Extranodal NK/T-cell Lymphoma, Nasal Type, Includes Cases of Natural Killer Cell and αβ, γδ, and αβ/γδ T-cell Origin

Department of Pathology, Division of Hematopathology, University of Pittsburgh School of Medicine, Pittsburgh 15213-2582, USA.
The American journal of surgical pathology (Impact Factor: 5.15). 02/2012; 36(4):481-99. DOI: 10.1097/PAS.0b013e31824433d8
Source: PubMed
ABSTRACT
Extranodal NK/T-cell lymphoma (ENKTL), nasal type, may be of NK or T-cell origin; however, the proportion of T-ENKTLs and whether they are of αβ or γδ type remains uncertain. To elucidate the cell of origin and detailed phenotype of ENKTL and assess any clinicopathologic associations, 67 cases of ENKTL from Thailand were investigated, together with 5 γδ enteropathy-associated T-cell lymphomas (EATLs) for comparison. In all, 70% of the ENKTL were T-cell receptor (TCR) β,γ and, in cases tested, δ negative (presumptive NK origin); 5% were TCR γδ, 3% were TCR αβ, 1% were TCR αβ/γδ, and 21% were indeterminate. Out of 17 presumptive NK-ENKTLs tested, 3 had clonal TCR rearrangements. All cases were EBV and TIA-1; >85% were positive for CD3, CD2, granzyme B, pSTAT3, and Lsk/MATK; and all were CD16. Presumptive NK-ENKTLs had significantly more frequent CD56 (83% vs. 33%) and CXCL13 (59% vs. 0%) but less frequent PD-1 (0% vs. 40%) compared with T-ENKTLs. Of the NK-ENKTLs, 38% were Oct-2 compared with 0% of T-ENKTLs, and 54% were IRF4/MUM1 compared with 20% of T-ENKTLs. Only αβ T-ENKTLs were CD5. Intestinal ENKTLs were EBV and had significantly more frequent CD30, pSTAT3, and IRF4/MUM1 expression but less frequent CD16 compared with γδ EATL. Significant adverse prognostic indicators included a primary non-upper aerodigestive tract site, high stage, bone marrow involvement, International Prognostic Index ≥2, lack of radiotherapy, Ki67 >40%, and CD25 expression. The upper aerodigestive tract ENKTLs of T-cell origin compared with those of presumptive NK origin showed a trend for better survival. Thus, at least 11% of evaluable ENKTLs are of T-cell origin. Although T-ENKTLs have phenotypic and some possible clinical differences, they share many similarities with ENKTLs that lack TCR expression and are distinct from intestinal γδ EATL.

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Extranodal NK/T-cell Lymphoma, Nasal Type, Includes
Cases of Natural Killer Cell and ab, gd, and ab/gd T-cell
Origin: A Comprehensive Clinicopathologic and
Phenotypic Study
Tawatchai Pongpruttipan, MD,*
w
Sanya Sukpanichnant, MD,
w
Thamathorn Assanasen, MD,
z
Pongsak Wannakrairot, MD,
z
Paisarn Boonsakan, MD,
y
Wasana Kanoksil, MD,
y
Kanita Kayasut,
MD,8 Winyou Mitarnun, MD,8 Archrob Khuhapinant, MD, PhD,
z
Udomsak Bunworasate, MD,#
Teeraya Puavilai, MD,** Anan Bedavanija, MD,
ww
Adriana Garcia-Herrera, MD,
zz
Elias Campo,
MD,
zz
James R. Cook, MD, PhD,
yy
John Choi, MD, PhD,88 and Steven H. Swerdlow, MD*
Abstract: Extranodal NK/T-cell lymphoma (ENKTL), nasal
type, may be of NK or T-cell origin; however, the proportion of
T-ENKTLs and whether they are of ab or gd type remains
uncertain. To elucidate the cell of origin and detailed phenotype
of ENKTL and assess any clinicopathologic associations, 67
cases of ENKTL from Thailand were investigated, together with
5 gd enteropathy-associated T-cell lymphomas (EATLs) for
comparison. In all, 70% of the ENKTL were T-cell receptor
(TCR) b,g and, in cases tested, d negative (presumptive NK
origin); 5% were TCR gd
+
, 3% were TCR ab
+
, 1% were TCR
ab/gd
+
, and 21% were indeterminate. Out of 17 presumptive
NK-ENKTLs tested, 3 had clonal TCR rearrangements. All
cases were EBV
+
and TIA-1
+
; >85% were positive for CD3,
CD2, granzyme B, pSTAT3, and Lsk/MATK; and all were
CD16
. Presumptive NK-ENKTLs had significantly more
frequent CD56 (83% vs. 33%) and CXCL13 (59% vs. 0%) but
less frequent PD-1 (0% vs. 40%) compared with T-ENKTLs. Of
the NK-ENKTLs, 38% were Oct-2
+
compared with 0%
of T-ENKTLs, and 54% were IRF4/MUM1
+
compared with
20% of T-ENKTLs. Only ab T-ENKTLs were CD5
+
.
Intestinal ENKTLs were EBV
+
and had significantly more
frequent CD30, pSTAT3, and IRF4/MUM1 expression but less
frequent CD16 compared with gd EATL. Significant adverse
prognostic indicators included a primary non-upper aero-
digestive tract site, high stage, bone marrow involvement, In-
ternational Prognostic Index Z 2, lack of radiotherapy, Ki67
>40%, and CD25 expression. The upper aerodigestive tract
ENKTLs of T-cell origin compared with those of presumptive
NK origin showed a trend for better survival. Thus, at least 11%
of evaluable ENKTLs are of T-cell origin. Although T-
ENKTLs have phenotypic and some possible clinical differ-
ences, they share many similarities with ENKTLs that lack TCR
expression and are distinct from intestinal gd EATL.
Key Words: extranodal NK/T-cell lymphoma, gd T-cell
receptor, natural killer cells, CXCL13, CD25
(Am J Surg Pathol 2012;36:481–499)
E
xtranodal natural killer/T-cell lymphoma (ENKTL),
nasal type, is defined in the 2008 WHO classification
as a “predominantly extranodal lymphoma characterized
by vascular damage and destruction, prominent necrosis,
cytotoxic phenotype and association with Epstein-Barr
virus (EBV).”
14
It is designated as an “NK/T-cell” lym-
phoma because, although most of the cases are believed
to be of “true” natural killer cell (NK) origin, “some” of
the cases are known to demonstrate a cytotoxic T-cell
phenotype.
14
The actual proportion of cases of T-cell
origin is not well established and, even when documented
to be of putative T-cell origin, whether the T cells are
of ab or gd type is often unknown. Furthermore, little
From the *Department of Pathology, Division of Hematopathology,
University of Pittsburgh School of Medicine, Pittsburgh; wDepart-
ment of Pathology; zDepartment of Medicine, Division of Hema-
tology; wwDepartment of Otolaryngology, Head and Neck Surgery,
Faculty of Medicine Siriraj Hospital, Mahidol University; #De-
partment of Medicine, Division of Hematology; zDepartment
of Pathology, Faculty of Medicine, Chulalongkorn University;
**Department of Medicine, Division of Hematology; yDepartment
of Pathology, Faculty of Medicine Ramathibodi Hospital, Bangkok;
8Department of Pathology, Faculty of Medicine, Prince of Songkla
University, Hat-Yai, Songkhla, Thailand; zzHematopathology Unit,
Center for Biomedical Diagnosis, Hospital Clinic, University of
Barcelona, Barcelona, Spain; yyPathology and Laboratory Medicine
Institute, Cleveland Clinic, Cleveland, OH; and 88Department of
Pathology, Children’s Hospital of Philadelphia, University of
Pennsylvania School of Medicine, Philadelphia, PA.
Conflicts of Interest and Source of Funding: The authors have disclosed
that they have no significant relationships with, or financial interest
in, any commercial companies pertaining to this article.
Correspondence: Steven H. Swerdlow, MD, Department of Pathology,
Division of Hematopathology, UPMC Presbyterian, 200 Lothrop
Street-Room G335, Pittsbugh, PA 15213-2582 (e-mail: swerdlowsh@
upmc.edu).
Supplemental Digital Content is available for this article. Direct URL
citations appear in the printed text and are provided in the HTML
and PDF versions of this article on the journal’s Website,
www.ajsp.com.
Copyright
r
2012 by Lippincott Williams & Wilkins
ORIGINAL ARTICLE
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attention has been focused on phenotypic variations
among the more numerous NK cell cases and their clin-
ical implications, even though NK cells, similar to
other lymphoid cells, are phenotypically heteroge-
neous.
36,78,103,104
Some of the phenotypic heterogeneity
such as expression of CD16 is well recognized,
36
but little
attention has been paid to the expression of many other
antigens, such as the B-cell–associated transcription fac-
tor Oct-2 or the T follicular helper-associated marker
CXCL13.
78,104
ENKTL is generally considered to be a very ag-
gressive lymphoma; however, some patients have more
indolent or therapy-responsive disease.
6,14,22
Although
there are some well-established mostly clinical prognostic
factors,
6,14,56
it remains unknown whether any pathologic
subsets of ENKTL should be distinguished for clinical or
therapeutic purposes and whether any of the antigens that
are only expressed by subsets of these cases have prog-
nostic implications.
Greater attention has been paid in recent years to
the relationship of varied T-cell subsets to specific T-cell
neoplasms. For example, it has been suggested that many
of the nonhepatic gd T-cell lymphomas, in addition to
those involving the skin, might be included under the
umbrella term of mucocutaneous gd T-cell lymphoma.
3,34
Whether any of the ENKTLs, another neoplasm classi-
cally of the innate immune system that frequently in-
volves mucosal or cutaneous sites, should also be
considered for inclusion in this potential category is un-
certain. This is an even more important question given the
recent finding that NK cell neoplasms of varied types
have molecular features similar to a group of non-
hepatosplenic gd T-cell lymphomas.
45
In contrast, other
recent studies have suggested that these nonhe patosplenic
gd T-cell lymphomas are heterogeneous.
32,101
Finally,
although much of our knowl edge about ENKTL is from
Asian countries, a large de tailed series of Thai cases has
not been published.
To address these issues, the clinicopathologic fea-
tures of 67 cases of ENKTL from Thailand were inves-
tigated, with particular attention paid to the expression of
T-cell receptor (TCR) proteins and the detailed pheno-
type of the neoplastic cells. In addition, the findings were
compared with those of enteropathy-associated T-cell
lymphomas of gd type (gd EATL) to address the question
as to whether intestinal ENKTL might represent an
EBV
+
variant of EATL.
MATERIALS AND METHODS
This study was approved by the Institutional Re-
view Boards of the University of Pittsburgh School of
Medicine and the Faculty of Medicine Siriraj Hospital,
Mahidol University.
Case Selection and Clinical Data Collection
Sixty-seven cases from Thailand that fulfilled the
WHO criteria for ENKTL were selected for review. None
of the cases in this study were included in a recently
conducted Thai ENKTL study.
71
Five cases of EATL
were also included for comparison purpose (3 type II and
1 type I gd EATL from the University of Pittsburgh
Medical Center prev iously reported in abstract form
101
and 1 type II EA TL from Siriraj Hospital). Clinical data
including age, sex, sites of involvement, Ann Arbor stage,
International Prognostic Index (IPI), any underlying im-
munodeficiency states, mode of treatment, duration of
follow-up, and patient status at time of last follow-up
were collected.
Histopathologic Assessment
Hematoxylin and eosin-stained sections of all cases
were reviewed by S.H.S. and T.P. The following features
were evaluated: tissue necrosis, ulceration with acute in-
flammation, angioinvasion, thrombosis, epithelial hyper-
plasia, epitheliotropism, nuclear irregularity (predominantly
round vs. irregular), prominent nuclear elongation (present
or absent), cytoplasmic granularity, prominent admixed
histiocytes, and granulomas. The average number of ad-
mixed neutrophils, eosinophils, and plasma cells was esti-
mated (0 to 10, 11 to 25, 26 to 75, and >75/HPF, 400,
field number 22). When possible, evaluation was performed
away from inflamed ulcer bases. Only limited morphologic
evaluation could be performed in cases with extensive ne-
crosis and/or crush artifact. Mitoses were counted in
10 HPFs (400, field number 22, fewer fields counted if
limited tissue).
Cases were divided into 5 groups on the basis of
nuclear size: (1) small cell predominant—small cells
>90%; (2) mixed small and larger cells—small cells 25%
to 90% plus larger cells >10%; (3) medium-sized cell
predominant—medium-sized cells >75%; (4) mixed
medium-sized and large cells—medium-sized cells 25%
to 75% plus large cells >25%; and (5) large cell
predominant—large cells >75% (with or without ana-
plastic cells). Groups 1 and 2 (subtypes with small cell
component) and groups 3 to 5 (subtypes without small cell
component) were combined for subsequent analyses.
Nuclear size was defined as follows: small—similar to that
of mantle zone lymphocytes; large—at least twice the size
of small lymphocytes; and medium—intermediate between
small and large.
Immunophenotypic and EBV-encoded RNA
(EBER) In Situ Hybridization (ISH) Studies
Three tissue microarray (TMA) blocks were con-
structed using a single 3 mm core from all cases from
which there was sufficient available tissue; tonsil and gd
EATL were included for controls. Most immunohistochemical
(IHC) stains were performed on the TMA. A limited number
of stains were performed on whole-tissue sections.
TCRg IHC staining was performed using clone 3.20
(Thermo Fisher Scientific, Rockford, IL) and the pre-
viously reported method.
77
TCRd IHC staining was per-
formed with antigen retrieval by incubating deparaffinized
slides with EnVision FLEX Target Retrieval Solution,
low pH (Dako), using a PT Link (Dako), followed by
a 90-minute incubation with antibody for the TCRd
constant region (clone 5A6.E9, Thermo Fisher Scientific).
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The immunostaining was performed at room temperature
using EnVision Mouse LINKER (Dako) for 15 minutes,
followed by EnVision FLEX+, Mouse (Dako), for 20 mi-
nutes, and 3-3
0
diamonibenzidine for 10 minutes. Blocking
of endogenous peroxidase was carried out for 5 minutes.
Only membranous staining was considered positive. De-
tails of clones, dilution, sources, and methods for all other
IHC stain are given in Supplemental Digital Content 1,
http://links.lww.com/PAS/A108. EBER ISH was also
performed.
48
All preexisting and new IHC and EBER ISH stains
were reviewed by S.H.S. and T.P. All stains except for
Ki67 and p53 were interpreted as follows: negative;
probably negative—minimal equivocal staining; probably
positive—weak or partial inde finite staining; partially
positive—definite positivity on <50% of presumptive
neoplastic cells; positive—Z 50% of positive cells; or
indeterminate—totally negative internal positive controls
or IHC stains with extensive staining in non-neoplastic
cells indistinguishable from the neoplastic population. To
aid in the distinction from possible non-neoplastic re-
activity, partial positivity for CD45, phosphorylated sig-
nal transducer and activator of transcription 3 (pSTAT3),
IRF4/MUM1, and CD25 required Z 10% positive neo-
plastic cells. For subsequent analyses, probably negative
results were considered negative, probably pos itive results
were considered positive, and indeterminate results were
considered as missing values.
The percentage of Ki67-positive and p53-positive
cells was estimated to the closest decile (or <10%). On
the basis of CD20 staining, the presence of B-cell lym-
phoid aggregates and lymphoid follicles was assessed, and
the percentage of other B cells was estimated as r5%, 5%
to 10%, 11% to 25%, 26% to 50%, 51% to 75%, and
>75%. The percentage of admixed reactive T cells was
also estimated using non-lymphoma–expressing T-cell
markers, such as CD5 for CD5-negative ENKTL. When
possible, evaluation of admixed reactive B cells and T cells
was performed away from clearly inflamed regions.
Polymerase Chain Reaction (PCR)-based TCR
Gene Rearrangement Studies
TCR gene rearrangement studies were conducted on
all 6 T-cell–type (T-ENKTL) cases and in the presumptive
NK-cell–type (NK-ENKTL) cases that had the most re-
maining tissue. DNA was extracted from paraffin-em-
bedded, formalin-fixed tissue using the Genomic DNA
Purification Kit (Gentra Systems, Minneapolis, MN).
PCR was performed in duplicate using fluorescently la-
beled TCRb and g primers (Invivoscribe, San Diego, CA)
following the manufacturer’s instructions and the recom-
mendations of the BIOMED-2 protocols.
92
Amplification
of internal control housekeeping genes was performed to
assess DNA quality; cases with size ladders <300 bp were
considered to have suboptimal DNA quality compromis-
ing interpretation. Two cases with r100 bp were consid-
ered inadequate for analysis. An additional 6 cases
underwent prior TCRg gene rearrangement analysis using
a previously reported method.
75
Statistical Evaluation
Statistical comparisons between groups were per-
formed using the 2-tailed Fisher exact test (GraphPad
Prism v. 5.03, San Diego, CA, and SPSS, v. 18.0, Chi-
cago, IL). Survival outcomes were compared on the basis
of clinical, pathologic, and all phenotypic features, using
the log rank (Mant el-Cox) test (SPSS, v. 18.0). To elim-
inate possible differences simply related to primary loca-
tion or cell of origin, survival analyses were also
performed comparing upper aerodigestive tract (UADT)
T-ENKTL with UADT NK-ENKTL, and separately for
the largest group of UADT NK-ENKTL. Mean ag e be-
tween groups was compared using 1-way ANOVA Bon-
ferroni adjustment (SPSS, v. 18.0).
RESULTS
Clinical Features
The most common primary sites were nasal and
pharynx/oral cavity (Table 1). About 2/3 of patients pre-
sented with low-stage disease, although 55% had known
secondary site(s) of involvement. Compared with the
ENKTL of the UADT, the non-UADT cases were asso-
ciated with high-stage disease (5/6 vs. 9/34, P =0.014) and
an IPI score of Z 2 (4/4 vs. 11/33, P =0.021).
Histopathology
The diagnostic biopsies demonstrated a variable,
but usually dense, lymphoid infiltrate with subtle-to-ex-
tensive necrosis in about 80% of cases and angioinvasion
in 45% (Table 2; Fig. 1). Epitheliotropism, when present,
was usually subtle or only focal. The neoplastic cells
showed a cytologic spectrum with very few cases having
predominantly small cells, most cases having predom-
inantly medium-sized cells, and others with at least sig-
nificant numbers of large cells. One large biopsy
demonstrated extensive areas of predominantly small
cells, together with a separate area of predominantly
medium-sized cells (included in the latter group). Most
cases had predominantly irregular and frequently elon-
gate nuclei. Both of these features were more commonly
found in UADT compared with non-UADT (42/54 vs. 3/
8, P = 0.03; 43/52 vs. 2/8, P = 0.002, respectively). His-
tologic sections in 28% of evaluable cases showed coarse
(2/61, Fig. 1F) or fine (15) eosinophilic cytoplasmic
granules (almost exclusively from 1 hospital). The number
of mitoses varied greatly with >60 mitoses/10 HPF in
8% of cases with a small cell component compared with
64% of other subtypes (P = 0.037).
In the 61 evaluable cases, admixed neutrophils, eo-
sinophils, and plasma cells were usually scant, although a
minority of cases had >10/HPF (20%, 5%, and 23%,
respectively). Four cases (7%) had extensive neutrophil
infiltration mimicking an acute inflammatory process,
2 were histiocyte-rich, and a granulom atous reaction was
identified in the subcutaneous fat in 1 case. One ab
T-ENKTL had 26 to 75 eo sinophils/HPF, 4 ENKTLs
had >75 plasma cells/HPF, and 3 ENKTLs had 26 to
75 plasma cells/HPF mimicking inflammatory processes.
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As identified with IHC, 41% of cases had >10%
admixed reactive T cells, including the 9% of cases also
with >10% admixed reactive B cells. Focal B-cell ag-
gregates were present in 28% of cases. Cases with >10%
admixed reactive T cells were associated with a mitosis
count of r60 mitoses/10 HPF (16/22 cases with >10%
T cells vs. 9/30 cases with r10% T cells had r60 mi-
toses/10 HPF; P = 0.004).
Phenotypic Features
TCR Expression
Five groups of ENKTLs were identified on the basis
of TCR protein expression. Forty-seven of 67 cases (70%)
were TCR silent and designated as presumptive NK type
(NK-ENKTL) on the basis of negativity for at least
TCRb and g, with TCRd also negative in 33 tested cases
(Fig. 2). Three cases (5%) were of gd T-cell type (gd
T-ENKTL) on the basis of positivity for both TCRg and
d but not TCRb (Fig. 3). Two cases (3%) were of ab
T-cell type (ab T-ENKTL) on the basis of positivity for
TCRb but not for TCRg or d (Fig. 4). Both cases of ab
T-ENKTL had rare larger cells with TCRg expression;
however, they were significantly fewer than the number of
EBER
+
or bF1
+
cells. One (1%) case expressed TCRb,
g,andd proteins (ab/gd T-ENKTL) (Fig. 5). Staining for
TCRg and TCRd was concordant in all cases and showed
similar staining patterns. Fourteen cases (21%) were
of not otherwise specified type because either TCRb or
g stains were not evaluable or could not be performed.
Other Phenotypic Features
All ENKTLs were positive for EBER ISH and TIA-1.
Many expressed CD3, CD2, granzyme B, pSTAT3,
Lsk/MATK, and to a somewhat lesser extent CD30 and
LMP-1 without documented differences between NK-
ENKTL and T-ENKTL, whereas all tested cases were
negative for CD16, CD20, EBV nuclear antigen-2 (EB-
NA2), and ALK (Table 3; Figs. 3–5). CD56 expression
was more common in cases of NK origin (83%) than in
thoseofT-cellorigin(33%)(P = 0.019). In some cases,
although the TMA core was CD56
,stainingofthe
whole section s howed CD56 positivity. Expression of
other antigens was more variable. Loss of CD7 or CD45
expression was commonly found in both NK-ENKTL
TABLE 1. Clinical Features* and Mode of Treatment of ENKTL Subtypes and gd EATL
ENKTL Defined by TCR Expression ENKTL Subgroups and cd EATL
Characteristics
Overall
ENKTLw NK cd Tz ab Tz ab/cd T
UADT
NK-
ENKTL
Cutaneous
NK-
ENKTL
Intestinal
ENKTL cd EATL
Age, median [range] (y) 44 [11-83] 44 [22-80] 52 [39-55] 23 [18-27] 34 43 [22-74] 59 [37-80] 47 [34-52] 73 [63-80]
Sex, M:F ratio 4.2: 1 5.7: 1 1: 2 1: 1 0: 1 5: 1 2: 0 1.5: 1 1.5: 1
No. of Cases/Total Cases (%)
Primary site
Nasal ± other
UADT
42/67 (63) 30/47 (64) 3/3 (100) 1/2 (50) 0 30/42 (71) 0 0 0
Non-nasal UADT 17/67 (25) 12/47 (26) 0 1/2 (50) 0 12/42 (29) 0 0 0
Large intestine 3/67 (4) 2/47 (4) 0 0 0 0 0 3/5 (60) 0
Small intestine 2/67 (3) 1/47 (2) 0 0 1/1 (100) 0 0 2/5 (40) 5/5 (100)
Skin 2/67 (3) 2/47 (4) 0 0 0 0 2/2 (100) 0 0
Deep soft tissue 1/67 (2) 0 0 0 0 0 0 0 0
No. other sites of involvement
None 18/40 (45) 14/30 (47) 2/3 (67) 1/2 (50) 0 14/26 (54) 0 0 1/3 (33)
1 sitey 15/40 (38) 10/30 (33) 1/3 (33) 1/2 (50) 0 9/26 (35) 0 2/4 (50) 2/3 (67)
2-4 sitesy 7/40 (18) 6/30 (20) 0 0 1/1 (100) 3/26 (22) 2/2 (100) 2/4 (50) 0
Ann Arbor stage
Low stage (1 and 2) 26/40 (65) 19/30 (63) 2/3 (67) 2/2 (100) 0 18/26 (69) 0 1/4 (25) 1/3 (33)
High stage (3 and 4) 14/40 (35) 11/30 (37) 1/3 (33) 0 1/1 (100) 8/26 (31) 2/2 (100) 3/4 (75) 2/3 (67)
IPI score
0 15/37 (41) 9/27 (33) 2/3 (67) 2/2 (100) 0 9/25 (36) 0 0 NA
1 7/37 (19) 7/27 (26) 0 0 0 7/25 (28) 0 0 NA
2-4 15/37 (41) 11/27 (41) 1/3 (33) 0 1/1 (100) 9/25 (36) 1/1 (100) 3/3 (100) NA
Modes of treatment
Chemoradiotherapy 14/42 (33) 9/32 (28) 2/3 (67) 1/2 (50) 0 9/28 (32) 0 0 0
Chemotherapy only 22/42 (52) 20/32 (63) 1/3 (33) 1/2 (50) 0 18/28 (64) 1/2 (50) 1/4 (25) 2/5 (40)
None of above 6/42 (14) 3/32 (9) 0 0 1/1 (100) 1/28 (4) 1/2 (50) 3/4 (75) 3/5 (60)
*None of the 40 patients with ENKTL and available information were HIV
+
. One patient with NK-ENKTL was receiving methotrexate for psoriasis, and the patient
with ab/gd T-ENKTL was receiving steroids for systemic lupus erythematosus.
wOverall, ENKTL includes ENKTL of NK, T, and NOS type. The ENKTL, NOS (cases in which either TCRb or g stains were not evaluable or could not be
performed), is only included in aggregate with the other ENKTLs in this table because it does not represent a unique category.
zThe UADT T-ENKT L (includes T-ENKTL except for the non-UADT double-positive case) had a female predominance in contrast to the UADT NK-ENKTL
(P = 0.057).
yKnown secondary sites of involvement included regional lymph nodes (28%), skin (13%), distant lymph nodes (8%), bone marrow (8%), and/or other organs
(3% to 7%).
NA indicates not available; NOS, not otherwise specified.
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and T-ENKTL. P53 expression of >10% was associated
with high-stage disease (7/7 high-stage vs. 6/14 low-stage
cases were p53
+
; P = 0.018), absence of a small cell com-
ponent (21/25 cases without vs. 0/6 cases with a small cell
component were p53
+
; P<0.001), and cutaneous lym-
phocyte antigen (CLA) expression (11/17 CLA
+
vs. 1/9
CLA
cases were p53
+
; P = 0.014). CLA
+
was found in 2
of 2 ENKTL cases with secondary skin involvement and in
5 of 19 cases without skin involvement.
CD30 expression was associated with CXCL13 (15/
20 CD30
+
cases wer e CXCL13
+
vs. 1/9 CD30
cases;
P = 0.003), IRF4/MUM1 (15/21 CD30
+
cases were
IRF4/MUM1
+
vs. 0/10 CD30
cases; P<0.001), and
possibly LMP1 (13/18 CD30
+
cases were LMP1
+
vs. 3/9
CD30
cases; P = 0.10) expression.
Molecular Genotypic Studies
Clonal PCR-based TCR gene rearrangement was
identified in 3 of 6 T-ENKTLs and 3 of 17 NK-
ENKTLs (Table 4). The DNA was of suboptimal quality
in 5 of 6 T-ENKTLs tested, including in all the negative
cases, and in 5 of 11 NK-ENKTLs tested, using the
BIOMED-2 primers. The 3 TCR-rearranged NK-ENKTL
cases (nasal—1; skin—1; and intestinal—1) were male,
none had a small cell component, and all had >60 mitoses/
10 HPF. They had the following phenotypic features:
CD56
+
(3), CD5
(3), IRF4/MUM1
+
(2), CXCL13
+
(1),
PD-1
(1), Oct-2
(1), and CD25
+
(1). None received
radiation therapy, and all died within 3 months.
Clinical Follow-up and Prognostic Indicators
The estimated median survival for 57 cases with
follow-up data was 5 months with a 1-year survival rate
of 35%. Statistically significant adverse prognostic in-
dicators included a primary non-UADT site (intestine—5;
skin—2; deep soft tissue—1), high-stage disease, bone
marrow involvement, IPI score Z 2, Ki67 >40%, and
CD25 expression (Table 5; Fig. 6). Among the UADT
ENKTL cases, the cases of NK versus T-cell origin
TABLE 2. Pathologic Features of Extranodal ENKTL Subtypes and gd EATL
ENKTL Defined by TCR Expression ENKTL Subgroups and cd EATL
Characteristic
Overall
ENKTL* NK cd Tw ab Tw ab/cd T
UADT
NK-
ENKTL
Cutaneous
NK-
ENKTLz
Intestinal
ENKTL cd EATLy
No. of Cases/Total Cases (%)
Size of lymphoma cells
Predominantly small
cells
3/62 (5) 2/45 (4) 1/3 (33) 0 0 2/40 (5) 0 0 0
Mixed small and
larger cells
9/62 (15) 4/45 (9) 1/3 (33) 1/2 (50) 0 4/40 (10) 0 0 0
Predominantly
medium-sized cells
23/62 (37) 18/45 (40) 0 1/2 (50) 0 17/40 (43) 0 1/5 (20) 5/5 (100)
Mixed medium-sized
and large cells
14/62 (23) 10/45 (22) 1/3 (33) 0 0 10/40 (25) 0 1/5 (20) 0
Predominantly large
cells
13/62 (21) 11/45 (24) 0 0 1/1 (100) 7/40 (18) 2/2 (100) 3/5 (60) 0
Predominantly irregular
nuclei
45/62 (73) 34/45 (76) 2/3 (67) 1/2 (50) 1/1 (100) 33/40 (83) 1/2 (50) 2/5 (40) 0/5 (0)
Frequent elongate nuclei 45/60 (75) 32/43 (74) 3/3 (100) 1/2 (50) 1/1 (100) 31/38 (82) 0/2 (0) 2/5 (40) 0/5 (0)
Necrosis 53/67 (79) 39/47 (83) 2/3 (67) 2/2 (100) 1/1 (100) 35/42 (83) 1/2 (50) 5/5 (100) 5/5 (100)
Ulcer edge with many
PMNs
42/62 (68) 31/45 (69) 2/3 (67) 2/2 (100) 1/1 (100) 28/40 (40) 1/2 (50) 4/5 (80) 5/5 (100)
Angioinvasion 30/67 (45) 22/47 (47) 1/3 (33) 2/2 (100) 1/1 (100) 17/42 (41) 2/2 (100) 4/5 (80) 5/5 (100)
Thrombosis 4/61 (7) 4/45 (9) 0/3 (0) 0/2 (0) 0/1 (0) 4/40 (10) 0/2 (0) 0/5 (0) 1/5 (20)
Epithelial hyperplasia 5/55 (9) 4/39 (10) 0/3 (0) 0/2 (0) 0/1 (0) 4/35 (11) 0/1 (0) 0/5 (0) 0/5 (0)
Epitheliotropism 23/55 (42) 17/39 (44) 0/3 (0) 2/2 (100) 0/1 (0) 16/35 (46) 1/1 (100) 0/5 (0) 19/46 (41)
Admixed reactive B cells
>10%
5/58 (9) 3/43 (7) 0/3 (0) 1/2 (50) 0/1 (0) 3/39 (8) 0/1 (0) 0/5 (0) 0/5 (0)
Admixed reactive T cells
>10%
23/56 (41) 13/42 (31) 1/1 (50) 2/2 (100) 0/1 (0) 13/38 (34) 0/1 (0) 1/5 (20) 0/5 (0)
Mitoses/10 HPF [median,
range]
[66, 2-248] [68, 2-248] [25, 17-42] [69, 8-103] [87] [68, 2-248] [73, 65-81] [77, 35-155] [92, 67-148]
Mitoses >60/10 HPF 29/56 (52) 23/41 (56) 1/2 (50) 0/3 (0) 1/1 (100) 18/36 (50) 2/2 (100) 4/5 (80) 4/4 (100)
*Overall, ENKTL includes ENKTL of NK, T, and NOS type.
wWhen compared with UADT NK-ENKTL, UADT T-ENKTL (includes T-ENKTL except for the non-UADT double-positive case) had more cases with a small cell
component (P = 0.042).
zWhen compared with UADT NK-ENKTL, the cutaneous NK-ENKTL had less frequent elongate nuclei (P = 0.046).
yWhen compared with intestinal ENKTL, gd EATL more often had predominantly medium-sized cell morphology (P = 0.048); when gd EATL were compared with
the gd T-ENKTL, they had elongate nuclei less frequently ( P = 0.018) and > 60 mitoses/10 HPF more frequently (P = 0.029).
NOS indicates not otherwise specified.
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showed a trend for worse survival (P = 0.092).
Significantly better survival was observed in patients who
received combined chemoradiation therapy, compared
with those who received only chemotherapy. All patients
receiving radiation had low-stage disease (and none had
CD25 expression), but the significance was retained when
FIGURE 1. ENKTL, histopathology. A, Note the dense lymphoid infiltrate with a large area of geographic necrosis. B, Prominent
angioinvasion of a muscular artery is present. C, Although the neoplastic cells are predominantly small, they have irregular and
elongate nuclear contours. D, The neoplastic cells are predominantly medium-sized but still irregular and elongate. E, Note the
prominent mitotic figures in this case with predominantly large cells. F, Some of the neoplastic cells demonstrate coarse eosi-
nophilic granules in their abundant pale cytoplasm (all cases of presumptive NK type except for C, which is of gd type, hema-
toxylin and eosin stains).
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FIGURE 2. NK-ENKTL, phenotypic features. A, The EBER ISH stain for EBV demonstrates numerous cells with nuclear positivity, as
well as cytoplasmic staining in cells undergoing mitosis. B, The neoplastic cells are positive for CD2, but (C) CD3 and (D) CD5
are only present on some presumptive normal T cells. E, CD56, (F) CD30, (G) CXCL13, and (H) IRF4/MUM1 are all positive.
The CXCL13 shows typical punctate staining. (A), EBER ISH; (B–H), immunoperoxidase stains, all with hematoxylin
counterstain.
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FIGURE 3. gd T-ENKTL, histopathologic and phenotypic features. A, The dense lymphoid infiltrate with a large area of geographic
necrosis is indistinguishable from an NK-ENKTL. B, Cytologically, this case with mixed medium-sized and large cells also has
irregular and elongate nuclei. C, The EBER ISH stain for EBV shows numerous positive nuclei. D, The neoplastic cells are also
positive for CD2 and (E) CD56 but (F) are negative for CD5. G, Although somewhat difficult to interpret, at least a significant
proportion of the atypical cells expressed TCRg and (H) TCRd. (A, B) hematoxylin and eosin stains; (C) EBER ISH with hematoxylin
counterstain, (D–H) immunoperoxidase with hematoxylin counterstain).
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FIGURE 4. ab T-ENKTL with clonal TCRg gene rearrangement, phenotypic features. A, There are many scattered EBV
+
neoplastic
cells, which are also positive for (B) CD3, (C) CD5, and (D) CD4, but negative for (E) CD8 and (F) CD56. G. There are many
scattered bF1
+
cells, including some larger cells, but (H) there are only infrequent TCRg
+
cells. (A) EBER ISH; (B–H) immuno-
peroxidase with hematoxylin counterstain.
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FIGURE 5. ab/gd T-ENKTL, intestinal: histopathologic and phenotypic features. A, The infiltrative mass involves only a very short
segment of bowel and yet is transmural and deeply ulcerated. B, There is prominent angioinvasion, which was most frequent
around the ulcer edge. C, The neoplastic cells are EBV, (D) TCRg, (E) TCRb, and (F) CLA
+
. (A, B) Hematoxylin and eosin stains; (C)
EBER ISH; (D–F) immunoperoxidase, all with hematoxylin counterstain.
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only low-stage cases and NK-UADT cases were analyzed.
Stage, bone marrow involvement, IPI score, and CD25
expression retained their significance in the UADT NK
group.
UADT ENKTL: Comparison of Those With
and Without TCR Expression
Patients with T-ENKTL showed a trend for a lower
M:F ratio (1.5:1 vs. 5:1, P = 0.057). The T-ENKTL also
included a higher proportion of cases with a small
cell component (3/5 vs. 6/40 UADT NK-ENKTLs;
P = 0.047) (Table 2) and showed a trend for a Ki67
proliferative index of r40% (4/4 vs. 8/18 UADT NK-
ENKTLs; P =0.096).
CD56 expression was more common in NK cases
(84%) compared with T-ENKTL (20%, P = 0.008), gd
T-ENKTL (33%, P = 0.091), and ab T-ENKTL (0%,
P = 0.036) cases (Table 3). Both ab T-ENKTL cases were
CD56
/CD5
+
, with one CD4
+
and the other CD8
+
. All
other cases were CD5
and CD4
. CXCL13 (58%),
IRF4/MUM1 (50%), CLA (50%), and Oct-2 (42%)
were present in a subset of NK- ENKTLs but not in any
T-ENKTLs (differences not significant; P = 0.098 for
CXCL13).
TABLE 3. Phenotypic Features of ENKTL Subtypes and gd EATL
ENKTL Defined by TCR Expression ENKTL Subgroups and cd EATL
Characteristic
Overall
ENKTL* NKw cd Tz ab Ty ab/cd T
UADT
NK-
ENKTL8
Cutaneous
NK-
ENKTL
Intestinal
ENKTLz cd EATL#
No. of Cases/Total Cases (%)
Pan T-cell antigens
CD2 32/36 (89) 24/28 (86) 3/3 (100) 1/1 (100) 1/1 (100) 21/24 (88) 1/1 (100) 3/4 (75) 3/5 (60)
CD3 65/67 (97) 45/47 (96) 3/3 (100) 2/2 (100) 1/1 (100) 40/42 (95) 2/2 (100) 5/5 (100) 5/5 (100)
CD5 3/66 (5) 0/47 (0) 0/2 (0) 2/2 (100) 0/1 (0) 0/42 (0) 0/2 (0) 0/5 (0) 0/5 (0)
CD7 15/33 (45) 13/27 (48) 1/3 (33) ND 0/1 (0) 10/23 (44) 0/1 (0) 3/4 (75) 5/5 (100)
T-/NK-cell antigen subset
CD4 1/60 (2) 0/47 (0) 0/3 (0) 1/2 (50) 0/1 (0) 0/42 (0) 0/2 (0) 0/5 (0) 0/5 (0)
CD8 6/63 (10) 4/47 (9) 0/3 (0) 1/2 (50) 1/1 (100) 3/42 (7) 1/2 (50) 1/5 (20) 2/5 (40)
CD16 0/31 (0) 0/25 (0) 0/3 (0) 0/1 (0) 0/1 (0) 0/22 (0) ND 0/4 (0) 3/5 (60)
CD56 44/56 (79) 35/42 (83) 1/3 (33) 0/2 (0) 1/1 (100) 32/38 (84) 2/2 (100) 3/4 (75) 4/5 (80)
TIA-1 65/65 (100) 47/47 (100) 3/3 (100) 2/2 (100) 1/1 (100) 42/42 (100) 2/2 (100) 5/5 (100) 5/5 (100)
Granzyme B 51/54 (94) 39/42 (93) 2/2 (100) 1/1 (100) 1/1 (100) 35/38 (92) 1/1 (100) 4/4 (100) 2/4 (50)
CXCL13 17/33 (52) 16/27 (59) 0/3 (0) 0/1 (0) 0/1 (0) 14/24 (58) ND 2/4 (50) 0/5 (0)
PD1 2/30 (7) 0/23 (0) 1/3 (33) 0/1 (0) 1/1 (100) 0/20 (0) ND 1/4 (25) 1/5 (20)
CLA 12/28 (43) 10/21 (48) 0/3 (0) 0/1 (0) 1/1 (100) 9/18 (50) ND 2/2 (50) 0/5 (0)
EBV-associated markers
EBER ISH 67/67 (100) 47/47 (100) 3/3 (100) 2/2 (100) 1/1 (100) 42/42 (100) 2/2 (100) 5/5 (100) 0/5 (0)
LMP1 18/32 (56) 13/25 (54) 2/3 (67) 1/2 (50) 1/1 (100) 11/21 (52) ND 3/4 (75) 0/5 (0)
EBNA2 0/35 (0) 0/28 (0) 0/3 (0) 0/1 (0) 0/1 (0) 0/25 (0) ND 0/4 (0) 0/5 (0)
Miscellaneous
CD45 18/29 (62) 14/23 (61) 1/3 (33) ND 1/ 1 (100) 13/20 (65) ND 2/4 (50) 5/5 (100)
CD45RA 0/34 (0) 0/27 (0) 0/3 (0) 0/1 (0) ND 0/24 (0) 0/1 (0) 0/2 (0) 2/5 (40)
CD30 41/57 (72) 32/43 (74) 1/3 (33) 1/1 (100) 1/1 (100) 27/38 (71) 2/2 (100) 5/5 (100) 0/5 (0)
ALK 0/42 (0) 0/33 (0) 0/3 (0) 0/1 (0) 0/1 (0) 0/28 (0) 0/2 (0) 0/4 (0) 0/5 (0)
CD25 11/31 (35) 9/25 (36) 0/3 (0) 1/1 (100) 1/1 (100) 8/22 (36) ND 2/4 (50) 0/5 (0)
pSTAT3 32/33 (97) 26/27 (96) 3/3 (100) 1/1 (100) 1/1 (100) 23/24 (96) ND 4/4 (100) 1/5 (20)
Lsk (MATK) 31/35 (87) 26/28 (93) 3/3 (100) 0/1 (0) 1/1 (100) 22/24 (92) 1/1 (100) 4/4 (100) 5/5 (100)**
IRF4/MUM1 17/35 (49) 15/28 (54) 0/3 (0) 0/1 (0) 1/1 (100) 12/24 (50) 1/1 (100) 3/4 (75) 0/5 (0)
Oct-2 8/26 (31) 8/21 (38) 0/3 (0) 0/1 (0) 0/1 (0) 8/19 (42) ND 0/3 (0) 1/5 (20)
CD20 0/67 (0) 0/47 (0) 0/3 (0) 0/2 (0) 0/1 (0) 0/42 (0) 0/2 (0) 0/5 (0) 0/5 (0)
Ki67 >40% 13/25 (52) 12/19 (63) 0/3 (0) 0/1 (0) 1/1 (100) 10/18 (56) ND 2/2 (100) 4/5 (80)
p53 >10% 21/31 (68) 18/25 (72) 1/3 (33) 0/1 (0) 1/1 (100) 15/22 (68) ND 4/4 (100) 5/5 (100)
*Overall, ENKTL includes ENKTL of NK, T, and NOS type.
wThe NK-ENKTL, when compared with T-ENKTL, had more frequent CXCL13 expression (P = 0.043) but less frequent PD1 expression (P = 0.026).
zThe gd T-ENKTL (all UADT), when compared with UADT NK-ENKT L, had less frequent CD56 expression (P = 0.092).
yThe ab T-ENKTL (all UADT), when compared with the UADT NK-ENKTL, had more frequent CD5 expression ( P = 0.001), more frequent CD4 expression
(P = 0.045), and less frequent CD56 expression (P = 0.036).
8The UADT T-ENKTL (only the ab/gd T-cell case excluded) had less frequent CD56 expression compared with UADT NK-ENKTL (P = 0.008).
zThe intestinal ENKTL, when compared with the gd EATL, had more frequent expression of CD30 (P = 0.008), pSTAT3 (P = 0.048), and IRF4/MUM1 (P = 0.048)
but less frequent CD16 expression (P = 0.048).
#The gd EATL, when compared with UADT NK-ENKTL, had more frequent expression of CD7 (P = 0.044), CD16 (P = 0.003), and CD45RA ( P = 0.025) but less
frequent expression of CXCL13 (P = 0.042), CD30 (P = 0.005), and pSTAT3 (P = 0.001). There are also trends for more frequent CD8 expression (P = 0.081) but less
IRF4/MUM1 expression (P = 0.059) in the gd EATL.
**Of the type II EATLs, 2 had strong Lsk expression in
˜
90% of cells, and 2 had weaker staining in 50% to 60%. The type I EATL had only approximately 20% weakly
Lsk
+
cells.
NOS indicates not otherwise specified.
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Comparisons of cd T-ENKTL and Intestinal
ENKTL With Enteropathy-associated T-cell
Lymphoma of cd Origin
Although meaningful comparisons are limited be-
cause of the small number of cases, gd T-ENKTL oc-
curred in younger individuals compared with gd EATL;
none were in the intestine, none had a monotonous me-
dium-sized neoplastic cell morph ology, and all were
EBV
+
. There were also some possible phenotypic and
clinical differences (Tables 3 and 5). Patients with an
intestinal ENKTL also were younger compared with
those with gd EATL (P = 0.039) and included 3 of 5 cases
that arose in the large intestine. In contrast to gd EATL,
80% of the intestinal ENKTL included at least a large cell
component and lacked prominent e pitheliotropism. Two
perforated intestinal ENKTLs had a small or grossly
undetectable tumor mass, extensive arterial invasion,
and deep ulceration (Fig. 5A), whereas the gd EATL
generally had larger expansile masses. In addition to their
invariable EBV positivity, compared with gd EATL, the
intestinal ENKTL had significantly more frequent CD30,
IRF4/MUM1, and pSTAT3 expression but less frequent
CD16 expression (Table 3).
DISCUSSION
ENKTL cases from Thailand are similar to those
reported from other geographic locations in terms of their
basic histopathology, age distribution, male predom-
inance, frequent low stage with predominantly UADT
disease, particularly in the nasal cavity, and with the most
common extranasal sites being the intestine and skin
(Supplemental Digital Contents 2 to 4, http://links.
lww.com/PAS/A109).
1,5–7,22,31,40,43,52,54,58,64,80,82
Similar
to most other Asian studies,
17,52,55
overall survival was
poor, unlike an American study that reported a 10-year
median survival.
22
A more recent study from the United
States that reported a 35% 5-year survival found a much
better survival in whites compared with ethnically Asian
patients.
1
Adverse prognostic indicators included in-
testinal/non-UADT presentations, high stage, bone mar-
row invo lvement, higher IPI scores, and lack of radiation
as part of the therapeutic regimen.
6,14,46
The more ag-
gressive non-UADT ENKTLs were significantly asso-
ciated with high-stage disease,
6
IPI score of Z 2, and
fewer cases with nuclear irregularity and elongate nuclei.
They also did not include any cases with a small cell
component.
The minority of patients whose ENKTL had a small
cell component showed a trend for a better prognosis.
Although the literature is not clear-cut, most studies have
not suggested a “small cell variant” to be at least an in-
dependent prognostic indicator.
7,54
More importantly,
cases with mostly small cells and cases that have prom-
inent neutrophils or many non-neoplastic small lympho-
cytes and/or plasma cells can mimic an acute or chronic
inflammatory process.
14
The impact of tumor-infiltrating
lymphocytes in ENKTL remains to be establ ished; how-
ever, cases with >10% of admixed reactive T cells were
more likely to have a lower mitotic count and showed a
trend for a lower Ki67. Rare cases showed coarse gran-
ules in the hematoxylin and eosin-stained sections with
finer granules in some others. Although it is well known
that granules are seen in ENKTL in Wright-Giemsa-
stained smears, it has been suggested that the lack of
granules in histologic sections of an NK-derived pro-
liferation is a feature that helps distinguish ENKTL from
“lymphomatoid gastropathy,”
86
an indolent NK-cell–de-
rived proliferation similar to “NK-cell enteropathy.”
61
Most prior studies have evaluated the cell of origin
in ENKTL strictly using PCR -based TCR gene re-
arrangement analysis,
6,52,64
even though TCR gene re-
arrangements can be present in normal B cells and
NK cells, in reactive proliferations, and in B-cell neo-
plasms.
33,37,53,70,88,93,94
The proportion of ENKTLs re-
ported to be of T-cell origin on the basis of PCR-based
studies ranges from 0% to 38%, with about 8% based on
a Southern blot analysis study.
6,27,40,52,59,64,66,82
A gene
expression profiling study reports that 11% of ENKTLs
are of gd T-cell type.
45
Very little information exists on TCR expression in
ENKTL, although a recent series reports 0 of 74
ENKTLs with either g or b TCR protein expression, even
though there were 18% CD5
+
cases and almost 10% had
a TCR-g rearrangement.
40
In contrast, 11% of the
evaluable ENKTLs in this study expressed one or more
TCRs, defining at least this subset as being of T-cell ori-
gin. They included 3 of gd type, 2 of ab type, and 1 ex-
pressed g , d, and b TCR proteins. Whether this latter case
represents aberrant coexpression of TCR proteins or
spurious staining is uncertain, but ab/gd double-positive
T-cell lymphomas (TCL) have been reported, including
TABLE 4. PCR-based TCR Gene Rearrangement Analysis of ENKTL of NK-cell and T-cell Types
No. of Cases/Total Cases (%)
PCR-based TCR Gene Rearrangement Analysis NK cd T ab T ab/cd T
Overall positive cases 3/17 (18%) 1/3 (33%) 1/2 (50%) 1/1 (100%)
TCRg gene 2/17 1/3 1/2 1/1
TCRb gene* 1/10 0/3 0/1 ND
Suboptimal DNAw 5/11 (45%) 2/3 (67%) 2/2 (100%) 1/1 (100%)
*TCRb gene rearrangement analysis was performed only in TCRg gene rearrangement-negative cases, except for the 1 TCRg-rearranged gdTCR
+
case.
wQuality of DNA was evaluated only in cases studied with BIOMED-2 primers. Cases with suboptimal DNA quality had internal control housekeeping gene amplicon
size ladders of 200 bp. Cases with size ladders <200 bp were excluded from this analysis. The 2 gdTCR
+
with suboptimal DNA were the 2 negative cases.
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4 TCLs of uncertain type, 3 cutaneous cytotoxic neo-
plasms with high-grade cytology, and 3 EBV
type II
EATLs.
13,69,97
The rapidly fatal ab/gd case reported here
was an EBV
+
, CD56
+
, CD8
+
, CD5
, CD7
perforated
ileal neoplasm with adenopathy but no skin disease. Al-
though TCR expression is better evidence of a T-cell
phenotype compared with PCR studi es, because TCR-
silent TCLs are also well known,
11,18,32,35
the possibility
that some of our presumptive NK cases represent CD5
“receptor silent” T-cell neoplasms must be seriously
considered and could potentially mask differences be-
tween the T and NK groups. It is of interest that one of
the presumptive NK-ENKTLs with a clonal TCR re-
arrangement had a b but not g rearrangement, which is
a very infrequent occurrence in neoplastic T cells.
92
The
limited number of TCR-silent, TCR-gene–rearranged
TABLE 5. Clinicopathologic Correlations With Overall Survival
Factors Total Cases 6-mo Survival (%) 1-yr Survival (%) Median Survival (mo) P (Log Rank)
Overall cases 57 47 35 5
Site of ENKTL
Nonintestinal 52 52 40 7 <0.001
Intestinal 5 0 0 0
Site of ENKTL
UADT 49 57 43 8 <0.001
Non-UADT 8 0 0 1
Stage
Low (I and II) 26 81 69 65 <0.001*
High (III and IV) 14 14 0 3
BM involvement
Present 3 0 0 1 <0.001
Absent 25 64 48 9
IPI score
0 15 100 93 NR 0.001 (vs. 1)*
1 7 57 43 7 0.043 (vs. 2-4)
2-4 15 20 7 3 <0.001 (vs. 0)*
Treatment (all stages)
Chemoradiotherapy 14 100 100 65 r0.001 (all comparisons)*
Chemotherapy only 22 41 18 4
None of the above 6 0 0 1
Treatment in stages I and II disease
Chemoradiotherapy 14 100 100 65 <0.001*
Chemotherapy only 10 70 40 8
Morphologic subtypes
With small cell component 9 78 53 NR 0.073w
Without small cell component 45 40 29 4
IRF4/MUM1 expression
Positive 17 29 24 4 0.054w
Negative 16 67 43 8
CD56 expression
Positive 37 46 29 4 0.064z
Negative 12 58 58 65
CD25 expression
Positive (Z10% cells) 10 20 0 2 0.001*
Negative 20 70 55 16
Ki67
>40% 12 42 17 4 0.037y
r40% 12 67 60 NR
Admixed reactive T cells
>10% 19 53 53 NR 0.0718
r10% 30 47 22 4
UADT ENKTL
NK origin 38 58 39 7
T origin, overall 5 80 80 NR 0.092 (vs. NK)
ab T-cell origin 2 100 100 NR 0.143 (vs. NK)
gd T-cell origin 3 67 67 NR 0.382 (vs. NK)
Intestinal lymphoma
Intestinal ENKTL 5 0 0 0 0.213
gd EATL 5 40 r20 2
*Analysis of the UADT ENKTL (NK-ENKTL plus T-ENKTL) or UADT NK-ENKTL groups also showed a significant survival difference.
wNo significant difference or trend was found when only the UADT ENKTL (NK-ENKTL plus T-ENKTL) or UADT NK-ENKTL groups were analyzed.
zAnalysis of the UADT ENKTL (NK-ENKTL plus T-ENKTL) but not of the UADT NK-ENKTL group also showed a similar trend.
yAnalysis of the UADT ENKTL (NK-ENKTL plus T-ENKTL) but not of the UADT NK-ENKTL group also showed a significant difference.
8Analysis of the UADT ENKTL showed a significance difference, and analysis of the UADT NK-ENKTL showed a similar trend.
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FIGURE 6. Overall survival. A, Patients with ENKTL with low-stage disease had superior survival compared with those with high-
stage disease (P = 0.001). B, Patients treated with combined chemoradiotherapy had a superior survival compared with those
treated only with chemotherapy or those not treated at all (P < 0.001). C, ENKTLs with an UADT primary site were associated with
a much better prognosis than those arising elsewhere (P < 0.001). D, Because of the major impact of the primary site, comparisons
limited to the UADT showed a trend for a better survival in patients with T compared with presumptive NK-cell origin (P < 0.10). E,
Among the patients with UADT NK-ENKTL, CD25 expression (P = 0.006), and (F) possibly r10% admixed reactive T cells, were
associated with an adverse prognosis (P = 0.053).
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cases precludes a meaningful comparison with the other
ENKTL; however, almost all of the findings are typical of
the other NK-ENKTL. Although the TCR-gene–
rearranged cases showed an adverse prognosis compared
with the presumptive NK-ENKTL without a TCR re-
arrangement, 2 of the 3 were non-UADT, all had a high
mitotic count, and none received radiation therapy. The
phenotypically defined T-ENKTL showed a trend for
better survival, perhaps related to a higher proportion of
cases with a small cell component and the possible in-
creased proportion of c ases with fewer Ki67-positive cells.
Study of more cases would be required to better assess the
relationship of outcome to TCR rearrangement or ex -
pression and determine the influence of precis e T-cell type
on survival. ENKTLs with or without TCR gene re-
arrangement are reported to have similar clinical, histo-
pathologic, and phenotypic features with no difference in
survival.
6,52,64
Decreased sensitivity using paraffin-embedded ma-
terial plus suboptimal DNA might explain why only 3 of
6 ENKTLs with TCR expression showed a clonal TCR
rearrangement. A BIOMED-2 study showed a yield of
0 and 11% for TCRb and TCRg primers, respectively,
when paraffin samples with a maximal control gene PCR
product of <300 bp were compared with matched fresh/
frozen samples and only a yield of 80% and 60% with
better quality DNA.
92
The T-ENKTL shared many phenotypic features
with NK-ENKTL. As in many other studies, all cases
were EBV
+
, although a few studies have reported 7% to
13% EBER-negative cases.
22,52,80
The EBER stain is of
considerable diagnostic utility and is also an aid in eval-
uating other IHC stains to avoid interpreting admixed
T cells as neoplastic cells. About half of the cases had an
EBV latency patte rn type II (LMP1
+
, EBNA2
) with the
remaining cases being type I.
12,20
Although generally
considered to have a latency II pattern, some report only
8% to 21% LMP1
+
cases.
16,29,31,40,68,82
The true fre-
quency of latency II cases may be underestimated because
of technical factors related to staining and the sometimes
small proportion of LMP1
+
cells in positive cases.
14
Almost all cases were CD3
+
, with CD2 and par-
ticularly CD7 present in a smaller proportion of cases,
consistent with the literature.
5–7,31,40,43,54,55,64,80,82
The
explanation for why some report only 73% to 82%
CD3
+
cases is uncertain.
6,64
Except for 2 ab T-ENKTL,
all the ENKTLs, including the gd-TCR
+
cases and a
reported ab T-ENKTL were CD5
.
42
Thus, absence of
CD5 is not a surrogate for an NK origin. Although most
others also report few, if any, CD5
+
ENKTL cases,
3 groups report 27% to 38% CD5
+
cases.
5,31,80
Similarly,
although the solitary CD4
+
ENKTL case was of ab
T-ENKTL origin, CD4
is also a feature of both NK and
T-cell cases with CD4 expression reported in 0% to 10%
of ENKTLs.
6,31,55,64,106
Although one series reported
75% ENKTLs to be CD8
+
, most series, consistent with
our results, report 9% to 22% positivity.
6,7,31,43,55,64,106
ENKTLs of all types usually have an activated cytotoxic
phenotype with uniform TIA-1 expression and frequent
granyzme B positivity.
6,31,43,52,64,80
Consistent with the
concept that ENKTLs often have an “activated” pheno-
type, almost 3/4 of cases expressed CD30, including both
NK an d T-cell cases. Other series have reported 19% to
63% of CD30
+
ENKTLs.
6,31,40,43,52,54,58,80
CD30 ex-
pression was associated with CXCL13 and IRF4/MUM1
expression. CXCL13, a chemokine that attracts B cells
into lymphoid follicles,
26,95
is largely known for its ex-
pression in T-follicular helper cells
25,83,84,95,104
but was
expressed in 59% of NK-ENKTLs and in 1 of 7 and 6
of 11 reported ENKTLs.
104,106
IRF4/MUM1, found in
about half of our cases, may also reflect “activation”
and also usually paralleled CD30 expression in a study
of peripheral TCL.
28,41
It has not been previously re-
ported in normal NK cells and, with the exception of a
recent Thai study, only in a single NK lymphoma.
63,71
CD25, the IL-2 receptor that is another activation marker
and a therapeutic targe t, has been reported only rarely in
ENKTL but was found in about 1/3 of our ENKTL.
49,50
Although not correlated with CD30, it did appear to
correlate with CXCL13 and IRF4/MUM1 at least among
the group as a whole (not all data shown). It is the only
phenotypic marker documented in this study with prog-
nostic implications. Caution is advised, however, as CD25
expression was associ ated with an absence of radiation
therapy, another adverse prognostic indicator.
pSTAT3, involved with oncogenesis and in creating
an immunosuppressive microenvironment in ENKTL as
well as being another potential therape utic target,
79
was
almost universally expressed in ENKTLs.
10,21,42,79,91
Similar to our findings, Lsk (MATK) was recently re-
ported to be a 100% sensitive marker for ENKTL and
type II EATL.
87
A recent gene expression profiling study
also demonstrated Lsk expression in both ENKTL and gd
peripheral TCL, but not in ab peripheral TCL.
45
Although not a distinguishing feature and, in contrast
to one previously published report,
43
it is of interest that
about 40% of ENKTLs lacked CD45 expression. Ex-
pression of the p53 tumor suppressor gene, previously
reported in 37% to 86% of ENKTLs, was found in about
2/3 of cases.
43,57,64,72,73
It was associated with higher stage
and CLA expression but not with survival differences.
Major phenotypic differences were also found be-
tween the T cell and other ENKTLs, in addition to their
TCR protein expression. CD56 expression, a hallmark of
ENKTL, reported in 37% to 100% of cases, was sig-
nificantly less common in T-ENKTL s.
6,43,52,58,80
At least
in part, the wide range of reported CD56
+
cases un-
doubtedly reflects technical factors. CD16, another NK-
associated antigen, was always negative here and in
2 other series but has been reported in up to 14% to 38%
of cases using flow cytometry or frozen-section
IHC.
31,49,55,82
In contrast to NK-ENKTLs and some other types
of TCLs, all T-ENKTLs were CXCL13
(discussed
above) and Oct-2
. Oct-2, a lymphocyte-specific tran-
scription factor associated with immunoglobulin produc-
tion regulation and promoted as a “B-cell marker,”
19
was
present in about 1/3 of the NK-ENKTLs. Very few Oct-2
+
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cases have been reported previously, except for a recent
smaller Thai study with 25% Oct-2
+
ENKTL.
62,71,78
PD-
1, a marker of T-follicular helper cells, which is also ex-
pressed in varied T-cell subsets and some activated NK
cells,
8,24,38,39,47,74,76,102 ,104
was only identified in 2 cases of T-
ENKTL, both gd ± ab TCR
+
. It has been reported in 1 of
8 “NK” lymphomas but in none diagnosed as ENKTL.
76,106
The only T-ENKTL that was CLA
+
or IRF4/
MUM1
+
was the ab/gd
+
case. CLA is expressed in
T lymphocytes (particularly cutaneou s T cells), NK cells,
high endothelial venules, peripheral monocytes, gran-
ulocytes, and a small proportion of memory B cells.
60,65,90
Among TCLs, it is more frequently expressed in those
involving the skin.
60,65
However, it has also been reported
in a subset of ENKTLs,
15,80,103
including half of the
UADT NK-ENKTL cases in our series. In contrast to
one study that found CLA to be associated with cuta-
neous involvement and an adverse prognosis,
103
we could
not document any prognostic implications.
ENKTL and EATL are both aggressive lymphomas
that share some clinicopathologic and immunophenotypic
features. Particularly, type II EATL, which is also more
common among Asians, can have a gd T-cell ori-
gin.
2,6,13,23,81,85,96,101
This raises the possibility that in-
testinal ENKTL is just an EBV
+
type II EATL. There
are, however, major differences, aside from EATL cases
being generally EBV
and uniformly of T-cell origin.
23,96
ENKTLs occur in both the small and large intestine,
whereas many EATLs occur in the small intestine.
23,51,52,81
Type II EATLs are typically composed of monomorphic
medium-sized cells, whereas ENKTLs not infrequently
have elongate and irregular nuclei and are more likely to
include larger cells. Virtually all type II EATLs have fo-
cally marked epitheliotropism
13,23,85,101
but not intestinal
ENKTL. Angioinvasion is often more prominent in the
ENKTL, which sometimes leads to small but perforated
intestinal masses.
The skin is another important site for non-UADT
ENKTL, although cutaneous ENKTLs constitute only a
small proportion of skin lymphomas or ENKTLs and are
often associated with extracutaneous disease at pre-
sentation or soon afterward.
4,30,98,103,105
Survival of pa-
tients with cutaneous ENKTL is poor, with a median/
mean survival of about 7 to 13 months.
4,9,105
Cases lo-
calized to the skin are report ed to have a better prognosis
in some but not all studies.
6,9,17,100,105
Consistent with the
results here, prior studies have demonstrated that cuta-
neous and UADT ENKTLs have many similarities, and
an array-based comparative genomi c hybridization study
of cutaneou s ENKTL revealed genetic abnormalities
9,44
that are also found in NK-cell malignancies, suggesting
more similarities than differences. However, they are re-
ported to cluster separately on the basis of their lower rate
of expression of CD54 and more frequent CLA ex-
pression, and one study reports 3 of 9 EBV
cas-
es.
4,67,103,105
Cutaneous ENKTLs, which in this and one
other study were not of gd type,
67
also share some sim-
ilarities with primary cutaneous gd TCL (PCgdTCL).
Both commonly present with ulcerated masses that
demonstrate angioinvasion, necrosis, a similar basic par-
affin section immunophenotype, and a poor prog-
nosis.
34,89,99
Although considered to be EBV
by most,
EBV
+
PCgdTCL cases have been reported.
34,67,89
In conclusion, at least ab out 10% of ENKTLs are
of T-cell origin, based on their TCR expression. Some
represent a type of mucosal gd TCL that is distinguished
partly on the basis of its EBV positivity, but others are of
ab type with one coexpressing g,d and b TCR. A minority
of the remaining cases may also be of T-cell origin but
TCR “silent.” Despite a different “cell of origin,” and
some phenotypic and possible clinical differences, the
many clinicopathologic similarities between ENKTL of
T-cell and NK-c ell type do not support distinguishing
these two groups for clinical purposes at this time.
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    • "Quintanilla et al reported 60% and 86% of p53-positive cases in Mexican and Peruvian populations, respectively [35], [41]. Pongpruttipan et al [29] found 68% of their cases from the Thai population to be positive, while Ye et al [42] found 33.3% of cases to be positive in the Chinese population. The latter two groups exhibited close correlation between p53 expression and both prognosis and advanced disease stage. "
    [Show abstract] [Hide abstract] ABSTRACT: NK/T-cell lymphoma (NKTCL) is the most frequent EBV-related NK/T-cell disease. Its clinical manifestations overlap with those of familial haemophagocytic lymphohistiocytosis (FHLH). Since PERFORIN (PRF1) mutations are present in FHLH, we analysed its role in a series of 12 nasal and 12 extranasal-NKTCLs. 12.5% of the tumours and 25% of the nasal-origin cases had the well-known g.272C>T(p.Ala91Val) pathogenic SNP, which confers a poor prognosis. Two of these cases had a double-CD4/CD8-positive immunophenotype, although no correlation was found with perforin protein expression. p53 was overexpressed in 20% of the tumoral samples, 80% of which were of extranasal origin, while none showed PRF1 SNVs. These results suggest that nasal and extranasal NKTCLs have different biological backgrounds, although this requires validation.
    Full-text · Article · Mar 2014 · PLoS ONE
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    • "Europeans have minor allele frequencies of 0.33, whereas African Americans have frequencies of 0.50, and Asians 0.42 for the allele of a single nucleotide polymorphism (rs 2392542) for TCRG.23 N-NK/T showed clonality for TCR in eight out of 12 (66.7%), which was higher than those of a Thai group (20/67, 30%) and a Taiwan study (0/8, 0%).17,18 Of seven ALCL cases, five (71.4%) had clonality for TCR, which was similar to data from a European study that reported clonality in 34 out of 43 (79.0%).12 "
    [Show abstract] [Hide abstract] ABSTRACT: A clonality test for immunoglobulin (IG) and T cell receptor (TCR) is a useful adjunctive method for the diagnosis of lymphoproliferative diseases (LPDs). Recently, the BIOMED-2 multiplex polymerase chain reaction (PCR) assay has been established as a standard method for assessing the clonality of LPDs. We tested clonality in LPDs in Koreans using the BIOMED-2 multiplex PCR and compared the results with those obtained in European, Taiwanese, and Thai participants. We also evaluated the usefulness of the test as an ancillary method for diagnosing LPDs. Two hundred and nineteen specimens embedded in paraffin, including 78 B cell lymphomas, 80 T cell lymphomas and 61 cases of reactive lymphadenitis, were used for the clonality test. Mature B cell malignancies showed 95.7% clonality for IG, 2.9% co-existing clonality, and 4.3% polyclonality. Mature T cell malignancies exhibited 83.8% clonality for TCR, 8.1% co-existing clonality, and 16.2% polyclonality. Reactive lymphadenitis showed 93.4% polyclonality for IG and TCR. The majority of our results were similar to those obtained in Europeans. However, the clonality for IGK of B cell malignancies and TCRG of T cell malignancies was lower in Koreans than Europeans. The BIOMED-2 multiplex PCR assay was a useful adjunctive method for diagnosing LPDs.
    Full-text · Article · Oct 2013 · The Korean Journal of Pathology
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    • "It is derived from either activated NK cells or, rarely, cytotoxic T-cells [17]. If a patient with NK or T-cell tumors has unusual reactions to treatment or unusual prognosis [18], it is better to differentiate the NK tumors from the T-cell tumors in our opinion. Simultaneously, several studies have demonstrated that it is necessary to elucidate the origin of NK/T cell lymphoma [18,19]. "
    [Show abstract] [Hide abstract] ABSTRACT: Extranodal natural killer (NK)/T-cell lymphoma, nasal type, is an uncommon lymphoma associated with the Epstein-Barr virus (EBV). It most commonly involves the nasal cavity and upper respiratory tract. Primary pulmonary NK/T cell lymphoma is extremely rare. If a patient with a NK or T-cell tumor has an unusual reaction to treatment or an unusual prognosis, it is wise to differentiate NK from T-cell tumors. The clinicopathologic characteristics, immunophenotype, EBV in situ hybridization, and T cell receptor (TCR) gene rearrangement of primary pulmonary NK cell lymphoma from a 73-year-old Chinese woman were investigated and the clonal status was determined using female X-chromosomal inactivation mosaicism and polymorphisms at the phosphoglycerate kinase (PGK) gene. The lesion showed the typical histopathologic characteristics and immunohistochemical features of NK/T cell lymphoma. However, the sample was negative for TCR gene rearrangement. A clonality assay demonstrated that the lesion was monoclonal. It is concluded that this is the first recorded case of genuine primary pulmonary NK cell lymphoma. The purpose of the present work is to recommend that pathologists carefully investigate the whole lesion to reduce the likelihood that primary pulmonary NK cell lymphoma will be misdiagnosed as an infectious lesion. In addition, TCR gene rearrangement and clonal analysis, which is based on female X-chromosomal inactivation mosaicism and polymorphisms at PGK and androgen receptor (AR) loci, were found to play important roles in differentiating NK cell lymphoma from T cell lymphoma. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5205300349457729
    Full-text · Article · Aug 2013 · Diagnostic Pathology
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