1. بسم الله الرحمن الرحيم IN THE NAME OF GOD 11/22/2018
Dr.Afshar Moghaddam

2. HBME- 1 immunostaining in reactive mesothelial versus metastatic adenocarcinoma cells in body serous fluids
11/22/2018
Dr.Afshar Moghaddam

3. Noushin Afshar Moghaddam MD* Mohsen Dehghani Zahedi MD
Authors
Noushin Afshar Moghaddam MD*
Mohsen Dehghani Zahedi MD
Hamidreza Heidarian MD
Reza Tahririan MD
*Correspondence:
Noushin Afshar Moghaddam, Pathology Department, Faculty of Medicine,
Isfahan University of Medical Science, Isfahan, Iran

4. Introduction

5. Introduction 1
Mesothelial cells are the only specific cells in serous cavities; whenever the serous membranes are irritated in a process of inflammation or longstanding effusion, these cells proliferate, shed in the fluid and show morphological changes in nucleus and cytoplasm. In some cases morphological differentiation of reactive mesothelial cells from adenocarcinoma in serous effusions is extremely difficult,1 so adoption of complementary methods will increase diagnostic accuracy.

6. Introduction 2
Nowadays immunocytochemistry (ICC) is one of the suggested methods which helps distinguishing between mesothelial and adenocarcinoma.2 ICC can be done on specimens like flow cytometry3, 4 but most laboratories prefer direct smear, cell block or cytospin; nevertheless, cellblock and cytospin had better results.5 Until late 20th century, immunocyochemical differentiation of mesothelial cells and adenocarcinoma was based on markers which were absent in mesothelial cells.

7. Introduction 3
In recent years immunoreactive antibodies against mesothelial cells have been introduced which show no reaction with carcinomatous cells.
Recommended mesothelial markers include: calretinin,6-8 thrombomodulin,9 cytokeratin5/6,10 and HBME-1.11, 12 HBME-1, a monoclonal anti-human antibody against mesothelial cells which reacts with unknown mesothelial cell surface antigen is commercially available. 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.13

8. The purpose of 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.

9. Material & Mehodes

10. Fifty two cytologic specimens of serous effusions processed by cell block technique were retrieved from the pathology archive of Alzahra Hospital (Isfahan).For the purpose of the study they were assigned to two groups as follows:
Group I:26 effusions containing reactive/benign mesothelial cells from patients with no evidence of malignancy based on cytomorphology, clinical and immaging data.
Group II: 26 effusions containing carcinomatous cells from patients whose diagnoses were established on routine histology.

11. The 3µm sections were cut and deparaffinized
The 3µm sections were cut and deparaffinized. Immunostaining with HBME-1 was performed using an Envision technique. Exposure of antigen to citrate buffer 1% (PH=6) was done in microwave for 20 minutes. Slides were incubated with HBME-1 anti-human monoclonal antibody, clone M3505 with 1/50 dilution (DAKO Co., Denmark) in room temperature. The staining intensity of cells was evaluated with high power field (×400) Zeiss microscope, in 0.46 millimeters dimension.

12. Positive cases were scored based on percentage of stained cells to 3 degrees: 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.14
In blocks with diagnosis of adenocarcinoma, the primary origin of tumor included: 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, ANOVA, Chi square, Leven and Scheffe.

20. RESULTS

21. The mean age of the patients in the reactive mesothelial and adenocarcinoma groups were 56.5± 12 and 55.5± 13 years, respectively.
The mean percentage of stained cells with HBME-1 in metastatic adenocarcinomatous cells was: ( ) and in reactive mesothelial cells was: ( ), (p=0.001).

24. Comparison of 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 (SD=20.82) and in metastatic adenocarcinoma was zero (p=0.001);
thin membranous pattern in reactive mesothelial cells was (SD=20.82) and in metastatic adenocarcinoma was (SD=42), (p=0.001);
cytoplasmic pattern in reactive mesothelial cells was zero and in metastatic adenocarcinoma was (44.49), (p=0.001);

28. Discussion

29. 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 such diseases as anemia, cirrhosis, systemic lupus erythematosus, pulmonary infarction, renal failure, and AIDS can help interpretation of these conditions; however, in many cases, especially outpatients, clinical data is 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.5

30. HBME-1 monoclonal antibody is the most challenging mesothelial marker; some researchers contend that this antibody reacts with an unknown antigen on mesothelial cell (villous) surface. Some authors believe the membranous pattern of staining in mesothelial cells versus cytoplasmic pattern in adenocarcinomas can distinguish the two In this study mean percentage of stained cells for HBME-1 in metastatic adenocarcinomas (25.75%) was lower than that of mesothelial cells (67.88%), (p=0.001).

31. 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 was seen in reactive mesothelial cells more frequently than in adenocarcinomas. Thus, the differential value of this marker improves by considering its pattern of staining.18
The comparative study of staining patterns and their intensity for HBME-1 by primary origins indicate that ovarian adenocarcinomas show a severe (3+) membranous pattern similar to that of reactive mesothelial cells.

32. Close 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 emberyonic derivation with mesothelial cells.19 So if ovarian metastatic adenocarcinomas show classic malignant morphologic changes like severe cellular atypism in crowded clusters or papillae, it will be easy to differentiate them from reactive mesothelials.
But, when individual cells are shedding, utility of the HBME-1 as a diagnostic aid is limited and other diagnostic means should be considered.

33. Findings of Ascoli et al
Findings of Ascoli et al. in their study entitled: ″Utility of HBME-1 immunostaining in serous effusions″ indicated that: immuoreactivity of reactive mesothelial cells was variable between %, most of which showed a membranous and a few cytoplasmic pattern. In the carcinoma group, 24 percent 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.13

34. Lozano et al. concluded that HBME-1 was negative in 80% of adenocarcinomas and positive in all reactive mesothelial cells.1
In a study done 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.20
Filho et al. found that 25% of ovarian adenocarcinomas were immunoreactive for HBME-1.21
Politi et al. contend that HBME-1 sensitivity and specificity in differentiation of reactive mesothelial cells from metastatic adenocarcinoma was 98% and 71%, respectively.22

35. 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.2,23 In this study we used cell blocks, in almost similar conditions with tissue biopsies, instead of cytologic smears, to reduce the mentioned technical problems.

36. Conclusion:
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 this kind of metastatic cells is limited.

37. Thank you for your attention
11/22/2018
Dr.Afsharmoghaddam