TCL1 expression in plasmacytoid dendritic cells (DC2s) and the related CD4⁺ CD56⁺ blastic tumors of skin

CD4⁺ CD56⁺ blastic tumor (BT) is a recently recognized highly aggressive tumor presenting in skin, usually with lymph node and bone marrow involvement, often terminating in a leukemic phase.¹  Based on lymphoid morphology, frequent expression of natural killer (NK) cell–associated antigens (eg, CD2, CD7), and the absence of T-cell receptor or surface CD3, these tumors had been considered to be of NK-cell origin^2-4  and were designated as blastic NK-cell tumors in the World Health Organization (WHO) classification.⁵ 

However, the CD4⁺ CD56⁺ BTs express markers typical of plasmacytoid dendritic cells (also known as DC2s), particularly CD123/interleukin-3 (IL-3) receptor δ-chain.^6,7  Based on the emergence of typical myelomonocytic leukemias in a subset of BT cases, we have suggested that these tumors may also have monocytic or multilineage potential.¹  This is consistent with the finding that DC2s are sometimes increased in patients with myelomonocytic leukemias and can be derived from monocytes in vitro under certain conditions and may undergo conversion in vivo to myeloid-type DCs.^8-11 

To investigate the histogenesis of CD4⁺ CD56⁺ BTs, we tested markers of various hematopoietic lineages. We report here that BTs and DC2s share the expression of CD123 and the lymphoid proto-oncogene product TCL1. TCL1 expression was previously thought to be restricted to B cells, some immature T cells, and most cases of T-cell prolymphocytic leukemia (T-PLL), in which it is activated as a consequence of chromosomal rearrangements involving 14q32.1.^12-16  TCL1 is a protein of unknown function that has recently been shown to bind in vitro to the AKT family of serine/threonine kinases and to promote AKT kinase activation through oligomerization and increased AKT transphosphorylation.^17-20 

The study was conducted according to the MD Anderson Cancer Center institutional review board–approved protocol, and cases were selected using WHO criteria for blastic NK-cell lymphoma, which required initial cutaneous presentation as well as CD4 and CD56 positivity and absence of surface CD3.⁵  TCL1 expression was analyzed by Western blot and/or immunohistochemistry using a TCL1 rabbit antiserum.¹³  TCL1 specificity was confirmed by Western blot using extracts of T-PLL cells with cytogenetically documented rearrangements of the TCL1 locus. Phosphorylated AKT (pAKT) was detected using 2 phospho-specific antisera directed against the Ser473 and Thr308 epitopes of AKT-1, -2, and -3 (pAKT; Cell Signaling Technology, Beverly, MA). Monoclonal antibodies were used to detect terminal deoxynucleotidyl transferase (TdT-NCL; Novocastra, Newcastle-upon-Tyne, England), cutaneous lymphocyte antigen (CLA, HECA-452; Pharmingen, San Diego, CA), CD3 (Dako, Carpinteria, CA), CD4 (1F6; Novocastra), CD20 (L26; Dako), CD45RA (4KB5; Dako), CD56 (1B6; Novocastra), CD68 (KP1; Dako), CD117/c-kit (104D2; Dako), and CD123 (6H6; eBiosciences, San Diego, CA).

Tumor cells were obtained by Ficoll-Hypaque density centrifugation of peripheral blood (PB). Cell lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane and detected using the enhanced chemiluminescence system (Amersham, Piscataway, NJ). BC-1 (B-cell large cell lymphoma line) and NALM-6 (B-cell lymphoblastic line) were used as negative and positive controls for TCL1, respectively, and Jurkat (T-lymphoblastic cell line) was used as the positive control for pAKT. Immunohisto-chemical staining was performed on formalin-fixed tissue sections using avidin-biotin-peroxidase reagents (LSAB⁺ kit; Dako) using a previously validated T-cell and NK cell lymphoma tissue array,²¹  with positive immunostaining results confirmed on whole-tissue sections.

Described previously were 6 BT cases;¹  here we added 6 additional cases having similar features, including initial cutaneous presentation with lymphoid morphology (Figure 1A, inset), expression of CD4 and CD56 (Figure 1A), and absence of CD3, myeloperoxidase, and CD117. CD68 was positive in 6 of 8 cases tested. All but one case lacked monoclonal T-cell receptor gamma gene rearrangements by polymerase chain reaction analysis. As previously described, we noted in 10 (83%) of 12 cases variable immunohisto-chemical positivity for TdT, which was strongest in tumor cells in 4 involved lymph node biopsy specimens (Figure 1B). Subsequently, 3 BT patients developed increasing blood myelomonocytic blasts terminating in acute myeloid leukemia (AML) 13, 15, and 22 months after initial cutaneous presentation. These phenotypically distinct blasts in each case were cytochemically positive for butyrate esterase and myeloperoxidase and were TdT-negative. Cytogenetic findings in 6 cases are listed in Table 1.

By immunohistochemistry, all cases (11/11 skin, 1/1 lymph node, 5/5 bone marrow) showed membrane expression of CD123/IL-3 receptor α-chain (Figure 1C, inset), which was variable in 4 cases. Of 12 BTs, 10 (83%) showed strong uniform nuclear and cytoplasmic expression for TCL1 (Figure 1C), including skin (10/12, 83%) and bone marrow (4/5, 80%) sites (Figure 1C-D). Western blot analysis of freshly isolated tumor cells from PB in one BT case confirmed TCL1 expression (Figure 1). All 3 cases with subsequent myeloid transformation showed strong TCL1 expression in the myelomonocytic bone marrow blasts (Figure 1E-F), with 1 of 3 tumors no longer expressing CD123 (Figure 1G).

We examined TCL1 expression in normal hematopoietic cell subsets, and tumors possibly related to BTs.^23,24  In normal spleen (n = 5), tonsil (n = 3), and bone marrow (n = 4), TCL expression was confined to CD20⁺ B cells and rare cells with dendritic morphology. In reactive lymph nodes (n = 5), aside from B cells the only other TCL1⁺ cells noted were clusters of plasmacytoid DC2s, which also expressed CD4 (dim), CD45RA (dim), CD68, and CD123. The clusters were variably positive for CLA and negative for CD3, CD20, and TdT (Figure 1K-M, and not shown), based on immunostaining of serial sections.

In a survey of true NK-cell tumors and other CD56⁺ myeloid neoplasms involving skin and extramedullary sites, as well as chronic myeloproliferative processes, and a variety of T-cell tumors, nearly all tumors were completely negative for TCL1 (Table 1). TCL1⁺ cases included most cases of T-PLL (30/40, 75%) and a single case each of precursor T-cell lymphoblastic lymphoma/leukemia, AML with monocytic differentiation, and adult T-cell leukemia/lymphoma, with each showing TCL1 staining in a subset of tumor cells (Table 1).

Given that TCL1 has been implicated in vitro in activation of AKT by phosphorylation, we compared the pattern of pAKT with that of TCL1.^17-20  Most TCL1⁺ BT cases (6/9; 66%) were negative for both Ser473 and Thr308 pAKT by immunostaining (Figure 1J). Absence of pAKT was also noted in a case of BT analyzed by Western blot (Figure 1N). Additionally, most cases of T-PLL were negative for pAKT, with low levels of pAKT detected by Western blot in 3 (30%) of 10 TCL1⁺ T-PLL (Figure 1N) and weak immunostaining noted in 2 (15%) of 13 TCL1⁺ T-PLL. Variable, often strong, pAKT immunostaining, however, was seen in a variety of TCL1^– T-cell lymphomas (44/79, 56%).

In conclusion, BTs and their myelomonocytic transformations highly express TCL1, a feature shared with plasmacytoid dendritic cells (DC2s) but not with myelomonocytic leukemia, true NK-cell tumors, or mature T-cell malignancies, except T-PLL. TCL1 expression in primary BT and T-PLL samples was infrequently associated with pAKT, suggesting other mechanisms of TCL1 action in vivo. CD123 and TCL1 represent useful markers to identify BTs and distinguish them from other CD56⁺ cutaneous tumors, including extranodal NK-cell lymphomas of nasal-type and myelomonocytic leukemias. Expression of TCL1 demonstrates another immunophenotypic linkage between normal DC2s and the immature blastoid (TdT⁺), lymphoid-appearing (TdT^–), and subsequent monocytic (butyrate esterase⁺) phases of CD4⁺ CD56⁺ blastic tumors.