, . 183: 432–439 (1997)

CYTOTOXIC PROTEIN EXPRESSION IN NATURALKILLER CELL LYMPHOMAS AND IN áâ AND ãä

PERIPHERAL T-CELL LYMPHOMAS

- 1, 2, 1, 3 1*1Département de Pathologie and ER 270, Hôpital Henri Mondor, 94010 Créteil, France

2Department of Pathology, University Hospital of Heraklion, Greece3Service d’Anatomie Pathologique, Institut Gustave Roussy, Villejuif, France

SUMMARY

Lymphomas with T-cell phenotype represent a heterogeneous group of diseases differing in histopathology, tumour site, and cell origin.They include peripheral T-cell lymphomas (PTCLs) derived from áâ cells, but also some recently recognized entities such as ãähepatosplenic lymphomas and natural killer (NK) cell lymphomas. Only a few studies have investigated the possibility that at least somePTCLs could be derived from lymphocytes with cytotoxic potential. In order to investigate this possibility, 60 cases of PTCL, including27 cases expressing the áâ T-cell receptor (TCRáâ), 15 TCRãä cases and 18 cases expressing neither TCR (TCR silent), as well as 14cases of NK-cell lymphomas, were studied by immunohistochemistry for the expression of TIA-1, perforin, and granzyme B proteins.Expression of TIA-1 is characteristic of cytotoxic cells regardless of their activation status, whereas expression of perforin andgranzymes is highly increased in activated cytotoxic cells and correlates with the induction of cytolytic activity. All NK-cell lymphomas(11 sinonasal, three systemic cases) expressed TIA-1, perforin, and granzyme B in most tumour cells. All ãä PTCLs (15 cases) expressedTIA-1 protein in most tumour cells, with a different cytotoxic antigen profile in hepatosplenic ãä PTCL (TIA-1+, perforin", granzymeB") and in non-hepatosplenic ãä PTCLs (three nasal, one skin, one lung), the latter expressing the three cytotoxic proteins. Of the 45cases of áâ and TCR silent PTCL, 15 (33 per cent) were considered to be derived from cytotoxic lymphocytes with expression of at leastone cytotoxic protein (TIA-1, 15/45; perforin, 10/41; granzyme B, 14/38) in tumour cells. This cytotoxic protein expression appeared tobe related to the site of localization, since 7/13 (54 per cent) extranodal and only 8/32 (25 per cent) nodal áâ and TCR silent PTCLsexpressed TIA-1, and to histology, since this pattern was observed in a proportion of anaplastic (6/8, 75 per cent) and pleomorphic (8/17,47 per cent) lymphomas, but not in AILD-type NHL (0/16). Taken together, our data suggest that NK-cell lymphomas andnon-hepatosplenic ãä PTCLs represent tumours of activated cytotoxic NK cells and ãä T cells, respectively; that hepatosplenic ãäPTCLs represent tumours of non-activated cytotoxic ãä T cells; and that a small proportion of áâ and TCR silent PTCLs, mostlyextranodal cases, or nodal anaplastic lymphomas, represent tumours of cytotoxic T cells. ? 1997 John Wiley & Sons, Ltd.

J. Pathol. 183: 432–439, 1997.No. of Figures: 3. No. of Tables: 5. No. of References: 43.

KEY WORDS—peripheral T-cell lymphomas; NK-cell lymphomas; TIA-1; perforin; granzyme B; cytotoxicity

INTRODUCTION

Peripheral T-cell lymphomas (PTCLs) are supposedto be derived from post-thymic T cells.1,2 They representa heterogeneous group of diseases with respect to histo-logy, site of origin, and cell origin. On histology, themost frequent subtypes are angioimmunoblastic(AILD), pleomorphic medium and large (PML), andanaplastic large cell (ALCL) lymphomas.3,4 MostPTCLs express the áâ T-cell receptor (TCR), whereasrare PTCLs express the ãä TCR.2 Most, if not all ãäPTCLs are extranodal lymphomas and among them,hepatosplenic ãä PTCLs constitute a distinct clinico-pathological entity with aggressive clinicalbehaviour.1,2,5–8 Besides áâ and ãä PTCLs, 20–30 percent of PTCLs express neither TCR and thus have beenreferred to as TCR silent PTCLs.2 We have recentlyshown9 that among the TCR silent PTCLs, there is agroup of extranodal, mainly sinonasal tumours which

can be considered as natural killer (NK) cell lymphomason the basis of the CD3 (Leu4)", CD2+, CD5",CD56+ phenotype and the absence of TCR ã generearrangements.Cytotoxic cells include NK cells and cytotoxic áâ and

ãä T lymphocytes (CTLs). These cells play a major rolein the defence against neoplastic processes and viralinfections.10,11 Two main mechanisms have beendescribed by which a cytotoxic cell may induce lysis ofits target. One involves granule exocytosis and the otherthe interaction between the Fas antigen and the Fasligand.10,11 The former mechanism involves granule-associated cytotoxic proteins, including the T-cell intra-cellular antigen-1 (TIA-1), perforin, and granzymes.12–15Perforin produces pores on the target cell membrane andcan induce necrosis of target cells by osmotic celllysis.11,12 In addition, perforin-produced pores can alsoallow the entry of other cytotoxic proteins such as TIA-1and granzymes, which trigger a process leading to DNAfragmentation and apoptosis of the target cells.13,14TIA-1, also recently renamed GMP-17, is a granulemembrane protein whose expression is restricted to

*Correspondence to: Dr Philippe Gaulard, Département dePathologie, Hôpital Henri Mondor, 94010 Créteil Cédex, France.

CCC 0022–3417/97/120432–08 $17.50 Received 28 February 1997? 1997 John Wiley & Sons, Ltd. Accepted 2 June 1997

CTLs, NK cells, and neutrophils.16 Biochemical studieshave shown that expression of the TIA-1 protein ischaracteristic of cytotoxic T and NK cells, regardless oftheir activation status.14–16 By contrast, although per-forin and granzymes have been detected at low levels inãä T cells and NK cells constitutively, their expression isinducible after activation in all cytotoxic cells, includingáâ T cells, and correlates with the induction of cytolyticactivity.10,17–19Recently, the development of monoclonal antibodies

against human TIA-1, perforin, and granzymes hasmade possible the immunohistological identification ofcells expressing these proteins.15,20,21 A few recentstudies have reported the expression of cytotoxic pro-teins in occasional extranodal PTCLs,22 includingsinonasal NK-cell lymphomas9,23 and ãä hepatospleniclymphomas,8 and in anaplastic lymphomas.24 Theexpression of cytotoxic proteins, however, has so far notbeen systematically investigated in relation to the expres-sion of TCR proteins in PTCLs. Thus, we have studiedby immunohistochemistry 60 cases of PTCLs including15 ãä PTCLs, as well as 14 cases of NK-cell lymphomas,for the expression of TIA-1, perforin, and granzyme Bproteins. The aims were to determine firstly whether theexpression of these proteins could be related to the TCRstatus of the PTCLs, to histology, and/or to tumour site,and secondly whether expression displays differentpatterns in PTCLs versus NK-cell lymphomas. Inaddition to their specificity, the antibodies against TIA-1and granzyme B cytotoxic antigens were also chosen fortheir ability to react in routinely fixed paraffin-embeddedtissue sections, since they recognize formalin-resistantepitopes, thus allowing identification of the neoplastic orreactive nature of the cells expressing these cytotoxicproteins.8,9,24

MATERIALS AND METHODS

Case selection

Sixty cases of peripheral T-cell lymphomas and 14cases of NK-cell lymphomas with available frozen tissuewere selected. The diagnosis was based on histologicaland immunohistological data available on both fixedand frozen tissue sections. Lymphoblastic lymphomasand mycosis fungoides were excluded. TCR silentPTCLs, ãä T-cell PTCLs, and NK-cell lymphomas wereretrieved from the files of the Department of Pathology,Henri Mondor Hospital. These included several cases ofreferred nasal/nasopharyngeal and ãä T-cell lymphomas.All cases of áâ PTCL with available frozen tissuewere collected over the last 30 months. All cases wereselected on the basis that the tumour cells expressed atleast one of the CD2, CD3, CD5 and CD7 T-cellantigens and were negative for B-cell markers (CD19,CD20, and CD22). Some of the immunophenotypicand/or genotypic data of 26 cases have been reportedelsewhere.2,5,6,9The PTCLs were divided into áâ lymphomas (27

cases), ãä lymphomas (15 cases), and ‘TCR silent’ lym-phomas (18 cases), according to the expression of the

TCR proteins using âF1 and äTCR-1 antibodies.2 Thediagnosis of TCR silent PTCL was considered whenlymphoma cells were negative for both âF1 and äTCR-1markers and did not have the phenotypic (CD5",CD56+) profile of NK-cell lymphomas.2,9 Among the18 such lymphomas, 16 of the 17 cases with availablegenomic data had clonal rearrangement of TCR ã (12cases) or â genes (four cases). Lymphomas were classi-fied according to the updated Kiel classification3 and thehepatosplenic ãä T-cell lymphomas were added accord-ing to the REAL classification.1 The histotypes and themain site of localization at presentation, where biopsywas performed for diagnosis, in relationship to the TCRstatus, are listed in Table I. In this table, TCR silentPTCLs and áâ PTCLs are grouped together, since wehave already shown that most TCR silent PTCLs withTCRâ and/or TCRã gene rearrangements, have thecommon histological, clinical, and genotypic features,including TCRä deletions, of áâ PTCLs.6In addition, 14 cases referred as NK cell lymphomas

were included. These had the TCR silent, CD2+,CD5", CD56+ phenotype, characteristic of normalNK cells and NK-cell lymphomas,9,25 and no clonalTCRã rearrangement was detected in the six casesstudied. They comprised 11 sinonasal tumours and threesystemic lymphomas with spleen, liver, and bonemarrow involvement associated in one case withabdominal intestinal tumour.

Tissue specimens

Fresh tissue specimens were obtained for diagnosis.One portion was immediately snap-frozen in liquidnitrogen and stored at "80)C for phenotypic andgenotypic studies. The others were fixed in bufferedformalin or Bouin’s fixative and paraffin-embedded.

Table I—Histological repartition according to the site ofbiopsy at presentation in NK-cell lymphomas and in ãä, áâ,and TCR silent peripheral T-cell lymphomas

NK-cell lymphomas (n=14)Sinonasal 11 11 PMLBone marrow 2 2 PMLIntestinal 1 1 PML

ãä T-cell lymphomas (n=15)Hepatosplenic 10 10 MMSNasal 3 3 PMLSkin 1 1 PMLLung 1 1 PML

áâ and TCR silent T-cell lymphomas (n=45)Lymph node 32 8 PML, 16 AILD, 7 ALCL, 1 LELSkin 8 5 PML, 1 ALCL, 2 PSCDigestive 4 3 PML, 1 PSCSinonasal 1 1 PML

AILD=angioimmunoblastic T-cell lymphoma; ALCL=anaplasticlarge cell lymphoma; LEL=lymphoepitheliod lymphoma;MMS=monomorphic medium-sized; PML=pleomorphic mediumand large cell lymphoma; PSC=pleomorphic small cell.

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Immunohistochemical staining

Cryostat sections were evaluated for B-cell, T-cell,and NK cell differentiation antigens using the alkalinephosphatase anti-alkaline phosphatase (APAAP) tech-nique.26 The monoclonal antibodies used are listedin Table II. Cytotoxic cell proteins were detectedusing monoclonal antibodies recognizing the T-cellintracellular antigen-1 (TIA-1),15 perforin, andgranzyme B.21 Expression of perforin was evaluated onfrozen tissue sections, whereas the mouse monoclonalantibodies TIA-1 and GrB-7, which recognize formalin-resistant epitopes of TIA-1 and granzyme B respectively,were used in paraffin-embedded tissue sections. Forbetter detection of TIA-1 and granzyme B, sectionswere pretreated using microwave oven heating (twocycles of 5 min in 0·01 citrate buffer, pH 6). Optimumlabelling for granzyme B was obtained by twice repeat-ing the bridge and APAAP complex. Rabbit anti-mouse immunoglobulins and APAAP complexes were

obtained from Dako. Each slide was examined by threeof us (MLB, PK, PG). Cytocentrifuged cells fromYT2C2 and YTIndi NK cell lines, as well as frozen andparaffin-embedded tissue sections of one NK-cell lym-phoma, which had a strong granular positivity forTIA-1, Grb-7, and perforin, were used as positivecontrols.A case was considered positive when there was

unequivocal staining of tumour cells, irrespective oftheir number. TIA-1 and granzyme B expression waseasy to interpret since the corresponding antibodies wereused on paraffin sections, thus allowing identification oftumour cells. In addition, most cases displayed stronglabelling on a sizeable proportion of neoplastic cells(>20 per cent). For perforin, which was studied onfrozen sections, a case was considered positive whenstaining was observed in at least 20 per cent of the cells.Cases with tumour cells negative, but with small reactivelymphocytes positive were classified as negative. Caseswere considered as non-interpretable when staining was

Table II—Monoclonal antibodies used for immunohistochemistry

Antigen Name Source

CD2 Leu 5 Becton Dickinson (Mountain View, CA, U.S.A.)CD3 Leu 4 Becton Dickinson (Mountain View, CA, U.S.A.)CD4 Leu 3 Becton Dickinson (Mountain View, CA, U.S.A.)CD5 Leu 1 Becton Dickinson (Mountain View, CA, U.S.A.)CD7 Leu 9 Becton Dickinson (Mountain View, CA, U.S.A.)CD8 Leu 2 Becton Dickinson (Mountain View, CA, U.S.A.)CD19 B4 Coulter (Hialeah, FL, U.S.A.)CD22 CD22 Dako SA (Glostrup, Denmark)CD56 NKH1 Coulter (Hialeah, FL, U.S.A.)TCRáâ âF1* T-cell Diagnostics (Woburn, MA, U.S.A.)TCRãä äTCR1† T-cell Diagnostics (Woburn, MA, U.S.A.)TIA-1 TIA-1 Coulter (Hialeah, FL, U.S.A.)Granzyme B GrB-7 Monosan (Uden, The Netherlands)Perforin Delta G9 T-cell Diagnostics (Woburn, MA, U.S.A.)

*âF1 recognizes a non-polymorphic epitope of the â chain of the áâ TCR heterodimer.†äTCR-1 recognizes a non-polymorphic epitope of the ä chain of the ãä TCR heterodimer.

Table III—Cytotoxic protein expression in NK-cell lymphomas and ãä T-cell lymphomas

TIA-1+ Granzyme B+ Perforin+ Overall+(%)

NK-cell lymphomas 14/14 13/13* 14/14 14/14 (100%)Sinonasal 11/11 11/11 11/11 11/11 (100%)Intestinal 1/1 1/1 1/1 1/1 (100%)Bone marrow 2/2 1/1* 2/2 2/2 (100%)

ãä T-cell lymphomas† 15/15 5/15 5/13* 15/15 (100%)Hepatosplenic 10/10 0/10 0/8* 10/10 (100%)Nasal 3/3 3/3 3/3 3/3 (100%)Skin 1/1 1/1 1/1 1/1 (100%)Lung 1/1 1/1 1/1 1/1 (100%)

Total 29/29 18/28 19/27 29/29 (100%)

*Slides for granzyme B expression were not interpretable in one case; slides for perforin expression were not availablein two cases.†All ãä T-cell lymphoma cases were CD4"/CD8".

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observed neither on tumour cells nor on reactive cells(i.e., no internal positive control).

RESULTS

NK-cell lymphomasAll cases of NK-cell lymphomas strongly expressed

TIA-1, perforin (14/14), and granzyme B (13/13) in thecytoplasm of virtually all tumour cells (Table III). Thestaining pattern for TIA-1 and granzyme B was granu-lar, with predominant paranuclear staining in the Golgiregion, whereas the pattern of perforin expression waseither similar or, in some instances, more diffuse andgranular (Figs 1A and 1B). A similar activated cytotoxicprofile (TIA-1+, granzyme+, perforin+) was observedboth in sinonasal and in systemic cases. All cases ofNK-cell lymphomas were classified as pleomorphicmedium and large lymphomas and had a CD4"/CD8" phenotype.

ãä T-cell lymphomas

The results of cytotoxic protein expression in ãä T-celllymphomas are summarized in Table III. All cases of ãäT-cell lymphomas (15/15) demonstrated TIA-1-positivetumour cells, whatever the site of localization (hepato-

splenic, nasal, skin, or lung) (Fig. 2A). Thirteen casesdisclosed strong staining in most, if not all, neoplasticcells, whereas in two hepatosplenic cases, only a pro-portion of tumour cells were shown to express thisprotein.The expression of granzyme B and perforin was de-

pendent on the site of localization. The five non-hepatosplenic cases expressed perforin and granzyme B,indicating an activated cytotoxic status. These lattercytotoxic proteins were found in most tumour cells, witha heterogeneous pattern in three cases. By contrast,perforin and granzyme B were detected in only a fewscattered, apparently reactive cells in all hepatospleniccases (Fig. 2B). Thirteen of the 15 cases of ãä T-celllymphomas had a CD4"/CD8" phenotype, the tworemaining being uninterpretable for CD4 or CD8staining. On histology, hepatosplenic cases weremonomorphic medium-sized, whereas the five non-hepatosplenic tumours were classified as pleomorphicmedium and large cell lymphomas.

áâ and TCR silent PTCLs

Forty-five cases of áâ and TCR silent PTCL ofvarious histotypes were tested (Table IV). In most cases,expression of TIA-1, granzyme B, and perforin was

Fig. 1—Cytotoxic protein expression in nasal NK-cell lymphoma (paraffin-embedded sample). (A) Tumour cells exhibit strong granularcytoplasmic staining with TIA-1. (B) Paraffin section immunostained with granzyme B demonstrates strong positivity in tumour cells andepitheliotropism

Fig. 2—Cytotoxic protein expression in hepatosplenic ãä T-cell lymphoma. (A) Tumour cells located in the red pulp of the spleen displaycytoplasmic TIA-1 positivity (paraffin section). (B) Tumour cells do not express perforin, whereas only a few apparently reactivelymphocytes reveal perforin immunoreaction (frozen section)

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restricted to small, apparently reactive cells, but 15/45cases (33 per cent), including 9/27 áâ and 6/18 TCRsilent PTCLs, expressed at least one cytotoxic granule-associated antigen in tumour cells. These 15 cases wereshown to express TIA-1 and all but one had an activatedcytotoxic antigen profile, since they contained neoplasticcells expressing either perforin (10/12) or/and granzyme(14/15). In all but one cases, TIA-1-expressing cellsoutnumbered perforin- and/or granzyme B-positivecells.

We divided the 45 cases according to site in nodal (32cases) and extranodal cases (13 cases) (Table V). Of the13 extranodal cases, 7 (54 per cent) disclosed character-istic granular cytoplasmic TIA-1 staining in virtually alltumour cells. All but one case (one anaplastic skinlymphoma) had pleomorphic morphology. They com-prised 3/4 digestive lymphomas (two CD4"/CD8"intestinal, one CD8+ gastric), 1/1 sinonasal CD8+lymphoma, and 3/8 skin lymphomas (one pleomorphicCD8+, one anaplastic CD4+, one pleomorphic CD4"/

Table IV—Cytotoxic protein expression in the 45 cases of áâ and TCR silent peripheral T-cell lymphoma

PatientNo.

Site oforigin Histology

TCRáâ

TCRãä CD4 CD8 TIA-1 Perforin Granzyme B

1 LN PML + " " " + " +2 LN PML + " + " " " "3 LN PML + " + " " " "4 LN PML + " + " " " "5 LN PML + " " + + + +6 LN PML + " " " + + +7 LN PML " " + " " " "8 LN PML " " + " " " "9 LN AILD + " + " " " na10 LN AILD + " + " " " "11 LN AILD + " + " " " "12 LN AILD + " + " " " "13 LN AILD + " " + " " "14 LN AILD + " + " " " na15 LN AILD + " + " " " na16 LN AILD + " + " " " "17 LN AILD " " + " " " "18 LN AILD " " + " " " na19 LN AILD " " " " " " na20 LN AILD " " + " " " "21 LN AILD " " + " " " na22 LN AILD " " + " " " "23 LN AILD " " + " " " "24 LN AILD " " + " " " "25 LN ALCL + " + " " " "26 LN ALCL + " " " + + +27 LN ALCL + " + " " ni "28 LN ALCL + " + " + + +29 LN ALCL " " " " + ni +30 LN ALCL " " + " + + +31 LN ALCL " " " + + + +32 LN LEL " " " " " " "

33 Stomach PML + " " + + " +34 Nose PML + " " + + + +35 Intestine PML + " " " + ni +36 Intestine PML " " " " " " "37 Intestine PSC " " " " + + "38 Skin PML " " " " + + +39 Skin ALCL " " + " + ni +40 Skin PML + " + " " " "41 Skin PSC + " + " " " "42 Skin PSC + " + " " " ni43 Skin PML + " + " " " "44 Skin PML + " " + + + +45 Skin PML + " + " " " "

Abbreviations are as in Table I; LN=lymph node."=no positivity in tumour cells; +=positivity in tumour cells; na=not available; ni=not interpretable.

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CD8"). All but the gastric case expressed perforin (4/5cases) and/or granzyme B (6/7 cases).Of the 32 nodal cases, only eight (25 per cent) revealed

cytotoxic antigen expression in tumour cells. These casesincluded 5/7 anaplastic lymphomas (Fig. 3A), as well asthree other nodal cases, classified as pleomorphicmedium and large cell lymphomas. Four of these eightpositive cases had a CD4"/CD8" phenotype; twowere CD4+, the two remaining being CD8+.In the remaining negative cases, a few small, appar-

ently reactive TIA-1-positive and usually less numerousgranzyme B-positive cytotoxic lymphocytes were seen(Fig. 3B). It is noteworthy that these cytotoxic smalllymphocytes were often numerous in most AILD-PTCLs, and include in four of these cases, a fewscattered lymphocytes with irregular and slightlyenlarged nuclei.

DISCUSSION

In the present study, we have shown that the expres-sion of the cytotoxic proteins can be related to NK-cell

origin and the TCR status of PTCL, as well as tohistology and clinical presentation. Indeed, we haveidentified cytotoxic proteins in all NK-cell and ãäT-cell lymphomas, whereas cytotoxic potential wasdemonstrated in only 33 per cent of áâ and TCR silentPTCLs. In the latter group, there was a striking ten-dency for the lymphomas derived from cytotoxic cellsto occur in extranodal sites, or to have anaplasticmorphology.In this study, all NK-cell lymphomas, irrespective of

sinonasal or extranasal clinical presentation, stronglyexpressed the three cytotoxic proteins in most tumourcells. This could be expected, since NK cells and NK celllines strongly express these proteins.12–14,18 Moreover,the strong expression of perforin and granzyme B inNK-cell lymphomas indicates that they representtumours of activated NK cells, since expression ofperforin and granzymes is greatly increased in activatedcytotoxic cells.17,18

ãä PTCLs are rare and relatively recently recognizedlymphomas, which include a peculiar clinicopathologicalentity named hepatosplenic ãä PTCL and other casesdeveloping preferentially in mucosal sites such as skin

Table V—Cytotoxic protein expression in áâ and TCR silent PTCLs, in relation to site of origin andhistology

TIA-1+ Granzyme B+ Perforin+ Overall+(%)

Extranodal n=13 7/13 6/12 4/11 7/13 (54%)Skin* n=8 3/8 3/7 2/7 3/8 (37%)Digestive n=4 3/4 2/4 1/3 3/4 (75%)Sinonasal n=1 1/1 1/1 1/1 1/1 (100%)

Nodal n=32 8/32 8/26 6/30 8/32 (25%)AILD n=16 0/16 0/10 0/16 0/16 (0%)Lennert n=1 0/1 0/1 0/1 0/1 (0%)PML n=8 3/8 3/8 2/8 3/8 (37%)ALCL n=7 5/7 5/7 4/5 5/7 (71%)

Total 15/45 14/38† 10/41† 15/45 (33%)

*Mycosis fungoides was excluded from the study.†Slides for granzyme B and perforin were not interpretable in one and four cases, respectively, and were not available

for granzyme B in six cases.

Fig. 3—TIA-1 expression in nodal áâ and TCR silent peripheral T-cell lymphoma. (A) Anaplastic TCR silent lymphoma: tumour cellsdisplay strong granular TIA-1 positivity. (B) Pleomorphic (TCR áâ+) medium and large cell lymphoma: only a few scattered, apparentlyreactive TIA-1-positive lymphocytes are present, whereas tumour cells are negative (paraffin section)

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and sinonasal regions.2,5,7,8 In the present study, we haveshown that all hepatosplenic ãä PTCLs expressed TIA-1protein, usually in most tumour cells, whereas perforinand granzyme B were observed in only a few apparentlyreactive cells in all cases. These results are in agreementwith a recent report.8 In contrast, the three cytotoxicproteins were expressed by most tumour cells in non-hepatosplenic ãä PTCLs (i.e., lung, skin, sinonasal).These data indicate that non-hepatosplenic ãä PTCLsrepresent tumours of activated cytotoxic ãä T cells,whereas hepatosplenic ãä PTCLs represent tumours ofnon-activated cytotoxic ãä T cells. The activation statusof non-hepatosplenic ãä PTCLs can be further substan-tiated by the expression of the CD30 molecule, which, incontrast, was not detected in hepatosplenic ãä PTCLs(data not shown). It is known that activation of cyto-toxic ãä T cells can be influenced by antigenic stimuliand/or cytokines. It has been reported that the numberand the cytotoxic activity of post-thymic ãä T cells canbe influenced by different antigenic stimuli: malariainfection in splenic ãä T cells,27 gluten in gut ãä T cells,28smoking in bronchial ãä T cells,29 or viruses such asEBV.30 In addition, cytokines stimulating (IL-2, IL-4,IL-6, IL-12) or counteracting (IL-10) the activation ofcytotoxic cells may also play a role in the heterogeneityof granzyme or perforin expression.12,17,31In contrast to the findings concerning NK-cell lym-

phomas and ãä PTCLs, cytotoxic proteins were detectedin only 15/45 cases (33 per cent) of áâ and TCR silentPTCLs. All were positive for TIA-1 and 14/15 expressedgranzyme B and/or perforin. Of these 15 TIA-1 cases,three had a CD4+ phenotype suggesting derivationfrom the subset of cytotoxic CD4+ cells.32,33 Indeed,CD4+ cells expressing cytotoxic proteins in vivo havebeen detected in follicular B-NHL34 and there is a recentreport of CD4+ large granular lymphoproliferationwith perforin-expressing tumour cells.33 In the remain-ing áâ and TCR silent PTCL cases, five had a CD8+phenotype and seven were CD4"/CD8". This is inkeeping with previous findings showing TIA-1 expres-sion in CD8+ and CD4"/CD8" T-cell malignan-cies.35 With respect to histology, the áâ and TCR silentPTCLs expressing at least one cytotoxic protein weremostly of anaplastic morphology (6/8) or, to a lesserextent, of pleomorphic small (1/3) or pleomorphicmedium and large cell types (8/17), but comprised noAILD lymphoma. These results suggest a high incidenceof cytotoxic protein expression in anaplastic large celllymphomas, as recently reported,24 but also in a pro-portion of pleomorphic PTCLs. Among the lymphomaswithout cytotoxic protein expression in the tumour cells,a peculiar pattern was observed in most AILD-PTCLswhere numerous apparently reactive, usually small lym-phocytes expressed TIA-1 and granzyme B, suggesting acytotoxic cell reaction. It could be hypothesized that thelatter could be related to the presence of EBV, which iscommonly found in some blastic cells of AILD-PTCL.36Finally, with respect to the clinical presentation, it wasnoteworthy that a cytotoxic profile was much morefrequently observed in extranodal áâ and TCR silentPTCLs (7/13, 54 per cent) than in nodal PTCLs (8/32, 25per cent).

Altogether, the present data show that most of thelymphomas with cytotoxic antigen expression occur inextranodal sites, especially mucosal and hepatosplenicsites. In mucosa, cytotoxic lymphomas appear to repre-sent tumours of activated cytotoxic T cells, either NK, ãäor áâ. It is suggested that these cells in mucosa can beregarded as the first line of immune defence.37 Asmentioned above, this activated cytotoxic profile ofmost non-B-cell lymphomas arising in sinonasal sites,lung, and digestive tract reinforces the putative role ofan antigen in the proliferation as well as the activation ofthe neoplastic T or NK cells. Such candidates forintestinal and sinonasal cytotoxic lymphomas could begluten and EBV, respectively.38,39 Indeed, 2/3enteropathy-associated T-cell lymphomas (EATCLs) inthe present series expressed TIA-1 and granzyme B, inkeeping with a previous study reporting that 77 per centof EATCLs are granzyme B-positive lymphomas andoriginate in activated cytotoxic intraepithelial T lym-phocytes (T-IELs).22 Furthermore, it has been shownthat T-IELs are cytotoxic T cells that are in the restingstate (TIA-1+, granzyme B") in the normal smallbowel as well as in giardiasis, but acquire an activatedcytotoxic profile in coeliac disease, as suggested by thegranzyme B positivity of their granules.40 In sinonasallymphomas, it was noteworthy that all cases in thepresent series originated from activated cytotoxic cells,either of NK (11/11), or Tãä (3/3) and/or Táâ (1/1) cellorigin, and that all of them were EBV-positive inmost tumour cells (data not shown).39 These findingsreinforce the role of EBV in the pathogenesis of suchlymphomas, since EBV, besides its oncogenic properties,can also induce activation and proliferation of cytotoxiccells.41The present findings indicate that the presence of a

cytotoxic phenotype is strongly associated with NK-celllymphomas, and that the TCR status of T-cell lympho-mas is related to the site of origin and to some histologi-cal subtypes, including anaplastic and pleomorphiclymphomas. Most of these tumours, including NK-celllymphomas,9,25,39 ãä hepatosplenic T-cell lymphomas,2,7and EATCLs,42 have a poor prognosis, and a poorresponse to the chemotherapy in use for aggressivelymphomas. Further investigations are needed in orderto search for a putative relationship between cytotoxicphenotype and resistance to therapy.43

ACKNOWLEDGEMENTS

We are indebted to the following pathologists: A.-C.Baglin, M. Bernier, J. Brière, S. Caulet-Maugendre,C. Duval, B. Epardeau, B. Fabiani, C. Lavignac, M.Lebezu, A. de Mascarel, E. Navratil, J. Quillard, T.Petrella, and M. Wassef for providing samples; M.-H.Delfau-Laurue for genomic studies; and A. Bensussanfor providing YT2C2 and YTIndi cell lines.

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