Demystifying the diagnosis and classification of lymphoma: a guide to the hematopathologist’s galaxy

Immunophenotypic evaluation

Immunophenotypic evaluation is essential because the lineage of lymphoma cells cannot be determined by morphology alone. The immunophenotype is the combination of proteins/markers (eg, CD20, CD3, TdT) expressed by cells. Usually, it is evaluated by immunohistochemistry and/or flow cytometry, which help determine the proportion of lymphoid cells that express a certain marker and its location and intensity within the cells. While immunohistochemistry is normally performed on formalin-fixed and paraffin-embedded tissue, flow cytometry can be evaluated only on fresh unfixed tissue. Flow cytometry has the advantage over immunohistochemistry of being faster and better at simultaneously identifying coexpression of multiple markers on multiple cell populations. However, certain markers can only be evaluated by immunohistochemistry.

The immunophenotypic analysis will in most cases reveal whether the lymphomas is of B-, T- or NK-cell origin, and whether a lymphoma subtype associated immunophenotype is present. Typical pan B-cell antigens include PAX5, CD19, and CD79a (CD20 is less broadly expressed throughout B-cell differentiation, although it is usually evident in most mature B-cell lymphomas), and typical pan T-cell antigens include CD2, CD5, and CD7. The immature or mature nature of a lymphoma can also be confirmed by evaluation of the immunophenotype. Immature lymphomas commonly express one or more of TdT, CD10, or CD34; T-lymphoblastic lymphoma cells may also coexpress CD1a. The majority of NHLs and all HLs are derived from (or reflect) B cells at different stages of maturation. Mature B-cell lymphomas are the most common type of lymphoma and typically, but not always, express pan B-cell markers as well as surface membrane immunoglobulin, with the latter also most useful in assessing clonality via a determination of light chain restriction. Some mature B-cell lymphomas tend to acquire markers that are either never physiologically expressed by normal mature B cells (eg, cyclin D1 in mantle cell lymphoma, or BCL2 in germinal center B cells in follicular lymphoma) or only expressed in a minor fraction (eg, CD5 that is characteristically expressed in small lymphocytic and mantle cell lymphoma). The most common mature B-cell lymphomas include diffuse large B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone lymphoma, Burkitt lymphoma, and lymphoplasmacytic lymphoma (Figures 2 and 3). Classical HLs are also lymphomas of B-cell origin that demonstrate diminished preservation of their B-cell immunophenotype (as evidenced by the dim expression of PAX5 but absence of most other pan B-cell antigens), expression of CD30, variable expression of CD15, and loss of CD45 (Figure 1). In contrast, nodular lymphocyte predominant HL shows a preserved B-cell immunophenotypic program and expression of CD45, typically without CD30 and CD15. Of note, the evaluation of the immunophenotype of the neoplastic cells in HL is routinely assessed by immunohistochemistry because most flow cytometry laboratories cannot reliably detect and characterize the low numbers of these cells.

Mature T-cell lymphomas generally express one or more T-cell markers, and tend to display a T-helper (CD4-positive) or cytotoxic (CD8-positive) immunophenotype and may show loss of markers expressed by most normal T-cells (eg, CD5, CD7; Figure 4). However, a subset of them may express markers not commonly detected in normal T cells, such as ALK. NK-cell lymphomas lack surface CD3 (expressing only cytoplasmic CD3) and CD5 but express some pan T-cell antigens (such as CD2 and CD7) as well as CD16 and/or CD56.

Patients with primary or acquired immune dysfunction are at risk for development of lymphoma and other less clearly defined lymphoproliferative disorders, the majority of which are associated with infection of the lymphoid cells with Epstein-Barr virus (EBV). Therefore, evaluation with chromogenic in situ hybridization for an EBV-encoded early RNA (EBER1) is routinely performed in these cases; it is thus essential that the hematopathologist be informed of the altered immune system of the patient. If lymphoma develops, they may be morphologically similar to those that appear in immunocompetent patients, which specifically in the post-transplant setting are known as monomorphic post-transplant lymphoproliferative disorders (PTLD). If the PTLD does not meet the criteria for any of the recognized types of lymphoma, it may be best characterized as a polymorphic PTLD.

Once the lineage (B-, T-, or NK-cell) of the mature lymphoma has been established, the sum (and on occasion the gestalt) of the clinical, morphologic, immunophenotypic and other findings will be considered for the subclassification of the neoplasm.

Cytogenetic and molecular evaluation

If the morphologic and immunophenotypic analysis is inconclusive or nondiagnostic, then molecular and/or cytogenetic testing may further aid in the characterization of the process. Some of available molecular tests include analyses for the rearrangements of the variable region of the immunoglobulin (IG) or T-cell receptor (TCR) genes and for mutations on specific genes. The identification of specific mutations not only confirms the clonal nature of the process but, on occasion, it may also help subclassify the lymphoma, whereas IG or TCR rearrangement studies are used to establish whether a lymphoid expansion is polyclonal or monoclonal. The molecular findings should not be evaluated in isolation, because not all monoclonal rearrangements are diagnostic of lymphoma, and not all lymphomas will show a monoclonal rearrangement. Other methodologies that can aid in the identification of a clonal process or specific genetic abnormalities include metaphase cytogenetics (karyotyping) and fluorescence in situ hybridization (FISH). If any cytogenetic abnormalities are found in sufficient numbers (and constitutional abnormalities are excluded), their identification indicates the presence of a clonal process. Also, some cytogenetic abnormalities are characteristic of certain lymphomas. However, they may be neither 100% diagnostically sensitive nor diagnostically specific, for example, the hallmark t(14;18)/IGH-BCL2 is not present in all follicular lymphomas and not all lymphomas with this translocation are follicular lymphomas. Whereas FISH is generally performed on a minimum of 200 cells, compared with typically 20 metaphase by “conventional” karyotyping, and is therefore considered to have higher analytical sensitivity, it evaluates only for the presence or absence of the abnormality being investigated with a given set of probes, and therefore other abnormalities, if present, will not be identified. The value of FISH cytogenetic studies is perhaps best illustrated in the need to diagnose double hit lymphomas, amongst other scenarios. The detection of certain mutations can aid in the diagnosis of certain lymphomas, such as MYD88 in lymphoplasmacytic lymphoma, prognosis of others, such as in follicular lymphoma and identify pathways that may be precisely therapeutically targeted.