Splenic marginal zone lymphoma

Clinical features 

Splenic marginal zone lymphoma shows a slight predilection for men compared to women (M/F = 1.3).6, 7, 8, 10, 11, 12, 13 The age range is variable (24–95) but most series report a median or average age of 63–69.8, 10, 11, 12, 13 The patients typically present with splenomegaly, B-symptoms, abdominal pain, and cytopenias.11, 13 A low level of monoclonal paraprotein can be detected in approximately half of the patients.5, 7 Lymphocytosis and circulating villous lymphocytes are seen in only two thirds of the patients whereas in most there is involvement of the bone marrow.11, 13 Other sites occasionally affected include the liver and, rarely, the abdominal lymph nodes.11 SMZL has an indolent clinical course with a mean overall survival of 8.5 years and a 5-year survival rate of 65%.11

Morphological features 

Peripheral blood cytology 

When the peripheral blood is involved, medium sized atypical lymphocytes are seen in peripheral blood smears. These usually account for more than 30% of all lymphocytes and appear larger than the cells of B-cell chronic lymphocytic leukemia (B-CLL). They often have round or oval nuclei with clumped chromatin and sometimes distinct nucleoli. There is often a moderate amount of cytoplasm with moderate degree of basophilia. However, the most striking feature is the presence of irregularities of plasma membrane consisting of thin short villi, which are typically unevenly distributed and polarised5, 6 (Fig 1). Although these appearances are characteristic of SMZL they are not by themselves diagnostic, as similar appearances can be seen in leukaemic phase of both follicular and mantle cell lymphoma.14, 15 Furthermore, in some cases peripheral blood cytology is indistinguishable from hairy cell leukemia or B-CLL.10

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Fig 1. SMZL, peripheral blood smear (Giemsa). Atypical lymphoid cells with polarized short villi (arrow).

Spleen 

The spleen is massively enlarged weighing well over 1000 g in most cases. Macroscopically, the infiltration appears diffuse with no identifiable tumor masses.13 Microscopically, the most striking feature is a nodular infiltrate centred on the pre-existing white pulp lymphoid follicles3 (Fig 2). The tumor has a biphasic pattern with an inner core of small lymphocytes and an outer margin of medium sized lymphocytes1 (Fig 2). The inner central zone of small lymphocytes resembles normal mantle zone cells with scanty cytoplasm and small irregular nuclei, clumped chromatin, and indistinct nucleoli. In contrast, the lymphocytes occupying the marginal zone appear similar to normal SMZL. These have well-defined clear cytoplasm and round-oval nuclei with a more open chromatin pattern and indistinct nucleoli. Variable numbers of scattered transformed blasts with prominent nucleoli are also present in the marginal zone. Occasionally plasma cell differentiation within the marginal zone or in the center of the nodules can be observed.16 In some nodules, residual reactive germinal centers in the center of the nodules are seen however a well-defined mantle zone separating the tumor cells from the follicle center is absent.

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Fig 2. SMZL, histological features (Hematoxylin-eosin). (A) Low power view showing marked marginal zone pattern. (B) Medium power view showing the central core of small lymphocytes (c), the larger cells occupying the marginal zone (MZ) and the red pulp infiltrate (RP). (C) High power view of the marginal zone component including occasional transformed blasts (arrow). (D) High power view of central small cell component. (E) High power view of red pulp involvement with intrasinusoidal infiltration. (F) Medium power view of bone marrow trephine biopsy showing nodular lymphoid infiltrate.

The reasons for the biphasic morphology of the same tumor clone are not known. The differences are likely to be a consequence of different signals present in different microenvironments of the follicle center and the marginal zone. This is analogous to follicular lymphoma where the neoplastic cells within the follicles have different morphological and immunophenotypic characteristics compared to those infiltrating the interfollicular areas.17

Red pulp infiltration is present to a varying degree in all cases. The majority of the cells in the red pulp comprise the small lymphocytic component and infiltrate the red pulp diffusely. Infiltration of sinuses is a prominent feature of most cases (Fig 2). Typically, there are also scattered small nodules of larger cells similar to those at the periphery of the follicles in the white pulp. In a minority of cases, diffuse red pulp infiltration can be so dense as to obscure the nodular white pulp component.

Immunophenotype 

The immunophenotype of SMZL is relatively uniform in tissue sections. The tumor cells have a mature B-cell immunophenotype and to a large extent mimic the phenotype of normal splenic marginal zone cell. The immunophenotype of both phases, the inner core of smaller cells, and the larger cells occupying the marginal zone are identical. They express CD20, cytoplasmic monotypic immunoglobulin, and Bcl-2 but typically lack CD5, CD10, Bcl-6, CD23, CD43, and cyclin D18, 21, 22, 23 (Fig 3). In contrast to normal splenic marginal zone cells, which mostly express IgM but not IgD, the majority cases of the SMZL cases stain for both IgM and IgD and the rest for IgM only.10 CD21 highlights follicular dendritic cell meshwork underlying the white pulp nodules. When residual germinal centers are seen in the center of the white pulp nodules, these characteristically express germinal center B-cell differentiation markers CD10 and Bcl-6, show a high proliferation fraction, but lack Bcl-2 (Fig 3). Usually, the small cell component of the SMZL rims the residual follicle center replacing the ‘mantle zone’ and no residual IgD positive polytypic mantle zone cells separating the follicle center from the tumor cells are seen. The proliferation fraction of SMZL is low with most Ki67 positive cells located at the periphery of the nodules, in the marginal zone. When a residual germinal center is present this imparts a so-called ‘target’ pattern of proliferation to the infiltrate (Fig 3).

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Fig 3. SMZL, immunophenotype (immunoperoxidase). (A) CD20: Both the white and red pulp are infiltrated by CD20+ cells. (B) Bcl-2: The tumor cells are positive for Bcl-2 whereas the central residual follicle center is negative. (C) Ki67: The proliferating cells are within the residual reactive follicle center or in the marginal zone, ‘Target pattern.’ (D) IgD: Both components of SMZL infiltrate are IgD+ whereas the central residual follicle center is negative. (E) Ig kappa light chain: Only some of the cells within the central residual follicle center are positive. (F) Ig lambda light chain: Both components of SMZL infiltrate and some of the cells within the central residual follicle center are positive.

Involved hilar lymph nodes and bone marrow show identical phenotypic features. In bone marrow biopsies, CD20 staining is very helpful in demonstrating intrasinusoidal distribution of tumor cells.

Flow-cytometric studies performed on circulating villous lymphocytes from cases defined as SLVL have shown a much more variable phenotype with expression of CD5 and even CD10 in many cases.24 The differences between the results of flow cytometry and immunohistochemistry can be partly explained by differences in immunophenotyping technique and, possibly, the compartment and microenvironment of the cells under study. However, because the detection of villous lymphocytes is somewhat subjective, it is conceivable that cases of SMZL defined on this basis as SLVL may have included other types of lymphoma with splenomegalic presentation and peripheral blood involvement.

Genetics 

In keeping with light chain restriction demonstrated by immunophenotyping, SMZL shows monoclonal rearrangement of immunoglobulin heavy chain genes.3, 25 The sequence analysis of the immunoglobulin heavy chain gene variable regions (VH) have showed 2 distinct groups, one with germline, unmutated VH sequences consistent with a naı̈ve B-cell origin, and the other with mutated germline VH sequences consistent with a postfollicular/memory B-cell origin.10, 26, 27, 28 Interestingly, the latter group appears to correspond to the cases that express IgM but lack IgD and it has been suggested that these cases could have been derived from true marginal zone cells.10

Cytogenetic, comparative genomic hybridization and loss of heterozygosity studies of SMZL have shown diverse abnormalities in chromosomes 1, 3, 5, 7, 8, and 17.29, 30, 31 Amongst these 7q31-32 allelic loss is seen in 40% of these cases and appears to be specific for SMZL.32 This abnormality is seen more frequently in cases with germline, unmutated VH sequences. Chromosomal translocation characteristic of other lymphomas including t(11;14), t(14;18) or t(11;18) have not been reported in histologically defined cases of SMZL.33, 34, 35 Abnormalities of p53 is seen on approximately 10% of the cases and may be associated with high-grade transformation.36, 37

Normal cell counterpart of SMZL and its relationship to other marginal zone lymphomas 

The normal cell counterpart of splenic marginal zone lymphoma is not currently known. The term “SMZL” was coined by Schmid et al3 who observed the morphological and immunophenotypic similarity between the outer marginal zone of the SMZL and normal splenic marginal zone. It later became apparent the tumor was biphasic and had an inner center of smaller cells.1, 8, 23 These smaller cells, unlike other marginal zone lymphomas, were not separated from the germinal center by a polytypic mantle zone and preferentially occupied the mantle zone and colonised the follicles. It was also realised that splenic follicular lymphomas or mantle zone lymphomas can show marginal zone differentiation, which is morphologically indistinguishable from the marginal zone component of the SMZL.22 This indicates that marginal zone phenotype, in isolation, can not be used to assign cell lineage. Furthermore most cases co-expressed IgD and IgM suggesting a naı̈ve (mantle zone?) B-cell origin. Analysis VH sequences, as described above, indicates that this entity may be originating 2 different B-cell subsets, one with memory B-cell features, the other with naive B-cell features.

The Revised European American Lymphoma (REAL) classification9 and the current WHO classification1 recognize 3 distinct entities as marginal zone lymphomas (MZL), nodal marginal zone lymphomas, extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT) (MALT lymphoma), and SMZL. This is rather unfortunate and has lead to inclusion of these tumors as a single disease entity to several biological and clinical studies.30, 38 The clinical, morphological, immunophenotypic, and genetic features of these 3 entities strongly indicate these are 3 unique, biologically unrelated diseases and cannot be considered under 1 umbrella despite their shared terminology.

Differential diagnosis 

The histological differential diagnosis of SMZL include a number of reactive and neoplastic conditions. The most important of these are reactive marginal zone hyperplasia, follicular lymphoma, mantle cell lymphoma, B-CLL, hairy cell leukemia, and MALT lymphoma. The differential diagnosis often requires examination of peripheral blood findings, histological features and immunophenotyping. The differential diagnosis of more common entities that cause difficulty in a clinical setting are summarized in Table 1, Table 2, others are discussed below.

Table 1. Histological Features Helpful in Differential Diagnosis of SMZL

		SMZL	FL	MCL	B-CLL
	Peripheral blood involvement	Common	Infrequent	Infrequent	Common
	Bone marrow involvement	Nodular, intrasinusoidal	Paratrabecular	Nodular, interstitial	Nodular, interstitial
	Splenic involvement	Nodular	Nodular	Nodular	Diffuse
	Marginal zone differentiation	Always	Occasionally	Occasionally	Absent
	Follicular colonisation	Frequent	Absent	Frequent	Absent
	Red pulp infiltration	Always	Frequent	Frequent	Always
	Reactive follicles	Frequent	Absent	Occasionally	Rare
	Cytology	Polymorphic, biphasic	Follicle centre cells	Monomorphous	Small lymphocytes, paraimmunoblasts

Abbreviations: SMZL, splenic marginal zone lymphoma; FL, follicular lymphoma; MCL, mantle cell lymphoma; B-CLL, B-cell lymphocytic lymphoma/chronic lymphocytic leukemia.

Table 2. Immunophenotypic Features Helpful in Differential Diagnosis of SMZL

		SMZL	FL	MCL	B-CLL
	CD20	+	+	+	+
	CD5	−	−	+	+
	CD43	−	−	+	+
	CD10	−	+	−	−
	CD23	−	−/+	−	−
	Bcl-6	−	+	−	−
	Bcl-2	+	+	+	+
	IgH expression	M+D, M	G, M, M+D, A	M+D	M+D
	Cyclin D1	−	−	+	−
	Ki67	Low, Target pattern	Low, Follicular	Low-medium, Follicular and interstitial	Low Proliferation, centers
	CD21	FDC++	FDC+++	FDC++	FDC−/+

Abbreviations: SMZL, splenic marginal zone lymphoma; FL, follicular lymphoma; MCL, mantle cell lymphoma; B-CLL, B-cell lymphocytic lymphoma/chronic lymphocytic leukaemia; IgH, immunoglobulin heavy chain; FDC, follicular dendritic cells.

The differential diagnosis from reactive marginal zone hyperplasia is not usually difficult. In hyperplasia, the marginal zones are expanded and there could be significant mitotic activity within the marginal zone. However, the normal architecture is preserved, there is no red pulp involvement and the marginal zones are separated from the hyperplasic reactive follicle with a well-defined mantle zone. If necessary, immunohistochemistry can be used to confirm the reactive nature of the proliferation.

One of the most important differential diagnoses remains the hairy cell leukaemia, which may clinically, cytologically, and phenotypically mimic SMZL. However, on histological sections, the diagnosis is straightforward. There is a distinctive bone marrow infiltration pattern and splenic involvement is diffuse with extensive infiltration of the red pulp with formation of characteristic blood sinuses and effacement of normal white pulp.

Gastric MALT lymphoma often shows cryptic infiltration of the marginal zone yet microscopically identifiable tumor involvement is a relatively rare event and is not a problem in daily practice.39 In most cases, there is appropriate clinical history of MALT lymphoma elsewhere and, histologically, the tumor cells occupy the true marginal zone outside the polytypic mantle zone. The characteristic biphasic morphology of SMZL is not seen and red pulp infiltration is not apparent.