JOURNAL OF PATHOLOGY, VOL.

164: 321-328 (1991)

PHENOTYPIC ANALYSIS OF MALIGNANT LYMPHOMA IN SIMIAN IMMUNODEFICIENCY VIRUS INFECTION USING ANTI-HUMAN ANTIBODIES ALAN D. RAMSAY, JUNE GIDDINGS, ARTHUR BASKERVILLE* AND MARTIN P. CRANAGE*

Department of Histopathology, University of Southampton, Southampton General Hospital, Tremona Road, Southampton, U.K.; *Public Health Laboratory Service, Centrefor Applied Microbiology and Research, Division of Pathology, Porton Down, Salisbury, Wiltshire, U . K . Received I November 1990 Accepted 7 February 1991

SUMMARY Primates infected with simian immunodeficiency virus (SIV) develop a condition similar to the human acquired immunodeficiency syndrome (AIDS). The close resemblance between the simian acquired immunodeficiency syndrome (SAIDS) and the human disease has led to the widespread use of SIV-infected monkeys as an animal model in the study of acquired immunodeficiency. We have investigated the use of standard anti-human antibodies for the immunohistochemical analysis of formalin-fixed, paraffin-embedded tissues from monkeys with SAIDS. With the exception of antibodies UCHLl (CD45RO), M T l (CD43), 4KB5 (CD45RA), and Ber H2 (CD30), our routine (human) lymphoma panel of markers worked successfully on the animal tissues. Using the anti-human antibodies, we were able to analyse the phenotypes of two cases of malignant lymphoma arising in a study group of 26 SIV-infected rhesus monkeys. Both of the cases stained with the antibodies WR16 (CD45RA) and L26 (CD20), and the B-cell lineage of the lymphomas was confirmed by the detection of IgAA immunoglobulin expression in one case, and IgM heavy chain in the other. We therefore report the successful use of anti-human antibodies in the immunohistochemical analysis of lymphomas arising in non-human primates infected with SIV. KEY wom-Simian immunodeficiency virus, simian acquired immunodeficiency syndrome, malignant lymphoma, paraffin immunohistochemistry, anti-human antibodies.

INTRODUCTION The morphology, genetic structure, and biology of the simian immunodeficiency virus (SIV) closely resemble those of the human immunodeficiency virus. SIV-infected non-human primates are therefore in common use as a model for the study of various aspects of the immunodeficiency syndrome. Lymph nodes in the simian acquired immunodeficiency syndrome (SAIDS) show histological changes that are analogous to those seen in the human acquired immunodeficiency syndrome (AIDS). Follicular and paracortical hyperplasia can Addressee for correspondence: Dr A. D. Ramsay, Department of Histopathology, Southampton General Hospital, Tremona Road, Southampton SO9 4XY, U.K.

0022-341 7/91/08032148 $05.00 0 1991 by John Wiley & Sons, Ltd.

occur early in SAIDS, resembling the persistent generalized lymphadenopathy (PGL) phase of HIV infection. As the simian disease progresses, there is diminution of both paracortex and follicular areas with eventual complete lymphoid depletion. Although the time course of the disease is shorter, a similar progression of changes appears to occur in both man and s rim ate.^ The development of malignant lymphoma, of both the non-Hodgkin’s and the Hodgkin’s variety, is a recognized feature of human AIDS.495The majority of these cases are non-Hodgkin’s lymphomas and have a B-cell phenotype. Many arise at extra-nodal sites, and a percentage exhibit histological and chromosomal similarities to Burkitt’s lymp h ~ m a . Malignant ~.~ lymphoma is probably less common in SAIDS, and in a study involving 26

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SIV-infected rhesus monkeys over a 2-year period, we have detected only two cases of lymphoma.3 There are no reports of immunohistochemical analysis of these SIV-associated lymphomas, as monoclonal antibodies raised against primate antigens are not routinely available. In this study, anti-human antibodies were used for the immunohistochemical analysis of paraffin-embedded tissue from the two SIV-associated malignant lymphomas.

individual crush-back cages under containment level 3 conditions, were fed a commercial pelleted diet supplemented with fruit, and were allowed water ad libitum. Virus

MATERIALS AND METHOD

Animals were injected intravenously with 2 ml of supernatant fluid from high passage SIV,,,25 1 virus grown on the C8166 human T-cell line. Details of virus origin and purification have been published elsewhere.’

A nimals The two cases reported were part of a group of 13 male and 13 female rhesus monkeys. All the animals were bred in the United Kingdom, were aged between 2 and 3 years at infection, and weighed between 2.9 and 5.0 kg. All animals were kept in

Autopsy and histopathology Autopsy was carried out immediately after euthanasiaand the following organsand tissues were taken and fixed in 10 per cent buffered neutral formalin: lymphomas, lungs, heart, liver, spleen, adrenals,

Table I-Details

of antibodies used

Antibody

Type

Trypsin

IgG, A, M

Oligoclonal rabbit anti-human Oligoclonal rabbit anti-human Monoclonal mouse anti-human Oligoclonal rabbit anti-human Oligoclonal rabbit anti-human Oligoclonal rabbit anti-cow Monoclonal mouse anti-human Polyclonal rabbit anti-CD3 peptide Monoclonal mouse anti-human Monoclonal mouse anti-human Monoclonal mouse anti-human Monoclonal mouse anti-human Monoclonal mouse anti-human Monoclonal mouse anti-human Monoclonal mouse anti-human

Y

Dako

Y

Dako

Y

Dr D B Jones, Southampton’*

Y

Dako

Y

Dako

Y

Dako”

N

Dr P Beverley, ICRF, London14

Y

Dako15

N

Prof S Poppema, Edmonton, Alberta, Canadat6

N

Dr K Gatter, Oxford”

N

Authors’ (ADR) laboratory”

N

Dako”

N

Becton-Dickinson”

Y

Dako2’

Y

Dr B Lane, ICRF, Clare Hall Laboratories”

K,

2.

MAC 387 a- 1-anti-trypsin

Muramidase SlOO

UCHLl (CD45RO) CD3 MT1 (CD43) 4KB5 (CD45RA) WR16 (CD45RA) L26 (CD20) Leu M I (CD15) Ber H2 (CD30) CAM 5.2

Source and Reference

IMMUNOHISTOCHEMISTRY OF SAIDS LYMPHOMAS

Fig. 1-Spinalcord from rhesus 19H (case A) showing infiltration by malignant lymphoma

kidneys, pancreas, stomach, ileum, caecum, colon, bladder, ovaries, uterus, vagina, testes, prostate, skeletal muscle, brown adipose tissue, thymus, mandibular, axillary, inguinal and mesenteric lymph nodes, salivary glands, brain, and spinal cord. After fixation, tissues were processed by standard methods and embedded in paraffin wax. Sections were cut at 5 pm and stained with haematoxylin and eosin. Selected sections were also stained by periodic acid Schiff (PAS), Gordon and Sweets's method for reticulin, Verhoeff van Gieson, Congo Red, Martius's Scarlet Blue, and Grocott's silver methenamine technique. Immunohistochemistry

Immunostaining of the paraffin sections was performed using the streptavidin-biotin complex technique.*Briefly, sections were deparaffinized in xylene and taken to 70 per cent alcohol. Endogenous peroxidase was inhibited by treating for 10 min in freshly prepared 0.5 per cent hydrogen peroxide in methanol.' After washing, those antibodies that did not require prior trypsin or protease digestion (see Table I) were treated with 1 per cent bovine serum albumin in Tris-buffered saline (TBS) for 30 min. If trypsinization was necessary, the slides were prewarmed to 37°C and treated with 0.1 percent trypsin in 0.1 per cent calcium chloride adjusted to H 7.8 for 10-30 min, followed by washing in TBS.l'h the case of the polyclonal CD3 antibody, pretreatment with protease at a concentration of 2 mg/l for 4-6 min replaced the trypsinization. The mouse mono-

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Fig. 2-High power view of lymphoma from the salivary gland region of rhesus 14H (case B), showingamixture ofimmunoblasts and plasmacytic cells

clonal, rabbit oligoclonal, or rabbit polyclonal anti-human antibody was then applied for 18-24 h (overnight) at 4°C. After three washes in TBS, biotinylated rabbit anti-mouse antibody, or goat anti-rabbit antibody where appropriate, was applied for 30 min, followed by a further three washes in TBS. Streptavidin-biotin complexes, prepared at least 30 min before use, were then layered on the slides for 30 min, and the diaminobenzidine (DAB) substrate was then applied for 10 min after further TBS washes." The slides were then rinsed in TBS and washed in running tap water for 5 min, counterstained with haematoxylin, dehydrated, cleared, and mounted using DPX mountant. Details of the antibodies used are given in Table I. An initial pilot study was carried out to determine which antibodies from our routine (human) diagnostic panel were useful for staining non-human primate tissues. To assess the specificities of these antibodies in non-human primates, a wide range of tissues from normal baboons, cynomologous and rhesus monkeys were stained using trypsin, protease, and neuraminidase pretreatment of the sections. Those antibodies found to be of value were then applied to sections of the lymphomas. RESULTS Virus isolation SIV was isolated at intervals during the infection and at autopsy by co-cultivation of samples with

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Table 11-Antibody Antibody

staining patterns in primate tissues Staining pattern in primate tissues

As in human tissues; plasma cells and cells in follicle centres well demonstrated K, 1 . As in human tissues MAC 387 Demonstrates macrophages in lymph nodes, tissue histiocytes, and alveolar macrophages. Resembles human staining pattern a- 1-anti-trypsin As in human tissues; granular cytoplasmic staining of macrophages Muramidase As in human tissues; granular cytoplasmic staining of macrophages Sl00 Stains chondrocytes and perineural cells; broadly resembles the human staining pattern UCHLl (CD45RO) Non-specific lymphoid cell marker; resembles human CD45RB antibodies As in human tissues; membrane staining of T-cells Polyclonal CD3 Non-specific lymphoid cell marker; resembles human CD45RB MTl (CD43) antibodies Non-specific lymphoid cell marker; resembles human CD45RB 4KB5 (CD45RA) antibodies WR16 (CD45RA) B-cell marker as in human tissues B-cell marker as in human tissues L26 (CD20) Resembles human pattern; neutrophils and some epithelia stain Leu M1 (CD15) Non-specific; weak cytoplasmic staining of lymphoid cells Ber H2 (CD30) Resembles human pattern; demonstrates columnar epithelia CAM 5.2 well

IgG, A, M

Table 111-Staining of primate lymphomas Antibody Heavy chain Light chain L26 (CD20) WR16 (CD45RA) Polyclonal CD3 Leu MI (CD15) Muramidase u- 1-anti-trypsin MAC 387 SlOO CAM 5.2

Case A ( 19H)

Case B (1 4H)

IgM (perinuclear) Not identified

IgA (cytoplasmic) 1 (cytoplasmic) +(focal)

Background T-cells

Background T-cells

++ ++

+

++

-

-

-

-

-

-

-

-

-

-

-

-

human cord-blood lymphocytes on the C8 166 human T-cell line. Virus was recovered from peripheral blood lymphocytes (PBL) of rhesus 19H (case A) and from PBL, lymphoma, brain, cerebrospinal fluid, and spleen of rhesus 14H (case B). Isolates were confirmed as SIV by reverse transcript-

ase activity, solid phase-gag immunoassay, and polymerase chain reaction.

PathologicalJindkiv ( a ) Macroscopic-Case A. 11; months after infection rhesus 19H, a female, developed sudden

IMMUNOHISTOCHEMISTRY OF SAIDS LYMPHOMAS

Fig. 3-WR16 (CD45RA) immunostaining of spinal cord lymphoma in case A. Streptavidin-biotin complex (SABC) immunoperoxidase method

Fig. 5-Case peroxidase

325

B, IgA heavy chain staining. SABC immuno-

enlarged rapidly and after a further 2 weeks, when the animal was autopsied after euthanasia, measured approximately 6 x 5 x 4 cm. The mass was dull white, firm, and encapsulated, and was attached posteriorly to the larynx and laterally to salivary gland, muscle, and skin. The spleen was of normal size, but most lymph nodes were smaller than normal. The lungs were extensively consolidated and Pneumocystis carinii pneumonia was confirmed histologically.

Fig. &Focal L26 (CD20) membrane staining of lymphoma cells in case B. SABC immunoperoxidase

paralysis of the legs. At autopsy after euthanasia there was a pink-brown mass 2cm long in the vertebral canal at the level of the fourth lumbar vertebra which was compressing the spinal cord. There was a similar 1 cm diameter mass in the cortex of the left kidney. The spleen was greatly enlarged (weight 46 g; normal range 8-1 1 g), as were inguinal, abdominal, and axillary lymph nodes. Case B. Rhesus 14H, a male animal, had remained normal for 20 months when a firm mass appeared between the mandibular rami. This

( b ) Microscopic-Case A (19H). Most of the enlarged lymph nodes and the spleen showed a picture of massive follicular hyperplasia, resembling the extended lymphadenopathy syndrome seen in man. The nodule in the kidney and the tumour involving the spinal cord was a high-grade nonHodgkin’s lymphoma showing prominent mitotic activity and a rather mixed cellular population. The cells included classical immunoblasts, smaller cells with an immunoblastic morphology, and cells with plasmacytic features, as well as scattered small lymphocytes (Fig. 1). The overall picture did not fit well into the standard lymphoma classifications, but was nearest to the Kiel category of ‘immunoblastic lymphoma with plasmablastic differentiati~n’.~~ Case B (14H). Histologically there was infiltration of nodes and salivary gland by a diffuse sheet of lymphoid cells. The majority of the cells had the features of immunoblasts, with pale, rounded nuclei, prominent central eosinophilic nucleoli, and a moderate amount of basophilic cytoplasm.

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A. D. RAMSAY ET A L .

pattern. The exceptions were the paraffin T-cell markers UCHLl and MTI, which lost their T-cell specificity; the antibody 4KB5, which lost its B-cell specificity; and Ber H2 (CD30), which showed only weak, non-specific cytoplasmic staining of lymphoid cells. The specific staining of the two cases of lymphoma is shown in Table 111. Both cases were positive with the B-cell markers L26 and WR16 (Figs 3 and 4), and negative with the non-lymphoid and macrophage markers. Case A showed p heavy chain staining, but no light chain was identified; case B had an IgAA phenotype (Figs 5 and 6). The CD3 antibody identified a background population of small and medium-sized T-cells amongst the neoplastic B-cells in both cases.

DISCUSSION

Fig. &(a) Kappa light chain immunostaining; (b) lambda light chain staining. Both case B, SABC immunoperoxidase

In addition, there were cells with plasmablastic and plasmacytic features together with scattered centroblastic forms. A minority population of small lymphocytes was present in the background. The picture was that of a diffuse high-grade lymphoma which resembled case A, but was more readily classifiable under Kiel as diffuse immunoblastic lymphoma with plasmablastic/plasmacytic differentiation (Fig. 2).

f r ) Imnzunohistochemistry-The results of the initial assessment of the staining specificites of the antibodies concerned are summarized in Table 11. Essentially, the majority of the antibodies used produced results resembling their human staining

Malignant lymphoma is reported to arise in AIDS with a frequency of approximately 3 4 per cent.5 Most of the non-Hodgkin's cases are B-cell derived, and there appears to be a predilection for extra-nodal sites.6 In SAIDS the frequency of lymphoma is less clear, a low sporadic occurrence being reported from some centres,24others observing as many as 43 cases over a 6-year period,25and a recent paper reporting an incidence of 38 per cent.26In our study of 26 monkeys, only the two cases described were noted. These cases are particularly interesting, since they are apparently the first to be induced by inoculation of monkeys with pure culture-grown SIV. Both cases were similar, involving extra-nodal sites and showing comparable morphological features. In AIDS, the favoured sites for extra-nodal lymphoma include bone marrow, the central nervous system, and the anorectal and it is of note that one of the primate lymphomas infiltrated the spinal cord. The salivary gland involvement in case B was considered to be due to local infiltration by a lymphoma originating in lymph nodes. Although the IgAA phenotype would be consistent with a mucosal origin, the tumour lacked the lymphoepithelial lesions and centrocyte-like cells that are indicative of a primary salivary gland lymph~ma.~' The spectrum of lymphoma seen in AIDS ranges from the Burkitt-like diffuse small non-cleaved lymphoma3' to more mature immunoblastic-plasmacytoid tumours that resemble the SAIDS neoplasms reported here.6

IMMUNOHISTOCHEMISTRY OF SAIDS LYMPHOMAS

The simian lymphomas were both B-cell derived, as indicated by their morphology and immunohistochemistry. The prominent plasmacytic change indicated a derivation from ‘mature’ B-cell, in contrast with the ‘early’ B-cell tumours of the Burkitt and Burkitt-like group. There is a recent report of Burkitt and Burkitt-like varieties of lymphoma arising in S A I D S . ~ ~ Lymphomas occurring in AIDS patients are frequently associated with the Epstein-Barr virus (EBV).32Although a herpes virus resembling EBV has been isolated from a rhesus monkey with lymp h ~ m aand , ~ preliminary ~ work by a Swedish group reports low titres of IgG against human EBVassociated viral capsid antigens in the serum of SIVinfected cynomologous monkeys developing lymphoma,26 there is as yet no definite association between SAIDS lymphomas and EBV. The effectiveness of most of the lymphoid antibodies used is presumably a reflection of antigenic similarity between primates and man. This similarity is not exact, however, since both the CD45RO antibodies, one of the CD45RA antibodies, and CD30 lost cell type specificity in the animal tissue. Although both CD45RA antibodies should identify the same antigen in man, they need not recognize the same epitope on that antigen, and a species difference between epitopes is likely to account for the differential staining pattern shown by the two CD45RA antibodies in the primate tissues. The loss of T-cell staining with CD45RO antibodies may also be due to different epitopes, or may reflect species differences in the CD45 sub-unit expression. Immunohistochemical comparison of human and primate staining may be of interest in the study of the evolution of the leucocyte common antigen molecule.34 The presence of a T-cell background population in both lymphomas is not unexpected, as variable numbers of reactive T-cells have been reported in human B-cell lymphoma^.^^ In the absence of gene rearrangement studies, it is impossible to be certain that the background population seen in these lymphomas does not constitute a co-existent T-cell neoplasm, but to our knowledge immunogenetic studies have not yet been performed on simian lymphoid tissue, although an assessment of the use of human genetic probes might yield interesting results. Our observations indicate that the lymphomas arising in experimental SIV infection share many of the features of those seen in human AIDS, and support the validity of the SAIDS animal model. The use of standard anti-human diagnostic antibodies in

327

the investigation of such SIV-induced lymphomas has not been previously reported, and our finding that the lymphoid malignancies arising in SAIDS can be phenotypically analysed by routine immunohistochemical methods further enhances the value of this animal model. REFERENCES I . King NW. Simian models of acquired immunodeficiency syndrome (AIDS): a review. Yer Purhol 1986; 2 3 345-353. 2 . Baskin GB, Murphey-Corb M, Watson EA, el a/. Necropsy findings in rhesus monkeys experimentally infected with cultured simian immunodificiency virus. (SIV)/Delta. Ye, Puthol 1988;25: 4 5 W 6 7 . 3. Baskerville A, Ramsay A, Cranage MP, er a/. Histopathological changes in simian immunodeficiency virus infection. J Purhol 1990; 162: 67 ~ 7 5 . 4. Ziegler JL, Beckstead JA, Volberding PA, et ul. Non-Hodgkin’s lymphoma in 90 homosexual men. N EngI J Med 1984; 31 1: 565-570. 5. Kaplan M H , Susin M, Pahwa SG, er 01. Neoplastic complications of HTLV-Ill infection. Am J Me(/ 1987;82: 389-395. 6. Knowles D M , Chamulak GA, Subar M, er ul. Lymphoid neoplasia associated with the acquired immunodeficiency syndrome (AIDS). Ann inirm Med 1988; 108: 744-753. 7. Cranage MP, Cook N, Johnstone P, er a/. SIV infection of rhesus macaques: in-vivo titration of infectivity and development ofanexperimental vaccine. In: Schellekens H, Horzinek MC, eds. Animal Models in AIDS. Amsterdam: Elsevier, 1990; 103-1 13. 8. Bonnard C, Papermaster DS, Kraekenbuhl JP. The streptavidinbiotin bridge technique: application in light and electron microscope immunocytochemistry. In: Polak JM, Varndell Im, eds. lmmunolabelling for Electron Microscopy. Amsterdam: Elsevier, 1984:95-111. 9. Streefkerk JG. Inhibition oferythrocyte pseudoperoxidase activity by treatment with hydrogen peroxide following methanol. J Hisrochem Cyrochem 1972; 20: 829-831 10. Mepham BL, FraterW, Mitchell BS.Theuseofproteoiyticenzyniesto improve immunoglobulin staining by the PAP technique. Histochem J 1979; 11: 345-357. I I . Graham RC Jr, Karnovsky MJ. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Hisrochem Cjmchrm 1966; 14: 291-302. 12. Brandtzaeg P, Jones DB, Flavell DJ, Fagerhol MK. Mac 387 antibody and detection of formalin resistant myelomonocytic LI antigen. J C/in ParhoI198X: 41: 963-970. 13. Vanstapel M-J, Gatter KC, de Wolf-Peelers C, Mason DY, Desmet V. New sites of human S-100 immunoreactivity detected with monoclonal antibodies. A m J CIin Pufhol1986; 8 5 16&168. 14. Norton AJ, Ramsay AD. Smith SH. Beverley PCL, lsaacson PG. Monoclonalantibody(UCHL1) thatrecognisesnormalandneoplastic T-cells in routinely fixed tissues. J Clin Puthol 1986; 3 9 399 4 0 5 . 15. Mason DY. Cordell J. Brown M, er ul. Detection of T cells in parafin waxembedded tissue usingantibodiesagainsta peptidesequence from the CD3 antigen. J Clin PurhoI 1989; 4 2 11941200. 16. Poppema S, Hollema H , Visser L, Vos H. Monoclonal antibodies (MTI, MT2, MBI, MB2, MB3) reactive with leukocyte subsets in paraffin-embedded tissue sections. Am J Parhol1987; 127: 418429. 17. Schwinzer R. Cluster report CD45iCD45R. In: Knapp W, eds. Leucocyte Typing IV. Oxford: Oxford University Press, 1990; 628--634. 18. Nesbitt AM. Jones DB, Moore K. Phenotypic changes in a C D 4 + lymphocyte suppressor subset correlate with a conversion from suppressor to helper inducer function. Immunology 1990; 6 9 65-70. 19. Mason DY, Comans-Bitter WM, Cordell JL, Verhoeven MJ, Van Dongen JJM. Antibody L26 recognisesan intracellular epitopeon the B-cell associated CD20 antigen. Am J Pafhol1990;1 3 6 1215-1222.

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