CD34 CD7 Leukemic Progenitor Cells May Be Involved in Maintenance

Vol. 11, 505–511, January 15, 2005 Clinical Cancer Research 505

CD34+CD7+ Leukemic Progenitor Cells May Be Involved in Maintenance and Clonal Evolution of Chronic Myeloid Leukemia

Nobuharu Kosugi,1 Yasuhiro Ebihara,2 INTRODUCTION Tatsutoshi Nakahata,2 Hiromitsu Saisho,3 Chronic myeloid leukemia (CML) results from a Shigetaka Asano,1 and Arinobu Tojo1 pluripotent stem cell acquiring the Philadelphia (Ph) chromo- Departments of 1Haematology/Oncology and 2Pediatric Haematology/ some, in which the ABL gene at 9q34 is juxtaposed to the Oncology, Institute of Medical Science, University of Tokyo, Tokyo, 5.8-kb limited region of the BCR gene at 22q11, resulting in Japan; and 3First Department of Internal Medicine, Chiba University the generation of 210 kDa protein, p210BCR-ABL (1). This ac- School of Medicine, Chiba, Japan tive tyrosine kinase is responsible for the clonal amplification of leukemia cells by inhibition of apoptosis and stimulation of ABSTRACT cell cycling (1–3). Ph-positive stem cells are initially capable Purpose: We analyzed CD34+ cells coexpressing CD7 in of producing almost all lineages of mature blood cells in CML chronic myeloid leukemia (CML) in chronic phase (CP) or (4–6), which evolves from chronic phase (CP) into blast cri- accelerated phase (AP) to clarify their role in progression or sis, sometimes via an accelerated phase (AP). During this regression of the disease during treatment. evolution, a fraction of Ph-positive stem/progenitor cells may Experimental Design: Enumeration of CD34+CD7+ cells be susceptible to additional genetic changes and finally mani- was done on bone marrow nucleated cells from normal donors fest maturation arrest at a given stage of the differentiation and CML patients. Fluorescence in situ hybridization analysis program. These genetic changes involve the inactivation of was done on sorted CD34+CD7+ and CD34+CD7 cells to tumor suppressor genes (7, 8) as well as the formation of examine the occupancy rate of each fraction by BCR-ABL+ unique transforming genes (9). In other instances, the clonal cells with or without additional cytogenetic abnormalities. evolution is often defined by the acquisition of additional + + Results: The proportion of CD34 CD7 cells was chromosomal aberrations, including trisomy 8, i(17q) and significantly affected by the treatment outcome and/or the double Ph (10). F disease status as follows: 20.5 10.4% in normal donors It is evidenced that normal stem/progenitor cells are still F (n = 22), 18.1 10.2% in CP with major cytogenetic present in the bone marrow and peripheral blood of CML F response (n = 14), 53.0 12.9% in CP at diagnosis (n = 18), patients. Treatment with IFN-a or imatinib mesylate not only 55.0 F 15.8% in CP with minor or no cytogenetic response induces hematologic remission, but also restores benign (n = 28), and 70.2 F 18.1% in AP (n = 6). The proportion of polyclonal hematopoiesis. IFN-a induces complete cytogenetic + + CD34 CD7 cells decreased in parallel with cytogenetic remission in 10% to 20% of CP patients (11) and so does improvement in individual patients. In six untreated CP imatinib in about 40% of CP patients who failed to respond to patients, the ratio of BCR-ABL+ cells was comparable be- IFN-a (12), although minimal residual disease usually persists tween each fraction. In three patients with major cytogenetic (13). In addition, long-term bone marrow culture results in the response, the ratio of BCR-ABL+ cells was remarkably lower dominant expansion of Ph-negative cells in CML-CP (14). in CD34+CD7 cells than in CD34+CD7+ cells. In three AP There are some reports that benign primitive progenitors in patients with additional cytogenetic abnormalities, extra CML-CP are enriched within the HLA-DRlow/ subfraction of signals were detected at a much higher rate in CD34+CD7+ CD34+ cells (15, 16). Van Den Berg et al. (17) reported the cells than in CD34+CD7 cells. enrichment of normal progenitors in the CD34+Thy1+Lin cell Conclusions: Our results suggest that CD34+CD7+ cells fraction in IFN-resistant/intolerant CML. Nevertheless, normal may be involved in maintenance and clonal evolution of and leukemic progenitors are not readily discriminated on the BCR-ABL+ cells in CML. basis of phenotypic characteristics. Little is known concerning which fraction of Ph-positive CD34+ cells contributes to maintenance and progression of CML. Recently, we (18) and Received 7/23/04; revised 10/11/04; accepted 10/14/04. Martin-Henao et al. (19) reported the high incidence of CD7 and Grant support: Tokyo Biochemical Research Foundation. CD34 coexpression on blast cells in blast crisis, and the latter The costs of publication of this article were defrayed in part by the group also showed that CD7 was expressed to a higher degree payment of page charges. This article must therefore be hereby marked on CD34+ cells from CML-CP patients than on those from advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. normal donors (19). More recently, Sempowski et al. (20) + + Note: T. Nakahata is currently at the Department of Pediatrics, Kyoto reported that the relative number of CD34 CD7 cells University School of Medicine, Kyoto 606-8507, Japan. significantly increased in CML patients compared with normal Requests for reprints: Arinobu Tojo, Department of Haematology/ donors. CD7 was initially recognized as an early differentiation Oncology, Research Hospital, The Institute of Medical Science, The marker of T/NK cells, but is known to be expressed on immature University of Tokyo, 4-6-1 Shirokanedai, Minato, Tokyo 108-8639, Japan. Phone: 81-3-5449-5633; Fax: 81-3-5449-5429; E-mail: progenitor cells capable of producing T, B, and myeloid lineages [email protected]. (21). Thus, CD7 may be a candidate marker for studying the D2005 American Association for Cancer Research. cellular origin of clonal evolution in CML.

In the present study, we found that the proportion of FISH Analysis of Sorted Cells. Aliquots of sorted CD34+CD7+ cells were significantly higher in untreated and CD34+ cell populations were spread onto glass slides, fixed, IFN-a-resistant CML than in normal donors and IFN-a-sensitive and then stained with May-Grunwald-Giemsa solution. The CML. Additional evidence suggests that CD34+CD7+ cells may probes used were as follows: LSI bcr/abl ES dual color be involved in maintenance and clonal evolution of Ph-positive translocation probe (Vysis, Woodcreek, IL); chromosome cells. 8 enumeration probe (CEP8; Vysis); and an AML1 probe, which was a mixture of overlapping P1 and cosmid clones MATERIALS AND METHODS (24). The labeled probes were hybridized to metaphase Patients and Samples. Bone marrow specimens were samples according to the standard procedures. Images of the obtained with informed consent from normal donors and patients hybridized signals were captured with a 4V,6-diamidino-2- with CML in CP or AP. Patient characteristics are summarized in phenylindole/green/orange triple-bandpass filter (Chromatech- Table 1. The disease status was classified according to the nology, Brattleboro, VT). For each slide, more than 100 + criteria of the International Bone Marrow Transplant Registry, interphase cells were analyzed. CD34 cells from normal and the cytogenetic response criteria were defined as described donors were used as a negative control and the frequency of previously (23). Bone marrow samples from CML patients were false-positive cells was estimated to be 0.2% to 3.5%. also subjected to standard G-banding analysis and fluorescence Clonal Methylcellulose Culture Assay. The recombinant in situ hybridization (FISH) analysis. human cytokines used were as follows: stem cell factor (Amgen Flow Cytometric Analysis. The following fluorescein Biologicals, Thousand Oaks, CA); interleukin-3 and erythro- isothiocyanate (FITC)–conjugated and phycoerythrin (PE)– poietin (Kirin Brewery, Tokyo, Japan); granulocyte colony- conjugated monoclonal antibodies (MAb) and their respective stimulating factor (G-CSF; Chugai Pharmaceutical, Tokyo, isotype-matched controls were used: 8G12-FITC (anti-CD34) Japan); and IFN-a2b (Schering-Plough Japan, Osaka, Japan). and mouse IgG1-FITC (Becton Dickinson, Sunnyvale, CA); These were added as follows: stem cell factor, 100 ng/mL; 3A1E-12H7-PE (anti-CD7) and mouse IgG2b-PE (Coulter, Inc., interleukin-3, 200 units/mL; erythropoietin, 2 units/mL; and G- 2 3 Hialeah, FL). Enumeration of CD34+ cells was done using a CSF, 10 ng/mL. IFN-a2b was added at 0, 10 , and 10 IU/mL, FACSCalibur (Becton Dickinson) according to the modified respectively. The culture medium consisted of a modified Ea- Milan/Mulhouse protocol (23), in which CD34+ cells were gle’s medium (Invitrogen Corp, Grand Island, NY), 0.9% identified as the CD34bright and low side light scatter population methylcellulose (ShinEtsu Chemical Co., Tokyo, Japan), 30% on a CD34 versus side light scatter dot plot (Fig. 1A, I). This fetal bovine serum (Hyclone, Logan, UT), 1% deionized bo- 5 CD34.SSC region was used to examine CD7 expression. The vine serum albumin (Sigma, St. Louis, MO), 5 10 mol/L 2- events of the isotype control were subtracted from the CD34/ mercaptoethanol, and the cytokine cocktail. Aliquots of the CD7 results (Fig. 1A, II and III). mixture containing 300 sorted cells were plated in 35 mm dishes j Immunomagnetic Separation of CD34+ Cells. Non- (Nunc, Roskilde, Denmark) and incubated at 37 Cin5%CO2. phagocytic mononuclear cells were prepared by density gradient Cultures were done in triplicate and scored on day 14. Individual centrifugation over Lymphoprep (Axis-Shield, Oslo, Norway) colonies were lifted with a micropipette, spread on glass slides, after mixing bone marrow samples with silica suspension (IBL, and analyzed by FISH with the BCR-ABL translocation probe, Fujioka, Japan). CD34+ cells were separated with a MACS Cell as described above. Isolation Kit and the MiniMACS cell separator (Miltenyi Biotec, Statistical Analysis. Results were expressed as means F Bergish Gladbach, Germany) according to the manufacturer’s SD. Significance levels were determined by two-sided Stu- instructions, and were stored in liquid nitrogen until use. dent’s t test with P < 0.05 considered statistically significant. Cell Sorting. Frozen CD34+ cells were thawed and labeled with anti-CD34 and anti-CD7 MAbs. Cells incubated RESULTS with their respective isotype-matched controls were used to + define the unlabeled population. Cells in the low side light CD7 Expression by CD34 Cells From Normal Donors and scatter and low-intermediate forward light scatter windows CML Patients + were sorted on a FACSVantage (Becton Dickinson) into The expression of CD7 on CD34 cells occurred over a CD34+CD7+or fractions, which were shown to be more wide range of intensities (Fig. 1A, III). We compared CD7 than 98% pure upon reanalysis. expression by setting a common gating threshold for both normal donors and CML patients. A significant difference in the proportion of CD34+ cells coexpressing CD7 was noted between normal donors and untreated/IFN-a-resistant CML patients + + Table 1 Patient characteristics (Fig. 1B). CD34 CD7 cells comprised only a minor population (20.5 F 10.42%) of CD34+ cells in normal donors (n = 22), Clinical diagnosis No. Median Male/ + age Female whereas CD7 was expressed on a major population of CD34 cells from CML-CP patients at diagnosis (52.95 F 12.89%) or Normal 22 38 (18-50) 11/11 in minor or no cytogenetic response (54.95 F 15.78%). Inter- CML-CP + + therapy () 18 48 (20-60) 15/3 estingly, the relative number of CD34 CD7 cells were signif- IFN-a (+) minor/no response 12 44 (25-63) 9/3 icantly reduced to normal levels (18.14 F 10.24%) in patients partial/complete response 14 49 (32-63) 10/4 achieving partial or complete cytogenetic response, but increased CML-AP 6 49 (30-59) 4/2 to 70.19 F 18.08% in AP patients (Fig. 1B).

Fig. 1 A, flow cytometric analysis of bone marrow CD34+ cells from patients with CML: I, CD34-FITC antigen staining versus side light scatter profile; II, CD34-FITC versus IgG2b-PE control staining profile; III, CD34-FITC versus CD7-PE antigen staining profile. B, relative proportions of a CD7+ subset within CD34+ cells in normal donors and patients with CML. Horizontal bars, the mean value in each group. Brackets connecting dot plots, statistically significant differences (P < 0.05). C, association between the relative proportion of CD34+CD7+ cells and the cytogenetic improvement in 15 CML patients at diagnosis and after 6 months of IFN-a (n = 12) or imatinib (n = 3) therapy. The percentages of Ph-positive metaphases (X-axis) and CD34+CD7+ cells (Y-axis) in the bone marrow are indicated at diagnosis (arrow-end) and after 6 months (arrowhead)in each patient. Minimum of 20 metaphase spreads were analyzed for each sample.

Correlation Between CD34+CD7+ Cell Population and complete hematologic remission (data not shown). Except for Cytogenetic Response two cases which failed to respond to IFN-a, the proportion of In a prospective study of 15 newly diagnosed patients, we CD34+CD7+ cells decreased in parallel with cytogenetic examined whether the relative number of CD34+CD7+ cells improvement, although in the remaining two cases with no would be affected by the patients’ hematologic and/or response, the proportion of CD34+CD7+ cells did not change cytogenetic responses to therapy. The results are shown in (Fig. 1C), suggesting that there is a correlation between the Fig. 1C. Twelve patients received IFN-a and the remaining proportion of CD34+CD7+ cells and the cytogenetic response three had imatinib from the start. All the patients achieved in each patient. In addition, the proportion of CD34+CD7+

cells at diagnosis was not likely to be a predictor of we examined the inhibitory effect of IFN-a on colony formation. therapeutic response. Some of the IFN-a-resistant cases listed IFN-a showed a weak inhibitory effect on erythroid bursts in a in Table 1 were moved to imatinib therapy and achieved dose-dependent manner, but a negligible effect on GM complete cytogenetic remission with concomitant decrease of progenitors. Although it seems that CD34+CD7+ cells were CD34+CD7+ cell population (data not shown). more resistant to IFN-a than CD34+CD7 cells, this did not reach statistical significance (Fig. 3). FISH Analysis of Sorted CD34+CD7+ and CD34+CD7 Cells Both normal and leukemic progenitors can reside in the bone marrow of CML-CP patients, and IFN-a can preferentially DISCUSSION eliminate leukemic progenitors in some patients but cannot Previous studies showed the presence of CD34+CD7+ cells prevent their clonal evolution in others. To gain insight into the in the fetal liver (25) and thymus (25), as well as fetal (26) and + + significance of the particular behavior of CD34 CD7 cells in adult bone marrow (27–29). In addition, CD7 is expressed not + CML, we did FISH analysis of sorted CD34 subpopulations only on T/NK leukemia cells, but also on a subset of acute using the BCR-ABL translocation probe to determine the myeloid leukemia cells (30). These observations suggest that occupancy rate of each fraction by Ph-positive cells (Table 2). CD34+CD7+ cells may include very primitive stem/progenitor Figure 2 shows a representative flow cytogram for sorting of cells capable of differentiating into T, B, and myeloid lineages, + + + + CD34 CD7 and CD34 CD7 cells from the enriched CD34 although in aggregate they are considered to be heterogeneous high cell population (patient 7 in Table 2). CD7 and CD7 frac- populations with respect to both differentiation stage and lineage tions were easily identified in such a population. At diagnosis commitment. In our clonal culture assays, both CD34+CD7+ and of CML-CP, BCR-ABL fusion signals were found equally in both CD34+CD7 cells showed similar cloning efficiency and + + + CD34 CD7 and CD34 CD7 cells, ranging from 60% to 90% produced various types of colonies. The differentiation potentials of total nuclei (patients 1 to 3, and 8 to 11). In three patients of these two CD34+ subsets may be overlapping, but there is showing a major cytogenetic response to IFN-a treatment, some evidence for loss of CD7 expression upon induction of + CD34 CD7 cells exhibited a much lower rate of the fusion differentiation of CD34+CD7+ cells (19). + + signals than CD34 CD7 cells (13.0 versus 65.0% in patient The present study was motivated by the recent finding that 4, 8.1 versus 43.7% in patient 5, and 3.3 versus 50.0% in pa- blast cells in nonlymphoid blast crisis preferentially coexpressed tient 8), indicating the preferential recovery of normal hemato- + CD34 and CD7 (18). Two other groups reported that CD7 was poiesis in the CD34 CD7 cell fraction. On the other hand, in expressed on a mean of 25.3% to 32.6% of CD34+ cells from three patients who acquired additional chromosomal aberrations CML-CP patients, and on a mean of 3.6% to 4.9% of CD34+ such as t(3;21) in patient 9 and trisomy 8 in patients 10 and 11, cells from normal donors (19, 20). There is somewhat of a dis- respectively, FISH analysis using the AML1 and CEP8 probes + + crepancy between these results, especially in the expression level showed that CD34 CD7 cells presented a much higher rate + + of CD7 on normal CD34 cells. The most likely reason may of extra signals than CD34 CD7 cells (48.1 versus 12.4% be the difference in fluorescent dye for labeling anti-CD7 MAb. in patient 9, 58.0 versus 7.7% in patient 10, and 70.8 versus 15.1% in patient 11). In patient 9, who also showed a minor cytogenetic response to IFN-a, 81.7% of CD34+CD7+ cells contained the BCR-ABL fusion signals, which was comparable to Table 2 FISH analysis of sorted CD34+ cells the pretreatment value (85.3%), whereas a smaller proportion of + Total Sorted CD34 CD7 cells showed fusion signals than before treatment nucleated cells CD34+ cells (34.2 versus 85.7%). BCR- Additional ABL (%) signal (%) Clonal Culture of Sorted CD34+CD7+ and CD34+CD7 Cells Patient Ph-positive Additional + + To examine whether the two CD34+ subpopulations possess no. IFN-a metaphase abnormality CD7 CD7 CD7 CD7 distinct biological features, sorted CD34+CD7+ and 1 20/20 62.7 59.1 CD34+CD7 cells were assayed for their capacity to form 2 20/20 66.9 71.3 3 19/20 (+Ph) 01/19 68.0 65.5 colonies in semisolid medium. In three separate experiments 4 13/20 13.0 65.0 using samples from untreated patients (Table 3, patients 1-3), 5 + 08/20 8.1 43.7 both CD34+CD7+ and CD34+CD7 cells produced various 6 + 01/20 4.0 4.4 types of colonies, including erythroid bursts, granulocyte- 7 + 00/20 0.0 1.9 macrophage (GM), and mixed colonies (Table 3). The cloning 8 20/20 88.0 91.1 + + + + 08/20 3.3 50.0 efficiency of CD34 CD7 and CD34 CD7 cells were variable, 9 20/20 85.7 85.3 but comparable in all three assays. However, we noted that + 17/20 t(3;21) 08/17 34.2 81.7 12.4* 48.1* CD34+CD7+ cells contained more erythroid blast-forming units 10 + 20/20 (+8) 03/20 88.4 78.5 07.7y 58.0y rather than CD34+CD7 cells, and that CD34+CD7 cells 11 + 20/20 (+8) 08/20 68.9 67.8 15.1y 70.8y contained more granulocyte-macrophage colony-forming units NOTE. Total bone marrow nucleated cells were subjected to a than CD34+CD7+ cells. Next, individual colonies were picked standard G-banding analysis. CD34+CD7 and CD34+CD7+ cells sorted + up and subjected to FISH analysis to dissect their clonality. A from purified CD34 cells were spread onto glass slides and analyzed using the BCR-ABL translocation probe. At least 100 (114 on average) total of 45 colonies including erythroid, GM, and mixed colonies interphase cells were analyzed. were all positive for the BCR-ABL fusion signals, indicating *Samples from patients 9 to 11 were also analyzed by AML1 probes. their leukemic origin (data not shown). Using the same samples, ySamples from patients 9 to 11 were also analyzed by CEP8 probes.

Fig. 2 Flow cytometric sorting of CD34+CD7 and CD34+CD7+ cells from the enriched CD34+ cell population in patient 7 (Table 2) before and after IFN therapy. CD7- FITC and CD34-PE antigen staining profiles are shown. CD34+CD7 cells (A) and CD34+CD7+ cells (B) within the lymphoid window on a forward light scatter versus side light scatter dot plot were sorted.

We used the MAb labeled by PE, but others used FITC which is FISH analysis of the sorted CD34+ subsets revealed the less fluorescent than PE. in vivo biological significance of CD34+CD7+ cells. CD34+CD7+ These observations raise the question of why cells revealed quite different behavior from CD34+CD7 cells, CD34+CD7+ cells expand in CML. In the present study, depending on the IFN-a response in each patient. CD34+CD7+ BCR-ABL fusion signals were detected in 60% to 90% of cells seem more resistant to IFN-a than CD34+CD7 cells, and CD34+ cells in untreated patients. This score is comparable to comprise the larger part of the AP clone with additional genetic the abnormal reference range (69-92%) reported by Dewald changes. Assuming that CD7 is programmed to be transiently et al. (31) for bone marrow nuclei from most untreated expressed in the stem/progenitor cell compartment and to be patients. Takahashi et al. (32) reported similar results for persistent only in the T/NK cell lineage, maintenance and clonal sorted CD34+CD7+ cells from patients with 100% Ph-positive evolution of Ph-positive cells may originate from CD34+CD7+ metaphases. Taken together, our results and those by other stem cells. Normann et al. (20) also pointed out that all the CML groups indicate that the majority of CD34+CD7+ cells are patients with signs of disease progression showed expansion of BCR-ABL+ in untreated CML patients. It is likely that p210 is CD34+CD7+ cells, although the clinical risk scores did not differ involved either directly or indirectly in this phenomenon. For at diagnosis irrespective of the proportion of CD34+CD7+ cells. example, p210 may confer a growth advantage to a CD7+ Very recently Jamieson et al. (34) reported that progression to subset of CD34+ cells by an unknown mechanism. Alterna- blast crisis was associated with expansion of the myeloid tively, p210 may activate the expression of certain genes progenitor fraction, which consists mainly of GM progenitors, encoding cytokines, etc., which may in turn up-regulate CD7 rather than expansion of hematopoietic stem cells in CML. They expression in leukemic as well as normal progenitors, by an showed that the h-catenin pathway was activated in leukemic autocrine or paracrine mechanism. In this respect, some GM progenitors (34). Although CD34+CD7+ cell fractions attention should be given to the report by Jiang et al. (33) that include not only GM but also erythroid and common myeloid both CD71+CD45RA+ and CD71CD45RA subsets of progenitors in untreated CML-CP, commitment of CD34+CD7+ leukemic CD34+ cells constitutively produce interleukin-3 cells might be restricted to the GM lineage in CML-AP. Fur- and G-CSF in CML. thermore, it is intriguing to explore the activation status of the h-catenin pathway in both CD34+CD7+ and CD34+CD7 cells because the human CD7 promoter contains putative TCF/LEF-1 Table 3 Colony formation by sorted CD34+ cells binding sites (-ACAAAGT-).4 Cloning CD7 is a 40-kDa membrane glycoprotein and a member of Type of colony (%) efficiency (%) the immunoglobulin superfamily (20). The CD7 ligand has been Patient recently identified, and this molecule, K12/SECTM1, was shown no. Cell type CFU-GM BFU-E CFU-Mix to be highly expressed by peripheral blood leukocytes (35). + + 1 CD34 CD7 43.2 54.6 2.2 35.0 Cross-linkage of CD7, either by its ligand K12/SECTM1 or by CD34+CD7 52.0 44.1 3.9 47.7 2 CD34+CD7+ 18.8 80.3 0.9 22.8 MAb, leads to activation of a certain tyrosine kinase and CD34+CD7 54.0 43.1 2.9 25.3 recruitment of the phosphatidylinositol-3 kinase pathway (36), 3 CD34+CD7+ 23.1 75.0 1.3 57.2 and contributes to protection from apoptosis through activation + CD34 CD7 56.8 41.8 1.5 54.2 of the Akt kinase (37). Signaling through CD7 may confer a Abbreviations: CFU-GM, granulocyte-macrophage colony-forming growth advantage to CD34+ stem/progenitor cells in vivo but not unit; BFU-E, erythroid blast-forming unit. Three hundred CD34+CD7+ and CD34+CD7 cells sorted from untreated CML-CP patients were cultured with stem cell factors, interleukin-3, G-CSF, and erythropoietin, respectively. Results are shown as the percentage of mean value in triplicate cultures. 4 N. Kosagi, A. Tojo, unpublished observation.

Fig. 3 Effects of IFN-a2b on growth of colonies derived from CD34+CD7+ cells (o) and CD34+CD7 cells (.)in untreated CML patients (patients 1-3 in Table 3). Results from three experiments are expressed as the mean percentage of control cultures without IFN.

in vitro. Supporting this hypothesis, Hou et al. (38) showed that of chronic myeloid leukemia: implication for new therapeutic strategies. cross-linking of CD7 on myeloblasts by MAb results in Ann Hematol 1999;78:49–64. production of GM-CSF. Then, in vivo repopulation assays using 4. Fialkow PJ, Jacobson RJ, Papayannopoulou T. Chronic myelocytic leukemia: clonal origin in a stem cell common to the granulocyte, immunodeficient mice, or coculture studies with various types of erythrocyte, platelet and monocyte/macrophage. Am J Med stromal cells, is required to characterize the biological features of 1977;63:125–30. + + a primitive subset of CD34 CD7 cells. 5. LeBien TW, Hozier J, Minowada J, Kersey JW. Origin of chronic Our observations indicate that IFN-a is not a potent growth myelocytic leukemia in a precursor of pre-B lymphocytes. N Engl J Med inhibitor of Ph-positive clonogenic cells. Similar to our re- 1979;301:144–7. sults, Despres et al. 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Talpaz M, Kantarjian H, Kurzrock R, Trujillo JM, Gutterman JU. few imatinib-treated cases were available in this study, the Interferon-a produces sustained cytogenetic responses in chronic behavior of CD34+CD7+ cells in these patients was similar to myelogenous leukemia Philadelphia chromosome-positive patients. that in IFN-a-sensitive cases. The results of the present study Ann Intern Med 1991;114:532–8. suggest that CD34+CD7+ cells play an important role in the 12. Kantarjian H, Sawyers C, Hochhous A, et al. Hematologic and maintenance and clonal evolution of Ph-positive cells, and that cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N Engl J Med 2002;346:645–52. the selective antileukemic effects of IFN-a in CML may not be 13. 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Nobuharu Kosugi, Yasuhiro Ebihara, Tatsutoshi Nakahata, et al.

Clin Cancer Res 2005;11:505-511.

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