Differences in heat sensitivity between normal and acute myeloid leukemic stem cells: feasibility of hyperthermic purging of leukemic cells from autologous stem cell grafts

OBJECTIVES: In autologous stem cell transplantation contamination of the graft with malignant cells is frequently noticed and necessitates the use of in vivo or in vitro purging modalities. The hematopoietic recovery after transplantation depends on the number of stem and progenitor cells in the transplant. Therefore, in the present study the effects of hyperthermic treatment on the human normal and acute myeloid leukemic (AML) stem cell compartment were investigated. METHODS: Normal bone marrow and AML blasts were heat treated up to 120 minutes at 43 degrees C. The surviving fractions of the different stem cell subsets were determined using in vitro methylcellulose and cobblestone area-forming cell (CAFC) clonogenic assays, as well as the in vivo NOD/SCID repopulating assay. The leukemic nature of the colonies from AML cells was confirmed by RT-PCR analysis. In order to increase the therapeutic index of the hyperthermic purging modality, the heat treatment was preceded by a 3-hour incubation at 37 degrees C with the ether lipid ET-18-OCH(3) (25 microg/mL). RESULTS: It could be demonstrated that normal progenitor cells are far more resistant to hyperthermia than leukemic progenitor cells (56%+/-7% vs 9.9%+/-2.6% survival after 60 minutes at 43 degrees C, respectively). Furthermore, normal hematopoietic stem cells appear to be extremely resistant to the heat treatment (94%+/-9% survival after 60 minutes at 43 degrees C). In contrast, in the leukemic stem cell compartment no significant differences in heat sensitivity between the stem cells and progenitor subsets could be observed (12.3%+/-2.9% vs 9.9%+/-2.6% survival after 60 minutes at 43 degrees C, respectively). The combined treatment resulted in a survival for normal progenitor and stem cells of 32%+/-6% and 85%+/-15% after 60 minutes at 43 degrees C, respectively. Under these conditions the number of leukemic stem cells was reduced to 1%+/-0.3%. After 120 minutes at 43 degrees C, no AML-colonies could be detected anymore. CONCLUSIONS: Our data demonstrate that leukemic stem cells have an increased hyperthermic sensitivity compared to their normal counterparts and that this difference can be further increased in combination with ET-18-OCH(3). These striking differences in heat sensitivity warrant the use of hyperthermia as a clinically applicable purging modality in autologous stem cell transplantation.     

Persistence of bcr-abl mRNA-expressing cells in long-term cultures established from chronic myeloid leukemic bone marrow or blood

Summary Long-term cultures (LTC) established from chronic myeloid leukemic (CML) bone marrow or blood contain bcr-abl mRNA-expressing cells, as demonstrated by the polymerase chain reaction (PCR). Bcr-abl sequences were detectable in cultures from all of nine patients investigated. In LTC from two patients, in which a cytogenetic conversion from Ph⁺ to Ph^– was noted at day 20 and 40, respectively, the PCR for bcr-abl remained positive, at least up to day 112 and 245, respectively, in vitro. The differences between the results of cytogenetic and PCR analyses was explained by the fact that the metaphases studied were obviously derived from spontaneously EBV-transformed bcr-abl mRNA-negative B cells, which may become the dominating cell type very early in LTC from bone marrow or blood. LTCs cloned for EBV-transformed B cells which no longer harbor admixed macrophages are bcr-abl mRNA negative. In conclusion, bcr-abl mRNA-expressing cells may show long-term persistence in LTC established from CML bone marrow or blood.     

Growth factors influence the sensitivity of leukemic stem cells to cytosine arabinoside in culture

We have proposed that the blasts in acute myeloblastic leukemia (AML) are renewal populations maintained by a small subpopulation of stem cells. The balance between self-renewal and differentiation in blast stem cells may be an important attribute contributing to treatment outcome. Cytosine arabinoside (ara-C) is included in most chemotherapeutic regimens for the treatment of AML. When ara-C survival curves are constructed, the drug appears to be more toxic when an assay is used that detects principally self-renewing divisions, compared with a procedure that depends on terminal divisions. AML blasts usually respond in culture to myelopoietic growth factors; their response often includes a change in self-renewal, differentiation, or both. These features of the model for AML blasts led to the prediction that growth factors would alter ara-C survival curves in a way that depended on the effects of the culture conditions on self-renewal and differentiation. Four AML blast populations were chosen to test this prediction on the basis of our ability to manipulate them by adding or withholding one or more growth factors. Highly significant changes were seen in the ara-C survival curves, depending on the growth factors present in the cultures as was predicted by the observed effects of the factors on renewal and differentiation.     

Rescue from programmed cell death in leukemic and normal myeloid cells.

Growth factor-independent clones of myeloid leukemic cells can regain a growth factor-dependent state during differentiation. Loss of viability in these differentiating leukemic cells in the absence of growth factor was associated with DNA fragmentation and morphologic changes typical of programmed cell death (apoptosis). The differentiating leukemic cells could be rescued from apoptosis by a hematopoietic growth factor such as interleukin-3 (IL-3) and by the tumor-promoting phorbol ester 12-O-tetra-decanoyl-phorbol-13-acetate (TPA), but not by the nonpromoting phorbol ester 4-alpha-TPA. IL-3 and TPA rescued differentiating myeloid leukemic cells by different pathways and also rescued normal myeloid precursor cells from apoptosis. The rescue of differentiating leukemic and normal myeloid cells by IL-3 or TPA was blocked by amiloride inhibitors of the Na+/H+ antiporter. We suggest that TPA may act as a tumor promoter by inhibiting programmed cell death.     

Effect of a congenital defect in hemopoiesis on myeloid growth and the stem cell (CFU) in an in vivo culture system.

W/Wv mice with congenitally defective CFU proliferation and their normal, congenic littermates were used as hosts for diffusion chamber (DC) implants. CFU growth in implanted allogenic CF1, or congenic +/+ marrow was significantly greater in W/Wv than in control hosts. When W/Wv mice were "cured" of their hemopoietic defect, CFU proliferation in the DCs decreased, but not to the control level. These observations have provided evidence for humoral control of CFU growth related to a genetic stem cell defect. Diffusion chamber myelopoiesis was also enhanced in W/Wv hosts. In comparison with their congenic controls, W/Wv mice were neutropenic and had decreased numbers of marrow myeloid elements. Thus, a humorally mediated feedback related to a defective myelopoiesis in the hosts might have accounted for increased DC myelopoiesis. However, a "spillover" effect from increased stem cell growth has not been excluded.     

Phenotypic heterogeneity of primitive leukemic hematopoietic cells in patients with chronic myeloid leukemia.

The peripheral blood of chronic myeloid leukemia (CML) patients with chronic-phase disease and elevated white blood cell (WBC) counts typically contains markedly increased numbers of a variety of neoplastic pluripotent and lineage-restricted hematopoietic progenitors. These include cells detected in standard colony assays as well as their more primitive precursors. The latter are referred to as long-term culture-initiating cells (LTC-IC) because of their ability to generate clonogenic cell progeny detectable after a minimum of 5 weeks incubation on competent fibroblast feeder layers. In this study, we have investigated a number of the properties of the LTC-IC and clonogenic cells present in the blood of such CML patients with high WBC counts. This included an analysis of the light scattering properties of these progenitors, as well as their expression of CD34 and HLA-DR, Rhodamine-123 staining, and in vitro sensitivity to 4-hydroperoxycyclophosphamide. In the case of LTC-IC, the production of different types of lineage-restricted and multipotent progeny was also analyzed. Most of the circulating LTC-IC and clonogenic cells in the CML patients studied (on average approximately 70% and approximately 90%, respectively) showed features of proliferating or activated cells. This is in marked contrast to the majority of progenitors in the blood of normal individuals and most of the LTC-IC in normal marrow, all of which exhibit a phenotype expected of quiescent cells. Interestingly, a significant proportion of the circulating clonogenic cells and LTC-IC in the CML samples studied (on average approximately 10% and approximately 30%, respectively) appeared to be phenotypically similar to normal circulating progenitors, although their absolute numbers were indicative of a neoplastic origin. Both phenotypes of circulating CML clonogenic cells and LTC-IC could be obtained at approximately 10% to 20% purity by differential multiparameter sorting. These findings suggest that expansion of the Philadelphia chromosome-positive clone at the level of the earliest types of hematopoietic cells results from the activation of mechanisms that enable some, but not all, signals that block the cycling of normal stem cells to be bypassed or overcome. In addition, they provide strategies for purifying these primitive leukemic cells that should facilitate further analysis of the mechanisms underlying their abnormal proliferative behavior.     

Autologous stem cell transplantation in chronic myeloid leukemia: a single center experience.

Between 1980 and 1996, we transplanted 72 patients with CML using blood stem cells collected at diagnosis before treatment and without any mobilization. The median age of patients at diagnosis was 47.5 years (range 20.5-59.5). The median numbers of nucleated cells and CFU-GM transplanted were 10 x 10(8)/kg and 97 x 10(4)/kg, respectively. The median duration to reach more than 0.5 x 10(9)/l neutrophils and 50 x 10(9)/l platelets was 12 (range 5-19) and 11 days (range 0-79), respectively. Twenty patients (group I) were transplanted in chronic phase either for resistance to IFN (14 patients) (group IA) or because the Sokal index was more than 1.2 (six patients) (group IB). All those patients had preparative regimen with busulfan (4 mg/kg/day x 4) and melphalan (140 mg/m2). They were treated with recombinant alpha-interferon (IFN) after transplant. The cumulative incidence of major cytogenetic response (MCR) at 12 months was 25 +/- 21% (95% CI), the 5-year survival was 75 +/- 42% (95% CI). These results (observed in patients with bad prognosis factors) are similar to those usually observed in CML patients treated by IFN, whatever the Sokal risk. Thus autologous transplantation is able to reproduce for poor prognosis patients the results observed in standard risk patients treated with IFN. This suggests that it could prolong survival. Fifty-two other patients (group II) were transplanted for CML in transformation (accelerated phase = 32; blast crisis = 20) after a preparative regimen containing either total body irradiation (TBI) or busulfan. The median survival was short (10.4 months) and only 21 patients survived more than 1 year. The survival was longer for patients transplanted in accelerated phase (vs blast crisis), those who were due to receive a double transplant (vs single) (34 patients), those who were treated with IFN after transplant (vs hydroxyurea) and for the patients who obtained a complete hematologic response.     

Tonsillar abscess formation due to herpes simplex type-1 in a severely immunocompromised stem cell transplant patient with chronic myeloid leukemia

Herpes simplex virus (HSV) causes life-threatening infections in immunocompromised patients such as transplant recipients and patients with hematologic malignancies. We herein describe the case of a patient with chronic myeloid leukemia blastic transformation who developed severe herpetic tonsillitis complicated by tonsillar abscess formation. Abscess formation was determined by computed tomography, whereas tonsillitis due to HSV was confirmed by pathologic and immunohistochemical examinations of the tonsillar biopsy. For molecular confirmation, HSV DNA was amplified by LightCycler PCR and type (HSV-1) determined by melting point analysis. The patient responded promptly to antiviral treatment and there were no signs of recurrent infection at the follow-up. To our knowledge, this case is unique for being the first case of tonsillar abscess formation due to HSV-1, also emphasizing the importance of herpetic infections in the differential diagnosis of oropharyngeal small-sized lesions in the immunocompromised patient population.     

Flow cytometric quantification and immunophenotyping of leukemic stem cells in acute myeloid leukemia

Leukemic stem cells (LSCs) are root of clonal growth in acute myeloid leukemia (AML) and responsible for the propagation of leukemic blasts (LBs). LSCs are considered as CD34?+?CD38- population among LBs and often express as CD123, CD44, or CD184, which are rarely expressed on normal hematopoietic stem cells and could be the potential therapeutic targets. Using multi-color flow cytometry, we analyzed the proportions of CD34?+?CD38- LSCs and expression of CD123, CD44, and CD184 on LSCs in 63 patients with AML. The median proportion of LSCs was 1.3?% (0.0-33.1?%) at the time of diagnosis. Of all patients, 74.6?% of them had CD123-positive LSCs, all patients had CD44-positive LSCs, and 85.7?% had CD184-positive LSCs, respectively. The proportions of LSCs were significantly lower in the complete remission (CR) group compared with non-CR group (P?=?0.006). The lower proportions of LSCs in CR group indicated that measurement of the proportion of LSCs might be helpful to predict the prognosis of AML.     

Allogeneic stem cell transplantation for patients with chronic myeloid leukemia: Risk stratified approach with a long-term follow-up

The use of allogeneic stem cell transplantation (SCT) for chronic myeloid leukemia (CML) was almost abandoned in recent years for very effective targeted therapy with tyrosine kinase inhibitors (TKIs). However, approximately one third of patients still need another treatment including SCT. 38 consecutive CML patients were treated (most in preimatinib era) with allogeneic SCT, using partial T cell depletion (TCD) and preemptive donor lymphocyte infusion (DLI), without post‐transplant graft‐versus‐host disease (GvHD) prophylaxis. Conditioning included busulfan, cyclophosphamide, antithymocytic globulin, and fludarabine followed by donor stem cell transfusion. With a median follow up of 90.5 months (1–134), 32 patients are alive. 97% engrafted. 5‐year leukemia free survival (LFS) and overall survival (OS) were 78.95% and 84.2%, respectively. All patients are in major molecular remission and 78% in complete molecular remission. Transplant‐related mortality (TRM) was 13%. Twenty‐four patients received DLI for residual disease. Acute GvHD, mostly Grades I‐II, occurred in 18% of patients post‐transplant and in 24% of patients receiving DLI. In conclusion, the risk‐adapted approach using only partial TCD and preemptive escalated dose of DLI precluded the need for immunosuppressive medications and reduced the risk of significant GvHD without compromising engraftment and long‐term disease control. Am. J. Hematol. 2012. © 2012 Wiley Periodicals, Inc.     

Flk-2 is a marker in hematopoietic stem cell differentiation: a simple method to isolate long-term stem cells.

Clonogenic multipotent mouse hematopoietic stem cells (HSCs) and progenitor cells are contained within the c-kit(+) (K) lineage(-/lo) (L) Sca-1(+) (S) population of hematopoietic cells; long-term (LT) and short-term (ST) HSCs are Thy-1.1(lo). c-kit is a member of the receptor tyrosine kinase family, a class of receptors that are important in the proliferation and differentiation of hematopoietic cells. To establish whether the Flk-2/Flt3 receptor tyrosine kinase was expressed on the most primitive LT-HSCs, we sorted highly purified multipotent stem and progenitor cells on the basis of Flk-2 surface expression and used them in competitive reconstitution assays. Low numbers of Flk-2(-) HSCs gave rise to long-term multilineage reconstitution in the majority of recipients, whereas the transfer of Flk-2(+) multipotent cells resulted in mostly short-term multilineage reconstitution. The KLS subset of adult mouse bone marrow was analyzed for Flk-2 and Thy-1.1 expression. Three phenotypically and functionally distinct populations were isolated: Thy(lo) Flk-2(-) (LT-HSCs), Thy(lo) Flk-2(+) (ST-HSCs), and Thy(-) Flk-2(+) multipotent progenitors. The loss of Thy-1.1 and gain of Flk-2 expression marks the loss of self-renewal in HSC maturation. The addition of Flk-2 antibody to the lineage mix allows direct isolation of LT-HSC from adult bone marrow as c-kit(+) lin(-) Sca-1(+) Flk-2(-) from many strains of mice. Fetal liver HSCs are contained within Flk-2(-) and Flk-2(+) KTLS cells.     

Prospective separation of normal and leukemic stem cells based on differential expression of TIM3, a human acute myeloid leukemia stem cell marker

Hematopoietic tissues in acute myeloid leukemia (AML) patients contain both leukemia stem cells (LSC) and residual normal hematopoietic stem cells (HSC). The ability to prospectively separate residual HSC from LSC would enable important scientific and clinical investigation including the possibility of purged autologous hematopoietic cell transplants. We report here the identification of TIM3 as an AML stem cell surface marker more highly expressed on multiple specimens of AML LSC than on normal bone marrow HSC. TIM3 expression was detected in all cytogenetic subgroups of AML, but was significantly higher in AML-associated with core binding factor translocations or mutations in CEBPA. By assessing engraftment in NOD/SCID/IL2Rγ-null mice, we determined that HSC function resides predominantly in the TIM3-negative fraction of normal bone marrow, whereas LSC function from multiple AML specimens resides predominantly in the TIM3-positive compartment. Significantly, differential TIM3 expression enabled the prospective separation of HSC from LSC in the majority of AML specimens with detectable residual HSC function.