Bart A. Nijmeijer

Monitoring of engraftment and progression of acute lymphoblastic leukemia in individual NOD/SCID mice

OBJECTIVE The aim of this study was to develop an animal model for human acute lymphoblastic leukemia (ALL) in which the kinetics and characteristics of leukemia can be sequentially monitored in individual mice. MATERIALS AND METHODS NOD/SCID mice were inoculated intravenously with primary ALL. Progression of leukemia was monitored throughout the development of disease by determination of absolute leukemic cell counts (LCC) in peripheral blood. RESULTS LCC as low as 10(4) leukemic cells/mL blood could be detected. ALL cells from 5 of 5 patients engrafted, and after identification of the first leukemic cells in peripheral blood, LCC increased exponentially. Leukemic cells showed specificity of homing to spleen and bone marrow, and LCC strongly correlated with the level of leukemic engraftment in these organs throughout disease progression, demonstrating that LCC are representative for overall leukemic burden. Cytogenetic analysis of leukemic cells recovered after six successive in vivo transfers revealed no major karyotypic changes as compared to primary cells, and selection of the dominant clones was observed. This selection process was reflected by an increase in the rate of leukemic progression as compared to the first inoculation, demonstrating the accuracy with which kinetics of leukemic progression can be studied by determination of LCC. CONCLUSIONS This model is suitable for detailed studies of kinetics and characteristics of ALL in vivo, and it may be useful for monitoring effects of novel therapeutic regimens.

Imatinib mesylate at therapeutic doses has no impact on folliculogenesis or spermatogenesis in a leukaemic mouse model

Imatinib should be avoided in women planning to become pregnant or during pregnancy, due to a higher risk of congenital malformations. However, it is not known whether imatinib affects future potential for fertility. Here we analysed ovaries and testes from adult mice receiving imatinib, focusing on testicular and ovarian functions. Seven male and 7 female mice were orally treated with 150 mg/kg body weight/day imatinib for two months. No effects on folliculogenesis or spermatogenesis could be observed postmortem by histological examinations, suggesting that, at least in two mouse models of imatinib treatment this tyrosine kinase inhibitor does not reduce fertility.

Differential Effects of Selective Inhibitors Targeting the PI3K/AKT/mTOR Pathway in Acute Lymphoblastic Leukemia

Purpose Aberrant PI3K/AKT/mTOR signaling has been linked to oncogenesis and therapy resistance in various malignancies including leukemias. In Philadelphia chromosome (Ph) positive leukemias, activation of PI3K by dysregulated BCR-ABL tyrosine kinase (TK) contributes to the pathogenesis and development of resistance to ABL-TK inhibitors (TKI). The PI3K pathway thus is an attractive therapeutic target in BCR-ABL positive leukemias, but its role in BCR-ABL negative ALL is conjectural. Moreover, the functional contribution of individual components of the PI3K pathway in ALL has not been established. Experimental Design We compared the activity of the ATP-competitive pan-PI3K inhibitor NVP-BKM120, the allosteric mTORC1 inhibitor RAD001, the ATP-competitive dual PI3K/mTORC1/C2 inhibitors NVP-BEZ235 and NVP-BGT226 and the combined mTORC1 and mTORC2 inhibitors Torin 1, PP242 and KU-0063794 using long-term cultures of ALL cells (ALL-LTC) from patients with B-precursor ALL that expressed the BCR-ABL or TEL-ABL oncoproteins or were BCR-ABL negative. Results Dual PI3K/mTOR inhibitors profoundly inhibited growth and survival of ALL cells irrespective of their genetic subtype and their responsiveness to ABL-TKI. Combined suppression of PI3K, mTORC1 and mTORC2 displayed greater antileukemic activity than selective inhibitors of PI3K, mTORC1 or mTORC1 and mTORC2. Conclusions Inhibition of the PI3K/mTOR pathway is a promising therapeutic approach in patients with ALL. Greater antileukemic activity of dual PI3K/mTORC1/C2 inhibitors appears to be due to the redundant function of PI3K and mTOR. Clinical trials examining dual PI3K/mTORC1/C2 inhibitors in patients with B-precursor ALL are warranted, and should not be restricted to particular genetic subtypes.

IL-7 receptor expression identifies suicide gene-modified allospecific CD8+ T cells capable of self-renewal and differentiation into antileukemia effectors.

In allogeneic hematopoietic cell transplantation (HSCT), donor T lymphocytes mediate the graft-versus-leukemia (GVL) effect, but induce graft-versus-host disease (GVHD). Suicide gene therapy-that is, the genetic induction of a conditional suicide phenotype into donor T cells-allows dissociating the GVL effect from GVHD. Genetic modification with retroviral vectors after CD3 activation reduces T-cell alloreactivity. We recently found that alloreactivity is maintained when CD28 costimulation, IL-7, and IL-15 are added. Herein, we used the minor histocompatibility (mH) antigens HA-1 and H-Y as model alloantigens to directly explore the antileukemia efficacy of human T cells modified with the prototypic suicide gene herpes simplex virus thymidine kinase (tk) after activation with different stimuli. Only in the case of CD28 costimulation, IL-7, and IL-15, the repertoire of tk(+) T cells contained HA-1- and H-Y-specific CD8(+) cytotoxic T cells (CTL) precursors. Thymidine kinase-positive HA-1- and H-Y-specific CTLs were capable of self-renewal and differentiation into potent antileukemia effectors in vitro, and in vivo in a humanized mouse model. Self-renewal and differentiation coincided with IL-7 receptor expression. These results pave the way to the clinical investigation of T cells modified with a suicide gene after CD28 costimulation, IL-7, and IL-15 for a safe and effective GVL effect.

Long–term culture of primary human lymphoblastic leukemia cells in the absence of serum or hematopoietic growth factors

OBJECTIVE B-lineage acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia in lymphatic blastic phase in adults have poor prognoses despite intensive chemotherapy. Novel targeted treatment modalities emerge, but their evaluation requires relevant in vitro models of lymphoblastic leukemia. Presently available cell lines do not fully represent this heterogeneous disease. Available in vitro culturing protocols do not support long-term proliferation of primary cells. We therefore aimed to develop a culture system that allows long-term proliferation of primary human B-lineage lymphoblastic leukemia. MATERIALS AND METHODS Primary lymphoblastic leukemia cells were cultured in a defined serum-free medium, in the absence or presence of human hematopoietic growth factors or serum. RESULTS In the defined serum-free medium, cells from 12 of 34 cases immediately proliferated in vitro. In the absence of hematopoietic growth factors and serum these cases proliferated for more than 1 year without signs of exhaustion. The culturing system supported different subtypes of lymphoblastic leukemia. Two chronic myeloid leukemia in lymphatic blastic phase, four bcr/abl-positive ALL, one etv6/abl-positive ALL, 2 e2a-pbx1-positive ALL, and one t(9;11)-positive ALL could be long-term expanded, as well as two ALL that displayed nontypical cytogenetics. Not all bcr/abl- or e2a-pbx1-positive ALL proliferated in vitro, demonstrating heterogeneity within these subtypes. The proliferating bcr/abl- and etv6/abl-positive cells displayed sensitivity to imatinib, demonstrating that their proliferation depended on the activity of these oncoproteins. CONCLUSION The serum-free culturing system may be a valuable instrument in the study of ALL cell biology, as well as in the evaluation of novel targeted therapeutics.

A mechanistic rationale for combining alemtuzumab and rituximab in the treatment of ALL.

B-lineage acute lymphoblastic leukemia (ALL) may express CD52 and CD20. Alemtuzumab (ALM) and rituximab (RTX) are therapeutic antibodies directed against CD52 and CD20, respectively, but showed limited activity against ALL in clinical trials. The mechanisms for the impaired responses remained unclear. We studied expression of CD52 and CD20 on ALL cells and found that most cases coexpressed CD52 and CD20. However, distinct CD52-negative (CD52(-)) subpopulations were detected in most cases as the result of defective glycophosphatidyl-inositol anchoring. Although ALM efficiently eradicated CD52-positive (CD52(+)) cells in NOD/scid mice engrafted with primary human ALL, CD52(-) subclones escaped therapy. In the same model, RTX showed limited activity resulting from occurrence of CD20 down-modulation. However, CD52(-) cells concurrently lacked the glycophosphatidyl-inositol-anchored complement regulators CD55 and CD59 and showed increased susceptibility to RTX-mediated complement-dependent cytotoxicity in vitro. At the same time, ALM was shown to inhibit down-modulation of CD20 in response to RTX by depleting the trogocytic capacity of phagocytic cells. Probably because of these complementary mechanisms, combined administration of ALM and RTX induced complete responses in vivo. Based on these data, we propose a mechanistic rationale for combined application of RTX and ALM in ALL.

Early Detection and Rapid Isolation of Leukemia-Reactive Donor T Cells for Adoptive Transfer Using the IFN-γ Secretion Assay

Purpose: The poor immunogenicity of most leukemias and the lack of specificity of the donor T cells limit the in vivo effectiveness of conventional donor lymphocyte infusions in many patients suffering from persistent or recurrent leukemia after allogeneic stem cell transplantation. These limitations may be overcome by the adoptive transfer of in vitro generated leukemia-reactive T cells. Although the potential clinical efficacy of this approach has been shown previously, lack of reproducibility of the procedure and the inability to show persistence and survival of the transferred T cells hampered further clinical application. The purpose of this study was to develop a new, broadly applicable strategy for the efficient generation and isolation of leukemia-reactive T cells with a better probability to survive and expand in vivo. Experimental Design: Myeloid and B-cell leukemias were modified into professional immunogenic antigen-presenting cells, and used to stimulate HLA-matched donor T cells. After two stimulations, responding donor T cells were isolated based on their secretion of IFN-γ and tested for their capacity to recognize and kill the primary leukemia. Results: Using one universal stimulation and isolation protocol for various forms of leukemia, T-cell populations containing high frequencies of leukemia-reactive T cells could reproducibly be generated and early isolated under mild stimulatory conditions. Isolated T cells still had high proliferative potential and their reactivity seemed to be restricted to cells of the patients hematopoiesis. Conclusion: We here show a new robust procedure for the generation and isolation of leukemia-reactive T cells for adoptive transfer.

Human allo-reactive CD4+ T cells as strong mediators of anti-tumor immunity in NOD/scid mice engrafted with human acute lymphoblastic leukemia

Adoptive immunotherapy with donor lymphocyte infusion (DLI) after allogeneic stem cell transplantation (alloSCT) may not only mediate Graft-versus-Leukemia (GvL) reactivity, but also induce Graft-versus-Host Disease (GvHD). As HLA-class II molecules are predominantly expressed on hematopoietic cells, CD4+ T cells may selectively mediate GvL reactivity without GvHD. Here, we assessed the capacity of human CD4+ T cells to act as sole mediators of GvL reactivity in a NOD/scid mouse model for human acute lymphoblastic leukemia and chronic myeloid leukemia in lymphoid blast crisis. Highly purified CD4+ DLI eradicated the leukemic cells. The anti-tumor immunity was mediated by a polyclonal CD4+ T cell response comprising cytokine-producing T-helper and cytolytic T-effector cells directed against the mismatched HLA-class II molecules of the patients. Furthermore, primary leukemic cells acquired an antigen-presenting cell (APC) phenotype in vivo after DLI, as well as in vitro after co-culture with leukemia-specific CD4+ T cells. In conclusion, our results show that CD4+ T cells can be strong mediators of anti-tumor immunity, and provide evidence that cross-talk between CD4+ T cells and leukemic cells is the basis for induction of leukemic cells with an APC phenotype. These data emphasize the clinical relevance of CD4+ T cell based immunotherapy as treatment modality for hematological malignancies after alloSCT.

Establishment and cytogenetic characterization of a human acute lymphoblastic leukemia cell line (ALL-VG) with ETV6/ABL1 rearrangement

Fusion kinases (FK) like BCR/ABL1 mediate leukemic transformation and represent therapeutic targets. Fusion of ETV6 (ETS translocation variant 6, previously known as TEL) to ABL1 due to t(9;12) has been observed in various hematological malignancies. ETV6/ABL1 and BCR/ABL1 FK display similar activity but they may not be identical in function. Here we present the generation of an ETV6/ABL1 positive human acute lymphoblastic leukemia (ALL) cell line, ALL-VG. The cell line expressed ETV6/ABL1 fusion transcripts and displayed sensitivity to imatinib with an IC(50) of 0.1 microM. Karyotyping did not reveal overt t(9;12), suggesting a cryptic translocation. Fluorescent in situ hybridization and array-based comparative genomic hybridization were performed to characterize the rearrangement. ETV6/ABL1 fusion was demonstrated to result from insertion of a duplicated 300 to 1300 kb region of 9q34 that contained the distal portion of the ABL1 gene, into the ETV6 locus on 12p13. With this insertion, an 1150 to 1750 kb region of 12p13 that contained the distal portion of the ETV6 gene as well the cyclin dependent kinase inhibitor (CDKN) 1B gene was lost. Furthermore, the cells displayed a del(9)(p21.1 approximately p23), typically associated with loss of CDKN2A and CDKN2B. The ALL-VG cell line may serve as a tool for the study of ETV6/ABL1.

Recruitment of leukemic cells from G0 phase of the cell cycle by interferons results in conversion of resistance to daunorubicin

Recruitment of leukemic cells from G 0 phase of the cell cycle by interferons results in conversion of resistance to daunorubicin