Diagnostic Algorithm of Common Mature B-Cell Lymphomas by Immunohistochemistry

The classic prototypes of CD5⁺/CD10⁻ B-cell lymphomas are small lymphocytic lymphoma (SLL) and mantle cell lymphoma (MCL). Small lymphocytic lymphoma and chronic lymphocytic leukemia (CLL) are 2 different presentations of the same disease. According to the International Workshop on Chronic Lymphocytic Leukemia, the diagnostic criteria for SLL are lymphadenopathy, absence of cytopenia because of bone marrow infiltration by CLL/SLL, and fewer than 5 × 10⁹/L peripheral blood B cells.¹ 

Although CLL is the most common leukemia affecting adults in Western countries,²  according to a recent study, CLL/SLL is the second most frequent B-cell malignancy, accounting for 18.6% of non-Hodgkin lymphomas in the United States.³  According to published data, 80% to 92% of CLLs stain positive for CD5,^4,5  but the exact percentage of SLL that is positive for CD5 is not known. Apart from CD5, SLL is typically positive for other B-cell antigens, such as CD19, CD20, CD22, CD23, CD79a, PAX5, and surface light-chain immunoglobulins. In addition, SLL is negative for CD10, CD81, and FMC-7, and negative to dimly positive for CD79b. Notably, compared with other non-CLL/SLLs, SLL/CLL is often dimly positive for B antigens, which are helpful in distinguishing CLL/SLLs from other CD5⁺ non-CLL/SLLs. In addition, the CD23⁺/FMC-7⁻ sets CLL/SLL apart from MCL in most, but not all, cases.

Similar to SLL/CLL, most (93%–95%) of MCLs are positive for the antigen CD5.^6,7  Besides CD5, MCL expresses all other B-cell antigens, including CD19, CD20, CD22, CD79a, CD79b, FMC-7, and PAX5. Mantle cell lymphoma is usually, but not always, negative for BCL6, CD10 (see below), and CD23. Almost all MCLs, including rare CD5⁻ ones, are positive for cyclin D1, also known as BCL1, CCND1, or PRAD1.^8,9  Cyclin D1 is a member of the cyclin D protein family.¹⁰  B cells from the mantle zone are negative for cyclin D1. Thus, MCL in situ is usually an incidental finding, and cyclin D1 IHC is required to recognize such rare cases (Figure 1, A).^11,12 

Cyclin D1⁺ MCL is easily diagnosed, regardless of CD5 immunoreactivity. However, diagnosis of cyclin D1⁻ MCL is challenging. Several studies have shown that SOX11,^13,14  cyclin D2, and/or cyclin D3^15,16 are positive in these cases. Importantly, neither cyclin D2 nor cyclin D3 is specific for MCL,¹⁷  but nuclear expression of SOX11 is a specific marker associated with MCL, according to Chen et al¹⁸  (Figure 1, B and C).

Lymphoplasmacytic lymphoma (LPL) is a rare type of non-Hodgkin lymphoma usually involving bone marrow, and less frequently, the lymph nodes and spleen. Although, in LPL, CD5 expression is anecdotal by IHC,¹⁹  positive expression of CD5 on the monotypic B cells ranges from 9% to 43% based on FC studies.^20,21 

Marginal zone B-cell lymphoma (MZBCL) has 3 morphologic types based on the sites involved, namely, nodal, splenic, and extranodal MZBCL of mucosa-associated lymphoid tissue (MALT lymphoma). In contrast to the higher rates of nodal MZBCLs expressing CD5 (8.6%),²²  less than 1% of MALT lymphomas are positive for CD5.²³  Splenic MZBCLs exhibit positive CD5 expression in about 20% of the cases.^24,25  CD5⁺ splenic MZBCL is closely related to its classic CD5⁻ counterpart, except for higher lymphocyte counts at diagnosis and more-diffuse bone marrow infiltration.²⁶ 

From the 2 types of diffuse large B-cell lymphomas (DLBCLs) expressing CD5, namely, de novo and transformed/secondary DLBCLs, only CD5⁺ de novo DLBCL, not otherwise specified (NOS), is reviewed in detail here. Approximately 10% of DLBCLs NOS are CD5⁺ de novo DLBCLs (Figure 2, A through D).²⁷  These lymphomas have higher rates of BCL2 expression (Figure 2, E) and recurrence in the central nervous system and are more likely to exhibit a nongerminal center B-cell phenotype^28,29  (Figure 2, F through H). Although there is no cytomorphologic difference between CD5⁺ and CD5⁻ DLBCL NOS, cyclin D2 has been reported to be highly specific for de novo CD5⁺ DLBCL.³⁰  For practical diagnostics, the pleomorphic variant of MCL and the DLBCL variant of the Richter transformation from CLL/SLL should be ruled out in CD5⁺ B-cell lymphomas with medium to large cell sizes. The CD5⁺/CD10⁻ B-cell lymphomas are summarized in Table 1.

Follicular lymphoma (FL) and Burkitt lymphoma (BL) are the 2 prototypical B-cell lymphomas expressing CD10. In FL, the extent of CD10 expression varies by grade, even within the same tumor. For example, CD10 is expressed in 80% of grade 1 versus 17% of grade 3 FLs.³¹  In addition, CD10 expression is greater in neoplastic cells from intrafollicular than from interfollicular regions.³²  CD10 is typically negative in FLs with marginal zone differentiation (Figure 3, A through F).³²  In addition to CD10, BCL2, an antiapoptotic protein, is useful in differentiating FL from reactive follicular hyperplasia, but expression of BCL2 depends on grade and location. For instance, although 85% to 90% of low-grade (grades 1 and 2) FLs express BCL2, only 50% of grade 3 FLs are positive for BCL2.³³  However, FLs testing negative for BCL2, based on standard antibodies for residues 41 to 54, can test positive for BCL2 when different antibodies are used.³⁴  Primary cutaneous follicle center lymphoma is typically negative for BCL2.³⁵ 

In our opinion, a minimal IHC panel for FL should include BCL2, CD3, CD10, and CD20; however, ideally, BCL6, CD5, and CD21 should be included as well. Recently, new germinal center (GC)-associated markers, such as GCET1, HGAL, and LMO2, have been introduced. Among these 3 markers, GCET1 shows the highest specificity for FL (60% of cases). Both HGAL and LMO2 lack specificity for FL, although they show stronger staining in FL compared with other types of mature B-cell lymphomas.³⁶ 

The classic immunohistochemical profile of BL is positive for BCL6, CD10, and other B-cell antigens without expression of BCL2,^37–40  although BL with positive BCL2 staining can still be diagnosed if other characteristic features of BL are present. Expression of CD38 and B-cell antigens, including CD20, CD79a, and CD79b, is generally strong in BL. In addition, Ki-67 and the Epstein-Barr virus (EBV) are helpful in their diagnosis as well. Ki-67 usually has a 100% proliferation index in BL. Epstein-Barr virus is typically positive in endemic BL but is only 20% to 30% positive in sporadic BL.⁴¹  In recent years, c-Myc IHC is more commonly used in routine diagnosis. Burkitt lymphoma has the highest level of immunoreactivity (60%) for c-Myc among aggressive B-cell lymphomas.⁴² 

Hairy cell leukemia (HCL) and MCL can occasionally be positive for CD10. CD10 expression can be detected by FC in approximately 10% to 20% of HCL cases.^43,44  A similar percentage is expected for IHC (Figure 4, A and B), although such studies have not, to our knowledge, been reported. However, none of the 127 MCLs studied by Gualco et al⁷  were found to have CD10 expression in 30% or more of the neoplastic cells. Akhter et al⁴⁵  reported CD10 expression in approximately 7% of MCLs using the same cutoff (30%) (Figure 5, A through C). According to the authors, the molecular basis of positive CD10 expression in MCL is related to a distinct GC signature rather than an immunophenotypical aberrancy, but there were no significant clinical or pathologic differences between CD10⁺ and CD10⁻ MCLs in their cohort.⁴⁵ 

Despite recent advances, DLBCLs NOS remain a group of aggressive, mature B-cell lymphomas with heterogeneous clinical, morphologic, immunophenotypic, genetic, and prognostic features. Although approximately 90% of DLBCLs NOS are negative for CD5,²⁷  10% to 40% of de novo DLBCLs NOS are positive for CD10.^46–48  Assessing CD10 expression in DLBCLs NOS by IHC has dual benefits. First, positive expression of CD10 in DLBCLs NOS should alert the pathologist to exclude a secondary DLBCL transformed from an underlying FL. Secondly, in the absence of transformation, positive CD10 makes DLBCL a de novo CD10⁺ DLBCL.

Positive CD10 expression in more than 30% of neoplastic cells classifies a patient with DLBCL as having a GC phenotype,³²  which has a better overall survival rate than that of patients with non-GC phenotype.⁴⁹  CD10⁺/CD5⁻ mature B-cell lymphomas are summarized in Table 1.

The prototypic CD5⁻/CD10⁻ mature B-cell lymphomas of small cell size are MZBCL, LPL, and HCL. Most DLBCLs NOS are also negative for both CD5 and CD10. In this section, we focus on MALT lymphoma because it is the most common form of MZBCL. Apart from all the B-cell–associated antigens expressed by neoplastic cells from MALT lymphoma, we have found that the addition of CD43 and κ and λ light chains to the panel of IHC is often useful in diagnosing B-cell malignancies including MALT lymphoma. This addition is particularly important for cases with scant amounts of diagnostic materials, such as those from core needle biopsies or those associated with plasmacytic differentiation. Positive CD43 expression can be detected in 20% to 40% of MALT lymphomas.⁵⁰  Aberrant coexpression of CD43 by neoplastic B cells in MALT lymphoma can be site/location dependent. For example, Arends et al⁵¹  found that CD43 was not helpful in discriminating gastric MALT lymphoma from chronic gastritis. Normal B cells from the terminal ileum, especially Peyer patches, are positive for CD43.⁵²  CD21 and another dendritic cell antigen, CD35, but not CD23, can aid in the diagnosis of MALT lymphoma in 2 ways. First, MALT lymphoma cells usually express CD21.⁵³  Second, the characteristics of follicular dendritic meshwork, including size, contour, uniformity, and density, serve as additional indicators of MALT lymphoma.⁵⁴  Sometimes MZBCL and SLL/CLL can exhibit plasmacytic differentiation. Plasmacytic differentiation is detected in approximately 33% of MALT lymphomas (Figure 6, A through F).⁵⁵  This feature not only assists in making the correct diagnosis but also helps in monitoring the disease status after treatment.

In contrast to MZBCL, LPL always contains a component of monotypic plasma cells (PCs), as the name indicates. Although MZBCL and LPL share many overlapping immunophenotypic features, the immunoglobulin heavy chain expressed by monotypic PCs from LPL is nearly always immunoglobulin (Ig) M, in contrast to the typical IgM expressed in MZBCL. Lymphoplasmacytic lymphoma is diagnosed by exclusion, and at times, MZBCL and LPL cannot be distinguished based on morphologic and immunophenotypic features; in those cases, a positive MYD88 L265P somatic mutation found in most LPLs can be employed.⁵⁶  Notably, as demonstrated by FC, the monotypic PCs derived from B-cell lymphoma have a similar immunophenotype to B cells and differ from those of PC myeloma.⁵⁷ 

Hairy cell leukemia is positive for all common B-cell antigens (CD19, CD20, CD79a, FMC-7, and PAX5), with characteristic expression of annexin A1 (Figure 7, A), CD11c, CD25, CD103, CD123, DBA-44 (CD72) (Figure 7, B), the Hector Battifora mesothelial epitope-1 (HBME-1), pERK, T-bet, and TRAP^58–61  but is typically negative for CD5 and CD10, although 10% to 20% of HCLs are positive for CD10.^43,45  With the exception of annexin A1, which is a specific marker for HCL,⁵⁸  the previously so-called HCL markers, including CD11c, CD103, DBA-44, and HBME-1, are not specific for HCL. They can be found in splenic, diffuse red pulp small B-cell lymphoma and the HCL variant.^60–62  Although annexin A1 is specific for HCL,⁵⁸  its expression in normal hematopoietic cells, especially in neutrophils, makes it unsuitable for detection of minimal, residual HCL, but T-bet was reported to be useful in those cases.⁶³  Weak cyclin D1 staining can be observed in most HCLs (Figure 7, C), but there is no rearrangement of the cyclin D1 gene at 11q13 locus.⁶⁴ 

After excluding CD5^+ 27 and/or CD10^+ 46–48 DLBCL NOS, approximately 50% to 70% of de novo DLBCLs NOS are negative for both CD5 and CD10. Similar to other mature B-cell lymphomas, DLBCL NOS is positive for B-cell–specific and the associated antigens with varying frequencies.⁶⁵  As mentioned, apart from CD10, 2 additional antigens, BCL6 and MUM1, are included in the designation of GC versus non-GC phenotypes of DLBCL in the Hans²⁹  algorithm with approximately 80% concordance with gene expression profiling. By adding GCET1 and FOXP1, Choi et al⁶⁶  increased the concordance of immunohistochemical classification of GC versus non-GC to 93%, as compared with gene expression profiling.

Other rare CD5⁻ and CD10⁻ mature B-cell lymphomas are CD5⁻ MCLs and CD10⁻ FLs. The CD5⁻ MCLs are very rare and account for only approximately 5% to 7% of MCLs.^6,7  Cytologically, CD5⁻ MCLs tend to display a marginal zone or the so-called monocytoid differentiation.^67,68  CD10⁻ FL was discussed previously. The CD5⁻/CD10⁻ mature B-cell lymphomas are summarized in Table 1.

Plasma cell neoplasms encompass plasmacytoma, plasma cell myeloma (PCM), monoclonal gammopathy of undetermined significance, and monoclonal immunoglobulin deposition diseases; the first 2 are reviewed here. Apart from CD38 and CD138, neoplastic PCs derived from PCM exhibit a different immunophenotype from PCs derived from B-cell lymphomas, according to Seegmiller et al.⁵⁷  Among all the antigens studied with FC, CD19 provided the best criterion for distinguishing between these 2 types of neoplastic PCs. In particular, neoplastic PCs from B-cell lymphomas are positive for CD19 and are almost always negative in neoplastic PCs from PCM.⁶⁹  In fact, less than 1% of PCM cases were positive for CD19.⁶⁹  According to the authors,^57,69  expression of CD20, CD45, and CD56 was detected in 9.3% to 27%, 8.8% to 41%, and 71.7% of neoplastic PCs from PCM, respectively, as compared with 32%, 91%, and 33%, respectively, in PCs from B-cell lymphomas.⁵⁷  The frequency of CD117 expression in PCs from PCMs detected with FC correlated perfectly with IHC according to Pruneri et al,⁷⁰  who reported 28.2% immunoreactivity of CD117 among PCMs. CD117 is very rarely expressed in B-cell lymphomas.⁷¹  Cyclin D1, a hallmark for MCL, is expressed in 30% to 35% of PCMs and in 0% to 17% of plasmacytomas.^9,72,73  As summarized in Table 2, the combination of BCL1, CD19, CD45, CD56, and CD117 is sufficient to distinguish PCs derived from PCMs and/or plasmacytomas from B-cell lymphomas, even in cases in which there is exuberant plasmacytic differentiation.⁷⁴ 

A recent report suggested that IgA-expressing nodal and extranodal plasmacytoma may represent a distinct form of extramedullary plasmacytoma characterized by young age, frequent nodal involvement, and low risk of progression to PCM.⁷⁵  Plasmablastic PCM shares similar immunohistochemical profiles with PCM, which are described below.

B-cell lymphomas in this category include plasmablastic PCM; plasmablastic lymphoma (PBL); primary effusion lymphoma (PEL), including the extracavitary variant; large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, and ALK⁺ large B-cell lymphoma.⁷⁶  The antigens CD38, CD138, and MUM1 are positive in all cases of plasmablastic PCM, PBL, and PEL.^77–79  However, plasmablastic PCM and PBL cannot be separated from each other based on an IHC panel that includes CD45, CD79a, CD56, and PAX5, according to Vega et al⁷⁷ ; however, EBV was found to be 100% positive in 9 cases of PBL but negative in 7 cases of plasmablastic PCM. In our opinion, CD19 should be included in this panel, which may aid in distinguishing these 2 lymphomas.

An extensive, large-scale immunohistochemical characterization of PEL has not, to our knowledge, been reported in the literature because PEL typically presents as body cavity effusions, rather than tissue masses, except for extracavitary PEL. The FC studies we conducted showed that CD38, CD71, and CD30 were positive in 100% of PELs.⁷⁸  Although PEL, including its solitary variant and large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, are both positive for HHV8, the latter is typically negative for CD138 and EBV,⁸⁰  which distinguishes this type of aggressive B-cell lymphoma from traditional PBL. ALK⁺ large B-cell lymphoma is a rare, aggressive B-cell lymphoma, which can be difficult to diagnose, and is typically negative for most of the common B-cell antigens but positive for PC markers such as CD138, VS38, EMA, and MUM1.^81,82  The immunophenotypic features of plasmablastic PCM, PBL, PEL, and ALK⁺ large B-cell lymphoma are summarized in Table 3.

We thank Helen Chifotides, PhD, for her secretarial assistance.