Immunohistochemical analysis for TNFAIP2 revealed moderate to strong staining of a subset of cells within reactive human tonsil, lymph node and spleen () and normal thymus (Figure 1S-A
). High power examination of these tissues indicated that the largest collections of positive staining cells were localized to reactive germinal centers of secondary follicles in the typical pattern of follicular dendritic cells (). The positive staining cells were elongated and spindled with small and inconspicuous nuclei. TNFAIP2 expression in these cells localized to the cytoplasm and, to a variable extent, the nuclei. Double labeling reactive tonsil for the follicular dendritic cell marker CD23 and TNFAIP2 revealed colocalization of these two markers (). Within germinal centers, macrophages filled with apoptotic debris (tingible bodies) weakly stained for TNFAIP2 (, arrows). Within the inter-follicular regions of the secondary lymphoid tissues, scattered small, spindled cells, consistent with interdigitating dendritic cells as well as scattered larger cells with oval nuclei and abundant cytoplasm consistent with macrophages stained for TNFAIP2 (). Double staining for the interdigitating dendritic cell marker S100 and TNFAIP2 showed colocalization of the proteins within cells (). In contrast to the dendritic cells and macrophages, the lymphoid cells within the germinal centers, colonizing the mantle zones, and in spleen, within the marginal zones, were uniformly negative for TNFAIP2 (). Double labeling tonsil with the B cell marker CD20 () or the T cell marker CD3 (not shown) with TNFAIP2 revealed distinct cell populations.
Figure 1 Reactive tonsil (A, 400×; B, 1000×) and spleen (C, 200×) stained for TNFAIP2 (brown coloration) and showing a distribution of positive staining consistent with dendritic cells and macrophages (B, red arrows= macrophages). Small (more ...)
To confirm that TNFAIP2 expression is absent in the non-neoplastic lymphoid cells comprising reactive tissues, we additionally stained biopsy samples of normal thymus (Figure 1S-A
), Kikuchi’s lymphadenitis (1S-B
), and lymph nodes with patterns and/or phenotypes consistent with toxoplasmosis (Figure 1S-C
), and acute EBV infection (Figure 1S-D
). In each case, TNFAIP2 staining highlighted macrophages and follicular and interdigitating dendritic cells but was negative in the reactive lymphoid cells.
The strong expression of TNFAIP2 by follicular and interdigitating dendritic cells as well as macrophages in normal secondary lymphoid tissues raised the possibility that TNFAIP2 might serve as a useful marker for neoplasms derived from these cell types. Follicular dendritic cell sarcoma (FDCS) and histiocytic sarcoma (HS) are exceedingly rare entities. However, we assembled a group of 7 tumors from our institution and found that the tumor cells in 4 of 4 cases of FDCS and 3 of 3 cases of HS were positive for TNFAIP2 (, respectively; ). These results suggest that TNFAIP2 may serve as a useful new marker for identifying these tumor types.
Figure 2 Cases of follicular dendritic cell sarcoma (A), histiocytic sarcoma (B), classical Hodgkin lymphoma (C; D), nodular lymphocyte predominant Hodgkin lymphoma (E), diffuse large B cell lymphoma, not otherwise specified (F), T cell/histiocyte rich large B (more ...)
TNFAIP2 expression in select lymphomas
We had previously observed that select markers of dendritic cells and macrophages, specifically fascin and galectin-1, are also expressed by the malignant RS cells of cHL.(7
) To determine whether TNFAIP2 is also expressed by this malignant cell type, we performed IHC on a large cohort of cHL cases. Weak, moderate, or strong staining of, at minimum, 50% of the RS cells of cHL with cytoplasmic and, to some extent, a nuclear pattern was observed in all examined cases (31 of 31 cases; ; ). These cases included 20 cases of nodular sclerosis Hodgkin lymphoma (NSHL), 5 cases of mixed cellularity Hodgkin lymphoma (MCHL), and 6 cases of cHL not otherwise specified. A subset of the positive staining cases was EBV+ although staining patterns did not correlate with EBV status (data not shown). The non-neoplastic macrophages that are generally part of the inflammatory background of cHL also stained for TNFAIP2 (). The non-neoplastic lymphoid cells were negative for antigen expression. We conclude that TNFAIP2 is a robust marker of the RS cells comprising cHL, although care must be taken to distinguish TNFAIP2 expression in RS cells from TNFAIP2 expression in intermixed macrophages.
The neoplastic LP cells (also known as L&H cells) and the non-neoplastic lymphoid infiltrate of NLPHL can resemble the RS cells and reactive background of cHL, respectively(22
). To determine whether LP cells express TNFAIP2 we stained a cohort of NLPHL cases and observed positive staining of >50% the neoplastic LP cells in all 12 cases (, ). Non-neoplastic macrophages are less frequent in NLPHL than in cHL and thus positive staining of the LP cells was straightforward to discern. As in cHL the small reactive lymphoid cells surrounding the scattered neoplastic cells were negative for the antigen (). We conclude that the malignant LP cells of NLPHL express TNFAIP2.
DLBCL is the most common large cell lymphoma and commonly identified by pan-B cell antigen expression by the tumor cells. We stained 45 cases of DLBCL for TNFAIP2 and detected no expression of the antigen in the tumor cells for 43 of 45 cases (). However the (non-neoplastic) macrophages and dendritic cells interspersed throughout the malignant B cells in most cases of DLBCL were positive for TNFAIP2 and these cells served as an appropriate internal control for staining (). We found no distinguishing clinical or pathologic features among the 2 positive staining cases of DLBCL which would suggest that these 2 cases were mis-classified (data not shown). Double immunohistochemical staining for CD20 and TNFAIP2 in select cases of DLBCL confirmed that TNFAIP2 expression was not localized to the malignant B cells in this tumor type (; data not shown). We conclude that TNFAIP2 is very rarely expressed by the malignant B cells of DLBCL.
Cases of diffuse large B cell lymphoma, not otherwise specified (A, B), and primary mediastinal (thymic) large B cell lymphoma (C, D) stained for CD20 (brown coloration) and TNFAIP2 (red coloration). All images were photographed at 1000×.
A specific subtype of diffuse large B cell lymphoma, termed T-cell/histiocyte rich large B cell lymphoma (T/HRLBCL), shows a mixed inflammatory background reminiscent of cHL. However in contrast to cHL, the malignant cells of T/HRLBCL resemble those of DLBCL, and like DLBCL, express pan-B cell antigens. We examined 3 cases of T/HRLBCL and found the malignant cells of each case to be positive for TNFAIP2 (). All three cases of TCRBCL occurred in patients without a known history of lymphoma, presented as rapidly developing adenopathy, and showed the typical morphologic and immunophenotypic features of the diagnostic entity. Although our finding suggests that this rare type of DLBCL may more closely resemble HL with respect to TNFAIP2 expression, additional, larger cohorts of cases will need to be studied to determine the frequency of TNFAIP2 expression in TCRBCL with more certainty.
The malignant B cells of PMBL morphologically and immunophenotypically resemble DLBCL, however this tumor has clinical, molecular and genetic features that more closely resemble cHL.(13
) We tested a large cohort of PMBL cases satisfying the major clinical, radiologic, and pathologic criteria for this entity and readily detected TNFAIP2 expression in the tumor cells in 27/31 cases (87%, ). As for cases of cHL, we found that biopsy tissue stained for TNFAIP2 had to be examined with care to ensure that expression of this antigen was localized to the malignant B-cells in addition the endogenous expression of the protein by the numerous, intermixed macrophages and dendritic cells (). Double staining for TNFAIP2 and CD20 in a subset of cases of PMBL confirmed the expression of TNFAIP2 in the tumor cells in this tumor type (). We conclude that TNFAIP2 is a robust marker of the malignant cells of PMBL.
Although the tumor cells of Burkitt lymphoma (BL) are generally intermediate in size, occasional cases show large cell size and thus might be considered within the differential diagnosis of a large cell lymphoma. We tested 18 cases of BL and found TNFAIP2 expression in 2 cases (11%). In the remaining cases, there was no staining of the tumor cells, despite positive TNFAIP2 expression by the intermixed tingible body macrophages that are characteristic of this tumor type (, and ). We also tested 4 cases of B-cell lymphoma, unclassifiable, with features intermediated between DLBCL and BL (int. DLBCL/BL). The tumor cells in these cases were uniformly negative for TNFAIP2 expression ().
The final major category of large cell lymphomas that we examined for TNFAIP2 expression was ALCL. ALCL is a tumor of T-cell origin, and like non-neoplastic T-cells, the malignant cells of ALCL were negative for TNFAIP2 expression in 18 of 19 of cases (). The ALCLs in our cohort included those with an ALK-rearrangement (10 cases) and without an ALK-rearrangement (9 cases) as determined by IHC or FISH (, data not shown).
Staining of an additional cohort of low grade B-cell lymphomas, including follicular lymphoma (FL, grades 1 and 2, n=18), mantle cell lymphoma (MCL, n=13), extranodal marginal zone lymphoma (MZL, n=14), small lymphocytic lymphoma (SLL, n=8) revealed expression of TNFAIP2 by the neoplastic cells in only few cases (; = positive staining MZL, = negative staining SLL). Staining of additional non-Hodgkin lymphomas, including angioimmunoblastic T-cell lymphoma (AIL-T, n=7), peripheral T cell lymphoma not otherwise specified (PTCL, NOS, n=10), extranodal NK/T cell lymphoma, nasal type (NK/T, n=5), and B and T lymphoblastic leukemia/lymphomas (B-LL, n=13; T-LL, n=10 respectively) reveal that TNFAIP2 is rarely, but occasionally expressed in other lymphoid neoplasms ().
Our data indicated that TNFAIP2 expression is largely restricted to the malignant cells of FDCS, HS, cHL, NLPHL, and PMBL and largely negative in the malignant cells of DLBCL and ALCL. Among these large cell lymphomas, PMBL and DLBCL are often difficult to distinguish by morphologic and immunophenotypic features alone. We therefore examined whether TNFAIP2 could serve as a more reliable diagnostic marker than the expression of TRAF1, nuclear cRel, or CD23 for resolving this differential diagnosis.(13
) Antibodies recognizing MAL, another antigen differentially expressed between PMBL and DLBCL, are not commercially available and therefore not tested.(2
) We found that the malignant cells in 23/31 cases (74%) of PMBL were positive for TRAF1, 17/30 cases (57%) of PMBL were positive for the expression and nuclear localization of cRel, and 9/28 cases (32%) of PMBL were positive for CD23 (). The combined expression of TRAF1 and nuclear cRel in the malignant cells was observed in 17/30 cases (57%) of PMBL - a value comparable to that observed in a prior study (13
). The overall immunophenotypic profile of the PMBL cases revealed that no marker was entirely sensitive for this tumor type (). However, among the tested biomarkers, TNFAIP2 proved to be the most sensitive (sensitivity= 87%) and specific (specificity= 96%) marker of tumor cells comprising PMBL relative to (non-mediastinal) DLBCL (). Among the same sets of cases, we found TNFAIP2 expression to be coordinately expressed with TRAF1 in the vast majority of PMBLs (). Four cases (13%) of PMBL in our cohort were negative for TNFAIP2, and of these cases, all were positive for TRAF1.
Phenotype of primary mediastinal large B cell lymphomas tested
Utility of select diagnostic markers for primary mediastinal large B cell lymphoma*