Search

PDF file - 339K, Flow cytometry histograms of serial levels of p-ERK and p-mTOR in patient samples.

Purpose: The clinical relevance of targeting the RAS/RAF/MEK/ERK pathway, activated in 70% to 80% of patients with acute myelogenous leukemia (AML), is unknown.Experimental Design: Selumetinib is an oral small-molecule inhibitor of MAP–ERK kinase (MEK)-1/2. Forty-seven patients with relapsed/refractory AML or 60 years old or more with untreated AML were enrolled on a phase II study. Patients were stratified by FLT3 ITD mutation status. The primary endpoint was response rate (complete, partial, and minor). Leukemia cells were analyzed for extracellular signal—regulated kinase (ERK) and mTOR phosphorylation.Results: Common drug-related toxicities were grade 1–2 diarrhea, fatigue, nausea, vomiting, and skin rash. In the FLT3 wild-type cohort, six of 36 (17%) patients had a response [one partial response, three minor responses, two unconfirmed minor responses (uMR)]. No patient with FLT3 ITD responded. NRAS and KRAS mutations were detected in 7% and 2% of patients, respectively. The sole patient with KRAS mutation had uMR with hematologic improvement in platelets. Baseline p-ERK activation was observed in 85% of patients analyzed but did not correlate with a response. A single-nucleotide polymorphism (SNP) rs3733542 in exon 18 of the KIT gene was detected in significantly higher number of patients with response/stable disease compared with nonresponders (60% vs. 23%; P = 0.027).Conclusions: Selumetinib is associated with modest single-agent antileukemic activity in advanced AML. However, given its favorable toxicity profile, combination with drugs that target other signaling pathways in AML should be considered. The potential association of SNP rs3733542 in exon 18 of the KIT gene with antileukemic activity of selumetinib is intriguing, but will require validation in larger trials. Clin Cancer Res; 20(2); 490–8. ©2013 AACR.

PDF file - 50K, Primers for KIT gene mutation testing.

Mutations in the all-trans retinoic acid (ATRA)-targeted ligand binding domain of PML-RARα (PRα/LBD+) have been implicated in the passive selection of ATRA-resistant acute promyelocytic leukemia clones leading to disease relapse.Among 45 relapse patients from the ATRA/chemotherapy arm of intergroup protocol C9710, 18 patients harbored PRα/LBD+ (40%), 7 of whom (39%) relapsed Off-ATRA selection pressure, suggesting a possible active role of PRα/LBD+. Of 41 relapse patients coanalyzed, 15 (37%) had FMS-related tyrosine kinase 3 internal tandem duplication mutations (FLT3-ITD+), which were differentially associated with PRα/LBD+ depending on ATRA treatment status at relapse: positively, On-ATRA; negatively, Off-ATRA. Thirteen of 21 patients (62%) had additional chromosome abnormalities (ACAs); all coanalyzed PRα/LBD mutant patients who relapsed off-ATRA (n = 5) had associated ACA. After relapse Off-ATRA, ACA and FLT3-ITD+ were negatively associated and were oppositely associated with presenting white blood count and PML-RARα type: ACA, low, L-isoform; FLT3-ITD+, high, S-isoform. These exploratory results suggest that differing PRα/LBD+ activities may interact with FLT3-ITD+ or ACA, that FLT3-ITD+ and ACA are associated with different intrinsic disease progression pathways manifest at relapse Off-ATRA, and that these different pathways may be short-circuited by ATRA-selectable defects at relapse On-ATRA. ACA and certain PRα/LBD + were also associated with reduced postrelapse survival. © 2012 by The American Society of Hematology.

Background In the present study, the prognostic impact of minimal residual disease during treatment on time to progression and overall survival was analyzed prospectively in patients with mantle cell lymphoma treated on the Cancer and Leukemia Group B 59909 clinical trial.Design and Methods Peripheral blood and bone marrow samples were collected during different phases of the Cancer and Leukemia Group B 59909 study for minimal residual disease analysis. Minimal residual disease status was determined by quantitative polymerase chain reaction of IgH and/or BCL-1/JH gene rearrangement. Correlation of minimal residual disease status with time to progression and overall survival was determined. In multivariable analysis, minimal residual disease, and other risk factors were correlated with time to progression.Results Thirty-nine patients had evaluable, sequential peripheral blood and bone marrow samples for minimal residual disease analysis. Using peripheral blood monitoring, 18 of 39 (46%) achieved molecular remission following induction therapy. The molecular remission rate increased from 46 to 74% after one course of intensification therapy. Twelve of 21 minimal residual disease positive patients (57%) progressed within three years of follow up compared to 4 of 18 (22%) molecular remission patients (P=0.049). Detection of minimal residual disease following induction therapy predicted disease progression with a hazard ratio of 3.7 (P=0.016). The 3-year probability of time to progression among those who were in molecular remission after induction chemotherapy was 82% compared to 48% in patients with detectable minimal residual disease. The prediction of time to progression by post-induction minimal residual disease was independent of other prognostic factors in multivariable analysis.Conclusions Detection of minimal residual disease following induction immunochemotherapy was an independent predictor of time to progression following immunochemotherapy and autologous stem cell transplantation for mantle cell lymphoma. The clinical trial was registered at ClinicalTrials.gov: NCT00020943.

Background The assessment of minimal residual disease (MRD) is a key component of prognosis and monitoring in acute lymphoblastic leukemia (ALL) and mantle cell lymphoma (MCL). Allele-specific oligonucleotide (ASO)-PCR can be used to assess MRD; however, this technique requires preparation of clonotype-specific primers for each patient, which is laborious and time-consuming. We demonstrated the utility of sequencing-based MRD assessment in ALL (Faham et al., Blood 2012). This quantitative approach relies on amplification and sequencing of immunoglobulin and T-cell receptor gene segments using consensus primers and can address some of the limitations associated with traditional MRD detection. Here we compared the ability of the sequencing and ASO-PCR methods to identify clonal cancer gene rearrangements at diagnosis and evaluated the concordance of MRD detection in bone marrow (BM) or blood samples from 37 patients (pts) with ALL and 22 pts with MCL entered onto prospective CALGB treatment trials, 10403 and 59909 (Alliance), respectively . Methods Using the quantitative ASO-PCR and sequencing assays, we analyzed diagnostic blood and BM samples from ALL pts for clonal rearrangements of immunoglobulin (IGH-VDJ, IGH-DJ, IGK) and T cell receptor (TRB, TRD, TRG) genes. We then assessed MRD at the IGH and/or TRG locus in 84 follow-up samples from ALL pts. Similarly, we analyzed samples from 22 MCL pts for immunoglobulin (IGH-VDJ, IGH-DJ, IGK) clonal gene rearrangements and measured MRD at the IGH and/or IGK locus in 114 follow-up samples. Sensitivity of the ASO-PCR vs sequencing was 1 X 10 4-5 vs 1 X 106, respectively. Concordance between sequencing and ASO-PCR MRD assessment on serial samples collected during and post-treatment was evaluated across all pts and within disease groups using concordance correlation coefficients for repeated measures (CCC-RM). Concordance in identification of detectable vs. undetectable MRD by both methods was also evaluated using Kappa statistics. Results Using the sequencing platform, high frequency clonal rearrangements were observed in at least two receptors in 97% and 95% of pts with ALL and MCL, respectively. Selected ALL samples were known to have IGH-VDJ or TRG clonal rearrangements by ASO-PCR; however, sequencing revealed additional clonal rearrangements in 36/37 (97%) pts with ALL. Good concordance was observed with identification of MRD positive vs. negative between the methods (K=0.62; p Conclusions This study demonstrates concordance between identification of detectable MRD in ALL and MCL by sequencing and ASO-PCR Methods. The sequencing approach offers improvements over ASO-PCR including the ability to monitor multiple clonal sequences, faster turnaround time (results in 1 week), and greater sensitivity. The clinical significance of this greater sensitivity remains to be tested prospectively and must be correlated with clinical results. Nevertheless, the sequencing method represents an alternative approach for clinical MRD monitoring which could fundamentally improve the ability to monitor disease progression and recurrence in patients with lymphoid malignancies. Disclosures: Carlton: Sequenta, Inc.: Employment, Equity Ownership. Weng:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

CALGB 50403 was a randomized phase II clinical trial to test the benefit of maintenance vs consolidation bortezomib therapy following aggressive chemo-immunotherapy and autologous stem cell transplant for adults < 60 years old with previously untreated mantle cell lymphoma (MCL). Eradication of minimal residual disease has been found to be an important biomarker of Progression Free Survival (PFS) in MCL. One goal of this study was to assess the kinetics of MRD during treatment of 50403: intensive chemo-immunotherapy induction; autologous stem cell mobilization and transplant; and post transplant therapy with rituximab followed by intensive bortezomib consolidation or prolonged bortezomib maintenance therapy. Methods 151 patients were enrolled in this study. 93 patients had adequate follow-up samples available and were screened for IgH/BCL-1 gene rearrangements using standardized primer sets (InVivoscribe). The sensitivity of this assay is from 1X104-105. 61/93 (66%) had an evaluable MRD marker and sequential samples for study of MRD kinetics. Patient specific oligonucleotides were generated from pre-treatment bone marrow for quantitative PCR (qPCR) amplification using previously published, standardized methods. The sensitivity of detection of MRD ranges from 1X104-105. 49 patients with sequential bone marrow samples at a minimum of 3 timepoints during and following treatment are included in this analysis. Presenting characteristics: the median age was 59 years (29-69); 32 (65%) were male; Stage IV MCL in 46 (94%); 16 (33%) had B-symptoms, 46 (94%) had PS 0 or 1; 17 (35%) had elevated LDH; MIPI risk group: Low 25 (51%), Intermediate 13 (26%) and high 11 (23%) ; 24 (49%) were randomized to maintenance bortezomib; 19 (39%) to bortezomib consolidation, and six were not randomized (12%). Results With a median f/u of 3.3 years from study registration, there have been 12 events amongst these 49 Patients: 10 with disease progression and 2 deaths from other causes (censored at time of death). PFS is defined as time from study entry until progression, or date of last follow-up. Early eradication of MRD following 2 cycles of intensive induction chemo-immunotherapy was significantly and independently associated with PFS (p =0.017), (Figure 1). None of the patients who achieved MRD negative status (n=15) following chemo-immunotherapy induction have progressed. Conversely, 10/32 patients with any level of MRD positivity at this time-point have suffered disease progression. No other clinical variables described above were associated with PFS or MRD status. MRD status prior to bortezomib randomization is also significantly associated with PFS, but to a lesser degree (p=0.05) while MRD measurements in the apheresis product did not predict for PFS. Conclusions Early achievement of undetectable MRD using qPCR for IgH gene rearrangements is highly prognostic for PFS for patients with MCL treated on CALGB 50403, with an observed 100% PFS amongst these patients. Longer follow-up is required to determine whether one of the two bortezomib arms improves treatment outcome compared to earlier CALGB studies in MCL. Nevertheless, these data suggest that the early introduction of novel agents into the 50403 treatment regimen may be a useful strategy for improving MRD eradication and PFS in MCL. Disclosures: Cheson: MedImmune: Research Funding.

6546 Background: MEK/MAPK and PI3K/Akt pathways have been demonstrated to be constitutively activated in the majority of AML patients and associated with poor prognosis, but inhibition of single pathway has only demonstrated modest clinical activity. Methods: The current study evaluated the efficacy of combination of orally available inhibitors to MEK (AZD6244, Astra Zeneca), PI3K/mTOR (NVP-BEZ235, Novartis), Akt (MK 2206, Merck) and Bcl-2 family members (ABT263, Abbott) in human AML cell lines and primary AML patient samples. Results: In MV 4;11 cells (harboring both MLL re-arrangement and FLT3-ITD mutation), AZD6244 or BEZ235 alone moderately decreased viable cells by 30-40%, but combination of these two had dramatic additive effect of cell killing by 70-80%. ABT263 plus AZD6244 or BEZ235 resulted in further additive cell killing effects. While MK2206 alone had little effect, combination of MK2206 with AZD6244 resulted in excellent synergy. Similar effects were observed in HL60 cells with complex karyot...

6521 Background: We hypothesized that targeting two mechanisms of epigenetic silencing with a potent HDAC inhibitor BEL plus the DNA methyltransferase inhibitor AZC would be additive or synergistic...

Abstract 1038 The relationships of inherent disease-related characteristics and of treatment to the nature of APL cell clones that emerge at disease relapse are poorly understood. We studied these relationships in 45 patients who relapsed after ATRA/chemotherapy treatment on the non-arsenic trixoxide arm of intergroup phase III trial C9710 (Powell, et al. Blood, Epub). Four variably-expressed APL cell clonal markers were assessed: PML-RARA ligand binding domain mutations (LBD-M), FLT3 internal tandem duplication mutations (ITD), FLT3 receptor tyrosine kinase mutations (D835; RTK-M) and additional chromosome abnormalities (ACA), i.e., in addition to the hallmark t(15;17). The methods for mutation analysis of PML-RARA and FLT3 have been reported and were RNA transcript-initiated supplemented by selected quantitative FLT3 mutation analyses initiated from DNA. Karyotype data were derived by standard cytogenetic methods. Four patients were excluded from analysis because the sample level of PML-RARA, as determined by quantitative RT-PCR, was insufficient to exclude possible false-negative mutation results. In 41 evaluable patients, the marker incidences were: LBD-M, 44%; ITD, 37%; RTK-M, 12%; ACA (22 tested), 59%. At presentation, the corresponding incidences were: LBD-M, 0% (by high-sensitivity testing a minor subclone of the relapse mutation was found in 2/7 patients tested); ITD, 43% (37 tested); RTK-M, 22% (37 tested); ACA, 24% (34 tested). The low frequency of RTK-M impeded further analysis. The other markers, using relapse determinations, were assessed for potential associations between the markers and with the following parameters: age, sex, presenting WBC count, PML-RARA type, time to relapse, relapse on or off ATRA treatment (on = taking or within 30 days of discontinuing) and post-relapse survival. The most essential positive findings are summarized below:Association of Marker with Parameter or Other MarkerCases with ParameterCases without Parameterp-valuePretreatment parameters: LBD-M with WBC count < 5,000/uL12/18 (67%)7/23 (30%)0.030 ITD with WBC count >10,000/uL10/15 (67%)5/26 (19%)0.006 ITD with S-form PML-RARA15/15 (100%)10/26 (38%) The results confirm reports, which tested pretreatment samples, of a strong association between FLT3ITD mutations present at relapse and high WBC count and S-form PML-RARA. At relapse, there was, also, divergent selection of ITD-harboring vs ACA-harboring clones. LBD-M occurred more frequently in patients with low presenting WBC counts. They, also, occurred in patients who relapsed while on ATRA, consistent with an ATRA selective role for clones harboring LBD mutations. A significant segregation of patients with LBD-M or ITD was observed after relapse off ATRA treatment (p = 0.019), while no segregation was observed in overall relapse patients (p = 0.346) or after relapse on ATRA (p = 0.316). In 2 patients who relapsed on ATRA, quantitative analysis indicated that LBD-M and ITD mutations must be present in the same APL cell clone. There were no significant survival differences related to any clonal marker (post-relapse survival = 66%). Although the small number of cases is cautionary, the overall data suggest that LBD-M may have an overriding, dominant APL clone selection role in relapse that occurs on ATRA therapy. Other inherent clinicopathologic characteristics of APL may predispose to divergent molecular pathways of disease progression and relapse in patients alternatively harboring clones with LBD-M or ITD after the termination of ATRA selection pressure. Disclosures: No relevant conflicts of interest to declare.

Abstract 3921 Autologous stem cell transplantation (ASCT) is an effective treatment strategy for Mantle Cell Lymphoma (MCL) that has been shown to improve disease free survival. However, data from recent trials suggest that the presence of minimal residual disease (MRD) contributes to relapse following current intensive treatment strategies, including ASCT. Thus, we sought to test a highly selective approach to eradication of MRD in stem cell collections from MCL patients using targeted small peptides that disrupt the aberrant cyclin/CDKs interactions that are involved in the pathogenesis of MCL. In the current study, the efficacy of a mimetic peptide to the CDK inhibitor p16INK4a to eliminate MRD was tested. Using (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) cell viability assays, we demonstrated that a transducible TAT-p16 mimetic peptide was able to induce cell death in the MCL cell line, Jeko-1, in a dose and time-dependent fashion (see Figure). A TAT-p16 mimetic was able to induce cell death in approximately 60% of Jeko-1 cells after 4 hours of incubation, and approximately 80% of Jeko-1 cells at 24 hours. In contrast, the same dose of TAT-p16 mimetic had no effect on cell survival when it was incubated with CD34+ rich apheresis samples from healthy donors. Furthermore, CFU-GM, BFU-E colony assays showed that there was no toxicity of the TAT-p16 mimetic when compared to untreated cells or cells treated with a control, scrambled TAT-p16 peptide. TAT-p16 mimetic did not impair erythroblast differentiation but induced mild apoptotic cell death in the erythroblast cells. The strategy was further tested using a CD34+ enriched apheresis sample collected from a patient with MCL with documented MRD who had been treated on a clinical trial of ASCT for MCL. Using Real-time quantitative PCR for IgH and BCL1 gene rearrangements, we demonstrated that the TAT-p16 mimetic was able to reduce MRD level by 40% compared with the scrambled TAT-p16 peptide. These data suggest that the use of a peptide-mimetic to p16 selectively and effectively can reduce MRD in MCL cells and in an apheresis (stem cell) sample from a patient with MCL. Treatment with the peptide allowed differentiation of CD34+ progenitor cells. A combinational approach using additional targeted cell cycle regulatory peptides that block p21CIP1/WAF1 and cyclin D2 is being explored to optimize the efficacy of this purging technique. Thus, this novel strategy may be an effective, selective and non-toxic method for eradication of MRD in MCL. Disclosures: No relevant conflicts of interest to declare.

Abstract 3978 It has been demonstrated that MEK/MAPK and PI3K/Akt are constitutively activated in the majority of AML cases and that their aberrant expression is associated with a poor prognosis. Targeted inhibition of either the MEK/MAPK or the PI3K/Akt pathway alone has only demonstrated mild to modest clinical activity, possibly due to feedback activation of compensatory pathways. Thus, preclinical studies have recently turned to targeted inhibition of both of these pathways simultaneously. In the current study, the efficacy of the combination of two orally available inhibitors to MEK (AZD6244, Astra Zeneca) and PI3K/mTOR (NVP-BEZ235, Novartis) was evaluated in AML cell lines and in primary AML patient samples. In MV 4;11 AML cells (harboring both the MLL re-arrangement and FLT3 internal tandem mutation), AZD6244 or BEZ235 alone moderately decreased viable cell numbers by 30–40% as measured by the MTS assay, a colorimetric assay for cellular growth and survival, but the combination of these two had a dramatic additive effect with a decrease of viable cell numbers by 70–80%. Similar effects were observed in AML cell lines with different cytogenetic and molecular abnormalities including THP-1 [t (6;11)], HL-60, KG-1 [del(5q)], and Kasumi-1 [t(8;21)]. Similar results were also obtained in leukemia cells from 3 patients with AML with different recurring cytogenetic abnormalities. Apoptotic cell death was determined by detection of Disclosures: No relevant conflicts of interest to declare.