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Natural Killer Cell Precursor Acute Lymphoma/Leukemia Presenting in an Infant.

Neoplasms of natural killer (NK) cell precursors comprise a rare subset of NK and NK-like T-cell malignancies and include myeloid/NK precursor acute leukemia and blastic (or blastoid) NK cell lymphoma/leukemia.[1-5] These entities are characterized by blastic morphologic features, an immunophenotype compatible with immature NK cell derivation ([CD56.sup.+] [CD57.sup.-], [CD16.sup.-]) and the absence of the Epstein-Barr virus (EBV) highly associated with mature NK cell neoplasms.[1-7] Propensity for extranodal sites, such as the skin, soft tissue, and mediastinum, with dissemination to lymph nodes and bone marrow delineates the aggressive clinical behavior of NK cell precursor neoplasms. Because of the blastlike appearance of the constituent cells, their differential diagnosis includes lymphoblastic lymphoma/acute lymphoblastic leukemia (LBL/ALL) and acute myeloid leukemia (AML). The diagnostic separation of precursor NK lymphoma/leukemia from LBL/ALL and AML is important because precursor NK lymphoma/leukemia may show poor or partial response to standard chemotherapeutic regimens used in the treatment of non-Hodgkin lymphomas and acute leukemias.[1,3] In addition, the predilection of NK cell precursor neoplasms for extranodal involvement increases their likelihood of misdiagnosis as nonhematolymphoid malignancies, including small, round, blue cell tumors.[8] We describe the clinical, histologic, immunophenotypic, and cytogenetic findings of an unusual case of NK precursor acute lymphoma/leukemia that occurred in an 11-month-old child with a clinical presentation and immunophenotype distinct from previously reported cases.


An 11-month-old white girl presented with intermittent fevers and cervical adenopathy of 6 weeks' duration. A prodrome of progressive adenopathy was present since 6 months of age. Her status was normal after spontaneous vaginal delivery at full term following an uncomplicated pregnancy. On computed tomography (CT) scans, mediastinal adenopathy impinging on the superior vena cava and trachea and posterior auricular, cervical, mesenteric, and ileofemoral adenopathy were detected. Thoracic and abdominal visceral involvement and hepatosplenomegaly were absent. There was no other pertinent history or family history. Serologic testing for EBV and cytomegalovirus were negative.

Biopsy specimens of a 9.0-cm cervical lymph node and the bone marrow revealed an undifferentiated malignant neoplasm. No circulating blasts were present on the peripheral blood smear despite marrow involvement. Initial immunophenotypic studies were done on paraffin sections of the lymph node because material was not available for flow cytometry. The atypical cells lacked reactivity for epithelial, mesenchymal, neuroendocrine, T-cell, B-cell, myeloid, and histiocytic differentiation. Weak staining for CD34 and CD99 and strong staining for CD43 were detected. These studies and the morphologic features were most consistent with a blastic lymphoma/leukemia, although definitive characterization of the neoplasm was not made at this time. An aggressive chemotherapy regimen combining induction therapy for acute leukemia and consolidation therapy for sarcoma was used (vincristine, adriamycin, cyclophosphamide, rifaphosphamide), with good response. The patient achieved clinical remission and 6 months later underwent an autologous bone marrow transplantation. One month after transplantation, the patient returned with fever, and the peripheral blood exhibited a white cell count of 19.8 x [10.sup.9]/L with 37% circulating blasts. Flow cytometry studies on peripheral blood detected blasts that expressed CD34, CD33, and CD56, although other surface T- and B-cell markers were negative. Subsequently, sections from the original lymph node were stained for CD56 and found to be positive. Cytoreductive chemotherapy was instituted; however, the patient continued to deteriorate and died 3 months after transplantation.


Morphologic Testing

The lymph node biopsy specimen was fixed in 10% formalin, whereas the bone marrow core biopsy specimen was fixed in Bouin solution and subjected to brief decalcification and stained with hematoxylin-eosin. Peripheral blood smears were stained with Wright-Giemsa.


Primary antibodies are listed in Table 1. Sections of 4 [micro]m were cut, deparaffinized, and hydrated in a graded series of alcohol. Antigen retrieval by microwave procedure in citric acid buffer (10 mM, pH 6.0) for 10 minutes preceded staining for CD3, CD8, CD20, CD30, CD43, CD45RB, CD56, CD68, CD79A, CD99, Ki67, TIA-I, and TdT. Microwave pretreatment for CD4 was carried out in EDTA buffer (1 mM, pH 8.0). Stains for CD56 and TdT were performed using a modified biotin-streptavidin method.[9] All other stains were performed on an automated machine (Ventana Medical Systems Inc, Tucson, Ariz).


Flow Cytometry

Direct dual-parameter flow cytometry was performed on a FACscan (Becton Dickinson, San Jose, Calif) using standard whole blood lysis techniques. Commercially available fluorescein isothiocyanate or phycoerythrin-conjugated monoclonal antibodies were used (Table 2). An analysis gate was selected to include the predominant population in the weak [CD45.sup.+]/low-side scatter region. The percentage of events reactive with each monoclonal antibody was determined, setting thresholds with isotypic controls. Positive expression was defined as greater than or equal to 20%. Cytoplasmic CD3, CD79a, and MPO were assessed by flow cytometry using CalTag Fix/Perm reagents (CalTag Laboratories, An Der Grub, Austria).


Cytogenetic Analysis

The bone marrow aspirate was cultured and chromosomes were analyzed using the GTW banding method.[10] Twenty-two chromosome metaphases were analyzed.

In Situ Hybridization for EBV EBER-1 RNA

In situ hybridization was performed using a 30-base oligonucleotide probe complimentary to a portion of the EBER-1 gene as previously described.[11]

Heteroduplex Analysis of T-Cell Receptor [Gamma] Gene Rearrangement

Amplification and heteroduplex polymerase chain reaction for T-cell receptor [Gamma] gene rearrangement was performed as previously described.[12] Polymerase chain reaction amplification was performed in a Perkin Elmer 2400 thermal cycler.


Morphologic Testing

The cervical lymph node showed effaced architecture and a sheetlike proliferation of dyscohesive cells with extracapsular extension. The neoplastic cells were medium sized with scant cytoplasm, round to slightly oval nuclei, inconspicuous or small nucleoli, and finely granular chromatin. Mitotic figures were numerous (3-6 per high-power field), including atypical mitotic profiles. No glandular elements, rosettes, or strap cells were present (Figure 1, A). The bone marrow biopsy specimen was hypercellular (95%), with areas of normal trilineage hematopoiesis interspersed with paratrabecular and interstitial infiltrates of atypical blastlike cells, comprising 20% of the marrow space (Figure 1, B). No circulating blasts were identified at this time. The peripheral blood smear after transplantation showed 37% blasts consistent with relapse and progression to leukemia (Figure 1, C).



The neoplastic cells exhibited strong staining for CD43 and CD56 and weak staining for CD34 and CD99. They lacked reactivity for all other markers tested (Table 1 and Figure 2).


Flow Cytometry

Posttransplantation peripheral blood sample showed a homogeneous population of cells, positive for CD34, CD56, CD33, and cytoplasmic CD3, whereas HLA-DR, surface CD3, CD5, CD7, and CD13 were not expressed. Cytoplasmic CD79a and MPO were also not expressed (Table 2 and Figure 3, A).


Cytogenetic Studies

Chromosomal analysis demonstrated an abnormal clone in 21 of 22 cells with the following karyotype: 46,XX,add(11)(p11.2),add(15)(q22),add(18)(q23). The rearrangements involving chromosomes 11, 15, and 18 were unbalanced and clonal. Additional nonclonal structural abnormalities were observed in 4 cells, 2 of which showed apparent artifactual chromosomal loss. Although consistent with a malignant process, no diagnostic specificity is attributable to this karyotype (Figure 3, B).

Studies for EBV and T-Cell Receptor Rearrangement

There was no evidence of EBV messenger RNA or of a clonal T-cell receptor gene rearrangement.


Several unusual parameters contributed to the difficulty in making a diagnosis in this infant: young age, multiple masses without organomegaly or circulating blasts, and biopsy findings reminiscent of a small, round, blue cell tumor. The undifferentiated appearance of the neoplastic cells evoked a broad morphologic differential diagnosis that included peripheral neuroectodermal tumor/Ewing sarcoma; neuroblastoma; Wilm tumor; congenital melanoma; rhabdomyosarcoma; desmoplastic, small, round cell tumor; and a blastic hematolymphoid malignancy. The initial panel of markers used for immunodiagnosis was extensive, although reactivity was present only for CD43, CD34, and CD99. But all markers for B-cell, T-cell, myeloid, and histiocytic differentiation, including CD45RB/leukocyte common antigen, were nonreactive. In addition, the patient's normal blood cell counts with absence of circulating blasts and partial nodular involvement rather than diffuse replacement of the marrow made the diagnosis of acute leukemia less likely. Although lack of TdT is unusual for LBL, given the undifferentiated morphologic features and lack of definitive markers, a blastic lymphoma was favored. The treatment choice for this patient reflected these diagnostic difficulties.

Flow cytometry on peripheral blood at the time of relapse showed blasts expressing CD34, CD56, CD33, and cytoplasmic CD3. The absence of all B- and T-lineage markers, including surface CD7, CD19, CD3, and cytoplasmic CD79a, did not support the diagnosis of early T- or B-cell precursor LBL/ALL. The definitive myeloid lineage antigen MPO was also absent. Cytoplasmic expression of CD3 subunits has been reported in precursor and activated NK cells.[13-16] In addition, the expression of CD56 but not CD57 on paraffin sections of the lymph node biopsy specimen indicates an immature NK phenotype. These results suggest an NK precursor acute leukemia.

CD56 expression has been reported in approximately 20% of AML, especially those cases associated with translocation t(8;21) or trisomy 8.[17] Leukemias postulated to arise from bipotential precursor cells (myeloid/NK cell acute leukemia) and a related entity with blastic cytologic features (myeloid/NK precursor acute leukemia) have also been reported.[2,18] Both these leukemias occur in a wide age range, spanning 18 to 72 years.[2,18] In addition, expression of CD56 in LBL[19-24] and in ALL[25-27] has also been reported. Most cases of LBL/ALL express TdT, whereas a few cases of NK precursor acute lymphoma/ leukemia are also known to express TdT.[3] These findings indicate significant morphologic and immunophenotypic overlap between LBL/ALL and NK precursor malignancies, and their relation is currently under debate.

Initial studies of blastic NK cell lymphoma/leukemia describe a distinct clinicopathologic entity of middle-aged and elderly patients with wide dissemination at presentation, absence of EBV, and an aggressive clinical course.[1,4,5,28,29] This entity has also been called acute undifferentiated leukemia by some authors.[6,30] Precursor NK malignancies, however, are rare in the pediatric age group. Three such cases occurring in children aged 4 to 7 years have been reported.[5,8,31] Transplacental transmission of an NK lymphoma has also been reported, although this case most likely represents a lymphoma derived from mature NK cells because no progenitor antigens were detected.[32] To our knowledge, a lymphoma arising from NK precursors occurring in infancy has not been previously reported. Of interest is the lack of expression of CD7 in our case, which favors a diagnosis of blastic NK precursor acute lymphoma/leukemia over myeloid/NK precursor acute leukemia. In contrast, the expression of CD33 and CD34 favors myeloid/NK precursor acute leukemia. The patient in this study shows some features that overlap with both myeloid/NK precursor acute leukemia and blastic NK precursor acute lymphoma/leukemia[2,3,5,8,31] and suggests that these entities may form a diagnostic continuum. The current case illustrates the need for a strong index of suspicion for recognition of this rare lymphoma/leukemia from solid tumors and other hematolymphoid neoplasms occurring in infancy that this disease may mimic.


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[3.] Suzuki R, Nakamura S. Malignancies of natural killer (NK) cell precursor: myeloid/NK cell precursor acute leukemia and blastic NK cell lymphoma/leukemia. Leuk Res. 1999;23:615-624.

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[7.] Kawano S, Tatsumi E, Yoneda N, et al. Novel leukemic lymphoma with probable derivation from immature stage of natural killer (NK) lineage in an aged patient. Hematol Oncol. 1995;13:1-11.

[8.] Gardiner CM, Reen DJ, O'Meara A. Recognition of unusual presentation of natural killer cell leukemia. Am J Hematol. 1995;50:133-139.

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[12.] Natkunam Y, Smoller BR, Zehnder JL, et al. Aggressive cutaneous NK and NK-like T-cell lymphomas: clinicopathologic, immunohistochemical, and molecular analyses of 12 cases. Am J Surg Pathol. 1999;23:571-581.

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[15.] Ho FC, Choy D, Loke SL, et al. Polymorphic reticulosis and conventional lymphomas of the nose and upper aerodigestive tract: a clinicopathologic study of 70 cases, and immunophenotypic studies of 16 cases. Hum Pathol. 1990;21: 1041-1050.

[16.] Lanier LL, Chang C, Spits H, et al. Expression of cytoplasmic CD3 epsilon proteins in activated human adult natural killer (NK) cells and CD3 gamma, delta, epsilon complexes in fetal NK cells: implications for the relationship of NK and T lymphocytes. J Immunol. 1992;149:1876-1880.

[17.] Suzumiya J, Takeshita M, Kimura N, et al. Expression of adult and fetal natural killer cell markers in sinonasal lymphomas. Blood. 1994;83:2255-2260.

[18.] Seymour JF, Pierce SA, Kantarjian HM, et al. Investigation of karyotypic, morphologic and clinical features in patients with acute myeloid leukemia blast cells expressing the neural cell adhesion molecule (CD56). Leukemia. 1994;8: 823-826.

[19.] Scott AA, Head DR, Kopecky KJ, et al. HLA-DR-, [CD33.sup.+], [CD56.sup.+], CD16-myeloid/natural killer cell acute leukemia: a previously unrecognized form of acute leukemia potentially misdiagnosed as French-American-British acute myeloid leukemia-M3. Blood. 1994;84:244-255.

[20.] Swerdlow SH, Habeshaw JA, Richards MA, et al. T lymphoblastic lymphoma with LEU-7 positive phenotype and unusual clinical course: a multiparameter study. Leuk Res. 1985;9:167-173.

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[22.] Koita H, Suzumiya J, Ohshima K, et al. Lymphoblastic lymphoma expressing natural killer cell phenotype with involvement of the mediastinum and nasal cavity. Am J Surg Pathol. 1997;21:242-248.

[23.] Ichinohasama R, Endoh K, Ishizawa K, et al. Thymic lymphoblastic lymphoma of committed natural killer cell precursor origin: a case report. Cancer. 1996;77:2592-2603.

[24.] Nakamura S, Koshikawa T, Yatabe Y, et al. Lymphoblastic lymphoma expressing CD56 and TdT. Am J Surg Pathol. 1998;22:135-137.

[25.] Nakamura F, Tatsumi E, Kawano S, et al. Acute lymphoblastic leukemia/ lymphoblastic lymphoma of natural killer (NK) lineage: quest for another NK-lineage neoplasm. Blood. 1997;89:4665-4666.

[26.] Brody JP, Allen S, Schulman P, et al. Acute agranular CD4-positive natural killer cell leukemia: comprehensive clinicopathologic studies including virologic and in vitro culture with inducing agents. Cancer. 1995;75:2474-2483.

[27.] Ichikawa M, Kawai H, Komiyama A, et al. Functional p75 interleukin-2 receptor expression on the fresh blast cells in childhood acute lymphoblastic leukemia with natural killer cell properties. Am J Hematol. 1991;36:259-264.

[28.] Pirruccello SJ, Bicak MS, Gordon BG, et al. Acute lymphoblastic leukemia of NK-cell lineage: responses to IL-2. Leuk Res. 1989;13:735-743.

[29.] Kobashi Y, Nakamura S, Sasajima Y, et al. Inconsistent association of Epstein-Barr virus with CD56 (NCAM)-positive angiocentric lymphoma occurring in sites other than the upper and lower respiratory tract. Histopathology. 1996;28: 111-120.

[30.] Nakamura S, Suchi T, Koshikawa T, et al. Clinicopathologic study of CD56 (NCAM)-positive angiocentric lymphoma occurring in sites other than the upper and lower respiratory tract. Am J Surg Pathol. 1995;19:284-296.

[31.] Ino T, Tsuzuki M, Okamoto M, et al. Acute leukemia with the phenotype of a natural killer/T cell bipotential precursor. Ann Hematol. 1999;78:43-47.

[32.] Nagata T, Higashigawa M, Nagai M, et al. A child case of [CD34.sup.+], [CD33.sup.-], [HLA-DR.sup.-] , [CD7.sup.+], [CD56.sup.+] stem cell leukemia with thymic involvement. Leuk Res. 1996;20:983-985.

Accepted for publication July 18, 2000.

From the Department of Pathology, Stanford University Medical Center, Stanford, Calif.

Reprints: Yasodha Natkunam, MD, PhD, Department of Pathology, Stanford University Medical Center, 300 Pasteur Dr, Stanford, CA 94305 (e-mail:
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Author:Natkunam, Yasodha; Cherry, Athena M.; Cornbleet, P. Joanne
Publication:Archives of Pathology & Laboratory Medicine
Geographic Code:1USA
Date:Mar 1, 2001
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