MLL Gene Rearrangement Research Articles

Genetic and clinical characterization of 45 acute leukemia patients with MLL gene rearrangements from a single institution

Chromosomal rearrangements affecting the MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemia. In this study, conventional cytogenetic, fluorescence in situ hybridization, and molecular genetic studies were used to characterize the type and frequency of MLL rearrangements in a consecutive series of 45 Portuguese patients with MLL-related leukemia treated in a single institution between 1998 and 2011. In the group of patients with acute lymphoblastic leukemia and an identified MLL fusion partner, 47% showed the presence of an MLL–AFF1 fusion, as a result of a t(4;11). In the remaining cases, a MLL–MLLT3 (27%), a MLL–MLLT1 (20%), or MLL–MLLT4 (7%) rearrangement was found. The most frequent rearrangement found in patients with acute myeloid leukemia was the MLL–MLLT3 fusion (42%), followed by MLL–MLLT10 (23%), MLL–MLLT1 (8%), MLL–ELL (8%), MLL–MLLT4 (4%), and MLL–MLLT11 (4%). In three patients, fusions involving MLL and a septin family gene (SEPT2, SEPT6, and SEPT9), were identified. The most frequently identified chromosomal rearrangements were reciprocal translocations, but insertions and deletions, some cryptic, were also observed. In our series, patients with MLL rearrangements were shown to have a poor prognosis, regardless of leukemia subtype. Interestingly, children with 1 year or less showed a statistically significant better overall survival when compared with both older children and adults. The use of a combined strategy in the initial genetic evaluation of acute leukemia patients allowed us to characterize the pattern of MLL rearrangements in our institution, including our previous discovery of two novel MLL fusion partners, the SEPT2 and CT45A2 genes, and a very rare MLL–MLLT4 fusion variant.

State of Chromosome 11q23 in T-ALL/LBL and Their Relation to Prognosis

The purpose of this study is to investigate the mixed lineage leukemia gene (MLL, located on chromosome 11q23) expression in T acute lymphoblastic leukemia (T-ALL)/lymphoblastic lymphoma (LBL) and its relationship to prognosis. Fifty cases of T-ALL/LBL with clinical data were selected from the Shanxi Cancer Hospital in China. The immunohistochemical EnVision method was used for the expression of CD3, CD7, CD10, CD20, CD23, CD43, CD45RO, CD99, terminal deoxynucleotidyl transferase, myeloperoxidase and ki67. Fluorescent in situ hybridization for MLL gene expression was performed on paraffin-embedded tissue. Among the 50 cases of T-ALL/LBL, the percentages of tumor cells expressing terminal deoxynucleotidyl transferase, CD99, CD3, CD7, CD10, CD43, and CD45RO were 92.0%, 96.0%, 72.0%, 92.0%, 34%, 60.0%, and 40.0%, respectively, whereas myeloperoxidase, CD20, and CD23 were all negative. A level of Ki67 expression >80% was found in 18 cases and ≤80% in 32 cases. The period of follow-up ranged from 1 to 108 months. The overall survival rate was 35.8%, with a median survival time of 330 days. Breakage of 11q23 was detected in 8 (16.00%) and amplification in 14 (28.00%) of the 50 cases. The rate of amplification in stage III-IV was higher than that in stage I-II (P<0.05). The prognosis in the 11q23 breakage group was worse than that in the nonbreakage group (P<0.05). The prognosis in the 11q23 amplification group was also worse than that in the nonamplification group (P<0.05). MLL gene rearrangement is a new subgroup concerned with prognosis in T-ALL/LBL. Both breakage and amplification of 11q23 in T-ALL/LBL might play important roles in the development and progression of T-ALL/LBL.

Minimal Residual Disease–Guided Treatment Deintensification for Children With Acute Lymphoblastic Leukemia: Results From the Malaysia-Singapore Acute Lymphoblastic Leukemia 2003 Study

To improve treatment outcome for childhood acute lymphoblastic leukemia (ALL), we designed the Malaysia-Singapore ALL 2003 study with treatment stratification based on presenting clinical and genetic features and minimal residual disease (MRD) levels measured by polymerase chain reaction targeting a single antigen-receptor gene rearrangement. Five hundred fifty-six patients received risk-adapted therapy with a modified Berlin-Frankfurt-Münster-ALL treatment. High-risk ALL was defined by MRD ≥ 1 × 10(-3) at week 12 and/or poor prednisolone response, BCR-ABL1, MLL gene rearrangements, hypodiploid less than 45 chromosomes, or induction failure; standard-risk ALL was defined by MRD ≤ 1 × 10(-4) at weeks 5 and 12 and no extramedullary involvement or high-risk features. Intermediate-risk ALL included all remaining patients. Patients who lacked high-risk presenting features (85.7%) received remission induction therapy with dexamethasone, vincristine, and asparaginase, without anthracyclines. Six-year event-free survival (EFS) was 80.6% ± 3.5%; overall survival was 88.4% ± 3.1%. Standard-risk patients (n = 172; 31%) received significantly deintensified subsequent therapy without compromising EFS (93.2% ± 4.1%). High-risk patients (n = 101; 18%) had the worst EFS (51.8% ± 10%); EFS was 83.6% ± 4.9% in intermediate-risk patients (n = 283; 51%). Our results demonstrate significant progress over previous trials in the region. Three-drug remission-induction therapy combined with MRD-based risk stratification to identify poor responders is an effective strategy for childhood ALL.

Abstract 1422: High FLT3 mRNA expression without FLT3-ITD in infants with acute myeloid leukemia

Abstract Introduction: When compared to older patients, infants with acute leukemia exhibit high frequency of MLL gene rearrangement (MLL-R), and are known to have higher vulnerability to intensive cytotoxic therapy, such as hematopoietic stem cell transplantation (HSCT). In contrast to acute lymphoblastic leukemia (ALL) in infants, there have been a few reports on acute myeloid leukemia (AML) in infants including our group (A. Shimada et al, ASH 2011). We and other group reported that MLL-R had the clinical impact in infant ALL, however, not in infant AML. Infant ALL with MLL-R expressed high FLT3 mRNA, on the other hand, the importance of FLT3 mRNA remains to be obscure in infant AML. Patients: Forty four infants with AML, aged less than 1 year at diagnosis, registered in JPLSG AML-05 from 2006-2010. Patients with Down syndrome were excluded. The most frequent FAB classification type was M7 (18/44, 40.9%), followed by M5 (15/44, 34.1%), and M4 (4/44, 9.0%). Total 16 of 44 (36.4%) infant AML patients had MLL-R, 9 patients were detected by G-banding method, and other 7 patients were by FISH analysis or RT-PCR. There was not t(8;21), t(15;17) except for one inv(16) patient. Methods: FLT3 mRNA expression and internal tandem duplication (ITD) were analyzed in the first diagnostic bone marrow samples by RQ-PCR and PCR. Results: No patients had FLT3-ITD, however, higher FLT3 mRNA expression was found in MLL-R patients (n=16) (median 623,150 copies/μgRNA) compared with MLL-wild type (WT) (n=28) (median 25,105) (p&amp;lt;0.005). This phenomenon was also observed in 12-23 months AML patients. Discussion: High FLT3 mRNA expression was reported in infant ALL with MLL-R and it can be a molecular target for FLT3 inhibitor. On the other hand, there was no report in infant AML. Our previous data suggested that SCT had the limited efficacy for infant AML, FLT3 inhibitor might be considered to be used for infant AML with MLL-R in future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1422. doi:1538-7445.AM2012-1422

Mature B‐cell acute lymphoblastic leukaemia associated with a rare MLL‐FOXO4 fusion gene

A 6-week-old female infant presented with pallor, hepatosplenomegaly and a leucocyte count of 329 9 10/l (91% blasts). Light microscopy of the peripheral blood film showed blasts with rounded nuclei and scanty cytoplasm. No intracytoplasmic vacuoles were present (top left). Immunophenotyping by flow cytometry using peripheral blood (bottom panels) showed expression of CD10, surface membrane immunoglobulin (mu heavy chain and kappa light chain) with no expression of nuclear terminal deoxynucleotidyl transferase or CD34, indicating mature B-cell leukaemia. Cytogenetic studies showed an abnormal karyotype, 46,XX, t(X;11)(q13;q23), suggesting translocation between the MLL gene on chromosome 11 and the FOXO4 gene on chromosome X (top middle). Interphase fluorescence in situ hybridization using dual-colour break-apart probes showed the presence of an MLL gene rearrangement (top right). She was treated with a protocol designed for precursor B-cell acute lymphocytic leukaemia. However, she succumbed to sepsis in the fifth week of induction therapy.

Heterogeneity in Infants with Acute Myeloid Leukemia: Retrospective Analysis of a Japanese Nationwide Survey

Abstract 1477 Introduction:When compared to older patients, infants with acute leukemia exhibit distinct cytogenetic features, such as higher prevalence of MLL gene rearrangement (MLL-R), and are known to have higher vulnerability to intensive cytotoxic therapy, such as hematopoietic stem cell transplantation. In contrast to acute lymphoblastic leukemia (ALL), there have been few reports on acute myeloid leukemia (AML) in infants. To develop more appropriate therapeutic strategies for infants with AML, it is necessary to elucidate the distinct clinical features of this subgroup. We therefore performed a retrospective analysis on infant AML in Japan. Patients: Infants with AML, aged less than 1 year at diagnosis, registered in any of the 6 Japanese AML clinical trials between 1991 and 2010 (TCCSG M91-13, TCCSG M96-14, AML99, CCLSG9805, CCLSG9805RE, and JPLSG AML-05) were included in this study. Patients with Down syndrome were excluded. Results: A total of 122 infant AML patients were included in the present analysis, which comprised approximately 10% of all pediatric AML patients. The most frequent FAB classification type was M5 (28.7%), followed by M7 (22.9%) and M4 (10.8%). About 30% of patients had 11q23 abnormalities/MLL -R, but there was no impact on prognosis. Several cases with normal karyotype were revealed to be MLL -R on FISH analysis or on MLL -fusion chimeric transcript analysis by RT-PCR. t(8;21), inv(16) and t(15;17) cases were very rare among the infant cohorts. Furthermore, 7.8% had t(1;22)(p13;q13), and 2.5% had t(7;12)(q36;p13). Genetic mutation results could be obtained in 11 cases in the AML99 study; only one case each was confirmed to have NRAS, KRAS or KIT gene mutation. No cases with FLT3-ITD were detected among the 11 cases in the AML99 or the 44 cases in the AML-05 study. Survival rate varied based on treatment received; 5-year OS rate was 58.3% to 71.4%, and 5-year EFS rate was 49.4% to 64.2%. Discussion: Survival rate in infant AML was identical to that in older pediatric AML. However, there was a possible underestimation of MLL -R patients based on sole chromosome analysis; the prevalence of MLL -R was less than 50% in infant AML patients, without any prognostic impact. Other well-known genetic alterations in pediatric AML also had no effect on outcome of infant AML. Infant AML is a heterogeneous subgroup of pediatric AML, and further studies, as well as novel biomarkers, will be necessary to fully understand its biology. Disclosures:No relevant conflicts of interest to declare.

Unique Familial MLL-rearranged Precursor B Cell Infant Acute Lymphoblastic Leukemia (ALL) in Non-Twin Siblings

Abstract 2417Leukemia is the commonest malignancy in infants, the most frequently occurring form of which is infant ALL. When ALL occurs in infants the disease is clinically aggressive and associated with poor outcome. MLL gene rearrangements producing transforming fusion oncoproteins are found in 75% of infant ALL and they are adverse prognostic factors. Infant ALL has never occurred in families except in monozygous twins, where concordance in leukemia occurrence is nearly 100%. Identical but non-germline genomic breakpoint junction sequences have pointed to an in utero origin of MLL gene rearrangements in these twin cases, where it is believed that metastasis of cells with the rearrangement occurs from one twin to the other via the placental circulation. Here we describe two highly novel siblings both deceased from precursor B cell infant ALL (ages at diagnosis: proband 160 d, sibling 121 d). Remarkably, the second of these two decedents is survived by a now 3 year-old monozygous twin who is as yet unaffected.MLLrearrangements in the leukemia blasts of both affected siblings were characterized by conventional cytogenetics and/or FISH, M-FISH, high resolution Illumina 610K Bead Chip SNP array and Southern blot analysis. MLL genomic breakpoint junction sequences and fusion transcripts were defined using panhandle PCR approaches, PCR with gene-specific primers and reverse transcriptase PCR. The twins were confirmed to be monozygous using the genotype calls from SNP array analysis of the peripheral blood and bone marrow of the unaffected and affected twins, respectively. Quantitative real-time PCR analysis of leukemia DNA was used to determine the allele specific sequences of the NQO1 (NADPH quinone oxidorecutase 1) gene, an inactivating polymorphism in which previously was implicated as a risk factor for MLL-rearranged infant ALL.The complex karyotype in the leukemia cells of the proband was 46, XX, der(2) t(2;3) (q3?;?), der(3) ?t(3;4;11), del(4) (q21), der(11) ?del(11) (p11.2) t(3;11) (?;q23).ish der(3) (5'MLL+), der(11) (3'MLL+) [14]/46, XX[8], suggesting extensive damage to the genome. Consistent with a three-way t(3;4;11) translocation, two alternately spliced 5'-MLL-MLLT2(AF-4)-3' fusion transcripts were identified, indicating disruption of the chromosome band 4q21 partner gene MLLT2. Also consistent with the three-way translocation, reverse panhandle PCR detected a 5'-partner-MLL-3' genomic breakpoint junction fusing 3' MLL to the upstream region of a highly novel chromosome 3 gene encoding a nucleotidyltransferase fold protein C3ORF31 (Accession no. NM_138807; Kuchta, 2009). Unlike in the proband, the ALL of the affected twin exhibited a simple t(4;11)(q21;q23) translocation, the reciprocal genomic breakpoint junctions of which fusing MLL and MLLT2 also have been characterized. Similar to non-familial infant ALL, the genomic breakpoint junctions in both infants contained sequence features of nonhomologous end joining DNA repair. The different MLL gene rearrangements in the leukemia cells in the affected siblings indicate that the translocations were not hereditary. Even though the NQO1 inactivating polymorphism is one genetic risk factor for infant ALL, the NQO1 genotype was wild-type in both affected siblings.Further studies in this uniquely afflicted family with two siblings who succumbed to infant ALL and a monozygous twin of one of the decedents surviving beyond infancy unaffected, will provide a one-time opportunity to capture novel mutations predisposing to the development of, and cooperating with, MLL translocations in infant ALL. The MLLT2 involvement in both cases has even further implications for the knowledge to be gained because the MLL-MLLT2 rearrangement occurs in 50% of infant ALL and adversely impacts outcome. Disclosures:Felix:Children's Hospital of Philadelphia: Methods and Kits for Analysis of Chromosomal Rearrangements Associated with Leukemia - U.S. Patent # 6,368,791 issued April 9, 2002.

Genetic Susceptibility Loci and Risk of Infant Leukemia by Subtype and MLL status: A Children's Oncology Group Report

Abstract 749 Introduction.Leukemia in infancy is extremely rare and etiologically distinct from leukemia arising in older children. Infant leukemia cases likely arise in utero, typically involve rearrangements of the mixed lineage leukemia (MLL) gene, and have a much poorer survival than older children with leukemia. Since genetic determinants of susceptibility to infant leukemia are likely different from those in older children, we aimed to evaluate genetic variants previously identified by genome wide association studies of childhood acute lymphoblastic leukemia (ALL). Methods.We genotyped candidate susceptibility loci in 171 Caucasian infants less than 12 months of age with acute leukemia (including 102 ALL, 67 acute myeloid leukemia (AML), and 2 biphenotypic cases) diagnosed during the period 1996–2006. Controls consisted of 384 healthy Caucasian blood donors. Associations between genetic polymorphisms in IKZF1, ARID5B, and CEBPE and infant leukemia were evaluated using logistic regression and were further stratified by leukemia subtype and presence or absence of MLL gene rearrangements. Results.We observed a statistically significant association with two copies of the IKZF1 rs11978267 variant allele in infant leukemia overall (Odds Ratio (OR)=2.3, 95% Confidence Interval (CI)=1.3-4.2), but not with the other susceptibility loci. The association with IKZF1 was concentrated in the AML group, with an increased risk associated with two copies of the variant allele (OR=3.9, 95%CI=1.8-8.4), but not one copy. The association was similarly strong in those with and without a MLL gene rearrangement. In contrast, a strong association with the IKZF1 variant was seen in infants with ALL lacking a MLL rearrangement (OR=5.1, 95%CI=1.8-14.5), but not in those with MLL rearrangements (OR=0.7, 95%CI=0.2-2.2). Moreover, infants with ALL and no MLL rearrangement had a strong association with a genetic variant in the gene, ARID5B (rs10821936, OR=7.2, 95%CI=2.5-20.6), which was also not seen in those with ALL and a MLL rearrangement. Discussion.IKZF1 (IKAROS)is expressed in early hematopoiesis, and its role in lymphopoiesis and lymphoid leukemia is well-described. IKAROS is also expressed in precursor myeloid cells, and our report is the first evidence that IKZF1 is important in the etiology of infant myeloid leukemia, irrespective of MLL gene rearrangements. Moreover, our data indicate striking differences in genetic susceptibility to infant ALL with and without rearrangements of the MLL gene. This knowledge could provide important new etiologic insights into this extremely rare but heterogeneous hematopoietic malignancy. Supported by NIH CA079940, T32 CA099936, K05 CA157439 and the Children's Cancer Research Fund, Minneapolis, MN. Disclosures:No relevant conflicts of interest to declare.

Cytogenetic Risk Remains a Major Predictor of Outcome in Pediatric AML and ALL Treated with Allogeneic Stem Cell Transplantation,

Abstract 3524 Background:Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) each represent a heterogeneous group of disorders resulting from diverse mechanisms of leukemogenesis. Cytogenetic abnormalities are considered reliable risk-stratification tools in both AML and ALL. Most studies assessing clinical outcome according to cytogenetic risk consider patients treated with conventional intensive chemotherapy. The impact of cytogenetics on outcome in patients treated uniformly with hematopoietic stem cell transplantation (HSCT), especially in the pediatric group, is not well studied. We evaluated whether allogeneic HSCT has an impact on outcome as predicted by cytogenetic risk at diagnosis. Methods:Bone marrow karyotyping and FISH data from 61 pediatric AML patients and 37 pediatric patients with ALL was reviewed and patients divided into two groups according to their cytogenetic risk: one group combined patients with favorable or intermediate risk and the second group comprised patients with adverse-risk cytogenetics. Cytogenetic risk classification was according to WHO and MRC cytogenetic criteria. The AML cohort included 47 (77%) patients treated in their first remission (CR1) while most non-CR1 AML patients were treated with HSCT in CR2. Of the ALL patients, 18 (49%) were treated with HSCT in CR1. The outcome of patients with either favorable/intermediate cytogenetic risk or adverse cytogenetics was compared for AML and ALL patients, first including all patients, then including only CR1 patients. The median age of patients was 8 years (range: 9 months to 13 years). Results:Patients with ALL and favorable / intermediate cytogenetics had significantly longer overall survival (OS) and event-free survival (EFS) as compared with the adverse-risk group (P=0.02 and P=0.03 for OS and EFS, respectively). A similar trend was observed when only CR1 ALL patients were evaluated, although their number is small. Notably, separation between the CR1 ALL and CR2 ALL groups was stronger when only Philadelphia chromosome positive and MLL positive patients were considered (P=0.01 for OS and EFS for all patients; P=0.003 for OS and EFS for CR1patients only). Also for pediatric AML, combined favorable / intermediate-risk cytogenetics was associated with significantly longer OS (P=0.03) and EFS (P=0.04) when all patients were included (Fig. 1). A similar trend was obtained when only CR1 AML patients were considered (P=0.03 for both OS and EFS). Conclusions:This study suggests that stratification of AML and ALL in pediatric patients according to their cytogenetic risk remains relevant even when treated with HSCT and irrespective if patients were treated in CR1 or CR2 after relapse. In particular, MLL gene rearrangements and the Philadelphia chromosome remain poor prognostic factors in pediatric ALL despite HSCT treatment. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Acute Bilineal Leukemia Is a Very Rare Entity in Childhood

Abstract 4871Recent WHO 2008 classification introduced a new category named Mixed Phenotype Acute Leukemia (MPAL) for leukemias in which primary lineage cannot be determined by morphology, cytochemistry and/or flow cytometry (FC). Acute bilineal leukemia (ABL) is a subtype of MPAL and is defined by presence of distinct myeloid and lymphoid clonal populations simultaneously at diagnosis. No epidemiological data on ABL have been published so far and there are also few data on the origin of distinct leukemic clones. We examined the incidence and biology of ABL cases among children with primary acute leukemia in the period between 1996 and 2011 in the Czech Republic. Morphology and FC were centrally evaluated in all patients. In total 1065 patients were diagnosed (919 ALL, 146 AML); out of them 3 patients were classified as ABL. Two cases had simultaneous presence of distinct B-cell precursor (BCP) and myeloid clones (BCP-My) at diagnosis, one patient had discrete T ALL and myeloid population (T-My). All ABL patients were screened for immunoglobulin (Ig) and T-cell receptor (TCR) clonality, BCR-ABL, MLL gene rearrangements and FLT3-ITD. All three patients had detectable clonality in lymphoid-specific Ig/TCR rearrangements. In pt1 (BCP-My), FLT3-ITD abnormality and in pt3 (BCP-My), BCR-ABL fusion gene were found. Both patients with BCP-My ABL had Ikaros (IKZF1) gene deletion. In pt2 complex karyotype with MLL gene translocation was identified. Using high speed cell sorting we evaluated the presence or absence of previously mentioned changes in separated subpopulations. In patients with BCP-My ABL, identical clonal Ig rearrangements were found in both lymphoid and myeloid clones. Also FLT3-ITD and BCR-ABL aberrations were present in both clones of respective patients. In pt2 (T-My) TCR gene rearrangement was absent in myeloid population. All three patients achieved complete remission (CR) by lymphoid-directed induction treatment followed by switch to myeloid-oriented blocks according Interfant 99, resp. 2006 protocol in pt 1 and 2 and ALL treatment combined with tyrosine kinase inhibitor (TKI) in pt3. Finally all patients underwent allogeneic hematopoietic stem cell transplantation (SCT) in first CR. Pt1 relapsed after SCT as “typical” cALL. However, the plasticity was maintained and the myeloid clone reappeared at day 28 of relapse therapy. Pt2 relapsed as AML with undetectable TCR gene rearrangements. Pt3 is in complete remission 23 months after SCT with detectable low-level MRD and mixed chimerism with repeated lowering MRD after re-administered TKI dasatinib. Conclusions:ABL is an extremely rare entity in childhood accounting for less than 0.3 % of all acute leukemia cases. Surprisingly, the same genetic changes can be identified in both clonal populations making the “true” ABL even rarer. FC in combination with morphology is the basic method for identifying ABL and should be followed by detailed genetic analysis. The prognosis of ABL patients in our cohort was poor, despite SCT preceded by the application of treatment modalities targeting both lymphoid and myeloid clones. Disclosures:No relevant conflicts of interest to declare.

The t(9;11) Confers Good Prognosis in AML Patients Treated with Stem Cell Transplantation As Compared to Non-t(9;11) and Other Adverse-Risk Abnormalities

Abstract 2527 Background:In contrast to most translocations affecting the MLL gene, the t(9;11) is not associated with a markedly poor prognosis. Several studies revealed a very favorable outcome in the pediatric patient group. In adult AML, the t(9;11) has also been associated with superior survival, at least compared to other 11q23 abnormalities. Therefore, 11q23 rearrangements in adult AML are now often dichotomized into t(9;11) and non-t(9;11), with the former being included in the intermediate-risk group and the latter in the adverse-risk group. The proposed European Leukemia Net (ELN) cytogenetic reporting criteria reflect this division. We investigated whether the outcome of AML patients treated with allogeneic hematopoietic stem cell transplantation (HSCT) with t(9;11) remains significantly different from the rest of the adverse-risk cytogenetic group. Methods:Conventional cytogenetics and FISH data from diagnostic bone marrow of 110 adult AML patients treated with HSCT was reviewed and patients classified according to the recommendations of the European Leukemia Net and included 32 with favorable risk, 60 in the intermediate-risk group, and 18 in the adverse-risk group. FISH confirmed MLL rearrangement in cases with apparent 11q23 abnormalities. We compared outcome of patients with t(9;11) to the group of patients with adverse-risk cytogenetics that included all MLL-positive non-t(9;11) among other cytogenetic abnormalities classified adverse-risk. Our study included 62 (56%) patients treated in first remission (CR1), while most non-CR1 AML patients were treated with HSCT in CR2. Patients were between 14 and 57 years, with median age of 25 years. Results:Of the 110 AML patients, 9 (8%) had MLL gene rearrangement. Of these patients only 5 (4.5% of all patients) had t(9;11). When all patients with MLL rearrangement were considered, there was no significant difference between this group and the patients with adverse-risk cytogenetics in overall survival (OS) or event-free survival (EFS). In contrast, when only patients with the t(9;11) were considered, the t(9;11) patients had significantly longer OS (P=0.02) and EFS (P=0.03) as compared with patients with adverse cytogenetics including all non-t(9;11) MLL-rearranged cases. The outcome of MLL-positive non-t(9;11) patients was similar to the group with adverse-risk cytogenetics. MLL rearrangements in the non-t(9;11) group included t(4;11)(q21;q23), t(6;11)(q27;q23) and a variant t(6;11;7)(q27;q23;q11.2), as well as t(11;17)(q23;q25). The survival for patients with t(9;11) remained significantly longer even when only patients treated with HSCT in first remission were considered, although numbers were small. All five patients with t(9;11) were treated with HSCT in CR1. Conclusions:The data supports the conclusion that MLL-positive t(9;11) AML patients should be classified differently from the rest of the MLL-rearranged cases and should be considered as part of the intermediate-risk group. This classification separating the t(9;11) cases from the rest of the MLL-positive cases should be maintained even when patients are treated with allogeneic HSCT. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Abstract B119: AF4 mimetic peptide SPK111 prolongs survival of MLL xenografted leukemias.

Abstract Acute leukemias with balanced translocations of the MLL gene are aggressive blood cancers with generally poor prognosis. Importantly, MLL leukemias are the most common type of leukemia in babies and account for 70% of the secondary leukemias that arise following treatment with topoisomerase II inhibitors. Only 40% of infants treated for acute lymphoblastic leukemia (ALL) with MLL gene rearrangements survive to the age of five while the survival rate is 90% for patients in the same age group whose disease lacks a translocated MLL gene. Hence, better treatment options for MLL-rearranged leukemias need to be explored. As results of reciprocal translocations, the 5' coding region of MLL is fused to the 3' coding region of one of many different genes. This results in transcripts that encode chimeric proteins. AF4 and AF9 proteins are the two most frequent fusion partners of MLL. They are components of the protein complex P-TEFb that promotes transcriptional elongation. Misdirection of the transcriptional elongation complex is one proposed mechanism by which oncogenic transformation occurs in MLL leukemias. Our laboratory previously identified the amino acids required for direct physical interaction between AF4 and AF9 and designed an AF4 mimetic peptide that disrupts the AF4 - AF9 interaction. Treatment of cell lines with MLL-AF4 and MLL-AF9 translocations with the mimetic peptide leads to necrotic cell death without affecting normal hematopoietic cells. In order to further establish the AF4 - AF9 interaction as an effective target for MLL leukemias, we tested effect of a modified peptide designed to enhance its stability in xenografted mice. The peptide, designated SPK-111 exherts effective antitumor activity without causing overt toxicity. It significantly improves the survival of NOD/SCID mice engrafted with human cell lines carrying MLL gene rearrangements when used as a single agent. Here, we describe the in vivo activity of the peptide and propose that it, or other derivatives, may represent a clinically important agent for the treatment of MLL leukemias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B119.

Acute myeloid leukemia of donor origin after allogeneic stem cell transplantation from a sibling who harbors germline XPD and XRCC3 homozygous polymorphisms

A 54-year-old woman was diagnosed with infiltrative ductal breast carcinoma. Two years after treatment, the patient developed an acute myeloid leukemia (AML) which harbored del(11q23) in 8% of the blast cells. The patient was submitted for allogeneic stem cell transplantation (aSCT) from her HLA-compatible sister. Ten months after transplantation, she relapsed with an AML with basophilic maturation characterized by CD45low CD33high, CD117+, CD13-/+, HLA Drhigh, CD123high, and CD203c+ blast cells lacking expression of CD7, CD10, CD34, CD15, CD14, CD56, CD36, CD64, and cytoplasmic tryptase. Karyotype analysis showed the emergence of a new clone with t(2;14) and FISH analysis indicated the presence of MLL gene rearrangement consistent with del(11q23). Interestingly, AML blast cell DNA tested with microsatellite markers showed the same pattern as the donor's, suggesting that this AML emerged from donor cells. Additionally, polymorphisms of the XPA, XPD, XRCC1, XRCC3 and RAD51 DNA repair genes revealed three unfavorable alleles with low DNA repair capacity.In summary, we report the first case of AML involving XPD and XRCC3 polymorphisms from donor origin following allogeneic stem cell transplantation and highlight the potential need for careful analysis of DNA repair gene polymorphisms in selecting candidate donors prior to allogeneic stem cell transplantation.

PREGNANCY, EXPOSURE TO PESTICIDES AND INFANT LEUKEMIA IN BRAZIL

Background and Aims: Infant leukemia (IL) is a rare hematological neoplasm associated with MLL gene rearrangements. Maternal exposures during pregnancy to pesticides, hormones, dipyrone, and topoisomerase-II DNA inhibitors and high birth weight were associated risk factors. Methods: This is a hospital-based multicenter case-control study. Mothers of 252 IL cases and 423 controls were interviewed and data were obtained regarding environmental maternal exposure during periconceptional, pregnancy and breast-feeding periods, including exposure to pesticides. Unconditional logistic regression was performed and odds ratios (OR) on the association between maternal pesticides exposure and IL, including their 95% confidence intervals (CI), were ascertained after adjustment to hormonal intake during pregnancy, mother’s age, mother’s level of education, birth weight and infant’s skin color. Results: An adjusted OR, 2.39, 95% CI, 1.63-3.51 was observed for the association between IL and maternal exposure to pesticides during pregnancy, being higher for acute myeloid leukemia (adjusted OR 3.50, 95% CI, 2.01-6.11). The use of pyrethroids during pregnancy revealed an OR 2.18 (95% CI, 1.44-3.29), while the use of other pesticides showed an OR 3.61 (95% CI, 1.69-7.73). The reported household use of pesticides revealed an adjusted OR 2.25 (95% CI, 1.48-3.43), and agriculture exposure showed an adjusted OR 9.26, (95% CI, 2.82-30.4). According to frequency of exposure, an adjusted OR, 2.48 (95% CI, 1.50 - 4.00) was seem among those reporting up to 1 contact weekly, and an adjusted OR 2.34 (95% CI, 1.30– 4.21) among those with &gt; 1 contact (p-trend &lt; 0.0001). Mixed exposure to different pesticides showed an adjusted OR, 3.83 (95% CI, 1.33-11.0) and the exposure to different chemical classes revealed an adjusted OR, 8.87 (95% CI, 1.55-50.6). Conclusions: The observed results seem to support the hypothesis that mothers’ household use of pesticides and other contaminants may be involved in the etiology of infant leukemia.

Systematic in-vitro evaluation of the NCI/NIH Developmental Therapeutics Program Approved Oncology Drug Set for the identification of a candidate drug repertoire for MLL-rearranged leukemia

Despite significant progress made in the overall cure rate, the prognosis for relapsed and refractory malignancies in children remains extremely poor. Hence, there is an urgent need for studies that enable the timely selection of appropriate agents for Phase I clinical studies. The Pediatric Oncology Experimental Therapeutics Investigators’ Consortium (POETIC) is systematically evaluating libraries of known and novel compounds for activity against subsets of high-risk pediatric malignancies with defined molecular aberrations for future clinical development. In this report, we describe the in-vitro activity of a diverse panel of approved oncology drugs against MLL-rearranged pediatric leukemia cell lines. Agents in the Approved Oncology Drug Set II (National Cancer Institute/National Institutes of Health Developmental Therapeutics Program) were evaluated by in-vitro cytotoxicity assays in pediatric acute lymphoblastic leukemia and acute myeloid leukemia cell lines with MLL gene rearrangements. Validation studies were carried out with patient leukemia cells in culture. Comparative analysis for toxicity against nonmalignant cells was evaluated in normal bone marrow stromal cells and normal human lymphocytes. Results from this study show that 42 of the 89 agents tested have measurable cytotoxicity against leukemia cells, and among these, 12 were effective against all five MLL-rearranged cell lines (IC50 [half maximal inhibitory concentration] < 1 μM). These 12 agents include cladribine, dactinomycin, daunorubicin, docetaxel, etoposide, gemcitabine, mitomycin C, mitoxantrone, teniposide, topotecan, triethylenemelamine, and vinblastine. We show that the Approved Oncology Drug Set II contains a number of agents with potent antileukemic activity in the tested cell lines. As approved drugs, these agents have been used in clinical settings for many years for other malignancies, thus their toxicity profile, pharmacokinetics, and other properties are readily available. Further evaluation of their use in future clinical trials for pediatric leukemia with MLL abnormalities should be considered.

The first case of blastic plasmacytoid dendritic cell neoplasm with MLL-ENL rearrangement

Blastic plasmacytoid dendritic cell neoplasm (BPDCN), previusly referred to as blastic NK-cell lymphoma or CD4+/CD56+ ematodermic neoplasm, is an extremely rare hematologic maligancy which is characterized by co-expression of CD4 and CD56 ithout any other lineage-specific markers [1]. The disease was ategorized as a type of NK-cell lymphoma, but is now classified as rare subgroup of acute myeloid leukemia (AML) and related preursor myeloid neoplasms in the WHO classification 4th edition, ecause the precursor of plasmacytoid dendritic cells (pDCs) were dentified as the origin of the malignancy [2]. MLL gene, located at chromosome 11 band q23, is one of the most ommon targets for translocations associated with various types of dult and pediatric acute leukemia. MLL gene rearrangements are ound in 2.8% of AML patients and are relatively common in infant cute lymphoblastic leukemia and secondary AML. AML with MLL earrangement carries a relatively worse outcome [3]. Here we report a patient with BPDCN with MLL-ENL fusion gene. o our knowledge, this is the first report of BPDCN with MLL-ENL earrangement. A 45-year-old male without remarkable medical history visted our hospital because of multiple disseminated purpuric skin odules over the trunk, the limbs, and the face. Lymphadenopahy was not noted. Laboratory examinations revealed neutropenia Neutrophils, 0.492 × 109/L) and anemia (hemoglobin concentraion, 11.2 g/dL). Histological examination of the skin lesion showed iffuse infiltration of medium to large sized agranular blastic cells nto the dermis and the subcutaneous adipose tissue (Fig. 1). Bone arrow and peripheral blood infiltration were also observed. The eoplastic cells were negative for peroxidase, and flow cytomery revealed that the neoplastic cells were positive for CD4, CD11c, D33, CD45RA, CD56, CD68, CD117, CD123, HLA-DR and negative or CD3, CD5, CD7, CD8, CD10, CD11b, CD13, CD19, CD20, CD34,

Differentially expressed miRNAs in cytogenetic and molecular subtypes of pediatric acute myeloid leukemia

miRNAs regulate gene expression, and thus play an important role in critical cellular processes. Aberrant miRNA expression patterns have been found in various types of cancer. So far, information about the expression of miRNAs in pediatric acute myeloid leukemia is limited. We studied expression of miR-29a, -155, -196a, and -196b by stem-loop based RT-qPCR in 82 pediatric acute myeloid leukemia patients selected to represent relevant cytogenetic and molecular subgroups. High miR-196a and -b expression was observed in patients carrying MLL gene rearrangements (P < 0.001), NPM1 mutations (P < 0.001), or FLT3-ITD in a cytogenetically normal background (P ≤ 0.02), compared to all other patients. In contrast, CEBPA mutated cases had a low expression of miR-196a and -b (P ≤ 0.001). Expression of miR-196a and -b was correlated with expression of neighboring HOXA and HOXB genes (Spearman's r = 0.46-0.82, P < 0.01). Expression of miR-155 was not related to cytogenetic features but high expression of miR-155 was observed in FLT3-ITD (P = 0.001) and NPM1-mutated cases (P = 0.04). Lower miR-29a expression was mainly observed in MLL-rearranged pediatric acute myeloid leukemia, specifically in cases carrying t(10;11) (P < 0.001). We show aberrant expression of specific miRNAs in clinically relevant cytogenetic and molecular subgroups of pediatric acute myeloid leukemia, suggesting a role for these miRNAs in the underlying biology in these specific subgroups.

P2-82 Pregnancy, exposure to pesticides and infant leukaemia in Brazil

Infant Leukaemia is a rare haematological neoplasm associated with MLL gene rearrangements. Maternal exposure during pregnancy to pesticides, hormones, dipyrone, and topoisomerase-II DNA inhibitors and birth weight were associated risk...

Increased MLL gene rearrangements in amniocytes from fetuses of mothers who smoke

We assess the possible genotoxic effect of maternal smoking on amniotic fluid cells, based on the presence of an increasing of structural abnormality of the 11q23 band bearing the MLL gene rearrangements. In this observational and prospective study cultured amniocytes were obtained from 20 control and 20 women who smoke (>10 cigarettes/day for >10 years and during pregnancy). We performed fluorescence in situ hybridization (FISH) analysis in amniocytes. Comparison of FISH data between smoker and control groups showed statistical significance for the MLL gene rearrangements. Epidemiologic studies, including a large series of patients, will be needed to determine whether the offspring of parents who smoke have an increased lifetime risk of leukemia.

Abstract 278: Role of AF4 and Dot1 in MLL-AF9 mediated leukemogenesis

Abstract Mixed lineage leukemia (MLL) is characterized by rearrangement of the MLL gene at chromosome band 11q23. MLL gene rearrangements give rise to chimeric proteins that contain the N-terminal portion of MLL fused to the C-terminal portion of over 50 different fusion partners. MLL chimeras do not retain the histone methyltransferase activity of wild type MLL and it is unclear how they deregulate the expression of MLL target genes. Our laboratory studies the two most common MLL fusion partners encountered in patients with leukemia, AF4 and AF9. We have previously shown that AF4 and AF9 heterodimerize and peptides that block this interaction are toxic to MLL leukemia cell lines. This supports the hypothesis that the interaction of MLL-AF9 with AF4 is required for leukemogenesis. However, we have also shown that AF4 and H3K79 methyltransferase, Dot1 have similar binding sites on AF9. Furthermore, we have found that Dot1 is required for immortalization of hematopoietic progenitor cells by MLL fusion genes. The work of Yokoyama and others demonstrated that AF4, ENL (a close homologue of AF9) and Dot1L do not exist in the same multimeric complex. To understand the role of AF4 and Dot1 in MLL-AF9 mediated leukemogenesis, we made point mutants of AF9 with differential binding for AF4 and Dot1. Based on our preliminary data and recent studies, we hypothesize that the binding of AF4 and not Dot1 to MLL-AF9 is required for MLL-AF9 mediated leukemogenesis. Nevertheless, Dot1 remains a critical factor in MLL-AF9 leukemia. We hypothesize that Dot1 recruitment is mediated by wild type AF9 rather than MLL-AF9. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 278. doi:10.1158/1538-7445.AM2011-278