Influence of Cytokines and Autologous Lymphokine-Activated Killer Cells on Leukemic Bone Marrow Cells and Colonies in AML

We have already shown that cytokine cocktails (IL-1 beta, IL-3, IL-6, SCF, GM-CSF) and/or lymphokine-activated killer (LAK) cells can reduce the amounts of clonal, CD34-positive mononuclear bone marrow cells (BM-MNC) in acute myeloid leukemia (AML). In addition, the influence of those cocktails and/or LAK cells on the clonogenic potential of AML BM-MNC was investigated. BM colonies cultured in agar during different stages of the disease were immunophenotyped in situ: 17 patients at diagnosis, 14 patients in complete remission, 8 patients at relapse, 8 healthy donors. A significant reduction in leukemic cells and colonies positive for CD34 after in vitro culture of BM-MNC with cytokine cocktails was achieved with all samples obtained at diagnosis (n = 8, p < 0.01), in 6 of 8 cases in complete remission but only in 2 of 6 cases at relapse. Cytokine cocktails stimulated granulopoiesis as well as B and T lymphopoiesis. Colonies with leukemic phenotype could never be detected in healthy BM. A significant reduction in leukemic colonies was achieved by coculture of BM-MNC (uncultured or cytokine precultured) with autologous LAK cells in all 4 cases at diagnosis and in 1 case at relapse. An additive effect of in vitro cytokine preincubation of BM samples on the leukemia-reducing effect of LAK cells could be demonstrated in all samples studied (p <0.001; diagnosis: n = 10, relapse: n = 3, complete remission: n = 7). Patients had a better prognosis if CD34-positive colonies in AML could be reduced by cytokine incubation (p = 0.03) or coculture with autologous LAK cells in vitro (p = 0.04). Our data show that cytokines as well as LAK cells alone and in combination can reduce, however not eliminate clonogenic AML cells. Such mechanisms might be responsible for maintaining stable remissions in AML. Copyright (C) 2001 S. Karger AG, Basel

Analogue peptides for the immunotherapy of human acute myeloid leukemia

Accepted manuscript. The final publication is available at: http://link.springer.com/article/10.1007%2Fs00262-015-1762-9The use of peptide vaccines, enhanced by adjuvants, has shown some efficacy in clinical trials. However, responses are often short-lived and rarely induce notable memory responses. The reason is that self-antigens have already been presented to the immune system as the tumor develops, leading to tolerance or some degree of host tumor cell destruction. To try to break tolerance against self-antigens, one of the methods employed has been to modify peptides at the anchor residues to enhance their ability to bind major histocompatibility complex molecules, extending their exposure to the T-cell receptor. These modified or analogue peptides have been investigated as stimulators of the immune system in patients with different cancers with variable but sometimes notable success. In this review we describe the background and recent developments in the use of analogue peptides for the immunotherapy of acute myeloid leukemia describing knowledge useful for the application of analogue peptide treatments for other malignancies

Chronic myeloid leukemia stem cells are not dependent on Bcr-Abl kinase activity for their survival

Recent evidence suggests CML stem cells are insensitive to kinase inhibitors and responsible for minimal residual disease in treated patients. We investigated whether CML stem cells, in a transgenic mouse model of CML-like disease or derived from patients, are dependent on Bcr-Abl. In the transgenic model, following re-transplantation, donor-derived CML stem cells in which Bcr-Abl expression had been induced and subsequently shut off, were able to persist in vivo and re-initiate leukemia in secondary recipients upon Bcr-Abl re-expression. Bcr-Abl knockdown in human CD34+ CML cells cultured for 12 days in physiological growth factors achieved partial inhibition of Bcr-Abl and downstream targets p-CrkL and p-STAT5, inhibition of proliferation and colony forming cells, but no reduction of input cells. The addition of dasatinib further inhibited p-CrkL and p-STAT5, yet only reduced input cells by 50%. Complete growth factor withdrawal plus dasatinib further reduced input cells to 10%, however the surviving fraction was enriched for primitive leukemic cells capable of growth in long-term culture initiating cell assay and expansion upon removal of dasatinib and addition of growth factors. Together these data suggest that CML stem cell survival is Bcr-Abl kinase independent and suggest curative approaches in CML must focus on kinase-independent mechanisms of resistance

Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia.

In acute myeloid leukaemia (AML), the cell of origin, nature and biological consequences of initiating lesions, and order of subsequent mutations remain poorly understood, as AML is typically diagnosed without observation of a pre-leukaemic phase. Here, highly purified haematopoietic stem cells (HSCs), progenitor and mature cell fractions from the blood of AML patients were found to contain recurrent DNMT3A mutations (DNMT3A(mut)) at high allele frequency, but without coincident NPM1 mutations (NPM1c) present in AML blasts. DNMT3A(mut)-bearing HSCs showed a multilineage repopulation advantage over non-mutated HSCs in xenografts, establishing their identity as pre-leukaemic HSCs. Pre-leukaemic HSCs were found in remission samples, indicating that they survive chemotherapy. Therefore DNMT3A(mut) arises early in AML evolution, probably in HSCs, leading to a clonally expanded pool of pre-leukaemic HSCs from which AML evolves. Our findings provide a paradigm for the detection and treatment of pre-leukaemic clones before the acquisition of additional genetic lesions engenders greater therapeutic resistance

"Society of Hematologic Oncology (SOHO) State of the Art Updates and Next Questions"-Treatment of ALL.

The outcome of adult acute lymphoblastic leukemia (ALL) has substantially improved by adopting pediatric-inspired regimens, and approximately half of the patients are nowadays cured. The evaluation of minimal residual disease currently represents the most important prognostic indicator, which drives treatment algorithms, which include allogeneic stem cell transplantation (allo-SCT) allocation. Indeed, for high-risk patients, allo-SCT should be pursued as soon as possible, whereas in standard-risk patients this procedure should be avoided also in light of related toxicity and because there are no significant benefits. Furthermore, better characterization of the molecular genetic events can drive therapeutic decisions: a historical example in this respect is represented by the use of tyrosine kinase inhibitors (TKIs) in Philadelphia chromosome-positive ALL; in the upcoming future, TKIs might be used also in other subgroups, such as breakpoint cluster region/Abelson 1-like cases and others with deregulated tyrosine kinases. Finally, the greatest progress is currently achieved with new immunotherapies targeting frequently expressed surface antigens in ALL. It is also a new chance for elderly ALL patients, so far spared from intensive chemotherapy and allo-SCT. These targeted therapies will substantially change this treatment algorithm and the great challenge is to find optimal sequence of the extended therapy options in an individual patient

Targeting self-renewal pathways in myeloid malignancies

A fundamental property of hematopoietic stem cells (HSCs) is the ability to self-renew. This is a complex process involving multiple signal transduction cascades which control the fine balance between self-renewal and differentiation through transcriptional networks. Key activators/regulators of self-renewal include chemokines, cytokines and morphogens which are expressed in the bone marrow niche, either in a paracrine or autocrine fashion, and modulate stem cell behaviour. Increasing evidence suggests that the downstream signaling pathways induced by these ligands converge at multiple levels providing a degree of redundancy in steady state hematopoiesis. Here we will focus on how these pathways cross-talk to regulate HSC self-renewal highlighting potential therapeutic windows which could be targeted to prevent leukemic stem cell self-renewal in myeloid malignancies

MEASURABLE RESIDUAL DISEASE AND LEUKEMIC STEM CELLS IN ACUTE MYELOID LEUKEMIA

Nearly all fit patients with acute myeloid leukemia (AML) receive intense chemotherapy, followed by consolidation therapy which can be either additional cycle(s) of chemotherapy, autologous stem cell transplantation or allogeneic stem cell transplantation. In this order, anti-leukemic efficacy increases together with toxicity. While, fortunately, most patients achieve complete remission, unfortunately, 40-50% of patients experience a relapse. Patients who relapse have a dismal prognosis since the relapse is mostly difficult to eradicate. A correct understanding of the risk to relapse is vital for selecting the correct therapy intensity. Risk stratification at diagnosis is based on factors such as age, white blood cell (WBC) count and genetic (mutations and cytogenetic aberrations) characteristics.1 This risk assessment at diagnosis does not suffice for an accurate estimation of patients that relapse, therefore, more specific and sensitive methods (both by flow cytometry and molecular techniques) are widely used to assess possible residual disease during and after therapy. When this residual disease (termed measurable residual disease or minimal residual disease, MRD) is present above a critical level, patients have a higher chance of experiencing a relapse. The overall aim of the studies described in this thesis is to investigate the role of measurable residual disease (MRD) and leukemic stem cells (LSC), and several initiatives to improve the MRD assessment to be used for relapse prediction for the individual patient. Chapter 2 covers a review on several aspects of LSCs in AML and its considered role in relapse progression. Moreover, it discusses how these relatively rare cells can be detected by flow cytometry, and furthermore discusses how this detection is currently used in clinical application. In chapter 3-4 we investigated if the LSC frequency harbors prognostic information for improved relapse prediction for AML. In chapter 3 we present the clinical significance of the presence and frequency of CD34+CD38- LSCs at time of diagnosis and in remission bone marrow in adult AML. In addition, the prognostic relevance of the combination of LSC-MRD and MFC-MRD is investigated. In chapter 4 we investigated whether detection of CD34+CD38- LSCs in BM of newly diagnosed pediatric AML bears similar prognostic relevance as shown in adult AML. In chapter 5-6 we elaborate on the importance of standardization of the flow cytometric MRD and LSC detection approaches. In chapter 5 we evaluated the technical and analytical feasibility of the previously designed eight‐color LSC single tube assay, as well as standardization of the process. In chapter 6 we present a new flow cytometric model for standardized and objective MRD calculation, retrospectively applied in a large clinical study. For this, we evaluate if the balance between neoplastic and normal progenitors in CR bone marrow has prognostic relevance. In chapter 7 we evaluate whether next-generation sequencing has clinical value for the prediction of relapse. Since measurements were simultaneously evaluated for MFC-MRD, we investigated whether NGS and MFC-MRD have independent and additive prognostic value. In addition, we studied whether MRD and LSC-MRD is a valid surrogate endpoint in AML. As shown in a recent clinical trial, the new therapeutic clofarabine has clinical beneficial effect in a subgroup of patients. In chapter 8 we investigated whether the prospectively defined MRD and LSC-MRD frequencies were different between patients with clofarabine and patients without clofarabine, and whether MRD levels mirrored the clinical outcome within this subgroup. Finally, in chapter 9 we summarize the results of this thesis and which implications these results may have for future AML relapse prediction. Furthermore, we evaluate the different techniques used in this thesis, discuss how each technique can be further optimized and elaborate on the optimal use for future clinical trials

In vivo imaging enables high resolution preclinical trials on patients' leukemia cells growing in mice.

Xenograft mouse models represent helpful tools for preclinical studies on human tumors. For modeling the complexity of the human disease, primary tumor cells are by far superior to established cell lines. As qualified exemplary model, patients' acute lymphoblastic leukemia cells reliably engraft in mice inducing orthotopic disseminated leukemia closely resembling the disease in men. Unfortunately, disease monitoring of acute lymphoblastic leukemia in mice is hampered by lack of a suitable readout parameter