Letters to the Editor 750 14 Caritas-Klinik St Theresia, Saarbru¨cken, Germany; untreated patients 61 years or older with acute myeloid leukemia. 15 Allgemeines Krankenhaus Altona, Hamburg, Germany; Leukemia 2004; 18: 1798–1803. 16Kreisklinik, Trostberg, Germany; 5 Rowe JM. Treatment of acute myelogenous leukemia in older 17Caritas Krankenhaus, Lebach, Germany; adults. Leukemia 2000; 14: 480–487. 18Department of Hematology and Oncology, University 6Bu¨chner T, Hiddemann W, Berdel WE, Wo¨rmann B, Schoch C, of Go¨ttingen, Germany and Fronatsch C et al. 6-Thioguanine, cytarabine and daunorubicin 19Central Unit of Biostatistics, German Cancer Research (TAD) and high-dose cytarabine and mitoxantrone (HAM) for Center, Heidelberg, Germany induction, TAD for consolidation and either prolonged maintenance E-mail: [email protected] by reduced monthly TAD or TAD-HAM-TAD and one course of intensive consolidation by sequential HAM in adult patients at all ages with de novo acute myeloid leukemia (AML): a randomized References trial of the German AML Cooperative Group. J Clin Oncol 2003; 21: 4496–4504. 1 Appelbaum F, Rowe J, Radich J, Dick J. Acute myeloid leukemia. 7 Goldstone AH, Burnett AK, Wheatley K, Smith AG, Hutchinson RM, Hematology (Am Soc Hematol Educ Program) 2001, 62–86. Clark RE, Medical Research Council Adult Leukemia Working Party. 2 Estey E, Do¨hner H. Acute myeloid leukemia. Lancet. Attempts to improve treatment outcomes in acute myeloid leukemia 3 Stone RM, Berg DT, George SL, Dodge RK, Paciucci PA, Schulman (AML) in older patients: the results of the United Kingdom Medical PP et al. Postremission therapy in older patients with de novo acute Research Council AML11 trial. Blood 2001; 98: 1302–1311. myeloid leukemia: a randomized trial comparing mitoxantrone and 8 Grimwade D, Walker H, Harrison G, Oliver F, Chatters S, Harrison intermediate-dose cytarabine with standard-dose cytarabine. Blood CJ et al. The predictive value of hierarchical cytogenetic classifica- 2001; 98: 548–553. tion in older adults with acute myeloid leukemia (AML): analysis of 4 Schlenk RF, Frohling S, Hartmann F, Fischer JT, Glasmacher A, del 1065 patients entered into the United Kingdom Medical Research Valle F et al. Phase III study of all-trans retinoic acid in previously Council AML11 trial. Blood 2001; 98: 1312–1320.

Preferential expression of a high number of ATP binding cassette transporters in both normal and leukemic CD34 þ CD38À cells

Leukemia (2006) 20, 750–754. doi:10.1038/sj.leu.2404131; Recently, an increasing number of ABC transporters has been published online 9 February 2006 associated with extrusion of xenobiotic compounds and chemotherapeutical agents and thus may be important for stem cell protection. However, the expression pattern of many ABC transporters on hematopoietic CD34 þ CD38À cells is currently In most cases of acute myeloid leukemia (AML) malignant stem unknown. In AML overexpression of ABC transporters involved cells are considered to be within the CD34 þ CD38À popula- in drug resistance might represent an important mechanism of tion. The involvement of CD34 þ CD38À cells in leukemogen- drug resistance.2 We recently demonstrated similar expression esis is suggested by the presence of cytogenetically aberrant and function of ABCG2 in CD34 þ CD38À cells in AML and cells in the stem cell compartment in AML, as demonstrated normal but observed significant mitoxantrone by PCR and FISH for leukemia specific translocations. Further- extrusion from these cells in the presence of ABCG2 inhibition, more, transplantation studies using NOD/SCID mice have suggesting the presence of additional drug transporters in these shown that cells with leukemic engraftment and self renewal cells. potential are exclusively found within the CD34 þ CD38À cell We assessed expression profiles of normal primitive population and not in more differentiated CD34 þ CD38 þ CD34 þ CD38À and more committed CD34 þ CD38 þ pro- populations.1 Incomplete eradication of these primitive cells genitor cells by quantitative real-time RT-PCR using micro may eventually lead to disease relapse. Multidrug resistance fluidic cards, covering all 45 transmembrane ABC transporters. induced by the expression of ATP binding cassette (ABC) Highly enriched CD34 þ CD38À and CD34 þ CD38 þ cells transporters may hamper the eradication of these primitive (purity above 90%) were isolated by FACS sorting either from leukemic cells. bone marrow (N ¼ 3) or from G-CSF stimulated peripheral blood ABC transporters represent the largest family of transmem- collections (N ¼ 8). brane involved in the transport of a wide variety of GAPDH-normalized levels of ABC transporter gene expres- substrates across biological membranes, including phospho- sion in the CD34 þ CD38À cell fraction are shown in Figure 1a. , ions, peptides, steroids, polysaccharides, amino acids, Out of the 45 ABC transporters the expression of 36 ABC organic anions, bile acids, drugs and other xenobiotics. transporters was detectable in CD34 þ CD38À cells. Twenty Currently, the ABC superfamily consists of 48 members; that ABC transporters were detectable in all 11 samples. The lower can be divided into seven subfamilies based on similarity in frequency of detection of the remaining 16 ABC transporters is structure (ABCA, B, C, D, E, F and G). Some of these probably due to the relatively low expression level of these transporters (ABCB1, ABCC1 and ABCG2) are expressed at high which will be reflected in a binary answer of the PCR.4 In levels on hematopoietic primitive cells.2 line with this, genes with the highest expression level where The physiological function of ABC transporters in stem detectable in all samples whereas genes with a lower expression cell biology is not well understood. A role for ABC transporters showed a lower frequency. in the protection from genetic damage by naturally occurring The five ABC transporters with the highest levels of expression xenobiotics has been suggested for hematopoietic stem cells.3 were ABCA2, ABCC1, ABCB2, ABCB7, and ABCB1, confirming This has recently been substantiated by the observation of the relatively high expression of known stem cell transporters increased sensitivity to xenobiotics in normal bone marrow cells ABCB1/MDR1 and ABCC1/MRP1. ABC transporter gene ex- in ABCB1 and ABCG2/BCRP knockout mice. pression levels were not significantly different in G-CSF

Leukemia Letters to the Editor 751 a 1.E-01

1.E-02

1.E-03

1.E-04 (ABC transporter / GAPDH)

1.E-05 Relative ABC transporter mRNA expression

1.E-06 ABCA2 ABCB7 ABCB2 ABCB1 ABCB3 ABCA7 ABCA5 ABCB8 ABCA3 ABCA1 ABCB6 ABCB9 ABCA9 ABCB4 ABCA6 ABCB5 ABCA8 ABCA4 ABCC1 ABCD4 ABCD3 ABCD1 ABCC4 ABCC5 ABCC6 ABCD2 ABCC2 ABCC8 ABCC3 ABCC7 ABCC9 ABCG2 ABCG1 ABCG4 ABCG5 ABCG8 ABCA13 ABCB10 ABCA10 ABCB11 ABCA12 ABCC13 ABCC10 ABCC11 ABCC12

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1 ABCA2 ABCB7 ABCB2 ABCB1 ABCB3 ABCA7 ABCA5 ABCB8 ABCA3 ABCA1 ABCB6 ABCB9 ABCA9 ABCB4 ABCA6 ABCB5 ABCA8 ABCA4 ABCC1 ABCD4 ABCD3 ABCD1 ABCC4 ABCC5 ABCC6 ABCD2 ABCC2 ABCC8 ABCC3 ABCC7 ABCC9 ABCG1 ABCG2 ABCG4 ABCG5 ABCG8 ABCB10 ABCA13 ABCA10 ABCB11 ABCA12 ABCC13 ABCC10 ABCC11 ABCC12

11111111111111 11111111111111111011 21111111110481072947648414 000000 0 0 0

0.1

Figure 1 Many ABC transporters are expressed in normal hematopoietic CD34 þ CD38À cells and they all show a higher expression in normal hematopoietic CD34 þ CD38À cells compared to more committed CD34 þ CD38 þ progenitor cells. (a) of 45 ABC transporters was investigated in CD34 þ CD38À hematopoietic cells from G-CSF mobilized peripheral blood and bone marrow (N ¼ 11). Levels of gene expression are depicted as natural logarithmic values on the y axis normalized for the expression of the GAPDH. Mean values of gene expression of positive samples ( þ 95% CI) are represented by bars. On the x axis ABC transporter gene name together with the frequency of expression (number of samples in which gene expression was detectable) is shown. Note that expression of 36 out of 45 ABC transporters is detected in the normal CD34 þ CD38À fraction. (b) Difference in level of gene expression between CD34 þ CD38À and CD34 þ CD38 þ cells is depicted on the y axis as mean fold difference ( þ 95% CI) in samples that expressed the ABC transporter in both fractions. Transporters for which differentially expression reached statistically significance (using Student’s t-test for paired samples, Po0.05) are indicated with an asterix. mobilized CD34 þ CD38À cells compared to CD34 þ CD38À To gain more insight in the biological relevance of ABC cells from normal bone marrow (data not shown). Except for transporter expression in CD34 þ CD38À cells, we compared ABCB5, that was detected in all G-CSF mobilized samples (8/8) this expression pattern to more committed CD34 þ CD38 þ but not in normal bone marrow (0/3). progenitor cells. In CD34 þ CD38 þ cells 38 ABC transporters

Leukemia Letters to the Editor 752 Table 1 Patient characteristics

Patient Age at Sex FAB % Blasts % CD34+ % CD34+ % CD34+ number diagnosis classification cells CD38À cells CD38+ cells

AML 1 55 M M0 89 89 3.1 21.1 2 26 F M1 85 85 1.3 15.1 3 59 F M1 76 54 8.0 11.1 4 72 F M1 64 64 1.2 16.5 5 71 F M4 87 87 0.5 27.3 6 67 M M0 90 90 1.4 44.7 7 10 M M2 82 82 1.5 33.6 8 67 F M2 86 86 25.6 11.1 9 72 M M4E 24 24 0.9 12.3 10 40 M M5B 55 48 1.7 17.4 11 62 F M5 82 19 2.0 2.1 12 57 F M4 28 5 0.2 0.4 13 50 F M5 90 7 F 5.1 Mean (range) 54 (10–72) 72 (24–90) 57 (5–90) 4.0 (0–25.6) 13.0 (0.4–44.7) Percentages of blasts and CD34+ cells are based on morphology and immunophenoptypic staining, respectively. CD34+CD38À cells were defined as the cells with CD38-PE fluorescence within the first decade of emission. The CD34+CD38+ cells were sorted from a gate positioning in the bulk of CD34+ cells separated by a decade from CD34+CD38À cells. The percentages of CD34+CD38À and CD34+CD38+ cells are the percentages of cells within the gates used in the sorting procedure relative to the total amount of cells.

were detectable. Strikingly, all ABC transporters detectable in In the leukemic CD34 þ CD38À cell fraction, 40 out of 45 the CD34 þ CD38À fraction showed a lower expression in the ABC transporters were detectable (Figure 2a). All genes that CD34 þ CD38 þ fraction (Figure 1b). In 22 of the 36 ABC were detectable in normal CD34 þ CD38À progenitor cells transporters this difference was statistically significant. Among were also detectable in leukemic CD34 þ CD38À cells, with the these genes are known transporters associated with hemato- exception of ABCA8 that was found only in one of the 11 poietic stem cells including ABCB1 (mean 7.57-fold difference, normal CD34 þ CD38À samples. The level of expression of 31 Po0.001), ABCC1 (mean 2.67-fold difference, P ¼ 0.004) and out of 35 ABC transporters was comparable in both normal and ABCG2 (mean 1.81-fold difference, P ¼ 0.016). Among the 10 leukemic CD34 þ CD38À cells. A statistically significant lower most differentially expressed ABC transporters in the normal expression in the CD34 þ CD38À cells in AML compared to the CD34 þ CD38À vs CD34 þ CD38 þ fraction 5 members of the same fraction in normal cells was found for four ABC ABCA family are listed (ABCA5, ABCA6, ABCA9, ABCA10 and transporters, ABCA3 (P ¼ 0.003), ABCA5 (P ¼ 0.003), ABCB1 ABCA2). Four of these genes, ABCA5, ABCA6, ABCA9 and (P ¼ 0.001) and ABCC1 (P ¼ 0.011). A significant difference in ABCA10, representing a phylogenetically distinct subgroup frequency of expression between normal and leukemic within the ABCA gene subfamily, are arranged in a cluster on CD34 þ CD38À cells was found for ABCA13 (10/11 vs 3/11, 17q24. Although the substrates and function of respectively; Po0.05) and ABCB5 (8/11 vs 1/11, respectively, these ABCA transporters are largely unknown, recent studies Po0.05). Finally, in the leukemic CD34 þ CD38À cell suggest that some of these transporters may function in the population five ABC transporters were detectable that were transmembrane transport of endogenous substrates, not found in their normal counterparts (ABCA4, ABCC7, such as phospholipids and essential fatty-acids, substrates ABCC9, ABCC11 and ABCG4). The frequency of detectable involved in the regulation of differentiation of hematopoietic expression of these transporters, however, was very low cells.5 Clearly, further research is warranted to elucidate the role (ABCA4; 2/11, ABCC7; 1/11, ABCC9; 4/11, ABCC11; 2/11 of these ABCA transporters in stem cell biology. The ABC and ABCG4; 1/11) and may reflect a binary answer of the PCR in transporter expression profiles of all individual samples are case of borderline expression levels, rather than indicating included in the Supplementary Information (Supplementary leukemia-specific expression of these genes. The ABC transpor- Figures 1 and 2). ter expression results of the individual AML patients are The preferential expression of ABC transporters in normal included in the Supplementary Information (Supplementary primitive cells and the possible implications of high expression Figures 3 and 4). in drug resistance, prompted us to investigate the expression of Similar to the expression pattern in normal bone marrow, these transporters in leukemic CD34 þ CD38À cells. ABC statistically significant higher expression in the CD34 þ CD38À transporter gene expression was assessed in CD34 þ CD38À cells compared to the CD34 þ CD38 þ cells was found for 27/ and CD34 þ CD38 þ cells from 12 untreated AML patients. In 40 ABC transporters in AML (Figure 2b). one leukemic patient sample, we were only able to sort the Currently, 13 ABC transporters are thought to be involved in CD34 þ CD38 þ fraction due to a very low percentage of transport of cytostatic drugs, and may, therefore, be involved in CD34 þ CD38À cells. Patient characteristics are shown in multidrug resistance. In addition to the expected transporters, Table 1. Others and we have previously shown that ABCB1, ABCC1 and ABCG2, all other ABC transporters CD34 þ CD38À cells in AML are predominantly of leukemic involved in drug transport were expressed in CD34 þ CD38À origin. GAPDH was used as a reference gene since we have cells in AML at varying frequencies. Six ABC drug transporters earlier demonstrated that this is an appropriate gene for were expressed in CD34 þ CD38À cells of all AML patients and normalization of target gene expression with similar expression statistically significant differential expression in CD34 þ CD38À levels in both normal and leukemic CD34 þ CD38À and cells compared to CD34 þ CD38 þ cells was found in 8/13 ABC CD34 þ CD38 þ cells.6 drug transporters.

Leukemia Letters to the Editor 753 a 1.E-01

1.E-02

1.E-03

1.E-04 (ABC transporter / GAPDH)

1.E-05 Relative ABC transporter mRNA expression

1.E-06 ABCA2 ABCB7 ABCB2 ABCB1 ABCB3 ABCA7 ABCA5 ABCB8 ABCA3 ABCA1 ABCB6 ABCB9 ABCA9 ABCB4 ABCA6 ABCB5 ABCA8 ABCA4 ABCC1 ABCD4 ABCD3 ABCD1 ABCC4 ABCC5 ABCC6 ABCD2 ABCC2 ABCC8 ABCC3 ABCC7 ABCC9 ABCG1 ABCG2 ABCG4 ABCG5 ABCG8 ABCB10 ABCA13 ABCA10 ABCB11 ABCA12 ABCC13 ABCC10 ABCC11 ABCC12

12121212121212 121212101212121212 9 512111210836 3112865931100220141 0 0

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1 ABCA9 ABCB4 ABCA6 ABCB5 ABCA8 ABCA4 ABCA2 ABCB7 ABCB2 ABCB1 ABCB3 ABCA7 ABCA5 ABCB8 ABCA3 ABCA1 ABCB6 ABCB9 ABCC2 ABCC8 ABCC3 ABCC7 ABCC9 ABCC1 ABCD4 ABCD3 ABCD1 ABCC4 ABCC5 ABCC6 ABCD2 ABCG4 ABCG5 ABCG8 ABCG2 ABCG1 ABCA13 ABCA10 ABCB11 ABCA12 ABCB10 ABCC11 ABCC12 ABCC13 ABCC10

12121212121212 121212101212121212 9 512111210836 3112865931100220141 0 0

0.1

Figure 2 ABC transporters are similarly expressed in leukemic and normal CD34 þ CD38À cells and virtually all expressed ABC transporters show a higher expression in hematopoietic CD34 þ CD38À cells in comparison to more committed CD34 þ CD38 þ progenitor cells in AML. (a) Gene expression of 45 ABC transporters was investigated in CD34 þ CD38À hematopoietic cells from AML patients at diagnosis (N ¼ 12). Levels of gene expression are depicted as natural logarithmic values on the y axis normalized for the expression of the housekeeping gene GAPDH. Mean values of gene expression of positive samples ( þ 95% CI) are represented by bars. On the x axis the ABC transporter gene name together with the frequency of expression (number of samples in which gene expression was detectable) is shown. (b) Difference in level of gene expression between CD34 þ CD38À and CD34 þ CD38 þ cells is depicted on the y axis as mean fold difference ( þ 95% CI) in samples that expressed the ABC transporter in both fractions. Transporters for which differentially expression reached statistically significance (using Student’s t-test for paired samples, Po0.05) are indicated with an asterix. All transporters, except for ABCB11 and ABCG4, are higher expressed in the CD34 þ CD38À fraction compared to the CD34 þ CD38 þ fraction.

The current study is the first reporting the ABC transporter transporters in (non-human) hematopoietic stem cells has been gene expression profile of human hematopoietic primitive cells described earlier for some transporters. Ramalho-Santos et al.7 using real-time RT-PCR but differential expression of ABC investigated the expression pattern in mouse (Sca þ cKit þ LinÀ)

Leukemia Letters to the Editor 754 hematopoietic stem cells in comparison to their differentiated Finally, the identification of previously unrecognized ABC counterparts using microarray expression profiling. They transporters in leukemic primitive cells offers the basis for future showed that eight ABC transporters were differentially ex- studies investigating the functional role of these ABC transpor- pressed. All these genes, except ABCB8, showed significantly ters in leukemia resistance and their usefulness as therapeuti- higher expression in normal CD34 þ CD38À cells in compar- cally targets to eradicate this crucial cell population in AML. ison to committed progenitor cells in our study. Similarly, gene expression profiling in fetal Sca þ AA4.1 þ Kit þ LinÀ liver 8 hematopoietic cells vs stem cell-depleted AA4.1À cells Acknowledgements identified 14 differentially expressed ABC transporters, all of which, when included, have higher expression in human The authors would like to thank late A Pennings and R CD34 þ CD38À cells in our study, except ABCG3 which has Woestenenk for excellent flow cytometry assistance. The Hema- no human ortholog. These comparisons indicate that the tology Data Base Center Nijmegen is thanked for providing reported expression profiles of ABC transporters in hematopoie- material and clinical data. tic (stem) cell populations show high concordance with the results from our study. In addition to the expected differentially EPLM de Grouw1,3, MHGP Raaijmakers2,3, JB Boezeman1, 1 1 2 expressed ABC transporters, we identified a large number of BA van der Reijden , LTF van de Locht , TJM de Witte , JH Jansen1 and RAP Raymakers2 ABC transporter genes, previously unknown to be expressed in 1 CD34 þ CD38À and CD34 þ CD38 þ cells, which displayed Central Hematology Laboratory, Nijmegen, higher expression in hematopoietic primitive cells compared to The Netherlands; 2Department of Hematology, Radboud University Nijmegen more committed progenitor cells. The higher sensitivity of real- Medical Centre, Nijmegen, The Netherlands; and time RT-PCR in comparison to cDNA and oligonucleotide 3These authors contributed equally to this article, Nijmegen, arrays, especially in low copy genes, may explain the The Netherlands identification of these genes in the current study. E-mail: [email protected] We demonstrate expression of all 13 ABC transporter members currently associated with drug resistance. The conserved expression of these drug resistance associated transporters after malignant transformation in AML could greatly References impede sensitivity of the leukemic CD34 þ CD38À cells to chemotherapeutical eradication, if mRNA expression levels reflect protein levels and function. If so, these data suggest that 1 Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat many members of the ABC transporter family may be involved Med 1997; 3: 730–737. in drug efflux mediated resistance of leukemic CD34 þ CD38À 2 Dean M, Fojo T, Bates S. Tumour stem cells and drug resistance. Nat cells. Modulation of multiple transporters might be required to Rev Cancer 2005; 5: 275–284. increase drug accumulation and induce chemotherapeutical 3 Chaudhary PM, Roninson IB. Expression and activity of P- eradication of primitive leukemic cells in AML. glycoprotein, a multidrug efflux pump, in human hematopoietic This has to be taken into account when interpreting the largely stem cells. Cell 1991; 66: 85–94. 4 Jung R, Ahmad-Nejad P, Wimmer M, Gerhard M, Wagener C, poor results of ABCB1 modulation using the potent inhibitor Neumaier M. Quality management and influential factors for the PSC833 on long-term disease outcome in clinical trials. It is detection of single metastatic cancer cells by reverse transcriptase important to stress that one of the main rationales for ABCB1 polymerase chain reaction. Eur J Clin Chem Clin Biochem 1997; 35: targeting is its prognostic value for remission induction in AML. 3–10. Based on the results of the current study, it is conceivable that 5 Rizzo MT. The role of arachidonic acid in normal and malignant ABCB1 may be a prognostic marker for AML cases that consist hematopoiesis. Prostaglandins Leukotriens Essential Fatty Acids 2002; 66: 57–69. predominantly of immature CD34 þ cells that co-express a high 6 Raaijmakers MH, van Emst L, de Witte T, Mensink E, Raymakers RA. number of ABC transporter molecules with putative involvement Quantitative assessment of gene expression in highly purified in drug extrusion and chemotherapy resistance. hematopoietic cells using real-time reverse transcriptase polymerase Additionally, the similar patterns of ABC transporter expres- chain reaction. Exp Hematol 2002; 30: 481–487. sion in normal and leukemic hematopoietic CD34 þ CD38À 7 Ramalho-Santos M, Yoon S, Matsuzaki Y, Mulligan RC, Melton DA. cells predict that modulation of these transporters in AML as a ‘Stemness’: transcriptional profiling of embryonic and adult stem cells. Science 2002; 298: 597–600. therapeutic strategy to target leukemic primitive cells will target 8 Phillips RL, Ernst RE, Brunk B, Ivanova N, Mahan MA, Deanehan JK residual normal hematopoietic primitive cells as well and is et al. The genetic program of hematopoietic stem cells. Science therefore likely to encounter increased bone marrow toxicity. 2000; 288: 1635–1640.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

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