Müllerian inhibiting substance type II receptor (MISIIR): A novel, tissue-specific target expressed by gynecologic cancers

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Gynecologic Oncology 108 (2008) 141 – 148 www.elsevier.com/locate/ygyno

Müllerian inhibiting substance type II receptor (MISIIR): A novel, tissue-specific target expressed by gynecologic cancers ☆ Jamie N. Bakkum-Gamez a , Giovanni Aletti a , Kriste A. Lewis a , Gary L. Keeney b , Bijoy M. Thomas a , Isabelle Navarro-Teulon c , William A. Cliby a,⁎ a

Department of Gynecologic Oncology, Mayo Clinic, Rochester, MN 55905, USA Department of Anatomic Pathology, Mayo Clinic, Rochester, MN 55905, USA INSERM, EMI 0227, Centre de Recherche en Cancérologie de Montpellier, Université Montpellier1, CRLC Val d'Aurelle-Paul lamarque, Montpellier, France b

c

Received 30 May 2007 Available online 7 November 2007

Abstract Objective. Müllerian inhibiting substance type II receptor (MISIIR) is expressed by ovarian, breast, and prostate cancers [Masiakos PT, et al. Human ovarian cancer, cell lines, and primary ascites cells express the human Mullerian inhibiting substance (MIS) Type II Receptor, bind, and are responsive to MIS. Clin Cancer Res 1999;5:3488–99; Hoshiya Y, et al. Mullerian inhibiting substance promotes interferon {gamma}-induced gene expression and apoptosis in breast cancer cells. J Biol Chem 2003;278:51703–12; Hoshiya Y, et al. Mullerian inhibiting substance induces NFkB signaling in breast and prostate cancer cells. Mol. Cell. Endocrinol. 2003;211:43–9. [1–3]]. We investigated the expression patterns of MISIIR in benign and malignant gynecologic tissues and benign non-gynecologic tissues to better assess the relevance of MISIIR as a target for new therapeutic and diagnostic approaches to gynecologic cancers. Secondarily, we examined the impact of MISIIR expression on overall survival (OS) and diseasefree survival (DFS) in a cohort of epithelial ovarian cancers (EOC). Methods. Reverse-transcription polymerase chain reaction (RT-PCR), immunoblotting, and immunohistochemistry (IHC) were used to determine MISIIR expression. EOC cell lines (10), primary EOCs (12), and tissue microarrays (TMAs) containing benign gynecologic (179) and non-gynecologic tissues (25), EOC (182), endometrial carcinomas (109), uterine sarcomas (98), and ovarian dysgerminomas (22) were examined for MISIIR expression. Clinical data were collected for a cohort of 182 EOCs. Results. Ninety-two percent of primary EOCs and 44% of EOC cell lines expressed MISIIR mRNA. We observed moderate or strong MISIIR expression via IHC in the majority of gynecologic cancers: EOC 69% (125/182), ovarian dysgerminomas 77% (17/22), endometrial cancers 75% (82/109), uterine malignant mixed Müllerian tumors (MMMT) 59% (30/51), uterine leiomyosarcomas (LMS) 52% (15/29), and endometrial stromal sarcomas (ESS) 22% (4/18). Over 74% of normal non-gynecologic tissues did not express MISIIR. There was a significant correlation between MISIIR expression and improved OS (p = 0.025, Chi square). Conclusions. In the largest study to date, we report that MISIIR is highly expressed by a wide variety of gynecologic cancers, including cancers currently without effective systemic therapies. Low levels of expression in select non-gynecologic tissues coupled with high expression in gynecologic malignancies make MISIIR an attractive target for novel therapeutics and tumor-directed imaging in the management of gynecologic cancers. Further investigation into the impact of MISIIR expression and OS is also warranted. © 2007 Elsevier Inc. All rights reserved. Keywords: Müllerian inhibiting substance type II receptor (MISIIR); Müllerian inhibiting substance (MIS); Epithelial ovarian cancer; Endometrial cancer; Uterine sarcoma; Ovarian dysgerminoma

Abbreviations: MISIIR, Müllerian inhibiting substance type II receptor; MIS, Müllerian inhibiting substance; EOC, epithelial ovarian cancer; OS, overall survival; DFS, disease-free survival; RT-PCR, reverse-transcription polymerase chain reaction; ICH, immunohistochemistry; TMA, tissue microarray; MMMT, malignant mixed Müllerian tumor; LMS, leiomyosarcoma; ESS, endometrial stromal sarcoma; AMH, anti-Müllerian hormone; TFG-β, transforming growth-factor β; ALK, activin-like kinase; SV40, simian virus 40; RT, room temperature; FFPE, formalin-fixed paraffin-embedded; bp, base pair; GCT, granulosa cell tumor; H&E, hematoxylin and eosin. ☆ Supported by a grant from the Minnesota Ovarian Cancer Alliance (MOCA) to WAC. ⁎ Corresponding author. 201 1st St. SW, Rochester, MN 55905, USA. Fax: +1 507 266 9300. E-mail address: [email protected] (W.A. Cliby). 0090-8258/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2007.09.010

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Introduction Ovarian cancer is the leading cause of gynecologic cancer death and the fifth leading cause of cancer-related death in women [4]. Approximately 60% of ovarian cancers are diagnosed in advanced stages [5]. Despite advances in surgical resection and chemotherapy, ovarian cancer continues to be difficult to treat and a majority of women will eventually succumb to the disease. More than 80% of ovarian cancers are of epithelial origin [6]. Such cancers arise from the coelomic epithelium surrounding the human ovary. This epithelium is of the same embryonic origin as several other female reproductive structures–the uterus, Fallopian tubes, cervix, and upper vagina–that arise from the primordial Müllerian duct [7]. In the 1940s, Dr. Alfred Jost laid the groundwork for the identification of Müllerian inhibiting substance (MIS). He showed that male embryos produced a testicular hormone other than testosterone that caused the primordial female reproductive tract to regress. This hormone, also known as anti-Müllerian hormone (AMH), was ultimately identified as a homodimeric glycoprotein ligand of the transforming growth-factor β (TGFβ) family and is now known as MIS [8,9]. MIS is produced in high levels by the fetal and postnatal testes but is undetectable in the fetal and postnatal ovary. During puberty, the level of MIS in females becomes detectable at low levels where it persists until menopause, at which time it decreases once again to undetectable levels [10]. As a member of the TGF-β family, MIS exerts its effect by binding to a heterodimeric transmembrane serine/threonine kinase cell surface receptor complex consisting of the MIS type I and type II receptors. MIS binds directly to the unique MISIIR which then binds a type I receptor. Current evidence suggests that these type I receptors are part of the activin-like kinase (ALK) family and may in fact be ALK2, ALK3, and ALK6 [11,12]. The heterodimeric type I–type II receptor complex then triggers a downstream signaling cascade of phosphorylation activating Smads 1, 5, and 8 [13]. The Smad signaling complex then enters the nucleus and interacts with transcription factors to induce gene expression [11], apoptosis [14], and, in male embryos, regression of the Müllerian duct [11]. Downstream effectors of the MIS receptor complex have been shown to cause growth inhibition in ovarian cancer cell lines and primary ovarian cancer ascites cells that express MISIIR [1]. In addition, two endometrial cancer cell lines, KLE and AN3CA, also appear to express MISIIR and their growth is inhibited by exposure to MIS [15]. MISIIR has become the focus of two approaches for ovarian cancer research. One area of research involves the use of the MISIIR promoter for the development of an animal model of ovarian cancer. In 2003, Connolly et al. demonstrated that, when an MISIIR promoter-controlled transforming region of the simian virus 40 (SV40) was injected into fertilized mouse eggs, approximately 50% of the female founders developed poorly differentiated ovarian carcinomas that spread via intraperitoneal patterns similar to human epithelial cancers [16]. The other approach attempts to capitalize on the potential tissue-specific nature of the receptor as an attractive target for immunomodula-

tion of ovarian cancer growth [1,17,18]. Utilization of antibodies against MISIIR in vitro [17] and utilization of the recombinant human ligand, rhMIS, in both in vitro and preclinical in vivo experiments [19,20] have shown promising anti-cancer therapeutic potential, and in 2006, Pieretti-Vanmarcke et al. showed that the addition of rhMIS to traditional ovarian cancer chemotherapeutic regimens appears to reduce the dose of chemotherapy required to achieve the same response as full-dose treatment [21]. A critical step in determining the relevance of MISIIR as a therapeutic target is the broad characterization of MISIIR expression in gynecologic cancers. To date studies of MISIIR expression in Müllerian tissues have been limited in scope and sample size. The primary purpose of this study was to determine the prevalence of MISIIR expression in a large cohort of gynecologic cancers and a wide variety of non-gynecologic normal tissues. A secondary goal of this study was to assess whether MISIIR expression in our EOC sample correlated with clinical presentation or prognosis. Materials and methods Ovarian cancer cell lines Established ovarian cancer cell lines IGROV-1, SKOV3ip, OVCAR8, OVCAR5, HEYC2 (ATCC, Manassas, VA), OV207, OV202, OV177, OV167, and OV17 were grown at 37 °C in 5% CO2. OV207, OV202, OV177, OV167, and OV17, previously characterized primary ovarian cancer cell lines originating at Mayo Clinic [22], were grown in AlphaMEM plus 20% fetal calf serum; IGROV-1, SKOV3ip, OVCAR8, and OVCAR5 were grown in RPMI with 10% fetal calf serum, and HEYC2 was grown in DMEM with 5% fetal calf serum.

Clinical specimens Mayo Clinic IRB approval was obtained for all studies. Primary ovarian cancer, endometrial cancer, benign endometrium, and benign non-gynecologic tissue specimens (which included esophagus, small intestine, stomach, liver, gall bladder, colon, tonsil, thyroid, adrenal, pancreas, ureter, bladder, kidney, lymph nodes, spleen, thymus, skeletal muscle, artery, peripheral blood cells, heart, lung, Fallopian tube, myometrium, cervix, breast, skin, placenta, hippocampus, testis, and prostate) were obtained from tissue repositories and discarded surgical waste from patients undergoing surgery at the Mayo Clinic. TMAs of EOC, endometrial cancer, and normal non-gynecologic tissues were constructed as previously described [23–25]. TMAs of benign proliferative, secretory, and atrophic endometrium, dysgerminomas, MMMT of the uterus, leiomyosarcoma, and ESS were also constructed for IHC analysis. Histologic sections of routinely formalin-fixed paraffin-embedded (FFPE) tissues were screened and areas of representative tumor and benign tissue were marked. Three tissue cores (0.6 mm in diameter) were taken from each tumor sample and manually placed in a new recipient paraffin block. Three cylinders of normal uterine myometrium or ovarian tissue adjacent to each tumor were also transferred to the recipient block when available. Four-micrometer sections were then cut from the TMA paraffin block and transferred to a slide for IHC staining.

Construction of MISIIR expression vectors The full-length coding sequence (bp 79–1800) of MISIIR [26] was amplified from a human fetal testis cDNA library (BioChain Institute, Inc., Hayward, CA). PCR primers (sense, 5′-GCTCTTCTCCTTCTGCTGCT-3′ and antisense, 5′GGGCATGTACATTGATGACAC-3′) were designed to amplify bp 43 to 1833 beginning in exon 1 and ending within exon 11 adding unique restriction sites for cloning. PCR using Taq DNA polymerase (Promega, Madison, WI) in a Bio-Rad iCycler (Hercules, CA) was run as follows: denaturation at 95 °C for 2 min

J.N. Bakkum-Gamez et al. / Gynecologic Oncology 108 (2008) 141–148 followed by 30 cycles of 94 °C for 1 min, 60 °C for 2 min, and 72 °C for 3 min with a final elongation step at 72 °C for 10 min. The resulting 1790 base pair band was extracted using the QIAquick Gel Extraction Kit (Qiagen, Inc., Valencia, CA) from a 1% agarose gel and sequenced using ABI PRISM Big Dye Terminator cycle sequencing ready reaction kit with AmpliTaq DNA Polymerase, FS version 1.1 and ABI PRISM 3730 DNA analyzer (96-capillary; Perkin-Elmer Applied Biosystems, Foster City, CA). It was then cloned into both pcDNA 3.1 (+) (pMISIIR) and pcDNA 3.1/mycHis C (p-myc-MISIIR) (Invitrogen, Carlsbad, CA) after digestion with XhoI and BamHI.

RT-PCR in EOC cell lines, primary EOC, normal endometrium, and primary endometrial cancers Primers were designed to amplify a 330 bp region of MISIIR from bp 1262 to bp 1592, beginning with exon 9 and extending into exon 11 (sense, 5′CTCTGGACCTACAGGATTGG-3′ and antisense, 5′-AGGCGCTGCTGTACACACTC-3′). EOC cell lines underwent mRNA extraction using the RNeasy kit (Qiagen Inc., Valencia, CA). Trizol was utilized to extract mRNA from solid tissue. All mRNA samples were converted to cDNA using SuperScript II (Invitrogen Corp, Carlsbad, CA). PCR using Amplitaq Gold (Applied Biosystems, Foster City, CA) in a Bio-Rad iCycler (Hercules, CA) was run as follows: denaturation at 95 °C for 5 min followed by 30 cycles of 94 °C for 45 s, 62 °C for 45 s, and 72 °C for 1 min with a final elongation step at 72 °C for 10 min. Products were separated on 1% agarose gel and sequenced as described above.

Antibodies The mouse monoclonal anti-myc antibody was purchased from Cell Signaling Technology, Inc. (Danvers, MA). The monoclonal anti-MISIIR mouse antibody (mAb12G4) used was described previously in Salhi et al. [27]. For the development of an anti-MISIIR rabbit polyclonal antibody, the peptide comprising residues 173–194 of MISIIR was identified as a candidate immunogenic site by antigenic analysis of the receptor protein using the PEPTIDESTRUCTURE ALGORITHM of the University of Wisconsin (Madison, WI) Genetics Computer Group (GCG) software. The corresponding 21-mer polypeptide was synthesized by the Mayo Clinic Proteomics Research Center Peptide Synthesis Facility. Rabbits were then inoculated with the 21mer polypeptide (Cocalico Biologicals, Inc, Reamstown, PA). Rabbit antiserum was screened for production of anti-MISIIR antibodies using Western blot analysis. The rabbit polyclonal antibody MC1828 was then immunopurified (SulfoLink, Pierce Biotechnology, Inc., Rockford, IL) using the MISIIR AA173–194 21-mer.

Transfection and stable cell line construction Two EOC cell lines that did not express MISIIR mRNA, OV167 and SKOV3ip, and the Chinese hamster ovary (CHO) cell line were transfected with MISIIR expression vectors. For transient lysate analysis, OV167 was transfected with either p-myc-MISIIR or empty pcDNA 3.1/myc-His C via electroporation at 330 V for two times 5-millisecond pulses (BTX830 electroporator). SKOV3ip and CHO cell lines were transfected using Fugene 6 (Roche) with either pMISIIR or empty pcDNA 3.1(+) to generate stable MISIIR positive and negative control cell line clones. Cells were challenged with G418 (400 μg/mL). Single clones were selected and screened for MISIIR expression via Western analysis with mAb12G4 (0.54 μg IgG/ml) and anti-mouse HRP (Kirkegaard and Perry Laboratories, Gaithersburg, MD) at 0.2 μg IgG/ml.

Western analysis of MISIIR expression Cell lysates were prepared by trypsinizing and washing the cells followed by incubation on ice in lysis buffer and sonication of 2 times 10 pulses (VirSonic Ultrasonic Cell Disrupter, Biopharma Process Systems, Ltd, Winchester, UK). Lysates were run on a 10% PAGE–SDS gel. Protein was then transferred to nitrocellulose and probed with either mAb12G4 (0.54 μg IgG/ml) or immunopurified MC1828 (0.4 μg IgG/ml) followed by secondary anti-mouse

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(0.2 μg IgG/ml) or anti-rabbit (0.2 μg IgG/ml) HRP antibody, respectively, and development with ECL (Amersham, Piscataway, NJ).

Immunocytochemistry Cells were fixed with 4% paraformaldehyde and washed with 1× PBS. They then underwent immunocytochemistry utilizing either mAb12G4 (54.4 μg/ml) and FITC labeled secondary goat-anti-mouse IgG antibody (20 μg/ml) (Sigma, St. Louis, MO) or MC1828 (40 μg/ml) and FITC labeled secondary goat-antirabbit IgG antibody (20 μg/ml) (Sigma-Aldrich, St. Louis, MO). Nuclei were stained with 20 μl/slide of DAPI (Vectashield, Vector Laboratories, Burlingame, CA) and cells were imaged with an LSM 510 confocal microscope (Carl Zeiss MicroImaging, Thornwood, NY).

Immunohistochemistry Formalin-fixed, paraffin-embedded (FFPE) TMAs were deparaffinized with 3 changes of xylene and rehydrated in a series of alcohols (100%, 95%, then 70% EtOH) and rinsed well in running distilled water. Slides were then placed in a preheated 1 mM EDTA, pH 8.0 retrieval buffer for 30 min then cooled in the buffer for 5 min followed by a 5-minute rinse in running distilled water. After heat inactivated epitope retrieval, slides were placed on the DAKO Autostainer for the following procedure (at RT). Sections were incubated with 3% H2O2 in ethanol for 5 min to inactivate the endogenous peroxides. Sections were incubated in a concentration of 54.4 μg/ml of mAb12G4 for 60 min. Sections were rinsed with TBST wash buffer. LSAB2 System-HRP (Cat. No. K0673, DAKO, Carpinteria, CA) was used for the detection method as follows: sections were incubated with a biotinylated link secondary antibody for 15 min and then rinsed with TBST. Slides were incubated with a tertiary agent Streptavidin–HRP for 15 min then rinsed with TBST. Sections were incubated in 3,3′-diaminobenzidine (DAB) (DAKO, Carpinteria, CA) for 5 min, counterstained with Modified Schmidts' Hematoxylin for 5 min, tap water rinsed for 3 min to blue sections, dehydrated through graded alcohols, cleared in 3 changes of xylene and then mounted with a Leica automated coverslipper. The use of patient specimens was approved by the Mayo Clinic IRB. Intensity of the IHC staining against MISIIR in the TMAs was scored independently by two of the authors (JNB, GLK) on a scale of increasing intensity, 0 (no staining), +1 (weak staining), +2 (moderate staining), and +3 (strong staining). Those tissues that received a score of 0 or +1 were classified as negative for MISIIR expression. Those tissues that received a score of +2 or +3 were classified as positive for MISIIR expression.

Clinical data Clinical data, including tumor stage, grade, debulking status, receipt of chemotherapy, time to recurrence, time to death and/or time to last follow-up were obtained for a subset of 144, primary, randomly selected patients included on the TMAs. Histologic subtype (serous, endometrioid, clear cell, or mucinous) was known for 88 of these patients. High grade was defined as grade 3 or 4. Advanced stage was defined as stage IIIA or higher. Optimal debulking was defined as b1 cm residual disease. Time to recurrence, time to death, and/or time to last follow-up (all in months) were obtained. MISIIR staining on TMA IHC was determined as above. A second subset of 38 primary, advanced stage (all were either stage IIIC or IV), high-grade (3 or 4) EOCs (“high-risk”), all optimally cytoreduced and treated with platinum-based chemotherapy, was identified and included on TMA IHC. These high-risk EOCs were dichotomized as either chemoresistant or chemosensitive disease. Chemoresistant tumors (22/38) were defined as recurrence within 12 months of completing chemotherapy. Chemosensitive tumors (16/38) were defined as recurrence greater than 12 months from the time of chemotherapy completion. Time to recurrence, time to death, and/or time to last follow-up (all in months) were obtained.

Statistical analysis Chi square, Fisher's exact test, Kaplan–Meier estimates, and Cox proportional hazards were used. A p value ≤ 0.05 was considered statistically significant. JMP6 statistical software was utilized in all analyses.

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Results MISIIR mRNA expression in gynecologic tissues Prior to large scale screening of several different gynecologic cancers for MISIIR expression, we analyzed mRNA from cell lines and primary tumors. Nine EOC cell lines (IGROV-1, SKOV3ip, HEYC2, OVCAR8, OVCAR5, OV17, OV167, OV202, and OV207), twelve primary EOCs, three normal endometrial samples, two grade I endometrial cancers, and two grade III endometrial cancers were evaluated qualitatively to determine the presence or absence of MISIIR mRNA. Forty-four percent (4/9) of EOC cell lines expressed MISIIR mRNA (IGROV-1, OVCAR8, OVCAR5, and OV207). SKOV3ip, OV17, OV167, OV202, and HEYC2 did not express MISIIR mRNA (Supplemental Fig. 1). The resulting 330 base pair fragment (bp 1262 to 1592) from RT-PCR was confirmed to be identical to the published MISIIR sequence [26] using automated sequencing. Ninety-two percent (11/12) of primary EOCs expressed MISIIR mRNA (Fig. 1). Additionally, both normal endometrium samples and 3 of 4 primary endometrial cancers expressed MISIIR. Polyclonal antibody validation

Fig. 2. MISIIR protein over-expression in transfected ovarian cell lines. (A) Myctagged expression of MISIIR in OV167 cells transfected with either p-myc-MISIIR or the empty vector. Western analysis was performed with the anti-myc monoclonal antibody. Fifty micrograms of total protein was loaded in each well. (B) MISIIR expression patterns in stable SKOV3ip-pMISIIR positive and negative clones and transiently transfected OV167-p-myc-MISIIR cells. Western analysis was performed with mAb12G4. Thirty-six micrograms of total protein was loaded in each well. MISIIR is 65 kDa in size.

plasm and on the cell membrane in SKOV3ip-pMISIIR clones (Figs. 4A and B). SKOV3ip stable clones then underwent immunocytochemical analysis with immunopurified MC1828. Cytoplasmic and cell surface expression of MISIIR in SKOV3ip-pMISIIR was again demonstrated. No expression was demonstrated in SKOV3ip negative clones (Figs. 4C and D).

First, myc-tagged MISIIR expression in transfected OV167 was confirmed by Western analysis with the anti-myc monoclonal antibody (Fig. 2A). OV167 transfectants then underwent Western analysis with mAb12G4 (Fig. 2B). Stable SKOV3ip-pMISIIR (Fig. 2B) and CHO-pMISIIR positive and negative expressing clones were screened and confirmed with Western analysis with mAb12G4. Stable SKOV3ip-pMISIIR and CHO-pMISIIR then underwent Western analysis with the immunopurified polyclonal antibody, MC1828 (Fig. 3). MISIIR migrates to 65 kDa. The specific 65 kDa band was competed away by incubating MC1828 with 1 mg/ml of the 21-mer MISIIR fragment (residues 173–194) initially inoculated into the MC1828 rabbit (Supplemental Fig. 2). Expression of MISIIR in stable SKOV3ip clones was then visualized by immunocytochemical analysis using mAb12G4. MISIIR expression was absent in SKOV3ip negative clones, whereas MISIIR expression is clearly demonstrated in the cyto-

Western analysis using mAb12G4 was performed on lysates from five EOC cell lines (OV17, OV177, OV202, OV207, and OVCAR8) and the OV167 cell line transiently transfected with either p-myc-MISIIR expression vector or empty vector (Fig. 5). Western analysis with mAb12G4 was performed on five additional ovarian cancer cell lines (parental OV167, parental SKOV3ip, IGROV-1, OVCAR5, and HEYC2) (data not shown). Of the ten EOC cell lines, 40% (OVCAR8, OV207, IGROV-1, and OVCAR5) expressed MISIIR at the protein level. These same cell lines were positive for MISIIR expression on RT-PCR (Supplemental Fig. 1).

Fig. 1. MISIIR mRNA expression in primary EOCs. RT-PCR yielded a 330 bp MISIIR fragment. Eight of the 12 primary tumors tested are shown. Of the 12 tumors tested, 11 were positive for MISIIR mRNA.

Fig. 3. Western analysis of stable clones using the polyclonal antibody, MC1828. MISIIR expression is identified by Western analysis utilizing MC1828 in both SKOV3ip-pMISIIR and CHO-pMISIIR stable clones. The 65 kDa band corresponding to MISIIR is present only in the positive clones.

MISIIR protein is expressed by epithelial ovarian cancer cell lines

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Fig. 4. Immunocytochemistry of stable SKOV3ip clones with both mAb12G4 and MC1828 (A) SKOV3ip-pMISIIR and (B) SKOV3ip negative stable clones. Both underwent immunocytochemistry with mAb12G4 and anti-mouse FITC. (C) SKOV3ip-pMISIIR and (D) SKOV3ip negative stable clones underwent immunocytochemistry with MC1828 and anti-rabbit FITC. MISIIR expression is seen on the cell surface and in the cytoplasm with both antibodies.

Large scale gynecologic cancer analysis for MISIIR protein expression TMAs containing tissue from primary ovarian cancers (dysgerminoma, clear cell, endometrioid, serous, and mucinous), benign ovaries, endometrial cancers, benign endometrium (proliferative, secretory, and atrophic), LMS, ESS, uterine MMMT, and benign myometrium, as well as normal nongynecologic tissues, underwent IHC staining with mAb12G4. Prior to TMA staining, the antibody was optimized for IHC using normal testis and ovarian granulosa cell tumor (GCT) as positive controls. We found that 69% (125/182) of the overall EOCs in our study expressed MISIIR. None of the five normal ovarian epithelium samples on the arrays expressed MISIIR protein via IHC (Fig. 6). When compared to the cohort of EOCs, there was

Fig. 5. Western analysis of EOC cell lines. MISIIR is expressed by OVCAR8 and OV207 cell lines. MISIIR is over-expressed by OV167 transfected with p-mycMISIIR and not expressed by OV202, OV177, and OV17. A total of 10 EOC cell lines were evaluated for MISIIR expression via Western blot and 4/10 (40%) expressed MISIIR. MISIIR is also expressed by IGROV-1 and OVCAR5.

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Fig. 6. MISIIR expression in benign ovarian epithelium and epithelial ovarian cancer (EOC). IHC with mAb12G4 shows no MISIIR expression in benign postmenopausal ovary and strong staining in serous EOC. (A) Postmenopausal ovary, 0.6×. (B) Postmenopausal ovarian surface epithelium, 20×. (C) Serous EOC, 1.25× and D, 20×.

a significantly higher level of MISIIR expression in the EOCs (p = 0.008, Fisher's exact test) than in normal ovarian surface epithelium. When stratified by histology, the difference in MISIIR expression between normal ovarian epithelium and EOC was statistically significant for all subtypes except clear cell carcinoma. The highest rate of expression among the ovarian histologies was observed in serous (76%) and mucinous (100%) EOCs. Dysgerminomas in our study were positive for MISIIR expression 77% of the time. Compared to normal ovarian stroma, which expressed MISIIR only 12.5% of the time, there was significantly higher expression of MISIIR in dysgerminomas (p = b 0.001, Chi square) (Table 1). Normal endometrium evaluated in this study was stratified as proliferative, secretory, or atrophic. Overall, only 28% (39/ 139) of the normal endometrium samples expressed MISIIR (Figs. 7A through F) with the highest rate of expression in atrophic endometrium (Table 1). Endometrial cancers in our study expressed MISIIR 75% (82/109) of the time (Figs. 7G and H). In comparison to each benign endometrium stratum, the rate of MISIIR expression in endometrial carcinoma was significantly higher (Table 1). Fifty-nine percent of uterine MMMT in our study expressed MISIIR. In comparison to normal myometrium, there was a higher rate of expression in MMMT (p b 0.001, Chi square). Just over half (52%) of LMS in this study expressed MISIIR, and again compared to normal myometrium, there was significantly higher expression in LMS (p = 0.004, Fisher's exact test). Only 22% of ESS were found to be positive for MISIIR expression, and there was no statistically significant difference in expression patterns between ESS and benign myometrium (p = 0.27, Fisher's exact test) (Table 1). In the normal tissue TMA, 74% of all tissues stained negative for MISIIR expression. Benign Fallopian tube, testis, prostate, and placenta were among the normal tissues that stained

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Table 1 MISIIR expression patterns in malignant and benign gynecologic tissues Tissue

MISIIR expression

Ovarian cancer (all EOCs) Serous Clear cell Endometrioid Mucinous Benign ovarian epithelium Dysgerminoma Benign ovarian stroma Endometrial cancer (all histologies) Benign endometrium Proliferative Secretory Atrophic Uterine malignant mixed Müllerian tumor Leiomyosarcoma Endometrial stromal sarcoma Benign myometrium

125/182 (69%) 22/29 (76%) 10/25 (40%) 17/31 (55%) 3/3 (100%) 0/5 (0%) 17/22 (77%) 3/24 (12.5%) 82/109 (75%) 39/139 (28%) 11/68 (17%) 16/47 (34%) 12/24 (50%) 30/51 (59%) 15/29 (52%) 4/18 (22%) 0/11 (0%)

p value 0.008 a, b 0.002 a, b 0.14 a, b 0.011 a, b 0.018 a, b – b0.001 c, d – b0.001 c, e – b0.001 c, f b0.001 c, f 0.018 c, f b0.001 c, g 0.004 a, g 0.27 a, g –

suggested improved OS for MISIIR expressing tumors; however, this was not statistically significant in either univariate (p = 0.08, Kaplan–Meier log rank) (Fig. 9A) or multivariate analysis (p = 0.098, Cox proportional hazards). Overall, there was no difference in recurrence between the MISIIR positive and negative EOCs (p = 0.25, Chi square; p = 0.49, Kaplan–Meier log rank) in the randomly selected cohort. Median time to recurrence for MISIIR positive EOCs was 37 months and median time to recurrence for the MISIIR negative EOCs was 35 months.

Histologic subtype was available for 88 of the 182 EOCs. a Fisher's exact test. b Chi square. c Compared to benign ovarian epithelium. d Compared to benign ovarian stroma. e Compared to overall benign endometrium. f Compared to endometrial cancer. g Compared to benign myometrium.

positive. Benign ovarian epithelium and stroma combined with benign endometrium and myometrium overall stained positive only 23% of the time. Those normal non-gynecologic tissue sites that stained positive included the parenchyma of the liver, the mucosa of the small bowel, the kidney tubules and glomeruli, adrenal gland, exocrine glands of the pancreas, breast ducts, and bronchiolar epithelium (Fig. 8A). Lower levels of staining were observed in tonsil and lymph nodes as well as arterial smooth muscle (Fig. 8B). Importantly, the normal tissues in this study were chosen randomly and were not stratified by gender or age which may impact the staining profile. Impact of MISIIR expression on EOC survival While the primary purpose of the study was to define expression patterns in gynecologic malignancies, we secondarily examined the association between MISIIR expression and clinical presentation and outcomes. In the randomly selected EOC TMAs, moderate to strong MISIIR expression was demonstrated in 69% (99/144) of the tumors. We found no correlation between MISIIR expression and stage or grade [early vs. advanced stage (p = 0.68, Chi square) or low vs. high grade (p = 0.85, Chi square)]. There was no difference in MISIIR expression between optimally and suboptimally debulked tumors (p = 0.19, Chi square) or between patients who received chemotherapy and those who did not (p = 0.71, Fisher's exact). A significant improvement in overall survival was observed in the randomly selected patients with tumors expressing MISIIR (p = 0.025, Chi square). Median time to death for the MISIIR positive cohort was 80 months compared to 55 months for the MISIIR negative cohort. Time-to-event survival analysis

Fig. 7. MISIIR expression in benign endometrium and endometrial carcinoma. (A) Only 17% of proliferative endometrium had moderate or strong expression of MISIIR. (B) Most proliferative endometrium specimens were weak or negative in MISIIR expression. (C) Only 34% of secretory endometrium had moderate or strong expression of MISIIR. (D) Most secretory endometrium specimens were weak or negative in MISIIR expression. (E) Half of all atrophic endometrium specimens expressed MISIIR and (F) half did not. (G) A high rate of MISIIR expression (75% overall) was observed in endometrial carcinoma. (H) Very few (27/109) endometrial carcinomas were weak or negative in MISIIR expression. All 20×.

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Fig. 8. MISIIR expression in non-gynecologic tissues. (A) Lung, weak MISIIR expression in epithelium, no MISIIR expression in lung parenchyma, 20×. (B) Artery, weak MISIIR expression in smooth muscle and endothelium; no MISIIR staining of peripheral blood cells, 20×.

In the high-risk TMA of 38 primary, advanced stage (IIIC or IV), high-grade (3 or 4) EOCs, all of which were optimally cytoreduced and treated with platinum-based chemotherapy, once again there was no statistically significant association between MISIIR expression and DFS (p = 0.12, Kaplan–Meier log rank). The median time to recurrence in the MISIIR positive EOCs was 26 months compared to 18 months in the MISIIR negative group. In this high-risk cohort, we also found no difference in OS (p = 0.4, Kaplan–Meier log rank) (Fig. 9B). Median time to death in the MISIIR positive EOCs was 60 months compared to 41 months for the MISIIR negative EOCs. In addition, there was no evidence of an association

Fig. 9. Kaplan–Meier overall survival curves for MISIIR positive and negative EOCs. (A) Randomly selected EOCs (n = 144) that express MISIIR had improved OS (p = 0.025, Chi square; p = 0.08 log rank). (B) No statistically significant difference in OS was observed in the high-risk cohort of EOCs, however, this cohort contained only 38 patients.

between MISIIR expression and response to chemotherapy in this high-risk group of EOCs (p = 0.37, Chi square). Discussion MIS and MISIIR appear to be involved in many facets of cellular growth, differentiation, and apoptosis. In embryonic development, MIS and its receptors play integral parts in early male gender differentiation [28,29]. In ovarian cancer, MIS exposure appears to be deleterious as evidenced by tumor regression following intraperitoneal MIS administration to mice containing tumors of human ovarian cancer cell line xenografts [19,20]. In addition, recent in vitro and in vivo experiments have shown that the combination of rhMIS and subclinical doses of chemotherapeutics yields an additive cytotoxic effect [21]. MIS and MISIIR are attractive targets for new therapies in EOC management. In this study, we assessed MISIIR expression in a wide range of malignancies that arise from the primordial Müllerian duct. MISIIR may not only be a potential target for EOC therapy; it may also facilitate the development of new treatment strategies for other gynecologic cancers. The importance of MISIIR as a target depends upon both common expression in tumors of interest and the degree of expression in normal tissues. We present the largest analysis of MISIIR expression in gynecologic cancers and benign tissues to date. We report that 64% of EOC expresses MISIIR with the most common expression seen in serous and mucinous tumors. In addition, we found high MISIIR expression in uterine carcinomas and sarcomas as well as in ovarian dysgerminomas. Many of these tumors represent significant treatment challenges in either the primary or recurrent setting and the need for novel therapies is great. In this study MISIIR expression was higher in EOC than in normal ovarian surface epithelium. Caution must be used as to whether this represents changes due to malignant transformation or simply the fragility of the single-cell layer ovarian surface epithelium, which led to low numbers of tissue cores containing sufficient amounts of surface epithelium. However, a similar increase in MISIIR expression was seen in endometrial carcinomas (75%) relative to benign endometrium (28% overall). This observation did not appear to be due to age or menopausal status. IHC staining revealed that 74% of all normal tissues express little or no MISIIR. Those normal non-gynecologic tissues expressing MISIIR included the liver parenchyma, epithelial

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lining of the lung, small intestine mucosa, adrenal, pancreas, and kidney. Utilization of therapeutic MIS or systemic immunotherapies against MISIIR could result in side effect profiles that involve these MISIIR-expressing organ systems. In addition, the normal tissues that express MISIIR could lead to false-positive results in receptor-based imaging. Further investigation into the vital organs that express MISIIR is warranted. These preliminary findings will need to be studied in a larger number of patients and stratified by gender. The secondary objective in this study was to determine whether a clinical correlation between MISIIR expression in EOC exists. Overall, there was no association between MISIIR expression and grade, stage, degree of cytoreduction achieved, or response of EOC to chemotherapy. This suggests broad applicability of MISIIR as a target in EOC. An improvement in overall survival was observed in the randomly selected EOCs that expressed MISIIR. While this improvement was not observed in the high-risk EOCs, small numbers (n = 38) in the high-risk cohort may contribute to this difference in findings. MISIIR is not associated with any improvement in DFS. However, the findings in the randomly selected EOCs suggest that MISIIR expression may be a marker for overall prognosis. Further investigation into survival differences is warranted. Our findings support other investigations indicating MISIIR as a tissue-specific receptor. Theoretically, this receptor is an attractive target for antibody-based detection [4] and therapy of EOC [1,14,16,17]. Our findings demonstrate that, in addition to EOC, several other important gynecologic malignancies including uterine carcinomas and sarcomas, which show poor response to traditional cytotoxic agents, frequently express MISIIR. This should expand the relevance of research into the therapeutic and diagnostic opportunities for this potential target. Acknowledgments We acknowledge the contributions of Kimberly Kalli, PhD, Karl Podratz, MD, PhD, Viji Shridahr, PhD, Amibola Famuyide, MD, and Shi Wen Jiang, MD of reagents used in this investigation. The Mayo Clinic Advanced Genomics Technology Center assisted in the IHC process. The AA173–194 peptide was synthesized by the Peptide Synthesis Facility of the Mayo Proteomics Research Center, Mayo Clinic, Rochester, MN. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ygyno.2007.09.010. References [1] Masiakos PT, et al. Human ovarian cancer, cell lines, and primary ascites cells express the human Mullerian inhibiting substance (MIS) Type II receptor, bind, and are responsive to MIS. Clin Cancer Res 1999;5:3488–99. [2] Hoshiya Y, et al. Mullerian inhibiting substance promotes interferon {gamma}-induced gene expression and apoptosis in breast cancer cells. J Biol Chem 2003;278:51703–12. [3] Hoshiya Y, et al. Mullerian inhibiting substance induces NFkB signaling in breast and prostate cancer cells. Mol Cell Endocrinol 2003;211:43–9.

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