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Keywords:

  • natural killer cell;
  • terminal deoxynucleotidyl transferase;
  • lymphoma;
  • myeloid leukemia;
  • biphenotypic;
  • lymphoblastic leukemia

Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

BACKGROUND

An unusual cutaneous tumor that has blastic morphology and coexpresses CD56 and terminal deoxynucleotidyl transferase (TdT) has been recently recognized and termed blastic natural killer cell lymphoma.

METHODS

The authors identified seven cases of such CD56+TdT+ blastic tumors presenting in skin at their institution. The authors correlated clinical course with histomorphology and immunophenotype.

RESULTS

All 7 patients (6 men, 1 woman, 52-85 years) presented with rapidly growing, frequently multiple cutaneous nodules. All patients had low level bone marrow involvement at diagnosis and frequently had lymph node involvement. Tumor cells were of intermediate size with irregular nuclear contours, fine chromatin, and indistinct small nucleoli. The expression of TdT varied between 5% and over 90% of the neoplastic cell population. Tumor cells were negative for surface CD3, CD5, and CD20 in all cases, but some patients showed expression of CD2 (three out of five), cytoplasmic CD3 (two out of seven), CD4 (six out of seven), and CD16 (three out of seven). Molecular studies showed absence of T-cell receptor gene rearrangements in all cases. All seven patients had rapid progression of disease, and six patients have died of their disease or complications. Three patients developed progressively increasing numbers of bone marrow blasts that had a myeloid immunophenotype and were negative for TdT and CD56. Two patients met criteria for acute myeloid leukemia at 11 and 22 months after presentation, respectively.

CONCLUSIONS

CD56+ TdT+ blastic tumor presenting in skin is a systemic malignancy likely of primitive/undifferentiated hematopoietic origin. Patients might subsequently develop tumors of myeloid or myelomonocytic phenotype, indistinguishable from acute myelogenous leukemia. Cancer 2002;94:2401–8. © 2002 American Cancer Society.

DOI 10.1002/cncr.10489

Natural killer (NK) cells are lymphocytes with cytotoxic potential that show variable expression of CD16, CD56, CD57, and cytotoxic granule proteins (e.g., TIA-1, perforin, and granzyme B).1, 2 Immature NK cells are typically positive for myeloid-associated antigens (e.g., CD13 and CD33) and show upregulation of CD56 and CD16 upon maturation. Tumors of NK origin include the well-characterized Epstein-Barr virus-positive sinonasal lymphoma (and its variants at other sites) and the NK large granular lymphocytic leukemia. Rarer entities include aggressive blastic NK leukemia, some of which coexpress myeloid antigens.

More recently described is a CD56+ cutaneous tumor of putative NK origin that shows partial expression of the lymphoid blast marker terminal deoxynucleotidyl transferase (TdT) and often expresses CD4 and/or CD68. Assignment of this tumor to the NK lineage was based on its lymphoid appearance, expression of some cytotoxic markers, and lack of clonal rearrangements of B-cell and T-cell antigen receptors.3, 4

We studied seven cases of such CD56+TdT+ blastic cutaneous tumors. We observed subtle bone marrow involvement in all cases at the time of diagnosis, and three patients showed subsequent rapid development of myelomonocytic neoplasms. These results suggest a relationship between these tumors and that CD56+TdT+ blastic cell tumors may actually represent a biphenotypic or primitive hematopoietic malignancy.

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Seven cases of CD56+TdT+ blastic cutaneous tumors were reviewed at the University of Texas, MD Anderson Cancer Center between 1998 and 2000. All patients initially presented with skin lesions. During this period, TdT immunostaining was typically performed on cutaneous hematopoietic tumors of indeterminate origin. Staging included examination of peripheral blood and bone marrow. Chest and abdomen computed tomography (CT) scans were performed in five patients and lymph node biopsies were done in three patients.

Immunophenotyping Studies

Immunophenotyping studies were performed by a combination of flow cytometry and immunohistochemistry of frozen or fixed, paraffin-embedded tissue sections of skin, lymph node, and bone marrow biopsy specimens. In three cases, frozen tissue was utilized to stain for CD2, CD3, CD4, CD7, CD8, CD19, CD20, CD25, and HLA-DR (antibodies from Dako, Carpinteria, CA). In all cases, paraffin section immunostains were done following heat-induced epitope retrieval. Paraffin sections were stained with monoclonal antibodies directed against CD2 (AB75, Novocastra, Newcastle upon Tyne, U.K.), CD4 (1F6, Novocastra), CD8 (C8/144B, Dako), CD20 (L26, Dako), CD56 (123C3, Monosan), and TdT (polyclonal, Dako). Tissue was also stained with polyclonal antibodies reactive with CD3 (Dako). In situ hybridization for Epstein-Barr virus was done with probes for Epstein-Barr virus encoded RNAs (EBER, Dako).

Flow cytometry immunophenotypic studies were performed using three-color analysis on a FACScan cytometer (Becton-Dickinson [BD], San Jose, CA). Lymphocytes were gated for analysis using CD45 expression and side scatter. The panel of antibodies included CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD13, CD19, CD20, CD23, CD16, CD56, T-cell receptor (TCR) α/β, TCRγ/δ, CD33 (BD), HLA-DR (Coulter, Hialeah, FL), TdT (Supertechs, Bethesda, MD) and CD45RO (Dako).

TdT staining was also performed by immunofluorescence on freshly-prepared bone marrow aspirate smears in some cases. Cytochemic studies were performed on bone marrow aspirate smears for myeloperoxidase (MPO) and α-naphthyl butyrate esterase (butyrate) using standard methods.

Molecular Studies

DNA was extracted from fresh tissue in three cases and from fixed, paraffin-embedded tissue in three cases. In the three cases with fresh tissue, Southern blot analysis of the TCR beta chain gene was performed using a cDNA probe for the constant region. In the remaining cases, analysis of the TCR gamma chain gene was done using four consensus GC-clamped V and J primers by a multiplex polymerase chain reaction (PCR) reaction method as described by Theodorou et al.5 The immunoglobulin heavy chain gene was analyzed by PCR using consensus V and J primers in five cases. Beta-globin gene was amplified as a control for the quality of DNA amplified in the PCR reactions.

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Clinical Features

The clinical features of the seven patients are summarized in Table 1. There were six men and one woman, with ages ranging from 52 to 85 years at the time of diagnosis. All presented with cutaneous lesions, accompanied by lymphadenopathy in five patients that was either regional (three patients) or generalized (two patients). One patient had splenomegaly. The skin lesions were nodular with the exception of one patient (Patient 4) who had both plaques and nodules. All had bone marrow involvement at staging, and one patient had morphologically evident low level peripheral blood involvement. No patients had hepatomegaly or systemic B symptoms. Serum lactate dehydrogenase levels were initially within normal range for all patients; two patients had elevation of serum β2-microglobulin to approximately twice the upper normal limit.

Table 1. Clinical Features of CD56+TdT+ Blastic Tumors
PatientAge/GenderSkin lesionsOther organs involvedTreatmentFollowup
  1. TdT: terminal deoxynucleotidyl transferase; BM: bone marrow; hyper-CVAD: hyperfractionated cyclophosphamide, vincristine, doxorubicine, and dexamethasone; POMP: mercaptopurine, methotrexate, vincristine, and prednisone; AML: acute myeloid leukemia; DOD: dead of disease; DOC: dead of complications; CHOP: cyclophosphamide, vincristine, doxorubicin, and prednisone; ATRA: taxol/topotecan, fludarabine, and all-trans retinoic acid; MPO: myeloperoxidase; WBC: white blood cell count.

156M∼2 cm noduleBM (20%)hyper-CVADRecurred with AML, 15 mos
Lymph nodePOMP maintenance troxacitabine/cytarabineDOD, 24 mos
261M∼2 cm nodulesBM (28%)hyper-CVAD, one dose onlyDOC, 6 mos (ARDS)
Lymph node
373M∼0.5–1.6 cm nodulesBM (5%)hyper-CVAD and methotrexate/cytarabineRecurred with AML, 22 mos
Lymph nodeSalvage treatmentDOD, 24 mos
455MUp to 10 cm plaques and nodulesBM (7%)CHOP × 1DOD, 3 months
Lymph nodeTaxol/topotecan
552MUp to 7 cm nodulesBM (3%)hyper-CVAD and methotrexate/cytarabineNED, 3 mos
685FUp to 4 cm nodulesBM (5%)CHOP, RTDOD, 7 mos
ATRA (liposomal)MPO+ blasts in BM
777MUp to 3 cm nodulesBM (6%)CHOP × 6DOD, 8 mos with leukemic phase (90,000 WBC)
Lymph nodeFludarabine
Spleen

Three patients received a full course of hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone (hyper-CVAD) that was alternated with four cycles of high-dose methotrexate (MTX) and cytarabine in one patient and followed by mercaptopurine, MTX, vincristine, and prednisone maintenance therapy in another.6 One patient underwent a single cycle of hyper-CVAD before developing a fatal complication (adult respiratory distress syndrome). Three patients initially received standard cyclophosphamide, vincristine, doxorubicin, and prednisone (CHOP) but were switched to taxol/topotecan, fludarabine, and all-trans retinoic acid, respectively, upon disease progression.

Three patients who were treated with full courses of hyper-CVAD had sustained remissions of their cutaneous disease for 13, 15, and 22 months after chemotherapy. However, one of these patients had progressive bone marrow dysplasia and monocytosis, beginning several months after initiating therapy, that eventually terminated in acute myeloid leukemia (AML)-M4 22 months after presentation. This patient received a variety of salvage therapies (including daunorubicin) but died two months later of progressive disease. A second patient, initially in complete remission, developed AML-M4 15 months after presentation (and 5 months after terminating maintenance therapy). This patient was reinduced with β-dioxolanecytidine (troxacitabine) and cytarabine (ara-C), then clofarabine, but died of refractory leukemia 24 months after initial presentation. The final patient was in remission after hyper-CVAD therapy thirteen months after presentation.

The three patients initially treated with CHOP died of their disease three, seven, and eight months following time of presentation, respectively. After an initial response in the first two cycles, two of these patients had progressive disease during their treatment; the third patient had progressive disease after completing six cycles of CHOP.

Morphologic Features of Initial Biopsy Specimens

Skin

All patients had diffuse, superficial, deep dermal infiltrates that showed preferential involvement of deep adnexal structures (Figs. 1 and 2). All but one patient showed a Grenz zone of separation from overlying epidermis. Two patients showed prominent tumor infiltration around dermal blood vessels with red blood cell extravasation and hemosiderin deposition consistent with low grade vascular damage.

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Figure 1. CD56+TdT+ blastic tumor terminating in acute myelogenous leukemia (Patient 1). A) Cutaneous tumor shows deep dermal perivascular tumor infiltrates. Tumor cells were positive for CD56 (not shown). B) Tumor cells in the lymph node are blastic in appearance. C) Staging bone marrow aspirate revealed scattered lymphoid forms with irregular nuclear contours. D, E) Tumor cells in skin were only partially positive for TdT but those in lymph node were diffusely positive. F) Development of acute myeloid leukemia-M4 15 months after presentation with poorly differentiated bone marrow blasts with associated trilineage dysplastic changes (arrows). G) Blasts were uniformly positive for myeloperoxidase and partially positive for butyrate esterase (not shown).

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thumbnail image

Figure 2. CD56+TdT+ blastic tumor terminating in acute myelogenous leukemia (Patient 3). A) Cutaneous tumor shows a dense dermal nodule of immature appearing lymphoid cells. CD56 is diffusely positive in tumor cells (inset). B) CD4 is diffusely positive in tumor cells. C, D) Following initial therapy, patient developed steadily increasing monocytes and blasts in bone marrow that terminated in acute myeloid leukemia -M4 22 months after initial presentation.

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The neoplastic infiltrate consisted of monomorphic small to intermediate sized cells resembling blastic lymphocytes. Cells exhibited nuclei with markedly irregular contours, fine chromatin with peripheral condensation, and small nucleoli. A high mitotic count, up to six per high power microscopic field, was noted in all cases. Angioinvasion and necrosis were absent.

Lymph node

Three patients had lymph node biopsy specimens showing partial to diffuse effacement by sheets of similar-appearing blastoid tumor cells (Figs. 1B and 3). In all specimens, there were areas of interfollicular infiltration with preserved lymphoid follicles (Fig. 3). Immature myeloid elements were not prominent.

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Figure 3. CD56+TdT+ blastic tumor in the lymph node (Patient 4) with an interfollicular pattern of infiltration. Tumor cells are lymphoid appearing with blastic nuclear features, including convoluted nuclear contours.

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Bone marrow findings at time of diagnosis

All cases showed evidence of bone marrow infiltration at the time of initial bone marrow staging. The degree of bone marrow involvement ranged from 3-28% of the overall cellularity and was manifested in core biopsy specimens by clusters of lymphoid-appearing blastoid cells. Immunostains (i.e., TdT and CD56) were often necessary to highlight tumor foci. The overall cellularity of the core biopsy specimens ranged from 65 to 95%, with erythroid hyperplasia with megaloblastoid maturation in five of seven patients. Mild trilineage dysplastic changes with increased monocytes were noted in one patient.

On aspirate smears, the neoplastic cells had moderately abundant cytoplasm with irregular, convoluted nuclei exhibiting open, immature chromatin without nucleoli (Fig. 1C). In two patients, the tumor cells contained azurophilic cytoplasmic granules. Cytochemic stains for MPO and butyrate esterase were negative in tumor cells on aspirate in four tested cases.

Immunophenotypic Results at the Time of Diagnosis

A summary of immunophenotypic findings derived from flow cytometric analysis and immunostains on the initial diagnostic samples and tumor recurrences is presented in Table 2.

Table 2. Phenotypic Features of CD56+TdT+ Blastic Tumorsa
Patient1123345667
2nd tumor AML-BM2nd tumor AMoL-BM2nd tumor myeloid BM
  • a

    Compilation of immunohistochemical and flow cytometric analysis results.

  • Tolt: terminal deoxynucleotidyl transferase; AML: acute myeloid leukemia; BM: phenotyping done on bone marrow aspirate material by flow cytometry; LN: immunostain on lymph node; S: immunostain on skin; wt: weakly or variably positive; LNF: flow cytometry on lymph node; SF: flow cytometry on skin; F: positive by flow cytometry; cyto: positive on cytochemical stain; IF: immunofluorescence.

TdT+95% LN+5% LN+15% S+7% LN+50% S+20% S+15% S
+30% S+20% S+2% S+BM
+BM (IF)+BM (IF)+BM
CD16−BM+SF/BM+BM+SF
CD56+S/LN+SF/BM+SF/S+BM/Sw+ S+S/BM+SF
+BM+LN+BM
TIA-1+S+S/BM+S−S−S
CD2+S−S−SF+BM+S−S−SF
+BM
sCD3−BM−LNF/SF−SF−BM−S−S−SF
−BM
cCD3−S/LN−S/BM+S+BM/S−S−S−S
−BM
CD4+−LNFw+ SF/S−BM+S+S+SF/S
+BMw+W+ BM
CD5−S−LNF/SF−SF−BM/S−S−S−S
−BM−BM
CD7−BM+LNF/SF−SF−BM−SF
w+ BM
CD8−S−LNF−SF/S−BM−S−S−S/SF
−BM−BM
CD10/19/20−S/LN−LNF/SF−SF/S−BM/S−S−S−SF/S
−BM−BM/S
CD13−BMw+−LNF−SF+−BM−SF
CD30−S−S−S−S−S−S−S
CD33+BM+−LNF−SF+−BM−SF
CD43+S−LN+S+S+S+S
CD45RO−S/LN−S+−S−S
CD68+S/LNv+−S−S
HLA-DR+BM++LNF+SF++SF
MPO−S/LN+(F)−S−S+(cyto)−S/BM−S−S+−S
−(cyto)
EBV−S−S/LN−S+S−S−S−LN
Karyotype46,XY47,XY,+845,XY,−746,XX

Flow cytometric immunophenotypic analysis was performed on six initial specimens from four patients (three skin, one lymph node, and two bone marrow). All cases were positive for CD56 and TdT and negative for the T-cell-specific markers surface CD3 and CD5 (four tested) as well as B-cell-specific markers (i.e., CD10, CD19, and CD20). All three tested cases were positive for HLA-DR. CD2 and CD4 were expressed in two of three and two of four cases, respectively.

Immunohistochemistry was performed on 15 initial specimens from all 7 patients (7 skin, 3 lymph node, 5 bone marrow). All cases stained positive for CD56 and TdT and were negative for CD20, CD30, and myeloperoxidase. CD56 was detected by immunostaining in all tumor cells (Fig. 2B). The proportion of neoplastic cells exhibiting unequivocal nuclear staining with TdT ranged from 5% to greater than 90%. In the three patients with concurrent lymph node and skin biopsy specimens, the level of TdT immunostaining by tumor cells was greater in lymph node than in skin (Fig. 1 and Table 2). Five cases were positive for CD4, four for CD43, and two for CD3 (cytoplasmic). TIA-1 expression was detected in three patients. Oligonucleotide in situ hybridization for EBER was expressed in tumor cells in one patient.

Bone Marrow Findings over the Course of the Disease

One patient who had regression of his skin lesions with hyper-CVAD developed AML-M4 15 months after presentation. The bone marrow blasts were strongly myeloperoxidase positive, partially positive for butyrate esterase (25%), and negative for TdT by immunofluorescence.

A second patient treated with hyper-CVAD, methotrexate, and cytarabine had evidence of remission in the bone marrow and skin but then developed increasing blasts, monocytosis, and dysplasia in follow-up bone marrow specimens beginning five months following the initiation of therapy. This culminated in the development of overt leukemia 22 months following the initial diagnosis (Fig. 2). The blasts were positive for MPO and butyrate esterase consistent with AML-M4 (not shown). A third patient who had progressive cutaneous and nodal disease had bone marrow aspiration and biopsy seven months after presentation that showed an aggressive myelodysplastic syndrome characterized by increased blasts (11%) positive for MPO by cytochemistry and negative for TdT by immunostain.

Leukemic recurrences in bone marrow from two patients were extensively characterized by flow cytometry on aspirate material as described in Table 2. Both tumors were positive for multiple myelomonocytic markers and negative for CD56 and TdT.

Molecular and Cytogenetic Studies

Molecular studies were performed on specimens from all patients. By PCR analysis, there was no evidence of monoclonal TCR-gamma chain rearrangements in the six skin biopsy and two bone marrow diagnostic specimens tested, nor was there evidence of immunoglobulin heavy chain gene rearrangements in the five skin biopsies tested. Southern blot analysis of the T-cell receptor beta chain gene showed no evidence of gene rearrangements in three cases.

Cytogenetic analysis revealed trisomy 8 in a bone marrow sample from one patient at time of diagnosis and monosomy 7 in multiple bone marrow samples from Patient 3, who showed emergence of myelomonocytic leukemia over a one year period.

DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

We described seven cases of CD56+TdT+ blastic tumors that presented as skin lesions but showed systemic dissemination to bone marrow and lymph node. All six patients with more than several months followup had an aggressive clinical course with progressive or recurrent disease, with five deaths due to disease. Thus, we confirm previous reports that CD56+TdT+ blastic cutaneous tumors present concurrently with bone marrow involvement and have a rapidly progressive course despite systemic polychemotherapy. The lymphoid morphology, immunophenotype, and absence of T-cell receptor gene rearrangements in the skin lesions would support a natural killer cell origin. However, the bone marrow blasts in three relapses showed evidence of myeloid differentiation with no immunofluorescent and/or immunohistochemical staining for TdT. Moreover, the subsequent tumors showed a myelomonocytic immunophenotype in all three patients. This finding and the presence of coexisting dysplastic changes in the bone marrow at initial diagnosis in five patients suggest that CD4+CD56+ blastoid tumors may be of primitive stem cell origin with mixed natural killer/myelomonocytic features.

Recognized several years ago, the CD56+TdT+ blastic cutaneous tumor has been termed by most investigators blastoid or blastic NK-cell lymphoma.4, 7 This immature, lymphoid appearing tumor is usually negative for Epstein-Barr virus (EBV), allowing distinction from the EBV-positive nasal-type NK lymphoma that occurs commonly in the sinonasal tract and less commonly in skin and other mucosal sites. Similar to the current series, previous CD56+TdT+ tumors presenting in the skin or lymph node have shown a uniformly aggressive clinical course.4, 8–15 Lymph node involvement is also commonly noted, and nearly all cases reported have been widely disseminated at the time of diagnosis or soon thereafter.

An interesting and consistent feature of this tumor is variable rather than uniform expression of TdT. Indeed, we generally noted a minority of cells positive for TdT, but one patient had a concurrent lymph node biopsy specimen where the tumor showed more uniform TdT expression. The frequent and variable coexpression of CD68 and CD4 and the variable absence of CD2 and/or cytoplasmic CD3 has lead to the suggestion that this tumor may be of myelomonocytic rather than NK origin.14, 16 The three patients who subsequently developed tumors with a myeloid phenotype are strong support for this hypothesis. Although these cases could represent emergence of an unrelated second leukemia, this interpretation appears unlikely given the short time interval between the two processes. The hyper-CVAD regimen, which four patients received, is widely used at our institution for high-grade lymphoma/leukemias and has not been associated with increased risk of therapy related leukemias.

Based on the current results, we propose that this tumor be regarded as a primitive or biphenotypic hematopoietic neoplasm. The designation raises its relationship to the previously described myeloid/NK precursor leukemias that express both myeloid antigens (e.g., CD13 and CD33) and CD56.3, 17, 18 TdT expression and cutaneous infiltration does not seem to be a feature of most of these cases.

Given their aggressive course, the optimal therapy for CD56+TdT+ blastic tumors remains unclear. In contrast to less intensive regimens, hyper-CVAD was capable of producing remissions of 13, 15 and 22 months, respectively, before two of these treated patients developed myelomonocytic leukemias. Three patients treated with less aggressive chemotherapy succumbed to their disease in less than six months. Recurrence of these tumors with a myeloid phenotype suggests that trial therapy with AML type regimens may be warranted.

REFERENCES

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES