Summary We examined the leukemic stem cell potential of blasts at different stages of maturation in childhood acute lymphoblastic leukemia. Human leukemic bone marrow was transplanted intrafemorally into NOD/scid mice. Cells sorted using the B precursor differentiation markers CD19, CD20 and CD34 were isolated from patient samples and engrafted mice before serial transplantation into primary or subsequent (up to quaternary) recipients. Surprisingly, blasts representative of all the different maturational stages were able to reconstitute and re-establish the complete leukemic phenotype in vivo. Sorted blast populations mirrored normal B precursor cells with transcription of a number of stage-appropriate genes. These observations have informed a model for leukemia-propagating stem cells in childhood ALL.

Open questions in the pathogenesis of childhood acute lymphoblastic leukemia (ALL) are which hematopoietic cell is target of the malignant transformation and whether primitive stem cells contribute to the leukemic clone. Although good-prognosis ALL is thought to originate in a lymphoid progenitor, it is unclear if this applies to high-risk ALL. Therefore, immature CD34 + CD19 À bone marrow cells from 8 children with ALL/t(9;22) and 12 with ALL/t(4;11) were purified and analyzed by fluorescence in situ hybridization, reverse transcription-PCR (RT-PCR), and colony assays. Fiftysix percent (n = 8, SD 31%) and 68% (n = 12, SD 26%) of CD34 + CD19 À cells in ALL/t(9;22) and ALL/t(4;11), respectively, carried the translocation. In addition, 5 of 168 (3%) and 22 of 228 (10%) myeloerythroid colonies expressed BCR/ABL and MLL/AF4. RT-PCR results were confirmed by sequence analysis. Interestingly, in some patients with ALL/t(4;11), alternative splicing was seen in myeloid progenitors compared with the bulk leukemic population, suggesting that these myeloid colonies might be part of the leukemic cell clone. Fluorescence in situ hybridization analysis, however, shows that none of these myeloid colonies (0 of 41 RT-PCRpositive colonies) originated from a progenitor cell that carries the leukemia-specific translocation. Thus, leukemic, translocation-positive CD34 + CD19 À progenitor/stem cells that were copurified by cell sorting were able to survive in these colony assays for up to 28 days allowing amplification of the respective fusion transcripts by sensitive RT-PCR. In conclusion, we show that childhood high-risk ALL/t(9;22) and t(4;11) originate in a primitive CD34 + CD19 À progenitor/stem cell without a myeloerythroid developmental potential. (Cancer Res 2005; 65(4): 1442-9)

IntroductionFor a long time it has been assumed that the leukemic transformation in childhood B-cell precursor acute lymphoblastic leukemia (ALL) occurs at the level of a lymphoid progenitor cell. 1 Presence and stability of clonal immunoglobulin and T-cell receptor gene rearrangements in ALL have been regarded as strong evidence to support this hypothesis.Contrasting this theory, molecular and functional analyses of purified immature progenitor cell populations provide increasing evidence for the involvement of a more primitive cell in the leukemic process. Cells with a CD34 ϩ CD38 Ϫ stem cell-like immunophenotype were shown to harbor the leukemia-specific T-cell receptor V␦2-D␦3 rearrangements 2 and were able to transfer Philadelphia chromosome-positive ALL onto immunodeficient nonobese diabetic/severe combined immunodeficiency mice. 3 In addition, CD34 ϩ CD19 Ϫ CD33 Ϫ CD38 Ϫ cells were found to contain variable percentages of leukemic blasts by fluorescence in situ hybridization (FISH), 4 indicating that there may be heterogeneity of stem cell involvement in ALL. This is in accordance with studies investigating clonal diversity at the level of immunoglobulin rearrangements. Several leukemic subclones with unrelated DJ rearrangements could be detected, particularly in patients with a poor prognosis. 5 These data suggest that at least in some patients with ALL the leukemia originates from a primitive progenitor/stem cell that has not yet initiated immunoglobulin gene rearrangement.However, these investigations may not reflect the situation in more common and prognostically favorable ALL subtypes, ie, in children with an overall event-free survival close to 80% at 5 years. 6 In the present study, we therefore focused on the childhood ALL subgroup characterized by the translocation t(12;21)(p13; q22). This chromosomal translocation leads to formation of the TEL/AML1 fusion oncogene and is the most common genetic aberration in childhood B-cell precursor ALL, occurring in approximately 20% to 25% of patients. 7 Presence of t(12;21) is usually regarded as a prognostically favorable marker 8 and correlates with in vitro sensitivity of the leukemic blasts to asparaginase, 9 vincristine, dexamethasone, and serum deprivation. 10 Due to the high incidence of t(12;21) in relapse patients 11 and a recent study in children with primary ALL showing no predictive value of t(12;21) regarding outcome, 12 the exact role of t(12;21) as a prognostic marker needs, however, further prospective evaluation. 13 To address the question of involvement of the immature progenitor/stem cell compartment in childhood ALL, flow cytometric analyses had been initiated to identify and distinguish uncommitted and early B-lineage progenitors. Expression of CD19 was found to be a good marker to distinguish CD34 ϩ CD19 ϩ leukemic from more immature CD34 ϩ CD19 Ϫ progenitor/stem cells in the bone marrow of patients with newly diagnosed B-cell precursor ALL. 14,15 CD19 is part of the CD19/CD21 complex that modulates B-cell-receptor signaling. 16 Its ex...

Immunosuppressive CD41CD25hi FoxP31 T cells (T reg cells) have been found at increased densities within the tumor microenvironment in many malignancies and interfere with protective antitumor immune responses. Osseous Ewing sarcomas (ESs) are thought to derive from a bone marrow (BM) mesenchymal cell of origin, and microscopic marrow involvement defines a subpopulation of patients at a high risk of relapse. We hypothesized that BM-resident T cells may contribute to a permissive milieu for immune escape of ESs. Using 6-color-flow cytometry, we investigated the pattern of immune cell subset distribution including NK cells, cd T cells, central and effector memory CD81 and CD41 T cells as well as T cells with regulatory phenotype (T reg cells) in BM obtained at diagnosis from 45 primary or relapsed ES patients treated within standardized protocols. Although patients at relapse had an inverted CD4:CD8 T-cell ratio, neither CD81 effector/memory T-cell subsets nor T reg cells significantly differed from patients at diagnosis. No significant associations of innate and effector/memory T-cell subpopulations with known risk factors were found, including age, gender, tumor site, primary metastases and histological tumor response. By contrast, T reg cells were found at significantly higher frequencies in patients with primary metastatic disease compared with localized ESs (5.0 vs. 3.3%, p 5 0.01). Thus, increased BM T reg cells in patients with metastasized ES may reflect an immune escape mechanism that contributes to the development of metastatic disease. Immunotherapeutic strategies will have to adequately consider the regulatory milieu within areas of Ewing tumor-immune interactions. ' 2009 UICC

Residual nonmalignant T cells in the bone marrow of patients with acute leukemias may be involved in active immune responses to leukemic cells. Here, we investigated the phenotypic signature of T cells present at diagnosis in 39 pediatric patients with acute lymphoblastic leukemia (ALL) treated within standardized ALL-BFM study protocols. Previously described age associations of lymphocyte subpopulations in the peripheral blood of healthy children were reproduced in leukemic bone marrow. Analysis of individual lymphocyte parameters and risk-associated variables using univariate linear regression models revealed a correlation of higher CD4/CD8 ratios at diagnosis with a favorable bone marrow response on day 15. Separate analysis of CD4+ cells with the CD4+CD25^hiFoxP3+ T_reg cell phenotype showed that the association was caused by non-T_reg CD4+ cells. The association of higher CD4/CD8 ratios with a favorable bone marrow response on day 15 of treatment persisted in a cohort extended to 69 patients. We conclude that CD4+ non-T_reg cells in leukemic bone marrow at diagnosis may have a role in early response to treatment. Prospective analysis of the CD4/CD8 ratio in a large cohort of pediatric patients is now needed. Moreover, future experiments will establish the functional role of the individual T cell subsets in immune control in pediatric ALL.