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ORIGINAL ARTICLE Table of Contents   
Year : 2009  |  Volume : 52  |  Issue : 1  |  Page : 25-28
Utility of fibronectin in immuocytochemial differentiation of reactive mesothelial cells from metastatic malignant cells in serous effusions


Lady Hardinge Medical College, New Delhi, India

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   Abstract 

Cytological evaluation of serous effusions often poses difficulties to the pathologists. This study was designed to assess the utility of fibronectin as a mesothelial cell marker and evaluate its use along with carcinoembryonic antigen (CEA) as a short panel to aid in the differentiation of reactive mesothelial cells from metastatic adenocarcinoma cells in serous effusions. Forty serous effusion cases from clinically benign cases and forty from clinically malignant cases with a known primary were included in the study. After routine cytological evaluation, the cases were categorized as benign, suspicious and malignant. All the cases were studied for fibronectin and CEA immunostaining using APAAP technique. In the present study, fibronectin emerged as a 100% specific and 93.4% sensitive marker for mesothelial cells. CEA immunostaining was seen in 80% cases of metastatic adenocarcinoma in serous effusions. Mesothelial cells did not show any CEA positivity. Short panel of CEA and fibronectin aided in reaching a correct diagnosis in three out of five cytologically suspicious cases.

Keywords: Carcinoembryonic antigen, fibronectin, immunocytochemistry, serous effusions

How to cite this article:
Agarwal C, Jain M. Utility of fibronectin in immuocytochemial differentiation of reactive mesothelial cells from metastatic malignant cells in serous effusions. Indian J Pathol Microbiol 2009;52:25-8

How to cite this URL:
Agarwal C, Jain M. Utility of fibronectin in immuocytochemial differentiation of reactive mesothelial cells from metastatic malignant cells in serous effusions. Indian J Pathol Microbiol [serial online] 2009 [cited 2019 Nov 12];52:25-8. Available from: http://www.ijpmonline.org/text.asp?2009/52/1/25/44957



   Introduction Top


Cytological analysis of body fluids has always been a diagnostic challenge to the pathologists. Many benign reactive processes can show significant atypical mimicking malignant changes. On the other hand, a few malignant conditions lack sufficient atypical changes for an unequivocal diagnosis of malignancy. Ancillary techniques such as electron microscopy, flow-cytometry and morphometry have been used to solve the ambiguity in cytological differential diagnosis. [1] However, these are of limited use owing to the high cost, availability at only a few specialized centers, requirement of highly skilled personnel and low sensitivity and specificity. The application of immunocytochemical techniques to serous effusions is well documented. In general, immunocytochemistry is more sensitive and specific than other methods and easy availability of reagents for use in most of the laboratories make it a good choice. [2]

A number of markers have been studied for adenocarcinoma cells; however, there is a scarcity of markers for the cells of mesothelial lineage. Fibronectin is a recently studied marker for mesothelial cells. [3],[4] Because there are only a few reports of experience with the immunostaining of fibronectin in cytological smears, we investigated its utility as a mesothelial cell marker and also studied the combined use of fibronectin and carcinoembryonic antigen (CEA) as a panel to distinguish metastatic malignant cells from reactive mesothelial cell proliferation in serous effusions.


   Materials and Methods Top


Eighty serous effusion fluid specimens obtained over a period of 1 year were included in the study. Forty specimens were from clinically benign cases (group 1) and forty from clinically and histologically diagnosed primary carcinomas (group 2). All the cases were subjected to conventional cytological examination as well as immunocytochemistry using monoclonal antibodies against fibronectin (1:200 dilution, Neomarkers) and CEA (1:50 dilution, Dakopatts) with alkaline phosphatase-anti-alkaline phosphatase (AP-AAP) technique and correlation was done between the two.

The fluids were cytocentrifuged and smears were prepared from the sediment. Ethanol (95%) fixed smears were stained with Papanicolaou stain and air-dried smears were stained with Giemsa and Hematoxylin and Eosin stains. For immunocytochemical study, air-dried smears were fixed in cold acetone (-10°C) for one hour and stored at -70°C until processing. Immunostaining was performed using the AP-AAP technique.

Using routine cytological criteria for evaluation, [5] the smears were classified as malignant when cells defined as malignant were found, as suspicious when atypical cells were found and as benign when reactive mesothelial cells or no malignant cells were observed. The immunostaining for fibronectin and CEA was considered positive when unequivocal staining was observed in cytoplasm and cell membrane in at least 20% cells.


   Results Top


The study included 80 fluid specimens. Forty cases were from patients without the evidence of malignancy and forty from patients with clinically and histologically diagnosed primary carcinomas. These two groups were labeled group 1 and group 2 respectively.

In group 1 (patients without malignancy), cytological findings were benign in 38 and suspicious in 2 cases. These two were proven tuberculous pleural effusion cases. In the second group (patients with malignancy), cytological findings were benign in 7, suspicious in 3 and malignant in 30.

Immunocytochemistry

The cytological smears of effusions in both the groups were subjected to immunocytochemistry using primary monoclonal antibody against CEA and fibronectin.

Fibronectin

In group 1 (patients without malignancy), unequivocal positivity with fibronectin was observed in 36/38 cytologically benign effusions [Figure 1]. The two cases that were fibronectin negative had sparse cellularity with only few lymphocytes. The intensity of staining was moderate to strong in all the cases.

In group 2, reactive mesothelial cells were present in 37/40 cases, all of which showed positive reaction with fibronectin. Fibronectin positivity was not noted in the carcinoma cells in any of cytologically malignant cases. Taking both the groups together, fibronectin showed l00% specificity and 93.4% sensitivity as a mesothelial cell marker.

Carcinoembryonic antigen

Positive immunostaining for CEA was obtained in 24/30 cytologically malignant effusions [Table 1]. Insofar as they could be assessed, the morphological features of these cells were compatible with carcinoma cells [Figure 2]. CEA was consistently absent in all the cytologically benign effusions. However, mild non-specific staining of inflammatory cells was present in few cases.

Suspicious cases

Two cases from group 1 and three cases from group 2 were reported to have suspicious cytological morphology. Suspicious cells in both the cases of group 1 showed positive fibronectin labeling and negative CEA staining, thereby confirming their mesothelial nature. CEA was negative in these cells of suspicious morphology. Out of the three suspicious cases of group 2, CEA positivity was obtained in one case. These suspicious looking cells, however, stained negative for fibronectin. This case, thus, was finally diagnosed as malignant. In the other two cases, the suspicious cells stained negative for both fibronectin and CEA. Thus, after the addition of immunocytochemical staining results, definite diagnoses could be reached in three out of the five cytologically suspicious cases.


   Discussion Top


Distinguishing carcinoma cells from reactive mesothelial cells is of central importance in the reliable cytological assessment of serous effusions. Because of a number of reasons, both artifactual and attributable to the nature of lesions, there could be a significant overlap between benign and malignant conditions. Immunocytochemical identification of carcinoma cells in effusions has won recognization over the past few decades as a worthwhile complement to conventional cytological diagnosis.

Many markers such as CEA, EMA, BerEP-4, B72.3 and Leu M1 have been studied for adenocarcinoma cells; [6] however, currently, no single reliable mesothelial cell-specific antibody exists. Vimentin is an intermediate filament present in the cells of mesenchymal derivation and as such could be a useful marker for distinguishing mesothelial cells from carcinomas. However, as many as 94% of the adenocarcinomas have shown positive with vimentin, [4] vimentin has proved to be of limited value in distinguishing epithelial and mesenchymal cells.

Fibronectin is a recently studied marker for mesothelial cells. [3],[4] It is a multifunctional adhesive protein whose primary role is to attach cells to a variety of matrices. It is 450-KDa glycoprotein consisting of two chains held together by disulfide bond. [7] It is a glycoprotein of connective tissue and has been identified intracellularly and extracellularly as well as in the growth medium from mesenchymal cells in vitro . [8],[9],[10] Several workers have studied fibronectin expression and organization in mesothelial and mesothelioma cells. Earlier studies involved the measurement of fibronectin concentration in various body fluids especially pleural fluid as an attempt to differentiate benign from malignant effusions. Fibronectin is produced by fibroblasts, monocytes and endothelial cells. [7] Fibronectin is thought to be directly involved in attachment, spreading and migration of cells. In addition, it serves to enhance the sensitivity of certain cells such as capillary endothelial cells to the proliferation effects of growth factors. [7]

Athanassiadou et al . were the first to show monoclonal fibronectin positivity in reactive mesothelial cells of serous effusions. [3] They demonstrated combined use of fibronectin and carcinoembryogenic antigen in the study of serous effusions. The combination of fibronectin positivity /CEA negativity was found to have 100% specificity and 92.3% sensitivity in patients with benign or reactive effusions and fibronectin negative/CEA positive to have 85.7% specificity and 80.7% sensitivity for malignancy.

Lee et al . used a panel consisting of cytokeratin, carcinoembryonic antigen, epithelial membrane antigen and fibronectin to distinguish between carcinoma and reactive mesothelial cells in serous effusions. [4] In their study, they found fibronectin to be a highly specific marker for mesothelial cells. This finding suggested that positive fibronectin staining would usually exclude carcinomas. However, fibronectin will not distinguish between neoplastic and reactive mesothelial states. In the present study, fibronectin emerged as a 100% specific and 93.4% sensitive marker for mesothelial cells. This finding suggests that fibronectin positivity in a cell excludes the possibility of it being a carcinoma cell. However, both benign and malignant mesothelial cells will give positive reaction with fibronectin; hence, it is not possible to distinguish them on the basis of Fibronectin staining. Recently other specific markers have been introduced for diagnosis of malignant mesothelioma, including thrombomodulin, calretinin, CD44, HBME-1, WT1 and keratin 5/6. [6] However, cross reactivity with other tumors in particular metastatic adenocarcinoma have been noticed. A small percentage of adenocarcinomas show positivity for calretinin (cytoplasmic staining in 10%-35% cases and nuclear staining in <5% cases). [11],[12] Thrombomodulin immunostaining is seen in 15% adenocarcinoma cases. [13] Cytokeratin 5/6 may also show intense staining in ovarian adenocarcinomas and squamous cell carcinomas and rarely in some pulmonary adenocarcinomas. Similarly, 8%-3% of ovarian adenocarcinomas may show positive results with WT-1. Results with HBME-1 to distinguish between mesothelial cells and adenocarcinoma cells is also controversial at present. [14] In our study, fibronectin positivity was seen only in the mesothelial cells and it did not show cross reactivity with the adenocarcinoma cells.

CEA is a high molecular weight glycoprotein, originally described as a specific marker for colorectal carcinoma by Gold and Fredman in 1965. [15] CEA expression has been found in the adenocarcinomas of the GIT., lung and less often in ovary, breast, kidney and liver. [16] CEA has been used as an immunocytochemical marker for metastatic adenocarcinoma cells in several studies. Percentage of adenocarcinomas with positive CEA staining has varied from 50%-100%. [2],[4] Most investigators have reported absence of CEA in benign exfoliated mesothelial cells, whereas others have reported weak peripheral reactivity in mesothelial cells and in an occasional case of mesothelioma. [2],[4] Such weak peripheral staining of some reactive mesothelial cells is explained in part by the trapping of reaction product by surface microvilli.

In the present study, carcinoma cells showed CEA expression in 80% cases. Reactive mesothelial cells did not show any reaction with anti-CEA. Fibronectin emerged as a highly sensitive and exceptionally specific marker for mesothelial cells. It should, therefore, be placed high in the panel of antibodies to differentiate mesothelial cells from metastatic adenocarcinoma cells.

A short panel of CEA and fibronectin was found to be extremely useful in the differentiation of metastatic adenocarcinoma cells from reactive mesothelial cells in the cytological smears prepared from body fluids, enabling one to make a definitive diagnosis in cytologically suspicious cases.

 
   References Top

1.Cajulis RS, Yu GH, Gokaslan T, Hidvegi DF. Modified interphase cytogenetics Technique as an adjunct in the analysis of atypical cells in body fluids. Diagn Cytopathol 1997;16:331-5.  Back to cited text no. 1    
2.Nance KV, Silverman JF. Immunocytochemical panel for the identification of malignant cells in serous effusions. Am J Clin Pathol 1991;95:867-74.  Back to cited text no. 2  [PUBMED]  
3.Athanassiadou P, Athanassiades P, Lazaris D, Kyrkou K, Petrakakou E Aravantinos D. Immunocytochemical differentiation of reactive mesothelial cells and adenocarcinoma cells in serous effusions with the use of carcinoembryonic antigen and Fibronectin. Acta Cytol 1993;38:718-22.  Back to cited text no. 3    
4.Lee JS, Nam JH, Lee MC, Park CS, Juhng SW. Immunocytochemical panel for distinguishing between carcinoma and reactive mesothelial cells in serous effusions. Acta Cytol 1996;40:631-6.  Back to cited text no. 4  [PUBMED]  
5.Koss LG. Effusions in the presence of cancer. In: Koss LG, editor. Diagnostic Cytology and its histopathological basis. 4th ed. Philadelphia: J.B Lippincott Company 1992; p. 1176-84.  Back to cited text no. 5    
6.Moran CA, Suster S. Tumours of lung and pleura. In: Fletcher CD, editor. Diagnostic histopathology of tumors. 2nd ed. Churchill Livigstone; 2001. p. 171-208.  Back to cited text no. 6    
7.Cotran RS, Kumar V, Collins T. Tissue repair: Cellular Growth, fibrosis and wound healing. In: Cotran RS, Kumar V, Collins T, editors. Robbins pathological basis of disease. 6TH ed. WB Saunders Company; 1999. p. 89-112.  Back to cited text no. 7    
8.Haynes RD. Fibronectins. Sci Am 1986;254:42-51.  Back to cited text no. 8    
9.Jaffe EA, Mosher DF. Synthesis of Fibronectin by cultured human endothelial cells. J Exp Med 1978;147:1779-91.  Back to cited text no. 9  [PUBMED]  [FULLTEXT]
10.Mosher DF. Fibronectin. Frog Hemost Thromb 1980;5:111-5.  Back to cited text no. 10    
11.Doglioni C, Tos AP, Laurino L, Iuzzolino P, Chiarelli C, Celio MR, et al . Calretinin: Novel immunocytochemical marker for mesothelioma. Am J Surg Pathol 1996;20:1037-46.  Back to cited text no. 11    
12.Riera JR, Astengo-Osuna C, Longmate JA, Battifora H. The immunohistochemical diagnostic panel for epithelial mesothelioma: A reevaluation after heat induced epitope retrieval. Am J Surg Pathol 1997;21:1409-19.  Back to cited text no. 12  [PUBMED]  [FULLTEXT]
13.Ordonez NG. Value of antibodies 44-3AG, SM3, HBME-l and thrombomodulin in differentiating epithelial pleural mesotheliomas from lung adenocarcinoma: A comparative study with other commonly used antibodies. Am J Surg Pathol 1997;21:1399-408.  Back to cited text no. 13    
14.Barr NJ, Wu NC, Taylor CR. Immunohistochemistry and diagnostic pathology. In: Bancroft JD, Gamble M, editors. Theory and Practice of Histological techniques. 5th ed. Churchill Livingstone; 2003. p. 537-52.  Back to cited text no. 14    
15.Gold P, Freedman SO. Demonstration of tumor specific antigen in human colonic carcinomata by immunological tolerance and absorbtion techniques. J Exp Med 1965;121:4398-62.  Back to cited text no. 15    
16.Mezger J, Stotzer O, Schilli G, Bauer S, Wilmanns W. Identification of carcinoma cells in ascetic and pleural fluid: Comparison of four panepithelial antigens with carcinoembryonic antigen. Acta Cytol 1992;36:75-81.  Back to cited text no. 16    

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Correspondence Address:
Chetna Agarwal
A-86, sector 27, Noida, Uttar Pradesh - 201 301
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.44957

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