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PARTICULARITÃÞI MORFOLOGICEªI IMUNOHISTOCHIMICE ALE LIMFOAMELOR CUTANATE

PRIMARE CU CELULE B – ALGORITM DIAGNOSTIC

MORPHOLOGICAL AND IMMUNOHISTOCHEMICALPARTICULARITIES OF PRIMARY CUTANEOUS B-CELL

LYMPHOMA – DIAGNOSTIC ALGORITHM

ROSEMARIE HERBECK*, D. BRÎNZEU**, ALIS DEMA***, ELENA LAZÃR***,C. SOLOVAN****, V. FEIER**, HORTENSIA IONIÞÃ****

Timiºoara

CAZURI CLINICECLINICAL CASES

DermatoVenerol. (Buc.), 55: 89-102

Summary

Introduction: Primary cutaneous B-cell lymphomas(CBCL) represent a unique group of extra-nodallymphomas, ranging between 20% and 25% amongprimary cutaneous lymphomas. The WHO-EORTCclassification for cutaneous lymphomas, published in 2005,includes three main entities of primary cutaneous B-celllymphomas (CBCL): primary cutaneous B-cell lymphomawith marginal zone (pcMZL), primary cutaneous folliclecentre lymphoma (pcFCL) and diffuse primary cutaneouslarge B-cell lymphoma, leg type (pcDLBCL).

Aim: To review the histological and phenol-typicalfeatures of the main entities of CBCL and to provide adiagnostic guideline for pathologists and dermato-pathologists.

Materials and methods: Histological review wasperformed on hematoxylin-eosin and immunohistochemicalstained sections from CBCL cases retrieved from the files ofthe Pathology Department of the University ofFrankfurt/Main, Germany, over a period of 3 years.

Results: Histological, pcMZL is characterized by anodular or diffuse infiltrate composed of small to medium-size lymphoid cells with indented nuclei and an abundant,

Rezumat

Introducere: Limfoamele cutanate primare cu celule B(LCPCB) reprezintã un grup particular de limfoameextranodale, cu o incidenþã între 20% ºi 25% din totalullimfoamele primare cutanate. Clasificarea WHO-EORTCpropusã în 2005 recunoaºte trei entitãþi principale alLCPCB: limfomul cutanat primar cu celule B de zonãmarginalã, limfomul cutanat primar centrofolicular ºilimfomul cutanat primar difuz cu celule B mari.

Scop: În aceastã lucrare ne propunem reevaluareaspectelor histologice ºi imunofenotipice ale LCPCB înscopul sintetizãrii unui protocol util în diagnosticulmorfopatologic.

Materiale ºi metode: S-au revizuit aspectele histologiceºi imunohistochimice ale cazurilor de limfoame cutanateprimare cu celule B diagnosticate în Departamentul dePatologie al Universitãþii Johann Wolfgang Goethe,Frankfurt/Main, Germania pe o perioadã de 3 ani.

Rezultate: Histologic limfomul cutanat primar cucelule B de zonã marginalã se prezintã sub forma unuiinfiltrat tumoral care intereseazã dermul ºi este dispusdifuz sau nodular ºi constituit din limfocite de talie micãsau medie. Imunohistochimic celulele tumorale sunt

* Departamentul de Anatomie Patologicã, Spitalul Clinic Judeþean de Urgenþã Timiºoara.** Disciplina de Dermatologie, Universitatea de Medicinã ºi Farmacie „Victor Babeº“ Timiºoara.*** Disciplina de Anatomie Patologicã, Universitatea de Medicinã ºi Farmacie „Victor Babeº“ Timiºoara.**** Disciplina de Hematologie, Universitatea de Medicinã ºi Farmacie „Victor Babeº“ Timiºoara.

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pale cytoplasma. They are immunohistochemical CD19+,CD20+, CD79a+, CD5-, CD10-, bcl-6- and bcl-2+. PcFCLis histological characterized by a nodular or diffuseinfiltrate represented by a relatively mono-morph cellularadmixture of centrocytes and centre-blasts. The tumor cellsexpress CD19, CD20, CD79a, bcl-6 and variably CD10 bynegativity for bcl-2. The microscopic aspect of PcDLBCL isa dense diffuse infiltrate, composed of centre-blasts and/orimmune-blasts with large nuclei, prominent nucleoli and ahigh mitotic activity. Immune-phenotype, the infiltrate isCD20+, CD79a+, bcl-6+, bcl-2+, MUM1+, CD5- andCD10-.

Conclusion: PcBCL are a heterogeneous group of B-cell lymphomas whose precise classification can be achievedonly after a complete synthesis of clinical, histopathologicaland immune-phenotype features.

Key words: Primary cutaneous B cell lymphomas,immunohistochemistry, guideline diagnostic.

CD19+, CD20+, CD79a+, CD5-, CD10-, BCL-6- ºi BCL-2+. Limfomul cutanat primar centrofolicular estecaracterizat histologic printr-un infiltrat nodular sau difuzalcãtuit din celule relative monomorfe. Celule tumoraleexprimã CD19, CD20, CD79a, BCL-6 ºi variabil CD10 ºisunt negative pentru BCL-2. Microscopic limfomul cutanatprimar difuz cu celule B mari se prezintã sub forma unuiinfiltrat difuz compus din limfocite B de talie medie saumare asemãnãtoare centroblaºtilor ºi / sau immunoblaºtilorcare imunohistochimic sunt CD20+, CD79a+, BCL-6+,BCL-2+, MUM1+, CD5- ºi CD10-.

Concluzie: LCPCB constituie un grup eterogen delimfoame cu celule B, a cãror încadrare poate fi realizatãnumai dupã o sintezã completã a caracteristicilor clinice,histopatologice ºi imunofenotipice.

Cuvinte cheie: Limfoame cutanate cu celule B,imunohistochimie, algoritm diagnostic.

Introducere

Limfoamele cutanate primare (LCP) fac partedin grupul de limfoame non-Hodgkin extra-nodale ºi sunt situate pe locul doi ca incidenþã, înurma celor localizate la nivelul tractului digestiv.Conform definiþiei, LCP se dezvoltã la nivelultegumentului cu absenþa leziunilor extracutanatela momentul diagnosticului spre deosebire delimfoamele cutanate secundare care reprezintãdeterminãri cutanate ale limfoamelor primarelimfonodale sau ale leucemiilor diseminate.

LCP cuprind un spectru larg de neoplaziilimfoproliferative, heterogene din punct devedere clinic ºi histologic. În funcþie de celule deorigine, se disting limfoame cu celule T, celuleNK sau celule B precum ºi alte malignitãþihematopoetice [1-3].

Limfoamele cutanate primare cu celule B(LCPCB) sunt mai puþin frecvente, cuprinzândaproximativ 20%-25% din totalul LCP [4]. Dupãani de confuzie privind terminologia ºiclasificarea acestora, Organizaþia Mondialã aSãnãtãþii – Organizaþia Europeanã pentruCercetare ºi Tratament al Cancerului (WHO/EORTC) propune în 2005 o clasificare carerecunoaºte trei entitãþi principale: limfomulcutanat primar cu celule B de zonã marginalã,limfomul cutanat primar centrofolicular ºilimfomul cutanat primar difuz cu celule B mari[5]. Recunoaºterea acestora ºi diferenþierea delimfoamele cutanate secundare prezintã o

Introduction

Primary cutaneous lymphomas (LCP) arepart of the group of non-Hodgkin’s extra-nodallymphoma and are ranked second in incidence,located behind the digestive tract ones. Asdefined, the LCP is developing in the skin withno extra-cutaneous lesions at the diagnosis timeunlike the secondary cutaneous lymphomaswhich present cutaneous determinations ofprimary lymph-node lymphoma or disseminatedleukemia.

LCP includes a wide spectrum of lympho-proliferative malignancies, heterogeneous fromclinical and histological point of view. Dependingon the cell of origin, one can distinguish T-celllymphomas, NK cells or B cells and otherhematopoietic malignancies [1-3].

Primary cutaneous B cell lymphomas(LCPCB) are uncommon, containing approxi-mately 20–25% of LCP [4]. After years ofconfusion on terminology and classification,World Health Organization – EuropeanOrganization for Research and Treatment ofCancer (WHO/EORTC) has proposed in 2005 aclassification that acknowledges three mainentities: primary cutaneous lymphoma withmarginal zone B cells, primary cutaneous centre-follicular lymphoma and primary cutaneousdiffuse large B cell lymphoma [5]. Thisclassification and differentiation of secondary

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deosebitã importanþã nu doar din punct devedere clinic dar ºi din punct de vedere terapeuticºi prognostic, fiind recunoscut faptul cã limfomulcutanat primar cu celule B de zonã marginalã ºilimfomul cutanat primar centrofolicular suntleziuni indolente care beneficiazã de o terapiemai puþin agresivã, diferitã de cea a limfoamelorlimfonodale [6]. În aceastã lucrare ne propunemreevaluare aspectelor histologice ºiimunofenotipice ale LCPCB în scopul sintetizãriiunui protocol util în diagnosticul morfopatologic.

Material ºi metodã

S-au revizuit cazurile de limfoame cutanateprimare cu celule B diagnosticate în Depar-tamentul de Patologie al Universitãþii JohannWolfgang Goethe, Frankfurt/Main, Germania peo perioadã de 3 ani. În acest scop s-au reevaluatatât preparatele în coloraþie uzualã (hemato-xilinã-eozinã) cât ºi cele marcate imuno-histochimic precum ºi datele actuale din litera-tura de specialitate.

Tehnica imunohistochimicãDin blocuri de þesut incluse în parafinã s-au

efectuate secþiuni cu grosimea de 4 µm. Acesteaau fost montate pe lame încãrcate pozitiv ºi apoideparafinate în 3 bãi succesive de xilen,rehidratate în bãi de alcool în concentraþiidescrescãtoare (100%, 96%, 80%, 70%) ºipurificate în apã distilatã. În scopul demascãriisitusurilor antigenice, secþiunile au fost supuseunui tratament termic prin fierbere în EDTA (pH8) pentru 1 min, utilizând oala sub presiune.Dupã rãcire la temperatura camerei, secþiunile aufost incubate timp de 30 de minute cu anticorpiiprimari. Furnizorii anticorpilor ºi diluþii utilizatesunt rezumate în tabelul I. În continuare, s-aaplicat protocolul sistemului de detecþie DakoREAL, Alkaline Phosphatase/RED, Rabbit/mouse.

Rezultate

Pe perioada evaluatã s-au identificat unnumãr de 8 pacienþi diagnosticaþi cu limfoamecutanate primare. Conform criteriilor de clasi-ficare WHO-EORTC pentru limfoame cutanate,pacienþii au fost încadraþi în urmãtoarelesubtipuri de limfoame cutanate primare: limfom

cutaneous lymphomas have great importance notonly clinically but also in terms of treatment andprognosis, being recognized that primarycutaneous marginal zone B cell lymphoma andprimary cutaneous lesions centre-follicularlymphoma are indolent, receiving a lessaggressive therapy, other than lymph-nodelymphoma [6]. In this paper we propose toreassess the histological and immune-phenotypeaspects of LCPCB and to synthesis a usefulprotocol in the morph-pathological diagnosis.

Materials and methods

There were reviewed the cases of primarycutaneous B cell lymphoma diagnosed in thePathology Department of the Johann WolfgangGoethe University, Frankfurt / Main, Germanyover 3 years period. To this aim there have beenreassessed as usual coloring preparations(hematoxylin-eosine) and those marked asimmunohistochemical and current data fromliterature.

Immunohistochemical technique

Of tissue included in paraffin blocks weremade 4 µm thick sections. They were mounted onpositively charged blades and then dewaxed in3 successive baths of xylene, rehydrated inalcohol baths with decreasing concentrations of(100%, 96%, 80%, 70%) and purified in distilledwater. In order to uncover antigenic sites, sectionswere heat-treated by boiling in EDTA (pH 8) for 1min, using the cooker pressure. After cooling toroom temperature, sections were incubated for 30minutes with primary antibodies. Antibodysuppliers and dilutions used are summarized inTable I. Next, the Dako REAL detection system,Alkaline Phosphatase / RED, Rabbit / mouseprotocol was applied.

Results

During evaluated period, there were identified anumber of 8 patients diagnosed with primarycutaneous lymphomas. According to WHO-EORTC criteria classification for cutaneouslymphomas, patients were classified into thefollowing subtypes of primary cutaneouslymphomas: Primary cutaneous B cell lymphoma

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cutanat primar cu celule B de zonã marginalã(n = 3; 37,5%), limfom cutanat primar centro-folicular (n = 3; 37,5%), limfom cutanat primardifuz cu celule B mari (n = 2; 25%). Carac-teristicile imunohistochimice ale celulelortumorale corespunzãtoare fiecãrui subtip delimfom cutanat primar cu celule B sunt sintetizateîn tabelul II.

of marginal zone (n = 3; 37.5%), centre-follicularprimary cutaneous lymphoma (n = 3; 37.5%),diffuse primary cutaneous lymphoma with largeB cell (n = 2; 25%). Immunohistochemical featuresof tumor cells for each subtype of primarycutaneous B cell lymphoma are summarized inTable II.

Tabelul II. Particularitãþile imunohistochimice ale LCPCB (+ = pozitiv; - = negativ; +/- = expresie variabilã, predominantpozitivã; -/+ = expresie variabilã,

predominant negativã)

CD20 CD79a Bcl-2 Bcl-6 CD10 MUM1 CD5 Cyclinã D1 CD3

Limfom cutanat + + + – – – – – –primar cu celuel Bde zonã marginalãLimfom primar + + – + ± – – – –cutanat centro-folicularLimfom primar + + + + ± + – – –cutanat difuz cucelule B mari

Table II. Immunohistochemical particularities of LCPCB (+ = positive; - = negative, +/- = variable expression, predominantlypositive, -/+= variable expression, predominantly negative)

CD20 CD79a Bcl-2 Bcl-6 CD10 MUM1 CD5 Cyclinã D1 CD3

Primary cutaneous + + + – – – – – –lymphoma withmarginal zone B cellsPrimary cutaneous + + – + ± – – – –centrofolicularlymphomaPrimary cutaneous + + + + ± + – – –diffuse lymphomawith large B cell

Tabelul I. Date privind anticorpii utilizaþi în evaluareaimunohistochimicã a LCPCB

Denumirea Clona Compania Diluþiaproducãtoare

CD20 L26 DAKO 1:200CD79a HM57 DAKO 1:200CD10 56C6 Novocastra 1:40

LaboratoriesBcl-6 PG-B6P DAKO 1:25Bcl-2 124 DAKO 1:50MUM1 MUM-1P DAKO 1:200CD5 4C7 Novocastra 1:10

LaboratoriesCyclina P2D11F11 Novocastra 1:10D1 LaboratoriesCD3 PS1 Novocastra 1:50

Laboratories

Tabel I. Data on the antibodies used inimmunohistochemistry assessment of LCPCB

Name Clone The manufacturing Dilutioncompany

CD20 L26 DAKO 1:200CD79a HM57 DAKO 1:200CD10 56C6 Novocastra 1:40

LaboratoriesBcl-6 PG-B6P DAKO 1:25Bcl-2 124 DAKO 1:50MUM1 MUM-1P DAKO 1:200CD5 4C7 Novocastra 1:10

LaboratoriesCyclina P2D11F11 Novocastra 1:10D1 LaboratoriesCD3 PS1 Novocastra 1:50

Laboratories

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Discuþii

Limfomul cutanat primar cu celule B de zonãmarginalã

Conform denumirii, originea acestui limfomeste postulatã la nivelul celulelor B din zonamarginalã a foliculilor limfoizi secundari.Infiltratul tumoral intereseazã dermul ºi estedispus difuz sau nodular ºi constituit dinlimfocite de talie micã sau medie. Nucleii suntuºor neregulaþi, cu cromatina moderat dispersatã,fãrã nucleoli vizibili, iar citoplasma este amplã,palid-eozinofilã, amintind aspectul centrocitelor[7]. Frecvent sunt intercalate plasmocite, iaracumularea de celule cu citoplasmã palid-eozinofilã poate crea un aspect monocitoid(Fig. 1). Examinarea la mãrire micã evidenþiazãun aspect cunoscut sub denumirea de „patterninvers” caracterizat prin prezenþa unor zonecentrale întunecate înconjurate de zone mailuminoase formate din celule palid-colorate.Infiltratul tumoral poate coloniza centriigerminativi, uneori distrugându-i complet,devenind astfel dificili de recunoscut [8]. În acestesituaþii, evidenþierea unei reþele de celuledendritice foliculare, prin imunomarcaj cumarker-ul CD21, faciliteazã recunoaºtereacentrilor germinativi restanþi [9]. Ocazional,infiltratul tumoral este dispus în jurul anexelortegumentare cu infiltrarea acestora ºi producerea

Discussions

Primary cutaneous B cell lymphoma ofmarginal zone

Under the name, origin of this lymphoma ispostulated in the marginal zone B cells folliclessecondary lymphoid. Tumor infiltrate dermis isinterested and is willing diffuse or nodular andcomposed of small or medium lymphocytes. Thenuclei are slightly irregular with moderatelydispersed chromatin without visible nucleoli, andcytoplasm is broad, pale eosinophilic, remindingof the centrocytes appearance [7]. There arefrequently interleaved plasma cells andaccumulation of cells with pale eosinophiliccytoplasm may create a monocitoid appearance(Fig. 1.). The examination of the small sizehighlights an issue known as „reverse pattern“characterized by the presence of dark central areasurrounded by brighter areas consisting of pale-colored cells. Tumor infiltration can colonizegerm centers, sometimes destroying themcompletely, thus becoming difficult to recognize[8]. In these situations, evidence of a dendritic cellnetworks by immune marking with the CD21marker facilitates the recognition of the locationof germ centers [9]. Occasionally, the tumorinfiltrate is arranged around skin annexes withtheir infiltration and production of lymphepithelial lesions in a similar manner to those

Fig. 1. Limfom cutanat primar cu celule B de zonã marginalãA. Proliferare tumoralã cu pattern nodular localizatã la nivelul dermului fãrã interesarea epidermului

B. Infiltrat cu aspect monocitoid format din limfocite mici sau medii cu citoplasmã abundentã, palid-eozinofilãFig. 1. Primary cutaneous B cell of marginal zone lymphoma

A. tumor proliferation with nodular pattern localized in the dermis without involving the epidermisB. the monocytoid infiltrate composed of small or medium lymphocytes with abundant cytoplasm, pale-eosinophilic

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de leziuni limfoepiteliale într-o manierã similarãcelor descris în limfomul de zonã marginalã detip MALT localizat la nivelul tractuluigastrointestinal [10].

Imunofenotipic limfomul cutanat primar cucelule B de zonã marginalã pãstreazãcaracteristicile limfocitelor B din zona marginalã,celulele tumorale fiind pozitive pentru CD19,CD20, CD22, CD79a ºi Bcl-2 ºi negative pentruBcl-6, CD5, CD10 ºi Cyclinã D1 (Fig. 2) [11].Prezenþa izolatã de limfocite Bcl-6 pozitive, cu unpattern de distribuþie similar celulelor dendriticefoliculare ºi invers faþã de expresia Bcl-2, permiteevidenþierea centrilor germinativi restanþi înurma înfiltrãrii tumorale (Fig. 3) [12].

Limfomul cutanat primar centrofolicular

Limfomul centrofolicular se dezvoltã de lanivelul limfocitelor centrului germinativ(centrocite ºi centroblaste) ºi prezintã un patternmorfologic variabil, cu aspect folicular, folicularºi difuz sau difuz în stadiile avansate. Infiltratultumoral este localizat la nivelul dermului,frecvent cu extindere spre hipoderm (Fig. 4).Foliculii neoplazici sunt dispuºi aglomerat,„spate în spate” ºi prezintã caracteristic uninfiltrat relativ monomorf, constituit în proporþiivariabile din celule de dimensiuni medii saumari, cu activitate mitoticã redusã ºi un numãrscãzut de macrofage cu corpi tingibili, cu absenþa

described in marginal zone lymphoma of MALTtype located in the gastrointestinal tract [10].

Primary cutaneous immune-phenotypiclymphoma with marginal zone B cell retain thecharacteristics of the marginal zone Blymphocytes, the tumor cells were positive forCD19, CD20, CD22, CD79a and Bcl-2 andnegative for Bcl-6, CD5, CD10 and Cyclin D1(Fig. 2.) [11]. The isolated presence of Bcl-6positive lymphocytes, with a similar distributionpattern of dendritic follicular cells and back to theBcl-2 expression, allows the highlight of the germcenters rests from tumor infiltration (Fig. 3.) [12].

Primary cutaneous lymphoma centrofolicular

The centre-follicular lymphoma is developingfrom the germinal center lymphocytes (centro-cyte and centre-blasts) lymphocytes and hasvariable morphological patterns, with follicular,follicular and diffuse or diffuse in advancedstages aspects. The tumor infiltrate is located inthe dermis, often extending to the hypodermis(Fig. 4.). Neoplastic follicles are busy willing,„back to back“ and present a relatively mono-morph infiltrate consisting in varying pro-portions of medium and large sized cells withlow mitotic activity and a low number ofmacrophages with the body pan, with noappearance of the sky starry created by them in

Fig. 2. Limfom cutanat primar cu celule B de zonã marginalãA. Imunomarcaj cu CD20 – expresie intensã la nivelul celulelor tumorale

B. Celule tumorale CD20 pozitive dispuse perianexialFig. 2. Primary cutaneous B cell of marginal zone lymphoma

A. Immune-marking with CD20 - intense expression in tumor cells levelB CD20 positive tumor cells arranged perianexially

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aspectului de cer înstelat creat de aceºtia în centriigerminativi netumorali. Frecvent zona de mantaeste mult redusã sau absentã (Fig. 5) [13].

Celulele tumorale exprimã markeri specificilimfocitelor B fiind pozitive pentru CD19, CD20,CD22 ºi CD79a. De asemenea sunt pozitivepentru markeri specifici limfocitelor de la nivelulcentrului geminativ, exprimând constant Bcl-6 ºi

non-tumor germ centers. Common zone is oftenmuch reduced or absent mantle (Fig. 5.) [13].Tumor cells express specific markers, the Blymphocytes are positive for CD19, CD20, CD22and CD79a. There are also positive for specificmarkers of lymphocytes from the germ levelcenter, constantly expressing Bcl-6 and variableCD10, and the cases with follicular growth

Fig. 4. Limfom cutanat primar centrofolicularA. Infiltrat limfoid cu pattern folicular ºi difuz

B. Infiltrat cu pattern folicular, cu foliculi dispuºi aglomerat, „spate în spate”, localizaþi la nivelul dermulicu extindere spre hipoderm

Fig. 4. Primary cutaneous centre-follicular lymphomaA. lymphoid infiltrate with follicular and diffuse patterns

B. infiltrate with follicular pattern, with follicles crowded “back to back”, located at dermulitis level extending to hypodermis

Fig. 3. Limfom cutanat primar cu celule B de zonã marginalãA. Expresie intensã a Bcl-2 cu celule izolate negative

B. Expresie negativã a celulelor tumorale pentru Bcl-6 (Bcl-2 ºi Bcl-6 prezintã un pattern invers de expresie, celulele izolateBcl-6 pozitive ºi Bcl-2 negative reprezentând resturi ale unui centru germinativ infiltrat tumoral)

Fig. 3. Primary cutaneous B cell of marginal zone lymphomaA strong expression of Bcl-2 with negative isolated cells

B. the negative expression of tumor cells for Bcl-6 (Bcl-2 and Bcl-6 shows a reverse pattern of expression, the Bcl-6 positive andBcl-2 negative isolated cells representing the rests of a germinal center tumor infiltration)

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variabil CD10; cazurile cu pattern de creºterefolicular fiind frecvent pozitive pentru CD10, iarcele cu creºtere difuzã frecvent negative (Fig. 6)[14]. Prezenþa celulelor Bcl-6 ºi CD10 pozitive înafara centrilor germinativi este înalt sugestivãpentru o proliferare tumoralã [15]. Este acceptatãprezenþa izolatã de celule Bcl-6 pozitive în ariileinterfoliculare, acest anticorp fiind exprimat ºi de

pattern are often positive for CD10, while thosewith diffuse growth often negative (Fig. 6.) [14 ].The presence of Bcl-6 cells and CD10 positiveexcept for germ centers is highly suggestive for atumor proliferation [15]. It is accepted the Bcl-6positive cells isolated presence in inter-follicularareas, this antibody being expressed and by a fewhighlighted T lymphocytes through CD3 positive

Fig. 6. Limfom cutanat primar centrofolicularA. Celule tumorale pozitive la imunomarcajul cu CD20

B. Reacþie imunohistochimicã pozitivã pentru CD10Fig. 6. Primary cutaneous centre-follicular lymphoma

A positive tumoral cells at the immune-marked with CD20B. Immune-histochemical positive reaction for CD10

Fig. 5. Limfom cutanat primar centrofolicular. Detaliu lamãrire mai mare a unui folicul neoplazic constituit în

proporþii variabile din celule de talie medie ºi mare, centrocit-ºi centroblast-like, cu absenþa macrofagelor cu corpi tingibili

Fig. 5. Primary cutaneous centre-follicular lymphomaDetail at a higher magnification of a neoplasic follicle

formed in varying proportions of medium and large sizedcells, centre-blast and centre-cit-like, with no pan bodied

macrophages

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un numãr redus de limfocite T evidenþiate prinpozitivare pentru CD3. Absenþa expresiei CD3 lanivelul limfocitelor Bcl-6 pozitive localizate înafara centrului germinativ ºi dispunerea acestoraîn grupuri susþine diagnosticul de limfomfolicular. Spre deosebire de limfoamele folicularelimfonodale, limfoamele centrofoliculare cutanateprimare sunt negative pentru Bcl-2, o moleculãantiapoptoticã neexprimatã de limfocitele B dincentrii germinativi normali dar prezentã constantîn transformãrile neoplazice (Fig. 7) [16]. Astfel,expresia Bcl-2 în celulele tumorale ale unuilimfom folicular cutanat este înalt sugestivãpentru originea limfonodularã a acestuia, cuimplicare cutanatã secundarã. Aceastãparticularitate, precum ºi absenþa translocaþiei (14;18) în cazurile de limfom centrofolicular cutanatprimar [17], prezentã în majoritatea cazurilor delimfom folicular limfonodal, indicã o patogenezãdiferitã pentru cele douã leziuni [18, 19].Negativitatea limfomului centrofolicular cutanatprimar pentru Bcl-2 îngreuneazã diferenþierea dehiperplazia limfoidã reactivã care este deasemenea negativã pentru acest marker. În acestecazuri distincþia între infiltratul limfoid reactivpoliclonal ºi infiltratul neoplazic monoclonal sebazeazã în special pe prezenþa sau absenþaexpresiei restricþionate a lanþurilor uºoare deimun-globuline κ ºi λ(Ig).

value for. The absence of CD3 expression in theBcl-6 positive cell lymphocytes level locatedoutside the germ center and in their groupsarrangement, supports the diagnosis of follicularlymphoma. Unlike lymph-node follicularlymphomas, the primary cutaneous centre-follicular lymphoma is negative for Bcl-2, anantiapoptotic molecule non-expressed by Blymphocytes from normal germ centers butconsistently present in neoplastic transformations(Fig. 7.) [16]. Thus, the Bcl-2 expression in tumorcells of a cutaneous follicular lymphoma is highlysuggestive for its lymph-nodular origin, withsecondary skin involvement. This feature and theabsence of translocation (14, 18) in primarycutaneous centre-follicular lymphoma cases [17],present in most follicular lymphoma lymph-nodecases, indicates a different pathogenesis for thetwo lesions [18, 19]. The primary cutaneouscentre-follicular lymphoma negativity for Bcl-2makes it difficult to differentiate from reactivehyperplasia lymphoid which is also negative forthis marker. In these cases the distinction betweenthe reactive polyclonal lymphoid infiltrate andmonoclonal neoplazic infiltrate, is basedprimarily on the presence or absence of restrictedexpression of a light chain of immune-globulinsκ and λ (Ig).

Fig. 7. Limfom cutanat primar centrofolicular. Celuletumorale negative pentru Bcl-2 cu pozitivarea limfocitelor

B netumorale dispuse perinodularFig. 7. Primary cutaneous centre-follicular lymphoma. Thenegative tumor cells for Bcl-2 with the positivity value of

B non-tumor lymphocytes perinodularly arranged

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Limfomul cutanat primar difuz cu celule Bmari

Limfomul cutanat primar difuz cu celule Bmari cuprinde doua subgrupe lezionale: varianta„leg type”, ºi varianta „others” care include toatecelelalte forme de manifestare care nu întrunesccriteriile variantei „leg type”. Cea mai frecventîntâlnitã este varianta „leg type” care, dupã cumsugereazã ºi numele, este localizatã cu predilecþiela nivelul piciorului. Totuºi, indiferent delocalizare, leziunile cu caracteristici morfologiceºi imunohistochimice definitorii pentru varianta„leg type” sunt incluse în aceastã categorie.

Varianta „leg type” se prezintã histologic subforma unui infiltrat limfoid cu pattern de creºteredifuz care ocupã dermul cu extindere sprehipoderm, frecvent fãrã infiltrarea epidermului.Infiltratul tumoral este monomorf, compus dinlimfocite B de talie medie sau mare asemãnãtoarecentroblaºtilor, cu nuclei mari neclivaþi ºi nucleolievidenþi ataºaþi membranei nucleare. Se potobserva de asemenea ºi imunoblaºti, celule cunucleu mare, veziculos ºi nucleoli situaþipredominant central. Spre deosebire de limfomulcentrofolicular nu se remarcã centrocite, unaspect util in diferenþiere formelor difuze alelimfomul centrofolicular de limfomul cutanatprimar difuz cu celule B mari. Nucleii suntpredominant de formã rotundã, cu cromatinadispusã grosier, în bulgãri ºi cu frecvente figurimitotice (Fig. 8) [20].

Imunofenotipic, celulele tumorale exprimãCD19, CD20, CD22 ºi CD79a, markeri careconfirmã origina de linie B . Bcl-6 este exprimat înmajoritatea cazurile, în timp ce markerii CD5,CD10 ºi Cyclina D1 sunt negativi. Pozitivitateaintensã pentru Bcl-2 ºi MUM-1 este ocaracteristicã importantã a acestui tip de limfom,indiferent de localizare (Fig. 9.) [21]. Acest aspectcontribuie, alãturi de morfologia infiltratului, ladiferenþierea dintre limfomul cutanat primarcentrofolicular cu pattern de creºtere difuz,negativ pentru Bcl-2 ºi MUM-1/IRF4 ºi limfomulcutanat primar difuz cu celule B mari [22].

Primary cutaneous lymphoma, diffuse largeB cell

The primary diffuse cutaneous lymphomawith large B-cell includes two lesion subgroups:„link type“ version and the „others“ variablewhich includes all other forms of expression thatdo not meet the „leg type” variant criteria. Themore common variant is the “leg type” which, asits name suggests, is located with predilection inthe foot level. However, regardless of thelocation, lesions with morphological andimmunohistochemical defining characteristics forthe “leg type” version are included in thiscategory.

Histological speaking the “leg type” versionfeatures as a lymphoid infiltrate form withgrowth diffuse pattern occupying the extendingdermis to the hypodermis, often withoutinfiltration of the epidermis. The infiltrate tumoris mono-morph, composed of medium or bigsized B-lymphocytes similar with centre-blasts,with large non-split nuclei and obvious nucleoliattached to membrane nucleus. There are alsoimmune-blasts, cells with large nucleus, vesicularand predominantly centrally located nucleolus.Unlike the centre-follicular lymphoma there areno centrocyte notes, a useful point in thedifferentiation in diffuse forms of centre-follicularlymphoma and of primary cutaneous diffuselymphoma with large B cell. Nuclei arepredominantly round with chromatin coarsearranges in lumps and frequent mytotic figures(Fig. 8.) [20].

Immune-phenotype tumor cells expressed asCD19, CD20, CD22 and CD79a, are markerswhich confirm the original B line. Bcl-6 isexpressed in most cases, while CD5, CD10 andCyclin D1 markers are negative. The intensepositivity for Bcl-2 and Mum-1 is an importantfeature for this type of lymphoma, regardless ofthe location (Fig. 9.) [21]. This aspect contributes,along with infiltrate morphology, to thedistinction between primary cutaneous centre-follicular lymphoma with diffuse growth pattern,negative for Bcl-2 and MUM-1/IRF4 and theprimary cutaneous diffuse lymphoma with largeB cell [22].

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Concluzii

În clasificãrile recente, încadrarea tumorilorlimfoide se bazeazã în special pe evaluareaimunohistochimicã integratã în contextul clinic ºimorfologic. Din punct de vedere clinic inte-reseazã localizarea ºi modul de evoluþie aleziunii, iar din punct de vedere histologic seevalueazã atât pattern-ul de creºtere al infil-

Conclusions

In recent classifications, the framing of thelymphoid tumors is based primarily onimmunohistochemical evaluation under clinicaland morphological context. In clinical terms ismade on the location and the development oflesion, while from the histological point of view isevaluated both the growth pattern of tumor

Fig. 8. Limfom cutanat primar difuz cu celule B mariA Proliferare limfoidã cu infiltrarea difuzã a dermului ºi distrugerea structurilor anexiale

B. Infiltrat tumoral monomorf, compus din limfocite de talie medie sau mareFig. 8. Primary cutaneous diffuse with large B cell lymphoma

A. The diffuse lymphoid proliferation with the diffuse infiltration of the dermis and the destruction of adnexal structuresB. mono-morph tumor infiltrate composed by medium or large size lymphocytes

Fig. 9. Limfom cutanat primar difuz cu celule B mariA. Infiltrat tumoral pozitiv pentru CD20

B. Pozitivitate intensã a celulelor tumorale pentru Bcl-2Fig. 9. Primary cutaneous diffuse with large B cell lymphoma

A the positive tumoral infiltrate for CD20B. intense positivity for Bcl-2 tumor cells

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tratului tumoral cât ºi aspectul morfologic alpopulaþiei de celule tumorale.

În concluzie sintetizãm markerii imuno-histochimici utili în diagnosticul limfoamelorcutanate primare cu celule B ºi propunemurmãtorul algoritm de diagnostic:1. Pentru confirmarea naturii tumorale ºi a

originii celulare a infiltratului:− originea de linie B a infiltratului celular

poate fi stabilitã prin pozitivarea pentruunul sau mai mulþi din urmãtorii markeri:CD19, CD20, CD22 ºi CD79a;

− natura monoclonalã (tumoralã) a infil-tratului limfoid se poate evidenþia prinidentificarea expresiei monotipice a lanþu-rilor uºoare de imunoglobuline.

2. În funcþie de aspectul morfologic se folosescurmãtorii anticorpi pentru confirmareavariantei de limfom cutanat primar cu celule B:a) Paternul morfologic folicular sau folicular ºi

difuz este întâlnit în limfomul cutanatprimar folicular ºi în limfomul cutanatprimar de zonã marginalã.

− expresia pozitivã pentru Bcl-6 ºi CD10 ºinegativã pentru Bcl-2 la nivelul structurilorfoliculare ºi în grupuri de celule careinfiltreazã difuz spaþiile interfoliculare estespecificã limfomului primar cutanat centro-folicular;

− expresia puternicã pentru Bcl-2, Bcl-6 ºiCD10 trebuie sã ridice întotdeauna suspi-ciunea unui limfom folicular sistemic cuimplicare cutanatã secundarã;

− expresia pozitivã pentru Bcl-2 ºi negativãpentru Bcl-6 ºi CD10 la nivelul infiltratuluieste sugestivã pentru limfomul primarcutanat de zonã marginalã;

− în cazuri selecþionate markerii CD5 ºiCyclinã D1 sunt utili în diagnosticuldiferenþial dintre limfomul primar cutanatde zonã marginalã (CD5-, Cyclinã D1-),limfomul cu celule din zona de manta cuinfiltrarea secundarã a tegumentului (CD5+,Cyclinã D1+) ºi determinarea cutanatã alimfomului/leucemiei limfocitice cu celuleB (CD5+, Cyclinã D1-).

b) Patternul morfologic difuz este caracteristiclimfomului cutanat primar difuz cu celule Bmari ºi formelor avansate ale limfomuluiprimar cutanat centrofolicular.

infiltrate and morphological aspect of tumor cellpopulation are evaluated.

In conclusion we gather useful immuno-histochemical markers in the diagnosis ofprimary cutaneous B cell lymphomas andsuggest the following diagnostic algorithm:1. To confirm the tumor cell nature and origin of

the infiltrate:– The B cell infiltrate origin line may be

determined by the positive value for one ormore of the following markers: CD19, CD20,CD22 and CD79a;

– Monoclonal nature (tumor) of the lymphoidinfiltrate can be showed through identifyingthe monotype expression of immuno-globulin light chains.

2. Depending on the morphological appearancethe following antibodies are used to confirmthe variant of primary cutaneous B celllymphoma:a) The morphological follicular pattern or

follicular and diffuse is found in primarycutaneous follicular lym-phoma and inprimary cutaneous marginal zonelymphoma.

– The positive expression for Bcl-6 and CD10and the negative one for Bcl-2 in thefollicular structures level and in groups ofcells that diffusely infiltrate the inter-follicular spaces is specific for the primarycutaneous centre-follicular lymphoma;

– The strong expression of Bcl-2, Bcl-6 andCD10 should always raise the suspicion of afollicular systemic lymphoma withsecondary skin involvement;

– The positive expression of Bcl-2 andnegative for Bcl-6 and CD10 in the infiltratelevel is suggestive for primary cutaneousmarginal zone lymphoma;

– In the selected cases, CD5 and Cyclin D1markers are useful in the differentialdiagnosis of the primary cutaneousmarginal zone lymphoma (CD5-, Cyclin D1-), the lymphoma with cells from the mantlezone with secondary skin infiltration (CD5+,Cyclin D1+) and the cutaneous lym-phoma/ leukemia B-cell lymphoma (CD5+, D1-Cyclinã) determination.

b) the diffuse morphological pattern ischaracteristic for the primary cuta-neous

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− expresia intensã a infiltratului pentru Bcl-2ºi MUM-1, cu expresie variabilã pentruBcl-6 ºi expresie negativã pentru CD10 suntdefinitorii pentru limfomul cutanat primardifuz cu celule B mari „leg type”;

− negativitatea infiltratului tumoral pentruBcl-2 ºi MUM-1 este sugestivã pentru formade creºtere difuzã a limfomului primarcutanat centrofolicular.

Intrat în redacþie: 20.10.2009

diffuse large B cell lymphoma and theadvanced forms of primary cutaneouscentre-follicular lymphoma.

– intense infiltrate expression for Bcl-2 andMum-1, with variable expression of Bcl-6and negative one for CD10 are characteristicfor primary cutaneous diffuse lymphomawith “leg type” large B-cell;.

– the tumor infiltrate negativity for Bcl-2 andMum-1 is suggestive for the diffuse growthform of primary cutaneous centre-follicularlymphoma.

Received: 20.10.2009

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Adresã de corespondenþã: Herbeck RosemarieMailing address: Spitalul Clinic Judeþean de Urgenþã Timiºoara

Bd. Iosif Bulbuca nr. 10, Cod 300736, Timiºoara, RomâniaTel.: 0722-574.300, E-mail: [email protected]

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