The MLL recombinome of acute leukemias in 2017.

Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients.

Abundant and equipotent founder cells establish and maintain acute lymphoblastic leukaemia.

High frequencies of blasts in primary acute lymphoblastic leukaemia (ALL) samples have the potential to induce leukaemia and to engraft mice. However, it is unclear how individual ALL cells each contribute to drive leukaemic development in a bulk transplant and the extent to which these blasts vary functionally. We used cellular barcoding as a fate mapping tool to track primograft ALL blasts in vivo. Our results show that high numbers of ALL founder cells contribute at similar frequencies to leukaemic propagation over serial transplants, without any clear evidence of clonal succession. These founder cells also exhibit equal capacity to home and engraft to different organs, although stochastic processes may alter the composition in restrictive niches. Our findings enhance the stochastic stem cell model of ALL by demonstrating equal functional abilities of singular ALL blasts and show that successful treatment strategies must eradicate the entire leukaemic cell population.

A phase I/II trial of AT9283, a selective inhibitor of aurora kinase in children with relapsed or refractory acute leukemia: challenges to run early phase clinical trials for children with leukemia.

Aurora kinases regulate mitosis and are commonly overexpressed in leukemia. This phase I/IIa study of AT9283, a multikinase inhibitor, was designed to identify maximal tolerated doses, safety, pharmacokinetics, and pharmacodynamic activity in children with relapsed/refractory acute leukemia. The trial suffered from poor recruitment and terminated early, therefore failing to identify its primary endpoints. AT9283 caused tolerable toxicity, but failed to show clinical responses. Future trials should be based on robust preclinical data that provide an indication of which patients may benefit from the experimental agent, and recruitment should be improved through international collaborations and early combination with established treatment strategies.

Long-term in vitro maintenance of clonal abundance and leukaemia-initiating potential in acute lymphoblastic leukaemia.

Lack of suitable in vitro culture conditions for primary acute lymphoblastic leukaemia (ALL) cells severely impairs their experimental accessibility and the testing of new drugs on cell material reflecting clonal heterogeneity in patients. We show that Nestin-positive human mesenchymal stem cells (MSCs) support expansion of a range of biologically and clinically distinct patient-derived ALL samples. Adherent ALL cells showed an increased accumulation in the S phase of the cell cycle and diminished apoptosis when compared with cells in the suspension fraction. Moreover, surface expression of adhesion molecules CD34, CDH2 and CD10 increased several fold. Approximately 20% of the ALL cells were in G0 phase of the cell cycle, suggesting that MSCs may support quiescent ALL cells. Cellular barcoding demonstrated long-term preservation of clonal abundance. Expansion of ALL cells for >3 months compromised neither feeder dependence nor cancer initiating ability as judged by their engraftment potential in immunocompromised mice. Finally, we demonstrate the suitability of this co-culture approach for the investigation of drug combinations with luciferase-expressing primograft ALL cells. Taken together, we have developed a preclinical platform with patient-derived material that will facilitate the development of clinically effective combination therapies for ALL.

The pre-B-cell receptor checkpoint in acute lymphoblastic leukaemia.

The B-cell receptor (BCR) and its immature form, the precursor-BCR (pre-BCR), have a central role in the control of B-cell development, which is dependent on a sequence of cell-fate decisions at specific antigen-independent checkpoints. Pre-BCR expression provides the first checkpoint, which controls differentiation of pre-B to immature B-cells in normal haemopoiesis. Pre-BCR signalling regulates and co-ordinates diverse processes within the pre-B cell, including clonal selection, proliferation and subsequent maturation. In B-cell precursor acute lymphoblastic leukaemia (BCP-ALL), B-cell development is arrested at this checkpoint. Moreover, malignant blasts avoid clonal extinction by hijacking pre-BCR signalling in favour of the development of BCP-ALL. Here, we discuss three mechanisms that occur in different subtypes of BCP-ALL: (i) blocking pre-BCR expression; (ii) activating pre-BCR-mediated pro-survival and pro-proliferative signalling, while inhibiting cell cycle arrest and maturation; and (iii) bypassing the pre-BCR checkpoint and activating pro-survival signalling through pre-BCR independent alternative mechanisms. A complete understanding of the BCP-ALL-specific signalling networks will highlight their application in BCP-ALL therapy.

Differential expression of miR-17~92 identifies BCL2 as a therapeutic target in BCR-ABL-positive B-lineage acute lymphoblastic leukemia.

Despite advances in allogeneic stem cell transplantation, BCR-ABL-positive acute lymphoblastic leukaemia (ALL) remains a high-risk disease, necessitating the development of novel treatment strategies. As the known oncomir, miR-17~92, is regulated by BCR-ABL fusion in chronic myeloid leukaemia, we investigated its role in BCR-ABL translocated ALL. miR-17~92-encoded miRNAs were significantly less abundant in BCR-ABL-positive as compared to -negative ALL-cells and overexpression of miR-17~19b triggered apoptosis in a BCR-ABL-dependent manner. Stable isotope labelling of amino acids in culture (SILAC) followed by liquid chromatography and mass spectroscopy (LC-MS) identified several apoptosis-related proteins including Bcl2 as potential targets of miR-17~19b. We validated Bcl2 as a direct target of this miRNA cluster in mice and humans, and, similar to miR-17~19b overexpression, Bcl2-specific RNAi strongly induced apoptosis in BCR-ABL-positive cells. Furthermore, BCR-ABL-positive human ALL cell lines were more sensitive to pharmacological BCL2 inhibition than negative ones. Finally, in a xenograft model using patient-derived leukaemic blasts, real-time, in vivo imaging confirmed pharmacological inhibition of BCL2 as a new therapeutic strategy in BCR-ABL-positive ALL. These data demonstrate the role of miR-17~92 in regulation of apoptosis, and identify BCL2 as a therapeutic target of particular relevance in BCR-ABL-positive ALL.

The MLL recombinome of acute leukemias in 2013.

Chromosomal rearrangements of the human MLL (mixed lineage leukemia) gene are associated with high-risk infant, pediatric, adult and therapy-induced acute leukemias. We used long-distance inverse-polymerase chain reaction to characterize the chromosomal rearrangement of individual acute leukemia patients. We present data of the molecular characterization of 1590 MLL-rearranged biopsy samples obtained from acute leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and novel TPGs identified. All patients were classified according to their gender (852 females and 745 males), age at diagnosis (558 infant, 416 pediatric and 616 adult leukemia patients) and other clinical criteria. Combined data of our study and recently published data revealed a total of 121 different MLL rearrangements, of which 79 TPGs are now characterized at the molecular level. However, only seven rearrangements seem to be predominantly associated with illegitimate recombinations of the MLL gene (≈ 90%): AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, ELL, partial tandem duplications (MLL PTDs) and MLLT4/AF6, respectively. The MLL breakpoint distributions for all clinical relevant subtypes (gender, disease type, age at diagnosis, reciprocal, complex and therapy-induced translocations) are presented. Finally, we present the extending network of reciprocal MLL fusions deriving from complex rearrangements.

Favorable outcome in infants with AML after intensive first- and second-line treatment: an AML-BFM study group report.

Infants <1 year of age have a high prevalence of prognostically unfavorable leukemias and a presumed susceptibility to treatment-related toxicities. A total of 125 infants with acute myeloid leukemia (AML) were treated in studies AML-BFM-98 (n = 59) and -2004 (n = 66). Treatment regimens of both studies were comparable, consisting of intensive induction followed by four courses (mainly high-dose cytarabine and anthracyclines). Allogeneic-hematopoietic stem-cell-transplantation (allo-HSCT) in 1st remission was optional for high-risk (HR) patients. Most infants (120/125=96%) were HR patients according to morphological, cytogenetic/molecular genetic and response criteria. Five-year overall survival was 66 ± 4%, and improved from 61 ± 6% in study-98 to 75 ± 6% in study-2004 (P(logrank) 0.14) and event-free survival rates were 44 ± 6% and 51 ± 6% (P(logrank) 0.66), respectively. Results in HR infants were similar to those of older HR children (1-<2- or 2-<10-year olds, P(logrank) 0.90 for survival). Survival rates of HSCT in 1st remission, initial partial response and after relapse were high (13/14, 2/8 and 20/30 patients, respectively). The latter contributes to excellent 5-year survival after relapse (50±8%). Despite more severe infections and pulmonary toxicities in infants, treatment-related death rate was identical to that of older children (3%). Our data indicate that intensive frontline and relapse AML treatment is feasible in infants, toxicities are manageable, and outcome is favorable.

Leukemic fusion genes MLL/AF4 and AML1/MTG8 support leukemic self-renewal by controlling expression of the telomerase subunit TERT.

MLL/AF4 and AML/MTG8 represent two leukemic fusion genes, which are most frequently found in infant acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), respectively. We examined the influence of MLL/AF4 and AML1/MTG8 fusion genes on the expression of TERT coding for the telomerase protein subunit, and subsequently telomerase activity in t(4;11)-positive ALL and t(8;21)-positive cell lines, respectively. MLL/AF4 suppression diminished telomerase activity and expression of TERT. Blocking pro-apoptotic caspase activation in conjunction with MLL/AF4 knockdown enhanced the inhibition of TERT gene expression, which suggests that MLL/AF4 depletion does not reduce TERT expression levels by inducing apoptosis. Knockdown of HOXA7, a direct transcriptional target of MLL/AF4 fusion gene, caused a reduction of telomerase and TERT to an extent similar to that observed with MLL/AF4 suppression. Chromatin immunoprecipitation of SEM cells, using ectopically expressed FLAG-tagged Hoxa7, indicates HOXA7 binding site in the TERT promoter region. Furthermore, suppression of the AML1/MTG8 fusion gene was associated with severely reduced clonogenicity, induction of replicative senescence, impaired TERT expression and accelerated telomere shortening. We thus present findings that show a mechanistic link between leukemic fusion proteins, essential for development and maintenance of leukemia, and telomerase, a key element of both normal and malignant self-renewal.

Understanding the cancer stem cell.

The last 15 years has seen an explosion of interest in the cancer stem cell (CSC). Although it was initially believed that only a rare population of stem cells are able to undergo self-renewing divisions and differentiate to form all populations within a malignancy, a recent work has shown that these cells may not be as rare as thought first, at least in some malignancies. Improved experimental models are beginning to uncover a less rigid structure to CSC biology, in which the concepts of functional plasticity and clonal evolution must be incorporated into the traditional models. Slowly the genetic programmes and biological processes underlying stem cell biology are being elucidated, opening the door to the development of drugs targeting the CSC. The aim of ongoing research to understand CSCs is to develop novel stem cell-directed treatments, which will reduce therapy resistance, relapse and the toxicity associated with current, non-selective agents.

Relapse, not regimen-related toxicity, was the major cause of treatment failure in 11 children with Down syndrome undergoing haematopoietic stem cell transplantation for acute leukaemia.

We report a retrospective analysis of 11 children with Down syndrome (DS) treated by SCT in eight German/Austrian SCT centres. Indications for transplantation were acute lymphoblastic leukaemia (N=8) and acute myeloid leukaemia (N=3). A reduced intensity conditioning (RIC) containing 2 Gy TBI was given to two patients, another five received a myeloablative regimen with 12 Gy TBI. Treosulphan or busulphan was used in the remaining four children. Four of eleven (36%) patients are alive. All of them were treated with a myeloablative regimen. One of the four surviving children relapsed 9 months after SCT and is currently receiving palliative outpatient treatment. The main cause of death was relapse (5/11). Two children died of regimen-related toxicity (RRT), one from severe exfoliative dermatitis and multiorgan failure after a treosulphan-containing regimen, the other from GvHD-related infections after RIC. Acute GvHD of the skin was observed in 10 of 10 evaluable patients, and chronic GvHD in 4 of 8. Our data show that DS patients can tolerate commonly used, fully myeloablative preparative regimens. The major cause of death is relapse rather than RRT resulting in an event-free survival of 18% and over all survival of 36%.

CD28 co-stimulation via tumour-specific chimaeric receptors induces an incomplete activation response in Epstein-Barr virus-specific effector memory T cells.

Expression of tumour antigen-specific chimaeric receptors in T lymphocytes can redirect their effector functions towards tumour cells. Integration of the signalling domains of the co-stimulatory molecule CD28 into chRec enhances antigen-specific proliferation of polyclonal human T cell populations. While CD28 plays an essential role in the priming of naive CD4(+) T cells, its contribution to effector memory T cell responses is controversial. We compared the function of the chRec with and without the CD28 co-stimulatory domain, expressing it in peripheral blood T cells or Epstein-Barr virus (EBV)-specific T cell lines. The chimaeric T cell receptors contain an extracellular single-chain antibody domain, to give specificity against the tumour ganglioside antigen G(D2). The transduced cytotoxic T lymphocytes (CTL) maintained their specificity for autologous EBV targets and their capacity to proliferate after stimulation with EBV-infected B cells. Intracellular cytokine staining demonstrated efficient and comparable antigen-specific interferon (IFN)-gamma secretion by CTL following engagement of both the native and the chimaeric receptor, independent of chimaeric CD28 signalling. Furthermore, tumour targets were lysed in an antigen-specific manner by both chRec. However, while antigen engagement by CD28 zeta chRec efficiently induced expansion of polyclonal peripheral blood lymphocytes in an antigen-dependent manner, CD28 signalling did not induce proliferation of EBV-CTL in response to antigen-expressing tumour cells. Thus, the co-stimulatory requirement for the efficient activation response of antigen-specific memory cells cannot be mimicked simply by combining CD28 and zeta signalling. The full potential of this highly cytolytic T cell population for adoptive immunotherapy of cancer requires further exploration of their co-stimulatory requirements.

Treatment of refractory CMV-infection following hematopoietic stem cell transplantation with the combination of foscarnet and leflunomide.

BACKGROUND: Treatment of cytomegalovirus (CMV) disease after allogeneic hematopoietic stem cell transplantation (HSCT) is limited by toxicities of current antiviral drugs and the occurrence of drug resistant strains. Leflunomide, an immunosuppressive agent used for treatment of rheumatoid arthritis, also has activity against CMV by impairing viral assembly. Here we report the control of refractory CMV disease by the combined use of foscarnet and leflunomide. PATIENTS AND RESULTS: A 1S-year-old boy with juvenile myelo-monocytic leukemia (JMML) received an allogeneic HSCT with bone marrow stem cells from a mismatched, unrelated donor (MMUD, recipient and donor CMV-positive). CMV-reactivation two months post transplantation (Tx) could only be controlled by the use of cidofovir. Because of secondary graft failure, the boy received a second HSCT with peripheral blood stem cells (PBSC) of the same donor after overall 6 months. CMV-infection was noticed three weeks later, associated with a considerable rise of both CMV-copy number and pp65-antigen. Since reinduction with cidofovir was ineffective and ganciclovir not warranted due to the history of graft failure, the child then received a combination of foscarnet/leflunomide, leading to a rapid decline of his CMV-copy number and to an afebrile state. Hematological, hepatic or renal toxicities were not observed. CONCLUSION: This case report suggests that leflunomide may be of use in the management of transplant recipients with CMV-infection refractory or intolerant to conventional antiviral therapy.

Lack of expression of the chondroitin sulphate proteoglycan neuron-glial antigen 2 on candidate stem cell populations in paediatric acute myeloid leukaemia/abn(11q23) and acute lymphoblastic leukaemia/t(4;11).

It has increasingly been acknowledged that only a few leukaemic cells possess the capability to renew themselves and that only these self-renewing leukaemic stem cells are able to initiate relapses. Therefore, these leukaemic stem cells should be the target cells for therapy and for minimal residual disease (MRD) detection. Because of its presence on blasts of 11q23-rearranged high-risk leukaemic patients, neuron-glial antigen 2 (NG2) is thought to be a valuable marker for detecting leukaemic stem cells. Six acute myeloid leukaemia (AML)/abn(11q23) and three acute lymphoblastic leukaemia (ALL)/t(4;11) samples were analysed by four-colour flow cytometry for NG2 expression on primitive cell populations. Candidate leukaemic cell populations were defined by the antigen profiles CD34+CD38- in AML and CD34+CD19-CD117+ in ALL. Surprisingly, in all patients these candidate stem cell populations were shown to lack expression of NG2. Instead, a correlation between the expression of the myeloid differentiation marker CD33 and increasing levels of NG2 on maturing cells could be demonstrated. Similarly, in ALL patients CD34+CD19+ cells showed a higher expression of NG2 mRNA compared with CD34+CD19-. Thus, NG2 appears to be upregulated with differentiation and not to be expressed on primitive disease-maintaining cells. This hampers the clinical use of NG2 as a therapeutic target and as a sensitive marker for MRD detection.

Adoptive cellular immunotherapy with CD19-specific T cells.

BACKGROUND: No effective therapeutic modalities exist for the treatment of relapsed high risk acute lymphoblastic leukemia (ALL). Adoptive cellular immunotherapy by transfusion of polyclonal donor lymphocytes is not always effective and is limited by cellular cross-reactivity with normal tissues, leading to development of clinical graft-versus-host disease (GVHD). METHOD: To develop an immunotherapeutic strategy for targeted elimination of residual leukemic blasts, human T cells were gene-modified to express CD19-specific chimeric receptors. RESULTS: Gene-modified T cells specifically lyse CD19-expressing lymphatic blast cells, however, they show a limited proliferative response to stimulation with CD19. Integration of the signal transduction domain of the costimulatory molecule CD28 enhances the proliferative properties of the gene-modified T cells. CONCLUSIONS: Adoptive transfer of gene-modified virus-specific T cells may provide a useful strategy for prevention and early treatment of ALL relapses following allogeneic stem cell transplantation.