doi:10.1182/blood-2005-06-2325 Prepublished online October 18, 2005; Preudhomme, Bryan D Young, Ama Z Rohatiner, T A Lister and Dominique Bonnet Daniel J Pearce, David Taussig, Kazem Zibara, Lan-Lan Smith, Christopher M Ridler, Claude implications for our understanding of the heterogeneity of AML AML engraftment in the NOD/SCID assay reflects the outcome of AML: (4217 articles) Neoplasia (3716 articles) Clinical Trials and Observations Articles on similar topics can be found in the following Blood collections http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml Information about subscriptions and ASH membership may be found online at: digital object identifier (DOIs) and date of initial publication. the indexed by PubMed from initial publication. Citations to Advance online articles must include final publication). Advance online articles are citable and establish publication priority; they are appeared in the paper journal (edited, typeset versions may be posted when available prior to Advance online articles have been peer reviewed and accepted for publication but have not yet Copyright 2011 by The American Society of Hematology; all rights reserved. 20036. the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by For personal use only. by guest on June 3, 2013. bloodjournal.hematologylibrary.org From
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AML engraftment in the NOD/SCID assay reflects the outcome of AML: implications for our understanding of the heterogeneity of AML
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doi:10.1182/blood-2005-06-2325Prepublished online October 18, 2005;
Preudhomme, Bryan D Young, Ama Z Rohatiner, T A Lister and Dominique BonnetDaniel J Pearce, David Taussig, Kazem Zibara, Lan-Lan Smith, Christopher M Ridler, Claude implications for our understanding of the heterogeneity of AMLAML engraftment in the NOD/SCID assay reflects the outcome of AML:
(4217 articles)Neoplasia � (3716 articles)Clinical Trials and Observations �
Articles on similar topics can be found in the following Blood collections
http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requestsInformation about reproducing this article in parts or in its entirety may be found online at:
http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprintsInformation about ordering reprints may be found online at:
http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtmlInformation about subscriptions and ASH membership may be found online at:
digital object identifier (DOIs) and date of initial publication. theindexed by PubMed from initial publication. Citations to Advance online articles must include
final publication). Advance online articles are citable and establish publication priority; they areappeared in the paper journal (edited, typeset versions may be posted when available prior to Advance online articles have been peer reviewed and accepted for publication but have not yet
Copyright 2011 by The American Society of Hematology; all rights reserved.20036.the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by
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59ψ tAML 147 NK Interm. Table 1: Summary of Patient’s Details. Mice were injected with 107 peripheral blood nucleated cells from the peripheral blood of AML patients. Murine marrows were analyzed six weeks post-transplant for the presence of human hematopoietic cells. AML engraftment was defined as the presence of human CD33+/CD45+ myeloid cells without an accompanying CD19+/CD45+ B-cell population. Patients marked with * were in relapse and patients marked with ** were given supportive care only. Patients marked with *** produced normal engraftment in NOD/SCID mice. Patients marked with a § underwent affymetrix analysis. Patients marked with Δ possessed a Flt3-ITD and patients marked with ⊥ had a mutated nucleophosmin gene.
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Patients marked with ψ were tested for Flt-3 mutations and were found to be negative. Prognosis risk group was defined as poor, intermediate or good via karyotype according to Grimwade et al 1998.10 FK = failed karyotype at diagnosis. # = An abnormal stemline clone was detected in 2 out of 10 cells examined, containing a complex rearrangement between chromosomes 2, 10 and 11 resulting in insertion of 11q material in 10p12 with a breakpoint at 11q23. Presentation white blood cell (WBC) count is given as 109 cells/L. Patients in whom the WBC was less than 2 x 109/L also had their bone marrow cells tested for engraftment capacity, with identical results to the peripheral blood data. All AML cases were assessed for NOD/SCID engraftment potential before any chemotherapy.
Engraftment in NOD/SCID mice reproduces AML
To confirm the leukemic nature of this myeloid (CD33+/CD45+/CD19neg)
NOD/SCID engraftment, we compared the morphological features identified during
diagnosis to the morphology of NOD/SCID engrafted cells. In all cases analyzed, the
morphology of NOD/SCID engrafted cells was very similar to the original sample. A
representative M2 AML is shown in Figure 1. Similar to the diagnosis smear, a high
proportion of the NOD/SCID engrafted cells were myeloblasts. Pathognomic AML
Auer rods were also detectable in NOD/SCID mice, confirming the leukemic nature
of these cells (arrowed in Figure 1B). Wherever possible, we also performed FISH to
detect characteristic genetic abnormalities in NOD/SCID engrafted cells (examples in
Figure 1C-F).
Figure 1: Confirmation of AML cell growth in NOD/SCID mice.
A B
C D E F
Auer rod
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Ten million cells were injected into NOD/SCID mice and marrows were analyzed for human, myeloid cell content six weeks later. (A) Diagnostic peripheral blood smear from an AML-M2 patient-10. (B) Murine marrow that was injected with cells from the same AML-M2 patient as Figure 1A. Myeloblasts, featuring Auer rods (arrowed) are present, indicating AML. (C) Dual fusion, dual colour fluorescent in situ hybridisation of a relapsed t[8,21] AML-M2 sample. Cells positive for the re-arrangement exhibit 1 green, 1 red and 2 orange spots. (D, E and F) Examples of NOD/SCID engrafted, FACSorted CD33+/CD45+ cells, exhibiting AML-M2 t[8,21] re-arrangement.
Figure 2: Gene expression analysis of engrafted AML cells. Dendrogram is shown from the unsupervised hierarchical cluster analysis of the 8 chips for the 2,260 genes passing the variation filter. Independent of karyotype, AML patients were grouped between before and after engraftment. This means that the AML in the original patient is very much related to the AML that has grown in the mouse. The samples corresponding to before and after engraftment were always adjacent to each other, reflecting a very close relationship between them.
Gene Expression profile is extremely similar between engrafted AML cells and
the original AML sample.
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Figure 3: Most cases of non-engrafting AML do not engraft in the B2-/-NOD/SCID model. Ten million mononuclear cells from 23 different AML patients were injected into both NOD/SCID and B2-/-NOD/SCID mice in paired experiments. Six weeks later, bone marrow engraftment was assessed via flow cytometry. AML engraftment was recorded if human CD33+/CD45+ myeloid cells were present without an accompanying CD19+/CD45+ B-cell population. 10 of 12 AML cases that failed to engraft in the NOD/SCID assay, did not engraft in the B2-/-NOD/SCID model.
ENGRAFTERS NON-ENGRAFTERS Patients ID % CXCR-4 on CD34+ Patients ID % CXCR-4 on CD34+
similar for cases capable and incapable of NOD/SCID engraftment (39.8 and 44.5 x
109/L, respectively) and hence, no statistically significant difference could be
detected.
Engraftment in NOD/SCID assay correlates with karyotypically defined
prognostic group.
The samples we describe here represent a broad spectrum of AML cases,
including de novo and therapy related leukemia (tAML) from FAB groups 0, 1, 2, 3, 4
and 5. We excluded eight patients from karyotypic analysis, as five were relapse
samples, two as the karyotype failed at diagnosis and one in which the karyotype was
not performed. All of the remaining poor prognosis patients we analyzed engrafted in
the NOD/SCID assay (5/5), whereas none of the previously untreated, good prognosis
patients did (0/11). Of the intermediate risk, de novo patients we analyzed, 50%
(18/36) engrafted. Using logistic regression analysis, the only factor that was
significantly associated with engraftment was the karyotypically defined prognosis
group reported in 2001 by Grimwade et al (see Table 3 for a summary; white cell
count p=0.85; FAB group p=0.302; risk group p=0.0002).10
Cytogenetics prognosis group
Capacity for
engraftment
Poor Intermediate Good
YES 5 18 0
NO 0 18 11
Table 3: De novo AML patients were organized into poor, intermediate and good prognosis risk groups according to karyotype definition. Four patients (7, 9 10, 2) were excluded due to relapse, one not done (Patient 22) and two karyotypes (Patients 4 and 5) failed at diagnosis. Engraftment correlates with poor prognosis and conversely all favorable prognosis patients did not engraft. There is no absolute correlation between two frequent mutations and NOD/SCID
engraftment
To examine these two groups of AML cases further, we examined two genes
that are frequently mutated in AML: Flt-3 and nucleophosmin. We detected the Flt3-
ITD mutation in 6 (out of 29 tested) of our AML samples, a proportion similar to the
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In conclusion, engraftment of AML in the NOD/SCID assay seems to be
dependent on an inherent ability of the cells, which correlates well with disease
prognosis.
ACKNOWLEDGEMENTS We thank the patients for providing samples and Dr J Amess for providing diagnostic data. We also thank Derek Davies, Gary Warnes, Ayad Eddaoudi and Kirsty Allen of the FACS Lab at Cancer Research UK for their invaluable expertise. This work would not have been possible without Julie Bee, Clare Millum and Ella Smallcombe of our Biological Resource Unit. Mathew Smith kindly performed mutation analysis. We also thank Spyros Skoulakis for his statistical analysis of the patients’ follow-up data.
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