UNIQUENESS OF EFFUSION IMMUNOCYTOCHEMISTRY

Intricacies associated with finding and locating the cells of interest in cell block sections of effusion fluids is a very important limiting factor, with the potential to adversely affect the final results. If it is not approached with special consideration, it may lead to improper immunocytochemical interpretation and, eventually, a suboptimal final result. Although it is not unique to effusion fluid cytology, it is quite common in this scenario to face the challenge associated with evaluation of coordinate immunoreactivity because of the higher frequency of small cell groups and solitary cells in effusion specimens. While processing and interpreting effusion fluid immunocytochemistry with objectivity, the following attributes should be considered:

a. The pattern of cancer cells in effusions varies from solitary scattered cells to small, cohesive groups. It may be more difficult to find the same cells or groups of cells in adjacent serial sections on different slides. Most of the individually scattered, abnormal cells, however, will be present in at least a few 4 μm thick serial sections (see Figures 5.3, 5.5, 5.8). It is important to know the sequence of these serial sections and to have them identically oriented on the slides to identify more precisely the same cell (or small group of cells) for evaluation (Figure 5.1). To achieve this, we routinely orient all serial sections identically on slides and label them sequentially (Figures 5.2, 5.3). This simple approach can help in some unexpected situations where the immunostaining pattern is not straightforward and calls for more careful scrutiny. If done routinely, it expedites and simplifies the immunocytochemical evaluation of effusion fluids.

Figure 5.3 Serial numbering and orientation of cell block sections.

Figure 5.5 Metastatic colonic adenocarcinoma, peritoneal fluid. The neoplastic cells (red arrow NC) are immunoreactive for pan-cytokeratin (B) and non-immunoreactive for vimentin (A), CD45 (C), calretinin (D), and WT-1 (E). They have nuclear immunoreactivity for CDX2 (F), which is consistent with a colonic primary. The reactive mesothelial cells [with immunoreactivity for calretinin (nuclear-cytoplasmic) (D), WT-1 (nuclear-cytoplasmic) (E), vimentin (A)] and inflammatory cells [with immunoreactivity for vimentin (A) and LCA (C)] in the background can be subtracted from neoplastic cells to deduce a diagnostic coordinate immunoreactivity pattern. LCA, leukocyte common antigen; NC, neoplastic cell; RM, reactive mesothelial cell; WT-1, Wilms’ tumor-1. [A–F, Immunostained cell block sections.]

Figure 5.8 Metastatic mammary adenocarcinoma, pleural effusion. Specimen with proliferation spheres. The neoplastic cells (red arrows NC) are immunoreactive for cytokeratin 7 (D) and BerEP4 (E). They are non-immunoreactive for vimentin (A), CD68 (B), and calretinin (C). Rare mesothelial cells (blue arrow RM) [with immunoreactivity for vimentin (A), calretinin (nuclear-cytoplasmic) (C), and cytokeratin 7 (D)] and inflammatory cells (arrowheads) [with immunoreactivity for vimentin (A) and CD68 (C)] in the background can be subtracted from the neoplastic cells to deduce a diagnostic coordinate immunoreactivity pattern. Cytokeratin 7 immunoreactivity with a clinical history of mammary carcinoma is consistent with a breast primary. NC, neoplastic cell; RM, reactive mesothelial cell. [A–E, Immunostained cell block sections.]

Figure 5.1 Basic immunopanel for evaluation by subtractive coordinate immunoreactivity pattern (SCIP) approach.

Figure 5.2 Metastatic colonic adenocarcinoma, peritoneal fluid. a. Serial sections of cell blocks oriented identically on all slides in a sequential order. The respective numbers identify their relative position with each other: 1, HE; 2, vimentin; 3, cytokeratin; 4, LCA (CD45); 5, calretinin; 6, WT-1; 7, CD-X2. b. Enlarged view showing slide numbers 4 and 5, emphasizing identical orientation (arrows) of cell block sections on all glass slides. Identical orientations of all serial sections facilitate immunocytochemical evaluation with the SCIP approach. If not properly organized in this fashion, it may be impossible in effusion specimens with a predominance of small cell groups and solitary cells to evaluate the coordinate immunoreactivity pattern in relation to various immunomarkers.

b. The most effective approach for diagnosis of metastatic effusions is the confirmation of a ‘second-foreign’ non-inflammatory population of cells other than mesothelial cells. This approach, however, will not help to distinguish between reactive and neoplastic mesothelial cells. The interpretation of epithelioid mesothelioma should be based on other features after demonstrating the abnormal cells to be mesothelial in nature (see Chapter 8). For reproducible results, it is important to select any immunopanel which will fundamentally identify most of the mesothelial and inflammatory cells to create the basic map for confirmation of a ‘second-foreign’ population by the subtractive coordinate immunoreactivity pattern (SCIP) approach, described later in this chapter (see Figure 5.1).

c. The challenge of distinguishing cells of epithelioid mesothelioma from reactive mesothelial cells has to be approached differently and must be based on the quantity (numerous vs a few) and the quality (numerous large groups vs a few small groups) of abnormal cells with proper clinical and radiologic correlation. Once it is determined that the cytologic features favor mesothelioma rather than reactive mesothelial cells, it is relatively simple, with the aid of immunocytochemistry to exclude adenocarcinoma (Figure 5.4).

Figure 5.4 Algorithm for immunocytochemical evaluation of effusions (in conjunction with Figure 5.1).

d. The final interpretation of any immunoprofile is the result of comparative evaluation of the database accumulated from the information in reported studies that were predominantly performed by using most of the commercially available antibodies on formalin-fixed paraffin-embedded tissue sections. The immunoreactivity pattern for a variety of immunomarkers may not remain the same when other fixation and processing protocols are used.⁵ Consequently, it is prudent to perform immunocytochemistry on formalin-fixed cell block sections only and to avoid other protocols such as the evaluation of various cytology smears (direct smears—wet fixed in alcohol or acetone; air-dried fixed with alcohol; air-dried smears rehydrated and post-fixed in formol alcohol; liquid-based cytology smears—SurePath or ThinPrep, Cytospin smears, etc.).^6,7

Thus, for reproducible results, a standardized protocol with steps comparable to the processing of formalin-fixed paraffin-embedded tissue sections is essential.^8–11 Deviating from such a practice may lead to suboptimal results with loss of reproducibility, which is frequently observed in clinical practice and related publications. The recommended approach is described in Chapters 14 and 15.

e. For the objective confirmation of an immunoprofile highlighting different types of cells in the effusion, it is important to see a coordinate pattern of immunoreactivity in the same cells (see Figure 5.1). This is not possible with cytology smears since the same cells cannot be followed on different smears. In contrast, however, serial sections of cell blocks allow evaluation of immunoreactivity by different immunomarkers in same cells in adjacent serial sections (coordinate immunoexpression).^12,13

f. The proteinaceous effusion fluid around suspended cells may contribute to unexpected non-specific immunoreactivity. The non-specific staining of adjacent inflammatory cells, especially when it is substantial, may hinder the evaluation of membranous immunostaining patterns.

g. Discrepant results between formalin-fixed paraffin-embedded tissue sections of surgical pathology material and effusion fluid cell block sections are not uncommon. The variables responsible for such discrepancies include sample size (tiny cell groups or single cells), selection of fixatives, antigen retrieval methods (i.e. heat-induced epitope retrieval, enzyme digestion, etc.), antibody clones used, antibody titer, and other variations in immunostaining protocols. Additional causes for variable results reported by different studies include variation in laboratory sensitivities and study size.^14,15 Furthermore, both the qualitative (pattern of immunostaining—membranous, cytoplasmic, nuclear, etc.) (Table 5.1) and quantitative criteria for interpretation of immunoreactivity may vary between pathologists and different institutions.¹⁶

Table 5.1 Characteristic immunostaining pattern with immunomarkers in effusion immunocytochemistry

PERSPECTIVE

Cell blocks are the preferred choice for immunocytochemical evaluation of effusions. However, if only very scanty material is present, it is not advisable to perform immunocytochemistry on such material. In such a setting an adequate additional specimen is recommended to be submitted. Because malignant effusions usually reaccumulate quickly, acquiring a new sample is generally not a problem. It is not uncommon to submit only a small fraction of a large volume of effusion fluid collected. Therefore, it may be specifically communicated in the request for resubmission of a new specimen with a comment: ‘Recommend submission of most of the drained effusion fluid (up to 1000 mL). Larger specimen volume facilitates retrieval of adequate cellular material in cell block sections for immunocytochemical evaluation.’

Cell blocks offer dual benefits of easy morphologic interpretation and better standardization, with results comparable to those with surgical pathology specimens. Multiple sections from a single cell block allow for a large number of immunomarkers to be evaluated. Additionally, the cell blocks may be archived and made available for other types of testing in the future.

The frequency of using cytology smears for immunocytochemical testing for different applications is increasing.^17–19 With the advent of heat-induced epitope retrieval techniques, the quality of immunostained cytology smears has improved remarkably. Immunocytochemical evaluation of effusion smears has also been reported and discussed^5,7 (see also Chapter 15). However, most experts do not recommend immunostaining of cytology smears of effusion fluids as a routine practice.⁵

On rare occasions, however, performing immunocytochemistry on effusion smears may be justified: e.g. patients with a known primary neoplasm showing a distinct immunoreactivity pattern. Immunostaining may be performed on cytology smears in such cases, if only a scant effusion specimen is available and cell blocks are not possible. However, applicable limitations, such as the inability to evaluate the coordinate immunoreactivity pattern in cytology smears and possible lack of cross-verification should be weighted prior to final interpretation.

Although straightforward positive–negative interpretation of immunostained sections is an artificial simplification, it has served its purpose in many situations. However, this approach needs to be refined in complex situations such as evaluation of cell block sections of effusion fluids, which are affected by many variables not applicable to other preparations. Some of these variables are:

e. The protocols for cell block preparation may vary and introduce remarkable variations at different steps, such as:

the fluid may not have been submitted fresh but in a fixative

these fixatives themselves may be of different types, such as ethanol or other proprietary fixatives, without any data on the exact composition

transportation conditions—time and temperature variation

delayed processing of unfixed specimen after collection

variation in methods of cell block preparation—agar gel method, picric acid method, thrombin clot, and so on.

For optimal results, the immunostained cell block sections of effusion fluids should be evaluated in a manner similar to hematoxylin and eosin (HE)-stained sections, where we consider numerous qualitative and quantitative morphologic features to reach a final interpretation. All aspects of individual and complementary immunomarkers should be considered collectively (see Figures 5.1 and 5.4), rather than applying a reflexive positive–negative approach.

CATEGORIZATION OF EFFUSION IMMUNOMARKERS

Many immunomarkers can be applied to distinguish reactive and neoplastic mesothelial cells from cells of metastatic neoplasms, which are predominantly adenocarcinomas. Immunomarkers for the evaluation of effusions secondary to lymphomas, melanomas, sarcomas, and unknown primaries are also discussed in relevant chapters. More details about immunomarkers related to the evaluation of neoplasms associated with serous cavities may be found in specific reviews.^6,20–23 Due to the continuous advances in this area with frequent updates, application of the basic principles described in this chapter to contemporary immunomarkers after relevant adjustments is recommended.

‘Positive’ mesothelial markers

Currently, calretinin, cytokeratin 5/6, and WT-1 appear to be the best immunomarkers for reactive and neoplastic mesothelial cells. Although calretinin and cytokeratin 5/6 are more sensitive than WT-1, immunoreactivity for all of these markers may be observed in a minority of carcinomas. WT-1 is slightly less sensitive, but is not expressed in lung adenocarcinomas and most other adenocarcinomas. WT-1 may be considered more specific in non-peritoneal settings because it is also immunoexpressed in ovarian/peritoneal carcinoma and desmoplastic small round cell tumor,²⁴ thus decreasing the specificity of WT-1 in the peritoneal fluid.

D2-40 was initially reported as a lymphatic endothelial marker with a membranous immunostaining pattern. Recently, it has been reported to be as sensitive as calretinin and more sensitive than cytokeratin 5/6 and WT-1 for the differential diagnosis of malignant mesothelioma and adenocarcinoma.²⁵ The report concluded that D2-40 is a sensitive ‘positive’ immunomarker for cells of mesothelial origin.²³ Podoplanin is another immunomarker reported to be a specific positive marker for mesothelial cells.^23,26,27 However, recently it has been reported that a commercially available mouse monoclonal antibody, D2-40, specifically recognizes human podoplanin.^27a,27b,27c These immunomarkers may be routinely included in the future as additional ‘positive’ immunomarkers for reactive and neoplastic mesothelial cells. However, as these immunomarkers are being evaluated, they also have shown some overlap with other neoplasms, especially ovarian neoplasms.^28,29

Because of the overlap between mesothelial and non-mesothelial cells, other immunomarkers such as thrombomodulin, mesothelin, HBME-1, N-cadherin, OV632, and CD44S, which were initially reported as mesothelial markers, are not recommended as a part of the routine diagnostic immunopanel for evaluation of effusion fluids. They are both less sensitive and less specific. Similarly, rabbit polyclonal antibody AMAD-2 with granular cytoplasmic immunoreactivity has been reported to be specific for mesothelial cells.³⁰ Vimentin and cytokeratin 7 are additional mesothelial immunomarkers with high sensitivity but lower specificity. Further refinement and standardization of the aforementioned immunomarkers may allow for some of them to be used as reliable ‘positive’ immunomarkers for mesothelial cells in the future.

‘Negative’ mesothelial markers

If there is a reliable immunomarker which is consistently negative in reactive and/or neoplastic mesothelial cells, it would be an ideal ‘negative’ mesothelial marker. mCEA, MOC-31, Ber-EP4. BG-8, and B72.3 are a few which may be included in this group. These appear to show a relatively improved sensitivity and specificity for the differential diagnosis of malignant mesotheliomas and adenocarcinomas. However, although rare, mesothelial cells may demonstrate unexpected cross-immunoreactivity with these immunomarkers.

Additional less-sensitive but relatively specific ‘negative’ mesothelial immunomarkers for the differential diagnosis of adenocarcinoma include thyroid transcription factor-1 (TTF-1), prostate-specific antigen (PSA), calcitonin, and estrogen receptor. In specific clinical settings, they may be useful for identifying unknown primary neoplasms (Table 5.2).

Table 5.2 Immunoreactivity patterns of ‘second population’ of neoplastic cells suggesting possible primary sites (modified from ref 13).

Possible primary site(s)	Immunoreactivity pattern of second population
Immunomarkers suggestive of a subset of specific tumors
Biliary tract, mucinous ovarian, and transitional cell ca	CK 20+, CK 7+
Breast, lung, endometrial, and non-mucinous ovarian ca	CK 20-, CK 7+
Thyroid, endometrial, and renal ca	Co-expression of Vim and CK
Breast and non-mucinous ovarian ca	ER/PR/Ar+
Immunomarkers for specific tumors
Breast (29)	CK7+, ER/PR/AR+ (even if ER/PR/CK7−), Mammaglobin+
Lung	CK7+TTF-1+, CK20−
Gastric	CK 7+, MUC5AC+, CK 20−
Small cell ca	Neuroendocrine (NE) markers+ (Synaptophysin, Chromogranin, CD56)
Merkel cell ca (30)	Globular CK20+, NE markers+
Thyroid ca	Vim+, CK+, Tff-1, Thyroglobulin+
Medullary ca (19)	Calcitonin+ TTF-1+
Prostate ca	PSAP+, PSA+, CEA−
Choriocarcinoma	HCG+
Colon ca (32)	CK 20+, CK7−, CDX2+(nuclear), diffuse pCEA+, MUC5AC−
Hepatocellular ca	pCEA+ canalicular pattern, AFP+, Hep Par 1+, MUC5AC−
Cholangiocarcinoma	CK 7+, MUC5AC+, pCEA− / diffuse (non-canalicular)+, AFP−
Seminoma	PLAP+
GIST	CD117+, CD34+
Desmoplastic small round cell tumor (24)	CK Globular+, Desmin Globular+, Vim+/−
Melanoma (22, 28)	S-100 protein +, MCW melanoma cocktail +, HMB45+, CK−, Calretinine-
Differential between two types of tumors
Prostatic adenocarcinoma versus transitional cell ca (31)	PSA/PSA/Leu 7 versus CK 7 & 20+, CK 903 (24bE12)+
Small cell ca lung versus Merkel cell ca	CK 7+, CK 20−, TTF-1 + versus CK 20+
Thyroid ca versus medullary ca of thyroid	Thyroglobulin+ versus Calcitonin+
Breast ca versus colon ca	ER/PR/AR/CK 7+ versus CK 20+, CDX2+
Ovarian mucinous ca versus appendiceal mucinous ca	CK 7+ versus CK 20+, CDX2+
Hepatocellular ca versus cholangiocarcinoma (32)	pCEA canalicular+, Hep Par 1+, Albumin+ versus CD5+, CD7+ MUC1+ CK 17+
Mesothelioma versus lung ca	Calretinin+ (nuclear) versus TTF-1+(nuclear)
Endocervical ca versus endometrial ca (74)	p16 (INK4)+, mCEA+, Vim− versus p16(INK4)−, mCEA+/−, Vim+
Stromal tumors versus leiomyosarcoma	CD10+ versus SMA+
Pancreatobiliary adenocarcinomas versus extra-pancreatobiliary nonmucinous adenocarcinomas (32)	MUC1+, CK17+ versus MUC2/CDX2+

ca, carcinoma; CK, cytokeratin; pCEA, polyclonal carcinoembryonic antigen; PSAP, prostate-specific acid phosphatase; PSA, prostate-specific antigen; Vim, vimentin; HCG, human chorionic gonadotropin, GIST, gastrointestinal stromal tumors.

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