Specific Antibodies and Their Diagnostic Combinations

Because of the multitude of IHC stains available to characterize lymphoid neoplastic processes, a preliminary differential diagnosis based on the histo-logical assessment typically drives the selection of markers to be applied in a particular case. Next, several specific antibodies and their combinations are discussed in the context of lymphoid proliferations, as well as specific diagnoses.

Fig. 17. Comparison of follicular hyperplasia and follicular lymphoma. (A) H&E and (B) Ki-67 proliferation rate of follicular hyperplasia. This contrasts with (C) H&E and (D) Ki-67 in a case of low-grade follicular lymphoma.

In lymphoid lesions, CD3 and CD20 immunostains should always be used in concert. This allows assessment of number and distribution of B- and T-cells. It is important not to order only a CD20 stain alone, because the number of T-cells can be significantly underestimated. Additional immunostains can be used to identify B-cells. These are particularly helpful in cases in which the neoplastic cells do not express CD20, or express it only weakly, such as chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), B-ALL, or HIV-associated B-cell lymphomas. In these cases, the most useful antibodies are CD79a and PAX-5, which are both pan-B-cell antigens. Another antibody used occasionally is CD74 (LN2).

Besides confirming the hematological nature of a poorly differentiated neoplasm, the use of CD45 is somewhat limited. If lymphoma is reasonable certain, the first step is to use the CD3/CD20 combination, as mentioned previously (see Subheading 3.3.1.). If other hematopoietic lesions (i.e., myeloid sarcoma, plas-macytoma) are being considered, then specific stains (or CD43; see Subheading 3.3.3.) should be used. CD45 is most useful in the context of classical Hodgkin lymphoma (CHL) as a negative finding.

It is important to remember that CD43 (and the formerly used pan-T-cell marker CD45RO) is not a true T-specific marker, as it will stain other hematopoietic elements, myeloid cells in particular. In cases where the expression of CD43 (and CD45RO) is greater than that of CD3, there are often macrophages or granulocytes present. However, in spite of its lack of specificity, the addition of CD43 to the CD3/CD20 combination adds considerable value to the diagnostic algorithm (20,34,35). CD43 is expressed on a variety of normal hematopoietic cells including: normal T-cells, most marrow-derived cells, and macrophages/histiocytes. It is not typically coexpressed on normal B-cells. It is often expressed by malignant B-cells, including several of the small cell types of lymphoma. CD43 is almost always expressed on mantle cell lymphoma (MCL) and SLL/CLL. It is expressed in a subset of marginal zone lymphoma (MZL) (20-40%) and is essentially never expressed in follicular lymphoma (FL). It is occasionally positive in cases of T-cell rich/histiocyte rich B-cell lymphomas, which may be helpful in its differential diagnosis with CHL. Moreover, CD43 may also provide additional useful information in several instances: (1) if positive in B-cells, then it is almost always lymphoma; (2) it is a confirmatory stain for T-cells and should parallel CD3 staining; and (3) it can help identify hematopoietic neoplasms that are CD3 negative and CD20 negative, such as myeloid sarcoma, mast cell disease, and plasma cell neoplasms.

3.3.4. CD15/CD30/CD45

This combination of immunostains is valuable in evaluating lymphoid neoplasms in which CHL is part of the differential diagnosis. CD45 positivity in large abnormally neoplastic cells greatly decreases the likelihood of CHL and favors other diagnostic considerations (37,38).

Conversely, CD15 positivity in large abnormally nucleolated cells is fairly specific and sensitive for CHL. Of course, there are rare exceptions, and no individual stain should be considered as absolutely diagnostic. CD15, however, is not always expressed in cases of CHL; even when it is positive, it may be only weak and in a few cells. Thus, a careful examination of Hodgkin/Reed-Sternberg cells for even weak positivity is recommended. CD15 will stain granulocytes, sometimes strongly, and so confirmation of the positive cell size and type is important. If a case is otherwise perfect for CHL, lack of CD15 should not prevent a diagnosis.

Table 1

Immunohistochemical Evaluation of Classical Hodgkin Lymphoma, Nodular Lymphocyte-Predominant Hodgkin Lymphoma, and T-Cell-Rich/Histiocyte-Rich Large B-Cell Lymphoma and Anaplastic Large Cell Lymphoma

Large neoplastic cells CHL NLPHL TCRBCL ALCL

aEMA staining often parallels ALK-1 staining. 6More often positive in mixed cellularity subtype.

CD30 is always positive in the malignant cells of CHL. However, other lymphoma entities may be positive as well. Besides ALCL (see Subheading 3.2.1.) examples include cases of T-cell rich/histiocyte rich B-cell lymphoma, and rarely, nodular lymphocyte predominant Hodgkin lymphoma (Table 1). This may lead to confusion in their differential diagnosis with CHL. In addition, many nonhematopoietic neoplasms stain for CD30. CD30 staining in reactive lymph nodes is often over interpreted. Both T and B immunoblasts/transformed cells can be positive. Plasma cells can be positive, as well. ALCL, a subtype of T-cell lymphoma, consistently expresses CD30 but it may lack staining for many T-cell antigens including CD3. Occasionally the differential diagnosis with cases of CHL (particularly CD15 negative) may be difficult. The expression of CD30 is also seen in rare cases of DLBCL associated with an anaplas-tic morphology. Invariably, these stain for some B-cell markers (i.e., CD20, CD79a, CD22, PAX-5) and lack T-cell markers. Because of a significant difference in prognosis and differences in biology, the distinction is important.

A subset of ALCL express anaplastic lymphoma kinase (ALK) gene product (39). This is most often a result of a characteristic translocation of chromosomes

Fig. 18. Anaplastic lymphoma kinase expression in a case of anaplastic large cell lymphoma. The presence of both nuclear and cytoplasmic staining is associated with the presence of the translocation between chromosome 2 and chromosome 5.

2 and 5. The t(2;5) causes a fusion of the ALK-1 gene on the short arm of chromosome 2 with the promoter region of the nucleophosmin (NPM) gene on the long arm of chromosome 5. When these genes are combined, they lead to overexpression of the ALK protein in both the nucleus and cytoplasm of the cell (Fig. 18). As in this case, overexpression of ALK in the cytoplasm, without nuclear expression, may be the result of variant cytogenetic translocation. Expression of the "ALK" tyrosine kinase protein product of the t(2;5) translocation or limitation of disease to the skin each predict excellent prognosis. Noncutaneous cases of ALK negative ALCL have a worse prognosis (40).

These lymphomas variably express cytotoxic molecules with the majority expressing TIA-1 and a subset expressing granzyme B or perforin. As is typical of T-cell lymphomas in general, pan-T markers are present (CD2, CD3, CD5, CD7) but loss of individual pan-T antigens is common. There is often loss of surface CD3 with only cytoplasmic CD3 expression.

Bcl-2 staining is common in many lymphomas (26). It is present in about 75% of all B-cell lymphomas, including: most FL, a subset of DLBCL, most MCL, most MZL, and most CLL/SLL. It is also positive in many T-cell lymphomas.

Table 2

Immunohistochemical Differentiation of Follicular Hyperplasia and Follicular Lymphoma

Table 2

Immunohistochemical Differentiation of Follicular Hyperplasia and Follicular Lymphoma

Stain

Follicular hyperplasia

Follicular lymphoma

Bcl-2

Negative

Positive

Bcl-6

Positive

Positive

CD10

Positive

Positive

Ki-67

High

Low

CD23 (in FDC)

Positive

Positive

The presence of bcl-2 staining is not equivalent to the presence of the 14;18 translocation of the bcl-2 gene with IgH heavy chain gene seen in FL and some DLBCL. Bcl-2 is expressed in cells that have a long life, such as normal mantle cells and T-cells.

A common consideration in practice is the differential diagnosis of follicular lymphoma vs follicular hyperplasia. Although there are several morphologic criteria that are valuable, immunohistochemical confirmation is always reassuring (Table 2). Bcl-2 is an antiapoptosis protein that is expressed in a variety of cells. In normal cells of the follicle center, it is not expressed, as benign germinal center cells need the capacity to undergo apoptosis as a function of clonal selection. In benign follicles, Bcl-2 protein is positive in the mantle zone cells but negative in follicle centers. Conversely, in follicular lymphoma, the B-cells of the follicle are positive for Bcl-2 in about 80% of cases (Fig. 19). It should be noted that 20% lack bcl-2 positivity.

Cyclin D1 is of particular importance in diagnosing MCL. Cyclin D1 overexpression correlates with a translocation of chromosomes 11 and 14 resulting in fusion of the cyclin D1 gene with the immunoglobulin heavy chain locus. Cyclin D1 is a very specific stain in the context of lymphoma; its variable nuclear staining being almost pathognomonic of MCL (Fig. 20). It should be noted that both plasma cell neoplasms and hairy cell leukemias and, rarely, CLL (40) can also express cyclin D1. Its expression is also fairly common in non-hematopoietic neoplasms, and one of its earlier names, PRAD-1 derives from its overexpression in parathyroid adenomas.

CD10 and bcl-6 are both stains that (almost always) indicate that the cells are of follicular origin (27,28). In cases where the architecture does not allow for

Fig. 19. Low-grade follicular lymphoma expressing bcl-2. (A) H&E section, (B) Bcl-2, and (C) CD3 expression. When interpreting a Bcl-2 stain it is important to compare results of a CD3 stain, as normal T cells are positive for Bcl-2.

interpretation of a follicular pattern, then either or both can be used to establish the follicular origin of cells.

The literature has several reports concerning prognostication in DLBCL using IHC (42,43). There have been several conflicting studies using CD10/bcl-2/bcl-6/MUM-1 on DLBCL and trying to correlate findings with the aggressiveness or treatability of DLBCL. Gene array studies have shown that there are three general types of DLBCL: (1) the germinal center type, (2) the activated B-cell type, and (3) a heterogeneous group provisionally called "type 3." In general, the germinal center type has a good prognosis and is highly curable, whereas the activated B-cell type tends to have a poor prognosis. Immuno-histochemical studies have tried to replicate the gene array results by using a few, well-known markers. Although it is likely that specific markers will eventually be validated for good vs poor prognosis in DLBCL, it has not yet become clear what markers are best for this determination.

Fig. 20. Cyclin D1 expression in a case of mantle cell lymphoma. Varying intensity of nuclear staining is the expected result for the cyclin D1 stain.

3.3.9. CD5/CD20/CD10/CD23

Besides expression in T-cells, CD5 is positive in CLL/SLL and MCL and only rarely on other small B-cell lymphomas. In conjunction with CD23 and CD10, CD5 can be helpful to differentiate among the various small B-cell lymphomas. MCL would be CD5+/CD23-, CLL/SLL would be CD5+/CD23+, and virtually all cases of FL or MZL are negative for CD5 and negative for CD23 (Table 3).

3.3.10. CD3/CD4/CD8 and Other T-Cell Markers

Besides CD3, other pan T-cell markers such as CD2, CD5, and CD7 are expressed early in T-cell differentiation and throughout the life of T-cells. Selective loss of T-cell markers is common in the neoplastic cells of T-cell lymphomas, and can support the diagnosis. CD4 and CD8 are markers of T-cell differentiation. Most T-cell lymphomas express CD4, although, there are occasional CD8-positive lymphomas. Some lymphomas, including precursor T-cell neoplasms, can be either "double-positive" (CD4+CD8+) or "double negative" (CD4-CD8-). CD1a, most commonly utilized to identify Langerhans cell histiocytosis, will also identify immature T-cells. To evaluate cytotoxic differentiation in T- and natural killer cell proliferations, CD56, TIA-1, perforin, granzyme-B, as well as CD4 and CD8 are helpful.

114 O'Malley and Orazi Table 3

Comparison of Immunohistochemical Results in Several B-Cell Lymphomas

CD20 CD3 Bcl-2 CD5 CD23 CD43 CyclinDl Other

Fig. 21. Follicular dendritic cells are highlighted by a CD21 stain in the case of low-grade follicular lymphoma.

3.3.11. CD21/CD35/CD23/S-100 as Dendritic Cell Stains

Follicular dendritic cells (FDC) are accessory cells that help make up the skeleton of normal follicles. Because of their antigen presenting capability, they play an important role in the ongoing immune response and B-cells maturation. CD21 stain is a useful stain for staining FDC, although CD35 works as well (some use a cocktail of both) (Fig. 21). CD23 staining has a slightly different staining pattern than the other two markers.

Neoplastic proliferation of FDC may occur in lymph nodes or elsewhere (these are termed FDC sarcomas); they can be identified by CD21 immunostaining. In addition, assessing the distribution of FDC can be occasionally useful in differentiating subtypes of lymphomas. In follicular lymphoma, FDC are typically retained in the nodular portions of the lymphoid proliferation, where they form tight networks within the neoplastic follicles. In contrast, other lymphomas may have reduced or diminished numbers of FDC. Irregularly expanded FDC networks are typically seen in cases of angioimmunoblastic T-cell lymphoma.

Interdigitating reticulum cell (IDC) sarcoma may simulate FDC. However, they can be reliably distinguished by immunostaining with CD21 (and/or CD35) and S-100 (the latter being only positive in IDC). Additionally, ultrastructural examination of the FDC reveals the presence of desmosomes, distinguishing the process from IDC, which lack this type of cell junction.

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