PGE2 production in oral cancer cell lines is COX-2-dependent.

It has been suggested that epithelial cyclooxygenase-2 (COX-2) promotes oral carcinogenesis and carcinoma malignancy through increased prostaglandin E(2) (PGE(2)) production. Although oral squamous cell carcinomas (OSCC) often express COX-2, they may also produce PGE(2) in a COX-1-dependent manner. We used 6 isolated cell lines to investigate which COX isoforms OSCC may use for PGE(2) production. COX-1 and -2 expression patterns divided the 6 OSCC cell lines into 3 distinct groups: both COX isoforms low, only COX-1 high, or both COX isoforms high. Multicolor immunohistofluorescence staining confirmed the COX-expression profiles in organotypic 3D cultures and the COX-2 dominance in OSCC tumors. Epidermal growth factor (EGF) stimulation induced COX-2 (but not COX-1) expression and increased PGE(2) production, which was attenuated by COX-2 (but not COX-1) specific inhibition or siRNA-mediated COX-2 gene knockdown. Thus, PGE(2) production in OSCC cell lines was COX-2-dependent.

Nerve growth factor receptor (p75 NTR) and pattern of invasion predict poor prognosis in oral squamous cell carcinoma.

AIMS: To evaluate the expression of p75 neurotrophin receptor (p75(NTR)) in oral squamous cell carcinoma (OSCC). The results were related to tumour node metastasis (TNM) stage, World Health Organization (WHO) grade, invasive front grading (IFG) and prognosis. METHODS AND RESULTS: Immunohistochemically, the expression of p75(NTR) was assessed in 53 T1-T2 OSCCs. Clinical data were recorded prospectively. The end-point was disease-free survival. All tumours expressed p75(NTR), and this expression, both in central/superficial tumour areas and at the invasive front, was associated with poor prognosis (P = 0.03 and P = 0.02) (log rank test). Tumours with marked cellular dissociation (IFG parameter) had more recurrences than tumours with collective tumour cell invasion (P = 0.03). In tumours showing both p75(NTR) at the invasive front and marked tumour cell dissociation, the average risk of recurrence was increased about 17 times (Cox regression analysis) compared with tumours with low p75(NTR) expression and collective invasion. Traditional prognostic systems were of no prognostic significance. CONCLUSION: p75(NTR) was expressed in all OSCCs. p75(NTR) expression and the pattern of invasion were significantly associated with a poor prognosis in OSCCs, and both were better prognostic factors than traditional prognostic parameters. The combination of p75(NTR) expression and the pattern of invasion strongly increased precision in the identification of tumours with poor disease-free survival.

COX-2 expression in striated muscle under physiological conditions.

OBJECTIVES: The role of cyclooxygenase-2 (COX-2) in disease has been extensively studied, (Annu Rev Med (2002) 53:35; N Engl J Med (2001) 345:433) but less information is available with respect to possible physiological functions of COX-2. Information on how and where COX-2 is expressed under physiological conditions may increase our understanding of its physiological role. Previous studies have revealed a COX-2 dependent production of prostanoids under physiological conditions, without entirely determining the source of this production. MATERIALS AND METHODS: To assess COX-2 expression under normal conditions, we analyzed tissue specimens that were removed from 30 healthy study subjects in conjunction with surgical procedure related to insertion of dental implants and from three patients which had muscle tissue from Quadriceps femoris muscle removed as part of surgical treatment of soft tissue sarcomas not directly affecting the muscle tissue. Immunohistochemistry and immunoblotting (Western blotting) was used to assess the presence of COX-2 protein. RESULTS: In 25 of 30 patients (83%), COX-2 protein was expressed in striated muscle, as assessed by immunohistochemistry. All cases had COX-2 expression verified by Western blotting. In none of the 25 subjects with COX-2 expression did we notice concomitant inflammation of the adjacent submucosal tissue. CONCLUSIONS: It is a novel finding that COX-2 is expressed in striated muscle under physiological conditions. COX-2 activity in striated muscle is a possible explanation for the hitherto unknown localization of prostanoids synthesis under physiological conditions.

The prognostic value of a histologic grading system, DNA profile, and MIB-1 expression in early stages of cervical squamous cell carcinomas.

This study evaluated the prognostic importance of a new grading system focusing on the invasive tumor front, DNA profile, and the proliferation marker MIB-1. A complete geographic series of 172 women treated with radical hysterectomy (Wertheim-Meigs) for FIGO stage I-II cervical carcinomas was the target population. The analyses were performed on 141 (82%) squamous cell carcinomas of the complete series. During the period of observation (mean 222 months), 17 recurrences (12.1%) were encountered. Prognostic factors for disease-free survival were lymph node status (P < 0.000001), radical surgical margins (P = 0.00004), and tumor size (P = 0.002). The complete score of the invasive front grading system (IFG), and the individual scores of two variables-pattern of invasion and host response-were all significantly (P = 0.002, P = 0.007, P = 0.0001) associated with pelvic lymph node metastases. Host response was the single most important factor in the IFG system, and it was superior to the complete score in predicting lymph node metastases. The total IFG score was also a significant (P = 0.003) prognostic factor for disease-free survival. DNA ploidy, S-phase fraction, and MIB-1 expression were nonsignificant factors in predicting pelvic lymph node metastases and disease-free survival of the patient. The IFG in the original or modified versions could predict low- and high-risk groups of tumors and therefore be of value in treatment planning for these patients.

The prognostic value of histopathologic grading parameters and microvessel density in patients with early squamous cell carcinoma of the uterine cervix.

The purpose of this study was to investigate the prognostic importance of clinical and histopathologic factors, including malignancy grading systems (MGS), partial index (PI), invasive front grading (IFG), and microvessel density. A complete geographic series of 172 early stage (FIGO I-II) cervical carcinomas treated by Wertheim-Meigs surgery during the period 1965-1990 was studied. The patients were followed up for at least 10 years. Significant prognostic factors for disease-free survival were lymph node status (P < 0.0000001), radical surgical margins (P = 0.00003), and tumor size (P = 0.008). In a multivariate Cox analysis it was shown that lymph node status was the single most important prognostic factor with regard to disease-free survival. The total MGS and the PI scores were highly significantly (P = 0.0001) associated with pelvic lymph node metastases and disease-free survival rate in squamous cell carcinomas. The MGS and the PI systems were superior to the IFG system in predicting lymph node metastases. The total IFG score was also a statistically highly significant (P = 0.003) prognostic factor with regard to disease-free survival in both univariate and multivariate analyses. Microvessel density was a nonsignificant prognostic factor. There was a highly significant (P = 0.002) association between vascular space invasion of tumor cells and the presence of lymph node metastases. In conclusion, histopathologic malignancy grading systems provide valuable prognostic information in patients with early stage squamous cell carcinomas of the uterine cervix.

Alphav integrin expression is a novel marker of poor prognosis in advanced-stage ovarian carcinoma.

PURPOSE: To analyze the possible correlation between expression of the alphav and beta1 integrin chains and survival in advanced-stage ovarian carcinomas, studying two patient groups with extremely different disease outcome. EXPERIMENTAL DESIGN: Sections from 56 primary ovarian carcinomas and metastatic lesions from 34 patients diagnosed with advanced-stage ovarian carcinoma (Fédération Internationale des Gynaecologistes et Obstetristes stages III-IV), divided into long-term (16) and short-term (18) survivors, were evaluated for expression of alphav and beta1 integrin chains using mRNA in situ hybridization. Protein expression was additionally studied in 52 specimens using immunohistochemistry. RESULTS: The mean values for disease-free survival and overall survival were 115 and 132 months for long-term survivors, as compared with 4 and 23 months for short-term survivors, respectively. Expression of alphav integrin mRNA was observed in carcinoma (18 of 56; 32%) and stromal (17 of 56; 30%) cells. beta1 integrin mRNA was similarly detected in carcinoma (25 of 56; 47%) and stromal (19 of 56; 34%) cells. No significant differences were observed when primary and metastatic lesions were compared (P > 0.05). Alphav integrin mRNA was present more often in carcinoma cells in tumors of short-term survivors (P = 0.017 for carcinoma cells). In univariate survival analysis for all cases, alphav integrin mRNA expression in tumor cells correlated with poor survival (P = 0.012). This finding retained its predictive power in a multivariate survival analysis, in which all of the molecules studied previously in this patient cohort were included (P = 0.031). Immunohistochemistry confirmed the differences in alphav integrin expression in tumor cells of short-term as compared with long-term survivors, whereas beta1 integrin protein expression was comparable in the two groups. CONCLUSIONS: To our best knowledge, this is the first evidence associating integrin expression with poor survival in ovarian carcinoma. Alphav integrin is, thus, a novel prognostic marker in advanced-stage ovarian carcinoma.

Ets-1 mRNA expression in effusions of serous ovarian carcinoma patients is a marker of poor outcome.

Ets-1 proto-oncogene is a transcription factor with a role in the activation of metastasis-associated molecules. We recently found that Ets-1 mRNA expression in solid tumors is a marker of poor prognosis in ovarian carcinoma. The objective of this study was to compare the expression of Ets-1 mRNA in effusions and primary and metastatic tumors of serous ovarian carcinoma patients and to evaluate its prognostic role in effusions. Sections from 67 malignant effusions and 90 primary and metastatic lesions were evaluated for expression of Ets-1 using mRNA in situ hybridization. Expression of Ets-1 mRNA was detected in carcinoma cells in 24 of 67 (36%) effusions. Expression in cancer cells was similar in peritoneal and pleural effusions. In solid lesions Ets-1 expression was detected in both tumor cells and stromal cells in 34 of 90 (38%) lesions. Ets-1 expression in tumor cells showed a strong association with that of stromal cells (p <0.001). Ets-1 expression in effusions showed an association with mRNA expression of basic fibroblast growth factor, previously studied in this patient cohort (p = 0.019). Ets-1 expression in solid lesions showed an association with mRNA expression of vascular endothelial growth factor (p <0.001 for both carcinoma and stromal cells), basic fibroblast growth factor (p = 0.007 for carcinoma cells, p = 0.006 for stromal cells), and interleukin-8 (IL-8) (p = 0.001 for tumor cells). Ets-1 mRNA showed upregulation in metastases when compared with effusion specimens (p = 0.028). In univariate survival analysis Ets-1 expression in carcinoma cells in effusions correlated with poor survival (p = 0.003). Our findings confirm the role of Ets-1 as a novel prognostic marker in advanced-stage ovarian carcinoma and extend it to effusion specimens. The elevated expression in solid metastases supports a central role in tumor progression as well. The association between Ets-1 mRNA expression and the expression of angiogenic genes, documented also in our previous study, points to the close link between these molecules, in agreement with the role of angiogenic genes in the transcriptional activation of Ets-1. The identical phenotype of carcinoma cells in pleural and peritoneal effusions provides further evidence for our theory that cells at these sites share similar genotypic and phenotypic profiles.

Ovarian carcinoma cells in serous effusions show altered MMP-2 and TIMP-2 mRNA levels.

The expression of matrix metalloproteinases (MMP) and their inhibitor TIMP-2 in serous effusions from patients with ovarian carcinoma and its association with clinico-pathological parameters were analysed. The findings in carcinoma cells in effusions were compared with corresponding primary and metastatic lesions. Sixty-six effusions and 96 tissue sections were stained for MMP-1, MMP-2 and MMP-9 applying immunohistochemistry (IHC) and analysed for MMP-2, MMP-9 and TIMP-2 expression using mRNA in situ hybridisation (ISH). MMP-2 and MMP-9 mRNA levels in 30 effusions were subsequently analysed using reverse transcription- polymerase chain reaction (RT-PCR). MMP and TIMP expression was detected in both carcinoma and mesothelial cells in effusions. The levels were consistently higher in malignant cells, significantly so for MMP-1 (P=0.016) and MMP-2 (P=0.036) proteins, as well as for TIMP-2 mRNA (P=0.008). In tissue sections, MMP-1, MMP-2 and MMP-9 protein expression was mostly localised to tumour cells, while MMP-2, MMP-9 and TIMP-2 mRNA were predominantly detected in stromal cells. Adenocarcinoma cells in effusions showed a significant upregulation of MMP-2 expression compared with primary tumours, with a concomitant downregulation of TIMP-2. RT-PCR demonstrated the presence of MMP-2 and MMP-9 in 28/30 and 0/30 specimens, respectively. MMP and TIMP are thus mainly synthesised by cancer cells in effusions, while stromal cells have a similar role in solid tumours. MMP-1 and MMP-2 production predominates over that of MMP-9 in effusions. Increased MMP-2 and reduced TIMP-2 levels are seen in ovarian carcinoma cells in effusions, possibly marking the acquisition of a metastatic phenotype.

Expression of cell cycle proteins in ovarian carcinoma cells in serous effusions-biological and prognostic implications.

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. METHODS: Fifty-five malignant effusions and 38 tumors (20 primary, 18 metastatic) were immunohistochemically stained for cyclin A, p27(kip1), and Ki-67. Staining extent (0-100% cells) and intensity (0-3 scale) were scored. Cyclin A and p27(kip1) expression was additionally studied in 29 malignant effusions using immunoblotting. Immunohistochemistry results in effusions were evaluated for possible association with clinicopathologic parameters. RESULTS: Nuclear immunoreactivity for all markers was detected on carcinoma cells in the majority of effusions using immunohistochemistry. Similarly, immunoblotting showed the presence of cyclin A and p27(kip1) in 29/29 and 25/29 specimens, respectively. Intense (3) immunoreactivity for Ki-67 was detected more often in peritoneal effusions, compared with those of pleural location (P = 0.036). Staining in primary and metastatic lesions was generally comparable to that of tumor cells in effusions. Staining for p27(kip1) was more diffuse in effusion specimens obtained prior to the institution of chemotherapy (P = 0.042). In an analysis of all effusions, an association was observed between the number of cells that were immunoreactive for Ki-67, cyclin A, and p27(kip1) (cyclin A-Ki-67: P = 0.008; p27(kip1)-Ki-67: P = 0.019; cyclin A-p27(kip1): P = 0.032). In survival analysis, the presence of more diffuse (P = 0.042) and intense (P = 0.019) staining for cyclin A correlated with prolonged overall survival. CONCLUSIONS: The expression of the studied cell cycle markers does not differ markedly between ovarian carcinoma cells in the pleural and peritoneal cavity, supporting our previous studies of several metastasis-associated molecules. The presence of cyclin-A-positive cell populations is associated with a more favorable disease outcome, possibly due to the targeting of proliferating cells by chemotherapeutic agents. However, the decline in the fraction of p27(kip1)-positive cells in posttreatment specimens may point to additional mechanisms involved in this selection.

Abnormal DNA content predicts the occurrence of carcinomas in non-dysplastic oral white patches.

The majority of oral squamous cell carcinomas (OSCCs) are preceded by visible changes in the oral mucosa, most often white patches. Although the histological finding of dysplasia in oral white patches signals increased risk of developing OSCC, this may also occur in non-dysplastic lesions. However, no reliable markers exist to predict the occurrence of OSCC in these patients. From a total of 263 patients diagnosed with oral white patches, biopsies from 45 patients were selected on the criteria that the patients had lesions histologically proven to be non-dysplastic. The lesions were analyzed with respect to their DNA content. The clinical outcome of the patients was known from the Cancer Registry of Norway, and these data were compared to the DNA content of their lesions. Among the 45 patients, five cases (11%) later developed an OSCC. Four of the cases that subsequently developed an OSCC were among the five aneuploid (abnormal) cases (P=0.001). One aneuploid lesion did not develop a carcinoma during a follow-up time of 120 months. The fifth case that subsequently developed an OSCC was diploid (normal), and developed into an OSCC after an observation time of 73 months (P=0.001). In conclusion, aberrant DNA content reliably predicts the occurrence of OSCC in patients that otherwise would be regarded as at very low risk. Normal DNA content indicates low risk.

Comparison of histological grading and large-scale genomic status (DNA ploidy) as prognostic tools in oral dysplasia.

Approximately one in ten oral white patches (leukoplakia) are histologically classified as dysplasia, with a well-documented potential for developing into oral squamous cell carcinoma (OSCC). Histological grading in oral dysplasia has limited prognostic value, whereas large-scale genomic status (DNA ploidy, nuclear DNA content) is an early marker of malignant transformation in several tissues. Biopsies from 196 patients with oral leukoplakia histologically typed as dysplasia were investigated. Inter-observer agreement among four experienced pathologists performing a simplified grading was assessed by Cohen's kappa values. For 150 of the 196 cases, it was also possible to assess large-scale genomic status and compare its prognostic impact with that of histological grading. Disease-free survival was estimated by life-table methods, with a mean follow-up time of 103 months (range 4-165 months). The primary considered end-point was the subsequent occurrence of OSCC. For grading of the total of 196 cases, kappa values ranged from 0.17 to 0.33 when three grading groups (mild, moderate, and severe dysplasia) were considered, and from 0.21 to 0.32 when two groups (low grade and high grade) were considered (p=0.41). For the 150 cases in which large-scale genomic status was also assessed, kappa values for the histological grading ranged from 0.21 to 0.33 for three grading groups and from 0.27 to 0.34 for two grading groups (p=0.47). In survival analysis, histological grading was without significant prognostic value for any of the four observers (p 0.14-0.44), in contrast to DNA ploidy (p=0.001). It is concluded that DNA ploidy in oral dysplasia has a practical prognostic value, unlike histological grading of the same lesions.

Caveolin-1 expression in advanced-stage ovarian carcinoma--a clinicopathologic study.

OBJECTIVE: The aim of this study was to analyze the correlation among the expression of caveolin-1, the protein constituent of caveolae, and disease outcome in advanced-stage ovarian carcinomas. METHODS: Sections from 76 primary ovarian carcinomas and metastatic lesions from 45 patients diagnosed with advanced-stage ovarian carcinoma (FIGO stages III-IV) were evaluated for caveolin-1 expression using immunohistochemistry. Patients were divided into long-term survivors and short-term survivors based on disease outcome. Twenty nonneoplastic fallopian tubes and ovaries were additionally studied. RESULTS: The mean follow-up period was 70 months. The mean values for disease-free survival and overall survival were 109 and 125 months for long-term survivors, compared to 3 and 21 months for short-term survivors, respectively. Caveolin-1 expression was localized to the cell membrane in 24/76 (32%) specimens and was detected in the cytoplasm in 52/76 (68%) cases. Both patterns were more often detected in metastases, when compared with primary tumors. In addition, membrane immunoreactivity was more often seen in tumor of short-term survivors. These differences did not reach statistical significance (P > 0.05). Combined membrane and cytoplasmic immunoreactivity was seen in 17/20 (85%) nonneoplastic lesions. Despite its role in tyrosine-kinase-mediated signal transduction in vitro studies, caveolin-1 expression in carcinomas showed no association with the protein expression of c-erbB-2 and epidermal growth factor receptor, evaluated in a previous study of this patient cohort. CONCLUSIONS: This study provides the first in vivo evidence of caveolin-1 membrane expression in human malignancies. Caveolin-1 is often expressed in advanced-stage ovarian carcinoma, but does not appear to be a powerful predictor of disease outcome in these tumors. The reduced expression level in carcinomas compared to nonneoplastic epithelium may point to a role for caveolin-1 as a tumor suppressor gene.

Expression of membrane-type 1, 2, and 3 matrix metalloproteinases messenger RNA in ovarian carcinoma cells in serous effusions.

We studied the levels of matrix metalloproteinase (MMP)-2, membrane-type (MT)1-MMP, MT2-MMP, and MT3-MMP in 43 malignant pleural and peritoneal effusions using reverse transcription-polymerase chain reaction (RT-PCR) and cellular localization of MT1-MMP in 66 effusion specimens and 85 corresponding primary and metastatic tumors using messenger RNA (mRNA) in situ hybridization (ISH). In 43 effusions, MMP-2 mRNA was detected in 37, MT1-MMP in 25, and MT2-MMP in 32. Expression of MT1-MMP and MT2-MMP was found in 21 specimens; in 16 MT-MMP-positive specimens, mRNA for only 1 of 2 enzymes was expressed. MT3-MMP mRNA was not detected. High levels of MMP-2 mRNA were detected more often in effusions with high MT1-MMP and/or MT2-MMP mRNA expression. Using ISH, MT1-MMP mRNA was localized to cancer cells in 27 of 58 malignant effusions; focal signals were detected in mesothelial cells in 7 of 42. MT1-MMP was localized to tumor cells in 32 of 85 primary and metastatic solid lesions, and stromal cells expressed MT1-MMP in 3. Tumor cell MT1-MMP expression in effusion specimens did not differ from primary or metastatic lesions. MT-MMP expression in tumor cells in effusions showed no association with effusion site or tumor type using ISH and RT-PCR.

Ets-1 messenger RNA expression is a novel marker of poor survival in ovarian carcinoma.

Ets-1 proto-oncogene is a transcription factor involved in several cellular functions, including the activation of several proteases participating in tumor invasion and metastasis. The objective of this study was to analyze the possible correlation between Ets-1 mRNA expression and survival in advanced-stage ovarian carcinomas, studying two patient groups with extremely different disease outcome. Sections from 66 primary ovarian carcinomas and metastatic lesions from 41 patients diagnosed with advanced-stage ovarian carcinoma (International Federation of Gynecologists and Obstetricians stages III and IV) were evaluated for expression of Ets-1 using mRNA in situ hybridization. Patients were divided into long-term (n = 17) and short-term (n = 24) survivors. The mean values for disease-free survival and overall survival were 116 and 133 months for long-term survivors, as compared to 3 and 21 months for short-term survivors, respectively. Expression of Ets-1 mRNA was detected in carcinoma cells and stromal cells in 28 of 66 (42%) and 22 of 66 (33%) lesions, respectively. Ets-1 expression showed an association with mRNA expression of vascular endothelial growth factor (P = 0.001 for carcinoma cells; P = 0.004 for stromal cells), basic fibroblast growth factor (P = 0.049 for carcinoma cells), and membrane type-1 matrix metalloproteinase (P = 0.045), which were previously studied in this patient cohort. Ets-1 mRNA was detected more often in both carcinoma and stromal cells in tumors of short-term survivors (P = 0.038 for carcinoma cells). In univariate survival analysis for all cases, Ets-1 expression in both tumor (P = 0.018) and stroma (P = 0.026) correlated with poor survival. These findings were reproduced in an analysis of primary tumors alone (P = 0.039 for tumor cells; P < 0.001 for stromal cells). Ets-1 mRNA expression in stromal cells retained its predictive power in a multivariate survival analysis in which all molecules studied previously in this patient cohort were included (P = 0.007). To our knowledge, this is the first evidence associating Ets-1 mRNA expression and poor survival in human epithelial malignancy. Ets-1 is thus a novel prognostic marker in advanced-stage ovarian carcinoma. The association between Ets-1 mRNA expression and the expression of membrane type-1 matrix metalloproteinase and angiogenic genes, first documented here in a study of patient material, points to the central role of this transcription factor in tumor progression in ovarian carcinoma.

[Diagnosis and treatment of oral precancerous lesions]. / Diagnostikk og behandling av forstadier til munnhulekreft.

BACKGROUND: Risk factors for oral carcinomas have been identified, but there are no reliable markers for assessing the clinical outcome in individual patients with oral precancerous lesions. DNA aneuploidy is now recognized as an early and significant event in carcinogenesis. METHODS: We identified 242 patients with oral red or white patches histologically verified as epithelial dysplasias and measured the nuclear DNA content (DNA ploidy) of the lesions to determine whether DNA ploidy could be used to predict the clinical outcome. Disease-free survival was assessed in relation to DNA ploidy and histological grade. The mean duration of follow-up was approximately eight years. RESULTS: Among 242 patients with verified epithelial dysplasia, a carcinoma developed in 48 (20%). 167 (69%) had diploid lesions, 20 (8%) had tetraploid lesions and 55 (23%) had aneuploid lesions. Of the 167 with diploid lesions, only four (1%) later developed an oral carcinoma. By contrast, 48 of 55 patients with aneuploid lesions (87%) later developed a carcinoma. INTERPRETATION: The DNA content (DNA ploidy) can be used to predict the risk for oral cancer in a wide range of oral precancerous lesions. By contrast, histological grading of the same lesions does not give any prognostic information. The clinical value of an early identification of oral lesions with malignant cell clones is substantiated by the fact that there are methods for early intervention.

E-cadherin and alpha-, beta-, and gamma-catenin protein expression is up-regulated in ovarian carcinoma cells in serous effusions.

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 alpha-, beta-, and gamma-catenin. Staining extent and intensity were scored. Effusion specimens were additionally analysed for calretinin immunoreactivity. Membrane immunoreactivity for E-cadherin and alpha-, beta-, and gamma-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 alpha-catenin expression, in both effusions and solid tumours, and for beta-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 alpha-, beta-, and gamma-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 beta-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.

E-Cadherin complex protein expression and survival in ovarian carcinoma.

OBJECTIVE: The aim of this study was to analyze the correlation between expression of E-cadherin complex proteins, epidermal growth factor receptor (EGFR), and c-erbB-2 and disease outcome in advanced-stage ovarian carcinomas. METHODS: Sections from 75 primary ovarian carcinomas (=37) and metastatic lesions (=38) from 45 patients diagnosed with advanced-stage ovarian carcinoma (FIGO stage III-IV) were immunostained and evaluated for staining pattern, extent, and intensity. Patients were divided in two groups based on disease outcome. Long-term survivors (21 patients) and short-term survivors (24 patients) were defined using a double cutoff of 36 months for disease-free survival (DFS) and 60 months for overall survival (OS). Mean follow-up period was 70 months. The mean values for DFS and OS were 109 and 125 months for long-term survivors, as compared to 3 and 21 months for short-term survivors, respectively. RESULTS: Comparison of all primary and metastatic lesions showed upregulation of gamma-catenin protein expression in the latter (P = 0.05). When segregated according to disease outcome, the expression of all studied proteins, with the exception of EGFR, was more diffuse in tumors of short-term survivors. The presence of cytoplasmic staining for c-erbB-2 was associated with poor survival in the entire cohort (P = 0.007), as well as in primary tumors alone (P = 0.003), in survival analysis. Similar results were seen in the evaluation of primary tumors for gamma-catenin (P = 0.002). CONCLUSIONS: gamma-Catenin, and possibly c-erbB-2, are valid markers of poor survival in advanced-stage ovarian carcinoma.

Carbohydrate antigen expression in primary tumors, metastatic lesions, and serous effusions from patients diagnosed with epithelial ovarian carcinoma: evidence of up-regulated Tn and Sialyl Tn antigen expression in effusions.

The object of this study was the investigation of carbohydrate antigen expression in malignant epithelial cells and benign mesothelial cells in serous effusions from patients diagnosed with epithelial ovarian carcinomas. In addition, to compare antigen expression in carcinoma cells in effusions with those of corresponding primary tumors and metastatic lesions. Sections from 63 malignant effusions from ovarian carcinoma patients and 15 reactive effusions were immunohistochemically stained, using 5 monoclonal antibodies for Lewis(y), Sialyl Lewis(x), Tn, and Sialyl Tn antigens. Tissue sections (n = 97) from corresponding primary ovarian carcinomas and metastatic lesions, as well as from 12 malignant mesotheliomas, were additionally stained using the above panel. Staining for the 4 antigens was seen in carcinoma cells in serous effusions in the majority of cases (range = 71% to 85%). In contrast, immunoreactivity was detected in mesothelial cells in only 6% to 23% of the specimens studied (P < .001 for all 5 markers). With the exception of B3 antibody against Lewis(y) antigen, malignant mesotheliomas stained negative, infrequently showing focal immunoreactivity. An up-regulation of Tn and Sialyl Tn expression was detected in carcinoma cells in effusions when compared with both primary tumors (P < .003 and P < .007, respectively) and metastatic lesions (P < .034 and .041, respectively). Cancer-associated carbohydrate antigens can thus be used as an adjunct in the differentiation between malignant epithelial and reactive mesothelial cells. Ovarian carcinoma cells in effusions show up-regulation of Tn and Sialyl Tn, possibly representing a transient phenotypic alteration facilitating metastasis.

N-Glycans influence the in vitro adhesive and invasive behaviour of three metastatic cell lines.

Alterations in cellular glycosylation may play a key role in metastatic behaviour of tumour cells. We studied three metastatic cell lines, LOX (malignant melanoma), FEMX (malignant melanoma) and MA-11 (mammary carcinoma). These cell lines have a very different metastatic behaviour in vivo, and different glycans have been postulated to be partly responsible for these differences. To further investigate the functional role of carbohydrates, these three cell lines have been treated with tunicamycin, an inhibitor of the biosynthesis of N-glycans and benzyl- alpha-N-acetylgalactosamine (benzyl-alpha-GalNAc; BnGalNAc), an inhibitor of mature O-linked glycans. Various in vitro adhesion and invasion assays were undertaken for functional studies. Tunicamycin significantly inhibited adhesion to laminin, but only slightly affected cell adhesion to collagen IV. The same compound significantly decreased cellular invasiveness through a Matrigel invasion chamber. Moreover, tunicamycin reduced homotypic aggregation of cells. BnGalNAc had generally little effect on cell behaviour in in vitro assay. The effects of the inhibitors were, however, to some extent cell line-specific. We conclude that N-glycans, but probably not mature O-glycans have important in vitro functions in cell adhesion to laminin, cell invasion through Matrigel and cellular aggregation in the studied cell lines. These results support the view that carbohydrates are functionally involved in several steps of the metastatic process.

Expression of carbohydrate antigens in advanced-stage ovarian carcinomas and their metastases-A clinicopathologic study.

OBJECTIVE: Up-regulated expression or loss of expression of various carbohydrate antigens on the surface of cancer cells has been associated with a metastatic phenotype and poor survival in epithelial malignancies of different origins. The object of this study was to investigate the expression of carbohydrate antigens in two groups of patients diagnosed with advanced-stage ovarian carcinoma-one with an extremely favorable outcome and the other with a uniformly poor survival. METHODS: Sections from 76 paraffin-embedded blocks (primary ovarian carcinomas and metastatic lesions) from 45 patients diagnosed with advanced-stage ovarian carcinomas (FIGO stages III-IV) were immunohistochemically stained using five monoclonal antibodies for Lewis(y) (Le(y))(two antibodies), Sialyl Lewis(x) (Slex), Tn, and Sialyl Tn (STn) antigens. Patients were divided in two groups based on outcome. Long-term survivors (21 patients) and short-term survivors (24 patients) were defined using a double cut-off of 36 months for disease-free survival (DFS) and 60 months for overall survival (OS). Staining results for primary tumors and metastases were analyzed separately. RESULTS: Mean follow-up period was 70 months. The mean values for DFS and OS were 109 and 125 months for long-term survivors and 3 and 25 months for short-term survivors. Staining for all four antigens was seen in the majority of cases (range = 72-96%) and tended to be comparable in primary tumors and their metastases. However, absence of immunoreactivity for STn was seen in 9/38 (24%) metastatic lesions and only 1/38 (3%) primary tumors. This finding did not reach statistical significance (P > 0.05). A combined pattern of membranous and cytoplasmic staining was predominant in the majority of cases. Enhanced staining for Le(y) and STn was detected in the invasive front of some tumors, while Slex and Tn immunoreactivity did not relate to cell location. Primary tumors and metastatic lesions of long-term survivors displayed immunoreactivity patterns that were comparable to those of short-term survivors. In the evaluation of survival curves, more diffuse staining for Slex showed marginal correlation with poor survival (P = 0.05), while a trend toward poorer survival was seen in tumors that were more extensively stained for Le(y) and Tn (P > 0.05). CONCLUSIONS: Le(y), Slex, STn, and Tn antigens are widely expressed in primary ovarian carcinomas and their metastases. Altered expression of Sialyl Tn is observed with tumor progression in a fraction of ovarian carcinomas. Expression of membrane carbohydrate residues is prevalent in tumors of both long-term and short-term survivors and does not appear to be a strong predictor of disease outcome. However, larger studies are needed to further elucidate the role of these molecules in ovarian carcinogenesis.