Currently, the WHO guidelines for the diagnosis of lymphoid malignancies require the integration of clinical, morphologic, immunophenotypic and genetic information for a definitive diagnosis. To date, no immunophenotypic or genetic marker specific to NMZL has been identified. Therefore, the diagnosis of this entity relies on the recognition and development of diagnostic criteria based solely on morphologic and immunoarchitectural patterns and the integration of clinical data at the time of presentation to render a diagnosis. Because of the rarity of NMZL, large series that thoroughly characterize their pathologic features are limited. In this study, we have morphologically and immunphenotypically characterized, to the best of our knowledge, the largest series of primary NMZL that meet the current WHO creteria for this entity. We define four distinct immunoarchitectural patterns of NMZL that we have designated diffuse, nodular/follicular, interfollicular and perifollicular, and describe their histologic and immunoarchitectural features.
Peripheral lymph node involvement can be seen in both extranodal and splenic MZL.9,17
Thus in our cohort, we excluded any case with extranodal disease, splenic or splenic hilar lymph node involvement. The distinction between of NMZL with clonal plasma cells and LPL involving lymph nodes is a problematic one and currently there are no unequivocal criteria to separate these entities. As such the morphologic and immunoarchitectural features need to be assessed in light of the clinical presentation. In the current study, we excluded cases with an increased monoclonal plasma cell component in the lymph node, extensive marrow involvement and clinical or radiologic features suggestive of LPL or plasma cell myeloma. Although plasma cells were noted in a large proportion of our cases (47%), they represented only a minor component f the neoplastic infiltrate in the lymph node. All cases presented in this report met strict diagnostic criteria for NMZL with primary lymph node presentation and absence of extranodal or splenic disease at the time of this study. It is however important to consider that some cases of NMZL may manifest as extranodal disease on follow-up and represent secondary involvement of lymph nodes.13
In accord with previous studies, a diffuse effacement of architecture (the diffuse pattern) was most commonly encountered with an associated vague nodular pattern in 45% of cases. In cases with both patterns, the diffuse pattern usually represented the major component and nodular pattern was a minor component occupying approximately 10–40% of the lesion. Traverse-Glehen and colleagues made a similar observation that cases with the diffuse pattern are closely associated with a nodular pattern.18
In contrast to previous studies,10,18
we identified well-formed follicles in 9% of cases, but did not observe any with the so-called “inverse follicular pattern”. This inverse follicular pattern, which has been described as a follicle with a dark-staining inner zone surrounded by a light-staining outer zone on H&E sections, is typically seen in a subset of splenic MZL and a small number of NMZL (2 of 21 cases) in one prior study.18
A problematic area in the diagnosis of NMZL with a well-defined follicular/nodular or diffuse pattern is its separation from low-grade follicular lymphoma. Germinal center-associated markers that highlight follicular lymphoma cells are typically absent in NMZL. However, subsets of follicular lymphoma may also lack germinal center-associated markers, making the distinction between these entities a diagnostic challenge. We encountered two such cases with a diffuse pattern that were originally diagnosed as NMZL based on the lack of CD10 staining. However, with the use of additional germinal center-associated markers, these cases were reclassified as diffuse follicle center lymphomas. In this context, BCL6, HGAL and LMO2 performed better than CD10 and showed more sensitivity and specificity for follicular lymphoma cells. In these two cases, CD21 immunostaining highlighted disrupted FDC meshworks, a finding that further confounded their separation from NMZL. Overall, CD21 highlighted disrupted FDC meshworks with follicular colonization in the majority (71%) of cases in our cohort regardless of the morphologic pattern, although it was more prominent in cases with a well-defined follicular/nodular pattern (4 of 5 cases; 80%). In a recent study of 15 NMZL with prominent follicular colonization, the difficulty in distinguishing NMZL from other low-grade lymphomas with a follicular pattern, are well illustrated.19
Our findings are in agreement with that study and further highlight the need for recognition of the extensive disruption of FDC meshworks and follicular colonization that is often seen in NMZL. Moreover, the presence of follicular colonization and disrupted FDCs in some cases and the complete absence of FDCs in other cases suggest a possible evolution of tumor pattern in NMZL. The cases that showed a complete absence of FDCs likely represent more advanced tumors that replace the lymph node architecture. We were unable to verify whether the attrition and eventual absence of FDC meshworks correlate with progression of disease in our cohort of cases as clinical follow-up information was limited. However, this concept warrants further examination on clinically well-characterized cases.
Immunohistologic staining for CD43 and BCL2 can be helpful in the diagnosis of NMZL, but shows significant variation among cases and in rates of positivity reported in previously published studies. Staining for CD43 is reported to range from 20–75% whereas the staining for BCL2 has been reported to range from 62–100%.12,13,18–22
Lai and colleagues previously showed that CD43 staining was positive in 20–40% of nodal and extranodal marginal zone lymphomas and staining for CD43 in our cohort was in the same range. However, BCL2 positivity was significantly lower in our cohort. A number of factors may be responsible for this difference including differences in methodology and antigen retrieval among cases sent in consultation. In addition, the inclusion criteria used in some studies whereby cases were selected based on their positivity of CD43 and/or BCL2 may also play into the higher number of positive cases reported in published series.12
Nonetheless, it is important to recognize that although immunohistologic stains for CD43 and BCL2 are helpful when present and aid in identifying the neoplastic B-cell infiltrates on which they are co-expressed, their expression is not essential for the diagnosis of NMZL. In addition, other B-cell non-Hodgkin lymphoma such as mantle cell lymphoma (frequently positive for both CD43 and BCL2) and follicular lymphoma (particularly, diffuse follicle center lymphoma) should also be carefully eliminated from the differential diagnosis.
Another important morphologic consideration in the diagnosis of NMZL is its relationship to diffuse large B-cell lymphoma (DLBCL), which is particularly problematic in NMZL with increased large transformed B-cells. Nathwani and colleagues previously described progression to DLBCL as those cases with >20% large cells.10
In our cohort, however, we found >20% large cells in 23% of cases: many of these cases exhibited increased numbers of scattered large cells, and in a minority of cases, up to 50% of large cells were present. These cases showed no significant correlation with a particular morphologic pattern and did not correspond to the single case that showed transformation to large cell lymphoma in a subsequent biopsy. In addition, none of our cases demonstrated sheets of large cells. Similar to Traverse-Glehen et al,18
our findings suggest that the presence of a large cell component in NMZL is more frequent than previously recognized.3,9,10
This finding of increased scattered large cells should not be confused with sheets of large cells, particularly if the latter is associated with an increase in proliferation (as measured by Ki-67 staining), as sheets of large cells, even if focal should raise concern for progression to large cell lymphoma. Kojima and colleagues recently reported a series of 65 cases of NMZL of which 20 cases had either >50% large cells or sheets of large cells and were classified as “DLBCL + MALT-type NMZL”; these cases were associated with a significantly worse outcome.12
Our cohort did not include cases with >50% large cells or sheets of large cells, and thus meet the current WHO definition of primary NMZL. Nevertheless, the criteria for the diagnosis of progression to large B-cell lymphoma from NMZL is not well established and currently there is no set cut-off for proliferation (as assessed by Ki-67) that is utilized to aid in this distinction.
B-cell lymphomas arise as a result of deregulation and clonal expansion of B-cells at distinct developmental stages, and NMZL are thought to arise from marginal zone B-cells of the lymphoid follicle. Previously, Conconi and colleagues observed various patterns of immunoglobulin heavy chain variable region (VH) gene mutations in NMZL and identified distinct subsets of B-cells including naïve (unmutated V regions) and germinal center and memory B-cells (undergone somatic hypermutation) from which NMZL were derived.23
These findings suggest that NMZL originate from different subsets of marginal zone cells and provide a plausible explanation for the immunoarchitectural heterogeneity of this group. Furthermore, although NMZL is thought to arise from marginal zone B cells of lymphoid follicles, aberrant expression of germinal center-associated markers has also been reported in NMZL. From a diagnostic standpoint, the expression of germinal center-associated markers in rare cases of NMZL and their lack in a subset of follicular lymphoma, make the separation of NMZL from diffuse follicle center lymphoma particularly challenging. In our experience, the use of additional germinal center-associated markers is helpful in ruling out a follicular lymphoma particularly if staining for CD10, a marker that is frequently employed in immunohistologic panels, is absent. In this study, we found that together with BCL6, HGAL and LMO2, two germinal center B-cell-associated markers that we recently characterized, are excellent adjuncts in the immunohistologic work-up of low grade B-cell lymphomas and especially those that exhibit a diffuse small lymphoid proliferation.
In summary, we describe a large series of primary NMZL that highlights the heterogeneity of this disease and exhibits a spectrum of morphologic and immunophenotypic variability. The four immunoarchitectural patterns we describe, together with their frequency and potential overlap with other lymphomas in the differential diagnosis, affords a greater understanding of the inherent heterogeneity of this lymphoma. The presence of large cells in a significant proportion of cases and the inclusion of germinal center-associated markers in their work-up support and extend previous observations. In addition, we found that CD21 staining was particularly useful in highlighting disrupted FDC meshworks and follicular colonization, a feature that lends support for the diagnosis of MZL. Recent gene expression profiling studies of splenic marginal zone lymphoma have pointed to specific abnormalities in that entity,24
which after further testing and validation, can be employed for the diagnosis of splenic MZL. Similarly, genomic and proteomic approaches to interrogate NMZL will be needed and will likely furnish candidate markers specific to the diagnosis. In the interim, awareness of the immunoarchitectural patterns of NMZL is important for rendering an accurate and reproducible diagnosis and to distinguish this entity from other lymphomas in its differential diagnosis.