Differentiation stage of natural killer cell-lineage lymphoproliferative disorders based on phenotypic analysis


Kiyoshi L. Mori, MD, PhD, Department of Haematology, Juntendo University School of Medicine, 2-1-1 Hongo Bunkyo-ku, Tokyo 113–8421, Japan. E-mail: klmori@ med.juntendo.ac.jp


In the normal developmental pathway of natural killer (NK) cells, pre-NK cells express CD161, immature NK cells express CD161 and CD56, and mature NK cells express CD161, CD56 and CD94. To identify the normal counterpart of NK cells from which neoplastic cells originate, surface antigens were analysed. Blastic NK-cell lymphoma/leukaemia lacked CD94 and CD161 but had CD56. Aggressive NK-cell leukaemia/lymphoma and nasal NK-cell lymphoma, although morphologically immature, expressed both CD56 and CD94 and strong NK activity. Cells from chronic NK lymphocytosis expressed CD56 and CD94.

Natural killer (NK) cells originate from bone marrow, and in vitro they can develop from NK precursor cells in the thymus (Sato et al, 1999) or liver (Jaleco et al, 1997). In vitro kinetic studies have led to a model for NK development (Bennett et al, 1996; Spits et al, 1998): pre-NK cells express CD161; immature NK cells express CD161 and CD56; and mature NK cells express CD161, CD94 and CD16.

Various types of NK-lineage lymphoproliferative disorders are known (Oshimi, 1996). Blastic NK-cell lymphoma/leukaemia is a rare disorder of probable precursor NK-cell origin. This disease presents mainly as skin lesions and eventual leukaemic change with a poor prognosis (Suzuki & Nakamura, 1999). Aggressive NK-cell leukaemia/lymphoma is characterized by high fever, hepatosplenomegaly, lymphadenopathy, a rapid progressive clinical course and poor prognosis (Imamura et al, 1990). At presentation, it is sometimes indistinguishable from chronic NK lymphocytosis. Nasal NK-cell lymphoma is characterized by the primary involvement of the nasal cavity and a poor prognosis in the advanced stage. Morphologically, aggressive NK-cell leukaemia/lymphoma and nasal NK-cell lymphoma seem to be immature. Chronic NK lymphocytosis is a rare disorder characterized by chronic proliferation of NK cells (Tefferi, 1996). These cases present with a chronic indolent clinical course. The normal counterparts from which NK cell proliferation originates have not been studied thoroughly. In this report, to clarify the origin of NK cell-lineage lymphoproliferation, differentiation markers were studied. Furthermore, the expression of killer immunoglobulin-like receptors (KIRs), CD158a, CD158b and p70 was also studied to delineate their expression in NK cell diseases.

Materials and methods

Criteria for admission Blastic NK-cell lymphoma/leukaemia was defined in a previous report (Suzuki & Nakamura, 1999), as was aggressive NK-cell leukaemia/lymphoma (Imamura et al, 1990), nasal NK-cell lymphoma (Jaffe et al, 1996) and chronic NK lymphocytosis (Oshimi et al, 1993; Tefferi, 1996).

Flow cytometric analysis Peripheral blood mononuclear cells (PBMC) or bone marrow mononuclear cells (BMMC) were isolated using Ficoll–Conray density gradient centrifugation. PBMC from aggressive NK-cell leukaemia/lymphoma and chronic NK lymphocytosis, BMMC from blastic NK-cell lymphoma/leukaemia and suspended lymphocytes from nasal NK-cell lymphoma were evaluated. The expression of cell-surface antigens was measured using flow cytometry (Cytoron Absolute; Ortho Diagnostic System, Raritan, NJ, USA) based on our previous report (Oshimi et al, 1993).

Monoclonal antibodies (mAbs) MAbs 3G8 (anti-CD16), HP-3B1(anti-CD94), EB6 (anti-CD158a), GL183 (anti-CD158b) and C1·7.1 (anti-C1·7) were purchased from Immunotech Coulter (Marseille, France). DX9 (anti-NKB1, p70) was from Becton Dickinson (Mountain View, CA, USA) and 3G10 (anti-CD161) was kindly provided by Dr Lopes-Botet.


Characteristics of the patients studied

Twenty-three patients with NK-cell proliferative disorders were entered into the study (Table IA). Four patients had blastic NK-cell lymphoma/leukaemia, six aggressive NK-cell leukaemia/lymphoma, three nasal NK-cell lymphoma and 10 chronic NK lymphocytosis.

Expression of cell surface markers

Cell surface markers are demonstrated in Table IB and Fig 1. CD161 was found in aggressive NK-cell leukaemia/lymphoma and chronic NK lymphocytosis (Fig 1A). All of the NK-lineage neoplasms highly expressed CD94 except for blastic NK-cell lymphoma/leukaemia (Fig 1B). CD16 was expressed in aggressive leukaemia/lymphoma and chronic NK lymphocytosis (Fig 1C). The majority of nasal NK-cell lymphomas and chronic NK lymphocytosis also expressed CD69, whereas aggressive NK-cell leukaemia/lymphomas often expressed it. Blastic NK-cell lymphoma/leukaemia did not express CD69 at all. KIR expression was tested. CD158a or CD158b was often expressed in various tumours. p70 was expressed in aggressive NK-cell leukaemia/lymphomas, nasal NK-cell lymphoma and chronic NK lymphocytosis. One blastic NK-cell leukaemia/lymphoma weakly expressed p70. C1·7 was highly expressed in aggressive NK-cell leukaemia/lymphoma, nasal NK-cell lymphoma, and chronic NK lympocytosis. One blastic NK-cell lymphoma/leukaemia weakly expressed C1·7.

Figure 1.

Percentages of NK cell differentiation markers expressed in blastic NK-cell lymphoma/leukaemia, aggressive NK-cell leukaemia/lymphoma, nasal NK-cell lymphoma and chronic NK lymphocytosis. The expression of cell-surface receptors was measured by immunofluorescence using the monoclonal antibodies anti-CD161 (A), anti-CD94 (B) and anti-CD16 (C).


In the normal developmental pathway, pre-NK cells express CD161, immature NK cells express CD161 and CD56, and mature NK cells express CD161, CD56 and CD94 (Table 1B) (Spits et al, 1998)

Table I.  (A) Patient characteristics. NT, not tested. Killing: 51Cr release assay for K562 target cells at an effector-to-target cell ratio of 20:1 with 4 h of incubation (normal activity range of peripheral blood mononuclear cells, 38 ± 20%).
Table I. (B) Cell surface markers expressed in NK-cell lineage lymphoproliferative disorders.
 Normal*NK cell lineage lymphoproliferative disorder
 pre-NKImmature NKmature NKBlastic NKAggressive NKNasal NKChronic NK
  1. *Another report led to a model of NK development (Spits etal, 1998).

  2. †Another report demonstrated nasal NK-cell lymphomas expressed CD16 (Suzumiya et al, 1994).


Chronic NK-lymphocytosis cells appear morphologically similar to mature lymphocytes. The expression of CD94 and CD16 antigens also confirms the origin to be mature NK cells. Though aggressive NK-cell leukaemia/lymphoma cells do not resemble mature lymphocytes morphologically, cell surface markers demonstrate that they originate from mature NK cells because they have CD94 and CD16 antigens. The presence of strong NK activity also supports their mature NK-cell origin. Nasal NK-cell lymphoma was also suggested to be a neoplasm originating from mature NK cells, because those cells demonstrate CD94. High NK activity in one nasal NK lymphoma also suggested a mature NK-cell origin. Blastic NK-cell lymphoma/leukaemia did not express any of these mature or immature NK-related markers except CD56. Because CD56 is often expressed on various types of neoplasms, the presence of CD56 made it difficult to determine their differentiation stage. However, the absence of CD94 and CD16 antigens indicates at least that blastic NK-cell lymphoma/leukaemia does not originate from mature NK cells.

Aggressive NK-cell leukaemia/lymphoma and chronic NK lymphocytosis expressed CD161, but blastic NK-cell lymphoma/leukaemia did not. Because only one third of normal peripheral NK cells express CD161 (Poggi et al, 1998), the absence of CD161 antigen on blastic NK-cell lymphoma/leukaemia cells did not exclude the possibility that they are immature.

No data are available for KIRs as differentiation markers of normal NK cells. KIRs are expressed on NK cells and cytotoxic T cells, and are functional cell-surface molecules. Chronic NK lymphocytosis usually expressed p70. Most nasal NK-cell lymphomas and aggressive NK-cell leukaemia/lymphomas also expressed p70, and one of the three blastic NK-cell lymphoma/leukaemia also expressed p70. CD158a and CD158b were often expressed on NK cells.

C1·7 is expressed on NK and CD8+ T cells. All NK-cell disorders except blastic NK-cell lymphoma/leukaemia highly expressed C1·7 antigen, and the detection of this antigen may also aid in the differentiation of mature NK-cell disorders.

CD69 is known as an activation marker of T and NK cells. CD69 was highly expressed on chronic NK lymphocytosis and nasal NK-cell lymphoma, but not on aggressive NK-cell leukaemia/lymphoma or blastic NK-cell lymphoma/leukaemia. Aggressive NK-cell leukaemia/lymphoma and chronic NK lymphocytosis are sometimes difficult to differentiate at presentation. From this study, the presence of CD69 as well as CD11b antigen seems to be a good marker for chronic NK lymphocytosis in addition to CD57 (Oshimi et al, 1993).


We thank Dr Lopes-Botet for providing monoclonal antibody 3G10.