expression of vascular endothelial growth factor in lymphomas and castleman's disease

, . 183: 44–50 (1997)

EXPRESSION OF VASCULAR ENDOTHELIAL GROWTHFACTOR IN LYMPHOMAS AND CASTLEMAN’S

DISEASE

- *, , , ,

Konsultations- und Referenzzentrum für Lymphknoten und Hämatopathologie supported by the Deutsche Krebshilfe(grant M25/89/St1) at the Institute of Pathology, Klinikum Benjamin Franklin, Free University of Berlin, Hindenburgdamm 30,

12200 Berlin, Germany

SUMMARY

Vascular endothelial growth factor (VEGF) is one of the main angiogenic cytokines in human solid tumours and inhibition ofVEGF-induced angiogenesis suppresses tumour growth. Some groups of malignant lymphoma, including peripheral T-cell lymphomasand Hodgkin’s disease, are characterized by a conspicuous proliferation of small vessels. To test the hypothesis that VEGF may also beinvolved in the angiogenesis in lymphomas and other lesions of the lymphoid system, VEGF expression was analysed in tissues,employing in situ hybridization with a 35S-labelled RNA probe specific for this cytokine. Significant expression of VEGF transcripts wasobserved in Hodgkin’s disease and peripheral T-cell lymphomas, particularly of the angioimmunoblastic type. In contrast, expression ofthis cytokine was minimal or absent in follicle centre lymphoma and chronic lymphocytic leukemia of B-cell type. VEGF was mainlyobserved in reactive non-lymphoid CD68-negative cells, which probably represent fibroblasts or myofibroblasts. In normal and ulceratedtonsils, VEGF was expressed in the squamous epithelium but only rarely found in the lymphoid tissue. Although infectious mononucleosistonsils contained high numbers of VEGF-positive cells in the interfollicular zone, expression of this cytokine was not found inEpstein–Barr virus (EBV)-infected cells, as determined by simultaneous in situ hybridization for VEGF and EBV-encoded small nuclearRNAs (EBER). In 5/8 cases of Castleman’s disease, germinal centres containing small vessels also showed expression of VEGF, incontrast to normal tonsillar germinal centres which are devoid of both vessels and VEGF transcripts. It is concluded that VEGF maybe involved in the induction of the angiogenesis of both peripheral T-cell lymphomas and Hodgkin’s disease, but not in low-grade B-celllymphomas. In contradistinction to solid tumours, in which this cytokine is commonly secreted by the tumour cells themselves, inmalignant lymphoma VEGF is not a product of neoplastic cells. Vascularization of germinal centres in Castleman’s disease may also bea consequence of abnormal local expression of VEGF. ? 1997 by John Wiley & Sons, Ltd.

J. Pathol. 183: 44–50, 1997.No. of Figures 2. No. of Tables 3. No. of References 29.

KEY WORDS—vascular endothelial growth factor (VEGF); lymphoma; in situ hybridization; angiogenesis; Hodgkin’s disease;Castleman’s disease

INTRODUCTION

The growth of both primary and metastatic tumoursis associated with the formation of new blood vessels, aprocess known as tumour angiogenesis.1,2 The tumourgrowth depends critically on this neovasculature,since inhibition of angiogenesis by angiostatin causesdormancy of primary tumours and metastatic deposits,a condition characterized by balanced tumour cell pro-liferation and apoptosis.3,4 These findings have attractedwidespread interest, since they suggest that inhibition ofangiogenesis may represent a new therapeutic strategyfor malignant tumours.Angiogenesis is enhanced by several cytokines

including basic fibroblastic growth factors, placentalgrowth factor, interleukin (IL)-8, vascular endothelial

growth factor/vascular permeability factor (VEGF), andothers.2,5,6 VEGF appears to be the most relevantcytokine in this respect, since its expression is closelycorrelated with vessel density in several human tumoursand inhibition of VEGF suppresses tumour growth.7–9 Itis produced by many tumour cell lines and by thetumour cells of human solid neoplasms, includingadenocarcinomas of the gastrointestinal tract,10 breastcarcinomas,11 melanomas,12 glial tumours,13 and evenvascular tumours such as angiosarcoma.14 In addition toits function as a proliferation factor for endothelialcells,6 it increases vascular permeability5 and may bechemotactic for monocytes.15In contrast to solid tumours, angiogenesis is not well

characterized in lymphoid proliferations. Malignantlymphomas are heterogeneous with respect to theirmicrovasculature. Peripheral T-cell lymphomas (TCLs)characteristically display many vessels with the mor-phology of high endothelial venules and this feature ismost prominent in peripheral T-cell lymphoma of angio-immunoblastic lymphadenopathy type (TCL-AILD).16Vascularity is also prominent in Hodgkin’s disease, but

*Correspondence to H.-D. Foss, MD.

Contract grant sponsor: Deutsche Forschungsgemeinschaft; Con-tract grant number: Ste 318/5-1.

Contract grant sponsor: Conselho Nacional de Pesquisas/CNPq,Brazil.

CCC 0022–3417/97/090044–07 $17.50 Received 3 September 1996? 1997 by John Wiley & Sons, Ltd. Revised 26 November 1996

Accepted 19 December 1996

not in most cases of low-grade B-cell lymphomas.17Vascularization of germinal centres is a characteristicand diagnostic feature of Castleman’s disease.18We speculated that VEGF may also be involved in

induction of angiogenesis in human lymphoproliferativediseases. To test this hypothesis, we investigated differ-ent lymphoma entities, reactive lesions, and cases ofCastleman’s disease for the presence of VEGF by iso-topic in situ hybridization (ISH) with a 35S-labelledRNA probe specific for this cytokine. In addition,expression of VEGF was correlated with vessel density.Sequential immunohistology and ISH were applied tocharacterize further the cytokine-expressing cells. AsEpstein–Barr virus (EBV) induces many cytokines,19 wealso investigated whether expression of VEGF occurredin EBV-infected cells. For this purpose, simultaneousisotopic and non-isotopic ISH for the detection ofVEGF and EBV-encoded small nuclear RNAs (EBER)was applied in Hodgkin’s disease, TCL-AILD, andinfectious mononucleosis.

MATERIALS AND METHODS

TissuesFormol-fixed/paraffin-embedded biopsy specimens

were taken from the archives of the Institute ofPathology, Klinikum Benjamin Franklin, Berlin, of thefollowing diseases: infectious mononucleosis (sevencases, all tonsils), normal or slightly hyperplastic tonsils(three cases), florid or ulcerative tonsillitis (four cases),Hodgkin’s disease (seven cases of nodular sclerosis sub-type, 13 cases of mixed cellularity subtype), chroniclymphocytic leukaemia of B-cell type (five cases), folliclecentre lymphoma grades 1 and 2 (three cases), lymph-nodal TCL unspecified (seven cases), TCL-AILD(five cases), and Castleman’s disease (eight cases). Allspecimens were obtained prior to the initiation oftherapy. The diagnosis of the lymphomas wasestablished according to the criteria of the REALclassification.20

Immunohistology

Four-micrometre sections of paraffin-embedded tissueblocks were stained by the immunoalkaline phosphatase(APAAP) method.21 The primary monoclonal anti-bodies were Ber-H2 (CD30), L26 (CD20), âF1 (T-cellantigen receptor â-chain), PGM-1 (CD68), C3D1(CD15), 1F8 (CD21), DFT1 (CD43), BU38 (CD23),C8/144 (CD8), JC70A (CD31), and Cs1–4, a cocktail offour antibodies specific for the EBV-encoded latentmembrane protein. With the exception of âF1, whichwas from T-Cell Sciences, (Cambridge, MA, U.S.A.),and C8/144, which was kindly provided by Dr Mason,Oxford, U.K., all antibodies were purchased fromDAKO, Glostrup, Denmark.

Plasmids

The VEGF cDNA probe was prepared by subcloningof the cytokine gene fragment from the plasmid pLen-

165, kindly provided by Scios Nova, Mountain View,CA 94043, U.S.A., in the run-off transcription vectorpAMP1 (Gibco-BRL-Life Technologies, Eggenstein,Germany).22 The nucleic acid sequence of the cytokineprobe was determined on the DNA sequencer 373(Applied Biosystems, Forster City, U.S.A.) and provedto conform to published data.22 After linearization ofthe pGEM constructs with appropriate restrictionenzymes, anti-sense and sense (control) RNA probeswere generated by run-off transcription with incorpor-ation of 35S-labelled nucleotides, yielding an averagespecificity of 1·3#109 cpm/ìg as described.23

In situ hybridization (ISH)

ISH was performed as described earlier.23 Prolongedexposure times (up to 8 weeks) were used to ensuremaximal sensitivity. Simultaneous double labellingfor EBER and VEGF was carried out as previouslyreported.24 Double labelling with sequential immuno-histology [for CD68 (PGM1)] and ISH (for VEGFtranscripts) was carried out as outlined previously.25

Evaluation and statistical analysis

For the evaluation of ISH, cells containing morethan 20 grains were scored positive. This correspondedin all cases to more than four times the backgroundsignal. Sections hybridized with the sense probe showedonly a weak non-specific background signal (notshown). Ten high-power fields were evaluated forcytokine transcripts and labelled cells were expressed per0·5 mm2. Counting of vessels was aided by immuno-histological demonstration of CD31. For statisticalanalysis, rank correlation according to Kendall wasused.

RESULTS

Non-Hodgkin’s lymphomas

VEGF transcripts were found in all cases of TCL,reactivity being most prominent in TCL-AILD (TableI). Labelled cells had bland oval pale nuclei resemblingmacrophages or activated fibroblastic cells and werelocalized around small vessels. Sequential immunohis-tology and ISH showed, however, that the majority ofthese cells were CD68 (PGM-1)-negative. Lymphocytes,including atypical presumably neoplastic lymphocytes,did not express VEGF. Simultaneous isotopic and non-isotopic ISH in five cases of TCL-AILD showed thatEBER-positive cells did not contain VEGF transcripts(Fig. 1a).Scattered reactive cells were labelled with the VEGF

probe in 3/8 cases of low-grade B-cell lymphomas(Table I). In the other cases, no VEGF-specific signalswere observed. Loss of significant amounts of RNAduring tissue processing in these cases was excluded bythe demonstration of specific signals with unrelatedcytokine probes (not shown).

45VEGF IN LYMPHOMAS

? 1997 by John Wiley & Sons, Ltd. , . 183: 44–50 (1997)

Classical Hodgkin’s disease

All cases of Hodgkin’s disease contained abundantVEGF-positive cells (Table II). As evidenced by ISH,simultaneous double labelling for VEGF and EBERin 13 cases, and sequential immunohistology and ISH,VEGF expression was restricted to reactive non-lymphoid CD68 (PGM-1)-negative cells, with the excep-tion of one case which contained one labelled tumourcell (Figs 1b, 1c, 1e, and 1f).

Association between vessel density and VEGF expressionin lymphomas

There was no evident correlation between VEGFexpression and vessel density when each lymphomasubgroup (TCL, B-cell lymphomas, Hodgkin’s disease)was analysed separately. However, a significant corre-lation was obtained when the non-Hodgkin’s lymphomacases (ô=0·56, P<0·0027) or all lymphoma cases(ô=0·43, P<0·001) were evaluated as combined groups.

Castleman’s disease

VEGF expression was observed in 5/8 cases ingerminal centres containing small vessels, but in onlyone case in the interfollicular zone (Figs 2a and 2b). Inthe remaining three cases, only completely atrophichyalinized germinal centres were found and these didnot contain VEGF transcripts. VEGF expression wasrestricted to non-lymphoid cells with the morphology offibroblasts.

Normal and infectious mononucleosis tonsils andulcerative tonsillitis

VEGF transcripts were not found in normal or hyper-plastic tonsils. In florid and ulcerative tonsillitis as wellas in infectious mononucleosis tonsils, prominent VEGFexpression was noted in the squamous epithelium in thevicinity of ulcerations or erosions (Fig. 2c). In addition,expression of VEGF was enhanced in the interfollicularzone in infectious mononucleosis when compared withnormal tonsils or cases of ulcerative tonsillitis (TableIII). EBER-positive cells did not contain VEGF tran-scripts, as demonstrated by simultaneous ISH for EBERand VEGF (Fig. 1d).

DISCUSSION

Tumour growth is angiogenesis-dependent3,4 andVEGF is one of the most powerful angiogenic factors.5–7Angiogenesis in many human tumours includingcarcinomas of different organs, gliomas, and vasculartumours is linked to the expression of VEGF.10–14 Inaddition, inhibition of VEGF has been shown to sup-press tumour growth.7 Since little is known about theprocess of angiogenesis in lymphoproliferative diseases,we investigated the expression of VEGF in such lesionsby ISH, correlated the results to vessel density, andfurther characterized the producer cells by combinedimmunohistology and ISH as well as combined isotopicISH and non-isotopic ISH for EBER.We found prominent expression of VEGF in TCL,

particularly of the angioimmunoblastic lymphadeno-pathy type, and in Hodgkin’s disease, but not in low-grade B-cell lymphomas. VEGF was not expressed inthe neoplastic cells of any of the above-mentionedlymphomas, with the exception of one labelled Reed–Sternberg cell in 1/20 cases of Hodgkin’s disease, but wasfound in non-lymphoid, predominantly CD68-negativecells with vesicular oval nuclei, which are most likely torepresent fibroblasts or myofibroblasts. EBER-positivecells in infectious mononucleosis and TCL-AILD, aswell as EBER-positive Hodgkin and Reed–Sternbergcells of Hodgkin’s disease, did not contain VEGF tran-scripts. Vascularized germinal centres in Castleman’sdisease displayed VEGF-specific signals, but notavascular germinal centres in normal tonsils.Prominent vascularity is a well-known feature of many

cases of TCL and this feature presents a histological clueas to the T-cell nature of the neoplasm.16 Among allTCLs, the angioimmunoblastic lymphadenopathy type

Table I—Expression of VEGF and vessel density in non-Hodgkin’s lymphomas

Case No. VEGF Vessels

TCL1 6 2202 8 1683 12 554 14 2805 17 1826 32 2227 138 ndMedian 14 201

TCL-AILD1 109·5 2202 184 4203 220 nd4 225 3605 255 280Median 220 320

CLL1 0 1152 0 1503 0 1724 1 1605 16 150

FCL1 0 862 0 1123 1 96Median B-NHL 0 132·5

The numbers indicated in the table refer to the number of VEGF-positive cells or CD31-positive vessels per 0·5 mm2. TCL=peripheralT-cell lymphoma unspecified; TCL-AILD=peripheral T-cell lym-phoma of angioimmunoblastic type; CLL=chronic lymphocytic leu-kaemia of B-cell type; FCL=follicle centre lymphoma; B-NHL=B-cellnon-Hodgkin‘s lymphoma; nd=not determined.

46 H.-D. FOSS ET AL.


Fig. 1—Simultaneous ISH for EBER and VEGF transcripts (a–d). Numerous EBER-negative cells contain VEGF transcripts in a case ofTCL-AILD (a). EBER-positive tumour cells of Hodgkin’s disease (b,c) do not contain VEGF, whereas reactive cells are labelled. Numerouscells showing VEGF-specific signals are found in the interfollicular zone of an infectious mononucleosis tonsil (d); again, these labelled cellsare not EBV-infected as is indicated by the absence of EBER. Sequential immunohistology for CD68 (PGM-1) and ISH for VEGF in a caseof Hodgkin’s disease shows that VEGF-containing cells do not express CD68 (e). ISH for VEGF in a case of Hodgkin’s disease discloses asingle labelled neoplastic cell in addition to labelled reactive cells (f)

47VEGF IN LYMPHOMAS


shows the most prominent vessels, which often arborizeand are enveloped by PAS-positive material.16 In view ofthe well-characterized role of VEGF as an angiogenicfactor,5–7,9 our results showing marked expression ofVEGF in the surroundings of vessels in these lym-phomas suggest that this cytokine may be involved inthe angiogenesis in TCL but not in low-grade B-celllymphoma. Similarly to peripheral TCL, VEGF is alikely candidate for the induction of angiogenesis inHodgkin’s disease. This hypothesis is supported by theobserved correlation between vessel density and VEGFexpression when all non-Hodgkin’s lymphoma cases orall lymphoma cases were analysed. The lack of corre-lation between the number of VEGF-positive cells andvessel density in each subgroup of lymphomas is mostlikely due to the small number of cases in each of thesesubgroups.In most carcinomas10,11 and gliomas,13 VEGF is

produced primarily by the tumour cells themselves,which points to a direct mechanism in the induction ofangiogenesis. Surprisingly, we observed the expressionof VEGF in only one tumour cell in 1/20 Hodgkin’sdisease cases and not at all in the tumour cells ofnon-Hodgkin’s lymphoma. The VEGF expressionproved to be almost entirely restricted to reactive cells,probably fibroblasts or myofibroblasts. This suggeststhat the process of angiogenesis differs in epithelial andlymphoid tumours and that the induction of angio-genesis through VEGF in malignant lymphomas is an

indirect process, involving reactive cells such as fibro-blasts. The reason for this difference is not clear; an

Table II—Expression of VEGF and vessel density in classicalHodgkin’s disease

VEGF Vessels

Nodular sclerosis1 72 nd2 77 1943 87 nd4 129 3205 132 nd6 156 nd7 220 220Median 129 220

Mixed cellularity1 15 2902 20 2033 25 2954 58 1965 61 3026 80 nd7 89 nd8 102 1649 102 17510 121 16211 132 30812 160 14813 181 ndMedian 89 200

The numbers indicated in the table refer to the number of VEGF-positive cells or CD31-positive vessels per 0·5 mm2. nd=not deter-mined.

Fig. 2—A germinal centre of a case of Castleman’s disease displayscells labelled with the VEGF probe (a, dark field; b, light field).Squamous epithelial cells surrounding an area of ulceration containVEGF transcripts (tonsil, c). ISH



inability of lymphocytes to produce VEGF appears anunlikely cause, since stimulated CD3-positive peripheralT-lymphocytes26 may express VEGF. It is, however,conceivable that neoplastic cells in TCL and Hodgkin’sdisease produce factors which induce VEGF in reactivecells.Castleman’s disease is a curious lymphoproliferative

disorder, histologically characterized by progressiveatrophy and vascularization of germinal centres, concen-tric arrangement of the mantle zone, and polyclonal ormonoclonal plasmacytosis.18 IL-6 has been implicated inthe pathogenesis of this disorder, probably by inducingplasmacytosis and systemic symptoms.27 The genesisof the peculiar vascularization of germinal centres inCastleman’s disease, however, has not been clarified.Our results showing prominent expression of VEGF invascularized germinal centres in Castleman’s disease butnot in normal, avascular germinal centres of tonsilsstrongly suggest that this feature may be due to theaction of VEGF. As in the case of IL-6, the mechanismof induction of VEGF remains to be elucidated.Increased vascularity is also a histological feature of

infectious mononucleosis and we have recently demon-strated that EBV-infected cells in this disorder expresscytokines such as lymphotoxin-á and tumour necrosisfactor-á,24 which may induce angiogenesis.28 In thisstudy we have shown enhanced expression of VEGFin the interfollicular zone of infectious mononucleosistonsils, the main area where viral infection takes place.29This points to an additional mechanism in the inductionof angiogenesis in this disease. In contrast to the above-mentioned cytokines, VEGF was not expressed inEBER-positive cells, either in lymphomas (Hodgkin’sdisease and TCL-AILD) or in infectious mononucleosis,suggesting that VEGF is not inducible by EBV ininfected lymphoid cells and that the induction of angio-

genesis in EBV-associated lymphoid disorders throughVEGF is indirect, involving non-infected cells. Forth-coming studies will show whether the same holds truefor EBV-associated epithelial lesions.In summary, our results suggest that VEGF is

involved in the induction of angiogenesis in TCL,particularly in TCL-AILD, and Hodgkins’ disease. Incontrast to solid tumours, angiogenesis in malignantlymphomas through VEGF may occur through an indi-rect mechanism involving fibroblastic cells as producercells. VEGF may also be linked to the vascularization ofgerminal centres in Castleman’s disease. Future studieshave to clarify the mechanisms which result in theexpression of VEGF in lymphoid lesions and will showwhether VEGF receptors are expressed in the diseasesinvestigated in this study.

ACKNOWLEDGEMENTS

This work contains part of the theses of I.A., G.D.,and H.K. We are indebted to Ms E. Berg and Mr L.Öhring for excellent technical assistance. We are gratefulto Scios-Nova, Mountain View, CA 94043, U.S.A. forproviding the pLen-165 plasmid containing a VEGFcDNA fragment. This work was supported by theDeutsche Forschungsgemeinschaft (grant Ste 318/5-1).I.A. was supported by a fellowship from the ConselhoNacional de Pesquisas/CNPq, Brazil.

REFERENCES1. Folkman J. How is blood vessel growth regulated in normal and neoplastic

tissue? GHA Clowes Memorial Award Lecture. Cancer Res 1986; 46:467–473.

2. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease.Nature Med 1995; 1: 27–31.

3. O’Reilly MS, Holmgren L, et al. Angiostatin: a novel angiogenesis inhibitorthat mediates the suppression of metastases by a Lewis’ lung carcinoma.Cell 1994; 79: 315–328.

4. O’Reilly MS, Holmgren L, Chen C, Folkman J. Angiostatin induces andsustains dormancy of human primary tumors in mice. Nature Med 1996; 2:689–692.

5. Dvorak HJF, Brown LF, Detmar M, Dvorak AM. Vascular permeabilityfactor/vascular endothelial growth factor, microvascular hyperpermeability,and angiogenesis. Am J Pathol 1995; 146: 1029–1039.

6. Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascularendothelial growth factor is a secreted angiogenic mitogen. Science 1989;246: 1306–1309.

7. Kim KJ, Li B, Winer J, et al. Inhibition of vascular endothelial growthfactor-induced angiogenesis suppresses tumor growth in vivo. Nature 1993;362: 841–844.

8. Millauer B, Shawver LK, Plate KH, Risau W, Ullrich A. Glioblastomagrowth inhibited in vivo by a dominant-negative Flk-1 mutant. Nature 1994;367: 576–579.

9. Kondo S, Asano M, Suzuki H. Significance of vascular endothelial growthfactor/vascular permeability factor for solid tumor growth, and its inhibi-tion by the antibody. Biochem Biophys Res Commun 1993; 194: 1234–1241.

10. Brown LF, Berse B, Jackman RW, et al. Expression of vascular per-meability factor (vascular endothelial growth factor) and its receptorsin adenocarcinomas of the gastrointestinal tract. Cancer Res 1993; 53:4727–4735.

11. Brown LF, Berse B, Jackman RW, et al. Expression of vascularpermeability factor (vascular endothelial growth factor) and its receptors inbreast cancer. Hum Pathol 1995; 26: 86–91.

12. Claffey KP, Brown LF, del-Aguilla LF, et al. Expression of vascularpermeability factor/vascular endothelial growth factor by melanoma cellsincreases tumor growth, angiogenesis, and experimental metastasis. CancerRes 1996; 56: 172–181.

13. Plate KH, Breier G, Weich HA, Risau W. Vascular endothelial growthfactor is a potential tumour angiogenesis factor in human gliomas in vivo.Nature 1992; 359: 845–847.

14. Hashimoto M, Ohsawa M, Ohnishi A, et al. Expression of vascularendothelial growth factor and its receptor mRNA in angiosarcoma. LabInvest 1995; 73: 859–863.

Table III—Expression of VEGF in reactive tonsils

Case No. Diagnosis* VEGF†

1 FH, IFH 02 FH 03 UT 04 FH 05 UT 4·56 UT 07 FH 0Median UT and FH 0

1 IM 3122 IM 2033 IM 1824 IM 2125 IM 1466 IM 2477 IM 210Median IM 210

*FH=follicular hyperplasia; IFH=interfollicular hyperplasia; UT=ulcerative tonsillitis; IM=infectious mononucleosis.†Values refer to the number of VEGF-positive cells in the inter-

follicular zone per 0.5 mm2.

49VEGF IN LYMPHOMAS


15. Barleon B, Sozzani S, Zhon D, Weich HA, Mantovani A, Marme D.Migration of human monocytes in response to vascular endothelial growthfactor (VEGF) is mediated via the VEGF receptor flt-1. Blood 1996; 87:3336–3343.

16. Warnke RA, Weiss LM, Chan JKC, Cleary ML, Dorfman RF. In: Rosai J,ed. Tumors of the Lymph Nodes and Spleen. Atlas of Tumor Pathology.Third Series. Fascicle 14. Washington, DC: Armed Forces Institute ofPathology, 1995; 259–276.

17. Burke JS. Hodgkin’s disease: histopathology and differential diagnosis. In:Knowles DM, ed. Neoplastic Hematopathology. Baltimore: Williams &Wilkins, 1992; 497–533.

18. Schnitzer B. Reactive lymphadenopathies. In: Knowles DM, ed. NeoplasticHematopathology. Baltimore: Williams & Wilkins, 1992; 427–459.

19. Benjamin D, Kofler G, Tschachler E. Human B-cell TNF-â microhetero-geneity. Lymphokine Cytokine Res 1992; 11: 45–54.

20. Harris NL, Jaffe ES, Stein H, et al. A revised European–Americanclassification of lymphoid neoplasms: a proposal from the InternationalLymphoma Study Group. Blood 1994; 84: 1361–1392.

21. Stein H, Gatter K, Asbahr H, Mason DY. Use of freeze-dried paraffinembedded sections for immunohistological staining with monoclonal anti-bodies. Lab Invest 1985; 52: 676–683.

22. Tischer E, Mitchell R, Hartman T, et al. The human gene for vascularendothelial growth factor. J Biol Chem 1991; 266: 11947–11954.

23. Milani S, Herbst H, Schuppan D, Hahn EG, Stein H. In situ hybridizationfor collagen types I, III and IV mRNA in normal and fibrotic ratliver. Evidence for predominant expression in non-parenchymal liver cells.Hepatology 1989; 10: 84–92.

24. Foss HD, Herbst H, Hummel M, et al. Patterns of cytokine expression ininfectious mononucleosis. Blood 1994; 83: 707–711.

25. Foss HD, Herbst H, Oelmann E, et al. Lymphotoxin, tumor necrosisfactor and interleukin-6 gene transcripts are present in Hodgkin andReed–Sternberg cells of most Hodgkin’s disease cases. Br J Hematol 1993;84: 627–635.

26. Freeman MR, Schneck FX, Gagnon ML, et al. Peripheral blood Tlymphocytes and lymphocytes infiltrating human cancers express vascularendothelial growth factor: a potential role for T cells in angiogenesis. CancerRes 1995; 55: 4140–4145.

27. Leger-Ravet MB, Peuchmaur M, Devergne O, et al. Interleukin-6 geneexpression in Castleman’s disease. Blood 1991; 78: 2923–2930.

28. Cavender DE, Edelbaum D, Ziff M. Endothelial activation induced bytumor necrosis factor and lymphotoxin. Am J Pathol 1989; 134: 551–560.

29. Niedobitek G, Herbst H, Young LS, et al. Patterns of Epstein–Barr virusinfection in non-neoplastic lymphoid tissue. Blood 1992; 79: 2520–2526.



expression of vascular endothelial growth factor in lymphomas and castleman's disease

Documents