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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.

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

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.

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.