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W. Wang et al.
for both CD123 and HLA-DR,16 but their level of CD123 expression is substantially lower than that of PDC.
More challenging is the evaluation of MRD status after treatment or staging assessment of BM specimens with a low tumor burden. This challenge is attributable to the immunophenotypic overlap between BPDCN cells and reactive PDC, especially, the CD56+ subset of reactive PDC. In fact, an initial panel designed for BPDCN MRD detection (panel #2, Table 1) failed to distinguish BPDCN from normal PDC. An immunohistochemical study with TCF4/CD123 was able to highlight PDC, but was inca- pable of differentiating BPDCN from reactive PDC. These problems prompted us to study the immunophenotype of reactive PDC, and explore the immunophenotypic differ- ence between neoplastic and reactive PDC.
Although both reactive PDC and BPDCN cells were uni- formly positive for CD123 and HLA-DR, BPDCN cells tended to have brighter HLA-DR and lower CD123 expression. While all reactive PDC were positive for CD33, 52% of BPDCN cases did not express this marker. All reactive PDC were positive for CD2 with a bimodal pattern, whereas only 19% of BPDCN cases were positive for CD2. With regards to other lymphoid antigens, CD7 expression in BPDCN was very frequent (64%), whereas CD5 was only observed in less than 5% of cases. CD303, a marker considered specific for PDC, was reported to be expressed in 90%,14 63%17 and 53%18 of BPDCN cases as determined by immunohistochemistry. According to flow cytometry analysis, CD303 was expressed in 75%15 and 64%19 of cases. Of note, various anti-CD303 antibodies have been used in previous studies, including clone DDX0043 (Dendritics, Dardilly, France),14,17 rabbit anti- cytoplasmic CD303,18 and AC144.15 In our study, using clone 201A from Biolegend, all reactive PDC were positive for CD303, whereas only 44% of BPDCN cases were pos- itive. Of the CD303+ BPDCN cases, many showed a decreased level of expression compared to that of the internal control-normal PDC. Recently, Huang and col- leagues reported decreased or absent CD303 expression in early stages of plasmacytotoid maturation in their series of myeloid neoplasms with PDC differentiation.20 The lower CD303 intensity in BPDCN might reflect the immaturity of tumor cells as they derived from less mature/precursors of PDC. Nonetheless, this altered level of expression of CD303 in BPDCN facilitates the identification of neoplas- tic cells in a background of normal PDC, and contributes to MRD detection.
We further confirmed that CD56 was normally expressed in a subset of normal PDC, ranging from 1.3% to 20% of total PDC. A similar observation was previous- ly made in peripheral blood and BM samples from healthy people.10-12 These CD56+ PDC have been pro- posed to be precursors as well as the cell of origin of BPDCN. We show here that the CD56+ subset of normal PDC are positive for CD2 and CD303, negative for CD7,10-12 and retained a high level of CD38. This immunophenotypic pattern is distinctively different from that of CD56+ BPDCN cells, which are often CD2– (81%),
CD7+ (64%), CD303– (56%), and show decreased or neg- ative CD38 expression (82%). Based on the immunophe- notypic differences, we designed a new flow cytometric panel composed of these markers. This panel was capable of detecting BPDCN cells to a level of 0.01% and, prospectively tested in 19 BM samples from seven patients, was able to reliably distinguish BPDCN cells from reactive PDC in all samples. Of note, every BM sam- ple contained reactive PDC, which served as internal con- trols for comparison. Other markers that could be explored in the future to distinguish BPDCN cells from normal PDC include CD5, CD13, CD22, and CD33. BCL2 is also potentially valuable as it is expressed in BPDCN but often negative in reactive PDC.5
In this study, the flow cytometry assay for MRD detec- tion was not compared to mutational analysis for a num- ber of reasons. First, not every case of BPDCN had detectable mutations using our current next-generation sequencing analysis covering 81 frequently mutated genes in myeloid/lymphoid neoplasms. Second, the mutations frequently found in BPDCN, such as TET2, ASXL1, TP53 and NRAS, are also commonly found in myeloid neoplasms. It is well known that myeloid neo- plasms such as myelodysplastic syndrome and chronic myelomonocytic leukemia frequently co-occur with BPDCN.8,21 Thus the detection of these mutations by next-generation sequencing cannot differentiate a BPDCN clone and a myeloid clone in such cases. Last, the sensitivity of next-generation sequencing is about 1%, which is unable to reach the 0.01% level of sensitiv- ity of flow cytometry.
In summary, we have provided the immunophenotypic characteristics of BPDCN in detail in this study. We have also defined “immunophenotypic aberrancies” of BPDCN in comparison with normal/reactive PDC. It is imperative to recognize that reactive PDC usually include a small subset of CD56+ cells, which should not be misinterpret- ed as BPDCN. These CD56+ PDC have an immunophe- notypic profile distinctively different from that of BPDCN, which allowed us to develop a flow cytometric assay that has a high sensitivity and specificity for the detection of MRD. Such laboratory tests are much in need in the era of targeted therapy and precision medi- cine. This flow cytometry panel is valuable for disease monitoring during treatment and also enables early detec- tion of relapse in BPDCN patients who have undergone allogeneic stem cell transplant, allowing for early inter- vention. The significance of positive MRD prior to and following stem cell transplantation is of great interest in BPDCN, and deserves future studies.
Disclosures
No conflicts of interest to disclose.
Contributions
WW and SAW designed and wrote the manuscript. TN per- formed the experiments. JX, SL, SL, JDK, RNM, JLJ, NP and LJM offered suggestions, wrote and reviewed the manuscript.
References
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2. Beird HC, Khan M, Wang F, et al. Features of non-activation dendritic state and immune deficiency in blastic plasmacytoid dendritic cell neoplasm (BPDCN). Blood Cancer J. 2019;9(12):99.
3. Facchetti F, Petrella T, Pileri S. Blastic plas-
macytoid dendritic cell neoplasm. In: Swerdlow S, Campo E, Harris N, et al., eds. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Revised 4th edition. Lyon, 2017:174-177.
4. Venugopal S, Zhou S, El Jamal SM, Lane
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