The cytologic diagnosis of malignant neoplasms in pleural and peritoneal effusions

This study presents data on 3,011 pleural and peritoneal effusion specimens that were examined over a three-year period (1982 to 1984). Totals of 812 (44%) of 1,846 pleural and 423 (36%) of 1,165 peritoneal specimens were positive for malignant cells. While 535 patients had malignant pleural effusions, 254 patients had malignant peritoneal effusions, and 57 had both malignant pleural and peritoneal effusions. The most common primary neoplasms causing malignant pleural effusions were carcinomas of breast (24%) and lung (19%) and lymphoreticular neoplasms (16%). The most common primary neoplasms causing malignant peritoneal effusions were carcinomas of ovary (32%) and breast (13%) and lymphoreticular neoplasms (7%). There was an average interval of more than 30 months between the histologic diagnosis of the primary neoplasm and the diagnosis of malignant effusions in patients with carcinoma of breast, lymphoreticular neoplasm and malignant melanoma. The average time until death following the diagnosis of a malignant effusions was five months or less, except for patients with carcinoma of the breast and carcinoma of the ovary. One hundred twenty-five patients (15%) presented with malignant effusions caused by neoplasms of unknown primary sites. The most common primary neoplasms that were later diagnosed were, in decreasing order of frequency, carcinoma of the ovary, carcinoma of the lung and lymphoreticular neoplasms.     

Gene expression signatures differentiate ovarian/peritoneal serous carcinoma from breast carcinoma in effusions

Ovarian/primary peritoneal carcinoma and breast carcinoma are the gynaecological cancers that most frequently involve the serosal cavities. With the objective of improving on the limited diagnostic panel currently available for the differential diagnosis of these two malignancies, as well as to define tumour‐specific biological targets, we compared their global gene expression patterns. Gene expression profiles of 10 serous ovarian/peritoneal and eight ductal breast carcinoma effusions were analysed using the HumanRef‐8 BeadChip from Illumina. Differentially expressed candidate genes were validated using quantitative real‐time PCR and immunohistochemistry. Unsupervised hierarchical clustering using all 54,675 genes in the array separated ovarian from breast carcinoma samples. We identified 288 unique probes that were significantly differentially expressed in the two cancers by greater than 3.5‐fold, of which 81 and 207 were overexpressed in breast and ovarian/peritoneal carcinoma, respectively. SAM analysis identified 1078 differentially expressed probes with false discovery rate less than 0.05. Genes overexpressed in breast carcinoma included TFF1, TFF3, FOXA1, CA12, GATA3, SDC1, PITX1, TH, EHFD1, EFEMP1, TOB1 and KLF2. Genes overexpressed in ovarian/peritoneal carcinoma included SPON1, RBP1, MFGE8, TM4SF12, MMP7, KLK5/6/7, FOLR1/3, PAX8, APOL2 and NRCAM. The differential expression of 14 genes was validated by quantitative real‐time PCR, and differences in 5 gene products were confirmed by immunohistochemistry. Expression profiling distinguishes ovarian/peritoneal carcinoma from breast carcinoma and identifies genes that are differentially expressed in these two tumour types. The molecular signatures unique to these cancers may facilitate their differential diagnosis and may provide a molecular basis for therapeutic target discovery.     

Characterization of glycoconjugates released in vitro by human ovarian carcinoma cells isolated from effusions.

Ovarian carcinoma cell clusters were isolated from patient effusions. The glycoconjugates released to culture medium in vitro were characterized by electrophoretic, immunoassay and gel filtration procedures. Metabolically radiolabelled glycoconjugates were heterodisperse with respect to molecular weight and this heterodispersity was independent of incubation time in vitro. This heterodispersity was also characteristic of mixed Mullerian tumor cells of endometrial origin whereas mesothelial cells released a discrete glycoconjugate of MW 65-70 kDa. Multiple Coomassie blue-stained polypeptides were released by the carcinoma cells. These polypeptides were not adsorbed serum components as assessed by immunodiffusion analyses. Periodic acid-Schiff-reactive macromolecules appeared only at the top of electrophoresis gels. The high molecular weight glycoconjugates synthesized by ovarian carcinoma cells precipitated with an effusion globulin fraction at low ionic strength, but the low molecular weight components (40-70 kDa) were soluble. Immunoprecipitation with anti-Ig failed to precipitate carcinoma glycoconjugates. Antisera raised against the released carcinoma macromolecules precipitated carcinoma glycoconjugates and normal ovarian polypeptides. Antisera raised against normal ovarian macromolecules precipitated ovarian polypeptides but reacted only slightly with carcinoma glycoconjugates. Immunodiffusion analyses showed the presence of alpha 1-acid glycoprotein and carcinoembryonic antigen (CEA)-like components in the carcinoma glycoconjugates. The presence of CEA-like glycoconjugates was confirmed by immunoprecipitation. The antigens and antisera for different histologic types of ovarian carcinoma were cross-reactive. The presence of beta 2-microglobulin suggested that some of the glycoconjugates were shed from the cell surface.     

Proliferative response of lymphocytes from ovarian cancer patients to autologous tumor cells

Summary Peripheral blood lymphocytes (PBL) from 43 patients with histologically confirmed ovarian carcinoma were stimulated in mixed lymphocyte-tumor culture (MLTC) with purified autologous tumor cells. Positive results, assessed as lymphocyte proliferation, were observed in 21 cases (48.8%). Lymphoid cells associated with ascitic fluid or infiltrating solid masses were in general less reactive than PBL as only 3/11 cases had positive MLTC. Tumor cells isolated from peritoneal effusions showed no significant difference in stimulatory potential as compared to the primary tumor. These results suggest that in an appreciable proportion of ovarian carcinoma patients (approximately 50%), lymphocytes have the potential to react to autologous tumor cells. Comprehension of the immunological mechanisms of antitumor resistance may have direct practical relevance for more effective treatment of neoplasms.     

Synthesis and release of glycoconjugates in vitro by human ovarian carcinoma cells isolated from effusions

Human ovarian carcinoma cells were isolated as multicellular aggregates from patient effusions. Freshly isolated cells were incubated with radioactive glycoconjugate precursors and the glycoconjugates released to culture medium were analyzed. The glycoconjugates appeared to contain both O- and N-linked oligosaccharides. Both fucosylation and sialylation of glycoconjugates occurred. The results of SDS-PAGE analysis showed the presence of a very heterogeneous array of glycoconjugates with no discrete components resolved. The results of glycoconjugate analyses were independent of carcinoma histology, differentiation, progression, and patient chemotherapy. In contrast, mesothelial cells synthesized a major glycoconjugate of molecular mass 65 KDa. The heterogeneous array of carcinoma-derived glycoconjugates may be transformation-related and may have utility in tumor diagnosis and prognosis.     

Isolation and phenotypic characterization of tumor cells of patients with a diagnosis of ovarian cancer

Ovarian cancer is the fifth leading cause of cancer-related deaths. It causes approximately 125,000 deaths per year worldwide; its diagnosis is made in advanced stages resulting in a high mortality rate. The objective of the study was optimizing the isolation of cells obtained from the solid tumor and ascitic fluid of patients with ovarian cancer and the phenotype with markers related to the epithelial–mesenchymal transition. For this, the solid tumor tissue was disaggregated and cultivated with different methodologies. As a result, cell growth was obtained and epi-immunofluorescence was performed using antibodies against E-cadherin, EpCAM, N-cadherin, vimentin, CD133, and CD44. The primary culture from the solid tumor was obtained using Dispase II and DMEM/F12. Finally, heterogeneity was detected in terms of the expression of mesenchymal and epithelial type markers in the two types of isolated cells. Additionally, CD133 and CD44 expression was detected, proteins associated with the tumor stem cells phenotype.     

Primary culture of ovarian surface epithelial cells and ascites-derived ovarian cancer cells from patients

Our laboratory has refined the technique for isolating primary cultures of normal human ovarian surface epithelial (OSE) cells by combining two different protocols involving the enzymatic and mechanical removal of OSE cells from ovarian biopsies. A simple protocol of obtaining primary epithelial ovarian cancer (EOC) cells from the ascites fluid removed from patients with high-grade ovarian cancer is also described. These methods allow for the direct application of many molecular and cellular analyses of normal versus cancer cells isolated freshly from patients, with the added potential for retrospective analyses of archived cells and tissues. Thus, we have included optional steps for the immediate preparation of ascites-derived EOC cells to be used for subsequent cytological analyses. Initial isolation of OSE or EOC cells can be completed in 1 h, and primary cells are further expanded in culture for several weeks.