Akiko Kudoh

The PI3K/mTOR dual inhibitor NVP-BEZ235 reduces the growth of ovarian clear cell carcinoma

Patients with clear cell carcinoma of the ovary (OCCC) have poor survival due to resistance to standard chemotherapy. OCCC has frequent activating mutations of the PIK3CA gene. The present study was conducted to clarify the efficacy of the inhibition of the PI3K-AKT-mTOR pathway in OCCC. We used 8 OCCC cell lines and 5 ovarian serous adenocarcinoma (OSAC) cell lines. The mutation status of the PIK3CA and KRAS genes was examined by direct sequencing. The IC50 values of NVP-BEZ235 (BEZ235) and temsirolimus were determined by WST-8 assay. Protein expression levels of PI3K-AKT-mTOR pathway molecules were examined by western blotting. Cell cycle distribution was analyzed by flow cytometry. Annexin V staining was used for detecting apoptosis. We also investigated the effects of BEZ235 on OCCC tumor growth in a nude mouse xenograft model. Four of the 8 OCCC cell lines showed a PIK3CA mutation while none of the 5 OSAC cell lines showed a mutation. The IC50 values of BEZ235 for the OCCC cell lines were lower than these values for the OSAC cell lines. The IC50 value of temsirolimus was higher than BEZ235 in the OCCC cell lines. The PIK3CA mutation was more frequently noted in OCCC than OSAC cells, but the sensitivity of these cell lines to BEZ235 or temsirolimus was not related to the mutation status. pHER3 and pAkt proteins were expressed more frequently in OCCC compared with OSAC. However, protein expression levels were distributed widely, and were not related to the sensitivity. Treatment with BEZ235 suppressed expression of pAkt, although treatment with temsirolimus did not. OCCC cells exhibited G1 phase arrest after treatment with BEZ235 and apoptosis with a higher concentration of the agent. BEZ235 significantly inhibited tumor growth in mice bearing OVISE and TU-OC-1 cell tumors. The present study indicated that the PI3K-AKT-mTOR pathway is a potential target for OCCC, and that BEZ235 warrants investigation as a therapeutic agent.

Abstract 3256: A novel therapy for mucinous adenocarcinoma of the ovary by using NVP-BEZ235 to inhibit PI3K and mTOR.

Objective: Mucinous adenocarcinoma of the ovary (MAC) has a poor prognosis. It resists conventional chemotherapy based on platinum or taxane. Recent molecular analyses of various types of ovarian tumors showed that overexpressed epidermal growth factor receptor (EGFR) family proteins and consequent activation of its downstream signaling Akt / mammalian target of rapamycin (mTOR) are found in 48% of tumors. These are associated with poor patients outcomes. We explored molecular-targeted therapy with NVP-BEZ235 (BEZ235), a dual inhibitor of phosphatidylinositol 3-kinase (PI3K) and mTOR, to treat MAC. Method: Seven cell lines of MAC (JHOM-1, JHOM-2B, MCAS, OMC-1, RMUG-L, RMUG-S, and TU-OM-1) were used. The sensitivity of these cells to BEZ235 was determined with the WST-8 assay. Mutation status of EGFR, KRAS, and PIK3CA genes was analyzed by direct sequencing. Cell cycle distribution was assessed by flow cytometry, and the expression of proteins in apoptotic pathways and molecules downstream of the PI3K/Akt/mTOR signaling pathways were determined by western blot analysis. We also examined the effects of BEZ235 on tumor growth in xenograft models with nude mice. Mice bearing subcutaneous tumors of RMUG-S or OMC-1 received the vehicle or BEZ235 (25 or 50 mg/kg/day) for 3 weeks. Tumor volume was measured using a caliper twice weekly. Results: The half-maximal inhibitory concentration (IC 50 ) values of each cell line ranged from 18 to 618 nM for BEZ235. All lines exhibited protein expressions of the PI3K/Akt/mTOR signaling pathways, but had no mutations in EGFR and PIK3CA genes. A point mutation of the KRAS gene at codon 12 in exon 2 was observed only in the MCAS cell line. The protein expression levels of phosphorylated (p) Akt, pp70S6K and p4E-BP1 were suppressed after exposure to BEZ235 in a dose-dependent manner. After treatment with BEZ235, the proportions of the cells in the G0/G1 phase increased, and the proportion in the S phase fraction decreased. Moreover, 24 h after treatment with BEZ235, the protein expression of cleaved PARP and cleaved caspase-9 were upregulated in both RMUG-S and TU-OM-1 cells. BEZ235 increased the number of apoptotic cells in both RMUG-S and TU-OM-1. Treatment of mice bearing OMC-1 or RMUG-S with BEZ235 at 25 or 50 mg/kg/day doses significantly suppressed tumor growth in xenograft models without severe weight loss. Conclusion: The PI3K/Akt/mTOR pathway is a potential therapeutic target for MAC using BEZ235 as a therapeutic agent. Citation Format: Akiko Kudoh, Tetsuro Oishi, Hiroaki Itamochi, Michiko Nonaka, Seiya Sato, Jun Naniwa, Shinya Sato, Muneaki Shimada, Naoki Terakawa, Junzo Kigawa, Tasuku Harada. A novel therapy for mucinous adenocarcinoma of the ovary by using NVP-BEZ235 to inhibit PI3K and mTOR. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3256. doi:10.1158/1538-7445.AM2013-3256

Recurrent colon perforation after discontinuation of bevacizumab for ovarian cancer

Bevacizumab (Bev) is an antiangiogenic drug used to treat various malignances, including ovarian cancer (OC). Bev is generally well-tolerated; however, it has a characteristic toxicity profile. In particular, gastrointestinal perforation (GIP) is a rare but serious side effect that can be lethal. A 55-year-old woman with recurrent OC had an episode of GIP during third-line chemotherapy comprising Bev and topotecan (TPT). Bev was discontinued while TPT was continued as monotherapy. Three months after discontinuation of Bev, the patient presented with left lower abdominal pain and was diagnosed with a second GIP. She had emergent surgery. One year later, she is still alive and healthy, and is continuing TPT. This is the first report of recurrent GIP after discontinuation of Bev. Our case suggests that physicians should be aware of GIP even after the discontinuation of Bev.

Significance of High-Risk Human Papillomavirus Testing for Atypical Glandular Cells on Cervical Cytology

Objectives: The aim of this study was to evaluate the diagnostic significance of high-risk human papillomavirus (hrHPV) testing for managing women with atypical glandular cells (AGC) and to explore the distribution of hrHPV genotypes. Methods: We analyzed cytologic and histopathologic diagnoses in patients referred to our institution due to AGC or atypical squamous cells of undetermined significance (ASC-US). All patients underwent hrHPV testing and genotyping, and positive (PPV) and negative predictive values (NPV) for cervical intraepithelial neoplasia (CIN) 2 or worse [CIN2+/adenocarcinoma in situ (AIS)+] were calculated. Results: Among 41 cases previously diagnosed with AGC, 22 (53%) were classified as CIN2+ (2 squamous cell carcinomas), whereas only 2 were AIS or adenocarcinoma. Twenty-seven (65.8%) cases in the AGC group were hrHPV positive. The most frequent genotypes in both the ASC-US and AGC groups were HPV16 and HPV52. The PPV of hrHPV testing for CIN2+/AIS+ was significantly higher in the AGC than in the ASC-US group (74.1 vs. 35.0%; p = 0.0005). The NPV for CIN2+/AIS+ was significantly lower in the AGC than in the ASC-US group (74.4 vs. 100%; p = 0.0441). Conclusion: In patients with AGC, both glandular and squamous lesions must be monitored. hrHPV testing is useful for detecting CIN2+/AIS+ in AGC.

Recurrent spontaneous pneumothorax following chemotherapy in a patient with ovarian granulosa cell tumor

Spontaneous pneumothorax (SP) associated with malignant disease is well known but rare. Diagnosing its etiology is important for therapy. We describe SP that recurred after chemotherapy in a patient with granulosa cell tumor. An SP supposedly developed secondary to regression of pulmonary metastasis and resulted in a bronchopleural fistula. The patient recovered after draining with a chest tube and pleurodesis. Chemotherapy was continued without further complications. We discuss several mechanisms for, and the clinical association of, the SP with metastasis in a granulosa cell tumor of the ovary.

Abstract 5298: Serum angiogenic factors may predict prognosis in patients with epithelial ovarian cancer

Objective: Angiogenesis is one of the processes that is critical for the growth, invasion and metastasis of solid tumors, including epithelial ovarian cancer (EOC). The vascular endothelial growth factor (VEGF) family is one of the major pathways involved in tumor angiogenesis, and VEGF expression is activated by hypoxia-inducible factor (HIF)-1α. The aim of this study was to determine whether serum levels of these angiogenic factors could be used as prognostic biomarkers in patients with EOC. Methods: A total of 133 patients with EOC who were treated at Tottori University Hospital between 2006 and 2012 were enrolled in this study. The study was approved by the Institutional Review Board of the School of Medicine of Tottori University. All patients gave written informed consent before the collection of specimens according to institutional guidelines. Serum samples were collected before initial surgery and levels of VEGF-A, -C and HIF-1α were analyzed by enzyme-linked immunosorbent assay (ELISA). We evaluated the association between the levels of VEGF-A, -C, and HIF-1α and clinicopathologic variables. We also determined the protein expression of VEGF-A, -C, and HIF-1α by immunohistochemical staining in paraffin-embedded tumors from EOC patients Results: The protein expressions of VEGF-A, -C, and HIF-1α were strongly observed in the cancer cells. Median levels of serum VEGF-A, -C and HIF-1α were 280, 4866 and 125 pg/ml, respectively. The levels of VEGF-A in patients at stage IV were significantly higher than those in patients at stage I. Both histologic subtypes and lymph node involvement were not related to levels of these angiogenic factors. We found a significant positive correlation between VEGF-A levels and the ascitic volume. The overall survival rate was significantly lower in FIGO stage III or IV patients with EOC. We set the cutoff value of these factors at the median levels of each angiogenic factors. The 5-year survival rate for patients with high VEGF-A levels was significantly lower than those with low levels (61.7% vs. 41.8%, P = 0.0147). Both HIF-1α and VEGF-C levels were not related to outcome of patients. Multivariate analysis revealed that serum VEGF-A level and FIGO stage were independent prognostic factors. Conclusion: These results suggest that serum VEGF-A may be a promising prognostic biomarker for EOC. Citation Format: HIROAKI KOMATSU, Tetsuro Oishi, Hiroaki Itamochi, Akiko Kudoh, Michiko Nonaka, Seiya Sato, Jun Chikumi, Shinya Sato, Muneaki Shimada, Junzo Kigawa, Tasuku Harada. Serum angiogenic factors may predict prognosis in patients with epithelial ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5298. doi:10.1158/1538-7445.AM2015-5298

Abstract 3740: NVP-BEZ235, a dual PI3K/ mTOR inhibitor, suppress tumorigenicity of ovarian clear cell carcinoma cells.

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 1 [Background] Ovarian clear cell carcinoma (CCC) has shown the resistance to the standard chemotherapy for epithelial ovarian cancer (EOC). Therefore, patients with CCC have a worse prognosis than those with ovarian serous adenocarcinoma (SAC), which is a dominant subtype in EOC. Recently, it has shown that CCC has a high frequency of activating mutations of PIK3CA. Thus, PI3K-Akt pathways may be an attractive target of therapy. NVP-BEZ235 (BEZ235) is a novel, orally bioavailable imidazoquinoline that potently and reversibly inhibits class 1 PI3K activity. BEZ235 also binds directly to the mTOR ATP-binding domain and directly inhibits its catalytic activity, thereby inhibiting both mTOR complex 1 (mTORC1) and mTORC2. There are few reports on the activity of BEZ235 against ovarian carcinoma cells. The purpose of the present study was to determine whether the drug targeting the PI3K-Akt-mTOR pathway suppresses cell proliferation and tumorigenicity of CCC. [Material& Methods] We used eight CCC cell lines (OVISE, SMOV-2, KK, TU-OC-1, OVTOKO, KOC-7c, OVMANA, and RMG-I) and five SAC cell lines (KOC-2s, KF, TU-OS-3, TU-OS-4, SHIN-3). IC50 to BEZ235 was determined on the basis of the dose-effect curves, using WST-8 assay. Protein expressions of Akt, phospho(p)-Akt, mTOR, pmTOR, p70S6K, pp70S6K, 4E-BP1, and p4E-BP1 were examined by western blotting. Cell cycle distribution was analyzed by flow cytometry. Apoptotic cells were assessed by Annexin V-FITC/PI double staining. We also investigated the effects of BEZ235 on tumor growth in a nude mice xenograft model. Mice bearing subcutaneous tumor of OVISE were received 3-weeks treatment with vehicle or BEZ235 (25mg/kg/day or 50mg/kg/day). The two perpendicular axes of the tumor diameter was measured in control and treated groups using a caliper twice weekly, and tumor volume was calculated, using the following formula: length x (width)*2 x (≥/6). [Results] IC50 to BEZ235 for CCC cell lines ranged from 44 to 777 nM (median:332.5nM) and those for SAC cell lines ranged from 779 to 25400 nM (median: 1004nM). We confirmed that all CCC cell lines exhibited protein expressions of pAkt and pmTOR. pAkt, p-p70S6K and p4E-BP1 expressions in CCC cells (OVISE and KK) were suppressed after exposure to BEZ235 in a dose-dependent manner. The both cell lines exhibited G1 cell cycle arrest after exposure to 10 or 100nM BEZ235, and apoptotic cells were induced by higher concentrations of the agent. Finally, treatment of the mice with BEZ235 at doses 25mg/kg/day or 50mg/kg/day significantly suppressed tumor growth in a xenograft model. [Conclusion] These results suggest that the PI3K-Akt-mTOR pathway is a potential therapeutic target for CCC and that NVP-BEZ235 is worth exploring as a therapeutic agent for CCC. 1 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3740. doi:1538-7445.AM2012-3740