Validation of a Human Acute Lymphoblastic Leukemia Mouse Model Using Next Generation RNA Sequencing,

Abstract 3576

Primary leukemia cells are known to be difficult to culture in vitro. Therefore, mouse xenograft models are commonly used to study human leukemia biology and to develop new therapeutics. Leukemia can be maintained and expanded through serial transplantations. Although many mouse models with different types of leukemia, mouse strains, and inoculation methods, have been used, there are very few studies validating the models.

We have established acute lymphoblastic leukemia (ALL) mouse models using primary ALL samples and NOD/SCID/IL2Rg null (NSG) mice. In order to increase engraftment efficiency, we transplanted leukemia cells into healthy adult mice via intra-bone marrow (BM) injection (1 to 100 million cells per mouse to tibia bilaterally). To our knowledge, the model with primary ALL samples in NSG mice via intra-BM injection is novel. A total of 9 samples, 2 T cell ALL and 7 precursor B (preB) ALL, were transplanted into cohorts of 3 to 8 mice each. Of these, 1 T cell ALL and 6 preB ALL samples engrafted and 1 T cell and 1 preB ALL sample failed to engraft. Leukemia engraftment was observed between 10 to 33 weeks after transplantation. In each cohort, each mouse developed leukemia at approximately the same time after leukemia transplantation. Necropsy showed significant hepatosplenomegaly and white BM in all mice, indicating leukemia infiltration. Cells were harvested from BM, spleen and other leukemia-infiltrated organs, and confirmed to be human origin by flow cytometry using anti-HLA-ABC and anti- human CD45 antibodies. Nearly 100% of BM was replaced with human leukemia cells.

One T cell and 1 preB ALL sample have been serially transplanted using the same method as the primary transplantation to quaternary and tertiary generations, respectively. We observed that the time to develop leukemia became shorter with each transplantation: 16 weeks for the first transplantation and 10 weeks for the tertiary transplantation in a T cell ALL sample. Some mice transplanted with a preB ALL sample developed chloroma. Interestingly, chloroma development was consistently observed with this sample, which was transferred through serial transplantation although the patient did not develop chloroma. Morphology and immunophenotype were similar to the original leukemia. There were some changes in CD expression patterns; however, immunophenotyping was consistent with the original leukemia.

To help understand phenotype progression in transplanted leukemia samples, we are currently comparing the transcriptome profiles of leukemia samples obtained from the patient, primary and quaternary mice for T cell ALL, and primary and tertiary mice for preB ALL samples (preB ALL patient sample not available). Next-generation sequencing (NGS)-based RNA-Sequencing (RNA-Seq) is in progress for this analysis. In addition to obtaining digital expression profiles, and differential gene expression, RNA-Seq analysis will reveal the complete repertoire of splice variants, point mutations, and fusion transcripts in the ALL samples. Complete results of these analyses for our mouse models with both T cell and preB ALL will be presented. Thus, this technique will provide the most detailed transcriptomic analysis and no validation studies have yet been reported on ALL samples sequentially engrafted NSG mice using RNA-Seq.