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iAMP21 xenografts
A
BC
Figure 4. Analysis of mutations affecting the RAS pathway. The key to the analytical methods used are shown top right. Blue boxes show non-synonymous RAS path- way mutations identified. White boxes summarize the estimated variant allele (VA) frequencies and methods of analysis used for patients’ and xenograft samples. (A) In patient 1 an NF1 mutation was clonal at presentation and in all xenografts. By contrast mutations of NRAS and KRAS showed marked fluctuations in VA fre- quency; NRAS (1), present in the patient as a sub-clone, was detected in both 1° xenografts but not in blasts from 2° and 3° animals. NRAS (2), identified by high depth targeted sequencing in 1% of patients’ sample reads, was undetected by whole exome sequencing in primografts but emerged as a dominant clone in 2°2a and all derivative 3° xenografts. A mutation of KRAS, although undetected in more than 6000 reads by targeted sequencing of the patient’s sample, marked the dominant clone present in primografts and 2°2b. Sanger sequence traces illustrate the relationship between the NF1, NRAS (2) and KRAS mutations in the two 2° xenografts, traces shown for 2°1a are also representative of 1°1a and 1°1b, traces shown for 2°1b are also representative of 3°1a-g. (B) In patient 3 an NRAS mutation identified in the patient remained clonal in all 1° and 2° xenografts. The Sanger sequencing trace shown for 1°3a is representative of all xenografts. (C) In patient 5 a FLT3 itd, detected as a minor sub-clone by exome sequencing of the presentation sample, became dominant in the 1° xenograft as demonstrated by the generation of two distinct exon 14 polymerase chain reaction (PCR) amplicons of equal intensity (first lane bottom right). In contrast only a single PCR product was amplified from the relapse sample of this patient (second lane bottom right). Mutations detected in the patients’ presentation samples have been previously published.13
not only of the physically deleted genes but also of TUSC1, positioned more than 3 Mb away from the deletion bound- aries.
Discussion
Compared with genetically engineered animal models, xenografts bring several advantages to the study of ALL, not least, their potential to fully recapitulate the spectrum of genomic abnormalities that occur within individual patients of a given genetic sub-type. This is particularly rel- evant to iAMP21-ALL, in which the primary abnormality is structurally complex, unique to each patient and impos- sible to reproduce in engineered animal models. As there are no cell lines carrying iAMP21, the xenografts presented here represent an important resource for future functional and pre-clinical studies.
Highlighting the potential of lentiviral constructs inte- grated into xenograft cells, we demonstrated their in vivo expression. However we observed considerable temporal and spatial variation in signal development that, as demon-
strated by analysis of spleen and CNS, was apparently related to heavy skewing of the ratios of transduced to non-transduced cells at specific sites. It seems likely that this skewing was caused largely by clonal expansion of small founder populations, particularly as tracking of spe- cific genomic abnormalities demonstrated aggressive expansion of minor sub-clones in xenografts. As a conse- quence, accurate analysis of disease burden by in-vivo imaging, will in future require enrichment for EGFP- expressing cells prior to engraftment.
Unexpectedly, light microscopy and transmission elec- tron microscopy together provided strong evidence that transplantation of NSG mice with iAMP21-ALL cells from two patients led to destruction of the bone marrow niche. As we saw no examples of similar morphology among iAMP21-ALL patients’ samples, it seemed likely that this phenomenon was xenograft-specific and a consequence of initiating a heavy leukemic burden at one site. Cells popu- lating the affected areas, although damaged, were hetero- geneous and showed little if any staining with human CD19 and CD45 suggesting they were of host origin. Histology was therefore consistent with destruction hav-
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