1. No category

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1994 84: 3785-3791
Granzyme B-expressing peripheral T-cell lymphomas: neoplastic
equivalents of activated cytotoxic T cells with preference for mucosaassociated lymphoid tissue localization
PC de Bruin, JA Kummer, P van der Valk, P van Heerde, PM Kluin, R Willemze, GJ
Ossenkoppele, T Radaszkiewicz and CJ Meijer
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Granzyme B-Expressing Peripheral T-cell Lymphomas: Neoplastic
Equivalents of Activated Cytotoxic T Cells With Preference
for Mucosa-Associated Lymphoid Tissue Localization
By Peter C. de Bruin, J. Alain Kummer, Paul van der Valk, Peter van Heerde, Philip M. Kluin, Rein Willemze,
Gert J. Ossenkoppele, Thaddaus Radaszkiewicz, and Chris J.L.M. Meijer
T-cell non-Hodgkin’s lymphomas can be considered the neoplastic equivalents of immunologically functional, site-restricted T lymphocytes. Liile is known
about theoccurrence
and clinical behavior of T-cell lymphomas thatare the neoplasticequivalentsofdifferent
functional T-cell subsets.
Here, w e investigated theprevalence, preferential site, immunophenotype, and clinicalbehaviorof
the neoplastic
equivalents of activated cytotoxic T cells (CTLs) in a group
of 140 nodal and extranodal T-cell lymphomas. Activated
CTLs were shown immunohistochemically with a monoclonal antibody against granzyme B, a major constituent of
the cytotoxicgranules of activatedT cells.Granzyme B-posiin mucosa-associtive T-cell lymphomas were mainly found
ated lymphoid tissue
(MALT; nose, 63% of the cases; gastrointestinal tract, 46%; and lung, 33%). Granzyme B-positive
cases with primary localization in MALT were more often
associated with angioinvasion ( P = .005),necrosis ( P = .002).
and histologic characteristics of celiac disease in adjacent
mucosa notinvolved with lymphoma. Eosinophilia was
more oftenobserved in granzyme B-negative cases ( P = .03).
Most cases belonged t o t hpleomorphic
e
medium-and largecell group of the Kiel classification. CD30 expression was
more often found in granzyme B-positivelymphomas of
MALT (P= .04). whereas CD56 expression was exclusively
found in nasal granzyme B-positive lymphomas. Immunophenotypically, most of the cases should be considered as
neoplastic equivalents of activatedCTLs based on thepresence of T-cell markers on tumorcells. In two cases of nasal
lymphoma, tumor cells probably were the neoplastic counterparts of natural killercells. The prognosis of the granzyme
B-positive gastrointestinal T-cell lymphomas was poor but
did not differ from granzyme B-negative gastrointestinal Tcell lymphomas. This indicates that, in peripheral T-cell
lymphomas, site of origin is more importantas a prognostic
parameter than derivation of activated CTLs.
0 1994 by The AmericanSociety of Hematology.
T
been detected in mast cell and macrophage cell line^.^','^
Although the precise role of granzymes in cell-mediated
cytotoxicity is poorly understood and more functions than
just DNA fragmentation can probably be attributed to granz y m e ~ ? ’ -the
~ ~ presence of these proteins can be usedto
identify activated CTLs and, consequently, T-cell lymphomas derived from activated CTLs.
Here, we investigated the prevalence, preferential site,
phenotype, and clinical behavior of T-cell lymphomas derived from activated CTLs using an MoAb against the serine
esterase granzyme B in a large series of T-cell lymphomas
from nodal and extranodal sites to see if differences could
be found in relation to granzyme B-negative T-cell lymphomas. The results show that granzyme B-positive lymphomas
are preferentially localized in mucosa-associated lymphoid
tissue (MALT), have a poor clinical behavior, andshow
some typical histologic characteristics.
-CELL MALIGNANCIES are rare and constitute about
15% to 20% of the non-Hodgkin’s lymphomas in the
United States and Western Europe.’ The only classification
with a detailed subdivision of T-cell lymphomas is the updated Kiel classification.2 In this classification, T-cell
lymphomas are classified based on morphologic criteria of
nodal T-cell lymphomas. However, its reproducibility has
been q~estioned.~
From homing studies with T cells, the
existence of tissue-restricted T-cell subsets that have their
own biologic behavior became apparent:” Thus, mucosal T
cells, skin T cells, and nodal T cells show organ-specific
homing mediated by organ-specific homing receptors that
bind with tissue-specific position markers on endothelial
cells, termed vascular addressins.8-12
Also, T-cell non-Hodgkin’s lymphomas derived from these different tissue-restricted T-lymphocyte subsets not only show differences in
adhesion molecule expression, but also show a different clinical behavior and p r o g n ~ s i s . ’ ~Therefore,
”~
we and others
proposed to include site of origin in a future classification
of T-cell lymphomas?’6
Until recently, it was impossible to differentiate most
functional subsets, eg, suppressor T cells and cytotoxic T
cells (CTLs) by membrane characteristics only. Therefore,
little is known about the occurrence, presentation, and clinical behavior of T-cell lymphomas that are the neoplastic
counterparts of different functional T-cell subsets. The production of monoclonal antibodies (MoAbs) against human
perforin and serine esterases, major components of the cytotoxic granules found in the cytoplasm of CTLs on activation,”.I8 have made the identification of CTLs by immunohistochemistry possible.” In humans, three serine proteases
have been identified in the cytoplasmic granules of activated
CTLs and natural killer (NK) cells at the protein level, granzyme A, granzyme B, and granzyme 3.20-22In mice, it was
shown that, with few exceptions, expression of granzyme
genes is restricted to NK cells and T cells with their thymic
precursor^.^^ In mature T cells, granzymes are expressed
only on activation.23In addition, granzyme B transcripts have
Blood, Vol 84, No 11 (December l ) , 1994: pp 3785-3791
MATERIALS AND METHODS
Patient selection. Paraffin wax-embedded specimens and, when
available, frozen tissue specimens of T-cell lymphoma cases were
selected from the files of theComprehensive Cancer Center Amster-
From the Departments of Pathology and Dermatology and Hematology, Free University Hospital, Amsterdam;the Netherlands Cancer Institute, Amsterdam; the Department of Pathology, University
Hospital, Leiden, The Netherlands; and the Department of Clinical
Pathology, University of Vienna, Vienna, Austria.
Submitted June 2, 1994; accepted July 29, 1994.
Address reprint requests to Peter C. de Bruin, MD, Department
of Pathology, Free University Hospital, de Boelelaan I l l 7, 1081
HV Amsterdam, The Netherlands.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore behereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8411-0010$3.00/0
3785
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3786
Table 1. Relation Between Histological Subtype, Site of Origin, and
Granzyme B Expression in T-cell Non-Hodgkin's Lymphoma
DE BRUIN ET AL
reactedwithamonomer
of 33-kDprotein.One of theseMoAbs
against granzyme B (GB9) gives
a strong staining result on formalinfixed,
paraffin
wax-embedded
tissue
and was subsequently used in
Granzyme B'*Fotal No.
this study. All nonlymphoid cells were completely negative for GB9
Subtype (n)
GI
Nose
Lung
Node
Skin
on tissue sections with the exception of polymorphonuclear (PMN)
leucocytes. However, on Western blot, granzyme proteins were not
MF (2)
012
detected in cell lysates of PMN leukocytes, indicating that staining
Le ( 4 )
014
of PMN leukocytes in tissue sections with GB9 is caused by crossAILD ( 2 )
012
reactivity with PMN leukocyte-associated serine proteases." ImmuPSC (7)
1l 3
314
nostaining with GB9 was performed after antigenretrieval'X of tissue
PMLC ( 8 4 )
15/37t
111 214
0133
019
(2 X 5 minutes at 100°C; maximum
sectionsinmicrowaveoven
IBL (8)
314
011
013
power, 700 W) with citrate buffer (0.I mol/L [pH 6.0]), for 1 hour
ALCL (25)
1I2
6 - 211
011
1l6
at room temperature. After blocking endogenous peroxidases, immuLBL (5)
015
noperoxidase staining was performed using a biotinylated horse antiUnclas ( 3 )
011
012
mouseantibody(VectorLaboratories,Burlingame,
CA) and the
Abbreviations: GI, gastrointestinal; MF, mycosis fungoides;Le, Lenstreptavidin-biotinhorseradishperoxidasecomplex(ABC;Dakonert's lymphoma; AILD, angioimmunoblastic lymphodenopathy with
patts, Glostrup, Denmark) as second and third step. The peroxidase
dysproteinemia; PSC, pleomorphic smallcell lymphoma; PMLC, pleoreaction was visualized using 3.3 diamino-benzidine-tetrahydrochlomorphicmediumandlarge
cell lymphoma; IBL, immunoblastic
ride/H20Z (Sigma, St Louis, MO).
lymphoma; ALCL, anaplastic largecell lymphoma; LBL, lymphoblastic
According to the number of tumor cells positive for granzyme B.
lymphoma; Unclas, unclassifiable.
cases were divided into four categories: ( I ) 0% to 10%; (2) 1 1 % to
* A lymphoma was considered to be granzyme B-positive when
20%; (3) 21% to 50%; and (4) more than SO% staining of the tumor
more than 50% of the tumor cells stained with granzyme B.
cell population by granzyme B. Only the latter were considered to
t Two cases had only 11% to 20% granzyme B-positive tumor cells.
be granzyme B-positive lymphomas to avoid any possible misinterpretation of admixedreactivecellsastumorcells.As
a control,
sections were incubated with an irrelevant primary antibody (AT11121) of the appropriate subclass(IgGI). Immunohistologic analysis
dam(Amsterdam,TheNetherlands;n
= 83);theLaboratoryof
for further characterization of granzyme B-positive cases and granPathology, University Hospital Leiden (Leiden, The Netherlands; n
zyme B-negative extranodal cases involved the
use of a panel of
= 4); the Dutch Cutaneous Lymphoma Working Group (Amsterdam,
MoAbs and polyclonal antibodies on frozen(n = 12) and/or paraffinThe Netherlands; n = 17); the Department of Pathology, Hospital
embedded tissue sections. They included on frozen sections leu 41
Henri Mondor (Crkteil, France; n = 1); and the Department of PaCD3. leu 3lCD4. leu 1 Ib/CD16, and leu 19KD.56 (Becton Dickinthology,UniversityofVienna(Austria;n
= 35).Therewere
63
son, Mountain View, CA); Riv 4/CD8 (Sanbio, Uden, the Netherprimary nodal T-cell lymphoma cases and 77 primary extranodalTlands); CLB-3AIKD7 (gift Central Laboratory of the Netherlands
celllymphomacases.ExtranodalT-celllymphomawasaccepted
Red Cross Blood Transfusion Service, Amsterdam, the Netherlands);
when patients presented with predominantor exclusive involvement
HML-IICD I03 (lmmunotech, Marseille, France);T-cell receptor b 1
of a certain extranodal region and had presenting symptoms related
(TCRbl : T Cell Diagnostics, Cambridge, MA). On paraffin-embedto that specific region. Cases were classified according to the updatedded sections they included UCHLI/CD4SRO, polyclonal CD3, and
Kiel classification (Table l). The T-cell nature was defined as presBerH2ICD30 (Dakopatts, Kopenhagen, Denmark); MTI/CD43 (Orence of one or more T-cell-specific and/or characteristic antigens
ganon Teknika, Oss, the Netherlands); leu7/CD57 (Becton andDickon the tumor cell population: on
paraffin wax-embedded sections,
inson); OPD4lCD4 (Dakopatts); and 144B/CD8 (kindly providedby
CD3, CD4SR0, and/or CD43; on frozen sections, when available
Dr D.Y. Mason, Oxford, UK). The immunohistochemical stainings
(n = 46), CD2, CD3, CD5, and/or CD7. B-cell (CD19, CD20, and/
wereperformedwithstandardtechniquesusinganavidin-biotin
or CD22) and histiocytic (CD68) markers had
to be negative on
horseradish peroxidase complex method, with or without modificatumor cells. Extranodal cases initially presented in the noseor parations as described for GB9, or using an indirect immunoperoxidase
nasal cavities (n = 8), in the lung (n = 3), in the gastrointestinal
method. The enzyme histochemical staining for naphtol-AS-D-chlotract (n = 46), or in the skin (n = 20). Clinical data were obtained
roacetate esterase (Leder)was performed according to standardlabofrom referring specialists. Complete remission (CR) was defined as
ratory techniques.
the total disappearance of all symptomsandclinicallydetectable
To identify the phenotype of granzyme B-expressing cells in nondisease that was present at the initiation of therapy and the appearneoplasticlymphoidtissue,sequentialfrozentissuesectionswere
ance of no new lesions for 3 months. Survival was calculated from
used for double stainings. Briefly, slides were incubated
with primary
the date of diagnosis to the date of death caused by the disease or
MoAbsdirectedagainstdifferent
CD markers(CD3,CD4,CD8,
to the date of last contact. Statistical analysis was performed
with
CD16, and CDS6) followedby the alkaline phosphataseanti alkaline
the BMDP Statistical Software (Los Angeles, CA), with P values
phosphatase (APAAP) method (APAAP, Dakopatts). Bound alkaline
of less than .OS being considered significant. Kaplan-Meier curves
phosphatase was then visualized by addition of naphtolAS-MX
phosphate (Sigma) and Fast Blue BB in 0.2 mol/L Tris-HCL, pH
the curveswereanalyzed
wereplotted,anddifferencesbetween
8.5. Endogenous alkaline phosphatase activity present in the tissues
with the Mantel-Cox statistic. To evaluate CD30 expression as a
wasblocked by addition of 1 mmol/Llevamisole to thereaction
prognostic parameter, a case was considered
to be CD30-positive
when more than SO% of the tumor cell population stained for BerH2. mixture. Subsequently, these slides were incubatedwith biotinylated
MoAbs against granzymes. Binding of these antibodies was detected
To study the distribution of granzyme B-positive cells in nonneousing the streptavidin-biotin peroxidase method. Endogenous peroxiplastic lymphoid tissue; nonneoplastic lymph nodes (n = 3); tonsils
(n = 3); and specimens from normal stomach, jejunum. and descend- dase activity was blocked by 0.1% sodium azide,0.3% H?02. Peroxidase activity was visualized using amino-ethyl-carbozole (Sigma).
ing colon were used.
Histology and immunohistochemistry. Sections were stained for
RESULTS
hematoxylin and eosin, Giemsa and periodic acid Schiff.
Granzyme B expressionand pathologic jindings. GranThe production and characterizationof MoAbs against granzyme
zyme B-positive neoplastic cells showed a diffuse granular
B has recentlybeendescribed."
All MoAbsagainstgranzymeB
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GRANZYME B EXPRESSION IN T-CELL LYMPHOMAS
3787
Fig 1. Primary gastrointestinalT-cell lymphoma, anaplastic large
cell lymphoma subtype is shown. Tumor cells are granzyme-B positive (brown, diffusegranular,cytoplasmic staining). (Hematoxylin
counterstain; original magnification x 400.)
Fig 3. Primary gastrointestinal T-cell lymphoma, PMLCsubtype
is shown. Tumor cells are granzyme B-positive; mitotic cells were
positive (arrowhead). (Hematoxylin counterstain; original magnification x 400.)
or dot-like perinuclearstaining. Overall, 29 of 140 (21%)
T-celllymphomas were foundto be granzymeB-positive
(ie, showed morethan 50% stainingof the tumorcell population; see Table 1). In an additional 2 primary gastrointestinal
cases, 11% to 20% of the tumor cells were GB9-positive.
No cases were found with 0% to 10% or 21% to 50% granzyme B-positive tumor cells.A total of27 of 77(38%)
primary extranodal T-cell lymphomas and 2 of 63 (3%) primary nodal T-cell lymphomas were granzyme B-positive in
more than 50% of the tumor cells. The extranodal granzyme
B-positive cases were predominantly found in the nose and
nasal cavities (5 of 8; 63%), gastrointestinal tract (20 of 46;
43%; see Fig l), and lung ( 1 of 3; 33%; see Fig2).The
remaining granzyme B-positive extranodal case was localizedinthe
skin(1 of 20; 5%). Mitotic cells wereoften
GB9-positivein granzyme B-positive cases (Fig 3). In 1
gastrointestinal lymphoma case, interpretation was difficult
because CD8-positive tumor cells were surrounded by nu-
merous granzyme B-positivesmall-to-intermediate
sized,
CD8-positive, possibly reactive, cells.
The relationshipbetween lymphomasubtype and granzyme Bexpression is depicted in Table I . Most cases belonged to the pleomorphic medium- and large-cell subtype
category.Pleomorphic small-cell,immunoblastic and, less
frequently, large-cell anaplastic granzyme B-positive T-cell
lymphomas were also observed.
in the
Necrosis and angioinvasion were more prominent
group of granzyme B-positive T-cell lymphomas, whereas
eosinophilia was slightlymoreprominentin
granzyme Bnegative cases. Epitheliotropism in the overlying mucosa of
neoplastic T cells did not differ between the granzyme Bpositive orgranzyme B-negative groups(Table2).
According to the criteria described by Chott and associates,”
theprimarygastrointestinal
lymphomas wereseparated in
enteropathy-associatedT-cell
lymphomas(EATCLs; n =
13), EATCL-like lymphoma without enteropathy (EATCLLLWE; n = 7 ) and T-cell lymphoma withoutenteropathy
andwithout features of EATCL(non-EATCL; n = 14).
Granzyme B-positive neoplastic cells were mainly associated
with EATCL. A total of 77% (n = IO) of the EATCL cases
versus 14% (n = 2) of the non-EATCL cases were granzyme
B-positive. In 57% (n = 4) of EATCL-LLWE cases, granzyme B expression was detected.
In the granzyme B-positive cases of which Giemsa-stained
imprint preparations were available(n = 4), numerousazurophilic granules were found in the cytoplasm of the neoplastic
Table 2. Histopathologic Findings in Gastrointestinal and Nasal
T-cell Lymphomas in Relation to Granzyme B Expression
Granzyme B ‘
Necrosis
Fig 2. Primary lung T-cell lymphoma, pleomorphic medium and
large cell(PMLC)subtypeis
shown. Tumorcells are granzyme Bpositive (arrowheads); macrophages with anthracotic pigment are
granzyme B-negative (arrows). (Hematoxylin counterstain; original
magnification x 400.)
Epitheliotropism
~
~
~
~ ~ ~ i ~ ~
16/23
(70%)
(26%)
7/27
,002
(38%)
(33%)
8/21
5/15
i13/22
(59%)
(19%)
5/26
.005~
~ (26%)
h1/24
i l i
.03
7/27
(4%)
Abbreviation: NS, not significant.
* x Z test.
Granzyme B
PValue*
NS
i
~
~
~
~
~
i
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DE BRUIN ET AL
3788
Fig 4. Primary nasal T-cell lymphoma, PMLC subtype, with cytotoxic granules in the cytoplasm of a tumor cell is shown by electron
microscopy. (Original magnification x 30,000.1 Inset shows cytotoxic
granules. (Original magnification x 90.000.)
cells. In a primary nasal granzyme B-positive case, the characteristic secondary lysosomes could be found using electron
microscopy (Fig 4).
Immunophenovpicanalysis.
The phenotypes of T-cell
lymphomas originating in gastrointestinal tract, nose,and
lung are listed in Table 3. CD30 expression was found significantly more often in granzyme B-positive cases than in
granzyme B negative cases. Moreover, there was a trend for
the CD4"cVCD8"cPphenotype to be present in granzyme Bpositive cases. Other markers demonstrable on paraffin waxembedded tissue were not associated with granzyme B expression. From the markers that can only be used on frozen
material (ie, TCRGI, HML-I, and CDS6), an association was
found between CD56 expression and granzyme B-positive
nasal T-cell lymphoma. However, the number of cases investigated was rather low (n = 12). Interestingly, no association
was found between the presence of TCRb I and granzyme B
expression in T-cell lymphomas. Granzyme B-positive cases
were always naphtol-AS-D-chloroacetateesterase-negative.
Both granzyme B-positive nodal cases were CD3-, CD4-,
CD8-, CD56-negative. The granzyme B-positive cutaneous
case was CD3-positive, but CD4- and CD8-negative.
Grartzyme B expression in control lymphoid tissues. The
results were the same as recently described." Granzyme Bpositive cells were detected in all lymphoid tissues tested.
In nonneoplastic lymph nodes and tonsillar specimens, granzyme B-positive cells were scarce. When found, they were
mostlyconfinedtothe
sinuses or medullary cords. In the
jejunum, with normal mucosa, onlysporadic small granzyme
B-positive cells were present in the lamina propria. Granzyme B-positive intraepithelial lymphocytes (IELs) were not
observed. A similar pattern was observed for normal mucosa
adjacent to or overlying the tumor tissue in gastrointestinal
lymphomas. Preliminary findings indicate that granzyme Bpositive IELs are more often found in patients with EATCL
or EATCL-LLWE in the nonlymphoma-involved, adjacent
mucosa (this study). In the control tissues tested, hardly any
of the granzyme B-positive cells stained with MoAb against
CD3. Most granzyme B-positive cells stained with MoAbs
against CD I6 or CDS6, indicating that the majority of granzyme B-positive cells were N K cells in these control
lymphoid tissues. The control-irrelevant primary antibody
did not stain lymphoid cells.
Clinicalfeatures. Patients were treated in different institutes in different countries, and therapy was, therefore, heterogeneous. The rnale:female ratio of patients with granzyme
B-positive tumors was I .25. The patients' ages ranged from
6 to 82 years (median, 54 years).
Patients withprimarynasal T-cell lymphomapresented
with localized disease (stage IE according to Ann Arbor) in
all but 1 case (stage IV according to Ann Arbor with cervical
lymph node and liver involvement). None were leukemic.
Of 5 patients with granzyme B-positive nasal tumors, 4 died
of disease between 1 and27 months (median, 4 months)
after initial presentation; I patient was alive with disease
with ongoing chemotherapy. Of 3 patients with granzyme
B-negative nasal T-cell lymphomas, 1 died of disease after
4 months (stage IV disease), 1 died of pneumonia without
evidence of disease after 14 months, and 1 is still alive after
53 months with disease in CR.
Patients with primary gastrointestinal T-cell lymphomas
most often presented with tumors of the small intestine. Most
Table 3. Phenotypes of T-cell Lymphomas of MALT
~~
GI Tract T-NHL
CD3CD4'
CD8'
CD4-ICD8CD30*
CD45RO'
CD43*
TCR61+
HML-l*
CD56'
Nasal T-NHL
Lung T-NHL
Total
GrB'
GrB-
GrB'
GrB-
GrB'
GrB-
GrB'
GrB-
19/20
5/20
5/20
10120
10119
15/20
14/19
1l3
213
012
22/26
12/26
8/26
6/26
6/25
17/26
21/26
016
518
012
214
115
115
315
014
415
314
014
012
515
3/3
213
013
1l3
013
113
1l3
111
011
011
011
011
011
111
111
011
1/l
ND
ND
ND
012
21/25
6/26
6/26
14/26
11/24
19/26
18/24
117
215
517
25/31
14/31
8/31
9/31
6/30
19/31
2313 1
117
5/9
013
012
012
212
012
112
1l2
ND
ND
ND
Abbreviations: GI, gastrointestinal; NS, not significant; GrB, granzyme B; T-NHL, T-cell non-Hodgkin's lymphoma; ND, not done.
xz test.
PValue.
NS
NS
NS
.06
.04
NS
NS
ND
ND
ND
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GRANZYME B EXPRESSION IN T-CELL LYMPHOMAS
patients presented with stage I or I1 disease (n = 31). When
more generalized disease was present (n = S), spread was
often apart from lymph nodes to other mucosal sites (lung,
stomach, and palatum). No apparent differences were noted
with regard to clinical presentation between granzyme Bpositive or granzyme B-negative cases. Median survival was
5 and 3.5 months for granzyme B-positive and granzyme
B-negative tumors, respectively, ( P = S ) . No significant
differences in survival were noted between patients with
CM-positive and CDS-positive or CDCnegative and CDSnegative gastrointestinal T-cell lymphomas ( P > .l). This
also accounted for CD30-positive gastrointestinal T-cell
lymphomas when compared with CD30-negative gastrointestinal T-cell lymphomas ( P = .15).
Both patients with nodal granzyme B-positive lymphomas
were young (6 and 17 years), with stage I and 111 disease
and with disease in CR of long duration (25 and 70 months,
respectively) after aggressive chemotherapy. The remaining
granzyme B-positive primary cutaneous lymphoma had a
favorable course (alive with disease in CR after 104 months).
DISCUSSION
We have shown that a substantial percentage (21%) of our
T-cell non-Hodgkin’s lymphomas are granzyme B-positive,
indicating that they probably are the neoplastic equivalents
of activated CTLs. Primary extranodal peripheral T-cell
lymphomas that made up a significant proportion of our
study group almost exclusively accounted for this substantial
percentage of granzyme B-positive T-cell lymphomas that
included 10 of 13 EATCL cases. Of the 29 granzyme Bpositive cases, 26 were localized in mucosal sites, ie, nose,
gastrointestinal tract, and lung. Only few were localized in
lymph nodes (n = 2) or skin (n = 1). Therefore, these
lymphomas have a preference for localization in MALT.
Histologically granzyme B-positive cases showed significantly more angioinvasion and necrosis and less infiltration
with eosinophils than granzyme B-negative lymphomas.
Most granzyme B-positive cases belonged to the pleomorphic subtype categories of the updated Kiel classification.
Moreover, granzyme B-positive lymphomas of the gut were
associated with histologic characteristics of celiac disease,
ie, villous atrophy and intraepithelial lymphocytosis in the
adjacent normal mucosa. The association between clinical
and/or histologic evidence of celiac disease and the occurrence of primary gastrointestinal T-cell lymphoma has been
~ell-established.”‘~~
Interestingly, most cases of T-cell
lymphoma with eosinophilia of the gastrointestinal tract have
not been associated with celiac disease.”
Activation of resting CTLs induces morphologic changes
with the occurrence of azurophilic granules3’ In granzyme
B-positive cases of which Giemsa stained cytologic slides
were available, azurophilic granules were found. Reports of
peripheral T-cell lymphomas with azurophilic granules are
scarce, and cases reported most often had extranodal disease
and often mucosal involvement.3641In general, cytotoxic
granules are enveloped by a lipid bilayer and contain small
internal vesicles and an electron-dense core surrounded by
a membrane!’*
Granules with identical morphology were
also found, by electron microscopy, in the cytoplasm of the
3789
granzyme B-positive tumor cells of the nasal T-cell
lymphoma investigated.
JAW levels of granzyme B are constitutively expressed by
NK cells but not by unstimulated CD3PO”/CD8P”s
or CD3POY
CD4ps unstimulated peripheral blood lyrnphocyte~!’~~This
might be the reason for the low number of CD3-positive
granzyme B-positive cells in control lymphoid tissue. NK
cells are CD3-negative, CD56-positive, and do not rean-ange
TCR
but are otherwise remarkably similar to T
cells with respect to the expression of other membrane antig e n ~The
. ~ T-cell
~
origin of our granzyme B-positive gastrointestinal and cutaneous and of part of the nasal cases was
confirmed by the presence of CD3. Two nasal granzyme Bpositive cases were CD56-positive and CD3-negative. These
might represent lymphomas of NK cell origin. In accordance,
non-major histocompatibility complex-restricted cytotoxicityby tumor cells has been described recently for CD3negative, CD56-positive nasal T-cell lymphoma with TCR
genes in germline c~nfiguration?~
Several studies have addressed the relation between CD56 expression and T-cell
lymphoma^.^'^.^' CD56-positive T-cell lymphomas were reported to have a propensity for extranodal sites, in particular
the upper aerodigestive tract,” which is in agreement with
our findings. Moreover, CD56-positive lymphomas were reported to share a very aggressive clinical behavior and resistance to conventional chemotherapy.40
Although our results do not provide conclusive evidence
as to whether granzyme B-positive lymphomas are neoplastic counterparts from IELs or activated CTLs in the lamina
propria of the small intestine?6 we slightly favor the first
option. In mice, IELs have been shown to express granzymes5’ and are predominantly y6 TCR-positive. In humans,
IELs are mostly a0 TCR-positive’* and probably also express granzyme proteins after activation. IELs are increased
in celiac disease, and granzyme B-positive IELs seemed to
be more prominent in the adjacent nonlymphoma involved
mucosa in cases classified as EATCL or EATCL-like
lymphoma without enteropathy. In addition, normal human
small intestine CD3TCD8”eg/CD4”g T lymphocytes comprise a minor fraction of IELs (6%) that are increased in
celiac di~ease.~’
This phenotype proved to be the dominant
phenotype in granzyme B-positive gastrointestinal T-cell
lymphomas.
We can only speculate on the association between signs
of celiac disease and the Occurrence of tumors derived from
activated CTLs. It has been assumed that the mucosal permeability for luminal antigens is increased in celiac disease
because of local and systemic IgG responses to gluten and
other dietary protein^.^' This then might exert a cytotoxic Tcell response. The continuous antigenic stimulation might
contribute to malignant transformation as has been proposed
for gastric B-cell lymphomas in association with the presence of Helicobater p y l ~ r i , ~ ~ . ~
In conclusion, granzyme B-positive T-cell lymphomas are
predominantly localized in MALT and differ from granzyme
B-negative T-cell lymphomas in increased frequency of angioinvasion and necrosis but less infiltration with eosinophils. Skin and lymph nodes are occasionally involved. Although in some nasal cases an NK cell nature can not be
excluded, most cases should be considered as neoplastic
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
3790
equivalents of activated CTLs. The prognosis of the granzyme B-positivegastrointestinalT-celllymphomas
is as
equally poor as the prognosis of granzyme B-negative gastrointestinal T-cell lymphomas, indicating that site of origin
ratherthanderivation
of activated CTLs predictsclinical
o u t c o m e of peripheral T-cell lymphomas.
DE BRUIN ET AL
Noorduyn LA, van Krieken JHJM,Kluin-Nelemans JC. WillemLe
R, Meijer CJLM: Differences in clinical behavior and imrnunophenotypebetweenprimarycutaneousandprimary
nodal anaplastic
large cell lymphoma of T-cell or null cell phenotype. Histopathol
23: 127. 1993
16. de Bruin PC,JiwaNM,OudejansJJ,
van der Valk P , van
Heerde P, Sabourin J-C, Csanaky G. GaulardPh, Noorduyn AL,
Willemze R, Meijer CJLM: Presence of Epstein-Barr virux in extraACKNOWLEDGMENT
nodal T-cell lymphomas:Differences
in relation tosite.
Blood
83:1612,1994
Theauthorsthankthepathologistsandclinicians
in the region
17. Lowrey DM, Hameed A, Lichtenheld R4, Podack ER: Isolaof the Comprehensive Cancer Center Amsterdam, Dr
Ph. Gaulard
tion and characterization of cytotoxic granules from human lympho(HBpital Henri Mundor, Creteil, France) who kindly provided tissue
material and clinical data, and
Dr D.Y. Mason (John Radcliffe Hospi- kine (interleukin 2) activated killer cells. Cancer Res 48:468 I, 1988
18. Lichtenheld MC, Olsen K J , Lu P, Lowrey DM, Hameed A.
tal, Oxford, UK) who kindly provided monoclonal antibody 144B/
Hengartner
H, Podack ER: Structure and function of human perforin.
CD8. J. Belien is thanked for the statistical analysis, and E. Boots
Nature 3352448, 1988
for his technical assistance with the electron microscopic pictures.
19. KummerJA,KampAM,
van Katwijk M,Brakenhoff JP,
Radosevic K, van LeeuwenAM, Borst J, Verwey CL,Hack CE:
REFERENCES
Productionandcharacterization
of monoclonalantibodies raised
against recombinant human granzymes A and B and showing cross
I . Pinkus GS, Said JW: Peripheral T-cell lymphomas,in’Knowles
reactions with the natural proteins. J Irnmunol Methods 163:77, 1993
DM (ed): NeoplasticHematopathology.Baltimore,MD,Williams
20. Krahenbuhl 0, Rey C, Jenne D, Lanzavecchia A. Groscurth
and Wilkens, 1992, p 837
P, Carrel S, Tschopp J: Characterizationofgranzymes A andB
G, Lennert K,
2. Stansfeld AG, Diebold J, Kapanci Y, Keltnyi
isolated from granules of cloned human cytotoxic T lymphocytes. J
Mioduszewska 0, Noel H, Rilke F, Sundstrom C, van Unnik JAM,
Irnmunol 141:3471, 1988
Wright DH: Updated Kiel classificationforlymphomas.Lancet
2 1. Hameed A, Lowrey DM, Lichtenheld M, Podack ER: CharacI :292, 1988
terization of three serine esterases isolated from human
IL-2 acti3. Hastrup N, Hamilton-DutoitS, Ralfkiaer E, Pallesen G: Periph1988
vated killer cells. J Immunol 141:3142,
eral T-cell lymphomas: An evaluation of the reproducibility of the
22. Poe M,BennettCD,BiddisonWE,BlakeJT,
Norton GP,
updated Kiel classification. Histopathol 18:99, 1991
Rodkey JA, Sigal NH, Turner RV, Wu JK, Zweerink HJ: Human
4. Cerf-Benussan N, Jarry A, Brousse N. Lisowska-Grospierre B,
cytotoxic lymphocyte tryptase. Its purification from granules and the
Guy-Grand D, Griscelli C: A monoclonal antibody (HML-1) defincharacterization of inhibitor and substrate specificity. J Biol Chem
ing a novel membrane molecule present on humanintestinal lympho263: I321 S, 1988
cytes. Eur J Immunol 17:1279, 1987
23. Garcia-Sanz JA, MacDonald HR, Jenne DE, Tschopp J , Nab5 . Picker LJ, Michie SA, Rott LS, Butcher EC: A unique phenoholz M: Cell specificity of granzyme gene expression. J Immunol
type of skin-associated lymphocytes in human: Preferential expres145:311 I . 1990
sion of the HECA-452 epitope by benign and malignant T-cell at
24. Brunet 1-F, Denjzot F, Suzm M, Haas W, Mencia-Huerta
cutaneous sites. Am J Pathol 136:1053, 1990
J-M, Berke G, Luciani MF, GolsteinP: CTLA- 1 and CTLA-3 serine
6.KishimotoTK,JutilaMA,ButcherEC:
Identification ofa
esterase transcripts are detected mostly in cytotoxic T cells, but not
humanperipherallymphnodehomingreceptor:Arapidlydownonly and not always. J Immunol 138:4102, 1987
regulatedadhesionmolecule.Proc
Natl AcadSciUSA87:2244,
2.5. Shiver JW, Lishan SU, Henkart PA: Cytotoxicity with target
I990
DNA breakdown by rat basophilic leukemia cells expressing both
7. Stein H, Dienemann D, Dallenbach F, Kruschwitz M: Periphcytolysin and granzyme A. Cell 71:315, 1992
1991
eral T-cell lymphomas. Ann Oncol 2:163,
26. Sayers TJ, Wiltrout TA, Sowder R, Munger WL, Smyth MJ,
8. Streeter PR, Berg EL, Rouse BTN, Bargatze RF, Butcher EC:
Henderson LE: Purification ofafactor
from thegranulesof
rat
A tissue specific endothelial cell molecule involved
in lymphocyte
natural killer cell line (RNK) that reduces tumor cell growth and
homing. Nature 33 1:4l. 1988
changes tumor morphology: Molecularidentity with a granule serine
9. Michl J, Qui Q-Y. Kuere HM:Homingreceptorsandadprotease (RNKP-I). J Immunol 148:292, 1992
dressins. Cum Opin Immunol 3:373, 1991
27. Froelich JC, Zhang X, Turbov J, Hudig D, Winkler U , Hanna
IO. Streeter PR, Rouse BTN, Butcher EC: Immunohistologic and
WL: Human granzyme B degrades aggrecan proteoglycan in matrix
functional characterization of a vascular addressin involved in lymsynthesized by chondrocytes. J Imrnunol 15 1:7 161, 1993
phocyte homing into peripheral lymph nodes. J Cell Biol 107:1853,
28. Shi S-R, Key ME, Kaka KL: Antigen retrieval in formalin1988
fixed, paraffin-embedded tissues: An enhancement method for imI I. Butcher EC: Cellular and molecular mechanisms that direct
munohistochemicalstaining based on microwaveovenheating of
leucocyte traffic. Am J Pathol 1363, 1990
tisue sections. J Histochem Cytochem 39:741, 1991
12. Picker LJ, Kishimoto TK, Smith CW, Warnock RA, Butcher
29. Chott A, DragosicsB,Radaszkiewicz
T: Peripheral T-cell
EC: ELAM-IisanadhesionmoleculeforskinhomingTcells.
lymphomas of the intestine. Am J Pathol 141: 1361, 1992
Nature 349:796, 1991
30. Kummer JA, Kamp AM, Tadema TM, Vos W, Meijer CJLM.
13. Pals ST, Meyer CJLM, Radaszkiewicz T: Expression of the
Hack CE: Localizationand identification of granzymes A and B
human peripheral lymphnode homing receptor (LECAM-I)in nodal
expressingcells in normalhumanlymphoidtissueand
peripheral
andgastrointestinalnon-Hodgkin’slymphomas.Leukemia5:628,
blood. Clin Exp Immunol (submitted)
1991
3 1 . Isaacson P, O’ConorNTJ,Spencer J , Bevan DH,Conolly
14. Picker LJ, Michie SA, Rott LS, ButcherEC: A unique phenoCE,Kirkham N, Pollock DJ, WainscoatJS,Stein H, Mason DY:
type of skin-associated lymphocytes in humans. Preferential expresMalignant histiocytosis of the intestine: A T-cell lymphoma. Lancet
sion of the HECA-452 epitope by benign and malignant T-cells at
2:688, 1985
cutaneous sites. Am J Pathol 1361053, 1990
32. Wright DH, Jones DB, ClarkH, Mead GM, Hodges E, Howell
15. de Bruin PC, Beljaards RC,
van Heerde P, van der Valk P,
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
G R A N N M E B EXPRESSION IN T-CELL LYMPHOMAS
WM: Is adult-onset coeliac disease due to a low grade lymphoma
of intraepithelial T lymphocytes? Lancet 337:1373, 1991
33. Alfsen GC, Beiske K, Bell H, Martin Per F: Low-grade intestinal lymphoma of intraepithelial T-lymphocytes with concomitant
enteropathy-associated T-cell lymphoma: Case report suggesting a
possible histogenetic relationship. Hum Pathol 20909, 1989
34. Sheperd NA, Blackshaw AJ, Hall PA, Bostad L, Coates PJ,
Lowe DG, Levison DA, Morson BC, Stansfeld AG: Malignant
lymphoma with eosinophilia of the gastrointestinal tract. Histopathology 11:115, 1987
35. Clark W, Ostergaard H, Gorman K, Torbett B: Molecular
mechanisms of CTL-mediated lysis: A cellular perspective. Immunol
Rev103:37, 1988
36. Sun T, Schulman P, Kolitz J, Susin M, Brody J, Koduru P,
Muuse W, Hombal S, Teichberg S, Broome J: A study of lymphoma
of large granular lymphocytes with modern modalities: report of two
cases and review of the literature. Am J Hematol 40:135, 1992
37. Longacre TA, Listrom MB, Spigel JH, Wilman CL, Dressler
L, Clark D: Aggressive jejunal lymphoma of large granular lymphocytes. Am J Clin Pathol 93:124, 1990
38. Kanavaros P, Lavergne A, Galian A. Boivin P, Ruskone
Fourmestreaux A, Cochand Priollet B, Flandrin G, Hautefeuille P:
A primary immunoblastic T malignant lymphoma of the small
bowel, with azurophilic intracytoplasmic granules. Am J Surg Pathol
12541, 1988
39. Chan JKC, Ng CS: Azurophilic granules in lymphomas immunoreactive for natural killer cell markers. Am J Clin Pathol
94: 120, 1990 (letter)
40. Wong KF, Chan JKC, Ng CS, Lee KC, Tsang WYW, Cheung
MMC: CD56 (NKH1)-positive hematolymphoid malignancies: an
aggressive neoplasm featuring frequent cutaneous/mucosalinvolvement, cytoplasmic azurophilic granules, and angiocentricity. Hum
Pathol 23:789, 1992
41. Chan JKC, Sin VC, Ng CS, Lau WH: Cutaneous relapse of
nasal T-cell lymphoma clinically mimicking erythema multiforme.
Pathology 21:164, 1989
42. Burkhardt JK, Hester S, Lapham CK,Argon Y: The lytic
granules of natural killer cells are dual-function organelles combining secretory and pre-lysosomal compartments J Cell Biol 111:2327,
1990
43. Peters PJ, Geuze HJ, van der Donk HA, Borst J: A new model
for lethal hit delivery by cytotoxic T lymphocytes. Immunol Today
11:28, 1990
44. Peter PJ, Geuze HJ, van de Donk HA, Stam NJ, Clevers HC,
Borts J: Molecules relevant for T-cell-target cell interaction are
present in cytologic granules of human T-lymphocytes. Eur J Immuno1 19:1469, 1989
45. Liu CC, Rafii S, Granelli-Piperno A, Trapani JA, Young
JD-E: Perforin and serine esterase gene expression in stimulated
human T-cells. J Exp Med 170:2105, 1989
379 1
46. Nakata M, Smyth MJ, Norihisa Y,Kawasaki A, Shinkai Y,
Okumura K, Yagita H: Constitutive expression of pore-fonning protein in peripheral blood y6 T cells: Implication for their cytotoxic
role in vivo. J Exp Med 172:1877, 1990
47. Koizumi H, Liu CC, Zheng LM, Joag SV, Bayne NK, Holoshitz, Young JD: Expression of performin and serine esterases by
human gammddelta T cells. J Exp Med 173449, 1991
48. Smyth MJ, Strobl SL, Young HA, Ortaldo JR, Ochoa AC:
Regulation of lymphokine-activated killer activity and pore forming
protein gene expression in human peripheral blood CD8' T lymphocytes. Inhibition by transforming growth factor-beta. J Immunol
146:3289,1991
49. Lu P, Garcia-Sanz JA, Lichtenfeld MG, Podack ER: Perforin
expression in human peripheral blood mononuclear cells: Definition
of an IL-2-independent pathway of perforin induction in CD8' T
cells. J Immunol 148:3354, 1992
50. Lanier LL, Phillips JH, Hackett J, Tutt M, Kumar V: Natural
killer cells: Definition of a cell type rather than a function. J Immunol
137:2735, 1986
51. Robertson MJ, Ritz J: Biology and clinical relevance of human natural killer cells. Blood 76:2421, 1990
52. Lanier LL, Spits H, Phillips JH: The developmental relationship between NK cells and T-cells. Immunol Today 13:392, 1992
53. Homer K, Ansari Q, Dawson B, Picker L, Vuitch F, McKenna
R: Aggressive natural killer cell lymphoma without leukemia. Mod
Pathol 7: 1 1IA, 1994 (abstr)
54. Ng CS, Chan JKC, Lo STH: Expression of natural killer cell
markers in non-Hodgkin's lymphomas. Hum Pathol 18:1257, 1987
55. Kern WF, Spier CM, Hanneman EH, Miller TP, Matzner M,
Grogan TM: Neural cell adhesion molecule-positive peripheral Tcell lymphoma: A rare variant with a propensity for unusual sites
of involvement. Blood 79:2432, 1992
56. Cerf-Benussan N, Schneeberger EE, Bhan AK: Immunohistologic and immunoelectronic microscopic characterization of the mucosal lymphocytes of human small intestine by the use of monoclonal
antibodies. J Immunol 130:2615, 1983
57. Guy-Grand D, Malassis-Seris M, Briottet C, Vasalli P: Cytotoxic differentiation of mouse gut thymodependent and independent
intraepithelial T lymphocytes is induced locally: Correlation between
functional assays, presence ofperforinand
granzyme transcripts,
and cytoplasmic granules. J Exp Med 173:1549, 1991
58. Brandtzaeg P, Halstensen TS, KettK, Krajci P, Kvale D,
Rognum TO, Scott H, Sollid LM: Immunobiology and immunopathology of human gut mucosa: Humoral immunity and intraepithelial
lymphocytes. Gastroenterology 97: 1562, 1989
59. Parsonnet J, Hansen S, Rodriquez L, Gelb AB. Warnke RA,
Jellum E, Orentreich N, Vogelman JH, Friedman G D Helicobucter
pylori infection and gastric lymphoma. N Engl J Med 330:1267,
1994
60. Isaacson DM: Gastric lymphoma and Helicobucter pylori. N
Engl J Med 330: 1310, 1994