ALBERT

Description: Abstract 1493 Background: The two most frequent mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor are internal tandem duplication (ITD) of the juxtamembrane region and mutations involving the D835/I836 residues of the tyrosine kinase domain (TKD). They are present in approximately 30% and 7% of AML cases respectively, and have been associated with higher relapse rate and worse overall survival. ITD and TKD mutations produce constitutive activation of the FLT3 receptor; however they appear to have different downstream effectors and different clinical significance. Currently, several FLT3 kinase inhibitors are in development and although some have shown promising clinical activity, responses tend to be transient and insufficient to induce a durable response. Sensitivity of different mutants to diverse FLT3 inhibitors is variable (Zhang et al. JNCI 2008; 100: 184). Aim: To determine the change in mutation status of patients with FLT3 ITD treated with FLT3 inhibitors. Methods: We analyzed 67 patients with FLT3 mutation positive AML, treated in our institution in clinical trials with different FLT3 inhibitors from October 2002 to July 2011 and in whom we obtained mutational assessment before and after treatment. Results: At baseline 58 (87%) patients had an ITD mutation, 5 (7%) had a D835/I836 mutation and 4 (6%) had combined ITD and codon D835/I836 mutations. Thirty three patients were male, median age was 55 years (range 18–87), and the median number of prior leukemia treatments was 2 (range 1–6). Karyotype was diploid in 36%, miscellaneous in 17%, complex in 14%, not done in 23%, and insufficient in 9% of patients. Concomitant NPM1, RAS, and CEBPA mutations were observed in 21%, 13%, and 4% of the patients, respectively. Patients remained on therapy for a median time of 50 days (range 18–561) before progression, 45 (67%) did not achieve response, 13 (19%) cleared their bone marrow blasts, 6 (9%) achieved a partial response, 4 (6%) a complete response and 1 (1%) patient died during treatment. None of the patients with D835/I836 or ITD+D835/I836 achieved a response. Treatment was always discontinued due to progressive disease, except in 9 (13%) patients, 6 who cleared bone marrow blasts and proceeded to hematopoietic stem cell transplantation and 3 who discontinued due to toxicity. One of them was in complete response but progressed within 4 months, and 2 who cleared their bone marrow blasts and were also bridged with other treatments to transplant. All patients in this cohort had assessment of their FLT3 mutation status at the time FLT3 inhibitor was discontinued, 14 (21%) patients progressed from a single ITD mutation to have combined ITD+D835/I836 mutations, 7 (10%) patients (6 ITD and 1 D835/I836) were negative for FLT3 mutation, and 46 (69%) patients had unchanged mutation status. At the time of this analysis, 10 patients were alive, 1 was lost to follow and 56 had died. The median survival in the entire group from the time of FLT3 inhibitor start was 5.3 months, and from time of treatment discontinuation 3.2 months. In those patients in whom the FLT3 mutation became negative the median survival was 7 months, in those with unchanged mutation 4.5 months, and in those with ITD-D835/I836 mutations the median survival was 6.1 months. Further therapy with another FLT3 inhibitor was attempted in 6 out of 14 patients with ITD-D835/I836 mutations, 5 failed to respond and 1 had a transient decrease in bone marrow blasts and peripheral white blood cell count (2 months) with a combination of a FLT3 inhibitor and a hypomethylating agent. Conclusion: There is evidence that a secondary TKD mutations arise after the use of FLT3 inhibitors in patients with single FLT3-ITD mutated AML, a phenomenon that is associated with a poor prognosis. Disclosures: Ravandi: Bayer: Research Funding; Onyx: Research Funding. Cortes:Ambit: Research Funding; Novartis: Research Funding.

Description: Abstract 1516 Background: Sorafenib is a potent inhibitor of FLT3 kinase with demonstrable clinical activity in patients with acute myeloid leukemia (AML) and FLT3-ITD mutation, but not those with FLT3-D835 mutation. Objectives: To determine the long term outcome of patients with AML treated with the combination of Sorafenib, cytarabine and idarubicin, and in particular those with FLT3-ITD mutation. Method: Between October 2007 to March 2010, 62 patients with newly diagnosed, previously untreated AML, were treated with Sorafenib 400 mg orally twice daily (BID) for 7 days, cytarabine 1.5 g/m2 by continuous intravenous (IV) infusion daily for 4 days (3 days if 〉 60 years of age), and idarubicin at 12 mg/m2 IV daily for 3 days on an IRB-approved clinical trial. After achieving remission, patients received up to 5 cycles of consolidation with sorafenib 400 mg BID continuously and abbreviated doses of cytarabine and idarubicin given at approximately one month intervals. Results: 62 patients were treated on the phase II portion of the study. Median age was 53 years (range, 18 – 66) and 12 (19%) patients were 〉 60 years. 23 (37%) had FLT3 mutations including 17 with FLT3-ITD, 4 with FLT3-D835, and 2 with both mutations. Cytogenetics was diploid in 36 (58%), chromosome 5 and 7 deletion in 4 (6%) and 1 (2%) respectively, complex in 8 (13%), miscellaneous in 13 (21%). Median white blood cell count (WBC) at diagnosis was 7.25 × 109/L (range, 0.6 – 225), and 28 (45%) patients had WBC 〉 10 × 109/L; among these, 12 (43 %) had FLT3-ITD. After induction, 49 (79 %) patients achieved CR and 5 (8%) CR with incomplete platelet recovery (CRp). 1 (2%) patient died before response assessment could be performed and 7 (11%) were non responders. Patients with FLT3-ITD were more likely to achieve a CR/CRp than patients without FLT3-ITD [18/19 (95%) patients vs. 36/43 (83%) patients, respectively (p=0.23)]. To date, 32 (59%) of the responders have relapsed including 10 of 18 (56%) patients with FLT3-ITD and 22 of 36 (61%) patients without FLT3-ITD (P=0.86). 35 patients received salvage therapy; 14 died after receiving salvage therapy, 11 achieved a second CR and 10 were refractory. After a median follow up of 40.6 months (range, 5.7 to 50 months), the median DFS and OS were 13 months and 29 months, respectively. Hematopoietic stem cell transplantation (HSCT) was performed in 34 (55 %) patients, including 23 in CR, and 11 with active disease. Stem cell source was from related donors in 15 (44%), unrelated donors in 9 (26%), cord blood in 7 (21%), and haploidentical donors in 3 (9%) patients. Overall, 35 (56%) patients have died; 16 (26%) had infectious complications, 12 (19%) multi-organ failure, 9 (15%) graft versus host disease, and 10 (16%) other causes with some patients having overlapping causes. Three-year disease free survival (DFS)(in patients achieving CR, n=54) and overall survival (OS) (n=62) were 34% and 47%, respectively (Figures 1 and 2). Conclusion: Combination of sorafenib, idarubicin and cytarabine is an effective regimen with durable responses in patients with newly diagnosed AML, particularly those with FLT3-ITD. Disclosures: Ravandi: Bayer: Research Funding; Onyx: Research Funding.

Description: Mycosis fungoides (MF) is a cutaneous T-cell lymphoma characterized by multifocal disease and protracted clinical course. The few studies that have assessed T-cell receptor (TCR) gene rearrangements (GRs) present at different anatomic sites in MF have generally reported a common clone. We used a previously validated 4-color polymerase chain reaction (PCR) assay to assess the size and V-family usage of TCR-γ GRs in 102 concurrent and/or sequential morphologically involved biopsy specimens (91 skin and 11 lymph nodes) from 39 MF patients. This assay detected TCR-γ clonal GRs in 89 samples (87%) from 36 patients (92%). In 24 patients (77%), an identical clonal GR was present in at least 2 skin samples. However, in one third of these patients, additional different clonal GRs were also noted. Four patients (13%) had clonal GRs that were distinct in different skin samples. In 3 patients (10%), no GR was detected in any sample. In a comparison of lymph node and skin samples, 8 patients had the identical clonal GRs at both sites, 2 patients had different clonal GRs, and 1 patient had no GR identified at either site. Independent of clinical stage, patients who had the same GR detected in multiple concurrent biopsy specimens at the time of diagnosis were more likely to have progressive disease than those who had different GRs (P = .04). Four-color TCR-γ PCR analysis can uncover multiple distinct clonal GRs in different samples consistent with multiclonal or oligoclonal disease in a significant proportion of MF patients. Demonstration of identical clonal GRs in multiple biopsy specimens at the time of diagnosis may provide prognostic information related to disease progression.

Description: We conducted this study to determine the feasibility and safety of cladribine followed by rituximab in patients with hairy cell leukemia including the vari-ant form (HCLv). Cladribine 5.6 mg/m2 given IV over 2 hours daily for 5 days was followed ∼ 1 month later with rituximab 375 mg/m2 IV weekly for 8 weeks. Responses were recorded and BM minimal residual disease (MRD) was evaluated after the completion of rituximab. Thirty-six patients have been treated including 5 with HCLv. Median age was 57 years (range, 37-89). All patients (100%) have achieved complete response (CR), defined as presence of no hairy cells in BM and blood with normalization of counts (absolute neutrophil count [ANC]〉 1.5 × 109/L, hemoglobin [Hgb] 〉 12.0 g/dL, platelets [PLT] 〉 100 × 109/L), as well as resolution of splenomegaly. There were no grade 3 or 4 nonhematologic adverse events directly related to the treatment. Only 1 patient (with HCLv) has relapsed; median CR duration has not been reached (range,1+-63+ months). Three patients with HCLv died including 1 with relapsed disease and 2 from unrelated malignancies. Median survival duration has not been reached (range, 2+-64+ months). Treatment with clad-ribine followed by rituximab is effective tk;4and may increase CR rate. This study was registered at www.clinicaltrials.gov as NCT00412594.

Description: Key Points The sensitivity and specificity of detecting the JAK2 p.V617F mutation in PB are both 100% compared with BM. The JAK2 p.V617F allele burden measured in PB is equivalent to that in BM aspirate (R2 = 0.991; P 〈 .0001).

Description: Abstract 2127 Background: The addition of tyrosine kinase inhibitors imatinib and dasatinib to combination chemotherapy regimens has improved the outcome of patients (pts) with Philadelphia-chromosme-positive (Ph+) acute lymphoblastic leukemia (ALL). We examined whether the dynamics of minimal residual disease (MRD) measured by multiparameter flow cytometry (MFC) or reverse transcription quantitative polymerase chain reaction (RQ-PCR) was different among pts treated with the combination of hyperCVAD with imatinib or dasatinib. Materials and Methods: From April 2001 to September 2006, 54 pts with newly diagnosed Ph+ ALL were treated with the combination of hyperCVAD and imatinib; from October 2006 to July 2009, 42 pts were treated with the hyperCVAD and dasatinib regimen. The median ages for the two groups were 50 and 51 years [ranges, (27 - 84) and (21 - 78)]. Fifty one (94%) and 40 (95%) achieved complete remission (CR) on the two regimen and were followed by serial bone marrow assessments for MRD. MFC was performed using 4 or 6 color combinations of antibodies to lymphoblast and myeloid antigens (e.g., CD10, CD13, CD15, CD19, CD20, CD22, CD25, CD33, CD34, CD38, CD58, CD66c, and CD81), with a sensitivity of 0.01%. RQ-PCR for BCR-ABL was performed using TaqMan primer/probes for the e1a2, e13a2 (b2a2), and e14a2 (b3a2) BCR-ABL transcripts in a single tube with normalization to total ABL transcripts. Results: The dynamics of the MRD by RQ-PCR and MFC over the initial 18 month period after achieving CR are shown in the attached figures 1 and 2, respectively. The decline in the median value of BCR-ABL/ABL was more rapid with dasatinib, with more patients reaching the level of

Description: Abstract 2623 Background: Monitoring minimal residual disease (MRD) using reverse transcriptase polymerase chain reaction (RT-PCR) for the PML-RARA fusion transcript in bone marrow (BM) samples has been used to predict relapse in patients (pts) with acute promyelocytic leukemia (APL). Rigorous, serial real-time quantitative PCR (RQ-PCR) has been shown to be the strongest predictor of relapse-free survival in APL (Grimwade D, JCO, 2009), and BM was more sensitive than peripheral blood (PB) for detecting pending relapse. However, the clinical utility of MRD monitoring and use of PB as a sample source for MRD detection remains to be established for APL pts treated with ATRA and ATO without chemotherapy. Purpose: To evaluate the reliability of using PB real-time quantitative PCR (RQ-PCR) in monitoring MRD in patients with APL treated with ATRA and ATO ± gemtuzumab ozogamicin (GO). Methods: From June 2007 to August 2011, 78 pts with newly diagnosed APL were treated on a clinical trial (NCT00413166) combining ATRA and ATO at our institution. High risk pts (WBC 〉 10 × 109/L) and pts with rising WBC also received GO on the first day or when WBC rose above 10, respectively. RQ-PCR for PML-RARα fusion transcript was performed on BM and PB after induction, every 3 months throughout consolidation (8 months of ATRA and ATO) for the first 2 years and then PB and/or BM analysis was performed every 6 months with follow up. The Spearman correlation and Paired Wilcoxon test were used to analyze the relation, generate the correlation coefficient and analyze the differences between BM and PB RQ-PCR values. Results: A total of 432 PB and 618 BM samples were obtained for RQ-PCR analysis. The median number of PB RQ-PCR and BM RQ-PCR obtained per patient were 6 (range, 0–16) and 8 (range, 0–14), respectively. Two hundred thirty six samples were obtained concomitantly from PB and BM (defined as obtained within a day of each other) from 63 patients. The median time to CR was 30 days (range, 9–63). The median time to complete molecular response (CMR) was 130 days (range, 20–271). Two patients died, 1 during induction (before assessing his response to treatment), the other one during 4th consolidation while in CR of unknown cause. Two pts have relapsed, one of them with CNS disease only, the other one with systemic then CNS relapse. Both patients had previously achieved CMR. The patient with CNS relapse remained RQ-PCR-negative on BM at the time of relapse (PB not available at time of relapse). The other relapsed patient had a positive PB RQ-PCR at time of relapse. There was a strong correlation (Spearman correlation, r=0.8, p

Description: Abstract 2562 Background: Massive parallel sequencing technology has elucidated the mechanism of molecular clonal evolution in relapsed AML patients (pts). However incidence and prognostic relevance of clonal evolution in real clinical setting is unknown. The aims of the current analysis are to: 1) evaluate incidence of clonal evolution both at cytogenetic and molecular level in relapsed and refractory AML pts using readily available molecular mutation panels, and 2) evaluate prognostic impact of both cytogenetic and molecular clonal evolution in such pts. Methods: We analyzed 87 pts with AML who relapsed after induction therapy or who were refractory to induction and received 1st salvage therapies. If available, results of conventional cytogenetic study and molecular mutation panel including FLT3 (D835 and ITD), KRAS, NRAS, IDH1/2, NPM1, CEBPA, JAK2 and C-KIT were compared between the initial diagnosis and at time of relapse or at disease progression. Cytogenetic clonal evolution (CyE) was defined as acquisition of new cytogenetic abnormalities. Molecular clonal evolution (ME) was defined when there was acquisition of new genetic mutation not present at initial diagnosis. Result: Median age of the analyzed group was 61years (range: 17–82); 37 (43%) pts were female. Sixty three (72%) pts had de novo AML and 24 (28%) had secondary AML (evolved from MDS). Nineteen (22%) pts had prior cancer history and had exposure to chemo and/or radiation therapy (ie. therapy related AML). Fifty three (61%) pts received conventional high dose cytarabine-based regimen for the induction, 17 (19%) received clofarabine-based, 6 (7%) received sapacitabine, and 3 (3%) received low dose cytarabine-based regimen. Thirty six (41%) pts achieved complete remission (CR) after induction with median 1st CR duration of 5.7 months (range: 1.0–16.4), while 51 (59%) pts were refractory to it. Baseline cytogenetics were diploid in 38 (44%) pts. Del 5q/−5was identified in 14 (16%) pts and del 7q/−7 was found in 21 (24%) pts. Complex cytogenetic abnormalities were seen in 26 (29%) pts. CyE was identified in 38 (44%) pts, of which 16 were seen at time of relapse and 22 at disease progression in refractory pts. Fifteen cases evolved from diploid and 23 developed from abnormal karyotype. Original founding cytogenetic abnormalities were persistently observed after evolution in all cases. Acquisition of trisomy 8 occurred in 6 (16%) pts, del7q/−7 in 4 (11%) and del5q/−5 in 2 (2%). Statistical association was not identified between development of CyE and other covariates (de novo vs. secondary AML, baseline cytogenetics, baseline molecular mutations, sex, therapy-related, baseline WBC, Hb, Plt, ME and presence of each molecular mutation). Initial molecular mutation was detected in 40 (46%) pts. FLT3 D835 mutation was detected in 4 (5%), FLT3-ITD in 14 (16%), NPM1 in 10 (12%), NRAS in 13 (15%), CEBPA in 5 (6%), IDH1 in 4 (5%), IDH2 in 5 (6%), c-KIT in 1 (1%) pts, respectively. ME was identified in 12 (14%) pts of which 3 were seen at time of relapse and 9 were seen at time of disease progression in refractory pts. Among the observed ME, acquisition of FLT3-D835 mutation was seen in 6 patients (4 in pts originally with ITD mutation), FLT3-ITD acquisition in 3, NRAS acquisition in 2 and KRAS acquisition in 1 pts, respectively. Statistical association was not identified between development of ME and other covariates that were tested for CyE. During the median follow up duration of 8.4 months (range 1–25), 47 (54%) pts were dead. Median overall survival (OS) of the total analyzed group was 10 months. Having CyE or ME did not affect OS (P = 0.47 and P = 0.70). Among the pts who relapsed after an initial CR (N= 36), a trend to worse OS was observed in pts with CyE (P = 0.08) but having ME did not affect OS (P = 0.65). In refractory AML pts (N= 51), having CyE or ME did not affect OS (P = 0.70 and P = 0.38) (Table 1). Conclusion: Approximately 44% and 14% of pts with relapsed/refractory AML experience CyE or ME, respectively, either at time of relapse or progression. Neither CyE nor ME have significant prognostic relevance but an unfavorable trend was observed in relapsed AML pts with CyE at relapse. Larger series are needed to confirm a possible association. Sequential cytogenetic and molecular mutation analysis may be important in relapsed/refractory AML pts upon disease progression especially molecularly targeted agents are considered. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 164 Background: The achievement of a major molecular remission (MMR) after imatinib therapy in pts with chronic myeloid leukemia (CML) in chronic phase (CP) predicts for decreased risk of events, but has little impact in overall survival (OS) among patients with complete cytogenetic response (CCyR). Deeper molecular responses (MR), including undetectable transcripts, are frequently sought in patients with CML treated with tyrosine kinase inhibitors (TKI), but the prognostic significance of these responses is not known. Objectives: To determine the long-term clinical significance of achieving deeper level of MR achieved after therapy with TKI for CML in CP. Methods: Pts were included in clinical trials for initial therapy for CML with one of the following modalities: imatinib 400mg/day (IM400), imatinib 800mg/day (IM800), nilotinib (NILO) and dasatinib (DASA). We defined the level of MR as MMR, MR4, MR4.5 and undetectable transcripts (UND), corresponding to an ABL/BCR-ABL ratio (International Scale) of ≤0.1%, ≤0.01%, ≤0.0032%, and undetectable transcripts (minimum sensitivity 4.5-log), respectively. Results: A total of 495 pts were treated: 83 pts with IM400, 204 with IM800, 106 with NILO and 102 with DASA. At presentation leukocyte counts were higher in the NILO group (41.5 vs 22.2, 27.5 and 27×109/L for IM400, IM800 and DASA pts). All other patient characteristics were equally distributed across the 4 treatment groups. After a median follow-up of 73 months (2 to 142), complete cytogenetic response (CCyR) was achieved in 88%. CCyR rates for IM400, IM800, NILO and DASA pts were 82%, 88%, 90% and 90%, respectively. Best level of MR for the entire population was:

Description: Abstract 2706 Background IDH1 and IDH2 gene mutations have been identified as novel, recurring molecular aberrations among patients with normal karyotype acute myeloid leukemia (AML). The potential impact of these mutations as well as an IDH1 single nucleotide polymorphism (SNP) on the outcome of the patients is being actively investigated. Materials and Methods Among 358 patients with AML treated from October 2004 to February 2010 on 4 consecutive protocols using high dose ara-C plus idarubicin induction therapy (IA alone, IA plus tipifarnib [IAT], IA plus sorafenib [IAS], and IA plus vorinostat [IAV]), pre-treatment samples were available for 170 patients [median age 53 years, (range, 17 – 73); 96% ≤ 65 years] for testing for IDH1R132, IDH2R172 and IDH2R140 mutations. All patients received an induction course of therapy followed by up to 5 reduced-dose consolidation cycles followed by maintenance therapy with T, S, or V for up to a year; patients treated with IA had no maintenance. We examined whether presence of mutations in either gene or the codon 105 SNP in IDH1 was associated with pre-treatment characteristics or outcome. We also sought whether treatment with any of the 4 regimens had an impact on the outcome of patients with IDH aberrations. Results Overall, IDH1 and IDH2 mutations were present in 12 (7%) and 24 (14%) patients, respectively, and IDH1G105 SNP in 24 (14%). Overall, 52 (30%) patients had IDH gene aberrations; 2 patients had concomitant IDH1R132 mutation and IDH1G105 SNP, 3 patients had IDH2R140 mutation and IDH1G105 SNP, 1 patient IDH2R172 mutation and IDH1G105 SNP, and 1 patient had IDH1R132 mutation, IDH1G105 SNP, and IDH2R140 mutation. There was a strong association with normal karyotype with 11 of 12 (92%) of IDH1 mutated, 18 of 24 (75%) of IDH2 mutated, and 18 of 24 (75%) of IDH1 SNP being diploid. There was no association between any of the aberrations and patient age, sex, therapy-related vs. de novo AML, presenting WBC, peripheral blood blasts, or FAB subtype. IDH1 mutation was associated with a higher presenting platelet count (median 99 vs. 50 × 109/L in IDH1 wild-type [WT], p