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With advances in the discovery of the clinical and molecular landscapes of prostate cancer (PCa), implementation of precision medicine-guided therapeutic testing in the clinic has become a priority. Patient derived organoids (PDOs) are three-dimensional (3D) tissue cultures that promise to enable the validation of preclinical drug testing in precision medicine and coclinical trials by modeling PCa for predicting therapeutic responses with a reliable efficacy. We evaluate the advances in 3D culture and PDO use to model clonal heterogeneity and screen for effective targeted therapies, with a focus on the technological advances in generating PDOs. Recent innovations include the utilization of PDOs both in original research and/or correlative studies in clinical trials to examine drug effects within the PCa tumor microenvironment (TME). There has also been a significant improvement with the utilization of various extracellular matrices and single cell assays for the generation and long-term propagation of PDOs. Single cell derived PDOs could faithfully recapitulate the original tumor and reflect the heterogeneity features. While most PDO use for precision medicine understandably involved tissues derived from metastatic patients, we envision that the generation of PDOs from localized PCa along with the incorporation of cells of the TME in tissue models would fulfill the great potential of PDOs in predicting drug clinical benefits. We conclude that single cell derived PDOs reiterate the molecular features of the original tumor and represent a reliable pre-clinical PCa model to understand individual tumors and design tailored targeted therapies., (© 2021. The Author(s).)

Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of prostate cancer with poor prognosis, and there is a critical need for novel therapeutic approaches. NEPC is associated with molecular perturbation of several pathways, including amplification of MYCN . Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase involved in the pathogenesis of neuroblastoma and other malignancies where it cooperates with N-Myc. We previously identified the first case of ALK F1174C-activating mutation in a patient with de novo NEPC who responded to the ALK inhibitor, alectinib. Here, we show that coactivation of ALK and N-Myc (ALK F1174C/N-Myc) is sufficient to transform mouse prostate basal stem cells into aggressive prostate cancer with neuroendocrine differentiation in a tissue recombination model. A novel gene signature from the ALK F1174C/N-Myc tumors was associated with poor outcome in multiple human prostate cancer datasets. ALK F1174C and ALK F1174C/N-Myc tumors displayed activation of the Wnt/β-catenin signaling pathway. Chemical and genetic ALK inhibition suppressed Wnt/β-catenin signaling and tumor growth in vitro in NEPC and neuroblastoma cells. ALK inhibition cooperated with Wnt inhibition to suppress NEPC and neuroblastoma proliferation in vitro and tumor growth and metastasis in vivo . These findings point to a role for ALK signaling in NEPC and the potential of cotargeting the ALK and Wnt/β-catenin pathways in ALK-driven tumors. Activated ALK and N-Myc are well known drivers in neuroblastoma development, suggesting potential similarities and opportunities to elucidate mechanisms and therapeutic targets in NEPC and vice versa. SIGNIFICANCE: These findings demonstrate that coactivation of ALK and N-Myc induces NEPC by stimulating the Wnt/β-catenin pathway, which can be targeted therapeutically., (©2021 American Association for Cancer Research.)

Robust patient-derived platforms that recapitulate the cellular and molecular fingerprints of glioblastoma are crucial for developing effective therapies. Here, we describe a chemically defined protocol for 3D culture and propagation of glioblastoma in 3D gliospheres, patient-derived organoids (PDOs), mouse brain orthotopic xenografts (PDOXs), and downstream drug and immunofluorescence assays. This simple-to-follow protocol allows assessing drug sensitivity, on-target activity, and combined drug synergy. Promising therapies can then be validated in PDOXs for translation in precision medicine oncology trials. For complete details on the use and execution of this protocol, please refer to Chadwick et al. (2020) and Patrizii et al. (2018)., Competing Interests: Rutgers University has patents pending related to single-cell-derived organoids. H.E.S. is the scientific founder of Celvive, Inc., (© 2021 The Authors.)

The EPHB4 receptor is implicated in the development of several epithelial tumors and is a promising therapeutic target, including in prostate tumors in which EPHB4 is overexpressed and promotes tumorigenicity. Here, we show that high expression of EPHB4 correlated with poor survival in prostate cancer patients and EPHB4 inhibition induced cell death in both hormone sensitive and castration-resistant prostate cancer cells. EPHB4 inhibition reduced expression of the glucose transporter, GLUT3, impaired glucose uptake, and reduced cellular ATP levels. This was associated with the activation of endoplasmic reticulum stress and tumor cell death with features of immunogenic cell death (ICD), including phosphorylation of eIF2α, increased cell surface calreticulin levels, and release of HMGB1 and ATP. The changes in tumor cell metabolism after EPHB4 inhibition were associated with MYC downregulation, likely mediated by the SRC/p38 MAPK/4EBP1 signaling cascade, known to impair cap-dependent translation. Together, our study indicates a role for EPHB4 inhibition in the induction of immunogenic cell death with implication for prostate cancer therapy.

Hepatic angiosarcoma (HAS) is a rare and highly lethal malignancy with few effective systemic treatments. Relatively little is known about the genetic abnormalities that drive this disease. As a result, there has been minimal progress towards applying targeted therapies to the treatment of HAS. We describe the first reported case of a patient with HAS that harbored a fusion of ROS1 with GOPC/FIG . Similar to other rearrangements involving ROS1 , the resulting fusion protein is believed to act as a major driver of carcinogenesis and may be subject to inhibition by drugs that target ROS1 such as crizotinib. We then queried the MSK-IMPACT clinical sequencing cohort and cBioportal datasets, demonstrating the previously unknown prevalence of ROS1-GOPC fusions in soft tissue sarcomas and hepatobiliary cancers. Amplification of these genes was also found to correlate with reduced overall survival. This is followed by a review of the role played by ROS1 rearrangements in cancer, as well as the evidence supporting the use of targeted therapies against the resulting fusion protein. We suggest that testing for ROS1 fusion and, if positive, treatment with a targeted therapy could be considered at the time of diagnosis for patients with angiosarcoma. This report also highlights the need for further investigation into the molecular pathophysiology of this deadly disease., Competing Interests: CONFLICTS OF INTEREST None.

As a kinase at the crossroads of numerous metabolic and cell growth signaling pathways, glycogen synthase kinase-3 beta (GSK-3β) is a highly desirable therapeutic target in cancer. Despite its involvement in pathways associated with the pathogenesis of several malignancies, no selective GSK-3β inhibitor has been approved for the treatment of cancer. The regulatory role of GSK-3β in apoptosis, cell cycle, DNA repair, tumor growth, invasion, and metastasis reflects the therapeutic relevance of this target and provides the rationale for drug combinations. Emerging data on GSK-3β as a mediator of anticancer immune response also highlight the potential clinical applications of novel selective GSK-3β inhibitors that are entering clinical studies. This manuscript reviews the preclinical and early clinical results with GSK-3β inhibitors and delineates the developmental therapeutics landscape for this potentially important target in cancer therapy.

Purpose: Small cell carcinoma of the prostate (SCCP) is an aggressive disease that can arise de novo or by transdifferentiation from prostate adenocarcinoma. Alterations in anaplastic lymphoma kinase ( ALK ) gene are involved in neuroblastoma, lung cancer, and other malignancies, but its role in SCCP has not been documented. We describe a patient with refractory de novo SCCP with ALK F1174C-activating mutation who obtained clinical benefit from treatment with ALK inhibitor. Experimental Design: Next-generation sequencing (NGS) was used to analyze primary and circulating tumor DNA (ctDNA). Prostate cancer databases were queried for alterations in ALK gene, mRNA, and its impact in clinical outcomes. In vitro prostate cell line/organoid models were generated by lentiviral-mediated expression of ALK and ALK F1174C and assessed for response to ALK inhibitors crizotinib and alectinib. Results: NGS analysis of the primary tumor and ctDNA of a 39-year-old patient with refractory SSCP identified ALK F1174C mutation. Treatment with second-generation ALK inhibitor alectinib resulted in radiographic stable disease for over 6 months, symptomatic improvement, and significant molecular response as reflected by declining ctDNA allele fraction. Analysis of prostate cancer datasets showed that ALK amplification was associated with poor outcome. In prostate cancer cells and organoids, ALK F1174C expression enhanced growth and induced expression of the neuroendocrine marker neuron-specific enolase. Alectinib was more effective than crizotinib in inhibiting ALK F1174C-expressing cell growth. Conclusions: These findings implicate ALK -activating mutations in SCCP pathogenesis and suggest the therapeutic potential of targeting ALK molecular alterations in some patients with SCCP. Clin Cancer Res; 24(12); 2732-9. ©2018 AACR ., (©2018 American Association for Cancer Research.)

The Vacuolar ATPase (V-ATPase) is a proton pump responsible for controlling the intracellular and extracellular pH of cells. The structure of V-ATPase has been highly conserved among all eukaryotic cells and is involved in diverse functions across species. V-ATPase is best known for its acidification of endosomes and lysosomes and is also important for luminal acidification of specialized cells. Several reports have suggested the involvement of V-ATPase in maintaining an alkaline intracellular and acidic extracellular pH thereby aiding in proliferation and metastasis of cancer cells respectively. Increased expression of V-ATPase and relocation to the plasma membrane aids in cancer modulates key tumorigenic cell processes like autophagy, Warburg effect, immunomoduation, drug resistance and most importantly cancer cell signaling. In this review, we discuss the direct role of V-ATPase in acidification and indirect regulation of signaling pathways, particularly Notch Signaling.

Extracellular matrix (ECM) critically impacts tumor progression and is influenced by both cancer and host tissue cells. While our understanding of cancer cell ECM remodeling is widespread, the importance of host tissue ECM, which provides initial congenial environment for primary tumor formation, is partly understood. Here, we report a novel role of epithelial cell-associated vacuolar ATPase 'a2' isoform (a2V) in regulating breast tissue ECM stiffness to control metastasis. Using a mammary gland-specific a2V-knockout model, we show that in the absence of a2V, breast tumors exhibit atypically soft tumor phenotype, less tumor rigidity, and necrotic tumor microenvironment. These tumors contain a decreased number of cancer cells at primary tumor site, but showed extensive metastases compared to control. Nanomechanical evaluation of normal breast tissues revealed a decrease in stiffness and collagen content in ECM of a2V-deleted breast tissues. Mechanistically, inhibition of a2V expression caused dispersed Golgi morphology with relocation of glycosyltransferase enzymes to early endosomes in mammary epithelial cells. This resulted in defective glycosylation of ECM proteins and production of compromised ECM that further influenced tumor metastasis. Clinically, in patients with cancer, low a2V expression levels in normal breast tissue correlated with lymph node metastasis. Thus, using a new knockout mouse model, we have identified a2V expression in epithelial cells as a key requirement for proper ECM formation in breast tissue and its expression levels can significantly modulate breast tumor dissemination. Evaluation of a2V expression in normal breast tissues can help in identifying patients with high risk of developing metastases., (© 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

PARP1/2 inhibitors are effective against BRCA2-deficient tumors. The PARP inhibitor (PARPi) olaparib received FDA breakthrough designation for treatment of metastatic castration-resistant prostate cancers (CRPC) carrying mutations in BRCA1/2 or ATM genes. Emergent resistance to PARPi has been associated with tumor-specific BRCA2 mutations that revert the normal open reading frame rescuing homologous recombination. We describe a case of metastatic CRPC with germline BRCA2 mutation with acquired resistance to olaparib related to biallelic BRCA2 reversion mutations of both the germline and somatic loss of function alleles detected by circulating tumor DNA testing. We also summarize a retrospective analysis of 1,534 prostate cancer cases with ctDNA analysis showing a 1.6% incidence of germline BRCA2 mutations. Within the germline BRCA2 -positive cases exposed to platinum chemotherapy or PARP inhibition, the prevalence of reversion mutations was 40%. This report documents the frequency of reversion mutations in a large cohort of prostate cancer patients carrying of BRCA mutations. It also shows the potential utility of ctDNA analyses for early detection of reversion mutation driving tumor resistance.

Bone metastases are present in up to 70% of advanced prostate and breast cancers and occur at significant rates in a variety of other cancers. Bone metastases can be associated with significant morbidity. The establishment of bone metastasis activates several immunosuppressive mechanisms. Hence, understanding the tumor-bone microenvironment is crucial to inform the development of novel therapies. This review describes the current standard of care for patients with bone metastatic disease and novel treatment options targeting the microenvironment. Treatments reviewed include immunotherapies, cryoablation, and targeted therapies. Combinatorial treatment strategies including targeted therapies and immunotherapies show promise in pre-clinical and clinical studies to overcome the suppressive environment and improve treatment of bone metastases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Among all tissues and organs, the mammary gland is unique because most of its development occurs in adulthood. Notch signaling has a major role in mammary gland development and has been implicated in breast cancer. The vacuolar-ATPase (V-ATPase) is a proton pump responsible for the regulation and control of pH in intracellular vesicles and the extracellular milieu. We have previously reported that a2V-ATPase (a2V), an isoform of 'a' subunit of V-ATPase, regulates processing of Notch receptor and alters Notch signaling in breast cancer. To study the role of a2V in mammary gland development, we generated an a2V-KO model (conditional mammary knockout a2V mouse strain). During normal mammary gland development, the basal level expression of a2V increased from puberty, virginity, and pregnancy through the lactation stage and then decreased during involution. Litters of a2V-KO mice weighed significantly less when compared with litters from wild-type mice and showed reduced expression of the lactation marker β-casein. Whole-mount analysis of mammary glands demonstrated impaired ductal elongation and bifurcation in a2V-KO mice. Consequently, we found disintegrated mammary epithelium as seen by basal and luminal epithelial staining, although the rate of proliferation remained unchanged. Delayed mammary morphogenesis in a2V-KO mice was associated with aberrant activation of Notch and TGF-β (transforming growth factor-β) pathways. Notably, Hey1 (hairy/enhancer-of-split related with YRPW motif) and Smad2, the key downstream mediators of Notch and TGF-β pathways, respectively, were upregulated in a2V-KO mice and also in human mammary epithelial cells treated with a2V siRNA. Taken together, our results show that a2V deficiency disrupts the endolysosomal route in Notch and TGF signaling, thereby impairing mammary gland development. Our findings have broader implications in developmental and oncogenic cellular environments where V-ATPase, Notch and TGF-β are crucial for cell survival.

Notch signaling pathways exert effects throughout pregnancy and are activated in response to TLR ligands. To investigate the role of Notch signaling in preterm labor, Notch receptors (Notch1-4), its ligand Delta-like protein-1, transcriptional repressor hairy and enhancer of split-1, and Notch deregulator Numb were assessed. Preterm labor was initiated on gestation d 14.5 by 1 of 2 methods: 1) inflammation-induced preterm labor: intrauterine injection of LPS (a TLR4 agonist) and 2) hormonally induced preterm labor: subcutaneous injection of mifepristone. Delta-like protein-1, Notch1, and hairy and enhancer of split-1 were elevated significantly, and Numb was decreased in the uterus and placenta of inflammation-induced preterm labor mice but remained unchanged in hormonally induced preterm labor compared with their respective controls. F4/80(+) macrophage polarization was skewed in the uterus of inflammation-induced preterm labor toward M1-positive (CD11c(+)) and double-positive [CD11c(+) (M1) and CD206(+) (M2)] cells. This process is dependent on activation of Notch signaling, as shown by suppression of M1 and M2 macrophage-associated cytokines in decidual macrophages in response to γ-secretase inhibitor (an inhibitor of Notch receptor processing) treatment ex vivo. γ-Secretase inhibitor treatment also diminished the LPS-induced secretion of proinflammatory cytokines and chemokines in decidual and placental cells cultured ex vivo. Furthermore, treatment with recombinant Delta-like protein-1 ligand enhanced the LPS-induced proinflammatory response. Notch ligands (Jagged 1 and 2 and Delta-like protein-4) and vascular endothelial growth factor and its receptor involved in angiogenesis were reduced significantly in the uterus and placenta during inflammation-induced preterm labor. These results suggest that up-regulation of Notch-related inflammation and down-regulation of angiogenesis factors may be associated with inflammation-induced preterm labor but not with hormonally induced preterm labor., (© Society for Leukocyte Biology.)

Nearly 65 years have passed since Peter Medawar posed the following question: "How does the pregnant mother contrive to nourish within itself, for many weeks or months, a fetus that is an antigenically foreign body." Now, understanding of reproductive immunology has demonstrated that the HLA antigens in the placenta are non-classical and do not induce rejection. In the placenta and in tumors, 50% or more of the cells are cells of the immune system and were once thought to be primed and ready for killing tumors or the "fetal transplant" but these cells are not potential killers but abet the growth of either the tumor or the placenta. We believe that these cells are there to create an environment, which enhances either placental or tumor growth. By examining the similarities of the placenta's and tumor's immune cells, novel mechanisms to cause tumors to be eliminated can be devised., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Development of resistance to platinum compounds significantly hinders successful ovarian cancer (OVCA) treatment. In tumor cells, dysregulated pH gradient across cell membranes is a key physiological mechanism of metastasis/chemo-resistance. These pH alterations are mediated by aberrant activation of key multi-subunit proton pumps, Vacuolar-ATPases (V-ATPases). In tumor cells, its 'a2' isoform (V-ATPase-V0a2) is a component of functional plasma-membrane complex and promotes tumor invasion through tumor-acidification and immuno-modulation. Its involvement in chemo-resistance has not been studied. Here, we show that V-ATPase-V0a2 is over-expressed in acquired-cisplatin resistant OVCA cells (cis-A2780/cis-TOV112D). Of all the 'a' subunit isoforms, V-ATPase-V0a2 exhibited an elevated expression on plasma membrane of cisplatin-resistant cells compared to sensitive counterparts. Immuno-histochemistry revealed V-ATPase-V0a2 expression in both low grade (highly drug-resistant) and high grade (highly recurrent) human OVCA tissues indicating its role in a centralized mechanism of tumor resistance. In cisplatin resistant cells, shRNA mediated inhibition of V-ATPase-V0a2 enhanced sensitivity towards both cisplatin and carboplatin. This improved cytotoxicity was mediated by enhanced cisplatin-DNA-adduct formation and suppressed DNA-repair pathway, leading to enhanced apoptosis. Suppression of V0a2 activity strongly reduced cytosolic pH in resistant tumor cells, which is known to enhance platinum-associated DNA-damage. As an indicator of reduced metastasis and chemo-resistance, in contrast to plasma membrane localization, a diffused cytoplasmic localization of acidic vacuoles was observed in V0a2-knockdown resistant cells. Interestingly, pre-treatment with monoclonal V0a2-inhibitory antibody enhanced cisplatin cytotoxicity in resistant cells. Taken together, our findings suggest that the isoform specific inhibition of V-ATPase-V0a2 could serve as a therapeutic strategy for chemo-resistant ovarian carcinoma and improve efficacy of platinum drugs., (Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The a2 isoform of vacuolar-ATPase (ATP6V0A2, referred to as a2V) is required for normal spermatogenesis and maturation of sperm. Treatment of male mice with anti-a2V disturbs the testicular cytokine/chemokine balance and leads to severe deficiencies of spermatogenesis. The aim of the present study was to investigate the role of a2V in male fertility and in the regulation of apoptotic pathways required for normal spermatogenesis in mice. To study the role of a2V single dose of anti-a2V monoclonal antibody or mouse IgG isotype (3μg/animal) was injected i.p. into males on alternate days for 10 days. The expression of sperm maturation-related molecules and pro-apoptotic molecules was measured by real-time PCR or immunohistochemistry in control and anti-a2V-treated testes. The caspase levels and their activity were measured by western blot and fluorometry. We found that the expression of the sperm maturation-related molecules SPAM1, ADAM1, and ADAM2 was significantly decreased in testes from anti-a2V-treated males. The expression of pro-apoptotic molecules (Bax, p53, and p21) and molecules involved in the intrinsic pathway of apoptosis (caspase-9, caspase-3, and PARP), which are crucial for normal spermatogenesis was significantly reduced in testes from anti-a2V-treated males compared with the control. The total ATP level was significantly lower in anti-a2V-treated testes. The data provide novel evidence showing that a2V can regulate the apoptotic pathways, an essential testicular feature, and is necessary for efficient spermatogenesis., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Triple Negative Breast Cancer (TNBC) is a subtype of breast cancer with poor prognosis for which no targeted therapies are currently available. Notch signaling has been implicated in breast cancer but the factors that control Notch in TNBC are unknown. Because the Vacuolar ATPase has been shown to be important in breast cancer invasiveness, we investigated the role of a2-subunit isoform of Vacuolar ATPase (a2V) in regulating Notch signaling in TNBC. Confocal microscopy revealed that among all the 'a' subunit isoforms, a2V was uniquely expressed on the plasma membrane of breast cancer cells. Both a2V and NOTCH1 were elevated in TNBC tumors tissues and cell lines. a2V knockdown by siRNA as well as V-ATPase inhibition by Bafilomycin A1 (Baf A1) in TNBC cell lines enhanced Notch signaling by increasing the expression of Notch1 intracellular Domain (N1ICD). V-ATPase inhibition blocked NICD degradation by disrupting autophagy and lysosomal acidification as demonstrated by accumulation of LC3B and diminished expression of LAMP1 respectively. Importantly, treatment with Baf A1 or anti-a2V, a novel-neutralizing antibody against a2V hindered cell migration of TNBC cells. Our findings indicate that a2V regulates Notch signaling through its role in endolysosomal acidification and emerges as a potential target for TNBC.

Notch signaling plays an important role in regulation of innate immune responses and trophoblast function during pregnancy. To identify the role of Notch signaling in preterm labor, Notch receptors (Notch1-4), its ligands (DLL (Delta-like protein)-1/3/4), Jagged 1/2) and Notch-induced transcription factor Hes1 were assessed during preterm labor. Preterm labor was initiated on gestation day 14.5 by intrauterine (IU) injection of peptidoglycan (PGN) and polyinosinic:cytidylic acid (poly(I:C). Notch1, Notch2, Notch4, DLL-1 and nuclear localization of Hes1 were significantly elevated in uterus and placenta during PGN+poly(I:C)-induced preterm labor. Ex vivo, Gamma secretase inhibitor (GSI) (inhibitor of Notch receptor processing) significantly diminished the PGN+poly(I:C)-induced secretion of M1- and M2-associated cytokines in decidual macrophages, and of proinflammatory cytokines (IFN-γ, TNF-α and IL-6) and chemokines (MIP-1β) in decidual and placental cells. Conversely, angiogenesis factors including Notch ligands Jagged 1/2 and DLL-4 and VEGF were significantly reduced in uterus and placenta during PGN+poly(I:C)-induced preterm labor. In vivo GSI treatment prevents PGN+poly(I:C)-induced preterm delivery by 55.5% and increased the number of live fetuses in-utero significantly compared to respective controls 48 hrs after injections. In summary, Notch signaling is activated during PGN+poly(I:C)-induced preterm labor, resulting in upregulation of pro-inflammatory responses, and its inhibition improves in-utero survival of live fetuses.

Tumor associated vacuolar H+-ATPases (V-ATPases) are multi-subunit proton pumps that acidify tumor microenvironment, thereby promoting tumor invasion. Subunit 'a' of its V0 domain is the major pH sensing unit that additionally controls sub-cellular targeting of V-ATPase and exists in four different isoforms. Our study reports an elevated expression of the V-ATPase-V0a2 isoform in ovarian cancer(OVCA) tissues and cell lines(A2780, SKOV-3 and TOV-112D). Among all V0'a' isoforms, V0a2 exhibited abundant expression on OVCA cell surface while normal ovarian epithelia did not. Sub-cellular distribution of V-ATPase-V0a2 confirmed its localization on plasma-membrane, where it was also co-associated with cortactin, an F-actin stabilizing protein at leading edges of cancer cells. Additionally, V0a2 was also localized in early and late endosomal compartments that are sites for modulations of several signaling pathways in cancer. Targeted inhibition of V-ATPase-V0a2 suppressed matrix metalloproteinase activity(MMP-9 & MMP-2) in OVCA cells. In conclusion, V-ATPase-V0a2 isoform is abundantly expressed on ovarian tumor cell surface in association with invasion assembly related proteins and plays critical role in tumor invasion by modulating the activity of matrix-degrading proteases. This study highlights for the first time, the importance of V-ATPase-V0a2 isoform as a distinct biomarker and possible therapeutic target for treatment of ovarian carcinoma.