According to the 2009 report of the Committee on Gynecologic Oncology of the Japan Society of Obstetrics and Gynecology, compared to other nations, the incidence of clear cell adenocarcinoma (CCA) is considerably higher in Japan, accounting for 23.4% of all ovarian cancers. Although many of the CCAs are detected as being at stage I, the advanced CCAs tend to take an unfavorable clinical course.
Paclitaxel + carboplatin (TC) therapy is the current first-line chemotherapy for epithelial ovarian cancer, but it only achieves a 60% to 70% response rate.1 Recently, the use of taxanes other than paclitaxel has been tried in the new regimens, but no significant difference of response was found between docetaxel + carboplatin (DC) therapy using docetaxel and TC therapy in a phase 3 comparative study, as shown by the following response ratios: 58.7% to DC therapy and 59.5% to TC therapy.2 Thus, there is no more effective treatment than TC therapy, and exploration of new regimens targeting an individual histological type is required. This is because, in particular, the response rate to TC therapy is low in CCA.
Von Hippel-Lindau (VHL) gene abnormality has often been detected in CCA of the ovary, and it has been suggested to be correlated with the prognosis.3,4 The primary function of VHL is degradation of hypoxia-inducible factor-1α (HIF-1α).5 Binding between VHL and HIF-1α causes the hydroxylation of proline residues (proline 402 and proline 564) in the oxygen-dependent degradation domain of HIF-1α by prolyl hydroxylase domain-containing protein, which allows HIF-1α to bind with E3 ubiquitin ligase that degrades it.6 Hypoxia-inducible factor-1α then translocates to the nucleus by binding to histone-deacetylase 7, where it forms the β-subunit and dimers, which then form a complex containing CREB binding protein (CBP)/p300 coactivator that produces factors such as erythropoietin (EPO) and vascular endothelial growth factor (VEGF).7
In previous comparative studies of CCA and other ovarian epithelial types, we have found increased nuclear expression of HIF-1α in CCA and have identified the presence of HIF-1α regulating factors,8,9 as well as suggesting a relationship between increased phosphorylation of the mammalian target of rapamycin (mTOR) and increased expression of HIF-1α independent of the oxygen concentration.10,11 The abnormalities of the P13K-Akt pathway upstream of mTOR have already been detected in patients with colon cancer12 and ovarian cancer.13 Phosphorylation of mTOR causes the release of eukaryotic translation initiation factor-4E from eukaryotic translation initiation factor-4E–binding protein (4E-BP1) and the translation of CAP-dependent messenger RNAs (mRNAs), including HIF-1α, which then results in the accumulation of HIF-1α as mentioned previously.14 Rapamycin is an mTOR inhibitor, which forms a complex with FKBP12 (FK506-binding protein) and binds to phosphorylated mTOR (p-mTOR) to arrest the cell cycle in G1 phase,15 and is known to have an antitumor effect via the induction of apoptosis.16 Recent studies have revealed that it also inhibits tumor angiogenesis by blocking production of VEGF through suppression of the mTOR/HIF pathway.17
In the present study, we investigated changes in the protein and mRNA expression of VHL and the Akt-mTOR-HIF-VEGF axis when CCA cells were exposed to mTOR inhibitors such as rapamycin and everolimus (a derivative of rapamycin). The antitumor effect of these mTOR inhibitors was investigated in vivo as well.
MATERIALS AND METHODS
A total of 49 cases with CCA, which were surgically resected at the Department of Obstetrics and Gynecology of Tokai University Hospital during the period from 1990 to 2006, were examined. This study was limited to usage of the materials of which the preoperative informed consent was confirmed by the patients. The clinicopathological stage was determined according to the International Federation of Gynecology and Obstetrics classification.10
Immunohistochemistry and Prognosis Analysis
The expressions of p-mTOR and HIF-1α were determined using an indirect peroxidase method (Table 1). The expressions were semiquantitatively assessed according to the following scoring scheme: negative; weak, 1+ (<10%); intermediate, 2+ (10%–50%); and strong, 3+ (>50%).10 The overall survival ratios were calculated by the Kaplan-Meier method, and the significance of difference in survival was analyzed by the log-rank test using the Statistical Package for the Social Science (SPSS) software version 21.0 for Windows. P < 0.05 was regarded as significant.
In Vitro Experiment
A human ovarian cancer cell line (primary CCA: RMG-1) was obtained from the Department of Obstetrics and Gynecology at Saint Marianna Medical University. RMG-1 cells were cultured in Ham F-12 medium with 10% fetal bovine serum at 37°C under a 5% carbon dioxide (CO2) atmosphere.
Rapamycin from Streptomyces hygroscopicus (Sigma, St. Louis, MO) was obtained in powder. For in vitro experiments, rapamycin was dissolved in 100% ethanol before addition to the cultured cells. Everolimus (Certican) was obtained as 5-mg tablets (Novartis Pharma AG, Basel, Switzerland). For in vitro experiments, everolimus was prepared in dimethyl sulfoxide before addition to the cultured cells.
Cultured CCA cells were treated with dimethyl sulfoxide (vehicle and positive control) or 400-, 800-, and 1200-nmol/L rapamycin and everolimus for 0, 6, 24, or 48 hours. Then, the cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer (20-mmol/L Tris-HCl, 150-mmol/L NaCl, 1-mmol/L EDTA, 1-mmol/L ethylene glycol tetraacetic acid, 1-mmol/L sodium orthovanadate, 1-mmol/L h-glycerophosphate, 2.5-mmol/L sodium pyrophosphate (PPi), 1-mmol/L4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 10-mg/mL aprotinin, 1-mg/mL leupeptin, and 1% Triton X-100) for 10 minutes at 4°C. Lysates were centrifuged at 12,000g at 4°C for 15 minutes, and the protein concentration of each supernatant was determined by using the Bio-Rad protein assay reagent. The suspensions were adjusted to a protein content of 2 μg/μL by lysate buffer (25-mmol/L Tris-HCl (pH 6.5), 5% glycerol, 1% sodium dodecyl sulfate, 0.05% bromophenol blue, and 1% β-mercaptoethanol). Then, SDS-PAGE was done, and a prestained protein marker ladder (Broad Range, Bio-Rad Laboratories, Hercules, CA) was run in the outside lane to confirm the molecular weights of mTOR, p-mTOR phosphoryrated-4E-BP1 (p-4E-BP1), HIF-1α, VEGF-A, EPO, and VHL (Table 1). The samples were then transferred to polyvinylidene fluoride membranes, which were blocked with 3% bovine serum albumin in 4% skim milk for 1 hour at 37°C. After 3 washes with 0.05% Tween 20 in 0.01-mol/L PBS, the membranes were incubated overnight at 4°C with the antibodies shown in Table 1. After 5 washes with 0.05% Tween 20 in 0.01-mol/L PBS, the membranes were incubated with biotin-labeled antimouse and antirabbit IgG (Nichirei Biosciences, Tokyo, Japan) for 1 hour at RT. Then, immunoreactive bands were developed using an enhanced Vectastain ABC kit (Vector Laboratories Inc, Burlingame, CA). The membranes were developed in 3′3-diaminobenzidine solution containing 0.006% hydrogen peroxidase (H2O2) for 3 to 5 minutes at room temperature (RT).
After treatment with everolimus at 400 nmol/L or 800 nmol/L for 24 hours, cytological preparations of cultured CCA cells fixed in 4% paraformaldehyde phosphate buffer solutions were prepared for immunocytochemistry. The endogenous peroxidase was blocked by incubation with 0.3% H2O2 in methanol for 30 minutes at RT. Next, the sections were washed in 0.01-mol/L PBS for 10 minutes and incubated with 4% normal goat serum phosphate buffer solution. For immunocytochemical detection, the sections were incubated overnight at 4°C with anti-mTOR, anti–p-mTOR, anti–HIF-1α, anti-VHL, and anti-Ki-67 (MIB-1) antibodies (Table 1). The sections were then washed and incubated with antimouse/rabbit IgG conjugated to horseradish peroxidase–labeled dextran polymer (EnVision kits, DAKO, Carpinteria, CA) or incubated with an avidin-biotin-peroxidase complex system (Vectastain ABC Elite Peroxidase Kit; Vector Laboratories) for 60 minutes at RT. After being washed 10 times in 0.01-mol/L PBS, the sections were developed in 3′3-diaminobenzidine solution containing 0.006% H2O2 for 3 to 5 minutes at RT. Finally, they were counterstained with hematoxylin before examination. The expressions were semiquantitatively assessed according to the following scoring scheme: negative; weak, 1+ (<10%); intermediate, 2+ (10%-50%); strong 3+ (>50%).
Real-Time Reverse Transcription Polymerase Chain Reaction Analysis
Cultured CCA cells were treated with 400-nmol/L everolimus for 24 hours. Messenger RNA levels were compared between treated and untreated groups. The levels of HIF-1α, VEGF, and VHL mRNA expression were quantitatively assessed by real-time reverse transcription polymerase chain reaction with the ABI Prism 7700 System (TaqMan PCR, Applied Biosystems, Foster City, CA), using assay-on-demand primers and the Hs00153153ml probe for HIF-1α, Hs03929036_s1 probe for VHL, and Hs00184451_m1 probe for VHL. Polymerase chain reaction was initiated at 95°C for 10 minutes, and this was followed by 50 cycles of amplification at 95°C for 15 seconds and 60°C for 1 minute. Human glyceraldehyde-3-phosphate dehydrogenase mRNA was amplified as a control using the Hs99999905ml probe. The relative expression of each mRNA was calculated by the ΔCt method.
Cell Proliferation Assay
The RMG-1 cells were cultured into 96-wells plates at 37°C under 5% CO2. After that, the cells were grown confluent, the medium was removed, and everolimus diluted with the medium (400, 800, and 1200 nmol/L) was added. After exposure for 6 hours, cell growth was evaluated by a colorimetric MTS proliferation assay performed with a CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI) according to the manufacturer’s instructions. After culture, 20 μL of combined MTS/PMS (20:1) reagent was added to the wells, and the cells were incubated for 4 more hours at 37°C under 5% CO2. Then, the absorbance at 490 nm was measured with a plate reader. Wells that contained medium only served as the blanks.
In Vivo Experiment
Everolimus was dissolved at the appropriate concentration in double-distilled water just before administration by gavage. Briplatin (cisplatin) was obtained in 10-mg vials (Bristol-Myers Squibb, Princeton, NJ) and was diluted with isotonic sodium chloride solution just before administration by gavage. TAXOL (taxol) was obtained in 30-mg vials (Bristol-Myers Squibb) and was dissolved in isotonic sodium chloride solution just before administration by gavage.
Administration Test Using a Subcutaneous Xenograft Model
BALB/c nu/nu mice (nude mice) at 6 weeks old were used in this study, which was performed with approval by the Animal Care Committee of the Tokai University for Experiment Animals. The nude mice were subcutaneously inoculated into the right flank with 1 × 107 RMG-1 cells in 200 μL of PBS. When the tumors reached approximately 100 mm3 in volume, the mice were assigned to 4 treatment groups of 6 animals each. The first group was treated with placebo, the second group was treated with everolimus (5 mg/kg), the third group was treated with TP (taxol at 12 mg/kg and cisplatin at 4 mg/kg), and the fourth group was treated with everolimus (5 mg/kg) + TP (taxol at 12 mg/kg and cisplatin at 4 mg/kg). Each treatment was given for 1 week. Both everolimus and placebo were administered orally, whereas taxol and cisplatin were given intravenously.
Quantitative data are presented as mean ± SD. Analyses were done using SPSS software version 21.0 for Windows.
- Association of HIF-1α and p-mTOR expression and prognosis (Fig. 1). Investigation of the correlation between HIF-1α positivity and the prognosis showed a relatively worse prognosis for nuclear-positive cases (data shown in detail in Miyazawa et al10). In addition, strong expression of p-mTOR was associated with a poor prognosis. However, no statistically significant difference of the prognosis was observed in both types of cancer.
- Expression of mTOR/HIF-1α and cofactors after exposure to mTOR inhibitors (rapamycin and everolimus; Fig. 2). Using Western blotting, no changes were observed in the expressions of Akt and phospho-Akt upstream of mTOR, or expression of mTOR itself, after the addition of both mTOR inhibitors, but there was a significant concentration-dependent decrease in the p-mTOR and the expression of the following downstream factors: p-4E-BP1, HIF-1α, VEGF, and EPO. In contrast, the expression of VHL increased in a concentration-dependent manner by the mTOR inhibitors. Similarly, the results of immunohistochemistry showed concentration-dependent changes after exposure to the mTOR inhibitors.
- Changes of mRNA for HIF-1α and cofactor after exposure to mTOR inhibitor (everolimus) (Fig. 3). A significant decrease in the expression of HIF-1α (0.42) and the downstream VEGF-A (0.34) was observed in the cells treated with the mTOR inhibitor (P < 0.05). In contrast, the expression of VHL mRNA increased (1.24), as was VHL protein expression.
- Effect of mTOR inhibitor (everolimus) on cell proliferation (Fig. 4). MTS assay showed a significant suppression of proliferation in a concentration-dependent manner (control, 1.00; everolimus [400 nmol/L], 0.66; everolimus [800 nmol/L], 0.60; everolimus [1200 nmol/L], 0.59). In addition, analysis of the Ki-67 labeling index revealed a significant decrease in the expression of Ki-67–positive cells after exposure to everolimus (control, 1.000; everolimus [400 nmol/L], 0.007; everolimus [800 nmol/L], 0.004).
- In vivo therapeutic effect of mTOR inhibitor (everolimus; Fig. 5). Apoptosis and necrosis were detected around the center of the tumors in the everolimus treated group rather than in the placebo group, and there was a decrease in tumor size. In the assessment of combination therapy with other antitumor agents, combined treatment with everolimus and TP caused a significant decrease in tumor size compared with the everolimus group and the TP group.
Our previous studies have revealed increased expression of HIF-1α in patients with ovarian CCA compared to those with other epithelial ovarian cancers8,9 and have identified a relationship between increased expression of HIF-1α and the mTOR/HIF pathway.10 Furthermore, the cases in which p-mTOR was overexpressed and HIF-1α expression was observed predominantly in the nucleus showed the tendency of poor prognosis (Fig. 1). To establish new treatments for CCA, the present study focused on the mTOR inhibitor, and we analyzed the expression of factors related to the mTOR/HIF pathway to clarify the effects of mTOR inhibitors. We also investigated the effect of the mTOR inhibitor on cell proliferation and analyzed its antitumor activity in the animal model.
The expression of mTOR/HIF pathway factors was assessed in the cultured CCA cells after treatment with mTOR inhibitors. We found no differences in the expression of upstream factors (Akt and phospho-Akt) or mTOR itself between the mTOR inhibitor-treated and untreated cultures. Therefore, it is considered that mTOR inhibitors might suppress downstream effectors by inhibiting the phosphorylation of mTOR. Immunocytochemical study also showed a significant decrease of p-mTOR and HIF-1α expressions in the mTOR inhibitor group compared to the untreated control group. In the analysis of expression of mRNAs for these factors, a significant decrease in the expression of HIF-1α and VEGF mRNA was found. These results suggest that the Akt-mTOR-HIF-1 pathway is activated in CCA. Accordingly, we considered that the mTOR inhibitors suppressed phosphorylation of the downstream effector 4E-BP1 in association with inhibition in the phosphorylation of mTOR and the translation of HIF-1α mRNA, resulting in decreased expression of HIF-1α and cofactors such as VEGF-A and EPO. There are several lines of evidence regarding the antitumor effects of mTOR inhibitors, and their antitumor activity is considered to be partially related to the suppression of tumor cell proliferation through suppression of HIF-1α and to inhibition of angiogenesis by suppression of VEGF.
Analysis of VHL, as playing a role of HIF-1α ubiquitin ligase, showed almost no expression in the untreated group, indicating that genetic abnormalities of VHL exist in the patients with CCA. Because mutation and deletion of the VHL gene were detected in 58% of the patients with CCA and renal clear cell carcinoma,17 it is assumed that abnormalities of VHL could have caused overexpression of HIF-1α leading to up-regulation of the proliferative factors (VEGF and EPO) via HIF-1. Based on these assumptions, it was considered that increased phosphorylation of mTOR and VHL genetic abnormalities caused overexpression of HIF-1α in the patients with CCA.
Interestingly, comparison of the Ki-67 labeling index data revealed a lower mean value in patients with CCA (18.4%) than in those with serous adenocarcinoma (38.8%), and the survival rate of patients with a Ki-67 labeling index less than 18.4% was previously reported to be low.18 Thus, mTOR inhibitors are considered to inhibit proteins that are important for driving the cell cycle forward by blocking the translation of various proteins (including cyclin) to arrest the cell cycle at the G1 checkpoint.19 In fact, by the analysis of MTS assay, a cell proliferation was significantly inhibited, depending on the concentration of everolimus (P < 0.0001). In addition, the Ki-67 labeling index significantly decreased when added with everolimus 800 nmol/L (P < 0.0001). Our results suggested that the everolimus has strong cytostatic effect.
To explore the potential for clinical application, we also evaluated the antitumor effect of everolimus in a mouse model of ovarian CCA. As a result, apoptosis and necrosis were detected at the center of tumors, and the treated tumors were smaller than the control tumors (Fig. 5A). In the study of combination therapy with other antitumor drugs, the combination of everolimus and TP caused a significant decrease in tumor size in comparison with everolimus or TP alone (Fig. 5B). Mabuchi at el20–22 reported a study of ovarian cancer in transgenic mice with MOVACR cell lines, which indicated a suppressive effect on tumor proliferation after the administration of everolimus. We previously analyzed the expression of cleaved caspase-3 among various apoptosis-related factors, and our in vitro study showed no expression of cleaved caspase-3 in the untreated group.23 Inhibition of mTOR is considered to have led to cell death and apoptosis. These results suggest that everolimus not only inhibits mTOR signaling but also promotes VHL expression and that everolimus exhibits antitumor activity through inhibition of HIF-1α expression.23 Further analysis of VHL expression could lead to the identification of a new mediator in the intracellular signaling system.
The efficacy of everolimus has primarily been studied in patients with renal cell carcinoma. The results of a global phase 3 study conducted in 2008 showed that the tumor was reduced in size after treatment with everolimus in 58% of patients versus 13% of the placebo group.24 Based on these results, everolimus was approved for use in Japan in January 2010. As the efficacy of everolimus for renal cell carcinoma was demonstrated by Amato et al,25 this drug is also expected to be effective for CCA.
At present, the Gynecologic Oncology Group of the United States has suggested the efficacy of adding the mTOR inhibitor (temsirolimus) as combination chemotherapy and maintenance therapy for patients with CCA at stage III/IV. The Gynecologic Oncology Group of Japan is also initiating a clinical study of everolimus for CCA. We would like to propose the use of p-mTOR as a potential biomarker in the clinical practice. However, additional and profound research to explore the biomarkers including VHL for selecting the therapeutic regimens needs to be done.
1. Sugiyama T, Kamura T, Kigawa J, et al. Clinical characteristics of clear cell carcinoma of the ovary
: a distinct histologic type with poor prognosis and resistance to platinum-based chemotherapy. Cancer
. 2000; 88: 2584–2589.
2. Vasey PA, Jayson GC, Gordon A, et al. Phase III randomized trial of docetaxel-carboplatin versus paclitaxel-carboplatin as first-line chemotherapy for ovarian carcinoma. J Natl Cancer Inst
. 2004; 96: 1682–1691.
3. Simsir A, Palacios D, Linehan WM, et al. Detection of loss of heterozygosity at chromosome 3p25-26 in primary and metastatic ovarian clear-cell carcinoma: utilization of microdissection and polymerase chain reaction in archival tissues. Diagn Cytopathol
. 2001; 24: 328–332.
4. Osada R, Horiuchi A, Kikuchi N, et al. Expression of hypoxia-inducible factor 1alpha, hypoxia-inducible factor 2alpha, and von Hippel-Lindau protein in epithelial ovarian neoplasms and allelic loss of von Hippel-Lindau gene: nuclear expression of hypoxia-inducible factor 1alpha is an independent prognostic factor in ovarian carcinoma. Hum Pathol
. 2007; 38: 1310–1320.
5. Semenza GL, Wang GL. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol
. 1992; 12: 5447–5454.
6. Jaakkola P, Mole DR, Tian YM, et al. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2
-regulated prolyl hydroxylation. Science
. 2001; 292: 468–472.
7. Kato H, Tamamizu-Kato S, Shibasaki F. Histone deacetylase 7 associates with hypoxia-inducible factor 1 alpha and increases transcriptional activity. J Biol Chem
. 2004; 279: 41966–41974.
8. Miyazawa M, Yasuda M, Fujita M, et al. Association of hypoxia-inducible factor-1 expression with histology in epithelial ovarian tumors: a quantitative analysis of HIF-1. Arch Gynecol Obstet
. 2009; 279: 789–796.
9. Yasuda M, Miyazawa M, Fujita M, et al. Expression of hypoxia inducible factor-1alpha (HIF-1alpha) and glucose transporter-1 (GLUT-1) in ovarian adenocarcinomas: difference in hypoxic status depending on histological character. Oncol Rep
. 2008; 19: 111–116.
10. Miyazawa M, Yasuda M, Fujita M, et al. Therapeutic strategy targeting the mTOR-HIF-1 alpha-VEGF pathway in ovarian clear cell adenocarcinoma
. Pathol Int
. 2009; 59: 19–27.
11. Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell
. 2012; 149: 274–293.
12. Johnson SM, Gulhati P, Rampy BA, et al. Novel expression patterns of PI3K/Akt/mTOR signaling pathway components in colorectal cancer. J Am Coll Surg
. 2010; 210: 767–776, 776–778.
13. Trinh XB, Tjalma WA, Vermeulen PB, et al. The VEGF pathway and the AKT/mTOR/p70S6K1 signalling pathway in human epithelial ovarian cancer. Br J Cancer
. 2009; 100: 971–978.
14. Clemens MJ. Translational regulation in cell stress and apoptosis. Roles of the eIF4E binding proteins. J Cell Mol Med
. 2001; 5: 221–239.
15. Pore N, Jiang Z, Shu HK, et al. Akt1 activation can augment hypoxia-inducible factor-1 alpha expression by increasing protein translation through a mammalian target of rapamycin-independent pathway. Mol Cancer Res
. 2006; 4: 471–479.
16. Mills GB, Lu Y, Fang X, et al. The role of genetic abnormalities of PTEN and the phosphatidylinositol 3-kinase pathway in breast and ovarian tumorigenesis, prognosis, and therapy. Semin Oncol
. 2001; 28: 125–141.
17. Lin F, Shi J, Liu H, et al. Immunohistochemical detection of the von Hippel-Lindau gene product (pVHL) in human tissues and tumors: a useful marker for metastatic renal cell carcinoma and clear cell carcinoma of the ovary
and uterus. Am J Clin Pathol
. 2008; 129: 592–605.
18. Itamochi H, Kigawa J, Sugiyama T, et al. Low proliferation activity may be associated with chemoresistance in clear cell carcinoma of the ovary
. Obstet Gynecol
. 2002; 100: 281–287.
19. Seufferlein T, Rozengurt E. Rapamycin inhibits constitutive p70s6k phosphorylation, cell proliferation, and colony formation in small cell lung cancer cells. Cancer Res
. 1996; 56: 3895–2897.
20. Mabuchi S, Altomare DA, Connolly DC, et al. RAD001 (Everolimus) delays tumor onset and progression in a transgenic mouse model of ovarian cancer. Cancer Res
. 2007; 67: 2408–2413.
21. Mabuchi S, Altomare DA, Cheung M, et al. RAD001 inhibits human ovarian cancer cell proliferation, enhances cisplatin-induced apoptosis, and prolongs survival in an ovarian cancer model. Clin Cancer Res
. 2007; 13: 4261–4270.
22. Mabuchi S, Kawase C, Altomare DA, et al. mTOR is a promising therapeutic target both in cisplatin-sensitive and cisplatin-resistant clear cell carcinoma of the ovary
. Clin Cancer Res
. 2009; 15: 5404–5413.
23. Harasawa M, Yasuda M, Hirasawa T, et al. Analysis of mTOR inhibition-involved pathway in ovarian clear cell adenocarcinoma
. Acta Histochem Cytochem
. 2011; 44: 113–118.
24. Motzer RJ, Escudier B, Oudard S, et al. Efficacy of everolimus in advanced renal cell carcinoma: a double-blind, randomised, placebo-controlled phase III trial. Lancet
. 2008; 372: 449–456.
25. Amato RJ, Jac J, Giessinger S, et al. A phase 2 study with a daily regimen of the oral mTOR inhibitor
RAD001 (everolimus) in patients with metastatic clear cell renal cell cancer. Cancer
. 2009; 115: 2438–2446.
Keywords:© 2013 by the International Gynecologic Cancer Society and the European Society of Gynaecological Oncology.
Ovary; Clear cell adenocarcinoma; mTOR inhibitor; HIF-1α