ALBERT

Description: Abstract 4904 Background: Rituximab is an IgG class CD20-directed monoclonal antibody used in the treatment of B-cell malignancies, including WM. We and others have previously demonstrated dependence for IgG class therapeutic antibodies on polymorphisms at FcγRIIIA-158. Approximately half of WM patients express V/V or V/F, and the remainder half express F/F at this polymorphic locus. Patients with WM expressing FcγRIIIA-158 V/V or V/F show improved rituximab single agent activity, as well as attainment of deeper responses (VGPR or CR) with combination Rituximab therapy. GA101 is a novel humanized anti-CD20 antibody with a glyco-engineered Fc domain that exhibits increased Fcg receptor binding and ADCC activity. Methods: In this study, we examined the in vitro activity of GA101 and Rituximab against WM cells, and also examined the activity of these antibodies in context of FcγRIIIA-158 polymorphisms. ADCC activity for GA101 and Rituximab was assessed using genotyped healthy donor derived NK cells against BCWM.1 WM cells, as well autologous NK cells against the patient's own lymphoplasmacytic cells. In vitro B-cell depletion and direct cell death induction assays were also performed. Results: We observed significantly greater ADCC activity against WM cells for GA101 versus Rituximab in both healthy donor, as well as autologous NK cell assays. GA101 mediated ADCC activity was particularly more robust versus Rituximab in patients expressing FcγRIIIA-158 F/F versus V/V or V/F (Figure 1). In addition, GA101 induced significant direct cell death against WM lymphoplasmacytic cells, as well as in vitro B-cell depletion assays in comparison to Rituximab, which exhibited little direct cell death induction activity. Nuclear translocation of apoptosis inducing factor (AIF) was observed following GA101 by immunofluorescence microscopy. Conclusions: GA101 is associated with enhanced ADCC activity relative to Rituximab by NK cells, particularly for those subjects expressing FcγRIIIA-158 F/F. In addition, GA101 demonstrated direct cell death in WM lymphoplasmacytic cells through an AIF mediated caspase-independent pathway. These studies provide the framework for the investigation of GA101 in WM, and suggest particular benefit for those patients who express FcγRIIIA-158 F/F. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3555 Introduction: Cytogenetic status represents a major prognostic factor for individuals with AML. However, approximately 50% of individuals have CN-AML and, within this cohort, genetic features have important prognostic and therapeutic implications. Mutations in the FLT3 receptor are found in 30–40% of patients with CN-AML. While the FLT3-internal tandem duplication (ITD) is associated with a poor DFS and OS when compared to CN-AML patients with a wtFLT3R, the prognostic significance of the FLT3-tyrosine kinase domain (TKD) mutation is less clear. There is considerable debate over the best postremission treatment for CN-AML patients who have a FLT3ITD. Our primary objective was to determine the relationship between different postremission therapies and outcome in CN-AML patients with FLT3 mutations. Methods: We retrospectively reviewed the outcomes of newly diagnosed AML patients at the Massachusetts General Hospital and Dana-Farber Cancer Institute/Brigham and Women Hospital between 2002–2008 who were younger than 60 years of age, had a normal karyotype, and had a FLT3ITD and/or TKD mutation at diagnosis. The method of Kaplan and Meier was used to estimate DFS and OS; groups were compared using the log-rank test. A p-value less than .05 was interpreted as statistically significant. Results: At diagnosis, there were 37 patients with an ITD mutation (7 also had a TKD mutation) and 19 patients with an isolated TKD mutation at diagnosis. Patients who received an allogeneic SCT were not included in the analysis. ITD positive patients with an allelic ratio (AR) of mutant to wt FLT3 〉.2 had a significantly worse DFS and OS than those with an AR

Description: Abstract 2689 Introduction: Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia (AML) characterized by promyelocytes in the blood and bone marrow, coagulopathy, and characteristic translocation between chromosomes 15q22 and 17q21. Internal tandem duplication (ITD) mutations within the FMS-like tyrosine kinase receptor (FLT3) are found in approximately 30% of patients with APL. While cytogenetically normal AML patients with ITD mutations (especially with high ratio of mutant: WT allele) have a poorer prognosis due to high relapse rate, the prognostic value of ITD in APL is debated. We analyzed the clinical outcome of patients with APL to determine the relationship between FLT3 receptor status and prognosis. Materials and Methods: We conducted a retrospective analysis of all adult APL patients 〈 age 60 who were newly diagnosed and tested for FLT3R mutations at Massachusetts General Hospital and Dana-Farber Cancer Institute/Brigham and Women's Hospital between 2002 and 2008. Patient characteristics, complete remission (CR) rates, disease free survival (DFS), and overall survival (OS) were assessed by medical record review under an IRB-approved protocol. CR, DFS and OS were defined according to standard criteria. Kaplan-Meier method was used to estimate median DFS and OS and log-rank p-values were presented. We assessed the associations of clinical characteristics and the type of mutation patients had using Kruskal-Wallis tests. A p-value less than 0.05 was interpreted as statistically significant. Results and Discussion: We identified a total of 26 patients with APL for whom FLT3 mutation status was known. There were 13 patients with wtFLT3, 9 with ITD (1 also had TKD), and the remaining 4 had an isolated TKD mutation. Of the 13 patients with wt FLT3APL, 4 had prior chemotherapy and/or radiation. No patients in the FLT3ITD APL or FLT3TKD APL groups had prior chemotherapy or radiation. As we had only 4 patients had an isolated FLT3TKD mutation, we restricted our analyses to patients who had either wt FLT3 or FLT3ITD+/− TKD. Of the wtFLT3 APL patients, 9/13 were enrolled and treated on CALGB 9710. Of the remaining 4 patients, two were treated according to the PETHEMA regimen, one with a history of breast cancer and significant anthracycline exposure was treated with ATRA and AsO3, and the last patient died before therapy could be initiated. Of the 9 patients with FLT3ITD APL, 5 were treated on CALGB 9710, 2 were treated according to 9710 but off protocol, and 2 with the PETHEMA regimen. Baseline clinical characteristics were similar between the two groups including CR rates [92 (12/13) versus 100% (9/9) for wt and ITD, respectively]. Patients with the ITD had a lower fibrinogen at presentation than those with wt (103 vs. 235 mg/dl, p=.04). While patients with ITD appeared to have a higher WBC, it was not statistically significant (p=.1). However, patients with ITD had an inferior DFS compared to wt (Figure 1) (p=.01) while there was no significant difference in OS between the two groups (p=.39). Of the 5 patients with ITD who relapsed, 3 received AsO3 reinduction and autologous SCT, 1 with CNS recurrence received myeloablative allogeneic SCT, and the fifth died in relapse without treatment. The observation that FLT3ITD APL patients have a reduced DFS raises the possibility that APL therapy may be improved for this group of patients, possibly by incorporating FLT3 inhibitors. Disclosures: DeAngelo: Deminimus: Consultancy. Stone:Novartis: Consultancy.

Description: Abstract 472 We and others recently identified locus rearrangements involving the type I cytokine receptor subunit CRLF2 (also known as thymic stromal lymphopoietin receptor (TSLPR)) in 5–7% of all adult and pediatric B-cell precursor acute lymphoblastic leukemia (B-ALL). CRLF2 rearrangement places full-length CRLF2 under alternate transcriptional control, and can either result from an intrachromosomal CRLF2-P2RY8 deletion or from a CRLF2-IGH translocation. Prognosis for CRLF2-rearranged B-ALL is particularly poor, suggesting that a sizable fraction of relapsed and ultimately fatal B-ALL harbor CRLF2 rearrangements. Approximately 50% of CRLF2-rearranged B-ALL harbor mutations in JAK2 (and rarely JAK1) that cluster around JAK2 Arg683. A separate 15% have a CRLF2 F232C mutation that promotes constitutive homodimerization and signaling. In the remaining cases, the driver of CRLF2 signaling is not known. To identify additional genetic alterations that contribute to CRLF2-mediated leukemogenesis, we performed next-generation sequencing on 3 CRLF2-rearranged B-ALL specimens, including one with CRLF2 F232C (#536). Exome libraries were assembled from bone marrow specimens with greater than 90% B-ALL involvement and from paired remission bone marrows. Transcriptome sequencing was also performed on cDNA isolated from the involved marrow of patient #536 to: 1) confirm the findings from exome sequencing, 2) focus on transcribed genes and 3) identify possible RNA editing events. Sequencing utilized the SOLiD (Sequencing by Oligonucleotide Ligation and Detection; Applied BioSystems) platform, with a target recovery of greater than 40 million (transcriptome) and 25 million (exome) uniquely mapping reads per sample. Mutations were considered high confidence if a minimum of 3 individual reads identified the same mutation within the involved specimen and the mutation was not identified in any reads from the remission specimen (minimum 10 reads at that base-pair). Paired exome sequencing of germline and involved marrow specimens identified 129, 297 and 630 high-confidence, non-synonymous, somatic mutations in the three samples. Of the total 1056 mutations, 1023 (96.9%) were missense and the remaining 33 (3.1%) were nonsense mutations. The expected mutation in CRLF2 F232C (#536) was recovered, as was a novel JAK2 mutation from a sample previously thought to be JAK2 wild-type (#002). Comparison between transcriptome and exome sequence markedly reduced the number of potential “driver” alterations, i.e., nonsynonymous coding mutations expressed in the tumor but not present in the remission exome. For example, from the 129 somatic alterations in the involved exome from #536, only 15 were recovered from the involved transcriptome. Sequence alterations affected genes known to be involved in histone modification, oxygen metabolism, and steroid responsiveness. To our knowledge, none of the identified mutations have previously been described. One mutation in a gene involved in E2F transcription was observed in two of three cases. In addition, 2,428 coding sequence mutations were identified from the involved marrow transcriptome of #536 that were not present in the involved marrow exome, possibly indicating a high rate of RNA editing. Additional sequence analysis and molecular epidemiology of the novel sequence alterations in CRLF2-rearranged and —unrearranged B-ALL is underway. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 177 Current therapy cures only a fraction of adults with AML, and it is clear that new treatments, specifically targeted to the genetic or epigenetic lesions that cause the disease, are needed to improve outcome. While monoclonal antibodies against cell surface antigens have been effective therapeutics in other hematologic malignancies, there has only been limited success in AML, partly due to the sharing of many antigens with normal stem cells. We have previously shown that alternative splicing (AS) of genes is common in AML cells when compared to normal CD34+ cells, and here we report efforts to identify alternatively spliced genes encoding surface proteins that could yield novel antibody targets. In order to identify genes that are differentially spliced between AML and normal progenitor cells (NPC), we extended our original genome-wide AS study of 27 AML patient samples to include 123 AML patient samples, 8 NPC's, and 11 AML cell lines. For this study, samples were hybridized to the Affymetrix Human Exon 10ST array. Results were analyzed using commercially available Xray software from Biotique Systems. We identified 217 genes that were differentially spliced in 〉 35% of patients with AML compared to NPC. An average of 30 differentially spliced genes was observed in each individual patient. Splicing events in any given patients ranged from 10 to 50. Of the 217 commonly spliced genes, 33 genes were found to encode trans-membrane proteins. Three genes, NOTCH2, FLT3 and CD13, were selected for further study. First, the exon array results were validated by RT-PCR, qRT-PCR, and DNA cloning/sequencing from 10 patients. In each case, at least one aberrant splice form was identified that is predicted to encode one or more alternative extracellular regions of the protein. These AS events did not otherwise change the open reading frame of these genes. Two different splice variants of NOTCH2 (NOTCH2-Va and -Vb) were detected in more than 80% (P=0.0001) AML patients, but at undetectable or minimal levels in NPC's. Similarly, we detected three novel aberrant splice variants of FLT3 and CD13, which we have designated as FLT3-Va, -Vb, and -Vc and CD13-Va, -Vb, -Vc, at least one of which was detected in all AML patient samples tested, alone or with full length transcripts. The breakdown for the frequency of expression for these variants in AML patients are as follows: FLT3 -Va (68/123; 55.3%; P=0.001); -Vb (62/123; 50.4%; P=0.003); -Vc (14/123; 11.4%; P=0.15) and CD13-Va (73/123; 59.3%; P=0.001); -Vb (57/123; 46.3%; P=0.005); -Vc (34/123; 27.6%; P=0.042). None of these variants were observed in BM or PB samples from normal donors. We evaluated the expression frequency of these splice variants in patient groups with different FAB subtypes, as well as in patients groups with cytogenetically normal or complex AML. This analysis identified frequent expression of NOTCH2-Va splice variant in patients (more than 85% patients) with M0, M1, M2, M5 and M6 AML, while FLT3-Va and -Vb variants were detected in more than 50 % of patients diagnosed with M1, M2 and M5 AML. CD13-Va and -Vb variants were identified in more than 60 % of M6 AML, while CD13-Va is expressed more than 80% of M1 AML. Interestingly, the NOTCH2-Va splice variant is expressed in nearly 90% of patients with cytogenetically complex AML, while NOTCH2-Va, FLT3-Va, -Vb and FLT3-Va were expressed more than 60% of patients with normal karyotype AML. Moderate expression frequency of other splice variants has been observed in AML patients with different subgroups analyzed. Overall, our results from genome-wide AS analysis suggest that alternative splicing is a common event in AML, with some splice variants being detected in a significant number of different subgroups of patients. The clinical consequences and significance of this finding, as well as frequency of novel variant transcripts in large population of AML is currently the focus of further investigation. Screening and correlation analysis of additional 300 patients samples obtained at time of diagnosis as well as during relapse are in progress. Certain of the more common splice variants may generate new targets for the development of novel therapeutics. Disclosures: No relevant conflicts of interest to declare.