American Journal of Pathology, Vol. 137, No. 6, December 1990
Copyright © American Association ofPathologists
Rapid Communication Malignant Lymphomas in Cynomolgus Monkeys Infected with Simian Immunodeficiency Virus Hans Feichtinger,* Per Putkonen,t Carlo Parravicini,* Su-Ling Li,* Ephata E. Kaaya,* Disa Bottiger, Gunnel Biberfeld,t and Peter Biberfeld* From the Immunopathology Laboratory,* Karolinska Institute, the Department of Immunology,t National
Bacteriological Laboratory, and the Department of Virology,* Karolinska Institute, Stockholm, Sweden
Malignant lymphomas were observed in 38% (9 of 24) of simian immunodeficiency virus (SIV)-infected cynomolgus monkeys (Macacafascicularis) 5 to 15 months after inoculation with SIV strain SMM3. Lymphomagenesis in the SIV-infected monkeys was not related directly to the SIV-infectious dose given. All SIV-infected animals developed severe immunodeficiency. No significant difference in immunodeficiency was observed between tumorbearing and non-tumor-bearing animals. In contrast, no lymphomas were observed in a comparable group of HIV-2-infected monkeys, which did not develop immunodeficiency; nor did the noninfected control monkeys. All 9 SIV-related tumors were high-grade B-cell lymphoblastic or pleomorphic lymphomas with extranodal, disseminated growth. Most tumors showed marked infiltration by monocytes and CD8+ T lymphocytes. Occasional tumor infiltrating cells showed immunohistochemical reaction for SIV. The cells of two tumors were established in vitro and shown to be of B-cellphenotype. The tumor cell cultures showed no reverse transcriptase activity and no evidence of virus infection by electron microscopy. Our observations indicate that SIV-induced immunodeficiency in cynomolgus monkeys also mimics HIV infection and AIDS in humans with regard to increased lymphomagenesis and type of lymphomas. (Am J Pathol 1990, 13 7: 1311-1315)
An increased incidence of malignant lymphomas in patients with acquired immune deficiency syndrome (AIDS) has been well documented.1 These studies indicate that human immunodeficiency virus (HIV)-associated lymphomas represent a selection of lymphoma types, with regard to histology, grade, and lymphocyte lineage derivation. Thus most of these lymphomas are lymphoblastic or follicle center-cell (FCC)-derived, of high malignancy and B-cell origin.1 2 In these tumors there is no evidence for a direct genomic association of HIV with neoplastic cells.3 This suggests an indirect, possibly multifactorial role of HIV in the tumorogenic process. A combination of polyclonal activation of B cells by HIV, Epstein-Barr Virus (EBV), and/or other activators and decreased immune surveillance has been proposed as lymphomagenic factors.4 Nonhuman, primate models of AIDS have been developed for studies of the pathogenesis of retrovirus-induced immunodeficiency and testing of new therapies and vaccines. Asian macaques infected with certain strains of simian immunodeficiency virus (SIVsm, SlVmac, and SlVmne) develop severe immunosuppressive disease showing many parallels to human AIDS.5-7 In contrast, human immunodeficiency virus type two (HIV-2), which is closely related to SIV, also infects these primates but has, until now, not been shown to cause immunologic abnormalities in cynomolgus monkeys (Macaca fascicularis).8 9 In experimental and natural SIV infection of Asian macaques in captivity, occasional malignant lymphomas have been reported.1013 In 24 cynomolgus monkeys experiSupported by the Swedish Medical Research Council (161-7740 and 16H-2380), Swedish Cancer Society (273-B90-21XC), Swedish Board for Technical Development, Swedish Agency for Research Cooperation with Developing Countries (SAREC), and ICSC World Laboratory, project no. MCD-309-2. Carlo Parravicini, MD was supported by the European Community Project Immunopathology and Immunology of HIV-related Diseases. Ephata E. Kaaya, MD and Edward Mgaya, MD were supported by SAREC and World-Lab Project MCD2-309-2. Accepted for publication October 3, 1990. Address reprlnt requests to Dr. Peter Biberfeld, Immunopathology Laboratory, Karolinska Institute, S-104 01 Stockholm 60, Sweden.
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mentally inoculated with SlVsm, we observed a remarkably high incidence (38%) of malignant lymphomas after approximately 1 year's follow-up. No lymphomas were found in comparable groups of HIV-2-infected cynomolgus monkeys and noninfected controls. We here report some of the basic characteristics of the observed lymphomas.
Materials and Methods Monkeys and SIV Infection The nine monkeys that developed malignant lymphoma were part of a larger study in which 24 cynomolgus monkeys of either sex, wild caught in the Philippines by different suppliers were intravenously inoculated with SlVsm propagated in human peripheral blood lymphocytes (PBL). As in previous studies,7 we used a SIV strain (SIV-SMM3) isolated from a naturally infected sooty mangabey monkey14 (provided by P. Fultz and H. McClure, Yerkes Primate Research Center, Atlanta, GA). The monkeys were inoculated with 10 to 1 05 animal infectious doses of SlVsm. At the day of SlVsm inoculation, the weights of these nine monkeys were 1870 to 6125 g, which corresponds to the weight of young adults (1.5 to 2 kg) and mature adults (more than 2 kg). Identical studies were performed on 26 cynomolgus monkeys infected with two different isolates of HIV-2 namely HIV-2&.L_wb9 (n = 12) and HIV2SBL-K135 (n = 14) and followed for up to 748 days.
Histo/lmmunopathology Biopsy and autopsy materials were partly fixed and processed for conventional histopathology of paraffin sections and partly frozen for immunohistochemical staining using various monoclonal antibodies (MAbs) and the alkalinephosphatase-anti-alkaline-phosphatase (APAAP) or peroxidase-anti-peroxidase (PAP) detection systems, as previously described.5-16 Frozen sections and cell suspensions were immunostained with the monoclonal antibodies to lineage-specific leukocyte markers and SIV antigens shown in Table 1. The lineage specificity of these antibodies was tested in control monkey tissues and cell suspensions (unpublished data). Lymphoma cell suspensions and cell cultures were characterized by immunoflow cytometry (Spectrum IlIl flow cytometer, Ortho Diagnostics, Raritan, NJ). The lymphomas were morphologically analyzed according to the Kiel classification.17 Cell Cultures Tumor tissues were minced and single-cell suspensions grown in Roswell Park Memorial Institute (RPMI) cul-
Table 1. Characterization of Monoclonal Antibodies Mab CD no. Source Specificity L26 n.c.* pan B Dakopatts Leu 16 20 pan B Becton/Dickinson B1 20 pan B Coulter OkT4 4 T4 Ortho Diagnostics Leu 2 8 T8 Becton/Dickinson KiM 6 68 Macrophage Boehringer Mannheim F8.11.13 45 Ra panleucocyte Sera Lab. SF4 n.c. anti-SIV p28 Biotech Res. Lab., Rockville Not clustered.
ture medium, 15% foctal calf serum (FCS), and antibiotics. Reverse transcriptase (RT) was determined as previously
described.7
Immune Status The absolute numbers of T-lymphocyte subsets in peripheral blood were determined by using FITC-conjugated anti-CD4 and anti-CD8 monoclonal antibodies (Table 1) and a Spectrum Ill flow cytometer. SIV serum antigenemia was determined using a commercial antigen capture enzyme-linked immunoassay (ELISA) Abbott Laboratories, North Chicago, IL).7
Results Of 24 SIV-infected cynomolgus monkeys observed for up to 464 days, 9 animals (38%) developed malignant lymphoma. The individual case histories including clinicopathologic stage, histology, and immunophenotype of the lymphomas as well as the immune status of the animals are summarized in Table 2. Eight of nine tumors were high grade by histology and six of nine were lymphoblastic and of Burkitt-like type (Figure 1a). One lymphoma (no. 2) with histologic characteristics of a lymphoplasmacytoid immunocytoma behaved clinically also like a rapidly growing high-grade tumor; two others (nos. 5 and 8) were pleomorphic, B-cell lymphomas, one of which presented as a primary tumor of the right testes; in this animal (no. 8), an orchiectomy was performed 2 weeks before autopsy. In all other animals, tumor spread was evaluated at autopsy performed within 1 week after tumor presentation and showed disseminated, mostly extranodal, visceral and cutaneous involvement (Table 2). Immunostaining indicated a B-cell phenotype of all lymphomas with a heterogeneous expression of Bi, Leu 16, L26, and F8.1 1.13 in most of the tumor cells (Figure 1 c). Immunohistochemically eight tumors showed a monoclonal expression of IgM heavy chains, five of lambda, and four of kappa light chains. The clonality also was confirmed by immunoflow cytometry in five tumors
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1313 AJP December 1990, Vol. 13 7, No. 6
Table 2. Features of SIV-associated Lymphomas in Cynomolgus Monkeys Immune status
No.
Virus dose*
Time of infection (days)
1
103-104
300
LB lymphoma, mainly extranodal in lungs, liver, kidney, gut: mesenteric LN.
2
10-102
255
3
105
174
LP immunocytoma in left orbita, submandibular region, nose, axillary lymph nodes, thoracic wall. LB lymphoma in right axillary and mesenteric LN.
4
10_102
390
5
n.t.
145
Histologyt and anatomic spread
LB lymphoma in right mandibular region, subcutaneously in the back and in mediastinum. Pleomorphic, high grade lymphoma, with generalized peritoneal, pericardial infiltrations and mesenteric LN involvement. LB lymphoma, with retro orbital and mediastinal localization
6
10_102
169
7
105
351
LB lymphoma with abdominal localization and intestinal infiltration.
8
10_102
464
Pleomorphic, high grade lymphoma of the right testis.
9
10-102
287
LB/IB: infiltration of gastrointestinal tract and mesenterium.
Immunophenotype of tumor cellst
CD4 count X 109/L§
CD4/CD8§
Kappa, mu, L26, CD20,
n.d.
n.d.
CD45RA. Lambda, mu, L26, CD45RA.
0.08 n.d.
0.52 n.d.
0.17 0.86
0.39 0.53
0.03 n.d.
0.07 n.d.
3.31
0.73
0.33
0.31
1.46
0.76
0.04 0.80
0.08 0.78
0.11 n.d.
0.04 n.d.
0.09 0.74
0.06 0.53
0.07
0.10
Lambda, mu, L26, CD45RA. Lambda, L26, CD45RA.
Lambda, mu, L26.
Kappa, mu, L26, CD45RA. Kappa, mu, L26, CD20, CD45RA. Kappa, mu, L26, CD20, CD45RA. Lambda, mu, L26, CD20, CD45RA.
Animal infectious doses given i.v.
t LB = lymphoblastic; LP = lymphoplasmacytoid; IB = immunoblastic. t As observed by immunostaining of frozen sections (see Material and Methods). § Cell counts and cell ratios were determined before infection and at autopsy. In a reference group of 1 17 healthy cynomolgus monkeys the values for CD4+-T cells were 0,53-2,81 X 109/l. ¶ 95% confidence limits of the geometric mean.
(nos. 2, 6, 7, 8, 9), of which single, tumor cell suspensions obtained. A common feature of the lymphoblastic tumors was an infiltration by macrophages and CD8 T cells. In some neoplasms, immunostaining for SIV-associated p28 was occasionally positive in tumor-infiltrating macrophages and multinucleated giant cells. However tumor cells never showed a positive reaction. In three cases (nos. 6, 7, 9) cultures of tumor cells were established. The cytology of these cells in early culture (1 week) is shown in Figure 1b. All cell cultures were negative when tested for reverse transcriptase (RT) activity. Most of the SIV-infected animals were monitored for blood CD4 and CD8 lymphocytes. As shown in Table 2, the animals with lymphomas developed a marked decrease in CD4 cells and CD4/CD8 cell ratios. However there was no significant difference in CD4 cell levels or CD4/CD8 cell ratios between tumor-bearing and tumorfree SIV-infected animals (data not shown). At necropsy, eight of nine animals were positive for circulating viral antigens as determined by ELISA. The histologic examination of tumor-free lymph nodes and spleen showed a broad spectrum of pathologic changes mainly in B-cell areas ranging from follicular hyperplasia to follicular involution and atrophy, similar to were
changes described in HIV-associated lymphadenopathy.15e16 By immunostaining, abundant viral antigen was found in follicles of all SIV-infected animals. Apart from the tumors, no distinctive histologic or immunohistologic features were found in animals with lymphomas as compared to tumor-free monkeys. No direct correlation was evident between lymphoma development and dose of SIV inoculated (Table 2) or estimated age of the animals. Of the 26 cynomolgus monkeys inoculated with HIV-2, all became infected, as shown by seroconversion and virus isolation. All of these animals have remained clinically healthy, without CD4-cell decrease. No animal has developed malignant lymphoma after 300 to 748 days of follow-up.
Discussion Malignant lymphomas are the most common, naturally occurring tumors in nonhuman primates, but the frequency is very low.18 Lymphomas and lymphoproliferative disorders have been reported in association with SIV infections in nonhuman primates.101319 However the high incidence of malignant lymphomas (38%) observed in the
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Figure 1. Characteristics of lympboma 7 (Table 2) in SIV-infected monkey. a: Hematoxylin and eosin-stained section showing morphology of predominantly lymphoblastic lympboma. b: Cytospin of rather monomorpbousproliferating lympboblasts. C: Immunostainedfrozen section. Most of the cells sbow positive reactionfor the B- cell ma rker L26 (magnification, X400for eachfigure).
present series of experimentally SIV-infected cynomolgus monkeys has not been documented. The lymphoma development was evidently associated with SIV disease because no tumors were observed in a comparable group of disease-free HIV-2-infected monkeys and noninfected controls. Thus the incidence of naturally occurring tumors in these noninfected control monkeys is relatively low, about 2 to 3 per 1000 animals as evaluated from approximately 4000 autopsies (unpublished data). All tumors in the present study were high-grade malignant lymphomas with a proved B-cell phenotype. Six of nine lymphomas were lymphoblastic with a Burkitt-like histology and all tumor-bearing animals had clinical and histopathologic evidence of severe immunodeficiency. This correlates with reports of individual lymphomas associated with virally induced immunodeficiency in monkeys that were highly malignant and some of B-cell origin.12 In lymphomas of HIV patients, the lymphoblastic Burkittlike type represents up to 25% of all lymphoproliferative
neoplasms.24 Accordingly EBV is suggested to play a major role in HIV-associated lymphomagenesis with presence of EBV-DNA in 28% of unselected non-Hodgkin lymphomas and up to 58% in selected lymphoblastic tumors.422 A B-lymphotropic virus related to but distinct from EBV has been detected in a lymphoma from an immunodeficient rhesus money.21 However, other lymphoproliferative disorders, including one lymphoma documented in an SIV-infected animal, were not associated with infection by any EBV-related virus.' In the present study, preliminary serologic data showed low titres (1.40 to 1:320) of IgG antibodies to human EBV-associated viral capsid antigens (VCA) in our series of SIV-infected animals, without any correlation between tumor status and titres (A. Linde, unpublished data). However preliminary results of Southern blot hybridizations indicate integration of EBVlike sequences in genomic DNA of some of the tumors. Further studies are in progress to identify and possibly isolate an EBV-related virus from these lymphomas.
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The present observations indicate that SIV-induced immunodeficiency in cynomolgus monkeys also is associated with a marked increase in malignant lymphomas, as in human AIDS. Furthermore most of the tumors showed characteristics analogous to HIV-related malignant lymphomas with regard to histology, immunophenotype, and clinical behavior. In the present series of animals, development of malignant lymphomas evidently was related to productive SIV infection and immunodeficiency. Thus monkeys infected with HIV-2, which does not induce immunodeficiency in these animals, did not develop lymphomas. However lymphomagenesis in SIV infection appeared not to be directly related to infectious dose or age of the monkeys. Evidently SIV-associated malignant lymphomas in cynomolgus monkeys provide a useful primate model for studies of lymphomagenesis.
References 1. Levine AM, Gill PS, Rasheed S: Human retrovirus-associated lymphoproliferative disorders in homosexual men. Prog Allergy 1986, 37:244-258 2. Knowles DM, Chamulak GA, Subar M, Burke JS, Dugan M, Wernz J, Slywotsky C, Pelicci G, Dalla-Favera R, Raphael B: Lymphoid neoplasia associated with the acquired immunodeficiency syndrome (AIDS). The New York University Medical Center experience with 105 patients (1981-1986). Ann Intern Med 1988, 108:744-753 3. Ziegler JL, McGrath MS: Lymphomas in HIV positive individuals. In Magrath II, ed. Non-Hodgkin's Lymphomas. Edward Arnold, London, 1990, pp 155-159 4. Ernberg I, Altiok E: The role of Epstein-Barr virus in lymphomas of HIV-carriers. APMIS 1989, (Suppl 8):58-61 5. Desrosiers RC, Ringler DJ: Use of simian immunodeficiency viruses for AIDS research. Intervirology 1989, 30:301-312 6. Gardner MB, Luciw PA: Animal models of AIDS. FASEB J
1989, 3:2593-2606 7. Putkonen P, Warstedt K, Thorstensson R, Benthin R, Albert J, Lundgren B, Oberg B, Norrby E, Biberfeld G: Experimental infection of cynomolgus monkeys (Macaca fascicularis) with simian immunodeficiency virus (SlVsm). AIDS 1989, 2:359365 8. Putkonen P, B1ottiger B, Warstedt K, Thorstensson R, Albert J, Biberfeld G: Experimental infection of cynomolgus monkeys (Macaca fascicularis) with HIV-2. AIDS 1989, 2:366373 9. Putkonen P, Thorstensson R, Benthin R, Hedstrom KG, Oberg B, Norrby E, Biberfeld G: Animal models for HIV infection and AIDS: HIV-2 and SlVsm infections of cynomolgus monkeys. In Schellekens H, Horzinek M, eds. Animal Models in AIDS. Amsterdam, Elsevier, 1990, pp 81-85 10. Daniel MD, Letvin NL, King NW, Kannagi M, Sehgal PK, Hunt RD, Kanki PJ, Essex M, Desrosiers RC: Isolation of T-cell tropic HTLV-111-like retrovirus from macaques. Science 1985, 228:1201-1204
11. Benveniste RE, Arthur LO, Tsai CC, Sowder R, Copeland TD, Henderson LE, Oroszlan S: Isolation of a lentivirus from a Macaque with lymphoma: Comparison with HTLV-I1I/LAV and other lenti viruses. J Virol 1986, 60:483-490 12. Baskin GB, Martin LN, Rangan SRS, Gormus BJ, MurpheyCorb M, Wolf RH, Solke KF: Transmissible lymphoma and simian acquired immunodeficiency syndrome in Rhesus monkeys. JNCI 1986, 77:127-139 13. Baskin GB, Murphey-Corb M, Watson EA, Martin LN: Necropsy findings in Rhesus monkeys experimentally infected with cultured simian immunodeficiency virus (SIV)/delta. Vet Pathol 1988, 25:456-467 14. Fultz PN, McClure H, Andersson DC, Swenson RB, Anand R, Srinivasan A: Isolation of a T-lymphotropic retrovirus from naturally infected sooty mangabeys monkeys (Cercocebus atys). Proc Natl Acad Sci USA 1986, 83:5286-5290 15. Biberfeld P, Porwit-Ksiazek A, Bottiger B, Morfeldt-Mansson L, Biberfeld G: Immunohistopathology of lymph nodes in HTLV3 infected homosexuals with persistent adenopathy or AIDS. Cancer Res 1985, 45(9 Suppl):4665-4670 16. Biberfeld P, ost A, Porwit A, Sandstedt B, Pallesen G, Bottiger B, Morfeldt-Mansson L, Biberfeld G: Histopathology and immunohistology of HTLV-111/LAV related lymphadenopathy and AIDS. Acta Pathol Microbiol Scand 1986, 95A:47-65 17. Gerard-Merchant R, Hamlin I, Lennert K, Rilke F, Stansfeld AG, Van Unnick JAM: Classification of non-Hodgkin's lymphomas. Letter to Editor. Lancet 1974, ii:406-408 18. McClure HM: Tumors in nonhuman primates: Observations during a six year period in the Yerkes Primate Center colony. Am J Phys Anthropol 1983, 38:425-430 19. Chalifoux LV, King NW, Daniel MD, Kannagi M, Desrosiers RC, Sehgal PK, Waldron LM, Hunt RD, Letvin NL: Lymphoproliferative syndrome in an immunodeficient Rhesus monkey naturally infected with an HTLV-111-like virus (STLV-111). Lab Invest 1986, 55(1):43-50 20. Desrosiers RC, Letvin NL, King NW, Hunt RD, Blake BJ, Arthur LO, Daniel MD: Three retroviruses infecting macaques at the New England Regional Primate Research Center. In Gallo RC, Haseltine W, Klein G, zur Hansen H, eds. Viruses and Human Cancer. New Ser., New York, Alan R. Liss, 1987, 43, pp 451-466 21. Rangan SRS, Martin LN, Bozelka, Wang N, Gormus BJ: Epstein-Barr virus-related herpesvirus from a Rhesus monkey (Macaca mulatta) with malignant lymphoma. Int J Cancer
1986, 38:425-432 22. Ernberg I: Epstein-Barr virus and acquired immunodeficiency syndrome. In Klein G, ed. Advances in Viral Oncology, Vol. 8. New York, Raven Press, 1989, pp 203-217
Acknowledgments The authors thank Edward Mgaya and Reinhold Benthin for their skilled assistance, and Dr. P. Fultz and Dr. H. McClure for providing the SIV isolate (SIV-SMM-3), which resulted from research sponsored by ARB, NIH, grant RR-001 65.
Copyright © American Association ofPathologists
Rapid Communication Malignant Lymphomas in Cynomolgus Monkeys Infected with Simian Immunodeficiency Virus Hans Feichtinger,* Per Putkonen,t Carlo Parravicini,* Su-Ling Li,* Ephata E. Kaaya,* Disa Bottiger, Gunnel Biberfeld,t and Peter Biberfeld* From the Immunopathology Laboratory,* Karolinska Institute, the Department of Immunology,t National
Bacteriological Laboratory, and the Department of Virology,* Karolinska Institute, Stockholm, Sweden
Malignant lymphomas were observed in 38% (9 of 24) of simian immunodeficiency virus (SIV)-infected cynomolgus monkeys (Macacafascicularis) 5 to 15 months after inoculation with SIV strain SMM3. Lymphomagenesis in the SIV-infected monkeys was not related directly to the SIV-infectious dose given. All SIV-infected animals developed severe immunodeficiency. No significant difference in immunodeficiency was observed between tumorbearing and non-tumor-bearing animals. In contrast, no lymphomas were observed in a comparable group of HIV-2-infected monkeys, which did not develop immunodeficiency; nor did the noninfected control monkeys. All 9 SIV-related tumors were high-grade B-cell lymphoblastic or pleomorphic lymphomas with extranodal, disseminated growth. Most tumors showed marked infiltration by monocytes and CD8+ T lymphocytes. Occasional tumor infiltrating cells showed immunohistochemical reaction for SIV. The cells of two tumors were established in vitro and shown to be of B-cellphenotype. The tumor cell cultures showed no reverse transcriptase activity and no evidence of virus infection by electron microscopy. Our observations indicate that SIV-induced immunodeficiency in cynomolgus monkeys also mimics HIV infection and AIDS in humans with regard to increased lymphomagenesis and type of lymphomas. (Am J Pathol 1990, 13 7: 1311-1315)
An increased incidence of malignant lymphomas in patients with acquired immune deficiency syndrome (AIDS) has been well documented.1 These studies indicate that human immunodeficiency virus (HIV)-associated lymphomas represent a selection of lymphoma types, with regard to histology, grade, and lymphocyte lineage derivation. Thus most of these lymphomas are lymphoblastic or follicle center-cell (FCC)-derived, of high malignancy and B-cell origin.1 2 In these tumors there is no evidence for a direct genomic association of HIV with neoplastic cells.3 This suggests an indirect, possibly multifactorial role of HIV in the tumorogenic process. A combination of polyclonal activation of B cells by HIV, Epstein-Barr Virus (EBV), and/or other activators and decreased immune surveillance has been proposed as lymphomagenic factors.4 Nonhuman, primate models of AIDS have been developed for studies of the pathogenesis of retrovirus-induced immunodeficiency and testing of new therapies and vaccines. Asian macaques infected with certain strains of simian immunodeficiency virus (SIVsm, SlVmac, and SlVmne) develop severe immunosuppressive disease showing many parallels to human AIDS.5-7 In contrast, human immunodeficiency virus type two (HIV-2), which is closely related to SIV, also infects these primates but has, until now, not been shown to cause immunologic abnormalities in cynomolgus monkeys (Macaca fascicularis).8 9 In experimental and natural SIV infection of Asian macaques in captivity, occasional malignant lymphomas have been reported.1013 In 24 cynomolgus monkeys experiSupported by the Swedish Medical Research Council (161-7740 and 16H-2380), Swedish Cancer Society (273-B90-21XC), Swedish Board for Technical Development, Swedish Agency for Research Cooperation with Developing Countries (SAREC), and ICSC World Laboratory, project no. MCD-309-2. Carlo Parravicini, MD was supported by the European Community Project Immunopathology and Immunology of HIV-related Diseases. Ephata E. Kaaya, MD and Edward Mgaya, MD were supported by SAREC and World-Lab Project MCD2-309-2. Accepted for publication October 3, 1990. Address reprlnt requests to Dr. Peter Biberfeld, Immunopathology Laboratory, Karolinska Institute, S-104 01 Stockholm 60, Sweden.
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mentally inoculated with SlVsm, we observed a remarkably high incidence (38%) of malignant lymphomas after approximately 1 year's follow-up. No lymphomas were found in comparable groups of HIV-2-infected cynomolgus monkeys and noninfected controls. We here report some of the basic characteristics of the observed lymphomas.
Materials and Methods Monkeys and SIV Infection The nine monkeys that developed malignant lymphoma were part of a larger study in which 24 cynomolgus monkeys of either sex, wild caught in the Philippines by different suppliers were intravenously inoculated with SlVsm propagated in human peripheral blood lymphocytes (PBL). As in previous studies,7 we used a SIV strain (SIV-SMM3) isolated from a naturally infected sooty mangabey monkey14 (provided by P. Fultz and H. McClure, Yerkes Primate Research Center, Atlanta, GA). The monkeys were inoculated with 10 to 1 05 animal infectious doses of SlVsm. At the day of SlVsm inoculation, the weights of these nine monkeys were 1870 to 6125 g, which corresponds to the weight of young adults (1.5 to 2 kg) and mature adults (more than 2 kg). Identical studies were performed on 26 cynomolgus monkeys infected with two different isolates of HIV-2 namely HIV-2&.L_wb9 (n = 12) and HIV2SBL-K135 (n = 14) and followed for up to 748 days.
Histo/lmmunopathology Biopsy and autopsy materials were partly fixed and processed for conventional histopathology of paraffin sections and partly frozen for immunohistochemical staining using various monoclonal antibodies (MAbs) and the alkalinephosphatase-anti-alkaline-phosphatase (APAAP) or peroxidase-anti-peroxidase (PAP) detection systems, as previously described.5-16 Frozen sections and cell suspensions were immunostained with the monoclonal antibodies to lineage-specific leukocyte markers and SIV antigens shown in Table 1. The lineage specificity of these antibodies was tested in control monkey tissues and cell suspensions (unpublished data). Lymphoma cell suspensions and cell cultures were characterized by immunoflow cytometry (Spectrum IlIl flow cytometer, Ortho Diagnostics, Raritan, NJ). The lymphomas were morphologically analyzed according to the Kiel classification.17 Cell Cultures Tumor tissues were minced and single-cell suspensions grown in Roswell Park Memorial Institute (RPMI) cul-
Table 1. Characterization of Monoclonal Antibodies Mab CD no. Source Specificity L26 n.c.* pan B Dakopatts Leu 16 20 pan B Becton/Dickinson B1 20 pan B Coulter OkT4 4 T4 Ortho Diagnostics Leu 2 8 T8 Becton/Dickinson KiM 6 68 Macrophage Boehringer Mannheim F8.11.13 45 Ra panleucocyte Sera Lab. SF4 n.c. anti-SIV p28 Biotech Res. Lab., Rockville Not clustered.
ture medium, 15% foctal calf serum (FCS), and antibiotics. Reverse transcriptase (RT) was determined as previously
described.7
Immune Status The absolute numbers of T-lymphocyte subsets in peripheral blood were determined by using FITC-conjugated anti-CD4 and anti-CD8 monoclonal antibodies (Table 1) and a Spectrum Ill flow cytometer. SIV serum antigenemia was determined using a commercial antigen capture enzyme-linked immunoassay (ELISA) Abbott Laboratories, North Chicago, IL).7
Results Of 24 SIV-infected cynomolgus monkeys observed for up to 464 days, 9 animals (38%) developed malignant lymphoma. The individual case histories including clinicopathologic stage, histology, and immunophenotype of the lymphomas as well as the immune status of the animals are summarized in Table 2. Eight of nine tumors were high grade by histology and six of nine were lymphoblastic and of Burkitt-like type (Figure 1a). One lymphoma (no. 2) with histologic characteristics of a lymphoplasmacytoid immunocytoma behaved clinically also like a rapidly growing high-grade tumor; two others (nos. 5 and 8) were pleomorphic, B-cell lymphomas, one of which presented as a primary tumor of the right testes; in this animal (no. 8), an orchiectomy was performed 2 weeks before autopsy. In all other animals, tumor spread was evaluated at autopsy performed within 1 week after tumor presentation and showed disseminated, mostly extranodal, visceral and cutaneous involvement (Table 2). Immunostaining indicated a B-cell phenotype of all lymphomas with a heterogeneous expression of Bi, Leu 16, L26, and F8.1 1.13 in most of the tumor cells (Figure 1 c). Immunohistochemically eight tumors showed a monoclonal expression of IgM heavy chains, five of lambda, and four of kappa light chains. The clonality also was confirmed by immunoflow cytometry in five tumors
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1313 AJP December 1990, Vol. 13 7, No. 6
Table 2. Features of SIV-associated Lymphomas in Cynomolgus Monkeys Immune status
No.
Virus dose*
Time of infection (days)
1
103-104
300
LB lymphoma, mainly extranodal in lungs, liver, kidney, gut: mesenteric LN.
2
10-102
255
3
105
174
LP immunocytoma in left orbita, submandibular region, nose, axillary lymph nodes, thoracic wall. LB lymphoma in right axillary and mesenteric LN.
4
10_102
390
5
n.t.
145
Histologyt and anatomic spread
LB lymphoma in right mandibular region, subcutaneously in the back and in mediastinum. Pleomorphic, high grade lymphoma, with generalized peritoneal, pericardial infiltrations and mesenteric LN involvement. LB lymphoma, with retro orbital and mediastinal localization
6
10_102
169
7
105
351
LB lymphoma with abdominal localization and intestinal infiltration.
8
10_102
464
Pleomorphic, high grade lymphoma of the right testis.
9
10-102
287
LB/IB: infiltration of gastrointestinal tract and mesenterium.
Immunophenotype of tumor cellst
CD4 count X 109/L§
CD4/CD8§
Kappa, mu, L26, CD20,
n.d.
n.d.
CD45RA. Lambda, mu, L26, CD45RA.
0.08 n.d.
0.52 n.d.
0.17 0.86
0.39 0.53
0.03 n.d.
0.07 n.d.
3.31
0.73
0.33
0.31
1.46
0.76
0.04 0.80
0.08 0.78
0.11 n.d.
0.04 n.d.
0.09 0.74
0.06 0.53
0.07
0.10
Lambda, mu, L26, CD45RA. Lambda, L26, CD45RA.
Lambda, mu, L26.
Kappa, mu, L26, CD45RA. Kappa, mu, L26, CD20, CD45RA. Kappa, mu, L26, CD20, CD45RA. Lambda, mu, L26, CD20, CD45RA.
Animal infectious doses given i.v.
t LB = lymphoblastic; LP = lymphoplasmacytoid; IB = immunoblastic. t As observed by immunostaining of frozen sections (see Material and Methods). § Cell counts and cell ratios were determined before infection and at autopsy. In a reference group of 1 17 healthy cynomolgus monkeys the values for CD4+-T cells were 0,53-2,81 X 109/l. ¶ 95% confidence limits of the geometric mean.
(nos. 2, 6, 7, 8, 9), of which single, tumor cell suspensions obtained. A common feature of the lymphoblastic tumors was an infiltration by macrophages and CD8 T cells. In some neoplasms, immunostaining for SIV-associated p28 was occasionally positive in tumor-infiltrating macrophages and multinucleated giant cells. However tumor cells never showed a positive reaction. In three cases (nos. 6, 7, 9) cultures of tumor cells were established. The cytology of these cells in early culture (1 week) is shown in Figure 1b. All cell cultures were negative when tested for reverse transcriptase (RT) activity. Most of the SIV-infected animals were monitored for blood CD4 and CD8 lymphocytes. As shown in Table 2, the animals with lymphomas developed a marked decrease in CD4 cells and CD4/CD8 cell ratios. However there was no significant difference in CD4 cell levels or CD4/CD8 cell ratios between tumor-bearing and tumorfree SIV-infected animals (data not shown). At necropsy, eight of nine animals were positive for circulating viral antigens as determined by ELISA. The histologic examination of tumor-free lymph nodes and spleen showed a broad spectrum of pathologic changes mainly in B-cell areas ranging from follicular hyperplasia to follicular involution and atrophy, similar to were
changes described in HIV-associated lymphadenopathy.15e16 By immunostaining, abundant viral antigen was found in follicles of all SIV-infected animals. Apart from the tumors, no distinctive histologic or immunohistologic features were found in animals with lymphomas as compared to tumor-free monkeys. No direct correlation was evident between lymphoma development and dose of SIV inoculated (Table 2) or estimated age of the animals. Of the 26 cynomolgus monkeys inoculated with HIV-2, all became infected, as shown by seroconversion and virus isolation. All of these animals have remained clinically healthy, without CD4-cell decrease. No animal has developed malignant lymphoma after 300 to 748 days of follow-up.
Discussion Malignant lymphomas are the most common, naturally occurring tumors in nonhuman primates, but the frequency is very low.18 Lymphomas and lymphoproliferative disorders have been reported in association with SIV infections in nonhuman primates.101319 However the high incidence of malignant lymphomas (38%) observed in the
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Figure 1. Characteristics of lympboma 7 (Table 2) in SIV-infected monkey. a: Hematoxylin and eosin-stained section showing morphology of predominantly lymphoblastic lympboma. b: Cytospin of rather monomorpbousproliferating lympboblasts. C: Immunostainedfrozen section. Most of the cells sbow positive reactionfor the B- cell ma rker L26 (magnification, X400for eachfigure).
present series of experimentally SIV-infected cynomolgus monkeys has not been documented. The lymphoma development was evidently associated with SIV disease because no tumors were observed in a comparable group of disease-free HIV-2-infected monkeys and noninfected controls. Thus the incidence of naturally occurring tumors in these noninfected control monkeys is relatively low, about 2 to 3 per 1000 animals as evaluated from approximately 4000 autopsies (unpublished data). All tumors in the present study were high-grade malignant lymphomas with a proved B-cell phenotype. Six of nine lymphomas were lymphoblastic with a Burkitt-like histology and all tumor-bearing animals had clinical and histopathologic evidence of severe immunodeficiency. This correlates with reports of individual lymphomas associated with virally induced immunodeficiency in monkeys that were highly malignant and some of B-cell origin.12 In lymphomas of HIV patients, the lymphoblastic Burkittlike type represents up to 25% of all lymphoproliferative
neoplasms.24 Accordingly EBV is suggested to play a major role in HIV-associated lymphomagenesis with presence of EBV-DNA in 28% of unselected non-Hodgkin lymphomas and up to 58% in selected lymphoblastic tumors.422 A B-lymphotropic virus related to but distinct from EBV has been detected in a lymphoma from an immunodeficient rhesus money.21 However, other lymphoproliferative disorders, including one lymphoma documented in an SIV-infected animal, were not associated with infection by any EBV-related virus.' In the present study, preliminary serologic data showed low titres (1.40 to 1:320) of IgG antibodies to human EBV-associated viral capsid antigens (VCA) in our series of SIV-infected animals, without any correlation between tumor status and titres (A. Linde, unpublished data). However preliminary results of Southern blot hybridizations indicate integration of EBVlike sequences in genomic DNA of some of the tumors. Further studies are in progress to identify and possibly isolate an EBV-related virus from these lymphomas.
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The present observations indicate that SIV-induced immunodeficiency in cynomolgus monkeys also is associated with a marked increase in malignant lymphomas, as in human AIDS. Furthermore most of the tumors showed characteristics analogous to HIV-related malignant lymphomas with regard to histology, immunophenotype, and clinical behavior. In the present series of animals, development of malignant lymphomas evidently was related to productive SIV infection and immunodeficiency. Thus monkeys infected with HIV-2, which does not induce immunodeficiency in these animals, did not develop lymphomas. However lymphomagenesis in SIV infection appeared not to be directly related to infectious dose or age of the monkeys. Evidently SIV-associated malignant lymphomas in cynomolgus monkeys provide a useful primate model for studies of lymphomagenesis.
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1989, 3:2593-2606 7. Putkonen P, Warstedt K, Thorstensson R, Benthin R, Albert J, Lundgren B, Oberg B, Norrby E, Biberfeld G: Experimental infection of cynomolgus monkeys (Macaca fascicularis) with simian immunodeficiency virus (SlVsm). AIDS 1989, 2:359365 8. Putkonen P, B1ottiger B, Warstedt K, Thorstensson R, Albert J, Biberfeld G: Experimental infection of cynomolgus monkeys (Macaca fascicularis) with HIV-2. AIDS 1989, 2:366373 9. Putkonen P, Thorstensson R, Benthin R, Hedstrom KG, Oberg B, Norrby E, Biberfeld G: Animal models for HIV infection and AIDS: HIV-2 and SlVsm infections of cynomolgus monkeys. In Schellekens H, Horzinek M, eds. Animal Models in AIDS. Amsterdam, Elsevier, 1990, pp 81-85 10. Daniel MD, Letvin NL, King NW, Kannagi M, Sehgal PK, Hunt RD, Kanki PJ, Essex M, Desrosiers RC: Isolation of T-cell tropic HTLV-111-like retrovirus from macaques. Science 1985, 228:1201-1204
11. Benveniste RE, Arthur LO, Tsai CC, Sowder R, Copeland TD, Henderson LE, Oroszlan S: Isolation of a lentivirus from a Macaque with lymphoma: Comparison with HTLV-I1I/LAV and other lenti viruses. J Virol 1986, 60:483-490 12. Baskin GB, Martin LN, Rangan SRS, Gormus BJ, MurpheyCorb M, Wolf RH, Solke KF: Transmissible lymphoma and simian acquired immunodeficiency syndrome in Rhesus monkeys. JNCI 1986, 77:127-139 13. Baskin GB, Murphey-Corb M, Watson EA, Martin LN: Necropsy findings in Rhesus monkeys experimentally infected with cultured simian immunodeficiency virus (SIV)/delta. Vet Pathol 1988, 25:456-467 14. Fultz PN, McClure H, Andersson DC, Swenson RB, Anand R, Srinivasan A: Isolation of a T-lymphotropic retrovirus from naturally infected sooty mangabeys monkeys (Cercocebus atys). Proc Natl Acad Sci USA 1986, 83:5286-5290 15. Biberfeld P, Porwit-Ksiazek A, Bottiger B, Morfeldt-Mansson L, Biberfeld G: Immunohistopathology of lymph nodes in HTLV3 infected homosexuals with persistent adenopathy or AIDS. Cancer Res 1985, 45(9 Suppl):4665-4670 16. Biberfeld P, ost A, Porwit A, Sandstedt B, Pallesen G, Bottiger B, Morfeldt-Mansson L, Biberfeld G: Histopathology and immunohistology of HTLV-111/LAV related lymphadenopathy and AIDS. Acta Pathol Microbiol Scand 1986, 95A:47-65 17. Gerard-Merchant R, Hamlin I, Lennert K, Rilke F, Stansfeld AG, Van Unnick JAM: Classification of non-Hodgkin's lymphomas. Letter to Editor. Lancet 1974, ii:406-408 18. McClure HM: Tumors in nonhuman primates: Observations during a six year period in the Yerkes Primate Center colony. Am J Phys Anthropol 1983, 38:425-430 19. Chalifoux LV, King NW, Daniel MD, Kannagi M, Desrosiers RC, Sehgal PK, Waldron LM, Hunt RD, Letvin NL: Lymphoproliferative syndrome in an immunodeficient Rhesus monkey naturally infected with an HTLV-111-like virus (STLV-111). Lab Invest 1986, 55(1):43-50 20. Desrosiers RC, Letvin NL, King NW, Hunt RD, Blake BJ, Arthur LO, Daniel MD: Three retroviruses infecting macaques at the New England Regional Primate Research Center. In Gallo RC, Haseltine W, Klein G, zur Hansen H, eds. Viruses and Human Cancer. New Ser., New York, Alan R. Liss, 1987, 43, pp 451-466 21. Rangan SRS, Martin LN, Bozelka, Wang N, Gormus BJ: Epstein-Barr virus-related herpesvirus from a Rhesus monkey (Macaca mulatta) with malignant lymphoma. Int J Cancer
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Acknowledgments The authors thank Edward Mgaya and Reinhold Benthin for their skilled assistance, and Dr. P. Fultz and Dr. H. McClure for providing the SIV isolate (SIV-SMM-3), which resulted from research sponsored by ARB, NIH, grant RR-001 65.
