Transmission and expansion of HOXB4-induced leukemia in two immunosuppressed dogs: implications for a new canine leukemia model

Abstract

Objective: There are currently no large animal models to study the biology of leukemia and development of novel antileukemia therapies. We have previously shown that dogs transplanted with homeobox B4 (HOXB4)-transduced autologous CD34(+) cells developed myeloid leukemia associated with HOXB4 overexpression. Here we describe the transmission, engraftment, and expansion of these canine leukemia cells into two genetically unrelated, immunosuppressed dogs.

Materials and methods: Two dogs immunosuppressed after major histocompatibility complex-haploidentical hematopoietic cell transplantation and exhibiting mixed donor-host chimerism were accidentally infused trace amounts of HOXB4-overexpressing leukemia cells from a third-party dog.

Results: Six weeks after infusion of HOXB4-overexpressing leukemia cells, these two dogs rapidly developed myeloid leukemia consisting of marrow and organ infiltration, circulating blasts, and, in one dog, chloromatous masses. Despite neither of these dogs sharing any dog leukocyte antigen haplotypes with the sentinel case, the HOXB4-transduced clones engrafted and proliferated without difficulty in the presence of immunosuppression. Chimerism studies in both dogs confirmed that donor and, in one case, host hematopoietic cell engraftment was lost and replaced by third-party HOXB4 cells.

Conclusions: The engraftment and expansion of these leukemia cells in dogs will allow studies into the biology of leukemia and development and evaluation of novel antileukemia therapies in a clinically relevant large animal model.

Figure 1

Timeline of events showing the course of transmission and expansion of HOXB4-overexpressing leukemia clones.

Figure 2

Peripheral blood mononuclear cell (MNC) chimerism studies demonstrate the rapid emergence and expansion of the 3^rd party HOXB4 leukemia in the DLA-haploidentical HCT model. MNC were isolated and chimerism was determined by VNTR analysis weekly after DLA-haploidentical HCT showing the ability of HOXB4 cells to engraft and overcome mixed donor-host chimerism. A) In G490, the expansion of the HOXB4 clone was rapid. Over the course of 10 days, this dog completely lost both host and donor grafts. B) In G542, the expansion of HOXB4 took place over 7 weeks. The dog maintained persistence of host engraftment but eventually lost its donor graft.

Figure 3

Blood and marrow necropsy samples from G542 demonstrate GFP+ cells of a myeloid lineage by FACS. A) Unstained peripheral blood showed the presence of two distinct populations. Because the vector expressed GFP, HOXB4 clones could be detectable within the FL1-FITC window. When peripheral blood was stained with biotinylated MAbs and incubated with streptavidin-PE, double-staining (GFP+/SAPE+) demonstrated that cells of a myeloid origin (CD14, DM5) contained the HOXB4 clone in 9% and 50% of its cells, respectively. Cells of a lymphoid origin (CD3, TCRαβ, and CD21) contained only minute numbers of cells that were GFP+/SAPE+. B) Marrow was stained with CD34-PE and demonstrated that all CD34+ progenitor cells expressed the GFP vector (1.5%).

Figure 4

The kinetics of A) white blood cell (WBC) and B) absolute monocyte counts (AMC) in relation to time to euthanasia indicate different trends of leukocytosis and monocytosis with leukemia progression in all 3 dogs (# indicates presumed time period G490 was infected with G374's cells; * indicates presumed time period G542 was infected with G374's cells) G374 exhibited a rapid burst then progressive leukocytosis and monocytosis approximately 50 days prior to euthanasia. G490 had stable counts that began to rise 2 weeks, then more rapidly several days, prior to euthanasia. G542 demonstrated a progressive rise in leukocytosis and monocytosis over the course of one month prior to euthanasia.

Figure 5

G490 demonstrated clinical manifestations of canine myeloid leukemia similar to human acute myeloid leukemia. A) G490 was euthanized on Day +80 after DLA-haploidentical HCT when a rapidly emerging rectal chloroma presented over the course of several days. B) Histopathological review of the chloroma demonstrated proliferating cells with morphology consistent with myelomonocytic leukemia. The chloroma was analyzed for chimerism using VNTR, demonstrating contributions from donor (37%), recipient (16%), and G374 (47%). C) Typical of clinical acute leukemias, there was hematogenous spread and presence of leukemic infiltrates within several organs, including liver and spleen (not shown). Here, leukemic infiltrates are seen invading the renal cortex.

Figure 6

Bone marrow morphology from both (A) G490 and (B) G542 show myelomonocytic leukemia. Bone marrow biopsies taken at the time of necropsy show diffuse sheets of monomorphic blasts having abundant cytoplasm and active mitoses, with morphology consistent with myelomonocytic leukemia (high magnification).

Figure 7

A) Identical integration site is verified in G542, G490 and G374 by clone tracking PCR All three dogs demonstrate the HOXB4 clone at 209 bp. G450 is a known negative control. B) In G542, Southern blot demonstrates the same two integration sites from the HOXB4 clone as seen in G374. When cut with BglII, 2.7K (c-myb site) and 3.76K (PRDM16 site) are seen in both dogs.

Figure 8

A) MLR demonstrates presence of T cell function after inoculation with HOXB4 clone. Prior to HCT, T cells are able to proliferate normally when exposed to allogeneic (G457, donor; G566, unrelated 3^rd party) and mitogenic (ConA) stimuli similar to controls. Two weeks after HCT, G542 has a suppressed reaction to the donor cells as tolerance to G457 emerges early after HCT but has a continued strong proliferative activity against 3^rd party and ConA as is normally seen. However, by Day +98, approximately 7 weeks after G542 was inoculated with the HOXB4 clone, T cells taken for G542 still show high degrees of proliferation not only to 3^rd party and ConA, but also to autologous cells and donor. The T cells thus maintain an ability to proliferate, but this dysregulated response is likely a result of contaminating HOXB4 cells. B) NK functional assays demonstrate ongoing robust activity after inoculation with HOXB4 clone. Prior to HCT, G542 had similar baseline NK functional activity as donor (G457) and unrelated 3^rd party (G566). By Day +15, as is normally seen early after DLA-haploidentical HCT, NK activity is more robust than baseline controls and is a reflection of rapid recovery of donor NK cells after HCT. By Day +98 after HCT, approximately 7 weeks after inoculation with HOXB4, there remains similar robust cytolytic activity of NK cells despite contamination with HOXB4 clones.