Cord blood NK cells engineered to express IL-15 and a CD19-targeted CAR show long-term persistence and potent antitumor activity.

Chimeric antigen receptors (CARs) have been used to redirect the specificity of autologous T cells against leukemia and lymphoma with promising clinical results. Extending this approach to allogeneic T cells is problematic as they carry a significant risk of graft-versus-host disease (GVHD). Natural killer (NK) cells are highly cytotoxic effectors, killing their targets in a non-antigen-specific manner without causing GVHD. Cord blood (CB) offers an attractive, allogeneic, off-the-self source of NK cells for immunotherapy. We transduced CB-derived NK cells with a retroviral vector incorporating the genes for CAR-CD19, IL-15 and inducible caspase-9-based suicide gene (iC9), and demonstrated efficient killing of CD19-expressing cell lines and primary leukemia cells in vitro, with marked prolongation of survival in a xenograft Raji lymphoma murine model. Interleukin-15 (IL-15) production by the transduced CB-NK cells critically improved their function. Moreover, iC9/CAR.19/IL-15 CB-NK cells were readily eliminated upon pharmacologic activation of the iC9 suicide gene. In conclusion, we have developed a novel approach to immunotherapy using engineered CB-derived NK cells, which are easy to produce, exhibit striking efficacy and incorporate safety measures to limit toxicity. This approach should greatly improve the logistics of delivering this therapy to large numbers of patients, a major limitation to current CAR-T-cell therapies.

The immunogenicity of virus-derived 2A sequences in immunocompetent individuals.

Genetic engineering of T cells for adoptive immunotherapy in cancer patients has shown significant promise. To ensure optimal antitumor activity and safety, the simultaneous expression of multiple genes is frequently required, and short viral-derived 2A sequences are increasingly preferred for this purpose. Concerns exist, however, that these virus-derived sequences may induce unwanted immune responses, and thus diminish persistence of the gene-modified cells after adoptive transfer. Whereas such responses were absent in immunocompromised recipients, potential immunogenicity in immunocompetent individuals remains a concern. We now address whether ex vivo T cell responses can be elicited against the most widely used 2A sequences (2A-Thosea asigna virus (TAV) or 2A-equine rhinitis virus (ERAV), specifically) in immunocompetent individuals. We used a potent ex vivo culture system previously validated to induce T cell responses even against weakly immunogenic antigens. Of the sixteen donors tested, only five released very low levels of interferon-γ in response to 2A-TAV peptide mixtures (single peptide specificity in three donors, adjacent self-antigen peptide specificity in one donor and nonspecific reactivity in one donor). None of them produced cytotoxic activity or responded to 2A-ERAV. These results suggest that exposure to viral-derived 2A sequences is unlikely to produce unwanted T cell responses in immunocompetent individuals and further supports their continued use for studies of human gene therapy.

Generation of multi-leukemia antigen-specific T cells to enhance the graft-versus-leukemia effect after allogeneic stem cell transplant.

Adoptive immunotherapy with ex vivo expanded T cells is a promising approach to prevent or treat leukemia. Myeloid leukemias express tumor-associated antigens (TAA) that induce antigen-specific cytotoxic T lymphocyte (CTL) responses in healthy individuals. We explored the feasibility of generating TAA-specific CTLs from stem cell donors of patients with myeloid leukemia to enhance the graft-versus-leukemia effect after stem cell transplantation. CTL lines were manufactured from peripheral blood of 10 healthy donors by stimulation with 15mer peptide libraries of five TAA (proteinase 3 (Pr3), preferentially expressed antigen in melanoma, Wilms tumor gene 1 (WT1), human neutrophil elastase (NE) and melanoma-associated antigen A3) known to be expressed in myeloid leukemias. All CTL lines responded to the mix of five TAA and were multi-specific as assessed by interferon-γ enzyme-linked immunospot. Although donors showed individual patterns of antigen recognition, all responded comparably to the TAAmix. Immunogenic peptides of WT1, Pr3 or NE could be identified by epitope mapping in all donor CTL lines. In vitro experiments showed recognition of partially human leukocyte antigen (HLA)-matched myeloid leukemia blasts. These findings support the development of a single clinical grade multi-tumor antigen-specific T-cell product from the stem cell source, capable of broad reactivity against myeloid malignancies for use in donor-recipient pairs without limitation to a certain HLA-type.

Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety.

T lymphocytes expressing a chimeric antigen receptor (CAR) targeting the CD19 antigen (CAR.19) may be of value for the therapy of B-cell malignancies. Because the in vivo survival, expansion and anti-lymphoma activity of CAR.19(+) T cells remain suboptimal even when the CAR contains a CD28 costimulatory endodomain, we generated a novel construct that also incorporates the interleukin-15 (IL-15) gene and an inducible caspase-9-based suicide gene (iC9/CAR.19/IL-15). We found that compared with CAR.19(+) T cells, iC9/CAR.19/IL-15(+) T cells had: (1) greater numeric expansion upon antigen stimulation (10-fold greater expansion in vitro, and 3- to 15-fold greater expansion in vivo) and reduced cell death rate (Annexin-V(+)/7-AAD(+) cells 10+/-6% for iC9/CAR.19/IL-15(+) T cells and 32+/-19% for CAR.19(+) T cells); (2) reduced expression of the programmed death 1 (PD-1) receptor upon antigen stimulation (PD-1(+) cells <15% for iC9/CAR.19/IL-15(+) T cells versus >40% for CAR.19(+) T cells); and (3) improved antitumor effects in vivo (from 4.7- to 5.4-fold reduced tumor growth). In addition, iC9/CAR.19/IL-15(+) T cells were efficiently eliminated upon pharmacologic activation of the suicide gene. In summary, this strategy safely increases the anti-lymphoma/leukemia effects of CAR.19-redirected T lymphocytes and may be a useful approach for treatment of patients with B-cell malignancies.

Selective elimination of a chemoresistant side population of B-CLL cells by cytotoxic T lymphocytes in subjects receiving an autologous hCD40L/IL-2 tumor vaccine.

Side-population (SP) analysis identifies precursor cells in normal and malignant tissues. Cells with this phenotype have increased resistance to many cytotoxic agents, and may represent a primary drug-resistant population in malignant diseases. To discover whether drug-resistant malignant SP cells are nonetheless sensitive to immune-mediated killing, we first established the presence of a malignant CD5(+)CD19(+) SP subset in the blood of 18/21 subjects with B-cell chronic lymphocytic leukemia (B-CLL). We examined the fate of these cells in six of these individuals who received autologous human CD40 ligand and interleukin-2 (hCD40L/IL-2) gene-modified tumor cells as part of a tumor vaccine study. Vaccinated patients showed an increase in B-CLL-reactive T cells followed by a corresponding decline in circulating CD5(+)CD19(+) SP cells. T-cell lines and clones generated from vaccinated patients specifically recognized B-CLL SP tumor cells. Elimination of SP cells is likely triggered by their increased expression of target antigens, such as receptor for hyaluronan-mediated motility (RHAMM), after stimulation of the malignant cells by hCD40L, as CD8(+) RHAMM-specific T cells could be detected in the peripheral blood of immunized patients and were associated with the decline in B-CLL SP cells. Hence, malignant B cells with a primary drug-resistant phenotype can be targeted by T- cell-mediated effector activity after immunization of human subjects.

Late donor cardiectomy after paediatric heterotopic cardiac transplantation.

BACKGROUND: Cardiac transplantation is a life-saving procedure in infants and children with advanced cardiomyopathy. However, it is greatly limited by shortage of paediatric donors and the complications of long-term immunosuppression, including post-transplant lymphoproliferative disorder (PTLD). We report the management of an infant who had heterotopic cardiac transplantation for advanced cardiomyopathy with secondary pulmonary hypertension who developed seemingly incurable PTLD. METHODS: An 8-month-old girl presented in 1994 with signs of severe heart failure, secondary to dilated cardiomyopathy. At age 11 months, the patient underwent a heterotopic cardiac transplantation. FINDINGS: The patient developed many episodes of PTLD associated with Epstein-Barr virus infection that were resistant to several therapies, including reduction of immunosuppression. Native heart recovery enabled removal of the donor heart 10.5 years after the original operation to allow complete cessation of immunosuppression. Her postoperative course was uncomplicated and the outcome was excellent. 3.5 years after surgery, the patient remains well, in complete remission from her PTLD, and has normal cardiac function. INTERPRETATION: This case shows several issues relating to the use of heterotopic cardiac transplantation in infants and the capacity of the heart to recover. It also provides new insights into the interaction between the immune system with several aspects of modern management of post-transplantation PTLD. FUNDING: None.

T cell therapies.

T cell therapies are increasingly used for the treatment of malignancies and viral-associated diseases. Initial studies focused on the use of unmanipulated T cell populations after allogeneic stem cell transplantation. More recently, the use of antigen-specific T cells has been explored. This chapter reviews the clinical experience with polyclonal Epstein-Barr virus (EBV)-specific cytotoxic T cells (CTL) for the treatment of EBV-associated malignancies. Strategies on how to improve the antitumor activity of EBV-specific CTL are being discussed. If effective, these strategies will have broad implications for T cell therapies for a range of human tumors with defined antigens.

Generation of autologous Epstein-Barr virus-specific cytotoxic T cells for adoptive immunotherapy in solid organ transplant recipients.

BACKGROUND: Epstein-Barr virus (EBV)-driven posttransplant lymphoproliferative disorders (PTLD) affect 2%-27% of solid organ transplant (SOT) recipients. Adoptive immunotherapy may have therapeutic potential in this setting, but there is little experience in generating autologous EBV-specific cytotoxic T-cell lymphocytes (EBV-CTLs) from SOT recipients, and their efficacy and persistence in an immunosuppressed environment is unknown. METHODS: EBV-CTLs were generated from eight SOT recipients, using weekly stimulations with autologous lymphoblastoid cell lines (LCLs) and interleukin-2. CTL phenotype and function were evaluated in the presence of therapeutic concentration of cyclosporin A or FK506. RESULTS: In all cases, CTLs expanded with normal kinetics. The majority was CD3+CD8+ (mean, 76%), with less than 3% of natural killer cells. All ex vivo-generated CTLs produced significantly higher killing of autologous LCLs than of HLA-mismatched LCLs (mean, 56% vs. 14% at 20:1 ratio). No lysis of autologous or allogeneic PHA blasts was observed. The CTL expansion rate was reduced in a concentration-dependent manner in the presence of immunosuppressive drugs; however, neither lytic activity nor phenotype was affected. CONCLUSIONS: Using methods that are approved for clinical application, EBV-CTLs can be generated from SOT recipients, even those with frank lymphoma, or who are receiving immunosuppressive drugs. These CTLs retain their function in the presence of immunosuppressive agents. Although in vivo efficacy, safety, and persistence can be assessed only in clinical trials, our results suggest that CTLs can be effective for the treatment of PTLD, even when immunosuppression cannot be reduced because of the high risk of graft rejection.

Adenovector-induced expression of human-CD40-ligand (hCD40L) by multiple myeloma cells. A model for immunotherapy.

OBJECTIVE: CD40L restores the antigen-presenting cell (APC) function of some B-cell tumors and induces professional APC maturation. We therefore evaluated the effects of transgenic CD40L expression on the behavior and immunogenicity of human multiple myeloma (MM) cells. MATERIALS AND METHODS: CD40L expression was induced in a CD40(+) (RPMI 8226) and a CD40(-) (U266B1) human myeloma cell line (HMCL) by adenoviral vector gene transfer. The viability and proliferative activity of control HMCL and HMCL/CD40L were determined by daily trypan blue staining and methyl-3H-thymidine incorporation. Mixed lymphocyte reaction (MLR) with allogeneic mononuclear cells (MNCs) and coculture of allogeneic dendritic cells (DCs) with HMCL expressing transgenic CD40L were used to evaluate the APC function of modified HMCL as well as the role of bystander DCs in inducing an anti-tumor immune response. RESULTS: CD40L expression significantly inhibited the growth of the CD40(+) HMCL and induced apoptosis. These effects were less evident for the CD40(-) HMCL. There was no upregulation of costimulatory molecules on either HMCL following CD40L expression. Both HMCL expressing transgenic CD40L induced maturation of bystander DCs and enhanced their ability to stimulate the proliferation of MNCs. DCs cultured with the poorly immunogenic RPMI 8226 expressing CD40L upregulated T-lymphocyte release of IFN-gamma and other Th1 cytokines (interleukin-2, tumor necrosis factor-alpha). CONCLUSIONS: Our data suggest that transgenic expression of CD40L exerts a dual effect favoring generation of an immune response to human MM. Where the tumor cells are CD40(+), the engagement of CD40 antigen by CD40L on tumor cells induces their apoptosis, allowing uptake of tumor-associated antigen by professional APC. Independently of tumor-cell expression of CD40, transgenic expression of CD40L on tumor cells allows them to stimulate CD40(+) APC, to increase their maturation and their capacity to stimulate cytotoxic T lymphocytes (CTL) that recognize the tumor-derived antigens the APC may have engulfed.

Selective expression and constitutive phosphorylation of SHC proteins [corrected] in the CD34+ fraction of chronic myelogenous leukemias.

The BCR/ABL fusion protein is a constitutively active tyrosine kinase that is responsible for the pathogenesis of chronic myelogenous leukemia (CML). Clinically, CML is characterized by a chronic phase (CP) that eventually terminates into a blast crisis (BC). BC transformation is associated with accumulation of CD34+ blasts. We investigated the expression and phosphorylation of Src-homology-2 and collagen-homology domains (SHC) [corrected] proteins in subpopulations of CML primary cells. Shc polypeptides are tyrosine kinase substrates that are constitutively tyrosine-phosphorylated in continuous cell lines of CML origin. High levels of Shc expression were found in the CD34+ cells from CML-BC, CML-CP and normal bone marrow. In contrast, CD34- fractions from CML-CP and normal bone marrow expressed low levels of p46Shc. Shc proteins were constitutively phosphorylated in the CD34+ fractions from CML cells (both CP and BC), but not in normal CD34+ cells. These data bear implications for the role of Shc in normal hemopoiesis and CML leukemogenesis: (a) dramatic changes of Shc expression during terminal differentiation of hemopoietic cells adds a further level of regulation to the signal transduction function of Shc; and (b) constitutive Shc tyrosine-phosphorylation in the rare CD34+ cells of CML-CP might contribute to the selection of this subpopulation during the blast crisis transformation of CMLs.

The use of cytotoxic t cells for the prevention and treatment of epstein-barr virus induced lymphoma in transplant recipients.

Epstein-Barr Virus Lymphoproliferative Diseases (EBV-LPD) can affect patients receiving allogeneic hemopoietic stem cell or solid organ transplant. They are caused by impairment of EBV specific CD8+ cytotoxic T-lymphocyte (CTL) response due to the immunosuppression that follows these procedures. Despite different therapies LPD can still have a rapid and lethal course. A promising solution is the application of adoptive immunotherapy approaches such as ex vivo generated EBV-specific CTLs for prevention and therapy. Their infusion has been demonstrated to be safe and effective in allogeneic hemopoietic stem cell transplant recipients and their use after allogeneic solid organ transplant is also under evaluation.

Effects of the tyrosine kinase inhibitor AG957 and an Anti-Fas receptor antibody on CD34(+) chronic myelogenous leukemia progenitor cells.

The hallmark of chronic myelogenous leukemia (CML) is the Philadelphia (Ph) chromosome that fuses genetic sequences of the BCR gene on chromosome 22 with c-ABL sequences translocated from chromosome 9. BCR/ABL fusion proteins have a dysregulated protein tyrosine kinase (PTK) activity exerting a key role in malignant transformation. Targeting the tyrosine kinase activity of BCR/ABL or using agents capable of triggering apoptosis might represent attractive therapeutic approaches for ex vivo purging. AG957, a member of the tyrphostin compounds, exerts a selective inhibition of p210(BCR/ABL) tyrosine phosphorylation. We report here that preincubation of CML or normal CD34(+) cells with graded concentration of AG957 (1 to 100 micromol/L) resulted in a statistically significant, dose-dependent suppression of colony growth from multipotent, erythroid, and granulocyte-macrophage progenitors as well as the more primitive long-term culture-initiating cells (LTC-IC). However, AG957 doses causing 50% inhibition (ID50) of CML and normal progenitors were significantly different for multilineage colony-forming units (CFU-Mix; 12 v 64 micromol/L; P =.008), burst-forming unit-erythroid (BFU-E; 29 v 89 micromol/L; P =.004), colony-forming unit-granulocyte-macrophage (CFU-GM; 34 v 85 micromol/L; P =.004), and LTC-IC (43 v 181 micromol/L; P =.004). In 5 of 10 patients, analysis of BCR/ABL mRNA on single progenitors by reverse transcription-polymerase chain reaction showed that AG957 at 50 micromol/L significantly reduced the mean (+/-SD) percentage of BCR/ABL-positive progenitors (92% +/- 10% v 33 +/- 5%; P =.001). Because AG957 treatment resulted in significantly higher percentages of apoptotic cells (30% v 9%) in the BCR/ABL-transfected 32DLG7 cells as compared with 32D-T2/93 cells (BCR/ABL-negative), we investigated the combined effects of AG957 with the anti-Fas receptor (Fas-R) monoclonal antibody CH11 that triggers apoptosis. As compared with AG957 alone, the sequential treatment of CML CD34(+) cells with AG957 (1 micromol/L) and CH11 (1 microgram/mL) increased CFU-Mix, BFU-E, and CFU-GM growth inhibition by 1.6-fold, 3-fold, and 4-fold, respectively. In contrast, the treatment of normal CD34(+) cells with AG957 and CH11 failed to enhance AG957-induced colony growth inhibition. We conclude that (1) AG957 inhibits in a dose-dependent manner CML CD34-derived colony formation by both primitive LTC-IC as well as committed CFU-Mix, BFU-E, and CFU-GM; (2) this growth inhibition is associated with the selection of a substantial amount of BCR/ABL-negative progenitors; and (3) the antiproliferative effect of AG957 is dramatically increased by combining this compound with the anti-Fas-R antibody CH11. These data may have significant therapeutic applications.

Primitive hematopoietic progenitors within mobilized blood are spared by uncontrolled rate freezing.

Uncontrolled-rate freezing techniques represent an attractive alternative to controlled-rate cryopreservation procedures which are time-consuming and require high-level technical expertise. In this study, we report our experience using uncontrolled-rate cryopreservation and mechanical freezer storage at -140 degrees C. Twenty-eight PBPC samples (10 cryovials, 18 freezing bags) from 23 patients were cryopreserved in a cryoprotectant solution composed of phosphate-buffered saline (80%, v/v) supplemented with human serum albumin (10%, v/v) and dimethylsulfoxide (10%, v/v). The cryopreservation procedure required on average 1.5 h. The mean (+/- s.e.m.) storage time of cryovials and bags was 344+/-40 and 299+57 days, respectively. Although cell thawing was associated with a statistically significant reduction of the absolute number of nucleated cells (vials: 0.3x10(9) vs. 0.2x10(9), P< or =0.02; bags: 14x10(9) vs. 11x10(9), P< or =0.0003), the growth of committed progenitors was substantially unaffected by the freezing-thawing procedure, with mean recoveries of CFU-Mix, BFU-E, and CFU-GM ranging from 60+/- 29% to 134+/-15%. Mean recoveries of LTC-IC from cryovials and bags were 262+/-101% and 155+/-27% (P< or =0.2), respectively. In 14 out of 23 patients who underwent high-dose chemotherapy and PBPC reinfusion, the pre-and post-freezing absolute numbers of hematopoietic progenitors cryopreserved in bags were compared. A significant reduction was detected for CFU-Mix (11 vs. 7.4x10(5)), but no significant loss of BFU-E (180 vs. 150x10(5)), CFU-GM (400 vs. 290x10(5)) and LTC-IC (15 vs. 16x10(5)) could be demonstrated. When these patients were reinfused with uncontrolled-rate cryopreserved PBPC, the mean number of days to reach 1x10(9)/l white blood cells and 50x10(9)/l platelets were 9 and 13, respectively. In conclusion, the procedure described here is characterized by short execution time, allows a substantial recovery of primitive and committed progenitors and is associated with prompt hematopoietic recovery following myeloablative therapy even after long-term storage.

Reverse transcription polymerase chain reaction is a reliable assay for detecting leukemic colonies generated by chronic myelogenous leukemia cells.

Single-colony karyotyping (SCK) and reverse-transcription polymerase chain reaction (RT-PCR) are two increasingly used techniques for the quantification of leukemic colonies generated by chronic myelogenous leukemia (CML) cell fractions purged or selected in vitro. Recently, the existence of Philadelphia (Ph) chromosome positive progenitors with a silent BCR-ABL gene has been reported, thus raising concerns on the use of RT-PCR for detecting BCR-ABL positive progenitors. In order to investigate this issue further, colonies (n = 204) generated by mononuclear (MNC) or CD34+ CML cells were individually harvested, divided into two aliquots and analyzed both at the cytogenetic level to detect the Ph chromosome, and the molecular level to detect BCR-ABL transcripts. The mean (+/- s.d.) percentages of colonies analyzable by either SCK or RT-PCR were 74 +/- 16% and 86 +/- 16%, respectively. A significant percentage of colonies (67 +/- 19%) could be successfully analyzed by both SCK and RT-PCR. Although the majority of these colonies (97 +/- 5%) were Ph-positive and BCR-ABL-positive, a negligible percentage (4%) of progenitors were Ph-positive but BCR-ABL-negative. In order to test the influence of colony size on the outcome of molecular analysis, the efficiency of our RT-PCR assay in detecting BCR-ABL transcripts was investigated by means of experiments in which the number of cells used to start RNA extraction was serially reduced. These experiments showed that at least 150 cells were necessary to achieve a reproducible amplification of BCR-ABL transcripts. By correlating the size of harvested colonies with the outcome of molecular analysis, it was evident that BCR-ABL-negative but Ph-positive colonies represented false negative results occurring when a number of leukemic cells below the detection limit of our RT-PCR assay was analyzed. In conclusion, our data demonstrate that individual CML colonies grown in semisolid culture assays can be indifferently analyzed by SCK or RT-PCR, and support an extensive use of a carefully standardized RT-PCR assay to estimate the leukemic burden within samples which have been purged and selected in vitro.

Use of granulocyte-macrophage colony-stimulating factor (GM-CSF) in combination with hydroxyurea as post-transplant therapy in chronic myelogenous leukemia patients autografted with unmanipulated hematopoietic cells.

BACKGROUND AND OBJECTIVE: Allogeneic bone marrow transplantation remains the only potentially curative treatment for CML, but more than 70% of patients will be ineligible for allogeneic marrow transplant either because they do not have a suitable HLA-matched related or unrelated donor or because they are more than 50 years old. Several experimental and clinical findings support a role for autologous stem cell transplantation (ASCT) in CML. It has been suggested that in the early phase following autografting the Ph-negative clone has a proliferative advantage over the Ph-positive clone. We hypothesized that post-transplant GM-CSF administration could reactivate the functional activity of quiescent normal progenitors and prolong the duration of the post-transplant proliferative advantage of Ph-negative over Ph-positive progenitors. In order to evaluate the effect of post-transplant GM-CSF administration, a pilot clinical study was performed in which CML patients resistant to IFN-alpha therapy were autografted with unmanipulated marrow or blood cells and given prolonged GM-CSF therapy post-transplant. METHODS: Five adult CML patients conditioned with the BAVC regimen were reinfused with either marrow (n = 2) or blood (n = 3) cells and given granulocyte-macrophage colony-stimulating factor (GM-CSF). Recombinant GM-CSF was initially administered at standard dosage (5 micrograms/kg/day) until a white blood cell count > or = 2 x 10(9)/L was achieved on two consecutive examinations, and thereafter at a low dose (1 microgram/kg/day) for 5 to 9 months. On a weekly basis, GM-CSF was discontinued and hydroxyurea (1,000 mg/d) was given for two days. RESULTS: Evidence of trilineage engraftment was observed in all cases. At autografting, 3 out of the 5 patients revealed 8-9% Ph-negative metaphases. During the initial phase of hematopoietic regeneration, direct cytogenetic analysis revealed 81% and 100% Ph-negative metaphases in two cases; nonleukemic hematopoiesis progressively decreased and was no longer detectable at +9 months. One patient showed cyclic Ph-negative hematopoiesis that appeared 3 months following autografting and peaked at +4 and +8 months. The fourth patient showed a low percentage (20%) of Ph-negative metaphases 1 month after ASCT, followed by a significant expansion of nonleukemic hematopoiesis, which could be detected up to month +13. No evidence of Ph-negative hematopoiesis could be detected in one patient. Three patients are in chronic phase 28, 30 and 31 months after autografting, respectively, and two patients evolved into blast crisis. INTERPRETATION AND CONCLUSIONS: This pilot study demonstrates that combined GM-CSF and hydroxyurea therapy seems to be effective in inducing and/or prolonging a transient period of Ph-negative hematopoiesis. The late appearance of Ph-negative hematopoiesis detected in two patients suggests an antileukemic activity of the combined GM-CSF/hydroxyurea therapy rather than an antileukemic effect of the conditioning regimen.

Selection of myeloid progenitors lacking BCR/ABL mRNA in chronic myelogenous leukemia patients after in vitro treatment with the tyrosine kinase inhibitor genistein.

Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characterized by a chimeric BCR/ABL gene giving rise to a 210-kD fusion protein with dysregulated tyrosine kinase activity. We investigated the effect of genistein, a protein tyrosine kinase inhibitor, on the in vitro growth of CML and normal marrow-derived multi-potent (colony-forming unit-mix [CFU-Mix]), erythroid (burst-forming unit-erythroid [BFU-E]), and granulocyte-macrophage (colony-forming unit-granulocyte-macrophage [CFU-GM]) hematopoietic progenitors. Continuous exposure of CML and normal marrow to genistein induced a statistically significant and dose-dependent suppression of colony formation. Genistein doses causing 50% inhibition of CML and normal progenitors were not significantly different for CFU-Mix (27 mumol/L v 23 mumol/L), BFU-E (31 mumol/L v 29 mumol/L), and CFU-GM (40 mumol/L v 32 mumol/L v 32 mumol/L). Preincubation of CML and normal marrow with genistein (200 mumol/ L for 1 to 18 hours) induced a time-dependent suppression of progenitor cell growth, while sparing a substantial proportion of long-term culture-initiating cells (LTC-IC) from CML (range, 91% +/- 9% to 32% +/- 3%) and normal marrow (range, 85% +/- 8% to 38% +/- 9%). Analysis of individual CML colonies for the presence of the hybrid BCR/ABL mRNA by reverse transcription-polymerase chain reaction (RT-PCR) showed that genistein treatment significantly reduced the mean +/- SD percentage of marrow BCR/ABL+ progenitors both by continuous exposure (76% +/- 18% v 24% +/- 12%, P < or = .004) or preincubation (75% +/- 16% v 21% +/- 10%, P < or = .002) experiments. Preincubation with genistein reduced the percentage of leukemic LTC-IC from 87% +/- 12% to 37% +/- 12% (P < or = .003). Analysis of individual colonies by cytogenetics and RT-PCR confirmed that genistein-induced increase in the percentage of nonleukemic progenitors was not due to suppression of BCR/ABL transcription. Analysis of nuclear DNA fragmentation by DNA gel electrophoresis and terminal deoxynucleotidyl transferase assay showed that preincubation of CML mononuclear and CD34+ cells with genistein induced significant evidence of apoptosis. These observations show that genistein is capable of (1) exerting a strong antiproliferative effect on CFU-Mix, BFU-E, and CFU-GM while sparing the more primitive LTC-IC and (2) selecting benign hematopoietic progenitors from CML marrow, probably through an apoptotic mechanism.