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iAMP21 xenografts
disease can be propagated through multiple generations of mice with high fidelity.
Intrachromosomal amplification of chromosome 21 (iAMP21) is an intriguing cytogenetic abnormality, defin- ing a specific subgroup of approximately 2% of cases of childhood precursor B-cell acute lymphoblastic leukemia (B-ALL). Chromosome 21 genomic profiles, although high- ly variable, always involve amplifications, flanked by regions of normal copy number or deletion.9,10 We have shown that the oncogenic potential of chromosome 21 is optimized through a combination of catastrophic sequence reorganization, driven by chromothripsis, deletion and amplification, resulting from dicentric chromosome forma- tion, breakage-fusion-bridge cycles and whole chromo- some arm duplications.11 This mechanism has the potential to produce a near infinite number of alternative derivative chromosomes 21. The structure of the iAMP21 chromo- some is stabilized by telomere acquisition or duplication, while a combination of protected amplified genes are pos- tulated to confer an overall growth advantage, leading to the development of ALL. Several lines of evidence indicate that iAMP21 is a stable, primary genetic change: (i) among 530 patients, iAMP21 was reported as a sub-clonal abnor- mality in only a single case;12 (ii) the iAMP21 chromosome morphology remains consistent between cells in the same patient; and (iii) the same chromosome structure is observed at diagnosis and relapse.9 A range of specific sec- ondary genetic abnormalities: CRLF2 activating rearrange- ments, X chromosome gain, deletions of RB1, ETV6, the long arm of chromosome 7 (7q) and 11q, and mutations of the RAS pathway frequently co-occur with iAMP21.9,12,13
This distinct iAMP21-ALL subgroup is clinically defined by older age (median 9 years), low white blood cell counts and a high risk of relapse on standard therapy.14-16 Intensive therapy significantly reduces the risk of relapse17,18 but asso- ciated morbidity highlights an urgent need for less toxic reg- imens. Development of rational targeted therapies requires understanding of the mechanism by which these rearrange- ments initiate leukemia. However the requisite tools for functional studies are lacking because no iAMP21-ALL cell lines exist and the complex nature of the abnormalities exclude their reproduction in engineered animal models. To address this shortfall, we transplanted primary leukemia cells from five iAMP21-ALL patients into NOD/LtSz-scid IL2Rg null (NSG) mice. In-vivo luminescent imaging, to track the physical development of ALL, was used to assess these xenografts as potential models for functional and pre-clinical studies. In addition, we characterized the disease morphol- ogy at the microscopic and ultrastructural levels and, as a first application, have performed extensive genomic analysis to investigate clonal heterogeneity of iAMP21-ALL, from which some intriguing findings have emerged.
Methods
Patients
Viable cells from children diagnosed with iAMP21-ALL, as previously defined,19 were provided by the Bloodwise Childhood Leukaemia Cell Bank. Ethical approval was obtained for all patients and informed consent was granted in accordance with the Declaration of Helsinki. Karyotypes and demographic details of the patients used to generate xenografts or as controls for histological analysis are presented in Online Supplementary Tables S1 - S3.
Xenografts and isolation of leukemia cells
Primografts were created by intrafemoral injection of patients’ cells into NSG mice, as previously described.1 Between 2x104 and 2x106 primograft bone marrow or spleen cells from each mouse were used in the same procedure to create secondary and tertiary xenografts (Online Supplementary Table S4). Xenografts were culled at end stage-disease as defined in the Online Supplementary Methods. Bone marrow cells flushed from femora and disaggregat- ed spleens were passed through 40 μm cell strainers. Leukemic cells used for all experimental work were purified from spleen preparations by separation over Ficoll-Paque [density 1.077 g/mL] (G.E. Health Care, Buckinghamshire, UK). Proportions of human and mouse cells and immunophenotypes of the human leukemia fractions were determined by flow cytometry as described in the Online Supplementary Methods.
Lentiviral transduction, in-vitro culture and in-vitro, in-vivo and ex-vivo imaging of xenograft cells
Detailed procedures are provided in the Online Supplementary Methods.
Histopathology and transmission electron microscopy
Detailed procedures are provided in the Online Supplementary Methods.
Single nucleotide polymorphism arrays
DNA extraction and SNP6.0 array hybridization and analysis were performed, as previously described.13 To define regions of chromosome 21 copy number evolution, single nucleotide poly- morphism copy number values were subtracted between second- ary xenografts 2°1B and 2°1A. Copy number abnormalities (CNA) in immunoglobulin or T-cell receptor regions, those not involving coding gene regions, present in patients’ remission samples or list- ed in the Toronto Database of Genomic Variants, are not report- ed. Genomic positions are those in the Hg19 database.
Fluorescence in situ hybridization and multiplex ligation-dependent probe amplification
Dual color FISH on 100-200 interphase cells was performed using fluorescently labeled BAC probes hybridizing to the RUNX1 (RP11-773I18) and APP (RP11-66H5 and RP11-15D13) genomic regions or commercially available probes to the CDKN2A/B genomic region and chromosome 9 centromere (CytoCell, Cambridge, UK), as previously described.20 Multiplex ligation-dependent probe amplification (MLPA) was performed using the SALSA MLPA 335 kit (MRC-Holland, the Netherlands), as previously described.21
Analysis of RAS pathway mutations and RNA sequencing
Detailed procedures are provided in the Online Supplementary Methods.
Results
Development of xenografts and characterization by in-vivo and ex-vivo imaging
Of six primary and two relapsed cases of iAMP21-ALL transplanted into femora of NSG mice, five, including one relapsed case, developed ALL derived from the human cells, in one or more animals (Online Supplementary Table S1). The mean time to development of end-stage disease in primografts was 30 weeks and splenomegaly was seen in all engrafted animals. Secondary and tertiary xenografts were established in three and one cases, respectively, and
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