SWR/J mice are susceptible to alkylator-induced myeloid leukemia

Therapy-related acute myeloid leukemia (t-AML) is a late complication following chemotherapy and/or radiation therapy. Approximately 10% of AML cases are therapy related and the incidence is rising. Alkylator-associated t-AML has a latency of 5–7 years, is often preceded by a myelodysplastic phase (t-MDS) and is frequently associated with loss of material from chromosomes 5 and/or 7 in humans. The survival of these patients is poor, motivating efforts to improve treatment and prevention strategies. It is not yet clear whether t-AML risk is influenced by host factors, or is solely a stochastic process. Previous studies have shown that inherited polymorphisms in drug detoxification (for example, p450 enzymes, phase II conjugation enzymes and NAD(P)H:quinoneoxidoreductase) and DNA repair (including homologous recombination and nucleotide excision) pathways may contribute to t-MDS/AML susceptibility, but confer only modestly increased risk in humans. Alkylators (for example, cyclophosphamide and ethyl-Nnitrosourea (ENU)) are often used to generate cooperating mutations in genetically engineered mouse models of leukemia. In standard laboratory strains (for example, C57BL/6 J, 129 Sv/J), alkylators promote thymic lymphomas efficiently, reducing the number of mice evaluable for myeloid neoplasms. We had previously demonstrated that susceptibility to alkylator-induced cancer has a genetic component in mice. For myeloid leukemia, 6 strains demonstrated variable degrees of susceptibility, whereas 14 were resistant (including C57 and 129 substrains). Of the 20 strains tested, SWR/J was the most susceptible. In the current study, we evaluated a large cohort of SWR/J mice and characterized the phenotype of tumors induced by the prototypical alkylator, ENU. SWR/J mice received either no treatment (‘N’ cohort, n1⁄4 22), hydrocortisone (HC) alone (‘HC’ cohort, n1⁄4 11), ENU alone (‘E’ cohort, n1⁄4 27), ENU followed by HC (‘EH’ cohort, n1⁄4 111) or HC followed by ENU (‘HE’ cohort, n1⁄4 107; see Supplementary Information). A spectrum of diseases was observed, including hematopoietic tumors of both myeloid and lymphoid origins, and invasive lung carcinoma (Supplementary Table 1). Lymphomas were characterized by infiltrating CD4þCD8þ cells (410%) in the bone marrow or spleen, large mediastinal masses and disruption of splenic architecture. Myeloid leukemias were identified by excess myeloblasts (420% of myeloid precursors) in the bone marrow or fixed tissues. Lung cancers were grossly visible and were phenotyped by histologic examination. Of the 245 mice treated with ENU, 205 were evaluable for cancer susceptibility (40 died immediately after ENU injection or were found postmortem). ENU treatment (with or without HC) resulted in 65 evaluable mice (31.7%) with lymphoma, 23 mice (11.2%) with myeloid leukemia and 192 mice (92.7%) with lung carcinoma (Supplementary Table 1). These diagnoses were not mutually exclusive; 5 mice had both lymphoma and myeloid leukemia, 20 had both myeloid leukemia and lung cancer, and 61 had both lymphoma and lung cancer. The lung cancer, lymphoma and leukemia incidences in this strain differ from our previously published results (60%, 0%, and 80%, respectively), likely owing to small cohort size in the previous study (n1⁄4 12 ENUtreated mice). Control mice developed rare lung cancers (1/22 and 1/11 evaluable mice from the untreated and HC only groups, respectively), but no spontaneous hematologic cancers. Previous studies in mice have shown that coadministration of steroids increased the frequency of radiation-induced leukemias. To test whether steroids could increase the incidence of ENU-induced leukemias and reduce the competing incidence of thymic lymphoma, we treated mice with HC either before or after ENU treatment. Neither regimen decreased the proportion of lymphomas, nor increased the proportion of leukemias, compared with ENU alone (Supplementary Table 1). It is not clear why steroids increased the incidence of radiation-induced leukemia and had no impact on ENU-induced leukemia, but this may be attributable to differences in the dose, schedule of administration or type of steroid used. The overall survival was significantly shorter in ENU-treated mice compared with control mice (N or HC, P1⁄4 0.002; Figure 1a). Two mice in the control groups died of lung cancer and the remainder were electively killed, whereas most of the ENU-treated mice were killed when moribund. For the mice treated with ENU, the median survival was longer in the EH cohort (331 days) compared with E or HE (290 and 291 days, respectively; Po0.0001). The cumulative probability of developing lymphoma was higher than leukemia (48.4% vs 23.9%), but the latency for these diseases was identical (134 days) following ENU exposure (Figure 1b). Lymphomas were characterized by splenomegaly, lymphadenopathy, leukocytosis, anemia and mediastinal enlargement (Supplementary Table 2 and data not shown). The bone marrow was infiltrated by CD4þCD8þ cells (53.4% of cases), or single positive CD8þ cells (1.7% of cases) or CD4þ cells (19% of cases). Myeloid and erythroid precursors were reduced in frequency (Supplementary Table 3). Histologically, the lymphoblasts were characterized by open chromatin, numerous nucleoli and vacuolization of the cytoplasm (Supplementary Figure 1). Myeloid leukemias were characterized by splenomegaly, leukocytosis and accumulation of immature myeloid precursors in the bone marrow (Supplementary Tables 2 and 3). The leukemias were uniformly Gr1þCD11bþ (Supplementary Table 2). The myeloblasts were characterized by abundant cytoplasmic granulation and frequent mitotic figures (Supplementary Figure 1). The bone marrow of six ENU-treated mice had significant dysplasia in the myeloid and/or erythroid lineages, of which two had myeloid leukemia, and all had significant anemia (mean hemoglobin1⁄4 11.3; Supplementary Table 4). Twenty-seven additional mice (all with concurrent lung cancer) did not meet criteria for myeloid leukemia, but had splenomegaly and excess myeloblasts (10–15%) in the bone marrow. Although we favor that these bone marrow proliferations are clonal in origin, a reactive proliferation cannot be excluded because of the co-occurrence of a non-hematopoietic tumor. Tumors from two donors with myeloid leukemia were transplanted into sublethally irradiated congenic recipients. Citation: Blood Cancer Journal (2013) 3, e161; doi:10.1038/bcj.2013.59 & 2013 Macmillan Publishers Limited All rights reserved 2044-5385/13