Michiko Nonaka

Combination chemotherapy of oxaliplatin and 5-fluorouracil may be an effective regimen for mucinous adenocarcinoma of the ovary: A potential treatment strategy

Resistance of ovarian mucinous adenocarcinoma to standard chemotherapy with paclitaxel and carboplatin is associated with poor prognosis, and an effective treatment is needed. The present study aimed to identify an effective chemotherapy for ovarian mucinous adenocarcinoma. Five human ovarian mucinous adenocarcinoma cell lines (MN‐1, OMC‐1, RMUG‐L, RMUG‐S, TU‐OM‐1) were used in this study. Sensitivity of the cells to the anticancer agents was determined by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay, and we assessed drug sensitivity by calculating the assay area under the curve for each agent. Protein expression was confirmed by Western blot analysis. We also examined the efficacy of combination chemotherapy on survival in a xenograft model of nude mice. The IC50 to anticancer agents ranged widely. The assay area under the curve indicated that two of five cell lines (MN‐1, TU‐OM‐1) were sensitive to oxaliplatin, 5‐fluorouracil and etoposide, and only one (TU‐OM‐1) was sensitive to 7‐ethyl‐10‐hydroxycamptothecin, which is an active metabolite of camptothecin. All cell lines were resistant to cisplatin and paclitaxel. The combination of oxaliplatin and 5‐fluorouracil resulted in additive or synergistic effects on all cell lines. The combination of oxaliplatin and 5‐fluorouracil significantly prolonged survival in a ovarian mucinous adenocarcinoma xenograft model of nude mice. Protein expression levels of the excision repair cross‐complementation group 1 were lower in oxaliplatin sensitive cell lines. Exposure to 5‐fluorouracil down‐regulated cross‐complementation group 1 expression in ovarian mucinous adenocarcinoma cells. We conclude that combination chemotherapy consisting of oxaliplatin and 5‐fluorouracil was an effective treatment for ovarian mucinous adenocarcinoma and may be a pivotal candidate for a novel treatment strategy. (Cancer Sci 2009; 100: 546–551)

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.

Preoperative and intraoperative assessments of depth of myometrial invasion in endometrial cancer.

Objective: Preoperative and intraoperative assessments of myometrial invasion (MI) are commonly used for planning surgical procedures such as dissection of the para-aortic node; however, the assessments often differ from the final diagnosis determined by pathological examination. The present study evaluated the accuracy of preoperative and intraoperative assessments of MI. Methods: A total of 191 patients with endometrial cancer, who underwent hysterectomy from 1995 to 2007 in Tottori University Hospital, were included in this study. One hundred seventy-four patients underwent endometrial curettage or Pipelle biopsy preoperatively. Histological grade was compared between preoperation and postoperation. Magnetic resonance imaging (MRI) was performed before surgery, and the depth of MI was assessed as 3 levels (no MI, <50%, and >50%). During surgery, the uterine wall was incised at the most invasive part, and then, intraoperative gross assessment was evaluated as less than or greater than 50%. Results: Histological evaluation revealed that 34 patients had no invasion, 97 had less than 50% MI, and 60 had greater than 50% MI. On MRI assessment, 135 patients had correct diagnoses, and the accuracy was 70.7%. Regarding the diagnosis of greater than 50% MI depth, the accuracy, the sensitivity, and the specificity of the MRI assessment were 83.2%, 75.0%, and 85.7%, respectively. Seventeen patients were overestimated, and 15 patients were underestimated by the MRI assessment. On intraoperative gross assessment, 162 patients had correct diagnoses, 8 patients were overestimated, and the remaining 21 patients were underestimated. The accuracy of the gross assessment was 84.8%, the sensitivity was 65.0%, and the specificity was 93.9%. The preoperative grading accuracy was 71.8% (125/174). A discrepancy between preoperative and postoperative grades was more frequent in a low-grade tumor. The incidence of underdiagnosis was significantly higher in patients with a grade 3 (G3) tumor than in those with a G1 or G2 tumor in both assessments. Conclusions: The present study suggests that gross assessment may be useful to determine MI of less than 50%, although patients with a G3 tumor were more frequently underestimated.

Activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway overcomes cisplatin resistance in ovarian carcinoma cells.

Objective This study was aimed to elucidate the roles of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3′-kinase (PI3K)/Akt pathways in regulating cytotoxicity induced by cisplatin (CDDP) in ovarian carcinoma cells. Methods We treated 7 ovarian cancer cell lines with CDDP alone or with CDDP and either a PI3K inhibitor (LY294002), a MEK inhibitor (PD98059), or a MEK/ERK activator (phorbol 12-myristate 13-acetate [PMA]) and assessed cell viability, expression of MEK/ERK and PI3K/Akt, cell cycle distribution, and apoptosis. We also investigated the effect of combination treatment on survival in a xenograft model. Results The cell lines showed half-maximal inhibitory concentrations (IC50) of CDDP from 2.4 to 26.9 µmol/L. KFr, a CDDP-resistant cell line developed from KF cells, showed an IC50 of CDDP of 9.6 µmol/L. Five of the cell lines with IC50 values of 9.6 µmol/L or greater were defined as CDDP-resistant. Cisplatin and LY294002 had an additive effect on inhibiting cell growth, and CDDP and PD98059 had and antagonistic effect on cell growth in all cell lines. In CDDP-resistant cells, CDDP and PMA dramatically suppressed the cell growth, up-regulated the expression of phosphorylated ERK and cleaved caspase-9, down-regulated the expression of checkpoint kinases, and increased the proportion of cells in the synthesis-phase fraction and apoptotic cells. The treatment of nude mice with CDDP and PMA prolonged survival in an ovarian cancer xenograft model. Conclusions The present study indicates that further study is warranted to determine the effectiveness of combination treatment with CDDP and PMA for platinum-resistant ovarian carcinoma.

Serum Vascular Endothelial Growth Factor-a as a Prognostic Biomarker for Epithelial Ovarian Cancer

Background Bevacizumab, which targets vascular endothelial growth factor (VEGF)-A, has recently been proven to be effective for the treatment of epithelial ovarian cancer (EOC). Thus, interest in VEGF-A has increased. There are few reports on concomitant detection of both ligands and its soluble receptors in serum samples, and the significance of serum VEGF-A in EOC is unclear, unlike the situation with tissue samples. We conducted the present study to explore the levels of serum VEGF family and its receptors and to evaluate their utility as prognostic biomarkers. Methods A total of 128 patients with EOC, who were consecutively treated at Tottori University Hospital between 2006 and 2012, were included. Blood samples were collected before initial surgery. Serum concentrations of VEGF-A, VEGF-C, VEGFR-1, and VEGFR-2 were analyzed by enzyme-linked immunosorbent assay. We also examined the mRNA and protein expression of VEGF-A in tumor tissue from 30 cases by real-time reverse transcription polymerase chain reaction and immunohistochemistry. Results The levels of VEGF-A in patients with stage III/IV disease were significantly higher than those with stage I/II disease (P = 0.0036). On the other hand, the level of VEGFR-2 in stage III/IV was significantly lower than that in stage I/II (P = 0.0026). With the cutoff value of VEGF/VEGFRs at the median level, the overall survival (OS) for patients with high VEGF-A levels was significantly lower than those with low levels (P = 0.015). Patients with high VEGFR-2 levels showed better prognosis than those with low VEGFR-2 levels (P = 0.023). Multivariate analysis revealed that International Federation of Gynecology and Obstetrics stage and serum VEGF-A were independent prognostic factors for OS [hazard ratio 2.01, 95% confidence interval (1.13–3.63), P = 0.017]. There was no significant correlation between mRNA or protein expression and serum levels of VEGF-A. Conclusions Serum VEGF-A is an independent prognostic factor for OS in patients with EOC, implying that serum VEGF-A is a prognostic biomarker for EOC. Further study to validate the data is needed.

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.

Abstract 3386: Serum VEGF-A may predict prognosis in patients with uterine cervical cancer

Objective: Angiogenesis is one of the processes that is critical for the growth, invasion and metastasis of solid tumors, including uterine cervical cancer (CC). The vascular endothelial growth factor (VEGF) family is one of the major pathways involved in tumor angiogenesis. The aim of this study was to determine whether serum levels of these angiogenic factors could be used as biomarkers in patients with CC. Methods: A total of 115 patients with International Federation of Gynecology and Obstetrics (FIGO) stage IB to IIB CC who were treated at Tottori University Hospital between 2006 and 2015 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 VEGFR1 were analyzed by enzyme-linked immunosorbent assay (ELISA). We evaluated the association between the levels of VEGF-A, -C, and VEGFR1 and clinicopathologic variables. Survival analysis was performed with 86 patients treated between 2006 and 2013. We also determined the mRNA expression VEGF-A, -C, and VEGFR1 by real-time RT-PCR in fresh frozen tumors and the protein expression by immunohistochemical staining in paraffin-embedded tumors from CC patients. Results: The mRNA and protein expressions of VEGF-A, -C, and VEGFR1 were strongly observed in the cancer cells. Median levels of serum VEGF-A, -C and VEGFR1 were 313, 8404 and 67.2 pg/ml, respectively. The levels of VEGF-A in patients with lymph node involvement were significantly higher than those in patients without it. On the contrary, the levels of VEGFR1 in patients with lymph node involvement were significantly lower than those in patients without it. Both histological types and FIGO stage were not related to levels of these angiogenic factors. We found a significant positive correlation between VEGF-A levels and the maximum tumor diameter. There was a significant negative correlation between VEGFR1 levels and the maximum tumor diameter. The overall survival rate was significantly lower in FIGO stage IIB than stage IB- IIA patients. 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 (92.9% vs. 72.8%, P = 0.014). Both VEGFR1 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 CC. Citation Format: Mayumi Sawada, Tetsuro Oishi, Hiroaki Komatsu, Hiroaki Itamochi, Michiko Nonaka, Seitya Sato, Jun Chikumi, Sinya Sato, Sinya Sato, Muneaki Shimada, Tasuku Harada. Serum VEGF-A may predict prognosis in patients with uterine cervical cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3386.

Abstract 3387: Serum VEGF-A 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. The aim of this study was to determine whether serum levels of these angiogenic factors could be used as biomarkers in patients with EOC. Methods: A total of 133 patients with EOC who were treated at Tottori University Hospital between 2006 and 2014 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 VEGFR1 were analyzed by enzyme-linked immunosorbent assay (ELISA). We evaluated the association between the levels of VEGF-A, -C, and VEGFR1 and clinicopathologic variables. We also determined the mRNA expression VEGF-A, -C, and VEGFR1 by real-time RT-PCR in fresh frozen tumors and the protein expression by immunohistochemical staining in paraffin-embedded tumors from EOC patients. Additionally, serum samples were collected before and after bevacizumab treatment, and levels of angiogenic factors were examined. Results: The mRNA and protein expressions of VEGF-A, -C, and VEGFR1 were strongly observed in the cancer cells. Median levels of serum VEGF-A, -C and VEGFR1 were 284, 4861 and 417 pg/ml, respectively. The levels of VEGF-A ans VEGFR1 in patients at stage III-IV were significantly higher than those in patients at stage I-II. 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 (60.7% vs. 45.5%, P = 0.029). Both VEGFR1 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. VEGF-A levels dramatically decreased after treatment with bevacizumab. Conclusion: These results suggest that serum VEGF-A may be a promising prognostic biomarker for EOC. Further study is needed to determine if could be predictive biomarker. Citation Format: Hiroaki Komatsu, Tetsuro Oishi, Hiroaki Itamochi, Mayumi Sawada, Michiko Nonaka, Seiya Sato, Jun Chikumi, Shinya Sato, Muneaki Shimada, Tasuku Harada. Serum VEGF-A may predict prognosis in patients with epithelial ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3387.