Naruaki Matsui

Granulosa cell tumor with activated mTOR-HIF-1α-VEGF pathway

The DNA‐binding activity of hypoxia‐inducible factor‐1 α (HIF‐1α) has been analyzed for various gynecological tumors. Among the tumors that were studied, there was a finding of a high level of DNA‐binding HIF‐1α activity, although it was limited to one case of adult type granulosa cell tumor (GCT). In this case a 60‐year‐old female had marked immunohistochemical expression of HIF‐1α. The expressions of the mammalian target of rapamycin (mTOR) and phosphorylated‐mTOR (p‐mTOR) were also marked, and vascular endothelial growth factor (VEGF) was moderately expressed. To compare the expression profiles, 11 consecutive cases with adult type GCT were used. All cases showed marked expressions of HIF‐1α and mTOR, but p‐mTOR expression was moderately to markedly observed in four of the 12 cases. VEGF was expressed in all cases in varying degrees. Based on the evidence that downregulation of the mTOR pathway due to treatment with rapamycin (everolimus) would suppress tumor cell growth, an experimental study using the GCT cell line was designed to clarify whether HIF‐1α and VEGF expressions decline. As a result, the expressions of p‐mTOR, HIF‐1α and VEGF were suppressed, but those of mTOR were not. It was concluded that mTOR‐targeted therapy may represent a promising strategy for some GCT with an activated mTOR‐HIF‐1α‐VEGF pathway.

Malignant transformation of atypical endometrial hyperplasia after progesterone therapy showing germ-cell tumor-like differentiation

A 31‐year‐old woman was treated for atypical endometrial hyperplasia (AEH) with high‐dose medroxyprogesterone acetate (MPA) therapy to preserve fertility. The AEH was found by repeated cytologic and histologic examinations to have completely disappeared with the therapy, but 3 years after her last follow up she required emergency surgery to treat severe genital bleeding. The hysterectomied uterus consisted mostly of poorly differentiated adenocarcinoma, G3 endometrioid type. Minor AEH was present in the exophytic area, in which some glands were cystically dilated. Part of the AEH had transformed into other histologic features with germ‐cell‐like differentiation, demonstrated by immunohistochemical positive reaction of placental alkaline phosphatase, alpha‐fetoprotein, and human chorionic gonadotrophin. Recurrent AEH had undergone malignant transformation, resulting in the development of well‐ and poorly differentiated adenocarcinoma and tumor exhibiting germ‐cell‐like differentiation. The patient died of a massive tumor extension 7 months after surgery. The AEH before MPA therapy and the recurrent tumors had genetically different characteristics based on evidence of a loss of heterozygosity, detected at D8S1132 (chromosomal locus, 8q22.1) in the latter but not in the former, by analysis of genetic alterations using microsatellite markers.

Endometrial intraepithelial carcinoma in association with polyp: review of eight cases

BackgroundThe uterine endometrial polyp (EMP) has a potential risk of developing malignant tumors especially in postmenopausal women. These malignancies include endometrial intraepithelial carcinoma (EIC).Patients and methodsEight patients with EIC in the EMP, who were postmenopausal with ages ranging from 49 to 76 years (av. 62), were cytologically reviewed in comparison with histological findings.ResultsThe endometrial cytological findings were summarized as follows: mucous and watery diathesis as a background lacking or with little necrotic inflammatory changes; micropapillary cluster formation; abrupt transition between carcinoma cells and normal cells; nuclear enlargement; high N/C ratio; and single or a few prominent nucleoli. Histologically, one case had EIC alone in the EMP; three cases had EIC with stromal invasion confined to the EMP; and four cases had EIC in the atrophic endometrium in addition to EIC in the EMP. Seven patients have taken a disease-free course after surgical resection, but one patient died 44 months following the initial diagnosis because of the massive tumor extending over her peritoneal cavity.ConclusionsEndometrial cytology may be helpful for the detection of early endometrial adenocarcinomas with serous features including EIC. Some early stage endometrial adenocarcinomas represented by EIC exceptionally take an aggressive clinical course irrespective of a lack of extrauterine lesions.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1651876760876449

Alterations of hypoxia-induced factor signaling pathway due to mammalian target of rapamycin (mTOR) suppression in ovarian clear cell adenocarcinoma: in vivo and in vitro explorations for clinical trial.

Objectives Before setting into the clinical trial using a combination of mammalian target of rapamycin (mTOR) inhibitors (rapamycin and everolimus) and other anticancer drugs, this study was conducted to confirm the efficacy of the new therapeutic strategy for ovarian clear cell adenocarcinoma (CCA), which targeted mTOR–hypoxia-induced factor (HIF) signal transduction system. Materials and Methods Using the cultured cells of CCA and animal models, alteration of mTOR-HIF cofactors and cell proliferation under the mTOR inhibitor–treated condition were analyzed. Results Mammalian target of rapamycin–HIF cofactors were inhibited dependent on concentration by mTOR inhibitor, resulting in suppression of the cultured CCA proliferation. However, von Hippel-Lindau was up-regulated at the messenger RNA level. In the nude mice with subcutaneously implanted CCA cells, apoptosis and necrosis were detected especially around the center of the tumors in the mTOR inhibitor–treated group more conspicuously than in the nontreated group. In the assessment of combination therapy with other antitumor agents, a combined treatment with mTOR inhibitor and chemotherapeutic agents caused a significant decrease in tumor size compared to the chemotherapeutic agents–only group. Conclusions Treatment by mTOR inhibitor is expected to down-regulate the cell proliferation of the CCA as a new therapeutic strategy.

Analysis of mTOR Inhibition-Involved Pathway in Ovarian Clear Cell Adenocarcinoma

This study was designed to clarify the mechanism of the mammalian target of rapamycin (mTOR)-hypoxia inducible factor-1 (HIF-1) pathway using the cultured cell strain derived from human ovarian clear cell adenocarcinoma (CCA). Everolimus (a derivative of rapamycin)-treated cells and non-treated cells did not show any difference in mTOR expression. But, phosphorylated-mTOR (p-mTOR) expression significantly decreased in the treated cells, and mTOR-related factors such as phosphorylated-4E-BP1 (p-4E-BP1), HIF-1α, and vascular endothelial growth factor (VEGF) in the downstream region of mTOR revealed a marked decrease in expression. The analysis of influences of the drug on the HIF-1α degradation system showed an increase in von-Hippel Lindau (VHL) expression in the treated cells. Increase of cleaved caspase-3, one of key factors involved in apoptosis, was also shown in the treated cells. In the next step, using nude mice implanted with RMG-1 cells, a decrease in tumor size was demonstrated in 4 of the 7 mice which were orally administered with everolimus. As a result, it was suggested that everolimus administration would be helpful as an anti-tumor therapy for CCA not only via down-regulation of p-mTOR but also degradation of HIF-1α by VHL and induction of apoptosis by cleaved caspase-3.

Sensitivity to CPT-11 and platinum derivatives of stage III/Dukes' C colorectal cancer with occult neoplastic cells in lymph node sinuses

Among 13 patients with recurrent colorectal cancer (recurrence group: RG), immunohistochemical expression of Topo-1 was high in 4 patients (30.8%) and low in 9 patients (69.2%), while the non-recurrence group (N-RG) (n=8) showed high expression in 1 patient (12.5%) and low expression in 7 patients (87.5%) (NS). Regarding immunohistochemical expression of Bax/ERCC-1, high Bax/low ERCC-1 expression was observed in 6 patients (46.2%) from the RG and other patterns of expression were seen in 7 patients (53.8%), while high Bax/low ERCC-1 expression was observed in 4 patients (50.0%) from the N-RG and other patterns were found in 4 patients (50.0%) (NS). PCR analysis of Topo-1 expression revealed high expression in 9 patients (75.0%) from the RG (n=12) and low expression in 3 patients (25.0%), while the N-RG (n=8) showed high expression in all 8 patients (100.0%) and low expression in none (NS). With respect to ERCC-1, PCR analysis revealed high expression in 7 patients (58.3%) from the RG (n=12) and low expression in 5 patients (41.7%), while the N-RG (n=8) showed high expression in 1 patient (12.5%) and low expression in 7 patients (87.5%) (p<0.05). These results suggest that tumor sensitivity to CPT-11 and platinum derivatives is similar in stage II colorectal cancer patients with ONCs.

Abstract A30: Comprehensive and serial analyses of tumor-stroma interactions in individual PDX/NOG models contribute to personalized chemotherapy

Xenografts derived from engrafting fresh surgical specimens directly into immunodeficient mice have recently enabled the development of more relevant in vivo models of human cancers. These patient-derived xenograft (PDX) models, established by the direct transfer of tumor tissue, retain similar morphologies, heterogeneities, and molecular signatures as the original cancers and, thus, may be used in promising personalized medicine for cancer. We previously reported the rapid and efficient establishment of PDXs using super immunodeficient NOG mice (PDX/NOG models). In the present study, we compared the gene-expression and cancer-stroma interaction profiles of PDX pairs metachronously established from the same patient due to therapeutic advances and investigated whether characteristic changes in these PDX pairs are influenced by chemotherapy. We also discussed the possibility of individual PDX/NOG model simulations for personalized cancer chemotherapy. More than 100 lines of cancer xenografts were established in our previous studies. In these cases, two pairs of PDX/NOG were established from the same patient who had undergone multiple operations or samplings and provided their samples twice. Genome sequencing and comprehensive analyses of tumor-stroma interactions (CAncer-STromal INteractome analysis, CASTIN) were performed on mRNA from two pairs of PDX/NOG. CASTIN showed tumor-stroma interactions in PDX/NOG comprehensively and quantitatively at the gene expression level by distinguishing gene arrangements in human tissue (Cancer) from mouse tissue (Stroma) as signal strength (ligand dependency (%)/receptor dependency (%)). Case 1: Sigmoid colonic cancer, adenocarcinoma, 58-year-old woman. She received chemotherapy with bevacizumab +FOLFOX and cetuximab+FOLFIRI between two operations. EGF (cancer)-EGFR (stroma) interaction strength was down-regulated from 7.3 (ligand dependency 59% / receptor dependency 81%) to 7.1 (37%/56%). VEGFA (cancer)-KDR (stroma) was up-regulated from 9.9 (77%/100%) to 121.6 (87%/100%). Case 2: Lung cancer with malignant pleural effusion, adenocarcinoma, 71-year-old man. He received chemotherapy with docetaxel+bevacizumab during two samplings. EGF (cancer)-EGFR (stroma) was down-regulated from 11.2 (85%/79%) to 5.1 (31%/71%). VEGFA (cancer)-KDR (stroma) was up-regulated from 45.2 (78%/94%) to 128.4 (68%/93%). Interaction changes in EGF-EGFR or VEGFA-KDR in PDX/NOG closely reflected the clinical effectiveness of the EGFR inhibitor (cetuximab) or VEGF-A inhibitor (bevacizumab). The CASTIN results of PDX/NOG appear to be reliable for clinical simulations of chemotherapy and will assist in the selection of the most sensitive anti-cancer drugs. Serial analyses of PDX/NOGs will disclose characteristic changes in cancer cells induced by the chemotherapy administered. It is considered suitable to establish serial PDX/NOGs for the same patients from malignant pleural effusion because these patients may repeatedly undergo therapeutic thoracentesis. The fast and efficient establishment of individual PDXs will contribute to personalized anti-cancer therapies. Citation Format: Tsuyoshi Chijiwa, Mizuha Haraguchi, Daisuke Komura, Akira Noguchi, Manabu Shiozawa, Makoto Katayama, Naoki Miyao, Naruaki Matsui, Yuichi Tateishi, Hiroshi Suemizu, Yoshiyasu Nakamura, Takayuki Isagawa, Hiroto Katoh, Shumpei Ishikawa, Masato Nakamura, Yohei Miyagi. Comprehensive and serial analyses of tumor-stroma interactions in individual PDX/NOG models contribute to personalized chemotherapy [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr A30.

Abstract 2813: An interactome analysis for personalized chemotherapy using PDX/NOG models of non-small cell lung cancer

Lung cancer is one of the most common malignant diseases in the world, and its prognosis is generally poor. It is crucial to elucidate the biological mechanisms underlying lung cancer and accelerate the development of new treatment strategies. Xenografts derived from engrafting fresh surgical specimens directly into immunodeficient mice have recently enabled the development of more relevant in vivo models of human cancers. These patient-derived xenograft (PDX) models, established by the direct transfer of tumor tissue, retain similar morphologies, heterogeneities, and molecular signatures as the original cancers, and, thus, may be used in promising personalized medicine for cancer. We previously reported the rapid and efficient establishment of PDXs using super immunodeficient NOG mice (PDX/NOG model). In the present study, we analyzed the gene expression and cancer-stroma interaction profiles of PDXs established from NSCLC patients. We also discussed the possibility of individual PDX/NOG model simulations for personalized cancer chemotherapy. Ten NSCLC lines of PDX/NOG (Age 43-78 years; 8 men and 2 women; 7 adenocarcinoma, 1 adenosquamous carcinoma, 1 squamous carcinoma, and 1 large cell carcinoma) were presented. In these cases, clinical information regarding chemotherapy for donor patients was retrieved where possible. Genome sequencing and comprehensive analyses of tumor-stroma interactions (CAncer-STromal INteractome analysis, CASTIN) were performed on mRNA. CASTIN showed tumor-stroma interactions in PDX/NOG comprehensively and quantitatively at the gene expression level by distinguishing gene arrangements in human tissue (Cancer) from mouse tissue (Stroma) as signal strengths (ligand dependency (%) / receptor dependency (%)). Interactions of EGF(cancer)-EGFR(stroma) were observed from 1.2 (15%/0%) to 11.5 (94%/80%) as widely-distributed. Interactions of VEGFA(cancer)-KDR(stroma) were observed from 49.1 (31%/51%) to 301.7 (83%/100%) as closely-distributed. These interactions of EGF-EGFR or VEGFA-KDR in PDX/NOG closely reflected the clinical effectiveness of an EGFR inhibitor (Cetuximab) or VEGF-A inhibitor (Bevacizumab). The CASTIN results of PDX/NOG appear to be reliable for clinical simulations of chemotherapy and will definitely assist in the selection of the most sensitive anti-cancer drug. The fast and efficient establishment of individual PDXs will contribute to personalized anti-cancer therapies. Citation Format: Tsuyoshi Chijiwa, Daisuke Komura, Mizuha Haraguchi, Akira Noguchi, Hidemitsu Sato, Hiroaki Ito, Haruhiko Nakayama, Makoto Katayama, Naoki Miyao, Naruaki Matsui, Yuichi Tateishi, Hiroshi Suemizu, Yoshiyasu Nakamura, Daisuke Furukawa, Takayuki Isagawa, Hiroto Katoh, Shumpei Ishikawa, Masato Nakamura, Yohei Miyagi. An interactome analysis for personalized chemotherapy using PDX/NOG models of non-small cell lung 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 2813. doi:10.1158/1538-7445.AM2017-2813

Use of epithelial‐specific antigen for cytological diagnosis of glandular lesions in the uterine cervix

To the Editor: It has been reported that endocervical adenocarcinoma accounts for 5–20% of all cervical carcinomas and its incidence has been increasing in recent years. Endocervical adenocarcinoma displays less radiosensitivity than squamous cell carcinoma and effective chemotherapy has not been established. Therefore, endocervical adenocarcinoma currently has a poor prognosis, although it is hoped that early detection and early treatment may result in prognostic improvement. However, the accuracy of cytological diagnosis for detecting endocervical adenocarcinoma is not always high due to problems with the cellular sampling method and with screening. Regarding cellular sampling, if the site from which the endocervical lesion cannot be observed by colposcopy, it can be difficult to obtain sufficient amounts of tumor cells. Regarding problems with screening, concomitant squamous epithelial lesions, observed in 40–60% of patients with endocervical adenocarcinoma, making it difficult to screening for adenocarcinoma cells. Additionally, the false-negative rate of cytological diagnosis for endocervical adenocarcinoma is reported to range from 12.8% to 55.0%. This data suggests that it is difficult to use cytological diagnosis for endocervical glandular lesion. It has been reported that the expression rate of epithelialspecific antigen (ESA) is high in histologically detected endocervical adenocarcinoma. The present study was performed to determine whether or not expression of ESA is specific for endocervical lesions in cytology specimens and histological specimens. The subjects of this study were a total of 61 patients, who underwent both cytological and histological diagnosis (biopsy and excised specimens) almost simultaneously at our hospital from 1990 to 2007. They included 20 patients with endocervical adenocarcinoma (EA), 10 patients with adenocarcinoma in situ (AIS), 11 patients with carcinoma in situ (CIS), and 20 patients with invasive squamous cell carcinoma (SCC). Epithelial-specific antigen (VU1D9, 1:100; Novocastra Laboratories, Newcastle upon Tyne, UK) was detected by the indirect immunohistochemical method (MAX-PO MALT; Nichirei Biosciences, Tokyo, Japan) using HE-stained and Papanicolaou-stained (Pap-stained) and decolorized specimens. When histological specimens were investigated, antigen activation was performed by pretreatment with 0.1% trypsin at 37°C for 30 min. In both histology specimens and cytology specimens, endogenous peroxidases were inactivated by incubation with 3% H2O2 for 30 min. Both the primary antibody and the secondary antibody were reacted with the specimens for 30 min at room temperature, after which color was developed with DAB. The percentage of ESA-positive cells among 500 tumor cells was calculated in both histology specimens and cytology specimens and was classified as follows: 0% = − ; ≤ 30% = 1+ ; 30–70% = 2+ ; ≥ 70% = 3+ . Evaluation of the ESA expression showed the positive reaction to the cell membrane or cytoplasm of epithelial cells. For statistical analysis, Student’s t-test was employed and P < 0.05 was considered to be statistically significant. Expression of ESA for histological findings, normal squamous cells were generally negative for ESA, although some of the basal cells were positive. Endocervical cells were mainly negative for ESA, though the basolateral membranes were partially positive (Fig. 1a). Squamous metaplasia partially showed weak expression of ESA. Expression of ESA in endocervical lesions was seen in 85.0% of EA lesions (17/20 patients) (Fig. 1b); 100.0% of AIS lesions (10/10 patients) (Fig. 1c); 18.2% of CIS lesions (2/11 patients); and 45.0% of SCC lesions (9/20 patients). Thus, expression of ESA was detected in all patients with AIS and nearly all patients with EA (sensitivity of 90.0% and specificity of 64.5%). The level of ESA expression in patients with EA was mainly 2+ (1+ in 25.0% of the patients; 2+ in 45.0%; and 3+ in 15.0%), although there were individual differences. All of the patients with AIS had ESA-positive tumors and strong expression was frequent (3+ in 70%). On the other hand, most of the patients with CIS had ESA-negative tumors (81.8%) (Fig. 1d), but 40% of the patients with SCC had ESA-positive tumors (1+ in 25.0% and 2+ in 15.0%). In cytological specimens, normal squamous cells, squamous metaplastic cells and endocervical cells were negative for ESA or slight cytoplasmic expression in endocervical cells (Fig. 2a). Expression of ESA in endocervical lesion was seen in 100.0% of EA lesions (20/20 patients) (Fig. 2b) and 100.0% of AIS lesions (10/10 patients) (Fig. 2c) while ESA was only positive in 18.2% of CIS lesions (2/11 patients) (Fig. 2d) and 30.0% of SCC lesions (6/20 patients). Thus, all of the patients with EA or AIS were ESApositive, whereas the ESA expression rate was low in squamous lesions (sensitivity of 100.0% and specificity of 74.2%). The level of ESA expression was strong in the patients with EA or AIS (3+ in 100.0% of the patients with EA and 80.0% of the patients with AIS) (Fig. 2a, b). On the other hand, weak ESA expression was seen in patients with CIS or SCC (1+ in doi:10.1111/pin.12379 bs_bs_banner

Possible role of thymidine phosphorylase in gynecological tumors as an individualized treatment strategy

Thymidine phosphorylase (TP) is structurally similar to platelet-derived endothelial cell growth factor, and it activates 5-fluorouracil (5-FU) prodrugs and also promotes angiogenesis. In the present study, the possibility of using TP expression as a biomarker for 5-FU prodrugs, and the significance of TP as an angiogenic factor, were investigated in patients with gynecological tumors. The subjects enrolled in the study were 188 patients with gynecological tumors who provided informed consent and underwent tumor resection at the Department of Obstetrics and Gynecology of Tokai University Hospital between February 2002 and January 2010. Measurement of the enzymatic activity of TP and dihydropyrimidine dehydrogenase (DPD) was performed by enzyme-linked immunosorbent assay. In addition, immunohistochemistry (IHC) analysis of microvessels by monochrome imaging, western blotting and reverse transcription-polymerase chain reaction were performed. The mean TP activity and the TP/DPD ratio were increased in squamous cell carcinoma of the cervix (306.9 and 2.2 U/mg protein, respectively) and adenosquamous carcinoma (317.6 and 1.4 U/mg protein, respectively) compared with benign tumors and other malignancies, including endometrial (uterine) carcinoma, ovarian serous adenocarcinoma and ovarian mucinous adenocarcinoma. However, these parameters were also elevated in other histological types of cancer such as clear cell adenocarcinoma of the ovary (115.2 and 2.1 U/mg protein, respectively), in which the microvessel area was the largest of all the histological types analyzed. Since high TP expression and a high TP/DPD ratio were identified in other tumors besides cervical cancer, it is possible that patients for whom 5-FU prodrugs are indicated could be selected appropriately if their TP activity is determined and their TP expression is analyzed by IHC prior to initiation of the treatment.