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Extranodal NK/T cell lymphoma and aggressive NK cell leukaemia: evidence for their origin on CD56+bright CD16−/+dim NK cells

Lima, Margarida

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Pathology - Journal of the RCPA: October 2015 - Volume 47 - Issue 6 - p 503–514
doi: 10.1097/PAT.0000000000000275
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Abstract

INTRODUCTION

The World Health Organization (WHO) classification of tumours of haematopoietic and lymphoid tissues stratifies neoplasms according to lineage and state of differentiation of the neoplastic cells.1 In the current schema, two distinct types of mature natural killer (NK) cell neoplasm are recognised: extranodal NK/T cell lymphoma, nasal type (ENKTL), and aggressive NK cell leukaemia (ANKL).2–4 Both diseases are aggressive conditions and their epidemiological, biological and clinical features are clearly different from those observed in the chronic lymphoproliferative disorders of NK cells (CLPD-NK).5

Extranodal NK/T cell lymphoma, nasal type and ANKL, are very uncommon in North America and Europe, but relatively frequent in Central and South American and Eastern countries.6–8 Aggressive NK cell leukaemia is even more rare than ENKTL (Fig. 1).8–10 Two variants of ENKTL have been considered, the nasal and the extranasal forms, the first being more frequent than the latter (Fig. 1).8,10,11–13 These tumours strongly associate with the Epstein–Barr virus (EBV), arising as a consequence of the inability of the immune system to control EBV infection and of the transforming potential of multiple EBV gene products.14,15 Only a few European series of ENKTL have been published to date,7,12 and reports of ANKL cases in European patients are sporadic.16–19 Recently, we have published a series of 12 Portuguese patients with aggressive NK cell neoplasms (10 ENKTL and 2 ANKL), with emphasis on flow cytometry.20

Clinically, nasal ENKTL usually presents with mid-facial and/or upper airway destructive lesions, whereas extranasal ENKTL often involves multiple organs and tissues at the time of the diagnosis (Table 1).6,10,20–22 Patients with ANKL frequently have hepatosplenomegaly, pancytopenia, abnormal liver function and markedly elevated serum lactic dehydrogenase levels, and they usually develop haemophagocytosis, disseminated intravascular coagulation and multiorgan failure (Table 1).8,23–25 In tissue biopsies, the tumour NK cells are mixed with inflammatory cells in both ENKTL and ANKL cases, frequently with angiocentricity, angioinvasion and ischaemic necrosis.26 ANKL patients typically have bone marrow (BM) involvement at the time of the diagnosis, and the neoplastic NK cells may also be observed in the peripheral blood (PB), where they appear as atypical ‘morphologically immature’ large granular lymphocytes.26,27

Fig. 1
Fig. 1:
Fig. 1. Frequency of extranodal NK/T cell lymphomas (ENKTL), nasal type, with nasal and extranasal presentations, and aggressive NK cell leukaemia (ANKL) in previously published series. (*) Seven additional cases presented with nodal disease and were considered unclassifiable.
Table 1
Table 1:
Table 1 Major clinical features of extranodal NK cell lymphoma, nasal type, and aggressive NK cell leukaemia6,8,10,20–28

Assuming that knowing the normal cell counterparts from which the neoplastic cells originate provides useful information to better understand the disease biology, the WHO classification postulates, to the extent possible, the normal cellular counterpart for each disease entity.1 However, the definition of the normal lymphoid compartments to which the malignant lymphoid cells correspond contains many uncertainties and NK cell neoplasms still lack obvious normal counterparts.2,4,5

Based on the immunophenotypic analysis of the neoplastic NK cells in a series of patients with aggressive NK cell neoplasms, we have recently postulated that ENKTL and ANKL cells do originate from viral-transformed, proliferating and activated mature CD56+bright NK cells.20 Herein we consolidate our hypothesis by reviewing the literature on the immunophenotype of ENKTL and ANKL cells, in comparison to those of normal PB mature CD56+ NK cell subsets and activated CD56+ NK cells from patients with viral infections and tumours.

SEARCHING FOR A NORMAL NK CELL COUNTERPART FOR AGGRESSIVE NK CELL NEOPLASMS

The WHO schema utilises histological, cytological, immunophenotypic and genotypic features in conjunction with clinical aspects to classify tumours of the haematopoietic and lymphoid tissues.1 In line, two major categories of lymphoid neoplasms have been considered: ‘precursor’ neoplasms, corresponding to lymphoblastic lymphoma and leukaemia, and ‘peripheral’ neoplasms, comprising the tumours originating from mature lymphoid cells. In addition, assuming that understanding lymphomas may be improved by knowing their relationship to the normal immune system, the WHO classification lists the putative normal counterparts of the tumour cells for each disease entity.

In most mature B cell neoplasms, the precise stage of the lymphoid maturation and terminal differentiation from which the tumour cells originate is relatively well known, and the correspondence with pre-germinal centre (GC), GC and post-GC stages is now relatively well established.28,29 With respect to peripheral T-cell lymphomas (PTCL), they are quite heterogeneous as far as histological, cytological, and immunophenotypic features are concerned and most PTCL types cannot yet be referred to specific normal counterparts.30–32 In that concerning the NK cell neoplasms, detailed information on the correspondent normal NK cells is still missing, being referred to in the current WHO classification as ‘activated NK cells’ and, less commonly, ‘cytotoxic T lymphocytes’ for extranodal ENKTL, ‘NK-cells’ for ANKL, and ‘mature NK cells’ for CLPD-NK.2,4,5

A decade ago, Mori et al. tried to establish the differentiation stage of the ENKTL, ANKL and CLPD-NK based on the phenotypic characteristics.33 By analysing four relevant cell surface molecules (CD161, CD56, CD94 and CD16) and defining three major stages of NK cell differentiation—pre-NK cells (only expressing CD161), immature NK cells (expressing only CD161 and CD56) and mature NK cells (expressing CD161, CD56 and CD94, and having variable CD16 expression)—they concluded that all three disease conditions originate from mature NK cells; nevertheless, they did not take into account the CD56+dim and the CD56+bright NK cell subsets, whose existence was not yet established at that time. To the best of our knowledge, a comprehensive approach to the immunophenotype of NK cell neoplasms has not been performed since then, and the normal NK cell populations from which they originate have not been yet identified.

Normal mature CD56+ NK cells

CD56+dim and CD56+brightNK cell subsets

Nowadays, it is well known that mature CD56+ NK cells comprise at least two NK cell populations—CD56+dim CD16+ (from now on designated CD56+dim) and CD56+bright CD16−/+dim (from now on designated CD56+bright)—with different phenotypes, functional characteristics and body distribution, probably corresponding to sequential stages of differentiation.34–40 In normal conditions, CD56+dim NK cells account for the majority of the CD56+ NK cells in PB and BM and the spleen; in contrast, CD56+bright NK cells predominate in secondary lymphoid tissues, such as lymph nodes (LN) and tonsils.40 In addition, NK cells are distributed in non-lymphoid / non-haematopoietic organs and tissues, most of them being enriched in CD56+bright NK cells, which also predominate in body cavity fluids, and there is evidence for their recirculation through to secondary lymphoid organs via the afferent lymph.41

By definition, NK cells do not express CD3 and the T-cell receptor (TCR) on their surface and they do not rearrange the TCR genes. However, activated NK cells may express the CD3 epsilon chain (CD3ε) in their cytoplasm, a signal transduction molecule that is also present in the cytoplasm of fetal thymus T/NK bipotential progenitor cells.42

In addition to expressing different amounts of CD56 (the neural cell adhesion molecule, NCAM) and CD16 (low-affinity receptor for the Fc portion of IgG, FcγRIII), CD56+dim and CD56+bright NK cells also differ on the expression of many other molecules. These include the leukocyte integrins, which act as adhesion molecules and complement receptors (CR) (e.g., CD11a, CD11b/CR3, CD11c/CR4), selectins (CD62L), co-stimulatory and co-inhibitory receptors (e.g., CD2, CD5, CD7, CD8, CD38), receptors for cytokines (e.g., CD25 and CD122, the alpha and beta chains of the interleukin 2 receptor, IL-2R) and growth factors (e.g., CD117, the receptor for the stem cell factor, SCF); as well as killer cell immunoglobulin-like (KIRs) (e.g., CD158a, CD158b, CD158e) and lectin type (KLRs) (e.g., CD94, NKG2/CD159, CD161) inhibitory and activating receptors, natural cytotoxic receptors (NCRs) (i.e., NCR1/CD335, NCR2/CD336, NCR3/CD337) and chemokine receptors (CKRs) (e.g., CXCR1/CD181; CXCR2/CD182; CXCR3/CD183; CCR5/CD195), among others. Altogether, these differences are crucial for their ability to form conjugates with target cells, affinity for the major histocompatibility complex (MHC) class I molecules, activation requirements and response to stimuli, homing potential into the lymphoid organs and/or migration to inflamed tissues.

General considerations about the NK cell immunophenotype

Normal mature CD56+dim and CD56+brightNK cells

Most of the CD56+dim NK cells present in the normal PB are CD2+, CD7+bright (a few CD2 cells may be found), CD5 (a subpopulation of CD5+dim NK cells may be present in some normal individuals), CD4, CD8−/+dim (a variable proportion of the CD56+dim cells do express dimly and heterogeneously the CD8 molecule, due to the presence of CD8αα chains), CD11a+bright, CD11b+, CD38+, CD45RA+bright, KIR+, and CD26, CD28, CD45RO and HLA-DR; in contrast, the expression of CD11c, CD57, CD62L, CD94 and CD161 is heterogeneous and variable.34,35,37–39

As normal CD56+dim NK cells, most of the CD56+bright NK cells that circulate in the normal PB are CD2+, CD7+bright (the levels of CD2 and CD7 expression are higher than those observed in normal CD56+dim NK cells), CD5, CD4, CD8−/+dim, CD11a+bright, CD11b+, CD38+, CD45RA+bright, and CD28, CD45RO and HLA-DR; however, these cells are CD26+, they express higher levels of the CD11c, CD62L and CD94 molecules, and they are KIR and CD57.34,35,37–39

Some CD56+ NK cells may express HLA-DR and/or CD45RO, the percentage of HLA-DR+ and/or CD45RO+ cells being higher in the CD56+bright NK cell population.34,35,37 Concerning the receptors for cytokines and growth factors, the CD56+bright NK cells do express receptors for IL-2, IL-7, IL-15, and SCF and they respond to these cytokines by phosphorylating STAT-5, ERK, and Akt.43 The IL-2/IL-15R beta chain (CD122) is expressed in both NK cell subsets, whereas the IL-2R alpha chain (CD25), which confers high affinity for IL-2, is present only in CD56+bright NK cells.44

Relationship between CD56+dim and CD56+bright NK cells

The precise relationship between these two major CD56+ NK cell subsets has been a matter of debate. Although some controversy still exists, it is currently accepted that under the influence of the appropriated stimuli, CD56+bright NK cells differentiate into CD56+dim NK cells. In accordance, upon in vitro activation with IL-2, IL-15 and to a lesser extent IL-12, peripheral blood CD56+bright KIR NK cells exhibited ‘de novo’ expression of KIRs on a significant proportion of cells; and although human CD56+bright NK cells collected from non-reactive LN display almost no KIRs and CD16 expression, NK cells derived from reactive LN, efferent lymph and PB express significant amounts of KIRs, implying that CD56+bright NK cells could acquire these molecules in the LN during inflammation and then circulate through the efferent lymph into PB as KIR+ NK cells.45 Also in line with these observations, upon IL-2 stimulation, normal CD56+bright NK cells up-regulate NCRs, express perforin, and acquire cytolytic properties, at the same time they express CD16 and KIRs, therefore converting into a phenotype similar to that of CD56+dim NK cells,36 and they are able to differentiate into CD56+dim NK cells when cultured in the presence of fibroblasts.46 On the other hand, CD56+dim NK cells acquire a CD56+bright-like phenotype during short term in vitro culture with IL-12 by up-regulating CD56 and down-regulating CD16 expression.47

Transitional NK cell populations

NK cell populations with phenotypic features intermediate between CD56+bright and CD56+dim NK cells have been reported.48–51 In accordance, CD56+brightCD16+ NK cells have been considered to represent a functional intermediate stage of NK cell differentiation.48 In addition, Yu et al. described a CD56+dim CD94+bright NK cell subset expressing CD2, CD62L, KIR, granzymes and perforin, producing interferon-gamma (IFN-γ) in response to monokines and exhibiting CD94-mediated redirected killing at levels intermediate between those observed in CD56+dim CD94+dim and CD56+bright CD94+bright NK cells.49 Moreover, Juelke et al. reported on a CD56+dim CD62L+ NK cell subset with the ability to produce IFN-γ and the capacity to kill.50 Finally, when studying the differentiation of CD56+bright CD94/CD159a (NKG2A)+ into CD56+dim CD94/CD159a+ NK cells, Béziat et al. found a transitional CD56+dim CD94/CD159a+ NK cell subset, expressing intermediate levels of CD62L, granzyme K, CD27, CD57, ILT2, FCRL6 and Siglec-9.51 Accordingly, it was proposed that the NK cell differentiation is based on a stepwise decrease of the CD94/NKG2A lectin type killer cell receptor and acquisition of KIRs.51

Activated CD56+ NK cells

Peripheral blood NK cells from patients with acute viral infections are usually CD56+dim CD16+.52,53 They correspond to recently activated NK cells and they display a pattern of expression of CD2 and CD7 similar to that referred for the normal CD56+dim NK cell subset. However, they typically have a higher fraction of CD2+ cells, higher levels of CD11a and CD38, as well as lower levels of CD11b and CD45RA expression, accompanied by a higher fraction of CD45RO+ and/or HLA-DR+ cells; in addition, they express dimly and heterogeneously the CD11c adhesion molecule and most of them are CD57 (CD11c+dim, CD57).52,53

Usually, most peripheral blood CD56+ NK cells from patients with chronic infections and tumours, which correspond to late activated NK cells, also have a CD56+dim CD16+ phenotype. These cells usually are CD2+bright and express heterogeneously low levels of the CD7, CD38 and CD11b molecules; moreover, the levels of CD57 expression are higher and the levels of CD11c expression are lower than those observed in normal PB CD56+dim NK cells (CD57+bright, CD11c−/+).52,53

ENKTL and ANKL tumour cells

Data on the immunophenotype of the neoplastic NK cells from five series of patients with ENKTL and seven series of patients with ANKL, totalling 411 and 114 patients, respectively, is summarised in Table 2 .9,10,23–25,54–59 The immunophenotype of the ENKTL cells was mainly derived from immunohistochemistry (IHC) studies performed in tissue biopsies, whereas ANKL cells were often characterised by flow cytometry.

Table 2
Table 2:
Table 2 Main immunophenotypic features of extranodal NK/T-cell lymphoma, nasal type,9,10,54–56 and aggressive NK cell leukaemia cells9,23–25,54,57–59 in accordance with previously published series
Table 2
Table 2:
Table 2 (Continued) Main immunophenotypic features of extranodal NK/T-cell lymphoma, nasal type,9,10,54–56 and aggressive NK cell leukaemia cells9,23–25,54,57–59 in accordance with previously published series

According to these series, and similarly to normal NK cells, tumour NK cells are surface CD3, TCR and do not rearrange the TCR genes; however, they often express the CD3ε chain in their cytoplasm, then staining positively in tissue biopsies using IHC. From the markers typically used to identify NK cells, CD56 is usually found to be brightly positive, whereas CD16 is dim and variably expressed, being negative in a considerable proportion of cases, and CD57 expression is almost never found. However, most series included atypical CD56 negative NK cell neoplasms. From the other T/NK cell associated markers analysed, CD2 is the most constantly present, being positive in the vast majority of the cases tested. In contrast, total or partial loss of CD7 expression is frequently observed, and only a few cases expressing the CD5 molecule are found. Dim and heterogeneous CD8 expression is observed in a relatively low proportion of cases, and the few cases reported as staining positive for CD4 may correspond to cases of CD4+ CD56+ plasmacytoid dendritic cell leukaemia, misdiagnosed as NK cell neoplasms. In addition, cytotoxic granule-associated proteins, such as T-cell restricted intracellular antigen (TIA-1), granzyme B, and perforin, are frequently positive.

In the series of 12 patients with aggressive NK cell neoplasms we have studied by flow cytometry (10 ENKTL and 2 ANKL), the neoplastic NK cells showed a high FSC and SSC as compared to normal residual lymphocytes and they strongly expressed the common leukocyte antigen, CD45, in all cases.20 Concerning the T-cell associated molecules, CD2 expression was observed in 83% of cases, whereas CD7 and CD8 were positive in only 42% and 22%, respectively, being usually dimly and heterogeneously expressed in a variable fraction of the neoplastic cells. In contrast, CD3, TCR, CD4 and CD5 were never found to be expressed. We also observed that CD56 was brightly expressed in most cases (92%), whereas CD16 was dimly positive in a variable fraction of the neoplastic NK cells in only a minority (8%) of cases, and CD57 was always negative.

Data on the expression of the CD26, CD27 and CD28 co-stimulatory molecules on the neoplastic NK cells is not available in the series reviewed. However, in our series, CD26 was found to be expressed in 83% of cases, whereas CD28 was dimly and heterogeneously expressed in 50% and CD27 was negative in all cases tested.20 Furthermore, we observed strong CD26 expression on the neoplastic NK cells from three additional patients with ANKL (data not shown).

Given the fact that ENKTL and ANKL do originate from transformed and usually EBV infected NK cells, it would be plausible to expect an activation related immunophenotype. Accordingly, HLA-DR and CD45RO expression was observed in the majority of the ENKTL and ANKL cases reported in the reviewed series, as well as in our series, in which the neoplastic NK cells were found to be HLA-DR+ in all cases analysed, as well as co-expressing CD45RA and CD45RO, the latter being positive in a variable fraction of cells.20 This is in contrast to the CD45RA+/CD45RO/HLA-DR pattern that is found in the vast majority of normal peripheral blood NK cells and similar to that found in reactive NK cells from patients with viral infections and tumours.52,53 The same observation could apply to the CD11a+bright CD11b-/+dim,het CD11c-/+dim,het pattern of expression of leukocyte integrins observed in the majority of ENKTL and ANKL cases tested in the reviewed series as well as in our series, as the CD11b and CD11c complement receptors were found to be dimly and heterogeneously expressed on circulating activated NK cells in acute viral infections.52,53 Loss of surface expression of CD7 observed on the neoplastic NK cells may also result from activation, as this cell surface molecule is usually weakly and heterogeneously expressed in chronically activated NK cells.52,53 Similarly, CD30 expression observed in some ENKTL and ANKL cases may also be activation-related. In fact, CD30, a receptor of the tumour necrosis factor receptor (TNFR) superfamily that is useful for the diagnosis of specific types of lymphomas, has long been recognised as activation-related molecule.60 Curiously, Mori et al. have found that two of three ENKTL and four of six ANKL cases were CD69+,33 and we observed a strong expression of this early activation marker in the only ENKTL we have tested.20

Lessons from killer cell receptors

Normal CD56+ NK cells

Killer cell receptors include NCRs, KLRs and KIRs and their patterns of expression differ on CD56+dim and CD56+bright NK cells.

The NCRs comprise three sets of molecules: CD335 (NCR1, NKp46), CD337 (NCR3, NKp30) and CD336 (NCR2, NKp44), and their ligands include pathogen- and cancer-associated molecules.61,62 Cells exposed to pathogens may exhibit pathogen derived and pathogen induced ligands, whereas transformed cells may display ‘self’ ligands which are derived from both the intracellular and membrane associated molecules. These expression patterns allow NK cells to discriminate between healthy and infected/tumour cells.63 Both CD56+dim and CD56+bright NK cells are CD335+ and CD337+, whereas CD336 is expressed only on activated NK cells.

Concerning the KLRs, the invariant CD94 molecule, which usually associates with CD159a (NKG2A) to form an inhibitory KLR complex, is preferentially expressed on CD56+bright NK cells, whereas CD161 is variably expressed on both NK cells subsets.34

Finally, KIRs (CD158a, b1/b2, c, d, e1/e2, f, h and z) are cell surface receptors specific for allelic forms of human leukocyte antigen (HLA) class I molecules. Upon engagement with HLA class I molecules, most KIRs have inhibitory functions, blocking NK cell activation and function. Thus, cells lacking HLA class I molecules are promptly killed by NK cells because of the predominant effect of several activating NK receptors, an important mechanism to control spreading of viral infected and tumour cells. Similar to HLA loci, KIR sequences are highly polymorphic and, moreover, KIR haplotypes greatly vary in the number of the type of genes they contain. KIRs are expressed by CD56+dim NK cells and subsets of cytotoxic T cells, whereas CD56+bright NK cells fail to express KIR molecules.39,64

ENKTL and ANKL tumour cells

To the best of our knowledge only six studies addressing the expression of the killer cell receptors in the neoplastic NK cell have been reported so far, either by using IHC, reverse transcriptase polymerase chain reaction (RT-PCR) or flow cytometry (Table 3).20,33,65–69 The CD94/CD159a (NKG2A) inhibitory KLR complex was found to be expressed in most ENKTL and ANKL cases, whereas CD161 was positive in only about one-third of the cases.20,33,65–67 In contrast, all the KIRs addressed in these studies (CD158a, CD158b, CD158e1, CD158i, CD158k) proved to have a limited expression in ENKTL and ANKL.20,33,65,66,68 However, these results should be interpreted with caution, not only because of the limited number of cases studied and different methods used, but also because clonal NK cells may exhibit a restricted KIR repertoire, limited to KIRs that may not be detected by monoclonal antibodies used. Data on the expression of the NCRs (i.e., CD335, CD336, CD337) on the neoplastic NK cells are limited to one study which revealed that ENKTL cells are CD335+ in most cases.69 The same applies to CD244, a killer cell receptor that belongs to the SLAM (Signaling Lymphocyte Activation Molecules) family, which was found to be expressed in all three ENKCL and six NKTL studied.33

Lessons from chemokines and chemokine receptors

Table 3
Table 3:
Table 3 Expression of killer cell receptors on the neoplastic NK cells from patients with extranodal NK cell lymphoma, nasal type, and aggressive NK cell leukaemia

Normal CD56+ NK cells

Chemokines and their receptors play an important role in determining lymphocyte homing into lymphoid tissues as well as in modulating the recruitment of inflammatory cells into sites of inflammation.70,71 Previous studies have shown that blood CD56+dim and CD56+bright NK cells have distinct repertoires of CKRs, which may dictate their homing and migratory properties.72,73 In accordance, CD56+bright NK cells are the only ones to express CD197 (CCR7) and they bear CD183 (CXCR3) at a much stronger density than CD56+dim NK cells, whereas the latter cells express CD181 (CXCR1) and the fractalkin receptor (CX3CR1) exclusively.70–73 The consequence of these different CKR repertoires is that the CD56+dim cells are potentially able to migrate to sites of acute inflammation, whereas the CD56+bright NK cell subset preferentially homes to secondary lymphoid organs, being also capable of migrating into peripheral tissues in chronic inflammatory conditions.

In physiological conditions, CD56+bright NK cells home into the LN where they localise in the parafollicular T-cell zones because they express CD197 (CCR7), which enables them to respond to locally produced CCL19/MIP-3β and CCL21/SLC chemokines.74 Within secondary lymphoid tissues, they interact with dendritic cells and T lymphocytes, leading to the T helper 1 (Th1) polarisation of adaptive immune responses.74 Under pathological conditions, CD56+bright NK cells migrate in response to inflammatory stimuli, with more CD56+bright NK cells distributing into chronically inflamed tissues, such as the skin, liver and lungs, a process that seems to be mediated by the interaction of the CD195 (CCR5) and CD183 (CXCR3) chemokine receptors and their ligands.75–84 These include CCL3 (Macrophage inflammatory protein-1alpha, MIP-1alpha) / CCL4 (MIP-1beta)/CCL5 (Regulated and normal T cell expressed and secreted, RANTES) for CD195 (CCR5), and CXCL9 (Monokine induced by gamma interferon, MIG)/CXCL10 (Interferon gamma-induced protein 10, IP-10)/CXCL11 (Interferon-inducible T-cell alpha chemoattractant, I-TAC or Interferon-gamma-inducible protein 9, IP-9) for CD183 (CXCR3). The same sets of CKR also dictate co-migration of NK cells and Th1 polarised T cells.75–84

Depending on the situation, NK cells may have a beneficial effect, for example, by combating tumour cells or virally infected cells, or rather be harmful, by perpetuating the inflammatory process and damaging the tissues through direct cytotoxicity or by the release of cytokines and chemokines which promote the recruitment of other inflammatory cells. In particular, CD56+bright NK cells have been involved in chronic inflammatory and allergic skin diseases,75–77 and migration of the CD56+bright NK cells for the inflamed joints and synovial fluids in patients with rheumatoid arthritis is facilitated by the CD183 (CXCR3) and CD195 (CCR5) axis.78–80 In addition, CD195 (CCR5) plays an important role in the accumulation of CD56+bright cells in virally infected sites, such as in the liver during hepatitis C virus infection, as well as in parasitic infections, and CD183 (CXCR3) is involved in migration and accumulation of CD56+bright NK cells in tumour tissues.81–83 Moreover, CD56+bright NK cells may also be recruited into antigen-stimulated LN in a CD183 (CXCR3)-dependent, CD197 (CCR7)-independent manner, where they provide an early source of IFN-γ that is needed for Th1 polarisation.84 Finally, a particular type of CD56+bright NK cell localises in the uterine decidua following the CD183 (CXCR3)/CXCL10 and/or the CD184 (CXCR4)/CXCL12 axis.85

ENKTL and ANKL tumour cells

If ENKTL and ANKL do in fact originate from normal CD56+bright NK cells as we propose, it will be plausible that their affinity for the extranodal organs and tissues is regulated by the same asset of CKRs. Unfortunately, CKRs were tested in only a few ENKTL and ANKL cases.56,86–89 In accordance with our hypothesis, at least two studies have found that skin infiltrating ENKTL tumour cells do express the CD183 (CXCR3) molecule.56,89 For instance, Schwartz et al. observed that 66 of 83 (80%) ENKTL were CD183 (CXCR3)+,56 a lower percentage of positive cases being reported by Ishida et al. (4/27, 14.8%).86 In addition, EBV+ ENKTL cell lines are CD183 (CXCR3)+, at same time they produce IP-10, a CXCR3 ligand, suggesting that these molecules may play an important role in tissue invasion by ENKTL cells through an autocrine mechanism.90 In ANKL it was observed that the leukaemic cells are CD195 (CCR5)+, as well as CD181 (CXCR1)+.87 The same authors have also found that not only serum levels of IL-8, the ligand for CXCR1, but also those of RANTES, MIP-1alpha and MIP-1beta, the natural ligands for CCR5, are significantly increased in ANKL patients, at the same time that ANKL cells and hepatocytes do express these chemokines. In CD183 (CXCR3) expression on ANKL cells, the only case studied was found to be positive for this inflammatory CKR.56

The pattern of CKR observed on ANKL cells could partially explain why these cells, as normal CD56+bright NK cells, localise in the BM and circulate in PB, at the same time they infiltrate the splenic red pulp, as well as the sinusoidal and interlobular regions of the liver, thereby justifying the hepatosplenomegaly and the hepatic failure observed in these patients.88 CD183 (CXCR3) and CD195 (CCR5) expression could also explain their tendency to infiltrate other organs and peripheral tissues.

Comparing ENKTL and ANKL tumour cells

Apparently, differences between ENKTL and ANKL cells were found for the expression of CD2 (ENKTL < ANKL), cytoplasmic CD3ε (ENKTL > ANKL), CD16 (ENKTL < ANKL), CD30 (ENKTL > ANKL), CD56 (ENKTL < ANKL), KIRs (ENKTL > ANKL) and cytotoxic granules proteins (perforin and, to a lesser extent, granzyme B; ENKTL > ANKL) (see also Tables 2 and 3).9,10,20,23,25,33,54–59,65,67

Some of these differences (i.e., higher percentage of CD2 and CD56 negative cases; lower percentage of CD16 positive cases) would point to a more immature phenotype of the ENKTL cells as compared to ANKL cells; however, the lower proportion of cases expressing KIRs and cytotoxic granule proteins (specially perforin) in the ANKL group, are not in agreement with this possibility. Other differences (higher percentage of cases expressing CD30 and CD3ε) are consistent with a more pronounced activation status of the tumour cells in ENKTL as compared to ANKL. However, these data should be compared and interpreted with caution and these differences need to be confirmed, as they may result at least in part from the methods used to characterise the neoplastic NK cells (mainly IHC in ENKTL and flow cytometry in ANKL), the samples studied (e.g., preferentially tissue biopsies in ENKTL and PB/BM samples in ANKL) and the limited number of cases tested for some markers (i.e., low number of ANKL cases tested for the expression of KIR and cytotoxic granule molecules). The possible effects of cell activation on the immunophenotype should also be taken into account.

CONCLUSIONS AND FUTURE PERSPECTIVES

Natural killer cell neoplasms are uncommon tumours with an aggressive clinical course, and their precise cellular origin has not been defined to date. This study clearly demonstrates that evidence has been accumulating suggesting that the immunophenotype of the ENKTL and ANKL cells mimics that of CD56+bright NK cells. In accordance, like normal CD56+bright NK cells, the neoplastic ENKTL and ANKL cells express brightly the CD56 adhesion molecule, the CD94 lectin type killer receptor and the CD26 dipeptidyl peptidase in most cases. At the same time, they often fail to express the alpha chain of the low affinity Fc receptor for IgG, CD16, and the killer immunoglobulin-like receptors CD158, and virtually never express the carbohydrate epitope CD57. In addition, neoplastic NK cells have an activation-related phenotype, as documented by a consistent expression of HLA-DR and CD45RO on a variable fraction of cells, as well as by the positivity for the cytoplasmic signalling molecule, CD3ε, and activation-related cell membrane protein of the TNFR family, CD30, in a considerable proportion of the cases reported; and by positivity for the early activation molecule, CD69, in the few cases tested. Other phenotypic differences, such as the tendency of the neoplastic cells to lose CD7 expression, can probably also be activation related, whereas high levels of the Ki-67 nuclear protein indicate a high proliferative rate.

Based on previously published data, we postulate that ENKTL and ANKL do arise from EBV infected activated mature CD56+bright NK cells. Discrete immunophenotypic differences between the ENKTL and ANKL tumour cells (e.g., higher fraction of CD16+ cases in ANKTL) point to a slightly more mature phenotype of ANKL cells, probably corresponding to a stage of differentiation intermediate between CD56+bright and CD56+dim NK cells.

Detailed immunophenotypic studies based on multicolour flow cytometry are needed to support our hypothesis and to better characterise the normal and the neoplastic NK cells, allowing for a precise correspondence between them (see Fig. 2 for a typical example).91 For instance, it will be important to test more ENKTL and ANKL cases for the expression of markers that are known to be differentially expressed on CD56+dim and CD56+bright NK cells, such as receptors for cytokines and growth factors (e.g., CD25, CD117). Further studies on the expression of CKRs will also be useful to better understand the tissue tropism of the neoplastic NK cells. Taking into account that the anatomical localisation and function of the neoplastic NK cells parallel their normal cell counterparts, this approach may ultimately contribute to better understanding of the clinical manifestations of these aggressive neoplasms.

Acknowledgements

Fig. 2
Fig. 2:
Fig. 2. Illustrative case of aggressive NK cell leukaemia (ANKL). Dot plots showing the immunophenotype of the neoplastic NK cells (red dots) from the bone marrow (BM) of a patient with ANKL, as compared to the normal residual NK cells (highlighted blue dots) present in the same sample; normal T (yellow dots) and B (pink dots) cells are also shown. NK cells comprise 32.7% of the nucleated BM cells (abnormal/neoplastic CD56+bright NK cells 31.0%; normal residual CD56+dim NK cells 1.7%). As shown in the dot plots in the first row, T cells and B cells were first defined as CD3+ (third dot plot, yellow dots) and CD19+ (fourth dot plot, pink dots), respectively. Then NK cells (CD3/CD56+ events) were classified as CD56+dim (blue dots) and CD56+bright (red dots) based on the different levels of CD56 expression (fifth dot plot). All these events were included in the forward (FSC) versus sideward (SSC) light scatter (first dot plot) and SSC versus CD45 (second dot plot) lymphocyte regions. Note that the neoplastic ANKL cells express brightly the CD56 adhesion molecule and the CD94 lectin type killer cell receptor, and they are positive for CD26; at the same time they fail to express CD16 and CD57. Moreover, they are CD3, CD4, CD5, CD7 (low and heterogeneous CD7 expression in only 9% of cells), CD8, CD11c and CD38+, and they stain positively for cytoplasmic granzyme B and perforin (perforin levels were slightly lower than that observed in normal residual CD56+dim NK cells). Enumeration and characterisation of BM lymphoid cell subsets was performed using the Euroflow LST tube (Cytognos, Spain), which contains a mixture of seven monoclonal antibodies (mAb) conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridin chlorophyll protein-cyanin 5.5 (PerCP-Cy5.5), phycoerythrin - protein-cyanin 7 (PE-Cy7), allophycocyanin (APC), allophycocyanin-H7 (APC-H7), pacific blue (PB) and pacific orange (PB): CD8 + anti-human immunoglobulin (Ig) lambda light chains, CD56 + anti-human Ig kappa light chains, CD5, CD19, CD3, CD38, CD20+CD4 and CD45.91Further immunophenotypic characterisation of the NK cells was performed using the EuroFlow NK-CLPD panel that comprises 8 colour combinations (FITC/PE/PE-Cy5.5/PE-Cy7/APC/APC-H7/PB/PO) of mAb specific for the following antigens: tube 1 (CD7/CD26/CD3/CD56/CD5/CD19/CD2/CD45), tube 2 (CD57/CD25/CD3/CD56/CD11c/CD19/CD16/CD45) and tube 3 (cytoplasmic perforin/cytoplasmatic granzyme B/CD3/CD56/CD94/CD19/HLA-DR/CD45).91 The sources and specificities of the mAb reagents used were previously described in detail.91 Cell staining was performed using a well-established stain-and-then-lyse method with FACSLysing (Becton Dickinson Biosciences, USA), according to the recommendations of the manufacturer. Combined staining for surface antigens and intracellular molecules was performed using the Fix & Perm reagent kit (Invitrogen, USA), according to the recommendations of the manufacturer. Data acquisition was performed immediately after completion of sample preparation, in a FACSCanto II flow cytometer (BD), using the FACSDiva software program (BD). For data analysis, the INFINICYT software program (Cytognos, Spain) was used.

The author thanks the medical doctors (Catarina Lau, Maria dos Anjos Teixeira), technicians (Ana Helena Santos, João Rodrigues, Lurdes Oliveira, Maria Luís Queirós, Marlene Santos, Marta Gonçalves, Sónia Fonseca), and research fellow (Magdalena Leander) of the Laboratories of Cytometry and Molecular Genetics of the Hospital de Santo António, Centro Hospitalar do Porto, Porto, Portugal, for support concerning NK cell immunophenotyping.

Conflicts of interest and sources of funding: The author states that there are no conflicts of interest to disclose.

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Keywords:

Aggressive NK-cell leukaemia; ANKL; CD56; ENKTL; extranodal NK/T cell lymphoma, nasal-type; flow cytometry; immunophenotyping; NK cell subsets

© 2015 Royal College of Pathologists of Australasia