Five decades of progress in hematopoietic cell transplantation based on the preclinical canine model

Abstract

The preclinical canine model has proved valuable for the development of principles and techniques of hematopoietic cell transplantation (HCT) applicable to human patients. Studies in random-bred dogs concerning the impact of histocompatibility barriers on engraftment and graft-versus-host disease, the kinetics of immunological reconstitution, the efficacy of various pre-transplant conditioning regimens, post-transplantation immunosuppression protocols, treatment of malignant diseases, and graft-versus-tumor effects have advanced HCT from an investigational therapy with uncertain clinical benefit half a century ago to an important treatment choice for thousands of patients treated annually in transplantation centers worldwide. More recent preclinical canine studies have resulted in the clinical translation of nonmyeloablative, minimally invasive transplantation protocols that have extended allogeneic HCT to include older human patients with malignant and non-malignant, acquired or inherited hematological disorders, and those with comorbid conditions. Here we review the contributions of the canine model to modern HCT and describe the usefulness of HCT for the treatment of canine hematological disorders.

The Beginnings of Hematopoietic Cell Transplantation (HCT)

The history of HCT began with attempts to allay the lethal effects of irradiation that were observed in the wake of the atomic bomb explosions toward the end of World War II. It was soon recognized that bone marrow was the most radiosensitive organ in the body, and that marrow transplantation could rescue radiation victims from the lethal effects of marrow aplasia. This discovery encouraged clinicians and scientists to explore more aggressive protocols in patients with life-threatening hematological malignancies and increase the intensity of cytotoxic therapies beyond levels that were marrow toxic in hopes of eradicating the underlying cancer; rescue from the otherwise lethal marrow aplasia would be accomplished by the intravenous infusion of bone marrow cells.

In 1957, Thomas and colleagues reported studies of marrow grafting in patients with leukemia,¹ concluding after two years² that marrow transplantation was for the most part unsuccessful, with almost all patients dying from either allograft failure or recurrent leukemia. The immunological mechanisms involved in graft rejection and graft-versus-host disease (GVHD) were not well understood at that time. Overall, the early clinical results were disappointing and raised the concern that the allogeneic barrier could never be overcome. Therefore, many clinicians abandoned the idea that marrow transplantation could be used for the treatment of hematological malignancies in human patients,³ even though graft-versus-leukemia effects had already been recognized.⁴

Preclinical Studies of HCT: Murine and Canine Models

The first studies aimed at elucidating the radiation protection phenomenon were conducted in inbred mice.^5–16 Early research showed that the protection of lethally irradiated mice could be achieved not only by shielding the spleen or one femur with lead during the radiation,⁶ but also by the intra-peritoneal⁷ or intra-venous¹⁶ infusion of syngeneic spleen or bone marrow after irradiation. Jacobson and colleagues⁶ hypothesized that the mouse spleen contained a humoral factor capable of stimulating the regeneration of blood-forming tissue, thereby advancing the “humoral” theory of hematopoietic reconstitution after irradiation. As an alternative to the humoral hypothesis, Barnes and colleagues⁸ suggested that the active principle in the spleen and bone marrow suspensions might be living cells that would act at least temporarily as a tissue graft, advancing the “cellular” theory which received little support at that time. The cellular hypothesis received strong support by Main’s and Prehn’s observation⁹ in mice that marrow donor skin grafts survived indefinitely in allogenic marrow recipients. Subsequently, Trentin and colleagues⁵ showed that the skin graft tolerance was specific for the donor strain. In 1956, three independent groups^10–12 confirmed the cellular theory by showing marrow repopulation through donor cells using various blood genetic markers. Further studies in inbred mice addressed other fundamental rules of marrow transplantation biology, including the observations that the mechanisms of graft rejection and GVHD¹³ were controlled by genetic factors¹⁴ that were in turn governed by the major histocompatibility complex (MHC).¹⁵ However, in 1967, the experimental hematologist Dirk W. van Bekkum drew attention to the fact that the first clinical trials in human patients with hematological malignancies failed mainly because “the clinical applications were undertaken too soon, most of them before even the minimum of basic knowledge required to bridge the gap between mouse and patient had been obtained.”¹⁷

In the following years, scientists directed their efforts at identifying more suitable preclinical animal models. Dogs appeared especially useful owing to their random-bred nature, large body size, longer life span, wide genetic diversity, and well-mixed gene pool; they are the only mammalian species besides humans to possess these qualities.¹⁸ Further, short gestation time and large litter sizes allowed studies of donor-recipient combinations that were matched or mismatched for MHC antigens, which are known as the dog leukocyte antigen (DLA) system, simulating human leukocyte antigen (HLA) matched donor-recipient pairs. Early experiments in the canine model using DLA-matched donor-recipient pairs and postgrafting immunosuppression^19,20 renewed a sense of confidence towards treating human patients with hematological diseases by allogeneic marrow transplantation. As a consequence, in late 1960s, the results of the preclinical canine HCT studies began to be translated to patients with aplastic anemia and hematological malignancies using HLA-matched sibling marrow donors.^21,22

Not surprisingly, studies in canines have provided an excellent basis for most of the HCT principles and techniques²³ that have been directly translated over the past decades to the clinical setting for a wide spectrum of human diseases.¹⁶ Dog models of total body irradiation (TBI),^17,24–31 chemical^32–35 and radioimmunological^36,37 myeloablation, cell dose requirements,^38–41 in vivo^42–47 and in vitro^48–51 graft manipulation, engraftment and GVHD,^52–54 as well as graft-versus-tumor effects^55–57 have been essential in order to solve the existing clinical problems of human HCT.

DLA Typing

Early canine allogeneic marrow transplantation studies showed occasional long-term stable engraftment, even though most animals succumbed to problems with GVHD and graft rejection.⁵⁸ From these experiments it was concluded that major efforts would need to be directed towards understanding and defining histocompatibility barriers between donors and recipients.

The use of canine species in the experimental HCT field involved a rigorous study of the DLA system.^19,59–68 In 1968, Epstein and colleagues⁵⁹ described a serologic canine histocompatibility typing system and demonstrated that compatibility between littermate donors and recipients played a fundamental role in reducing the risks of both graft rejection and GVHD. Further, long-term donor engraftment was shown to occur in both unrelated and related recipients, which were matched either by serologic typing alone or by serology and mixed leukocyte culture non-reactivity.^19,61 Thus, the dog was the first random-bred species in which the impact of in vitro donor-recipient matching on HCT outcomes was demonstrated.¹⁹

Antigenic canine histocompatibility polymorphisms were first studied by serological^59,61 and cellular typing in mixed leukocyte culture systems.^60,61 It was not until later that the term DLA (dog leukocyte antigen) was introduced, and with it, the recognition that the histocompatibility complex could be divided into class I and class II regions. Subsequently, understanding of the molecular organization of the DLA region provided tools for molecular histocompatibility typing, which was facilitated by identification of convenient microsatellite polymorphisms within class I and class II regions that were inherited in a Mendelian fashion.⁶⁸ As a consequence, molecular assessment of DLA class I and class II microsatellite marker polymorphisms,^62,63 combined with DLA class II DRB1 allele sequencing,^65,67 enabled high resolution histocompatibility testing of canine families and rapid selection of DLA-identical donors.

Graft Collection

Initial canine HCT studies involved the use of bone marrow as the source of hematopoietic progenitor cells obtained by aspiration from the humeral and femoral bones.²³ Marrow cells stored at −80° C in dimethyl sulfoxide were capable of recovering 80% of the hematopoietic colony forming units in vitro⁶⁹ and induced hematopoietic reconstitution of lethally irradiated dogs.^20,48

Subsequent experiments in the canine model showed that either autologous^40,49,70 or allogeneic^50,51,71,72 peripheral blood stem cells could be successfully used as substitutes for marrow grafts, leading to recovery of normal hematopoiesis after otherwise lethal marrow ablation. Quantitative studies of peripheral blood mononuclear cell (PBMC) grafting demonstrated that approximately ten-fold more PBMCs than marrow cells were necessary to ensure stable engraftment.^39,40 To induce expansion and/or peripheralization of hematopoietic progenitors from the marrow space into the peripheral blood, various mobilizing agents, such as cyclophosphamide (Cy);⁴² recombinant cytokines, including recombinant canine (rc) granulocyte colony-stimulating factor (G-CSF),^43,45 rc stem cell factor (SCF, c-kit ligand),^43,45 rc granulocyte-macrophage colony-stimulating factor,⁴⁴ or the recently described CXCR4 antagonist AMD3100,⁴⁷ were evaluated in the canine model. In addition, to facilitate PBMC collection, numerous apheresis-based techniques have been adapted to canines.^{19,23,72–78}

Currently, over 90% of all autologous HCT protocols for adult human patients with hematological malignancies include the use of mobilized PBMC grafts as a source of stem cells because of higher cell yields, decreased procedural risks, faster engraftment, and shorter hospitalization compared to the use of marrow grafts. However, human patients with aplastic anemia⁷⁹ and those given grafts from unrelated donors⁸⁰ showed better outcomes after allogeneic marrow rather than PBMC grafts. Although the major disadvantage of allogeneic PBMC grafts was associated with a higher incidence of chronic GVHD,⁸¹ an advantage in survival was seen in human patients with advanced hematological cancers.⁸²

Conditioning Regimens

Conditioning regimens given before HCT serve to suppress the recipient’s immune system for graft acceptance while eradicating the underlying disease. Extensive preclinical canine studies investigating various conditioning regimens of chemotherapy, TBI, monoclonal/polyclonal antibody administration, or combinations thereof have set the stage for successful HCT trials in human patients.

Chemotherapy

In 1957, Thomas and colleagues established that irradiation itself did not eliminate leukemia in human patients; it was postulated that eradication of underlying malignancy might by possible by addition of chemotherapy.¹

Encouraging results of pre-transplant conditioning with Cy in mice and rats⁸³ prompted investigation of high-dose Cy conditioning regimens in dogs and rhesus monkeys, in place of TBI.^32,33,83 It has been shown that dogs given a single Cy dose of 100 mg/kg could be rescued from lethal immunosuppression by administration of either autologous³³ or allogeneic³² marrow grafts from DLA-matched donors, leading to sustained engraftment and mixed donor-recipient hematopoietic chimerism. High dose Cy conditioning regimens were subsequently translated to human patients with aplastic anemia undergoing allogeneic HCT.⁷⁹

Following studies with busulfan in the rat,⁸³ pre-HCT conditioning with dymethyl myleran (DMM), an intravenously injectable homologue of busulfan known for its marked myelosuppressive activities, was extensively evaluated in both healthy dogs^34,35 and in dogs with malignant lymphoma.⁸⁴ In healthy dogs, DMM given at a dose of 7.5 mg/kg induced profound marrow aplasia which was reversed by autologous marrow grafting.³⁴ In the allogeneic HCT setting, despite the achievement of a mixed chimeric state, the level of immunosuppression using DMM at a dose of 10 mg/kg as a single-agent led to successful engraftment in 50% of the dogs, while the reminder rejected their grafts and died from pancytopenia; however, more consistent engraftment was achieved when either antithymocyte serum (ATS), produced by immunization of rabbits with puppy thymocytes, or a combination of ATS and procarbazine was given in addition to DMM.³⁵ In dogs with malignant lymphoma, DMM was less effective in inducing complete remissions compared to TBI conditioning at similar marrow toxic doses.^84,85

Total body irradiation

Conditioning regimens using TBI have been extensively studied in both healthy^{17,24–31,86} and tumor-bearing dogs^{56,57,85,87–89} as part of engraftment, toxicity, and malignant disease treatment studies. It was recognized that bone marrow hematopoietic stem cell progenitors were very vulnerable and reacted uniformly to the harmful effects of irradiation. Therefore, for the treatment of lymphoid malignancies, TBI conditioning provided the advantage of eradicating non-cycling malignant cells as effectively as eradicating cycling cells.

Thomas and colleagues were the first to demonstrate prompt recovery of bone marrow and lymphoid tissues in lethally irradiated dogs given intravenous infusions of freshly isolated or cryopreserved autologous or allogeneic marrow cells.^24,86,90 All dogs that were not given a marrow infusion after a TBI dose of 400 cGy died from complications of marrow failure.²⁴

In the absence of post-grafting immunosuppression, stable allogenic DLA-identical littermate marrow grafting and full donor chimerism were consistently obtained only after conditioning with TBI doses higher than 900 cGy, delivered at a dose rate of 7 cGy/min.²⁹ However, engraftment of DLA-identical littermate marrow was consistently achieved with a single dose of 450 cGy when the exposure rate was increased to 70 cGy/min.⁹¹ Several studies investigating the impacts of dose rate on engraftment and toxicity showed that the exposure rate and total dose were the most important parameters for acute toxicity associated with TBI. It was determined that the overall TBI dose tolerated by healthy dogs given autologous marrow grafts could be increased from 10 to 14 Gy by reducing the exposure rate from 10 to 5 cGy/min. Generally, acute toxicity correlated with the TBI dose rate, while chronic toxicity was related to the total TBI dose.^26,27

In order to lower TBI-related toxicity rates seen in human patients, but to retain a strong anti-tumor effect and promote sustained engraftment, a new approach was investigated in the canine model by using higher TBI doses given in a fractionated fashion.^30,31,91 This strategy was based on the principle that hematopoietic cells were less capable of undergoing DNA repair after multiple, fractionated TBI doses compared to other tissues that would thus be affected to a lesser extent. As a consequence, long-term complications were significantly less in dogs given fractionated versus single TBI doses. When total doses of 12 to 21 Gy were given in multiple fractions of 1.5 to 2 Gy at intervals of 3 to 6 hours, with dose rates ranging from 2 to 20 cGy/min, an advantage of fractionated doses regarding acute toxicities was noted only at the highest rate of 20 cGy/min.³⁰

Other studies investigated the effect of post-TBI administration of hematopoietic growth factors on engraftment of DLA-identical littermate marrow grafts.^92,93 Post-TBI administration of recombinant human G-CSF or rcSCF significantly accelerated recovery of neutrophils and monocytes without altering platelet recovery and without significantly increasing the risks of graft failure or GVHD; however, lymphocyte recovery was more rapid in G-CSF-treated dogs. Although growth factors did not improve engraftment of DLA-identical grafts, they slightly improved survival.

Sustained engraftment of DLA-nonidentical grafts was seen only after preparative regimens with higher TBI doses than those used in the DLA-identical HCT setting. TBI doses of 1500 to 1800 cGy were generally tolerated only when given in fractions.^26,28,94,95 When a TBI dose of 1800 cGy was given in three fractions of 600 cGy delivered at 2.1 cGy/min at 48 hours intervals, the immunosuppression achieved was sufficient to allow sustained engraftment of unrelated DLA-incompatible marrow grafts.⁹⁵ Furthermore, attainment of sustained engraftment of histoincompatible marrow grafts following infusion of donor buffy coat cells or lymphocytes^19,96 was an important observation that promoted the use of PBMC grafts. Other manipulations, such as the addition of monoclonal antibodies to TBI,^97–100 have been also successful in assuring sustained engraftment of DLA-nonidentical grafts, as described below.

Monoclonal and polyclonal antibodies

The efforts to minimize TBI-related toxicities prompted the investigation of novel conditioning regimens in the canine model, including the use of monoclonal antibodies, polyclonal antibodies, or fusion peptides targeted towards specific T-cell populations or T-cell costimulatory pathways.

A beneficial effect in overcoming resistance to DLA-mismatched marrow grafts was noted in 50% of the dogs given anti-class II monoclonal antibodies in addition to TBI and post-grafting immunosuppression.⁹⁷ In the same model, the administration of S5 monoclonal antibody directed against the CD44 antigen led to sustained engraftment in 75% of the transplanted dogs.¹⁰⁰ Further canine studies explored the immunosuppressive effects of radio-labeled monoclonal antibodies, such as the ¹³¹I-labeled/S5³⁶ or bismuth-213-labeled anti-CD45¹⁰¹/anti-TCRαβ³⁷ conjugates, capable of inducing lethal marrow suppression³⁶ or selective T-cell ablation,³⁷ respectively.

A targeted nonmyeloablative strategy focused on induction of T-cell anergy was tested in dogs given DLA-identical littermate marrow grafts after conditioning with CTLA4-Ig, a fusion peptide that blocked T-cell costimulation through the B7-CD28 signaling pathway, and an otherwise inadequate TBI dose of 100 cGy; when this approach was combined with post-grafting immunosuppression by MMF and CSP, stable mixed hematopoietic chimerism was achieved in 70% of the dogs.¹⁰² These results have encouraged further efforts in the preclinical canine model towards conditioning strategies that would further reduce the TBI dosing or even eliminate the need for TBI, thereby further reducing or even avoiding both short- and long-term side effects from radiation.

Immunosuppression after HCT to Prevent GVHD

The preclinical canine model has served to investigate various post-grafting immunosuppressive regimens consisting of single drugs or combinations of immunosuppressive agents;^{52–54,102–117} these therapies might suppress the immune system, either by affecting all immune reactive tissues in general or by specifically affecting only replicating immune reactive cells. Parameters such as the clinical effectiveness, engraftment, GVHD prevention, immune reconstitution, and undesirable side effects in long-term survivors have all been used to determine whether a given immunosuppressive regimen could be safely translated from the preclinical setting to clinical protocols.

The main factors that influenced the success in crossing the immunological barriers between donor and recipient were the degree of histocompatibility matching between donor and recipient, and the efficacy of pre-transplant conditioning and post-transplant immunosuppression regimens. In both dogs and humans, even when donors and recipients were rigorously MHC-matched, GVHD prevention remained a difficult goal; marrow grafting from MHC-identical donors carried the risk of acute and/or chronic GVHD, despite the administration of post-grafting immunosuppression.^52,103 It was first recognized in dogs that GVHD was due to sensitization of donor T lymphocytes not only to disparate major, but also minor histocompatibility antigens of the recipients.²⁸ Once activated, lymphocytes attacked host tissues, predominantly the hematopoietic system, skin, gut, and liver.^52,118 Omission of immunosuppression after grafting has been associated with a high incidence of acute GVHD and adverse effects on survival.⁵⁹

Studies in unrelated DLA-nonidentical dogs have resulted in valuable strategies for GVHD prevention^{53,54,103,106–109,111,113} and treatment.^104,105 In this model, in the absence of post-grafting immunosuppression, acute and rapidly fatal GVHD occurred within 2 weeks of myeloablative HCT.⁵³ Significant delay in the onset of GVHD and prolongation of survival were obtained when postgrafting immunosuppression was given as single-agent therapy with methotrexate (MTX),¹⁰³ cyclosporine (CSP),¹⁰⁶ azathioprine,⁵³ succinylacetone,¹⁰⁸ FK-506 (tacrolimus),¹⁰⁹ or as combinations of these agents.^{107,109,111,119} Based on canine studies, post-HCT immunosuppression regimens consisting of either MTX, CSP, or a brief course of MTX combined with either CSP or FK506, were successfully translated to human patients for GVHD prophylaxis; antithymocyte globulin (ATG) was used to treat acute GVHD once it was established.^52,54 Furthermore, it was found that postgrafting immunosuppression could be discontinued generally after 3–6 months of treatment due to instauration of mutual graft-versus-host tolerance.

Clear evidence of therapeutic graft-versus-tumor effects^1,56 mediated by allogeneic effector T-cells that destroy recipient’s tumor cells after recognizing and reacting to disparate tumor-associated antigens, prompted the exploration in the canine model of less aggressive, nonmyeloablative HCT regimens or so called “minitransplants”. These regimens relied solely upon host immunosuppression to facilitate stable mixed chimerism, prevention of both allograft rejection and GVHD, and eradication of hematological malignant disease via a graft-versus-tumor response, following sublethal, minimal-invasive TBI conditioning.^102,110,115 Mycophenolate mofetil (MMF),¹¹⁷ a purine synthesis inhibitor, was used in combination with other immunosuppressive drugs,⁵⁴ leading to reduced frequency and severity of acute allograft rejection and improved survival in dogs given either DLA-identical^102,110,112 or unrelated DLA-nonidentical^53,111 marrow grafts. In the DLA-identical model, pre-transplant conditioning with a nonmyeloablative dose of 200 cGy TBI and post-HCT immunosuppression with MMF and CSP induced stable mixed chimerism and prevented both allograft rejection and GVHD.¹¹⁰ This protocol has been successfully translated to human patients who were not eligible for conventional myeloablative transplantation due to advanced age or comorbidities.¹²⁰

The Impact of Preceding Blood Transfusions on HCT Outcomes

Early canine HCT studies drew attention to the fact that transfusions given before marrow grafts might jeopardize engraftment; it was hypothesized that this phenomenon might be associated to sensitization of recipients to non-DLA-associated polymorphic minor histocompatibility antigens.¹²¹ It was furthermore shown that DLA-identical recipients that received either three whole blood transfusions from the marrow donor or nine whole blood unrelated transfusions before TBI conditioning rejected 100% and 40%, respectively, of the marrow grafts.²⁸ These studies demonstrated that several minor polymorphic loci, which were undetected by the usual in vitro histocompatibility typing, were involved in the occurrence of transfusion-induced sensitization. It was almost two decades later when the sensitizing cells responsible for transfusion-associated graft rejection were identified as being dendritic cells contained in the transfusion product.¹²² These observations prompted the exploration of treatments designed to eliminate or inactivate the cells responsible for the induction of this phenomenon. The incidence of graft rejection was lessened by reducing antigen-presenting mononuclear cells through the use of buffy coat-poor blood transfusion products; transfusion-induced sensitization was successfully overcome by using a combination of an alkylating agent, procarbazine, and ATS as pre-HCT conditioning, or prevented by in vitro treatment of blood transfusions with ultraviolet light or 2000 cGy gamma radiation.^28,123,124 These findings were then translated into the clinic, leading to improved management of the multiply-transfused patients with aplastic anemia or other nonmalignant diseases who were candidates for marrow transplantation.^125–127

Hematopoietic Reconstitution and Side Effects after HCT

Hematopoietic reconstitution

Following myeloablative HCT, granulocyte counts achieved normal levels approximately by days 12; during the early post-irradiation period, dogs might require whole blood or platelet transfusions. However, following nonmyelablative HCT, life threatening declines of peripheral blood cell counts generally did not occur.¹¹⁰ Although dogs with successful engraftment were profoundly immunodeficient for 200 to 300 days after myeloablative HCT, long-term survivors recovered their immune function and were not susceptible to increased incidences of infection.¹²⁸

Conditioning regimen-induced side effects

The main long-term side effects after high-dose TBI conditioning in dogs were pancreatic insufficiency and atrophy leading to maldigestion and malnutrition, keratitis, pneumonitis, change in coat color, cataracts, and sterility; in addition, a five-fold increased incidence of spontaneous carcinomas and sarcomas was reported. These findings were not seen in a smaller number of dogs conditioned with either Cy or busulfan.¹²⁹

Acute side effects were associated to Cy administration, including anorexia, hematuria, vomiting, and diarrhea. Based on long-term surveillance for more than ten years after HCT, canine recipients conditioned with Cy regained fertility and sired normal litters.¹³⁰

Side effects induced by immunosuppression after HCT

The limiting toxicity of MTX in dogs was gastrointestinal, as evidenced by diarrhea and vomiting; however, mouth ulceration or so-called mucositis, which is a major side effect in human patients, was rarely seen in dogs. The side effects associated to MMF administration in dogs were gastrointestinal, consisting mainly of diarrhea.¹³¹

The administration of calcineurin inhibitors in dogs was also associated with gastrointestinal side effects, including an increased incidence of intussusception. CSP also caused liver and kidney function changes, although these appeared to be less common in dogs than in humans at therapeutic levels. Following long-term CSP administration, dogs exhibited problems with papilloma infection; in addition, dogs presented changes in the skin and gums, as well as increased hair growth and blood pressure, all of which were reversible upon discontinuation of CSP administration.¹³² In order to reduce toxicities, CSP blood levels should be monitored and the dosing adjusted to keep levels within the therapeutic range.

HCT for Canine Malignant Diseases

Another advantage of the dog model consisted of the availability of animals with spontaneous malignant diseases, resembling closely those found in humans. In 1974, Weiden and colleagues established that dogs with malignant lymphoma had significant impairments of the humoral and/or cellular immune reactivity, similar to those seen in human patients.¹³³ In addition, it was shown that canine malignant lymphoma was responsive to combination chemotherapy.¹³⁴ Many other malignant conditions of the dog have well recognized counterparts in humans.¹³⁵ These observations heightened the value of the canine cancer model in the investigational setting of HCT, including graft-versus-tumor effects, in a randomly bred species.

The attempts to cure spontaneous canine malignancies by marrow transplantation were pioneered by the Seattle marrow transplantation group.^{55–57,84,85,87–89,136}. The dogs involved into those studies were referred by practicing veterinarians with the consent of the dogs’ owners and the survivors returned to their owners at the completion of the studies. Autologous grafts were given experimentally in dogs with malignant lymphoma,^{57,84,85,87–89,136} leukemia,⁸⁵ and solid tumors;⁸⁵ the latter included mammary gland, adenocarcinoma, squamous cell carcinoma, undifferentiated carcinoma, mastocytoma, leiomyosarcoma, osteosarcoma, and melanoma. These early preclinical trials investigated different conditioning regimens consisting of either TBI,⁸⁵ chemotherapy,⁸⁴ or combinations thereof.^{57,87–89,136} Studies showed that 25% of the dogs with malignant lymphoma in chemotherapy-induced remission given high doses of TBI, and either autologous cryopreserved marrow^57,85,87 or freshly isolated PBMC¹³⁶ grafts, became disease-free long-term survivors; however, the solid tumor-bearing dogs showed little antitumor responses, consistent with the known greater radioresistance of solid non-hematological tumors.⁸⁵

Three important allogeneic marrow transplantation studies were carried out by the Seattle transplantation group in dogs with malignant lymphoma,^55–57 leukemia,⁵⁶ and miscellaneous solid tumors.^55,56 The graft-versus-lymphoma effect of allogeneic grafts was demonstrated by prolonged disease-free survivals (p < 0.005) after allogeneic compared to autologous HCT using similar conditioning regimens.⁵⁶ However, allogeneic grafts were complicated by high rates of fatal GVHD⁵⁵ and toxicities related to postgrafting immunosuppression.⁵⁷ No suggestion of significant graft-versus-tumor effects in dogs with non-hematological solid tumors was obtained, and all transplanted dogs died with evidence of persistent tumors.⁵⁶

Overall, it was established that the mortality rate in dogs with hematological malignancies given either autologous or allogeneic HCT was adversely affected by a compromised clinical status at the time of transplantation.^56,85 From these studies it was predicted that HCT could be more effective if performed in patients not only with minimal tumor burden, but before deterioration of their general condition; clinical translation of these observations resulted in improved transplantation outcomes.¹³⁷

HCT for Canine Nonmalignant Diseases

The therapeutic potential of allogeneic DLA-identical HCT has been investigated for a wide range of canine non-malignant hematological and non-hematological disorders, such as congenital immunological and enzymatic deficiencies, and Duchenne muscular dystrophy.

Allogeneic marrow grafts from DLA-identical littermates corrected cyclic neutropenia (Grey Collie disease)¹³⁸ and X-linked severe combined immunodeficiency¹³⁹ with full reconstitution of neutrophil, and T and B cell functions, respectively. Allogeneic HCT studies were also performed in Basenji dogs with hemolytic anemia due to pyruvate kinase deficiency; these studies demonstrated reversal of iron overload after successful transplantation¹⁴⁰ and determined the level of donor chimerism needed to prevent hemolysis.^141,142 Using DLA-identical marrow grafts for the treatment of lysosomal storage diseases, such as α-L-iduronidase deficiency,¹⁴³ mucopolysaccharidosis I,¹⁴⁴ and fucosidase deficiency,¹⁴⁵ the disease-related pathologies were partially corrected. However, the outcomes in dogs with ceroid lipofuscinosis (Batten’s disease),¹⁴⁶ GM1 gangliosidosis,¹⁴⁷ hemophilia,¹⁴⁸ and Duchenne muscular dystrophy¹⁴⁹ were not improved following allogeneic HCT. For the treatment of Duchenne muscular dystrophy, stable hematopoietic chimerism induced tolerance in the immune system of recipients to both dystrophin and cells from HCT donors, thereby setting the stage for ongoing investigations of additional therapeutic strategies such as gene and muscle stem cell therapies.

HCT as a Therapy for Pet Dogs with Hematological Malignancies

Cancer is one of the leading causes of death in dogs, showing a one-log higher incidence than in humans.¹⁵⁰ Among canine cancers, hematological malignancies are rarely curable by radio/chemotherapy.¹⁵¹ Although more than 90% response rates were achievable after various combination chemotherapy regimens,¹⁵² remissions were short due to the development of multi-drug resistance and recurrence of disseminated disease.¹⁵¹ Other therapeutic alternatives for canine malignant lymphoma, such as the use of high dose half body irradiation given either alone¹⁵³ or after preceding remission inductions with chemotherapy,¹⁵⁴ led to measurable decreases in tumors size; however, besides radiation-induced side effects, the median remission durations were not significantly prolonged compared to those achieved following conventional combination chemotherapy.¹⁵⁴

The preclinical research developed in dogs and summarized in this review has the potential for improving cancer therapies in pet dogs. Hematopoietic stem cell transplantation can cure canine malignant hematological conditions that have been considered fatal.^{57,87,136,155,156} Molecular DLA typing methods offer a high-resolution technology for donor selection among single- and multi-generation canine families,^63,65,156 which is a pre-requisite for lower GVHD rates and improved transplantation outcomes. The canine genome map^18,64,157 has contributed to identification of new microsatellite repeat sequences that have extended the panel of markers for evaluation of donor chimerism.¹⁵⁸ The availability of safe PBMC mobilizing agents and apheresis devices for dogs offers a minimally invasive alternative for graft collection compared to the traditional bone marrow harvest. Less toxic nonmyeloablative HCT regimens have been recently developed in the canine model, which permit graft-versus-tumor effects while controlling GVHD, and which could be applied to veterinary patients.

The continuous advances in veterinary medical oncology, radiation oncology, and transfusion medicine, the expansion of canine blood bank networks, and rapid progress in canine immunogenetics, all promise to improve HCT-based therapies for canine patients with cancer. In the clinical veterinary field, the treatment of canine malignant lymphoma in chemotherapy-induced remission by autologous marrow transplantation has already been reported.¹⁵⁵ We have recently described the successful treatment of a pet dog with malignant T-cell lymphoma in chemotherapy-induced remission by TBI and allogeneic rcG-CSF-mobilized PBMC transplantation.¹⁵⁶

From Past to Present and Future

As a consequence of the numerous experimental studies in random-bred dogs, HCT has advanced from an investigational therapy that was declared without perspective in the 1960s, to an important treatment option that produces amazing clinical results. In 1990, Dr. Donnall E. Thomas was awarded the Nobel Prize in Medicine for his pioneering work in marrow transplantation. He emphasized that “marrow grafting could not have reached clinical application without animal research, first in inbred rodents and then in outbred species, particularly the dog.”¹⁵⁹

The efforts continue in the preclinical canine model to improve the HCT regimens for direct translation to human patients. Research has been directed towards designing specific antibodies against costimulatory molecules with key roles in the development of the immunological reactions after allogeneic HCT.¹⁶⁰ Potential antileukemic effects of donor-derived natural killer cells¹⁶¹ are currently explored in the nonmyeloablative HCT setting. The observation that in some human patients graft-versus-tumor effects are associated with GVHD, whereas in others remission can be achieved without GVHD, has led to investigation of strategies to separate graft-versus-tumor effects from GVHD through adoptive transfer of selected T cell populations.¹⁶² The use of adoptive immunotherapy could make allogeneic HCT more effective to recognize tumor-specific antigens¹⁶³ and perhaps extend its application to the treatment of metastatic and non-hematopoietic tumors that arise in organs such as the breast, prostate, pancreas, or colon. In this regard, dogs with spontaneous tumors could become ideal candidates for preclinical trials of anti-tumor vaccination. Furthermore, research involving the recently recognized immune regulatory T cells that arise after HCT could lead to a better understanding of mechanisms of graft rejection and GVHD, and improve strategies to overcome these immunological barriers.¹⁶² Promising preclinical results of kidney grafting in a canine mixed hematopoietic chimera model¹⁶⁴ suggest that HCT may serve in the future as an immunologic platform for solid organ grafting without the need for lifelong immunosuppression. Finally, the approach of transferring cloned genes into the hematopoietic stem cells to correct specific genetic defects may be a promising avenue for the therapy of genetic diseases.¹⁶⁵

Not only in human history but also in the history of medicine, the dog has been loyal to human beings. It is now well recognized in the HCT field that the dog has contributed to a legacy that saves thousands of patients annually, proving once again the paradigm of “man’s best friend”. The remarkable progress in the clinical applications of preclinical HCT protocols established in canines, and the rapid development of the biological sciences predict sustained advances over the coming years in both the human and veterinary oncology fields.