Limits...
An advanced preclinical mouse model for acute myeloid leukemia using patients' cells of various genetic subgroups and in vivo bioluminescence imaging.

Vick B, Rothenberg M, Sandhöfer N, Carlet M, Finkenzeller C, Krupka C, Grunert M, Trumpp A, Corbacioglu S, Ebinger M, André MC, Hiddemann W, Schneider S, Subklewe M, Metzeler KH, Spiekermann K, Jeremias I - PLoS ONE (2015)

Bottom Line: Adequate model systems are required for preclinical studies to improve understanding of AML biology and to develop novel, rational treatment approaches.Neither serial transplantation nor genetic engineering markedly altered sample characteristics analyzed.Transgene expression was stable in PDX AML cells.

View Article: PubMed Central - PubMed

Affiliation: Group Apoptosis, Department of Gene Vectors, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany; German Cancer Consortium (DKTK), Heidelberg, Germany; German Cancer Research Center (DKFZ), Heidelberg, Germany.

ABSTRACT
Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease with poor outcome. Adequate model systems are required for preclinical studies to improve understanding of AML biology and to develop novel, rational treatment approaches. Xenografts in immunodeficient mice allow performing functional studies on patient-derived AML cells. We have established an improved model system that integrates serial retransplantation of patient-derived xenograft (PDX) cells in mice, genetic manipulation by lentiviral transduction, and essential quality controls by immunophenotyping and targeted resequencing of driver genes. 17/29 samples showed primary engraftment, 10/17 samples could be retransplanted and some of them allowed virtually indefinite serial transplantation. 5/6 samples were successfully transduced using lentiviruses. Neither serial transplantation nor genetic engineering markedly altered sample characteristics analyzed. Transgene expression was stable in PDX AML cells. Example given, recombinant luciferase enabled bioluminescence in vivo imaging and highly sensitive and reliable disease monitoring; imaging visualized minimal disease at 1 PDX cell in 10000 mouse bone marrow cells and facilitated quantifying leukemia initiating cells. We conclude that serial expansion, genetic engineering and imaging represent valuable tools to improve the individualized xenograft mouse model of AML. Prospectively, these advancements enable repetitive, clinically relevant studies on AML biology and preclinical treatment trials on genetically defined and heterogeneous subgroups.

No MeSH data available.


Related in: MedlinePlus

Engraftment and retransplantation of AML cells in NSG mice conserves genetic alterations of the primary sample.Primary AML patient samples and matched PDX cells, reisolated out of the BM (CD45 chimerism 80–99%) after first passage in NSG mice (PDX-0) or after 1 or 2 re-transplantation cycles (PDX-1/-2), were characterized by targeted resequencing of 43 AML-related genes (S1 Table). Plots depict variant allele frequencies for each driver gene mutation found within the sample. a/b/c/d/f: PDX cells of three to five mice injected in parallel were analyzed. *: primary cells were frozen and thawed before injection. BCOR (BCL-6 corepressor); CEBPA (CCAAT/enhancer binding protein alpha); DNMT3A (DNA (cytosine-5)-methyltransferase 3 alpha); FLT3 (Fms-related tyrosine kinase 3); ITD (internal tandem duplication); KRAS (Kirsten rat sarcoma viral oncogene homolog); NPM1 (nucleophosmin-1); NRAS (neuroblastoma RAS viral oncogene homolog); SRSF2 (serine/arginine-rich splicing factor 2); TET2 (tet methylcytosine dioxygenase 2); TP53 (tumor protein p53). Raw data is depicted in S2 Table.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4368518&req=5

pone.0120925.g002: Engraftment and retransplantation of AML cells in NSG mice conserves genetic alterations of the primary sample.Primary AML patient samples and matched PDX cells, reisolated out of the BM (CD45 chimerism 80–99%) after first passage in NSG mice (PDX-0) or after 1 or 2 re-transplantation cycles (PDX-1/-2), were characterized by targeted resequencing of 43 AML-related genes (S1 Table). Plots depict variant allele frequencies for each driver gene mutation found within the sample. a/b/c/d/f: PDX cells of three to five mice injected in parallel were analyzed. *: primary cells were frozen and thawed before injection. BCOR (BCL-6 corepressor); CEBPA (CCAAT/enhancer binding protein alpha); DNMT3A (DNA (cytosine-5)-methyltransferase 3 alpha); FLT3 (Fms-related tyrosine kinase 3); ITD (internal tandem duplication); KRAS (Kirsten rat sarcoma viral oncogene homolog); NPM1 (nucleophosmin-1); NRAS (neuroblastoma RAS viral oncogene homolog); SRSF2 (serine/arginine-rich splicing factor 2); TET2 (tet methylcytosine dioxygenase 2); TP53 (tumor protein p53). Raw data is depicted in S2 Table.

Mentions: AML is a genetically heterogeneous group of diseases, and various gene mutations have been shown to be associated with treatment response and patient outcome [39]. Furthermore, AML patients may harbor multiple, genetically related disease subclones [40,41]. The benefit of using patient-derived cells in preclinical in vivo models depends on their ability to faithfully recapitulate the genotypic heterogeneity of the disease. Thus, we performed comprehensive analyses as quality controls, as proposed recently [41]. Using a targeted resequencing approach that covers 43 genes with known roles in AML pathogenesis (S1 Table), we studied six primary patient samples and matched xenografts. Per sample, we identified two to six mutations (Fig. 2, Fig. 3F; S2 Table). Each patient sample contained mutations with a variant allele frequency (VAF) of close to 50%, indicating that these variants were present in most cells, assuming heterozygosity. All of these mutations, which mark the founding clone of the AML cell population, were preserved in the PDX cells recovered from mice after amplification, consistent with data published previously [41]. In each primary sample, we detected additional mutations with a significantly lower VAF, suggesting the presence of subclones within the AML cell population [41,42]. Most of these mutations were also detected within the respective PDX cells. In two cases, we observed an increase in the VAF of mutations in PDX compared to primary cells, indicating that the subclone carrying the mutation had an engraftment or growth advantage (AML-361 and AML-393; Fig. 2 and Fig. 3F). Four samples showed evidence of polyclonal engraftment. In these cases, variants found in the patient at a low VAF were detected in PDX cells with similar, low allelic frequencies, that were highly consistent between multiple mice transplanted in parallel (AML-372, AML-373, AML-407, and AML-412; Fig. 2). In two samples, small subclones (VAFs of the subclone-specific mutations below 10%) were detected in the patient sample but were undetectable in mice (AML-373 and AML-412). These subclones may have had a relative engraftment or proliferation disadvantage in our model. In one sample from a patient with a FLT3-ITD (60bp length), we detected a second, additional FLT3-ITD (96 bp length) in PDX cells obtained from three mice injected in parallel (VAF 2–4%). This second mutation was not detectable in the primary specimen (AML-373). Since the same, additional FLT3-ITD emerged in all three mice that were inoculated with the patient’s cells in parallel, we conclude that a small subclone carrying this variant was present in the patient sample, albeit below our limit of detection. After xenotransplantation, the subclone carrying the 96bp FLT3-ITD expanded relative to the 60bp FLT3-ITD-mutated clone, and thus had an engraftment or growth advantage in our model. Our data are in line with published results describing the challenge of reliably mimicking rare subclones upon xenotransplantation [41].


An advanced preclinical mouse model for acute myeloid leukemia using patients' cells of various genetic subgroups and in vivo bioluminescence imaging.

Vick B, Rothenberg M, Sandhöfer N, Carlet M, Finkenzeller C, Krupka C, Grunert M, Trumpp A, Corbacioglu S, Ebinger M, André MC, Hiddemann W, Schneider S, Subklewe M, Metzeler KH, Spiekermann K, Jeremias I - PLoS ONE (2015)

Engraftment and retransplantation of AML cells in NSG mice conserves genetic alterations of the primary sample.Primary AML patient samples and matched PDX cells, reisolated out of the BM (CD45 chimerism 80–99%) after first passage in NSG mice (PDX-0) or after 1 or 2 re-transplantation cycles (PDX-1/-2), were characterized by targeted resequencing of 43 AML-related genes (S1 Table). Plots depict variant allele frequencies for each driver gene mutation found within the sample. a/b/c/d/f: PDX cells of three to five mice injected in parallel were analyzed. *: primary cells were frozen and thawed before injection. BCOR (BCL-6 corepressor); CEBPA (CCAAT/enhancer binding protein alpha); DNMT3A (DNA (cytosine-5)-methyltransferase 3 alpha); FLT3 (Fms-related tyrosine kinase 3); ITD (internal tandem duplication); KRAS (Kirsten rat sarcoma viral oncogene homolog); NPM1 (nucleophosmin-1); NRAS (neuroblastoma RAS viral oncogene homolog); SRSF2 (serine/arginine-rich splicing factor 2); TET2 (tet methylcytosine dioxygenase 2); TP53 (tumor protein p53). Raw data is depicted in S2 Table.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4368518&req=5

pone.0120925.g002: Engraftment and retransplantation of AML cells in NSG mice conserves genetic alterations of the primary sample.Primary AML patient samples and matched PDX cells, reisolated out of the BM (CD45 chimerism 80–99%) after first passage in NSG mice (PDX-0) or after 1 or 2 re-transplantation cycles (PDX-1/-2), were characterized by targeted resequencing of 43 AML-related genes (S1 Table). Plots depict variant allele frequencies for each driver gene mutation found within the sample. a/b/c/d/f: PDX cells of three to five mice injected in parallel were analyzed. *: primary cells were frozen and thawed before injection. BCOR (BCL-6 corepressor); CEBPA (CCAAT/enhancer binding protein alpha); DNMT3A (DNA (cytosine-5)-methyltransferase 3 alpha); FLT3 (Fms-related tyrosine kinase 3); ITD (internal tandem duplication); KRAS (Kirsten rat sarcoma viral oncogene homolog); NPM1 (nucleophosmin-1); NRAS (neuroblastoma RAS viral oncogene homolog); SRSF2 (serine/arginine-rich splicing factor 2); TET2 (tet methylcytosine dioxygenase 2); TP53 (tumor protein p53). Raw data is depicted in S2 Table.
Mentions: AML is a genetically heterogeneous group of diseases, and various gene mutations have been shown to be associated with treatment response and patient outcome [39]. Furthermore, AML patients may harbor multiple, genetically related disease subclones [40,41]. The benefit of using patient-derived cells in preclinical in vivo models depends on their ability to faithfully recapitulate the genotypic heterogeneity of the disease. Thus, we performed comprehensive analyses as quality controls, as proposed recently [41]. Using a targeted resequencing approach that covers 43 genes with known roles in AML pathogenesis (S1 Table), we studied six primary patient samples and matched xenografts. Per sample, we identified two to six mutations (Fig. 2, Fig. 3F; S2 Table). Each patient sample contained mutations with a variant allele frequency (VAF) of close to 50%, indicating that these variants were present in most cells, assuming heterozygosity. All of these mutations, which mark the founding clone of the AML cell population, were preserved in the PDX cells recovered from mice after amplification, consistent with data published previously [41]. In each primary sample, we detected additional mutations with a significantly lower VAF, suggesting the presence of subclones within the AML cell population [41,42]. Most of these mutations were also detected within the respective PDX cells. In two cases, we observed an increase in the VAF of mutations in PDX compared to primary cells, indicating that the subclone carrying the mutation had an engraftment or growth advantage (AML-361 and AML-393; Fig. 2 and Fig. 3F). Four samples showed evidence of polyclonal engraftment. In these cases, variants found in the patient at a low VAF were detected in PDX cells with similar, low allelic frequencies, that were highly consistent between multiple mice transplanted in parallel (AML-372, AML-373, AML-407, and AML-412; Fig. 2). In two samples, small subclones (VAFs of the subclone-specific mutations below 10%) were detected in the patient sample but were undetectable in mice (AML-373 and AML-412). These subclones may have had a relative engraftment or proliferation disadvantage in our model. In one sample from a patient with a FLT3-ITD (60bp length), we detected a second, additional FLT3-ITD (96 bp length) in PDX cells obtained from three mice injected in parallel (VAF 2–4%). This second mutation was not detectable in the primary specimen (AML-373). Since the same, additional FLT3-ITD emerged in all three mice that were inoculated with the patient’s cells in parallel, we conclude that a small subclone carrying this variant was present in the patient sample, albeit below our limit of detection. After xenotransplantation, the subclone carrying the 96bp FLT3-ITD expanded relative to the 60bp FLT3-ITD-mutated clone, and thus had an engraftment or growth advantage in our model. Our data are in line with published results describing the challenge of reliably mimicking rare subclones upon xenotransplantation [41].

Bottom Line: Adequate model systems are required for preclinical studies to improve understanding of AML biology and to develop novel, rational treatment approaches.Neither serial transplantation nor genetic engineering markedly altered sample characteristics analyzed.Transgene expression was stable in PDX AML cells.

View Article: PubMed Central - PubMed

Affiliation: Group Apoptosis, Department of Gene Vectors, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany; German Cancer Consortium (DKTK), Heidelberg, Germany; German Cancer Research Center (DKFZ), Heidelberg, Germany.

ABSTRACT
Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease with poor outcome. Adequate model systems are required for preclinical studies to improve understanding of AML biology and to develop novel, rational treatment approaches. Xenografts in immunodeficient mice allow performing functional studies on patient-derived AML cells. We have established an improved model system that integrates serial retransplantation of patient-derived xenograft (PDX) cells in mice, genetic manipulation by lentiviral transduction, and essential quality controls by immunophenotyping and targeted resequencing of driver genes. 17/29 samples showed primary engraftment, 10/17 samples could be retransplanted and some of them allowed virtually indefinite serial transplantation. 5/6 samples were successfully transduced using lentiviruses. Neither serial transplantation nor genetic engineering markedly altered sample characteristics analyzed. Transgene expression was stable in PDX AML cells. Example given, recombinant luciferase enabled bioluminescence in vivo imaging and highly sensitive and reliable disease monitoring; imaging visualized minimal disease at 1 PDX cell in 10000 mouse bone marrow cells and facilitated quantifying leukemia initiating cells. We conclude that serial expansion, genetic engineering and imaging represent valuable tools to improve the individualized xenograft mouse model of AML. Prospectively, these advancements enable repetitive, clinically relevant studies on AML biology and preclinical treatment trials on genetically defined and heterogeneous subgroups.

No MeSH data available.


Related in: MedlinePlus