nasal t cell lymphomas

7/27/2019 Nasal T Cell Lymphomas

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1993 81: 2688-2695

GaulardP Kanavaros, MC Lescs, J Briere, M Divine, F Galateau, I Joab, J Bosq, JP Farcet, F Reyes and Ppeculiar phenotype and with Epstein-Barr virusNasal T-cell lymphoma: a clinicopathologic entity associated with

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reserved.Copyright 2011 by The American Society of Hematology; all rights900, Washington DC 20036.weekly by the American Society of Hematology, 2021 L St, NW, SuiteBlood (print ISSN 0006-4971, online ISSN 1528-0020), is published

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N a s a l T - c e l l L ym p ho m a: A C l i n i c o p a t h o l o g i c E n t i t y A s s o c i a t e d W i t h P e c u l i a rP h e n o t y p e a n d W i t h E p s t e i n - B a r r V i r u sBy Panagiotis Kanavaros, Marie-Claude Lescs, Josette Briere, Marine Divine, Franqoise Galateau, I rh e Joab,

Jacques Bosq, Jean-Pierre Farcet, Felix Reyes, and Philippe GaulardRecent evidence has shown that most nasal lymphomas(NL) are associated with a T-cell phenotype and are thuscalled nasal T-cell lymphomas (NTCL), but litt le informationis available about the T-cell receptor (TCR) expression. Thepresence of Epstein-Barr virus (EBV) genome has been re-cently reported in NTCL in Oriental populations in whichNL and EBV-associated umors are more common and inoccasional Occidental cases. This prompted us to investi-gate lymphoma biopsies from 7 non-Oriental patients withNTCL for the expression of natural killer (NK) and T-cellantigens, including TCR proteins, for the presence of EBV-encoded atent membrane protein (LMP) using immunohis-tochemistry and for the presence of EBV DNA and Epstein-Barr early region (EBER) RNA using in situ hybridization(ISH). Six cases displayed a CD3- , TCRaB-, TCRy6-,CD2+, CD7+, CD5-, CD4-, CD8-, CD56+ phenotype,suggesting hat these tumors may be peripheral T-cell lym-

ALIGNANT LYMPHOMAS involving the nasalM cavity are unusual in Western in con-trast to Oriental countries, in which they represent the secondmost frequent group of extranodal lymphomas after gas-trointestinal lymphoma^.^^ Nasal lymphomas include tumorsclinically presenting as a lethal midline granuloma and oftendisplay histologic features of pleomorphic malignant tumorsvariably associated with angiocentricity, angioinvasion, andnecrosis.24-8-10 hen the latter features are present, the termpolymorphic reticulosis (PR) has been used,' but the alter-native term of angiocentric immunoproliferative lesion (AIL)has been more recently proposed by Ja!E.11.12n recent years,there has been growing evidence that most cases are relatedcases of nasal lymphomas in Oriental populations can be con-sideredas nasal T-cell lymphomas (NTCL) because they com-monly express phenotypic markers of T cells."' Interestingly,immunohistochemicalstudies showed peculiar phenotypic fea-tures such as expression of natural killer (NK)cell-relatedmarkers and extensive T-cell antigen loss including absence ofexpression of a@T-cell receptor (TCR).',I5 In Western patients,recent studies2" indicate that most nasal lymphomasaso exhibit

to premalignant or malignant T-celldisorders.',3,6,'0,'2-'4ost

From the Dkpartements de Pathologie, d'HPmatologie Clinique,d'lmmunologie and INSERM U91, H6pital Henri Mondo r, Crkteil;the DPpartements de Pathologie, H6p ital Laen nec, Paris; CH U C6tede Nacre, Caen; and the Dipartement de Pathologie and CNRS 1301,Institut G ustave Rous sy, Villeju$ France.Submitted September 3, 1992; accepted December 14 , 1992.Supported in part by grants fro m UniversitP Paris- Val-de-M arneand C.N.A.M.T.S.Address reprint requests to Philippe Gaulard, MD, DPpartementde Pathologie, H6pital H enri Mondo r, 94010 CrPteil ckdex, France.Th e publication costs o this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked"advertisement" in accordance with 18 U.S.C. section 1734 solely toindicate this act.0 99 3 by The American Society o Hematology.0006-4971/93/8110-0013$3.00/0

phomas (PTCL)with extensive loss of T-cell antigens andexpressionof the NK-cell (CD56) antigen or, alternatively,NK-cell neoplasias. The remaining case was a y6 PTCL, asshown by the CD3+, TCRy6' phenotype and the biallelicy and 6 TCR gene rearrangements. Using ISH, EBER RNAtranscripts were detected in tumor cells in all cases andEBV DNA was shown in the 6 tested cases. In all cases,tumor cells expressed LMP. These findings support theconcept that NTCL constitute a distinct group of lymphomasthat, in addition to their peculiar clinical features, exhibitan unusual TCR "silent" CD56+ or TCRy6+ phenotype andharbor he EBV. In view of the LMP transforming potential,these data suggest that EBV may play a role in the patho-genesis of NTCL.0 993by The American Society of Hematology.

T-cell phenotype, although one of them reported predominanceof B-cell tumors.' However, the expression of TCR and NK-cell-related antigens, has not been reported so far.Epstein-Barr virus (EBV) is closely associated with benignand malignant lymphoproliferations such as infectiousmononucleosis, African Burkitt's lymphomas (BL), and B-cell lymphoproliferative disorders arising in immunocom-promised individuals.I6 More recently, EBV has been linkedto many cases of Hodgkin's disease (HD)'7-22 nd, much lessfrequently, to B- or T-cell non-Hodgkin's lymphomas (NHL)arising in patients without overt preexisting immunodefi-ciency. 8.23-25 Interestingly,recent studies indicate that orientalNTCL are associated with EBV, because viral genomes havebeen detected by Southern blot in 9 NTCL occurring inChinese patients26and by DNA in situ hybridization (ISH)in 5 NTCL occumng in Japanese patients.' In the latter,tumor cells also expressed the EBV-encoded latent membraneprotein (LMP). The presence of EBV genomes has also beenidentified in the tumor cells of a few occidental nasal lym-phomas with a T-cell p h e n ~ t y p e . ~ ~ . ~ ~owever, to our knowl-edge, studies for the detection of EBV-encoded protein LMP,which isknown for its transforming and oncogenic propertiesin vitro, have not been reported in Occidental NTCL.These data prompted us to investigate 7 cases of NTCLoccurring in 6 French patients and 1 Mauritanean patient toanalyze extensively the expression of T-cell differentiationand activation antigens in these tumors and to determinetheir relationship with EBV. We particularly focused on theexpression of TCR antigens, NK-related antigens, and EBV-encoded LMP protein and on the detection of EBV DNA andEpstein-Barr early region (EBER) RNAs. To identify NTCL asa group of EBV-associated T-cell lymphomas distinct from otherperipheral T-cell lymphomas (PTCL), we also studied 2 1 nodalPTCL for the expression of the EBV-LMP protein.

PATIENTS AND METHODSPatients and Clinical Data

We defined NTCL as a histologically recognizable lymphoma ex-hibiting one or more T-cell markers on immunohistochemical2688 Blood, Vol 81, No 10 (May 15), 1993: pp 2688-2695


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EBV AND T-CELL RECEPTORS IN NAS AL LYMP HOMA 26 89

grounds, involving predominantly or exclusively the nasal cavity,and in w hich patients had presenting sympto ms related to nasal dis-ease. According to this definition, 7 cases of NTC L, for which frozentissue was available, were selected in 6 French patients and I Maur-itanean patient and included in this study. A summary of clinicaldata is shown in Table 1. Clinical data of 1 patient (case 1) have beenrepor te d e l s e ~h e re , ~s well as part of the immunohistochemicaland genom ic results in this patient? One patient (case 4) had alreadyreceived local radiotherapy 24 years before for a nasal reticulosar-coma that, after careful histologic and immuno histologic review,was shown to be a p leomorphic large-cell lymp hom a of T-cell type.Multiagent chemotherapy was administered in all cases, and wasassociated with local radiation therapy in cases 2 and 6. Persistentcomplete remission was observed in only 1 (case 6) of the 4 caseswith significative follow-up.Methods

Fresh tissue samples were obtained for diag-nosis from the nasal cavity (6 ), from th e skin ( I ) , and from a lymphnode (1). Fresh and /or paraffin-em bedded tissues of sites of recurrenc ewere also obtained in patients 2,4, and 5 . On e portion was paraffin-embedded and the others were snap-frozen in liquid nitrogen after30 minutes of incubation in gum-sucrose and stored at -70C. Inaddition, frozen sections from 21 randomly selected nodal PTCLwere also analyzed as a control for the expression of the EBV-encodedLMP protein. Paraffin-embedded tissue specimens werestained with hematoxylin-eosin, giemsa, and Gordon Sweet. Lym-phomas were classified according o the W orking Formulation (WF )30and the updated G e l cla~sification.~Immunohistochemical methods. Cryostat sectio ns were evaluatedfor T-, B-, and NK-cell differentiation antigens, for the activationCD25 and CD30 markers, as well as for the EBV-encoded LMP,using the alkaline phosphatase antialkaline phosphatase (APAAP)t e ~ h n i q u e . ~ he mo noclonal antibodies (Mo Abs) used in this study

Tissue specimens.

Histologic studies.

and their commercial sources are the following: Leu6/CDI, Leu5/CD2, Leu4/CD3, Leu3/CD4, Leu 1/CD5, Leu9/CD7, Leu2/CD8, a ndILR2/CD 25 (Becton Dickinson, Mountain View, CA); B4/CD19 a ndNK H 1/CD56 (Coulter, Hialeah, FL); BerH2/CD30, CD2 I , CD22,and anti-LMP-I/CS 1-4 (Dako SA, Glostrup, Denmark); and HML l(Immunotech SA, Marseille, France). LM P an d C D30 antigens werealso tested in paraffin-embedded sections using the same APAAPtechnique, because the corresponding MoAb s recognize epitopes re-sistant to fixation. Rabbit an timouse Igs and APAAP complexes wereobtained from Dako. For LMP protein, o ptim um labeling requiredenhancement by twice repeating the bridge and APAAP complexincubation steps. As positive controls for LMP immunoreactivity,the Raji cell line and one case of infectious mononucleosis were used.The negative control consisted in substitution of the primary antibodywith nonimmune serum. EBVgenomes were detected using ISH on cryostat sections with a biotin-ylated EBV DNA probe corresponding to the 3.1-kb BamHIW in-ternal repeat fragment of the EBV genome (EN ZO Diagnostics, NewYork, NY). The DISH was performed as described previo~sly.~~Briefly, cryostat sections previously fixed in 10%formalin in phos-phate-buffered saline (PBS), pH 7.6, were digested with pepsin, de-hydrated, and air-dried. The probe was diluted in a m ixture containing50% deionized formamide, 25% dextran sulfate, 0.1X SSC, and 250mg/mL sonicated salmon sperm DN A. Sections were prehybridized,denatured at 95 C for 30 minutes, an d then hybridized overnight at42C w ith the hybridization mixture containing 1 pg/mL of the probe.After several washes with 0. I X SSC in 50% formamide, the slideswere incubated with streptavidin alkaline-phosphatasecomplex (DakoSA). Alkaline phosphatase activity was visualized by incubation ina solution containing bromochloroindolylphosphate (BCIP) and ni-troblue tetrazolium (NBT) (D ako SA) for 2 hours at pH 9.6. Theslides were briefly counterstained with methyl g reen.For RISH, the fluorescein-conjugated EBV (EBER 1 and 2) oli-gonucleotides (Dako SA) complem entary to nuclear RNAs p ortions

DNA and RNA in situ hybridization (DISH and RISH).

Tab le 1. Cl in i ca l and H is to log ic Fea tu res n the Pa t i en t s W i th N T C LLocal izationat Class if ication Angio- Angio-

Case Age/Sex Symp toms Presentation (WF/Kiel) centr ic i ty invas ion Necros is Treatment, Outcome1 3 0 / M RMS Nose (nasal DM/PML + + + Chemot herapy (CHOP).

septum ), palate, hepatosp lenom egaly, DODcervical lymphnode

afer 6 mo (hepatic fai lure)2 44/F RMS Nose, palate DLC/PML - - + Chemotherapy (ACVB) + RT;

nodules; DOD after 15 m o

dead of pseudomonassepticem ia after 3 m o

Nose (R), skin (R) DLC/PML + - + local recurrence and skin3 34/F RMS, fever, dys pnea Nose, skin, lung DLC/PML + + + Chemotherapy (MA COP-6);4 64/F RMS. history of Nose (1966) DLC/PML + + + Chemot herapy; al ive with- + LED after 18 m o

~ Chemotherapy (ACVB) + RT;sinus, ethmo id DM/PML + + + local recurrence; al ive wit h

LED after 18 moa f te r 1 5 mowith LED after 7 mo

NTCL 24 yr before Nose (1990) DLC/PML -treated by RT -17/ M RMS Nose (NF), maxil lary DM/PML -

Nose (R)6 35 / M RMS Nose (NF) DLC/PML + + + Chemotherapy + RT; NED7 34/F RMS Nose (NF), maxil lary DM/PML - - + Chemot herapy (ACVB); al ive

sinusAbbreviat ions:R, relapse; RMS, recurrent maxil lary sinusitis; NF, nasal foss ae; DM . diffuse m ixed; DLC, diffuse large cell ; PML, pleomo rphic medium

and large; DOD. dead of disease; RT, radio therapy; NED, no evidence of disease; LED, local evolutive disease; WF, Wo rking Formulation; Kiel, updatedKiel classification.


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2690 K A N A V A R O S ET A L

of the EBER I and 2 genes that are actively transcribed in latentlyinfected cells were used.28Deparaffinized sections were rehydratedand pretreated with pro teinaseK, dehyd rated, air-dried, and hybrid-ized for 2 hours at 37C with the fluorescein isothiocyanate-conju-gated (FIT C) EBER oligonucleotides in hyb ridization solution con-sisting of 30% formam ide, 10% dextran sulphate, 0.1% sodiumpyrop hosph ate, 0.2% polyvinyl pyrrolidon e, 0.2% ficoll, 5 mmol/LNa2 EDTA, and 50 m mol/L T ris/HCl, pH 7.5. After washing inTBS, pH 7.6, containing 0.1% Triton X-100, the following imm u-nohistochemical detection system was used: mouse anti-FITC, rabbitantimouse Ig, and APAAP complexes (Dak o SA). The visualizationof the reaction was performed as for DISH using BCIP and NBT.As positive controls for DISH and RISH , the Raji cell line, I case ofEBV-positive BL, 2 cases o f EBV-positive B lymphoproliferationsoccurring in transplant patients, and 1 case of infectious mononu-cleosis were used. As negative controls, EBV-negative ymphoid tissuesand the hyb ridization mixture without EBV probe (DI SH) or EBERoligonucleotides (RISH) were used. Negative controls for each casewere run in parallel in every experim ent. Th e EBER signal was abol-ished after RNAase treatm ent c onfirm ing the specificity o f the re-action.

RESULTSHistology

The histologic findings are summarized in Table I . T h elymphoid proliferations were classified as diffuse large celllympho ma (cases 2, 3,4 , a n d 6) or diffuse mixed lymphoma(cases I , 5, an d 7) according to the WF.30 n K iel classification,all cases were pleom orphic med ium- an d large-cell lymph o-m a ~ . ~ 'n allcases, histologic examin ation o f biopsy specim enstaken from the nasal cavity and other sites showed pleo-mo rphic lymp hoid proliferation comp osed mainly of large-an d medium -sized lympho id cells intermingled with variablenumbers of small lymphoid cells, plasma cells, histiocytes,an d polynuclear granu locytes. Areas of necrosis were observedin all cases. Angiocentricity and angioinvasion (Fig 1) werefound in nasal biopsies in cases I , 3, 5, a n d 6 and in the skinin case 2. Epitheliotropism was detected in the nasal biopsiesin cases I , 3, 4, an d 5, and in the skin in case 2. In the twoskin biopsies available (case 2 at relapse and case 3 at pre-sentation), the atypical lymphoid infiltrate was identical totha t observed in th e nasal biopsies. It is noteworthy th at incase 4 the pat ient had a past history of a nasal lymphoidtumor that was treated by local radiotherapy (Table 1) andhad remained free of disease 24 years before she again de-veloped a nasal lymph oid malignancy. Review of the histologyshowed that the ini t ia l tum or was a pleomorphic lymphom a,and paraffin section immunohistochemistry suggested a T-cell phenotype because tum or cells were UC HL I (CD45RO)positive an d L26 (CD20) negative.Immunohistochemistry

Imm unohistochem ical staining results are recorded in Ta-ble 2. MoA bs recognizing the CDI9 an d CD22 B-cell antigen sand Ig light chains labeled only a few, apparently reactivelymphocytes. By contrast, all cases expressed at least the CD2an d CD7 T-cell antigens. Of the 7 lymphomas, 6 showed anhomogeneous phenotype, ie , CD3-, TCRaP-, TCR$,CD2+,CD7+,CD5-, CD4-, CD8-. Th e remaining case (case1) was a CD3+,TCRyG+ lymp hom a that also expressed CD2

an d CD7 antigens, but was CD5-. T h e NKH 1 (CD56)antigenwas detected on t um or cells in all cases (Fig 2). T h e CD25an d CD30 antigens were expressed o n a sizable proportionof tum or cells in all cases. The H M L- 1 ant ibody did no t s ta intumor cells in any case. CD21 antigen was expressed on asubstantial proportion of tumor cells in case 6, whereas inthe oth er cases, scattered, apparently n onneop lastic CD2 1+cells were found.E B V Studies

The results of EBV studies are summarized in Table 3.Evidence of EBV was found by ISH and by imm unohis to-chemistry in all cases. By DISH, the EBV genome was de-tected w ithin th e tu mo r cells in the 6 cases studied. Positivecells were numerous in 4 cases. Positive cells clearly sur-rounded residual negative mucous glands in most cases,whereas in case 2, positive intraep ithelial tu m or cells werepresent. By RISH, a large number of cells containing theEBV-EBER( 1-2) m RN As was identified in all cases (Fig 3).The positive cells included large neoplastic cells, sometimesshowing multilobated nuclei. Intensively positive cells rep-resented the great majority of th e entire tum or cell populationin 4 cases. The skin biopsy in case 3 displayed only a fewpositive cells surrou ndin g vessels. A pe rivascular pattern ofreactivity was also observed in case 7. By contrast, epithelialcells were negative, bu t occasional EBV-EB ER-po sitive cells,consistent with infiltration of the epithelium by tumor cells,were found in epithelial structures in cases 1, 2, 4, 5, a n d 6.In the 2 patients (cases 2 a n d 5) with recurrent material , anidentical large number of EBV-positive cells was observed atpresentat ion and at re lapse. Comp arison of the DISH a n dRISH staining patterns disclosed, in most cases, that RISHgave stronger reactivity a nd tha t the R ISH-positive cells out-numb ered those detected by DISH, as well as those expressingthe EBV-encoded L M P protein.By frozen section immunohistochemistry, a substantialproportion of tu mo r cells were found to express LM P in casesI , 2,4,6, and 7 (Fig 4), whereas in cases 3 a n d 5, a few cellswere stained. Using paraffin section im mu nohisto chem istryfo r LMP, 6 of the 7 cases were fou nd to be positive, with thestaining present exclusively i n morphologically neoplasticcells. In ad dition, com parison of adjacent tissue sections dis-closed an overlapping staining pattern for LMP and CD30.Interestingly, LMP was expressed in tumor cells in nasalbiopsies at presentation and at relapse in cases 2 a n d 5. Inaddition, LMP was also found in tumor cells in skin speci-me ns in cases 2 an d 3 as well as in cervical lymph node incase I . In the patient with a history of NT CL 24 years before(case 4), EBER mRN A a nd L MP were a lso found in tumo rcells in the paraffin-embedded nasal biopsy t ha t was retro-spectively studied. By contrast, L M P was found in only 1 ofth e 2 1 nodal PTCL studied on frozen sections.

DISCUSSIONT h e 7 NT CL in our s tudy had the cl inical presentat ion oflethal midline granuloma and displayed histologic featuresof pleomo rphic tumo rs. Angiocentricity, angio invasion, andepitheliotropism were frequent although not constant find-ings, whereas areas of necrosis were constantly present. Dis-


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Fig 1. Pleomorphic large-celllymphoma with angioinvasion(case 6). Atypical lymphoid cellsinfiltrate the wa ll of a small ar-tery (nasal biopsy with hema-toxylin-eosin stain; o riginalmagnification X 200).

Fig 2. Immunohistology oncryostat sections of NTCL. Inf il-trating lymphoid cells surround-ing residual mucosal glandsstrongly express CD56 antigen(case 2, APAAP technique;original magnification X 320).

Fig4. Detection of EBV LMP expression n NTCL(case 6). Immunohistology shows numerous neo-plastic LMP-positive cells (cryostat section, APAAPtechnique; original magnification X 200).


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2692 KAN AVAR O S ET A L

Table 2. Immunohistochemical Features in NTCLCase CD 2 CD 3 CD 4 CD5 CD 7 C D 8 CD21 C D 2 5 CD30 CD56 TCRaa TCR7I

1 Nose + +1 LN + +2 Nose + -2 Nose, skin (R) + -3 Skin + -4 Nose + -5 Nose +6 Nose +7 Nose +

---

NINI

- +/- +/-- +/- +/-- +/- +/-- +/- +/-- +/- +/-- +/- +/-- +/- +/++ +/ - +- +/- +/ -

All cases were negative for CD19 and CD22 pan-B antigens and for CD1.Abbreviations: LN. lym ph node; R . relapse; NI. not interpretable; (+), weak positivity; +, mos t tumor cel ls positive; +I-, sizable proportio n of tumor

cells positive; -, tumo r cel ls negative.

semination to other extranodal sites (skin and lung) was foundin 2 of our cases. These clinicopathologic features are inkeeping with those reported in cases of NTCL in Orientaland Western population^.^-^.^-^ NTCL have been related tothe recently described entity AIL,1 ~ 1 2 ~ 1 4hich are angiocen-tric, polymorphous lymphoreticular infiltrates that involveprimarily the midline structures and the lung. The term AILhas been proposed as a generic concept unifying entities pre-viously defined as polymorphic reticulosis and lymphomatoidgranulomatosis that are now considered to be similar if nota single disease process related to a neoplastic T-cell disor-der.6,9,2-4lthough most NTCL are characterized histolog-ically by angiocentricity, this feature was not found in somecases described herein and elsewhere.In the present study, immunohistology showed that NTCLhad peculiar phenotypic features. Indeed, 6 of the 7 casesexhibited the CD2+, CD7+, CD3-, CD5-, CD4-, CD8-,TCRafl-, TCRys-, CD56+ phenotype. This phenotype raisesquestions about the cell origin ofthese tumors. One possibility

Table 3. EBV Status in NTCLCase

ImmunohistochemistryDISH RlSH LMP

1 Nose1 Cervical lymp h node2 Nose2 Nose (relapse)3 Nose3 Skin4 Nose (1966)4 Nose (1990)5 Nose5 Nose (relapse)6 Nose7 Nose

++++++ND++++

NDND++++

++ND

++++++ND+

+++++ +++++

+++++

++ +++++++++++++++

++++

Abbre viations: ND, not done; +, a few cells positive; ++ , many cellsLMP was also shown to be present on tum or cells in paraffin-embed-

positive; +++, mo st cel ls pos itive.ded specimens in cases 2 through 7.

is that they are derived from the T-cell lineage but displayextensive loss of T-cell antigens, including absence of TCRexpression and expression of NK-related CD56 antigen. Thishypothesis could be supported by the finding that loss of T-cell antigens is a common feature of PTCL.29*34,35n addition,we and others have reported that about 20% of PTCL do notexpress detectable TCR proteins and belong to the TCR si-lent group of PTCL.29.36,37oreover, the NK-related CD56antigen may be found in T cells. We have previously reportedcases of PTCL with CD3-, TCR silent, CD56 phenotypethat were found to exhibit TCR @,y, or 6 re a ~ a n g e me n t .~ ~Alternatively, these tumors may represent true NK-cell neo-plasias, as suggested previously. 4,5,26 This can be supportedby the recent findings that some cases of NTCL may notshow TCR @, y, or 6 rearrangement^'^.^^ and by the conceptthat NK cells could represent primitive forms of T cells notrequiring TCR a@or y6 for cell recognition and thereforenot showing classical TCR gene rearrangement^.^'Regardless of the cell origin of these tumors, this peculiarCD2+, CD3-, TCR silent, CD56+ phenotype seems to beclosely associated to the NTCL. Indeed, CD2+, CD3-,TCRa@-, CD56+ phenotype was much more frequentlyfound in nasal lymphomas than in nodal lymphomas in Ori-ental population^.',^^ In Western populations, a CD2+,CD3-phenotype was also frequently reported in the few cases ofNTCL studied in frozen section^.^^,*^-^^ Taken altogether, theabove phenotypic data indicate that NTCL display distinctimmunophenotypic profile independently of the racial/geo-graphical distribution.

The remaining case was a CD3+, TCRyG+, CD56 lym-phoma. The clinical, histologic, immunohistochemical, andgenotypic features of this case, along with those of other casesofy6PTCL, have been extensively described p r e v i o u ~ l y . ~ ~ ~ ~ ~ ~Detailed phenotypic analysis of the y6 TCR status in thiscase of nasal Ty6 lymphoma showed that the tumor cellsexpressed the pan-TCR y6marker TCR-61 29 These data werefurther substantiated by Southern blot analysis showing bial-lelic y and 6 TCR rearrangements involving the Jy 1 and theJ6 1 genes.36Unfortunately, DNA analysis could not be per-formed in the other cases in which no sufficient material wasavailable. Interestingly, 6-TCR rearrangement has been re-cently reported in a case of CD2+, CD3-, TCR silent West-


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EBV AND T-CELL RECEPTORS IN NASAL LYMPHOMA 2693

Fig 3. RNA ISH in NTCL.EBER transcripts were found inthe majority of tumor cells in thenasal biopsy rom 1990 (case4,paraffin sections; or iginal mag-nification X 200).

ern NTCL.I4 and we have previously observed that. in aCD2+ . CD 3+ , TCRyb. CD5 6 lymph oma. tu mo r progres-sion was associated with phenotypic changes resulting in aCD2. CD3-. T CR silent. CD56 lymp homa with the sam e6 gene rearrangement as in iti all ~.~ ased on these data. wecould speculate that so me NT CL with the above phenotypecould correspond to y b tumors that have lost CD3 andTC Ry b expression during th e course of the disease.

In the present study, by using DNA and RNA ISH. theEBV DNA an d the EBV-encoded EBER m RN A, respectively,were detected in t um or cells in all tested cases of NTCL. Th isis in keeping with previous findings in Orientalx.6 an d W est-ern NTCL.. In the present stud y, EBV geno mes were alsoshown at d istant sites of spread a nd d etected independentlyof the presence of angio centric lesions. Take n together. thesedata show a close association between EBV an d NT CL an dindicate that this association is not restricted to the racial/geographical distribution. Th ese dat a were furthe r substan-tiated by our immu nohistochemical detection of LM P in allNTCL. irrespective of tumor site. Interestingly, using paraffinsections, LM P was demonstrated morphologically in tum orcells. These results show that EBV genomes in NT CL maybe transcribed an d translated t o proteins. indicating that EBVis not just a silent passenger in the tumor cells. Further-more. the expression of LM P by tum or cells isan impor tantfinding because LM P is thoug ht to play a crucial role in EBV-induced in vitro transformation and has been detected inhu ma n malignancies:-5 LM P has transform ing activitieswhen introduced in to rodent fibroblastoid cell lines and ren-de rs these cells tum origenic in nu de mice: In epithelia l cells,LM P induces inhibition of differentiation and morphologict r a ns f ~ r m a t ion . ~ , ~ n B-cell lines. LM P transfection ind ucesgene deregulation a nd morphologic ~h an ge s. 4~nd preventsprogrammed cell death by inducing the expression of bcl-2gene In hum an malignancies, LM P has been found

in undifferentiated nasoph aryngeal B-celllympho-prolifcrations in immunosuppressed individuals.4large-cell NHL . and in angio-immunoblastic lymph adenop-athy.I By contras t. we observed L M P expression in only 1of the 2 I nodal PTCL. As a result of the present study andof a previous Oriental report. NTC L mu st be added to thisseries of lymphoid malignancies associated with LMPexpression.The present findings in NTCL supports recent data thatexpression of LMP correlates with CD30 expression inNHL .5 Indeed, the pattern of CD 30 expression was foundto overlap with that of L MP in paraffin sections in all 7 NTCL.It could be suggested that EBV induc es the expression of theactivation molecule CD 30 and L M P may play a crucialrole in this process. This can be further supported by thefindings that LMP has been detected in Reed-Sternbergcells HD, which characteristically express the CD30 anti-

Th e mechanism by which EBV can gain entry into tum orcells of NTCL is not clear. Indeed. the CR2 EBV receptor(CD2 ), which isexpressed in abou t 30%of huma n peripheralblood T-lymphocytes.5 was detected on tumor cells in I ofthe 7 NTCL in this study and has been so far reported inonly I T-NHL. Thus. it is possible that in most EBV-har-boring T-NHL. the EBV infection has occurred during tran-sient CD 2 expression before clonal expansion of the trans-formed cells. Alternatively. molecules oth er than CD 2 mayalso play the role of EBV receptor on T cells.The clinical significance of the unusual T-cell phenotypeof NTCL as well as of their close association with EBV iscurrently unclear. It has been shown that patien ts with NT CLhave a relatively sh ort me dian survival.54Thi s could fit withour data because 3 of the 7 patients with NT CL died ofdiseasewithin 3 to 15 months after diagnosis. Further studies are

i n HD.48.49 .n C D3 0 positive anaplastic5 an d n onanaplastics

gen.4x-5


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2694 KAN AVAR O S ET AL

ne ce s sa ry t o e va lua te whe the r the presence of E B V a n d / o rLM P a n t ige n in t umor c e l l s ha s an unfa vora b le prognosticsignificance. In th is respect, PTCL c o n t a i n i n g E B V D N Ah a v e been recent ly reported as clinically aggressive lympho-m a ~ . ~ ~Our immunoh i s to log ic and ISH f indings provide furtherevidence that the so-ca lled N TC L can be regarded as a distinctg r o u p of l y m p h o m a s that, in addition to their peculiar clinicaland his tologic fea tures , exhibi t an unusual TC R silentC D 5 6 + , or TC R y6 phe no type an d are closely associated withEBV. Th e de tec t ion of LMP in tumor cells of NTCL indicatestha t EB V is n o t a silent passenger a n d suggests t ha t t hevirus may play a role in the pathogenesisof these lymphomas .

ACKNOWLEDGMENTTh e authors are grateful to C . Ducos for technical assistance andto L. Vimeux for secretarial work. They are especially indebted toDrs I. Abdalsamad and Bonnitre for referring specimens for thisstudy and to Drs C. Haioun, M. Simon, and X. Troussard for their

clinical assistance. The anti-LMP MoAb and the EBER probe werekindly provided by Dako SA France.REFERENCES

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