Claudin upregulation in ovarian carcinoma effusions is associated with poor survival

Claudins are tight junction proteins that are highly expressed in ovarian carcinoma (OC). The objective of this study was to analyze the anatomic site-related expression and clinical role of claudins in OC. Effusions (n = 218), corresponding primary tumors (n = 81), and solid metastases (n = 164) (total = 463 tumors) were immunostained for claudin-1, claudin-3, claudin-4, and claudin-7. Results were analyzed for association with anatomic site, clinicopathologic parameters, and survival. All 4 claudins were expressed in >85% of tumors at all anatomic sites. However, staining extent of all except claudin-4 was significantly higher in effusions compared with both primary carcinomas and solid metastases (P < .001). In univariate survival analysis of the entire cohort, higher claudin-3 (P = .038) and claudin-7 (P = .035) expression in effusions correlated with shorter overall survival (OS), with similar results for claudin-7 in analysis of progression-free survival (P = .026). In separate analysis for patients with prechemotherapy effusions, higher claudin-7 expression correlated with shorter OS (P = .045). For patients with postchemotherapy effusions, higher claudin-1 (P = .018) and claudin-3 (P = .009) expression correlated with shorter OS. In multivariate survival analysis of the entire cohort, claudin-7 expression was an independent predictor of poor progression-free survival (P = .017). Claudin-3 independently predicted poor OS for patients with postchemotherapy effusions (P = .012). With the exception of claudin-4, claudins are upregulated in OC effusions compared with solid tumors, in agreement with our previous data for cadherins and integrins in this cancer type, suggesting a prosurvival role for these surface molecules. Claudin-3 and claudin-7 expression in effusions independently predicts poor survival in OC.     

Immunohistochemistry in the diagnosis of malignant mesothelioma

Summary The histological diagnosis of malignant mesothelioma of the pleura, especially the distinction from peripheral adenocarcinoma of the lung, may be difficult. The immunohistochemical reports previously published on this subject show diverging results mainly because a variety of antibodies and staining techniques have been used by the different authors. To obtain comparable and reproducible results standard techniques and commercialized antibodies should be applied in routine pathology. In order to investigate the value of immunohistochemistry for the separation of the two entities formalin fixed and paraffin embedded blocks of 47 mesotheliomas and 22 adenocarcinomas were investigated with the PAP technique and commercially available antibodies to carcino-embryonic antigen (CEA), keratin, vimentin, epithelial membrane antigen (EMA), pregnancy specific antigen (SP₁), S-100 protein and monoclonal antibody lu-5 (mAB lu-5). CEA positivity was found in all 22 adenocarcinomas examined, but only 2/47 (4%) of all mesotheliomas showed a positive result. SP₁ was positive in 13/22 (59%) of the adenocarcinomas, whereas only 3/47 (6%) mesotheliomas were positive for this marker. No significant difference in the rate of positive cases in the adenocarcinoma and mesothelioma group could be found with the other above mentioned antigens. The results of our study indicate that especially CEA, but also SP₁ are valuable markers in the diagnosis of malignant mesothelioma.     

Immunocytochemistry of malignant mesothelioma: OV632 as a marker of malignant mesothelioma.

In pleural or ascitic effusions the cytomorphological distinction of adenocarcinoma cells, reactive mesothelial cells, and malignant mesothelioma cells often causes a diagnostic dilemma. The value of immunocytochemistry was investigated on cytological smears of 24 well-established cases of malignant mesothelioma, a selected series of 31 metastatic adenocarcinomas, and 20 smears of patients without known malignancy. In these smears we scored the immunoreactivity with a panel of four monoclonal antibodies. In addition to antibodies for epithelial membrane antigen (EMA) and carcinoembryonic antigen (CEA), the monoclonal antibody MOC31 and the ovarian carcinoma specific antibody OV632 were incorporated in the panel. With none of these four antibodies was immunostaining of reactive mesothelial cells found. CEA- and MOC31-positive tumour cells were frequent in metastatic adenocarcinomas, but occurred rarely in malignant mesotheliomas. EMA-positive tumour cells were found in all metastatic adenocarcinomas (100 per cent) and in most malignant mesotheliomas (83 per cent). In addition to the expected reactivity of OV632 with ovarian carcinomas, 22 of 24 malignant mesotheliomas contained immunopositive tumour cells, while only a small proportion of non-ovarian adenocarcinomas reacted with this antibody. This selective staining of malignant mesothelioma cells, but not reactive mesothelial cells, with OV632 now permits the positive identification of malignant mesothelioma cells in male patients.     

Expression of HLA-G in malignant mesothelioma and clinically aggressive breast carcinoma

The aim of the present study was to evaluate HLA-G expression in breast carcinoma and malignant mesothelioma (MM). Malignant breast carcinoma effusions (46) and corresponding solid tumors (39) and 104 MM (26 effusions, 78 solid tumors) were analyzed using immunohistochemistry (IHC). HLA-G protein and mRNA expression were further studied using immunoblotting (IB) and RT-PCR. HLA-ABC expression was analyzed using flow cytometry (FCM). IHC showed predominantly focal HLA-G expression in 12 of 46 (26%) breast carcinoma effusions and 16 of 39 (41%) solid lesions. In MM, 20 of 78 (26%) solid lesions and 14 of 26 (54%) effusions were focally HLA-G positive. Expression in MM was higher in effusions (p=0.008). IB showed more frequent HLA-G expression in MM compared with breast carcinoma effusions, while RT-PCR showed HLA-G mRNA expression in both tumors. FCM showed conserved HLA-ABC expression in 15 of 15 effusions. Breast cancer patients with HLA-G-positive tumor cells had shorter disease-free survival (mean 37 vs 85, median 25 vs 31 months), though not significantly (p=0.14). In conclusion, HLA-G is focally expressed in MM and breast carcinoma, while HLA-ABC expression is conserved. However, the up-regulated expression of HLA-G in MM effusions and its possible association with shorter disease-free survival in advanced stage of breast carcinoma suggest a possible role in immune response evasion in some tumors.     

Expression of Wilms tumor-1 protein and CD 138 in malignant mesothelioma of the tunica vaginalis

Malignant mesothelioma (MM) of the tunica vaginalis is a very rare tumor which can be difficult to diagnose histologically. We report an 83-year-old patient with MM of tunica vaginalis of the left testis. To the best of our knowledge, this is the first reported case of MM of the tunica vaginalis expressing Wilms tumor-1 protein, CD138, the expression of which could help in confirming the histopathological diagnosis and in targeting therapy.     

Soluble mesothelin-related peptides levels in patients with malignant mesothelioma

Soluble mesothelin-related peptides (SMRP) are a potential tumor marker for malignant mesothelioma. The aim of this study was to determine the differences in SMRP levels in patients with malignant mesothelioma before treatment and in various responses to treatment and to investigate whether SMRP level could be useful in evaluating tumor response to treatment. The study included patients with malignant mesothelioma treated at the Institute of Oncology Ljubljana between March 2007 and December 2009. Blood samples were collected before treatment and/or in various responses to treatment. SMRP levels were determined using ELISA assay based upon a combination of two monoclonal antibodies. Mann-Whitney test was used to determine the differences in SMRP levels in various responses to treatment. Median SMRP was 2.80 nmol/L (range 0.00-34.80) before treatment, 0.00 nmol/L (range 0.00-0.00) in complete response, 0.48 nmol/L (range 0.00-4.40) in partial response, 1.65 nmol/L (range 0.00-20.71) in stable disease and 7.15 nmol/L (range 0.44-31.56) in progressive disease. Pre-treatment SMRP levels were significantly higher than in stable disease, partial response and complete response (p=0.006), as were SMRP levels in progressive disease compared to stable disease, partial response and complete response (p< 0.001). Our findings suggest that SMRP may be a useful tumor marker for detecting the progression of malignant mesothelioma and evaluating tumor response to treatment.     

Role of fragile histidine triad protein expression in pathogenesis of malignant pleural mesothelioma

AIM:To investigate the relationship of fragile histidine triad (FHIT) and Ki-67 expression with clinicopathological variables of patients with malignant pleural mesothelioma (MPM). METHODS: Formalin-fixed, paraffin-embedded tissue sections of 30 asbestos induced MPM (epithelial and biphasic) patients were examined for FHIT and Ki-67 expression using immunohistochemical techniques and results were compared with clinicopathological variables. RESULTS: Immunohistochemical study results were as follows: 12 (40%) cases showed low FHIT expression and 18 (60%) showed high expression. There was no significant relationship between FHIT and age, gender or histological subtypes (p > 0.05). Ki-67 expression was 'low' in 13 (43.3%) cases and 'high' in 17 (56.7%) cases. No correlation could be demonstrated between Ki-67 expression and age, gender or histological subtypes (p > 0.05). No significant association was observed between FHIT and Ki-67 expression in MPM. CONCLUSION: The results support the role of FHIT as a tumour suppressor gene in asbestos induced MPM. There is no significant correlation between FHIT and cell proliferation marker expressions in malignant pleural mesothelioma. Therefore, it can be concluded that loss of FHIT does not interfere with tumour proliferation. This can be accepted as evidence for an early role of FHIT loss in carcinogenesis; however, it needs to be strengthened by further studies.     

Cytopathology of malignant mesothelioma: a stepwise logistic regression analysis.

Twenty-four cytologic features, previously reported to be useful in the distinction of malignant mesothelioma, adenocarcinoma, and benign mesothelial proliferation in serous effusions were assessed. Forty-four cases of malignant mesotheliomas, 46 cases of metastatic adenocarcinomas, and 30 cases of benign mesothelial proliferations were examined for these parameters. When these cytologic features were subjected to a stepwise logistic regression analysis, five features were selected to distinguish malignant mesothelioma from adenocarcinoma. These were true papillary aggregates, multinucleation with atypia, cell-to-cell apposition, acinus-like structures, and balloon-like vacuolation, the latter two features being characteristic of adenocarcinoma. The four variables selected to distinguish malignant mesothelioma from benign mesothelial proliferations were nuclear pleomorphism, macronucleoli, cell-in-cell engulfment, and monolayer cell groups, the latter being a feature of benign proliferations. Using these selected variables, the logistic model correctly predicted 95.4% of cases of malignant mesothelioma versus 100% of adenocarcinoma and 100% of malignant mesotheliomas versus 90% of benign mesothelial proliferations. The results of regression analysis suggest that many of the previously described cytologic features are not important diagnostic discriminators.     

Reduced Fhit protein expression in human malignant mesothelioma

Human malignant mesothelioma (MM) is an aggressive neoplasm related to occupational exposure to asbestos and characterised by a long latency time. Multiple chromosomal deletions and DNA losses have been revealed in MM by studies performed with karyotypic, comparative genomic hybridisation and loss of heterozygosity (LOH) analyses. Among frequently deleted chromosomal sites, LOH at chromosome 3p has been detected in MM, suggesting the presence of one or several tumour suppressor genes that have an important role in development of the disease. The FHIT (fragile histidine triad) tumour suppressor gene, located at 3p14.2, has been proposed to be a target to major human lung carcinogens, such as tobacco smoke and asbestos. Although many studies have indicated decreased Fhit protein expression in a variety of malignancies, there is no report of FHIT gene aberrations or Fhit protein abnormalities in MM. We examined expression of the Fhit protein and LOH at the FHIT gene in malignant mesothelioma. Altogether, 13 paraffin embedded MM tumours were analysed for Fhit protein expression, and 21 fresh tumours and 10 cell cultures for LOH at the FHIT gene with two intragenic microsatellite markers. All tumours showed less intense immunostaining than normal bronchial epithelium or mesothelium. Fhit expression was absent or reduced in 54% (7 of 13) of the tumours, with the weakest staining observed in poorly differentiated areas. Allele loss was seen in 3 of 10 (30%) of the MM cell lines, but only in 1 of the 21 fresh tumours studied, suggesting concealment of LOH by normal cells present in MM tumours. In conclusion, our present data indicate a frequent decrease of Fhit protein expression, thus supporting the significance of FHIT inactivation in development of MM.     

The use of vimentin antibodies in the diagnosis of malignant mesothelioma

Summary An immunohistochemical investigation of vimentin, an intermediate filament, was carried out on formalin fixed paraffin embedded sections of 44 malignant mesotheliomas of the pleura and 24 pulmonary adenocarcinomas, in order to assess its value in differential diagnosis. Seventy-five percent of the malignant mesotheliomas showed positive staining for vimentin. An unexpected finding was the presence of vimentin tin in 46% of the pulmonary adenocarcinomas. In either case there was no correlation between the presence of vimentin and the histological types or grades of differentiation. The overall level of vimentin staining was significantly greater in the malignant mesotheliomas than in the pulmonary adenocarcinomas but no single antibody dilution was found to provide clear cut separation of the two groups. Vimentin does not appear to be a simple discriminatory marker of malignant mesothelioma.     

Heparanase and basic fibroblast growth factor are co-expressed in malignant mesothelioma

Heparanase is an endoglycosidase that degrades heparan sulfate (HS) in the extracellular matrix (ECM) and cell surfaces, and fulfills a significant role in cancer metastasis and angiogenesis. We evaluated the expression of heparanase and its possible association with the expression of angiogenic molecules in malignant mesothelioma (MM), and analyzed whether expression of these proteins is site-related (pleural vs peritoneal MM, solid lesions vs effusions). Sections from 80 MM (56 biopsies, 24 effusions) were analyzed for heparanase protein expression using immunohistochemistry (IHC). Sixty MM were of pleural origin, and 20 were peritoneal. Effusion specimens consisted of 6 peritoneal and 18 pleural effusions, while biopsies consisted of 14 peritoneal and 42 pleural lesions. Fifty-four specimens were additionally evaluated for expression of basic fibroblast growth factor (bFGF), interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) proteins using IHC. Microvessel density (MVD) was studied in 28 biopsies using an anti-CD31 antibody. mRNA expression of heparanase (HPSE-1), VEGF and the VEGF receptor KDR was analyzed in 23 effusions using RT-PCR. Heparanase protein expression was seen in 69/80 (86%) tumors. Of these, 35 showed combined membrane and cytoplasmic expression, 30 cytoplasmic expression, and four exclusively membrane expression. Both total (P= 0.001) and cytoplasmic (P = 0.002) expression was significantly higher in solid tumors compared to effusions. Protein expression of VEGF, IL-8 and bFGF was seen in 21/54 (39%), 22/54 (41%) and 44/54 (81%) specimens, respectively. Protein expression of bFGF was significantly higher in solid tumors (P 0.001) and correlated with heparanase expression (P = 0.005). HPSE-1 and VEGF mRNA expression was detected in all 23 effusions using RT-PCR, while KDR mRNA was found in 12/23 MM. KDR mRNA expression correlated with that of both HPSE-1 (P = 0.005) and VEGF (P = 0.001). Our results document frequent expression of heparanase in MM, in agreement with the biological aggressiveness of this tumor. The co-expression of heparanase with bFGF is in agreement with the role of the former in releasing bFGF from the ECM. The concomitant reduction in protein expression of both molecules in effusions as compared to solid tumors, supports the hypothesis of a reduced need for pro-angiogenic stimuli in effusions, and may aid in defining tumor progression in this setting.     

Pathology of malignant mesothelioma

The diagnosis of malignant mesothelioma can pose several problems to the surgical pathologist. First, the morphological appearances of the tumour are known to be diverse with mimicry of a range of both reactive and neoplastic conditions. Second, due to the relative inaccessibility of the serosa, biopsy material is often scanty and fragmentary, producing a plethora of interpretive ambiguities. Third, adjunct techniques such as mucin histochemistry and immunohistochemistry, whilst useful in excluding malignant mesothelioma have little role in confirming the diagnosis. The accurate diagnosis of diffuse malignant mesothelioma is important for two reasons: (1) In relation to prognosis as it has an almost invariable fatal outcome, which contrasts with the other mesothelial neoplasms such as the benign adenomatoid tumour and the borderline malignant tumours, namely the well-differentiated papillary mesothelioma and multicystic mesothelioma; (2) In relation to occupational-related compensation claims following asbestos exposure. This review summarizes the aetiology of asbestos-induced neoplasia, possible mechanisms of tumour development and highlights potential diagnostic pitfalls.     

The influence of the diagnostic technique on the histopathological diagnosis in malignant mesothelioma

Summary In the histopathology of malignant mesotheliomas three different types (epithelial, connective tissue and mixed type) are distinguished. Some authors believe all tumours to be of mixed type, but consider that due to inadequate sampling or small biopsies this may be missed frequently. In this study the relationship between the histopathological diagnosis and the amount of tissue examined was investigated. In a series of 124 cases of malignant pleural mesothelioma a high percentage of mixed type tumours was found (55%). In cases where the decisive diagnostic procedure had been an Abrams biopsy (the small-specimen technique) mixed-type histology was found in 36%. If thoracoscopy, thoracotomy or autopsy (the large-specimen techniques) had delivered a definite diagnosis, mixed-type histology was found in 63%. Apparently diagnosing the mixed-type variety depends on the amount of tumour tissue obtained. However, the assumption that all mesotheliomas are of mixed type cannot be confirmed.     

Localized malignant mesothelioma of the pleura

We describe a case of malignant pleural mesothelioma appearing as a solitary pleural tumor in a 56-year-old Japanese man with no history of exposure to asbestos. A chest radiograph revealed an isolated extrapulmonary mass in the left hemithorax. The patient underwent tumor resection, but the tumor later recurred on the contralateral pleura. The patient developed cerebral metastases and died 16 months after the initial surgery. The resected tumor was sessile with broad-based pleural attachment. Microscopically, the tumor was composed of interlacing fascicles of plump spindle cells intermixed with few polygonal cells. Most of the tumor cells showed positive immunoreactivity for cytokeratins (AE1 and AE3) and vimentin. Many of the tumor cells were positive for epithelial membrane antigen, and a few were positive for desmin. In contrast, the tumor cells were consistently negative for carcinoembryonic antigen, epithelial antigen BerEP4, calretinin, S-100 protein, neuron-specific enolase, muscle actin antigen HHF35, alpha-smooth muscle actin antigen and CD34. Ultrastructurally, the tumor cells had diffusely distributed cytoplasmic intermediate filaments, desmosome-like junctions, and a few microvilli. Some tumor cells contained cytoplasmic tonofilaments. Immunohistochemical and ultrastructural findings supported the mesothelial nature of the tumor, and led us to diagnose this tumor as a sarcomatoid localized malignant mesothelioma.     

Fibulin-3表达对恶性胸膜间皮瘤细胞的影响

目的:研究纤蛋白3(fibulin-3)基因过表达/沉默对人恶性胸膜间皮瘤(malignant pleural mesothelioma,MPM)细胞系SMC-1的影响,为MPM的治疗寻找新的方法。方法:分别构建fibulin-3过表达和短发夹RNA(short hairpin RNA,shRNA)载体,并将SMC-1细胞分别转染空白对照载体(control)、过表达载体(Exp)、过表达对照载体(Exp-NC)、干扰载体(shRNA1、shRNA2、shRNA3和shRNA4)和干扰对照载体(shRNA-NC)。应用流式细胞术检测细胞周期及凋亡,real-time PCR法和Western blot法检测过表达/干扰前后fibulin-3的表达情况。结果:SMC-1细胞转染相应载体后,流式细胞术结果显示,与control组相比,fibulin-3+Exp组的G_2期细胞数量增加(P0.05),shRNA2组的G_2期数量减少(P0.05);凋亡检测结果显示,与control组相比,Exp组的细胞凋亡率下降(P0.05),而shRNA2组的细胞凋亡率上升(P0.05)。分子水平检测结果显示,与control组相比,Exp组fibulin-3和间皮素(mesothelin)的mRNA和蛋白表达水平上调(P0.05),各shRNA组fibulin-3和mesothelin的mRNA和蛋白表达水平下调(P0.05)。结论:本实验构建了fibulin-3基因的过表达及沉默载体,证实转染Exp和shRNA能有效改变fibulin-3的表达水平及SMC-1细胞的生长,为以fibulin-3为靶点的RNA沉默手段应用于临床MPM的治疗提供了实验依据。     

Immunoreactivity for p53 and mdm2 and the detection of p53 mutations in human malignant mesothelioma

Previous immunohistochemical studies on malignant mesothelioma with antibodies recognizing both the wild and the mutant types of the p53 protein have shown immunoreactivity in 25–70% of cases. This study was designed to determine whether there is immunoreactivity for p53 and mdm2 protein in malignant mesothelioma and to correlate p53 expression with the detection of mutations in p53 at DNA level. In 10 of 15 cases there was immunoreactivity for p53. In 6 of these cases immunoreactivity for mdm2 was also detected. In one p53-immunonegative case, a mutation of the p53 gene resulting in a stop codon was found. These results suggest that mdm2 might be involved in the inhibition of p53 in malignant mesothelioma. Also, these data suggest the existence of other proteins than mdm2 that may associate with p53.This work was supported by the Belgian Cancer Association (Vereniging voor Kankerbestrijding) and the Flemish Biotechnology Programme of the Ministry of Economy     

Monoclonal antibody Ber-EP4: Its use in the differential diagnosis of malignant mesothelioma and carcinoma in cell blocks of malignant effusions and FNA specimens

Formal sublimate-fixed cell blocks derived from 129 malignant pleural (and some peritoneal) effusions, 8 benign effusions with reactive mesothelial cells, and 23 FNA specimens, were immunostained with monoclonal antibody Ber-EP4 to assess its ability to distinguish malignant mesothelioma (MM) from carcinoma. Only 2 of 44 (4%) well-characterized MM were Ber-EP4⁺, while none of 8 benign mesothelial proliferations reacted with the antibody. Fifty-seven percent of 23 pulmonary adenocarcinomas (AC) and 60% of 43 pulmonary carcinomas of all other histological types were Ber-EP4⁺. Of 40 metastatic AC originating from breast, gastrointestinal tract, ovary, endometrium, and kidney, 80% were Ber-EP4⁺. The predictive value of positive Ber-EP4 staining in distinguishing AC from MM was 96%. The predictive value of a negative Ber-EP4 in excluding MM was 70%, when the differential diagnosis was adenocarcinoma. These results suggest that Ber-EP4 is helpful in differentiating MM and AC if used together with other discriminating antibodies. © 1994 Wiley-Liss, Inc.     

Expression and clinical role of the bric-a-brac tramtrack broad complex/poxvirus and zinc protein NAC-1 in ovarian carcinoma effusions

We recently identified NAC-1, member of the bric-a-brac tramtrack broad complex/poxvirus and zinc domain family, as an overexpressed gene in ovarian serous carcinoma and found more frequent NAC-1 protein expression in recurrent compared to primary tumors. In the present study, we assessed the clinical significance of NAC-1 expression in ovarian carcinoma effusions. Formalin-fixed, paraffin-embedded sections from 176 effusions (137 peritoneal, 39 pleural) and 197 corresponding solid tumors (69 primary tumors, 128 solid metastases) were analyzed for NAC-1 expression using immunohistochemistry. Staining intensity and extent results were analyzed for possible association with clinicopathologic parameters and survival. Nuclear NAC-1 immunoreactivity was found in carcinoma cells in 98% of (173/176) effusions, 94% (65/69) of primary tumors, and 95% (121/128) of metastases. Staining intensity and extent were significantly higher in effusions compared with matched solid tumors (P = .002 for intensity, P = .003 for extent compared with primary tumors; P < .001 for both intensity and extent compared with metastases). Furthermore, NAC-1 expression intensity was significantly higher in specimens obtained after the administration of chemotherapy (P = .002) and correlated with shorter progression-free survival (PFS) in analysis of 62 patients with post-chemotherapy effusions (P = .039). International Federation of Gynecology and Obstetrics stage (IV versus III) was the only clinical parameter associated with PFS in this group (P = .004). In Cox analysis, only the International Federation of Gynecology and Obstetrics stage was an independent predictor of shorter PFS (P = .009). In conclusion, NAC-1 expression is higher in ovarian carcinoma cells in effusions compared with their solid tumor counterparts. NAC-1 is up-regulated in tumor cells after chemotherapy, suggesting a role for this protein in tumor progression and in the development of chemotherapy resistance in ovarian cancer.