REVIEW Characteristics and Analysis of Normal and Leukemic Stem Cells

Characteristics and analysis of normal and leukemic stem cells: current concepts and future directions C Brendel and A Neubauer

Department of Hematology/Oncology/Immunology at the Universita¨tsklinikum of the Philipps-Universita¨t Marburg, Baldingerstrasse, 35043 Marburg, Germany

Acute myeloid leukemias (AML) are considered to be clonal dis- differentiated progeny, co-express the progenitor cell antigen orders involving early hematopoietic progenitor cells. The CD34 and lineage markers. These cells are capable of colony recent advances in characterization of early stem cells give rise to the question whether it is possible to distinguish healthy formation in short-term assays termed clonogenic assays such progenitors from cells of the leukemic clone in leukemia as colony-forming units (CFUs) or burst-forming units (BFUs). patients. Differences and similarities in phenotype, genotype In the case of myeloid differentiation they typically carry the and biology are described for leukemic cells and normal hema- CD33 and CD13 antigen. tological progenitors. Recent new insights into human stem The leading concept has been that stem cells harbor the cell development offer the perspective that distinction between CD34 antigen. This led to widespread use of CD34-enriched benign and malignant progenitors might be possible in the future at a very early stage of maturation. Leukemia (2000) 14, cell populations in human transplantation. To distinguish the 1711–1717. small amount of real stem cells with self-renewal capacity Keywords: stem cell; acute myeloid leukemia; CD34; antigen; from the majority of CD34-positive cells that have already mutation; cytogenetic aberration acquired some degree of differentiation is still the focus of intense interest since the discovery of the CD34 antigen.2 Different model systems are applied to define stem cells Introduction with pluripotent hematopoietic potential. Many groups use irradiated non-obese diabetic mice with severe combined For decades, morphology and histochemical staining have immunodeficiency syndrome (NOD/SCID mice) as recipients been the gold standards for definition of AML according to for human candidate progenitor cells. Others apply in vitro the FAB (French–American–British Cooperative Group) classi- cell culture systems to identify the cells of interest such as fication.1 Immunophenotyping and molecular genetics long-term culture-initiating cells (LTC-ICs) and limiting brought new insights with considerable therapeutic impli- dilution assays. The sheep in utero transplantation system cations into the biology of these diseases. Besides standard seems to be most reliable, because the human progenitor cells chemotherapy hematopoietic stem cell transplantation is an are transferred into the sheep fetus in utero before the important treatment option for AML. If HLA matched related development of the ovine immune system.3,4 There is still or unrelated donors are not available, autologous trans- controversy about suitability and comparability of these model plantation has to be considered as an additional treatment systems for the definition of pluripotent stem cells.5 choice. Therefore, distinction between healthy and malignant Largely through application of in vitro assays, the surface stem cells has become a major focus of interest and various antigen expression pattern of human hematopoietic stem cells + − purging techniques have been proposed in order to improve has been defined as CD34 CD38 . Several other antigens remission rates and duration. have been described to accompany CD34 expression. Their presence or absence has been reported to define different stem cell populations. The CD45 antigen is expressed on primitive Current concept of hematopoietic stem cell development hematopoietic stem cells as well as on all nucleated peripheral blood cells. If the CD34 cells carry the CD90 antigen It is a well established model that blood cells derive from (Thy-1), they are thought to represent early progenitor cells pluripotent progenitor cells with the capacity for self-renewal with superior proliferation and differentiation potential com- − and differentiation. More differentiated cells are generally pared to CD90 cells.6,7 The same is the case for the AC133 considered to be more committed to a certain lineage with antigen8 and the data suggest that the CD82 antigen may also concomitant loss of self-renewal capacity. Although cell mor- be expressed on stem cells.9 Recently, the KDR receptor also phology provides good information about the developmental known as vascular endothelial growth factor receptor 2 stages of bone marrow cells, immunophenotyping of blood (VEGFR2) has been reported to be a new marker that cells is of great help in identifying lineage determination distinguishes pluripotent hematopoietic stem cells from lin- especially in early progenitor cell compartments. Differen- eage-committed stem cells.10 In contrast to this finding, other tiated cells display distinct patterns of antigen expression, like groups report that VEGFR may define a subtype of hematopo- CD11b, CD13, CD33, CD14, CD15 and CD66 antigens on ietic progenitor cells with endothelial features.11 The signal- myelomonocytic cells. These lineage markers (lin+) are not regulatory proteins (SIRPs) have been described very recently expressed on early hematopoietic cells. Cells of intermediate as candidate stem cell proteins that are co-expressed on bone + + + differentiation level, which are still capable of giving rise to marrow CD34 , AC133 and CD90 cells.12 Expression of CD117 antigen (c-kit),13 a receptor tyrosine kinase, is low on early human CD34 cells and subsequently becomes more 14 Correspondence: A Neubauer; Fax: +49-6421-286-6358 detectable as the maturation process continues. This pattern Received 6 April 2000; accepted 20 June 2000 of antigen expression is also true for HLA-DR15 and CD38,16 Review C Brendel and A Neubauer 1712 Flt-3 receptor and interleukin-6 (IL-6) receptor signal transduc- and inversions of chromosome 16 have been described most ing element (gp 130),17 as well as CD71, the transferrin recep- commonly in AML. Quite often AML patients harbor a leuk- tor.18 It varies with the source of progenitor cells.16 emic clone with multiple cytogenetic aberrations. The dogma of the CD34-positive multipotent stem cell has During the past years, there has been extensive research on been questioned for about 3 to 4 years. Several groups the molecular background of these chromosomal aberrations reported that the earliest hematopoietic progenitor cell has a which elucidated the understanding of leukemogenesis. The CD34−lin− phenotype19–21 characterized by the CD45 antigen t(8;21), which characteristically occurs in FAB M2, results in and lack of expression of c-kit, Thy-1 and lineage markers. the AML1-ETO fusion protein. AML1 is the α-subunit of core Expression of CD38 seems to be variable.22 At first sight these binding factor (CBF) that heterodimerizes with the β-subunit findings appear to be in contrast to the established model of and functions as a transcription factor which plays a key role CD34+ CD38− phenotype of pluripotent progenitor cells. in hematopoiesis. The chimeric protein retains the ability to Other recent publications confirmed the idea of CD34-nega- heterodimerize and bind DNA, but potentially inhibits tran- tive earliest stem cells and present intriguing new data about scription.31,32 It also acts as a transactivator for other genes33 progenitor cell development, demonstrating the reversibility and has been shown to inhibit terminal granulocytic differen- of CD34 expression on pluripotent hematopoietic cells. This tiation in cell lines.34 was confirmed in the murine system23 as well as in humans.24 In inversion 16 or less common t(16;16) the β-subunit of A new model was consequently proposed: CD34 expression CBF is altered due to fusion with the tail domain of a smooth may represent a reversible activation state of hematopoietic muscle myosin heavy chain (SMMYHC), resulting in stem cells, as revised by Goodell.25 multimerized complexes of CBFα and CBFβ subunits. These During the past year some groups reported that adult human complexes are sequestered preferentially therefore interfering stem cells have the ability to reconstitute human bone marrow with DNA binding of CBF and its transactivation function.35 even across the tissue border. They showed that stem cells Inversion of chromosome 16 is associated with AML FAB sub- from the adult brain retained the youthful ability to become type M4eo. several different kinds of tissues. It has been demonstrated that The FAB M3 leukemia has a unique translocation pattern, gene-marked adult neural stem cells were able to engraft the the t(15;17), which results in fusion of the retinoic acid recep- bone marrow of sublethally irradiated mice and may thus tor (RARα) to the promyelocytic leukemia gene (PML). DNA function as hematopoietic progenitor cells capable of differen- binding of this alterated receptor is inappropriate causing a tiating into myeloid and lymphoid progeny.26 Additionally, differentiation blockage in myeloid cells. non-hematopoietic cells isolated from murine adult skeletal However, for other less common translocations occurring muscle seemed to exhibit even more bone marrow in AML such as t(3;21), t(12;21), t(9;22), etc, loss or gain of reconstituting hematopoietic activity as compared to whole function from genes involved in those lesions have also been bone marrow.27 These data evoked controversy of the view described intensively.35–39 Excellent reviews on the genes that the developmental potential of stem cells is restricted to involved in AML with those common translocations providing the differentiated elements of the tissue in which they reside. extensive information about the molecular details and the Figure 1 summarizes these data and hypotheses about normal consecutive biologic effects are presented by Appelbaum et stem cell phenotype and maturation. al40 and Friedman.35 In addition, there are many reports about gene alterations in AML without any defined gross chromosomal abnormality Characterization of the malignant cell clone in acute such as ras-oncogene mutations,41–43 p53 mutations,44,45 p- myeloid leukemia glycoprotein expression,46 internal tandem dublication of flt3 receptor,47–49 c-myc amplification,50,51 hox overexpression52 Acute myeloid leukemia (AML) is thought to be a clonal dis- or nm23 expression.53 These gene alterations are neither spe- order of poorly differentiated hematopoietic progenitor cells. cific for AML FAB subtypes nor do they probably represent The current concept of malignancy is based on the idea that causative lesions. But since some typical translocations like altered gene function either confers a growth advantage to a t(8;21) and inv 16 may not be sufficient to induce the full cell or prevents apoptosis. Activation of oncogenes or loss of malignant potential, these gene mutations may be secondary function of tumor suppressor genes, disturbance of DNA hits in the process of multistep-carcinogenesis54,55 (revised in repair genes, alteration of cell cycle genes, etc, results in fac- Ref. 35). Some translocations and gene alterations have sig- tor-independent uncontrolled growth and lack of contact inhi- nificant prognostic value indicating a more favorable or fatal bition. The idea of a clonal evolution process of malignancy outcome.42,56 is an important part of that concept, meaning that no single event transforms a cell but additional genetic alterations accumulate and synergize in growth promotion or apotosis Differentiation between healthy and malignant progenitor inhibition.28 This concept is called ‘multistep-carcinogenesis’. cells in acute myeloid leukemia Many different kinds of mutations are known to result in altered gene function, eg point mutations, deletions or Although extensive research has provided a better understand- inversions. These aberrations are often observed in solid ing of how chromosomal aberrations can disturb gene func- tumors. In leukemia and lymphomas, however, gross chromo- tion resulting in growth advantage or block of differentiation, somal alterations like balanced translocations occur quite fre- it is still difficult to distinguish normal from leukemic stem quently, resulting in fusion of unrelated genes and sub- cells. Normal human stem cells and malignant leukemic blast sequently yielding chimeric proteins with altered functions. cells exhibit similar morphological, biological and phenotypic At least 60% of de novo AML harbor cytogenetic abnor- features. It is not always possible to distinguish malignant malities.29 High-resolution banding techniques suggest that blasts from healthy progenitors solely by morphology. About almost all patients have cytogenetic abnormalities.30 Translo- 20% to 50% of the acute myeloid leukemias show aberrant cations between chromosome 8 and 21, between 15 and 17 antigens,57 but almost all aberrant antigen combinations can

Leukemia Review C Brendel and A Neubauer 1713

Figure 1 Summarization of recent hypotheses of human stem cell development. Model of CD34 expression in the human bone marrow during maturation progress. The earliest stem cell is probably not tissue-determined, quiescent and with self-renewing capacity. Possibly through cell matrix interaction it can acquire a tissue specific phenotype and function. This early hematopoietic progenitor cell with ability for self- renewal seems to be CD34− CD45+ exhibiting variable expression of CD38. On activation this cell can acquire a CD34+ phenotype and give rise to differentiated progeny. It displays a strong CD34 expression which is reversible if an activation equilibrium occurs. The activated CD34+ cell is partially capable of self-renewal and long-term engraftment, especially if it has a certain pattern of co-expressed antigens indicating an immature developmental stage like CD90+,DR−. As maturation proceeds the co-antigen expression pattern changes and the clonogenic potential improves while self-renewal capacity persists. It should be emphasized that maturation in reality is a continuing process unlike this diagram, which suggests a stepwise maturation with defined stages. Antigen expression is probably a gradually increasing or decreasing process reflecting the fluent maturation progress. also be detected in a small percentage of healthy individuals. human signal-regulatory protein (SIRP), described as a stem Aberrant antigen expression may also vary during the course cell marker very recently, is a serious candidate protein for of AML treatment and is therefore not suitable as a single distinction between hematopoietic progenitor cells and reliable marker of the malignant cell clone. myeloid blasts because the latter cells do not express that pro- In the absence of aberrant antigen combinations, malignant tein or express it in a significantly reduced manner.12 myeloid cells in AML often display a very similar surface pro- Although the malignant leukemic blasts in AML generally tein expression pattern as compared with their healthy CD34 arise from very early CD34+ progenitor cells, the FAB subtypes counterpart. Until now there has been almost no antigen of acute myeloid leukemia seem to have different described that has proved to be expressed on malignant blast differentiation levels. The CD13 and CD33 antigens and mye- cells exclusively. The AC133 antigen is neither specific for loperoxidase that characterize normal myeloid progenitor non-AML blast cells nor for myeloid blast cells.58,59 The CD82 cells are abundantly expressed on almost all AML cells, but antigen is also expressed on hematopoietic progenitor cells are variable in FAB M0, which is considered to be a rather and on AML cells.9 The Flt3-receptor tyrosine kinase, which undifferentiated leukemia. Most of the leukemic cells of this is up-regulated on normal activated stem cells, has high m- FAB subtype exhibit a CD34+ CD38− antigen expression and RNA and protein expression levels in AML cells.60 However, lack other progenitor cell antigens.62 The c-kit antigen the internal tandem duplication within the juxtamembrane expression is present in about 60% of AML in general but region of that receptor occurs exclusively in AML cells. It has differs between FAB subtypes and karyotype, AML M0 and M1 been reported that the expression pattern of adhesion mol- leukemias show more pronounced c-kit-antigen expression as ecules is different in cells from AML patients and normal stem compared with M5 leukemias.63 The M2 leukemia which fre- cells, but the differences are quantitative and not absolutely quently harbors the t(8;21) and even the promyeolocytic FAB specific.61 Whether the KDR receptor also occurs on myeloid M3 leukemia with t(15;17) have recently been characterized leukemia cells has not been investigated until now. The as diseases of multipotent progenitor cells with lineage-nega-

Leukemia Review C Brendel and A Neubauer 1714 tive phenotype (CD34+ CD38−).64–66 AML M4 and M5 leuke- genetic analysis because of higher proliferative potential of the mias are thought to display a more mature phenotype often malignant cell type in cell culture compared to healthy blood carrying a lineage-positive monocytic antigen pattern. Lineage cells. In some specific lesions, interphase FISH seems to be restriction may also occur in AML M6 and M7, which is superior in detecting the cytogenetic aberration. Although underscored by morphology and flow cytometry (eg glyco- FISH is a widely accepted sensitive standard technique for the phorin and glycoprotein IIb/IIIa expression, respectively). detection of specific cytogenetic aberrations in semi-quantitat- Nevertheless, Dicks group demonstrated the immature nature ive manner, false positive signals can appear due to artifacts of acute myeloid leukemia cells,67 and Blair et al68 charac- when analyzing three-dimensional cells from a two-dimen- + − − − terized AML cells as CD34 HLA-DR CD71 CD90 15 which sional perspective. Therefore, cut-off levels have to be defined represents an early progenitor cell phenotype, however, most for each probe on healthy individuals. If the cell of interest of their examined AML cases were M4 and M5 leukemias. has been exposed to stressful events such as sorting, culturing There is one report on differentiative and proliferative etc, cut-off levels may be significantly higher and may vary capacities of leukemic blast, that demonstrates the potential considerably due to a higher amount of dead cells within a for self-renewal of leukemic stem cells in different AML fraction or squeezed nuclei resulting in interpretation failure.75 subtypes.69 This study strongly supports the hypothesis of a To avoid these difficulties, polymerase chain reaction (PCR) primitive stem cell being involved in acute leukemic trans- analysis can be applied as an equal or even superior tech- formation, although heterogeneous maturation characteristics nique. It is a well established method for sensitive detection were observed in different AML FAB subtypes. Bonnet and of specific gene rearrangements such as bcr-abl, t(15;17) or Dick69 proved that all cells capable of initiating human AML t(8;21). For detection of inversion 16 it seems to be more + − in NOD-SCID mice were exclusively CD34 CD38 . They also reliable than standard cytogenetic analysis.76 Despite the high demonstrated that some features of cell growth remain similar sensitivity, standard PCR analysis is not suitable for exact in normal and malignant blast cells. quantification. New technologies for REAL-time PCR analysis + + According to the data of Blair’s group CD34 CD90 pro- may partially overcome that problem. However, only defined genitor cells were thought to be benign cells in AML. In order genetic alterations can be sensitively detected by PCR and to analyze this particular antigen pattern in AML in detail, we FISH technique and require well defined probes that are avail- used multiparameter flow cytometry sorting, cytogenetics and able only for the most common genetic lesions. Since AML FISH techniques. To this end, we have shown that in second- + + patients display a variety of chromosomal defects, which are ary AML the early CD34 CD90 cells also carry the malignant often not detectable either with FISH or PCR analysis, combi- 70 clone, meaning that in this disease leukemic cells resemble nation of both techniques with standard cytogenetic analysis the earliest CD34 cell progenitor phenotype. The patients is recommended. examined in this study had progressive disease with poor Different genetic alterations within leukemic cells seem to response to chemotherapy treatment. The discordant findings have very unique biological features reflecting the heterogen- could be explained with the different study populations eity of AML cases. All patients whose blasts harbor t(8;21) and (primary vs secondary AML, different FAB subtypes). It is also + inv16 (CBF leukemias) have a favorable outcome.77,78 One very likely that the malignant cell clone predominates the could speculate that in those leukemias the cells are more earliest stem cell compartments only in later or progressive susceptible to chemotherapy but there is no proven expla- stages as is the case in chronic myeloid leukemia.71 However, nation for the benefit of those rearrangements. Promyelocytes this controversy reflects the difficulty of defining early and in t(15;17) M3 leukemia respond to retinoic acid treatment more mature acute myeloid leukemia cases solely by with differentiation. This treatment improves survival in AML immunophenotype. Acute myeloid leukemias that predomi- − + patients with this particular lesion. Another type of acute nantly display a CD34 CD33 phenotype might also harbor + + myeloid leukemia, familial AML with monosomy 7, has been early malignant clones within the CD34 CD90 population. shown to affect very early multipotent progenitor cells and to This clone will probably expand during disease progression 79 due to loss of the ability to differentiate further or some growth have extraordinarily poor patient survival. In addition, AML advantage after chemotherapy treatment. In CML it has been patients with a history of myelodysplastic syndrome and demonstrated that the early malignant bcr-abl-positive CD34+ monosomy 7 often have a fatal outcome. One could assume cells stimulate their growth through autocrine IL-3 and G-CSF that chromosome 7 harbors a gene important in apoptotic release. These cells lose this capacity during maturation and pathways but until now such a target gene has not been differentiation.72 A similar mechanism could be assumed for identified. Another example of biological significance of cer- AML cells. tain genetic lesions is the Flt3 gene. Its alteration can modify 47 The discordance of our findings as compared with the litera- the proliferative ability of leukemic cells. ture can also be explained in part by different methods As mentioned above, biological features of leukemic and 69 applied while addressing the question of developmental stages normal cells in AML are often similar. Leukemic cells can of stem cells. Most investigators make use of karyotypic abnor- form short-term colonies and have long-term proliferative + malities in AML cells in order to identify the malignant clone potential in vitro.15 Similar to normal CD34 progenitors some applying either conventional G-banding cytogenetic analysis, AML cells can be forced to differentiate into dendritic cells80 interphase fluorescence in situ hybridization (FISH) analysis and can also be stimulated to differentiate according to their or PCR-based molecular genetic techniques. Several pitfalls lineage, for example with CD44 antibody in M1/2 to M5 have been demonstrated for these techniques. In certain leukemia.81 cytogenetic lesions such as monosomy 7 or t(15;17) standard To date, it can be stated that combined analyses of stem cell G-banding analysis obscures the high number of aberrant phenotypic antigen expression, genetic lesions and biological karyotypes within blood cell fractions, probably due to differ- features in AML provide useful tools to detect differences ent proliferative abilities of the malignant clone in vitro.73,74 between normal and malignant progenitor cells that are of One could assume that with other leukemic karyotypic lesions clinical benefit possibly providing data for new sorting false positive results could be obtained with the standard cyto- strategies in this disease.

Leukemia Review C Brendel and A Neubauer 1715 Perspectives References 1 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gral- It has been reported that in chronic myeloid leukemias (CML) nick HR, Sultan C. Proposals for the classification of the acute or myelodysplastic syndrome (MDS) sorting of benign stem leukaemias. French–American–British (FAB) co-operative group. 71,82 Br J Haematol 1976; 33: 451–458. cells with a certain phenotype could be accomplished. 2 Civin CI, Strauss LC, Brovall C, Fackler MJ, Schwartz JF, Shaper These diseases are also considered to be clonal disorders of JH. Antigenic analysis of hematopoiesis. III. A hematopoietic pro- multipotent progenitor cells.83 But to date for autologous genitor cell surface antigen defined by a monoclonal antibody transplantation, no reliable purging method exists for raised against KG- 1a cells. J Immunol 1984; 133: 157–165. obtaining leukemia free grafts. 3 Zanjani ED, Almeida-Porada G, Livingston AG, Flake AW, Ogawa − The current model system of CD34− phenotype of the earli- M. Human bone marrow CD34 cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells est progenitor cell and switch of the resting stem cell from − + (see comments). Exp Hematol 1998; 26: 353–360. CD34 to CD34 phenotype due to external activation stimuli 4 Zanjani ED, Almeida-Porada G, Livingston AG, Porada CD, gives rise to the question whether this cell might be free from Ogawa M. Engraftment and multilineage expression of human leukemic genetic lesions providing a source for healthy stem bone marrow CD34− cells in vivo. Ann NY Acad Sci 1999; 872: cells in autologous transplantation. This hypothesis seems 220–231; discussion 231–232. likely, because the very early progenitor cell is probably a 5 Larochelle A, Vormoor J, Hanenberg H, Wang JC, Bhatia M, Lapi- dot T, Moritz T, Murdoch B, Xiao XL, Kato I, Williams DA, Dick dormant cell, less prone to gene altering events that can occur JE. Identification of primitive human hematopoietic cells capable in proliferating cells. If chemotherapy agents or radiation alter of repopulating NOD/SCID mouse bone marrow: implications for the recovery state of the bone marrow, the stem cells attain an gene therapy. Nature Med 1996; 2: 1329–1337. activation state upon external proliferation stimuli. One could 6 Murray L, Chen B, Galy A, Chen S, Tushinski R, Uchida N, Negrin imagine that through activation the prior dormant stem cells R, Tricot G, Jagannath S, Vesole D, Barlogie B, Hoffman R, Tsuka- respond with multiple surface receptor up-regulation and moto A. Enrichment of human hematopoietic stem cell activity in + the CD34+Thy− 1+Lin− subpopulation from mobilized peripheral therefore become CD34 cells. If some of these activated + blood. Blood 1995; 85: 368–378. CD34 cells enter the cell cycle in order to reconstitute the 7 Humeau L, Bardin F, Maroc C, Alario T, Galindo R, Mannoni P, bone marrow cell pool they become susceptible targets for Chabannon C. Phenotypic, molecular, and functional characteriz- DNA damaging agents such as benzene or alkylating sub- ation of human peripheral blood CD34+/THY1+ cells. Blood stances. These cells are more prone to becoming leukemic 1996; 87: 949–955. than dormant cells. The effort of detecting a leukemic-free cell 8 de Wynter EA, Buck D, Hart C, Heywood R, Coutinho LH, Clayton + A, Rafferty JA, Burt D, Guenechea G, Bueren JA, Gagen D, Fair- compartment within the CD34 population might therefore bairn LJ, Lord BI, Testa NG. CD34+AC133+ cells isolated from be frustrating. cord blood are highly enriched in long-term culture-initiating − In addition to their dormant state, CD34 hematopoietic cells, NOD/SCID-repopulating cells and dendritic cell progenitors. stem cells possibly have potent mechanisms protecting them Stem Cells 1998; 16: 387–396. from genetic damage like efflux pumps for toxic agents. There- 9 Burchert A, Notter M, Menssen HD, Schwartz S, Knauf W, Neu- fore, the dye efflux definition of CD34− stem cells seems bauer A, Thiel E. CD82 (KAI1), a member of the tetraspan family, 22 is expressed on early haemopoietic progenitor cells and up-regu- likely. The expectation that these cells might display a com- lated in distinct human leukaemias. Br J Haematol 1999; 107: partment free of leukemic cell clones is very promising but 494–504. needs to be proven in future studies. If these early progenitors 10 Ziegler BL, Valtieri M, Porada GA, De Maria R, Muller R, Masella could be identified and mobilized in vivo for collection, only B, Gabbianelli M, Casella I, Pelosi E, Bock T, Zanjani ED, Peschle very few cells might be sufficient for engraftment in stem cell C. KDR receptor: a key marker defining hematopoietic stem cells. transplantation settings and might provide a new source of Science 1999; 285: 1553–1558. 11 Gehling UM, Ergun S, Schumacher U, Wagener C, Pantel K, Otte leukemic-free graft with reduced risk of leukemic relapse. − + M, Schuch G, Schafhausen P, Mende T, Kilic N, Kluge K, Schafer According to that model, CD34 cells will turn into CD34 B, Hossfeld DK, Fiedler W. In vitro differentiation of endothelial cells that up-regulate growth factor receptors, kit-ligand and cells from AC133-positive progenitor cells (in process citation). other surface antigens in response to external stimuli, resulting Blood 2000; 95: 3106–3112. in augmented self-renewal and differentiation potential. 12 Seiffert M, Cant C, Chen Z, Rappold I, Brugger W, Kanz L, Brown This new perspective for autologous bone marrow trans- EJ, Ullrich A, HJ Bh. Human signal-regulatory protein is expressed on normal, but not on subsets of leukemic myeloid cells and plantation without risk of malignant cell contamination mediates cellular adhesion involving Its counterreceptor CD47. becomes even more likely if one considers the data about Blood 1999; 94: 3633–3643. bone marrow repopulating potential of non-hematologic stem 13 Ashman LK, Cambareri AC, Levinsky RJ, Juttner CA. Expression of cells from brain or muscle tissue. It is not expected that these the YB5.B8 antigen (c-kit protooncogene product) in normal tissues will be affected by the malignant leukemic cell clone human bone marrow. Blood 1991; 78: 30–37. thus possibly providing a safe source for leukemic-free bone 14 Sogo S, Inaba M, Ogata H, Hisha H, Adachi Y, Mori S, Toki J, Yamanishi K, Kanzaki H, Adachi M, Ikehara S. Induction of c- marrow reconstituting cells useful for stem cell trans- kit molecules on human CD34+/c-kit , low cells: evidence for plantation. We assume that these exciting new data will CD34+/c-kit , low cells as primitive hematopoietic stem cells. challenge scientists and physicians to co-operate in further Stem Cells 1997; 15: 420–429. investigations in the area of stem cell characterization. 15 Blair A, Hogge DE, Sutherland HJ. Most acute myeloid leukemia progenitor cells with long-term proliferative ability in vitro and in vivo have the phenotype CD34(+)/CD71(−)/HLA-DR. Blood 1998; 92: 4325–4335. 16 Roy V, Miller JS, Verfaillie CM. Phenotypic and functional charac- Acknowledgements terization of committed and primitive myeloid and lymphoid hematopoietic precursors in human fetal liver. Exp Hematol 1997; 25: 387–394. This work was supported in part by grants of the Deutsche 17 Kollet O, Aviram R, Chebath J, Ben-Hur H, Nagler A, Shultz L, Forschungsgemeinschaft (Ne 310/6–3), and the Jose´ Carreras Revel M, Lapidot T. The soluble interleukin-6 (IL-6) receptor/IL-6 Stiftung. fusion protein enhances in vitro maintenance and proliferation of

Leukemia Review C Brendel and A Neubauer 1716 human CD34(+)CD38(−/low) cells capable of repopulating severe GF. Molecular and phenotypic spectrum of de novo Philadelphia combined immunodeficiency mice. Blood 1999; 94: 923–931. positive acute leukemia. Int J Mol Med 1999; 4: 665–667. 18 Gross S, Helm K, Gruntmeir JJ, Stillman WS, Pyatt DW, Irons RD. 40 Appelbaum FR, Gilliland DG, Tallmann MS. The biology and Characterization and phenotypic analysis of differentiating CD34+ treatment of acute myeloid leukemia. American Society of Hema- human bone marrow cells in liquid culture. Eur J Haematol 1997; tology Education Program Book 1998, pp 15–43. 59: 318–326. 41 Pedersen-Bjergaard J, Janssen JWG, Lyons J, Philip P, Bartram CR. 19 Bhatia M, Bonnet D, Murdoch B, Gan OI, Dick JE. A newly disco- Point mutations of the ras protooncogenes and chromosome aber- vered class of human hematopoietic cells with SCID-repopulating rations on acute nonlymphoblastic leukemia and preleukemia activity (see comments). Nature Med 1998; 4: 1038–1045. related to therapy with alkylating agents. Cancer Res 1988; 48: 20 Osawa M, Hanada K, Hamada H, Nakauchi H. Long-term lym- 1812–1817. phohematopoietic reconstitution by a single CD34− low/negative 42 Neubauer A, Dodge R, George SL, Davey FR, Silver RT, Schiffer hematopoietic stem cell. Science 1996; 273: 242–245. CA, Mayer R, Ball ED, Wurster-Hill D, Bloomfield CD, Liu ET. 21 Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Iso- Prognostic importance of mutations in the ras protooncogenes in lation and functional properties of murine hematopoietic stem de novo acute myeloid leukemia. Blood 1994; 83: 1603–1611. cells that are replicating in vivo. J Exp Med 1996; 183: 1797– 43 Schmidt CA, Przybylski G, Vogel D, Ludwig WD, Oettle H, Neu- 1806. bauer A, Siegert W. ras point mutations occur in acute myeloid 22 Goodell MA, Rosenzweig M, Kim H, Marks DF, DeMaria M, Par- leukemia with illegitimate T-cell receptor delta gene rearrange- adis G, Grupp SA, Sieff CA, Mulligan RC, Johnson RP. Dye efflux ment. Leukemia 1994; 8: 102–105. studies suggest that hematopoietic stem cells expressing low or 44 Seliger B, Papadileris S, Vogel D, Hess G, Brendel C, Storkel S, undetectable levels of CD34 antigen exist in multiple species. Nat- Oertel J, Kolbe K, Huber C, Huhn D, Neubauer A. Analysis of p53 ure Med 1997; 3: 1337–1345. and mdm-2 gene in acute myeloid leukemia. Eur J Haematol 23 Sato T, Laver JH, Ogawa M. Reversible expression of CD34 by 1996; 57: 230–240. murine hematopoietic stem cells (see comments). Blood 1999; 94: 45 Slingerland JM, Minden MD, Benchimol S. Mutation of the p53 2548–2554. gene in human acute myelogeneous leukemia. Blood 1991; 77: 24 Nakamura Y, Ando K, Chargui J, Kawada H, Sato T, Tsuji T, Hotta 1500–1507. T, Kato S. Ex vivo generation of CD34(+) cells from CD34(−) hema- 46 Marie JP, Legrand O. MDR1/P-GP expression as a prognostic fac- topoietic cells. Blood 1999; 94: 4053–4059. tor in acute leukemias. Adv Exp Med Biol 1999; 457: 1–9. 25 Goodell MA. Introduction: focus on hematology. CD34(+)or 47 Rombouts WJ, Broyl A, Martens AC, Slater R, Ploemacher RE. CD34(−): does it really matter? (comment). Blood 1999; 94: Human acute myeloid leukemia cells with internal tandem dupli- 2545–2547. cations in the Flt3 gene show reduced proliferative ability in 26 Bjornson CR, Rietze RL, Reynolds BA, Magli MC, Vescovi AL. stroma supported long-term cultures. Leukemia 1999; 13: 1071– Turning brain into blood: a hematopoietic fate adopted by adult 1078. neural stem cells in vivo (see comments). Science 1999; 283: 48 Kiyoi H, Naoe T, Nakano Y, Yokota S, Minami S, Miyawaki S, 534–537. Asou N, Kuriyama K, Jinnai I, Shimazaki C, Akiyama H, Saito K, 27 Jackson KA, Mi T, Goodell MA. Hematopoietic potential of stem Oh H, Motoji T, Omoto E, Saito H, Ohno R, Ueda R. Prognostic cells isolated from murine skeletal muscle (in process citation). implication of FLT3 and N-RAS gene mutations in acute myeloid Proc Natl Acad Sci USA 1999; 96: 14482–14486. leukemia. Blood 1999; 93: 3074–3080. 28 Loeb LA. Cancer cells exhibit a mutator phenotype. Adv Cancer 49 Xu F, Taki T, Yang HW, Hanada R, Hongo T, Ohnishi H, Kobaya- Res 1998; 72: 25–56. shi M, Bessho F, Yanagisawa M, Hayashi Y. Tandem duplication 29 Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harri- of the FLT3 gene is found in acute lymphoblastic leukaemia as son G, Rees J, Hann I, Stevens R, Burnett A, Goldstone A. The well as acute myeloid leukaemia but not in myelodysplastic syn- importance of diagnostic cytogenetics on outcome in AML: analy- drome or juvenile chronic myelogenous leukaemia in children. Br sis of 1,612 patients entered into the MRC AML 10 trial. Blood J Haematol 1999; 105: 155–162. 1998; 92: 2322–2333. 50 Alitalo K, Saksela K, Winqvist R, Alitalo R, Keski-Oja J, Laiho M, 30 Yunis JJ, Brunning RD, Howe RB, Lobell M. High-resolution chro- Ilvonen M, Knuutila S, Delachapelle A. Acute myelogenous leuk- mosomes as an independent prognostic indicator in adult acute emia with c-myc amplification and double minute chromosome. nonlymphocytic leukemia. New Engl J Med 1984; 311: 812–818. Lancet 1985; 11: 1035–1038. 31 Frank R, Zhang J, Uchida H, Meyers S, Hiebert SW, Nimer SD. 51 Baer MR, Augustinos P, Kinniburgh AJ. Defective c-myc and c- The AML1/ETO fusion protein blocks transactivation of the GM- myb RNA turnover in acute myeloid leukemia cells. Blood 1992; CSF promoter by AML 1B. Oncogene 1995; 11: 2667–2674. 79: 1319–1326. 32 Meyers S, Lenny N, Hiebert SW. The t(8;21) fusion protein inter- 52 Thorsteinsdottir U, Sauvageau G, Hough MR, Dragowska W, feres with AML-1B-dependent transcriptional activation. Mol Cell Lansdorp PM, Lawrence HJ, Largman C, Humphries RK. Over- Biol 1995; 15: 1974–1984. expression of HOXA10 in murine hematopoietic cells perturbs 33 Rhoades KL, Hetherington CJ, Rowley JD, Hiebert SW, Nucifora both myeloid and lymphoid differentiation and leads to acute G, Tenen DG, Zhang DE. Synergistic up-regulation of the myeloid- myeloid leukemia. Mol Cell Biol 1997; 17: 495–505. specific promoter for the macrophage colony-stimulating factor 53 Okabe-Kado J, Kasukabe T, Honma Y. Differentiation inhibitory receptor by AML1 and the t(8;21) fusion protein may contribute factor Nm23 as a prognostic factor for acute myeloid leukemia. to leukemogenesis. Proc Natl Acad Sci USA 1996; 93: 11895– Leuk Lymphoma 1998; 32: 19–28. 11900. 54 Okuda T, Cai Z, Yang S, Lenny N, Lyu CJ, van Deursen JM, Harada 34 Ahn MY, Huang G, Bae SC, Wee HJ, Kim WY, Ito Y. Negative H, Downing JR. Expression of a knocked-in AML1-ETO leukemia regulation of granulocytic differentiation in the myeloid precursor gene inhibits the establishment of normal definitive hematopoiesis cell line 32Dc13 by ear-2, a mammalian homolog of Drosophila and directly generates dysplastic hematopoietic progenitors. Blood seven-up, and a chimeric leukemogenic gene, AML1/ETO. Proc 1998; 91: 3134–3143. Natl Acad Sci USA 1998; 95: 1812–1817. 55 Castilla L, Bodine D, Garrett L, Liu P. Leukemia development by 35 Friedman AD. Leukemogenesis by CBF oncoproteins. Leukemia the induction of a second ‘hit’ in chimeric mice containing CBF- 1999; 13: 1932–1942. beta-MYH11 ES cells. Blood 1998; 92: 214a. 36 Zent C, Rowley JD, Nucifora G. Rearrangements of the 56 Bloomfield CD, Lawrence D, Byrd JC, Carroll A, Pettenati MJ, Tan- AML1/CBFA2 gene in myeloid leukemia with the 3;21 translo- travahi R, Patil SR, Davey FR, Berg DT, Schiffer CA, Arthur DC, cation: in vitro and in vivo studies. Leukemia 1997; 11 (Suppl. 3): Mayer RJ. Frequency of prolonged remission duration after high- 273–278. dose cytarabine intensification in acute myeloid leukemia varies 37 Rubnitz JE, Pui CH, Downing JR. The role of TEL fusion genes in by cytogenetic subtype. Cancer Res 1998; 58: 4173–4179. pediatric leukemias. Leukemia 1999; 13: 6–13. 57 DeVita VTJ, Hellman S, Rosenberg SA. Cancer – Principles and 38 Look AT. Oncogenic transcription factors in the human acute Practice of Oncology, 4th edn. JB Lippincott Co: Philidelphia, leukemias. Science 1997; 278: 1059–1064. 1993. 39 Lim LC, Heng KK, Vellupillai M, Tan LT, Boey BC, Lau LC, How 58 Buhring HJ, Seiffert M, Marxer A, Weiss B, Faul C, Kanz L, Brugger

Leukemia Review C Brendel and A Neubauer 1717 W. AC133 antigen expression is not restricted to acute myeloid benign primitive hematopoietic progenitors in chronic myelogene- leukemia blasts but is also found on acute lymphoid leukemia ous leukemia on the basis of HLA-DR antigen expression. Blood blasts and on a subset of CD34+ B-cell precursors (letter). Blood 1992; 79: 1003–1010. 1999; 94: 832–833. 72 Jiang X, Lopez A, Holyoake T, Eaves A, Eaves C. Autocrine pro- 59 Horn PA, Tesch H, Staib P, Kube D, Diehl V, Voliotis D. duction and action of IL-3 and granulocyte colony-stimulating fac- Expression of AC133, a novel hematopoietic precursor antigen, on tor in chronic myeloid leukemia. Proc Natl Acad Sci USA 1999; acute myeloid leukemia cells (letter). Blood 1999; 93: 1435–1437. 96: 12804–12809. 60 Drexler HG, Meyer C, Quentmeier H. Effects of FLT3 ligand on 73 Sokolic RA, Ferguson W, Mark HF. Discordant detection of proliferation and survival of myeloid leukemia cells. Leuk Lym- monosomy 7 by GTG-banding and FISH in a patient with phoma 1999; 33: 83–91. Schwachman–Diamond syndrome without evidence of myelod- 61 De Waele M, Renmans W, Jochmans K, Schots R, Lacor P, Trulle- ysplastic syndrome or acute myelogenous leukemia. Cancer mans F, Otten J, Balduck N, Vander Gucht K, Van Camp B, Van Genet Cytogenet 1999; 115: 106–113. Riet I. Different expression of adhesion molecules on CD34+ cells 74 Tanaka K, Arif M, Eguchi M, Shintani T, Kumaravel TS, Asaoku in AML and B- lineage ALL and their normal bone marrow H, Kyo T, Dohy H, Kamada N. Interphase fluorescence in situ counterparts. Eur J Haematol 1999; 63: 192–201. hybridization overcomes pitfalls of G-banding analysis with spe- 62 Costello R, Mallet F, Chambost H, Sainty D, Arnoulet C, Gastaut cial reference to underestimation of chromosomal aberration JA, Olive D. The immunophenotype of minimally differentiated rates. Cancer Genet Cytogenet 1999; 115: 32–38. acute myeloid leukemia (AML-MO): reduced immunogenicity and 75 Feuring-Buske M, Haase D, Buske C, Hiddemann W, Wormann high frequency of CD34+/CD38− leukemic progenitors. Leukemia B. Clonal chromosomal abnormalities in the stem cell compart- 1999; 13: 1513–1518. ment of patients with acute myeloid leukemia in morphological 63 Schwartz S, Heinecke A, Zimmermann M, Creutzig U, Schoch C, complete remission. Leukemia 1999; 13: 386–392. Harbott J, Fonatsch C, Loffler H, Buchner T, Ludwig WD, Thiel E. 76 Ritter M, de Kant E, Huhn D, Neubauer A. Detection of DNA Expression of the C-kit receptor (CD117) is a feature of almost all methylation in human leukemias by differential polymerase chain subtypes of de novo acute myeloblastic leukemia (AML), including reaction. Leukemia 1995; 9: 915–921. cytogenetically good-risk AML, and lacks prognostic significance. 77 Bloomfield C, Shuma C, Regal L, Philip P, Hossfeld D, Hagemeijer Leuk Lymphoma 1999; 34: 85–94. A, Garson O, Peterson B, Sakurai M, Alimena G, Berger R, Rowley 64 Wittebol S, Raymakers R, van de Locht L, Mensink E, de Witte T. J, Ruutu T, Mitelman F, Dewald G, Swansbury J. Long-term sur- In AML t(8;21) colony growth of both leukemic and residual nor- vival of patients with acute myeloid leukemia. Cancer 1997; 80: mal progenitors is restricted to the CD34+, lineage-negative frac- 2191–2198. tion. Leukemia 1998; 12: 1782–1788. 78 Byrd JC, Dodge RK, Carroll A, Baer MR, Edwards C, Stamberg J, 65 Miyamoto T, Nagafuji K, Akashi K, Harada M, Kyo T, Akashi T, Qumsiyeh M, Moore JO, Mayer RJ, Davey F, Schiffer CA, Bloom- Takenaka K, Mizuno S, Gondo H, Okamura T, Dohy H, Niho field CD. Patients with t(8;21)(q22;q22) and acute myeloid leuk- Y. Persistence of multipotent progenitors expressing AML1/ETO emia have superior failure-free and overall survival when repeti- transcripts in long-term remission patients with t(8;21) acute mye- tive cycles of high-dose cytarabine are administered (in process logenous leukemia. Blood 1996; 87: 4789–4796. citation). J Clin Oncol 1999; 17: 3767–3775. 66 Edwards RH, Wasik MA, Finan J, Rodriguez R, Moore J, Kamoun 79 Kwong YL, Ng MH, Ma SK. Familial acute myeloid leukemia with M, Rennert H, Bird J, Nowell PC, Salhany KE. Evidence for early monosomy 7: late onset and involvement of a multipotential pro- hematopoietic progenitor cell involvement in acute promyelocytic genitor cell (in process citation). Cancer Genet Cytogenet 2000; leukemia. Am J Clin Pathol 1999; 112: 819–827. 116: 170–173. 67 Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres- 80 Cignetti A, Bryant E, Allione B, Vitale A, Foa R, Cheever MA. Cortes J, Minden M, Paterson B, Caligiuri MA, Dick JE. A cell CD34(+) acute myeloid and lymphoid leukemic blasts can be initiating human acute myeloid leukemia after transplantation into induced to differentiate into dendritic cells. Blood 1999; 94: SCID mice. Nature 1994; 367: 645–648. 2048–2055. 68 Blair A, Hogge DE, Ailles LE, Lansdorp PM, Sutherland HJ. Lack 81 Charrad RS, Li Y, Delpech B, Balitrand N, Clay D, Jasmin C, of expression of Thy-1 (CD90) on acute myeloid leukemia cells Chomienne C, Smadja-Joffe F. Ligation of the CD44 adhesion mol- with long-term proliferative ability in vitro and in vivo. Blood ecule reverses blockage of differentiation in human acute myeloid 1997; 89: 3104–3112. leukemia (see comments). Nature Med 1999; 5: 669–676. 69 Bonnet D, Dick JE. Human acute myeloid leukemia is organized 82 Saitoh K, Miura I, Takahashi N, Miura AB. Fluorescence in situ as a hierarchy that originates from a primitive hematopoietic cell. hybridization of progenitor cells obtained by fluorescence- Nature Med 1997; 3: 730–737. activated cell sorting for the detection of cells affected by chromo- 70 Brendel C, Mohr B, Schimmelpfennig C, Muller J, Bornhauser M, some abnormality trisomy 8 in patients with myelodysplastic syn- Schmidt M, Ritter M, Ehninger G, Neubauer A. Detection of cyto- dromes. Blood 1998; 92: 2886–2892. genetic aberrations both in CD90 (Thy-1)-positive and (Thy-1)- 83 Janssen JWG, Buschle M, Layton M, Drexler HG, Lyons J, van den negative stem cell (CD34) subfractions of patients with acute and Berghe H, Heimpel H, Kubanek B, Kleihauer E, Mufti GJ, Bartram chronic myeloid leukemias. Leukemia 1999; 13: 1770–1775. CR. Clonal analysis of myelodysplastic sybdromes. Evidence for 71 Verfaille CM, Miller WJ, Boylan K, McGlave PB. Selection of multipotent stem cell origin. Blood 1989; 73: 248–254.

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