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infant pro-B acute lymphoblastic leukemia with MLL-AF4 rearrangement

REVIEW

Insights into the cellular origin and etiology of the infant pro-B acute lymphoblastic leukemia with MLL-AF4rearrangement

C Bueno,R Montes,P Catalina,R Rodr?′guez and P Menendez

Andalusian Stem Cell Bank,Centro de Investigacio ′n Biome ′dica,Consejer?′a de Salud-Universidad de Granada,Granada,Spain

Infant acute lymphoblastic leukemia (ALL)involving mixed-lineage leukemia (MLL)fusions has attracted a huge interest in basic and clinical research because of its prenatal origin,mixed-lineage phenotype,dismal prognosis and extremely short latency.Over 90%of infant ALLs are pro-B ALL harboring the leukemic fusion MLL-AF4.Despite the fact that major achievements have provided a better understanding about the etiology of infant MLL-AF4tALL over the last two decades,key questions remain unanswered.Epidemiological and genet-ic studies suggest that the in utero origin of MLL rearrange-ments in infant leukemia may be the result of prenatal exposure to genotoxic compounds.In fact,chronic exposure of human embryonic stem cells (hESCs)to etoposide induces MLL rearrangements and makes hESC more prone to acquire subsequent chromosomal abnormalities than postnatal CD34tcells,linking embryonic exposure to topoisomerase II inhibitors to genomic instability and MLL rearrangements.Unfortunately,very little is known about the nature of the target cell for transformation.Neuron-glial antigen 2expression was initially claimed to be speci?cally associated with MLL rearrangements and was recently shown to be readily expressed in CD34tCD38t,but not CD34tCD38àcells suggesting that progenitors rather than stem cells may be the target cell for transformation.Importantly,the recent ?ndings showing that MLL-AF4rearrangement is present and expressed in mesen-chymal stem cells from infant patients with MLLAF4tALL challenged our current view of the etiology and cellular origin of this leukemia.It becomes therefore crucial to determine where the leukemia relapses come from and how the tumor–stroma relationship is de?ned at the molecular level.Finally,MLL-AF4leukemogenesis has been particularly dif?cult to model and bona ?de MLL-AF4disease models do not exist so far.It is likely that the current disease models are missing some essential ingredients of leukemogenesis in the human embryo/fetus.We thus propose modeling MLL-AF4tinfant pro-B ALL using prenatal hESCs.

Leukemia (2011)25,400–410;doi:10.1038/leu.2010.284;published online 7December 2010

Keywords:MLL-AF4;infant leukemia;hESCs;mesenchymal stem cells;cellular origin;etiology

Etiology of the infant pro-B acute lymphoblastic leukemia harboring the MLL-AF4fusion gene

Over 90%of the leukemias diagnosed in newborns/infants (o 1year old)are pro-B stage acute lymphoblastic leukemias (ALLs)harboring the leukemic fusion gene mixed-lineage leukemia (MLL)-AF4.1,2The MLL gene located in chromosome 11q23fuses to generate chimeric genes with over 100partners

in human leukemia.3,4This MLL-AF4pro-B ALL represents a very rare leukemia as compared with other pediatric ALL affecting later differentiation stages (pre-B),which are typically seen in older children (3–10years old).5MLL-AF4tinfant pro-B ALL is associated with dismal prognosis (5-year disease-free survival lower than 20%)and very brief latency.This raises the question of how this disease can evolve so quickly,particularly if additional secondary mutations are required.MLL-rearranged leukemias commonly have activating FLT3mutations 6,7and around 50%of the cases have additional chromosomal abnormalities.2,8,9

Over the last decades,major achievements have provided a better understanding about the etiology of infant MLL-AF4tALL.Elegant studies on identical twins with concordant MLL-AF4tleukemia and retrospective analyses of the clonotypic MLL-rearranged sequences of blast cells from young patients in their neonatal blood spots revealed a in utero origin of the MLL rearrangements.10,11Importantly,compelling information indicates that the MLL-AF4does not suf?ce to promote leukemogenesis on its own and additional secondary genetic insults are required.3,9

Epidemiological and genetic studies support the contention that the in utero origin of MLL rearrangements in infant leukemia may be the result of exposures,during pregnancy,to genotoxic compounds present in the maternal diet intake capable of inducing breaks in the MLL locus in the fetus but not in the mother who has functional DNA repair mechanisms in place.12,13The MLL rearrangements may in fact be the result of transplacental exposures to substances that alter the function of DNA topoisiomerase II,a DNA repairing enzyme highly expressed during embryonic development.14–20Among these genotoxic compounds,the etoposide (VP16)is the best studied.Etoposide is a topoisomerase II inhibitor commonly used in chemotherapy cocktails and is responsible for 5–15%of the therapy-related acute leukemias.12,13,21,22Exposure of cells to topoisomerase II inhibitors increases the frequency of illegiti-mate recombination events,23a physiologic activity that may be related to both cytotoxicity and leukemogenicity of etoposide.Recent studies 24,25suggest that high dietary intake of bio?avo-noids,an abundant source of topoisomerase-II inhibitors in the diet,could cause breaks in MLL and possibly in other partner genes,therefore having an important role in the generation of the preleukemic clone in infancy and in the development of therapy-related acute leukemia.26It has been shown that exposure to high doses of etoposide experimentally induces MLL breaks in mouse embryonic stem cells (ESCs),15fetal liver-derived CD34thematopoietic stem cells (HSCs)19and in cord blood (CB)-derived CD34tHSCs.14,17,18However,the effects of etoposide earlier during human embryonic development remain to be determined.Human ESCs (hESCs)hold the promise to become a powerful tool for drug screening and toxicity but also to determine the spatial-temporal onset of diseases that are known to arise in utero .16,27–29

Received 6September 2010;revised 8October 2010;accepted 27October 2010;published online 7December 2010

Correspondence:Dr P Menendez,Andalusian Stem Cell Bank,

Instituto de Investigacio

′n Biome ′dica,Parque Tecnolo ′gico de la Salud,Avda del Conocimiento,Granada 18100,Spain.E-mail:pablo.menendez@juntadeandalucia.es

Leukemia (2011)25,400–410

https://www.wendangku.net/doc/cc3096067.html,/leu

Etoposide induces MLL rearragements in hESCs

and CB-CD34thematopoietic stem/progenitor cells We have recently used hESCs as a model to test the effects of etoposide on human early embryonic development.16We wanted to address whether (i)very low doses of etoposide promote MLL rearrangements in hESCs and hESC-derived hematopoietic cells;(ii)MLL rearrangements are suf?cient to confer hESCs with a selective proliferation/survival advantage and;(iii)whether continuous exposure to very low doses of etoposide predisposes hESCs to acquire other chromosomal abnormalities.Interestingly,exposure to a single low dose of etoposide induced a pronounced cell death in undifferentiated hESCs but not in postnatal CD34tHSCs.The striking vulnerability of hESCs to etoposide-induced cell death is in line with previous studies con?rming the crucial role of both DNA topoisomerase II-a and -b in human developing tissues.20

A single low dose of etoposide induced MLL breaks in

B 2–3%of the hESCs and CB-derived CD34tHSCs (Figures 1a and b).This datum is similar to that reported in fetal and CB-derived CD34tHSC.14,17–19Interestingly,however,hESCs are much more susceptible than mouse ESCs to etoposide-induced MLL breaks.15,16Etoposide concentrations as high as 100m M barely induced MLL rearrangements in just one out of 62,500mouse ESCs,15whereas relatively low concentrations (0.2–0.5m M )induced MLL gene fusions in an average of three out of 100hESCs (a 1800-fold increase).16Physiological doses of etoposide were used by Bueno et al .16because the etoposide concentration in the plasma of cancer patients treated with this drug ranges between 1and 2m M ,30

a concentration far below the 100m M employed in previous studies.

It has been suggested that early prenatal HSCs may be the target for MLL fusions.19,27Therefore,using conditions pre-viously optimized to promote hematopoietic differentiation from hESCs,16,29,31–41Bueno et al assessed to what extent etoposide induces MLL breaks at two different developmental stages during human embryonic hematopoietic development.Early (day t15)hESC-derived hematopoietic derivatives seem to be slightly more susceptible to etoposide-induced MLL breaks than late (day t22)fully differentiated hESC-derived hematopoietic derivatives (2.9vs 1.6%,respectively).16This suggests that postnatal CD34tand late hESC-derived hematopoietic cells are less vulnerable to etoposide-induced MLL rearrangements than undifferentiated hESCs or earlier hESCs-derived hemato-poietic cells.

Human ESCs are slightly more vulnerable to etoposide-induced MLL rearrangements than CB-derived CD34tHSCs.After long-term culture,the proportion of hESCs harboring MLL rearrangements diminishes and neither cell cycle variations (S-phase:64.4vs 63.1%,respectively)nor genomic abnormal-ities are observed in the etoposide-treated hESCs,suggesting that MLL rearrangements are insuf?cient to confer hESCs with a selective proliferation/survival advantage and that additional secondary cooperation mutations may be missing.In fact,continuous exposure to very low doses of etoposide induced MLL rearrangements and primed hESCs (Figure 1c)but not CB-derived CD34tHSCs (Figure 1d)to acquire other major karyotypic abnormalities.Thus,chronic exposure

Figure 1Effects of etoposide exposure in hESCs and CB-derived CD34tHSCs.MLL rearrangements were identi?ed,quanti?ed and characterized by inverse PCR (data not shown)and ?uorescence in situ hybridization using a split-apart MLL probe in hESCs (a )and CD34tHSCs (b )treated with a single dose of etoposide (0.2or 0.5m M ).Upon continuous exposure to very low doses of etoposide (0.02m M )chromosomal abnormalities were detected by conventional G-banding,which could be further con?rmed by spectral karyotyping in hESCs (c )but not in CB-derived CD34tcells (d ).

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developmentally early stem cells to etoposide induces MLL rearrangements and makes hESCs more prone to acquire other chromosomal abnormalities than postnatal CD34tcells,link-ing embryonic exposure to inhibitors of the topoisomerase II to genomic instability and MLL gene rearrangements. Environmental exposures and delayed infection early in life as a plausible etiological mechanism for childhood leukemias

The etiology and pathogenesis of MLL-rearranged pediatric leukemias differs from other MLL-germline leukemias harboring other fusion genes such as E2A-PBX1,BCR–ABL or TEL-AML1. Much speculation and epidemiological endeavor in identifying the underlying causal mechanisms in childhood leukemia have been simplistic42and the implicit premise that‘the cause’should be attributable solely to‘an exposure’is to ignore the complexity of biology.Leukemia,if not all cancers,is the result of a combination of crucial exposures,modifying in?uences, inherited susceptibility and chance.42A plethora of candidate environmental exposures have been proposed.The only established causal exposure for childhood leukemia is ionizing radiation.This unambiguous conclusion is derived from data on Japanese atomic bomb survivors from1945who were acutely exposed to up to4200mSv43and,at a much lower dose level (B10mSv),from historical data on diagnostic exposure of the fetus from X-ray pelvimetry during pregnancy.44Unfortunately, most of these environmental exposures lack a biological rationale or consistent epidemiological evidence.Although there might not be a single or exclusive cause,an abnormal immune response to common infection(s)has emerged as a plausible etiological mechanism for childhood leukemias.42The most recent epidemiological surveys conclude that although con?icting data exist,the weight of evidence overall is supportive of both population mixing and delayed infection in infancy as being signi?cant causal factors.45Time-space clustering is also compatible with the delayed-infection hypothesis,but more-speci?c epidemiological predictions of this hypothesis relate to the timing of infections or opportunities for infection early in life.46,47The patterns of exposure,timing of infections in the?rst year of life and the immunological response to such challenges will have multifactorial determinants.These will include breastfeeding practices and mothers’immune status,social factors such as hygiene conditions and interactions with other children of the same or older age,community factors such as population density,mobility,age and infectious history.46,47Unfortunately,we have no algorithm to compute the overall likelihood of infection and immunological response under these highly variable circumstances.The simplest prediction of the hypothesis is that patients with childhood leukemia might be expected to have fewer recorded common infections in the?rst year of life,and less social contact and the potential for infectious exposure outside their home F for example,through day-care attendance.46,47

Cellular origin of the infant MLL-AF4tpro-B ALL

In infant ALL wherein t(4;11)MLL-AF4is very common(490%), the fusion gene arises in utero.10,48,49However,very little is known about the nature of the target cell for transformation in the embryo/fetus and the mechanisms accounting for its B-cell lineage af?liation.Unfortunately,mouse models and transformed cell lines have been used with only modest success to model the effects of MLL-AF4and the disease phenotypes achieved do not faithfully mimic those seen in the actual infant disease.Moreover,MLL-AF4protein seems toxic when retro-virally overexpressed in mouse or human stem cells.It could be argued that a cell in a wrong developmental or hierarchical position had been targeted in these experiments.Alternatively, MLL-AF4might have a detrimental effect when expressed under the long terminal repeat retroviral promoter at levels much higher than required to be oncogenic.Despite the target cell where MLL-AF4arises being unde?ned,HSCs and hematopoie-tic progenitor(HPC)cells represent likely targets for transforma-tion:the infant MLL-AF4tpro-B ALL displays a pro-B or pro-B/ monocyte phenotype and?uorescence in situ hybridization studies in isolated cell subsets indicate that MLL-AF4may be already present in a very primitive CD34tCD19àcell subset.50

New perspectives in the association between the expression of neuron-glial antigen2(NG2)and the presence of MLL rearrangements acute leukemias

The NG2molecule and its human homolog was?rst reported on oligodendrocyte progenitor cells.51NG2is recognized by the 7.1monoclonal antibody.52The physiological role of this molecule remains to be elucidated.53The expression pattern of NG2in leukemia is controversial.NG2expression was initially claimed to be speci?cally associated with MLL gene rearrangements.54In fact,NG2has gradually been incorporated in diagnostic panels for immunophenotyping of leukemic patients because of its potential predictive value for MLL rearrangements in childhood and adult acute myeloid leukemias.54–58Moreover,commonly seen in the clinic are leukemic patients harboring MLL rearrangements but lacking NG2expression(Table1).55,57,59Conversely,there are a proportion of cases in which expression of NG2is clearly detected in the absence of MLL rearrangements.56,57More recently,it has been suggested that7.1expression could be speci?cally associated with only two speci?c subtypes of leukemia harboring either the translocations t(4;11)(q21;q23) or t(9;11)(p13;q23),which encode for the leukemic fusion genes MLL-AF4and MLL-AF9,respectively,but not for other MLL rearrangements.19Importantly,we and many others have reported the existence of acute leukemias and plasmacytoid dendritic cell(pDC)leukemias(450%)lacking MLL rearrange-ments but expressing NG2(Table1).60–62

Based on the controversial data about the clinical relevance of NG2expression together with the existence of NG2-expressing acute leukemias lacking MLL rearrangements,in particular pDC leukemias,60,61we wanted to gain further insight into the biological association between NG2expression and MLL rearrangements.We analyzed whether the expression of NG2 may depend on the particular gene(s)paired to MLL when it is rearranged and we also explored the hypothesis that the expression of NG2in leukemias lacking MLL rearrangements, such as NG2tpDC leukemias,may be due to the existence of a minor subset of CD34thematopoietic stem/progenitor cells readily coexpressing NG2wherein the leukomogenesis process may be initially triggered.62Our experimental data support the clinical?nding of both human leukemias with balanced MLL rearrangements coexpressing NG2and human leukemias harboring balanced MLL gene translocations but lacking NG2expression.Several cellular and molecular mechanisms,intrinsic molecular determinants and extrinsic signals may contribute to the controversial correlation between MLL rearrangements and NG2regulation.Extensive in vitro data rule out the possibility that potential hits/mutations secondary to

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MLL translocations are required for triggering NG2expression as we used fully transformed/immortalized cell lines derived from patients with overt disease,therefore carrying a paramount of cooperating mutations and genetic insults.The possibility that NG2expression could be associated with non-balanced MLL rearrangements such as deletions or inversions has previously been ruled out.54,55Although unlikely,the possibility should not be excluded that NG2expression could be linked to MLL internal duplications,which are not prospectively analyzed in human leukemias at diagnosis.62

We therefore hypothesized that NG2expression may be dependent on the cell of origin where a speci?c leukemic abnormality initially occurs.For instance,NG2might only be regulated when the leukemic abnormality arises either in a lineage-speci?c progenitor(HPC)or in a more immature,less committed stem cell(HSC).In order to con?rm this hypothesis,a rare subset of the CD34tcells is expected to coexpress NG2. When a large number of CB-derived CD34tcells were analyzed by?ow cytometry,63,64coexpression of NG2was readily observed in a subset of CD34tCD38tHPCs from CB (2.1%)(Figure2a),bone marrow(BM)(0.83%)(Figure2b)and mobilized-PB(1.3%)62suggesting that HPCs rather than HSCs may be the target cell for transformation.To verify that this CD34tCD38tNG2tcell subset truly represents HPCs,this population was enriched by?uorescence-activated cell sorting from CB and the cells plated in methylcellulose assays. Importantly,multilineage(CFU-G,CFU-M,CFU-Mix,burst-forming unit erythroid)hematopoietic colonies were obtained in in vitro colony-forming unit(CFU)assays,62suggesting that HPCs rather than HSCs may be the target cell for transformation. The expression of NG2in60%of pDC leukemias lacking MLL rearrangements have recently been reported61(and our unpub-lished observations).We therefore addressed whether pDC CD34tprecursors coexpress NG2.Interestingly,we found that 12.2%of the pDC precursors(CD34t/CD45t/CD38t/ CD123high/HLADRt)65–67readily coexpress NG2(Figure2c). This suggests that,regardless of the status of the MLL locus, the NG2antigen may be expressed in pDC leukemias if the leukomogenesis process is initially triggered in a pDC CD34tprecursor readily expressing NG2,which might act as a leukemic-initiating cell.In fact,based on the observation of NG2tcell lines and NG2thuman primary leukemias lacking MLL rearrangements,our data illustrate that the leukemic abnormality underlying NG2expression does not necessarily need to be a MLL rearrangement.It would be worthwhile to purify both NG2tand NG2àcell subsets from acute leukemias harboring MLL rearrangements and transplant them into immunode?cient mice68,69in order to de?ne which cell subset is more enriched in leukemia-initiating cells.

MLLAF4rearrangement is present and expressed in mesenchymal stem cells(MSCs)from infant patients suffering from MLLAF4tpro-B ALL

Of note,it is also becoming evident that the MLL-AF4tdisease may be even more complex than previously suspected.A very recent study by Stam et al.70evaluated by whole-genome gene expression pro?ling a large cohort of ALL with or without MLL rearrangements.These researchers have provided convincing evidence that MLL-rearranged infants,MLL germline infants and MLL germline non-infant children can be distinguished based on gene expression pro?ling.MLL germline infants clustered closely to MLL-rearranged infants although they could easily be separated.70The close clustering of infant ALL regardless of the MLL status shows gene expression similarities that are likely to be crucial for the biology of these entities.Worth noting,this study also anticipates that infants with HoxA9negative MLL-rearranged ALL may have a much higher likelihood of relapse70 The possibility that human MLL-AF4tleukemia has a different cellular origin than other MLL fusion leukemias is strengthened by this study showing the presence of MLL-AF4but not other MLL fusions in BM MSCs71

There is an increasing body of evidence suggesting that balanced chromosomal translocations resulting in leukemic-speci?c fusion genes associated to childhood leukemias(MLL-AF4,TEL-AML1,AML1-ETO,BCR–ABL,E2A-PBX1,and so on) might be present in MSCs/stroma from the BM of these pediatric patients.The rationale for this hypothesis is based on the following:

There is a considerable proportion of acute leukemias in which the blasts lack the expression of the pan-hematopoietic marker CD45,indicating a potential prehematopoietic origin of the leukemia.

A considerable proportion of tumors secondary to the primary

leukemia are non-hematopoietic mesenchymal tumors (osteosarcomas or soft-tissue sarcomas),72suggesting that the cytotoxic treatment may be effective against the leukemic blasts inside the BM and the hematopoietic tumoral clone whereas unable to destroy the tumor-associated MSCs which seem to somehow escape from the chemotherapy-related cytotoxic effects.

It is well-established that upon an allogeneic transplantation MSCs are more resistant to chemotherapy treatment that the leukemic blasts as suggested by the fact that the BM stroma is commonly receptor-derived and barely donor-derived.73–76 The presence of MSCs harboring these leukemic fusion genes would explain,at least in part,the higher sensibility of the molecular techniques such as quantitative reverse transcrip-tase PCR over?ow cytometry methods for the detection of minimal residual disease because by?ow cytometry we just analyze hematopoietic cells.77

The detection of the BCR/ABL oncogene and lymphoma-speci?c genetic aberrations in endothelial cells from chronic myelogen-ous leukemia and B-cell lymphoma patients suggests that endothelial cells may be part of the neoplastic clone78–80and that hemangioblasts rather than HSCs appear to be target cells for the?rst oncogenic hit,which could occur during the?rst

Table1Published clinical data supporting the lack of association between MLL rearrangements and NG2expression

Reference Diagnosis MLL rearranged/

NG2à

(%cases)

MLL germline/ NG2+ (%cases)

Hilden et al.55Infant AML45F Mauvieux et al.59Pediatric AML34F

Adult AML64F Wuchter et al.57Childhood ALL14F

Childhood AML23F

Adult AML3661 Schwartz et al.56Childhood ALL165–10 Bueno et al.61DC leukemia F60 Our clinical lab data AML F50

ALL F60

Adult AML/ALL4F Abbreviations:ALL,acute lymphoblastic leukemia;AML,acute myeloid leukemia;DC,dendritic cell leukemia;NG2,neuron-glial antigen2.Cellular origin and etiology of the infant pro-B ALL

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steps of ESC differentiation and/or in hemangioblasts persisting in adults.81

The cellular organization and relationships among precursors that initiate embryonic angiogenesis and hematopoiesis in humans have been characterized.82A bipotent primitive hemangioblast derived from hESCs is uniquely responsible for endothelial and hematopoietic development.82There is com-pelling evidence that several of the common chromosome translocations (that is,MLL-AF4,TEL-AML1,AML1-ETO)that are seen in pediatric leukemia often originate in utero during embryonic/fetal development,10,48,49suggesting that such a rearrangement may arise in a mesodermal prehematopoietic precursor/bipotent hemangioblast capable of giving rise to both hematopoietic and endothelial lineages.Furthermore,the BM hematopoietic microenvironment has a role in the pathogenesis of a variety of hematological malignancies including acute leukemia,multiple myeloma,lymphomas or myelodysplastic syndrome 80,83–86and controversy does exist about whether the BM stroma may or may not be part of the tumoral clone.

Based on this background we wanted to ascertain whether common childhood-associated leukemic fusion genes and hyperdiploidy are present in BM MSCs from 38children diagnosed with cytogenetically different acute leukemias.87Fusion genes were absent in BM MSCs of childhood leukemias carrying TEL-AML1,BCR–ABL,AML1-ETO,

MLL-AF9,MLL-AF10,MLL-ENL or hyperdiploidy.However,MLL-AF4was detected by ?uorescence in situ hybridization and inverse PCR (Figure 3a,Table 2)and expressed by real-time PCR (Figure 3b)in BM MSCs from all cases of MLL-AF4tB-ALL.87,88All MLL-AF4tMSCs were consistently euploid,precluding the possibility of cell fusion between a MSC and a leukemic blast.87Importantly,monoclonal VD(J)H immunoglobulin gene rearran-gements were performed.Whereas monoclonal immunoglobu-lin gene rearrangements were consistently detected in MLL-AF4tleukemic blasts,no monoclonal rearrangements could be detected in BM MSCs from any MLL-AF4tB-ALL patient,ruling out potential contamination of the MSC cultures by leukemic cells and suggesting a close early developmental relationship between MSCs and the leukemic blasts rather than plasticity or dedifferentiation of B-ALL blasts.87

Some remarkable differences between these two recent studies caught our attention.87,88Menendez et al .87failed to detect other chimeric mRNAs such as TEL-AML1,BCR–ABL,AML1-ETO,MLL-AF9,MLL-AF10,MLL-ENL or hyperdiploidy.In contrast,Shalapour et al .88clearly demonstrated the presence of TEL-AML1and MLL-ENL in BM MSCs.Whereas we could not detect monoclonal immunoglobulin gene rearrangements in the MLL-AF4-expressing MSCs,Shalapour et al .88demonstrated the presence of monoclonal immunoglobulin gene rearrangements in three out of eight patients.This leukemia–stroma relationship

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Figure 2Flow cytometry analysis of NG2(7.1)antigen in normal CD34tprogenitors and pDC precursors.(a )Expression of NG2in gated CB-derived CD34tcells.Analyses of as many as 2?105CD34tcells revealed that 2.1±2.4%of the CD34tcells coexpress NG2tcells.All these CD34tNG2tcells are CD38t,therefore representing HPCs.An irrelevant isotype-matched antibody was used as a negative control (inset panel).(b )NG2is also expressed in approximately 1%of BM-derived CD34tHPCs.(c )The pDC precursors express NG2.Six color high-speed ?ow cytometry analysis showing the expression of NG2(green dots)in a population of pDC precursors (CD45tCD34tCD38tCD123ttHLADR t).

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seems therefore to be far more complex than previously described and further prospective experiments are still highly demanded.Importantly,endogenous or ectopic expression of MLL-AF4,TEL-AML1or MLL-AF9exerted no effect on MSC culture homeostasis,indicating that these fusion genes themselves are not suf?cient for MSC transformation and their expression in MSCs is compatible with a mesenchymal phenotype,suggesting a differential impact in the hematopoietic system and mesen-chyme.Together,these ?ndings suggest that MSCs may be in part tumor-related,highlighting an unrecognized role of the BM milieu on the pathogenesis of MLL-AF4tB-ALL.The absence of monoclonal rearrangements in MLL-AF4tBM MSCs precludes the possibility of cellular plasticity or dedifferentiation of B-ALL blasts and suggests that MLL-AF4might arise in a population of prehematopoietic precursors.87–89

The above data supporting the expression of MLL-AF4in BM-MSCs from infant ALL challenge our current view and pave the way for further experiments aimed at answering key questions such as where do the leukemia relapses come from?Should the current methods for minimal residual disease

detection be revisited?Do some chromosomal abnormality aberrations prevent or facilitate the establishment of MSC cultures?How is the tumor–stroma relationship de?ned at the molecular level?Is it possible that genetically aberrant MSCs interfere with normal physiological immunesurveillance (that is,augmenting suppression of T-cell effector function or inhibiting DC maturation and proliferation)?

Modeling MLL-AF4tinfant pro-B ALL using hESCs

MLL-AF4leukemogenesis has been particularly dif?cult to model 90and bona ?de MLL-AF4disease models do not exist as of yet.Our little understanding of transformation by MLL fusions and their mode of action come from murine models.Unfortunately,however,in vivo leukemias do not recapitulate the actual human disease.Some success has been achieved recently in the Kersey laboratory by ESC knock in,91but the resultant disease differs signi?cantly from that seen in infant ALL in two respects:(i)the latency is exceptionally protracted;(ii)

the

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Figure 3BM MSCs from infants with MLL-AF4tpro-B ALL harbor and express the MLL-AF4fusion gene.(a )Fluorescence in situ hybridization performed in patient-derived MSCs (top row)and leukemic blasts (bottom row)(n ?38).Leukemia-speci?c fusion genes were always observed in the leukemic https://www.wendangku.net/doc/cc3096067.html,ing a split-apart probe,MLL rearrangements are identi?ed by the presence of one red signal,one green signal and one yellow signal (germline).Using locus-speci?c probes,the fusions TEL-AML1,AML1-ETO and BCR–ABL are determined by the presence of yellow fusion signals (and the derivative chromosome)whereas cells without the translocation have two green (either BCR,TEL or ETO)and two red signals (either ABL or AML1).The white arrows depict the rearranged allele.Bar,100m m.(b )Representative quantitative reverse transcriptase PCR experiments performed in duplicate from two patients showing MLL-AF4transcript expression in MSCs from infants with MLL-AF4tpro-B ALL.

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disease is classi?ed as either myeloproliferative or mature/follicular B.Similarly,Rabbitts’s group has developed and employed the invertor conditional technology to create a mouse model of MLL-AF4,in which a ?oxed AF4complementary DNA was knocked into MLL in the opposite orientation for transcrip-tion.Cell-speci?c Cre expression was used to generate MLL-AF4expression.The mice developed exclusively B-cell lineage neoplasias,but of a more mature phenotype than normally observed in childhood leukemia.92Alternatively,it has been very recently suggested that the presence of both reciprocal MLL fusion proteins (MLL-AF4and AF4-MLL)93or AF4-MLL alone 94confers biological properties known from t(4;11)leukemia,suggesting that each of the two fusion proteins contribute speci?c properties and,in combination,also synergistic effects to the leukemic phenotype.This reproducible but confusing in vitro and in vivo data suggest that these mouse models are missing some essential ingredients of leukemogenesis in the human embryo/fetus.

Potential differences may include particular stem/progenitor cell targets,level of transgene expression or the impact of etiological exposure factors or other events necessary for conversion of MLL-AF4expression.For instance,it has been suggested that the remarkably brief latency of infant ALL might be due to the MLL-AF4-driven preleukemic cells being conti-nuously exposed to the same putative transplacental chemical carcinogens that included the fusion gene itself.3,16–19,21Clearly this ‘etiological’component is missing from the mouse models in relation to rapid acquisition of essential secondary mutations.It is plausible that MLL-AF4speci?cally exerts its function in human cells,indicating that questions regarding target cells,secondary hits and latency have to be addressed using prenatally-derived human stem cells.

Leukemia is generally studied once the full transformation events have already occurred and therefore,the mechanisms by which MLL-AF4transforms to a preleukemic state followed by rapid transition to overt ALL are not amenable to analysis with

Table 2Presence of leukemic fusion genes (and hyperdiploidy)in BM-MSCs from a cohort of infants/children with cytogenetically distinct acute leukemia Patient ID Diagnosis Cytogenetics (fusion gene)Age (months)

Fusion gene

in MSC (%positive MSCs)

Positive cases in each diagnostic group

1PreB-ALL t(12;21)TEL-AML130NO F 2PreB-ALL t(12;21)TEL-AML124NO F 3PreB-ALL t(12;21)TEL-AML136NO F 4PreB-ALL t(12;21)TEL-AML142NO F 5PreB-ALL t(12;21)TEL-AML170NO F 6PreB-ALL t(12;21)TEL-AML184NO 0/12(0%)

7PreB-ALL t(12;21)TEL-AML142NO F 8PreB-ALL t(12;21)TEL-AML196NO F 9PreB-ALL t(12;21)TEL-AML160NO F 10PreB-ALL t(12;21)TEL-AML148NO F 11PreB-ALL t(12;21)TEL-AML130NO F 12PreB-ALL t(12;21)

TEL-AML1

54NO F 13M2-AML t(8;21)AML1-ETO 144NO 0/1(0%)14PreB-ALL t(9;22)BCR–ABL 72NO F 15PreB-ALL t(9;22)BCR–ABL 48NO F 16PreB-ALL t(9;22)BCR–ABL 60NO 0/5(0%)17PreB-ALL t(9;22)BCR–ABL 72NO F 18PreB-ALL t(9;22)

BCR–ABL

72NO F 19T -ALL Hyperdiploid 30NO F 20PreB-ALL Hyperdiploid 48NO F 21PreB-ALL Hyperdiploid 24NO F 22PreB-ALL Hyperdiploid 36NO F 23PreB-ALL Hyperdiploid 36NO 0/10(0%)

24PreB-ALL Hyperdiploid 48NO F 25PreB-ALL Hyperdiploid 142NO F 26PreB-ALL Hyperdiploid 24NO F 27PreB-ALL Hyperdiploid 60NO F 28PreB-ALL Hyperdiploid 18NO F 29Pro-B-ALL t(4;11)MLL-AF46YES (4%)F 30Pro-B-ALL t(4;11)MLL-AF411YES (7%)4/4(100%)

31Pro-B-ALL t(4;11)MLL-AF46YES (7%)F 32Pro-B-ALL t(4;11)

MLL-AF4

4YES

(8%)

F 33M5-AML t(9;11)MLL-AF9180NO F 34M5-AML t(9;11)MLL-AF912NO F 35M2-AML t(9;11)MLL-AF97NO 0/6(0%)36M5-AML t(10;11)MLL-AF1015NO F 37PreB-ALL t(11;19)MLL-ENL 7NO F 38

AML

t(11;19)MLL-ENL 9

Abbreviation:ALL,acute lymphoblastic leukemia;AML:acute myeloid leukemia,BM-MSC,bone marrow mesenchymal stem cell;B-ALL,B-cell acute lymphoblastic leukemia;T -ALL,T -cell acute lymphoblastic leukemia.Note that all the MLL-AF4+ALL patients are infants (o 11months old).Infantile pro-B-ALL cases are shown in bold.

Cellular origin and etiology of the infant pro-B ALL

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patient samples.Two fundamental aspects need to be addressed are(i)the nature of the target cell for transformation and(ii) the mechanisms accounting for the MLL-AF4-B-cell lineage af?liation.MLL-AF4arises prenatally during embryonic/fetal hematopoiesis as evidenced by the analysis of MLL clonotypic breakpoints in leukemic cells of monozygotic twins with ALL10 and the detection of genomic MLL-AF4fusion sequences in archived neonatal blood spots of infants who developed ALL.11 Moreover,the exceptionally high concordance rate of leukemia in monozygotic twin infants,approaching100%,48,49suggests that all necessary genetic events required for leukemogenesis,are accomplished prenatally.49The nature of the cells initially transformed by MLL-AF4in utero is unknown.Infant ALL with MLL-AF4fusion has a pro-B/monocyte phenotype but immuno-selection and?uorescence in situ hybridization studies indicate that the fusion gene is present in a more primitive CD34t/ CD19àcell subset.95No in vivo xenogenic transplantation assay for this leukemia has been reported to date.The leukemia stem cell(LSC)may itself be distinct from the cell in which the MLL-AF4has a preleukemic impact and this in turn,may also differ from the cell in which MLL-AF4arises.Therefore,the hierarchical (stem cells vs progenitor cells)and ontogeny position(prenatal, neonatal vs somatic hematopoiesis),need to be considered when de?ning the‘target’cell in MLL-AF4pathogenesis.

The hESCs were?rst derived by Thomson and collea-gues29,37,96and are envisioned to become a potentially power-ful tool for modeling different aspects of human disease that cannot otherwise be addressed by patient sample analyses or mouse models.Regarding cancer biology,there are different types of childhood mesenchymal tumors,other than acute leukemias,wherein clinically signi?cant manifestations can arise in utero.The fact that cellular transformation manifests as a blockage or altered cell differentiation suggests that hESCs differentiation could become a promising human system for characterizing the emergence of early transformation events that drive cell transformation rather than normal lineage speci?cation.27,28

On the basis of this hypothesis,we have recently begun to explore the developmental impact of MLL-AF4in human embryonic and neonatal stem cell fate.Our preliminary data based on the development of transgenic MLL-AF4-expressing hESCs suggest that this fusion gene may have developmental effects not only in the hESC-derived hematopoietic cells but also on more primitive prehematopoietic precursors such as the hemangioblasts as the ectopic expression of MLL-AF4seems to skew the hematopoietic vs the endothelial differentiation arising from these MLL-AF4-expressing hESCs(data not shown). Experiments are also underway to address whether MLL-AF4is capable of transforming hECSs or their progeny on its own or,in contrast,it requires additional secondary cooperating mutations such as FLT3-activating mutations.6,97,98

Con?ict of interest

The authors declare no con?ict of interest.

Acknowledgements

PM’s group is funded by the CSJA(0029/2006to PM)and CICE (P08-CTS-3678to PM)de la Junta de Andaluc?′a,the FIS/FEDER to PM(PI070026&PI100449)and CB(CP07/00059)and the MICINN to PM(PLE-2009-0111).PM and CB have been partially supported by the International Leukemia Foundation Josep Carreras(ED-Thomas-05).RR is supported by the Spanish Association against Cancer(AECC).We are indebted to Dr Isidro

Prat and Dr Mar?′a del Carmen Hernandez from the Malaga

Cord Blood Bank for provision of CB units and Prof Mel Greaves,

Dr Gustavo J Mele′n,Dr Javier Garc?′a-Castro,Dr Ramo′n

Garc?′a-Castro and Dr Alberto Orfao for their critical insights and

fruitful discussions.

References

1Caslini C,Alarcon AS,Hess JL,Tanaka R,Murti KG,Biondi A.

The amino terminus targets the mixed lineage leukemia(MLL)

protein to the nucleolus,nuclear matrix and mitotic chromosomal

scaffolds.Leukemia2000;14:1898–1908.

2Eguchi M,Eguchi-Ishimae M,Knight D,Kearney L,Slany R,

Greaves M.MLL chimeric protein activation renders cells

vulnerable to chromosomal damage:an explanation for the very

short latency of infant leukemia.Genes Chromosomes Cancer

2006;45:754–760.

3Eguchi M,Eguchi-Ishimae M,Greaves M.Molecular pathogenesis

of MLL-associated leukemias.Int J Hematol2005;82:9–20.

4Krivtsov AV,Armstrong SA.MLL translocations,histone modi?ca-

tions and leukaemia stem-cell development.Nat Rev Cancer2007;

7:823–833.

5Pui CH.Acute lymphoblastic leukemia in children.Curr Opin

Oncol2000;12:3–12.

6Ono R,Nakajima H,Ozaki K,Kumagai H,Kawashima T,Taki T

et al.Dimerization of MLL fusion proteins and FLT3activation

synergize to induce multiple-lineage leukemogenesis.J Clin Invest

2005;115:919–929.

7Taketani T,Taki T,Sugita K,Furuichi Y,Ishii E,Hanada R et al.

FLT3mutations in the activation loop of tyrosine kinase domain

are frequently found in infant ALL with MLL rearrangements and

pediatric ALL with hyperdiploidy.Blood2004;103:1085–1088.

8Bardini M,Spinelli R,Bungaro S,Mangano E,Corral L,Cifola I

et al.DNA copy-number abnormalities do not occur in infant ALL

with t(4;11)/MLL-AF4.Leukemia2010;24:169–176.

9Moorman AV,Hagemeijer A,Charrin C,Rieder H,Secker-Walker LM.

The translocations,t(11;19)(q23;p13.1)and t(11;19)(q23;p13.3):a

cytogenetic and clinical pro?le of53patients.European11q23

Workshop participants.Leukemia1998;12:805–810.

10Ford AM,Ridge SA,Cabrera ME,Mahmoud H,Steel CM,Chan LC et al.In utero rearrangements in the trithorax-related oncogene in

infant leukaemias.Nature1993;363:358–360.

11Gale KB,Ford AM,Repp R,Borkhardt A,Keller C,Eden OB et al.

Backtracking leukemia to birth:identi?cation of clonotypic gene

fusion sequences in neonatal blood spots.Proc Natl Acad Sci USA

1997;94:13950–13954.

12Alexander FE,Patheal SL,Biondi A,Brandalise S,Cabrera ME, Chan LC et al.Transplacental chemical exposure and risk of infant

leukemia with MLL gene fusion.Cancer Res2001;61:2542–2546.

13Ross JA,Potter JD,Reaman GH,Pendergrass TW,Robison LL.

Maternal exposure to potential inhibitors of DNA topoisomerase II

and infant leukemia(United States):a report from the Children’s

cancer group.Cancer Causes Control1996;7:581–590.

14Barjesteh van Waalwijk van Doorn-Khosrovani S,Janssen J, Maas LM,Godschalk RW,Nijhuis JG,van Schooten FJ.Dietary

?avonoids induce MLL translocations in primary human CD34+

cells.Carcinogenesis2007;28:1703–1709.

15Blanco JG,Edick MJ,Relling MV.Etoposide induces chimeric Mll gene fusions.FASEB J2004;18:173–175.

16Bueno C,Catalina P,Melen GJ,Montes R,Sanchez L,Ligero G et al.Etoposide induces MLL rearrangements and other chromo-

somal abnormalities in human embryonic stem cells.Carcino-

genesis2009;30:1628–1637.

17Libura J,Slater DJ,Felix CA,Richardson C.Therapy-related acute myeloid leukemia-like MLL rearrangements are induced by

etoposide in primary human CD34+cells and remain stable after

clonal expansion.Blood2005;105:2124–2131.

18Libura J,Ward M,Solecka J,Richardson C.Etoposide-initiated MLL rearrangements detected at high frequency in human

primitive hematopoietic stem cells with in vitro and in vivo long-

term repopulating potential.Eur J Haematol2008;81:185–195.

Cellular origin and etiology of the infant pro-B ALL

C Bueno et al

407

Leukemia

19Moneypenny CG,Shao J,Song Y,Gallagher EP.MLL rearrange-ments are induced by low doses of etoposide in human fetal hematopoietic stem cells.Carcinogenesis2006;27:874–881.

20Zandvliet DW,Hanby AM,Austin CA,Marsh KL,Clark IB,Wright NA et al.Analysis of foetal expression sites of human type II DNA topoisomerase alpha and beta mRNAs by in situ hybridisation.

Biochim Biophys Acta1996;1307:239–247.

21Felix CA.Secondary leukemias induced by topoisomerase-targeted drugs.Biochim Biophys Acta1998;1400:233–255.

22Rowley JD,Olney HJ.International workshop on the relationship of prior therapy to balanced chromosome aberrations in therapy-related myelodysplastic syndromes and acute leukemia:overview report.Genes Chromosomes Cancer2002;33:331–345.

23Chatterjee S,Trivedi D,Petzold SJ,Berger NA.Mechanism of epipodophyllotoxin-induced cell death in poly(adenosine diphosphate-ribose)synthesis-de?cient V79Chinese hamster cell lines.Cancer Res1990;50:2713–2718.

24Spector LG,Xie Y,Robison LL,Heerema NA,Hilden JM,Lange B et al.Maternal diet and infant leukemia:the DNA topoisomerase II inhibitor hypothesis:a report from the children’s oncology group.

Cancer Epidemiol Biomarkers Prev2005;14:651–655.

25Strick R,Strissel PL,Borgers S,Smith SL,Rowley JD.Dietary bio?avonoids induce cleavage in the MLL gene and may contribute to infant leukemia.Proc Natl Acad Sci USA2000;97: 4790–4795.

26Ross JA,Potter JD,Robison LL.Infant leukemia,topoisomerase II inhibitors,and the MLL gene.J Natl Cancer Inst1994;86: 1678–1680.

27Bueno C,Garcia-Castro J,Montes R,Menendez P.Human embryonic stem cells:a potential system for modeling infant leukemia harboring MLL-AF4fusion gene.Drug Discov Today:Dis Models2008;4:53–60.

28Lensch MW,Daley GQ.Scienti?c and clinical opportunities for modeling blood disorders with embryonic stem cells.Blood2006;

107:2605–2612.

29Menendez P,Bueno C,Wang L,Bhatia M.Human embryonic stem cells:potential tool for achieving immunotolerance?Stem Cell Rev2005;1:151–158.

30Edick MJ,Gajjar A,Mahmoud HH,van de Poll ME,Harrison PL, Panetta JC et al.Pharmacokinetics and pharmacodynamics of oral etoposide in children with relapsed or refractory acute lympho-blastic leukemia.J Clin Oncol2003;21:1340–1346.

31Catalina P,Cobo F,Cortes JL,Nieto AI,Cabrera C,Montes R et al.Conventional and molecular cytogenetic diagnostic methods in stem cell research:a concise review.Cell Biol Int2007;31: 861–869.

32Catalina P,Montes R,Ligero G,Sanchez L,de la Cueva T,Bueno C et al.Human ESCs predisposition to karyotypic instability:is a matter of culture adaptation or differential vulnerability among hESC lines due to inherent properties?Mol Cancer2008;7:76. 33Cobo F,Navarro JM,Herrera MI,Vivo A,Porcel D,Hernandez C et al.Electron microscopy reveals the presence of viruses in mouse embryonic?broblasts but neither in human embryonic?bro-blasts nor in human mesenchymal cells used for hESC main-tenance:toward an implementation of microbiological quality assurance program in stem cell banks.Cloning Stem Cells2008;

10:65–74.

34Cortes JL,Sanchez L,Catalina P,Cobo F,Bueno C,Martinez-Ramirez A et al.Whole-blastocyst culture followed by laser drilling technology enhances the ef?ciency of inner cell mass isolation and embryonic stem cell derivation from good-and poor-quality mouse embryos:new insights for derivation of human embryonic stem cell lines.Stem Cells Dev2008;17:255–267. 35Cortes JL,Sanchez L,Ligero G,Gutierrez-Aranda I,Catalina P, Elosua C et al.Mesenchymal stem cells facilitate the derivation of human embryonic stem cells from cryopreserved poor-quality embryos.Hum Reprod2009;24:1844–1851.

36Gutierrez-Aranda I,Ramos-Mejia V,Bueno C,Mun?oz-Lo′pez M, Real JP,Macia A et al.Human induced pluripotent stem cells develop teratoma more ef?ciently and faster than human embryonic stem cells regardless the site of injection.Stem Cells 2010;28:1568–1570.

37Menendez P,Bueno C,Wang L.Human embryonic stem cells:a journey beyond cell replacement therapies.Cytotherapy2006;8: 530–541.38Menendez P,Wang L,Chadwick K,Li L,Bhatia M.Retroviral transduction of hematopoietic cells differentiated from human embryonic stem cell-derived CD45(neg)PFV hemogenic precur-sors.Mol Ther2004;10:1109–1120.

39Montes R,Ligero G,Sanchez L,Catalina P,de la Cueva T,Nieto A et al.Feeder-free maintenance of hESCs in mesenchymal stem cell-conditioned media:distinct requirements for TGF-beta and IGF-II.Cell Res2009;19:698–709.

40Ramos-Mejia V,Melen GJ,Sanchez L,Gutierrez-Aranda I,Ligero G,Cortes JL et al.Nodal/Activin signaling predicts human pluripotent stem cell lines prone to differentiate towards the hematopoietic lineage.Mol Ther2010;18:2173–2181.

41Ramos-Mejia V,Mun?oz-Lo′pez M,Garc?′a-Pe′rez JL,Menendez P.

iPSC lines which do not silence the expression of the ectopic reprogramming factors may display enhanced propensity to genomic instability.Cell Res2010;20:1092–1095.

42Greaves M.Infection,immune responses and the aetiology of childhood leukaemia.Nat Rev Cancer2006;6:193–203.

43Preston DL,Kusumi S,Tomonaga M,Izumi S,Ron E,Kuramoto A et al.Cancer incidence in atomic bomb survivors.Part III.

Leukemia,lymphoma and multiple myeloma,1950–1987.Radiat Res1994;137:S68–S97.

44Doll R,Wakeford R.Risk of childhood cancer from fetal irradiation.Br J Radiol1997;70:130–139.

45McNally RJ,Eden TO.An infectious aetiology for childhood acute leukaemia:a review of the evidence.Br J Haematol2004;127: 243–263.

46Greaves MF.Speculations on the cause of childhood acute lymphoblastic leukemia.Leukemia1988;2:120–125.

47Greaves MF.Aetiology of acute https://www.wendangku.net/doc/cc3096067.html,ncet1997;349: 344–349.

48Greaves MF,Maia AT,Wiemels JL,Ford AM.Leukemia in twins: lessons in natural history.Blood2003;102:2321–2333.

49Greaves MF,Wiemels J.Origins of chromosome translocations in childhood leukaemia.Nat Rev Cancer2003;3:639–649.

50Enver T,Tsuzuki S,Brown J,Hong D,Gupta R,Ford T et al.

Developmental impact of leukemic fusion genes on stem cell fate.

Ann N Y Acad Sci2005;1044:16–23.

51Stallcup WB,Cohn M.Correlation of surface antigens and cell type in cloned cell lines from the rat central nervous system.Exp Cell Res1976;98:285–297.

52Smith FO,Rauch C,Williams DE,March CJ,Arthur D,Hilden J et al.The human homologue of rat NG2,a chondroitin sulfate proteoglycan,is not expressed on the cell surface of normal hematopoietic cells but is expressed by acute myeloid leukemia blasts from poor-prognosis patients with abnormalities of chromo-some band11q23.Blood1996;87:1123–1133.

53Levine JM,Nishiyama A.The NG2chondroitin sulfate proteogly-can:a multifunctional proteoglycan associated with immature cells.Perspect Dev Neurobiol1996;3:245–259.

54Behm FG,Smith FO,Raimondi SC,Pui CH,Bernstein ID.

Human homologue of the rat chondroitin sulfate proteoglycan, NG2,detected by monoclonal antibody7.1,identi?es childhood acute lymphoblastic leukemias with t(4;11)(q21;q23)or t(11;19) (q23;p13)and MLL gene rearrangements.Blood1996;87: 1134–1139.

55Hilden JM,Smith FO,Frestedt JL,McGlennen R,Howells WB, Sorensen PH et al.MLL gene rearrangement,cytogenetic11q23 abnormalities,and expression of the NG2molecule in infant acute myeloid leukemia.Blood1997;89:3801–3805.

56Schwartz S,Rieder H,Schlager B,Burmeister T,Fischer L,Thiel E.

Expression of the human homologue of rat NG2in adult acute lymphoblastic leukemia:close association with MLL rearrange-ment and a CD10(à)/CD24(à)/CD65s(+)/CD15(+)B-cell pheno-type.Leukemia2003;17:1589–1595.

57Wuchter C,Harbott J,Schoch C,Schnittger S,Borkhardt A, Karawajew L et al.Detection of acute leukemia cells with mixed lineage leukemia(MLL)gene rearrangements by?ow cytometry using monoclonal antibody7.1.Leukemia2000;14:1232–1238. 58Zangrando A,Intini F,te Kronnie G,Basso G.Validation of NG2 antigen in identifying BP-ALL patients with MLL rearrangements using qualitative and quantitative?ow cytometry:a prospective study.Leukemia2008;22:858–861.

59Mauvieux L,Delabesse E,Bourquelot P,Radford-Weiss I, Bennaceur A,Flandrin G et al.NG2expression in MLL rearranged

Cellular origin and etiology of the infant pro-B ALL

C Bueno et al 408

Leukemia

acute myeloid leukaemia is restricted to monoblastic cases.

Br J Haematol1999;107:674–676.

60Almeida J,Bueno C,Alguero MC,Sanchez ML,Canizo MC, Fernandez ME et al.Extensive characterization of the immuno-phenotype and pattern of cytokine production by distinct subpopulations of normal human peripheral blood MHC II+/ lineage-cells.Clin Exp Immunol1999;118:392–401.

61Bueno C,Almeida J,Lucio P,Marco J,Garcia R,de Pablos JM et al.

Incidence and characteristics of CD4(+)/HLA DRhi dendritic cell malignancies.Haematologica2004;89:58–69.

62Bueno C,Montes R,Martin L,Prat I,Hernandez MC,Orfao A et al.

NG2antigen is expressed in CD34+HPCs and plasmacytoid dendritic cell precursors:is NG2expression in leukemia depen-dent on the target cell where leukemogenesis is triggered?

Leukemia2008;22:1475–1478.

63Bueno C,Montes R,Menendez P.The ROCK inhibitor Y-27632 negatively affects the expansion/survival of both fresh and cryopreserved cord blood-derived CD34+hematopoietic progeni-tor cells.Stem Cell Rev2010;6:215–223.

64Menendez P,Redondo O,Rodriguez A,Lopez-Berges MC, Ercilla G,Lopez A et https://www.wendangku.net/doc/cc3096067.html,parison between a lyse-and-then-wash method and a lyse-non-wash technique for the enumeration of CD34+hematopoietic progenitor cells.Cytometry1998;34: 264–271.

65Matarraz S,Lopez A,Barrena S,Fernandez C,Jensen E,Flores J et al.The immunophenotype of different immature,myeloid and B-cell lineage-committed CD34+hematopoietic cells allows discrimination between normal/reactive and myelodysplastic syndrome precursors.Leukemia2008;22:1175–1183.

66Menendez P,Caballero MD,Prosper F,Del Canizo MC,Perez-Simon JA,Mateos MV et al.The composition of leukapheresis products impacts on the hematopoietic recovery after autologous transplantation independently of the mobilization regimen.Trans-fusion2002;42:1159–1172.

67Menendez P,Perez-Simon JA,Mateos MV,Caballero MD, Gonzalez M,San-Miguel JF et al.In?uence of the different CD34+and CD34-cell subsets infused on clinical outcome after non-myeloablative allogeneic peripheral blood transplant-ation from human leucocyte antigen-identical sibling donors.

Br J Haematol2002;119:135–143.

68Bueno C,Montes R,de la Cueva T,Gutierrez-Aranda I, Menendez P.Intra-bone marrow transplantation of human CD34(+)cells into NOD/LtSz-scid IL-2rgamma(null)mice permits multilineage engraftment without previous irradiation.Cyto-therapy2010;12:45–49.

69Levac K,Menendez P,Bhatia M.Intra-bone marrow transplanta-tion facilitates pauci-clonal human hematopoietic repopulation of NOD/SCID/beta2m(à/à)mice.Exp Hematol2005;33: 1417–1426.

70Stam RW,Schneider P,Hagelstein JA,van der Linden MH, Stumpel DJ,de Menezes RX et al.Gene expression pro?ling-based dissection of MLL translocated and MLL germline acute lympho-blastic leukemia in infants.Blood2010;115:2835–2844.

71Kumar A,Kersey J.Infant ALL:diverse origins and outcomes.

Blood2010;115:2835.

72Kersun LS,Wimmer RS,Hoot AC,Meadows AT.Secondary malignant neoplasms of the bladder after cyclophosphamide treatment for childhood acute lymphocytic leukemia.Pediatr Blood Cancer2004;42:289–291.

73Garcia-Castro J,Balas A,Ramirez M,Perez-Martinez A,Madero L, Gonzalez-Vicent M et al.Mesenchymal stem cells are of recipient origin in pediatric transplantations using umbilical cord blood, peripheral blood,or bone marrow.J Pediatr Hematol Oncol2007;

29:388–392.

74Koc ON,Peters C,Aubourg P,Raghavan S,Dyhouse S,DeGasperi R et al.Bone marrow-derived mesenchymal stem cells remain host-derived despite successful hematopoietic engraftment after allogeneic transplantation in patients with lysosomal and perox-isomal storage diseases.Exp Hematol1999;27:1675–1681.

75Rieger K,Marinets O,Fietz T,Korper S,Sommer D,Mucke C et al.

Mesenchymal stem cells remain of host origin even a long time after allogeneic peripheral blood stem cell or bone marrow transplantation.Exp Hematol2005;33:605–611.

76Stute N,Fehse B,Schroder J,Arps S,Adamietz P,Held KR et al.

Human mesenchymal stem cells are not of donor origin in patients

with severe aplastic anemia who underwent sex-mismatched

allogeneic bone marrow transplant.J Hematother Stem Cell Res

2002;11:977–984.

77Bruggemann M,Schrauder A,Raff T,Pfeifer H,Dworzak M, Ottmann OG et al.Standardized MRD quanti?cation in European

ALL trials:proceedings of the Second International Symposium on

MRD assessment in Kiel,Germany,18–20September2008.

Leukemia2010;24:521–535.

78Fang B,Zheng C,Liao L,Han Q,Sun Z,Jiang X et al.Identi?cation of human chronic myelogenous leukemia progenitor cells with

hemangioblastic characteristics.Blood2005;105:2733–2740.

79Gunsilius E,Duba HC,Petzer AL,Kahler CM,Grunewald K, Stockhammer G et al.Evidence from a leukaemia model for

maintenance of vascular endothelium by bone-marrow-derived

endothelial https://www.wendangku.net/doc/cc3096067.html,ncet2000;355:1688–1691.

80Streubel B,Chott A,Huber D,Exner M,Jager U,Wagner O et al.

Lymphoma-speci?c genetic aberrations in microvascular endothe-

lial cells in B-cell lymphomas.N Engl J Med2004;351:250–259.

81Prindull G.Hemangioblasts representing a functional endothelio-hematopoietic entity in ontogeny,postnatal life,and CML

neovasculogenesis.Stem Cell Rev2005;1:277–284.

82Wang L,Li L,Shojaei F,Levac K,Cerdan C,Menendez P et al.

Endothelial and hematopoietic cell fate of human embryonic stem

cells originates from primitive endothelium with hemangioblastic

properties.Immunity2004;21:31–41.

83Blau O,Hofmann WK,Baldus CD,Thiel G,Serbent V, Schumann E et al.Chromosomal aberrations in bone marrow

mesenchymal stroma cells from patients with myelodysplastic

syndrome and acute myeloblastic leukemia.Exp Hematol2007;

35:221–229.

84Corre J,Mahtouk K,Attal M,Gadelorge M,Huynh A,Fleury-Cappellesso S et al.Bone marrow mesenchymal stem cells are

abnormal in multiple myeloma.Leukemia2007;21:1079–1088.

85Lopez-Villar O,Garcia JL,Sanchez-Guijo FM,Robledo C,Villaron EM,Hernandez-Campo P et al.Both expanded and uncultured

mesenchymal stem cells from MDS patients are genomically

abnormal,showing a speci?c genetic pro?le for the5q-syndrome.

Leukemia2009;23:664–672.

86Walkley CR,Qudsi R,Sankaran VG,Perry JA,Gostissa M,Roth SI et al.Conditional mouse osteosarcoma,dependent on p53loss

and potentiated by loss of Rb,mimics the human disease.Genes

Dev2008;22:1662–1676.

87Menendez P,Catalina P,Rodriguez R,Melen GJ,Bueno C,Arriero M et al.Bone marrow mesenchymal stem cells from infants with

MLL-AF4+acute leukemia harbor and express the MLL-AF4fusion

gene.J Exp Med2009;206:3131–3141.

88Shalapour S,Eckert C,Seeger K,Pfau M,Prada J,Henze G et al.

Leukemia-associated genetic aberrations in mesenchymal stem

cells of children with acute lymphoblastic leukemia.J Mol Med

2010;88:249–265.

89Borkhardt A.Where do the leukaemia relapses come from?J Mol Med2010;88:219–222.

90Daser A,Rabbitts TH.The versatile mixed lineage leukaemia gene MLL and its many associations in leukaemogenesis.Semin Cancer

Biol2005;15:175–188.

91Chen W,Li Q,Hudson WA,Kumar A,Kirchhof N,Kersey JH.

A murine Mll-AF4knock-in model results in lymphoid and

myeloid deregulation and hematologic malignancy.Blood2006;

108:669–677.

92Metzler M,Forster A,Pannell R,Arends MJ,Daser A,Lobato MN et al.A conditional model of MLL-AF4B-cell tumourigenesis using

invertor technology.Oncogene2006;25:3093–3103.

93Gaussmann A,Wenger T,Eberle I,Bursen A,Bracharz S,Herr I et https://www.wendangku.net/doc/cc3096067.html,bined effects of the two reciprocal t(4;11)fusion proteins

MLL AF4and AF4MLL confer resistance to apoptosis,cell

cycling capacity and growth transformation.Oncogene2007;26:

3352–3363.

94Bursen A,Schwabe K,Ruster B,Henschler R,Ruthardt M, Dingermann T et al.The AF4.MLL fusion protein is capable of

inducing ALL in mice without requirement of MLL.AF4.Blood

2010;115:3570–3579.

95Hot?lder M,Rottgers S,Rosemann A,Schrauder A,Schrappe M, Pieters R et al.Leukemic stem cells in childhood high-risk

ALL/t(9;22)and t(4;11)are present in primitive lymphoid-restricted

CD34+CD19-cells.Cancer Res2005;65:1442–1449.

Cellular origin and etiology of the infant pro-B ALL

C Bueno et al

409

Leukemia

96Thomson JA,Itskovitz-Eldor J,Shapiro SS,Waknitz MA,Swiergiel JJ,Marshall VS et al.Embryonic stem cell lines derived from human blastocysts.Science1998;282:1145–1147.

97Kottaridis PD,Gale RE,Frew ME,Harrison G,Langabeer SE, Belton AA et al.The presence of a FLT3internal tandem duplication in patients with acute myeloid leukemia(AML) adds important prognostic information to cytogenetic risk group and response to the?rst cycle of chemotherapy:analysis of854

patients from the United Kingdom Medical Research Council AML 10and12trials.Blood2001;97:752–759.

98Kottaridis PD,Gale RE,Langabeer SE,Frew ME,Bowen DT, Linch DC.Studies of FLT3mutations in paired presentation and relapse samples from patients with acute myeloid leukemia: implications for the role of FLT3mutations in leukemogenesis, minimal residual disease detection,and possible therapy with FLT3inhibitors.Blood2002;100:2393–2398.

Cellular origin and etiology of the infant pro-B ALL

C Bueno et al 410

Leukemia