Therapeutic strategy targeting the mTOR–HIF-1α–VEGF pathway in ovarian clear cell adenocarcinoma

Malignant tumors usually involve a relatively hypoxic state, which induces overexpression of hypoxia-inducible factor-1α (HIF-1α) to satisfactorily enable the tumor to survive. Thus, inhibition of the mammalian target of rapamycin (mTOR) pathway including HIF-1α is expected to play a major role in suppression of tumor cell growth, having recently drawn much attention as an anti-cancer therapeutic strategy for various malignant tumors. In the present study, which compared clear cell adenocarcinoma (CLA) of the ovary with serous adenocarcinoma (SEA), the immunohistochemical expression of mTOR, phosphorylated-mTOR (p-mTOR), HIF-1α, and vascular endothelial growth factor (VEGF) was examined in surgically resected specimens of 29 SEA and 47 CLA. There were no significant differences in expression of mTOR, HIF-1α and VEGF between SEA and CLA, but it was noted that p-mTOR expression was more prominent in CLA than SEA. Then, using the cell lines of CLA (RMG-1 and W3uF), an experimental study was designed to clarify whether tumor suppression due to downregulation of mTOR activity could represent a promising therapeutic strategy for CLA. After treatment of an analogue of rapamycin (everolimus), expression of mTOR, p-mTOR, HIF-1α and VEGF was examined on western blot. As a result, although mTOR expression remained unchangeable, expression of p-mTOR, HIF-1α and VEGF was shown to be sharply depressed. The same expression alterations were demonstrated in the xenograft model treated with everolimus. In conclusion, mTOR-targeted therapy through usage of drugs such as everolimus may be more effective for CLA of the ovary because of its significant expression of p-mTOR.     

Relation between hyaluronan synthesis and cell morphology in ovarian clear cell carcinomas

Ovarian clear cell carcinomas often show a spherule‐like mucoid stroma. In ascitic fluid, they form spheroids with a hollow acellular space. In spite of the absence of stromal cells, both the mucoid stroma and hollow spheroids contain abundant extracellular matrix, and one of the major components is hyaluronan. It has been suggested that tumor‐derived hyaluronan plays a significant role in the formation of these structures. To clarify this, a hyaluronan inhibition assay was performed on HAC‐2, a clear cell carcinoma cell line, in vitro. When hyaluronan synthesis was inhibited by 4‐methylumbelliferone, HAC‐2 failed to show the spherule‐like accumulation of hyaluronan or hollow spheroids. Inhibition of hyaluronan synthesis was associated with the reduction of cell growth. Analysis of 28 archival ascites cytology specimens showed that clear cell carcinomas expressed hyaluronan more frequently than serous carcinomas (11 of 14 vs 3 of 14, respectively, P < 0.05). All of these facts indicate that tumor‐derived hyaluronan is essential for the formation of the mucoid stroma or hollow spheroids, and that hyaluronan is also involved in the regulation of cell growth in ovarian clear cell carcinomas. The inhibition of hyaluronan synthesis could be a potential adjunctive therapy for refractory clear cell carcinomas outside the ovary.     

Ovarian malignant mixed germ cell tumor with clear cell carcinoma in a postmenopausal woman

Malignant germ cell tumors of the ovary are very rare and account for about 2-5% of all ovarian tumors of germ origin. Most patients are adolescent and young women, approximately two-thirds of them are under 20 years of age, occasionally in postmenopausal women. But clear cell carcinoma usually occurs in older patients (median age: 57-year old), and closely related with endometriosis. Here we report a case of a 55-year old woman with right ovarian mass that discovered by B ultrasonic. Her serum levels of human chorionic gonadotropin (hCG) and α-fetoprotein (AFP) were elevated. Pathological examination revealed the tumor to be a mixed germ cell tumor (yolk sac tumor, embryonal carcinoma and mature teratoma) with clear cell carcinoma in a background of endometriosis. Immunohistochemical staining showed SALL4 and PLAP were positive in germ cell tumor area, hCG, CD30 and OCT4 were positive in epithelial-like cells and giant synctiotrophoblastic cells, AFP, AAT, CD117 and Glyp3 were positive in yolk sac component, EMA and CK7 were positive in clear cell carcinoma, CD10 was positive in endometrial cells of endometriotic area. She was treated with surgery followed by seven courses of chemotherapy. She is well and serum levels of hCG and AFP have been decreased to normal levels.     

Mixed angiosarcoma, clear cell adenocarcinoma and mature teratoma elements in an ovarian tumor: a case report and literature review

Malignant transformation of a mature teratoma in the ovary is a rare event, with an approximate rate of only 1-2%. Here, we report an ovarian tumor with a unique combination of epithelial and non-epithelial malignant components, including mature teratoma elements. A 59 year-old postmenopusal woman underwent total hysterectomy and bilateral salpingo-oophorectomy to remove a huge solid mass of the right ovary. The ovarian tumor was 16 × 12 × 4.5 cm in dimensions, composed of red-brown and greyish-white tissue with several cystic areas. Microscopically, atypical cells immunopositive for both CD31 and CD34 formed irregular ectatic vascular patterns with a high MIB-1 labeling index in red-brown areas. In contrast, tubule-cystic and papillary structures were lined by HNF-1β-immunopositive atypical cuboidal and hobnail cells with clear cytoplasm in greyish-white areas. In addition, normal-looking epithelial and stromal components, including mature squamous, cuboidal and ciliated epithelial cells, and adipose tissues, were observed in red-brown areas, suggesting an ovarian tumor combining angiosarcoma, clear cell adenocarcinoma, and mature teratoma features. We could demonstrate identical X-chromosome inactivation patterns among all three components by human androgen receptor gene (HUMARA) assays, pointing to complex inter-relationships regarding their pathogenesis. These observations suggest that a malignant tumor composed of two characteristic phenotypes arose in mature teratoma.     

Gastric-type mucinous adenocarcinoma of the uterine cervix with neoadjuvant therapy mimicking clear cell carcinoma

Gastric-type mucinous adenocarcinoma, an uncommon subtype of cervical carcinoma, is characterized by a distinct morphology and immunophenotype. Herein, we report a case of a 71-year-old woman who received neoadjuvant radiotherapy and chemotherapy after cervical biopsy revealed moderately differentiated invasive endocervical adenocarcinoma. Subsequently, the outside patient underwent radical hysterectomy with bilateral salpingo-oophorectomy. The post-neoadjuvant therapy hysterectomy specimen showed tumor cells with clear cytoplasm, hyperchromatic nuclei with irregular contours, which mimicked clear cell carcinoma. However, immunohistochemical staining showed that these tumor cells were positive for carcinoembryonic antigen, cytokeratin 7 (diffuse), and cytokeratin 20 (patchy), After review of the pretreatment cervical biopsy specimen, the tumor was favored to represent a gastric-type mucinous adenocarcinoma of the cervix. Pathologists should be aware of this rare tumor and its post-neoadjuvant therapy morphologic changes, which can make diagnosis more challenging.     

雷帕霉素对食管鳞癌细胞裸鼠移植瘤生长及mTOR/4EBP1信号通路的影响

 目的 体内观察雷帕霉素对食管鳞癌细胞生长及mTOR/4EBP1信号通路的影响。方法 给BALB/c-nude小鼠皮下注射食管鳞癌细胞株（EC9706）,建立移植瘤模型,用雷帕霉素及顺铂对肿瘤进行治疗;用RT-PCR及Western blot等观察细胞形态及信号通路中各因子的变化。结果 与对照组相比,雷帕霉素对肿瘤生长具有显著抑制作用（P<0.05）,联合顺铂处理组的抑瘤作用更明显;雷帕霉素在体内降低了mTOR和p-4EBP1的表达,并使4EBP1的表达水平升高。结论 雷帕霉素在体内能明显抑制食管鳞癌细胞EC9706裸鼠移植瘤的生长并抑制mTOR/4EBP1信号通路的活性。     

Low expression of miR-99b promotes progression of clear cell renal cell carcinoma by up-regulating IGF1R/Akt/mTOR signaling

Objective: Dysfunctions of microRNAs have been implicated in the progression of clear cell renal cell carcinoma (ccRCC). Here, we investigated the roles of miR-99b and miR-99b* in ccRCC development. Methods: The expression levels of miR-99b and miR-99b* in tumor and tumor-adjacent tissues from ccRCC patients were quantified by quantitative Real-Time PCR (qRT-PCR). MicroRNA mimics and inhibitors were employed to evaluate the functions of miR-99b and miR-99b*. The effects of miR-99b on the proliferation and migration of ccRCC cells were analyzed by MTT and wound-healing assays, respectively. The effect of miR-99b on the expression of its target gene IGF1R and mTOR was determined by western blotting and qRT-PCR. Results: The abundances of miR-99b and miR-99b* were lower in ccRCC tissues than in the tumor-adjacent tissues from patients. Similarly, the expression of these two microRNAs was higher in the normal kidney HK-2 cells than in the ccRCC cell lines. Moreover, miR-99b and miR-99b* inhibited the proliferation and migration of ccRCC cells. MiR-99b was found to down-regulate IGF1R and mTOR expression, likely through targeting their mRNAs to induce degradation. Consistently, the mRNA levels of IGF1R and mTOR were higher in ccRCC tissues than in the tumor-adjacent tissues, and Akt, a downstream factor of IGF1R, was highly activated correspondingly in ccRCC tissues. Conclusion: The low expression of miR-99b and miR-99b* contributes to ccRCC development and miR-99b acts as an onco-suppressor by suppressing IGF1R and mTOR expression to down-regulate IGF1R/AKT/mTOR signaling.     

Immunocytochemistry for hepatocyte nuclear factor-1beta (HNF-1beta): a marker for ovarian clear cell carcinoma

Recent microarray studies have shown that the expression of hepatocyte nuclear factor-1beta (HNF-1beta) was significantly up-regulated in clear cell carcinoma (CCC) of the ovary. HNF-1beta may be a useful marker for CCC in peritoneal fluid cytology. We designed an experimental study using three CCC cell lines to evaluate the influence of alcohol fixation or air drying on immunocytochemistry for HNF-1beta. Each cell line was cultured on chamber slides or transplanted into the abdominal cavity of nude mice, then the slides or ascites smears of nude mice were immunostained with or without microwave-mediated epitope retrieval. Immunoreactivity with HNF-1beta, which was either alcohol-fixed or air-dried, was noticeably improved after microwave heating. In contrast, two serous adenocarcinoma cell lines never showed immunoreactivity. Based on these results, 21 archival Papanicolaou-stained slides of peritoneal fluid (5 CCCs, 12 serous, 2 mucinous, and 2 endometrioid adenocarcinomas) were decolorized and immunostained under heating pretreatment. Five of 5 CCCs were distinctively positive for HNF-1beta, whereas all non-CCC ovarian cancers were negative for this protein. The present study clearly demonstrated that heating epitope retrieval improved the immunoreactivity for a nuclear protein in alcohol-fixed or air-dried cytology specimens. HNF-1beta is likely to be helpful for the diagnosis of CCC in the peritoneal fluid.     

IGFBP-1 is expressed specifically in ovarian clear cell adenocarcinoma

Sugita S, Morishita Y, Kano J, Furuya S, Shiba‐Ishii A & Noguchi M
(2011) Histopathology 58 , 729–738
IGFBP‐1 is expressed specifically in ovarian clear cell adenocarcinoma Aims: To investigate the specific expression of insulin‐like growth factor binding protein‐1 (IGFBP‐1) in ovarian clear cell adenocarcinoma (CCA). Methods and results: Immunohistochemistry and in situ hybridization for IGFBP‐1 were performed in normal endometrium, placenta, and 100 surgically resected cases of ovarian cancer including 31 CCAs and 69 non‐CCAs. Immunohistochemistry for hepatocyte nuclear factor‐1 (HNF‐1β) was also examined in all cases. Specific expression of IGFBP‐1 was confirmed in secretory endometrium, decidua of placenta and Arias‐Stella glands of miscarriage material. Among ovarian cancers, almost all cases of CCA showed expression of both IGFBP‐1 protein and mRNA, but non‐CCA hardly expressed IGFBP‐1. There was a significant difference between CCA and non‐CCA in the expression of IGFBP‐1 protein and mRNA. No correlation was found between the rate of IGFBP‐1 expression and pathological T and N factors of the tumour–node–metastasis (TNM) classification. All CCA cases except for one exhibited expression of HNF‐1β protein, whereas only 15.9% of non‐CCAs did so. Conclusion: The expression of IGFBP‐1 in CCA is more specific than that of HNF‐1β. IGFBP‐1 shows expression by decidual endometrium and Arias‐Stella glands, and CCA also exhibits characteristic expression. These results indicate that IGFBP‐1 is a immunohistochemical marker for CCA.     

Clinicopathologic study on clear cell hepatocellular carcinoma

In order to study the clinicopathologic characteristics of the clear cell variant of hepatocellular carcinoma (HCC), 215 consecutive cases measuring less than 5 cm in diameter were reviewed. The cases were divided into clear cell HCC (20 cases); focal clear cell HCC (77 cases); and non-clear cell HCC (118 cases). Clinical and pathological findings were compared among these groups. Clear cell HCC was moderately differentiated in 80% of cases and the incidence was not related to tumor size. The male to female ratio was 2.3:1, lower than the 6.9:1 of non-clear cell HCC. The association rate with liver cirrhosis was 90%, higher than the 59.3% of non-clear cell HCC. Three- and five-year survival rates, and no recurrence time were 54.5%, 33.3%, and 564 days, respectively, lower than the findings of 74.3%, 46.1%, and 770 days for non-clear cell HCC. But there is no significant difference in prognosis between both groups. Ultrastructurally, clear cells showed abundant glycogen storage and a variable degree of fat vacuoles, with a marked reduction of the number and size of organelles in the 8 cases examined. Non-clear cells of focal clear cell and non-clear cell HCC showed a moderate degree of glycogen storage in 85.7% and 28.6% of the seven cases examined from each group, with significant difference. It was concluded that clear cell HCC occurs mostly in the moderately differentiated form and is characterized by high female prevalence, high rate of association with liver cirrhosis, and has no significant difference in prognosis compared with non-clear cell HCC.     

多囊性肾透明细胞癌的病理学诊断

目的：探讨多囊性肾透明细胞癌的临床病理特点。方法：对1例多囊性肾透明细胞癌进行了免疫组化染色,并进行文献复习。结果：本例右肾肿物18年。大体见肿物由多发不等的囊腔组成。镜下囊内壁主要由单层立方或柱状上皮被覆,部分为多层并有乳头形成。瘤细胞胞质透亮,无明显异型性。癌细胞免疫表型cytokeratin、CEA和vimentin呈阳性表达。本例诊断为多囊性肾透明细胞癌。结论：多囊性肾透明细胞癌是一种罕见的肾癌病理类型。临床上主要采用根治切除术。本瘤的生物学行为属于低度恶性肿瘤。     

Vesical clear cell adenocarcinoma V. Nephrogenic adenoma: a diagnostic problem

Following the diagnosis of nephrogenic adenoma in a bladder lesion, which was later interpreted as early clear cell adenocarcinoma, the morphological and immunocytochemical features of these two lesions were reviewed to see if differences could be established for future diagnostic application. The architecture, extent, cell type, nuclear pleomorphism, presence of mitotic figures and glycogen content were recorded in 28 nephrogenic adenomas and the clear cell carcinoma. Similarly, the immunoreactivity for CAM 5.2, LP34, EMA and CEA of 10 nephrogenic adenomas and the clear cell carcinoma were compared. Proliferation rate in five nephrogenic adenomas and the carcinoma was assessed by antibody M1B1. Many of the features showed differences in degree or extent (clear cell change, nuclear pleomorphism, CAM 5.2 and CEA positivity). The only features distinct to clear cell carcinoma were the presence of solid islands, mitoses greater than 1/10 HPF (HPF area = 0.4 mm²) and M1B1 counts in excess of 29/200 in clear cell carcinoma (range 30/200-83/200). Only the high M1B1 count was present in the first biopsy of the clear cell carcinoma.     

AM激活mTOR信号通路对人卵巢癌细胞增殖的影响

目的探讨肾上腺髓质素(Adrenomedullin,AM)通过激活mTOR(哺乳动物雷帕霉素靶蛋白)信号途径对人卵巢癌细胞HO8910增殖的影响。方法应用AM及mTOR拮抗剂(雷帕霉素)诱导卵巢癌细胞,CCK8法检测AM及雷帕霉素对卵巢癌细胞增殖的影响,进一步采用Western印迹法,测定AM对mTOR磷酸化的激活作用结果 AM可促进卵巢癌细胞的增殖,并呈剂量依赖关系及受体依赖关系,而这种促增殖作用可被mTOR信号通路的拮抗剂雷帕霉素所抑制;随着AM浓度的增加,卵巢癌细胞mTOR磷酸化水平随之升高。结论AM介导的mTOR信号通路促进卵巢癌细胞增殖,为卵巢癌的分子靶向治疗提供新的思路。     

蛋白激酶B抑制剂哌立福辛对卵巢透明细胞癌ES2细胞系的杀伤作用

目的探讨蛋白激酶B抑制剂哌立福辛对卵巢透明细胞癌ES2细胞系的杀伤作用。方法体外培养人卵巢透明细胞癌ES2细胞,以不同药物浓度(0、7、9、11、13和15μmol/L)哌立福辛处理细胞24 h,用细胞计数试剂盒CCK-8检测细胞的存活率;DAPI染色观察细胞核的形态;用annexin V-FITC/PI凋亡检测试剂盒及流式细胞术检测细胞的凋亡;实时荧光定量PCR法检测Ki-67和MCM2 mRNA表达。结果哌立福辛能够降低细胞存活率(P0.05),在一定程度上呈浓度依懒性;哌立福辛作用于细胞后,荧光显微镜下可见大量的核碎裂及凋亡小体;随着哌立福辛浓度的增加,细胞的凋亡率增加(P0.01);药物作用48 h后,Ki-67和MCM2 mRNA表达水平明显降低(P0.01)。结论哌立福辛对人卵巢透明细胞癌ES2细胞有明显的杀伤作用,并且可以负性调节Ki-67和MCM2mRNA的表达。     

Ovarian clear cell carcinoma with plasma cell‐rich inflammatory stroma

We report a case of clear cell carcinoma (CCC) of the ovary with plasma cell‐rich inflammatory stroma, a recently proposed subtype of CCC, and present the cytological findings. The patient was a 48‐year‐old woman, who was incidentally found to have a right ovarian tumor during the preoperative work‐up for an early‐stage adenocarcinoma of the uterine cervix. Cytological examination of an imprint smear of the ovarian tumor and peritoneal washing revealed solid cell clusters of irregular, often dendritic shapes, which were intermingled with many inflammatory cells. “Raspberry bodies” were not found. Histopathological examination of the extirpated ovarian tumor showed the features of CCC with plasma cell‐rich inflammatory stroma. This subtype of ovarian CCC poses cytological and histological diagnostic problems, and its differentiation from dysgerminoma is often difficult, because it mostly lacks the hyaline or mucoid stroma. Irregularly shaped clusters of large polyhedral cells, coarsely clumped nuclear chromatin, and plasma cell‐rich inflammatory infiltrates suggest CCC, but the cytological differences between dysgerminoma and CCC are often subtle, and immunohistochemical examinations for cytokeratin 7 or epithelial membrane antigen may be necessary. Diagn. Cytopathol. 2017;45:128–132. © 2016 Wiley Periodicals, Inc.     

Cutaneous clear cell neoplasms: a systematic review

Neoplasms of the skin with clear cell morphology consist of a broad variety of primary and metastatic lesions with epithelial, mesenchymal and melanocytic differentiation. The clear cell changes are attributable to a variety of causes from artifact to accumulation of intracellular substances such as glycogen, mucin, and/or lipids. The clear cell morphology is easily identified by the pathologist, however is it not specific. Additional salient histologic findings may be subtle and therefore ancillary studies such as immunohistochemistry and molecular studies may be necessary to help elucidate a diagnosis. This review features cutaneous neoplasms characterized by clear cell changes, organized by histogenesis. It discusses the differential diagnosis of these lesions with a focus on helpful immunohistochemical and molecular studies which may aid in the clinicopathologic evaluation of these challenging lesions.