To the editor:

Recently, Cox et al1  described that only acute lymphoblastic leukemia (ALL) blasts with a CD19CD133+ immune-phenotype are able to propagate human leukemia in immune-deficient nonobese diabetic severe combined immunodeficiency (NOD/scid) mice. We welcome this contribution to the rapidly evolving debate on leukemic stem cells.

Published data on ALL stem cells are conflicting. While some groups have detected self-renewing blasts as being restricted to the primitive CD19 or CD38 stem cell compartment,1-3  others found repopulating cells only in the more mature CD19+ lymphoid compartment4  or within aberrant CD19+ lymphoid cells that lack CD38 expression.5  To make the picture even more confusing, we have recently shown that a wide variety of blasts at different stages of maturation (CD34+ and CD34, CD19 and CD19+, CD20 and CD20+) are able to propagate leukemia in natural killer (NK) cell–depleted NOD/scid and NOD/scid gamma (NSG) mice after intrafemoral injection.6  Unlike the mechanism suggested by Cox et al,1  our data cannot be explained by prior passage of the human cells in the mice as primary sorted cells show the same engraftment pattern (Table 1). Although this may have been an obvious concern, we believe that we are not creating in vivo cell lines with an altered stem cell hierarchy.

Table 1

Engraftment of purified ALL cells in primary mice*

Patient IDCell doseImmune phenotype of sorted cellsEngraftment level (%)Posttransplantation survival, wk
L754 100 000 CD20low 63 24 
   40 32 
   33 
  CD20high 59 23 
   21 29 
WB51 62 400 CD20low 23 15 
   28 15 
   32 15 
 10 800 CD20high 37 16 
   39 19 
 4000 CD19low 25 19 
   28 20 
 31 200 CD19high 27 15 
   38 16 
   43 16 
   35 17 
No. 1 100 000 CD34highCD19low 26 
   76 32 
No. 9 30 000 CD34highCD19low 93 
   99 16 
 200 000-250 000 CD34highCD19high 97 
   47 13 
   96 14 
   99 15 
No. 12 10 000 CD34highCD19high 73 15 
  CD34lowCD19high 56 15 
Patient IDCell doseImmune phenotype of sorted cellsEngraftment level (%)Posttransplantation survival, wk
L754 100 000 CD20low 63 24 
   40 32 
   33 
  CD20high 59 23 
   21 29 
WB51 62 400 CD20low 23 15 
   28 15 
   32 15 
 10 800 CD20high 37 16 
   39 19 
 4000 CD19low 25 19 
   28 20 
 31 200 CD19high 27 15 
   38 16 
   43 16 
   35 17 
No. 1 100 000 CD34highCD19low 26 
   76 32 
No. 9 30 000 CD34highCD19low 93 
   99 16 
 200 000-250 000 CD34highCD19high 97 
   47 13 
   96 14 
   99 15 
No. 12 10 000 CD34highCD19high 73 15 
  CD34lowCD19high 56 15 
*

le Viseur et al6  and K. Wilson and K. Rehe, unpublished data, March 2009.

Similar to our own results, Kong et al7  have recently shown that both CD34+CD19+CD38 and CD34+CD19+CD38+ blasts in infant ALL/t(4,11) are able to transfer leukemia onto NSG mice. Most importantly, work out of Civin's laboratory as presented at the 2008 Annual Meeting of the American Society of Hematology showed that, after intravenous injection, as few as 10 unselected ALL cells were sufficient to reinitiate the leukemia in NSG mice.8 

How can it be explained that many groups seem to come up with a different phenotype for ALL stem cells? It is interesting to note that studies identifying rare leukemic stem cells with an immature immune phenotype use more conventional NOD/scid mice as recipients, whereas studies observing leukemia-initiating potential in a wider variety of populations use either NK cell–depleted NOD/scid mice or one of the newer genetically engineered NOD/scid mouse strains with deletion of the IL2 receptor gamma chain (eg, NSG mice). Work from Bonnet's laboratory has elegantly demonstrated that conventional NOD/scid mice can clear human cells coated with certain antibodies, such as anti-CD38.9  Consequently, these models may not only underestimate the frequency of human stem cells but also wrongly imply a hierarchy between different populations. Similar observations have recently been made in several solid tumor models (reviewed in Dick10 ): while earlier xenograft assays had suggested the presence of only rare cancer stem cells, recent results using NSG mice showed stem cell frequencies as high as 1 in 4 tumor cells.

Much of the confusion could therefore be around methodology. It will be paramount to standardize the xenotransplantation approaches and to use optimized mouse models that offer the least hostile microenvironment for human cells.10  With these newer and more sensitive models, we may be surprised to find that many more populations have stem cell properties then previously thought. We look forward to continuing this debate.

Approval for these studies was obtained in Münster from the combined Ethics Committee of the Medical Faculty of Münster University and the regional chamber of physicians (Registration number 31 VVormoor) and in Newcastle upon Tyne from the Newcastle and North Tyneside Ethics Committee 2 (REC reference number 06/Q0906/79). Informed consent was obtained in accordance with the Declaration of Helsinki.

Acknowledgments: This work was supported by grants from the Deutsche José Carreras Leukämie-Stiftung eV (Münich, Germany), the North of England Childhood Cancer Research Fund (Newcastle upon Tyne, United Kingdom), and the JGW Patterson Foundation (Newcastle upon Tyne, United Kingdom).

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Josef Vormoor, Northern Institute for Cancer Research, Newcastle University, Sir James Spence Institute, 4th Fl, Royal Victoria Infirmary, Newcastle upon Tyne, NE1 4LP, United Kingdom; e-mail: [email protected].

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