Variant immunoarchitectural patterns of FL can be difficult to distinguish from those of other B-cell lymphomas and reactive conditions. Three markers that are routinely used in the diagnosis of FL CD10, BCL2 and BCL6 show variability of expression and are often diminished or absent in the interfollicular and diffuse components.(27
) Similarly, cytogenetic and molecular studies aimed at detecting t(14;18) can also show variability, further confounding the diagnosis of FL. Therefore, additional markers that are better able to uniformly and reliably stain all components of follicular lymphoma infiltrates would improve the separation of FL from other B-cell lymphomas, and consequently, lead to more appropriate clinical management of patients. Moreover, identifying an abnormal interfollicular or diffuse distribution of cells labeling for a germinal center B-cell marker may be helpful in supporting a diagnosis of lymphoma in a difficult case. Our previous studies had shown that HGAL and LMO2 proteins are expressed with high specificity in normal germinal center B-cells and in subsets of germinal center-derived B-cell lymphomas.(22
) We therefore, sought to address whether they would also add sensitivity to the diagnosis of FL, particularly when variant morphologic and immunophenotypic features confound the diagnosis. Our findings show that both HGAL and LMO2 are sensitive markers for the diagnosis of FL, and that HGAL is superior to all other markers tested in this study (CD10, BCL2, BCL6 and LMO2), in detecting the interfollicular and diffuse components of FL.
Variant architectural patterns and cytologic features of neoplastic infiltrates in follicular lymphoma can result in diagnostic difficulty. Although the presence of diffuse areas in low-grade follicular lymphoma is not associated with an adverse outcome, diffuse areas together with increased large cells, are two important histologic parameters that offer information regarding possible transformation of FL. Furthermore, marginal zone differentiation of FL causes a morphologic overlap with marginal zone lymphomas that secondarily colonize normal follicles; in cases such as these, careful comparisons of the expression patterns of germinal center markers and the morphology need to be taken into consideration to separate FL from marginal zone lymphoma. The floral variant can also present with extensive disruption of the follicular architecture, resulting in morphologic overlap with other small B-cell lymphomas and reactive conditions such as progressive transformation of germinal centers. In addition, the variation in cytologic features also raises other entities in the differential diagnosis: for example, although typically tingible body macrophages are absent in neoplastic follicles with few exceptions, their presence raises a reactive follicular hyperplasia in the differential diagnosis. Similarly, signet ring features are likely to cause confusion with plasmacytoid neoplasia as well as undifferentiated carcinoma. The example of FL with Hodgkin/Reed-Sternberg-like cells raised the differential diagnosis of a lymphocyte-rich classical Hodgkin lymphoma, lymphocyte predominant Hodgkin lymphoma as well as a composite lymphoma that is composed of follicular and Hodgkin lymphoma. These variant follicular architectures and cytologic features are important to recognize as they often complicate the diagnosis of FL, particularly in small biopsies.
Foremost among markers used in FL diagnosis is CD10, which is expressed in normal and neoplastic follicles, in addition to granulocytes, B- and T-cell precursors and in follicular helper T-cells.(13
) Its expression in the interfollicular neoplastic infiltrate of FL, when present, can serve as a valuable discriminatory feature.(6
) In addition, CD10 is extremely helpful in the separation of FL from other small B-cell lymphomas, particularly when a follicular growth pattern is lacking or adequate tissue to evaluate follicular architecture is lacking (fine needle aspirates, needle cores, small or fragmented biopsies). It is also the most frequently used (and often the only) marker in flow cytometry panels employed to detect a lymphoma of follicle center derivation. Differentiating a marginal zone lymphoma with colonized follicles associated with residual CD10-positive follicle center cells from an FL lacking CD10 can be difficult. (6
) Equally problematic is a FL with a significant diffuse component or a diffuse follicle center lymphoma (not included in this study), which can morphologically mimic a marginal zone lymphoma. The lack of CD10 does not preclude FL, nor does its expression eliminate a marginal zone lymphoma, as CD10-positive marginal zone lymphomas also rarely occur.(8
) In addition, CD10 is less frequently expressed in grade 3 FL and in FL involving the bone marrow or peripheral blood.(8
) Rare cases of mantle cell lymphomas can also show partial positivity for CD10 (3 of 127 cases).(10
In the current study, CD10 was positive in 77% of FL cases with the following breakdown among grades: grade 1 (70%), grade 2 (92%), grade 3A (70%) and 3B (44%) (p value=0.006). It was absent in the interfollicular component of 23% of our FL cohort. The overall CD10 positivity of 80% is within the range of what is reported in other studies in the literature.(2
)In the study by Eshoa and collegues, CD10 expression was detected in 70–100% of FL grades 1 and 2, whereas it was detected only in 20% of grade 3 FL.(8
) Our findings are similar and suggest that CD10 expression is frequently weak to absent in grade 3 FL as well as in the interfollicular component of FL.
Virtually all B-cell neoplasms with the exception of Burkitt lymphoma express BCL2;(18
) however, its down-regulation in normal germinal center B-cells can be exploited in the separation of reactive from neoplastic follicles. Given its frequent expression in most B-cell lymphomas, its presence should be interpreted with caution when follicular architecture is lacking. Additionally, normal mantle zone B-cells, primary follicles and normal T-cells express BCL2,(31
) and therefore, additional stains for IgD, IgM and pan T-cell markers such as CD3 or CD5 should be used to eliminate these possibilities as well as partial expression of BCL2 (sometimes reduced to perinuclear rim of staining) in neoplastic B-cells. Furthermore, although most BCL2-negative FL lack t(14;18), a mutated BCL2
gene may also lead to a false-negative immunostaining result due to alteration of the BCL2 epitope recognized by the most commonly used antibodies.(26
) Overall, up to 10% of FL lack expression of BCL2; this protein is expressed in 85–90% of grades 1 and 2 FL whereas only 50% of grade 3 FL express this protein.(18
) In the current study, BCL2 was positive in 78% of cases, with 92% and 78% in grades 1 and 2 FL, and 65% and 62% in grade 3A and 3B, respectively. As with CD10, these results are in agreement with previously published literature.(2
BCL6 is expressed in normal germinal center B-cells and in a significant proportion of FL, including grade 3B FL that often lack CD10 and BCL2.(3
) In addition, its expression has been recently reported in CD10-negative, MUM1/IRF4-positive FL that frequently present in the elderly as high grade (3A or 3B) FL in the absence of the t(14;18) translocation.(16
) BCL6 is a useful adjunct in the diagnosis of CD10 and/or BCL2-negative FL. Its expression is best interpreted in conjunction with a CD3 or CD5 stain as BCL6 is also expressed in a subset of follicular T-cells. In the current study, only a third of the cases had enough material to evaluate BCL6 staining. Similar to CD10 and BCL2, our results are in agreement with the published literature: BCL6 was expressed in 43% and 67% of grades 1 and 2 FL and in 33% and 50% of grade 3A and 3B FL; it was absent in the interfollicular component of 14% of cases tested.
Our previous studies on HGAL had showed that this protein is specifically expressed in the cytoplasm of germinal center B-cells, but was absent in mantle and marginal zone B-cells and in the interfollicular and paracortical regions in normal tonsils and lymph nodes.(22
) Its high degree of specificity for germinal center B-cells make it an ideal marker for the detection of germinal center-derived B-cell lymphomas. HGAL is expressed in the majority of FL regardless of grade (97% in grades 1 and 2 FL and 95% in grade 3 FL in our prior study (22
); 94% in grades 1 and 2 FL and 96% in grade 3A and 88% in grade 3B FL, in the current study). Therefore, unlike CD10, BCL2 and BCL6, HGAL is capable of staining the majority of grade 3A and 3B FL. In our prior study, HGAL expression was found in only 4% of marginal zone lymphomas and was absent in mantle cell and T-cell lymphomas.(22
) In the current study, HGAL staining showed the highest degree of overall sensitivity for FL among the markers we tested. In addition, all cases that lacked CD10 and BCL2, expressed HGAL. Furthermore, HGAL was also found to be the most sensitive marker for the detection of the interfollicular as well as the diffuse components of FL. These results suggest that HGAL is a highly beneficial addition to an immunodiagnostic panel of markers in the work-up of small B-cell lymphomas, not only because of its high specificity and sensitivity in detecting FL, but also because of its efficacy in detecting the interfollicular and diffuse components of FL. Our results attest to HGAL as a superior marker in the diagnosis of problematic FL cases and for differentiating FL from other mimics with a follicular architecture.
Previously, we showed that the transcription factor LMO2 was expressed in normal germinal center B-cells and in a subset of lymphomas derived from those cells in addition to bone marrow hematopoietic precursors and endothelial cells.(9
) LMO2 protein expression also has an important role in the prognostication of diffuse large B-cell lymphomas in patients treated with anthracycline-based chemotherapy with and without the anti-CD20 antibody, rituximab.(21
) Its important role in angiogenesis and erythropoiesis has also been elucidated previously.(30
) It is weakly expressed in mantle zone B-cells but not in mantle cell or marginal zone lymphomas.(23
) In the current study, LMO2 was expressed in 70% of FL cases, which is in the same range as we had previously reported in an independent series of FL cases (50% of FL regardless of grade).(23
) Because LMO2, like HGAL, is capable of staining grade 3 FL at a similar frequency as low grade FL, its efficacy in detecting CD10- and BCL2-negative FL was almost comparable to that of HGAL (6 of 8, 75%, in contrast to 100% for HGAL). These data suggest that LMO2 is also a useful adjunct in the diagnosis of FL. Although its overall sensitivity is less than that of HGAL, its performance was comparable to CD10 and BCL2 and superior to BCL6. Since LMO2 appears not to be down-regulated in higher grade FL or the interfollicular and diffuse components of FL, its utility in variant immunoarchitectural patterns of FL and in cases that lack CD10 and BCL2, is similar to that of HGAL. One added advantage of LMO2 is its crisp nuclear localization that allows for easier interpretation of the stain on paraffin sections in comparison to the diffuse cytoplasmic staining pattern of the HGAL protein.