Xenotransplantation of primary AML samples into immunodeficient mice (PDX models) represents a unique opportunity for pre-clinical testing on a group of primary human samples that possess defined genetic lesions. However, given our recent recognition that multiple genetically distinct subclones can exist in AML, there is a risk that there may be selection for sub-clones from the xenotransplanted sample that might not fully represent the patient’s disease.

We sought to establish a collection of genetically defined AML samples capable of engraftment in immunodeficient mice. We transplanted 30 AML patient samples; within 150 days (median 91 days) post transplantation 12 samples produced human CD45+ CD33+ CD19- CD3- engraftment in one or multiple NSG mice. Median patient sample amplification in 25 mice was 21 fold.

Genomic DNA and total RNA was isolated from 7 AML patient samples (3 diagnostic samples from patients who remain in remission; 2 diagnostic samples from patients who later relapsed, 2 diagnostic samples from patients with refractory disease) and 14 matched xenotransplanted samples (2 mice per patient sample). Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 272 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >499x for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. The mutations found in the 7 diagnostic samples were consistently identified in the 14 engrafted AML samples, but with some cases showing variation in allele frequency between diagnostic and engrafted samples. This finding shows that the human disease that engrafted in mice mimics the genetic makeup of the disease found in patients.

We then assessed for allele frequency (AF) changes from diagnostic to xenografted sample as a measure of clonal progression. Clonal progression was defined as emergence of a clone carrying a novel genetic variant in the xenografted sample as compared to the diagnostic patient sample. Five patient samples (from 10 mice) did not show emergence of novel genetic lesions. In this group 2 patients had refractory disease and 3 patients remain in remission. Two patient samples (from 4 mice) demonstrated apparent emergence of novel genetic lesions not detected in diagnostic patient samples. Both of these patients have relapsed since the diagnostic samples were acquired. In the first case, both xenotransplanted mice engrafted with disease carrying NRAS N12S mutation (AF 0.05 and 0.09), which subsequent evaluation revealed to be present below the limit-of-detection (AF 0.004) in the clinical isolate obtained from patient presentation. We are currently conducting the same analysis on the relapsed sample from this patient. In the second case, both mice engrafted with disease carrying PTPN11 E76V (AF 0.03 and 0.0016) while the patient diagnostic sample did not contain any evidence of the alteration at 718x unique sequence coverage.

Of note, one xenografted sample had an IDH1 R132C and another had IDH2 R140Q mutation, both of which have previously been shown to play a role in AML pathogenesis. Available AML cell lines do not carry IDH1/2 mutations, making it challenging to test IDH1/2 inhibitors in pre-clinical settings. These xenografted samples offer an opportunity to test such inhibitors.

Overall we conclude that the xenotransplanted samples possess the diversity of genetic abnormalities found in diagnostic AML samples and thus can be used to assess efficacy of novel targeted therapies. We would like to further investigate a model in which the absence of clonal progression in xenografted samples would predict a better patient outcome, while emergence of novel clones might indicate an increased potential for relapse. We are currently expanding the study to include more diagnostic, xenotransplanted and relapsed samples to assess the associations between the ability of a sample to engraft in mice with clinical outcome and genetic/epigenetic lesions.


Armstrong:Epizyme Inc.: Has consulted for Epizyme Inc. Other.

Author notes


Asterisk with author names denotes non-ASH members.