Leukemia (2014), 1–4 © 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu
LETTER TO THE EDITOR
CD34+CD38−CD58− cells are leukemia-propagating cells in Philadelphia chromosome-positive acute lymphoblastic leukemia Leukemiaadvance online publication, 19 August 2014; doi:10.1038/leu.2014.228
Prognosis of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) has improved with the use of tyrosine kinase inhibitors but most persons relapse. Some persons with Ph+ALL develop resistance to tyrosine kinase inhibitors but others relapse because of the persistence of quiescent leukemia stem cells (also termed leukemia-propagating cells (LPCs)). LPCs are defined by their ability to initiate human leukemia and to proliferate and self-renew in immune-deficient mice.1–4 LPCs in persons with acute myeloid leukemia have diverse phenotypes but most are CD34+CD38−.1,4 Recently, persons with acute myeloid leukemia and high LPCs frequencies in the bone marrow and persons whose bone marrow cells have a gene expression profile typical of LPCs are reported to have worse clinical outcomes following therapy with anti-leukemia drugs.5,6 In persons with Ph+ALL, CD34+CD38− cells were identified as LPCs in the non-obese diabetic/severe combined immunodeficient (NOD/SCID) xenograft assay,7 but the clinical relevance of this finding, if any, is unknown. CD58 is reported to be overexpressed in leukemia blasts and might be used as a marker of minimal residual disease in persons with B-cell ALL.8 Higher proportion of CD58+ cells is reported to correlate with better outcomes in B-ALL.9 In adults with Ph+ALL, there are no data on the potential prognostic importance of differences in CD58 expression patterns in CD34+ CD38− cells. We hypothesized candidate LPCs may be further enriched in the CD34+CD38−CD58− bone marrow fraction possibly translating to unfavorable prognosis. Sixty-three consecutive newly diagnosed adults with Ph+ALL were prospectively studied at Peking University Institute of Hematology from 1 January 2010 to 31 December 2012 (Supplementary Figure 1). Inclusion criteria included: (1) age 18–60 years; (2) diagnosis of ALL based on the 2008 World Health Organization criteria; (3) detection of the Ph-chromosome and/or BCR-ABL mRNA; (4) no contraindication to therapy with imatinib or an allotransplant. The study was approved by the Ethics Committee of Peking University People’s Hospital and written informed consent was obtained from all subjects before study entry in accordance with the Declaration of Helsinki. Induction chemotherapy included 1 cycle of a CODP regimen (cyclophosphamide, 750 mg/mE+2, day 1; vincristine 1.4 mg/mE +2, days 1, 8, 15, 22; daunorubicin, 40 mg/mE+2, days 1–3; prednisone, 1 mg/kg/day, days 1–21). Subjects achieving complete remission (CR) received 8 cycles of consolidation therapy including hyper-CVAD B (cycles 1, 3, 5 and 7; methotrexate, 1 g/mE+2, d 1; cytarabine, 1 g/mE+2, q12h, days 2–3) alternating with the hyperCVAD A (cycles 2, 4, 6 and 8; cyclophosphamide, 300 mg/mE+2, q12h, days 1–3; doxorubicin, 60 mg/mE+2, day 4; vincristine, 1.4 mg/mE+2, days 4, and 11; dexamethasone, 40 mg/day, days 1–4 and 11–14). Subjects also received imatinib (400 mg/day) during induction and consolidation therapy. After two cycles of Accepted article preview online 25 July 2014
consolidation, subjects with a suitable donor including an human leukocyte antigen-matched sibling, an human leukocyte antigen-matched unrelated donor or a human leukocyte antigenhaplotype identical-related donor were advised to receive an allotransplant.10–12 Subjects in the chemotherapy cohort received 6 more cycles of consolidation chemotherapy including the Hyper-CVAD B program alternating with the Hyper-CVAD A program followed by maintenance therapy (6-mercaptopurine, 60 mg/mE+2 daily; methotrexate, 20 mg/mE+2 weekly; monthly vincristine (4 mg/day, day 1)/prednisone (1 mg/kg/day, days 1–7) pulse) for 2 years. Multi-parameter flow cytometry analyses of CD58-FITC (Beckman-Coulter, Brea, CA, USA)/CD10-PE/CD19-APC-Cy7/CD34PerCP/ CD45-Vioblue/ CD38-APC (BD Biosciences, San Jose, CA, USA) on gated leukemia blasts was performed using a multi-color MACSQuant Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany). Fluorescence-minus-one controls were used to determine positive events for CD34, CD38 and CD58. There was considerable heterogeneity in expression of CD38 and CD58 (Supplementary Figure 2). Samples with ⩾ 20% blasts expressing the relevant CD antigen were considered positive. CD34+ blasts with ⩾ 20% CD38 expression were defined as a CD34+CD38+ phenotype, whereas CD34+ blasts with o 20% CD38 expression were classified as the CD34+CD38− phenotype. CD58 expression was calculated as a percent in the CD34+CD38+ population or CD34+CD38− population. CD34+CD38+ blasts with ⩾ 20% CD58 expression were defined as the CD34+CD38+CD58+ phenotype, whereas CD34+CD38+ blasts with o 20% CD58 expression were classified as the CD34+CD38+CD58− phenotype. Similarly, CD34+ CD38− blasts with ⩾ 20% CD58 expression were determined to be the CD34+CD38−CD58+ phenotype, whereas CD34+CD38− blasts with o20% CD58 expression were classified as the CD34+ CD38−CD58− phenotype. Based on blast phenotypes at diagnosis, subjects were further divided into the CD34+CD38−CD58− cohort (N = 13) and other phenotype cohort (N = 50, including subjects with CD34+CD38−CD58+, CD34+CD38+CD58− or CD34+CD38+ CD58+ phenotypes and subjects with the above defined four fractions concurrently). The clinical characteristics of the two phenotype groups did not differ significantly (Table 1). Median follow-up was 24 months (range, 6–43 months) for all subjects and 30 months (range, 8–43 months) for survivors. The CD34+CD38−CD58− cohort had a lower proportion of CR after the first course of chemotherapy (62% vs 90%; P = 0.03). Median time to achieve CR in the CD34+ CD38−CD58− cohort was significantly longer compared with the other phenotype cohort (median, 56 days vs 32 days; P = 0.04). Significantly, higher levels of BCR/ABL mRNA were detected in subjects in remission in the CD34+CD38−CD58− cohort than persons in remission in the other phenotype cohort especially after the third cycle of therapy. Cumulative incidence of relapse at 3 year in the CD34+CD38−CD58− cohort was significantly higher compared with cumulative incidence of relapse in the other phenotype cohort (60% (54–65%) vs 19% (18–19%); P = 0.02). Three-year leukemia-free survival of subjects in the other
Letter to the Editor
2 Table 1.
Characteristics of Ph+ALL subjects with CD34+CD38−CD58− or other phenotype at diagnosis CD34+CD38− CD58−
Other phenotype
P-value
13 36 (18–60) 6/7
50 38.5 (18–60) 30/20
0.5 0.5
Blood cell count at diagnosis Median WBC (×10E+9/l) (range)
32.5 (2.9–147)
30.1 (2.3–359)
0.5
Median BM blasts at diagnosis (%) (range) Median CD34+CD38−CD58− cells by BM blasts at diagnosis (%) (range) BCR-ABL (P190/P210) With/without additional cytogenetic abnormalities CR rate after 1 course of induction (%) Median time to achieve CR1 (days) (range)
85.3 (67–98) 82.3 (56–88.2) 8/5 2/11 61.5 56 (21–90)
79.0 (20.6–95.3) 6.5 (1.2–18.8) 30/20 16/34 90 31.5 (15–85)
0.09 o0.0001 1.00 0.32 0.03 0.04
5 8 (3/5)
12 38 (15/23)
BCR-ABL/ABL (%) during therapy After 1st cycle After 2nd cycle After 3rd cycle
19.3 (0–91.3) 16.3 (0–150) 11.9 (0–100.4)
5.7 (0–8.1) 5.18 (0–22.4) 1.2 (0–15)
0.05 0.02 0.02
Survival status BCR/ABL+at CR BCR/ABL+after the 3rd cycle CCR Relapse NRM
13 12 5 7 1
34 26 36 9 5
0.03 0.01 0.05 0.01 1.00
Characteristics Number of subjects Median age (range) Gender (male/female)
Post-consolidation therapy Chemo+IM Allo-HSCT (MSD/HID)+IM
0.3
Abbreviations: Allo-HSCT, allogeneic hematopoietic stem cell transplantation; BM, bone marrow; CR, complete remission; chemo, chemotherapy; CCR, continuous complete remission; HID, human leukocyte antigen-mismatched/haploidentical donors; IM, imatinib; MSD, human leukocyte antigen-matched sibling donors; NRM, non-relapse mortality; Ph+ALL, Philadelphia chromosome-positive acute lymphoblastic leukemia; WBC, white blood cell.
phenotype cohort was significantly higher than in subjects in the CD34+CD38−CD58− cohort (69% (53–81%) vs 33% (9–60%); P = 0.04). The CD34+CD38−CD58− cohort also had worse 3-year survival than the other phenotype cohort (32% (6–62%) vs 71% (55–82%)), but this difference was not significant (P = 0.07) (Supplementary Figure 3). In multivariate analyses, the CD34+CD38−CD58− phenotype was an independent risk factor correlated with likelihood of achieving CR (P = 0.03, odds ratio (OR) = 0.4 (0.2–0.9)), relapse (P = 0.03, OR = 3.4 (1.1–10.5)), leukemia-free survival (P = 0.01, OR = 3.1 (1.3–7.4)) and survival (P = 0.03, OR = 2.8 (1.1–6.9)) (Supplementary Table 1). Because of differences in clinical outcomes between subjects with and without CD34+CD38−CD58− phenotype, we studied the ability of cells from Ph+ALL subjects with a CD34+CD38−CD58−, CD34+CD38−CD58+, CD34+CD38+CD58− and CD34+CD38+CD58+ phenotypes to initiate leukemia in a murine xenograft assay. The six subjects were classified into the other phenotype group because the CD34+CD38−CD58− fraction was detected in only a few blasts (Supplementary Table 2). Bone marrow mononuclear cells were stained with mouse anti-human CD58-FITC (BeckmanCoulter) and CD34-PE/CD19-APC-Cy7/CD45-PerCP/CD38-APC/CD3, CD4,CD8-PE-Cy7 monoclonal antibodies (BD Biosciences) and sorted using the FACS Aria II (Becton Dickinson, San Jose, CA, USA). In the viable CD3−CD4−CD8− bone marrow mononuclear cells, CD34+CD38−CD58−, CD34+CD38−CD58+, CD34+CD38+CD58− and CD34+CD38+CD58+ fractions were sorted (Supplementary Figure 4). Purity of each fraction was 497%. The anti-CD122 (interleukin-2 receptor β (IL-2Rβ))-conditioned NOD/SCID xenograft assay was performed by intra-bone marrow injection.13,14 Doses were 1 × 10E+3, 1 × 10E+4 and 1 × 10E+5/mouse. We found different engraftment kinetics in the blood of primary and Leukemia (2014) 1 – 4
secondary recipients when 1 × 10E+3, 1 × 10E+4 or 1 × 10E+5 CD34+CD38−CD58− cells were transplanted. The efficiently engrafted human leukemia cells in all recipients transplanted with CD34+CD38−CD58− cells were phenotypically and clonally derived from the donor subjects analyzed by multi-parameter flow cytometry and BCR/ABL mRNA. Human leukemia cells were also detected infiltrating into liver, kidney and brain of primary and secondary murine recipients transplanted with CD34+ CD38−CD58− cells by hematoxylin and eosin staining and immune histochemistry with rabbit anti-human CD34 and CD19 (Abcam, Cambridge, MA, USA) (Figure 1). In contrast, CD34+CD38−CD58+, CD34+CD38+CD58− and CD34+CD38+CD58+ cells transplanted at the same or higher doses of 1 × 10E+6 and 1 × 10E+7 cells failed to engraft. These data suggests Ph+ALL LPCs are derived from the CD34+CD38−CD58− cells. Self-renewal capacity of CD34+CD38−CD58− cells was studied by serial transplants in mice. High levels of human CD45+CD19+ engraftment were observed in all of the secondary recipients of CD45+CD34+CD38−CD58− cells. In contrast, when 1 × 10E+3, 1 × 10E+4, 1 × 10E+5 CD45+CD34+CD38−CD58+ or CD45+CD34+CD38+ fractions from the same CD34+CD38−CD58− primary recipients were transplanted, no human engraftment was detected in secondary recipients. These findings suggest that CD34+CD38−CD58− cells not only initiate human leukemia but also self-renewal. Limiting dilution analyses were performed to estimate LPCs frequencies in the above four cell fractions. We calculated a median frequency of 1 LIC in 128 CD34+CD38−CD58− cells (95% confidence interval, 11–626). No LPCs were found in the CD34+ CD38−CD58+, CD34+CD38+CD58− or CD34+CD38+CD58+ fractions even at higher injection doses. © 2014 Macmillan Publishers Limited
Letter to the Editor
3
Figure 1. Comparison of human Ph+ALL engraftment in the anti-CD122-conditioned NOD/SCID recipients transplanted with CD34+ CD38−CD58− vs other phenotype (CD34+CD38−CD58+, CD34+CD38+CD58− or CD34+CD38+CD58+) of Ph+ALL cells. (A) May–Giemsa staining and fluorescence in situ hybridization (FISH) analyses of leukemic blasts in the original Ph+ALL subjects. (B) The recipient transplanted with CD34+CD38−CD58− Ph+ALL cells at 12-week posttransplantation (+) exhibited splenomegaly and suppression of erythropoiesis in the femur. (C) Recipients transplanted with the other phenotype of Ph+ALL cells (black) exhibited higher overall survival (OS) compared with the primary recipients (blue) and the secondary recipients (red) transplanted with CD34+CD38−CD58− Ph+ALL cells, as estimated by the Kaplan–Meier method (Po0.0001 for comparison of recipients within a given graft dose and for all recipients combined). No significant difference was noted between the primary and secondary recipients transplanted with the CD34+CD38−CD58− Ph+ALL cells. (D) Low-magnification images of human Ph+ALL engraftment in bone sections (upper panels) of the CD34+CD38−CD58− cells transplanted recipients; engraftment was further confirmed by morphologic and cytogenetic analyses as well as hematoxylin and eosin (HE) and immunohistochemical staining with antihuman CD19 and CD34 antibodies at high magnification (lower panels). In contrast, no human engraftment was demonstrated in the recipients transplanted with other phenotype cells. (E) CD34+CD38−CD58− Ph+ALL cells infiltrated into recipient organs. HE staining and antihuman CD19 and CD34 antibody labeling of the brain, liver, spleen and kidney of a recipient transplanted with CD34+CD38−CD58− cells compared with a recipient transplanted with other phenotype cells.
A previous report suggested Ph+ALL LPCs are found in the CD34+ CD38− population.7 Our data indicate these LPCs are found in the CD34+CD38−CD58− population. Subjects with the CD34+ CD38−CD58− phenotype had the worst clinical outcomes. We also found CD34+CD38− cells are heterogeneous. CD34+CD38−CD58− human Ph+ALL cells but not CD34+CD38−CD58+ can initiate Ph+ALL and self-renewal in anti-CD122-conditioned NOD/SCID mice. Archimbaud et al.9 reported a correlation between less CD58 expression on ALL blasts with worse survival. Similarly, we found worse outcomes in subjects with a CD34+CD38−CD58− phenotype. The conventional NOD/SCID mouse assay with intravenous injection is widely used to assay human hematopoietic stem cells and LPCs.1,4 Recent improvements include depletion of natural killer cells with anti-CD122 antibody and direct intra-medullary © 2014 Macmillan Publishers Limited
injection.13,14 Using this improved assay, we found candidate Ph+ALL LPCs in the CD34+CD38−CD58− fraction. Based on these data, we suggest the adverse clinical outcomes associated with the CD34+CD38−CD58− phenotype consistent with biological studies demonstrating that LPCs are quiescent and relatively resistant to chemotherapy.15 In conclusion, our study suggested that Ph+ALL LPCs are enriched in the CD34+CD38−CD58− phenotype which translates to adverse clinical outcomes.
CONFLICT OF INTEREST The authors declare no conflict of interest.
Leukemia (2014) 1 – 4
Letter to the Editor
4
ACKNOWLEDGEMENTS Professor Robert Peter Gale kindly reviewed the typescript. The work was supported by the National Natural Science Foundation of China (81370638 and 81230013), the National Clinical Priority Specialty, the Beijing Municipal Science and Technology Program (grant no. Z141100000214011), and Peking University People’s Hospital Research and Development Funds (RDB2012-23).
AUTHOR CONTRIBUTIONS X-JH designed the study and supervised the analyses and typescript preparation. YK performed the research, analyzed and interpreted the data, performed statistical analyses and wrote the typescript. All other authors participated in the collection of patient data. All of the authors agreed to submit the final typescript.
Y Kong1, Y-J Chang1, Y-R Liu1, Y-Z Wang1, Q Jiang1, H Jiang1, Y-Z Qin1, Y Hu1,2, Y-Y Lai1, C-W Duan3, D-L Hong3 and X-J Huang1,2 1 Peking University People’s Hospital, Peking University Institute of Hematology, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Beijing, China; 2 Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China and 3 Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai, China E-mail: [email protected] REFERENCES 1 Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997; 3: 730–737. 2 Cobaleda C, Sanchez-Garcia I. B-cell acute lymphoblastic leukaemia: towards understanding its cellular origin. Bioessays 2009; 31: 600–609. 3 Kong Y, Yoshida S, Saito Y, Doi T, Nagatoshi Y, Fukata M et al. CD34+CD38+CD19+ as well as CD34+CD38-CD19+ cells are leukemia-initiating cells with self-renewal capacity in human B-precursor ALL. Leukemia 2008; 22: 1207–1213. 4 Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 1994; 367: 645–648.
5 Eppert K, Takenaka K, Lechman ER, Waldron L, Nilsson B, van Galen P et al. Stem cell gene expression programs influence clinical outcome in human leukemia. Nat Med 2011; 17: 1086–1093. 6 Gentles AJ, Plevritis SK, Majeti R, Alizadeh AA. Association of a leukemic stem cell gene expression signature with clinical outcomes in acute myeloid leukemia. JAMA 2010; 304: 2706–2715. 7 Cobaleda C, Gutierrez-Cianca N, Perez-Losada J, Flores T, Garcia-Sanz R, Gonzalez M et al. A primitive hematopoietic cell is the target for the leukemic transformation in human Philadelphia-positive acute lymphoblastic leukemia. Blood 2000; 95: 1007–1013. 8 Chen JS, Coustan-Smith E, Suzuki T, Neale GA, Mihara K, Pui CH et al. Identification of novel markers for monitoring minimal residual disease in acute lymphoblastic leukemia. Blood 2001; 97: 2115–2120. 9 Archimbaud E, Thomas X, Campos L, Magaud JP, Dore JF, Fiere D. Expression of surface adhesion molecules CD54 (ICAM-1) and CD58 (LFA-3) in adult acute leukemia: relationship with initial characteristics and prognosis. Leukemia 1992; 6: 265–271. 10 Huang XJ, Zhu HH, Chang YJ, Xu LP, Liu DH, Zhang XH et al. The superiority of haploidentical related stem cell transplantation over chemotherapy alone as postremission treatment for patients with intermediate- or high-risk acute myeloid leukemia in first complete remission. Blood 2012; 119: 5584–5590. 11 Yan CH, Liu DH, Liu KY, Xu LP, Liu YR, Chen H et al. Risk stratification-directed donor lymphocyte infusion could reduce relapse of standard-risk acute leukemia patients after allogeneic hematopoietic stem cell transplantation. Blood 2012; 119: 3256–3262. 12 Xiao-Jun H, Lan-Ping X, Kai-Yan L, Dai-Hong L, Yu W, Huan C et al. Partially matched related donor transplantation can achieve outcomes comparable with unrelated donor transplantation for patients with hematologic malignancies. Clin Cancer Res 2009; 15: 4777–4783. 13 Mazurier F, Doedens M, Gan OI, Dick JE. Rapid myeloerythroid repopulation after intrafemoral transplantation of NOD-SCID mice reveals a new class of human stem cells. Nat Med 2003; 9: 959–963. 14 Tanaka T, Tsudo M, Karasuyama H, Kitamura F, Kono T, Hatakeyama M et al. A novel monoclonal antibody against murine IL-2 receptor beta-chain. Characterization of receptor expression in normal lymphoid cells and EL-4 cells. J Immunol 1991; 147: 2222–2228. 15 Ishikawa F, Yoshida S, Saito Y, Hijikata A, Kitamura H, Tanaka S et al. Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region. Nat Biotechnol 2007; 25: 1315–1321.
Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)
Leukemia (2014) 1 – 4
© 2014 Macmillan Publishers Limited
LETTER TO THE EDITOR
CD34+CD38−CD58− cells are leukemia-propagating cells in Philadelphia chromosome-positive acute lymphoblastic leukemia Leukemiaadvance online publication, 19 August 2014; doi:10.1038/leu.2014.228
Prognosis of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) has improved with the use of tyrosine kinase inhibitors but most persons relapse. Some persons with Ph+ALL develop resistance to tyrosine kinase inhibitors but others relapse because of the persistence of quiescent leukemia stem cells (also termed leukemia-propagating cells (LPCs)). LPCs are defined by their ability to initiate human leukemia and to proliferate and self-renew in immune-deficient mice.1–4 LPCs in persons with acute myeloid leukemia have diverse phenotypes but most are CD34+CD38−.1,4 Recently, persons with acute myeloid leukemia and high LPCs frequencies in the bone marrow and persons whose bone marrow cells have a gene expression profile typical of LPCs are reported to have worse clinical outcomes following therapy with anti-leukemia drugs.5,6 In persons with Ph+ALL, CD34+CD38− cells were identified as LPCs in the non-obese diabetic/severe combined immunodeficient (NOD/SCID) xenograft assay,7 but the clinical relevance of this finding, if any, is unknown. CD58 is reported to be overexpressed in leukemia blasts and might be used as a marker of minimal residual disease in persons with B-cell ALL.8 Higher proportion of CD58+ cells is reported to correlate with better outcomes in B-ALL.9 In adults with Ph+ALL, there are no data on the potential prognostic importance of differences in CD58 expression patterns in CD34+ CD38− cells. We hypothesized candidate LPCs may be further enriched in the CD34+CD38−CD58− bone marrow fraction possibly translating to unfavorable prognosis. Sixty-three consecutive newly diagnosed adults with Ph+ALL were prospectively studied at Peking University Institute of Hematology from 1 January 2010 to 31 December 2012 (Supplementary Figure 1). Inclusion criteria included: (1) age 18–60 years; (2) diagnosis of ALL based on the 2008 World Health Organization criteria; (3) detection of the Ph-chromosome and/or BCR-ABL mRNA; (4) no contraindication to therapy with imatinib or an allotransplant. The study was approved by the Ethics Committee of Peking University People’s Hospital and written informed consent was obtained from all subjects before study entry in accordance with the Declaration of Helsinki. Induction chemotherapy included 1 cycle of a CODP regimen (cyclophosphamide, 750 mg/mE+2, day 1; vincristine 1.4 mg/mE +2, days 1, 8, 15, 22; daunorubicin, 40 mg/mE+2, days 1–3; prednisone, 1 mg/kg/day, days 1–21). Subjects achieving complete remission (CR) received 8 cycles of consolidation therapy including hyper-CVAD B (cycles 1, 3, 5 and 7; methotrexate, 1 g/mE+2, d 1; cytarabine, 1 g/mE+2, q12h, days 2–3) alternating with the hyperCVAD A (cycles 2, 4, 6 and 8; cyclophosphamide, 300 mg/mE+2, q12h, days 1–3; doxorubicin, 60 mg/mE+2, day 4; vincristine, 1.4 mg/mE+2, days 4, and 11; dexamethasone, 40 mg/day, days 1–4 and 11–14). Subjects also received imatinib (400 mg/day) during induction and consolidation therapy. After two cycles of Accepted article preview online 25 July 2014
consolidation, subjects with a suitable donor including an human leukocyte antigen-matched sibling, an human leukocyte antigen-matched unrelated donor or a human leukocyte antigenhaplotype identical-related donor were advised to receive an allotransplant.10–12 Subjects in the chemotherapy cohort received 6 more cycles of consolidation chemotherapy including the Hyper-CVAD B program alternating with the Hyper-CVAD A program followed by maintenance therapy (6-mercaptopurine, 60 mg/mE+2 daily; methotrexate, 20 mg/mE+2 weekly; monthly vincristine (4 mg/day, day 1)/prednisone (1 mg/kg/day, days 1–7) pulse) for 2 years. Multi-parameter flow cytometry analyses of CD58-FITC (Beckman-Coulter, Brea, CA, USA)/CD10-PE/CD19-APC-Cy7/CD34PerCP/ CD45-Vioblue/ CD38-APC (BD Biosciences, San Jose, CA, USA) on gated leukemia blasts was performed using a multi-color MACSQuant Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany). Fluorescence-minus-one controls were used to determine positive events for CD34, CD38 and CD58. There was considerable heterogeneity in expression of CD38 and CD58 (Supplementary Figure 2). Samples with ⩾ 20% blasts expressing the relevant CD antigen were considered positive. CD34+ blasts with ⩾ 20% CD38 expression were defined as a CD34+CD38+ phenotype, whereas CD34+ blasts with o 20% CD38 expression were classified as the CD34+CD38− phenotype. CD58 expression was calculated as a percent in the CD34+CD38+ population or CD34+CD38− population. CD34+CD38+ blasts with ⩾ 20% CD58 expression were defined as the CD34+CD38+CD58+ phenotype, whereas CD34+CD38+ blasts with o 20% CD58 expression were classified as the CD34+CD38+CD58− phenotype. Similarly, CD34+ CD38− blasts with ⩾ 20% CD58 expression were determined to be the CD34+CD38−CD58+ phenotype, whereas CD34+CD38− blasts with o20% CD58 expression were classified as the CD34+ CD38−CD58− phenotype. Based on blast phenotypes at diagnosis, subjects were further divided into the CD34+CD38−CD58− cohort (N = 13) and other phenotype cohort (N = 50, including subjects with CD34+CD38−CD58+, CD34+CD38+CD58− or CD34+CD38+ CD58+ phenotypes and subjects with the above defined four fractions concurrently). The clinical characteristics of the two phenotype groups did not differ significantly (Table 1). Median follow-up was 24 months (range, 6–43 months) for all subjects and 30 months (range, 8–43 months) for survivors. The CD34+CD38−CD58− cohort had a lower proportion of CR after the first course of chemotherapy (62% vs 90%; P = 0.03). Median time to achieve CR in the CD34+ CD38−CD58− cohort was significantly longer compared with the other phenotype cohort (median, 56 days vs 32 days; P = 0.04). Significantly, higher levels of BCR/ABL mRNA were detected in subjects in remission in the CD34+CD38−CD58− cohort than persons in remission in the other phenotype cohort especially after the third cycle of therapy. Cumulative incidence of relapse at 3 year in the CD34+CD38−CD58− cohort was significantly higher compared with cumulative incidence of relapse in the other phenotype cohort (60% (54–65%) vs 19% (18–19%); P = 0.02). Three-year leukemia-free survival of subjects in the other
Letter to the Editor
2 Table 1.
Characteristics of Ph+ALL subjects with CD34+CD38−CD58− or other phenotype at diagnosis CD34+CD38− CD58−
Other phenotype
P-value
13 36 (18–60) 6/7
50 38.5 (18–60) 30/20
0.5 0.5
Blood cell count at diagnosis Median WBC (×10E+9/l) (range)
32.5 (2.9–147)
30.1 (2.3–359)
0.5
Median BM blasts at diagnosis (%) (range) Median CD34+CD38−CD58− cells by BM blasts at diagnosis (%) (range) BCR-ABL (P190/P210) With/without additional cytogenetic abnormalities CR rate after 1 course of induction (%) Median time to achieve CR1 (days) (range)
85.3 (67–98) 82.3 (56–88.2) 8/5 2/11 61.5 56 (21–90)
79.0 (20.6–95.3) 6.5 (1.2–18.8) 30/20 16/34 90 31.5 (15–85)
0.09 o0.0001 1.00 0.32 0.03 0.04
5 8 (3/5)
12 38 (15/23)
BCR-ABL/ABL (%) during therapy After 1st cycle After 2nd cycle After 3rd cycle
19.3 (0–91.3) 16.3 (0–150) 11.9 (0–100.4)
5.7 (0–8.1) 5.18 (0–22.4) 1.2 (0–15)
0.05 0.02 0.02
Survival status BCR/ABL+at CR BCR/ABL+after the 3rd cycle CCR Relapse NRM
13 12 5 7 1
34 26 36 9 5
0.03 0.01 0.05 0.01 1.00
Characteristics Number of subjects Median age (range) Gender (male/female)
Post-consolidation therapy Chemo+IM Allo-HSCT (MSD/HID)+IM
0.3
Abbreviations: Allo-HSCT, allogeneic hematopoietic stem cell transplantation; BM, bone marrow; CR, complete remission; chemo, chemotherapy; CCR, continuous complete remission; HID, human leukocyte antigen-mismatched/haploidentical donors; IM, imatinib; MSD, human leukocyte antigen-matched sibling donors; NRM, non-relapse mortality; Ph+ALL, Philadelphia chromosome-positive acute lymphoblastic leukemia; WBC, white blood cell.
phenotype cohort was significantly higher than in subjects in the CD34+CD38−CD58− cohort (69% (53–81%) vs 33% (9–60%); P = 0.04). The CD34+CD38−CD58− cohort also had worse 3-year survival than the other phenotype cohort (32% (6–62%) vs 71% (55–82%)), but this difference was not significant (P = 0.07) (Supplementary Figure 3). In multivariate analyses, the CD34+CD38−CD58− phenotype was an independent risk factor correlated with likelihood of achieving CR (P = 0.03, odds ratio (OR) = 0.4 (0.2–0.9)), relapse (P = 0.03, OR = 3.4 (1.1–10.5)), leukemia-free survival (P = 0.01, OR = 3.1 (1.3–7.4)) and survival (P = 0.03, OR = 2.8 (1.1–6.9)) (Supplementary Table 1). Because of differences in clinical outcomes between subjects with and without CD34+CD38−CD58− phenotype, we studied the ability of cells from Ph+ALL subjects with a CD34+CD38−CD58−, CD34+CD38−CD58+, CD34+CD38+CD58− and CD34+CD38+CD58+ phenotypes to initiate leukemia in a murine xenograft assay. The six subjects were classified into the other phenotype group because the CD34+CD38−CD58− fraction was detected in only a few blasts (Supplementary Table 2). Bone marrow mononuclear cells were stained with mouse anti-human CD58-FITC (BeckmanCoulter) and CD34-PE/CD19-APC-Cy7/CD45-PerCP/CD38-APC/CD3, CD4,CD8-PE-Cy7 monoclonal antibodies (BD Biosciences) and sorted using the FACS Aria II (Becton Dickinson, San Jose, CA, USA). In the viable CD3−CD4−CD8− bone marrow mononuclear cells, CD34+CD38−CD58−, CD34+CD38−CD58+, CD34+CD38+CD58− and CD34+CD38+CD58+ fractions were sorted (Supplementary Figure 4). Purity of each fraction was 497%. The anti-CD122 (interleukin-2 receptor β (IL-2Rβ))-conditioned NOD/SCID xenograft assay was performed by intra-bone marrow injection.13,14 Doses were 1 × 10E+3, 1 × 10E+4 and 1 × 10E+5/mouse. We found different engraftment kinetics in the blood of primary and Leukemia (2014) 1 – 4
secondary recipients when 1 × 10E+3, 1 × 10E+4 or 1 × 10E+5 CD34+CD38−CD58− cells were transplanted. The efficiently engrafted human leukemia cells in all recipients transplanted with CD34+CD38−CD58− cells were phenotypically and clonally derived from the donor subjects analyzed by multi-parameter flow cytometry and BCR/ABL mRNA. Human leukemia cells were also detected infiltrating into liver, kidney and brain of primary and secondary murine recipients transplanted with CD34+ CD38−CD58− cells by hematoxylin and eosin staining and immune histochemistry with rabbit anti-human CD34 and CD19 (Abcam, Cambridge, MA, USA) (Figure 1). In contrast, CD34+CD38−CD58+, CD34+CD38+CD58− and CD34+CD38+CD58+ cells transplanted at the same or higher doses of 1 × 10E+6 and 1 × 10E+7 cells failed to engraft. These data suggests Ph+ALL LPCs are derived from the CD34+CD38−CD58− cells. Self-renewal capacity of CD34+CD38−CD58− cells was studied by serial transplants in mice. High levels of human CD45+CD19+ engraftment were observed in all of the secondary recipients of CD45+CD34+CD38−CD58− cells. In contrast, when 1 × 10E+3, 1 × 10E+4, 1 × 10E+5 CD45+CD34+CD38−CD58+ or CD45+CD34+CD38+ fractions from the same CD34+CD38−CD58− primary recipients were transplanted, no human engraftment was detected in secondary recipients. These findings suggest that CD34+CD38−CD58− cells not only initiate human leukemia but also self-renewal. Limiting dilution analyses were performed to estimate LPCs frequencies in the above four cell fractions. We calculated a median frequency of 1 LIC in 128 CD34+CD38−CD58− cells (95% confidence interval, 11–626). No LPCs were found in the CD34+ CD38−CD58+, CD34+CD38+CD58− or CD34+CD38+CD58+ fractions even at higher injection doses. © 2014 Macmillan Publishers Limited
Letter to the Editor
3
Figure 1. Comparison of human Ph+ALL engraftment in the anti-CD122-conditioned NOD/SCID recipients transplanted with CD34+ CD38−CD58− vs other phenotype (CD34+CD38−CD58+, CD34+CD38+CD58− or CD34+CD38+CD58+) of Ph+ALL cells. (A) May–Giemsa staining and fluorescence in situ hybridization (FISH) analyses of leukemic blasts in the original Ph+ALL subjects. (B) The recipient transplanted with CD34+CD38−CD58− Ph+ALL cells at 12-week posttransplantation (+) exhibited splenomegaly and suppression of erythropoiesis in the femur. (C) Recipients transplanted with the other phenotype of Ph+ALL cells (black) exhibited higher overall survival (OS) compared with the primary recipients (blue) and the secondary recipients (red) transplanted with CD34+CD38−CD58− Ph+ALL cells, as estimated by the Kaplan–Meier method (Po0.0001 for comparison of recipients within a given graft dose and for all recipients combined). No significant difference was noted between the primary and secondary recipients transplanted with the CD34+CD38−CD58− Ph+ALL cells. (D) Low-magnification images of human Ph+ALL engraftment in bone sections (upper panels) of the CD34+CD38−CD58− cells transplanted recipients; engraftment was further confirmed by morphologic and cytogenetic analyses as well as hematoxylin and eosin (HE) and immunohistochemical staining with antihuman CD19 and CD34 antibodies at high magnification (lower panels). In contrast, no human engraftment was demonstrated in the recipients transplanted with other phenotype cells. (E) CD34+CD38−CD58− Ph+ALL cells infiltrated into recipient organs. HE staining and antihuman CD19 and CD34 antibody labeling of the brain, liver, spleen and kidney of a recipient transplanted with CD34+CD38−CD58− cells compared with a recipient transplanted with other phenotype cells.
A previous report suggested Ph+ALL LPCs are found in the CD34+ CD38− population.7 Our data indicate these LPCs are found in the CD34+CD38−CD58− population. Subjects with the CD34+ CD38−CD58− phenotype had the worst clinical outcomes. We also found CD34+CD38− cells are heterogeneous. CD34+CD38−CD58− human Ph+ALL cells but not CD34+CD38−CD58+ can initiate Ph+ALL and self-renewal in anti-CD122-conditioned NOD/SCID mice. Archimbaud et al.9 reported a correlation between less CD58 expression on ALL blasts with worse survival. Similarly, we found worse outcomes in subjects with a CD34+CD38−CD58− phenotype. The conventional NOD/SCID mouse assay with intravenous injection is widely used to assay human hematopoietic stem cells and LPCs.1,4 Recent improvements include depletion of natural killer cells with anti-CD122 antibody and direct intra-medullary © 2014 Macmillan Publishers Limited
injection.13,14 Using this improved assay, we found candidate Ph+ALL LPCs in the CD34+CD38−CD58− fraction. Based on these data, we suggest the adverse clinical outcomes associated with the CD34+CD38−CD58− phenotype consistent with biological studies demonstrating that LPCs are quiescent and relatively resistant to chemotherapy.15 In conclusion, our study suggested that Ph+ALL LPCs are enriched in the CD34+CD38−CD58− phenotype which translates to adverse clinical outcomes.
CONFLICT OF INTEREST The authors declare no conflict of interest.
Leukemia (2014) 1 – 4
Letter to the Editor
4
ACKNOWLEDGEMENTS Professor Robert Peter Gale kindly reviewed the typescript. The work was supported by the National Natural Science Foundation of China (81370638 and 81230013), the National Clinical Priority Specialty, the Beijing Municipal Science and Technology Program (grant no. Z141100000214011), and Peking University People’s Hospital Research and Development Funds (RDB2012-23).
AUTHOR CONTRIBUTIONS X-JH designed the study and supervised the analyses and typescript preparation. YK performed the research, analyzed and interpreted the data, performed statistical analyses and wrote the typescript. All other authors participated in the collection of patient data. All of the authors agreed to submit the final typescript.
Y Kong1, Y-J Chang1, Y-R Liu1, Y-Z Wang1, Q Jiang1, H Jiang1, Y-Z Qin1, Y Hu1,2, Y-Y Lai1, C-W Duan3, D-L Hong3 and X-J Huang1,2 1 Peking University People’s Hospital, Peking University Institute of Hematology, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Beijing, China; 2 Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China and 3 Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai, China E-mail: [email protected] REFERENCES 1 Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997; 3: 730–737. 2 Cobaleda C, Sanchez-Garcia I. B-cell acute lymphoblastic leukaemia: towards understanding its cellular origin. Bioessays 2009; 31: 600–609. 3 Kong Y, Yoshida S, Saito Y, Doi T, Nagatoshi Y, Fukata M et al. CD34+CD38+CD19+ as well as CD34+CD38-CD19+ cells are leukemia-initiating cells with self-renewal capacity in human B-precursor ALL. Leukemia 2008; 22: 1207–1213. 4 Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 1994; 367: 645–648.
5 Eppert K, Takenaka K, Lechman ER, Waldron L, Nilsson B, van Galen P et al. Stem cell gene expression programs influence clinical outcome in human leukemia. Nat Med 2011; 17: 1086–1093. 6 Gentles AJ, Plevritis SK, Majeti R, Alizadeh AA. Association of a leukemic stem cell gene expression signature with clinical outcomes in acute myeloid leukemia. JAMA 2010; 304: 2706–2715. 7 Cobaleda C, Gutierrez-Cianca N, Perez-Losada J, Flores T, Garcia-Sanz R, Gonzalez M et al. A primitive hematopoietic cell is the target for the leukemic transformation in human Philadelphia-positive acute lymphoblastic leukemia. Blood 2000; 95: 1007–1013. 8 Chen JS, Coustan-Smith E, Suzuki T, Neale GA, Mihara K, Pui CH et al. Identification of novel markers for monitoring minimal residual disease in acute lymphoblastic leukemia. Blood 2001; 97: 2115–2120. 9 Archimbaud E, Thomas X, Campos L, Magaud JP, Dore JF, Fiere D. Expression of surface adhesion molecules CD54 (ICAM-1) and CD58 (LFA-3) in adult acute leukemia: relationship with initial characteristics and prognosis. Leukemia 1992; 6: 265–271. 10 Huang XJ, Zhu HH, Chang YJ, Xu LP, Liu DH, Zhang XH et al. The superiority of haploidentical related stem cell transplantation over chemotherapy alone as postremission treatment for patients with intermediate- or high-risk acute myeloid leukemia in first complete remission. Blood 2012; 119: 5584–5590. 11 Yan CH, Liu DH, Liu KY, Xu LP, Liu YR, Chen H et al. Risk stratification-directed donor lymphocyte infusion could reduce relapse of standard-risk acute leukemia patients after allogeneic hematopoietic stem cell transplantation. Blood 2012; 119: 3256–3262. 12 Xiao-Jun H, Lan-Ping X, Kai-Yan L, Dai-Hong L, Yu W, Huan C et al. Partially matched related donor transplantation can achieve outcomes comparable with unrelated donor transplantation for patients with hematologic malignancies. Clin Cancer Res 2009; 15: 4777–4783. 13 Mazurier F, Doedens M, Gan OI, Dick JE. Rapid myeloerythroid repopulation after intrafemoral transplantation of NOD-SCID mice reveals a new class of human stem cells. Nat Med 2003; 9: 959–963. 14 Tanaka T, Tsudo M, Karasuyama H, Kitamura F, Kono T, Hatakeyama M et al. A novel monoclonal antibody against murine IL-2 receptor beta-chain. Characterization of receptor expression in normal lymphoid cells and EL-4 cells. J Immunol 1991; 147: 2222–2228. 15 Ishikawa F, Yoshida S, Saito Y, Hijikata A, Kitamura H, Tanaka S et al. Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region. Nat Biotechnol 2007; 25: 1315–1321.
Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)
Leukemia (2014) 1 – 4
© 2014 Macmillan Publishers Limited
