Prognostic significance of p16/cdkn2a loss in pleural malignant mesotheliomas

Homozygous deletion of p16/CDKN2A is the most common genetic abnormality in malignant mesotheliomas. The aim of this study was to determine prognostic significance of p16/CDKN2A loss in malignant pleural mesotheliomas (MPM) as defined by immunohistochemistry and fluorescence in situ hybridization (FISH). High-density tissue microarrays were constructed from archival formalin-fixed paraffin-embedded samples of 48 MPM. Long survival (LS) was defined as survival greater than 3 years from the time of diagnosis, and short survival was defined as less than 3 years from the time of diagnosis. Both loss of p16 protein expression by immunohistochemistry and homozygous deletion of p16 by FISH were associated with adverse prognosis. Female gender, positive p16 immunoexpression, and lack of p16/CDKN2A deletion significantly predicted the survival for the LS group. Statistical analysis showed a very strong correlation of immunohistochemistry and FISH data. Cases positive for p16 immunoexpression and negative for 9p21 deletion showed the best survival time. Our study is the first to demonstrate decreased frequency of homozygous deletion of 9p21 and loss of p16 immunoreactivity in pleural mesotheliomas from patients with long-term survival of greater than 3 years in contrast to patients with rapidly fatal mesotheliomas. A possible implementation of these tests into preoperative prognostication of MPM and therapeutic decisions should be considered.     

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.     

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.     

Chromosomal alterations in early stages of malignant mesotheliomas

In a case of a 67-year-old man with two different early stages of a predominantly epithelioid mesothelioma (“mesothelioma in situ”, “early-stage mesothelioma”), chromosomal imbalances were determined by comparative genomic hybridisation (CGH), a molecular cytogenetic technique to detect chromosomal gains and losses in tumour cells. In the case of the mesothelioma in situ cells, nine different chromosomal alterations could be detected (losses on 3p, 5q, 6q, 8p, 9p, 15q, 22q, Y; gain on 7q), whereas the early-stage mesothelioma showed the same defects except for the gain on 7q. The simultaneous losses of 6q, 9p and 22q, as well as other chromosomal regions, correlate well with the most common defects previously found in 90 cases of more-advanced-stage mesotheliomas using CGH. These data demonstrate that initial chromosomal defects in early stages of mesotheliomas can be detected by conventional CGH in combination with laser microdissection. The molecular cytogenetic findings support the histological diagnosis of a pleural mesothelioma. The surprisingly high number and extent of genomic alterations found in the examined case probably reflects the genomic instability in the tumour cells and indicates a “genetic chaos” even in earlier stages of malignant mesotheliomas.     

Keratins in malignant mesotheliomas and pleural adenocarcinomas: comparative immunohistochemical analysis with polyclonal and monoclonal antibodies

The distribution of intra-cellular keratins was studied in normal pleural mesothelium, malignant mesotheliomas and adenocarcinomas. This study was performed on deparaffinized sections of tissue fixed in Bouin's solution by indirect immunofluorescence with a monoclonal antibody (KL1) and a conventional keratin antiserum (AKS). Discrepancies were detected using one antibody or the other. Cells from normal mesothelium and 18 cases of malignant mesotheliomas (papillary, tubulary, solid epithelial type) were strongly labelled only by KL1. The 2 cases of sarcomatoid type were negative with both antibodies. In contrast 5 metastatic adenocarcinomas and 5 lung adenocarcinomas were weakly positive or negative with both antibodies. These data confirm the presence of cytokeratins in epithelial differentiation process. Although a clear-cut distinction between mesotheliomas and adenocarcinomas was not possible using these keratin antibodies. Our data point out the importance of reactivity pattern of the antibody used in such investigations.     

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.     

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。     

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.     

Primary malignant gonadal mesotheliomas and asbestos

AIMS: The clinicopathological, immunohistochemical and aetiological aspects, with respect to asbestos, of seven primary gonadal mesotheliomas (three intratesticular, four ovarian) are described and compared. These tumours are extremely rare, poorly described and the knowledge of their natural history is very limited. METHODS AND RESULTS: The cases were collated from the UK Health and Safety Executive Mesothelioma Register over a 24-year period (1968-91). Primary mesotheliomas of the tunica vaginalis and ovary comprised 0. 09% (10 cases) and 0.03% (three cases) of mesothelioma deaths, respectively. No primary intratesticular (non-tunica vaginalis) malignant mesotheliomas have been described. In this study, we present seven (three intratesticular, four ovarian) primary malignant gonadal mesotheliomas. In both genders the tumours show a similar age distribution (with median onset in the sixth decade), a similar association with asbestos (in approximately 50% cases), a diverse histological spectrum (with predominantly tubulopapillary epithelial subtype tumours) and an immunophenotype that is comparable with malignant pleural and peritoneal mesothelioma. The clinical course appears variable (mean, 26 months; range, 9-50 months). All tumours in the study presented as localized masses and their prognosis appeared more favourable than that of diffuse pleural and peritoneal cases. CONCLUSIONS: An awareness of the existence of these rare forms of malignant mesothelioma is important to prevent misdiagnosis. Immunohistochemistry has an important role in confirmation of the diagnosis. The accurate diagnosis of primary gonadal mesothelioma has potentially important medicolegal compensation considerations as a significant proportion of these cases are associated with asbestos.     

Ultrastructural features of diffuse malignant mesotheliomas

The distinction of malignant mesothelioma from tumors metastatic to the serosal membranes can often be made based on the results of histochemical or immunohistochemical studies. However, in some cases, these techniques are inadequate to make a firm diagnosis. In these instances, electron microscopic studies with the observation of a constellation of characteristic ultrastructural findings may permit an unequivocal diagnosis of mesothelioma.     

Intermediate filament expression in mesotheliomas: leiomyoid mesotheliomas are not uncommon

In this study we examined intermediate filament expression in 45 formalin-fixed mesotheliomas. Immunostaining for cytokeratin was found in 86%, for vimentin in 71%, and for desmin in 4%; none stained for glial fibrillary acidic protein or neurofilament. The two biphasic mesotheliomas which expressed desmin also expressed smooth muscle actin but were negative for myoglobin. This, together with the ultrastructural findings, was taken as unequivocal evidence of a leiomyoid form of mesothelioma which might easily be confused with leiomyosarcoma. Both of these tumours co-expressed cytokeratin, exemplifying the value of cytokeratin immunostaining in the distinction between mesothelioma and sarcoma. Consistent non-expression of glial fibrillary acidic protein in mesotheliomas may help to distinguish them from nerve sheath tumours.     

Localized pleural malignant mesothelioma

Pleural malignant mesothelioma (PMM) is a rare tumor and it is commonly seen in the form of multiple nodules or a diffuse tumor. A localized tumor mass in the pleura is extremely rare. Only seven cases have been reported. In this report, we present an additional case of localized PMM and describe the immunohistochemical and flow cytometric findings. A 61-year-old woman, without a history of smoking or asbestos exposure, presented with a severe pain in her right shoulder and arm. Chest radiography showed a solitary mass in the right upper lung field. Computed tomography showed a 5 cm right upper lung mass. Magnetic resonance imaging showed that the mass extended to the wall of the thorax. The patient underwent surgery for total removal of the tumor. Pathology revealed a localized malignant mesothelioma. Immunohistochemical analysis showed that the tumor was strongly and diffusely positive for cytokeratins with high and low molecular weight, and focally positive for vimentin and epithelial membrane antigen (EMA), but it was negative for carcinoembryonic antigen, Factor VIII, α -fetoprotein and Leu-M1. Flow cytometry showed an aneuploid DNA content in the tumor. The final diagnosis was localized malignant mesothelioma (epithelial type). The patient showed signs of local recurrence 5 months after surgery, and radiotherapy was given.     

Immunoreactivity for cadherins, HGF/SF, met, and erbB-2 in pleural malignant mesotheliomas

AIMS: To assess the immunoreactivity of malignant mesotheliomas for N- and E-cadherins, hepatocyte growth factor/scatter factor (HGF/SF) and the tyrosine kinase receptors, met and erbB-2. METHODS AND RESULTS: Pleural malignant mesotheliomas were stained using a standard indirect immunoperoxidase method applied to paraffin sections. Malignant mesotheliomas were immunoreactive for N-cadherin (26/29; 90%), met (29/29; 100%) and erbB-2 (28/29; 97%). Focal immunoreactivity was present for E-cadherin in epithelioid or mixed tumours (14/25; 56%), and for HGF/SF (9/24; 38%). CONCLUSIONS: Expression of N-cadherin supports the diagnosis of malignant mesothelioma and use of appropriate antibodies would be a useful addition to a diagnostic antibody panel. Focal staining for E-cadherin does not exclude mesothelioma. Signalling pathways mediated via met and erbB-2 may play a role in the growth and spread of malignant mesotheliomas.     

BAP1 immunohistochemistry and p16 FISH results in combination provide higher confidence in malignant pleural mesothelioma diagnosis: ROC analysis of the two tests

Differentiation of malignant pleural mesothelioma (MPM) from benign mesothelial proliferation remains problematic. Loss of nuclear staining of BRCA1‐associated protein 1 (BAP1; detected using immunohistochemistry (IHC)) and homozygous deletion (HD) of p16 (detected using fluorescence in situ hybridization (FISH)) are useful for differentiation of MPM from reactive mesothelial hyperplasia (RMH), but the correlation between BAP1 expression loss and p16 HD has not been fully described. We performed BAP1 IHC and p16‐specific FISH for 40 MPM and 20 RMH cases, and measured proportions of cells showing BAP1 expression and p16 HD for each case. The diagnostic accuracy for MPM and the cut‐off values of the two methods were assessed using receiver operating characteristic (ROC) analysis. BAP1 expression loss, p16 HD and coexistence of both were present in 27 (67.5 %), 27 (67.5 %) and 17 (42.5 %) MPM cases, respectively. Three MPM cases (7.5 %) and all 20 RMH cases had neither BAP1 loss nor p16 HD. There was no correlation between the results of the two methods. Their combination showed higher sensitivity (92.5 %, 37/40) and estimated probability than BAP1 IHC and p16‐specific FISH used alone. BAP1 IHC and p16‐specific FISH have independent diagnostic value, and have increased reliability when used in combination, for MPM diagnosis.     

A new insight into the histogenesis of 'mesodermomas'—malignant mesotheliomas

A myogenic phenotype was induced in cultures of human mesothelial cells treated for 72 h with atrazine, a triazine derivative. Immunoreactivity for both myosin and myoglobin was detected in a large number of these cells, irrespective of their polygonal or spindle morphology, whereas no expression of desmin was observed. These findings support the embryological identity of mesothelium and mesoderm, the former being, in the post-embryonic stage, potentially capable of differentiation along the same lineages which the latter normally displays during embryogenesis. In the light of this concept it can be assumed that primary malignancies arising from the mesothelium have the competence to express the pluripotent nature of embryonic mesoderm, and hence the term mesodermoma is appropriate for this group of tumours, including mesotheliomas in a classical sense. A postulated mechanism for the phenotypic change of mesothelial cells is also outlined, involving atrazine conversion to 5-aza-chloro-cytidine, a probable DNA hypomethylating and gene activating agent, like its analogue 5-azacytidine.