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// Masako Ishikawa 1 , Kentaro Nakayama 1 , Kohei Nakamura 1 , Ruriko Ono 1 , Kaori Sanuki 1 , Hitomi Yamashita 1 , Tomoka Ishibashi 1 , Toshiko Minamoto 1 , Kouji Iida 1 , Sultana Razia 1 , Noriyoshi Ishikawa 2 and Satoru Kyo 1 1 Department of Obstetrics and Gynecology, Shimane University School of Medicine, 6938501 Izumo, Japan 2 Department of Organ Pathology, Shimane University School of Medicine, 6938501 Izumo, Japan Correspondence to: Kentaro Nakayama, email: kn88@med.shimane-u.ac.jp Keywords: ovarian clear cell carcinoma; ovarian endometrioid carcinoma; ARID1A ; POLE ; liquid microdissection Received: September 05, 2017 Accepted: February 13, 2018 Epub: February 22, 2018 Published: March 13, 2018 ABSTRACT Aim: Endometriosis-related ovarian neoplasms (ERONs) have recently attracted considerable attention; however, the prevalence and patterns of ARID1A and POLE mutations in ERONs have not been studied in detail. The aim of this study was to investigate not only the carcinogenesis of ERONs, but also the prognostic significance of several gene mutations in this cohort. We used DNA purified from only tumor epithelial cells, from which fibroblasts were removed, using a specific method we called “liquid microdissection”. Methods: Tissue samples from 22 ovarian carcinomas (13 endometrioid, and nine clear cell) were used. Tumor cells were isolated using a cell sorting system and DNA was purified from tumor epithelial cells. Nucleotide sequencing was conducted to analyze the mutational status of ARID1A, p53, PTEN, POLE, PIK3CA , and KRAS . Results: In ERONs, the frequencies of somatic mutations in ARID1A, p53, POLE, PTEN, PIK3CA , and KRAS were 19/20 (95.0%), 7/19 (36.8%), 9/22 (40.9%), 13/19 (68.4%), 3/19 (15.8%), and 1/9 (11.1%). The frequency of ARID1A mutations was significantly higher than that reported previously. Kaplan-Meier survival analysis revealed that mutations in all genes, including POLE , were not associated with patient prognosis in our Japanese cohort. Conclusions: Our results suggest that the frequency of ARID1A mutations in ERONs may be higher than that previously reported. In addition, the “liquid microdissection” method that we chose for DNA purification could be used to obtain high-quality sequencing results. The findings suggest that ARID1A mutations represent the basis of ERON carcinogenesis; other subsequent gene mutations may result in the progression of carcinogenesis.

The aim of the present study was to determine the prognostic value of the Glasgow prognostic score (GPS) in endometrial carcinoma (EC). Patients with EC who underwent surgery at the Shimane University Hospital between January 1997 and December 2013 were enrolled (n=118). The associations between pretreatment GPS and clinical parameters, including age, histological type, International Federation of Gynecology and Obstetrics stage, tumor grade, carbohydrate antigen 19-9 and carcinoembryonic antigen levels, progression-free survival (PFS), and overall survival (OS), were investigated. Survival analysis was performed with the Kaplan-Meier method, and prognostic factors were evaluated with Cox's proportional hazards regression model. A high pretreatment GPS was associated with advanced clinical stage, histological type and tumor grade (P

In recent years, it has become evident that tumor cells have immune escape mechanisms, and immune checkpoint inhibitor therapy (anti-PD-1/PD-L1 antibody) has shown benefit in various cancers. In endometrial tumors with microsatellite-instability (MSI), somatic mutations have the potential to encode ‘’non-self’’ immunogenic antigens, and lymphocytes have been shown to infiltrate the tumor. Therefore, immune checkpoint inhibitor therapy might be effective in endometrial cancers with MSI. Expression of mismatch repair (MMR) proteins (MLH1, PMS2, MSH2, and MSH6), the presence of tumor-infiltrating lymphocytes (CD8+), and PD-1/PD-L1 expression were assessed by immunohistochemistry in 149 patients with endometrial cancer. We examined whether tumors with MSI had an enhanced immune microenvironment and whether MSI could be a predictor of the therapeutic effect of PD-1/PD-L1 immunotherapy in endometrial cancer. Loss of MMR protein expression was identified in 42 (28.2%) of 149 patients (MSI group) with endometrial cancer. There was no significant relationship between MSI status and age (p = 0.193), histological grade (p = 0.097), FIGO stage (p = 0.508), pelvic lymph node metastasis (p = 0.139), or depth of myometrial invasion (p = 0.494). However, the presence of tumor-infiltrating lymphocytes (CD8+) and PD-L1/PD-1 expression were significantly higher in the MSI group compared to the microsatellite-stable group (p = 0.002, p = 0.001, and p = 0.008, respectively). These results suggest that immune checkpoint inhibitors (anti-PD-1/PD-L1 antibody) could be effective in endometrial cancers with MSI. The presence of MSI may be a biomarker for good response to PD-1/PD-L1 immunotherapy in endometrial cancer.

Haematogenous metastases of breast cancer tumors has previously been demonstrated to frequently occur at the sites of the lung, bones, liver and brain, however presence in the uterine remains a rare occurrence. Metastatic carcinoma of the uterus usually originates from other genital sites, most frequently from the ovaries. The current review presents the first reported case of lobular breast carcinoma metastasizing to an endometrial polyp, the cervix and a leiomyoma simultaneously. The patient (58 years, female) first presented with abnormal uterine bleeding. Invasive ductal carcinoma had previously been diagnosed in her right breast, with lobular and ductal cancer cells observed to be present in her lymph nodes. A hysteroscopic procedure to examine the postmenopausal bleeding revealed an endometrial polyp, which was subsequently resected. The morphology and immunohistochemical studies confirmed the diagnosis of metastasis of lobular breast carcinoma to an endometrial polyp. An 18F fludeoxyglucose positron emission tomography/computed tomography (PET-CT) scan performed following the diagnosis, revealed a slightly increased uptake in the myoma, which is often observed in benign uterine leiomyoma. The patient then underwent a total abdominal hysterectomy with bilateral salpingo-oophorectomy and partial colectomy. Pathology results demonstrated that the uterine leiomyoma and cervix shared the same histopathological features as those presented by the primary lobular breast carcinoma. Although rare, breast tumors may metastasize to an endometrial polyp, cervix and leiomyoma concurrently in patients, therefore physicians may now consider the potential of the diagnosis of metastatic spread to the genital tract, in a patient with abnormal uterine bleeding and a history of lobular breast cancer. Gynecologists planning a laparoscopic hysterectomy for a patient with a history of lobular breast carcinoma may consider abdominal rather than laparoscopic hysterectomy, as lobular carcinoma is difficult to detect. The use of PET-CT may be beneficial for the identification of an unexpected mass.

Ureterolysis is a surgical method with a high level of difficulty, which may be necessary when performing total laparoscopic hysterectomy (TLH) for large cervical myoma, despite the benign nature of this tumor. The aim of the present study was to introduce techniques that are commonly applied in malignant tumor surgery in order to safely perform TLH for large cervical myoma. Between 2014 and 2016, TLH was performed at the Shimane University Hospital (Izumo, Japan) in 153 patients with benign tumors, including 25 cases with a large uterus (uterine weight ≥500 g). The surgical methods applied in 3 of these large uterine cervical myoma cases were investigated in detail, including techniques devised by our department. TLH was performed without enucleating myomectomy in all 3 cases; however, all 3 cases required ureterolysis, transection of the anterior layer of the vesicouterine ligament and isolation of the ureter. In conclusion, although radical laparoscopic hysterectomy is commonly performed for cervical cancer at our department, techniques used for malignant tumor surgery may prove useful for benign cases with a high level of difficulty.

This study aimed to describe the long-term outcomes of patients with menorrhagia treated with microwave endometrial ablation (frequency, 2.45 GHz), as well as to identify factors associated with recurrence or re-surgery. This retrospective cohort study was conducted from 2007 to 2015 at Shimane University Hospital in Japan. Patients with severe menorrhagia and a desire to preserve their uterus were included in the study. Clinical factors associated with recurrence of menorrhagia or re-surgery were analyzed with a multivariable logistic regression model. Of 160 microwave endometrial ablation candidates, 100 had uterine myomas, 20 adenomyosis, 26 functional excessive menstruation, and 12 endometrial polyps. In the full cohort, age (

Ovarian low-grade serous carcinoma is thought to begin as a serous cystadenoma or adenofibroma that progresses in a slow stepwise fashion. Among the low-grade serous carcinomas, there is a high frequency of activating mutations in the KRAS or BRAF genes; however, it remains unclear as to how these mutations contribute to tumor progression. This is the first report to track the histopathological progression of serous adenofibroma to low-grade serous carcinoma. Each stage was individually analyzed by pathological and molecular genetic methods to determine what differences occur between the distinct stages of progression.

The utility of hydrogen stable isotope ratio measurement by IR-MS for establishing the origin of ephedrine and pseudoephedrine (ephedrines), precursors of methamphetamine, was evaluated. There are two kinds of commercial semisynthetic ephedrines, one produced from molasses and the other from pyruvic acid. While the semisynthetic ephedrines derived from pyruvic acid cannot be discriminated from biosynthetic ephedrines and synthetic ephedrines based on delta(13)C and delta(15)N values, they could be identified from the delta(2)H values. The low deuterium content of biosynthetic ephedrines (delta(2)H: -193 to -151 per thousand) allows a clear distinction from synthetic ephedrines (delta(2)H: -73 to -30 per thousand), semisynthetic ephedrines derived from pyruvic acid (delta(2)H: +75 to +148 per thousand) and semisynthetic ephedrines derived from molasses (delta(2)H: -74 to +243 per thousand). The wide range of delta(2)H values of semisynthetic ephedrines is therefore very useful for the detailed classification of ephedrines, in combination with the measurement of delta(13)C and delta(15)N values as described in our previous work. This study was carried out on a limited number of samples reflecting the various routes of ephedrines manufacture. But it has become apparent that this stable-isotope analysis is an appropriate means by which to screen for manufacturing process of ephedrines. This approach should be useful for worldwide precursor control of methamphetamine.

[Introduction] In recent years, tumor cells have immune escape mechanism and immune checkpoint inhibitor therapy (anti PD-1/ PD-L1 antibody) has shown benefit in various cancers. Somatic mutations have the potential to encode ‘’non-self’’immunogenic antigens and lymphocytes infiltrate tumor cells in Microsatellite-instable (MSI) endometrial cancers. Therefore, immune checkpoint inhibitor therapy might be effective in MSI endometrial cancers. [Method] Mismatch repair protein (MLH1, PMS2, MSH2, and MSH6), tumor-infiltrating lymphocytes (CD8), and PD-1/PD-L1 expression were assessed by immunohistochemistry in 60 patients with endometrial cancer. We examined whether MSI status have enhanced immune microenvironment and become the therapeutic effect predictor of PD-1/PD-L1 immunotherapy in endometrial cancer. [Results] Loss of mismatch repair protein (MSI group) was identified in 8 (13.3 %) of 60 patients with endometrial cancer. Expression of tumor-infiltrating lymphocytes (CD8) and PD-L1/PD-1 were significantly higher in MSI group compared to MSS group (p=0.001, p=0.044 and p=0.013). [Conclusion]These results suggested that immune checkpoint inhibitor (anti PD-1/PD-L1 antibody) is effective in endometrial cancers with MSI. MSI testing is likely to be a biomarker for PD-1/PD-L1 immunotherapy in endometrial cancer. Citation Format: Hitomi Yamashita, Kentaro Nakayama, Noriyoshi Ishikawa, Toshiko Minamoto, Tomoka Ishibashi, Kohei Nakamura, Kaori Sanuki, Ruriko Ono, Masako Ishikawa, Takeshi Isobe, Satoru Kyo. Microsatellite instability is a potential biomarker for immune checkpoint inhibitor in endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2621. doi:10.1158/1538-7445.AM2017-2621

Purpose: Recent studies suggest that fallopian tube secretory epithelial cells (FTSECs) are potential cells-of-origin for high-grade serous ovarian carcinoma (HGSOC). Several genetic alterations are involved in HGSOC carcinogenesis, but the minimal requirement for tumor initiation remains unclear. Therefore, we sought to identify oncogenic mutations indispensable for HGSOC carcinogenesis in a stepwise model using immortalized FTSECs. Experimental Design: We established an in vitro stepwise carcinogenesis model using immortalized FTSECs overexpressing cyclin D1, CDK4R24C, and hTERT, and mimicked select genetic abnormalities identified as gene alterations essential for carcinogenesis, including p53, c-Myc, or RAS/PI3K pathway mutations. Results: Our analyses revealed two distinct patterns of gene alterations essential for HGSOC carcinogenesis: p53/KRAS/AKT and p53/KRAS/c-Myc. Dominant-negative p53 expression alone or in combination with oncogenic KRAS (KRASV12), constitutively active AKT (CA-AKT), or c-Myc in immortalized cells failed to induce a tumorigenic phenotype; however, overexpression of either CA-AKT or c-Myc in combination with dominant-negative p53 and KRASV12 was sufficient to confer tumorigenic potential. Importantly, all transformed FTSECs formed tumors in xenograft mice, which were grossly, histologically, and immunohistochemically similar to human HGSOC. Interestingly, mice harboring tumors with c-Myc amplifications displayed extensive metastases, consistent with the increased dissemination observed in their human counterparts. C-Myc is associated with cell proliferation in vitro; therefore, this genetic abnormality may promote HGSOC progression. Conclusions: Collectively, our data showed that aberrant p53/KRASV12/c-Myc or p53/KRASV12/PI3K-AKT signaling is the minimal requirement for FTSEC carcinogenesis. Moreover, the model generated with this evidence will likely facilitate analysis of early events in HGSOC carcinogenesis. Citation Format: Kohei Nakamura, Kentaro Nakayama, Tohru Kiyono, Noriyoshi Ishikawa, Toshiko Minamoto, Tomoka Ishibashi, Kaori Sanuki, Hitomi Yamashita, Ruriko Ono, Kouji Iida, Hiroki Sasamori, Razia Sultana, Mohammad Mahmud Hossain, Masako Ishikawa, Satoru Kyo. In vitro reconstitution of high-grade serous ovarian carcinoma from primary fallopian tube secretory epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5755. doi:10.1158/1538-7445.AM2017-5755

High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecologic malignancy in the world. Recent studies suggest that fallopian tubal secretory epithelial cells (FTSECs) are candidates for the origin of HGSOC. Several genetic alterations involved in the carcinogenesis have been reported in HGSOC, but their minimal requirement and the specific combinations remained unclear. To clarify these points, we established an in vitro stepwise carcinogenesis model using immortalized FTSECs with overexpressed cyclinD1/cdk4 and hTERT, in which we additionally mimicked selected genetic abnormalities frequently observed in clinical samples. Mutational analysis using clinical samples of HGSOCs identified frequent p53 mutations (96%). Furthermore, RAS/PI3K pathway is important signaling pathway and was deregulated in 45% of HGSOCs. PI3K/AKT pathway is also important pathway in HGSOCs. Thus, we introduced dominant negative form of p53 alone or in combination with oncogenic mutant KRAS allele into immortalized cells, but both failed to exhibit tumorigenic phenotypes. Additional introduction of genetic factors were attempted, and overexpression of c-Myc or phosphorylated Akt (p-Akt) were independently found to confer tumorigenic phenotypes. Importantly, all transformed FTSECs exhibited high-grade serous carcinomas that were grossly, histologically, and immunohistochemically similar to human HGSOCs. Thus, p53/KRAS/c-Myc or p53/KRAS/PI3K-AKT pathways were determined to be minimal required for carcinogenesis. The strength of our study is the use of immortalized FTSECs that have original characteristics of fallopian tube as well as the introduction of genetic mutations clinically observed in human HGSOCs. This experimental model provides a foundation for future studies of early events in carcinogenesis of HGSOCs and the identification of candidate targets for drug development and secreted biomarkers for early detection. It is also likely that a similar model can be broadly applied to studying the normal biology of other epithelial cells where precursor lesions and malignant counterparts are not entirely understood. Citation Format: Kohei Nakamura, Kentaro Nakayama, Tohru Kiyono, Noriyoshi Ishikawa, Masako Ishikawa, Hiroshi Katagiri, Toshiko Minamoto, Tomoka Ishibashi, Emi Sato, Kaori Sanuki, Hitomi Yamashita, Kouji Iida, Razia Sultana, Satoru Kyo. Establishment of in vitro carcinogenic model of high-grade serous ovarian carcinoma using immortalized fallopian tubal secretory epithelial cell. [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 5160.

Objective The aim of the present study was to evaluate the prognostic value of pretreatment plasma dimerized plasmin fragment D (D-dimer) levels in endometrial carcinoma after adjusting for venous thromboembolism (VTE). Study design The relationships between the following clinical parameters from 110 patients were investigated: age, histological type, the International Federation of Gynecology and Obstetrics (FIGO) stage, tumor grade, pretreatment plasma D-dimer level, platelet count, fibrinogen, CA19-9, and CEA levels, progression-free survival (PFS), and overall survival (OS). A survival analysis was performed using the Kaplan–Meier method, and prognostic factors were assessed using Cox's proportional hazards regression model. Results High pretreatment D-dimer levels were detected in 32% of cases. High D-dimer levels correlated with an advanced tumor stage, histological type, and tumor grade ( P =0.001, P =0.021, P =0.044). A multivariate analysis identified high D-dimer levels as an independent prognostic factor for OS ( P =0.013), whereas the histological type, but not D-dimer levels had independent prognostic value for PFS ( P =0.225). Conclusion High pretreatment D-dimer levels have an impact on prognoses independently of VTE, and also have potential as markers to predict surgical outcomes in patients with endometrial carcinoma. Pretreatment D-dimer levels may contribute to the identification of high-risk populations for treatment decisions.