Reactive Mesothelium Research Articles

Comparative immunochemical study of lectin‐binding sites and cytoskeletal filaments in static and reactive mesothelium and adenocarcinoma

In cytological preparations, reactive mesothelial cells (RMC) in serous effusions are sometimes difficult to distinguish from adenocarcinoma cells (AC). RMC and AC can be distinguished by lectin-binding patterns, but the pattern of binding of lectins to normal mesothelium is not well defined. We investigated the expression of cytoskeletal filaments, cytokeratin (CK) and vimentin (VM), and the cell surface binding pattern of 10 lectins (HPA, SBA, ABA, DSA, PNA, RCA-I, UEA-I, LTA, WGA and ConA) in the serosa of 48 adenocarcinoma specimens. We also investigated the usefulness of six lectins (HPA, SBA, RCA-I, UEA-I, LTA and WGA) in identification of RMC and AC in 16 serous effusions. DSA reactivity was significantly higher (P < 0.05) in static mesothelial cells (SMC) than in RMC. Reactivity for LTA and ConA was significantly lower (P < 0.05) in SMC than in RMC. Anti-CK and anti-VM immunoreactivity was always positive in RMC and almost negative in SMC. In serous effusions, HPA, SBA and UEA-I binding was evident in 100, 88 and 81% of AC, respectively. Little to no binding of HPA, SBA or UEA-I was detected in RMC. Our results suggest that the morphological differences between SMC and RMC are likely to be due to differences in cytoskeletal composition, with accompanying changes in cell-surface lectin-binding patterns. HPA, SBA and UEA-I are likely to be useful markers for identification of RMC and AC in cytology.

Immunocytochemistry in the differential diagnosis of serous effusions

The distinction between pleural mesothelioma (MS), reactive mesothelium (RM), and adenocarcinoma (AC) in serous effusions continues as a diagnostic problem in pathology. Immunohistochemistry can help, especially in surgical samples, but the optimum panel of antibodies has yet to be reported. The application of these antibodies to serous effusions has displayed variable results. The aim of this study was to evaluate the usefulness of eight monoclonal antibodies in the differential diagnosis of MS, RM, and AC in serous effusions. A total of 44 cytologic specimens of serous effusions (26 pleural, 15 peritoneal, and 3 pericardial) from 30 ACs, 3 MSs, and 11 RMs, previously stained with Papanicolaou stain, were selected retrospectively from our files and stained with HBME-1, thrombomodulin, calretinin, MOC-31, Ber-EP4, E-cadherin, CEA, and CD-15. The immunoreactions were evaluated independently by two pathologists. A stepwise logistic regression analysis was applied to the data to select an appropriate panel of antibodies. Statistical significance was found with HBME-1, thrombomodulin, MOC-31, Ber-EP4, and CD-15, when comparing both AC versus MS, and AC versus any type of mesothelial proliferation (MS or RM). Using HBME-1, 80% of ACs were negative whereas all three MSs reacted strongly with P = 0.003. A P = 0.02 was reached with thrombomodulin with 76.5% of ACs showing no immunoreactivity. Ber-EP4 and MOC-31 displayed good results with a P < 0.001 and 0.01, respectively. CD-15 reached a P = 0.034. No differences were found using the other antibodies. Ten ACs, all 3 MSs, and 10 RMs were double immunostained with HBME-1 and/or MOC-31 and Ber-EP4 successfully. Immunohistochemical studies performed on Papanicolaou stained cytologic smears proved to be useful in the differentiation between metastatic AC and mesothelial proliferation. HBME-1, thrombomodulin, MOC-31, Ber-EP4, and CD-15 were the most useful. In selected cases, it appeared that double immunostaining aided the differential diagnosis. Cancer (Cancer Cytopathol)

P53 Immunohistochemistry for Distinguishing Reactive Mesothelium from Low Grade Ovarian Carcinoma

To determine the utility of immunohistochemical staining for p53 in cell block material for distinguishing reactive mesothelium from borderline or low grade ovarian carcinoma. Paraffin-embedded cell blocks from paracentesis and pelvic wash fluid of 44 cases of ovarian carcinoma and 20 cases containing only reactive mesothelium were immunostained for p53 using monoclonal antibody DO-7. Tumor grades ranged from borderline to high grade and were serous papillary (33), clear cell (3), mucinous (2), endometrioid (2), mixed serous papillary/clear cell (3) and undifferentiated (1). The three authors independently evaluated the staining, including estimation of the percentage and intensity of positive nuclear staining. A separation of positive from negative cases was seen when staining intensity was considered the critical parameter; moderate to strong staining was considered truly positive. Seventy-three percent (8/11) of borderline tumors, 80% (8/10) of low grade tumors and 65% (15/23) of intermediate to high grade tumors showed moderate to strong positivity. Percentage of staining was a less-reliable parameter as 25% of negative cases were positive by this assessment. p53 Immunohistochemistry, using monoclonal antibody DO-7 combined with standard morphologic evaluation, may be useful in distinguishing benign reactive mesothelium from borderline or low grade ovarian carcinoma.

Mesothelial hyperplasia with reactive atypia: Diagnostic pitfalls and role of immunohistochemical studies—a case report

The cytomorphologic features of highly reactive mesothelial cells can be difficult to distinguish from malignant cells. We report on an unusual case of mesothelial hyperplasia in a pericardial effusion. The specimen contained bizarre-shaped cells and large tissue fragments in a patient with a history of lung carcinoma. The atypical cells were negative for CEA and LeuM-1 and positive for cytokeratins (AE1/3) and HBME-1. Strong HBME-1 positivity supported a mesothelial origin of the atypical cells and led to the diagnosis of reactive mesothelium. While HBME-1 cannot be used as the sole marker to establish an mesothelial origin; its use in a immunohistochemistry panel may be useful in individual cases to distinguish reactive mesothelial cells from carcinoma in effusion cytology. Diagn. Cytopathol. 2000;22:113–116. © 2000 Wiley-Liss, Inc.

Value of the Mesothelium-Associated Antibodies Thrombomodulin, Cytokeratin 5/6, Calretinin, and CD44H in Distinguishing Epithelioid Pleural Mesothelioma from Adenocarcinoma Metastatic to the Pleura

Until recently, the standard approach of most laboratories in distinguishing epithelioid pleural mesothelioma from metastatic adenocarcinoma has been a negative result from a panel of adeno-carcinoma-associated antibodies. However, several “mesothelium-associated” antibodies have been proposed as useful in this situation, and we have applied four of these putative mesothelioma markers—thrombomodulin, cytokeratin 5/6, calretinin, and CD44H—to a series of 61 epithelioid pleural mesotheliomas and 63 metastatic adenocarcinomas with known primary sites (lung = 19; breast = 21; ovary = 6; colon = 10; kidney = 4; uterus, epididymis, pancreas = 1 case each). Of the mesotheliomas, 55 of 61 (90%) stained for thrombomodulin, 56 of 61 (92%) for cytokeratin 5/6, 47 of 51 cases (92%) were positive for calretinin, and 39 of 43 (91%) were positive for CD44H. Of the metastatic adenocarcinomas, 12 of 63 (19%) cases were positive for thrombomodulin, 9 of 63 (14%) were positive for CK5/6, and 27 of 60 (45%) were positive for CD44H. With calretinin, only 1 case of 59 (2%) showed positive nuclear staining. All four antibodies stained reactive mesothelium; thrombomodulin also stained endothelium; and CD44H variably stained lymphocytes, macrophages, and fibroblasts. We conclude that all four antibodies show high sensitivity for epithelioid mesothelioma, but only calretinin (98%), cytokeratin 5/6 (86%), and thrombomodulin (81%) show sufficient specificity for practical use in this situ-ation.

Presence of simian virus 40 sequences in malignant mesotheliomas and mesothelial cell proliferations

Malignant mesotheliomas (MMs) are pleural-, pericardial-, or peritoneal-based neoplasms usually associated with asbestos exposure. Mesothelial cells are biphasic and may give rise to epithelial and sarcomatous MMs. In addition, benign or atypical proliferations of mesothelial cells may occur in response to many stimuli. There have been recent reports of simian virus 40 (SV40) DNA large T antigen (Tag) sequences in pleural MMs. To further understand the relationship between SV40, MMs, and mesothelial proliferations, we studied 118 MMs from multiple sites in Germany and North America, including 93 epithelial pleural, 14 sarcomatous or mixed pleural MMs, and 11 peritoneal MMs. In 12 pleural MMs, adjacent noninvasive tumor foci were identified and studied separately. Information about asbestos exposure (detailed history and/or microscopic examination for asbestos bodies) was available from 43 German patients. In addition, 13 examples of reactive mesothelium and 20 lung cancers from the United States were tested. DNA was extracted from frozen tumor and adjacent nontumorous tissues or after microdissection of archival formalin-fixed, paraffin-embedded microslides. Two rounds of PCR were performed with primers SVFor 3 and SVRev, which amplify a 105 bp region specific for SV40 Tag. The specificity of the PCR product was confirmed in some cases by sequencing. Our major findings were: 1) Specific SV40 viral sequences were present in 57% of epithelial invasive MMs, of both pleural and peritoneal origin. No significant geographic differences were found, and frozen and paraffin-embedded tissues were equally suitable for analysis. 2) There was no apparent relationship between the presence of SV40 sequences and asbestos exposure. 3) SV40 sequences were present in the surface (noninvasive) components of epithelial MMs. 4) SV40 sequences were not detected in MMs of sarcomatous or mixed histologies. 5) Viral sequences were present in two of 13 samples (15%) of reactive mesothelium. 6) Lung cancers lacked SV40 sequences, as did non-malignant tissues adjacent to MMs. Our findings demonstrate the presence of SV40 sequences in epithelial MMs of pleural and peritoneal origin and their absence in tumors with a sarcomatous component. Viral sequences may be present in reactive and malignant mesothelial cells, but they are absent in adjacent tissues and lung cancers. J. Cell. Biochem. 76:181–188, 1999. © 1999 Wiley-Liss, Inc.

Presence of simian virus 40 sequences in malignant mesotheliomas and mesothelial cell proliferations.

Malignant mesotheliomas (MMs) are pleural-, pericardial-, or peritoneal-based neoplasms usually associated with asbestos exposure. Mesothelial cells are biphasic and may give rise to epithelial and sarcomatous MMs. In addition, benign or atypical proliferations of mesothelial cells may occur in response to many stimuli. There have been recent reports of simian virus 40 (SV40) DNA large T antigen (Tag) sequences in pleural MMs. To further understand the relationship between SV40, MMs, and mesothelial proliferations, we studied 118 MMs from multiple sites in Germany and North America, including 93 epithelial pleural, 14 sarcomatous or mixed pleural MMs, and 11 peritoneal MMs. In 12 pleural MMs, adjacent noninvasive tumor foci were identified and studied separately. Information about asbestos exposure (detailed history and/or microscopic examination for asbestos bodies) was available from 43 German patients. In addition, 13 examples of reactive mesothelium and 20 lung cancers from the United States were tested. DNA was extracted from frozen tumor and adjacent nontumorous tissues or after microdissection of archival formalin-fixed, paraffin-embedded microslides. Two rounds of PCR were performed with primers SVFor 3 and SVRev, which amplify a 105 bp region specific for SV40 Tag. The specificity of the PCR product was confirmed in some cases by sequencing. Our major findings were: 1) Specific SV40 viral sequences were present in 57% of epithelial invasive MMs, of both pleural and peritoneal origin. No significant geographic differences were found, and frozen and paraffin-embedded tissues were equally suitable for analysis. 2) There was no apparent relationship between the presence of SV40 sequences and asbestos exposure. 3) SV40 sequences were present in the surface (noninvasive) components of epithelial MMs. 4) SV40 sequences were not detected in MMs of sarcomatous or mixed histologies. 5) Viral sequences were present in two of 13 samples (15%) of reactive mesothelium. 6) Lung cancers lacked SV40 sequences, as did non-malignant tissues adjacent to MMs. Our findings demonstrate the presence of SV40 sequences in epithelial MMs of pleural and peritoneal origin and their absence in tumors with a sarcomatous component. Viral sequences may be present in reactive and malignant mesothelial cells, but they are absent in adjacent tissues and lung cancers.

Diagnostic Utility of Sialosyl-Tn in Discriminating Carcinomatous Cells from Benign Mesothelium in Body Cavity Effusions

Detecting malignant cells in the setting of reactive mesothelium can be difficult. Several techniques have been tried but without widespread acceptance. Sialosyl-Tn (STn) is an aberrantly glycosylated precursor of the MN blood group antigen frequently expressed in carcinomas and dysplastic epithelium. We investigated the STn monoclonal antibody for its clinical utility as an isolated stain to discriminate benign mesothelium from malignant cells. Cell block material from 72 cases of body cavity fluids were immunostained for STn using the avidin-biotin complex method without antigen retrieval. Slides were incubated overnight at 4 degrees C in a humidified chamber. Strong immunoreactivity was noted in 31/40 (77%) carcinomatous cases. Only moderate staining was noted in 1 of 28 (4%) benign effusions and weak staining in 5 (18%) additional benign cases. Specificity was 100%, sensitivity 78%, positive predictive value 100% and negative predictive value 76%. No staining was noted in four noncarcinomatous malignant effusions. STn may have diagnostic value in this cytologic setting as part of a diagnostic panel but not as an isolated stain.

Desmin Expression in Reactive Mesothelium

Desmin is a marker of smooth and striated muscle, but evidence is accumulating that it may be expressed by human mesothelium. The aim of this study was to describe desmin expression in normal, reactive, and hyperplastic peritoneal mesothelium, and to evaluate its potential use as a marker for differentiating between epithelial and mesothelial proliferations. We immunohistochemically studied 27 tissue specimens (from 22 patients) with reactive mesothelium, including omentum (n = 14), fallopian tubes (n = 7), ovaries (n = 3), ascitic fluid (n = 1), and peritoneal washings (n = 2). Ovarian surface epithelium (OSE) from 9 cases and 28 ovarian surface epithelial tumors was evaluated for comparison. The desmin expression pattern in the mesothelium, which was similar to but less consistent than that of cytokeratins, was evident in flat and reactive mesothelium, including hyperplastic mesothelial sheets and mesothelium entrapped in clefts. Mesothelial pseudoglandular structures, present in three cases, were predominantly negative for desmin. Desmin expression was observed in the OSE in 4 of 9 cases but not in any mullerian-derived epithelium or mullerian type tumor. Thus, in contrast to cytokeratins, desmin discriminated mesothelial cells from mullerian type epithelia. Compared with vimentin, desmin discriminated mesothelial cells from other tissues except muscle cells. We conclude that desmin may be used in addition to cytokeratins and vimentin as a marker of peritoneal mesothelium.

Immunoreactivity of pleural malignant mesotheliomas to glutathione S-transferases.

Malignant mesotheliomas show a highly variable aggressiveness, but it is difficult to predict the outcome in the individual case at the time of diagnosis. Glutathione S-transferases are detoxification enzymes that have been correlated with the prognosis in some tumours. We have therefore assessed the value of GST expression as a prognostic parameter in mesotheliomas. The reactivities to GST-pi, -alpha and -mu antibodies were studied in histological sections from altogether 88 cases. Most of them showed distinct cytoplasmic reactivity to one or more of the GST antibodies tested. This high prevalence is in good agreement with the low responsiveness of mesotheliomas to chemotherapy. However, there was no prognostic value in detecting GST immunoreactivity, and it gave no information of value for distinguishing between neoplastic and reactive mesothelium.

Complementary value of five carcinoma markers for the diagnosis of malignant mesothelioma, adenocarcinoma metastasis, and reactive mesothelium in serous effusions.

Cytological slides of serous fluids of 41 malignant mesotheliomas, 88 metastatic adenocarcinomas, and 25 reactive effusions were immunostained with the antibodies anti-CEA, MOC-31, Leu-M1, Ber-EP4, and B72.3. Most mesotheliomas and all reactive fluids failed to stain with these antibodies. The sensitivity of the five markers to detect carcinoma cells differed remarkably. Especially MOC-31, Ber-EP4, and B72.3 stained with a high number of carcinoma cases and the complemetary value of Ber-EP4 and B72.3 to immunostain carcinoma cells was impressive: 94% of the metastatic adenocarcinoma cases reacted with Ber-EP4 or B72.3 in contrast to 1 of 41 malignant mesotheliomas.

Complementary value of five carcinoma markers for the diagnosis of malignant mesothelioma, adenocarcinoma metastasis, and reactive mesothelium in serous effusions

Complementary value of five carcinoma markers for the diagnosis of malignant mesothelioma, adenocarcinoma metastasis, and reactive mesothelium in serous effusions

CD44H expression in reactive mesothelium, pleural mesothelioma and pulmonary adenocarcinoma.

Malignant mesotheliomas are known to produce hyaluronic acid, in contrast to most pulmonary adenocarcinomas which produce neutral mucin. CD44H is the major cell surface receptor for hyaluronic acid. The aim of this study was to investigate immunohistochemically the expression of this antigen in reactive mesothelium, pleural mesothelioma and pulmonary adenocarcinoma and to assess its diagnostic utility in distinguishing the two tumours. Diffuse and intense membranous CD44H immunoreactivity was seen in 15 of 20 (75%) mesotheliomas and in all 20 biopsies of reactive mesothelium. In contrast, focal (< 10% tumour) expression of CD44H was seen in only three of 20 (15%) pulmonary adenocarcinomas. We advocate the use of CD44H as a positive mesothelial marker for incorporation alongside other established immunohistochemical markers used to distinguish mesothelioma from adenocarcinoma.

Parathyroid hormone‐related protein antigen localization distinguishes between mesothelioma and adenocarcinoma of the lung

The distinction between pleural malignant mesothelioma and pulmonary adenocarcinoma remains a problem in diagnostic histopathology. Parathyroid hormone-related protein (PTHrP) has been demonstrated in the neoplastic cells of malignant mesotheliomata, using a polyclonal antiserum raised to synthetic PTHrP(1-16). In a series of 44 malignant mesotheliomata and 44 cases of pleural adenocarcinomata, PTHrP was localized immunohistochemically in 84 per cent of the mesotheliomata and in 11 per cent of the pleural adenocarcinomata. Normal and reactive mesothelium did not contain detectable PTHrP. The presence of PTHrP in a very high percentage of malignant mesotheliomata indicates the value of including it in the panel of antibodies utilized in the differential diagnosis of mesothelioma.

Immunoreactivity for bcl-2 protein in malignant mesothelioma and non-neoplastic mesothelium.

Immunohistochemical study of bcl-2 protein immunoreactivity in human non-neoplastic mesothelium (44 cases) and in malignant mesothelioma (62 cases) using a murine monoclonal antibody (clone 124) showed cytoplasmic immunoreactivity for bcl-2 protein in five cases of malignant mesothelioma. Non-neoplastic mesothelium was not immunoreactive. Immunoreactivity for bcl-2 protein does not add useful prognostic information in malignant mesothelioma since survival times of bcl-2 positive and bcl-2 negative cases did not differ. Nevertheless, the detection of bcl-2 protein in malignant mesothelioma might be useful for the differentiation from reactive mesothelium.

Proliferation in malignant mesothelioma as determined by mitosis counts and immunoreactivity for proliferating cell nuclear antigen (PCNA)

In order to assess its discriminating and prognostic value, we studied immunoreactivity for proliferating nuclear cell antigen (PCNA) in human malignant mesothelioma (31 cases) and in human non-neoplastic mesothelium (33 cases with reactive mesothelium and 20 cases of normal mesothelium) using the murine monoclonal antibody PC10. We also compared it with mitosis counts expressed as the mitotic volume index (MV index). There were differences between malignant mesothelioma, reactive mesothelium, and normal mesothelium for percentage of PCNA immunoreactive cells (mean +/- SD; 27 +/- 9, 9.5 +/- 5.1, and 3.6 +/- 1.6, respectively) and for their MV index (20.3 +/- 4.5, 9.4 +/- 2.1, and 3.6 +/- 0.6, respectively). The median actuarial survival was 10.1 months for patients with less than 25 per cent PCNA immunoreactive cells, 9.4 months for patients with less than 20 mitoses per mm2 of tumoural tissue, 5.9 months for patients with more than 25 per cent PCNA immunoreactive cells, and 5.3 months for patients with more than 20 mitoses per mm2 of tumoural tissue. Our results suggest that PCNA immunoreactivity is useful in discriminating between neoplastic and non-neoplastic mesothelium and that it may have prognostic value in malignant mesothelioma.

Pathology of mesothelioma

As a diagnostic pathologist, “art” seems an appropriate word to describe my response to the problems presented by pleural biopsies. There was a time when “sport” might have been the right word, but I have not yet reached the point where I can honestly describe my activities as a “science”. What are the problems the pathologist is called upon to solve? 1. Benign or malignant? (a) Reactive mesothelium or epithelial mesothelioma (b) Fibrosis or desmoplastic mesothelioma (c) Organizing pleurisy or mixed mesothelioma 2. What kind of malignancy is it? (a) Adenocarcinoma or mixed mesothelioma (b) Undifferentiated carcinoma, melanoma or epithelial mesothelioma (c) Sarcoma primary or secondary, especially MFH or sarcomatous mesothelioma (d) Lymphoma or lymphohistiocytoid mesothelioma Of these, I find 1, especially l(b) much more difficult than 2 and it is here that “science” in the form of new techniques has failed to come to the assistance of “art”. Flow cytometry is not a technique of which 1 have any firsthand experience, but I had expectations that it might be able to separate reactive from malignant mesothelial cells After all, it does seem to be a more sensitive prognostic test than histology in lung cancer [ 11. However, Tierney et al. [2] found that with mesothelial lesions there is so much overlap that it is useless as a diagnostic test. As always, this is likely to be particularly so in those cases in which the reactive cells are atypical or the mesothelioma cells well differentiated which are just the ones setting the problem which “art” alone finds difficult to solve. While some metastatic tumours are wonderfully separated by being all aneuploid, some reactive cases are aneup$id and some mesotheliomas are diploid. Nuclear organizing regions (NORs) are chromosomal segments in which ribosomal RNA is encoded. Silver staining identifies acidic proteins associated with sites of rRNA transcription. Theoretically, a human diploid cell has 20 NORs in pairs but after mitosis they usually clump together to form a nucleolus. If the cell is aneuploid, there are more chromosomes and more NORs and if cell proliferation is rapid, clumping together is not

Expression of p53 in reactive mesothelium

Expression of p53 in reactive mesothelium

P53 immunostaining in the distinction between benign and malignant mesothelial proliferations using formalin-fixed paraffin sections.

The distinction between reactive mesothelium and mesothelioma in pleural biopsy specimens is notoriously difficult, and conventional immunohistochemical markers have provided no relief. The object of this study was to examine the frequency of immunohistochemically detectable p53 overexpression in routinely processed, paraffin-embedded tissue from pleural mesotheliomas and from pleura showing reactive mesothelial hyperplasia, using a polyclonal antibody to formalin-resistant p53 epitopes, and to consider the diagnostic utility of this antibody in the distinction between mesothelioma and reactive mesothelium in pleural biopsy specimens. Immunostaining was enhanced by pepsin predigestion prior to the application of the primary antibody. Positivity occurred in 10/16 epithelial mesotheliomas, 9/19 biphasic mesotheliomas, 2/12 sarcomatous mesotheliomas but in none of 20 reactive pleura. Immunostaining was particularly intense in some of the biopsy specimens, which may be due to the rapidity with which these small pieces of tissue were fixed. In conclusion, this study suggests that p53 immunostaining can help to distinguish epithelial or biphasic mesothelioma from reactive mesothelial hyperplasia in formalin-fixed, paraffin-embedded pleural biopsy specimens.

Immunoreactivity for p53 protein in malignant mesothelioma and non-neoplastic mesothelium.

The presence of p53 protein in non-neoplastic pleural mesothelium (40 cases) and in human malignant mesothelioma (36 cases) was assessed immunohistochemically using the antibodies DO7, CM-1, and PAb240. In a quarter of the malignant mesotheliomas, there was nuclear immunoreactivity for p53 protein with both the DO7 and CM-1 antibodies. There were no statistically significant differences between the various mesothelioma subtypes (P > 0.05). No immunoreactivity was found with the PAb240 antibody, suggesting absence of mutant-type p53 protein. Nonneoplastic mesothelium was not immunoreactive with any of the antibodies. We conclude that there is immunoreactivity for p53 protein in some mesotheliomas. p53 protein immunoreactivity could be used to discriminate between neoplastic and reactive mesothelium.