The ovarian reserve represents the stock of quiescent primordial follicles in the ovary which is gradually depleted during a woman's reproductive lifespan, resulting in menopause. Müllerian inhibiting substance (MIS) (or anti-Müllerian hormone/AMH), which is produced by granulosa cells of growing follicles, has been proposed as a negative regulator of primordial follicle activation. Here we show that long-term parenteral administration of superphysiological doses of MIS, using either an adeno-associated virus serotype 9 (AAV9) gene therapy vector or recombinant protein, resulted in a complete arrest of folliculogenesis in mice. The ovaries of MIS-treated mice were smaller than those in controls and did not contain growing follicles but retained a normal ovarian reserve. When mice treated with AAV9/MIS were paired with male breeders, they exhibited complete and permanent contraception for their entire reproductive lifespan, disrupted vaginal cycling, and hypergonadotropic hypogonadism. However, when ovaries from AAV9-MIS-treated mice were transplanted orthotopically into normal recipient mice, or when treatment with the protein was discontinued, folliculogenesis resumed, suggesting reversibility. One of the important causes of primary ovarian insufficiency is chemotherapy-induced primordial follicle depletion, which has been proposed to be mediated in part by increased activation. To test the hypothesis that MIS could prevent chemotherapy-induced overactivation, mice were given carboplatin, doxorubicin, or cyclophosphamide and were cotreated with AAV9-MIS, recombinant MIS protein, or vehicle controls. We found significantly more primordial follicles in MIS-treated animals than in controls. Thus treatment with MIS may provide a method of contraception with the unique characteristic of blocking primordial follicle activation that could be exploited to prevent the primary ovarian insufficiency often associated with chemotherapy.M üllerian inhibiting substance (MIS), also known as antiMüllerian hormone (AMH), has long been appreciated for its role in sex differentiation and reproduction, and sensitive ELISAs measuring blood levels are used in fertility clinics around the world as a measure of ovarian reserve (1-6). MIS plays important roles in the development of the gonad and the differentiation of the urogenital ridge. In the male fetus, MIS produced by the developing testes causes regression of the Müllerian duct (7). In the female fetus, MIS may play a role in early follicle assembly in the gonad by primordial germ cells (not to be confused with primordial follicles), since mice overexpressing MIS are devoid of germ cells shortly after birth (8), and, similarly, ex vivo incubation of fetal ovaries with MIS results in the inhibition of follicle assembly (9). These data highlight a role of MIS during fetal development that is distinct from its regulatory role of folliculogenesis postnatally.In the adult, MIS is produced predominantly by the cumulus (less so by the mural) granulosa cells of secondary and early antral...

Mullerian Inhibiting Substance (MIS) has been shown to inhibit ovarian cancer cells both in-vitro and in-vivo. Furthermore, recent evidence suggests that MIS may effectively target a putative ovarian cancer progenitor cell population enriched by a panel of CD44+, CD24+, Ep-CAM+, and E-cadherin-cell surface markers. In order to accommodate clinical testing of MIS in ovarian cancer patients, the production of recombinant human MIS must be optimized to increase yield and purity. Here we show that, compared to wild type, the substitution of the MIS leader sequence to that of human serum albumin, combined with a modification of the endogenous cleavage site from RAQR/S to a furin/kex2 RARR/S consensus site results in high expression, increased C-terminus cleavage and a reduction in unwanted cryptic internal cleavage products when produced in CHO cells. Purified MIS containing these alterations retains its capacity to induce regression of the Mullerian duct in fetal rat embryonic urogenital ridge assays.

To improve ovarian cancer patient survival, effective treatments addressing chemoresistant recurrences are particularly needed. Mullerian inhibiting substance (MIS) has been shown to inhibit the growth of a stem-like population of ovarian cancer cells. We have recently engineered peptide modifications to human MIS [albumin leader Q425R MIS (LRMIS)] that increase production and potency in vitro and in vivo. To test this novel therapeutic peptide, serous malignant ascites from highly resistant recurrent ovarian cancer patients were isolated and amplified to create low-passage primary cell lines. Purified recombinant LRMIS protein successfully inhibited the growth of cancer spheroids in vitro in a panel of primary cell lines in four of six patients tested. Adeno-associated virus (AAV) -delivered gene therapy has undergone a clinical resurgence with a good safety profile and sustained gene expression. Therefore, AAV9 was used as a single i.p. injection to deliver LRMIS to test its efficacy in inhibiting growth of palpable tumors in patient-derived ovarian cancer xenografts from ascites (PDXa). AAV9-LRMIS monotherapy resulted in elevated and sustained blood concentrations of MIS, which significantly inhibited the growth of three of five lethal chemoresistant serous adenocarcinoma PDXa models without signs of measurable or overt toxicity. Finally, we tested the frequency of MIS type II receptor expression in a tissue microarray of serous ovarian tumors by immunohistochemistry and found that 88% of patients bear tumors that express the receptor. Taken together, these preclinical data suggest that AAV9-LRMIS provides a potentially well-tolerated and effective treatment strategy poised for testing in patients with chemoresistant serous ovarian cancer.varian cancer is an enigmatic disease with 70% recurrence, and it is almost invariably fatal, even after complete response to chemotherapy and debulking i.p. surgery. Newer agents, such as angiogenesis inhibitors (1, 2) or ADP-ribose polymerase inhibitors (3), have only provided short-term improvement to survival outcomes. A dire need exists to develop novel therapeutic strategies addressing those highly chemoresistant recurrences that drive mortality. Epithelial ovarian cancers have long been known as "Mullerian" tumors, because histopathology of serous cystadenocarcinoma, endometrioid, and mucinous carcinomas recapitulates the embryonic Mullerian duct, the anlagen of the fallopian tube, uterus, cervix, and upper vagina (4). Hence, we turned to Mullerian inhibiting substance (MIS; also known as anti-Mullerian hormone), an evolutionarily conserved endogenous hormone of fetal and adult gonads (5), as a therapeutic against Mullerian-derived ovarian cancer. Recombinant human MIS (rhMIS) produced using a genomic clone (6) and purified from CHO serum-free media (7, 8) caused regression of fetal Mullerian ducts ex vivo and inhibited mouse ovarian cancer cell lines generated by Misr2-Cre directing T antigen in vitro and in vivo (9, 10). Similar effects were evident in established h...

CD44 is a transmembrane hyaluronic acid receptor gene that encodes over 100 different tissue-specific protein isoforms. The most ubiquitous, CD44 standard, has been used as a cancer stem cell marker in ovarian and other cancers. Expression of the epithelial CD44 variant containing exons v8-10 (CD44v8-10) has been associated with more chemoresistant and metastatic tumors in gastrointestinal and breast cancers, but its role in ovarian cancer is unknown; we therefore investigated its use as a prognostic marker in this disease. The gene expression profiles of 254 tumor samples from The Cancer Genome Atlas RNAseqV2 were analyzed for the presence of CD44 isoforms. A trend for longer survival was observed in patients with high expression of CD44 isoforms that include exons v8-10. Immunohistochemical (IHC) analysis of tumors for presence of CD44v8-10 was performed on an independent cohort of 210 patients with high-grade serous ovarian cancer using a tumor tissue microarray. Patient stratification based on software analysis of staining revealed a statistically significant increase in survival in patients with the highest levels of transmembrane protein expression (top 10 or 20%) compared to those with the lowest expression (bottom 10 and 20%) (p = 0.0181, p = 0.0262 respectively). Expression of CD44v8-10 in primary ovarian cancer cell lines was correlated with a predominantly epithelial phenotype characterized by high expression of epithelial markers and low expression of mesenchymal markers by qPCR, Western blot, and IHC. Conversely, detection of proteolytically cleaved and soluble extracellular domain of CD44v8-10 in patient ascites samples was correlated with significantly worse prognosis (p<0.05). Therefore, presence of transmembrane CD44v8-10 on the surface of primary tumor cells may be a marker of a highly epithelial tumor with better prognosis while enzymatic cleavage of CD44v8-10, as detected by presence of the soluble extracellular domain in ascites fluid, may be indicative of a more metastatic disease and worse prognosis.

Purpose: Ovarian cancer is the most lethal malignancy of the female reproductive tract; as such, effective treatments are needed to improve patient survival. MIS has previously been shown to inhibit growth of a stem-like subset of the total cell population in ovarian cancer cell lines (Wei et al, 2010; Meirelles et al, 2012). We have recently engineered novel peptide modifications to human MIS (LR-MIS), which increase production and potency in vitro and in vivo (Pepin et al, 2013), and inserted it into an AAV vector. AAV delivered gene therapy has undergone a clinical resurgence with a good safety profile and sustained gene expression (Zhang et al, 2011). Therefore, we assessed the efficacy of a single intraperitoneal (ip) dose of AAV9-LR-MIS to inhibit tumor growth of an established cell line for subsequent evaluation in patient-derived ovarian cancer xenografts (PDX). Methods: 6 week old female nude mice were treated with a single ip injection of 3e11 or 1e12 virions of AAV9 LR-MIS or GFP. Serum levels of human MIS were monitored weekly by ELISA. After serum steady state was achieved, the established ovarian cancer cell line, OVCAR5 was xenografted subcutaneously at 1e6 cells per animal. Tumors treated with AAV were measured twice weekly. Additionally, Patient ascites were processed to isolate the cancer cell population, which was specifically amplified in vitro to create low passage primary cell lines. Results: AAV9-LR-MIS treatment resulted in elevated and sustained blood concentrations of MIS (Fig1A) in nude mice without any signs of toxicity, and significantly inhibited OVCAR5 tumor growth (p=0.0003) (Fig1B) in two separate experiments. AAV9-GFP revealed that the pancreas, omentum, gut mesentery, and body wall muscles were the primary sites of infection. Treatment with MIS induced downregulation of CCND1 in tumors. A panel of 15 primary ovarian cancer cell lines, all expressing the MIS type II receptor were generated from patient ascites and cells are being tested for their ability to grow as PDX in Nude, NOD/SCID, and NSG mice. Figure 1.AAV-LR-MIS treatment results in stable expression of MIS and inhibition of tumor growth of OVCAR5 xenografts. A) Weekly MIS ELISA of serum from mice after single doses of 1e10 to 1e12 virions. B) Tumor growth of 1e6 OVCAR5 cells xenografted into nude mice (N=5) pretreated with 3e11/mouse (1.5e13/kg) LR-MIS or GFP virus (p=0.0003 by two-way ANOVA).Figure 1. AAV-LR-MIS treatment results in stable expression of MIS and inhibition of tumor growth of OVCAR5 xenografts. A) Weekly MIS ELISA of serum from mice after single doses of 1e10 to 1e12 virions. B) Tumor growth of 1e6 OVCAR5 cells xenografted into nude mice (N=5) pretreated with 3e11/mouse (1.5e13/kg) LR-MIS or GFP virus (p=0.0003 by two-way ANOVA). Conclusions: AAV9 virally delivered human LR-MIS can produce adequate sustained levels of MIS that inhibited the growth of the human ovarian cancer cell line, OVCAR 5. AAV9 LR MIS provides a well-tolerated, low toxicity treatment of ovarian cancer, which can now be evaluated in a phase 0 trial using PDX models being prepared from human ovarian cancer ascites. Citation Format: David Pepin, Amanda Sosulski, Katherine Hendren, Li Hua Zhang, Fotini Nicolaou, Dan Wang, Guangping Gao, Patricia K Donahoe. An adeno-associated virus (AAV) 9 vector delivering modified human mullerian inhibiting substance (MIS) as a gene therapy for ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS25.

BACKGROUND Trauma is the leading cause of nonobstetric death during pregnancy and is associated with an increased risk of maternal and fetal mortality. In an effort to improve the delivery of care to pregnant trauma patients, we developed an institutional multidisciplinary quality initiative designed to improve response times of nontrauma specialists and ensure immediate availability of resources. We hypothesized that implementation of a perinatal emergency response team (PERT) would improve time to patient and fetal evaluation and monitoring by the obstetrics (OB) team and improve both maternal and fetal outcomes. METHODS We performed a 6-year (2012–2018) retrospective cohort analysis of consecutive pregnant trauma patients presenting to our university-affiliated, level I trauma center. Patients in the pre-PERT cohort (before April 2015) were compared with a post-PERT cohort. Variables analyzed included patient demographics, mechanism of injury, Injury Severity Score, and level of trauma activation. The main outcome measure was time to OB evaluation. Secondary outcomes included time to cardiotocometry, and mortality. RESULTS Of 92 pregnant trauma patients, there were 50 patients (54.3%) in the pre-PERT cohort and 42 (45.7%) in the post-PERT group. Blunt injuries predominated (98.9%), with the most common mechanism being motor vehicle collisions (76.1%), followed by assaults (13%) and falls (6.5%). The mean time to obstetrical evaluation was 44 minutes in the pre-PERT cohort compared with 14 minutes in the post-PERT cohort (p = 0.001). There was a significant decrease in level I (highest acuity) trauma activations pre-PERT and post-PERT (46% vs. 21%, p = 0.01), and the time to cardiotocography was significantly decreased post-PERT implementation (72 vs. .37 min, p = 0.01) CONCLUSION Implementation of a multidisciplinary PERT improves time to evaluation by the OB team and time to cardiotocometry in the pregnant trauma patient. LEVEL OF EVIDENCE Retrospective review, level IV.

Mullerian Inhibiting Substance (MIS) has been shown to inhibit ovarian cancer cells both in-vitro and in-vivo. MIS inhibits a putative ovarian cancer progenitor cell population enriched by a panel of CD44+, CD24+, Ep-CAM+, and E-cadherin- cell surface markers. In order to accommodate clinical testing of MIS in ovarian cancer patients, the production of recombinant human MIS must be optimized to increase yield and purity. Here, we show that the substitution of the MIS leader sequence to that of human serum albumin (HSAL), combined with a modification of the endogenous cleavage site from RAQR/S to a furin/kex2 RARR/S consensus site results in high expression, increased c-terminus cleavage and a reduction in unwanted cryptic internal cleavage products when produced in CHO cells. Purified MIS containing the HSAL modification displays increased potency to induce regression of the Mullerian duct in fetal rat embryonic urogenital ridge assays, reflecting the increased cleavage. The modifications herein described, which may be applicable to other TGF-beta family members, will facilitate production of active recombinant c-terminal MIS for clinical trials in ovarian cancer. Citation Format: David Pepin, Mien Hoeng, Fotini Nicolaou, Katherine Hendren, Leo Benedict, Amanda Sosulski, Ahmad Al-Moujahed, Demetrious Vavvas, Patricia K. Donahoe. An albumin leader sequence coupled with a cleavage site modification enhances the yield of recombinant c-terminal Mullerian inhibiting substance. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A42.

CD44 is a transmembrane hyaluronic acid receptor gene that encodes over 100 different tissue specific protein isoforms. The most prevalent, standard CD44 (CD44s), has been used as one of a panel of markers identifying a stem cell enriched population in ovarian cancer. While expression of the CD44 epithelial specific variant containing exons v810 (CD44v810) has been associated with more resistant and metastatic tumors in gastrointestinal and breast cancers, its role in highgrade serous ovarian cancer is unknown. The gene expression profiles from a cohort of 254 tumor samples, of The Cancer Genome Atlas RNAseqV2 ovarian serous cystadenocarcinoma collection, were analyzed for expression of different CD44 isoforms. A trend for longer survival was observed in patients with tumors with high expression of CD44 isoforms that include exons V8-10. Immunohistochemical analysis of tumors using an antibody recognizing CD44v810 (rat antiCD44v9 RV3) was performed on an independent cohort of 210 patients with high-grade serous ovarian cancer using a tumor tissue microarray. Patient stratification based on unbiased software analysis of staining revealed a statistically significant increase in survival in patients with the highest levels of protein expression compared to those with the lowest expression with a median survival time in the top 10% of 1203 days versus 751 days for the bottom 10% (p= 0.001). Immunohistochemical expression of CD44v810 transmembrane protein in primary ovarian cancer cell lines was correlated with a predominantly epithelial phenotype characterized by high expression of epithelial markers and low expression of mesenchymal markers by principal component analysis. Conversely, detection of proteolytically cleaved and secreted extracellular domain of CD44v810 (solCD44v810) in patient ascites samples was correlated with significantly worse prognosis. Therefore, presence of transmembrane CD44v8-10 on the surface of primary tumor cells may be a marker of a highly epithelial tumor with better prognosis while enzymatic cleavage of CD44v8-10, as detected by presence of the soluble extracellular domain in ascites fluid, may be indicative of a more metastatic disease with worse prognosis. Citation Format: David Pepin, Amanda Sosulski, Heiko Horn, Lihua Zhang, Caroline Coletti, vinod Vathipadiekal, Cesar M. Castro, Michael J. Birrer, Osamu Nagano, Hidekuyi Saya, Kasper Lage, Patricia K. Donahoe. Cell surface expression of CD44v8-10 is associated with good prognosis while cleaved extracellular domain is a marker of poor prognosis in high-grade serous epithelial ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B26.