ANIMAL MODEL OF HUMAN DISEASE
Acute Lvmphoblastic Leukemia
Animal Model: Transplantable Guinea Pig L2C Leukemia Contributed by: Leonard S. Kaplow, MD, Chief, Laboratory Service, Veterans Administration Medical Center, West Haven, CT 06516 and Professor of Pathology and Laboratory Medicine, Yale University School of Medicine, New Haven, CT 06510, and Eli Nadel, MD, Professor of Pathology and Assistant Vice President for Medical Research, St. Louis University Medical Center, 1402 S. Grand Blvd., St. Louis, MO 63104.
Guinea pig (GP) L2C leukemia is a unique transmissible disease similar to human acute lymphoblastic leukemia (ALL). The fulminant course. blood picture, organ involvement, immunologic features, and response to therapy closely resemble the human counterpart. Two viral particles are associated with the disease and/or the host. Evidence, howvever, points to a cellular graft mechanism as the mode of transmission. Bioogic Features
L*C leukemia arose spontaneously in an inbred female strain 2 GP in the laboratory of Lorenz at NCI in the 1940s and has since been continuously passaged in susceptible animals and is being actively studied by- at least 10 groups. Significant differences exist among the five L2C lines under study.' A review of the literature, recent developments, and transplantation history (1954-1976) was published by Nadel.2 The disease is 100% transmissible in inbred strain 2 GPs and F1 strain 2i Hartley hybrids. Inbred newborn strain 13 and some random-bred newvborns of other strains are also highly susceptible. However, in out-of-strain animals, transmissibility falls rapidly with age but is enhanced in adults by priming with corticosteroids. Transmission is easily accomplished by injection of fresh or thawed frozen (with 10% dimethvlsulfoxide at -70 C for up to 3 years) leukemic whole blood or tissue brei (spleen, Iymph nodes) by the intraperitoneal route (preferable) or subcutaneously. The latter produces a localized sarcomatous tumor at the site of inoculation, Publication sponsored b%- the Registrv of Comparative Pathology of the Armed Forces Institute of by Public Health Sen-ice Grant RR 00301 from the Div ision of Research Resources. US Department of Health, Education and W'elfare, under the auspices of Universities Associated for Research and Education in Pathology, Inc. Supported in part by the Medical Research Sen-ice of the V'eterans Administration and bv the McBride-Lose Foundation, St. Louis Address reprint requests to Leonard S. Kaplow, NID, Chief. Laborators Sen-ice 113, V'eterans Administration Medical Center, West Haven, CT 06516. 0002-9440/79/041 1-0273$01.00 273
Pathology and supported
( American Association of Pathologists
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American Journal of Pathology
KAPLOW AND NADEL
which is usually followed by leukemia. It is preferable to inject 106 fresh or 5 X 10 6 stored cells, although transmission was reported with intraperitoneal inoculation with as little as 50 or less leukemic cells.3 Since the preterminal white blood cell counts usually range from 150,000 to 600,000/cu mm, inoculation with 0.1 ml of fresh or 0.5 ml of stored blood will insure transfer. Leukemic cells appear in the circulation 5 to 7 days after inoculation and increase rapidly. Death occurs 10 to 18 days after inoculation. Few or no clinical symptoms of disease appear until 24 to 48 hours before death, when sluggishness and loss of righting reflex may be observed. The leukemic cell is a large (18 to 40 ,u) primitive cell with high nuclear: cytoplasmic ratio. The nucleus is usually round or oval but may be indented or convoluted. The chromatin structure is sieve-like, and one or more large nucleoli may be evident. The cytoplasm appears light grayblue to dark blue with Wright's stain and may contain a few or many small round vacuoles which do not stain for fat or carbohydrate (Figure 1A). Inclusion bodies are not seen; PAS stain is negative; and cytochemical staining shows no evidence of peroxidase, alkaline phosphatase, or esterase activity. Massive lymphadenopathy and splenomegaly are evident. Most other parenchymal tissues are usually affected (Figure iB); however, bone marrow and thymus show lesser involvement. All five LC lines have receptors for the third component of complement 4 and surface IgM immunoglobulin.5 Scanning electron microscopy (EM) shows numerous villous-like surface projections.2 Thus, they have the characteristics of B (bone-marrow-derived) lymphocytes and represent a rare animal B-cell tumor. By transmission EM the cells have primitive features with sparse endoplasmic reticulum. Endogenous viral particles may be seen within cisternae of rough endoplasmic reticulum and in lesser numbers within the cytoplasm and extracellular spaces. These resemble C-type viral particles but also were described as being A or B type. Regardless of morpholai00
Bll1|; 0r~r
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_M;JL
z!d
.'
-.Xt
B-Infiltration of Figure 1A-Leukemic blast cells in peripheral blood at height of disease. C-"Starry sky" appearance of lymph node replaced heart parenchyma by leukemic cells. by leukemic infiltrate. (A, x 1600; B and C, x 800) (with photographic reduction of 34%)
Vol. 95, No. 1 April 1979
ACUTE LYMPHOBLASTIC LEUKEMIA
275
A
Figwe 2-Electron photomicrograph soing viral particles associated with GP L,C leukemia. A-lntracisternal C-type-like partile in cytoplasm of leukemic blast cell. BHigh-power view of C-type partice. (Modified from Hsiung GD, Kaplow LS: BibI Haematol 36:582, 1970) C-KIdney monolayer tissue culture cell showing intranuclear DNA herpeslike viral particles. (Modified from Hsiung GD, Kaplow LS, Booss J: Am J Epidemiol 93:302, 1971) D-High-power view of empty herpes-like viral particle. E-Single-coated herpes virion. F-Don ble-coated mature herpes virion with dense nucleid. (A, x-19,700; B, x115,000; C, x16,300; D, Et and F, x120,000) (with photographic reduction of 25%)
ogy, they fall into the oncornavirus group (Retraviridae family).6'7 Viral particles in tissue culture of leukemic cells may be greatly increased in number by addition of halogenated pyrirnidines.8'9 Similar but fewer particles may be seen in tissues of normal GPs, particularly in fetal and lymphoid tissue. In addition, a herpes-like virus was also isolated from leukocytes and solid tissues of both leukemic and nonleukemic strain 2 animals by in vitro tissue culture techniques.1O This DNA herpesvirus was never seen in noncultured GP tissues. The relationship of these two viruses (Figure 2) to each other and to the leukemic process remains uncertain. Transfer of the disease by cell-free filtrates has not been confirmed. Recent evidence' indicates that all [LC cell lines, despite 30 years of transplantation, contain the female karyotype regardless of the sex of the host animal, making cell-free transmission untenable. Comparison Wih Huan Disease
[LC blast cells are morphologically and cytochemically similar to human stem cells or lymphoblasts, although the latter may exhibit PAS positivity not seen with the animal model. The intracytoplasmic vacuoles and the characteristic histologic "starry sky" appearance (Figure 1C) of lymph nodes associated with Burkitt's lymphoma may also be seen in cells
276
KAPLOW AND NADEL
American Journal of Pathology
and tissues of leukemic GPs. There are also morphologic and biochemical similarities between the GP herpesvirus and human EB virus of Burkitt's lymphoma.ff 1 Murphy and LoBuglio summarized many of the similarities between acute human and L2C leukemias.12 Usefulness of the Model
Strain 2 GPs are particularly suitable for experimental studies of leukemia. They are attractive and docile, with little or no odor and of sufficient size to provide adequate samples of solid tissues or leukocytes (108 cells/20 ml). The fulminant course of the disease and the response to therapy make them especially useful for drug trials. Although all five L2C cell lines have some common cytogenetic and immunologic markers, only four possess tumor-specific transplantation and Ta antigens. These circumstances provide an unusual opportunity for research in the field of experimental immunotherapy. Availability
Strain 2 GPs are available from NIH (Bethesda, Md). L2C cell lines may be obtained through the courtesy of active investigators in the field. Interested individuals should refer to the issue of Federation Proceedings (36:2247-2332, 1977) which contains the presentations at a recent comprehensive workshop on Guinea Pig L2C Leukemia. References 1. Whang-Peng J: Cytogenetic studies in L2C leukemia. Fed Proc 36:2255-2259, 1977 2. Nadel EM: History and further observations (1954-1976) of the L2C leukemia in the guinea pig. Fed Proc 36:2249-2254, 1977 3. Gross L, Dreyfuss Y: Immunization of guinea pigs against L2C leukemia with live leukemic cells by intradermal inoculation: Early and late effects. Fed Proc 36:22722276, 1977 4. Shevach EM, Ellman L, Davie JM, Green I: L2C guinea pig lymphatic leukemia: A "B" cell leukemia. Blood 39:1-12, 1972 5. Finkelman FD, Shevach EM, Vitetta ES, Green I, Paul WE: Guinea pig immune response-related histocompatibility antigens: Partial characterization and distribution. J Exp Med 141:27-41, 1975 6. Hsiung GD: Virological studies of guinea pig leukemia: An overview with reference to herpesvirus and oncornavirus. Fed Proc 36:2285-2289, 1977 7. Vernon ML, Knipscher RC, More NS, Ruch DG, Green I, Rhim JS: Morphology of the guinea pig leukemia associated viruses. Fed Proc 36:2297-2304, 1977 8. Hsiung GD: Activation of guinea pig C-type virus in cultured spleen cells by 5bromo-2'-deoxyuridine. J Natl Cancer Inst 49:567-570, 1972 9. Nayak DP, Davis AR: Endogenous guinea pig retravirus and L2C leukemia. Fed Proc 36:2305-2309, 1977 10. Hsiung GD, Kaplow LS: Herpeslike virus isolated from spontaneously degenerated tissue culture derived from leukemia-susceptible guinea pigs. J Virology 3:355-357, 1969 11. Rhim JS, Vernon ML, Green I: In vitro studies on L2C leukemia: Isolation of guinea pig herpes virus from mink cells after cocultivation with leukemic cells. Fed Proc 36:2328-2332, 1977 12. Murphy SG, LoBuglio AF: L~C leukemia: A model of human acute leukemia. Fed Proc 36:2281-2285, 1977
Acute Lvmphoblastic Leukemia
Animal Model: Transplantable Guinea Pig L2C Leukemia Contributed by: Leonard S. Kaplow, MD, Chief, Laboratory Service, Veterans Administration Medical Center, West Haven, CT 06516 and Professor of Pathology and Laboratory Medicine, Yale University School of Medicine, New Haven, CT 06510, and Eli Nadel, MD, Professor of Pathology and Assistant Vice President for Medical Research, St. Louis University Medical Center, 1402 S. Grand Blvd., St. Louis, MO 63104.
Guinea pig (GP) L2C leukemia is a unique transmissible disease similar to human acute lymphoblastic leukemia (ALL). The fulminant course. blood picture, organ involvement, immunologic features, and response to therapy closely resemble the human counterpart. Two viral particles are associated with the disease and/or the host. Evidence, howvever, points to a cellular graft mechanism as the mode of transmission. Bioogic Features
L*C leukemia arose spontaneously in an inbred female strain 2 GP in the laboratory of Lorenz at NCI in the 1940s and has since been continuously passaged in susceptible animals and is being actively studied by- at least 10 groups. Significant differences exist among the five L2C lines under study.' A review of the literature, recent developments, and transplantation history (1954-1976) was published by Nadel.2 The disease is 100% transmissible in inbred strain 2 GPs and F1 strain 2i Hartley hybrids. Inbred newborn strain 13 and some random-bred newvborns of other strains are also highly susceptible. However, in out-of-strain animals, transmissibility falls rapidly with age but is enhanced in adults by priming with corticosteroids. Transmission is easily accomplished by injection of fresh or thawed frozen (with 10% dimethvlsulfoxide at -70 C for up to 3 years) leukemic whole blood or tissue brei (spleen, Iymph nodes) by the intraperitoneal route (preferable) or subcutaneously. The latter produces a localized sarcomatous tumor at the site of inoculation, Publication sponsored b%- the Registrv of Comparative Pathology of the Armed Forces Institute of by Public Health Sen-ice Grant RR 00301 from the Div ision of Research Resources. US Department of Health, Education and W'elfare, under the auspices of Universities Associated for Research and Education in Pathology, Inc. Supported in part by the Medical Research Sen-ice of the V'eterans Administration and bv the McBride-Lose Foundation, St. Louis Address reprint requests to Leonard S. Kaplow, NID, Chief. Laborators Sen-ice 113, V'eterans Administration Medical Center, West Haven, CT 06516. 0002-9440/79/041 1-0273$01.00 273
Pathology and supported
( American Association of Pathologists
274
American Journal of Pathology
KAPLOW AND NADEL
which is usually followed by leukemia. It is preferable to inject 106 fresh or 5 X 10 6 stored cells, although transmission was reported with intraperitoneal inoculation with as little as 50 or less leukemic cells.3 Since the preterminal white blood cell counts usually range from 150,000 to 600,000/cu mm, inoculation with 0.1 ml of fresh or 0.5 ml of stored blood will insure transfer. Leukemic cells appear in the circulation 5 to 7 days after inoculation and increase rapidly. Death occurs 10 to 18 days after inoculation. Few or no clinical symptoms of disease appear until 24 to 48 hours before death, when sluggishness and loss of righting reflex may be observed. The leukemic cell is a large (18 to 40 ,u) primitive cell with high nuclear: cytoplasmic ratio. The nucleus is usually round or oval but may be indented or convoluted. The chromatin structure is sieve-like, and one or more large nucleoli may be evident. The cytoplasm appears light grayblue to dark blue with Wright's stain and may contain a few or many small round vacuoles which do not stain for fat or carbohydrate (Figure 1A). Inclusion bodies are not seen; PAS stain is negative; and cytochemical staining shows no evidence of peroxidase, alkaline phosphatase, or esterase activity. Massive lymphadenopathy and splenomegaly are evident. Most other parenchymal tissues are usually affected (Figure iB); however, bone marrow and thymus show lesser involvement. All five LC lines have receptors for the third component of complement 4 and surface IgM immunoglobulin.5 Scanning electron microscopy (EM) shows numerous villous-like surface projections.2 Thus, they have the characteristics of B (bone-marrow-derived) lymphocytes and represent a rare animal B-cell tumor. By transmission EM the cells have primitive features with sparse endoplasmic reticulum. Endogenous viral particles may be seen within cisternae of rough endoplasmic reticulum and in lesser numbers within the cytoplasm and extracellular spaces. These resemble C-type viral particles but also were described as being A or B type. Regardless of morpholai00
Bll1|; 0r~r
0
_M;JL
z!d
.'
-.Xt
B-Infiltration of Figure 1A-Leukemic blast cells in peripheral blood at height of disease. C-"Starry sky" appearance of lymph node replaced heart parenchyma by leukemic cells. by leukemic infiltrate. (A, x 1600; B and C, x 800) (with photographic reduction of 34%)
Vol. 95, No. 1 April 1979
ACUTE LYMPHOBLASTIC LEUKEMIA
275
A
Figwe 2-Electron photomicrograph soing viral particles associated with GP L,C leukemia. A-lntracisternal C-type-like partile in cytoplasm of leukemic blast cell. BHigh-power view of C-type partice. (Modified from Hsiung GD, Kaplow LS: BibI Haematol 36:582, 1970) C-KIdney monolayer tissue culture cell showing intranuclear DNA herpeslike viral particles. (Modified from Hsiung GD, Kaplow LS, Booss J: Am J Epidemiol 93:302, 1971) D-High-power view of empty herpes-like viral particle. E-Single-coated herpes virion. F-Don ble-coated mature herpes virion with dense nucleid. (A, x-19,700; B, x115,000; C, x16,300; D, Et and F, x120,000) (with photographic reduction of 25%)
ogy, they fall into the oncornavirus group (Retraviridae family).6'7 Viral particles in tissue culture of leukemic cells may be greatly increased in number by addition of halogenated pyrirnidines.8'9 Similar but fewer particles may be seen in tissues of normal GPs, particularly in fetal and lymphoid tissue. In addition, a herpes-like virus was also isolated from leukocytes and solid tissues of both leukemic and nonleukemic strain 2 animals by in vitro tissue culture techniques.1O This DNA herpesvirus was never seen in noncultured GP tissues. The relationship of these two viruses (Figure 2) to each other and to the leukemic process remains uncertain. Transfer of the disease by cell-free filtrates has not been confirmed. Recent evidence' indicates that all [LC cell lines, despite 30 years of transplantation, contain the female karyotype regardless of the sex of the host animal, making cell-free transmission untenable. Comparison Wih Huan Disease
[LC blast cells are morphologically and cytochemically similar to human stem cells or lymphoblasts, although the latter may exhibit PAS positivity not seen with the animal model. The intracytoplasmic vacuoles and the characteristic histologic "starry sky" appearance (Figure 1C) of lymph nodes associated with Burkitt's lymphoma may also be seen in cells
276
KAPLOW AND NADEL
American Journal of Pathology
and tissues of leukemic GPs. There are also morphologic and biochemical similarities between the GP herpesvirus and human EB virus of Burkitt's lymphoma.ff 1 Murphy and LoBuglio summarized many of the similarities between acute human and L2C leukemias.12 Usefulness of the Model
Strain 2 GPs are particularly suitable for experimental studies of leukemia. They are attractive and docile, with little or no odor and of sufficient size to provide adequate samples of solid tissues or leukocytes (108 cells/20 ml). The fulminant course of the disease and the response to therapy make them especially useful for drug trials. Although all five L2C cell lines have some common cytogenetic and immunologic markers, only four possess tumor-specific transplantation and Ta antigens. These circumstances provide an unusual opportunity for research in the field of experimental immunotherapy. Availability
Strain 2 GPs are available from NIH (Bethesda, Md). L2C cell lines may be obtained through the courtesy of active investigators in the field. Interested individuals should refer to the issue of Federation Proceedings (36:2247-2332, 1977) which contains the presentations at a recent comprehensive workshop on Guinea Pig L2C Leukemia. References 1. Whang-Peng J: Cytogenetic studies in L2C leukemia. Fed Proc 36:2255-2259, 1977 2. Nadel EM: History and further observations (1954-1976) of the L2C leukemia in the guinea pig. Fed Proc 36:2249-2254, 1977 3. Gross L, Dreyfuss Y: Immunization of guinea pigs against L2C leukemia with live leukemic cells by intradermal inoculation: Early and late effects. Fed Proc 36:22722276, 1977 4. Shevach EM, Ellman L, Davie JM, Green I: L2C guinea pig lymphatic leukemia: A "B" cell leukemia. Blood 39:1-12, 1972 5. Finkelman FD, Shevach EM, Vitetta ES, Green I, Paul WE: Guinea pig immune response-related histocompatibility antigens: Partial characterization and distribution. J Exp Med 141:27-41, 1975 6. Hsiung GD: Virological studies of guinea pig leukemia: An overview with reference to herpesvirus and oncornavirus. Fed Proc 36:2285-2289, 1977 7. Vernon ML, Knipscher RC, More NS, Ruch DG, Green I, Rhim JS: Morphology of the guinea pig leukemia associated viruses. Fed Proc 36:2297-2304, 1977 8. Hsiung GD: Activation of guinea pig C-type virus in cultured spleen cells by 5bromo-2'-deoxyuridine. J Natl Cancer Inst 49:567-570, 1972 9. Nayak DP, Davis AR: Endogenous guinea pig retravirus and L2C leukemia. Fed Proc 36:2305-2309, 1977 10. Hsiung GD, Kaplow LS: Herpeslike virus isolated from spontaneously degenerated tissue culture derived from leukemia-susceptible guinea pigs. J Virology 3:355-357, 1969 11. Rhim JS, Vernon ML, Green I: In vitro studies on L2C leukemia: Isolation of guinea pig herpes virus from mink cells after cocultivation with leukemic cells. Fed Proc 36:2328-2332, 1977 12. Murphy SG, LoBuglio AF: L~C leukemia: A model of human acute leukemia. Fed Proc 36:2281-2285, 1977
