Small B-Cell Lymphomas

Morphology

In approximately 80% of cases, SLL completely effaces the node architecture, often with invasion of the capsule and extension into the pericapsular fat ( Fig. 7.1 ). In the remainder, neoplastic cells infiltrate the interfollicular areas, sparing small atrophic lymphoid follicles and leaving the sinuses patent. The neoplastic cells in SLL are small (6–12 µm) lymphocytes with round nuclear contours, distinctly condensed chromatin, inconspicuous nucleoli, and sparse agranular cytoplasm ( Fig. 7.2 ). Although small lymphocytes predominate, almost all cases of SLL involving lymph nodes contain admixed intermediate sized cells termed paraimmunoblasts . They are approximately 1.5 times the size of the neoplastic small lymphocytes, have partially condensed chromatin, and small, distinct, central nucleoli ( Fig. 7.3 ). Paraimmunoblasts can be distributed singly but, more often (90% of cases), they form ill-defined clusters imparting a pale, vaguely nodular pattern at low magnification. The collections of paraimmunoblasts are termed proliferation centers , growth centers , or pseudofollicles . Proliferation centers are not present in reactive conditions, and, among the small B-cell lymphomas discussed in this chapter, they only occur in CLL/SLL. In some instances, proliferation centers can be prominent and even begin to fuse ( Fig. 7.4 ). Expansion of proliferation centers should not be mistaken for large cell lymphoma transformation (see below). However, studies do show an association of prominent, coalescing proliferation centers with adverse prognosis, unfavorable cytogenetic features (17p deletion), and high Ki-67 proliferation index.

In those instances, when the lymph node architecture is partially preserved, proliferation centers can be located in the interfollicular areas ( Fig. 7.5 ), or they can encircle the residual lymphoid follicles. These phenomena are thought to represent early stages of lymph node involvement and are morphologically quite subtle. The interfollicular pattern can be easily overlooked, whereas the perifollicular pattern can be mistaken for marginal zone lymphoma (MZL). One study has suggested that absence of proliferation centers in subtle infiltrates may be an indicator of a so-called tissue-based MBL in patients with minimal nodal disease who have a low rate of progression.

In the involved spleen, a uniform expansion of the white pulp creating grossly visible nodules in a miliary fashion is seen early in the course of the disease. However, at later stages the red pulp becomes involved ( Fig. 7.6 ), and the white pulp is obliterated, producing a diffuse pattern of involvement encompassing the entire splenic parenchyma. The microscopic features are similar to the lymph node with infiltration by small lymphocytes and admixed paraimmunoblasts. When the disease becomes disseminated, essentially any extranodal site can be involved and the same morphologic features are seen, but proliferation centers are not nearly as frequent in extranodal sites as in lymph nodes. Sometimes the infiltrate is sparse and the distinction from lymphoid hyperplasia can be challenging. Special studies to demonstrate monoclonality and to confirm the typical immunophenotype of SLL are prudent in this context.

Immunophenotype

The typical immunophenotype of SLL cells is CD5+, CD10−, CD19+, CD20+ (dim), CD22 (dim), CD23+, CD43+, CD79b−/dim+, CD200+, LEF1+, SOX11−, FMC7−/dim+, and surface immunoglobulin (sIg) light chain dim+. Some cases may not have detectable sIg. Cyclin D1 is not expressed, although the large cells within proliferation centers may be positive for cyclin D1 by immunohistochemistry in up to 20% of cases and can also express MYC protein. Notably these cases lack both t(11;14)(q13;q32) involving CCND1/IGH and rearrangements involving the MYC gene. The most reliable markers in paraffin section immunohistochemistry are CD5, CD10, CD20, and cyclin D1. CD23 is also helpful but can be difficult to demonstrate on small lymphocytes in some cases. Proliferation centers are often nicely highlighted by immunostaining for CD23. Newer markers that have helped aid in the diagnosis of challenging cases include CD200 and LEF1. CD200 is typically assessed by flow cytometry. This marker has a high sensitivity in distinguishing CLL/SLL from MCL and may have prognostic value in CLL. LEF1, lymphoid enhancer-binding factor 1, is normally expressed in pro-B cells and mature T cells but is downregulated in mature B cells. However, it is expressed in neoplastic B cells in many cases of CLL/SLL, and has shown up to 92% specificity and 70% sensitivity for the diagnosis. LEF1 is typically performed by immunohistochemistry, but may also be performed by flow cytometry. This described immunologic profile of SLL is identical to that of CLL (see Chapter 12 for details). A reported difference is the expression of lymphocyte function–associated antigen (LFA-1), an adhesion molecule, on SLL but not CLL cells. By contrast, CLL cells express the chemokine receptors CCR-1 and CCR-3 that are absent from SLL cells.

CD38 and ZAP-70 expression have become accepted as poor prognostic markers in CLL/SLL. It is worth noting here that both ZAP-70 and CD38 expression can be assessed by immunostaining, and at least in the case of ZAP-70 there is good correspondence between its detection by flow cytometry and immunohistochemistry.

Genetics

Clonal immunoglobulin gene rearrangements are detectable in almost all cases. The molecular genetic features are covered in the section on CLL. There are no specific karyotypic abnormalities in SLL. As with CLL, cases of SLL variably show trisomy 12, del(6q), del(11q), del(13q), and del(17p). These genetic abnormalities, though common in CLL/SLL, have been described in many other lymphoma types. In practice they should be considered prognostic markers (see section on CLL) rather than disease-defining markers for CLL/SLL. (See Table 12.4 for common recurrent mutations in CLL/SLL.)

Transformation

Transformation into an aggressive lymphoma (Richter syndrome) occurs in approximately 5% of SLL cases. Affected patients present with rapidly growing masses, elevated serum lactate dehydrogenase (LDH), and B symptoms. Most commonly the transformation is in the form of diffuse large B-cell lymphoma, which may or may not be clonally related to the B-cell SLL. The morphologic features are similar to those for diffuse large B-cell lymphoma in general. They are composed of a monomorphous population of large lymphoid cells, resembling centroblasts or immunoblasts that grow in a diffuse pattern, effacing the architecture of the involved tissue ( Fig. 7.7 ), including overrunning any residual SLL/CLL in the sample.

Some SLL cases contain prominent, almost coalescing proliferation centers that give the superficial impression of a large B-cell lymphoma. Recognition of the spectrum of small, medium, and larger lymphocytes present in these cases and appreciation for the manner in which the prominent proliferation centers appear to blend into adjacent small lymphocyte-rich areas helps distinguish this phenomenon from overt transformation. As mentioned above, some studies have found a correlation between prominent proliferation centers and more aggressive disease in CLL. Such cases, however, should not be considered transformation to diffuse large B-cell lymphoma.

Finally, rare cases of SLL are complicated by transformation to classic Hodgkin lymphoma (CHL), usually of nodular sclerosis or mixed cellularity type. Single cell polymerase chain reaction (PCR) studies have shown that the Reed-Sternberg cells in these cases are clonally related to the preexisting CLL clone in some cases but not in others. Epstein-Barr virus (EBV) can usually be detected in the Reed-Sternberg–like cells. The prognosis of patients with CHL complicating SLL/CLL is worse than the typical SLL but still better than that for patients with large B-cell lymphoma transformation. Importantly, as discussed below, transformation to CHL must be distinguished from SLL/CLL with Hodgkin-Reed Sternberg–like cells.

Morphology

The gross pathology of MCL in lymph nodes is not distinctive. This disorder produces nodal enlargement characterized by homogeneous, tan cut surfaces with or without small nodules. When MCL involves the spleen, it produces splenic enlargement, usually greater than 1000 g with small, 1- to 3-mm, miliary, white nodules scattered throughout the splenic parenchyma. In a peculiar pattern of involvement in the gastrointestinal (GI) tract, MCL can also produce numerous sessile and pedunculated polyps throughout the entire length of the small and large intestine in a pattern termed multiple intestinal lymphomatous polyposis .

MCLs are characterized by three growth patterns: mantle zone, nodular, and diffuse, in increasing order of frequency. In the mantle zone pattern, prominent mantle zones composed of the neoplastic cells surround reactive germinal centers that can be either normal sized or small and atrophic ( Fig. 7.8A and 7.8B ). When the germinal centers are normal size, distinguishing MCL from follicular hyperplasia can be a challenge unless one recognizes that the architecture of the internodular areas is effaced by the expanded “cloud” of neoplastic mantle zone cells. Numerous coalescing nodules of tumor cells devoid of germinal centers are characteristics of nodular pattern MCL ( Fig. 7.9 ), whereas a diffusely growing lymphoma cell population that effaces the underlying architecture of the involved tissue defines the diffuse pattern ( Fig. 7.10 ). Hyalinized small blood vessels course through the lymphoma infiltrates in a substantial fraction of diffuse pattern MCLs and can be a useful low-power morphologic clue to the diagnosis of this tumor type.

In the prototypic case, MCL is composed of a highly monomorphous population of small lymphocytes with spheroidal nuclei containing irregularities, cleaves, and grooves. The chromatin is clumped, nucleoli are inconspicuous, and the cytoplasm is sparse ( Fig. 7.11 ). Proliferation centers and intermixed centroblasts or immunoblasts are absent. In addition, MCLs often contain intermixed, singly distributed epithelioid macrophages. Because this cell population is unusual in other lymphoma of small B-lymphocytes, its presence is a useful clue to the diagnosis of MCL. Plasma cell differentiation has also been described in MCL. These cases contain small lymphocytes morphologically and phenotypically identical to those of standard MCL cases that are mixed with varying numbers of plasma cells that are monotypic for the same light chain as the MCL lymphocytes.

In a small subset of cases, the neoplastic mantle cells have “monocytoid” characteristics. These include somewhat larger nuclei and less clumped chromatin than standard MCL cases together with voluminous clear or lightly eosinophilic cytoplasm ( Fig. 7.12 ). This cytology can mimic exactly the cytology of nodal and splenic MZLs, highlighting the importance of ancillary studies in supporting the diagnosis of MCL.

One can recognize three other cytologic variants of MCL. In the first, the neoplastic cells resemble lymphoblasts. They are medium sized with irregular nuclei containing stippled chromatin, small nucleoli, and imperceptible cytoplasm ( Fig. 7.13A ). They are often associated with numerous mitotic figures. In the second, the neoplastic cells resemble centroblasts with round nuclei, multiple nucleoli often attached to nuclear membranes, and more voluminous amphophilic cytoplasm. In the third, the nuclei of the neoplastic cells are pleomorphic, vary in size and shape, are often hyperchromatic, and have variably prominent nucleoli ( Fig. 7.13B ). The WHO lymphoma classification groups these last three cytologic variants into the category blastoid/pleomorphic variant of MCL. Blastoid/pleomorphic MCLs can occur de novo, in which case they are associated with increased risk for adverse outcome. Alternatively, standard MCLs with progression can acquire blastoid cytologic characteristics.

In ISMCN, the architecture of the lymph node or tissue remains intact and undistorted, and it is usually only by the serendipitous recognition of small clusters of cyclin D1+ cells in nonexpanded mantle zones that this phenomenon is recognized ( Fig. 7.14 ).

Involvement of Extranodal Sites

Mantle cell lymphomas frequently involve extranodal sites. The bone marrow is positive in 70% of patients. Paratrabecular, nodular, and/or interstitial infiltrates are found in involved bone marrow biopsy specimens ( Fig. 7.15 ). In contrast to the marked monomorphism of the tumor cells of typical MCLs in fixed tissue specimens, the neoplastic cells in air-dried Wright-Giemsa stained bone marrow aspirate and blood smears are quite polymorphous ( Fig. 7.16 ). Nuclear size and shape vary, the chromatin has a reticulated pattern, and nucleoli can be quite prominent. Peripheral blood involvement by MCL can mimic B-cell chronic lymphocytic leukemia both by absolute lymphocyte count and cytology. The cases previously thought to be B-cell prolymphocytic leukemia with the t(11;14)(q21;q32) are now considered to represent leukemic phase MCLs. In the spleen, MCL involves the white pulp, and a subset of cases will demonstrate marginal zone differentiation toward the periphery of the white pulp nodules. As mentioned above, MCL is one of the lymphoma types that can cause multiple intestinal lymphomatous polyposis ( Fig. 7.17 ).

Phenotype

Mantle cell lymphoma cells express pan B-lymphocyte antigens such as CD19, CD20 ( Fig. 7.18A ), CD79a, CD79b, and PAX-5. They are positive for IgM or IgM plus IgD and exhibit immunoglobulin light chain restriction. More MCL cases express lambda immunoglobulin light chain than kappa immunoglobulin light chain. Greater than 95% are positive for CD5 ( Fig. 7.18B ) but negative for other T-cell antigens, such as CD3 ( Fig. 7.18C ), and most either completely lack CD23 expression or show weak CD23 marking in a subset of the tumor cells ( Fig. 7.18D ). They typically lack or have low-intensity expression of CD200 and are most often LEF1-negative, both of which may aid in the distinction from CLL/SLL. They are usually also negative for CD10 and BCL6. Exceptions to this typical phenotype exist. CD5− MCL cases have been described. They can be recognized on the basis of typical morphologic features and expression of cyclin D1 and will have the classic t(11;14)(q13;q32) CCND1/IGH as described below. A very small number of MCLs is positive for the follicle center cell marker BCL6, and MCL is one of the types of B-cell lymphomas that can co-express both CD5 and CD10. As a consequence of the t(11;14)(q13;q32) (see below), the nuclei of the majority of MCLs are positive for cyclin D1 ( Fig. 7.18E ), a marker demonstrated best by paraffin section immunohistochemistry. More recently, cyclinD1-negative MCLs have been described, which can pose a diagnostic challenge to the pathologist. Expression of SOX11 by immunohistochemistry is seen in a high proportion (>90%) of MCL and appears useful in recognition of cyclin D1 negative MCL. Interestingly, SOX11-negative cases seem to represent a subtype of MCL, which has a more indolent course, leukemic/non-nodal involvement, and mutated IGHV . SOX11 is not expressed in other small B-cell lymphomas or DLBCL (including CD5+ DLBCL). It can be expressed in some cases of Burkitt lymphoma and lymphoblastic lymphoma, but these entities are not typically in the differential diagnosis of MCL.

Genetics

Almost all cases of MCL contain a balanced translocation involving the cyclin D1 gene (CCND1) on chromosome 11q13. The breakpoints occur in a region of CCND1 5′ to the coding region of the gene so that intact cyclin D1 protein can be produced as a result of the translocation. In most cases, the immunoglobulin heavy chain gene (IGH) on chromosome 14q32 is the partner in the translocation, but in a very small subset of cases the kappa immunoglobulin light chain gene (2p11) or the lambda immunoglobulin light chain gene (22q11) are the partner loci. At IGH , the translocation involves the VDJ joining regions of the gene, and CCND1 comes under the regulatory control of the IGH enhancer sequences, causing CCND1 overexpression. Because the morphology and phenotype of MCL overlap with other small B-cell lymphomas, demonstrating cyclin D1 positivity in the neoplastic cells by paraffin section immunohistochemistry or detecting CCND1/IGH fusion by fluorescence in situ hybridization ( Fig. 7.19 ) is typically considered an essential confirmatory finding for this diagnosis.

Cyclin D1–negative cases of MCL have been identified as those cases that have typical morphologic and phenotypic attributes (CD5+, CD23− B-cell lymphoma) of MCL often combined with positivity for cyclin D2 or cyclin D3 and absence of staining of the tumor for p27. These criteria are based on gene expression profiling (GEP) experiments that demonstrated identical GEP signatures of cyclin D1+ and cyclin D1− cases defined in this way. More recently, SOX11 immunohistochemistry has aided in further recognition of this subset of cases. A subset of cyclin D1− MCL cases harbor translocations involving cyclin D2. Cyclin D3 rearrangements have also been evaluated, although they are rare and appear less specific for the diagnosis of MCL. This is an area that requires additional study and clinical analysis due to the rarity of these cases.

In addition to CCND1 translocations, MCLs typically contain a high number of other non-random chromosome abnormalities. These include gains of 3q26, 7p21, and 8q24 and losses of 1p13-p31, 6q23-q27, 9p21, 11q22-q23, 13q11-q13, 13q14-q34 and 17p13-pter. ATM gene mutations occur in a substantial subset of cases, and trisomy 12 is a relatively frequent abnormality in this disease. Blastoid variants are also characteristically tetraploid, and progression from standard variants to aggressive variants can be associated with acquisition of MYC amplification or translocation, P16 deletion or hypermethylation, and/or TP53 mutation/deletion.

On a molecular level, clonal immunoglobulin heavy and light chain gene rearrangements can be demonstrated in virtually all cases of MCL. As alluded to earlier, most cases of MCL have unmutated or minimally mutated IGHV . However, it is now accepted that MCL arises along two different pathways, with the more common type having the characteristic nodal, aggressive presentation and unmutated IGHV . A distinct group, however, shows mutated IGHV and follows a more indolent course. This subgroup tends to be SOX11− and have non-nodal presentations.

Next-generation sequencing (NGS) studies suggest that in addition to translocations involving CCND1 , there are recurrent point mutations in the CCND1 gene in 35% of MCL cases. Additional driver mutations are commonly identified in the ATM and TP53 genes. NOTCH1 and NOTCH2 mutations are associated with poor prognosis. These along with other recurrently mutated genes may provide prognostic markers as well as targets for therapy. As with most other lymphomas, NGS testing is not routinely performed clinically, and the best way to integrate the results of these studies with other clinicopathologic features is still being evaluated.