| Abstract|| |
Background: The cytological diagnoses of serous effusions are usually made by routine cytomorphology with certainty. However, overlapping cases sometimes exist between reactive mesothelial and adenocarcinoma cells. We tried to evaluate the diagnostic utility of HBME-1 in distinguishing between reactive mesothelial cells and adenocarcinoma in serous effusions. Materials and Methods: Fifty-two cytologic specimens processed by cell-block technique were retrieved from the archive and were assigned to two groups: Group I: 26 effusions containing reactive/benign mesothelial cells; Group II: 26 effusions containing carcinoma cells. Immunostaining with HBME-1 was performed using an Envision technique. The staining intensity of cells, according to proportion of immunoreactive cells, was scored as: 0 (negative), 1+ (<10%), 2+ (10-50%), and 3+ (≥50%); and the predominant staining pattern of positive cells were determined. Statistical analysis and tests of significance were performed using SPSS software. Results: The calculated mean values (in percentile) for stained cells in adenocarcinoma and reactive mesothelial cells were 25.57 and 67.88, respectively ( P = 0.001). Thin membranous, thick membranous, thick and thin membranous, cytoplasmic and combined patterns of staining in adenocarcinoma cells were respectively 4 cases (21.1%), 0 case, 0 case, 8 cases (42.1%), and 7 cases (36.8%), and in reactive mesothelial cells, these were 7 cases (26.9%), 1 case (3.8%), 18 cases (69.2%), 0 case, and 0 case, respectively. The intensity of staining in majority (88.5%) of reactive mesothelial cells was scored 3+, but the distribution varied in the other group. Conclusions: The staining pattern and intensity for HBME-1 is a useful panel for differentiation of adenocarcinoma and mesothelial cells.
Keywords: Cytology, HBME-1 antibody, metastatic adenocarcinoma, reactive mesothelial cell, serous effusion
|How to cite this article:|
Rahmani A, Dehghani MZ, Afshar NM, Heidarian H, Tahririan R. HBME-1 immunostaining in reactive mesothelial versus metastatic adenocarcinoma cells in serous fluid. Indian J Pathol Microbiol 2011;54:460-3
|How to cite this URL:|
Rahmani A, Dehghani MZ, Afshar NM, Heidarian H, Tahririan R. HBME-1 immunostaining in reactive mesothelial versus metastatic adenocarcinoma cells in serous fluid. Indian J Pathol Microbiol [serial online] 2011 [cited 2020 Jan 27];54:460-3. Available from: http://www.ijpmonline.org/text.asp?2011/54/3/460/85075
| Introduction|| |
Mesothelial cells are the only specific cells in serous cavities; when serous membranes are irritated in the process of inflammation or longstanding effusion, these cells proliferate, shed in the fluid, and show morphological changes in the nucleus and cytoplasm. In some cases, morphological differentiation of reactive mesothelial cells from adenocarcinoma in serous effusions is extremely difficult 1 ; therefore, adoption of complementary methods will increase diagnostic accuracy. At present, Immunocytochemistry (ICC) is one of the suggested methods, which helps in distinguishing between mesothelial cells and adenocarcinoma. , ICC can be performed on specimens such as flow cytometry but most laboratories prefer direct smear, cell block or cytospin; nevertheless, cellblock and cytospin had shown better results. ,, Until late 20 th century, immunocytochemical differentiation of mesothelial cells and adenocarcinoma was based on markers, which were absent in mesothelial cells. In recent years, immunoreactive antibodies against mesothelial cells have been introduced, which show no reaction with carcinoma cells. Recommended mesothelial markers include Calretinin, thrombomodulin, cytokeratin5/6, and HBME-1. ,,,,,, HBME-1, a monoclonal anti-human antibody against mesothelial cells, which reacts with unknown mesothelial cell surface antigens, is available on commercial basis. ,, HBME-1 immunoreactivity in normal tissues is limited to mesothelial cells, bronchial epithelium, endocervix, and cartilage. In tumors, however, it has been reported in epithelial mesothelioma, certain adenocarcinomas, and cartilaginous tumors. 
The purpose of this study was to evaluate diagnostic utility of ICC staining with HBME-1 monoclonal antibody for distinguishing mesothelial from adenocarcinomatous cells in benign and malignant effusions.
| Materials and Methods|| |
Paraffin blocks and hematoxylin and eosin (H and E)-stained slides of peritoneal and pleural fluid cell blocks were retrieved from cytology archive of Alzahra Hospital, Medical University of Isfahan. From among 856 slides, which were screened to ascertain their diagnosis, 26 blocks with a diagnosis of metastatic adenocarcinoma and their confirmatory biopsy, and 26 blocks containing reactive mesothelial cells without a history or any clinical or imaging documents in favor of malignancy, were selected. For ICC staining with HBME-1 monoclonal antibody, 3-mm-thin sections were obtained from selected blocks, and then the specimens were subjected to deparaffinization and hydration. The antigen was exposed to citrate buffer 1% (pH = 6) in a microwave for 20 minutes. Slides were incubated with HBME-1 anti-human monoclonal antibody, clone M3505 with 1/50 dilution (DAKO Co., Denmark) at room temperature. The staining intensity of cells was evaluated with high-power field (΄400) Zeiss microscope, in 0.46-mm dimension. Positive cases were scored based on percentage of stained cells to 3°: 1+, less than 10%; 2+, 10-50%; and 3+, more than 50%. The staining patterns were classified as membranous (thin and thick), cytoplasmic, and combined.  In blocks with diagnosis of adenocarcinoma, the primary origin of tumor included the following: ovary (10 cases), lung (7 cases), breast (5 cases), and alimentary tract (4 cases). For data analysis, SPSS software (version: 15) was used for t test, analysis of variance (ANOVA), Chi-square, Leven and Scheffe.
| Results|| |
The mean age of patients in the reactive mesothelial groups was 56.5 years and in the adenocarcinoma group was 55.5 years . The mean percentage of stained cells with HBME-1 in metastatic adenocarcinomatous cells was: 25.57% (14.9-36.23%) and in reactive mesothelial cells was: 67.88% (61.95-73.82%) (P = 00.001). A comparison of intensity and patterns of staining of the two groups is shown in [Table 1] and [Table 2].
|Table 1: Comparison of mean and SD of staining intensity of metastatic adenocarcinomas by specific origin|
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|Table 2: Comparison of staining patterns for HBME-1 in the two groups of reactive mesothelial cells and metastatic carcinoma|
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Comparison of the means of percentages of different patterns of staining between the two groups revealed that the mean of:
- thick membranous pattern in reactive mesothelial cells was 18.26 (SD = 20.82) and in metastatic adenocarcinoma, it was zero (P = 0.001);
- thin membranous pattern in reactive mesothelial cells was 81.73 (SD = 20.82) and in metastatic adenocarcinoma, it was 32.69 (SD = 42) (P = 0.001);
- cytoplasmic pattern in reactive mesothelial cells was zero and in metastatic adenocarcinoma, it was 40.38 (44.49) (P = 00.001).
Cytoplasmic and membranous patterns of staining are shown in [Figure 1] and [Figure 2].
|Figure 1: Membranous pattern of staining with HBME-1 monoclonal anti body in reactive mesothelial cells (IHC, x400)|
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|Figure 2: Cytoplasmic pattern of staining with HBME-1 monoclonal anti body in some of the metastatic adenocarcinoma cells (IHC, x400)|
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Descriptive statistics as well as comparison of intensity and patterns of staining of metastatic adenocarcinomatous cell by their primary origin are shown in [Table 3],[Table 4],[Table 5], respectively.
|Table 3: Comparison of mean and SD of staining intensity of metastatic adenocarcinomas by specific origin|
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|Table 4: Comparison of HBME-1 staining intensity in metastatic adenocarcinomas by specific origins|
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|Table 5: Comparison of HBME-1 staining patterns in metastatic adenocarcinomas by specific origin|
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| Discussion|| |
In some cases, reactive mesothelial cells may imitate malignant cells due to their severe nuclear changes, including enlargement and irregularity of nuclei with coarse chromatin and conspicuous nucleoli. Clinical data with respect to diseases such as anemia, cirrhosis, systemic lupus erythematosus, pulmonary infarction, renal failure, and AIDS can help in the interpretation of these conditions. However, in many cases, especially outpatients, clinical data are not easily available. Surprisingly, malignant cells in adenocarcinoma, the most common malignancy of serous membranes, can mimic mesothelial cells; therefore, complementary diagnostic procedures are mandatory in these cases.  HBME-1 monoclonal antibody is the most challenging mesothelial marker; some researchers contend that this antibody reacts with an unknown antigen on mesothelial cell surface. Some authors believe that the membranous pattern of staining in mesothelial cells versus cytoplasmic pattern in adenocarcinomas can distinguish the two. ,, In this study, the mean percentage of stained cells for HBME-1 in metastatic adenocarcinomas (25.75%) was lower than that of mesotheliomas (67.88%) (P = 0.001). With regard to staining intensity, severe reaction (3+) was seen in most reactive mesothelial cells. Cytoplasmic pattern of staining was absent in reactive mesothelial cells, but membranous patterns were seen in reactive mesothelial cells more frequently than in adenocarcinomas. Thus, the differential value of this marker improves by considering its pattern of staining. 
A comparative study of staining patterns and their intensity for HBME-1 by primary origins indicates that ovarian adenocarcinomas show a strong (3+) membranous pattern similar to that of reactive mesothelial cells. Intimacy of the origin of cells in ovarian adenocarcinoma and mesothelial cells can explain this condition. Ovarian adenocarcinomas originate from the germinal epithelium of ovary, which is considered to have identical embryonic derivation with mesothelial cells.  Therefore, if ovarian metastatic adenocarcinomas show classic malignant morphologic changes such as severe cellular atypia in crowded clusters or papillae, it will be easy to differentiate them from reactive mesothelial cells. But, when individual cells are shedding, utility of the HBME-1 as a diagnostic aid is limited and other diagnostic means should be considered.
Ascoli et al. in their study indicated that immuoreactivity of reactive mesothelial cells was variable between 20% and 100% , most of which showed a membranous pattern and a few showing a cytoplasmic pattern. In the carcinoma group, 24% reacted with HBME-1, most of which (83%) were ovarian adenocarcinomas and mainly showed a membranous pattern. In other carcinomas, immunoreactivity was much lower: Breast (11%), lung (7%), and alimentary tract (14%), with a cytoplasmic pattern. 
Lozano et al.  concluded that HBME-1 was negative in 80% of adenocarcinomas and positive in all reactive mesothelial cells. In a study conducted by Fetsch et al., all reactive mesothelial cells were immunoreactive for HBME-1, with a dominant membranous pattern and moderate cases (61%) to severe cases (12%) staining intensity. Filho et al.  found that 25% of ovarian adenocarcinomas were immunoreactive for HBME-1.
Politi et al.  contend that HBME-1 sensitivity and specificity in differentiation of reactive mesothelial cells from metastatic adenocarcinoma was 98% and 71%, respectively.
Variation in immunoreactivity for HBME-1 and the overlapping of its staining patterns in reactive mesothelial cells and adenocarcinoma are shown in many studies. Probable causes of discrepancy of results are: sample size and type, fixation, method of antibody demaskation, antibody clones, and pathologists who interpret the immunoreactivity. , In this study, we used cell blocks, in almost similar conditions with tissue biopsies, instead of cytologic smears, to reduce the previously mentioned technical problems.
| Conclusions|| |
The pattern and intensity of staining with HBME-1 is useful in differentiating reactive mesothelial cells from adenocarcinoma. Due to similarity of immunoreactivity of HBME-1 in ovarian metastatic adenocarcinoma and reactive mesothelial cells, the utility of this marker per se in peritoneal fluids containing such metastatic cells is limited.
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Department of Pathology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]