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1JClin Pathol 1995;48:18-21
Expression of the bcl-2 protein in B celllymphomas arising from mucosa associatedlymphoid tissue
E Navratil, P Gaulard, P Kanavaros, J Audouin, J Bougaran, N Martin, J Diebold,D Y Mason
AbstractAim-To determine whether lymphomasarising from mucosa associated lym-phoid tissue (MALT) express the bcl-2
Department of protein.
Pathology, CHU Methods-Forty two cases of MALT BHenri Mondor, cell lymphomas, 20 low grade neoplasmsCreNteil, France and 22 high grade tumours, were studied.P Gaulard Immunohistological staining was per-N Martin formed on paraffin wax embedded tissueDepartment of using a monoclonal antibody specific forPathology, University the bcl-2 protein.Hospital, Heraklion, Results-Al of the low grade lymphomasPKanavaros gave positive results on staining, withDepartment of clear cytoplasmic labelling for bcl-2 pro-Pathology, Hotel Dieu tein in the small neoplastic cells, some ofHospital, Paris, which formed characteristic lympho-France epithelial lesions. A striking feature wasJ Diebold that larger bcl-2 negative cells wereDepartment of observed in nine of these tumours. TheyPathology, CMC were either scattered singly among thePorte de Choisy, small neoplastic cells or formed smallParis, France clusters, suggesting that they could rep-
Deparentof resent early areas of transformation toPathology, John high grade neoplasia. Germinal centresRadcliffe Hospital, in the vicinity ofthe tumours lacked bcl-2Oxford protein and hence contrasted clearly withD Y Mason
the neoplastic cells. In some cases thisCorrespondence to:Dr P Gaulard, Departement permitted germinal centres, which werede Pathologie, CHU Henri not obvious on conventional histologicalMondor, 51 Avenue duMarechal de Lattre de staining, to be recognised. In 20 of the 22Tassigny, 94010 Creteil, cases of high grade B cell lymphoma theFrance.
large neoplastic cells were bcl-2 negative;Accepted for publication10 May 1994 the rema i two cases, however, con-
Table 1 Bcl-2 reactivity ofMALT lymphomas: Low grade MALT lymphomas
HistologyBcl-2 staining
Small Centroblasts Reactive SmallSite neoplastic cells or immunoblasts follicles neoplastic cells
Stomach Centrocyte-like Absent Present + + +Stomach Centrocyte-like Absent Present + + +Stomach Centrocyte-like Clusters Present + +Stomach Centrocyte-like Rare Present + + +Stomach Centrocyte-like Clusters Absent + + +Stomach Centrocyte-like Absent Present + + +Stomach Lymphoplasmocytoid Rare Present + + +Stomach Lymphoplasmocytoid Rare Present + + +Stomach Lymphoplasmocytoid Rare Present + + +Stomach Lymphoplasmocytoid Rare Present + +Colon Centrocyte-like Absent Present + + +Thyroid Centrocyte-like Rare Absent + + +Thyroid Lymphoplasmocytic Absent Absent + +Lung Centrocyte-like Rare Present + +Lung Centrocyte-like Absent Present + + +Lung Centrocyte-like Absent Absent + + +Lung Centrocyte-like Absent Present + +Lung Centrocyte-like Absent Absent + +Salivary gland Centrocyte-like Absent Present + + +Salivary gland Centrocyte-like Absent Present + + +
Staining intensity for bcl-2 is indicated on a scale from negative (-) to strongly positive (++ +).
tained a proportion of large neoplasticbcl-2 positive cells. In four of the 22 casesof high grade tumours a low grade com-ponent was found which expressed bcl-2in all cases.Conclusion-Bcl-2 protein is expressedin low grade, but not in most high grade,MALT lymphomas. In view ofrecent dataindicating that most high grade nodallymphomas express bcl-2, these findingssuggest that MALT lymphomas may regu-late bcl-2 gene expression differently tonodal lymphomas.( Clin Pathol 1995;48:18-21)
Keywords: Bcl-2 protein expression, MALT lym-phoma.
Low grade B cell lymphomas originatingin mucosa associated lymphoid tissue(MALT)1-6 account for a large proportion ofextranodal low grade B cell lymphomas. Theyarise in the gastrointestinal tract but also inother organs which contain MALT, includingthe lungs and the thyroid and salivary glands.4These lymphomas often take the form of a dif-fuse infiltration of neoplastic cells, possessinga centrocyte-like morphology, often associ-ated with prominent reactive germinal centresand lymphoepithelial lesions. Low grade Bcell MALT lymphomas are characterised byan indolent clinical course and prolongedlocalisation of the tumour to the original sitewithout dissemination.4The bcl-2 protein is mainly expressed in
the inner mitochondrial membrane and canextend cell survival by blocking programmedcell death ("apoptosis").7 It is absent fromreactive germinal centres but is oftenexpressed in neoplastic germinal centres infollicular B cell lymphomas,8 usually in associ-ation with the 14;18 chromosomal transloca-tion. The expression of bcl-2 protein has alsobeen reported in many types of nodal non-Hodgkin's lymphomas, independently of thepresence of the 14;18 translocation.910However, the absence of bcl-2 expression inmost cases of MALT high grade B celllymphoma has been reported" and thisprompted us to compare bcl-2 protein expres-sion in low and high grade MALT lym-phomas. As labelling was performed onparaffin wax embedded tissue, we were alsoable to assess the relation between bcl-2expression and cell morphology.
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Expression of bcl-2 protein in B cell lymphomas arisingfrom MALT
Table 2 Bcl-2 reactivity ofAMALTlymphomas: High grade MALTlymphomasassociated with a low grade component
Histology Bcl-2 staining
Small Large Reactive Small LargeSite neoplastic cells neoplastic ceUls follicles neoplastic cells neoplastic cells
Stomach Centrocyte-like Centroblast-like Present + +Stomach Lymphoplasmocytoid Immunoblastic Present + + *Ileum Centrocyte-like Centroblast-like Present + + -*Thyroid Lymphoplasmocytoid Immunoblastic Present *
*Rare positive cells.Staining intensity for bcl-2 is indicated on a scale from negative (-) to strongly positive
Table 3 Bcl-2 reactivity ofMALLTlymphomas: High grade MALT lymphomas
HistologyBcl-2 staining
Small Large Reactive LargeSite neoplastic cells neoplastic cells follicles neoplastic cells
Stomach Absent Centroblast-like PresentStomach Absent Centroblast-like PresentStomach Absent Centroblast-like PresentStomach Absent Centroblast-like PresentStomach Absent Centroblast-like PresentStomach Absent Centroblast-like PresentStomach Absent Centroblast-like Present -
Stomach Absent Centroblast-like PresentStomach Absent Centroblast-like Present -
Stomach Absent Centroblast-like PresentStomach Absent Centroblast-like PresentColon Absent Centroblast-like PresentColon Absent Centroblast-like Present -
Ileum Absent Centroblast-like PresentIleum Absent Centroblast-like Present -
Ileum Absent Centroblast-like Present -
Ileum Absent Centroblast-like Present -
Ileum Absent Centroblast-like Present -
Staining intensity for bcl-2 is indicated on a scale from negative (-) to strongly positive
MethodsAll tissue specimens were obtained followingsurgical resection. The material consisted of46 cases of B cell lymphoma of MALTorigin, including gastric, colorectal, ileal,pulmonary, salivary gland, and thyroid glandlymphomas (tables 1-3). The B cell origin ofthese tumours was assessed by their reactivitywith the L26 (CD20) monoclonal antibody ortheir monotypic immunoglobulin expressionon frozen sections. All samples had been fixedin Bouin's fixative or formalin and embedded
~~ '!'!'Figure IMPdmonary low grade B cellMALT lymphoma. Surface epithelium invaded bycentrocyte-like lymphoma cells stainedfor bcl-2.
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in paraffin wax. These sections were then cutat 4 ,um and stained with haematoxylin andeosin and Giemsa stains. Immunostaining wasperformed on deparaffinised sections by thealkaline phosphatase anti-alkaline phos-phatase (APAAP) technique,'2 using a mono-clonal antibody (bcl-2/100) generated againsta synthetic peptide.10 Of the 46 cases, fourwere excluded after initial review because ofunsatisfactory bcl-2 staining (no staining ofany cells, including reactive cells), leaving 42in which there was clear staining of neoplasticand/or normal cells. Twenty of these caseswere classified histologically as low grade and22 as high grade tumours. In four of the 22high grade tumours a low grade componentwas found.
ResultsIn all 20 cases (table 1) the neoplastic cellswere bcl-2 positive (figs 1 and 2). In 15 casesthe tumour cells were of centrocyte-like mor-phology. In four cases most of the tumourcells were of lymphoplasmocytoid morphol-ogy and the remaining case was a lymphoplas-mocytic tumour. In 14 cases the bcl-2 positiveneoplastic cells were more strongly stainedthan reactive mantle zone lymphocytes, whilein the remaining six cases their labelling wasof comparable intensity. Lymphoepitheliallesions were seen in many cases, comprisinglabelled centrocyte-like cells infiltrating sur-face epithelium (fig 1). In seven of the 20cases a small number of large centroblast-likebcl-2 negative neoplastic cells were scatteredamong the centrocyte-like neoplastic cells,and in two further cases small clusters of suchcells could be identified morphologicallyamongst the smaller neoplastic cells (fig 3).
In 15 cases reactive secondary follicles werepresent in the vicinity of the neoplasm, and ineach instance the germinal centre cells werebcl-2 negative (fig 2). In some cases thisenabled residual germinal centres to be recog-nised which were not discernible on conven-tional histological staining.The 22 cases in this category were mainly
composed of large neoplastic cells resemblingcentroblasts (21 cases) or immunoblasts (onecase). In 20 of the 22 cases of high grade Bcell lymphomas the large neoplastic cells werebcl-2 negative (fig 4), whereas the remainingtwo cases contained a few large bcl-2 positivecells. In four of the 22 cases of high gradetumours a low grade component was found,and three of these expressed the bcl-2 protein(fig 5). In the remaining case most of the neo-plastic cells with lymphoplasmocytoid mor-phology were bcl-2 negative. In each negativecase small reactive lymphocytes scatteredamongst the neoplastic cells provided aninternal positive control (fig 4).
DiscussionThe present study indicates that the bcl-2protein is expressed by the neoplastic cellsin all low grade B cell lymphomas of MALTorigin. Although rearrangements of the
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Navratil, Gaulard, Kanavaros, Audouin, Bougaran, Martin, et al
Figure 2 Gastric low grade B cellMALT lymphoma. Centrocyte-like lymphoma cellssurrounding a negative reactive follicle.
Figure 3 Gastric low grade B cellMALT lymphoma. Centrocyte-like lymphoma cellsstainedfor bcl-2 contrasting with scattered centroblastic and immunoblastic large negativecells.
Figure 4 Gastric high grade B cell lymphoma. Neoplastic large cels negative for bcl-2,contrasting with the presence of bcl-2 positive small normal lymphocytes.
Figure S High grade MALT lymphoma associated withlow grade B cell MIALT lymphoma. Centrocyte-likelymphoma cells stainedfor bcl-2 surrounding a reactivegerminal centre. 0 = A bcl-2 negative area composed oflarge "transformed" lymphoid cells.
bcl-2 gene have been rarely detected in lym-phomas of MALT origin,13 expression ofthe bcl-2 protein in MLALT lymphomas is notunexpected, as bcl-2 expression has beenreported in many node based non-Hodgkin'slymphomas which lack t(14;18). Bcl-2 proteinexpression also occurs in Reed-Stemberg cellsin some cases of Hodgkin's disease as well as innormal B and T lymphocytes.9 10 17 18
Occasionally, the diffuse infiltrate of smallcentrocyte-like cells, which tends to invademucosal epithelium to form characteristiclymphoepithelial lesions in low grade B cellMALT lymphomas, may transform into ahigh grade large cell neoplasm.y9 It was there-fore of interest that large bcl-2 negative cellswere seen in the present series, either scat-tered or in small clusters amongst small bcl-2positive cells in nine of 20 of the low gradetumours. This indicates that low gradeMALT lynmphomas have a tendency to losebcl-2 protein when they progress to highgrade tumours. A parallel can be drawn withour previous findings in centroblastic/centro-cytic follicular lymphomas, in which largenon-cleaved cells (centroblasts) may be nega-tive for bcl-2 while small cleaved cells (centro-cytes) express this protein strongly."The tendency of high grade MALT lym-
phomas to lose bcl-2 protein expression hasbeen confirmed in the present series as in20 the large neoplastic cels lacked bcl-2 andin the two remaining cases only a proportionof large tumour cells expressed bcl-2. Thiscontrasts with the bcl-2 positive resultsreported in most cases of nodal large B cell
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Expression of bcl-2 protein in B cell lymphomas arisingfrom MALT
lymphoma"'8 but is in keeping with the previ-ous observation that most large cell MALTlymphomas are bcl-2 negative.'120 This sug-gests that MALT lymphomas differ fromnodal lymphomas in the regulation of bcl-2gene expression.'4 The absence of detectablebcl-2 protein in most high grade MALT lym-phomas could reflect a breakdown in bcl-2gene expression at the transcriptional or thepost-transcriptional level. The possibility ofpost-transcriptional breakdown in bcl-2 geneexpression has been considered based ondiscordant bcl-2 messenger RNA (mRNA)and bcl-2 protein expression in reactivelymph nodes.202' In addition, stimulatoryand inhibitory mechanisms of bcl-2 geneexpression at the transcriptional and post-transcriptional level have been described inhuman proliferating lymphocytes and lym-phonodal lymphomas.2223 Further studiesshowed that proliferating normal lymphoidcells fail to express the bcl-2 protein.'0Therefore, the absence of bcl-2 proteinexpression in most high grade MALT lym-phomas may be related to their high rate ofproliferation. In this respect Isaacson et a124have shown that colonised follicles in MALTlymphomas are bcl-2 negative. This findingmay be related to the high proliferation rate incolonised follicles, or to physiological down-regulation of bcl-2 in the follicular environ-ment.24The clinical and prognostic importance of
the absence of bcl-2 protein expression inhigh grade MALT lymphomas when com-pared with high grade lymphonodal lym-phomas has not been established. However,high grade MALT lymphomas exhibit a betterprognosis than high grade lymphonodal lym-phomas.25 This might be related to findingsthat bcl-2 positive high grade lymphomashave a shorter disease free survival than bcl-2negative ones'8 and that a B leukaemic cellline with elevated bcl-2 protein levels exhibits astrikingly long survival when cultured in thepresence of antineoplastic drugs.26
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