Yoshiro Otsuki

Loss of ARID1A expression is related to shorter progression-free survival and chemoresistance in ovarian clear cell carcinoma.

Recently, the ARID1A gene has been identified as a novel tumor suppressor in ovarian clear cell carcinoma. The prognostic significance of the loss of ARID1A expression is not known. The current study was designed to evaluate whether ARID1A was a prognostic factor for progression, survival, and chemoresistance in ovarian clear cell carcinoma. A total of 60 patients, who were surgically treated for primary ovarian clear cell adenocarcinoma, were enrolled. Surgical specimens were examined for ARID1A protein expression by immunohistochemistry. The correlations between the loss of ARID1A expression and clinicopathological characteristics, prognosis, and chemosensitivity were investigated. Loss of ARID1A expression was identified in 9 (15.0%) of 60 ovarian clear cell carcinoma samples. Loss of ARID1A staining intensity (0+) was more frequently found in cells of clear cell carcinomas than in high-grade serous carcinomas (P<0.01). Loss of ARID1A expression was significantly correlated with advanced FIGO stage and high CA125 levels (P=0.02, 0.01). There were no significant correlations between loss of ARID1A expression and patient age, status of residual tumor, Ki-67 labeling index, or the status of endometriosis. Loss of ARID1A correlated with shorter progression-free survival of patients with clear cell carcinomas treated with platinum-based chemotherapy (P<0.01). Loss of ARID1A expression tended to correlate with shorter overall survival in patients with ovarian clear cell carcinomas treated with platinum-based chemotherapy. When data were stratified for the multivariate analysis, only the loss of ARID1A expression remained a significant (P=0.03) predictor of reduced progression-free survival. Of the 60 patients with ovarian clear cell carcinomas, 14 patients had measurable residual tumor after primary cytoreductive surgery. Tumors with loss of ARID1A expression were more likely to be chemoresistant than tumors with positive ARID1A expression (100.0 vs 40.0%, P=0.04). This study demonstrates that loss of ARID1A in ovarian clear cell carcinoma is a negative prognostic factor in patients treated with platinum-based chemotherapy. Measurement of ARID1A expression may be a method to predict resistance to platinum-based chemotherapy in patients with ovarian clear cell carcinoma.

EGFR gene amplification is related to adverse clinical outcomes in cervical squamous cell carcinoma, making the EGFR pathway a novel therapeutic target

Background:The aim of this study was to investigate the patterns of epidermal growth factor receptor (EGFR) overexpression, EGFR gene amplification, and the presence of activating mutations in the tyrosine kinase domain of this gene in squamous cell carcinomas and adenocarcinomas/adenosquamous carcinomas of the uterine cervix.Methods:The EGFR expression, amplification, and mutation in cervical carcinomas were assessed by immunohistochemistry, fluorescence in situ hybridisation, and PCR–SSCP, respectively, and correlated with clinical data collected by a retrospective chart review. A functional assessment was performed by inactivating EGFR in cervical cancer cells with the potent inhibitor AG1478.Results:Immunohistochemical analysis revealed that 6 out of 59 (10.2%) cervical squamous cell carcinomas showed significant amplification of the EGFR locus, whereas none of the 52 adeno/adenosquamous cell carcinomas had detectable EGFR amplification (P<0.05). The EGFR amplification significantly correlated with shorter overall survival (P=0.001) in cervical squamous cell carcinomas. Multivariate analysis showed that EGFR gene amplification was an independent prognostic factor for overall survival (P=0.011). None of the squamous cell carcinomas (0%: 0 out of 32) had detectable oncogenic mutations in EGFR exons 18 through 21. The frequencies of KRAS and BRAF mutations were very low in both squamous and adeno/adenosquamous cell carcinomas. Sensitivity of cervical cancer cells to AG1478 depended on the presence of EGFR overexpression. AG1478-induced EGFR inactivation in cell lines with EGFR overexpression significantly suppressed tumour development and progression in a mouse xenograft model.Conclusion:Our data suggest that EGFR signalling is important in a subset of cervical squamous cell carcinomas and that anti-EGFR therapy may benefit patients who carry the 7p11.2 amplicon in their tumours.

Clinicopathologic and biological analysis of PIK3CA mutation in ovarian clear cell carcinoma

Somatic mutations of PIK3CA (phosphoinositide-3-kinase) have recently been shown playing an important role in the pathogenesis of ovarian clear cell carcinoma. In this study, the frequency of PIK3CA mutations and the relationship of PIK3CA mutations with clinicopathologic and biological variables were investigated in ovarian clear cell carcinomas from Japanese patients. Mutational analysis of PIK3CA was performed in 56 primary ovarian clear cell carcinomas from Japanese women. The relationship of these mutations with various clinicopathologic and biological variables (phosphorylated AKT and phosphorylated mTOR (mammalian target of rapamycin) expression by immunohistochemistry) was determined. To clarify the roles of PI3K/AKT activation in ovarian clear cell carcinomas harboring PIK3CA mutations, we inactivated the PI3K/AKT/mTOR pathway in ovarian carcinoma cells with LY294002, temsirolimus and NVP-BEZ235. Missense mutations of PIK3CA were found in 16 (28.6%) of 56 ovarian clear cell carcinomas, but no mutation was found in 15 ovarian high-grade serous carcinomas. PIK3CA mutations were significantly associated with a favorable overall survival of patients with ovarian clear cell carcinoma (P < .05). There was no significant association between PIK3CA mutations and phosphorylated AKT or phosphorylated mTOR immunointensity status. No relationship was found between PIK3CA mutation status and sensitivity to PI3K/AKT/mTOR inhibitors in ovarian clear cell carcinoma cells. No association of PIK3CA mutations was found between positive phosphorylated AKT and positive phosphorylated mTOR, which suggests that the PI3K/AKT/mTOR pathway may be activated by other molecular mechanisms. Although PIK3CA mutations were associated with a more favorable prognosis, they did not predict the sensitivity of ovarian clear cell carcinoma cells to PI3K/AKT/mTOR inhibitors.

Prognostic and therapeutic impact of the chromosome 20q13.2 ZNF217 locus amplification in ovarian clear cell carcinoma

The goal of this study was to examine the clinical significance of ZNF217 amplification and assess whether ZNF217 could be a potential therapeutic target in ovarian clear cell carcinoma (OCCC).

Notch3 Overexpression as Potential Therapeutic Target in Advanced Stage Chemoresistant Ovarian Cancer

This study examined the clinical significance of Notch3 expression and assessed its usefulness as a potential therapeutic target in chemoresistant ovarian cancer. Notch3 expression was assessed with immunohistochemical examination, and clinical variables were collected with a retrospective chart review. Notch3 siRNA or γ-secretase inhibitor was used to assess Notch3 function in ovarian cancer cell lines. Notch3 overexpression correlated with shorter progression-free/overall survival in patients with advanced stage (stage III, IV) ovarian carcinoma treated with platinum and taxane. Three of 5 patients showed increased Notch3 immunostaining in recurrent tumors compared with corresponding primary tumors. Notch3 overexpression was observed in both the cisplatin-resistant KFr13 and cisplatin/paclitaxel-resistant KFr13Tx cells. Inactivation of Notch3 by γ-secretase inhibitor or siRNA decreased cell proliferation and induced apoptosis in the KFr13 and KFr13Tx cells. Our findings suggest that Notch3 expression may be related to chemoresistance and that the Notch3 pathway may represent a novel therapeutic target for advanced stage chemoresistant ovarian cancer.

Expression of nuclear Notch3 in cervical squamous cell carcinomas and its association with adverse clinical outcomes

OBJECTIVE The aim of this study was to clarify the functional role of Notch3 in human cervical carcinomas. METHODS Notch3 expression in cervical cancer was assessed by immunohistochemistry, and data on clinical variables were collected by retrospective chart review. We used dual-color fluorescence in situ hybridization (FISH) to analyze DNA copy number alterations in cervical cancer. Inactivation of Notch3 and knocking down Notch3 gene were done using gamma-secretase inhibitor and Notch 3 specific SiRNA to asses Notch3 function in cervical cancer either in vivo or in vitro. RESULTS Immunohistochemical analysis revealed that Notch3 was significantly overexpressed in cervical squamous cell carcinomas compared with adenocarcinomas. In contrast to normal cervical tissue and cervical intraepithelial neoplasms [CINs], squamous cell carcinomas demonstrated higher nuclear Notch3 immunoreactivity. Notch3 amplification was not found in any cervical carcinomas using FISH analysis. Notch3 nuclear expression was significantly correlated with Jagged-1, a putative Notch3 ligand, and Pbx1b, a potential Notch3 downstream target (P<0.05).Patients with cervical carcinomas positive for nuclear Notch3 expression had significantly shorter overall survival than their peers whose tumors did not express nuclear Notch3. Inactivation of Notch3 decreased cell proliferation and induced apoptosis in ME180 and SKGIIIb cell lines that overexpressed Notch3. Injection of a gamma-secretase inhibitor into ME180 cell tumors established on mice, demonstrated a reduction in tumor growth. CONCLUSION Our findings suggest that Notch3 might play important role for the proliferation and survival of Notch3 overexpressing tumors and that inactivation of Notch3 may represent a new therapeutic avenue for cervical squamous cell carcinomas.

Fatty acid synthase expression associated with NAC1 is a potential therapeutic target in ovarian clear cell carcinomas.

Background:This study examined the clinical significance of NAC1 and the expression level of its potential downstream target fatty acid synthase (FASN) in ovarian clear cell carcinomas (OCCCs), and evaluated the NAC1/FASN pathway as a potential therapeutic target.Methods:NAC1 and FASN expression and NACC1 gene amplification were assessed in ovarian cancers by immunohistochemistry, fluorescence in situ hybridisation, and clinical data collected by a retrospective chart review. C75, a FASN inhibitor, was used to assess whether this pathway represented a therapeutic target in OCCC.Results:High NAC1 expression was most frequent in clear cell tumours (40.0%:24/60). NACC1 gene amplification was identified in none of the 58 OCCCs. The frequency of NACC1 gene amplification was significantly higher in the high-grade serous histology than in the clear cell histology (P<0.01). NAC1 expression was significantly correlated with FASN expression in both OCCC samples and OCCC cell lines. Either high NAC1 expression or high FASN expression significantly correlated with shorter progression-free and overall survival (P=0.002 and 0.0048). NAC1 overexpression stimulated FASN expression, and NAC1 silencing using siRNA decreased FASN expression in OCCC cell lines. Profound growth inhibition was observed in C75-treated carcinoma cells with FASN overexpression when compared with the response in carcinoma cells without FASN expression.Conclusion:These findings indicate that NAC1/FASN overexpression is critical to the growth and survival of a subset of OCCC. The FASN silencing by the C75-induced phenotypes depends on the expression status of the targeted cell line. Therefore, NAC1/FASN pathway-targeted therapy may benefit selected OCCC patients.

Frequent loss of tumor suppressor ARID1A protein expression in adenocarcinomas/adenosquamous carcinomas of the uterine cervix.

Objectives Expression of ARID1A (the adenine, thymine-rich interactive domain 1A), a putative tumor suppressor, has recently been shown to be lost in several tumor types. This study investigated whether ARID1A expression was also lost in cervical squamous cell carcinomas and adenocarcinomas/adenosquamous carcinomas. Methods A total of 91 patients with cervical carcinoma were enrolled. Cervical carcinoma specimens were examined for ARID1A protein expression by immunohistochemistry. The correlations between the loss of ARID1A expression and clinicopathological characteristics, and prognosis were investigated. Results Using immunohistochemistry, the frequency of loss of ARID1A expression in adenocarcinomas/adenosquamous carcinomas (31.1% [14/45]) was significantly higher than that in squamous cell carcinomas (6.5% [3/46]; P = 0.0017). There was no significant association between the loss of ARID1A expression and International Federation of Gynecology and Obstetrics staging, lymphovascular space invasion, lymph node metastasis, age, and Ki-67 LI in cervical adenocarcinomas/adenosquamous carcinomas. Loss of ARID1A expression was not correlated with shorter overall/disease-free survival in cervical adenocarcinomas/adenosquamous carcinomas. Conclusions In conclusion, this study provides the first evidence of the frequent loss of ARID1A protein expression in cervical adenocarcinomas/adenosquamous carcinomas. No significant differences between ARID1A positive and negative cases were observed with respect to any clinicopathological features examined.

Hepatocellular carcinoma with mesothelioma-like dissemination

Reported herein is a case of hepatocellular carcinoma (HCC) with unusual peritoneal dissemination masquerading as peritoneal mesothelioma. A 61‐year‐old man was clinically found to have multiple tumors in his abdominal cavity; peritonitis carcinomatosa was suspected. An autopsy revealed numerous tumors of various sizes in the abdominal serosa, omentum, and diaphragm. No signs of tumor, fibrosis, or cirrhosis were found in the liver, except for a small nodule in the hepatic triangular ligament. Histologically, the tumor cells proliferated in thick trabeculae or in sheets and formed a few canaliculi and tubules with homogenously brown contents in their lumina, which stained positively with Hall stain. Immunohistochemically, these tumors were positive for hepatocyte, α‐fetoprotein (AFP) and low‐molecular‐weight cytokeratin; were focally positive for pan‐cytokeratin and epithelial membrane antigen (EMA); and were negative for high‐molecular‐weight cytokeratin, vimentin, and calretinin. Carcinoembryonic antigen (CEA) produced a bile canalicular immunohistochemical staining pattern. Thus, the tumor was diagnosed as an HCC (Edmondson II type) of the triangular ligament with massive peritoneal dissemination. The origin of this tumor and its differential diagnosis (malignant mesothelioma, hepatoid adenocarcinoma, and hepatoid yolk sac tumor) are discussed.

Loss of autophagy-related protein Beclin 1 may define poor prognosis in ovarian clear cell carcinomas

The aim of the present study was to clarify the role of autophagy in cisplatin (CDDP) sensitivity in OCCCs and the role of Beclin 1 in OCCC progression. Autophagy was measured using: i) western blot analysis of LC3 and p62 and ii) microscopic observation of GFP-LC3 puncta. Autophagy was suppressed using chloroquine and Beclin 1 siRNA. Surgical specimens were examined for Beclin 1 protein expression by immunohistochemistry. The correlations between the loss of Beclin 1 expression and clinicopathological characteristics, prognosis and chemosensitivity were investigated. Inhibition of autophagy by chloroquine or Beclin 1 siRNA did not enhance the sensitivity of the ES2 and TOV-21G OCCC cell lines to CDDP. Loss of Beclin 1 expression was observed in 38.3% (23/60) of the analyzed tumors. There was no significant correlation between loss of Beclin 1 expression and FIGO stage, CA125 levels, patient age, status of endometriosis, Ki-67 labeling index, chemotherapy regimen or status of residual tumor. However, negative expression of Beclin 1 was associated with a shorter progression-free survival in comparison to positive Beclin 1 expression in OCCC who received cytoreductive surgery, followed by a standard platinum-based chemotherapy regimen (P=0.027, log-rank test). Beclin 1-negative tumors were no more resistant to primary adjuvant chemotherapy than were Beclin 1-positive tumors (50.0 vs. 66.7%, P=0.937). Beclin 1 knockdown using siRNA increased cell growth but not cell migration and invasion in ES2 and TOV-21G OCCC cell lines. Autophagy defects caused by loss of Beclin 1 are not related to chemoresistance and metastasis, but may be associated with malignant phenotype and poor prognosis of OCCC.