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Eph receptor tyrosine kinases and their cell-surfacebound ligands, the ephrins, play key roles in diverse biological processes. Eph receptors comprise the largest family of receptor tyrosine kinases consisting of eight EphA receptors (with five corresponding ephrinA ligands) and six EphB receptors (with three corresponding transmembrane ephrinB ligands). Originally identified as neuronal pathfinding molecules, EphB receptors and ephrinB ligands are later proved to be crucial regulators of vasculogenesis and embryogenesis. More studies indicate that Eph receptors are involved in angiogenesis and tumorigenesis. This study aimed to investigate the expression of EphB2 and EphB4 in breast carcinomas. Semiquantitative RT-PCR and immunohistochemistry were used to examine the expression patterns of EphB2 and EphB4. Clinicopathological and survival correlations were statistically analyzed in a series of 94 breast carcinomas, 9 normal specimens and 4 breast carcinoma cell lines. 1(1%), 16(17%), 29(31%), 48(51%) of the 94 tumors were negative, weak, moderate and strong EphB2 protein expression, respectively. 6(6%), 27(29%), 28(30%), 33(35%) of the tumors were negative, weak, moderate and strong EphB4 expression, respectively. Both EphB2 and EphB4 RT-PCR products could be detected in all specimens. Increased EphB2 protein expression was negatively associated with overall survival, and there was a trend that increased EphB2 protein expression was correlated with shorter disease free survival, while EphB4 protein expression was associated with histological grade and stage. EphB4 membrane staining was increased with S phase fraction and associated with DNA aneuploidy. These findings indicate that both EphB2 and EphB4 are involved in the development of breast cancer and that both molecules could be potential predictive markers.

NKX3.1is an androgen regulated gene that is largely specific to the prostate for expression and it is predicted to encode a homeobox protein. Null alleles of NKX3.1in mice results in impaired prostate development as well as hyperplasia and dysplasia of the prostate. In addition, the NKX3.1gene maps to a region of high loss of heterozygosity in prostate cancer in humans, suggesting that NKX3.1might have a direct role in prostate carcinogenesis, possibly functioning as a tumor suppressor protein. Previous studies of the levels of NKX3.1mRNA or protein in prostate cancer specimens have resulted in conflicting findings.

The events that mediate tumor progression in ovarian carcinoma are poorly understood to date. This review summarizes our results studying metastasis-associated molecules in advanced-stage ovarian carcinomas, details the co-expression of mRNA of these genes, and discusses their prognostic role. Fifty-five primary and metastatic FIGO stage III–IV ovarian carcinomas were analyzed for the expression of αv and β1 integrin subunits, the matrix metalloproteinases MMP-2, MMP-9, and MT1-MMP, the MMP inhibitor TIMP-2, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), interleukin-8 (IL-8), PEA3 and Ets-1 using mRNA in situ hybridization. Tumor and adjacent stromal cell expression was scored. The association between integrin subunit expression and the expression of MMP, TIMP-2, angiogenic genes, PEA3 and Ets-1 was statistically analyzed. αv integrin subunit mRNA expression in carcinoma cells showed significant association with that of MMP-2 and IL-8 in this cellular compartment, while the presence of β1 integrin subunit mRNA showed similar association with that of PEA3, Ets-1, IL-8, bFGF and MMP-2. Expression of β1 integrin subunit mRNA in stromal cells was associated with that of TIMP-2 and Ets-1 in this compartment. In addition, significant intercellular associations were found between αv integrin subunit mRNA expression in carcinoma cells and stromal cell expression of Ets-1, as well as between stromal cell expression of αv integrin subunit and labeling for IL-8 in carcinoma cells. The presence of β1 integrin subunit mRNA in carcinoma cells showed a significant association with that of Ets-1, IL-8 and bFGF in stromal cells, while the presence of β1 integrin subunit mRNA in stromal cells was associated with tumor PEA3 mRNA expression. To the best of our knowledge, this is the first evidence for coordinated autocrine and paracrine expression of members of these four families of metastasis-associated genes in human cancer. The results of this analysis support experimental data regarding cross-talk between carcinoma cells and peritumoral fibroblasts. They also suggest the existence of a putative activation sequence of metastatic genes, involving the β1 (and possibly αv) integrin subunits, IL-8, PEA3, Ets-1 and MMP in ovarian carcinoma.

We compared the efficiency of immunophenotyping using flow cytometry (FCM) and a combination of morphologic and immunocytochemical studies for detecting malignant cells in 92 effusions. Cytologic results were as follows: carcinoma cells, 43 specimens; benign, 42 specimens; suggestive of nonepithelial malignancy, 7 specimens. After immunocytochemical analysis, 5 benign specimens were reclassified as malignant and 4 malignant epithelial specimens as benign. With FCM, cells positive for Ber-EP4, B 72.3, AH6, and HB-TN were detected in 28 to 36 (64%-82%) of 44 carcinomas but only 2 to 12 (5%-29%) of 41 benign specimens. Significant association was seen for coexpression. Ber-EP4 and AH6 were the most sensitive; Ber-EP4 was the most specific. The presence of cells positive for 3 of 4 markers strongly suggested malignancy (34/44 carcinoma specimens [77%]; 3/41 reactive specimens [7%]). The presence of cells positive for all 4 markers was diagnostic of malignancy (17/44 malignant specimens [39%]; 0/41 reactive effusions [0%]). FCM and immunocytochemical resultsfor Ber-EP4 expression showed excellent association. FCM is a powerful tool for diagnosing difficult effusions and can quantify coexpression of various markers in fresh specimens. By using established cellular markers coupled with biological markers, FCM also has great promise for experimental purposes.

We studied the role of caveolin-1 in tumor progression and prognosis in serous ovarian carcinoma and the association between caveolin-1 and MDR1 expression. The study involved immunohistochemical analysis for caveolin-1 and P-glycoprotein (P-gp) expression in 75 effusions and 90 solid lesions from ovarian and primary peritoneal carcinoma; in situ hybridization for MDR1 messenger RNA (mRNA) expression in 62 effusions and all 90 tumors; and reverse transcription-polymerase chain reaction (RT-PCR) for caveolin-1 mRNA expression in 23 effusions. Immunohistochemical analysis localized caveolin-1 to the cell membrane in 43 effusions and 24 tumors. P-gp membrane expression was detected in 14 effusions and 11 tumors; MDR1 mRNA, in 20 effusions and 30 tumors. Caveolin-1 mRNA was expressed in 19 effusions. Caveolin-1 protein expression showed no association with that of P-gp protein or MDR1 mRNA. The expression of all markers was similar in carcinoma cells in pleural and peritoneal effusions. Caveolin-1 is a novel diagnostic marker for effusions; expression is moderately elevated in tumor cells in effusions, possibly owing to altered signal transduction and metabolism in cancer cells at this site. Expression seems MDR1 independent.

Objective.The aim of this study was to investigate the expression of cell cycle proteins in ovarian carcinoma cells in serous effusions and respective solid tumors.

The aims of this study were firstly, to investigate the expression of E-cadherin complex proteins in ovarian carcinoma cells in serous effusions and in primary and metastatic lesions; and secondly to study the value of these four proteins and calretinin, a mesothelial marker, in the differential diagnosis of ovarian carcinoma cells from reactive mesothelial cells in effusions. Sixty-seven malignant effusions and 97 corresponding primary (n=36) and metastatic (n=61) lesions were immunohistochemically stained for E-cadherin and α-, β-, and γ-catenin. Staining extent and intensity were scored. Effusion specimens were additionally analysed for calretinin immunoreactivity. Membrane immunoreactivity for E-cadherin and α-, β-, and γ-catenin was detected on carcinoma cells in the majority of the effusions, but rarely on reactive mesothelial cells (p<0.001 for all markers). Calretinin immunoreactivity was confined to mesothelial cells (p<0.001). An association was seen between E-cadherin and α-catenin expression, in both effusions and solid tumours, and for β-catenin in solid tumours (range p<0.001 to p=0.014). Up-regulation of all four cadherin complex proteins was seen in carcinoma cells in effusions, when compared with corresponding primary tumours (range p<0.001 to p=0.028). As with effusions, metastatic lesions showed up-regulation of α-, β-, and γ-catenin when compared with primary carcinomas (p=0.002–0.015). Carcinoma cells in effusions showed in addition elevated levels of E-cadherin when compared with metastatic lesions (p<0.001). Staining results in effusions showed no association with effusion site, tumour type or histological grade. Immunoblotting on 29 malignant effusions confirmed the presence of all four proteins in the majority of samples and co-precipitation of E-cadherin and β-catenin was seen in ten specimens examined. E-cadherin complex proteins are widely expressed in ovarian carcinoma cells. Together with calretinin, they form a powerful battery of markers for the cytological diagnosis of carcinoma cells in effusions. The up-regulation of E-cadherin complex proteins in serous effusions and metastatic lesions may mark an early metastatic phenotype and possibly mediates survival of tumour cells at these sites through the inhibition of apoptosis. Copyright © 2000 John Wiley & Sons, Ltd.

The aims of this study were firstly, to investigate the expression of E‐cadherin complex proteins in ovarian carcinoma cells in serous effusions and in primary and metastatic lesions; and secondly to study the value of these four proteins and calretinin, a mesothelial marker, in the differential diagnosis of ovarian carcinoma cells from reactive mesothelial cells in effusions. Sixty‐seven malignant effusions and 97 corresponding primary (n=36) and metastatic (n=61) lesions were immunohistochemically stained for E‐cadherin and α‐, β‐, and γ‐catenin. Staining extent and intensity were scored. Effusion specimens were additionally analysed for calretinin immunoreactivity. Membrane immunoreactivity for E‐cadherin and α‐, β‐, and γ‐catenin was detected on carcinoma cells in the majority of the effusions, but rarely on reactive mesothelial cells (p<0.001 for all markers). Calretinin immunoreactivity was confined to mesothelial cells (p<0.001). An association was seen between E‐cadherin and α‐catenin expression, in both effusions and solid tumours, and for β‐catenin in solid tumours (range p<0.001 to p=0.014). Up‐regulation of all four cadherin complex proteins was seen in carcinoma cells in effusions, when compared with corresponding primary tumours (range p<0.001 to p=0.028). As with effusions, metastatic lesions showed up‐regulation of α‐, β‐, and γ‐catenin when compared with primary carcinomas (p=0.002–0.015). Carcinoma cells in effusions showed in addition elevated levels of E‐cadherin when compared with metastatic lesions (p<0.001). Staining results in effusions showed no association with effusion site, tumour type or histological grade. Immunoblotting on 29 malignant effusions confirmed the presence of all four proteins in the majority of samples and co‐precipitation of E‐cadherin and β‐catenin was seen in ten specimens examined. E‐cadherin complex proteins are widely expressed in ovarian carcinoma cells. Together with calretinin, they form a powerful battery of markers for the cytological diagnosis of carcinoma cells in effusions. The up‐regulation of E‐cadherin complex proteins in serous effusions and metastatic lesions may mark an early metastatic phenotype and possibly mediates survival of tumour cells at these sites through the inhibition of apoptosis. Copyright © 2000 John Wiley & Sons, Ltd.

Differential diagnosis between lymphomas and reactive lymphoid proliferations often requires ancillary techniques and morphologic evaluation. Flow cytometry (FCM) and polymerase chain reaction (PCR) can aid the detection of monoclonal B-cell populations. In the present study, the sensitivity and specificity of these two methods in the study of cytology specimens were compared. Eighty-six cytologic specimens from 81 patients (lymph nodes, solid organs, and body cavities) were evaluated. These specimens were taken from three groups of patients: those who underwent an initial evaluation for suspected lymphoma; those who were previously diagnosed with B-cell lymphoma and were now evaluated for possible disease recurrence; and those who were diagnosed with a nonhemato-logic malignancy. Histologic diagnosis was available for 51 samples. All samples were tested by FCM for the detection of monoclonality using kappa:lambda ratio and for clonal immunoglobulin heavy chain (IgH) gene rearrangements using a single-round PCR after cytologic evaluation. Tissue morphology, FCM and PCR results, and clinical findings in specimens without histologic diagnosis were correlated. Histologic evaluation (N = 51) revealed 44 specimens with B-cell malignancy. Twenty of the 44 lymphoma specimens (45) were accurately diagnosed in cytologic smears, 18 (41) were classified as suspicious of lymphoma, and 6 (14) were diagnosed as reactive. FCM had superior sensitivity compared with PCR (77 vs. 64). Fifty-six percent of specimens with B-cell malignancy were FCM+/PCR+, 23 were FCM+/PCR-, 14 were FCM-/PCR+, and 7 were FCM-/PCR-. The combined use of FCM and PCR resulted in a diagnosis of B-cell lymphoma in 41 (93) of 44 B-cell lymphoma specimens and increased the sensitivity of fine needle aspiration by 48. Both FCM and PCR aid in the diagnosis of lymphoid lesions in cytology specimens, and both can detect monoclonal B-cell populations that may be interpreted in cytology smears as reactive, even by experienced cytologists. Although FCM had higher sensitivity than PCR test in the present study, their combined use should be considered because of a relatively large number of specimens that were detected as monoclonal only with PCR.

Objective.The objective of this study was to evaluate the prognostic significance of clinical and histopathologic factors, including a new grading system focusing on the invasive tumor front.

Abstract: The detection of malignant cells in pleural, peritoneal, and pericardial fluids of cancer patients marks the presence of metastatic disease and is associated with a grave prognosis. We evaluated five epithelial markers for the detection of cancer cells in 94 fresh pleural, peritoneal and pericardial effusions. Eighty-four of the samples were regarded as adequate for analysis after evaluation of cytological smears, including 61 samples from patients known to have gynaecological neoplasms. The other 23 samples were from patients with various non-gynaecological malignancies or tumours of unknown origin. Our control cases were 10 fallopian tubes not affected by any malignancy and 12 malignant mesotheliomas. Cell blocks from all cases were stained for CA-125, BerEP4, carcinoembryonic antigen (CEA), BG8 (Lewis Y blood antigen), and B72.3 (TAG-72). Fifty-one of 84 cases were diagnosed as malignant or suggestive of malignancy in cytological smears and/or cell block sections. However, staining for epithelial markers highlighted the presence of malignant cells in 7 additional cases. When membrane staining was evaluated, the sensitivity of the markers studied in detecting malignant cells was as follows: CA-125: 88%, BerEP4: 78%, CEA: 26%, BG8: 86%, B72.3: 79%. Membrane positivity for CEA, B72.3 and BerEP4 was not detected in reactive mesothelial cells. However, membranous staining in mesothelial cells was evident in 13% and 31% of cases with the use of BG8 and CA-125, respectively. Weak cytoplasmic staining for CEA was observed in mesothelial cells in 2 cases. When Ber-EP4, B72.3, and BG8 staining results in cancer cells were combined, the following sensitivity levels were observed: BG8+B72.3: 91%; BG8+Ber-EP4: 90%; B72.3+Ber-EP4: 93%; BG8+ Ber-EP4+B72.3: 95%. The detection of malignant cells in effusions is facilitated by the use of immunocytochemistry using a wide panel of antibodies. BerEP4 and B72.3 appear to be the best markers when both sensitivity and specificity are considered, followed by BG8, while CEA and CA-125 have a limited role in the detection of metastases from gynaecological tumours owing to the low sensitivity of the former and the low specificity of the latter. Analysis of all staining results should be based on a thorough morphological examination.