Panobinostat and decitabine prior to donor lymphocyte infusion in allogeneic stem cell transplantation.

Outcome after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is adversely affected by relapse to a considerable degree. To exploit the graft-versus-leukemia effect more effectively, we assessed the feasibility of early initiation of epigenetic therapy with panobinostat and decitabine after allo-HSCT and before donor lymphocyte infusion (DLI) in poor-risk patients with acute myeloid leukemia (AML) or refractory anemia with excess blasts with International Prognostic Scoring System score ≥1.5. A total of 140 poor-risk patients with AML aged 18 to 70 years were registered, and 110 proceeded to allo-HSCT. Three dose levels were evaluated for dose-limiting toxicities, including panobinostat monotherapy 20 mg at days 1, 4, 8, and 11 of a 4-week cycle (PNB mono group) and panobinostat combined with either decitabine 20 mg/m2 (PNB/DAC20 group) or decitabine 10 mg/m2 (PNB/DAC10 group) at days 1 to 3 of every 4-week cycle. After phase 1, the study continued as phase 2, focusing on completion of protocol treatment and treatment outcome. PNB mono and PNB/DAC10 were feasible, whereas PNB/DAC20 was not related to prolonged cytopenia. Sixty of 110 patients who underwent transplantation were eligible to receive their first DLI within 115 days after allo-HSCT. Grade 3 and 4 adverse events related to panobinostat and decitabine were observed in 23 (26%) of the 87 patients, and they received epigenetic therapy. Cumulative incidence of relapse was 35% (standard error [SE] 5), and overall survival and progression-free survival at 24 months were 50% (SE 5) and 49% (SE 5). Post-allo-HSCT epigenetic therapy with panobinostat alone or in combination with low-dose decitabine is feasible and is associated with a relatively low relapse rate. The trial was registered at the European Clinical Trial Registry, https://www.clinicaltrialsregister.eu, as ECT2012-003344-74.

Immunophenotypic Detection of Measurable Residual (Stem Cell) Disease Using LAIP Approach in Acute Myeloid Leukemia.

Half of the patients with acute myeloid leukemia (AML), who achieve complete remission after chemotherapy treatment, will ultimately experience a relapse. Measurable residual disease (MRD) is an important post-treatment risk factor in AML, because it gives additional information about the depth of the remission. Within MRD, the small population of leukemic stem cells (LSCs) is thought to be at the base of the actual relapse. In this protocol, the flow cytometric detection of MRD and LSCs herein is outlined. We give a detailed overview of the sampling procedures for optimal multiparameter flow cytometry assessment of both MRD and LSC, using leukemia associated immunophenotypes (LAIPs) and LSC markers. Moreover, an overview of the gating strategies to detect LAIPs and LSC markers is provided. This protocol serves as guidance for flow cytometric detection of measurable residual (stem cell) disease necessary for proper therapeutic decision making in AML patients. © 2019 The Authors. Basic Protocol 1: Immunophenotypic LAIP detection for measurable residual disease monitoring Basic Protocol 2: Immunophenotypic detection of CD34+CD38- leukemic stem cells.

CD34+CD38- leukemic stem cell frequency to predict outcome in acute myeloid leukemia.

Current risk algorithms are primarily based on pre-treatment factors and imperfectly predict outcome in acute myeloid leukemia (AML). We introduce and validate a post-treatment approach of leukemic stem cell (LSC) assessment for prediction of outcome. LSC containing CD34+CD38- fractions were measured using flow cytometry in an add-on study of the HOVON102/SAKK trial. Predefined cut-off levels were prospectively evaluated to assess CD34+CD38-LSC levels at diagnosis (n = 594), and, to identify LSClow/LSChigh (n = 302) and MRDlow/MRDhigh patients (n = 305) in bone marrow in morphological complete remission (CR). In 242 CR patients combined MRD and LSC results were available. At diagnosis the CD34+CD38- LSC frequency independently predicts overall survival (OS). After achieving CR, combining LSC and MRD showed reduced survival in MRDhigh/LSChigh patients (hazard ratio [HR] 3.62 for OS and 5.89 for cumulative incidence of relapse [CIR]) compared to MRDlow/LSChigh, MRDhigh/LSClow, and especially MRDlow/LSClow patients. Moreover, in the NPM1mutant positive sub-group, prognostic value of golden standard NPM1-MRD by qPCR can be improved by addition of flow cytometric approaches. This is the first prospective study demonstrating that LSC strongly improves prognostic impact of MRD detection, identifying a patient subgroup with an almost 100% treatment failure probability, warranting consideration of LSC measurement incorporation in future AML risk schemes.

The interference of genetic associations in establishing the prognostic value of the immunophenotype in acute myeloid leukemia.

BACKGROUND: In acute myeloid leukemia controversy exists about the role of immunophenotyping of the blasts at diagnosis as a potential prognostic factor. METHODS: We retrospectively analyzed immunophenotypic marker expression on blasts in relation to genetic aberrancies and survival data of 684 patients. All patients were included in different studies from the HOVON/SAKK Consortium. RESULTS: Markers CD2, CD7, CD11b, CD19, CD22, and CD56 all appeared to be associated with one or more established prognostic genetic aberrancies. In the overall population, differences in univariate survival analyses were observed for CD2, CD11b, and CD22. After correcting these survival differences for currently used risk profile data, only CD11b expression remained of prognostic value for poor overall survival (OS) and event-free survival (EFS). CD11b expression turned out to be an independent factor for poor OS and EFS in the subgroup of patients who lacked cytogenetic and molecular aberrancies. CONCLUSIONS: In this study, we demonstrate that associations between immunophenotypic markers and genetic aberrancies interfere with the prognostic properties of immunophenotypic markers. Such may account for most of the previously reported prognostic impact of these markers. Only CD11b expression remained as an independent prognostic marker. © 2017 International Clinical Cytometry Society.

Graft-Versus-Leukemia Effect of Allogeneic Stem-Cell Transplantation and Minimal Residual Disease in Patients With Acute Myeloid Leukemia in First Complete Remission.

PURPOSE: The detection of minimal residual disease (MRD) in patients with acute myeloid leukemia (AML) may improve future risk-adapted treatment strategies. We assessed whether MRD-positive and MRD-negative patients with AML benefit differently from the graft-versus-leukemia effect of allogeneic hematopoietic stem-cell transplantation (alloHSCT). METHODS: A total of 1,511 patients were treated in subsequent Dutch-Belgian Hemato-Oncology Cooperative Group and the Swiss Group for Clinical Cancer Research AML trials, of whom 547 obtained a first complete remission, received postremission treatment (PRT), and had available flow cytometric MRD before PRT. MRD positivity was defined as more than 0.1% cells with a leukemia-associated immunophenotype within the WBC compartment. PRT consisted of alloHSCT (n = 282), conventional PRT by a third cycle of chemotherapy (n = 160), or autologous hematopoietic stem-cell transplantation (n = 105). RESULTS: MRD was positive in 129 patients (24%) after induction chemotherapy before proceeding to PRT. Overall survival and relapse-free survival were significantly better in patients without MRD before PRT compared with MRD-positive patients (65% ± 2% v 50% ± 5% at 4 years; P = .002; and 58% ± 3% v 38% ± 4%; P < .001, respectively), which was mainly because of a lower cumulative incidence of relapse (32% ± 2% compared with 54% ± 4%; P < .001, respectively). Multivariable analysis with adjustment for covariables showed that the incidence of relapse was significantly reduced after alloHSCT compared with chemotherapy or autologous hematopoietic stem cell transplantation (hazard ratio [HR], 0.36; P < .001), which was similarly exerted in both MRD-negative and MRD-positive patients (HR, 0.38; P < .001; and HR, 0.35; P < .001, respectively). CONCLUSION: The graft-versus-leukemia effect of alloHSCT is equally present in MRD-positive and MRD-negative patients, which advocates a personalized application of alloHSCT, taking into account the risk of relapse determined by AML risk group and MRD status, as well as the counterbalancing risk of nonrelapse mortality.

CD45RA, a specific marker for leukaemia stem cell sub-populations in acute myeloid leukaemia.

Chemotherapy resistant leukaemic stem cells (LSC) are thought to be responsible for relapses after therapy in acute myeloid leukaemia (AML). Flow cytometry can discriminate CD34(+) CD38(-) LSC and normal haematopoietic stem cells (HSC) by using aberrant expression of markers and scatter properties. However, not all LSC can be identified using currently available markers, so new markers are needed. CD45RA is expressed on leukaemic cells in the majority of AML patients. We investigated the potency of CD45RA to specifically identify LSC and HSC and improve LSC quantification. Compared to our best other markers (CLL-1, also termed CLEC12A, CD33 and CD123), CD45RA was the most reliable marker. Patients with high percentages (>90%) of CD45RA on CD34(+) CD38(-) LSC have 1·69-fold higher scatter values compared to HSC (P < 0·001), indicating a more mature CD34(+) CD38(-) phenotype. Patients with low (<10%) or intermediate (10-90%) CD45RA expression on LSC showed no significant differences to HSC (1·12- and 1·15-fold higher, P = 0·31 and P = 0·44, respectively). CD45RA-positive LSC tended to represent more favourable cytogenetic/molecular markers. In conclusion, CD45RA contributes to more accurate LSC detection and is recommended for inclusion in stem cell tracking panels. CD45RA may contribute to define new LSC-specific therapies and to monitor effects of anti-LSC treatment.

Absence of leukaemic CD34+ cells in acute myeloid leukaemia is of high prognostic value: a longstanding controversy deciphered.

Primary resistance and relapses after initial successful treatment are common in acute myeloid leukaemia and therefore outcome remains poor. More accurate risk group stratification and effective personalized risk adapted treatment are necessary to improve outcome. In the last two decades, controversial results have been published concerning the prognostic relevance of CD34 expression. In this study of 706 acute myeloid leukaemia patients, we established a new flow cytometric-based CD34-definition, without use of cut-off values. We discriminated CD34-positive (n = 548) and CD34-negative (n = 158) patients by the presence or absence of neoplastic CD34+ cells, respectively. CD34-status was defined using aberrant immunophenotypes and validated using molecular phenotypes. This new definition of CD34 enables strong prediction of treatment outcome in the entire patient group and in several risk subgroups. Previously observed discrepancies in prognostic impact of CD34 protein expression using cut-offs (5-20%) can now entirely be explained by considering the number of CD34-negative cases. In the total patient group, the absence of neoplastic CD34-positive cells is paralleled by low levels of minimal residual disease, suggesting relative therapy sensitivity and explaining longer survival. Overall, we present CD34 surface expression as a relatively simple, powerful and independent predictor of clinical outcome, now warranting incorporation in acute myeloid leukaemia risk stratification.

Leukemic stem cell frequency: a strong biomarker for clinical outcome in acute myeloid leukemia.

INTRODUCTION: Treatment failure in acute myeloid leukemia is probably caused by the presence of leukemia initiating cells, also referred to as leukemic stem cells, at diagnosis and their persistence after therapy. Specific identification of leukemia stem cells and their discrimination from normal hematopoietic stem cells would greatly contribute to risk stratification and could predict possible relapses. RESULTS: For identification of leukemic stem cells, we developed flow cytometric methods using leukemic stem cell associated markers and newly-defined (light scatter) aberrancies. The nature of the putative leukemic stem cells and normal hematopoietic stem cells, present in the same patient's bone marrow, was demonstrated in eight patients by the presence or absence of molecular aberrancies and/or leukemic engraftment in NOD-SCID IL-2Rγ-/- mice. At diagnosis (n=88), the frequency of the thus defined neoplastic part of CD34+CD38- putative stem cell compartment had a strong prognostic impact, while the neoplastic parts of the CD34+CD38+ and CD34- putative stem cell compartments had no prognostic impact at all. After different courses of therapy, higher percentages of neoplastic CD34+CD38- cells in complete remission strongly correlated with shorter patient survival (n=91). Moreover, combining neoplastic CD34+CD38- frequencies with frequencies of minimal residual disease cells (n=91), which reflect the total neoplastic burden, revealed four patient groups with different survival. CONCLUSION AND PERSPECTIVE: Discrimination between putative leukemia stem cells and normal hematopoietic stem cells in this large-scale study allowed to demonstrate the clinical importance of putative CD34+CD38- leukemia stem cells in AML. Moreover, it offers new opportunities for the development of therapies directed against leukemia stem cells, that would spare normal hematopoietic stem cells, and, moreover, enables in vivo and ex vivo screening for potential efficacy and toxicity of new therapies.