Immunophenotypic characterization of reactive and neoplastic plasmacytoid dendritic cells permits establishment of a 10-color flow cytometric panel for initial workup and residual disease evaluation of blastic plasmacytoid dendritic cell neoplasm

Abstract

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematopoietic neoplasm whose immunophenotype remains incompletely characterized, particularly in terms of distinction from reactive plasmacytoid dendritic cells (PDCs). This limitation complicates detection of low-level involvement by BPDCN as well as minimal residual disease (MRD) assessment following therapy. We conducted the current study to characterize the immunophenotype of BPDCN in a cohort of 39 patients, and compared it to reactive PDCs. We found that, in addition to CD56 expression (97%), BPDCN showed a number of aberrancies, including decreased/negative CD38 (82%), positive CD7 (64%), negative CD2 (81%), negative CD303 (56%), increased HLA-DR (69%) and decreased CD123 (78%). Although BPDCN cells were characterized by CD56 expression, reactive PDCs consistently included a CD56-positive subset, ranging 1.3%-20% (median 4.5%) of total PDCs, challenging MRD detection. These CD56+ reactive PDCs, however, were consistently positive for CD2 and CD303, brightly positive for CD38, and negative for CD7, distinctively different from BPDCN. Based on these findings, we set up a 10-color flow cytometry assay for BPDCN and validated it to a sensitivity of 0.01%. This panel was prospectively tested in 19 bone marrow samples from 7 BPDCN patients, and it effectively distinguished BPDCN cells from background reactive PDCs in all cases. In summary, by understanding the immunophenotype of reactive and neoplastic PDCs, BPDCN can be effectively detected by flow cytometry to a very low level using a panel of markers in addition to CD56, and such assay can be used for initial bone marrow workup as well as MRD detection after therapy.

Figure 1.

The immunophenotype of blastic plasmacytoid dendritic cell neoplasm. The figure summarizes the immunophenotype of blastic plasmacytoid dendritic cell neoplasms in 39 patients. Different colors represent different levels of expression.

Figure 2.

The location of blastic plasmacytoid dendritic cell neoplasm cells on CD45/SSC plots. (A): Most (92%) cases of blastic plasmacytoid dendritic cell neoplasm (BPDCN) show CD45 expression at a level similar to or slightly higher than that detected in granulocytes. (B) A small subset (8%) of cases shows lower CD45 expression. The red population represents BPDCN cells.

Figure 3.

Differential immunophenotypic characteristics of blastic plasmacytoid dendritic cell neoplasm and reactive plasmacytoid dendritic cells. Blastic plasmacytoid dendritic cell neoplasm (BPDCN) cells often show increased HLA-DR, decreased CD123, decreased CD303, decreased CD38, and positive CD56 expression. Pink: basophils; blue: reactive plasmacytoid dendritic cells (PDC); red, neoplastic PDC; and gray: monocytes. (A) Both basophils and PDC are bright for CD123. Basophils are negative whereas PDC are positive for HLA-DR. In comparison to reactive PDC (blue), neoplastic PDC (red) often show decreased CD123 and increased HLA-DR expression. Monocytes (gray) are also positive for CD123 and HLADR, but their CD123 level is much lower than that of PDC. (B) Neoplastic PDC are positive for CD56 and negative for CD303. CD303 is positive in reactive PDC. (C) Neoplastic PDC often show decreased CD38 expression when compared to reactive PDC. (D) Reactive PDC are positive for CD33, and approximately half of BPDCN cases are negative for CD33.

Figure 4.

Representative cases of reactive and neoplastic CD56⁺ plasmacytoid dendritic cells. Gray: CD56^– reactive plasmacytoid dendritic cells (PDC); blue: CD56⁺ reactive PDC; red: CD56⁺ neoplastic PDC. (A) CD56⁺ reactive PDC are consistently positive for CD2 and CD303, negative for CD7. CD38 expression is bright. (B) In contrast, neoplastic CD56⁺ neoplastic PDC are often negative for CD2 with decreased to negative CD303 expression. CD7 expression is often positive and CD38 expression level is often decreased. Focusing on CD56^– PDC (gray) in both panels (A) and (B), these cells are positive for CD303 and CD38. CD2 shows a bimodal pattern of expression (both negative and positive cells present). A small subset of reactive PDC is CD7⁺ and these CD7⁺ reactive PDC are negative for CD56.

Figure 5.

A case of reactive plasmacytoid dendritic cell proliferation positive for CD56 by immunohistochemistry. (A) A skin biopsy shows small clusters of plasmacytoid dendritic cells (PDC), some with plasmacytoid morphology in a self-limited skin lesion, likely caused by an insect bite. The insert shows the low-power view of the skin biopsy. (B) Double staining for CD123/TCF4 highlights scattered and loosely clustered PDC. (C) CD56 immunostaining shows that many PDC are positive.

Figure 6.

Immunostain and flow cytometric analysis of a case of blastic plasmacytoid dendritic cell neoplasm before and after transplantation. (A, B) Immunostain using a dual-color TCF4/CD123 double stain showed scattered plasmacytoid dendritic cells (PDC) in both samples, before (A) and after (B) transplantation. (C, D) Flow cytometric analysis showed that a subset of PDC (red) in the pre-transplant sample (C) was aberrant (decreased CD38, negative CD2, decreased CD303 expression) whereas all PDC in the post-transplant sample (D) showed a normal immunophenotype. CD56⁺ reactive PDC are highlighted blue in (C) and (D).