Tubal Origin of 'Ovarian' Low-grade Serous Carcinoma

Jie Li; Nisreen Abushahin; Shujie Pang; Li Xiang; Setsuko K Chambers; Oluwole Fadare; Beihua Kong; Wenxin Zheng


Mod Pathol. 2011;24(11):1488-1499. 

In This Article

Materials and Methods

Case Selection

Slides of adnexal tissues, all resected between 2000 and 2010 from a total of 178 patients, were used in this study. All were obtained from the archived files of the pathology department at the University of Arizona College of Medicine following approval from its institutional review board. The first group comprised 128 adnexal serous tumors, including serous cystadenomas (n=48), serous borderline tumors (n=42), and low-grade serous carcinomas (n=38), that had all previously been categorized as being of ovarian origin based on conventional criteria, that is, predominant localization of disease. These cases will hereafter be referred to as 'ovarian'. The diagnosis of the ovarian tumors was typically established by the morphologic examination only, with rare cases requiring immunohistochemical confirmation. The histologic diagnoses were verified by concurrent pathologic review by two co-authors (NA and WZ) using a variation on published criteria.[17] The second group consisted of 98 ovaries and fallopian tubes from 50 patients who had previously undergone a total hysterectomy and bilateral salpingo-oophorectomy for uterine diseases or for adnexal pathologic changes that were ultimately deemed to be non-neoplastic. Patients' age in the second group was matched to the first group. All other relevant clinical information was extracted from the pathology reports and clinical records as necessary.

Morphologic Analysis

Among the 98 ovaries, 48 showing ovarian surface epithelium covering at least 10% of the ovarian surface and at least 1 ovarian epithelial inclusions in a single tissue section were studied. The number of ovarian epithelial inclusions in each section (case) was counted. The morphologic features of ovarian surface epithelium and ovarian epithelial inclusions (columnar versus attenuated/low cuboidal) were documented for each case. The number of secretory and ciliated cells within the tubal fimbria epithelium was evaluated by light microscopy.

Immunohistochemical Analyses

Regular Immunohistochemical Method Immunohistochemical studies, using antibodies to PAX8, calretinin, tubulin, and MIB1, were performed. PAX8 is a member of the PAX gene family, consisting of nine well-described transcription factors (PAX 1–9).[18] PAX8 expression is present in the secretory, but not ciliated epithelial cell population of the normal human fallopian tube,[19] and may play a role in differential diagnosis of ovarian epithelial cancers from histologic mimics.[19–23] Calretinin is a recognized mesothelial cell marker, which has been successfully applied to differentiate ovarian serous carcinomas from mesotheliomas, although a few ovarian high-grade serous carcinomas can have weak and focal positivity.[24–26] Tubulin identifies cellular surface cilia, and is therefore an appropriate marker to identify ciliated cells.[27] The MIB1 antibody is directed against the proliferation marker Ki-67, which is present in the G1, S, M, and G2 phases of the cell cycle. Proliferative endometrial tissue sections served as positive controls for PAX8 and MIB1 staining, fallopian tube sections for tubulin, and normal mesothelium for calretinin. Negative controls were carried out by replacing primary antibodies with class-matched mouse immunoglobulin G on parallel sections. The subcellular staining localization is nuclear for PAX8 and MIB1, cytoplasmic for calretinin, and apical/glycocalyceal for tubulin.

Dual Immunohistochemical Staining To better highlight the different cell populations within the ovarian epithelial inclusions, especially as the lining cells were occasionally morphologically indifferent, we performed dual immunohistochemical staining with both PAX8 and calretinin in representative ovarian epithelial inclusion sections. The dual-staining procedure has previously been described elsewhere.[28] The specific signal for PAX8 was visualized by incubation with peroxidase followed by incubation with Diaminobenzidine as chromogen creating a brown nuclear staining product. Then, calretinin immunohistochemical was performed by using alkaline phosphatase generating a bright red product in cytoplasm.

Scoring To potentially gain some insight into the histogenesis of the ovarian tumors based on the differential distribution (qualitative and quantitative) of secretory cells and ciliated cells in the ovaries (ovarian surface epithelium and ovarian epithelial inclusions) and fallopian tubes, the secretory-to-ciliated cell ratios in these tissues were determined. Ciliated cells were counted using routine light microscopy, by counting at least 500 cells or all available cells if the total number of cells was <500. The secretory-to-ciliated cell ratio was determined for selected ovarian epithelial inclusions in each case (see below). Immunohistochemical determinations of the secretory-to-ciliated cell ratio were based on the PAX8- positive/tubulin-positive ratio in a representative focus. The proliferative index in each case was determined by selecting the most proliferative region in the slide of interest, selecting a microscopic field, counting the number of cells displaying nuclear MIB1 positivity and the number of cells in the background, and extrapolating the findings to the whole slide.

Statistical Analysis

The data were analyzed by standard contingency table methods and nonparametric Mann–Whitney U-tests using the Eproliferative index LOG (Epicenter Software, Pasadena, CA, USA) and Stat View (SAS Institute, Cary, NC, USA) computer package programs. Fisher's exact tests were used to calculate two-sided P-values.


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