T-lineage Acute Lymphoblastic Leukemia Research Articles

Involvement of the cerebrospinal fluid cells in children with acute lymphoblastic leukemia: prognostic implications.

In this issue of the Revista Brasileira de Hematologia e Hemoterapia, Cancela et al.(1) analyzed the incidence and the risk factors for central nervous system (CNS) relapse in children and adolescents with acute lymphoblastic leukemia (ALL) treated using the GBTLI-ALL 99 protocol and found that a leukocyte count > 50 x 109/L at diagnosis was the only significant factor associated with a high incidence of CNS relapse. Leukemic infiltration of the CNS is defined as five or more leukocytes/mm3 and blast cells in cerebrospinal fluid (CSF) or cranial nerve palsy(2). It is a well-established prognostic factor in children with ALL(2-6). The cumulative incidence of CNS relapse (combined or isolated) presently varies from 2.6 to 9.5%(7). No clinical or morphological CNS involvement is evident at diagnosis in about 60% of patients who eventually develop CNS relapse(3-6,8-10). Similar to systemic chemotherapy in the treatment of systemic ALL, there is a growing concern to identify factors associated with increased risk of leukemia infiltration of the CNS and to consequently adapt the protocol aimed at the treatment and prophylaxis of neurological involvement(11). Several risk factors have been associated with a higher incidence of initialinvolvement or relapse in CNS, such as high risk genetic abnormalities [t(9;22) and mixed lineageleukemia (MLL) rearrangements], T-lineage ALL and high peripheral leukemic-cell burden(7,12). Some studies have also demonstrated that patients with any identifiable blast cells in CSF, or CSF contamination by blastic cells during traumatic lumbar puncture, present an increased risk of CNS relapse(13-15). Others however have not found this association(16-18). Conventional cytological analysis has proved useful, but the analysis of cells in CSF, especially with low cell counts, is more difficult than is widely admitted and it is not always conclusive(19). Molecular involvement detected by more sensitive and specific techniques such as PCR and direct sequencing has been shown in around 45% of pediatric patients; the prognostic impact of this molecular involvement seems to be dependent of the intensity of treatment(20,21). It is possible that morphological involvement of the CNS would represent one of the extremes of a clinical spectrum ranging from gross to minimal residual disease involvement of the CNS and that molecular involvement could reflect biologically more aggressive disease. The explanation for these discrepancies is probably related to the efficacy of systemic and CNS-directed therapy in different treatment regimens, suggesting that the poor prognosis associated with these variables can be overcome by more effective therapy(21).

Detection of Common Chimeric Fusion Transcripts in Acute Lymphoblastic Leukemia (ALL) Patients Using Multiplex Reverse Transcriptase Polymerase Chain Reaction Assay: A North Indian Tertiary Care Centre Experience

Abstract 4234 Introduction:Chromosomal abnormalities constitute an important parameter to identify prognostically relevant subgroups in acute lymphoblastic leukemia (ALL). Many western studies report the incidence of common chimeric fusion transcripts as 30–35%, however the data regarding the Indian patients is very scarce. Aims and Objectives:The aim of the present study was to detect presence of common chimeric fusion transcripts of t(12;21), t(9;22), t(1;19) and t(4;11) in adult and pediatric ALL cases using the Multiplex RT-PCR assay. Materials and Methods:This prospective study included 100 consecutive ALL cases diagnosed during last one year; 54 patients were <12 years of age. Diagnosis of ALL was made on the basis of bone marrow (BM) examination and flowcytometric immunophenotypic (FCM-IP) analysis. Approximately 2 ml of peripheral blood (PB) sample or 0.5 ml of BM sample was collected in EDTA. RNA extraction and cDNA synthesis was performed using commercial kits, according to standard protocols. RT-PCR assay was carried out using primers specific to the fusion transcripts of TEL-AML1 t(12;21); BCR-ABL t(9;22); E2A-PBX t(1;19) and MLL-AF4 t(4;11). The results of RT-PCR and FCM-IP were blinded and compared later. Results:Out of the 100 cases enrolled in the study, 54% were pediatric and 46% adult cases. Among the pediatric ALL cases, 49 were diagnosed as B-lineage ALL, 3 T-lineage ALL and 2 Biphenotypic ALL. Whereas among the adult ALL cases, 30 were diagnosed as B-lineage ALL, 12 T-lineage ALL and 4 Biphenotypic ALL. A total of 26% showed positivity for various fusion transcripts: 13% each pediatric and adult cases being positive. Of the 12 positive pediatric B-lineage ALL cases, 8 were positive for TEL-AML1 transcripts, 1 for BCR-ABL transcripts, 1 for E2A-PBX transcripts and 2 for MLL-AF4 transcripts. Out of the 11 positive adult cases, 7 were positive for BCR-ABL transcripts, 2 were positive for TEL-AML1 transcripts and 1 each for E2A-PBX transcripts and MLL-AF4 transcripts. BCR-ABL transcripts were detected in 1 out of 2 pediatric biphenotypic ALL and 2 out of 4 adult biphenotypic ALL cases. None of the pediatric cases was positive for the MLL-AF4 transcripts. None of the 4 transcripts were detected in any T-lineage ALL. Conclusion:Results of our study are comparable to those of other western and Asian studies, with TEL-AML1 transcripts being the most common fusion transcripts seen in pediatric B-ALL cases and BCR-ABL in adult cases. However two Indian studies had reported much lower incidence of TEL-AML1 and MLL-AF4 transcript in B-ALL cases. Identification of good prognostic groups and rationalization of therapy assumes great importance in our set ups as many patients are economically under-privileged. Relationship of these transcripts to the prognosis of our ALL patients will be evaluated in future. Disclosures:No relevant conflicts of interest to declare.

Phenomenon of Oligoclonality in Adult Patients with Acute Lymphoblastic Leukemia

Abstract 4895 Introduction:Minimal residual disease (MRD) in adult patients with acute lymphoblastic leukemia (ALL) can be analyzed by flow cytometry, fluorescent in situ hybridization (FISH) if there are chromosomal abnormalities, polymerase chain reaction (PCR) for chimerical transcripts and immunoglobulin heavy chain (IgH) and T-cell receptor gamma (TCRG) genes rearrangements. IgH and TCRG rearrangements are the for monitoring of MRD, because it is possible to find specific clonal tumor marker in 80–90% patients. According to the data of different research groups phenomenon of oligoclonality (more than one clonal rearrangement) is identified in 16–80% cases depending on how much molecular markers are used (Scrideli CA, 2001; Gameiro P, 2002; Thorsten Raff, 2006; Csinady E, 2009; Katsibardi K, 2011). Methods and materials:We studied bone marrow samples from 59 patients with new diagnosed B-lineage ALL (43 patients), T-lineage ALL (15 patients) and one patient with bilinear B-T-ALL. Ph-positive ALL was diagnosed in 6 patients, t(4;11) – in one patient. Patients were treated by standard ALL-protocols based on 8 weeks induction remission, 3–5 consolidation courses and maintenance treatment during 2 years. Patients with Ph-positive B-ALL were treated by the same chemotherapy with imanitinib and one patient with mature B-ALL was treated by NHL-BFM-90 chemotherapy. Detection of IgH and TCRG genes rearrangements were carried out by polymerase chain reaction using standard family-specific primers. Synthesis of patient specific primers was based on defined nucleotide sequence of clonal PCR-product. Monitoring MRD was realized by nested PCR using patient specific primers. Results:IgH and/or TCRG genes rearrangements were detected in 50 of 59 patients (84,7%). IgH complete rearrangements were found in 31of 43 patients (72,1%) with B-lineage ALL, IgH incomplete rearrangements were detected in 12 of 43 patient (27,9%) with B-lineage ALL and in one of 15 patient with T-lineage ALL. TCRG rearrangements were found in 13 of 15 patients (86,7%) with T-lineage ALL and in 24 of 43 patients (55,8%) with B-lineage ALL. Seventeen (34%) of 50 patients had one clonal marker. So, phenomenon of oligoclonality was revealed in 33 patients (66%). Two clonal rearrangements were detected in 23 cases, 3 rearrangements were detected in 7 cases and 3 patients had 4 rearrangements. In 3 of 27 patients, in whom all markers were followed, monitoring of MRD showed different pattern of tumor subclones elimination. Monitoring of MRD in patients who had also chromosomal abnormalities such as t(9;22), t(4;11) was carried out by FISH and PCR in Real-time. In one case the patient had 3 IgH rearrangements and chromosomal abnormality t(4;11) at the diagnosis. Later at various stages of chemotherapy we observed a different clearance of IgH rearrangements, however, when cytogenetic remission was achieved and MLL-AF4 expression was absent there were two of three IgH markers. This fact confirms the presence of several tumor subclones, their different clearance during the treatment and suggests persistence of tumor in this patient. Conclusion:In ALL patients with several subclones MRD monitoring should carried out with all markers, as we proved different subclone clearance suggesting different cells sensitivity to cytostatic drugs. Disclosures:No relevant conflicts of interest to declare.

Flow Cytometric Leukemic Blasts Detection in Cerebrospinal Fuid of Children with Acute Leukemias

Abstract 1449Central nervous system (CNS) involvement is one of the important risk factors in childhood acute leukemia (AL). Tumor cells detection in cerebrospinal fluid (CSF) is one of the main signs of CNS lesion. Traditionally blasts presence in CSF is assessed by conventional cytomorphology (CM) of cytospin slides. However, sensitivity of this method is relatively low. Flow cytometry (FC) having a higher sensitivity could provide better diagnostic applicability for CSF blasts detection. Aim. To compare results of tumor cells detection in CSF of children with AL by flow FC and CM. Methods. 183 samples from 52 boys and 31 girls aged from 5 months to 15 years with different types of acute lymphoblastic leukemia (ALL) (77 patients), acute myeloid leukemia (AML) (5 patients) and acute biphenotypic leukemia (1 patient) were investigated. 17 positive samples obtained by traumatic lumbar puncture were excluded from analysis because tumor blasts were also detected in peripheral blood. Comparison between FC and CM data was performed in 166 samples. Among these samples 61 was taken at the time of initial diagnostics, 34 – during AL follow-up, 17 – at relapse and 54 – during relapse monitoring. Monoclonal antibodies panels were constructed according to immunophenotype of tumor cells in bone marrow. Results. In 24 out of 166 samples (14.5%) tumor cells were detected by CM. In all these cases blasts were also found by FC, while FC allowed finding blasts in other 35 samples. Thus the total number of FC-positive samples was 59 out of 166 (35.5%). This frequency was significantly higher than rate of CM-positive cases (g < 0.0001). Among initial diagnostics samples there were 20 FC-positive and only 10 CM-positive patients (32.8% vs. 16.1%, p=0.0585). At relapse 9 (52.9%) patients were FC-positive, while 6 (35.3%) were CM-positive (p=0.4897). In both B-lineage and T-lineage ALL, analyzed separately, FC detected blasts in CSF frequently than CM (p=0.0098 and p=0.0002 respectively). Absolute blast count in 1 ml in CSF samples, positive by both methods was significantly higher than in samples, positive only by FC (median = 418, range 8–158171 and median = 34, range 5–2762 respectively, g = 0.0002). Thus FC allows detecting tumor cells in CSF much more frequently than conventional CM, which could be explained mainly by higher FC sensitivity. Moreover FC is applicable also for qualitative and quantitative monitoring of CNS lesion. Nevertheless prognostic impact of FC CSF investigation is questionable. Among 13 patients in whom discordant results were obtained in initial diagnostics samples and at relapse, only for one patient risk stratification could have been changed. For all other patients there were other risk factors, that decreased significance of FC leukemic blast detection in CSF. Conclusion. Flow cytometry allows more frequent detection of tumor blasts in CSF of children with AL, while prognostic significance of these findings is still unclear and needs to be confirmed in large prospective trials. Disclosures:No relevant conflicts of interest to declare.

Focal BTG1 Deletions Occur in Specific Precursor B-Cell Acute Lymphoblastic Leukemia Subtypes At Defined Hotspots Due to Aberrant V(D)J Recombination

Abstract 399Recurrent deletions in genes affecting key cellular pathways are a hallmark of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). To gain more insight into the mechanism underlying these deletions, we have studied the occurrence and nature of abnormalities in one of these genes, the B cell translocation gene 1 (BTG1), in a large cohort of pediatric BCP- and T-lineage ALL cases.The BTG1 gene was found to be exclusively affected by genomic microdeletions, which were detected in 65 out of 722 BCP-ALL patient samples (9%), but not in 109 T-ALL cases. Genomic sequencing of the entire BTG1 open reading frame (n=135) and the main coding second exon (n=158), as well as bisulphite sequencing of its promoter (n=25) provided no evidence for the presence of BTG1 point mutations or promoter methylation, respectively. Eight distinct deletion sizes (ranging from 101 to 557 kb) were identified, which all clustered at the telomeric site within a 30 bp stretch in the second and last exon of the BTG1 gene. The deletions resulted in almost identical truncations of the open reading frame and a concomitant loss of two conserved C-terminal protein interaction domains. Truncated BTG1 fusion transcripts specific for each type of deletion could be detected exclusively in the deletion-positive BCP-ALL cell lines and primary BCP-ALL samples, whereas wild-type BTG1 mRNA was expressed in both deletion-positive and -negative cases. The presence of V(D)J recombination signal sequences at both sites of virtually all deletions strongly suggests RAG1/RAG2-mediated recombination as the responsible mechanism. In line with these observations, BCP-ALL, but not T-ALL, cell lines showed increased levels of histone H3 trimethylation at lysine 4 (H3K4me3) at the BTG1 gene locus. This epigenetic mark is associated with actively transcribed loci and acts as a docking site for RAG2 binding, thereby facilitating V(D)J recombination.BTG1 deletions were found to be unevenly distributed between the different cytogenetic subgroups, being present in 19% (n=27/142) of the ETV6-RUNX1 (TEL-AML) and 26% (n=6/23) of the BCR-ABL1 positive cases, and in only 3% (n=5/160) of the hyperdiploid cases (P<0.001, P=0.003 and P=0.002, respectively). In addition, targeted copy number analysis of recurrently affected genes in ALL revealed that cases with BTG1 deletions more frequently harbor deletions of ETV6, RB1 and EBF1 (P=0.007, P<0.001 and P<0.001, respectively). Using a sensitive PCR-based screening assay, we identified (multiple) additional BTG1 deletions at the subclonal level in 19 of the 65 deletion-positive BCP-ALL cases (29%), and in 21 of the 89 deletion-negative BCP-ALL cases (24%), but not in the T-ALL cases (n=77) or bone-marrow samples from healthy donors (n=26). Similar to the clonal BTG1 deletions, these subclonal events were enriched in the ETV6-RUNX1 subgroup and absent in the hyperdiploid cases.In conclusion, our results indicate that BTG1 deletions act as ‘driver’ mutations in specific BCP-ALL subtypes, in which they can arise independently in multiple subclones in a locus that appears to be prone to aberrant RAG1/RAG2-mediated recombination events. Disclosures:No relevant conflicts of interest to declare.

MicroRNA Profiling Reveals Aberrant MicroRNA Expression in Adult ETP-ALL and Functional Studies Demonstrate a Role of Mir-222 in Acute Leukemia,

Abstract 3458 Introduction:Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup in pediatric acute T-lymphoblastic leukemia (T-ALL). ETP-ALLs originate from early thymic progenitors that retain stem cell features and are characterized by a specific immunophenotype and by a specific gene expression profile. Recently, we have characterized ETP-ALL as subgroup of early T-ALL in adults. To unravel the underlying mechanisms for the aberrant and distinct gene expression profile in ETP-ALL we now explored the expression of microRNAs (miRNAs) as gene regulators in ETP-ALL and further investigated their functional role in acute leukemia. Patients and methods: We screened expression levels of 667 miRNAs in newly diagnosed T-ALL patients (n=14: ETP-ALL n=8, non-ETP early T-ALL n=3, thymic T-ALL n=3) and 2 healthy controls (CD3 selected T cells) using a Taqman Low Density Array (TLDA; Applied Biosystems). The expression levels of miR-221 and miR-222 were validated in a large cohort of adult T-ALL (n=178: ETP-ALL n=66 and typical T-ALL n=112 including early-, thymic-, and mature T-ALLs) and healthy controls (n=6) by real-time-PCR (Taqman microRNA assays; Applied Biosystems). These patients were enrolled on the GMALL study 07/03. Functional analysis was performed by AMAXA transfection of pSUPER vector containing the miR-221 and miR-222 DNA sequences in Jurkat (T-lineage ALL) and KG1a (AML) cells. Viability was determined by cell proliferation reagent WST-1, and BrdU ELISA. Apoptosis was measured by annexin-V/7AAD staining with subsequent flow cytometric analysis. Results: In our screen 229 (34%) of the 667 miRNAs represented by the TLDA were expressed at detectable levels in at least two-thirds of the T-ALL samples and the controls. Of these we identified 55 miRNAs to be differentially expressed in T-ALL samples compared to healthy controls: 49 miRNAs were upregulated and 6 miRNAs were downregulated in the patient samples. Of these, miR-221 and miR-222 were the only miRNAs significantly upregulated in the ETP-ALL compared to the combined early and thymic T-ALL subgroups (8.0-fold, P<0.01; 5.0-fold, P<0.01, respectively). Therefore, we further validated miR-221 and miR-222 expression in a larger set of 178 T-ALL samples and observed a 3.3-fold higher expression of miR-221 (P<0.0001) and 3.9-fold higher expression of miR-222 (P<0.0001) in ETP-ALL (n=66) compared to typical T-ALL (n=112). For the correlation of miR-221 and miR-222 expression with molecular features, samples were divided into groups (low and high) according to the median of the miRNA expression levels. High miR-222 expression was associated with an immature phenotype of early T-ALL (P<0.01) and cell surface expression of CD34 (mean: 32%) compared to low miR-222 expressers (mean: 8%; P<0.01). Moreover, high miR-222 expression was associated with expression of myeloid markers CD33 (mean: 23% compared to 4% in low miRNA expressers, P<0.01) and CD13 (mean: 18% vs. 4%, P<0.01). Similar results were obtained for miR-221. In vitro studies revealed that overexpression of miR-222 inhibited proliferation of Jurkat (58% reduction of proliferation compared to cells transfected with the empty vector, P<0.001) and KG1a cells (50% reduction of proliferation compared to control cells, P<0.01) determined by WST-1. A reduction of the DNA synthesis was detected by BrdU incorporation after miR-222 overexpression in Jurkat (37% reduction compared to control cells) and in KG1a cells (54% reduction compared to control cells). Overexpression of miR-222 induced a 1.3- (P=0.02) and 3.0-fold (P<0.01) increase in apoptosis in Jurkat and KG1a cells, respectively. No significant changes were observed for miR-221 transfected cells in these functional assays. Conclusion: In summary, our study revealed aberrant expression of miRNAs in ETP-ALL, with miR-221 and miR-222 as the most overexpressed miRNAs. Functional analysis demonstrated that miR-222 impaired the proliferation and induced apoptosis, indicating a potential role for miR-222 in acute leukemia. Importantly, miR-222 targets such as the proto-oncogene c-KIT and the ETS1oncogene are contained in the gene expression profile of ETP-ALL. Thus, aberrant expression of miR-222 may directly impact leukemogenesis by altering expression of key oncogenes in acute leukemia. Disclosures:Goekbuget:Micromet: Consultancy.

EZH2 Mutations Can Be Detected in 23% of t(10;11)(p13;q14)/PICALM-MLLT10 Positive Acute Leukemias,

Abstract 3440The t(10;11)(p13;q14)/PICALM-MLLT10 (CALM-AF10) rearrangement is most frequently associated with T-lineage acute lymphoblastic leukemia/lymphoma (T-ALL), and is rarely observed in AML. The EZH2 gene, located on 7q36.1, is a highly conserved histone H3 lysine 27 methyltransferase that influences stem cell renewal. EZH2 mutations were observed in 10% of patients with myelofibrosis, myelodysplastic/myeloproliferative neoplasms, or chronic myelomonocytic leukemia. In a previous study, we had investigated AML patients for EZH2 deletions using FISH. 6/20 (30%) of cases had been detected to carry a deletion. Additionally, we had screened these 6 cases for molecular mutations in EZH2 (transcript-ID: ENST00000320356) using an amplicon-based deep-sequencing assay, and one of the 6 patients was harboring both an EZH2 deletion and an EZH2 mutation. More interestingly, this double-mutated case was carrying a PICALM-MLLT10 rearrangement. Therefore, in this study, we were interested to investigate an expanded cohort of 13 cases (T-lineage ALL and AML) harboring a PICALM-MLLT10 rearrangement. Our cohort comprised 12 adults and one pediatric patient (7 males, 6 females) and was characterized by a predominant T-cell origin: 11 patients had T-ALL, 1 patient had mixed phenotype T/myeloid acute leukemia, and 1 patient had AML. EZH2 alterations were detected in 3/13 (including the index case). In more detail, the EZH2 mutation carriers were characterized as follows: Patient #1 (male, 26 years, AML) had a splice site mutation in exon 14 with a mutation load of 13% in a cysteine-rich region. Patient #2 (male, 19 years, T-ALL) harbored a missense mutation (Phe136Leu) with a mutation load of 93%. Patient #3 (female, 53 years, T-ALL) showed three concomitant EZH2 missense mutations in exon 5: His120Gln, Tyr124His, and Gly150Arg. The mutation load detected was 17% for each alteration. A fourth patient had a 1459G>A base substitution (corresponding to Ala487Thr) which to our knowledge had not been described before. However, this alteration had to be interpreted as germline as it was still detectable in the remission state. In contrast, in an independent cohort of 12 patients with PICALM-MLLT10 negative T-ALL (7 females, 5 males) analyzed for comparison no EZH2 mutation was detected. Interestingly, in patients #2 and #3, the mutations were located in exon 5 in the region which interacts with the DNMT1, DNMT3A, and DNMT3B DNA methyltransferase genes (D1). Moreover, DNMT3A mutations were recently identified in patients with AML and MDS in association with poor outcomes. Therefore, we additionally performed investigation for DNMT3A mutations in all 13 patients with PICALM-MLLT10 positive leukemias but detected no mutation. To investigate further molecular associations, we analyzed these cases also for RUNX1 mutations and FLT3-ITD, but we did not detect any mutation in these molecular genes. Further, we compared the gene expression profiles of 8 patients with PICALM-MLLT10 positive T-ALL to the profiles of 21 PICALM-MLLT10 negative T-ALL patients. Hierarchical clustering revealed a distinct gene expression signature of the PICALM-MLLT10 positive cases. Significant upregulation was found for HOXA5 and HOXA9 genes. Other differentially overexpressed HOX were HOXA3, A4, A6, A7, A10. Genes with a function for cell differentiation and regulation of apoptosis (ZAK) as well as for signal transduction (AKT3) were significantly underexpressed. Subsequently, we compared the gene expression profiles of 2 EZH2 mutated patients to 6 EZH2 wild-type patients in the PICALM-MLLT10 positive cohort. By hierarchical clustering, both EZH2 mutated cases showed a distinct gene expression signature. Increased expression was observed for genes with a role for the regulation of transcription (ZNF207, KDM5B, or CASZ1) or for intracellular transport (SARB1). In summary, we detected EZH2 mutations in 3/13 cases in this series of PICALM-MLLT10 positive malignancies, comprising mostly T-ALL, but also AML or mixed phenotype acute leukemia. This further emphasizes a cooperative effect of EZH2 mutations with the PICALM-MLLT10 fusion in acute leukemias of different lineages. Disclosures:Grossmann:MLL Munich Leukemia Laboratory: Employment. Artusi:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Expression of Ikaros Isoform 6 in Chinese Children With Acute Lymphoblastic Leukemia

Ikaros is a zinc-finger transcription factor that plays an important role in the differentiation and proliferation of lymphocytes. Dominant-negative Ikaros isoform 6 (Ik6), one of its common subtypes, is overexpressed in leukemia patients and is associated with unfavorable prognosis in childhood B-cell progenitor acute lymphoblastic leukemia (ALL). This study was to identify specific isoforms, especially Ik6, in Chinese pediatric patients with ALL. The mRNA expression of Ikaros was detected in 88 children with previously untreated ALL by nested reverse transcription-polymerase chain reaction (RT-PCR). Sequencing of the PCR products was performed to identify specific isoforms. The expression of fusion genes was determined by using multiplex RT-PCR. The functional isoforms Ik1, Ik2/3, and dominant negative isoforms Ik4, 6, 8, 9, 10 identified by nested RT-PCR were further confirmed by sequence analysis. In the 88 cases, the Ik6 was found to be overexpressed in 8 of 70 cases of B-lineage ALL and in 1 of 18 cases of T-lineage ALL patients. Among Ik6 B-lineage ALL patients, 3 had expression of BCR/ABL fusion gene and 1 had HOX11 expression. Ik6 overexpression was independent of age, white blood cell count at diagnosis, risk group, and expression of the fusion genes currently measured in China except BCR/ABL (P<0.01). And it was strongly associated with elevated levels of minimal residual disease at day 28 (P<0.01). Ik6 can be included as a high-risk factor at diagnosis. In developing countries with limited resources, it can be economically detected by nested RT-PCR.

CREST maps somatic structural variation in cancer genomes with base-pair resolution

We developed 'clipping reveals structure' (CREST), an algorithm that uses next-generation sequencing reads with partial alignments to a reference genome to directly map structural variations at the nucleotide level of resolution. Application of CREST to whole-genome sequencing data from five pediatric T-lineage acute lymphoblastic leukemias (T-ALLs) and a human melanoma cell line, COLO-829, identified 160 somatic structural variations. Experimental validation exceeded 80%, demonstrating that CREST had a high predictive accuracy.

Childhood T-lineage acute lymphoblastic leukemia: Management and outcome at a tertiary care center in North India

To assess the clinical features, prognostic factors and outcome of childhood T-ALL in comparison with B-lineage ALL, treated with a uniform treatment regimen (MCP 841). Pediatric oncology division of a tertiary care institution in Northern India. Retrospective analysis of clinical data and survival outcome. 60 children with T-ALL and 139 with B- lineage ALL, and less than 15 years of age treated over 15 years. T-ALL was observed in 30%. High risk features at presentation (age >10 years, WBC >50,000/mm3, mediastinal mass, and CNS leukemia) were significantly more frequent in T-ALL as compared to B-lineage ALL (P=0.049, P<0.001, P<0.001 and P=0.02, respectively). Fifty five of 60 T-ALL patients (91.7%) achieved complete remission after induction therapy. There were 3 induction and 10 remission deaths while 11 (18.3%) relapsed. The overall survival and event-free survival of T-lineage ALL (61.5±7.6 and 49.9±7.4, respectively) were similar to that of B-lineage patients (68.7±4.7 and 47.1±5.1, respectively). National Cancer Institute risk groups emerged as significant prognostic factor for event free survival only in B-lineage patients. Even though high risk features were significantly more frequent in T-ALL, survival outcome was similar to that of B-lineage patients. None of the routinely described prognostic parameters significantly impacted survival.

Nelarabine May Be Safely Incorporated Into a Phase III Study for Newly Diagnosed T-Lineage Acute Lymphoblastic Leukemia: A Report From the Children's Oncology Group

AbstractAbstract 865With dose-intensified, multi-agent therapy, 80 to 85% of patients with T-lineage acute lymphoblastic leukemia (T-ALL) are cured. Nelarabine (Compound 506U78; IND# 52611) has shown impressive single agent clinical activity in relapsed T-ALL, but has been associated with significant neurotoxicity. We previously showed that Nelarabine could be added safely to an intensive multi-agent chemotherapy backbone in the COG AALL00P2 pilot study without excess neurotoxicity. The Phase III AALL0434 T-ALL study was designed to assess the safety and efficacy of adding Nelarabine to a COG-augmented BFM chemotherapy regimen. In the Safety Phase of the study, 600 patients with newly-diagnosed T-ALL were accrued from January 2007 to June 2010. Patients with high-risk T-ALL, defined by bone marrow (BM) minimal residual disease (MRD) level >1% or >5% BM blasts at day 29 of Induction, were randomized to receive backbone chemotherapy +/− five 5-day courses of Nelarabine 650 mg/m2/day once in Consolidation, once in Delayed Intensification and at the start of the first three Maintenance cycles. Patients were also randomly assigned to treatment arms that contained either high-dose methotrexate (HD-MTX) plus leucovorin rescue or Capizzi style escalating intravenous MTX without rescue during Interim Maintenance. Fifty-seven patients were assigned to the High-risk arm and were equally randomized to each of the MTX regimens +/− Nelarabine; their progress was followed for at least 8 weeks after receiving cranial radiation in the Delayed Intensification Phase (week 34) to assess for neurotoxicities. Toxicities were compared between patients who were randomized to regimens with Nelarabine (n= 28) and without Nelarabine (n= 29). There were no differences in the incidence of pre-defined targeted neurotoxicities of peripheral motor neuropathy (10 Nelarabine vs 6 no Nelarabine p= 0.38), sensory neuropathy (10 Nelarabine vs 7 no Nelarabine p = 0.78) and central neurotoxicity (0 Nelarabine vs 3 no Nelarabine; p=0.11 [two-tailed Fisher's Exact Test]). Additional toxicities including febrile neutropenia (24 vs 29 p= 0.058), and AST/ALT elevations (24 vs 20; p=0.36) were similar in the regimens with/without Nelarabine. The only difference in toxicities between the regimens was increased AST and ALT (unrelated to Nelarabine) within the escalating MTX treatment arms, as compared to arms containing HD-MTX (34 vs 10 p=0.003). Adverse events were reported for 8 of 28 patients on Nelarabine-containing and 7 of 29 patients on the non-Nelarabine containing treatment arms. There were two CNS adverse events reported for the 29 patients treated without Nelarabine. There were no CNS adverse events on the Nelarabine treatment arms. There were 3 PNS adverse events reported among the 28 patients that received Nelarabine: two events in the same patient included abdominal pain and constipation, and one patient had a transient Grade 2 motor neuropathy following a course of Nelarabine. These results show that the addition of Nelarabine to the backbone chemotherapy is safe and feasible. The AALL0434 Efficacy phase has now opened, and both intermediate and high-risk patients will be randomly assigned to all four treatment arms. The COG experience with Nelarabine demonstrates that this agent may have significant toxicity in heavily pre-treated T-ALL patients, but may be well-tolerated in newly diagnosed patients. AALL0434 will determine whether or not Nelarabine treatment improves outcome of children, adolescents and young adults with newly diagnosed T-ALL. Disclosures:Borowitz:genzyme: Research Funding; becton-dickinson: Research Funding; Alexion: Consultancy; beckman-coulter: Research Funding. Hunger:bristol myers squibb: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; eisai: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Antileukemic Activity of the HSP70 Inhibitor Pifithrin-μ

AbstractAbstract 3306 Introduction:Heat shock protein (HSP) 70 is aberrantly expressed in acute leukemias and other hematologic and solid malignancies, promoting tumor cell survival and therapy resistance. Recently, the small molecule pifithrin-μ (2-phenylethynesulfonamide) has been identified as a direct inhibitor of inducible HSP70, showing antiproliferative activity in different cell lines of solid tumors. Here, we analysed the in vitro antileukemic effect of pifithrin-μ in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) cell lines, as well as in primary AML blasts. In addition, incubations of pifithrin-μ with cytarabine, the histone deacetylase inhibitor SAHA, the HSP90 inhibitor 17-AAG, and the multikinase inhibitor sorafenib were performed to evaluate the potential use of combination therapies with pifithrin-μ in acute leukemias. Methods:Leukemic cell lines KG-1a (AML), K562 (CML in blast crisis), K562r (cytarabine-resistant K562), NALM-6 (B-lineage ALL), TOM-1 (B-lineage ALL, BCR-ABL pos.), Jurkat (T-lineage ALL), BE-13 (T-lineage ALL) and 9 bone marrow cell samples from newly diagnosed or relapsed AML patients were exposed to pifithrin-μ. Cell viability of all cell lines listed above was quantified by WST-1 assay. Subsequent functional analyses were performed on KG-1a and NALM-6 cells. Apoptosis was determined by annexin-V/7-AAD staining and subsequent flow cytometric analysis. Activated caspase-3 was detected by flow cytometry. Levels of the cell signaling kinase Akt were measured by intracellular staining and FACS analysis. Coincubations of pifithrin-μ with cytarabine, SAHA, 17-AAG or sorafenib were performed in KG-1a, NALM-6 and TOM-1, using WST-1 assays to analyse cytotoxic effects of combination therapies. Results:Pifithrin-μ at low micromolar concentrantions significantly inhibited viability of all acute leukemia cell lines tested, with IC50 values ranging from 2.5 to 12.7 μM independent of the differentiation lineage. Importantly, viability of both cytarabine-sensitive and -resistant K562 cells was effectively inhibited by pifithrin-μ. The median IC50 of primary AML blasts was 8.8 μM, ranging from 5.7 to 11.8 μM with no obvious differences regarding patients' clinical or genetic characteristics.Apoptosis was induced in a time- and dose-dependent fashion with a rate of specific apoptosis of 46% at 4 μM pifithrin-μ for NALM-6 and 36% at 40 μM pifithrin-μ for KG1a. In NALM-6, treatment with 3 μM pifithrin-μ for 24 hours resulted in a significant increase in the cleaved, active form of caspase-3, whereas in KG1a no increase in active caspase-3 was detected. Intracellular concentrations of Akt were markedly reduced after 12 hours incubation of NALM-6 with pifithrin-μ.In NALM-6, KG-1a, and TOM-1 combination treatment of pifithrin-μ at concentrations below the IC50 with either SAHA, 17-AAG or sorafenib resulted in a significant decrease of cell viability compared to corresponding monotherapy. Thus in NALM-6 combination of 2 μM pifithrin-μ with 0.6 μM SAHA inhibited viability by 73%, compared to 22% and 0% inhibition for either drug alone (p<0.05). Combination of 2 μM pifithrin-μ with 2 μM 17-AAG led to 58% inhibition, in contrast the monotherapy inhibited cell viability only in 31% for either drug alone. In NALM-6 and TOM-1, the combination of pifithrin-μ with cytarabine decreased viability significantly (47% and 55%, respectively), whereas the single agents were less effective (22% for 2 μM pifithrin-μ, 24% for 9 nM cytarabine in NALM-6; 26% for 3.5 μM pifithrin-μ and 41% for 40 nM cytarabine in TOM-1). Conclusion:This is, to our knowledge, the first report of the antileukemic effects of the HSP70 inhibitor pifithrin-μ. The inhibitor is highly active against all AML and ALL cell lines tested, including cytarabine resistant cell lines as well as primary leukemic cells. Effectivity of pifithrin-μ could even be increased in combination treatment with other antileukemic agents. Targeting HSP70 might be a promising new therapeutic approach for the treatment of acute leukemias to overcome drug resistance. Thus, our data might build a framework for future clinical trials. Disclosures:No relevant conflicts of interest to declare.

Targeting the Leukemia-Associated Hypoxic Microenvironment with Hypoxia-Activated Prodrug PR-104

AbstractAbstract 868Interactions between leukemia cells and the bone marrow (BM) microenvironment are known to promote leukemia cell survival and confer resistance to drugs commonly used in the management of this disease. We investigated the protective role of hypoxia in the BM microenvironment. We observed a marked expansion of hypoxic niches in the bone marrow of immunodeficient mice engrafted with the acute lymphoblastic leukemia (ALL) cell line Nalm6 and with primary ALL cells, as detected by the reductive 2-nitroimidazole compound pimonidazole (PIM), which forms stable adducts in hypoxic regions. We further demonstrated induction of the chemokine receptor CXCR4, and of the enzyme carbonic anhydrase 9 (CAIX), both targets of Hypoxia-Inducible Factor 1α (HIF-1a), in hypoxic areas of BM from mice harboring the Nalm6 xenografts. Furthermore, we used a CML blast crisis model to evaluate the time course of hypoxia expansion in the BM. In C57Bl6/J mice engrafted with murine HSCs co-expressing BCR/ABL and Nup98 we observed a time-dependent increase in PIM positive areas which coincided with the presence of GFP positive cells. In line with these findings, HIF-1α was highly expressed in BM biopsies from newly diagnosed ALL patients (n=15) but was significantly reduced when the patients achieved complete remission (CR). Culture under hypoxic conditions (1% O2) conferred resistance of pre-B ALL cells REH, Nalm-6, and of AML OCI-AML3 leukemic cells against several chemotherapeutic agents including vincristine, methotrexate and idarubicin (% of Annexin V(+) cells at 21% vs 1%O2; REH plus 1ng/ml vincristine:88.5+/−2.3 vs 18.1+/−10.3; REH plus 0.25uM etoposide: 86.4 +/−7 vs 14.4+/−8.6; Nalm6 plus 10ng/ml methotrexate: 33.4+/−2.7 vs 4.6+/−1; OCI-AML3 plus 50ng/ml idarubicin: 28.45+/−5 vs 12.76+/−1) . Taken together, these results provide rationale for examining the potential of hypoxia-activated pro-drugs to eliminate leukemia progenitor cells within hypoxic niches. To this end, we tested the hypoxia-activated prodrug PR104, a dinitrobenzamide nitrogen mustard that is reduced to its active metabolites under hypoxic conditions (Patterson et al., Clin Can Res 2007). In vitro, PR-104 induced cell death in three different leukemia cell lines (Nalm6, REH and the AML line Molm13) selectively under hypoxic (pO2 1%) conditions. The anti-leukemic efficacy of PR-104 as a single agent was next examined in several in vivo leukemia models. Administration of PR-104 prolonged survival and decreased leukemia burden of 1) NOD/Scid/IL2Rg-KO (NOG) mice injected with cells from primary refractory FLT3-mutated AML; 2) NOG mice injected with leukemic cells from an infant with MLL-rearranged B-lineage ALL; and 3) NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice injected with Nalm6-luciferase ALL cells. Next, we evaluated anti-tumor effects of PR-104 at clinically relevant doses (200, 100 and 50 mg/kg) and at maximal tolerated dose (550 mg/kg) in two ALL xenograft models (a T-lineage ALL and a B-cell precursor ALL) (Figure 1, studies supported by NCI NO1CM42216 and by PPTP contract NO1-CM91001-03). Compared to vehicle control, PR-104 significantly delayed progression of the T-ALL xenografts at all doses tested and at three of the four doses (550, 200 and 100 mg/kg) of B-cell pre-ALL, which resulted in significantly increased event-free survival (EFS) of mice in the treatment groups.Altogether, these findings strongly suggest that targeting hypoxia is feasible. If successful, this approach may significantly impact leukemia therapy and ultimately improve patient survival. This concept is currently being tested in an ongoing Phase I clinical trial of PR-104 in relapsed/refractory AML patients. [Display omitted] Disclosures:Wilson:PROACTA: Equity Ownership. Konopleva:PROACTA: PI on clin trial funded by Proacta.

Longer Time to the Start of Continuation Therapy Is Associated with Improved Survival In High Risk Pediatric Acute Lymphoblastic Leukemia (ALL): A Report From the Children's Oncology Group (COG)

AbstractAbstract 3223 Background:Delays in treatment are common in pediatric ALL and may occur more often in high risk ALL (HR-ALL). Delays during maintenance therapy have adverse impact on survival, but the significance of delays prior to maintenance is unknown (Schmiegelow, et al, American Journal of Pediatric Hematology Oncology 1990 and Dibenedetto et al, Pediatric Hematology Oncology 1994). We studied the effect of delays in therapy prior to maintenance by analyzing data from two HR-ALL clinical trials, CCG1961 and POG9906. The objective was to determine the association between time to maintenance (TTM) from starting induction therapy and event-free survival (EFS) in children with high-risk ALL. Methods:CCG1961 enrolled 2078 patients from September 1996 to May 2002 with B- and T-lineage ALL with high-risk features, defined as those aged 1–9 years with initial white blood cell count (WBC) ≥ 50,000/μl, any patient aged ≥ 10 years and ≤ 21 years, and those with central nervous system (CNS) leukemia or overt testicular leukemia. Rapid early responders (RERs), those with < 25% blasts in the bone marrow on day 7 of therapy, were randomized to receive standard or augmented BFM therapy with either a single interim maintenance and delayed intensification phase (SDI) or two interim maintenance and delayed intensification (DDI) courses as outlined in Table 1. Slow early responders (SERs) were randomized to augmented BFM with DDI with either doxorubicin or idarubicin and cyclophosphamide in the DI courses. POG9906 enrolled 276 patients from March 2000 to April 2003 with high-risk B-precursor ALL, defined as all patients with CNS disease, testicular disease, MLL rearrangement, age ≥ 16 years, or WBC ≥ 100K. In addition, it included selected patients aged 12–15 with high-risk disease based on the Shuster criteria for age and initial WBC. Patients with favorable cytogenetic features (ETV6-RUNX1 fusion or trisomy of chromosomes 4 and 10) were excluded unless they had CNS or testicular leukemia. Patients were treated with augmented BFM with DDI. Patients who made it to start of maintenance are included in this report. TTM was calculated as time from start induction until start of maintenance therapy. Expected TTM for those with B-lineage ALL is shown in Table 1. TTM was dichotomized by plotting the Akaike information criterion (AIC) versus all possible cutoff values of TTM and identifying the cut-point which best discriminated between groups who did and did not fail. The identified threshold was then examined as a predictor of EFS using the Kaplan Meier method and log rank test. The outcomes were 5-year EFS for CCG1961 and 4-year EFS for POG9906. Results:Among the B-lineage patients, longer TTM was significantly associated with improved EFS in CCG1961 Std BFM DI and SER Aug BFM Doxo arms, as well as on POG 9906. For example, in Std BFM DI, 5-year EFS was 83% in those with longer TTM and 71.9% in those with shorter TTM (P=.036). Among the T lineage cohort enrolled on CCG1961, similar results were seen with longer TTM being significantly associated with improved EFS in Std BFM DDI and SER Aug BFM Ida, and a trend toward improved EFS with longer TTM in each of the other arms. Conclusions:These data suggest that longer TTM may be associated with superior EFS in pediatric high-risk ALL. Multiple factors may contribute to this association. For instance, pharmacogenetic differences resulting in more treatment toxicity and delays may also be associated with more potent anti-leukemic effects, or patients with infectious complications may have an endogenous immune response that also has an anti-leukemia effect. Further research is needed to validate the association between TTM and EFS and to characterize the timing and causes of these delays in protocol therapy. Disclosures:No relevant conflicts of interest to declare.

Akt Activation Is Important In KRAS-Mediated Multistep Leukemogenesis

AbstractAbstract 4200Activating mutations of Ras are found in approximately 30% of all human malignancies, and 85% of these mutations are in the K-ras isoform. These mutations dysregulate cell cycle progression, proliferation and apoptosis, and have been implicated in both initiation and maintenance of tumors. While mutant K-ras thus represents an attractive therapeutic target, attempts to develop a Ras inhibitor have been unsuccessful to date. K-Ras has three canonical downstream effector pathways: MEK/Erk, PI3K/Akt, and Ral. One or more of these pathways may represent an alternative drug target for Ras-driven malignancies, but it is not yet understood how each of these pathways contributes to leukemogenesis. Partial loss-of-function Ras mutations have been identified that render oncogenic K-Ras (K-RasG12D) unable to interact with one or more downstream effectors. Our lab has previously demonstrated that one such partial loss-of-function mutation, K-RasG12D,Y64G, does not activate the PI3K/Akt pathway. Mice transplanted with hematopoietic progenitor cells transduced with MSCV vectors encoding K-RasG12D,Y64G develop an aggressive T-lineage acute lymphoblastic leukemia (T-ALL) with a median survival of 112 days (Shieh and Shannon, Blood (ASH Annual Meeting Abstracts) 2007 110: Abstract 1617).To determine if the “missing” Ras effector pathway is deregulated during multistep tumorigenesis, we generated cell lines from K-RasG12D,Y64G leukemias (n=6). Western blot analysis revealed low Ras levels and absent PTEN protein expression in 5 of 6 K-RasG12D,Y64G leukemia cell lines. Quantitative PCR analysis revealed reduced PTEN mRNA levels in these cell lines, which was not due to somatic Pten mutations. As expected, cell lines without detectable PTEN showed high pAkt levels that persisted during serum and cytokine deprivation. One K-RasG12D,Y64G leukemia cell line was remarkable because it contained high levels of Ras and retained PTEN expression. DNA sequence analysis of this cell line unexpectedly revealed both the Y64G substitution and a de novo in frame insertion of two amino acids (arginine and aspartic acid) within the switch II domain of K-Ras, between codons 69 and 70. This insertion was also identified in the primary T-ALL. Murine fetal liver cells engineered to express K-RasG12D,Y64G, 69RN70 induced a dramatic pattern of hypersensitive progenitor growth characterized by cytokine-independent colony formation and large and aberrant CFU-GM morphology in the presence of GM-CSF that was indistinguishable from cells expressing K-RasG12D. Phospho-FACS analysis of these cells revealed markedly increased expression of pAkt when compared to cells expressing K-RasWT or K-RasG12D,Y64G, and similar to pAkt levels in cells expressing K-RasG12D. Preliminary in silico structural analysis of K-RasG12D,Y64G,69RN70 suggests that this novel insertion may restore a critical salt bridge between K-Ras and PI3Kγ.These data suggest that K-RasG12D oncogenes defective in PI3K signaling are still able to cause dysregulated growth of hematopoietic cells in vitro and in vivo via the acquisition of additional mutations that restore signaling through the PI3K pathway, and strongly support simultaneously targeting multiple downstream effector pathways as a general therapeutic strategy for the substantial fraction of human cancers that contain RAS mutations. Disclosures:Bollag:Plexxicon, Inc: Employment, Equity Ownership, Patents & Royalties.

Integrated Genetic and Epigenetic Analysis of Childhood Acute Lymphoblastic Leukemia Reveals a Synergistic Role for Structural and Epigenetic Lesions In Determining Disease Phenotype

AbstractAbstract 537Acute lymphoblastic leukemia (ALL), the commonest childhood malignancy, is characterized by recurring gross and submicroscopic structural genetic alterations that contribute to leukemogenesis. Disordered epigenetic regulation is a hallmark of many tumors, and while analysis of DNA methylation of limited numbers of genes or ALL samples suggests epigenetic alterations may also be important, a large-scale integrative genome-wide analysis evaluating DNA methylation in ALL has not been performed. Here, we report an integrated epigenomic, transcriptional and genetic analysis of 167 childhood ALL cases, comprising B-progenitor ALL with hyperdiploidy (N=26), ETV6-RUNX1 (N=27), TCF3-PBX1 (N=9), BCR-ABL1 (N=19), rearrangement of MLL (MLLr) (N=20), rearrangement of CRLF2 (N=11, CRLF2r), deletion of ERG (N=11), miscellaneous or normal karyotype (N=14), and T-lineage ALL (N=30), including 4 MLLr cases and 8 cases with early T-cell precursor immunophenotype. Genome-wide profiling of structural DNA alterations was performed for all cases using Affymetrix 500K and SNP 6.0 arrays. Affymetrix U133A gene expression profiling data was available for 154 cases. Genome-wide methylation profiling was performed using the HELP microarray assay, which measures methylation at approximately 50,000 CpGs distributed among 22,722 Refseq promoters. Methylation data was compared to that of normal pro-B (CD34+CD19+sIg-), pre-B (CD34-CD19+sIg-) and mature B (CD34-CD19+sIg+) cells FACS-sorted from bone marrow of 6 healthy individuals. Unsupervised hierarchical clustering of the top 4043 most variable methylation probesets identified 9 B-ALL clusters with significant correlation to specific genetic lesions including ETV6-RUNX1, MLLr, BCR-ABL1, CRLF2r, TCF3-PBX1 and ERG deletion. T-ALLs and hyperdiploid B-ALLs also defined specific DNA methylation clusters. Supervised analysis including limma and ANOVA identified distinct DNA methylation signatures for each subtype. Notably, the strength of these signatures was subtype dependent, with more differentially methylated genes observed in ALL cases with genetic alterations targeting transcriptional regulators (e.g. ETV6-RUNX1 and MLLr) and fewer genes in cases with alterations deregulating cytokine receptor signaling (e.g. CRLF2r). Aberrant DNA methylation affected specific and distinct biological processes in the various leukemia subtypes implicating epigenetic regulation of these pathways in the pathogenesis of these different forms of ALL (e.g. TGFB and TNF in ERG deleted leukemias; telomere and centriole regulation in BCR-ABL1 ALL). Aberrantly methylated genes were also enriched for binding sites of known or suspected oncogenic transcription factors that might represent cooperative influences in establishing the phenotype of the various B-ALL subtypes. Most importantly, an integrated analysis of methylation and gene expression of these ALL subtypes demonstrated striking inversely correlated expression of the corresponding gene transcripts. The methylation signatures of each subtype exhibited only partial overlap with those of normal B cells, indicating that the signatures do not simply reflect stage of lymphoid maturation. In a separate approach, we discovered that 81 genes showed consistent aberrant methylation across all ALL subtypes, including the tumor suppressor PDZD2, HOXA5, HOXA6 and MSH2. Inverse correlation with expression was confirmed in 66% of these genes. These data suggest the existence of a common epigenetic pathway underlying the malignant transformation of lymphoid precursor cells. Integrative genetic and epigenetic analysis revealed hypermethylation of genes on trisomic chromosomes that do not show increased expression, suggesting that epigenetic silencing may control genes within amplified regions and explain why only selected genes are overexpressed. Finally, analysis of individual genes targeted by recurring copy number alterations in ALL revealed a subset of genes also targeted by abnormal methylation, with corresponding changes in gene expression (e.g. ERG, GAB1), suggesting that such genes are inactivated far more frequently than suggested by genetic analyses alone. Collectively, the data support a key role of epigenetic gene regulation in the pathogenesis of ALL, and point towards a scenario where genetic and epigenetic lesions cooperatively determine disease phenotype. Disclosures:No relevant conflicts of interest to declare.

Targeting Human B-Precursor Acute Lymphoblastic Leukemia Cells with Recombinant Human CD19 Ligand

AbstractAbstract 599CD19 is a 95-kDa B-lineage restricted receptor molecule that functions as a key regulator of transmembrane signals in both B-cells and B-cell precursors. Here we report the cloning and characterization of a novel high-mobility group (HMG)-box protein as the membrane-associated natural ligand of human CD19 receptor (CD19-L) on human immature as well as mature lymphoid cells. We cloned the gene encoding CD19-L from a human thymus cDNA library by yeast two-hybrid screening using the cDNA encoding human CD19 extracellular domain (AA 1 to 273) (CD19ECD) fused to the GAL4 DNA binding domain as the bait plasmid. The cDNA for the surface membrane-associated CD19-L protein is 2290-bp in length encoding a 487-aa protein with a predicted molecular mass of 54-kDa. The comparison of the amino acid sequence of CD19-L protein with reported sequences in Genebank Database revealed that CD19-L is a new member of the HMG-box protein family. CD19-L contains two leucine-rich hydrophobic nuclear export signal (NES) motifs associated with unconventional ER- and Golgi-independent transport of nuclear/cytoplasmic secretory proteins to the surface membrane. Expression of the CD19-L gene expression is limited to the lymphocyte compartment within the human lymphohematopoietic system and particularly abundant in T-lineage cells. CD19-L displays abundant expression on immature double-positive (DP) thymocytes as well as leukemic T-cell precursors from T-lineage ALL patients corresponding to immature double-negative (DN) pro-thymocyte and DP cortico-thymocyte stages of human T-cell ontogeny. CD19-L is also expressed on B-lineage lymphoid cells at all stages of human B-cell ontogeny, including fetal liver derived biphenotypic CD2+CD19+ pro-B/T cells, pro-B cells, pre-B cells and mature B-cells. Soluble recombinant human CD19-L protein produced in a baculovirus expression system exhibited exquisite specificity for the extracellular domain of CD19 and had profound effects on apoptosis-related signaling and gene expression in CD19+ human leukemia cells. Engagement of CD19 co-receptor on B-lineage ALL cells with soluble CD19-L protein perturbed CD19-associated signaling network and triggered tyrosine phosphorylation of CD19 in a time-dependent fashion with peak phosphorylation occurring within 1–5 min. CD19-phosphorylation was associated with rapid and transient activation of SYK tyrosine kinase. Treatment of B-lineage ALL cells with 100 ng/mL CD19-L for 24 h corrupted the regulation of gene expression and altered the expression levels of 13 genes directly involved in regulation of apoptosis. We next examined the ability of CD19-L to induce apoptosis in leukemic B-cell precursors from chemotherapy-resistant CD19-positive human B-lineage ALL cell lines NALM-6 (pre-B ALL), RS4;11 (MLL-AF4+ Pro-B ALL), and ALL-1 (BCR-ABL+ Pre-pre-B ALL). CD19-L (but not control proteins CD19ECD or CD19ICD) caused apoptosis in each of these 3 ALL cell lines. As CD19-L specifically targets CD19ECD, excess CD19ECD protein was able to compete with surface CD19 receptor for CD19-L binding and thereby prevent CD19-L induced apoptosis. Excess CD19 intracellular domain protein (CD19ICD) that was included as a negative control did not affect CD19-L induced apoptosis. CD19-L was also capable of causing apoptosis in chemotherapy-resistant primary leukemic cells from relapsed CD19+ B-lineage ALL patients. This collection of experimental data provides compelling evidence that CD19-L is a potent biotherapeutic new agent candidate against CD19+ lymphoid malignancies. The identification of human CD19-L may lead to therapeutic innovation for B-lineage ALL as well as other B-lineage lymphoid malignancies by providing an effective alternative to CD19-directed monoclonal antibody-based biotherapeutic agents that have encountered several limitations in clinical settings. Disclosures:No relevant conflicts of interest to declare.

Clinical relevance of FLT3 receptor protein expression in Indian patients with acute leukemia

FLT3 is a receptor tyrosine kinase that plays an important role in the pathogenesis of leukemia. The present study aimed to evaluate the role of FLT3 protein in patients with acute leukemia. FLT3 protein was quantified by flow cytometry on leukemic blasts using CD135 antibody in 160 patients with acute leukemia. We demonstrated FLT3 protein expression (>20%) in 82% of acute myeloid leukemia (AML), 60% of B-lineage acute lymphoblastic leukemia (B-ALL), 23% of T-lineage acute lymphoblastic leukemia (T-ALL) and 80% of biphenotypic leukemia. Further, FLT3 expression was seen to be significantly higher in AMLM2, M4, and M5 than in AMLM3. In B-ALL, FLT3 was found to be higher in pro-B-ALL and lower in early B-ALL. A CD34 expression >20% was associated with FLT3 positive B-ALL. When correlated with disease status, all patients in the relapsed AML group had FLT3 > 20% at diagnosis. Unlike AML, the relapsed group of B-ALL showed a lower incidence of FLT3 than the remission group. In summary, our data imply that there is frequent overexpression of the FLT3 protein in AML and B-ALL patients of Indian origin. In future, the FLT3 protein level may be used to select patients for whom FLT3 inhibitor therapy may be indicated.

PHF6 mutations in T‐lineage acute lymphoblastic leukemia

<i>PHF6</i> mutations in T‐lineage acute lymphoblastic leukemia

Identification of new markers discriminating between myeloid and lymphoid acute leukemia

Background: The heterogeneity of acute myeloid leukemia (AML) with respect to biology and clinical course resides in the fact that patients belonging to the same group show marked differences in their response to chemotherapy, necessitating a refinement of AML classification.Methods: In order to define molecular markers for AML, we performed microarray analysis on peripheral blood cells from two M5 AML patients, and selected four differentially expressed genes to validate their expression by real-time quantitative PCR (RT-PCR).Results: We have shown that two downregulated genes in AML, those encoding guanine nucleotide-binding protein γ11 (GNG11) and amphiregulin (AREG), are also downregulated in B-lineage acute lymphoblastic leukemia (B-ALL) and T-lineage acute lymphoblastic leukemia (T-ALL) patients. A second gene, that encoding ceruloplasmin (CP), is upregulated in AML but not in B-ALL and T-ALL. The level of expression of these genes varies from one patient to another.Conclusion: Since the number of patients studied is limited, further studies are needed with a larger series of patients to evaluate the potential utility of GNG11, AREG and CP as molecular markers for AML subtype classification. Our study is the first to analyze these genes in AML, B-ALL, T-ALL and chronic leukemia (myeloid and lymphoid) patients by RT-PCR. This rapid and sensitive method could be used to screen these genes in different types of leukemia.