ALBERT

Description: Acute myeloid leukemia (AML) has a different clinical and biologic behavior in patients at older age. To gain further insight into the molecular differences, we examined a cohort of 525 adults to compare gene expression profiles of the one-third of youngest cases (n = 175; median age 31 years) with the one-third of oldest cases (n = 175; median age 59 years). This analysis revealed that 477 probe sets were up-regulated and 492 probe sets were down-regulated with increasing age at the significance level of P 〈 .00001. After validation with 2 independent AML cohorts, the 969 differentially regulated probe sets on aging could be pointed to 41 probe sets, including the tumor-suppressor gene CDKN2A (encoding p16INK4A). In contrast to the induced p16INK4A expression that is associated with physiologic aging, p16INK4A is down-regulated in AML samples of patients with increasing age. However, this was only noticed in the intermediate- and unfavorable-risk group and not in the favorable-risk group and the molecularly defined subset “NPM1 mutant without FLT3-ITD.” Multivariate analysis revealed p16INK4A, besides cytogenetic risk groups, as an independent prognostic parameter for overall survival in older patients. We conclude that, in addition to altered clinical and biologic characteristics, AML presenting at older age shows different gene expression profiles.

Description: Despite considerable progress has been made in elucidating the new diagnostic and prognostic markers over the past decades, acute myeloid leukemia (AML) remains a life threating malignancy in children. Previous studies defined AML patients into three prognostic groups (adverse, favorable and intermediate) based on cytogenetics. However, kinomics based signaling pathways have not been established based on these prognostic classes. This study addresses the kinomic profiling of these three prognostic groups to identify signaling pathways which could be novel druggable targets. Primary blood and bone marrow samples of 93 pediatric AML patients (adverse 15%, favorable 20% and intermediate 65%) were included in this study. PepChipTMKinomics microarray system was used to study the kinome profile of AML patients. Pairwise comparisons among three prognostic groups on 992 target peptides from the kinome array reported 118 significantly different phosphorylated peptides (P

Description: Introduction: Philadelphia-positive chronic myelogenous leukemia (CML) is a rare disease in children and constitutes approximately 3-5% of all childhood leukemias. With tyrosine kinase inhibitors (TKI), the frequency of accelerated phase (AP) or blast crisis (BC) is remarkably reduced, estimated to 1% to 1.5% per year in adults compared with more than 20% per year in the pre-TKI era. But no data are available among children. Purpose: We described the characteristics, the treatment and the outcome of 21 children with CML, who evolved in accelerated phase and/or blast crisis under TKI. Results: From 2001 to april 2015, 415 European patients were enrolled in the CML pediatric database. Twenty-one patients (5.1%), in chronic phase (CP) treated by TKI, presented AP or BC. The median age of AP/BC cohort was 13.2 years (range: 4.5-16.9 years) with a sex ratio M/F at 2. At CML diagnosis, 15 patients (71%) had high risk Sokal Score with a median score of 1,4 (range: 0,16-2,4). All patients harbored t(9;22)(q34;q11) but one had a complex translocation t(1;9;22)(q12;q34;q11) and another one presented additional inv(3)(q21q26). Imatinib was the first line TKI for all patients. Before AP or BC, only five patients (24%) obtained a complete cytogenetic response (CCyR) and three achieved MMR. For incomplete molecular response or progression to accelerated phase, 8 patients (38%) were switched to dasatinib. Median duration of TKI before AP or BC was 11 months (range: 3 months-56.5 months). Six patients evolved to AP with a median interval of 8.7 months (range: 1 months-24 months), leading to blast crisis for 4 patients with a median time of 3.5 months (range: 0.3-5.4 months). Among the 2 patients remaining in AP, imatinib was increased for one and the other was switched for dasatinib, all before hematopoietic stem cells transplantation (HSCT). One patient died of post-transplant complication and the other one is still alive in complete molecular response without TKI. Nineteen patients presented BC, including 4 after AP. Thirteen patients (62%) presented ALL, five (24%) AML and one a bi phenotypic leukemia. Central nervous system (CNS) was involved for two patients with ALL, one isolated, one combined. At AP or BC, nine patients (43%) presented new additional cytogenetic abnormalities. Eighteen patients with BC were treated according to AML or ALL protocols, combined with second generation TKI for twelve patients. Only one patient underwent preparative regimen, without intensive chemotherapy before HSCT. Ten patients reached complete remission. Four patients died before HSCT, by progressive disease for 2 and by fatal infection for 2. Overall, 15 patients in BC were transplanted. Before HSCT, median molecular response was 0.2% (range: 0-29%) and only four patients had a complete molecular response. After transplant, seven patients received second generation TKI. Four patients died, including three related to transplant toxicity. Thirteen patients were alive, but one with ALL BC relapsed 26 months post-transplant and was waiting for second HSCT. With a median follow-up of 4.4 years, 4-year overall survival was 59% (66% for ALL BC versus 40% for AML BC). Conclusion: Incidence of AP/BC after imatinib for CP CML is at 5%, in the CML pediatric database. Despite second generation TKI, combined with HSCT, outcome remains poor. Post-transplant indication of TKI is heterogenic. Recommendations would be useful for practice. Figure 1. Figure 1. Disclosures Suttorp: Novartis, Bristol Meyer Squib: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Background: Pediatric acute myeloid leukemia (Pedi-AML) accounts for approximately 15% of childhood acute leukemias and the overall survival is 60-70%. Although cytogenetics provides risk stratification, most recurrent genetic events lack agents that specifically target them. Since genetic events are revealed by the expression and activation status of proteins, we hypothesized that we could define recurrent protein expression signatures that could suggest targets for individualized therapies. Methods:Custom Reverse Phase Protein Arrays (RPPA) with 95 Pedi-AML samples and 10 normal CD34+ bone marrow samples were performed and probed with 194 antibodies to determine protein expression levels and activation status. Proteins were divided in protein functional groups (ProFnGrp) to analyze them in the context of other proteins. Progeny clustering was performed to determine the optimal number of protein clusters and principal component analysis was used to visualize the distribution between protein clusters and normal CD34+ cells. Protein networks were generated using known literature associations combined with computational derived edges from the dataset. Associations between clinical features, outcomes and protein clusters were determined. Hierarchical clustering (Figure 1) was performed on a compilation of all protein clusters into one binary matrix to identify recurrent protein expression signatures (PrSIG) that comprised similar combinations of protein constellations(PrCON). A list of proteins that were over or under expressed was made for each signature. Results: For each ProFnGrp 3 to 6 distinct clusters were recognized. All but 2 ProFnGrp (Cell Cycle, Protein Kinase C) had at least one protein cluster that was similar to normal CD34+ samples and all ProFnGrp had clusters specific to the leukemic state. Protein expression levels were overlaid onto the networks and revealed distinct expression and activation states. Hierarchical clustering showed strong co-correlation between protein clusters from various ProFnGrp and suggested 12 PrCON. Patients that expressed similar recurrent combinations of PrCON compiled 8 PrSIG. Heterogeneity was observed for overall survival (OS) and complete remission duration (RD). High OS rates were seen in PrSIG 5 compared to PrSIG 1 (8/12 vs. 3/11 alive, Median NR vs. 54 wks, P=0.09) and high relapse free survival was observed for PrSIG 3 compared to PrSIG 1 (6/6 vs. 3/8 CCR, (Median RD NR vs 39 wks, P=0.04). Rearrangements of 11q23 were overrepresented in PrSIG 2 (7/16, 43.8%) and 7 (5/19, 26.3%) and underrepresented in PrSIG 4 (1/20, 5%) and 6 (0/5, 0%) (P=0.027). Complex karyotype was highly present in PrSIG 8 (4/6, 67%) compared to the overall population (16/95, 16.7%). Hispanic ethnicity was high in PrSig 7 & 8 (53 & 67%) and low in PrSIG 1, 2 4 & 5 (20%, 25%, 5%, 8%). PrSIG membership was also correlated with FAB classification (P=0.025, M0 with PrSIG 4 and M5 with PrSIG 5) as well as with WBC, blast count, HGB and PLT. As we had both Pedi-AML and acute lymphoblastic leukemia (ALL) samples on this RPPA array (see accompanying ALL abstract), this enabled comparison between protein expression levels in both diseases. Combined re-analysis suggested 12 PrSIG and 12 PrCON; most PrSIG and PrCON were dominant for ALL or AML and only 1 PrSIG and 3 PrCON showed overlap between the two diseases. Identification of significantly altered proteins for each PrSIG can be used to select targeted therapies for combination. For example, for PrSIG1 combined inhibition of RPTOR and RICTOR may be useful as both are overexpressed. Conclusion: As hypothesized we demonstrated the existence of protein expression signatures in Pedi-AML based on strong correlations between different protein clusters. Signatures were correlated with therapeutic outcome, as well as with cytogenetics, Hispanic ethnicity, and laboratory features. Recognition of altered proteins within the signatures suggested combinations of targets that could facilitate personalized therapy. Shared protein constellations between AML and ALL indicate joint protein deregulations that could be targetable in both diseases. Figure 1. Hierarchical clustering shows recurrent patterns of 12 PrCON (y-axis) that form 8 recurrent PrSIG (x-axis). The association with Hispanic ethnicity is shown. Figure 1. Hierarchical clustering shows recurrent patterns of 12 PrCON (y-axis) that form 8 recurrent PrSIG (x-axis). The association with Hispanic ethnicity is shown. Disclosures No relevant conflicts of interest to declare.

Description: The proteasome degrades unneeded and damaged proteins. Tumor cells highly depend on increased protein production and their degradation suggesting that malignant cells with a high proliferation index will be more sensitive to proteasome inhibition. The addition of the proteasome inhibitor Bortezomib (Velcade, 'BTZ') to standard pediatric AML chemotherapy (cytarabine, daunorubicin and etoposide, 'ADE') depleted leukemia-initiating cells in a phase 2 clinical trial in pediatric AML (pedi-AML) patients. A randomized phase 3 clinical trial was then conducted by the Children's Oncology Group (COG) comparing ADE and ADE+BTZ treatment regimes in pedi-AML (AAML1031). To determine if there were specific protein expression profiles that correlated with response to BTZ-containing chemotherapy, we analyzed key components of the proteome of pedi-AML that participated in the trial using reverse phase protein arrays (RPPA). RPPA-based expression of 293 validated antibodies was tested in 500 leukemia samples and compared to expression in CD34+ samples from healthy individuals (n=30). Among all proteins, FOXO3 expression was identified as the protein with the highest influence on outcome in the ADE group. The expression of FOXO3 was prognostic for event-free survival (EFS) in both univariate (HR = 0.56, 95% CI = 0.34-0.90, P = 0.017) and multivariate (HR = 0.55, 95% CI = 0.34-0.88, P = 0.013) analysis. All patients were divided into two clusters (low and high) based on their FOXO3 expression level using median FOXO3 expression in normal CD34+. Kaplan-Meier survival analysis showed poor OS (3 year OS 65.3% vs. 73.9%, P = 0.03) and EFS (3 year EFS, 42.8% vs. 55%, P = 0.01) in low FOXO3 expressors (n=119) compared to patients with high FOXO3 expression (n=291) (fig. 1A). Notably, the poor prognostic effect of low FOXO3 for OS was seen in ADE (3 year OS 60% vs. 72.3%, P = 0.03), but not in ADE-BTZ (3 year OS 70.3% vs. 75.3%, P = 0.23) (fig. 1B). This suggests that in particular patients with low FOXO3 may be eligible candidates for BTZ-addition. To validate our findings, we performed knockdown (KD) of FOXO3 using a short hairpin approach in OCI-AML3 (p53WT) and THP-1 (p53null) cell lines. KD FOXO3 in OCI-AML3 had a short-term growth advantage compared to controls (Day 4, P = 0.004), but not KD FOXO3 THP-1 cells suggesting a role for p53 in the FOXO3 functional pathway. KD FOXO3 cells were more resistant to doxorubicin and etoposide combination therapy than controls (P = 0.04), confirming our clinical observations. Since therapeutic regimes in AML are currently shifting towards Bcl-2 inhibition by Venetoclax (ABT-199, 'ABT'), we were eager to test whether BTZ and ABT could act in synergy, and if this is related to FOXO3 expression. Single low dose BTZ and ABT did not reduce cell numbers after 3 days, but were effective when used in combination (

Description: Background: Heat shock factor 1 (HSF1) is a key-component of the heat shock response and plays a major role in cancer biology. The heat shock response is a highly conserved mechanism that is important for cell survival under stressful conditions. It facilitates normal protein folding and guards the proteome from misfolding and aggregation. Bortezomib (BTZ) is a reversible proteasome inhibitor and was recently tested in a phase 3 clinical trial for patients with de novo pediatric acute myeloid leukemia (pedi-AML). This trial compared standard therapy (cytarabine/daunorubicin/etoposide (ADE)) to ADE plus BTZ (ADE+B). As HSF1 and BTZ act in opposing circumstances, our goal was to globally assess the phosphorylation of HSF1 at serine 326 (HSF1.pS326) and to determine if baseline HSF1.pS326 was prognostic of clinical response to ADE+B and if we could identify a subset of patients that benefitted from ADE+B. Methods: Reverse phase protein array (RPPA) probed with 222 total antibodies and 69 specific post-translationally modified proteins was used to determine the protein expression levels in 505 bulk leukemic pedi-AML samples compared to 20 CD34+ samples from healthy pediatric donors. Patients participated in the Children's Oncology Group (COG) AAML1031 Phase 3 clinical trial that compared ADE to ADE+B. Survival curves were generated using the Kaplan-Meier method. Results: HSF1.pS326 protein expression levels in the pedi-AML patient samples. Based on the HSF1.pS326 expression levels, patients were classified into two clusters; high HSF1 326 (n = 216) and low HSF1.326 (n = 289). The median of the normal CD34+ samples was used as cut-off point. Expression of HSF1.pS326 was not prognostic for outcome in patients treated with ADE (event-free survival (EFS), p = 0.51) (Fig. 1A). In patients that were treated with ADE+B however, HSF1.pS326 levels were highly prognostic, with low HSF1.pS326 associated with a better EFS (p = 0.009) (Fig. 1B). Patients with high HSF1.pS326 did not benefit from BTZ addition (EFS, p = 0.77) (Fig. 1C), whereas patients with low HSF1.pS326 levels significantly improved (EFS, p = 0.004) (Fig. 1D). Between the two patient clusters, the NPM1 mutation state was higher in patients with high HSF1.pS326 levels (p = 0.042), and the CEBPA mutation state was higher in patients with low HSF1.pS326 (p = 0.011). No other patient or disease characteristics were different between the two clusters. To functionally validate our findings, we overexpressed wild type or mutant HSF1 in 293T cells and assessed its effect on BTZ sensitivity. The mutant HSF1 constructs contained amino acid substitutions at Ser326, resulting in either a phospho-mimic HSF1 (S326E), or a phospho-dead HSF1 protein (S326A). Cell viability analysis showed clear resistance to BTZ in cells with increased phosphorylation (HSF1 S326E) (Fig. 1E - blue curve). Cells that expressed reduced phosphorylation of HSF1 (HSF1 S326A), however were highly sensitive to BTZ (Fig. 1E, red curve). This confirms our observations from the pedi-AML patients. Conclusions: Using a proteomics approach, we identified a subgroup of pedi-AML patients that benefitted from BTZ addition to ADE therapy. We hypothesize that patients with low HSF1 activation are more susceptible to protein cell stress, and protein cell stress susceptibility is amplified by the addition of BTZ with ADE. The use of HSF1.pS326 as potential biomarker could identify patients that would benefit from ADE+B therapy. Patients with high HSF1 could potentially benefit from ADE+B in combination with a HSF1 (phospho) inhibitor, suggesting a novel combinational therapy. Figure 1. Kaplan Meier survival curves for EFS based on HSF1.pS326 expression. A. HSF1.pS326 did not affect outcome in ADE treated patients (high HSF1.PS326 shown in red, low HSF1.pS326 in blue). B. HSF1.pS326 was highly prognostic for EFS ADE+B. C. Patients with high HSF1.pS326 did not benefit from BTZ (ADE shown in blue, ADE+B in green). D. Patients that expressed low HSF1.pS326 did significantly better after ADE+B. E. To assess the effect of high and low HSF1.pS326 on BTZ sensitivity, 293T cells were transfected with two HSF1.pS326 mutants that mimicked high (blue) and low (red) HSF1.pS326 patients, in addition to wild type HSF1 (orange) and an empty vector as control (green). Cell viability assays showed resistance to BTZ in the phospho-mimic (blue) mutant, whereas the phospho-dead (red) mutant was sensitive to BTZ. Figure 1. Figure 1. Disclosures Kolb: Roche- Genentech: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees.