheFarmacologiche Mario Negri, Ranica, and Luca Porcu for helping in statis-tical analysis.

Grant SupportThe work was supported by the Italian Association for Cancer Research

(IG14536 to G. Damia and IG14532 and IG12182 to R. Giavazzi) and Fonda-zione CARIPLO (no. 2011-0617 to M. Cesca). P. Ostano was supported by agrant from Lauretana SPA. F. Ricci is a recipient of a fellowship fromFondazione Umberto Veronesi, Milan, Italy and A. Decio is a recipient ofa fellowship from Italian Foundation for Cancer Research (FIRC).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received January 31, 2014; revised September 4, 2014; accepted September 29,2014; published OnlineFirst October 10, 2014.

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Ricci et al.




2014;74:6980-6990. Published OnlineFirst October 10, 2014.Cancer Res Francesca Ricci, Francesca Bizzaro, Marta Cesca, et al. Clinicopathology and Genetic AlterationsPatient-Derived Ovarian Tumor Xenografts Recapitulate Human

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