Failure Of Induction Chemotherapy Research Articles

Dysregulated arginine metabolism in precursor B-cell acute lymphoblastic leukemia in children: a metabolomic study

BackgroundPrecursor B-cell acute lymphoblastic leukemia (B-ALL) is the most common cancers in children. Failure of induction chemotherapy is a major factor leading to relapse and death in children with B-ALL. Given the importance of altered metabolites in the carcinogenesis of pediatric B-ALL, studying the metabolic profile of children with B-ALL during induction chemotherapy and in different minimal residual disease (MRD) status may contribute to the management of pediatric B-ALL.MethodsWe collected paired peripheral blood plasma samples from children with B-ALL at pre- and post-induction chemotherapy and analyzed the metabolomic profiling of these samples by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). Healthy children were included as controls. We selected metabolites that were depleted in pediatric B-ALL and analyzed the concentrations in pediatric B-ALL samples. In vitro, we study the effects of the selected metabolites on the viability of ALL cell lines and the sensitivity to conventional chemotherapeutic agents in ALL cell lines.ResultsForty-four metabolites were identified with different levels between groups. KEGG pathway enrichment analyses revealed that dysregulated linoleic acid (LA) metabolism and arginine (Arg) biosynthesis were closely associated with pediatric B-ALL. We confirmed that LA and Arg were decreased in pediatric B-ALL samples. The treatment of LA and Arg inhibited the viability of NALM-6 and RS4;11 cells in a dose-dependent manner, respectively. Moreover, Arg increased the sensitivity of B-ALL cells to L-asparaginase and daunorubicin.ConclusionArginine increases the sensitivity of B-ALL cells to the conventional chemotherapeutic drugs L-asparaginase and daunorubicin. This may represent a promising therapeutic approach.

The Kinetics of FLT3L during Induction Chemotherapy for Pediatric Acute Lymphoblastic Leukemia Suggests a Novel Clinical Diagnostic and Treatment Strategy

Acute lymphoblastic leukemia (ALL), one of the most common malignancies, has been reported to account for nearly one-third of all pediatric cancers. Failure of induction chemotherapy is the leading cause of relapse and mortality in pediatric patients treated for ALL. Monitoring the therapeutic efficacy of induction chemotherapy and improving its success rate are critical to the clinical management of ALL. FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is expressed on progenitor cells and ALL blasts. FLT3 ligand (FLT3L) is detectable during homeostasis and increases in hypoplasia due to genetic defects or treatment with cytoreductive agents. Here, we continuously monitored changes in FLT3L levels during induction chemotherapy in children with ALL to provide a more sensitive assessment of MRD, similar to NGS. The study included 54 children with ALL whose peripheral blood FLT3L levels were measured by ELISA weekly during induction chemotherapy. Weekly sampling revealed a significant difference in the kinetics of FLT3L response between children in complete remission (CR) and partial remission (PR), at day 14 of treatment (CR:519±10pg/ml vs PR:54±10pg/ml, P＜0.001). Furthermore, the achievement of CR in these patients was associated with higher FLT3L (150±10pg/ml vs 32±10pg/ml, P＜0.05) at day 28. Clinical prediction models show that combining minimal residual disease (MRD) status with FLT3L measurements better predicts whether patients will achieve CR after induction chemotherapy. Moreover, FLT3L levels were positively correlated with DC and T cells and negatively correlated with WBC, PLT and LDH during induction chemotherapy in children with ALL. Therefore, we hypothesized that higher levels of FLT3L might improve antitumor immunity by activating DC and T cells. However, further research is needed. In conclusion, measurement of FLT3L in children with ALL during induction may provide added value in assessing early treatment response to chemotherapy, the impact on long term outcome needs further study.

High WT1 expression predicted induction chemotherapy failure in acute myeloid leukemia patients with non-favorable cytogenetic risk.

The prognostic significance of WT1 expression at diagnosis in acute myeloid leukemia (AML) remains obscure, and subgroup analysis is the way for clarification. We previously reported the results in t(8;21) AML. In this study, 437 consecutive adult AML patients with non-favorable cytogenetic risk were enrolled. All patients were tested WT1 transcript levels using real-time quantitative PCR at diagnosis; AML-related common fusion genes, KMT2A-PTD, FLT3-ITD, NPM1, CEBPA and TP53 mutations were simultaneously tested. 92.4% of patients overexpressed WT1 compared to normal bone marrow. The existence of FLT3-ITD, NPM1 mutation and the absence of CEBPA biallelic mutation were significantly related to higher WT1 expression. The cutoff value for WT1 was determined by performing receiver operating characteristic curve analysis in regard to complete remission (CR) achievement and was used to categorize patients into low-expression (WT1-L) and high-expression (WT1-H) groups. In the entire cohort, WT1-H was significantly associated with a lower 1-course and 2-course CR rate (P < 0.0010 and P = 0.0060) but was not related to relapse-free survival (RFS). Multivariate analysis showed that WT1-H was an independent adverse prognostic factor for both 1-course and 2-course CR achievement. Subgroup analysis was further performed. WT1-H had a significant adverse impact on CR achievement within intermediate-cytogenetic risk, high-cytogenetic risk, ELN-defined-intermediate-risk, normal karyotype, KMT2A rearrangement, FAB-M2, FAB-M5 and NPM1 mutation (+) subgroups, whereas it had no impact within ELN-defined-low-risk, ELN-defined-high-risk, FAB-M4, FLT3-ITD mutation (+) and CEBPA biallelic mutation (+) subgroups. Moreover, WT1-H patients had a significantly lower RFS rate than WT1-L patients within both FAB-M5 and KMT2A rearrangement subgroups (P = 0.010 and 0.028), whereas WT1 had no impact on RFS within other subgroups mentioned above (all P > 0.05). Therefore, high WT1 expression at diagnosis independently predicted induction chemotherapy failure in AML patients with non-favorable cytogenetic risk, and it was related to relapse just within FAB-M5 and KMT2A rearrangement subgroups.

Abstract 2870: Genetic mechanisms of primary chemotherapy resistance in pediatric acute myeloid leukemia

Abstract Survival for children with acute myeloid leukemia (AML) remains low, with a primary induction remission failure rate of 10-15%. Although some mutations have been identified that are associated with increased relapse risk, the genomic landscape of pediatric AML resistant to primary chemotherapy is not well characterized. As part of the TARGET initiative (Therapeutically Applicable Research To Generate Effective Treatments), we assembled a cohort of 28 pediatric AML patients with primary chemotherapy resistance and failure of induction chemotherapy, uniformly treated as part of the COG AAML0531 study. We analyzed whole-genome DNA, mRNA, and miRNA sequence data obtained at diagnosis and after induction chemotherapy. At least three genetically distinct groups were apparent in this cohort, including those with NUP98 rearrangements, somatic mutations of WT1 in the absence of NUP98 variants, and additional recurrent variants including those in KMT2C and MLLT10. Comparisons of mutations before and after chemotherapy revealed both common and distinct gene expression programs. On chemotherapy exposure, these leukemias exhibited diverse forms of clonal evolution. We observed selection for mutant alleles of FRMD8, DHX32, PIK3R1, SHANK3, MKLN1, as well as persistence of WT1 and TP53 mutant clones, and elimination or contraction of FLT3, PTPN11, and NRAS mutant clones. These findings suggest diverse genetic mechanisms for primary chemotherapy resistance in pediatric AML, which should guide new approaches for its diagnosis and therapy. Citation Format: Nicole McNeer, John Philip, Heather Geiger, Rhonda E. Ries, Vincent-Philippe Lavallee, Michael Walsh, Minita Shah, Kanika Arora, Anne-Katrin Emde, Nicolas Robine, Todd A. Alonzo, E. Anders Kolb, Alan S. Gamis, Malcom Smith, Daniela Se Gerhard, Jaime Guidry-Auvil, Soheil Meshinchi, Alex Kentsis. Genetic mechanisms of primary chemotherapy resistance in pediatric acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2870.

Genetic mechanisms of primary chemotherapy resistance in pediatric acute myeloid leukemia.

Acute myeloid leukemias (AML) are characterized by mutations of tumor suppressor and oncogenes, involving distinct genes in adults and children. While certain mutations have been associated with the increased risk of AML relapse, the genomic landscape of primary chemotherapy resistant AML is not well defined. As part of the TARGET initiative, we performed whole-genome DNA and transcriptome RNA and miRNA sequencing analysis of pediatric AML with failure of induction chemotherapy. We identified at least three genetic groups of patients with induction failure, including those with NUP98 rearrangements, somatic mutations of WT1 in the absence of apparent NUP98 mutations, and additional recurrent variants including those in KMT2C and MLLT10. Comparison of specimens before and after chemotherapy revealed distinct and invariant gene expression programs. While exhibiting overt therapy resistance, these leukemias nonetheless showed diverse forms of clonal evolution upon chemotherapy exposure. This included selection for mutant alleles of FRMD8, DHX32, PIK3R1, SHANK3, MKLN1, as well as persistence of WT1 and TP53 mutant clones, and elimination of FLT3, PTPN11, and NRAS mutant clones. These findings delineate genetic mechanisms of primary chemotherapy resistance in pediatric AML, which should inform improved approaches for its diagnosis and therapy.

Effect of High VEGF-C mRNA Expression on Achievement of Complete Remission in Adult Acute Myeloid Leukemia

Although vascular endothelial growth factor-C (VEGF-C) is known to be expressed in acute myeloid leukemia (AML) blasts, the relevance of VEGF-C in the clinical setting remains to be fully explored. We examined the effect of VEGF-C on achievement of complete remission (CR) in adult de novo AML and immune cell population profiles according to VEGF-C mRNA expression. In comparison of VEGF-C expression between the no-CR and CR groups, the CR group showed a trend toward higher levels of plasma VEGF-C (P = .088), whereas mRNA expression of VEGF-C was downregulated (P = .008). Next, patients with continuous data for VEGF-C were divided into two groups (low vs. high) by a ROC curve analysis. The low- versus high-level groups for plasma VEGF-C (RR of 0.20, P = .030), mRNA expression of VEGF-C (RR of 18.75, P = .003), and the ratio of plasma level to mRNA expression (RR of 0.05, P = .007) were potential predictors of CR on univariate analysis. After adjusting for potential clinical factors including genetic group, multivariate analyses revealed that high VEGF-C mRNA expression was an independent risk factor for failure of induction chemotherapy. Furthermore, patients with high VEGF-C mRNA expression had a lower frequency of NKT and CD8+ cells and showed a trend for a lower frequency of NK cells. These results suggest that interruption of VEGF-C signaling might be a potential therapeutic target for antileukemic treatment in AML patients.

Ratio of Primary Bone Marrow Cells after Induction Chemothrapy for Two Weeks in the Patients with Ph- ALL and Its Influence on Complete Remission and Overall Progrosis

To investigate the influence of bone marrow blasts ratio after induction chemotherapy for 2 weeks in patients with Ph- ALL, and it's influence on complete remission (CR) and overall prognosis. A total of 172 patients with Ph- ALL in our hospital from March 2012 to February 2016 were selected. The bone marrow blast ratio was analyzed by the receiver-operating characteristic curve (ROC) in patients after induction chemotherapy for 2 weeks, at same time its influence on CR and overall prognosis of Ph- ALL patients was evaluated. The cutoff value of CR was 0.075, its area under ROC was 0.763; the comparison of area under ROC with Az=0.5 showed statistically significant difference, therefore 172 patients with Ph- ALL were grouped according to bone marrow blast ratio after induction chemotherapy for 2 weeks: 104 cases (60.5%) with bone marrow blast ratio <0.075, 68 cases (39.5%) with bone marrow blast ratio ≥0.075. The Ph- ALL patinets with bone marrow blast ratio <0.075 who achieved CR and finally achieved CR after induction chemotherapy for 4 weeks acconnted for 89 (85.6%) and 99(95.2%) respectively, which were significantly higher than those in Ph- ALL patients with bone marrow blast ratio≥0.075, [29(42.6%) and 52 (76.5%)](P<0.05). In addition, the influencing factor clinically reducing the OS and DFS rate of patients and enhancing the ralapse rate of patients were mainly chemotherapy, the failure of induction chemotherapy (patients did not achieve CR after induction therapy for 4 weeks), the bone marrow blast ratio≥0.075 after induction treatment for 2 weeks, and CNSL at diagnosis and so on, while the enhaced WBC count at diagnosis was poor factor affecting the DFS rate of patients. After induction chemotherapy for 2 weeks, the elevated bone marrow blast ratio in Ph- ALL patients will be infavourable to CR, and the overall prognosis is poor.

Outcome of Elderly Patients after Failure to Hypomethylating Agents Given As Frontline Therapy for Acute Myeloid Leukemia (AML): Single Institution Experience

Background: AML outcome in elderly patients unfit for intensive chemotherapy continues to be challenging. Hypomethylating agents (HMA) can be effective in these patients but responses are short-lived (Fenaux et al, JCO 2009; Dombret et al Blood 2015). Even though HMAs achieve superior response rates to supportive care alone, complete remissions (CR) are rarely achieved and often quickly lost when they occur. The majority of patients will either have stable disease or progress through therapy. We hereby describe the outcome of these patients at our institution after they fail HMAs.Methods: A total of 56 AML patients' data from Feb 2007 to June 2015 at Mayo Clinic were reviewed after appropriate IRB approval was obtained. All cases had their bone marrow slides reviewed at our institution. Patients were considered for our study if they received HMA as frontline therapy for their AML. Patients who received HMAs after induction chemotherapy failure or for their MDS were excluded. Response was identified based on IWG AML criteria (Cheson et al, JCO 2003). Prognostic factors were analyzed by univariate and multivariate analyses. Survival estimates were calculated using Kaplan-Meier curves and univariate and multivariate analyses was based on log-rank testing using JMP software version 10.Results:i)Characteristics at Diagnosis: We identified 56 AML patients treated with HMA as frontline therapy with median age of 76 years (range, 59-91), 68% of which were males. Cases were classified as AML with myelodysplasia related changes in 25%, and therapy related myeloid neoplasm in 2%, and not otherwise specific in 73%. Out of 56 cases, 23% were secondary to MDS, 2% ET (Essential Thrombocytopenia), 5% PV (Polycythemia Vera), 5% MF (Myelofibrosis), 4% from CMML (Chronic Myelomonocytic Leukemia); while 61% were de novo AML. Per cytogenetics data, 2% were considered favorable risk, 62% intermediate, and 36% poor (34% had complex karyotype).ii)Clinical outcome to Frontline HMA: Out of 56 patients, 15(27%) patients received azacitidine (AZA) and 41 (73%) received decitabine (DAC) with a median number of cycles of 4 (range 1-62). Median number of cycles was higher for AZA vs DAC (8 vs 4, p =0.3). CR was found in 10 (18%), CR with incomplete count recovery in 3 (5%), partial remission (PR) in 3 (5%) patients, with overall response of 28% and median response duration of 10 months (range, 1-78 months). Thirteen (81%) out of 16 responders relapsed. Therefore, only 53 patients were included in primary or secondary failure analysis.iii)Clinical outcome to Frontline HMA failure: Out of 53 patients, 12 (23%) received subsequent treatments. Out of the 12 patients, 3 (25%) received another HMA (switch), 2 (17%) subcutaneous low dose cytarabine, 4 (33%) intensive chemotherapy, while 3 (25%) received either therapy on a clinical trial or targeted therapy. None of the 12 patients who got first salvage therapy achieved remission. Five out of the 12 patients received second salvage therapy, 2 (40%) of which achieved CR. Only one patient had third salvage therapy and did not respond. No patient was able to proceed to allogenic hematopoietic cell transplantation. Median overall survival (mOS) for this group was 2 months. Median OS for patients who received subsequent salvage therapies was better than those who did not receive any subsequent therapy after failing HMA (9.5 vs 2 months, p = 0.0009).Conclusions: Elderly patients diagnosed with AML who are unfit for intensive chemotherapy have a poor prognosis. HMA can get some patients into remission but most patients will ultimately fail. Outcome for patients who have primary or secondary (relapse) failure is very poor with a median OS of 2 months. Switch of HMA did not result in any response. Response to salvage therapy continues to be low but offering salvage therapies seem to improve OS. Our study provides historical data for future novel therapies, which are sorely needed for these patients. [Display omitted] DisclosuresAl-Kali:Celgene: Research Funding; Onconova Therapeutics, Inc.: Research Funding.

Deficiency of Current Acute Myeloid Leukemia (AML) Response Criteria to Predict Response to Hypomethylating Agent Therapy: The Value of Long-Lasting Stable Disease

Background: International Working Group (IWG) has set criteria in 2003 for response assessment in patients with AML; at which time intensive chemotherapy was the main therapy (Cheson, JCO 2003). Hypomethylating agents (HMA) were approved shortly after for the treatment of elderly AML (Fenaux, JCO 2009; Dombret et al Blood 2015). Although few patients achieve a deep response, the majority will have stable disease (SD), some of which could last several months while patients continue to receive HMA and gain clinical benefit. We hereby describe outcomes for patients who achieve SD with HMA for the treatment of elderly unfit AML patients.Methods: A total of 56 AML patients’ data from Feb 2007 to June 2015 at Mayo Clinic were reviewed after appropriate IRB approval was obtained. All cases had their bone marrow slides reviewed at our institution. Patients were considered for our study if they received HMA as frontline therapy for their AML. Patients who received HMAs after induction chemotherapy failure or for their MDS were excluded. Response was identified based on 2003 IWG AML response criteria. Patients with SD were categorized into short-lasting (<6 cycles of HMA, cohort 1) or long-lasting (≥6 cycles of HMA, cohort 2) SD.Prognostic factors were analyzed by univariate and multivariate analyses. Survival estimates were calculated using Kaplan-Meier curves and univariate and multivariate analyses was based on log-rank testing using JMP software version 10.Results:i)Characteristics: Fifty six AML patients were treated with HMA as frontline therapy with median age of 76 years (range, 59-91), 68% of which were males. Therapy related AML (T-AML) was seen in 2% of the cases. Cytogenetics were favorable risk in 2%, intermediate in 62%, and poor in 36%. Complete remission (CR) was found in 10 (18%) patients, CR with incomplete count recovery in 3 (5%), partial remission (PR) in 3 (5%) patients. Forty patients were non-responders (SD).ii)Characteristics of HMA SD cohorts: Upon comparison of cohort 1 versus cohort 2 there was no statistical significance in median age, white blood count, absolute neutrophil count, platelets, peripheral blood and bone marrow blasts, bone marrow cellularity and sex. Median number of cycles was expectedly higher in cohort 2 vs 1 (9 vs 2, p<0.0001). Intermediate cytogenetic risk was more common in cohort 2 vs 1 (90% vs 43%, p=0.006), while poor risk was more common in cohort 1 vs 2 (57% vs 10%, p=0.006). Only 10% had complex karyotype in cohort 2 compared to 50% in cohort 1 (p=0.017). NPM1 mutation was absent in all cases, while FLT3 mutation was present 30% in cohort 1 and 25% in cohort 2 (p = 0.9). Decitabine was more commonly given in cohort 1 vs cohort 2 (80% vs 50%, p =0.07).iii)Clinical outcome of HMA SD cohorts: Median overall survival was higher in long-lasting SD patients (cohort 2) vs short-lasting SD (18 vs 4 months, p = 0.0002). Upon multivariate analysis, both age (p <0.0001) and long-lasting SD (p =0.01) were statistically significant for overall survival but not cytogenetics risk grouping nor type of HMA administered.Conclusions: Standard response criteria for AML by IWG were developed for intensive chemotherapy and do not apply to newer agents, specifically HMA. Stable disease (SD) could be meaningful to some elderly AML patients when treated with HMA. Intermediate cytogenetics and azacitidine use were more frequent in patients with long-lasting SD. Long-lasting SD did confer a survival benefit in elderly AML patients. Newer response criteria for assessing novel agents' efficacy are needed in this population. [Display omitted] DisclosuresTibes:Italfarmaco S.p.A.: Consultancy, Honoraria. Foran:pfizer: Honoraria; Millennium Pharmaceuticals, Inc.: Research Funding; medscape: Honoraria; karyopharm: Honoraria; novartis: Honoraria; boehringer ingelheim: Research Funding; agios: Research Funding; Cellerant: Research Funding. Al-Kali:Novartis: Research Funding; Celgene: Research Funding.

Genomic and Proteomic Analysis of Primary Chemoresistance and Induction Failure in Acute Myeloid Leukemia

Despite intense efforts, the cure rates of children and adults with AML remain unsatisfactory. Resistance to cytotoxic chemotherapy is the dominant cause of treatment failure. To investigate the molecular basis of primary chemoresistance, we used targeted gene sequencing using the Foundation One Heme platform to profile 405 genes in DNA and 265 genes in RNA known to be somatically mutated in hematologic malignancies in 107 primary specimens obtained at diagnosis from children and adults with cytogenetically normal AML. Comparative analysis revealed mutations of SETBP1 and ASXL1 to be enriched in patients with failure of induction chemotherapy as compared to those who achieved remission (8 out of 50 versus 1 out of 57, p = 0.01). However, the low prevalence of these alterations indicated that other genomic or functional mechanisms must mediate chemoresistance. To identify these mechanisms, we mapped kinase signaling cascades activated in chemoresistant AML cells using functional mass spectrometry proteomics. Phosphopeptides were enriched in primary leukemic blasts isolated from cytogenetically normal AML patients, with relative abundances in each patient determined from iTRAQ 8-plex reporter ions in the associated MS/MS fragment ion spectra. This analysis identified phosphorylation of the leukemogenic transcription factor MEF2C at S222 in the majority of cases. We found MEF2C to be overexpressed in cytogenetically normal AML, as well as MLL-rearranged leukemias, and to be associated with increased risk of relapse (Laszlo et al, ASH submitted). Using reporter assays, we found that phosphorylation of MEF2C S222 was both necessary and sufficient to fully transactivate MEF2C promoter elements (Fig. 1). Expression of mutant S222A MEF2C that cannot be phosphorylated, but not phosphomimetic S222D, induced mitochondrial apoptosis in human AML and genetically-engineered MLL-AF9 mouse leukemia but not in normal mouse hematopoietic progenitor cells. In syngeneic mouse transplant models, we found that MEF2C phosphorylation was required for the maintenance of MLL-AF9 leukemias in vivo. Transcriptome and proteome analysis of gene expression changes and composition of MEF2C transcriptional complexes induced by phosphorylation mutants showed that aberrant leukemia cell survival is caused at least in part by alterations in the expression of apoptotic regulators. Using a recombinant kinase screen, we identified MARK kinases as specific enzymes that phosphorylate MEF2C S222. Treatment of primary patient specimens and human AML cell lines with the MARK kinase inhibitor MRT199665 induced MEF2C downregulation and apoptosis in cells with MEF2C but not those lacking MEF2C expression (Fig. 2). Thus, aberrant MEF2C phosphorylation is associated with primary chemoresistance and induction failure, is required for enhanced leukemia cell survival, and confers susceptibility to MARK inhibition therapy. In addition, genome and proteome profiles should provide additional biomarkers and targeted therapeutic strategies to overcome chemoresistance in AML. [Display omitted] [Display omitted] DisclosuresHe:Foundation Medicine, Inc.: Employment, Equity Ownership. Balasubramanian:Foundation Medicine, Inc.: Employment. Zhong:Foundation Medicine, Inc.: Employment, Equity Ownership. Pavlick:Foundation Medicine, Inc.: Employment. Yilmazel:Foundation Medicine, Inc.: Employment. Stone:Merck: Consultancy; AROG: Consultancy; Abbvie: Consultancy; Juno: Consultancy; Amgen: Consultancy; Celator: Consultancy; Agios: Consultancy; Karyopharm: Consultancy; Pfizer: Consultancy; Roche/Genetech: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Celgene: Consultancy; Novartis: Research Funding. Byrd:Acerta Pharma BV: Research Funding. Levine:CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees. Armstrong:Epizyme, Inc: Consultancy.

KDM7A Is Targeted By MiR-155 in Acute Myeloid Leukemia and Impacts Differentiation

Acute myeloid leukemia (AML) is a heterogeneous disease that develops secondary to the acquisition of mutations that disrupt cell differentiation, proliferation and survival. MicroRNAs (miRNAs or miRs) are short non-coding RNA molecules that modulate post-transcriptional gene expression by either cleaving or repressing translation of target mRNA transcripts. Differential expression of miRNAs has been identified in AML and noted to correlate with specific disease characteristics, cytogenetic abnormalities and prognosis.MiR-155 expression is upregulated in both adult and pediatric patients with cytogenetically normal AML (CN-AML) and correlates with adverse clinical outcomes. Specifically, we have shown that high miR-155 expression is associated with an increased incidence of induction chemotherapy failure and inferior overall and event free survival. However, how miR-155 up-regulation contributes mechanistically to adverse clinical outcomes is poorly understood.In prior work, we correlated the expression of predicted or validated miR-155 target genes with miR-155 expression in a gene expression profiling (GEP) dataset of pediatric AML samples. We identified 22 candidates with inversely correlated expression by GEP for further validation in diagnostic bone marrow specimens from children with the highest miR-155 expression levels (n=9) vs. children with the lowest miR-155 expression levels (n=9). Although the expression of miR-155 inversely correlated with 9 target genes, only expression of the putative target KDM7A demonstrated a statistically significant difference in expression between low and high miR-155 expressing cases (p = 0.03).KDM7A is a lysine-specific histone demethylase enzyme that may play a role in regulating differentiation by impacting transcriptional elongation. Computational software programs, i.e. TargetScan, identified two predicted miR-155 binding sites in the KDM7A 3'UTR. To evaluate whether miR-155 directly binds to the KDM7A 3'UTR, we cloned two regions of the KDM7A 3'UTR containing predicted miR-155 binding sequences into luciferase reporter vectors and then mutated the binding sites by site-directed DNA mutagenesis. We validated that both predicted binding sites in KDM7A 3'UTR were direct miR-155 targets using HEK293T cells. Next, we examined the impact of miR-155 overexpression in K562 cells, an acute leukemia cell line that express very low levels of endogenous miR-155, and can be differentiated along the erythroid lineage after hemin exposure. KDM7A RNA expression was decreased 16-fold in miR-155 versus control lentivirally transduced K562 cells as detected by qPCR. KDM7A protein expression was also decreased in miR-155 versus control expressing K562 cells as measured via Western blot. These data demonstrate that KDM7A is a previously uncharacterized target of miR-155.Next, we explored the effect of differential KDM7A expression on cell differentiation, and cell death and apoptosis after exposure to daunorubicin chemotherapy. For this work we used GFP-labeled miR-155 and YFP-labeled KDM7A lentiviral constructs and labeled control constructs. To examine differentiation we used benzidine staining of hemin-exposed K562 cells transduced with empty control vector (ECV), miR-155, KDM7A, or both constructs. The lowest percentage of benzidine staining, consistent with limited erythroid differentiation, was seen in K562 cells with miR-155 overexpression compared to ECV (28.2% vs. 39.8% positive). This effect on blocked erythroid differentiation was fully reversed with overexpression of KDM7A in miR-155 overexpressing cells (41.4% positive). Confirming these observations, we also observed decreased benzidine staining in hemin exposed K562 cells that were transduced with KDM7A shRNA versus control (28.9% versus 42.7%). Together, these data support that KDM7A plays a role in cell differentiation that is in part controlled by miR-155 expression. Preliminary data also support that re-expression of KDM7A in miR-155 overexpressing cells promotes cell death after exposure to daunorubicin. Further work is ongoing. In conclusion, we have identified a new target of miR-155, KDM7A. Our data suggest that KDM7A plays a role in cell differentiation and that decreased KDM7A expression in AML cells that overexpress miR-155 contributes to blocked differentiation, and may also contribute to resistance to chemotherapy. DisclosuresNo relevant conflicts of interest to declare.

Prediction Of Therapeutic Resistance In Adult Acute Myeloid Leukemia: Analysis Of 4,550 Newly Diagnosed Patients From MRC/NCRI, HOVON/SAKK, SWOG, and MD Anderson Cancer Center

BackgroundPrimary failure of induction chemotherapy or disease recurrence after short remission duration (“therapeutic resistance”) remains the principal problem in adult acute myeloid leukemia (AML). Although cytogenetic and molecular abnormalities have proven useful in the identification of subsets of patients with distinct disease risks, it is unclear to what degree therapeutic resistance can be predicted for individual patients. Patients and MethodsWe used information on patients with newly diagnosed AML other than acute promyelocytic leukemia receiving curative-intent treatment on trials conducted by the U.K. Medical Research Council/National Cancer Research Institute (MRC/NCRI; 1988-2010; n=2,615), the Dutch-Belgian Cooperative Trial Group for Hematology/Oncology and the Swiss Group for Clinical Cancer Research (HOVON/SAKK; 1987-2008; n=1,098), the U.S. cooperative group SWOG (1987-2009; n=428), and MD Anderson Cancer Center (2000-2011; n=409). Achievement of a complete remission (CR) with the initial 1-2 courses of induction chemotherapy was defined as therapeutic success. Patients who failed to achieve CR were defined as primary refractory for the purpose of this analysis; patients who experienced treatment-related mortality (i.e., death within 28 days of treatment initiation) were excluded from this analysis. We used logistic regression analyses to assess the relationship between individual covariates and various measures of therapeutic resistance. The following pre-treatment covariates were used in the regression modeling: age at diagnosis, gender, white blood cell (WBC) count, platelet count, bone marrow blast percentage, disease type (primary vs. secondary), cytogenetic risk, FLT3/NPM1 status, and treatment site. We then used the area under the receiver operator characteristic curve (AUC) to quantify a model's ability to predict therapeutic resistance; in this approach, an AUC of 1 indicates perfect prediction while an AUC of 0.5 indicates no prediction; AUC values of 0.6-0.7, 0.7-0.8, and 0.8-0.9 are commonly considered as poor, fair, and good, respectively. ResultsA total of 4,550 patients (median age: 52 years [range: 15-90 years]) were included in this study. A CR to the initial 1-2 courses of induction chemotherapy was achieved in 3,597 (79.1%) of patients, whereas 953 (20.9%) were primary refractory; 1,304/4,497 patients (29.0%) with sufficient follow-up time were either primary refractory or had a relapse-free survival (RFS) of 3 months or less after CR achievement, 1,774/4,445 patients (39.9%) with sufficient follow-up time were either primary refractory or had a RFS of 6 months or less after CR achievement, and 2,523/4,386 patients (57.5%) with sufficient follow-up time were primary refractory or had a RFS of 12 months or less after CR achievement. Increasing age (p<0.001) and WBC (p<0.001), secondary disease (p<0.001), FLT3/NPM1 status (p<0.001), and cytogenetic risk (favorable or intermediate vs. adverse, p<0.001) were independently associated with being primary refractory to induction chemotherapy in a combined analysis of all patients. In the total patient cohort, a bootstrap-corrected multivariate model predicting primary refractoriness yielded an AUC of 0.79; removal of FLT3 and NPM1 from the model minimally, but statistically significantly, decreased the AUC (0.77). Between individual treatment sites, these AUCs varied from 0.82/0.81 to 0.69/0.67. Prediction of therapeutic resistance, as defined as primary refractoriness or relapse after short remission duration, was more difficult. Specifically, when analyzing the entire study cohort, the AUCs for models predicting primary refractory disease or relapse within 3 months were 0.76/0.74 (with/without inclusion of FLT3/NPM1 data) and further decreased to 0.76/0.73 and 0.75/0.71 for models predicting primary refractory disease or relapse within 6 or 12 months, respectively. ConclusionOur ability to predict therapeutic resistance based on routinely available clinical covariates, even with inclusion of commonly used molecular data on FLT3 and NPM1, is relatively limited. This finding would support the continued use of randomization to assign patients between standard and investigational therapies, and argues for the integration of early treatment response measures (e.g. minimal residual disease) to optimize prediction of therapeutic resistance. Disclosures:No relevant conflicts of interest to declare.

Palliative radiotherapy in locally advanced head and neck cancer after failure of induction chemotherapy: Comparison of two fractionation schemes

Context:Among patients with locally advanced head and neck squamous cell cancers (LAHNSCC), the prognosis after nonresponse or progression despite induction chemotherapy (IC) is dismal, and further treatment is often palliative in intent. Given that nonresponse to chemotherapy could indicate subsequent radioresistance, we intended to assess the outcomes with two different fractionation schemes.Aims:To compare the outcomes of two fractionation schemes- ’standard’ (consisting 3GyX5 daily fractions for 2 consecutive weeks) versus ‘hybrid’ (6GyX3 fractions on alternate days during the 1st week, followed by 2GyX5 daily fractions in the 2nd week).Settings and Design:Prospective randomized controlled two-arm unblinded trial.Materials and Methods:Patients with locally advanced oropharyngeal, laryngeal, and hypopharyngeal cancers treated with a minimum of two cycles of taxane, platinum, and fluorouracil-based IC were eligible if residual disease volume amounted <30 cm3. Kaplan-Meier survival curves were compared by the log-rank test. Response rates were compared using the unpaired t-test. Quality of life (QOL) was measured via patient reported questionnaires.Results:Of the initially enrolled 51 patients, 45 patients (24 from standard arm, and 21 from the hybrid arm) were eligible for analysis. Despite being underpowered to attain statistical significance, there still seemed to be a trend towards improvement in progression-free (Hazard ratio (HR) for progression: 0.5966; 95% CI 0.3216-1.1066) and overall survival (HR for death: 0.6062; 95% CI 0.2676-1.3734) with the hybrid arm when compared to the standard arm. Benefits were also observed with regards to response rates and QOL. Rate of complications were similar in both arms.Conclusions:In comparison to the routinely used palliative fractionation scheme of 30 Gray (Gy) in 10 fractions (Fr), the use of hybrid fractionation which integrates hypofractionation in the 1st week, followed by conventional fractionation in the 2nd week, could possibly offer better response rates, QOL increments, and potential survival benefits among LAHNSCC patients even after failing to respond to IC.

Clonal Evolution and Devolution Following Chemotherapy in Adult Acute Myelogenous Leukemia.

Abstract 2487 Introduction:Despite significant advances in the understanding of the biology of adult acute myelogenous leukemia (AML), overall survival remains poor due chiefly to the high rate of relapse after achieving complete remission as well as primary failure of induction chemotherapy. Efforts to further unravel the mechanisms leading to relapse and primary refractory disease are critical in order to guide the development of effective and durable treatment strategies for AML. To that end, this study seeks to elucidate the clonal relationship of AML in various disease phases. Methods:We employed SNP 6.0 array-based genomic profiling of acquired copy number aberrations (aCNA) and copy neutral LOH (cnLOH) together with sequence analysis of recurrently mutated genes to characterize paired AML genomes. We analyzed 28 AML sample pairs from patients that achieved complete remission with chemotherapy and subsequently relapsed (median remission duration 272 days [range 25 – 1249 days]) and 11 sample pairs from patients with persistent disease following induction chemotherapy. AML cell samples were isolated with a Ficoll gradient, negatively selected using Miltenyi microbead columns, and then further purified with flow cytometric cell sorting. Processed DNA isolated from highly purified AML blasts and paired buccal DNA was hybridized to Affymetrix SNP 6.0 arrays. aCNA were visually identified using the dChip program in paired data displays and corroborated by algorithmic lesion scoring, and cnLOH was detected using internally developed software. In addition, 11 genes known to be recurrently mutated in AML (CEBPA, DNMT3A, IDH1, IDH2, RUNX1, BCORL1, NPM1, NRAS, KRAS, FLT3 and TP53) were resequenced in all 39 presentation samples to identify somatically acquired mutations. Genes found mutated in individual AML cases were subsequently tested for the persistence of the mutation in paired samples. Results:For the 28 paired specimens in the relapsed cohort, comparison of aCNA and cnLOH occurrences, gene mutation patterns and karyotypes revealed 6 cases that carried no aCNA/cnLOH at either presentation or relapse, but at presentation carried at least 1 gene mutation, all of which but one were stable in relapse (1 case lost a RUNX1 mutation but carried a t(8;21) in both disease stages); 11 cases that were characterized by the presence of aCNA/cnLOH at presentation, of which 55% (6 of 11) gained additional aCNA/cnLOH at relapse; 6 cases without aCNA/cnLOH at presentation that gained aCNA/cnLOH at relapse, of which 2 concurrently lost a FLT3-ITD or CEPBA mutation; and 5 cases that carried no informative genomic events. For the 11 paired specimens in the persistent AML cohort, the same comparison revealed 2 cases without aCNA/cnLOH before or after chemotherapy and stable gene mutations; 5 cases with aCNA/cnLOH at presentation that carried the same genomic lesions and gene mutations before and after chemotherapy; 3 cases with aCNA/cnLOH present at enrollment that lost some but not all of these aCNA/cnLOH and gained none after initial induction therapy; and 1 additional case that lost a FLT3-ITD.Comparative analysis of these patterns demonstrates that relapsed AML invariably represents reemergence or evolution of an antecedent clone. Furthermore, all individual aCNA or cnLOH detected at presentation persisted at relapse indicating that this lesion type is proximally involved in AML evolution. Analysis of informative paired persistent AML disease samples uncovered at least two coexisting dominant clones of which at least one was chemotherapy sensitive and one resistant. Conclusion:This detailed genomic analysis supports the conclusion that incomplete eradication of AML founder clones rather than stochastic emergence of fully unrelated novel clones underlies AML relapse and persistence with direct implications for clinical AML research. Disclosures:No relevant conflicts of interest to declare.

Response of chemoradiation therapy after induction chemotherapy failure in locally advanced head and neck squamous cell carcinoma (LA-HNSCC).

5552 Background: Although induction chemotherapy (IC) for LA-HNSCC has shown high response rates, some patients do not respond to IC. Surgery has been the usual choice for the non-responding patients, but practically, not all of these patients are eligible for operation. The purpose of this study was to evaluate the efficacy of definitive radiation therapy (RT) for the HNSCC patients who failed IC. Methods: We have retrospectively analyzed the outcome of patients with LA-HNSCC who were initially treated with IC at Seoul National University Hospital between Jan. 2006 and Dec. 2010. Chemotherapeutic regimens used were 5-FU/cisplatin, docetaxel/5-FU/cisplatin, and docetaxel/cisplatin with or without cetuximab. After IC, patients were treated with RT, concurrent chemoradiation (CCRT), or surgery, either alone or with postoperative RT. Treatment modality was chosen on multi-disciplinary basis of our institution. Response was evaluated using RECIST criteria. Results: A total of 225 LA-HNSCC patients treated with IC were analyzed. Median age was 54 (range 24-77). Primary tumor locations were oral cavity (39.5%), nasal cavity/paranasal sinus (13.1%), oropharynx (13.1%), nasopharynx (13.1%), larynx (7.9%) and hypopharynx (13.1%). Among the 225 patients, 38 (16.9%) patients did not respond to IC [stable diseases (SD) = 23 (10.2%), progressive disease (PD) = 15 (6.7%)], and their median overall survival was 14.5 months. Among the 38 non-responders, 14 (36.8%) patients were undertaken surgery and 22 (57.8%) unresectable or inoperable patients were treated with either definitive RT or CCRT. Responses to subsequent definitive RT or CCRT for 20 evaluable patients were as follows: complete response = 6 (30.0%), partial response = 7 (35.0%), SD = 3 (15.0%), PD = 4 (20.0%) Overall response rate to definitive RT or CCRT in non-responder to IC was 65.0% (95%CI: 44.1%- 85.9%). Nine out of 15 patients who showed PD to IC received definitive RT or CCRT and interestingly, 3 (33.3%) patients responded to definitive RT or CCRT. Conclusions: A significant portion of HNSCC patients who failed to IC can benefit from subsequent definitive RT or CCRT. Further prospective study is warranted.

Absence of Biallelic TCRγ Deletion Predicts Early Treatment Failure in Pediatric T-Cell Acute Lymphoblastic Leukemia

To identify children with T-cell acute lymphoblastic leukemia (T-ALL) at high risk of induction chemotherapy failure by using DNA copy number analysis of leukemic cells collected at diagnosis. Array comparative genomic hybridization (CGH) was performed on genomic DNA extracted from diagnostic lymphoblasts from 47 children with T-ALL treated on Children's Oncology Group Study P9404 or Dana-Farber Cancer Institute Protocol 00-01. These samples represented nine patients who did not achieve an initial complete remission, 13 who relapsed, and 25 who became long-term, event-free survivors. The findings were confirmed in an independent cohort of patients by quantitative DNA polymerase chain reaction (DNA-PCR), an assay that is well suited for clinical application. Analysis of the CGH findings in patients in whom induction chemotherapy failed compared with those in whom induction chemotherapy was successful identified the absence of biallelic TCRgamma locus deletion (ABD), a characteristic of early thymocyte precursors before V(D)J recombination, as the most robust predictor of induction failure (P < .001). This feature was also associated with markedly inferior event-free (P = .002) and overall survival (P < .001) rates: 25% versus 58% and 25% versus 72%, respectively. Using a rapid and inexpensive quantitative DNA-PCR assay, we validated ABD as a predictor of a poor response to induction chemotherapy in an independent series of patients. Lymphoblasts from children with T-ALL should be evaluated at diagnosis for deletion within the TCRgamma locus. Patients lacking biallelic deletion, which confers a high probability of induction failure with contemporary therapy, should be assigned to alternative therapy in the context of a prospective clinical trial.

Successful allogeneic stem cell transplantation in two patients with acute myelogenous leukaemia and invasive aspergillosis by antifungal combination therapy

Invasive aspergillosis (IA) is an important cause of infectious morbidity and mortality in patients who undergo haematopoietic stem cell transplantation (HSCT). History of IA before allogeneic HSCT is still challenging because of the high risk of recurrence after HSCT. Recent advances in early-stage diagnosis and new, more effective classes of antifungal agents have improved the management of IA in the HSCT recipients. We report two cases with acute myelogenous leukaemia after primary failure of induction chemotherapy with the patients developing pulmonary IA. They responded well to a combination of voriconazole (VCZ) and micafungin, resulting in a remarkable reduction of pulmonary IA lesions at short intervals. Thereafter, antifungal therapy was switched to liposomal amphotericin B (L-AmB), followed by conditioning regimen for allogeneic HSCT, because of the possibility of VCZ altering the metabolism of chemotherapeutic agents and calcineurin inhibitors. Successful engraftment was achieved without severe adverse side-effects or aggravation of IA after HSCT. Combining VCZ with micafungin followed by L-AmB throughout HSCT could be advantageous in stabilising IA in HSCT patients.

Expression and Prognostic Significance of MDR and IAP-Family Genes in Patients with Acute Myeloid Leukemia

Introduction. Refractory Acute Myeloid Leukemia (AML) is associated with very bad prognosis. One of the major reason of induction chemotherapy failure is the expression of genes and proteins responsible for multidrug resistance (Multidrug Resistance 1 - MDR1, Multidrug Resistance-Related Protein 1 - MRP1, Breast Cancer Resistance Protein-BCRP, Lung Cancer Related Protein - LRP) and apoptosis (IAP-family genes: XIAP, cIAP1, cIAP2, survivin). The aim of the study was the evaluation of the influence of MDR and IAP-family genes expression on results of chemotherapy in patients (pts) with AML.Material and methods. The study was performed in 45 pts (20 females and 25 males; ranged from 18 to 82 years - mean 51.2) with newly diagnosed AML. There were following types acc. FAB classification: M0–1, M1–5, M2–13, M3–4, M4–16, M5–6 and cytogenetic risk groups: favorable-8, intermediate-29 and unfavorable-8 pts. The genes expression in leukemic cells was detected by RT-PCR method. All pts were treated with induction and consolidation therapy acc. Polish Adult Leukemia Group (PALG).Results. The expression of MDR1 gene was detected in 47%, MRP1 in 21%, BCRP in 10%, LRP in 26% and IAP-family genes in 78% of pts. Expression of MDR1 was significantly higher in M5 (p=0.02), but expression of cIAP1 and cIAP2 in M4 type (p=0.01). There was significant difference in MDR1 expression between unfavorable and good risk cytogenetic group (p=0.03), but there was none difference between cytogenetic groups in expression of IAP-family genes. There was significant difference in MDR1 expression between pts who achieved complete remission (CR) and pts without CR (p=0.01). When the expression of MDR1 gene was associated with the expression of BCRP and LRP, CR was never obtained. In all pts with complex genetic changes (MDR1+MRP1, MDR1+BCRP+LRP, MDR1+MRP1+BCRP+LRP) CR after one course of induction chemoterapy was not obtained. There was significant difference between CR rate after one course of chemotherapy in pts without MDR genes expression or expression only one gene and in pts with expression of two or more genes (p=0.02). There was no correlation between IAP-family genes expression and CR rate. Despite of differences in post-consolidation therapy (bone marrow transplantation in some pts) expression of MDR1 and survivin genes correlated with overall surival rate (p=0.05 and 0.03, respectively).Conclusions. Results of our studies on expression of MDR and IAP-family genes in pts with AML showed that expression of MDR genes better predicted response on induction chemotherapy than IAP genes. After confirmation on larger group of pts these findings may have important significance in defining of prognosis in AML and individualization of induction and postremission therapy, eventually with new generation of MDR modulators.

Perspectives of proteomics in acute myeloid leukemia

Acute myeloid leukemia (AML) is a frequent hematological malignancy. Despite enormous therapeutic efforts that range from various cytotoxic agents to allogeneic stem cell transplantation, overall survival of patients with AML remains unsatisfying. The poor survival rates are mainly due to therapy-related mortality, failure of induction chemotherapy and early relapses. Therefore, novel therapeutic agents that are more efficient and better tolerated are eagerly sought after. For existing therapeutic strategies, there is a lack of markers that are capable of reliably predicting prognosis or the therapeutic response prior to treatment. There is hope that elucidation of the AML-specific proteome will prompt the discovery of novel therapeutic targets and biomarkers in AML. Modern mass-spectrometry instrumentation has acheived excellent performance in terms of sensitivity, resolution and mass accuracy; however, so far, the contribution of proteomics to the care of patients with AML is virtually zero. This might be partly because mass spectrometry instrumentation and protein fractionation still lack true high-throughput capabilities with highest levels of reproducibility, thus hampering large-scale translational studies with clinical samples. Since mass-spectrometry instruments are very intricate devices, their successful operation will hinge on the willingness and ability of mass-spectrometry experts and clinical researchers to adopt new views, learn from each other and cooperate in order to ultimately benefit the patient suffering from AML. This review highlights some clinical problems circumventing the treatment of patients with AML. Furthermore, it provides a brief overview of the technical background of standard proteomics approaches and describes opportunities, challenges and pitfalls of proteomic studies with regards to AML.

One cycle of induction chemotherapy (IC) to select for organ preservation for patients (PTS) with advanced squamous carcinoma of the oral cavity (SCCOC)

5555 Background: A previous trial at the University of Michigan demonstrated that one cycle of IC can select pts with advanced laryngeal cancer for organ preservation. Methods: Pts with advanced SCCOC received one cycle of cisplatin and 5-fluorouracil. Those who achieved 50% reduction of their tumor received concurrent chemoradiation; those with less than 50% response underwent salvage surgery. Chemoradiation consisted of 70 Gy (2 Gy fraction daily x 7 weeks) with concurrent cisplatin 100 mg/m2 every 3 weeks x 3 cycles. After chemoradiation, pts with a complete histologic response received 2 cycles of paclitaxel 175 mg/m2; those with residual tumor had salvage surgery. Endpoints were survival and rate of organ preservation. Results: Between 7/24/2000 and 3/29/2004, 18 pts were enrolled, and 18 were eligible. Sixty-one percent were male and 39% were female. Mean age was 59 (range 37–87). Stage III -28% and stage IV -72%. Two patients died immediately after IC, prior to direct laryngoscopy. Following 1 cycle of IC, 16 pts underwent evaluation. Nine pts (56%) had a greater than 50% response and received chemoradiation. All 9 underwent evaluation for histologic response. Three pts had persistent disease: 1 had salvage surgery, 1 was surgically unresectable due to locally advanced disease, and 1 refused surgery. Six pts (67%) were complete histologic responders (CHR). Five CHR underwent neck dissections following chemoradiation. Adjuvant paclitaxel was given to 3 CHR - 2 received 2 cycles and 1 received 1 cycle. Seven pts (44%) had less than 50% response to IC. Of these, 4 had surgical resection; 1 had surgery off study; 1was no longer a surgical candidate and 1 died prior to surgery. The median time to follow-up is 30 months. Nine pts are dead, 7 from local/regional disease (3 after chemoradiation, 2 after surgery following IC, and 2 following IC), and two from other non-cancer related causes. The 3-year overall survival is 47% (95% CI [23.4%, 71.4%]) and the 3-year disease-free survival is 68.2% (95% CI [38.1, 98.3]). Conclusion: Given the poor response to IC and high failure rates following chemoradiation, this approach to organ preservation is not recommended for pts with advanced SCCOC. No significant financial relationships to disclose.