Flow cytometry has been widely proved its diagnostic significance in detecting BM involvement in patients with B-cell non-Hodgkin lymphoma (B-NHL). One of the key points for flow cytometry is to set up an elaborate gating strategy to identify monoclonal B cells as much as possible. Because lymphoma B cells most often show as monoclonal B cells, e.g. B cells with Ig light chain restriction. If the number of infiltrating lymphoma cells is high, with a simple gating strategy (CD45++, CD19+, CD20+), monoclonal lymphoma B cells would show up. On the other hand, aberrant expression of other markers (such as CD10) and light scatter properties are also pivotal parameters to make the diagnosis, which could also be used to set up the gate. However, under the following circumstances, the identification of monoclonal B cells is not straightforward. First, surface Ig is not detectable in some cases of DLBCL [20, 21]. Second, it is very difficult to separate a minimal number of lymphoma cells when mixing up with florid reactive B cells. Third, CD10 is invalid in most cases of the non-GBC subtype. Fourth, the histologic discordance between the BM specimens and lymphoid tissues is not uncommon in B-NHL [10, 22, 23]; thus, the immunophenotype of the primary site (such as lymph node) may not be helpful to set up the lymphoma cell gate. Fifth, the monoclonal B cells do not equal malignancy [24–26]. Sixth, under rare circumstances, if more than one B-cell lymphoma population is present (composite lymphoma or bi-clonal B-cell lymphoma), the underlying lymphoma cells would masquerade as a normal ratio of Ig light chain . To solve these problems and enhance the sensitivity of flow cytometry, we need to explore new markers. Recently, there have been some studies focusing on researching new markers in DLBCL. CD81 is a germinal center marker in both normal mature B cells and DLBCL . Non-GCB DLBCL expresses higher levels of CD39 and CD95 in comparison with follicular lymphoma and Burkitt lymphoma .
Previously, there are some studies about the expression of CD54 in patients with B-NHL. Low expression of CD54 on lymphoma cells from primary sites is related to an advanced stage, extranodal involvement, BM infiltration, poor therapeutic response, and worse survival in aggressive B-cell lymphoma . Loss of CD54 on lymphoma cells is related to decreased tumor-infiltrating T cells in patients with DLBCL . In all those studies, the expression of CD54 was detected by using IHC, so it is difficult for us to compare with their results due to different detection methods. Recently, a study using flow cytometry shows that CD54 is significantly higher in DLBCL than Burkitt lymphoma . So far, there are no studies to evaluate the expression profile of CD54 on normal B cells, and the difference of CD54 between normal and lymphoma B cells.
In this research, we clarify that normal hematogones and mature B cells express a low level of CD54. While DLBCL lymphoma cells from 52% of BM specimens highly express CD54. This means that abnormal expression of CD54 on lymphoma cells is a frequent phenomenon in DLBCL patients with BM involvement. In patients with the GCB subtype, CD10 is routinely used to gate on lymphoma cells. However, there is no such marker in the non-GCB subtype. Our research shows that lymphoma cells from around 70% of BMs involved by non-GCB DLBCL abnormally expressed a high level of CD54. Therefore, CD54 is especially useful to detect lymphoma cells in patients with the non-GCB subtype and might be used as a backbone marker to gate on lymphoma cells.
Normal plasma cells highly express CD54. We then clarified that the high expression of CD54 on lymphoma cells is not related to plasmacytoid differentiation. When designing flow cytometry panels, we combined CD54 with markers for plasma cells (e.g. CD38 and CD138) and B cells (e.g. CD19, CD20) in one tube. And we highly recommend combining those markers together to accurately set up the B-cell gate and avoid interference from plasma cells. Since the delay in processing specimens and freeze/thaw could result in the decrease of CD138 expression [33, 34], and the majority of the normal plasma cells are positive for CD19, we highly recommend finishing the detection in time.
In this study, most of the cases with BM involvement were diagnosed by BM biopsy. However, there were 5 cases whose BM involvement was diagnosed by flow cytometry and confirmed by Ig heavy chain rearrangement. As shown in Supplementary 2, the number of lymphoma cells infiltrating BMs might be too low to be detected by BM biopsy (0.03% − 0.71%). Among them, four cases highly expressed CD54. This also hints that the detection sensitivity of CD54 could attain 0.03%.
In the future, we will enroll more patients, and detect the expression of CD54 in lymphoid nodes, spleen, and other lymphoid tissues. We will also study the prognostic significance of CD54.