Immunocytochemistry of effusions: Processing and commonly used immunomarkers

SPECIMEN PROCESSING

• As in surgical pathology, formalin-fixed paraffin-embedded cell-block section, although not ideal, are preferred for immunocytochemical evaluation. The use of other fixatives or preservatives may alter antigenic properties and cause misleading non-reproducible results.

Although cell-block sections are preferred and considered optimum, depending on the practical situation, immunocytochemistry may be performed on different types of cytology smears, including direct smears, Cytospin smears,^[3-6] and liquid-based cytology preparations (SurePath and ThinPrep), etc.^[4-6] However, with this approach, various limiting factors, such as possible interference with immunoreactivity, increased background staining, and lack of opportunity to evaluate coordinate immunoexpression in the same cells (NOT just similar cells), should be considered in the final interpretation of results. • The results obtained are generally compared to those reported in the literature, which are predominantly based on immunocytochemistry with formalin-fixed paraffin-embedded tissue sections. This is the most important caveat to be considered when using cytology smears for immunocytochemistry of effusions, since the results obtained may not be reproducibly comparable to those that are published.

CYTOLOGY SMEARS

When the sample is scanty, immunocytochemistry may have to be performed on smears. Direct smears, Cytospin smears, or liquid-based cytology preparations (SurePath and ThinPrep) may be standardized for immunostaining, as indicated below.

CELL-BLOCKS

• Most published studies comparing immunophenotypes are based on results obtained with formalin-fixed paraffin-embedded tissue sections. Because of this, formalin-fixed paraffin-embedded cell-blocks are recommended for immunocytochemical evaluation of effusions.^[6,23] Substituting formalin or altering any of the processing steps may adversely affect the immunoreactivity pattern and lead to non-representative results.^[24] For an individual case, the immunoreactivity may be enhanced by a particular approach with excellent crisp immunostaining, but this may not be comparable with the results in the literature and may be responsible for false-positive interpretation. At the other end of the gamut is loss of immunoreactivity by the modified protocol, which leads to false-negative results.

The pattern of malignant cells in effusions varies from the small groups of cells to the scattered isolated cells. Singly scattered abnormal cells in 3–4 μm serial sections may be difficult to locate in subsequent sections. • To overcome this, 3–4 µm thick serial sections should be oriented and mounted in an identical manner on glass slides.^[1,2] The slides should be labeled serially, so that each section can be related to another by referring to the serial numbers on the slides. This simple SCIP approach is especially rewarding in unexpected situations especially when immunostaining pattern is not straightforward and needs careful scrutiny. ^[1,2]

Sections of cell-blocks (3–4 μm) are mounted on charge-coated slides and immunostained with a routine protocol following standard precautions and guidelines for quality control.^[15]

CELL-BLOCK VERSUS CYTOLOGY SMEARS [Direct Smears/Cytospins/Liquid based cytology preparations (SurePath and ThinPrep)] FOR IMMUNOCYTOCHEMISTRY

The most significant limitation with cytology smears is the inability to evaluate coordinate immunoreactivity, since the same cells cannot be present on more than one smear. On the other hand, serial sections of cell-blocks allow such evaluation of multiple immunomarkers in serial sections of the same cells.

Although cytologic preparations have been used successfully for immunostaining, they are not recommended for the immunocytochemical evaluation of effusions.^[3] Complicating factors associated with effusion fluid immunocytochemistry include non-specific and unexpected immunoreactivity due to the protein-rich fluid in which the cells are floating, and large three-dimensional cell groups which may entrap immunostains, leading to false-positive results. Crushed, degenerated, or necrotic cells may show non-specific immunostaining.

IMMUNOMARKERS

Over the past few years, the number and spectrum of antibodies available for immunocytochemistry have increased dramatically.^[15,25] In addition, other technical advances have also enhanced the role of immunocytochemistry. The technique of heat-induced antigen epitope retrieval has remarkably improved immunostaining for a wide range of antibodies.^[26–28] Automation and ready-to-use kits have further improved the reliability and the reproducibility of immunostaining. Quality assurance programs, participation in external proficiency testing, laboratory inspections (reinforcing appropriate documentation, equipment maintenance, procedure updating) and the requirement of qualified testing personnel have further improved the technical aspects of immunostaining.^[29-31] In addition, recent availability of rabbit monoclonal antibodies has furthered the refinements in this technology.^[32]

GROUPING OF IMMUNOMARKERS FOR EFFUSION IMMUNOCYTOCHEMISTRY

The different immunomarkers used for effusions may be grouped by their predominant pattern of immunoreactivity:

Predominantly adenocarcinoma immunomarkers

BerEP4

Claudin-4

EMA—cytoplasmic

B72.3

mCEA

CA19.9

CD15 (not effective for effusions)

Miscellaneous adenocarcinoma immunomarkers

HMFG-2

MOC-31

Other organ-specific immunomarkers such as TTF-1, PSA, CDX2, GATA3, PAX8, PAX2, NKX3.1, etc.

Predominantly mesothelial immunomarkers

Calretinin

Vimentin

WT-1

Cytokeratin 5/6

Cytokeratin 7

EMA (membranous with microvilli)

HBME-1 (membranous with microvilli)

D2-40 (podoplanin): new— promising role

Mesothelin: new role evolving

Miscellaneous mesothelium-associated immunomarkers

CD44

Thrombomodulin

Antimesothelial cell antibody

N-cadherin

OV632

Loss of immunomarkers in mesothelial cells as diagnostic of mesothelioma

BAP1 (nuclear)

MTAP (cytoplasmic)

Miscellaneous with conflicting reports

E-cadherin

HBME-1

RECENT UPDATE: Dual-Color Immunostaing to Improve Scip Approach^[23,33,34]

Recently we evaluated dual-color immunostaining of effusion fluid cell-block sections.^[33,34] Based on the categorization of different effusion immunomarkers mentioned above, various pairs of two immunomarkers may be applied. A few combinations mentioned below were evaluated (Table 1):

1. BerEP4 (brown) followed by Vimentin (red) [Figure 1]

2. Vimentin (brown) followed by cytokeratin 7 (red)

3. Calretinin (brown) followed by BerEP4 (red)

4. Calretinin (brown) followed by cytokeratin 20 (red)

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Figure 1:

Dual-color immunohistochemistry with BerEP4 (brown) as first, followed by vimentin (red) immunostaining. Metastatic adenocarcinoma cells (NC) are non-immunoreactive for vimentin with immunoreactivity for BerEP4 (brown) on the background of vimentin (red) immunoreactive inflammatory cells and reactive mesothelial cells. (Pleural fluid, history of pancreatic adenocarcinoma), (a, 20X, b, 100X).

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Combination A (for immunostaining protocol, please see below) showed excellent results for detecting and identifying second foreign population of metastatic adenocarcinoma cells in most cases as brown immunoistained cells on the red immunoistained background (reactive mesothelial cells and inflammatory cells) (Figure 1).^[33] Similar to BerEP4 brown immunoreactivity, the metastatic adenocarcinoma cells may also be standardized by replacing Claudin 4 as brown immunostaining step with vimentin red immunostaining.

Combination B correlated with the immunoreactivity pattern observed with single-color immunostaining. The original immunoreactivity pattern of the second immunomarker was compromised in combinations C and D. BerEP4 showed decreased immunostaining intensity with a negative result in some cases. Immunoreactivity for cytokeratin 20 was completely lost in combination D. This interference is under investigation, but initial findings suggest that the concentration of the second antibody (related to alkaline phosphatase system with red chromogen) should be higher than that used for the peroxidase system with 3,3’-Diaminobenzidine (DAB) as the chromogen.

On a scale of 1 (easy) to 5 (difficult), the average difficulty of interpretation was 2.95 (range 1–5) utilizing the single-color method.^[34] However, interpretation with the dual-color method was very easy with an average difficulty score of only 1. This difference was statistically significant (two tailed p value <0.0001, paired t test). Higher scores of difficulty were observed in cases with scant tumor cells or predominantly single tumor cells, when the SCIP approach^[2] with conventional one-color immunocytochemistry was applied. The interpretation of some of the specimens in this category was impossible, if the sections were not oriented identically on all slides and the serial relationship was not properly identified on individual slides.

The studies^[1,2] concluded that dual staining facilitated easy identification of the foreign populations of malignant cells in effusion fluids. However, slightly decreased or complete loss of immunoreactivity for BerEP4 and complete loss for Cytokeratin (CK) 20 suggested a need for standardization of any combination to be applied with any particular protocol. The protocol used in this study is detailed below.

Dual-color immunostaining protocol [example BerEP4 (brown)with vimentin (Red)]:

Note:

• Staining performed on automatic Ventana BenchMark ULTRA IHC/ISH platform.

• Unless indicated all reagents are from Ventana-Roche.

• Slide is rinsed with Reaction Buffer ( pH 7.6).

• Coverslip (Ultra LCS) solution is oil base solution, to prevent evaporation during high temperature incubation.

1. Deparaffinize the section with EZPrep solution (1:10) at 72^oC.

2. Apply Cell Conditioner # 1 and incubate for 36 minutes at 95^oC.

3. Apply one drop of UV INHIBITOR (part of ultraView Universal DAB Detection Kit), incubate for 4 minutes.

4. Apply one drop of First Primary Antibody (Prep kit #11 BerEp4 , Dako), incubate for 32 minutes at 36^oC.

5. Apply one drop of UV Horseradish Peroxidase (HRP) Universal Multimer (part of DAB Kit), incubate for 8 minutes.

6. Apply one drop of UV DAB (part of DAB Kit) and one drop of UV DAB H2O2 (part of DAB Kit), incubate for 8 minutes.

7. Apply one drop UV COPPER (part of DAB Kit), incubate for 4 minutes.

8. Apply one drop of Second Primary Antibody (Vimentin), incubate for16 minutes at 36^oC.

9. Apply one drop of UV RED UNIV MULT (part of ultraView Universal Alkaline Phosphatase (AP) Red Detection Kit), incubate for 12 minutes.

10. Apply one drop of UV Red Enhancer (part of AP Red Kit), incubate for 4 minutes.

11. Apply one drop of UV Fast Red A (part of AP Red Kit) and UV Red Naphthol (part of AP Red Kit), incubate for 8 minutes.

12. Apply one drop of UV Fast Red B (part of AP Red Kit), incubate for 8 minutes.

13. Apply one drop of Hematoxylin II (counterstain), incubate for 8 minutes.

14. Apply one drop of Bluing Reagent (post counterstain), incubate for 4 minutes.

15. Remove oil in Coverslip (Ultra LCS) solution by rinsing in soapy water (100 ml of DAWN Professional Liquid Concentrate Detergent (P&G Professional www.PGPRO. com) in 250 ml Tap water).

16. Dehydrate quickly in two times in 95% alcohol and two times in 100% alcohol.

17. Clear quickly with 10 dips in xylene (three times).

18. Coverslip with xylene on Tissue-Tec coverslipper.

The metastatic cells, located in relation to different components of the effusion fluid, could be evaluated further with additional immunomarkers (Table 2) in conjunction with pertinent clinical history.

Methods applying more than two immunocolors have also been reported.^[35-37] Based on the reported literature, a variety of diagnostic combinations may be evaluated. As observed, a protocol-related interference in immunoreactivity pattern is a possibility. Because of this, such combinations and their immunostaining protocols should be standardized and evaluated prior to their clinical application.

IMMUNOMARKERS COMMONLY USED IN EFFUSION IMMUNOCYTOCHEMISTRY

Some of the immunomarkers commonly applied to effusion immunocytochemistry are described below briefly in alphabetical order. The details of some of these as used in our laboratory are set out in Table 3.

ANTIMESOTHELIAL CELL ANTIBODY

A polyclonal antibody against cytoplasmic protein of mesothelial cells was reported to be immunoreactive with all epithelioid mesotheliomas and non-immunoreactive with all of the adenocarcinomas.^[38] Further studies, however, are not found in the English language literature.

B72.3 (TAG-72)

Clones/synonyms: BRST-3, TAG-72, CC49

This is an antibody that reacts with a tumor-associated oncofetal antigen. A wide variety of adenocarcinomas, including those of lung, breast, gastrointestinal (GI) tract, ovary, pancreas, and endometrium, are immunoreactive to B72.3.^[42] Melanomas, sarcomas, and leukemia/lymphomas do not show immunoreactivity for B72.3. It is negative in most non-neoplastic tissues, but normal secretory endometrium is immunoreactive.^[38,43-46] The predominant immunostaining pattern with B72.3 is cytoplasmic (Figure 2,3c,d).^[42]

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Figure 2:

B72.3 immunoreactivity pattern (metastatic mammary adenocarcinoma, pleural fluid). Metastatic adenocarcinoma cells (red arrow NC) show a cytoplasmic immunoreactivity pattern. [a, Immunostained cell-block section (a, 40X).]

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Effusion studies show B72.3 immunoreactivity in 44–80% of adenocarcinomas,^[45–47] but mostly negative or rarely positive (<10%) in malignant mesotheliomas and reactive mesothelial cells.^[38,45,46] These results are comparable to those with surgical pathology tissue specimens.^[49-52]

BerEP4 (HUMAN EPITHELIAL ANTIGEN)

Clones/synonyms: BER-EP4 Dako

This antibody reacts with two glycoproteins along the cell surface and in the cytoplasm of epithelial cells^[44-46,116] and does not show immunoreactivity with mesothelial cells, and nerve, glial, muscle, lymphoid, or mesenchymal tissue.^[116] Compared to all other antiepithelial antibodies, only rare cases of epithelioid mesothelioma may show immunoreactivity with BerEP4. The immunoreactivity pattern is predominantly membranous (Figure 4). In our experience BerEP4 is more sensitive than B72.3 (see Figure 3).

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Figure 3:

Comparison of immunoreactivity with BerEP4 and B72.3 (metastatic mammary adenocarcinoma, pleural fluid). (a,b) Most of the adenocarcinoma cells (red arrows NC) show strong membranous BerEP4 immunoreactivity. (c,d) Compared to this, B72.3 immunoreactivity is absent in most of these neoplastic cells (blue arrows NC) with immunoreactivity in rare neoplastic cells (arrowheads). [a–d, Immunostained cell-block section (a,c, 20X; b,d, 40X).]

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Figure 4:

BerEP4 immunoreactivity pattern (metastatic mammary adenocarcinoma, pleural fluid). (a) The neoplastic cells in proliferation spheres (red arrow NC) show membranous immunostaining (arrowhead in a) with a honeycomb-like pattern. (b) Solitary adenocarcinoma cells (red arrow NC) show a membranous immunostaining pattern along the cell membrane (arrowhead in inset). [a,b, Immunostained cell-block section (a,b, 40X).]

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In effusion studies, BerEP4 typically shows negative immunostaining in epithelioid mesothelioma and reactive mesothelial cells. It shows immunoreactivity to adenocarcinoma cells in 32–96% of cases.^{[45,46,53,86]} A large study on effusions reported 83% immunoreactivity in adenocarcinoma—93% ovarian, 88% GI tract, 81% lung, and 73% breast.^[53] Other studies have reported some overlap of immunoreactivity with focal immunostaining in 26% of epithelioid mesothelioma and strong–diffuse immunostaining in 92% adenocarcinomas.^[52] Strong–diffuse immunostaining with BerEP4 points to adenocarcinoma rather than malignant mesothelioma.

CALRETININ AB1 49, DC8

Clones/synonyms: Dak Calret1

Calretinin is a neuron-specific calcium-binding protein. It is strongly expressed in neural tissues and certain non-neural cell types such as mesothelial cells.^{[9,61,78,87,120-123]} Antibodies to calretinin show strong nuclear (and cytoplasmic) immunoreactivity in benign and neoplastic mesothelial cells. The cytoplasmic and nuclear immunostaining has been described as a ‘fried-egg’ appearance (Figures 5, 6).^[62] Adenocarcinomas are usually non-immunoreactive or show weak cytoplasmic immunoreactivity in rare cells.^{[61,78,122,123]}

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Figure 5:

Calretinin immunoreactivity pattern (pleural fluid). Reactive mesothelial cells (blue arrows). The effusion also contains metastatic mammary carcinoma cells (red arrow NC). The diagnostic immunoreactivity pattern is nuclear (arrowhead in inset), which is usually associated with concurrent cytoplasmic immunoreactivity (blue arrow RM). Cytoplasmic immunoreactivity alone should be considered non-specific for interpretation of mesothelial cells with calretinin. [Immunostained cell-block section (40X).]

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Figure 6:

Calretinin immunoreactivity pattern (epithelioid mesothelioma, pleural fluid). Mesothelioma cells (arrow in a) show nuclear (arrowhead 1) immunoreactivity, usually with cytoplasmic immunostaining (arrowhead 2), imparting the so-called ‘fried-egg’ appearance. In the absence of concurrent nuclear immunostaining, cytoplasmic immunoreactivity alone should be considered non-specific for mesothelial interpretation. [a,b, Immunostained cell-block section (a, 40X; b, 100X zoomed).]

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Similar to other immunomarkers, different types and sources of antibodies with variable processing protocols may lead to inconsistent results. Some of the studies used polyclonal antibodies from diverse sources.^[50,52,71] A few studies evaluating this immunomarker concluded calretinin to be unreliable.^[58,87,92] However, with the advent of purified and monoclonal antibodies, calretinin immunostaining has demonstrated better results in distinguishing between malignant mesothelioma and adenocarcinoma in histologic and cytologic preparations.^[52,121,124] A recent study evaluating polyclonal and monoclonal calretinin antibodies demonstrated similar results.^[63]

Studies on tissue have reported immunoreactivity in 92–100% of malignant mesotheliomas, with only the rare adenocarcinoma demonstrating nuclear immunoreactivity.^[61,78] Many studies with cell-blocks, Cytospins, and de-stained PAP-stained smears of effusions have demonstrated encouraging results with reactive and neoplastic mesothelial cells with calretinin immunoreactivity (nuclear and cytoplasmic) in 88–100% of mesotheliomas and 80– 100% of reactive effusions.^{[9,62,64,123,125]} We find calretinin to be an excellent immunomarker for evaluation of effusion cytopathology if properly applied and interpreted. We use monoclonal antibody for calretinin (Clone Dak Calret 1, Dako Corporation, Carpinteria, CA; dilution: 1:400, for 30 minutes at room temperature, after heatinduced epitope retrieval in 10 mmol/L citrate buffer, pH 6.0, for 35 minutes with 20 minutes cooling time at room temperature).^[124] As a part of an optimal immunopanel, calretinin is extremely valuable for distinguishing singly scattered cells of adenocarcinomas from reactive mesothelial cells.

CDX2

Clones/synonyms: 7C7/D4, CDX-2-88

CDX2 is a homeobox gene that encodes an intestinespecific transcription factor alpha-methylacyl-CoA racemase (AMACR/P504S). It has fairly restricted expression in selective neoplasms. It is associated with an adenomatous polyposis coli (APC) mutation and activation of the Wnt pathway. [66] CDX2 immunostaining may be useful in discriminating between colorectal carcinomas and other adenocarcinomas of non-gastrointestinal origin.^[67,69] With diagnostic nuclear immunostaining pattern, CDX2 has shown promising results (Figure 7) with effusions (personal experience).

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Figure 7:

CDX2 immunoreactivity pattern (metastatic colonic adenocarcinoma, peritoneal fluid). The adenocarcinoma cells show nuclear immunoreactivity (arrow NC). Compare with non-immunoreactive nuclei with blue hematoxylin counterstain (arrowhead). [Immunostained cell-block section (40X).]

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CEA CARCINOEMBRYONIC ANTIGEN)

Clones/synonyms: 11-7, 12.140.10, A115, A5B7, CEJ065, CEMO10, COL-1, D14, IL-7, M773, PARLM1, T84.66, TF3H8-1, ZC23

Immunoreactivity with the monoclonal antibodies to CEA is relatively specific for adenocarcinoma. It is typically nonimmunoreactive in reactive and malignant mesothelioma cells.^[45,74] With the exception of granulocytes, non-malignant tissues do not show significant immunoreactivity.^[74] The immunostaining associated with colorectal and other carcinomas of lung, breast, and stomach^{[38,43-46,56,72]} is cytoplasmic and membranous (Figure 8).

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Figure 8:

Monoclonal CEA (mCEA) immunoreactivity pattern (metastatic ovarian carcinoma, peritoneal fluid). Metastatic adenocarcinoma cells show cytoplasmic (c) and membranous (m) immunostaining. [Immunostained cell-block section (100X).]

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Since the immunoreactivity varies considerably with the type of antibody used,^[45] it is critical to select the proper CEA antibody when ordering the immunostain: pCEA (polyclonal antibodies to CEA) are more sensitive, but they demonstrate cross-reactivity with CEA-related substances and show strong immunostaining of neutrophils and macrophages.^[45,52] Although this cross-reactivity phenomenon is desirable when evaluating bile canaliculi in hepatocellular carcinoma, it may be misleading in the differential diagnosis of effusions. This cross-reactivity by pCEA may have resulted in the reports documenting immunoreactivity for CEA in mesothelial cells; CEA immunoreactivity is usually not observed in malignant mesothelioma with mCEA.^[71,72]

Tissue studies report mCEA immunoreactivity in 85–94% of adenocarcinomas and virtually negative immunoreactivity in malignant mesothelioma.^[49-51] Non-mucinous ovarian adenocarcinomas are usually non-immunoreactive for CEA.^[49,50]

Reactive mesothelial cells and malignant mesothelioma cells in effusions are typically non-immunoreactive for CEA,^{[38,45,46,72]} but immunoreactivity rates for adenocarcinomas are 21–79%. This rate for effusions is lower than that reported for tissue studies. This discrepancy may be due to the type of antibodies used in different studies since monoclonal antibodies are less sensitive than polyclonal antibodies.^[45,46,56,7]

Studies has reported that pCEA (polyclonal CEA) antibody can be applied for evaluation of hepatocellular lesions. It demonstrates canalicular immunostaining pattern in hepatocytes (both neoplastic and non-neoplastic) in Paraffin-embedded cell-blocks (but may show diffuse cytoplasmic immunostaining in non-hepatocytic lesions). mCEA antibody has no value in the differential diagnosis of hepatocellular carcinoma.

CYTOKERATINS

These intermediate-sized monofilaments form part of the cytoskeleton. They are present in the cytoplasm and along the cytoplasmic membrane in nearly all true epithelial structures (both neoplastic and non-neoplastic).^{[44,49,79-81]}

Broadly, they are divided into low and high molecular weight cytokeratins. • Non-neoplastic mesothelial cells and meso- thelioma cells are immunoreactive for both low and high molecular weight cytokeratins. Adenocarcinomas are usually immunoreactive for low molecular weight keratin.^[45] Since the type of cytokeratin expressed shows significant overlap (such as the immunoreactivity of spindle cell type of malignant mesothelioma for only low molecular weight cytokeratin), it is not a reliable means for distinguishing mesotheliomas from adenocarcinomas.^[49,70]

A cocktail of antibodies immunoreactive to most of the cytokeratins or an antibody immunoreactive to the common epitope present in all cytokeratins may be included in a panel to rule out other neoplasms such as lymphoma and melanoma. Cytokeratin immunoreactivity would also establish immunointegrity of the cells under scrutiny.^[49,81] A murine monoclonal antibody, mAB Lu-5, has been reported as a formaldehyde-resistant cytokeratin marker. The epitope is located on most of the cytokeratin polypeptides of both the acidic (type I) and basic (type II) subfamilies. It has a cytoplasmic immunostaining pattern and has been recommended as a first-order ‘pan-epithelial marker’ to distinguish epithelial neoplasms from other non-epithelial neoplasms such as sarcoma, lymphoma, and melanoma.^[128,129] In our experience, however, some neoplasms that were otherwise immunoreactive for one of the cytokeratin antibodies did not show immunoreactivity with Lu-5. A cocktail of cytokeratin AE1/AE3 and CAM5.2 shows better results (personal experience) (SFigure 9).

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Figure 9:

Pan-cytokeratin immunoreactivity pattern (pleural fluid). Reactive mesothelial cells with cytoplasmic immunostaining (arrow in inset). Some reactive mesothelial cells may show a concentric immunostaining pattern around the nucleus, which is better appreciated by adjusting fine focus. [Immunostained cell-block section (40X).]

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Other specific types of cytokeratins, such as CK 5/6, CK 7, and CK 20, may have a role in evaluation of effusions. Studies have reported the application of CK 5/6 for distinguishing epithelioid mesothelioma from adenocarcinomas.^[50,52,78] CK 5/6 immunoreactivity was observed in 92–100% of malignant mesotheliomas,^[52,78] with only 0–14% of adenocarcinomas showing weak focal immunoreactivity.^[52,78] However, it has not been widely reported for effusions. CK 7 and CK 20 may have a role in different situations, including evaluation of an unknown primary neoplasm.^[82] The pattern of immunostaining with cytokeratin 7 may help to identify reactive and neoplastic mesothelial cells (Figure 10). Similarly, as compared to adenocarcinoma cells, mesothelial cells may show a concentric immunostaining pattern (better appreciated by adjusting fine focus) for pancytokeratin around the nucleus (Figure 9).

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Figure 10:

Cytokeratin 7 immunoreactivity pattern (epithelioid mesothelioma, pleural fluid). Neoplastic mesothelial cells with cytoplasmic immunostaining. Note the bushy microvilli (arrowhead). [Immunostained cell-block section (100X zoomed).]

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D2-40 (SEE ALSO PODOPLANIN)

D2-40 is a novel monoclonal antibody to M2A antigen, a relative molecular mass (Mr) 40 000 O-linked sialoglycopro tein that reacts with a fixation-resistant epitope. M2A antigen expression was reported in testicular germ cell tumors^[130] and lymphatic endothelium.^[131] Recently, D2-40 immunoreactivity has been reported as an immunomarker for reactive and neoplastic mesothelial cells. It has been recommended for the differential diagnosis of mesothelioma from adenocarcinoma.

The observed sensitivity was similar to calretinin but better than cytokeratin 5/6 and WT-1.^[83–85] Recently, some studies have reported D240 expression in primary peritoneal serous carcinomas and ovarian tumors.^[132] One of the meta-analysis, concluded that D2-40 immunostaining alone may not be sufficient to diagnose malignant melanoma.^[127] Recently, it is reported that commercially available mouse monoclonal antibody D2-40 in fact recognizes human podoplanin (see below).^[143-145]

EPITHELIAL MEMBRANE ANTIGEN (EMA)

Clones/synonyms: 214D4, E29, GP1.4, MC5

EMA, one of the human milk fat globule membrane proteins, is associated with a wide variety of neoplastic and nonneoplastic epithelia.[38,44,45,86,104,136,137] Both mesothelial and non-mesothelial epithelial elements are immunoreactive for EMA. Various squamous and glandular epithelia and lymphoid neoplasms (i.e. anaplastic large cell lymphomas) may show membranous immunoreactivity. Mesothelial cells in effusions may show weak immunoreactivity.

EMA immunoreactivity in effusions is reported in 75–100% of epithelioid mesothelioma, 91% of adenocarcinomas, and 6% of reactive mesothelial cells.^[38,45] Comparable results are reported with EMA immunoreactivity in 89.5% of epithelioid mesothelioma and 100% of adenocarcinomas in surgical pathology specimens.

The pattern of immunostaining is the key to interpretation. Epithelioid mesothelioma shows a characteristic immunoreactivity pattern, with EMA demonstrating a ‘thick’ membranous immunostaining along the periphery of cell clusters highlighting the long microvilli (Figures 11,18a,b)^[70,104] Adenocarcinoma cells are also immunoreactive for EMA, but the immunostaining pattern is usually cytoplasmic (see Figure 2a).^[70,104] Due to relative difficulty in evaluating immunostaining patterns, EMA usually is not a favored addition to the panel of immunomarkers in effusions for distinguishing between epithelioid mesothelioma and adenocarcinoma.^[52]

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Figure 11:

EMA immunoreactivity pattern (epithelioid mesothelioma, pleural fluid). Mesothelioma cells with membranous (arrow) and cytoplasmic immunostaining. Note the microvilli (arrowhead). [Immunostained cell-block section (100X zoomed)].

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After evaluating clones E29 and Mc5 of EMA antibodies, E29 has been reported to distinguish between non-neoplastic reactive mesothelial cells and neoplastic mesothelioma cells. In this study, 75% of malignant mesotheliomas showed immunoreactivity for EMA with clone E29, but reactive mesothelial cells were non-immunoreactive in all cases.^[138]

GATA3

Clones/synonyms: L50-823, Cell Marque

GATA3 is one of the 6 GATA family of zinc-finger transcription factors. It is involved in cell development and differentiation including luminal differentiation of breast epithelium, development of collecting system/urothelium, and trophoblastic differentiation. Studies have reported GATA3 expression in neoplasms of both urothelium and breast.^[91,139] It is reported to be either absent or expressed rarely in other epithelial tumors, except salivary gland and parathyroid tumors.^[139]

Studies have reported diffuse, moderate to strong nuclear GATA3 immunoreactivity (Figure 12) in breast carcinomas^[140] and adenocarcinomas of the urinary bladder including those with signet ring features.^[89-91,139]

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Figure 12:

GATA 3: Metastatic adenocarcinoma mammary primary (history of infiltrating duct carcinoma of breast). Nuclear immunoreactivity for GATA 3 in metastatic carcinoma cells (NC). (Pleural fluid).

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HBME-1

HBME-1 immunoreactivity is observed along the cell membrane of mesothelial cells (Figures 13, 18c-e).^{[55,87,93,94]} Normal cells other than mesothelial cells are not immunoreactive for HBME-1. However, sarcomas, chordomas, and lymphomas, along with papillary and follicular carcinomas of the thyroid, show HBME-1 immunoreactivity.^[50,93]

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Figure 13:

HBME-1 immunoreactivity pattern (epithelioid mesothelioma, pleural fluid). Mesothelioma cells with membranous (arrow in a) and cytoplasmic immunostaining. Note the microvilli (arrowhead in b). [a,b, Immunostained cell-block section (a, 100X; b, 100X zoomed).]

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The characteristic immunoreactivity pattern with epithelioid mesothelioma is thick membranous immunostaining (see Figures 13,18c-e), but with adenocarcinomas it is usually cytoplasmic, and if membranous it is flimsy and thin (see Figure 2b).^[55,94] Desmoplastic/sarcomatous variants of mesotheliomas are usually non-immunoreactive for membranous immunostaining pattern with HBME-1. This is considered to be due to the loss of microvilli on the cell surface of these variants.^[93]

Many studies performed on tissue specimens have reported overlapping results in epithelioid mesothelioma and adenocarcinomas,^{[50,52,87,92]} concluding that HBME-1 is less efficient than the other markers for definitive identification of mesothelioma.^[92] Similarly, a study on effusions reported immunoreactivity in 89% of mesotheliomas and 65% of adenocarcinomas, with thick and thin membranous immunostaining patterns in both entities.^[55] Thus, HBME-1 appears to be of limited application for evaluation of effusion specimens.

IMMUNOMARKERS FOR INFLAMMATORY CELL COMPONENT

CD68

Clones/synonyms: KP-1, PG-M1

CD45

Clones/synonyms: 1.22/4.14, 2B11+PD7/26, 2D1, LCA, PD7, PD7/26, T29/33

LCA (leukocyte common antigen, CD45) or PGM1 (CD68) or a mixture of LCA and PGM1 may be used as an immunomarker for inflammatory cells^[65] and also for lymphoma leukemia cells. Both LCA (Figure 14) and PGM1 (Figure 15) show a cytoplasmic immunoreactivity pattern. Since LCA (CD45) is the stronger antibody (see Figure 14), it does not require the antigen retrieval step. In comparison, PGM1 (CD68) needs the antigen retrieval step. To cover all inflammatory cells in the effusions for creation of a basic map for evaluation with the SCIP approach, LCA and PGM1 may be combined. However, the titer of LCA antibody will have to be adjusted to accommodate the antigen retrieval used for PGM1. In our experience, PGM1 usually lacks the non-specific immunoreactivity frequently observed with KP1, and so we prefer PGM1 over KP1 as our CD68 antibody.

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Figure 14:

LCA (CD45) immunoreactivity pattern (pleural fluid). Reactive mesothelial cells (blue arrow RM) with chronic infl ammatory cells (red arrows). The infl ammatory cells show a strong cytoplasmic immunoreactivity pattern obscuring the nucleus (arrowhead in inset). [Immunostained cell-block section (40X).]

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Figure 15:

CD68 (PGM1) immunoreactivity pattern (metastatic mammary adenocarcinoma with proliferation spheres (red arrow NC), pleural fluid). Histiocytes show CD68 immunoreactivity (blue arrows H). In our experience, PGM1 does not show non-specific immunostaining usually associated with KP1. Inset, histiocytes (blue arrow H) with cytoplasmic immunoreactivity pattern around the nucleus. [Immunostained cell-block section (40X).]

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MESOTHELIN

MOC-31

MOC-31 is an antibody raised by using the GLS-1 cell line of small cell carcinoma of lung. This antigen is a human cell surface glycoprotein related to an epithelial antigen present on the most neoplastic and non-neoplastic epithelia.^[52] It has demonstrated immunoreactivity with the majority of adenocarcinomas, but only rarely with mesotheliomas and reactive mesothelium.^{[50,52,92,141]}

MOC-31 has thus been recognized as one of the ‘negative’ mesothelioma markers for the differential diagnosis of adenocarcinoma and mesothelioma. It reacts with a 38 kDa protein associated with a wide variety of adenocarcinomas; 62 of 63 adenocarcinomas of various origins were positive and five epithelioid mesotheliomas were all negative for MOC31.^[97] Many other studies have also demonstrated the role of MOC-31 in the differential diagnosis of mesothelioma.^{[50,52,92,97,141]} The cytoplasmic and membranous immunoreactivity pattern of MOC-31 is usually strong and diffuse with the adenocarcinomas (Figure 16), in contrast to a weak and focal immunoreactivity in mesotheliomas. MOC-31 has been described as a useful immunomarker for distinguishing between epithelioid mesothelioma and pulmonary adenocarcinoma involving the pleura.

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Figure 16:

MOC-31 immunoreactivity pattern (metastatic mammary carcinoma, pleural fluid). The adenocarcinoma cells show predominantly membranous (m) with cytoplasmic (c) immunoreactivity. [Immunostained cell-block section (100X).]

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NKX3.1

NKX3.1 protein is product of NKX3.1 gene located on chromosome 8p21.2. It functions as a haploinsufficient tumor suppressor gene. It is an androgen-regulated homeodomain gene expressed predominantly in prostate epithelium.^[99]

Studies have reported NKX3.1 immunostaining to be highly sensitive and specific for prostatic adenocarcinoma as compared to urothelial carcinoma.^[100] The study evaluated nuclear NKX3.1 immunoexpression in adenocarcinomas of the urinary bladder, and cancers from various sites including the breast, colon, salivary gland, stomach, pancreas, thyroid, and central nervous system, adrenal cortex, kidney, liver, lung, and testis. With sensitivity of 98.6% (68/69 cases positive) for metastatic prostatic adenocarcinomas (as compared to 94.2% (65/69 for PSA and 98.6% for PSAP) and specificity of 99.7% (1/349 non-prostatic tumor, 1 case of lobular carcinoma of breast was positive) is a valuable adjunct to distinguish prostatic primary from other metastatic carcinomas.^[99,100]

PAX2^{[101,142,143]} and PAX8^[101-103]

PAX2 and PAX8 belong to the paired box gene (PAX) family (with 9 members PAX1 through PAX9) of transcription factors which paly crucial role in renal and thyroid development. Recently, expression of both PAX2 and PAX8 has been reported in malignant neoplasms of Mullerian and renal origin. Neither PAX2 nor PAX8 immunoexpression is noted in metastatic adenocarcinomas from breast, lung, gastrointestinal tract, and pancreaticobiliary primaries.^[142] Nuclear immunoreactivity for of PAX8 (Figure 17) is reported in clear cell, papillary, thymic tumors, and chromophobe renal cell carcinoma (RCC).^[102,142] Direct comparison of PAX2 and PAX8 demonstrated that PAX8 (88 of 99 cases; 89%) is a more sensitive for metastatic RCC than PAX2 (75 of 99 cases; 76%).^[143] However, a small subset of metastatic RCCs express only PAX2 and so both PAX8 and PAX2 should be included in a panel.^[143]

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Figure 17:

PAX 8: Metastatic adenocarcinoma Mullerian primary (patient had ovarian mass). Nuclear immunoreactivity for PAX8 in metastatic carcinoma cells (NC). (Ascitic fluid).

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However, PAX2 and PAX8 immunoexpression is not restricted only to RCC.^[142] They are expressed in Mullerian clear cell carcinoma, nephrogenic adenoma, and parathyroid parenchymal lesions. PAX8 but not PAX2 is expressed in thyroid follicular neoplasms and well differentiated neuroendocrine tumors.^[143] PAX8 immunoexpression in lung cancers regardless of tumor subtype is rare.^[103]

SATB2

SATB2 is AT-rich sequence DNA-binding protein with 733 amino acids demonstrating significant degree of evolutionary conservation. The difference between mouse and human protein is only three amino acids. It binds nuclear matrix attachment regions and is involved in chromatin remodeling and transcriptional regulation.^[144]

SATB2 expression is feature of glandular cells of the distal gastrointestinal tract. This feature is preserved in colorectal cancers. Studies reported SATB2 immunoexpression as sensitive and highly specific immunomarker for colorectal cancer. SATB2 immunoexpression is reported in more than 95% of colorectal carcinoma.^{[106,107,144]}

PODOPLANIN (see also D2-40)

Podoplanin is a mucin-type glycoprotein that is reported to be specific for the endothelium of lymphatics. It was also observed to be expressed in reactive mesothelial cells. Later it was evaluated in mesothelioma in tissue sections. All five malignant mesotheliomas demonstrated podoplanin immunoreactivity along the cell membrane. All other 118 neoplasms (including 93 adenocarcinomas, 4 squamous cell carcinomas, 6 gastrointestinal stromal tumors, and 5 endocrine tumors) were negative for podoplanin.^[105] It was concluded that podoplanin has the potential to be an excellent tumor marker with high specificity and sensitivity. Other studies on tissue sections have reported encouraging results.^[84,85] However, strong immunoexpression of podoplanin in granulosa cells of normal ovarian follicles and in cells of ovarian dysgerminoma and granulosa cell tumors has been reported. Although it is primarily absent from normal human epidermis, strong podoplanin immunoexpression is reported in 22 of 28 squamous cell carcinomas.^[111] The mouse monoclonal antibody, D2-40, recognizes human podoplanin.^[133-135] The role of these immunomarkers as an additional positive mesothelial cell marker is evolving.

THROMBOMODULIN (CD141)

THYROID TRANSCRIPTION FACTOR-1 (TTF-1)

Clones/synonyms: 8G7G3/1, SC-13040, SPT-24

TTF-1 is a tissue-specific transcription factor expressed in normal lung and thyroid tissue.^[108] Additional studies^[109,110] have demonstrated a high specificity of TTF-1 for lung and thyroid carcinomas with a nuclear immunoreactivity pattern (Figure 18). It has demonstrated its role in distinguishing between mesotheliomas and pulmonary adenocarcinomas.^[60] Seventy-five percent of pulmonary and 100% of thyroid adenocarcinomas demonstrated immunoreactivity for TTF-1; however, all other adenocarcinomas and mesotheliomas were nonimmunoreactive.^[60]

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Figure 18:

TTF-1 immunoreactivity pattern (metastatic pulmonary carcinoma, pleural fluid). The solitary adenocarcinoma cells as the predominant population (arrows NC) show nuclear immunoreactivity (arrowheads in inset). [Immunostained cell-block section (40X).]

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VIMENTIN

Clones/synonyms: 3B4, RPN1102, V10, V9, V1M-3B4

A comparative study of 11 immunohistochemical markers concluded that vimentin is one of the two best immunomarkers for distinguishing between epithelioid mesotheliomas and adenocarcinomas, with cytoplasmic immunoreactivity in mesothelial cells (Figure 19).^[112] However, other studies did not find vimentin a useful immunomarker in the diagnosis of mesothelioma.^{[71,86,113,114]} If it is understood that adenocarcinomas from some primary sites such as endometrium and kidney^[22] are also immunoreactive for vimentin, it is a good immunomarker to distinguish between mesothelioma and adenocarcinoma. This highlights the significant variations in approach to immunocytochemical evaluation of effusions. We find vimentin to be one of the important components of a basic immunopanel for interpretation of the SCIP.^[2]

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Figure 19:

Vimentin immunoreactivity pattern (peritoneal wash). Reactive mesothelial cells (arrow RM) show cytoplasmic immunoreactivity pattern (arrowhead in inset). [Immunostained cell-block section (40X).]

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WT-1 (WILMS’ TUMOR GENE PRODUCT)

Clones/synonyms: 6F-H2, C-19

WT-1 has a nuclear immunoreactivity pattern (Figure 20). Tissue studies demonstrated WT-1 immunoreactivity in 72% of mesotheliomas, 100% of non-neoplastic pleural mesothelium, and 83% of ovarian adenocarcinomas. Immunoreactivity was observed rarely with all other types of adenocarcinomas.^{[50,52,60,87]} Thus, this immunomarker may be more specific in non-peritoneal effusions, especially in males. Studies with effusions are indicated to verify the role of this immunomarker. WT-1 immunoreactivity is also observed with desmoplastic small round cell tumors (DSRCT).^[115]

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Figure 20:

WT-1 immunoreactivity pattern (metastatic colonic adenocarcinoma, peritoneal fluid). Reactive mesothelial cells (arrow RM) show nuclear immunoreactivity (arrowhead in inset) with some cytoplasmic immunostaining. Rare adenocarcinoma cells demonstrating nuclear immunoreactivity for CDX2 were also seen in other section. [Immunostained cell-block section (40X).]

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LIST OF ABBREVIATIONS (In alphabetic order)

AdCa – Adenocarcinoma

AP – Alkaline phosphatase

Ca – Carcinoma

CK – Cytokeratin

DAB – 3,3’-Diaminobenzidine

DSRCT – Desmoplastic small round cell tumors

GI – Gastrointestinal

HIER – Heat induced epitope retrieval

HRP – Horseradish peroxidase

LBC – Liquid-based cytology

PAP – Papanicolaou

RTU – Ready to use

SCIP – Subtractive coordinate immunoreactivity pattern

m – Monoclonal

Min – Minutes

p – Polyclonal

RCC – Renal cell carcinoma

UV – UltraView