A 7-year-old neutered male Golden Retriever that weighed 37.5 kg (82.5 lb) was clinically evaluated at Bellingham Veterinary & Critical Care in February 2004. The dog had a 20-day history of anorexia, lethargy, and vomiting and had 10% weight loss during the preceding 2 months.
Clinical examination revealed an enlarged spleen without peripheral lymphadenopathy. Hematologic examination revealed pancytopenia and mild anemia. No atypical cells were identified in a blood specimen. Ionized and total serum calcium concentrations were high (1.82 mmol/L and 14.9 mg/dL, respectively), and parathyroid hormone concentration (1.5 pmol/L) was low. In addition, parathyroid hormone–related protein concentration was slightly high (1.2 pmol/L). Serum alkaline phosphatase activity (205 U/L) was high, as was BUN concentration (32.1 mg/dL). Thoracic and abdominal radiography confirmed splenomegaly. Magnetic resonance imaginga revealed an enlarged mesenteric lymph node and generalized homogeneously increased signals from the bone marrow of the ilium and vertebral bodies. On the basis of these findings, splenectomy was performed, followed by mesenteric lymph node, liver, kidney, duodenum, and skin biopsies. In addition, bone marrow biopsy specimens were obtained from the iliac crest.
Histologic examination of the spleen and biopsy specimens confirmed multicentric lymphoma in the spleen, mesenteric lymph node, and bone marrow. Monomorphic, medium to large lymphocytes with irregular nuclear membrane contours expanded the periarterial lymphoid sheaths of the splenic white pulp and encroached on the follicular areas. The atypical cells invaded the splenic red pulp and trabeculae multifocally. Extramedullary hemopoiesis was also observed in the splenic red pulp. Infiltration of similar monomorphic lymphocytes expanded the paracortical regions of the mesenteric lymph node, compressed lymphoid follicular regions, and invaded lymph node trabeculae, but spared the capsule. In this location, the atypical lymphocytes included up to 6 mitotic figures/hpf (40 × objective). The bone marrow contained isolated nests of atypical lymphocytes. Histologic examination of liver, kidney, duodenum, and skin did not reveal involvement by lymphoma.
Immunophenotyping of the splenic and mesenteric lymph node infiltrates confirmed that the lymphoma was of T-cell origin (CD3+). Genomic DNA was extracted from paraffin-embedded sections of the mesenteric lymph node and spleen for T-cell (T-cell receptor γ locus) and Bcell antigen receptor rearrangement (immunoglobulin heavy chain locus) analysis.1 Duplicate PCR reactions were performed for each tissue analyzed and revealed consistent clonal T-cell receptor γ rearrangements in the spleen and mesenteric lymph node. Clonal immunoglobulin heavy chain rearrangements were not observed. The morphologic, immunohistochemical, and molecular clonality results confirmed that the atypical lymphoid infiltrates consisted of a clonal T-cell population. These findings, combined with the clinical signs, were consistent with a stage V (b) T-cell malignant lymphoma, according with the World Health Organization Staging System of Canine Lymphoma.2
Induction of remission was undertaken by administration of cyclophosphamide (50 mg/m2, PO, q 48 h), vincristine (0.56 mg/m2, IV, weekly), cytosine arabinoside (100 mg/m2, SC, q 12 h), and prednisone (50 mg/m2, PO, q 24 h; COAP protocol).3
After 2 weeks of COAP chemotherapy, the dog developed pancytopenia, anemia, anorexia, and acute bilateral pyoderma on the forelimbs, and transfusion support was initiated by use of dog erythrocyte antigen 1.1-negative compatible packed RBCs. In an attempt to prevent sepsis and bleeding, chemotherapy was discontinued and filgastrimb (5 μg/kg [2.3 μg/lb], SC, q 12 h) was administered for 5 days. Chemotherapy was then reinstituted with cyclophosphamide (50 mg/m2, PO, once) and prednisone (20 mg/m2, PO, q 48 h), and a 2-month remission was achieved.
Relapse was made evident by the appearance of lymphoblasts (2%) with high nuclear-to-cytoplasmic ratio in the blood, accompanied by increased serum calcium concentration (14.1 mg/dL). In addition, the dog developed anemia; lymphocytopenia (280 cells/μL); azotemia (BUN, 52 mg/dL); and increased serum activities of alkaline phosphatase (435 U/L), alanine aminotransferase (110 U/L), and aspartate aminotransferase (64 U/L). A second remission induction with cyclophosphamide (50 mg/m2, PO) and prednisone (20 mg/m2, PO, q 48 h) was attempted; however, after a single dose of cyclophosphamide, whole-blood transfusion was required for severe thrombocytopenia. One week later, a single dose of Escherichia coli L-asparaginase (20,000 U/m2, IM) was given. A second remission was achieved; atypical lymphocytes disappeared from the blood, and hypercalcemia resolved.
To find a suitable HCT donor, DLA typing of 29 family members from 4 generations of dogs located in 3 countries was performed at the Fred Hutchinson Cancer Research Center. Blood samples from each dog were obtained for DNA extraction via standard protocols.4 The DNA samples were tested for highly polymorphic microsatellite markers5,6 in the DLA class I (FH 2200) and DLA class II (FH 2202) regions by use of a variable number tandem repeat-PCR method5 and specific 2200 and 2202 primer pairs.7,8,c Results were analyzed by use of electrophoresis on 6% polyacrylamide gels prepared with a sequencing system kit.d Interpretation of the banding patterns suggested that 5 family members were DLA matched for both DLA class I and class II regions with the affected dog. For confirmation, the DLA class II DRB1 hypervariable region4,9 was amplified by use of an SSCP-PCR method10,11 and specific IC and ID primers.11,c Results were analyzed by electrophoresis on 0.5% mutation detection enhancement gelse that revealed identical banding patterns for those dogs identified as matched by segregation analysis of microsatellite polymorphisms. The interpretation of DRB1 allele banding patterns was not sufficient for histocompatibility assessment because different DRB1 alleles could have the same size and identical banding patterns. Therefore, DRB1 allele sequencing was performed by use of a genetic analyzerf to identify potential allelic disparities on the basis of nucleotide alignments. Among the 30 dogs from which sequences were analyzed with the software,g 3 DRB1 alleles were identified: alleles 6, 15, and 30 (Figure 1). The affected dog shared the same DLA-DRB1 allelic sequence profile with the 5 dogs identified to be matched on the basis of the segregation analysis of microsatellite polymorphisms and SSCP gel banding patterns. Among the DLA-matched relatives, a 13-month-old male that weighed 29.5 kg (65 lb) was selected as the HCT donor. The donor's blood group was DEA1.1 negative.
Four-generation genealogy and genotyping profiles of family members of a dog with T-cell lymphoma receiving an HCT from a DLA-matched related donor. A—Affected dog's dam's first litter. B—Affected dog's dam's second litter. 1-30 = Dogs for which blood samples were evaluated for highly polymorphic microsatellite markers within DLA class I and class II regions, and DLA class II DRB1 allele sequencing. A3 = Affected dog. A15, A17, A21, B29, and B30 = Dogs matched with the affected dog on the basis of results of segregation analysis of the polymorphisms and DRB1 allele sequencing. A17 = Dog selected as donor.
Citation: Journal of the American Veterinary Medical Association 228, 5; 10.2460/javma.228.5.728
The HCT was performed 18 weeks after diagnosis and 1 month after the second induction chemotherapy, while the dog was still in remission. Two weeks before transplantation, 240 mL of blood was collected from the intended donor and stored for the purpose of priming the apheresis machine. To avoid transmission of any potential blood-borne pathogens to the recipient, the donor was screened for Dirofilaria immitis, Ehrlichia canis, Ehrlichia equi, Babesia canis, Borrelia burgdorferi, and Rickettsia rickettsi.
For mobilization of PBMCs, the donor was given recombinant canine granulocyte colony–stimulating factorh (5 μg/kg [2.3 μg/lb], SC, q 12 h, for 5 consecutive days). Leukapheresis was performed with a 12-F, 20-cm dual-lumen central venous catheter12,i and a continuous flow blood separator,j with acid citrate dextrose solution as an anticoagulant. The PBMC collection lasted 4 hours with the donor under general anesthesia while body temperature, respiratory rate, and pulse rate were monitored. Electrolytes and the hematologic profile were evaluated every hour by use of a portable clinical analyzer.k To avoid anticoagulant-induced calcium depletion, 10% calcium gluconate was administered throughout the apheresis procedure via IV pump injection, at 10 mL/h. The donor's body temperature was maintained by use of microwave heating pads. The apheresis product contained 92 × 103 nucleated cells/μL and 0.36 × 103 CD34+ cells/μL. The CD34+ cell count was analyzed via flow cytometry by use of a canine anti-CD34 biotin–conjugated monoclonal antibody.13,l The affected dog was given 2 fractions of 4 Gy total body irradiation with an interfraction interval of 3 hours, delivered at 7 cGy/min from a 4-MV clinical linear accelerator.m Immediately after irradiation, the dog was given an IV infusion of 440 mL of freshly isolated PBMCs containing 3.6 × 106 CD34+ cells/kg (1.6 × 106 CD34+ cells/lb; Figure 2).
Myeloablative HCT schema of a dog with T-cell lymphoma. *Days before or after HCT. CSP = Cyclosporine. D = Donor. rcG-CSF = Recombinant canine granulocyte colony–stimulating factor. G-PBMC = rcG-CSF mobilized PBMCs. R = Recipient. TBI = Total body irradiation.
Citation: Journal of the American Veterinary Medical Association 228, 5; 10.2460/javma.228.5.728
Post-HCT immunosuppression was attained by administration of cyclosporine (5 mg/kg, PO, q 12 h), from the day before HCT until day 35 after HCT. Cyclosporine blood concentration was evaluated twice weekly by use of a monoclonal whole-blood assay.n To achieve therapeutic concentrations ranging from 400 to 600 ng/mL, as measured by the monoclonal whole-blood assay, the cyclosporine dose was adjusted to 4.5 to 5.5 mg/kg (9.9 to 12.1 mg/lb). The CBC was assessed daily until the WBC concentration exceeded 1,000 cells/μL and platelet concentration exceeded 30,000 platelets/μL, when frequency of CBC was reduced to once per week. Serum biochemical analyses were performed daily to monitor liver and kidney functions.
Supportive treatment after HCT included administration of lactated Ringer's solution (13 mL/kg [5.9 mL/lb], SC, q 12 h) and 20% fat emulsion° in lactated Ringer's solution (6.5 mL/kg/h [3 mL/lb/h], continuous rate infusion) administered via IV catheter, which was maintained for 4 days after HCT; thereafter, the dog was given nutrition PO. Antimicrobials, consisting of neomycin sulfate (6 mg/kg [2.7 mg/lb], PO, q 8 h), polymyxin B sulfate (25,000 U/kg/d [11,364 U/lb/d], PO, q 8 h), and enrofloxacin (10 mg/kg [4.5 mg/lb], SC, q 24 h), were administered until the WBC concentration exceeded 1,000 cells/μL. Lactobacillus GGp (80 mg, PO, q 12 h) was administered as a balanced probiotic. During the first week after HCT, the dog lost 12% of baseline body weight, all of which was regained during the following month. On day 14 after HCT and hospitalization, the dog was released to its owners, with continuation of administration of the cyclosporine, enrofloxacin, and Lactobacillus GG.
Hematopoietic engraftment was evident as manifested by sustained increases in granulocyte and platelet counts after the postradiation nadir and detection of donor cells in the peripheral blood by use of microsatellite marker polymorphism analyses.8,14 Granulocyte count was within reference range by day 10 and platelet count by day 22 (Figure 3), and the dog did not require transfusion. For analysis of donor engraftment, granulocyte and lymphocyte fractions were separated from the recipient's blood by use of a gradient method, and genomic DNA was extracted via standard protocols.4 A PCR-based assay was performed with a 2001 primer pair.8,14,c The donor contribution to recipient hematopoiesis was quantified by estimating the proportion of donor-specific DNA among host DNA after autoradiography. This revealed mixed hematopoietic chimerism by week 2 after transplantation and, subsequently, progression to full donor chimerism in the granulocyte and lymphocyte fractions, which has been maintained for at least 58 weeks (Figure 4).
Blood concentrations of WBCs and platelets after HCT in a dog with T-cell lymphoma.
Citation: Journal of the American Veterinary Medical Association 228, 5; 10.2460/javma.228.5.728
Chimerism analyses of blood from a dog with T-cell lymphoma receiving an HCT from a DLA-matched related donor. DSB = Donor specific bands. RSB = Recipient-specific bands.
Citation: Journal of the American Veterinary Medical Association 228, 5; 10.2460/javma.228.5.728
On day 55 after HCT, serum activities of alanine aminotransferase (233 U/L) and aspartate aminotransferase (106 U/L) increased. At the same time, an erythematous rash developed, affecting the plantar region of the paws, periocular and perioral regions, dorsal and lateral nasal skin, pinnae of the ears, abdomen, and dorsal region of both forelimbs. Histologic examination of multiple skin biopsy specimens revealed diffuse lymphocytic infiltrates and apoptotic cells in the follicular epithelium and occasionally in the glandular epithelium, confirming graft versus host disease. Treatment was initiated with cyclosporine (7.5 mg/kg [3.4 mg/lb], PO, q 12 h) and enrofloxacin (10 mg/kg, SC, q 24 h). The skin lesions improved after 20 days of cyclosporine administration, which was continued at the same dose for 5 months and then gradually tapered by 5%/wk until month 13 after HCT, when it was discontinued. While receiving cyclosporine, the dog was without clinical signs with the exception of sporadic vomiting and diarrhea without weight loss. Clonal rearrangement studies of blood T-cell antigen receptor genes1 and serum thymidine kinase assays15 performed after HCT have revealed no evidence of residual lymphoma.
Discussion
Systemic chemotherapy is presently the treatment of choice for canine lymphoma, with remission rates approaching 58% to 96%.2 However, most dogs succumb to recurrence of their disease, typically by 12 months after diagnosis16 because of development of multidrug resistance. When the COAP protocol alone is used for the treatment of dogs with lymphoma, a median survival time of 25 weeks has been reported.3
The canine species has served as an excellent random-bred preclinical model for human HCT,17–19 providing a valuable basis for many of the principles and techniques of transplantation. Increasing knowledge of the molecular immunogenetics of the canine major histocompatibility complex has provided unique tools for rapid identification of suitable DLA-matched family members, which could serve as hematopoietic donors.4–6,8,10,11,20–23 Treatment of spontaneous lymphoma in dogs with a high dose of total body irradiation and chemotherapy followed by autologous24–30 or allogeneic30–32 marrow transplantation has been reported. Among dogs in chemotherapy-induced remission that were given autologous marrow grafts, approximately 25% became disease-free long-term survivors.26,29,30 However, allogeneic grafts were complicated by high rates of fatal graft versus host disease31 and other complications, although there was evidence of beneficial graft versus lymphoma effects.33 Since those early studies, outcomes of allogeneic HCT have improved, in part because of improved donor-recipient selection by use of molecular DLA typing5,11 and in part because of better prevention of graft versus host disease,34,35 better supportive care, and substitution of PBMCs for bone marrow.36 The dog described here has remained in remission for more than 15 months after HCT and 19 months since the initial diagnosis and has been thriving, with complete donor chimerism.
| COAP | Cyclophosphamide, vincristine, cytosine arabinoside, prednisone |
| DLA | Dog leukocyte antigen |
| SSCP | Single-stranded conformational polymorphism |
| HCT | Hematopoietic cell transplantation |
| PBMC | Peripheral blood mononuclear cell |
Northwest Radiologists, Bellingham, Wash.
Neupogen, Amgen, Thousand Oaks, Calif.
Invitrogen Corp, Carlsbad, Calif.
Ultra Pure Sequagel Sequencing System Kit, National Diagnostics, Atlanta, Ga.
MDE Gel Solution, Cambrex Bio Science Rockland Inc, Rockland, Me.
ABI PRISM 3100, Applied Biosystems, Foster City, Calif.
SeqEd, Applied Biosystems, San Jose, Calif.
Supplied by Amgen Corp, Thousand Oaks, Calif.
Arrow International, Reading, Pa.
COBE Spectra apheresis system, Gambro BCT, Lakewood, Colo.
I-STAT CG8+, I-STAT Corp, East Windsor, NJ.
1H6 canine anti-CD34 antibody, Biologics Facility, Fred Hutchinson Cancer Research Center, Seattle, Wash.
Clinac 4/80, Varian Associates, Palo Alto, Calif.
Abbot Laboratories, Abbot Park, Ill.
Liposyn, Intralipid 20%, Baxter Healthcare Corp, Clintec Nutrition Division, Deerfield, Ill.
Culturelle, ConAgra Foods, Omaha, Neb.
References
- 1↑
Burnett RC, Vernau W, Modiano JF, et al. Diagnosis of canine lymphoid neoplasia using clonal rearrangements of antigen receptor genes. Vet Pathol 2003; 40: 32–41.
- 2↑
Vonderhaar MA, Morrison WB. Lymphosarcoma. In: Cancer in dogs and cats: medical and surgical management. Baltimore: The Williams & Wilkins Co, 1998; 667–695.
- 3↑
Kitchell BE, Dhaliwal RS. Section 6. Hematology, oncology and immunology. CVT update: anti cancer drugs and protocols using traditional drugs. In: Bonagura JD, ed. Kirk's current veterinary therapy XIII: small animal practice. Philadelphia: WB Saunders Co, 1995; 465–473.
- 4↑
Wagner JL, Burnett RC, Works JD, et al. Molecular analysis of DLA-DRBB1 polymorphism. Tissue Antigens 1996; 48: 554–561.
- 5↑
Wagner JL, Burnett RC, DeRose SA, et al. Histocompatibility testing of dog families with highly polymorphic microsatellite markers. Transplantation 1996; 62: 876–877.
- 6
Burnett RC, Francisco LV, DeRose SA, et al. Identification and characterization of a highly polymorphic microsatellite marker within the canine MHC class I region. Mamm Genome 1995; 6: 684–685.
- 7
Mellersh CS, Langston AA, Acland GM, et al. A linkage map of the canine genome. Genomics 1997; 46: 326–336.
- 8
Francisco LV, Langston AA, Mellersh CS, et al. A class of highly polymorphic tetranucleotide repeats for canine genetic mapping. Mamm Genome 1996; 7: 359–362.
- 9
Kennedy LJ, Carter SD, Barnes A, et al. Interbreed variation of DLA-DRB1, DQA1 alleles and haplotypes in the dog. Vet Immunol Immunopathol 1999; 69: 101–111.
- 10
Wagner JL, Creer SA, Storb R. Dog class I gene DLA-88 histocompatibility typing by PCR-SSCP and sequencing (brief communication). Tissue Antigens 2000; 55: 564–567.
- 11↑
Wagner JL, Works JD, Storb R. DLA-DRB1 and DLA-DQB1 histocompatibility typing by PCR-SSCP and sequencing (brief communication). Tissue Antigens 1998; 52: 397–401.
- 12↑
Lee R, Storb R, Little M-T, et al. Percutaneous central duallumen catheter for apheresis in the canine. J Invest Surg 2002; 15: 337–341.
- 13↑
McSweeney PA, Rouleau KA, Wallace PM, et al. Characterization of monoclonal antibodies that recognize canine CD34. Blood 1998; 91: 1977–1986.
- 14
Yu C, Ostrander E, Bryant E, et al. Use of (CA)n polymorphisms to determine the origin of blood cells after allogeneic canine marrow grafting. Transplantation 1994; 58: 701–706.
- 15↑
von Euler H, Einarsson R, Olsson U, et al. Serum thymidine kinase activity in dogs with malignant lymphoma: a potent marker for prognosis and monitoring the disease. J Vet Intern Med 2004; 18: 696–702.
- 16↑
Kraegel SA. Appendices, appendix A: cancer-lymphoma. In: Ettinger SJ, Feldman EC, eds. Textbook of veterinary internal medicine: diseases of the dog and cat. Vol. 2. 5th ed. Philadelphia: WB Saunders Co, 2000; 1923.
- 17
Deeg HJ, Storb R. Bone marrow transplantation in dogs. In: Makowka L, Cramer DV, Podesta LG, eds. Handbook of animal models in transplantation research. Boca Raton, Fla: CRC Press Inc, 1994; 255–285.
- 18
Wagner JL, Storb R. Preclinical large animal models for hematopoietic stem cell transplantation. Curr Opin Hematol 1996; 3: 410–415.
- 19
Thomas ED, Storb R. The development of the scientific foundation of hematopoietic cell transplantation based on animal and human studies. In: Thomas ED, Blume KG, Forman SJ, eds. Hematopoietic cell transplantation. 2nd Ed. Boston: Blackwell Science, 1999; 1–11.
- 20
Vriesendorp HM, Westbroek DL, D'Amaro J, et al. Joint report of 1st International Workshop on Canine Immunogenetics. Tissue Antigens 1973; 3: 145–163.
- 21
Sarmiento UM, Storb RF. Characterization of class II alpha genes and DLA-D region allelic associations in the dog. Tissue Antigens 1988; 32: 224–234.
- 22
Burnett RC, DeRose SA, Wagner JL, et al. Molecular analysis of six dog leukocyte antigen class I sequences including three complete genes, two truncated genes, and one full-length processed gene. Tissue Antigens 1997; 49: 484–495.
- 23
Graumann MB, DeRose SA, Ostrander EA, et al. Polymorphism analysis of four canine MHC class I genes. Tissue Antigens 1998; 51: 374–381.
- 24
Weiden PL, Storb R, Lerner KG, et al. Treatment of canine malignancies by 1200 R total body irradiation and autologous marrow grafts. Exp Hematol 1975; 3: 124–134.
- 25
Weiden PL, Storb R, Shulman H, et al. Dimethyl myleran and autologous marrow grafting for the treatment of spontaneous canine lymphoma. Eur J Cancer Clin Oncol 1977; 13: 1411–1415.
- 26
Weiden PL, Storb R, Deeg HJ, et al. Prolonged disease-free survival in dogs with lymphoma after total-body irradiation and autologous marrow transplantation consolidation of combinationchemotherapy-induced remissions. Blood 1979; 54: 1039–1049.
- 27
Weiden PL, Storb R, Deeg HJ, et al. Total body irradiation and autologous marrow transplantation as consolidation therapy for spontaneous canine lymphoma in remission. Exp Hematol 1979; 7(suppl5): 160–163.
- 28
Deeg HJ, Appelbaum FR, Weiden PL, et al. Autologous marrow transplantation as consolidation therapy for canine lymphoma: efficacy and toxicity of various regimens of total body irradiation. Am J Vet Res 1985; 46: 2016–2018.
- 29
Appelbaum FR, Deeg HJ, Storb R, et al. Cure of malignant lymphoma in dogs with peripheral blood stem cell transplantation. Transplantation 1986; 42: 19–22.
- 30
Appelbaum FR, Deeg HJ, Storb R, et al. Marrow transplant studies in dogs with malignant lymphoma. Transplantation 1985; 39: 499–504.
- 31↑
Epstein RB, Graham TC, Storb R, et al. Studies of marrow transplantation, chemotherapy and cross-circulation in canine lymphosarcoma. Blood 1971; 37: 349–359.
- 32
Weiden PL, Storb R, Sale GE, et al. Allogeneic hematopoietic grafts after total-body irradiation in dogs with spontaneous tumors. J Natl Cancer Inst 1978; 61: 353–357.
- 33↑
Weiden PL, Storb R, Deeg HJ. Antitumor effect of marrow transplantation in randomly bred species: studies in dogs with spontaneous lymphoma. In: Okunewick J, Meredith R, eds. Graft-versusleukemia in man and animal models. Boca Raton, Fla: CRC Press Inc, 1981; 127–138.
- 34
Storb R, Thomas ED. Graft-versus-host disease in dog and man: the Seattle experience (review). Immunol Rev 1985; 88: 215–238.
- 35
Storb R, Yu C, Wagner JL, et al. Stable mixed hematopoietic chimerism in DLA-identical littermate dogs given sublethal total body irradiation before and pharmacological immunosuppression after marrow transplantation. Blood 1997; 89: 3048–3054.
- 36↑
Sandmaier BM, Storb R, Santos EB, et al. Allogeneic transplants of canine peripheral blood stem cells mobilized by recombinant canine hematopoietic growth factors. Blood 1996; 87: 3508–3513.
