Bimodal expression of potential drug target CLL-1 (CLEC12A) on CD34+ blasts of AML patients.

OBJECTIVES: This study aims to retrospectively assess C-lectin-like molecule 1 (CLL-1) bimodal expression on CD34+ blasts in acute myeloid leukemia (AML) patients (total N = 306) and explore potential CLL-1 bimodal associations with leukemia and patient-specific characteristics. METHODS: Flow cytometry assays were performed to assess the deeper immunophenotyping of CLL-1 bimodality. Cytogenetic analysis was performed to characterize the gene mutation on CLL-1-negative subpopulation of CLL-1 bimodal AML samples. RESULTS: The frequency of a bimodal pattern of CLL-1 expression of CD34+ blasts ranged from 8% to 65% in the different cohorts. Bimodal CLL-1 expression was most prevalent in patients with MDS-related AML (P = .011), ELN adverse risk (P = .002), NPM1 wild type (WT, P = .049), FLT3 WT (P = .035), and relatively low percentages of leukemia-associated immunophenotypes (P = .006). Additional immunophenotyping analysis revealed the CLL-1- subpopulation may consist of pre-B cells, immature myeloblasts, and hematopoietic stem cells. Furthermore, (pre)-leukemic mutations were detected in both CLL-1+ and CLL-1- subfractions of bimodal samples (N = 3). CONCLUSIONS: C-lectin-like molecule 1 bimodality occurs in about 25% of AML patients and the CLL-1- cell population still contains malignant cells, hence it may potentially limit the effectiveness of CLL-1-targeted therapies and warrant further investigation.

Pre-Clinical Evaluation of the Proteasome Inhibitor Ixazomib against Bortezomib-Resistant Leukemia Cells and Primary Acute Leukemia Cells.

At present, 20-30% of children with acute leukemia still relapse from current chemotherapy protocols, underscoring the unmet need for new treatment options, such as proteasome inhibition. Ixazomib (IXA) is an orally available proteasome inhibitor, with an improved safety profile compared to Bortezomib (BTZ). The mechanism of action (proteasome subunit inhibition, apoptosis induction) and growth inhibitory potential of IXA vs. BTZ were tested in vitro in human (BTZ-resistant) leukemia cell lines. Ex vivo activity of IXA vs. BTZ was analyzed in 15 acute lymphoblastic leukemia (ALL) and 9 acute myeloid leukemia (AML) primary pediatric patient samples. BTZ demonstrated more potent inhibitory effects on constitutive ß5 and immunoproteasome ß5i proteasome subunit activity; however, IXA more potently inhibited ß1i subunit than BTZ (70% vs. 29% at 2.5 nM). In ALL/AML cell lines, IXA conveyed 50% growth inhibition at low nanomolar concentrations, but was ~10-fold less potent than BTZ. BTZ-resistant cells (150-160 fold) displayed similar (100-fold) cross-resistance to IXA. Finally, IXA and BTZ exhibited anti-leukemic effects for primary ex vivo ALL and AML cells; mean LC50 (nM) for IXA: 24 ± 11 and 30 ± 8, respectively, and mean LC50 for BTZ: 4.5 ± 1 and 11 ± 4, respectively. IXA has overlapping mechanisms of action with BTZ and showed anti-leukemic activity in primary leukemic cells, encouraging further pre-clinical in vivo evaluation.

Harnessing Gene Expression Profiles for the Identification of Ex Vivo Drug Response Genes in Pediatric Acute Myeloid Leukemia.

Novel treatment strategies are of paramount importance to improve clinical outcomes in pediatric AML. Since chemotherapy is likely to remain the cornerstone of curative treatment of AML, insights in the molecular mechanisms that determine its cytotoxic effects could aid further treatment optimization. To assess which genes and pathways are implicated in tumor drug resistance, we correlated ex vivo drug response data to genome-wide gene expression profiles of 73 primary pediatric AML samples obtained at initial diagnosis. Ex vivo response of primary AML blasts towards cytarabine (Ara C), daunorubicin (DNR), etoposide (VP16), and cladribine (2-CdA) was associated with the expression of 101, 345, 206, and 599 genes, respectively (p < 0.001, FDR 0.004-0.416). Microarray based expression of multiple genes was technically validated using qRT-PCR for a selection of genes. Moreover, expression levels of BRE, HIF1A, and CLEC7A were confirmed to be significantly (p < 0.05) associated with ex vivo drug response in an independent set of 48 primary pediatric AML patients. We present unique data that addresses transcriptomic analyses of the mechanisms underlying ex vivo drug response of primary tumor samples. Our data suggest that distinct gene expression profiles are associated with ex vivo drug response, and may confer a priori drug resistance in leukemic cells. The described associations represent a fundament for the development of interventions to overcome drug resistance in AML, and maximize the benefits of current chemotherapy for sensitive patients.

Relationship between CD34/CD38 and side population (SP) defined leukemia stem cell compartments in acute myeloid leukemia.

Leukemic stem cells (LSCs), defined by CD34/CD38 expression, are believed to be essential for leukemia initiation and therapy resistance in acute myeloid leukemia. In addition, the side population (SP), characterized by high Hoechst 33342 efflux, reflecting therapy resistance, has leukemia initiating ability. The purpose of this study is, in both CD34-positive and CD34-negative AML, to integrate both types of LSC compartment into a new more restricted definition. Different CD34/CD38/SP defined putative LSC and normal hematopoietic compartments, with neoplastic or normal nature, respectively, were thus identified after cell sorting, and confirmed by FISH/PCR. Stem cell activity was assessed in the long-term liquid culture stem cell assay. SP fractions harbored the strongest functional stem cell activity in both normal and neoplastic cells in both CD34-positive and CD34-negative AML. Overall, inclusion of SP fraction decreased the size of the putative CD34/CD38 defined LSC compartment by a factor >500. For example, for the important CD34+CD38- LSC compartment, the median SP/CD34+CD38- frequency was 5.1 per million WBC (CD34-positive AML), and median SP/CD34-CD38+ frequency (CD34-negative AML) was 1796 per million WBC. Improved detection of LSC may enable identification of therapy resistant clones, and thereby identification of novel LSC specific, HSC sparing, therapies.

RNA-based FLT3-ITD allelic ratio is associated with outcome and ex vivo response to FLT3 inhibitors in pediatric AML.

Controversy exists whether internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3-internal tandem duplication [ITD]) allelic ratio (AR) and/or length of the ITD should be taken into account for risk stratification of pediatric acute myeloid leukemia (AML) and whether it should be measured on RNA or DNA. Moreover, the ITD status may be of relevance for selecting patients eligible for FLT3 inhibitors. Here, we included 172 pediatric AML patients, of whom 36 (21%) harbored FLT3-ITD as determined on both RNA and DNA. Although there was a good correlation between both parameters ARspearman = 0.62 (95% confidence interval, 0.22-0.87) and ITDlengthspearman = 0.98 (95% confidence interval, 0.90-1.00), only AR ≥ 0.5 and length ≥48 base pairs (bps) based on RNA measurements were significantly associated with overall survival (AR: Plogrank = .008; ITDlength: Plogrank = .011). In large ITDs (>156 bp on DNA) a remarkable 90-bp difference exists between DNA and RNA, including intron 14, which is spliced out in RNA. Ex vivo exposure (n = 30) to FLT3 inhibitors, in particular to the FLT3-specific inhibitor gilteritinib, showed that colony-forming capacity was significantly more reduced in FLT3-ITD-AR ≥ 0.5 compared with ITD-AR-low and ITD- patient samples (P < .001). RNA-based FLT3-ITD measurements are recommended for risk stratification, and the relevance of AR regarding eligibility for FLT3-targeted therapy warrants further study.

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.

Gene expression profiles associated with pediatric relapsed AML.

Development of relapse remains a problem for further improvements in the survival of pediatric AML patients. While virtually all patients show a good response to initial treatment, more patients respond poorly when treated at relapse. The cellular characteristics of leukemic blast cells that allow survival of initial treatment, relapse development and subsequent resistance to salvage treatment remain largely elusive. Therefore, we studied if leukemic blasts at relapse biologically resemble their initial diagnosis counterparts. We performed microarray gene expression profiling on paired initial and relapse samples of 23 pediatric AML patients. In 11 out of 23 patients, gene expression profiles of initial and corresponding relapse samples end up in different clusters in unsupervised analysis, indicating altered gene expression profiles. In addition, shifts in type I/II mutational status were found in 5 of these 11 patients, while shifts were found in 3 of the remaining 12 patients. Although differentially expressed genes varied between patients, they were commonly related to hematopoietic differentiation, encompassed genes involved in chromatin remodeling and showed associations with similar transcription factors. The top five were CEBPA, GFI1, SATB1, KLF2 and TBP. In conclusion, the leukemic blasts at relapse are biologically different from their diagnosis counterparts. These differences may be exploited for further development of novel treatment strategies.

Clinical relevance of molecular aberrations in paediatric acute myeloid leukaemia at first relapse.

Outcome for relapsed paediatric acute myeloid leukaemia (AML) remains poor. Strong prognostic factors at first relapse are lacking, which hampers optimization of therapy. We assessed the frequency of molecular aberrations (FLT3, NRAS, KRAS, KIT, WT1 and NPM1 genes) at first relapse in a large set (n = 198) of relapsed non-French-American-British M3, non-Down syndrome AML patients that received similar relapse treatment. We correlated molecular aberrations with clinical and biological factors and studied their prognostic relevance. Hotspot mutations in the analysed genes were detected in 92 out of 198 patients (46·5%). In 72 of these 92 patients (78%), molecular aberrations were mutually exclusive for the currently analysed genes. FLT3-internal tandem repeat (ITD) (18% of total group) mutations were most frequent, followed by NRAS (10·2%), KRAS (8%), WT1 (8%), KIT (8%), NPM1 (5%) and FLT3-tyrosine kinase domain (3%) mutations. Presence of a WT1 aberration was an independent risk factor for second relapse (Hazard Ratio [HR] = 2·74, P = 0·013). In patients who achieved second complete remission (70·2%), WT1 and FLT3-ITD aberrations were independent risk factors for poor overall survival (HR = 2·32, P = 0·038 and HR = 1·89, P = 0·045 respectively). These data show that molecular aberrations at first relapse are of prognostic relevance and potentially useful for risk group stratification of paediatric relapsed AML and for identification of patients eligible for personalized treatment.

High-frequency type I/II mutational shifts between diagnosis and relapse are associated with outcome in pediatric AML: implications for personalized medicine.

Although virtually all pediatric patients with acute myeloid leukemia (AML) achieve a complete remission after initial induction therapy, 30%-40% of patients will encounter a relapse and have a dismal prognosis. To prevent relapses, personalized treatment strategies are currently being developed, which target specific molecular aberrations. To determine relevance of established AML type I/II mutations that may serve as therapeutic targets, we assessed frequencies of these mutations and their persistence during disease progression in a large group (n = 69) of paired diagnosis and relapse pediatric AML specimens. In 26 of 42 patients (61%) harboring mutations at either stage of the disease, mutation status changed between diagnosis and relapse, particularly in FLT3, WT1, and RAS genes. Presence or gain of type I/II mutations at relapse was associated with a shorter time to relapse (TTR), whereas absence or loss correlated with longer TTR. Moreover, an adverse outcome was found for patients with activating mutations at relapse, which was statistically significant for FLT3/ITD and WT1 mutations. These findings suggest that mutational shifts affect disease progression. We hence propose that risk stratification, malignant cell detection, and selection of personalized treatment should be based on status of type I/II mutations both at initial diagnosis and during follow-up.

Strategies for the analysis of in vitro radiation sensitivity and prediction of interaction with potential radiation modifying agents.

PURPOSE: To better predict radiation-drug interactions in in vitro model systems, thorough assessment of the effects of in vitro exposure is required. The aim of this article is to show that both clonogenic capacity and cellular proliferation, which represent important different elements of tumour conduct, can be considered when assessing in vitro radio sensitisation. METHODS: A model was designed that can predict radiation-drug interactions based on changes in clonogenic capacity and cell proliferation by radiation modifying agents. RESULTS: Using this mechanistical model, the effect of combined exposure to radiation and potential drugs can be tested on both established cell lines and primary cells. In addition, we could obtain more information about the mechanisms underlying the radiation-drug interaction by assessing the results of in vitro exposure on tumour cell proliferation and clonogenic capacity according to our model. CONCLUSIONS: The significance of our model is not to replace the clonogenic gold standard but to give additional information about the radiation-drug combination by determining cell proliferation. Moreover, the advantage is that the interaction can also be predicted in cases where a clonogenic assay is not possible. Additional research into the biological effect of potential radio-sensitisers is warranted for future (pre)clinical studies.