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cancers
Review
Aggressive NK Cell Leukemia: Current State of
the Art
Siba El Hussein , L. Jeffrey Medeiros and Joseph D. Khoury *
Department of Hematopathology, the University of Texas MD Anderson Cancer Center,
Houston, TX 77030, USA; sel2@mdanderson.org (S.E.H.); ljmedeiros@mdanderson.org (L.J.M.)
*Correspondence: Jkhoury@mdanderson.org
Received: 26 August 2020; Accepted: 29 September 2020; Published: 9 October 2020
Simple Summary:
Aggressive natural killer cell leukemia (ANKL) is a rare, lethal disease that
presents many diagnostic and therapeutic challenges. Recent studies have shed new light on the
salient features of its molecular pathogenesis and provided further insight into the clinicopathologic
spectrum of this disease. This review presents a state-of-the-art overview of ANKL, spanning its
historical evolution as a distinct entity, pathobiology, and potential therapeutic vulnerabilities.
Abstract:
Aggressive natural killer (NK) cell leukemia (ANKL) is a rare disease with a grave prognosis.
Patients commonly present acutely with fever, constitutional symptoms, hepatosplenomegaly,
and often disseminated intravascular coagulation or hemophagocytic syndrome. This acute clinical
presentation and the variable pathologic and immunophenotypic features of ANKL overlap with
other diagnostic entities, making it challenging to establish a timely and accurate diagnosis of
ANKL. Since its original recognition in 1986, substantial progress in understanding this disease
using traditional pathologic approaches has improved diagnostic accuracy. This progress, in turn,
has facilitated the performance of recent high-throughput studies that have yielded insights into
pathogenesis. Molecular abnormalities that occur in ANKL can be divided into three major groups:
JAK/STAT pathway activation, epigenetic dysregulation, and impairment of TP53 and DNA repair.
These high-throughput data also have provided potential therapeutic targets that promise to improve
therapy and outcomes for patients with ANKL. In this review, we provide a historical context of the
conception and evolution of ANKL as a disease entity, we highlight advances in diagnostic criteria to
recognize this disease, and we review recent understanding of pathogenesis as well as biomarker
discoveries that are providing groundwork for innovative therapies.
Keywords: leukemia; NK cell; molecular; immunohistochemistry; biomarker
1. Introduction
Aggressive natural killer (NK) cell leukemia (ANKL) is a rare, fulminant disease with a dismal
prognosis that presents many diagnostic challenges. Since its original description in the 1980s,
the diagnostic approach to ANKL has evolved slowly, in large part due to disease rarity and the
complexity of clinical and pathologic presentations. An additional major challenge has been a lack
of a specific immunophenotypic or molecular signature characteristic of ANKL. Most early studies
attempted to provide a systematic approach to the diagnosis ANKL by relying on clinical and
morphologic correlation, coupled with ancillary tools such as flow cytometry and conventional
karyotyping. Although robust, these tools did not uncover specific biomarkers, partly because
traditional approaches lack the versatility inherent in next-generation sequencing, particularly in the
context of an NK cell neoplasm.
Cancers 2020,12, 2900; doi:10.3390/cancers12102900 www.mdpi.com/journal/cancers
Cancers 2020,12, 2900 2 of 15
In recent years, high-throughput genomic analyses at the DNA and RNA levels has contributed
greatly to our understanding of ANKL and provided novel grounds for potential targeted therapies.
This review is intended to provide an overview of ANKL, as defined in the current World
Health Organization (WHO) classification [
1
], spanning its historic origin, clinical and laboratory
manifestations, biology, and novel therapeutic approaches, with emphasis on advances in our
understanding of this rare malignancy in light of recent advances. This review is based on a review of
the literature using the term “aggressive NK cell leukemia”.
2. Historical Overview
The recognition of ANKL as a separate entity was presaged by the disease group referred to
as NK-type lymphoproliferative disease of granular lymphocytes (NK-LDGL). Entailing abnormal
proliferation of large granular lymphocytes (LGLs) expressing a characteristic NK cell immunophenotype
(CD3
−
/CD56+/CD16+), NK-LGDL was understood to encompass heterogeneous clinical manifestations
ranging from indolent, asymptomatic conditions to aggressive, fulminant diseases. As a body of
informative work underscoring some characteristics of NK-LGDL emerged [
2
–
5
], gradual attention was
turned to separating indolent forms from those with an acute clinical course. Thus eventually emerged
from NK-LDGL the entities of NK-LGL, chronic lymphoproliferative disorder of NK cells (CLPD-NK),
and ANKL.
The term ANKL was first mentioned in a publication by Fernandez et al. in 1986 [
6
]. The authors
described a case of a 70-year-old man with multiple episodes of intestinal perforation of unknown
cause, splenomegaly, and bone marrow infiltration by cells described as being morphologically similar
to large granular lymphocytes (LGLs), but lacking T cell markers. The patient had a rapidly progressive
clinical course and died within two months of hospital admission. At autopsy, he had extensive
involvement of the bone marrow, spleen, lymph nodes, and jejunum by LGL-like cells. Conventional
cytogenetic analysis revealed clonal aberrations, leading the authors to conclude that the disease
was neoplastic. The authors also postulated that this disease represented an aggressive form of NK
cell leukemia.
In 1988, Imamura et al. reported two cases of NK cell leukemia/lymphoma that lacked clonal
rearrangements of the T-cell receptor (TRB,TRG) and immunoglobulin genes [
7
]. The authors noted that
the neoplastic cells in these two cases expressed CD56 (NKH-1/Leu-19) and lacked CD57 (HNK-1/Leu-7).
Two years later, the same authors compared four cases of ANKL to seven other similar cases reported
in the literature [
6
,
8
–
11
]. Imamura and colleagues concluded that this disease emanates from “a third
lineage of lymphoid cells” and used the term “aggressive natural killer cell leukemia/lymphoma” [
12
].
ANKL was subsequently reported to arise in two patients with “lethal midline granuloma”,
currently known as extra-nodal NK/T cell lymphoma, nasal type (ENKTL), raising the possibility
that ANKL disease could represent a terminal event in the evolution of ENKTL [
13
]. This report was
followed by another, which described four cases of ANKL with dissemination to various organs in a
manner akin to lymphomas [
14
]. These reports raised controversy regarding the relationship between
ANKL and ENKTL, which arguably remains largely unresolved [15,16].
In 1994, the Revised European–American classification of lymphoid neoplasms recognized ANKL
as an entity separate from ENKTL, and such separation was adopted in the WHO classification
system [17,18].
3. Clinical Features
ANKL patients have a median age of 40 years, and there is no gender predilection. The disease has a
higher incidence in Asian populations, but ANKL can also arise in other groups [
19
]. The most common
presenting signs and symptoms include fever, B-symptoms, hepatosplenomegaly, lymphadenopathy,
disseminated intravascular coagulopathy, and hemophagocytosis. Epstein-Barr virus (EBV) infection
has been observed in a subset of ANKL cases. Despite intensive chemotherapy, patients with ANKL
have a very poor prognosis, with a median survival of less than two months [
20
,
21
]. Several prognostic
Cancers 2020,12, 2900 3 of 15
factors have been proposed, including patient age, serum lactate dehydrogenase level, and serum total
bilirubin level [
22
], but these factors have not been validated [
23
]. The clinicopathologic features of
ANKL have been elucidated in several case series [19–21,24–27].
4. Morphologic Features
Histologically, bone marrow involvement by ANKL can be prominent or subtle, with two main
patterns of infiltration: interstitial and sinusoidal [
19
]. The neoplastic cells are generally medium-sized,
possess a moderate amount of cytoplasm, and exhibit highly irregular nuclei with condensed chromatin
and conspicuous nucleoli. Apoptosis and focal necrosis are common findings; however, geographic
necrosis is typically not observed in ANKL, unlike ENKTL [
19
] (Figure 1A). Microscopic examination
of peripheral blood and bone marrow smear preparations usually demonstrate neoplastic cells that
are intermediate to large in size, with moderate amounts of basophilic agranular cytoplasm often
containing punched-out vacuoles. The neoplastic cells typically have highly irregular nuclear contours
and an open chromatin pattern with prominent nucleoli.
Cancers 2020, 12, x FOR PEER REVIEW 3 of 16
dehydrogenase level, and serum total bilirubin level [22], but these factors have not been validated
[23]. The clinicopathologic features of ANKL have been elucidated in several case series [19–21,24–
27].
4. Morphologic Features
Histologically, bone marrow involvement by ANKL can be prominent or subtle, with two main
patterns of infiltration: interstitial and sinusoidal [19]. The neoplastic cells are generally medium-
sized, possess a moderate amount of cytoplasm, and exhibit highly irregular nuclei with condensed
chromatin and conspicuous nucleoli. Apoptosis and focal necrosis are common findings; however,
geographic necrosis is typically not observed in ANKL, unlike ENKTL [19] (Figure 1A). Microscopic
examination of peripheral blood and bone marrow smear preparations usually demonstrate
neoplastic cells that are intermediate to large in size, with moderate amounts of basophilic agranular
cytoplasm often containing punched-out vacuoles. The neoplastic cells typically have highly
irregular nuclear contours and an open chromatin pattern with prominent nucleoli.
Figure 1. Aggressive natural killer (NK) cell leukemia involving bone marrow. (A) Trephine biopsy
showing an extensive infiltrate of neoplastic lymphocytes in interstitial and less pronounced
sinusoidal infiltration patterns. (B) CD2 immunohistochemistry, positive in neoplastic cells. (C)
Perforin A immunohistochemistry, positive in neoplastic cells. (D) Epstein–Barr virus encoded small
RNA (EBER1/2) expression in neoplastic cells. (E) Flow cytometry immunophenotyping performed
on an aspirate sample demonstrated an aberrant population of lymphocytes (pink highlight), positive
for CD2, CD8, CD16, and CD56. These cells were negative for CD3 and CD5. In addition, they were
negative for CD4, CD57, T cell receptor (TCR)-alpha/beta, and TCR-gamma/delta.
5. Immunophenotypic Features
When assessing normal NK cells by multiparameter/multicolor flow cytometry
immunophenotyping, 90% were CD56dim/CD16bright, and 10% were CD56bright/CD16dim [27]. It is
thought that the CD56dim/CD16bright group represents a more mature stage of NK cell differentiation,
but prior to mature NK cells with expression of CD57 and killer immunoglobulin-like receptors
(KIRs). The CD56bright/CD16dim group represents an earlier stage of differentiation [19,28,29].
Following this rationale, NK cell neoplasms can be divided into two broad groups. The first group is
CD56bright/posit ive/CD16dim/negative, suggesting failure of differentiation [30]. This group, which
encompasses ANKL and ENKTL, retains a cytokine-secreting function that underlies the cytokine
storm that occurs characteristically in patients with these two diseases [30]. The second group is
Figure 1.
Aggressive natural killer (NK) cell leukemia involving bone marrow. (
A
) Trephine biopsy
showing an extensive infiltrate of neoplastic lymphocytes in interstitial and less pronounced sinusoidal
infiltration patterns. (
B
) CD2 immunohistochemistry, positive in neoplastic cells. (
C
) Perforin A
immunohistochemistry, positive in neoplastic cells. (
D
) Epstein–Barr virus encoded small RNA
(EBER1/2) expression in neoplastic cells. (
E
) Flow cytometry immunophenotyping performed on an
aspirate sample demonstrated an aberrant population of lymphocytes (pink highlight), positive for
CD2, CD8, CD16, and CD56. These cells were negative for CD3 and CD5. In addition, they were
negative for CD4, CD57, T cell receptor (TCR)-alpha/beta, and TCR-gamma/delta.
5. Immunophenotypic Features
When assessing normal NK cells by multiparameter/multicolor flow cytometry immunophenotyping,
90% were CD56
dim
/CD16
bright
, and 10% were CD56
bright
/CD16
dim
[
27
]. It is thought that
the CD56
dim
/CD16
bright
group represents a more mature stage of NK cell differentiation,
but prior to mature NK cells with expression of CD57 and killer immunoglobulin-like receptors
(KIRs). The CD56
bright
/CD16
dim
group represents an earlier stage of differentiation [
19
,
28
,
29
].
Following this rationale, NK cell neoplasms can be divided into two broad groups. The first
group is CD56
bright/positive
/CD16
dim/negative
, suggesting failure of differentiation [
30
]. This group,
which encompasses ANKL and ENKTL, retains a cytokine-secreting function that underlies the
cytokine storm that occurs characteristically in patients with these two diseases [
30
]. The second group
Cancers 2020,12, 2900 4 of 15
is CD56
dim/negative
/CD16
bright/positive
, encompassing chronic lymphoproliferative disorder of NK cells
(CLPD-NK), and characterized by neoplastic cells with a cytotoxic function [30,31] (Table 1).
Table 1.
An integrative clinicopathologic overview, comparing multiple aspects of NK cell proliferative disorders.
Diagnosis Etiology Immunophenotype Functionality Genomic Landscape
Transient increase
in NK cells
Autoimmune disorders,
viral infections
CLPD* Unknown stimulus,
possibly viral
CD8+(uniform
positivity), CD16+,
CD56+(dim); Loss of
CD2, CD7 and CD57
Cytotoxic function Activating mutations in the
STAT3 SH2 domain
ENKTL* Strong association
with EBV infection
CD2+, CD56+; Loss of
sCD3, CD4, CD8, CD16
and CD57
Cytokine secretion
function
DDX3X, JAK/STAT signaling
pathway (STAT3, STAT5B,
JAK3, and PTPRK), KIT,
CTNNB1, TP53, PRDM1,
ATG5, AIM1,FOX03,
and HACE1, RAS, MYC,
KMT2D/MLL2, ARID1A,
EP300, ASXL3, CDKN2A,
CDKN2B, CDKN1A, FAS
ANKL* Strong association
with EBV infection
CD2+, CD16+, CD56+;
Loss of sCD3, CD4, CD7,
CD8 and CD57
Cytokine secretion
function
JAK/STAT signaling pathway,
TP53, TET2, CREBBP, ASXL1,
ASXL2, BRINP3, PRPF40B
*CLPD: chronic lymphoproliferative disorder; *ENKTL: extra-nodal NK/T cell lymphoma; *ANKL: aggressive NK
cell leukemia.
The main flow cytometry features of ANKL are prominent forward scatter (FSC), denoting
an increased cell size when compared to non-neoplastic background lymphocytes, and expression
of CD56 and CD94 [
19
]. CD56 is a neural cell adhesion molecule that assists in infiltration and
invasion. CD94 is a molecule that forms heterodimers with either NKG2A (CD159a) or NKG2C
(CD159c). The lectin-like CD94/NKG2A receptor is inhibitory and recognizes the human leukocyte
antigen (HLA) class Ib molecule HLA-E as its predominant ligand [
32
,
33
], while the CD94/NKG2C is
activating/triggering [
34
,
35
]. While Zambello et al. have demonstrated that in the vast majority of
NK-LGDL cases (16/18; 88.9%) CD94 is associated with NKG2A [
5
], to our knowledge the functional
associations of CD94 in ANKL has not been characterized yet. ANKL cells appear to consistently
express CD2, in addition to cytoplasmic CD3 (epsilon chain), CD16, and cytotoxic molecules, such as
granzyme B, TIA1, and perforin A. Expression of CD7 or CD8 is heterogeneous, whereas CD3 (surface),
CD4, CD5, CD57, and T cell receptors (TCR) are usually absent in ANKL [
36
]. The KIR (killer cell
immunoglobulin-like receptor) proteins (CD158a/b/e), known to be present only in peripheral blood
CD56
dim
NK cells, are mostly negative in ANKL [
19
,
36
]. In practical terms, neoplastic NK cell
populations can be separated from lymphocytes using a CD45/SSC gating strategy. In the case of
neoplastic NK cells overlapping with lymphocytes, using a CD56/CD3 gating strategy is helpful to
identify the aberrant cells [36] (Figure 1B–E and Table 2).
Table 2. Immunophenotypic characteristics of aggressive NK cell leukemia.
Consistently Positive Markers Frequently Positive Markers Frequently Negative Markers
CD2 CD7 CD3
Cytoplasmic CD3 epsilon CD8 CD4
CD16 CD5
CD56 CD57
CD94 KIR (CD158a–e)
Granzyme B T-cell receptors
TIA
Perforin
One challenge to establishing an early diagnosis of ANKL is the common presence of only a
small number of neoplastic NK cells in the bone marrow [
19
]. In one study of ANKL, the proportion
Cancers 2020,12, 2900 5 of 15
of abnormal NK cells in the bone marrow was reported to be <5% in half of the patients [
27
].
Flow cytometry immunophenotypic analysis is particularly useful in the early detection of ANKL,
usually with a high specificity [
27
]. As ANKL progresses, the number of neoplastic cells in the bone
marrow increases. In one study, the rate of detection of aberrant NK cell populations increased
from 86.8% to 97.4% when second and third bone marrow samples were assessed as the disease
progressed [
27
]. Thus, it is advisable to obtain several specimens from the blood, bone marrow, or other
tissue sites and to conduct consecutive flow cytometry analyses when positivity is not detected and
the patient is suspected clinically to have a neoplasm such as ANKL [27] (Figure 1E).
Homing of neoplastic NK cells to body cavities has been outlined recently in NK cell
neoplasms, including ANKL, creating a differential diagnosis with primary effusion lymphoma [
37
–
40
].
This differential diagnosis can be addressed successfully by using flow cytometry immunophenotyping
when morphologic examination is impeded or is of limited value [36].
6. The Role of Epstein–Barr Virus
The first cases of ANKL reported by Fernandez et al. and Imamura et al. were EBV-negative or
EBV was not assessed [
6
,
12
]. Kawa-Ha et al., in 1989, were the first to highlight that neoplastic NK cells
in the “aggressive” and “chronic” forms of “lymphoproliferative disease of granular lymphocytes”
(currently, ANKL and T cell LGL, respectively) could carry EBV DNA, thus implicating EBV in the
pathogenesis of these diseases [
41
]. EBV-positive cases are positive for EBV-encoded RNA (EBER) and
are negative for latent membrane protein type 1 (LMP-1) in some reports, suggesting a type I latency
pattern of infection [
24
,
25
]. This latency pattern is believed to provide an advantage to neoplastic NK
cells by enabling them to evade host virus-specific cytotoxic T cell activity [42,43].
Although ANKL has an established association with EBV infection, EBV-negative ANKL is also
widely recognized [
19
–
21
,
44
–
48
]. In contrast with EBV-positive ANKL, EBV-negative cases occur
more frequently in older patients and arise equally in Asian and non-Asian
populations [44,45]
.
No morphologic or immunophenotypic differences have been shown in EBV-positive versus
EBV-negative ANKL [
45
]. Although some reports have suggested a more indolent behavior of
EBV-negative ANKL [
46
,
49
,
50
], recent articles have shown that these cases are associated with an
aggressive clinical behavior, similar to EBV-positive cases [44,45].
The absence of EBV in a subset of ANKL cases can potentially lead to a delayed diagnosis,
with adverse clinical consequences. When evaluating a potential case of ANKL, one should not use
EBV-negative status as a criterion to exclude the diagnosis. Further investigation of these neoplasms is
needed to unmask NK cell lineage. Performing immunohistochemical (IHC) analysis can be helpful
because CD56 and cytotoxic molecules are reported to be positive in most ANKL cases [44,45].
7. Cytogenetic Features
Conventional cytogenetic abnormalities in ANKL have been reported in few studies, and these
abnormalities commonly include del (6) (q21q25) and del (11q) [
24
,
25
,
51
]. Another study found an
association between ANKL and chromosome 7 abnormalities, and also reported chromosome 6q
deletion associated with ENKTL [
52
]. More recently, array-based comparative genomic hybridization
(aCGH) analysis in a cohort of patients with ANKL and ENKTL showed that gains of 1q23.1–q23.2 and
1q31.3–q44, as well as losses of 7p15.1–q22.3 and 17p13.1, are more frequent in ANKL than ENKTL [
53
].
In contrast, cases of ENKTL more frequently had gains of 2q and losses of 6q16.1–q27, 11q22.3–q23.3,
5p14.1–p14.3, 5q34–q35.3, 1p36.23–p36.33, 2p16.1–p16.3, 4q12, and 4q31.3–q32.1 [
53
]. Others have
reported that loss of 6q16.1–q27 is a common finding in NK cell malignancies [
54
], detected in both
ANKL [24,25,51] and ENKTL, but more frequently in ENKTL [53].
8. Molecular Pathogenesis and Genomic Landscape
Abundant data are available with regards to genetic pathways involved in the pathogenesis of
ENKTL. These abnormalities include mutations in the JAK/STAT [
55
–
59
], AKT [
59
], and NF-
κ
B [
59
]
Cancers 2020,12, 2900 6 of 15
signaling pathways. Additional abnormalities include recurrent chromosomal aberrations in ENKTL
including a 6q21 deletion [
60
] (silencing the tumor suppressors PRDM1 and FOXO310), and mutations
in the RNA helicase gene DDX3X [
61
]. However, apart from copy number aberration analyses [
24
,
25
,
53
],
a deeper understanding of the genetic alterations in ANKL was missing until recently. The advent of
next generation sequencing methods has provided an unprecedented impetus for understanding the
molecular pathogenesis of ANKL. Three recent studies [
19
,
62
,
63
] provided a comprehensive genetic
analysis of ANKL via next generation sequencing (Table 3). While the findings in these studies show
some differences that are likely the result of differing methodologies, they share many common threads
that provide insight into the molecular landscape of ANKL. Major genetic findings in these studies are
discussed below:
Table 3.
Breakdown of the common mutations reported in the three most recent genomic studies of
aggressive NK cell leukemia.
Mutations Huang et al. [63]
(29 Patients)
Dufva et al. [62]
(14 Patients)
El Hussein et al. [19]
(6 Patients)
JAK/STAT
STAT3, STAT5B, STAT5A,
JAK2, JAK3, STAT6,
SOCS1, SOCS3 and
PTPN11 (48%)
STAT3 (21%) JAK1, JAK3, STAT3
(66.6%)
RAS/MAPK – (29%) (16.7%)
Epigenetic modifiers
TET2 (28%), CREBBP
(21%), KMT2D (21%),
BCOR (3%)
SETD2, KMT2D and
BCOR (50%), TET2 (7%)
TET2 (16.7%), CREBBP
(16.7%), GFI1 (16.7%)
RNA helicase (DDX3X) (7%) (21%) –
Cell cycle regulation and
DNA damage repair TP53 (34%) TP53 (7%)
TP53 (50%), ASXL1
(33.3%), ASXL2 (33.3%),
BRINP3 (16.7%)
mRNA splicing – – PRPF40B (16.7%)
8.1. JAK/STAT Signaling Pathway
Four JAKs (JAK1, JAK2, JAK3, TYK2) and seven STATs (STAT1, STAT2, STAT3, STAT4, STAT5a,
STAT5b, STAT6) are used by more than 50 cytokines and growth factors [
64
]. Extracellular binding of
cytokines or growth factors with their corresponding trans-membrane receptors induce conformational
changes in receptor-bound JAK proteins, creating a distance between their kinase domains and
inhibitory pseudo-kinase domains [
65
]. Trans-activated JAKproteins subsequently phosphorylate STAT
proteins, resulting in dimerization, nuclear translocation, and direct DNA binding [
65
]. STATs disperse
throughout the genome and regulate transcription of both protein-coding and non-coding genes [
66
].
In addition, all STATs recognize the same DNA sequence, known as the GAS motif [
64
]. However,
STATs may antagonize the action of one another by competing for the binding of the same genomic
site [
64
]. For example, in T cells, dendritic cells, and cancer cell lines, STAT3-driven IL-17 transcription
is blocked by STAT5 [67–69].
Dufva et al. analyzed 14 patients of ANKL using whole-exome sequencing [
62
]. They showed
frequent genetic mutations in the JAK/STAT (21% of cases exhibited STAT3 mutations) and RAS-MAPK
signaling pathways [
62
]. Similarly, Huang et al. analyzed eight patients with ANKL by whole-genome
sequencing (WGS) and 29 ANKL (including the eight patients analyzed by WGS) patients by targeted
sequencing [
63
]. They noted that mutations in molecules of the JAK/STAT system, namely, STAT3,
STAT5B,STAT5A,JAK2,JAK3,STAT6,SOCS1,SOCS3, and PTPN11, were seen in 48% of patients,
with 17% of the cases harboring STAT3 mutations [
63
]. El Hussein et al. analyzed six ANKL patients,
finding mutations in JAK1 in one patient (who also harbored a mutation in STAT3), JAK3 in one
patient, and STAT3 in three patients [
19
]. In general, mutations in the JAK/STAT pathway were
mutually exclusive.
Cancers 2020,12, 2900 7 of 15
Huang et al. [
63
] and Dufva et al. [
62
] found that most of the STAT3 and STAT5B mutations
were localized to exons 20 and 21 encoding the Src homology 2 (SH2) domain, which mediates STAT
protein dimerization [
63
]. This domain also constitutes the hotspot containing activating mutations in
NKTCL [
55
,
61
]. Other JAK/STAT-related mutations reported were 9p copy number gain-containing
JAK2 and a point mutation in the protein tyrosine phosphatase (PTP) PTPRK, as well as mutations
in PTPN4 and PTPN23 [
62
]. Of these molecules, PTPRK is a tumor suppressor shown to negatively
regulate STAT3, and is commonly deleted in NKTCL.
Huang et al. suggested that EBV-encoded small RNAs (EBERs) that are highly expressed
in some cases of ANKL induce the release of massive amounts of IL-10, a well-known upstream
activator of the JAK/STAT pathway [
70
]. This stimulation in turn stimulates STAT3 phosphorylation,
leading to downstream MYC activation [
63
]. Importantly, Huang and colleagues also observed
that JAK/STAT-mutated and -unmutated leukemic NK cells showed a similar expression pattern
in MYC-driven programs [
63
]. This finding suggests that STAT3 signaling can be activated
by over-production of IL-10 or through other unknown mechanisms, independent of genetic
mutations [
63
]. This observation has been supported by immunohistochemical analysis, which has
shown phosphorylated STAT3 (p-STAT3; indicative of activation) is significantly higher in neoplastic
cells residing in the bone marrow of both JAK/STAT wild-type and mutated ANKL cases as compared
with controls [63].
The high frequency of mutations involving the JAK/STAT signaling pathway prompted Huang et al.
to analyze the plasma levels of inflammatory cytokines in patients with ANKL [
63
]. They found
significantly high serum levels of IL-10 in ANKL patients [
63
]. The authors concluded that IL-10 plays
a major role in upstream activation of the JAK/STAT pathway in ANKL, leading to increased expression
of MYC [
63
]. The authors subsequently proposed an IL-10–STAT3–MYC transcription regulation model
involved in the pathogenesis of ANKL [
63
]. On another note, IL-10 treatment of cell lines in the same
study preferentially stimulated JAK/STAT wild-type cells, but not the STAT3 Y640F-mutant ANKL
cells, causing an increase pathway activity and cellular proliferation [
63
]. This result suggests that
IL-10 upregulation plays a more prominent role in JAK/STAT unmutated ANKL patients [63].
8.2. Epigenetic Dysregulation
Half of the cases in the Dufva et al. study harbored mutations in epigenetic regulatory molecules
such as TET2 and CREBBP, and four (28%) patients had mutations in the RNA helicase DDX3X [
62
].
Huang and colleagues also identified mutations in epigenetic modification-related genes, including
TET2 (28%), CREBBP (21%), and MLL2 (21%) [
63
]. In contrast, DDX3X and BOCR, commonly found in
ENKTL [
61
,
71
], were less frequently mutated in ANKL [
63
]. Our group also has found mutations in
TET2,CREBBP, and GFI1 in ANKL cases [19].
8.3. TP53 Alterations and DNA Repair
Huang et al. identified TP53 mutations in 34% of cases of ANKL [
63
], whereas Dufva and
colleagues identified TP53 mutations in 1 out of 14 patients [
62
]. In our study, we found TP53 mutations
in three of six patients, and demonstrated uniform absence of p53 protein expression in four of eight
ANKL cases assessed by immunohistochemistry [
19
]. This finding is in keeping with observations in
an earlier aCGH study by Nakashima et al., who reported 17p13.1 losses more frequently in ANKL
than ENKTL cases [53].
Dufva and colleagues noted the lack of a DNA double-strand break repair-associated signature
in ANKL cases when compared with ENKTL, CLPD-NK, and T-LGL, suggesting a divergence in
underlying mutational processes between ANKL and ENKTL [
62
]. However, no mutations specific
to ANKL were uncovered in any of the recent studies [
62
,
63
]. Moreover, several genes that have
been identified in ENKTL cases, such as DDX3X [
61
], STAT3 [
55
,
61
], BCOR [
71
], and KMT2D [
61
],
were also identified in ANKL [
62
,
63
]. From these observations, we suggest that one can infer that the
pathogenesis of ANKL and NKTCL is closely related. Nevertheless, mutations in genes such as DDX3X
Cancers 2020,12, 2900 8 of 15
and TP53 were reported to contribute to a poorer prognosis in ENKTL [
61
], whereas the distribution of
these same mutations was not homogeneous in recent studies of ANKL (Table 1). This observation
suggests that mutations DDX3X and TP53 play a minor role in ANKL and refutes the hypothesis
that ANKL evolves from ENKTL. It is also worth mentioning that although a connection between
EBV status and mutational signature was suggested in past studies [
44
], this theory appears to have
weakened because the investigation of additional ANKL cases have been frequently EBV-negative.
Several gene mutations identified in EBV-positive cases also have been identified in EBV-negative
cases, such as STAT3 [
45
], TP53 [
44
], TET2 [
44
], and DDX3X [
44
]. Therefore, although EBV plays an
undeniable role in the pathogenesis of a subset of cases of ANKL, other epigenetic factors may exert
similar alterations on the genomic level, resulting in ANKL independent of EBV.
9. Examples of Clinicogenomic Data Integration
An example of genomic and clinical data integration is illustrated by the work of Tang et al. [
22
].
These authors identified 29 cases of classic ANKL with a fulminant presentation and eight cases of ANKL
with “subacute clinical course”. The latter group was defined as patients who manifested infectious
mononucleosis-like symptoms for more than 90 days, before full-blown manifestations of ANKL
developed. This subacute group is important to recognize because it can be mistaken it for a self-limited
infectious disease, likely delaying intervention with adverse clinical consequences [
22
]. By applying
ultra-high multiplex PCR technology, they identified mutations in TP53 (most commonly), genes in the
JAK/STAT RAS-MAPK signal transduction systems, in the transcription factors NF-
κ
B1 and NF-
κ
B1A,
and in the epigenetic regulatory molecules TET2 and CREBBP [
22
]. TP53 was mutated significantly
less often in “subacute” ANKL (none were detected) in comparison to fulminant ANKL [
22
]. However,
gene mutations in the JAK/STAT pathway were similar between the two groups, suggesting that the
key driving mechanisms are similar between the classic and subacute variants of ANKL [22].
El Hussein et al. published an integrative genomic and immunophenotypic landscape study of
12 cases of ANKL, of which six cases had available next generation sequencing data [
19
]. The authors
also identified mutations in the JAK/STAT (JAK1,JAK3) and RAS/MAPK pathways (KRAS), as well as in
epigenetic modifiers (TET2,CREBBP, and GFI1), cell cycle regulation, and DNA damage repair (TP53,
ASXL1,ASXL2, and BRINP3) and mRNA splicing factors (PRPF40B) (Table 3). In addition, the authors
coupled these genomic findings with immunophenotypic analysis by immunohistochemistry, showing
frequent loss of p53 expression, in addition to overexpression of BCL-2 and MYC in ANKL cases. To date,
this study includes the largest number of cases at a single institution analyzed by next-generation
sequencing performed from a clinical perspective, as other studies, although highly valuable,
were performed more from translational and investigational perspectives.
10. Immune Checkpoint Status
Gao et al. reported CD274 (PD-L1) overexpression in two of three ANKL cases [
44
]. Furthermore,
our group has shown PD-L1 expression in two of eight ANKL cases [
19
]. In a small group of patients with
ANKL and ENKTL, JAK2 gain was associated with gains of the neighboring immune evasion-associated
CD274 (PD-L1) and CD273 (PD-L2) genes [
62
]. In addition, sensitivity of ENKTL to PD-1 inhibition
was recently outlined [
72
]. Taking all of these data into account, PD-1 inhibition represents a promising
approach for ANKL treatment. This may be particularly true in EBV-positive cases of ANKL, as EBV
infection plays a role in PD-L1 stimulation [73].
11. Treatment of ANKL
11.1. Chemotherapy in ANKL
No consensus chemotherapeutic regimen has been established to manage patients with ANKL as
the rarity of this disease has precluded prospective clinical trials [
23
]. Current knowledge of therapy
is based on small clinical studies focused on chemotherapeutic approaches and clinical outcomes.
Cancers 2020,12, 2900 9 of 15
These studies have shown a role for anthracycline-containing chemotherapy regimens as ANKL
patients have shown a complete response [
20
]. Subsequently, a role for L-asparaginase in treating
ANKL was recognized. Treating ANKL cell lines with L-asparaginase results in apoptosis [
74
],
and L-asparginase has been included in various chemotherapy regimens to treat ANKL patients, such
as the SMILE (dexamethasone, methotrexate, ifosfamide, etoposide, and L-asparaginase), AspaMetDex
(L-asparaginase, methotrexate, and dexamethasone), or VIDL (etoposide, ifosfamide, dexamethasone,
and L-asparaginase) regimens, resulting in improved outcomes [
75
–
77
], However, no study has
compared these regimens in the setting of ANKL [
27
,
76
,
78
]. In addition, one study suggested that the
gemcitabine, cisplatin, and dexamethasone (GDP) regimen was efficacious in selected patients [
48
].
A complete response, including negativity for EBV DNA in the blood after treatment, is associated
with a better outcome, including overall survival [
76
]. However, prognosis remains poor. Patients who
achieve a complete remission (CR) after chemotherapy (including L-asparaginase) rarely survive more
than one year without further treatment.
11.2. Allogeneic Hematopoietic Cell Transplantation in ANKL
Allogeneic hematopoietic stem cell transplantation (SCT) improves outcome in ANKL patients.
In a study of eight patients who did not achieve complete remission (CR) before allogeneis SCT,
four patients reached CR, and two survived for several years [
75
]. Subsequent studies also have shown
the significant efficacy of allogeneic SCT for ANKL patients [
22
,
76
]. A total of 21 ANKL patients
enrolled in the International Bone Marrow Transplantation Registry (IBMTR) database underwent
allogeneic SCT, with most receiving L-asparaginase-containing chemotherapy before proceeding to
transplant [
79
]. Patients with a CR prior to allogeneic SCT showed a significantly better survival after
two years than those without a CR (38 vs. 0%) [
79
]. This study demonstrated that transplant can
provide durable disease control in a subset of ANKL patients who achieve CR before transplantation.
However, 76% of all patients died in the long run, mostly due to ANKL [
79
]. In summary, allogeneic
SCT in the setting ANKL might help extend survival in some patients, but success appears rather
limited, and novel therapies are needed for ANKL patients.
11.3. Novel Therapeutic Applications
Vulnerability of NK cells to targeted therapies could be better assessed through RNA sequencing
and drug sensitivity profiling of normal NK cells, as well as cells derived from NK cell neoplasms,
opening the gate to the practice of precision medicine in ANKL patients.
11.3.1. BCL2 Inhibitors
Dufva et al. investigated drug sensitivity profiling of nine NK cell lines, including three ANKL
and two ENKTL cell lines [
62
]. They concluded that NK cells show particular sensitivity to inhibition
of IL2–JAK–STAT signaling compared with other hematopoietic cells. The authors also reported high
efficacy of the JAK inhibitor ruxolitinib and the BCL2 family inhibitor navitoclax across all cell lines
(neoplastic and non-neoplastic) [
62
]. In addition, they observed that the BCL2 inhibitor venetoclax
and mTOR inhibitors were effective only against malignant NK cell lines [
62
]. Venetoclax was less
effective as a single agent compared with the more broad-spectrum BCL2 family inhibitor navitoclax,
but venetoclax exerted a synergistic effect with ruxolitinib more consistently and across all cell lines,
especially with the addition of the Aurora kinase (AURK) inhibitor alisertib [
62
]. In keeping with these
findings, El Hussein et al. demonstrated BCL2 protein overexpression in six of eight ANKL cases,
underscoring potential therapeutic applicability of BCL2 inhibitors in clinical settings [19].
11.3.2. Heat Shock Protein 90 (HSP90) Inhibitors
Other effective drug classes reported by Dufva et al. were heat shock protein 90 (HSP90)
inhibitors, Polo-like kinase (PLK) and cyclin-dependent kinase inhibitors, as well as histone deacetylase
inhibitors [
62
]. On the other hand, most NK cell lines were resistant to MEK inhibitors [
62
].
Cancers 2020,12, 2900 10 of 15
Glucocorticoids were highly effective against healthy NK cells compared to other cell types, but induced
responses only in few cell lines, implying glucocorticoid resistance in a subset of malignant NK cells [
62
].
Consistent with the role of HSP90 in JAK/STAT regulation [
80
], JAK and HSP90 inhibitors as well as
the Nedd8 activating enzyme (NAE) inhibitor pevonedistat were more effective in IL-2 stimulation than
resting NK cells, suggesting that the sensitivity of NK cells to JAK and HSP90 inhibition results largely
from inhibition of IL-2-derived JAK/STAT activation.
The role of the polycomb repressive complex 2 (PRC2) has been elucidated in the biology of T and
NK cell lymphomas [
81
–
84
]. PRC2 is a multiprotein complex composed of several core components
including EZH2, a histone methyltransferase responsible for epigenetic silencing of target genes via
trimethylation of H3K27. Increased or unchecked PRC2 activity hypermethylates H3K27, leading to
repression of tumor suppressor genes [
44
,
85
]. In one study, EZH2 and its’ effector H3K27me3 were
shown to be overexpressed by IHC in all three EBV-negative ANKL patients [
44
]. As we have discussed
above, activation of the JAK/STAT pathway is suggested to play a major role in overexpression of
MYC [
63
]. MYC, in turn, has been shown to interact with PRC2 through EZH2 and other cofactors such
as SUZ12/EED, and is responsible for inducing histone modification of H3K27me3 [
86
–
88
]. This axis
potentially contributes to the pathogenesis of NK cell neoplasms in general, and ANKL in particular.
This observation also provides new insights into potential druggable targets in ANKL through the
utilization of EZH2 inhibitors.
11.3.3. Other Approaches
Consideration of specific targeted therapies using ex vivo drug screening on patient-derived
xenografts might offer another approach for experimental therapies in patient with ANKL. Such an
approach has shown promise in patients with T cell neoplasms [
89
]. One example might be targeting
CD38 with daratumumab, as CD38 has already been shown to be expressed in ANKL [12,20].
12. Conclusions
Although the outcome of patients with ANKL is poor, and our understanding of ANKL has
evolved only slowly over time, substantial recent progress has been made in deciphering the molecular
alterations orchestrating the oncogenesis of ANKL. Pharmacologic experiments with therapeutic
agents tailored to optimize attack on leukemic NK cells and target recently characterized pathways in
ANKL, such as JAK/STAT, hold promise for the future. Implementing the data collected from anecdotal
observations into clinical trials, coupled with the creation of a multi-institutional genomic bank of
ANKL samples that can be analyzed by high throughput molecular methods, represents steps on the
journey towards understanding the biology of and designing effective therapies for this disease.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
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