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1
Seelisch, Jennifer, Matthew Zatzman, Federico Comitani, Fabio Fuligni, Ledia Brunga, Patrick A. Brown, Adam Shlien, and Sumit Gupta. "Resolving driver events in MLL-r negative high-risk infant ALL." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): 10030. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.10030.
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10030 Background: Infant acute lymphoblastic leukemia (ALL) is the only subtype of childhood ALL whose outcome has not improved over the past two decades. The most important prognosticator is the presence of rearrangements in the Mixed Lineage Leukemia gene (MLL-r), however, many patients present with high-risk clinical features but without MLL-r. We recently identified two cases of infant ALL with high-risk clinical features resembling MLL-r, but were negative for MLL-r by conventional diagnostics. RNA sequencing revealed a partial tandem duplication in MLL (MLL-PTD). We thus aimed to determine if MLL-PTD, other MLL abnormalities, or other genetic or transcriptomic features were driving this subset of high-risk infant ALL without MLL-r. Methods: We obtained 19 banked patient samples from the Children’s Oncology Group (COG) infant ALL trial (AALL0631) from MLL wildtype patients as determined by FISH and cytogenetics. Utilizing deep RNA-sequencing, we manually inspected the MLL gene for MLL-PTD, while also performing automated fusion detection and gene expression profiling in search of defining features of these tumors. Results: 3 additional MLL-PTDs were identified, all in patients with infant T-cell ALL, whereas both index cases were in patients with infant B-cell ALL. Gene expression profiling analysis revealed that all five MLL-PTD infants clustered together. Eight infants (7 with B-cell ALL) were found to have Ph-like expression. Five of these 8 infants were also found to have an IKZF1/JAK2 expression profile; one of these five had a PAX5-JAK2 fusion detected. Two infants (including the one noted above) had novel PAX5 fusions, known drivers of B-cell leukemia. Additional detected fusions included TCF3-PBX1 and TCF4-ZNF384. Conclusions: MLL-PTDs were found in both B- and T-cell infant ALL. Though Ph-like ALL has been described in adolescents and young adults, we found a substantial frequency of Ph-like expression among MLL-WT infants. Further characterization of these infants is ongoing. If replicated in other infant cohorts, these two findings may help explain the poor prognosis of MLL-WT ALL when compared to children with standard risk ALL, and offer the possibility of targeted therapy for select infants.
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Stam, Ronald W., Monique L. Den Boer, Monique Passier, Stephen E. Sallan, Scott A. Armstrong, and Pieters Pieters. "MLL Rearranged Infant Acute Lymphoblastic Leukemia Is Characterized by Silencing of the Putative Tumor Suppressor Gene FHIT." Blood 104, no. 11 (November 16, 2004): 525. http://dx.doi.org/10.1182/blood.v104.11.525.525.
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Abstract MLL gene rearranged acute lymphoblastic leukemia (MLL) is an aggressive type of acute lymphoblastic leukemia (ALL) with a relatively poor response to treatment, predominantly occurring in infants (<1 years of age). Since conventional chemotherapy is failing in >50% of these patients, identification of novel therapeutic targets to direct therapy against is demanded. For this, understanding this disease and finding biological and molecular features characterizing MLL may be crucial. Here we report that infant MLL is characterized by silencing of the putative tumor suppressor fragile histidine triad (FHIT) gene. Gene expression profiling showed that FHIT expression is significantly reduced in MLL patients (n=20) as compared to patients diagnosed with conventional ALL (n=24) bearing germ line MLL genes. Using quantitative real-time PCR we confirmed FHIT mRNA expression to be low in MLL gene rearranged infant ALL (infant MLL) as compared to both infants and older children (non-infants) not carrying chromosomal abnormalities involving the MLL gene. Infant MLL patients (n=35) significantly (p<0.001) expressed ~15-fold less FHIT mRNA compared to infant ALL patients (n=8) and 11-fold less than non-infant ALL patients (n=22). Methylation specific PCR (MSP) analysis demonstrated that the FHIT promoter region is hypermethylated in all of the 36 infant MLL cases tested, resulting in strongly reduced FHIT mRNA as well as FHIT protein expression. In contrast, the incidence of FHIT promoter methylation in both infant and non-infant ALL patients carrying germ line MLL genes was markedly lower, with a frequency of 63% (5/8) and 56% (24/43) respectively. Suppression of FHIT expression could be reversed by exposing leukemia cells bearing MLL gene rearrangements to the demethylating drug 5′-aza-2′-deoxycytidine. These findings imply that MLL rearranged ALL is characterized by FHIT promoter hypermethylation. Currently experiments are being conducted to determine the biological significance of FHIT silencing in this type of cancer.
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Sutton, Rosemary, Tamara Law, Nicola C. Venn, Carol Wadham, Emily VA Mould, Murray D. Norris, Rishi S. Kotecha, et al. "Comparison of MRD Levels and Gene Expression Patterns in MLL-R Versus Non-MLL Infant ALL." Blood 128, no. 22 (December 2, 2016): 1740. http://dx.doi.org/10.1182/blood.v128.22.1740.1740.
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Abstract Introduction Acute lymphoblastic leukaemia (ALL) in infants has poor overall survival despite being characterized by very few genetic aberrations per case. The most common genetic change, present in over 75% of cases, is the rearrangement of the mixed lineage leukaemia (MLL/KMT2A) gene (MLL-R) that also occurs in AML and mixed phenotype acute leukaemia (MPAL). Because infant ALL is rare and distinctive, most Australian and New Zealand patients are enrolled on specific clinical trials. In earlier infant ALL trials, MRD was not used for risk stratification firstly because of the difficulty of finding sensitive markers for specific immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangements and the secondly because, in infant ALL, Ig/TCR markers are often present in sub-clones that can be lost at relapse due to clonal selection. MRD testing is performed in the current Interfant 06 trial preferentially using markers based on the genomic breakpoint sequences of MLL gene rearrangements. The treatment of infant ALL remains very challenging with relatively poor survival rates attributable to both toxicity and relapse. The objectives of this study were therefore to analyse MRD data for Interfant 06 patients enrolled at ANZCHOG centres and to perform a pilot experiment evaluating gene expression for key genes in infant ALL samples on a microfluidics platform, as a basis for identifying potential targeted therapies. Methods MRD was measured in bone marrow DNA from Interfant 06 patients enrolled since 2006, using sensitive Real-time Quantitative PCR (PCR-MRD) patient-specific assays to detect either MLL gene rearrangements (in 17) and/or conventional immunoglobulin and T-cell receptor (Ig/TCR) markers (21). Gene expression levels for 90 genes important in childhood cancers were measured by Taqman-based microfluidic assays in duplicate using cDNA from 2 micrograms of total RNA from 10 infant ALL samples (5 MLL, 5 non-MLL) at diagnosis (6) or relapse (4). Results Patient-specific PCR-MRD tests were developed for 27 out of 28 Australian and New Zealand infant ALL patients. 17/18 MLL-R ALL patients had MLL-R assays and 21/28 patients had Ig/TCR MRD tests, with only 1 (non-MLL) patient having no MRD markers. There was a wide range of MRD responses to induction therapy (Figure 1). Bone marrow MRD at the end of induction was high (>1x10-3) in 44% of MLL-R ALL infants compared to 25% of non-MLL ALL infants and 15% of older children enrolled on ANZCHOG ALL8. In 8/13 MLL-R patients who had both types of marker, MRD levels were higher when measured by their MLL-R marker than by their Ig/TCR marker. In a set of 90 genes selected for expression analysis, higher levels were found for 17 genes in 2 or more of the 10 infant ALL samples evaluated. These more highly expressed genes included potential or known drug targets BCL2, ERBB2, ERBB4, ILRA2, CSF1R and PARP1. Conclusions The quantitation of MRD based on MLL rearrangements in ALL is effective and can also be used to monitor response to therapy in infant ALL as well as MLL-R cases of AML and MPAL. The combined application of MLL-R and Ig/TCR markers allowed 97% of infant ALL patients to be MRD monitored with a sensitive marker. In most patients with both type of MRD marker, higher levels of MRD were detected in end of induction samples using the MLL versus Ig/TCR tests. One interpretation is that Ig/TCR genes are rearranged after the MLL rearrangement in ALL sub-clones that are both more mature and more chemo-sensitive. This finding also confirms the current consensus that disease-related MLL-R markers provide better risk assessment than Ig/TCR markers. Our Fluidigm analysis has shown that quantitative measurement of multiple gene expressions is feasible on small RNA samples and can be used to rapidly screen for specific expression of genes coding for drug targets in ALL patients. Support: NHMRC Australia APP1057746, Sporting Chance Cancer Foundation. Figure 1. Comparison of MRD response to induction therapy in MLL-R infant ALL compared with non-MLL infant ALL (Interfant 06) and older children (ANZCHOG ALL8). Figure 1. Comparison of MRD response to induction therapy in MLL-R infant ALL compared with non-MLL infant ALL (Interfant 06) and older children (ANZCHOG ALL8). Disclosures No relevant conflicts of interest to declare.
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Stumpel, Dominique J. P. M., Pauline Schneider, Eddy H. J. van Roon, Judith M. Boer, Paola de Lorenzo, Maria G. Valsecchi, Renee X. de Menezes, Rob Pieters, and Ronald W. Stam. "Specific promoter methylation identifies different subgroups of MLL-rearranged infant acute lymphoblastic leukemia, influences clinical outcome, and provides therapeutic options." Blood 114, no. 27 (December 24, 2009): 5490–98. http://dx.doi.org/10.1182/blood-2009-06-227660.
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Abstract MLL-rearranged infant acute lymphoblastic leukemia (ALL) remains the most aggressive type of childhood leukemia, displaying a unique gene expression profile. Here we hypothesized that this characteristic gene expression signature may have been established by potentially reversible epigenetic modifications. To test this hypothesis, we used differential methylation hybridization to explore the DNA methylation patterns underlying MLL-rearranged ALL in infants. The obtained results were correlated with gene expression data to confirm gene silencing as a result of promoter hypermethylation. Distinct promoter CpG island methylation patterns separated different genetic subtypes of MLL-rearranged ALL in infants. MLL translocations t(4;11) and t(11;19) characterized extensively hypermethylated leukemias, whereas t(9;11)-positive infant ALL and infant ALL carrying wild-type MLL genes epigenetically resembled normal bone marrow. Furthermore, the degree of promoter hypermethylation among infant ALL patients carrying t(4;11) or t(11;19) appeared to influence relapse-free survival, with patients displaying accentuated methylation being at high relapse risk. Finally, we show that the demethylating agent zebularine reverses aberrant DNA methylation and effectively induces apoptosis in MLL-rearranged ALL cells. Collectively these data suggest that aberrant DNA methylation occurs in the majority of MLL-rearranged infant ALL cases and guides clinical outcome. Therefore, inhibition of aberrant DNA methylation may be an important novel therapeutic strategy for MLL-rearranged ALL in infants.
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Stam, Ronald W., Pauline Schneider, Jill A. P. Hagelstein, Marieke H. van der Linden, Dominique J. P. M. Stumpel, Renee X. de Menezes, Paola de Lorenzo, Maria G. Valsecchi, and Rob Pieters. "Gene expression profiling–based dissection of MLL translocated and MLL germline acute lymphoblastic leukemia in infants." Blood 115, no. 14 (April 8, 2010): 2835–44. http://dx.doi.org/10.1182/blood-2009-07-233049.
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Abstract Acute lymphoblastic leukemia (ALL) in infants (< 1 year) is characterized by a poor prognosis and a high incidence of MLL translocations. Several studies demonstrated the unique gene expression profile associated with MLL-rearranged ALL, but generally small cohorts were analyzed as uniform patient groups regardless of the type of MLL translocation, whereas the analysis of translocation-negative infant ALL remained unacknowledged. Here we generated and analyzed primary infant ALL expression profiles (n = 73) typified by translocations t(4;11), t(11;19), and t(9;11), or the absence of MLL translocations. Our data show that MLL germline infant ALL specifies a gene expression pattern that is different from both MLL-rearranged infant ALL and pediatric precursor B-ALL. Moreover, we demonstrate that, apart from a fundamental signature shared by all MLL-rearranged infant ALL samples, each type of MLL translocation is associated with a translocation-specific gene expression signature. Finally, we show the existence of 2 distinct subgroups among t(4;11)–positive infant ALL cases characterized by the absence or presence of HOXA expression, and that patients lacking HOXA expression are at extreme high risk of disease relapse. These gene expression profiles should provide important novel insights in the complex biology of MLL-rearranged infant ALL and boost our progress in finding novel therapeutic solutions.
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Stam, Ronald W., Monique L. Den Boer, Pauline Schneider, Jasper de Boer, Jill Hagelstein, Stephen E. Sallan, Hugh J. M. Brady, Scott Armstrong, and Rob Pieters. "Rearranged Infant Acute Lymphoblastic Leukemia." Blood 112, no. 11 (November 16, 2008): 1913. http://dx.doi.org/10.1182/blood.v112.11.1913.1913.
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Abstract MLL rearranged Acute Lymphoblastic Leukemia (ALL) represents an unfavorable and difficult to treat type of leukemia that often is highly resistant to glucocorticoids like prednisone and dexamethasone. As the response to prednisone largely determines the clinical outcome of pediatric ALL patients, overcoming resistance to these drugs may be an important step towards improved prognosis. Here we compared gene expression profiles between prednisone-resistant and prednisone-sensitive pediatric ALL patients to obtain gene expression signatures associated with prednisone resistance for both childhood (>1 year of age) and MLL rearranged infant (<1 year of age) ALL. Merging both signatures in search for overlapping genes associated with prednisone resistance in both patient groups we, found that elevated expression of MCL-1 (an anti-apoptotic member of the BCL-2 protein family) appeared to be characteristic for both prednisone-resistant ALL samples. To validate this observation, we determined MCL-1 expression using quantitative RT-PCR in a cohort of MLL rearranged infant ALL samples (n=23), and confirm that high-level MCL-1 expression significantly confers glucocorticoid resistance both in vitro and in vivo. Finally, down-regulation of MCL-1 in prednisone resistant MLL rearranged ALL cells by RNA interference (RNAi) markedly sensitized these cells to prednisone. Therefore we conclude that MCL-1 plays an important role in glucocorticoid resistance and that MCL- 1 suppressing agents co-administered during glucocorticoid treatment may be beneficial especially for MLL rearranged infant ALL patients.
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Stam, Ronald W., Monique L. Den Boer, Pauline Schneider, Jasper de Boer, Jill Hagelstein, Maria G. Valsecchi, Paola de Lorenzo, et al. "Association of high-level MCL-1 expression with in vitro and in vivo prednisone resistance in MLL-rearranged infant acute lymphoblastic leukemia." Blood 115, no. 5 (February 4, 2010): 1018–25. http://dx.doi.org/10.1182/blood-2009-02-205963.
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Abstract MLL-rearranged acute lymphoblastic leukemia (ALL) represents an unfavorable type of leukemia that often is highly resistant to glucocorticoids such as prednisone and dexamethasone. Because response to prednisone largely determines clinical outcome of pediatric patients with ALL, overcoming resistance to this drug may be an important step toward improving prognosis. Here, we show how gene expression profiling identifies high-level MCL-1 expression to be associated with prednisolone resistance in MLL-rearranged infant ALL, as well as in more favorable types of childhood ALL. To validate this observation, we determined MCL-1 expression with quantitative reverse transcription–polymerase chain reaction in a cohort of MLL-rearranged infant ALL and pediatric noninfant ALL samples and confirmed that high-level MCL-1 expression is associated with prednisolone resistance in vitro. In addition, MCL-1 expression appeared to be significantly higher in MLL-rearranged infant patients who showed a poor response to prednisone in vivo compared with prednisone good responders. Finally, down-regulation of MCL-1 in prednisolone-resistant MLL-rearranged leukemia cells by RNA interference, to some extent, led to prednisolone sensitization. Collectively, our findings suggest a potential role for MCL-1 in glucocorticoid resistance in MLL-rearranged infant ALL, but at the same time strongly imply that high-level MCL-1 expression is not the sole mechanism providing resistance to these drugs.
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Popov, Alexander, Grigory Tsaur, Tatiana Verzhbitskaya, Olga Streneva, Egor Shorikov, Leonid Saveliev, and Larisa Fechina. "Immunophenotypic Investigation of Infant Acute Lymphoblastic Leukemia." Blood 120, no. 21 (November 16, 2012): 2547. http://dx.doi.org/10.1182/blood.v120.21.2547.2547.
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Abstract Abstract 2547 Acute lymphoblastic leukemia (ALL) in children less than 1 year old is the relatively rare disease with specific biological features and poor outcome. It is also characterized by high incidence of MLL gene rearrangements. Immunophenotype of infants' leukemia varies due to presence or absence of MLL-rearrangements. Aim of the study. description of immunophenotype in infant acute lymphoblastic leukemia. Methods. Totally 421 cases of pediatric acute leukemia (AL) were studied. 81 patients (39 boys and 42 girls) aged from 5 days to 11 months were included in the study group. Their data was compared to 332 cases of acute leukemia in older children. Tumor cells immunophenotyping was performed by 6–8-color flow cytometry. Detection of various types of MLL-gene rearrangements was done by simultaneous application of chromosomal banding analysis, fluorescence in-situ hybridization, reverse-transcriptase polymerase chain reaction (PCR) and long-distance inverse PCR. Results. There were 54 (66.7%) ALL cases in the study group. ALL was found less frequently in infants than in older children (66.7% and 88.5% respectively, p<0.0001) while percentage of acute myeloid leukemia cases was higher in infants (27.2% and 10.0% respectively, p=0.0001). EGIL immunophenotypes distribution also differed between infants and older children. BI-ALL was the most common immunological ALL type in infant ALL (55.6% vs 3.3% in older age group, p<0.0001), while BII-ALL was notably less frequent compared with other age groups (33.3% and 77.1% respectively, p<0.0001). T-lineage ALL was also less frequent in infants (3.7% vs 14.3% in older age group) although difference did not achieve statistical significance (p=0.0536). Totally infant ALL were mainly presented by B-cell precursor ALL (BCP-ALL) – 51 patients (94.4%). Various types of MLL-rearrangements were found in 40 (74.1%) patients (pts) out of 54 infants ALL cases. Among them 21 pts (52.5%) carried MLL-AF4 fusion gene, 8 pts (20.0%) – MLL-MLLT1, 5 pts (12.5%) – MLL-MLLT3, 3 pts (7.5%) – MLL-EPS15, 1 pt (2.5%) – MLL-MLLT10, 1 pt (2.5%) – MLL-AFF3 and 1 pt (2.5%) had MLL-rearrangement with unidentified partner gene. Significant immunophenotypic differences were observed in patients with and without MLL gene rearrangements. Number of cases in those tumor cells expressed CD10, CD20, CD45, CD133, CD15, NG2 significantly varied between MLL-positive and MLL-negative groups (p=0.0001, p<0.0001, p=0.0008, p=0.0018, p=0.0306 and p<0.0001 correspondingly). NG2-positivity represented the highest overall correct prediction (OCP) rate for presence of MLL-rearrangements (95.5%). Diagnostic accuracy of CD20-negativity and CD45-positivity was slightly lower (87.5% and 86.3% respectively) while OCP for CD10-negativity (78.4%), CD133-positivity (75.0%) and CD15-positivity (66.7%) was not sufficient enough. Nevertheless CD10-positive BCP-ALL with MLL-rearrangements differed from CD10(+) cases in MLL-germline group. CD10 homogeneous expression was noted in 10 out of 11 MLL-germline patients and in 1 of 10 MLL-rearranged cases (p=0.0011). Although there were found no significant differences in CD22-positive patients' number, CD22(+)-cells percentage was significantly lower in MLL-positive cases (median 89.9%, range 25.2–99.7% and median 99.9%, range 96.0–99.9% respectively, p=0.0026). Thus CD20-negativity, CD10-negativity/low expression, high CD45, CD15, CD65 and NG2 expression, decreased CD22-expression are immunophenotypic signatures of MLL-rearranged infant ALL, although NG2 has the highest diagnostic efficacy. Interestingly there were no markers able to distinguish MLL-AF4-positive cases from patients carrying other types of MLL-rearrangements. Even NG2 expression intensity did not differ between these groups (p=0.2720). Except BCP-ALL pts, two cases of T-lineage ALL and one mature B-ALL (without other Burkitt lymphoma features) were found. Conclusion. Thus immunophenotype of ALL in children less than 1 year old differs significantly from patients of older age groups. Infants' B-cell precursor ALL immunophenotype varies greatly due to the presence of MLL gene rearrangements. Complex diagnostic immunophenotyping of infants' ALL allows predicting presence of MLL rearrangements and NG2 is the most applicable single marker. Disclosures: No relevant conflicts of interest to declare.
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Urtishak, Karen A., Blaine W. Robinson, Jaclyn A. Biegel, Kim E. Nichols, Julie W. Stern, and Carolyn A. Felix. "Unique Familial MLL-rearranged Precursor B Cell Infant Acute Lymphoblastic Leukemia (ALL) in Non-Twin Siblings." Blood 118, no. 21 (November 18, 2011): 2417. http://dx.doi.org/10.1182/blood.v118.21.2417.2417.
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Abstract Abstract 2417 Leukemia 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.
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Stumpel, Dominique Jpm, Pauline Schneider, Eddy HJ van Roon, Judith M. Boer, Renee X. Menezes, Rob Pieters, and Ronald W. Stam. "Specific Promoter CpG Island Methylation Patterns Identify Different Subgroups of MLL - Rearranged Infant Acute Lymphoblastic Leukemia, and Define Clinical Outcome." Blood 112, no. 11 (November 16, 2008): 596. http://dx.doi.org/10.1182/blood.v112.11.596.596.
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Abstract At present, long-term survival rates in childhood acute lymphoblastic leukemia (ALL) easily exceed 80%. However, the prognosis for infants (<1 year) with ALL barely reaches 50%. Infant ALL is characterized by chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene that occur in about 80% of the cases. The most frequent MLL translocations in infant ALL include t(4;11), t(11;19) and t(9;11). In about 20% of the infant ALL cases no MLL rearrangements are observed. Recent gene-expression profiling characterized MLL-rearranged ALL as a unique type of leukemia, that is genetically clearly separable from other ALL subtypes. As epigenetic modifications affect gene-expression, we hypothesized that the specific gene-expression profiles associated with MLL-rearranged ALL may well be driven by epigenetic changes. The best-studied epigenetic event in hematological malignancies constitutes the transcriptional silencing of (tumor suppressor) genes by promoter CpG island hypermethylation. To explore the DNA methylation patterns underlying MLL-rearranged infant ALL, we applied Differential Methylation Hybridization (DMH) using both 9k (Huang, 2002) and 244k CpG island microarrays (Agilent) on primary infant ALL samples carrying t(4;11) (n=21), t(11;19) (n=17), t(9;11) (n=6) or wild-type MLL genes (n=13). The resulting DNA methylation patterns were compared with the patterns found in healthy pediatric bone marrow samples (n=8). In addition, relapse material from three infants with MLL-rearranged ALL was included and compared with the corresponding patient sample obtained at diagnosis. Both CpG island microarray platforms demonstrate that t(4;11) and t(11;19) characterize extensively hypermethylated leukemias, whereas t(9;11)-positive and translocation-negative infant ALL epigenetically resemble normal bone marrow. When the CpG array data (Agilent) were compared with available gene expression profiles (Affymetrix), we found that 95% of the genes from the top 100 of genes most significantly hypermethylated in t(4;11)- or t(11;19)-positive infant ALL were indeed down-regulated. Using the t(4;11)-positive cell line models SEMK2 and RS4;11, we demonstrate that the majority of these hypermethylated genes could be demethylated by the demethylating agent zebularine. Among t(4;11)- and t(11;19)-positive infant ALL samples, two subgroups could be identified displaying either more or less pronounced methylation patterns. Heavy methylation appeared to be associated with a significantly reduced relapse-free survival (p=0.03). Encouraged by these data, we analyzed relapse samples from t(4;11)- and t(11;19)-positive infant ALL patients, and found that these samples were even more extensively hypermethylated than the corresponding initial infant ALL samples. We here present, for the first time to our knowledge, a global view of the methylome in infant patients with MLL-rearranged ALL. We demonstrate that severe promoter CpG hypermethylation is present in t(4;11)- and t(11;19)-positive infant ALL. Of main therapeutic interest is our finding that the degree of DNA methylation among t(4;11)- and t(11;19)-positive infant ALL patients is related to relapse-free survival. Therefore, MLL-rearranged infant ALL patients with heavily methylated leukemias in particular should be considered candidates for therapies including inhibitors of DNA methylation in order to reverse the malignant phenotypes of these leukemias, and improve prognosis. Since MLL-rearranged infant ALL patients are even more hypermethylated at relapse, inhibition of aberrant DNA methylation might also be of vital importance at this stage of disease. Based on these data, we propose to initiate clinical trials using demethylating agents for patients with relapsed MLL- rearranged infant ALL. Meanwhile, we are investigating the in vitro cytotoxicity of various demethylating agents in our laboratory to pave the way for future clinical trials.
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Kang, Huining, Carla S. Wilson, Richard C. Harvey, I.-Ming Chen, Maurice H. Murphy, Susan R. Atlas, Edward J. Bedrick, et al. "Gene Expression Profiling Reveals Genes Predictive of Outcome In Infant Acute Lymphoblastic Leukemia (ALL) and Distinctive Age-Related Gene Expression Profiles (< 90 Days vs. > 90 Days): A Children's Oncology Group Study." Blood 116, no. 21 (November 19, 2010): 412. http://dx.doi.org/10.1182/blood.v116.21.412.412.
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Abstract Abstract 412 ALL arising in infants is a highly refractory disease. Overall event-free survival (EFS) remains poor and infants with MLL rearrangements (MLL-R) or those <90 days of age are known to have particularly poor outcomes. To identify genes predictive of event-free survival (EFS) that might serve as new diagnostic and therapeutic targets, we completed gene expression profiling (Affymetrix HG_U133Plus2) in 97 infant ALL cases registered to COG Clinical Trial P9407. Of these 97 infants, 78 were most recently uniformly treated on P9407 cohort 3. In the 97 cases, median age at diagnosis was 166 days (range 1–365) and increased age at diagnosis was significantly associated with improved EFS (P = 0.001). 89/97 infants had MLL-R, of which 49 had an AF4 partner gene (MLL-AF4 (AFF1)). Infants <90 days of age (P=.0001) and those with MLL-R (MLL-AF4, MLL-ENL (MLLT1), MLL-Other) had a significantly decreased EFS, while infants with MLL-AF9 (MLLT3) or cases lacking MLL-R had a significantly better EFS (P=0.014). From modeling expression profiles and multivariate analyses, a number of genes were identified that had a significant effect on EFS and were independent of patient age or MLL-R status, including: TACC2 and IRX2 (from modeling the entire cohort of 97 cases); RORA, IGJ, ZEB1, YES1 (cohort 3 modeled alone); and IRX1, IRX2, ST3GAL6, HLA-DQB1, STAB1, NEGR1, IRX5 (MLL-AF4 cases modeled alone). The significant effect of MEIS1 and KCNK12 expression on EFS was lost after consideration of MLL-R status, while the significance of many genes (particularly in the HOXA family) was not independent of patient age in multivariate analyses. Assessment of the expression levels of two genes alone at diagnosis: TACC2 and IRX2 in the entire cohort of 97 cases (P<0.0001; Fig. A), or, NEGRI and IRX2 in the MLL-AF4 cases (P<0.0001; Fig. B), were highly predictive of outcome on current treatment regimens. Distinctive and strikingly different gene expression profiles were also seen in infant ALL cases <90 days of age vs. >90 days of age (in the overall cohort and in the MLL-AF4 cases). Specifically evaluating the impact of patient age treated as a continuous variable revealed a striking transition in expression profiles at 90 days with a differential expression pattern involving many genes encoding histone-related, heat shock family, or immune response regulators (including HLA-DRB4, IL1R2, HSPA1A///1B). These distinctive profiles may reflect different transformed stem/precursor cells or susceptibilities to leukemic transformation at different patient ages, altered marrow microenvironments, or altered immune status; high expression of the heat shock proteins in particular among the youngest infants may reflect a more limited immune surveillance capacity. Given the rarity of infant ALL, this study represents one of the largest uniformly treated groups of infant leukemia to undergo gene expression profiling. In these studies we have identified genes that are highly predictive of outcome at diagnosis, in all infant ALL and in MLL-AF4 cases. Further analysis of these expression profiles, coupled with validation studies in other infant ALL cohorts, may allow for the identification of novel therapeutic targets among the genes discovered herein and ultimately for the development of more effective therapies. Disclosures: Felix: None: Patent not licensed.
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Qazi, Sanjive, and Fatih M. Uckun. "Augmented Expression of the IL3RA/CD123 Gene in MLL/KMT2A-Rearranged Pediatric AML and Infant ALL." Onco 2, no. 3 (August 25, 2022): 245–63. http://dx.doi.org/10.3390/onco2030014.
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Here, we evaluated transcript-level IL3RA/CD123 expression in mixed lineage leukemia 1 (MLL) gene/KMT2A-rearranged (MLL-R+) vs. MLL-R− pediatric AML as well as infant ALL by comparing the archived datasets of the transcriptomes of primary leukemic cells from the corresponding patient populations. Our studies provide unprecedented evidence that IL3RA/CD123 expression exhibits transcript-level amplification in MLL-R+ pediatric AML and infant ALL cells. IL3RA was differentially upregulated in MLL-AF10+ (2.41-fold higher, p-value = 4.4 × 10−6) and MLL-AF6+ (1.83-fold higher, p-value = 9.9 × 10−4) but not in MLL-AF9+ cases compared to other pediatric AML cases. We also show that IL3RA/CD123 expression is differentially amplified in MLL-AF4+ (1.76-fold higher, p-value = 2.1 × 10−4) as well as MLL-ENL+ infant ALL (1.43-fold higher, p-value = 0.055). The upregulated expression of IL3RA/CD123 in MLL-R+ pediatric AML and infant ALL suggests that CD123 may be a suitable target for biotherapy in these high-risk leukemias.
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Toia, Liana M., Erica Lynne Braverman, Jinno Antonio Magno, and Jessica C. Shand. "HMGB1 Interacts with the MLL-AF4 Fusion Complex to Regulate Pro-Leukemic Gene Transcription in Infant Acute Lymphoblastic Leukemia." Blood 132, Supplement 1 (November 29, 2018): 2602. http://dx.doi.org/10.1182/blood-2018-99-110833.
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Abstract Acute lymphoblastic leukemia (ALL) in infants carries a poor prognosis and is characterized by cytogenetic rearrangements producing abnormal MLL fusion genes. Clinically effective targeting of the MLL fusion heterocomplex remains challenging, and therapeutic options remain limited. We have observed that the reduced isoform of HMGB1, a chromatin architectural protein that stabilizes DNA and facilitates transcription, is selectively over-expressed in the nuclei of infant MLL-ALL cells. In this study, we generated an HMGB1 siRNA knockdown in primary MLL-ALL cells from 3 infants to test our hypothesis that HMGB1-MLL interactions regulate pro-leukemic gene expression and represent a rational therapeutic target. CD19-selected leukemic blasts were isolated from the cryopreserved bone marrow or peripheral blood specimens of 3 infants with cytogenetically confirmed MLL-AF4 rearrangements. HMGB1 knockdown was confirmed by comparing HMGB1 mRNA and protein expression, by qPCR and Western Blot, in cells transfected with HMGB1 vs. control sequence siRNA. First, determined whether HMGB1 knockdown affected expression of the MLL fusion gene itself, by comparing MLL-AF4 mRNA and protein levels 72 hours after siRNA transfection. HMGB1 knockdown produced a 2.8 (± 0.55)- fold decrease in MLL-AF4 mRNA expression by qPCR (p<0.05), with a corresponding decrease in MLL-AF4 fusion protein expression by Western Blot, in each of the 3 specimens. Next, we determined whether HMGB1 binds functionally relevant regions of the MLL gene. We developed an electrophoretic mobility assay (EMSA) to compare the mobility of lysates from control vs. HMGB1 siRNA-treated infant MLL-ALL cells when mixed with biotinylated oligonucleotides spanning the transcriptionally active domains of MLL1. In each of 3 primary infant MLL-ALL cells, we detected a consistent gel-shift pattern on SDS-PAGE, in wild-type and control siRNA lysates, with oligonucleotides spanning exons 6-9- where many MLL-AF4 fusions occur. The gel-shift was completely abrogated in HMGB1 siRNA lysates. We then compared the expression of MLL target genes involved in leukemic transformation, by qPCR, in infant MLL-ALL cells treated with HMGB1 vs. control siRNA. We observed a significant (p<0.01) reduction in expression of MEIS1 (5.8 ± 2.2-fold decrease), HOXA7 (4.3 ± 0.4-fold decrease) and HOXA9 (3.7 ± 1.5-fold decrease) in infant MLL-ALL cells treated with HMGB1 vs. control siRNA. These data confirmed a role for HMGB1 in MLL gene/target gene regulation at the DNA level. Finally, we considered whether HMGB1, as a scaffold protein, could interact directly with the MLL fusion heterocomplex at the protein level. We immunoprecipitated HMGB1 from the nuclear fraction of wild-type primary infant MLL-ALL cells (n=3 patients), then probed the pull-down for N-terminal MLL (MLLn), C-terminal MLL (MLLc), the MLLn-AF4 fusion, the MLLn-ENL fusion, and the MLL-associated histone 3 methyltransferase DOT1L. MLLn and MLLn-AF4 were strongly detected in all HMGB1 immunoprecipitates. Individual and sequential co-immunoprecipitation of HMGB1 with MLL-AF4 and DOT1L in revealed loss of known complex formation between MLL-AF4 and DOT1L following HMGB1 knockdown. This was accompanied by a 3.4 (± 0.9)-fold decrease in DOT1L mRNA expression (p<0.001) by qPCR and a complete loss of histone 3k79me2 protein expression by Western blot. Taken together, these data suggest a central role for the fully reduced isoform of HMGB1, found in high abundance in infant ALL nuclei, in the formation of the MLL-AF4 transcription complex- including for the stable recruitment of DOT1L and H3K79me2, and in the regulation of MLL target genes such as HOXA9 and MEIS1. We are currently conducting chromatin immunoprecipitation and sequencing studies to identify methylation marks, particularly at H3K79me2, impacted by HMGB1 knockdown in infant ALL cells. We hope these studies will directly inform the development of small molecule inhibitors that specifically disrupt the binding sites and capacities of HMGB1 with MLL, which could synergize with the effects of methyltransferase inhibitors to more completely silence leukemic gene expression in infant ALL and improve the prognosis of this devastating disease. Disclosures No relevant conflicts of interest to declare.
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Zhang, Alena Y., Blaine W. Robinson, Kajia Kao, Kathryn Behling, Meenakshi Devidas, Todd Alonzo, Susana C. Raimondi, et al. "Cell Death Regulatory Gene Expression Correlates with MLL Rearrangement Status and Prognostic Clinical Covariates in Acute Leukemia in Infants." Blood 112, no. 11 (November 16, 2008): 2255. http://dx.doi.org/10.1182/blood.v112.11.2255.2255.
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Abstract Infants with acute leukemia, especially neonates, comprise a unique high-risk population prone to chemotherapy resistance and treatment complications. Basal levels of expression of cell death and cell survival factors vary greatly in different types of cancer and are key determinants of response to treatment. Until now, expression of these factors had not been evaluated in detail in acute leukemia of infants. In this study the basal expression of pro-death and pro-survival genes in the intrinsic apoptosis pathway, apoptosis execution genes and mitochondrial maintenance genes was quantified in primary leukemia cells in a large cohort of infants, and relationships between gene expression and MLL status and clinical covariates were analyzed. Methods: Bone marrow or peripheral blood samples from the time of diagnosis were examined in 103 infants (age 1–363 d, median 177 d) with acute leukemia (63 ALL, 40 AML). White blood cell (WBC) counts ranged from 4.4–1676×103/μL (median 127.6×103/μL). MLL status was characterized by standard cytogenetics, FISH, Southern blot analysis, reverse transcriptase PCR and/or panhandle PCR approaches. The ALL cases were classified as MLL-AF4+ (n= 31), other MLL rearrangement positive (other MLL+) (n= 22) or MLL rearrangement negative (MLL−) (n= 10). Due to heterogeneity in partner genes, the AML cases were classified as MLL+ (n= 22) or MLL− (n= 18). Quantitative real time PCR analysis of total RNAs was performed using a custom TaqMan® low-density array with one or more assays, each in quadruplicate, for 35 cell death regulatory genes and 3 housekeeping genes, and ΔCT values (average cycle threshold values normalized to 18S rRNA) were computed. The Behrens-Fisher nonparametric multiple comparison test in the npmc package for R was used to determine the significance of the difference in median −ΔCT values among the three ALL groups and between the two AML groups (p<0.001 considered as significant). Relationships were sought between −ΔCT values and clinical covariates (age, sex, WBC count, CNS status, events) by computing Spearman correlation coefficients and their levels of significance. Results: In ALL, expression of the following genes was greater in MLL-AF4+ than MLL− cases: anti-apoptotic BCL-2 and MCL-1; pro-apoptotic BAX and BID; apoptosis execution APAF1, CASP3 and CASP9; and mitochondrial maintenance COX5A, CYC1, UQCR1 and UQCR. For some of these genes, expression was also greater in other MLL+ than MLL− cases. In AML, expression of pro-apoptotic BAK and BIK was higher in MLL+ than MLL− cases. For the sample set as a whole, expression of the following genes was positively correlated with WBC count (r>0.3; p<0.01): anti-apoptotic BCL-2 and BCL-W; pro-apoptotic NOXA; apoptosis execution CASP8; and mitochondrial maintenance HCCS. The correlation between WBC count and BCL-2 was the strongest (r=0.40;p=4E-5). Only the expression of pro-apoptotic BIM was correlated with age at diagnosis and the correlation was not particularly strong (r = −0.246;p=0.01). Conclusions: These results suggest that infant ALL and AML differ with respect to the expression of genes that regulate cell death. Furthermore, cell death regulatory gene expression is affected more by MLL translocations in infant ALL than infant AML. This finding may explain the greater impact of MLL translocations on prognosis in infant ALL than AML. The abundance of specific pro- as well as anti-apoptotic BCL-2 family mRNAs in MLL+ infant ALL is also relevant to the identification of novel therapeutic targets in this pathway. The positive correlation between anti-apoptotic BCL-2 expression and WBC count is biologically plausible and consistent with the aggressive nature of acute leukemia in infants.
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Bardini, Michela, Lilia Corral, Eleonora Mangano, Roberta Spinelli, Grazia Fazio, Ingrid Cifola, Andrea Biondi, Cristina Battaglia, and Giovanni Cazzaniga. "Infant ALL Patients Carrying t(4;11) Have a Different Genotypic Profile Than Older ALL Children." Blood 110, no. 11 (November 16, 2007): 1436. http://dx.doi.org/10.1182/blood.v110.11.1436.1436.
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Abstract Mice models and prenatal studies indicate that in childhood ALL the individual genetic lesions alone are insufficient to generate a full leukemic phenotype, and cooperating oncogenic lesions are required. Recently, multiple genome-wide studies on childhood ALL (1–18 years) identified deletions at several loci, mainly affecting genes that play a critical role in regulating B cell development and differentiation. By contrast, the prenatal and postnatal steps in the pathogenesis of Infant ALL (less than 1 year at diagnosis) are not defined. Infant ALL is a very aggressive disease, with t(4;11)/MLL-AF4 fusion representing the major subgroup. Although the very short latency period suggests that leukemogenic events occur prenatally, mice models indicates that MLL-AF4 alone is not sufficient to induce leukemia, and additional mutations may occur. Also unclear is whether the molecular pathways needed for lymphoid cell differentiation are altered in cases with an MLL rearrangement and, if so, whether these alterations differ between the leukemia of infants and older children. Aim of this study was to detect MLL-cooperating aberrations, undetectable by conventional techniques, by using genome-wide single nucleotide polymorphism (SNP) genome wide analysis (100K SNP human mapping, Affymetrix). More specifically, we searched for Loss of Heterozygosity (LOH) associated or not to copy number alteration. The identification of these lesions could help identifying leukemia pathogenesis, as well as providing the basis for targeted therapy. We have analyzed 28 cases of Infant ALL with t(4;11) at diagnosis and their corresponding samples at remission, when available (n=18). SNP data were analyzed by using dChip software, and confirmed by CNAG 2.0. A more dense SNP array analysis (250K) has been applied in selected cases to confirm LOH and precisely dissect the affected chromosomal regions. Compared to older childhood ALL patients, a far limited number of deletions/amplifications has been found; only 2/28 patients showed deletions, namely 1p36.33-p36.31 in 1 patient and 3p11.1-p12.2 plus 7q22.1-q22.2 in another patient, while 26/28 Infant ALL did not present any visible structural variation. Different from older children, several segmental copy-number neutral (CNN) LOH have been detected by dChip. The extension and prevalence of the affected regions was variable; among them 6p21.32 (4/28 cases), 7q31.33-q32.1 (3/28), 8q21.12-q21.3 (2/28), 8q24.11 (2/28) and 14q21.2 (2/28). Overall, these results confirm that Infant ALL with t(4;11)/MLL-AF4 fusion represents a biologically unique disease, different from other type of leukemia occurring in older children. While in older children a multistep mechanism (with the involvement of several genes) is required for the full leukemic phenotype, MLL rearrangements per se might play a major role on the leukemogenesis. By this approach it could not be excluded that different mechanisms could cooperate with MLL in transforming cells, including point mutations. The functional role of CNN-LOH still needs to be understood: they could either reflect the duplication of oncogenic mutations, or be related to epigenetic mechanisms.
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Stumpel, Dominique J. P. M., Pauline Schneider, Eddy H. J. van Roon, Judith M. Boer, Renee X. Menezes, Rob Pieters, and Ronald W. Stam. "Distinct Methylation Patterns among Infants with MLL Rearranged Acute Lymphoblastic Leukemia." Blood 110, no. 11 (November 16, 2007): 2794. http://dx.doi.org/10.1182/blood.v110.11.2794.2794.
Full textAbstract:
Abstract Acute Lymphoblastic Leukemia (ALL) in infants (i.e. children <1 year of age) is characterized by a high incidence of rearrangements of the MLL gene (∼80%) which is associated with a poor prognosis. The most frequent MLL rearrangements in infant ALL are translocations t(4;11), t(11;19) and t(9;11). Recently, gene expression profiling has established MLL rearranged leukemia as a unique type of leukemia (denoted MLL), that is clearly distinguishable from other ALL subtypes. Currently, these gene expression profiles are slowly revealing important genetic properties underlying this aggressive type of leukemia, however, any epigenetic data on MLL are still lacking. Therefore, the present study was designed to unravel the MLL-specific methylation patterns underlying infant MLL by applying differential methylation hybridization (DMH) using CpG island microarrays containing ∼9000 CpG island probes, in duplicate. Primary infant ALL samples carrying t(4;11) (n=21), t(11;19) (n=17) and t(9;11) (n=6) were compared to infant ALL (n=13) and non-infant pediatric ALL (n=15) samples without MLL rearrangements. In addition, healthy pediatric bone marrow samples (n=9) were included as a reference. Compared to healthy controls, 656 CpG island probes were identified as significantly hypermethylated in t(4;11) positive samples, and 131 CpG island probes in t(11;19) positive samples (p<0.01, false discovery rate <5%). Interestingly, t(11;19) positive ALL patients shared 95% of their methylated probes with t(4;11) patients, suggesting a common methylation pattern which is completely absent in both infant and non-infant ALL patients lacking MLL rearrangements. Remarkably, displaying only a single probe significantly methylated as compared to healthy bone marrow, this common methylation pattern is also absent in t(9;11) positive ALL patients, indicating that based on genome-wide methylation, these patients represent a distinct entity clearly distinguishable from other MLL subgroups. Moreover, the fact that t(4;11) patients exhibit 532 methylated CpG island probes that were not found to be methylated in t(11;19) patients, demonstrates that these patients also exhibit a t(4;11) specific set of methylated genes. Identification of the genes represented by these CpG island probes and subsequent validation of the results obtained in this study is currently being performed (using pyrosequencing and methylation specific PCR analyses). In conclusion, these data reveal that different types of MLL rearranged infant ALL show distinct genome-wide methylation patterns. Specifically, infant ALL patients carrying t(4;11) and t(11;19) are characterized by severe CpG island hypermethylation, as compared to both t(9;11) positive infant ALL patients, as well as pediatric ALL patients lacking MLL rearrangements. Therefore, t(4;11) and t(11;19) patients in particular may well be suitable candidates for DNA methylation inhibiting therapeutic intervention. Finally, these promising results for the first time provide epigenetic insights into the complex biology of infant MLL, and clearly warrant further investigation currently being performed at our laboratory.
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Harrison, Christine J., Anthony V. Moorman, Paola de Lorenzo, Rob Pieters, Zoann E. Dreyer, Nyla A. Heerema, Andrew J. Carroll, et al. "Cytogenetics and Outcome Of Infants With Acute Lymphoblastic Leukemia and Absence Of MLL Rearrangements." Blood 122, no. 21 (November 15, 2013): 1349. http://dx.doi.org/10.1182/blood.v122.21.1349.1349.
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Abstract Acute lymphoblastic leukemia (ALL) in infants less than one year of age is characterized by a high frequency of rearrangements of the MLL gene (MLL-R) and poor prognosis. Infants with no MLL rearrangements (MLL-G) have a better outcome than those with MLL-R. To better understand the association of chromosomal abnormalities and outcome among MLL-G infants, we carried out a detailed cytogenetic investigation of patients from two infant ALL trials: Interfant-99 and Children’s Oncology Group (COG)-P9407. Among 162 MLL-G patients, an abnormal karyotype was detected in 90/128 (70%) patients with a successful result. They were categorised according to cytogenetic risk group (good, intermediate and poor) as previously defined for childhood ALL. Compared with childhood ALL (1-18 years) using data from the UKALL97/99 treatment trial, the frequency of good risk cytogenetic abnormalities among MLL-G infants was significantly lower (12% v 60%, p<0.01), while the frequency of poor risk abnormalities (excluding MLL translocations) was similar (8% v 10%). While ETV6-RUNX1 fusion is present in 25% of childhood ALL, no ETV6-RUNX1 cases were found among 75 patients tested by FISH or RT-PCR. High hyperdiploidy was the most prevalent abnormality, although the frequency was also much lower than childhood ALL (12% v 38%, p<0.01). Other established translocations were observed in a small number of cases: t(9;22)(q34;q11)/BCR-ABL1 (n=2), t(1;19)(q23;p13)/TCF3-PBX1 (n=3) and the infant specific, t(7;12)(q36;p13)/ETV6-HLXB9 fusion (n=1). Interestingly the incidence of t(9;22) and t(1;19) among MLL-G infants was not markedly different from childhood ALL: 1.6% v 2.6% and 2.3% v 3.5%, respectively. Abnormalities of the short arm of chromosome 9 (9p) were observed in 14 (11%) cases at a similar incidence to childhood ALL. Chromosome 15 abnormalities were found in 12 patients. This more frequent occurrence of 15q abnormalities in infant ALL has been previously noted. It is well established that MLL-R infants are younger than their MLL-G counterparts. Classification of MLL-G patients by cytogenetic risk group showed further correlation with age. The majority of cytogenetic good risk and all poor risk patients were >9 months old, whereas half of the cytogenetic intermediate risk patients were <9 months. In addition, there was evidence of outcome heterogeneity according to cytogenetic risk group. Event free survival at 4 years was 93% (SE 6.9), 66% (SE 4.7) and 50% (SE 15.8) for good, intermediate and poor risk groups, respectively, similar to that observed in childhood ALL when the same cytogenetic classification was applied. Low FLT3 expression has been associated with an excellent outcome in infants with ALL treated on COG-P9407, and the majority (10/11) of cases with low expression were MLL-G. However, low FLT3 expression was not simply a reflection of MLL-G status, as 7 MLL-G patients had high FLT3 expression. We have shown that low expression in MLL-G patients is not associated with a particular cytogenetic risk group. Interestingly, none of the MLL-G patients with low expression had an event, whereas all 5 of the events occurred in the high expressers. We have confirmed a unique cytogenetic profile among infants with MLL-G ALL. We demonstrate that the MLL-G infants share the same cytogenetic abnormalities as older children with ALL, but the distribution of abnormalities differs. Generally infants with MLL-G ALL are older, have low FLT3 expression and have an improved outcome compared to their MLL-R counterparts. Despite small numbers of MLL-G infants, when classified into the same good, intermediate and poor risk cytogenetic subgroups as childhood ALL, their pattern of outcome was very similar to that observed in older children. However, their overall worse outcome likely reflects the differences in distribution of good and poor risk abnormalities: lower incidence of the good risk abnormality: high hyperdiploidy, absence of ETV6-RUNX1, and higher incidence of poor risk abnormalities. Nevertheless, these data suggest that some MLL-G infants, especially those with good risk cytogenetics, may benefit from treatment on childhood protocols, which are generally less intensive and less toxic than infant ALL regimens. Disclosures: No relevant conflicts of interest to declare.
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Shand, Jessica C., Jinno Magno, Michael Weinstein Winter, and Liana M. Toia. "MLL-Rearranged Infant ALL, but Not Standard-Risk ALL, Induces Inflammasome Signaling and Immune Activation." Blood 124, no. 21 (December 6, 2014): 3660. http://dx.doi.org/10.1182/blood.v124.21.3660.3660.
Full textAbstract:
Abstract T cell-based therapy for infants with MLL-rearranged B-ALL (MLL-ALL) represents a promising approach in this high-risk, poor-prognosis subset. Factors in the immune microenvironment that influence T-cell function, however, are poorly understood in MLL-ALL. We have observed complete inhibition of healthy donor T-cell proliferation by ALL cells from MLL-ALL, but not standard risk ALL (SR-ALL) patients. T-cell function is restored in the presence of bone marrow macrophages. In this study, we hypothesized that MLL-ALL cells selectively induce the inflammasome- a cytokine signaling complex formed by macrophages in response to damage-associated molecular patterns (DAMPs) released by leukemic cells. In the canonical inflammasome pathway, DAMPs engage macrophage Toll-like receptors, which activate caspase-1 and drive production of T-cell activating cytokines such as IL1-β. First, we determined whether ALL cells obtained from MLL-ALL vs. SR-ALL patients could produce high mobility group box 1 (HGMB1), a prototypical DAMP associated with tumor cell stress and macrophage PRR activation, particularly after anthracycline therapy. CD19+ cells were isolated from the diagnostic bone marrow specimens of patients with MLL-ALL (n=1), SR-ALL (n=4), and healthy donor PBMC (n=2) and treated with either control media or 2uM doxorubicin. Prior to treatment both MLL-ALL and SR-ALL, but not healthy donor cells, expressed HMGB1 protein as measured by Western immunoblot (29 kDa, GADPH internal control). Following doxorubicin treatment, HMGB1 protein was increased in MLL-ALL but not SR-ALL. Soluble HMGB1, measured by ELISA, was significantly increased in MLL-ALL (12.6 + 3.7 ng/ml untreated vs. 51.4 + 1.9 treated) compared to SR-ALL cells (5.0 + 0.8 ng/ml untreated, 4.3 + 1.5 treated, p<0.05). Soluble HMGB1 was not detectable in healthy donor cells before or after treatment. Next, we determined whether the increased HMGB1 secretion observed in MLL-ALL vs. SR-ALL cells was associated with macrophage caspase-1 activation. Control media and 2 uM doxorubicin treated-and-washed MLL-ALL, SR-ALL and healthy control CD19+ cells were cultured alone or 1:1 with allogeneic primary human macrophages for 24 hours. The active isoform of caspase-1 (20 kD tetramer) was detectable by immunoblot only in macrophages exposed to doxorubicin-treated MLL-ALL. Mean levels of IL1-β, measured by ELISA, were significantly increased in cocultures of doxorubicin-treated MLL-ALL + macrophages (1255 pg/ml + 322) compared to untreated MLL-ALL (820 pg/ml + 183, p=<0.05) and doxorubicin-treated SR-ALL + macrophages (494 pg/ml + 85 p<0.01). Macrophages cultured alone did not produce significant levels of IL1-β before or after doxorubicin treatment. Finally, we wished to determine whether expression of Toll-like receptor 4, a surface receptor for HMGB1 known to signal caspase-1, was increased on macrophages exposed to doxorubicin-treated MLL-ALL vs. SR-ALL. Folowing coculture with untreated and doxorubicin treated MLL-ALL and SR-ALL cells, macrophage TLR4 mRNA and surface expression was quantified by qPCR and flow cytometry, respectively. Both mRNA and surface expression of TLR4 was high at baseline, and did not change significantly after coculture. Unexpectedly, however, the MLL-ALL cells themselves displayed a significant increase in TLR4 mRNA (20-fold compared to endogenous beta-glucuronidase, p<0.0001; 12-fold compared to untreated MLL-ALL, p<0.05 by ΔΔCt) and surface expression (median fluorescence intensity 1005 + 89 vs. 367 + 32 respectively, p<0.05) following doxorubicin treatment. This phenomenon was not observed in SR-ALL or healthy CD19+ cells and is consistent with other tumor models of autocrine immune activation by HMGB1. Taken together, these data indicate that primary MLL-ALL cells, but not SR-ALL cells, are capable of inducing a caspase-1 associated inflammasome that enhances both innate and T-cell immune activation following anthracycline therapy. Studies are currently underway with additional primary MLL-ALL samples to determine how strategic augmentation of this pathway may impact the anti-leukemia killing capacity of therapeutic T-cells. Disclosures No relevant conflicts of interest to declare.
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Kerstjens, Mark JB, Emma MC Driessen, Merel Willekes, Sandra S. Mimoso Pinhancos, Rob Pieters, and Ronald Stam. "MEK Inhibition Is a Promising Therapeutic Strategy for MLL-Rearranged Infant ALL Patients Carrying RAS Mutations." Blood 124, no. 21 (December 6, 2014): 919. http://dx.doi.org/10.1182/blood.v124.21.919.919.
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Abstract Background: Acute Lymphoblastic Leukemia (ALL) in infants is characterized by a high incidence (~80%) of chromosomal rearrangements of the Mixed Lineage Leukemia (MLL) gene, fusing the N-terminal portion of MLL to the C-terminal region of one of its translocation partner genes. MLL-rearranged infant ALL patients are challenged by a very poor prognosis (i.e. 30-40% 5-year EFS), hence the need for better risk stratification and improved therapeutic solutions is evident. We recently screened a relatively large cohort (n=109) infant ALL patients (all enrolled in INTERFANT treatment protocols) for the presence of KRAS and NRAS mutations and found that the incidence of such mutations ranges between 14-24%, depending on the type of MLL translocation. Moreover, these mutations were found to represent independent predictors of exceedingly poor prognosis; patients carrying RAS mutations essentially stand no chance in surviving their malignancy, as all RAS mutation-positive MLL-rearranged infant ALL patients deceased within 3 years from diagnosis. Aims: Here we aimed to identify a therapeutic strategy to improve the prognosis of MLL-rearranged infant ALL patients carrying RAS mutations. Methods: For this, 8 small molecule inhibitors against different RAS-pathway components were selected and initially tested for anti-leukemic activity against the MLL-rearranged ALL cell lines SEM and RS4;11 (RASwt) and KOPN8 (RASmut) using MTS cell viability assays. Next, primary MLL-rearranged infant ALL samples (n=20) all carrying MLL translocation t(4;11) (giving rise to the MLL-AF4 fusion protein) either with (n=6) or without (n=14) RAS mutations were exposed to these inhibitors in MTT cytotoxicity assays. In addition, we assessed the RAS activity in RAS mutated and wild-type MLL-rearranged infant ALL cells, and performed immunoblotting analysis of downstream MEK and ERK both in the absence and presence of the MEK inhibitors. Results: We found that the MEK inhibitors MEK162, Selumetinib and Trametinib effectively reduced the viability of KOPN8 cells (RASmut), whereas SEM and RS4;11 cells (RASwt) largely remained unaffected. In line with this, MLL-AF4+ infant ALL patient samples carrying RAS mutations were significantly more sensitive to these MEK inhibitors when compared with patients carrying wild-type RAS genes: LC50 values for MEK162 were 0.04 vs. 26.9 µM (p<0.01), for Selumetinib 0.04 and 23.7 µM (p<0.01), and for Trametinib 0.01 vs. 26.5 µM (p<0.01), respectively. Furthermore, the presence of RAS mutations in primary MLL-rearranged infant ALL samples was associated with significantly increased RAS activity, as determined by immunoprecipitation of GTP-bound RAS. Remarkably, however, enhanced RAS activation did not manifest itself in the form of increased phosphorylation of the downstream ERK protein, while a slight increase of MEK phosphorylation was observed. Yet, MEK inhibitor exposure in both KOPN8 and SEM cells resulted in nearly complete abrogation of ERK phosphorylation, without affecting total ERK protein levels, suggesting that the loss of ERK activation plays an important role in the observed anti-leukemic effects. Furthermore, MEK162 and Selumetinib seemed to induce accumulation of phosphorylated MEK, while MEK phosphorylation was hardly affected by Trametinib. Interestingly, a subgroup of patient samples (n=5) with wild-type RAS also showed sensitivity towards MEK inhibition, similar to the primary cells with RAS mutations. However, this observation could not be explained by increased RAS activation, nor by the phosphorylation levels of either MEK or ERK. Conclusions: Our data show that MEK inhibition represents a promising therapeutic approach for MLL-rearranged infant ALL patients carrying additional RAS mutations. Furthermore, the mechanism of action provoked by these MEK inhibitors seems to involve abrogation of ERK phosphorylation, but the initial level of ERK phosphorylation did not correlate with MEK inhibitor sensitivity, and has no predictive value. Currently we are in the process of testing the efficacy of the above mentioned MEK inhibitors in vivo using a xenograft mouse model, while further elucidation of the molecular mechanisms underlying the anti-leukemic effects of these inhibitors in MLL-rearranged ALL cells is in progress. Disclosures No relevant conflicts of interest to declare.
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Szczepanski, Tomasz, Lukasz Sedek, Paola de Lorenzo, Martin Schrappe, Richard Ratei, Goran Gustaffson, Liisa Hovi, et al. "Prognostic Value of Immunophenotype In Infant ALL – Results of the INTERFANT'99 Study." Blood 116, no. 21 (November 19, 2010): 2700. http://dx.doi.org/10.1182/blood.v116.21.2700.2700.
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Abstract Abstract 2700 Objective: Infant acute lymphoblastic leukemia (ALL) is characterized by distinct biological and clinical features. In this study, we aimed at detailed evaluation of infant ALL immunophenotype based on the large cohort of patients treated homogeneously on INTERFANT'99 protocol. Patients and Methods: We were able to obtain sufficient flow cytometry based immunophenotypic data from 286 (130 boys and 156 girls) of 483 infant ALL cases treated on INTERFANT'99 protocols. The distribution of patient and leukemia characteristics as well as clinical outcome were very similar to the entire study cohort (Pieters et al, Lancet 2007; 370: 240–250). The positivity for a certain antigen was defined at the cut-off value of >20% of cells expressing this antigen. In B-cell precursor ALL (BCP-ALL), evaluated antigens included CD10, CD20, CD34, myeloid markers (CD13, CD33, CD15 and/or CD65) and NG2 antigen assessed with 7.1 antibody. The strength of association between characteristics (patient's age and gender, initial white blood count [WBC], CNS involvement, presence of MLL gene rearrangements, prednisone response [PR]) and antigen expression was analyzed with Fisher exact test adjusted for multiple comparisons. Univariate analysis of outcome by immunophenotype was based on event free survival (EFS) and compared by log-rank test. Multivariate analysis was performed using Cox model in MLL-rearranged cases. P values < 0.05 were considered to be significant. Results: Altogether, 272 cases of infant BCP-ALL and 14 of infant T-ALL were analyzed. Most BCP-ALL cases were CD10 negative (66%), CD20 negative (88%), CD34 positive (60%), and NG2 positive (58%). Co-expression of myeloid antigens was less frequent and concerned CD13 in 8%, CD33 in 17%, CD15 in 31%, and CD65 in 28%. CD10 negativity and NG2 expression were significantly more frequent in younger infants, patients with initial high leukocytosis and with MLL rearrangements. CD34 expression was observed more frequently in younger infants, while CD20 positivity was significantly associated with lower initial leukocytosis and absence of MLL rearrangement. For myeloid antigen expression, the only significant association was more frequent CNS involvement in infants with CD13+ and/or CD33+ BCP-ALL. Analysis of outcome showed significantly better prognosis for CD10 positivity (4-year EFS of 56.3 as compared to 37.2 for CD10 negative BCP-ALL), absence of NG2 expression (4-year EFS of 63.9 as compared to 34.9 for NG2 positive BCP-ALL) and CD33 negativity (4-year EFS of 47.6 as compared to 27.7 for CD33 positive BCP-ALL). There was also a trend towards better outcome in CD20 positive infant BCP-ALL. While CD10 negativity and NG2 positivity were associated with well-known high-risk features, prognostic significance of CD33 expression is somewhat more difficult to explain. Of note, CD10 negativity was not associated to a worst outcome in MLL-rearranged patients. Moreover, in this subset, multivariate analysis showed that none of the markers analyzed had independent prognostic significance, after adjustment for sex, age at diagnosis, WBC and PR. In rare infant T-ALL cases, we observed equal distribution to different immunophenotypic subgroups as defined by EGIL classification. Conclusion: Infant BCP-ALL has distinct immunophenotypic features. However, immunophenotype has no independent prognostic relevance when MLL, age, WBC, PR and other factors are included. Disclosures: No relevant conflicts of interest to declare.
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Ross, Julie A., Amy M. Linabery, Crystal Blommer, Erica Langer, Logan G. Spector, Joanne M. Hilden, Nyla A. Heerema, Richard L. Tower, and Stella M. Davies. "Genetic Susceptibility Loci and Risk of Infant Leukemia by Subtype and MLL status: A Children's Oncology Group Report." Blood 118, no. 21 (November 18, 2011): 749. http://dx.doi.org/10.1182/blood.v118.21.749.749.
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Abstract 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.
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Stumpel, Dominique JPM, Pauline Schneider, Lidija Seslija, Hikari Osaki, Owen Williams, Rob Pieters, and Ronald Stam. "Connectivity Mapping Identifies HDAC Inhibitors as Suitable Candidates for the Treatment of t(4;11)-Positive Acute Lymphoblastic Leukemia In Infants." Blood 116, no. 21 (November 19, 2010): 3222. http://dx.doi.org/10.1182/blood.v116.21.3222.3222.
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Abstract Abstract 3222 To date, MLL-rearranged infant Acute Lymphoblastic Leukemia (ALL) remains the most aggressive type of childhood leukemia characterized by a high rate of early relapses and a grim prognosis. This type of leukemia arises from chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene. The genesis of such translocations seems to be initiated in utero. Hence, MLL-rearranged ALL is typically diagnosed in infants at or shortly after birth. The most common MLL translocation among infant ALL patients, occurring in about 50% of the cases, is t(4;11) generating the oncogenic fusion product MLL-AF4. MLL-rearranged ALL is distinguishable from other ALL subtypes by unique gene-expression profiles. While inappropriate gene activation seems to be largely driven by specific histone modifications, we recently showed that gene inactivation in MLL-rearranged infant ALL can be explained by aberrant promoter hypermethylation. Focusing on genome-wide DNA methylation, we here uncovered that MLL-rearranged infant ALL is characterized by the activation of particular set of (proto-onco)genes as a result of abnormal promoter hypomethylation. In search for therapeutic agents capable of targeting these potential cancer-promoting genes, we applied connectivity mapping on a gene expression signature based on the genes most significantly hypomethylated in t(4;11)-positive infant ALL as compared with healthy bone marrows. Connectivity map analyses revealed that histone deacetylase (HDAC) inhibitors represent the most suitable compounds to reverse this gene signature. We show that six different HDAC inhibitors effectively induce leukemic cell death in primary t(4;11)-positive infant ALL cells in vitro. These cytotoxic responses were accompanied by down-regulation of MYC, SET, RUNX1 and RAN, and were preceded by degradation of the oncogenic MLL-AF4 fusion product. Our data shows that DNA methylation profiles allow the identification hypomethylated proto-oncogenes, and underlines the essential role for epigenetic de-regulation in MLL-rearranged ALL. Furthermore, we show for the first time that connectivity mapping can indirectly be applied on DNA methylation patterns, providing a rationale for HDAC inhibition in t(4;11)-positive leukemias. Given the presented potential of HDAC inhibitors to target important proto-oncogenes including the leukemia-driving MLL fusion in vitro, these agents should urgently be tested in in vivo models and subsequent clinical trials. Disclosures: No relevant conflicts of interest to declare.
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Gill Super, HJ, PG Rothberg, H. Kobayashi, AI Freeman, MO Diaz, and JD Rowley. "Clonal, nonconstitutional rearrangements of the MLL gene in infant twins with acute lymphoblastic leukemia: in utero chromosome rearrangement of 11q23." Blood 83, no. 3 (February 1, 1994): 641–44. http://dx.doi.org/10.1182/blood.v83.3.641.641.
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Abstract Rearrangements of chromosome band 11q23 are common in infant leukemias, comprising more than 70% of the observed chromosome abnormalities in children less than 1 year of age. The MLL gene, which is located at the 11q23 breakpoint in infant, childhood, and adult acute leukemias, has been cloned and has homology to the Drosophila trithorax gene. The breakpoints in MLL are restricted to an 8.3-kilobase pair (kb) region of the gene that is involved in translocations with as many as 29 other chromosomal regions in a number of phenotypically distinct acute leukemias. We have detected an identical, clonal, nonconstitutional rearrangement of the MLL gene in peripheral blood cells from a pair of female infants twins with acute lymphoblastic leukemia (ALL) and a t(11;19)(q23;p13.3). The detection of nonidentical IGH rearrangements suggests that the MLL rearrangement took place in a B-cell precursor or hematopoietic stem cell in one twin which was transferred in utero to the other fetus resulting in ALL with an identical aneuploid karyotype in both infants. We speculate that the other MLL-related infant leukemias may also develop in utero, and that the rearrangements may occur consistently in stem cells or early precursor cells, accounting for the frequency of mixed-lineage leukemia in infants.
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Gill Super, HJ, PG Rothberg, H. Kobayashi, AI Freeman, MO Diaz, and JD Rowley. "Clonal, nonconstitutional rearrangements of the MLL gene in infant twins with acute lymphoblastic leukemia: in utero chromosome rearrangement of 11q23." Blood 83, no. 3 (February 1, 1994): 641–44. http://dx.doi.org/10.1182/blood.v83.3.641.bloodjournal833641.
Full textAbstract:
Rearrangements of chromosome band 11q23 are common in infant leukemias, comprising more than 70% of the observed chromosome abnormalities in children less than 1 year of age. The MLL gene, which is located at the 11q23 breakpoint in infant, childhood, and adult acute leukemias, has been cloned and has homology to the Drosophila trithorax gene. The breakpoints in MLL are restricted to an 8.3-kilobase pair (kb) region of the gene that is involved in translocations with as many as 29 other chromosomal regions in a number of phenotypically distinct acute leukemias. We have detected an identical, clonal, nonconstitutional rearrangement of the MLL gene in peripheral blood cells from a pair of female infants twins with acute lymphoblastic leukemia (ALL) and a t(11;19)(q23;p13.3). The detection of nonidentical IGH rearrangements suggests that the MLL rearrangement took place in a B-cell precursor or hematopoietic stem cell in one twin which was transferred in utero to the other fetus resulting in ALL with an identical aneuploid karyotype in both infants. We speculate that the other MLL-related infant leukemias may also develop in utero, and that the rearrangements may occur consistently in stem cells or early precursor cells, accounting for the frequency of mixed-lineage leukemia in infants.
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Popov, Alexander, Grigory Tsaur, Tatiana Verzhbitskaya, Olga Streneva, Egor Shorikov, Leonid Saveliev, and Larisa Fechina. "Presence of MLL Gene Rearrangements in Infant Acute Leukemia Could be Predicted By Tumor cells’ Immunophenotype." Blood 124, no. 21 (December 6, 2014): 5359. http://dx.doi.org/10.1182/blood.v124.21.5359.5359.
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Abstract Acute leukemia (AL) in children less than 1 year old is the relatively rare disease with specific biological features and poor outcome. It is also characterized by high incidence of MLL gene rearrangements. Immunophenotype of infants’ leukemia varies due to presence or absence of MLL gene rearrangements. Aim of the study –description of immunophenotype in infant acute lymphoblastic and acute myeloid leukemia (ALL and AML respectively) due to presence of MLL gene rearrangements. Methods. Totally 540 cases of pediatric AL were studied. 113 patients (59 boys and 54 girls) aged from 5 days to 11 months were included in the study group. Their data was compared to 427 cases of acute leukemia in older children. Tumor cells immunophenotyping was performed by 6-8-color flow cytometry. Detection of various types of MLL-gene rearrangements was done by fluorescence in-situ hybridization, reverse-transcriptase polymerase chain reaction (PCR) and long-distance inverse PCR. Results. ALL was found less frequently in infants than in older children (68.1% and 86.9% respectively, p<0.001) while percentage of acute myeloid leukemia cases was higher in infants (27.4% and 11.5% respectively, p<0.001). Significant immunophenotypic differences were observed in patients with and without MLL gene rearrangements in both ALL and AML. Number of ALL cases in those tumor cells expressed CD10, CD20, CD45, CD133, CD15, CD65 NG2 significantly varied between MLL-positive and MLL-negative groups (p<0.001, p<0.001, p=0.002, p<0.001, p=0.004, p=0.019 and p<0.001 respectively). NG2-positivity represented the highest overall correct prediction (OCP) rate for presence of MLL-rearrangements (90.6%). Diagnostic accuracy of CD20-negativity and CD45-positivity was lower (81.2% and 81.9% respectively) while OCP for CD10-negativity (76.4%), CD133-positivity (76.5%) CD15-positivity (67.7%) and CD65-positivity (53.7) was not sufficient enough. Nevertheless CD10-positive BCP-ALL with MLL-rearrangements differed from CD10(+) cases in MLL-germline group. CD10 homogeneous expression was noted frequently in MLL-germline cases than in MLL-rearranged ones (p=0.001). Although there were found no significant differences in CD22-positive patients’ number, CD22(+)-cells percentage was significantly lower in MLL-positive cases (median 89.9%, range 25.2-99.7% and median 99.9%, range 96.0-99.9% respectively, р=0.003). Thus CD20-negativity, CD10-negativity/low expression, high CD45, CD15, CD65, CD133 and NG2 expression, decreased CD22-expression are immunophenotypic signatures of MLL-rearranged infant ALL, although NG2 has the highest diagnostic efficacy. Interestingly CD10-negativity and positivity for CD34, CD15 and CD65 could be able to distinguish MLL-AF4-positive cases from patients carrying other types of MLL-rearrangements. Number of AML cases in those tumor cells expressed CD99, CD133, CD15, CD65, CD4, CD11b, CD61, NG2 varied between MLL-positive and MLL-negative groups ((p=0.019, p=0.012, p=0.002, p=0.004, p=0.005, p<0.001, p=0.015 and p<0.001 respectively). Thus CD61-negativity, high CD99, CD15, CD133, CD65, CD11b, CD4 and NG2 expression were immunophenotypic signatures of MLL-rearranged infant AML, although CD11b and NG2 had the highest diagnostic efficacy (95.0% and 89.7% respectively). Conclusion. Thus immunophenotype of AL in children less than 1 year old differs significantly from patients of older age groups. Infants’ ALL and AML immunophenotype varies greatly due to the presence of MLL gene rearrangements. Complex diagnostic immunophenotyping of infants’ AL allows predicting presence of MLL rearrangements while NG2 and CD11b are the most applicable single markers for ALL and AML respectively. Disclosures No relevant conflicts of interest to declare.
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van der Linden, Marieke Hendrika, Merel Willekes, Eddy Van Roon, Pauline Schneider, Rob Pieters, and Ronald Stam. "MLL Fusion Driven Activation of CDK6 Potentiates Proliferation in MLL-rearranged Infant ALL." Blood 120, no. 21 (November 16, 2012): 1426. http://dx.doi.org/10.1182/blood.v120.21.1426.1426.
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Abstract Abstract 1426 MLL-rearranged acute lymphoblastic leukemia represents a highly aggressive and clinically unfavorable type of childhood leukemia, displaying unique gene expression signatures. Nevertheless, the overwhelming number of differentially expressed genes made it difficult to elucidate the actual “drivers” of the leukemia. However, recent advances demonstrated that MLL fusion proteins recruit the histone methyltransferase DOT1L, leading to H3K79 methylation. Hence, genomic regions displaying aberrant enrichment of H3K79 methylation are prone to mark genes transcriptionally activated by the MLL fusion protein itself. Based on this concept, two independent studies recently identified gene signatures consisting of genes likely to represent direct MLL fusion targets. Yet, functional validation of such genes so far remains unacknowledged. In the present study we confirmed that CDK6 (cyclin-dependent protein kinase 6) represents a direct target of MLL-AF4 in t(4;11)-positive ALL cells. In contrast to its functional homologue CDK4, ChIP-sequencing analysis showed the presence of both MLL and AF4, as well as H3K79 methylation at the CDK6. Moreover, CDK6 mRNA appeared significantly (p<0.001) higher expressed in primary MLL- rearranged infant ALL patient samples when compared with other childhood ALL subtypes without translocations of the MLL gene. Next, using RNA interference, we performed MLL-AF4 and MLL-ENL knockdown experiments in ALL cell lines bearing these corresponding fusion transcripts, resulting in CDK6 down-regulation, whereas CDK4 expression was unaffected. These results emphasize that CDK6 is indeed a genuine transcriptional target of the MLL fusion protein itself. Moreover, direct knockdown of CDK6 itself significantly inhibited proliferation in MLL-rearranged ALL cells, whereas knockdown of CDK4 virtually had no effect on the cell cycle in these cells. Taken together we conclude that CDK6 up-regulation in MLL-rearranged ALL is directly mediated by the MLL fusion itself and provides these cells with a proliferation advantage. Disclosures: No relevant conflicts of interest to declare.
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Urtishak, Karen A., Wang Li-San, David T. Teachey, Tasian K. Sarah, Jeffrey S. Barrett, I.-Ming L. Chen, Susan R. Atlas, et al. "PI3K/AKT/mTOR Signaling Is a Significant Druggable Pathway In Infant Acute Lymphoblastic Leukemia (ALL)." Blood 122, no. 21 (November 15, 2013): 1669. http://dx.doi.org/10.1182/blood.v122.21.1669.1669.
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Abstract Introduction Infant acute lymphoblastic leukemia (ALL) is an orphan disease with unmet need for safe effective therapies. This is an urgent problem because conventional chemotherapies are ineffective and have life-threatening toxicities in infants. Although the MLL rearrangements occurring in 75% of cases are associated with poor outcome, survival is inferior whether MLL is rearranged or not. We recently reported that infant ALL proved sensitive to obatoclax mesylate (GeminX Pharmaceuticals; now an indirect, wholly owned subsidiary of Teva Pharmaceutical Industries Ltd.) in vitro regardless of poor prognostic features including MLL gene rearrangement. Moreover, we showed that the leukemia cell killing by obatoclax involved apoptosis, necroptosis and autophagy (Urtishak et al., Blood 2013). Therefore, the recent pharmaceutical abandonment of obatoclax led us to search for similarly acting drugs, the Results of which identified the well-known antipsychotic thioridazine as a candidate for potential repurposing. Methods Correlative analyses were performed between basal gene expression profiles at leukemia diagnosis and single agent obatoclax EC50 values from MTT assays in 47 cases of infant ALL from the Children's Oncology Group P9407 trial (25 MLL-AF4; 8 MLL-ENL; 7 other MLL-rearranged; 7 MLL-germline) in order to find a priori determinants of obatoclax sensitivity; significant genes were further studied by Ingenuity Pathway Analysis (IPA). A search for similarly acting compounds was conducted by Connectivity Map analysis of gene expression profiles of MLL-AF4 ALL cell lines after obatoclax treatment. MTT assays without and with cell death pathway inhibition, Western blot and flow cytometric cell death assays, and phosphoflow cytometric signaling analyses were utilized to investigate activity and target modulation by potential candidates. Results IPA identified significant correlations between basal gene expression of the mTOR and downstream intersecting eIF4/p70S6K signaling programs and obatoclax EC50 in all 47 primary cases of infant ALL, as well as in the subset of the 25 cases with MLL-AF4 rearrangements. Consistent with the relevance of this pathway in leukemia cell killing that was suggested by the basal gene expression profiles in the primary cases, the Connectivity Map analysis of obatoclax-treated cell lines for compound matching returned a number of highly ranked PI3K/AKT/mTOR signal transduction inhibitors as potential obatoclax substitutes. Three of the compounds (LY294002, wortmannin, thioridazine) were not only cytotoxic in MLL-AF4 ALL cell lines, but also they abrogated PI3K/AKT/mTOR signaling as indicated by robust inhibition of phosphorylated S6. Of these compounds, the phenothiazine derivative thioridazine, which has been used clinically for decades as a neuroleptic, was of high interest because of potential advantages of drug repurposing for more rapid drug advancement. Moreover, detailed flow cytometric and Western blot analyses, and MTT assays of thioridazine in the presence of cell death pathway inhibitors validated activation of all three cell death mechanisms in the MLL-AF4 ALL cell lines similarly to obatoclax. Conclusions Thioridazine is a well-known antipsychotic drug that also has recently recognized properties as a PI3K/AKT/mTOR signaling inhibitor and as an inhibitor of other pathways relevant to cancer. In MLL-AF4 ALL cell lines characterized by the most common chromosomal translocation in infant ALL, single-agent thioridazine is highly cytotoxic, robustly inhibits PI3K/AKT/mTOR signaling and, moreover, like obatoclax, demonstrates activity as a multi-cell-death pathway agonist. Further preclinical studies now are warranted to determine the extent to which thioridazine inhibits PI3K/AKT/mTOR signaling and causes leukemia cell killing in primary infant ALL cells in vitro and in vivo. The repurposing strategy that this drug may allow could have promise to streamline drug development in infant ALL where the need for new therapies is so urgent. Disclosures: No relevant conflicts of interest to declare.
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Sanjuan-Pla, Alejandra, Clara Bueno, Cristina Prieto, Pamela Acha, Ronald W. Stam, Rolf Marschalek, and Pablo Menéndez. "Revisiting the biology of infant t(4;11)/MLL-AF4+ B-cell acute lymphoblastic leukemia." Blood 126, no. 25 (December 17, 2015): 2676–85. http://dx.doi.org/10.1182/blood-2015-09-667378.
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Abstract Infant B-cell acute lymphoblastic leukemia (B-ALL) accounts for 10% of childhood ALL. The genetic hallmark of most infant B-ALL is chromosomal rearrangements of the mixed-lineage leukemia (MLL) gene. Despite improvement in the clinical management and survival (∼85-90%) of childhood B-ALL, the outcome of infants with MLL-rearranged (MLL-r) B-ALL remains dismal, with overall survival <35%. Among MLL-r infant B-ALL, t(4;11)+ patients harboring the fusion MLL-AF4 (MA4) display a particularly poor prognosis and a pro-B/mixed phenotype. Studies in monozygotic twins and archived blood spots have provided compelling evidence of a single cell of prenatal origin as the target for MA4 fusion, explaining the brief leukemia latency. Despite its aggressiveness and short latency, current progress on its etiology, pathogenesis, and cellular origin is limited as evidenced by the lack of mouse/human models recapitulating the disease phenotype/latency. We propose this is because infant cancer is from an etiologic and pathogenesis standpoint distinct from adult cancer and should be seen as a developmental disease. This is supported by whole-genome sequencing studies suggesting that opposite to the view of cancer as a “multiple-and-sequential-hit” model, t(4;11) alone might be sufficient to spawn leukemia. The stable genome of these patients suggests that, in infant developmental cancer, one “big-hit” might be sufficient for overt disease and supports a key contribution of epigenetics and a prenatal cell of origin during a critical developmental window of stem cell vulnerability in the leukemia pathogenesis. Here, we revisit the biology of t(4;11)+ infant B-ALL with an emphasis on its origin, genetics, and disease models.
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Tsaur, Grigory, Alexander Popov, Tatyana Nasedkina, Olga Kalennik, Anatoly Kustanovich, Olga Aleinikova, Tatyana Riger, et al. "Minimal Residual Disease Monitoring by Quantification of Fusion Gene Transcripts In Infant with MLL-rearranged Acute Lymphoblastic Leukemia Treated by MLL-Baby Protocol." Blood 116, no. 21 (November 19, 2010): 2731. http://dx.doi.org/10.1182/blood.v116.21.2731.2731.
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Abstract Abstract 2731 Background. Despite many attempts worldwide treatment results for infants with MLL rearrangements and especially MLL-AF4 remain unsatisfied. MLL-Baby protocol was developed for infant acute lymphoblastic leukemia (ALL). In this treatment approach conventional chemotherapy is augmented by administration of all-trans retinoic acid (ATRA). This treatment program is successfully applied for infant ALL within Russian Federation and Republic of Belarus (L. Fechina et al, ASH 2007 #2828). Minimal residual disease (MRD) is a strong tool for risk-adapted treatment. Majority of infants carry MLL rearrangements, so in this group MRD monitoring by quantification of fusion gene transcripts (FGt) is fast, easy and cost-effective approach. Objective. To evaluate the prognostic significance of MRD monitoring by FGt measurements in MLL-rearranged infant ALL, enrolled into MLL-Baby study. Methods. Twenty three infants with defined MLL translocation partner genes who received at least two ATRA courses were included in the current study. Median of follow-up period in the observed group was 41 months. Presence of MLL rearrangements was detected by FISH and confirmed by long-distance inverse PCR (C. Meyer et al, 2005). MRD detection in bone marrow (BM) was performed by real-time quantitative PCR and qualitative nested reverse-transcriptase PCR as previously described (A. Borkhardt et al.,1994, J. van Dongen et al., 1999, N. Palisgaard et al., 1998, J. Gabert et al, 2003) with sensitivity 1E–05. MRD-negativity was defined as absence of FGt in both assays. Among 23 infants there were 13 MLL-AF4-positive patients (pts), 4 MLL-MLLT10-positive pts, 3 MLL-EPS15-positive pts, 2 MLL-MLLT1-positive pts and one MLL-MLLT3-positive patient. BM samples were obtained at the time of diagnosis, on day 15 of remission induction (time point (TP) 1), at the end of remission induction (TP2) and after each course of ATRA administration (TP3-TP9). Informed consent was obtained in all cases. Results. All patients were MRD-positive at TP1. At TP2 two MLL-MLLT10-positive patients became MRD-negative. At TP3 other 4 pts (3 MLL-AF4-positive and 1 MLL-MLLT1-positive) converted to MRD-negativity. By TP4 18 pts were MRD-negative, while FGt were detected in 5 pts. 2 pts became MRD-negative before protocol II (at TP9), while 3 pts never achieved MRD-negativity. Retrospectively, we compared prognostic significance of MRD at each TP. TP4 was the earliest TP when discriminative data was obtained. According to the qualitative MRD results at this TP pts were divided into MRD-positive and MRD-negative groups. The first group consisted of 18 pts with different MLL translocation partner genes, while the second group included 5 MLL-AF4-positive pts, who remained MRD-positive at TP4. Groups did not differ in age at diagnosis, sex distribution, initial WBC count, immunophenotype, type of MLL partner gene, number of blast cells at day 8 of dexamethasone prophase, BM status on day 15, CNS disease, and achievement of hematological remission at day 36. Number of relapses was significantly higher in the second group (p=0.017). Odds ratio was 20.00 with 95% CI 1.61–247.99. In the first group there were 3 relapses (in one MLL-AF4-positive case and two MLL-EPS15-positive cases) while in the second group 4 relapses occurred. Cumulative incidence of relapse for pts who achieved MRD-negativity by TP4 was 0.17, for MRD-positive pts 0.80 (p=0.005). 7-years event-free survival in the first group was 0.82±0,09, in the second group 0.20±0.17 (p=0.008) (fig 1). Conclusions. MRD monitoring by FGt measurements has significant prognostic value in infants with MLL-rearranged ALL treated by MLL-Baby protocol. In our series achievement of MRD-negativity by TP4 corresponds to favorable outcome in infant ALL with MLL rearrangements treated by MLL-Baby protocol. Persistence of MRD-positivity at TP4 allows to define group with high incidence of relapse. Disclosures: No relevant conflicts of interest to declare.
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De Groot, Anne Pieta, Yoriko Saito, Eiryo Kawakami, Mari Hashimoto, Yuki Aoki, Rintaro Ono, Ikuko Ogahara, et al. "Targeting Critical Kinases and Anti-Apoptotic Molecules Overcomes Steroid Resistance in Infant MLL-Rearranged Leukemia." Blood 134, Supplement_1 (November 13, 2019): 3885. http://dx.doi.org/10.1182/blood-2019-129719.
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Acute lymphoblastic leukemia (ALL) with rearrangement of the mixed-lineage leukemia (MLL) gene frequently affects infants and is associated with a poor prognosis. Standard treatment protocol for infant MLL-rearranged ALL (MLL-ALL) includes glucocorticoids (GCs). However, resistance to GCs remains a major problem. Therefore, it is important to find new treatment strategies that overcome GC resistance in MLL-ALL. To identify novel therapeutic targets to overcome GC resistance in MLL-ALL, we compared transcriptional profiles of normal cord blood CD34+CD38- hematopoietic stem and progenitor cells and GC-resistant infant MLL-ALL leukemia-initiating cells (LICs). We found enrichment of the Src family kinases (SFKs) and Fms-like tyrosine kinase 3 (FLT3) signaling pathways in the LICs. We hypothesized that activation of these kinases may contribute to GC resistance in MLL-ALL cells. Using our previously developed infant MLL-ALL patient-derived xenograft (PDX) models (Aoki et al., Blood, 2015), we recapitulated human GC-resistance in vivo, characterized by increased hCD45+ chimerism in the peripheral blood after treatment with dexamethasone (% peripheral blood human MLL-ALL at pre-treatment: 34.1±5.2% vs. post treatment 51.3±6.6%, n=26). In order to investigate if inhibition of SFKs and FLT3 can overcome GC-resistance, we treated the MLL-ALL PDX models with our previously developed FLT3-SFK multiple kinase inhibitor RK-20449 (Saito et al., Science Translational Medicine, 2013). Combination treatment with dexamethasone and RK-20449 successfully eliminated human MLL-ALL cells from the peripheral blood (dexamethasone alone: 47.2±7.2% vs. combination: 5.6±2.8% hCD45+ cells, p=6.91E-7), bone marrow (dexamethasone alone: 72.2±4.6% vs. combination: 19.5±4.8% hCD45+ cells, p=1.79E-10), and spleen (dexamethasone alone: 52.6±4.8% vs. combination: 11.7±3.5% hCD45+ cells, p=3.75E-9). In addition, combination treatment eliminated infiltrated MLL-ALL from kidney and liver of the MLL-ALL PDX models. Bcl-2 homology domain 3 (BH3) profiling demonstrated that MLL-ALL cells resistant to RK-20449 treatment were dependent on the anti-apoptotic Bcl-2 protein for their survival. Additional inhibition of Bcl-2 by ABT-199 led to complete elimination of MLL-ALL cells in vitro and in vivo in all MLL-ALL patient cases (Figure1). Taken together, in this study we demonstrated that inhibition of SFKs and FLT3 by RK-20449 overcomes GC-resistance in MLL-ALL. Further, we identified Bcl-2 dependence as a mechanism of treatment resistance in MLL-ALL. Therefore, we believe that the combined inhibition of kinase and anti-apoptotic pathways may lead to effective treatment options for highly resistant infant MLL-ALL. Aoki, et al., Identification of CD34+ and CD34- leukemia-initiating cells in MLL-rearranged human acute lymphoblastic leukemia. Blood, 2015: p. 967-980. Saito, Y., et al., A Pyrrolo-Pyrimidine Derivative Targets Human Primary AML Stem Cells in Vivo. Science Translational Medicine, 2013: p. 1-15. Disclosures No relevant conflicts of interest to declare.
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Ikawa, Y., N. Sugimoto, S. Koizumi, A. Yachie, and Y. Saikawa. "Promoter DNA methylation of CD10 in infant acute lymphoblastic leukemia with MLL/AF4 fusion gene." Journal of Clinical Oncology 27, no. 15_suppl (May 20, 2009): 10045. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.10045.
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10045 Background: Infant ALL displays distinct biologic and clinical features with a poor prognosis. The CD10-negative immunophenotype of infant ALL is a hallmark and provides a predictable signature of MLL rearrangements. While CD10 negativity reflects an earlier stage of B-cell development, complete IgH gene rearrangements (VDJH) show more mature IgH status. Discordance between immunophenotype and genotype of infant ALL suggests an aberrant process in immunophenotypic steps of differentiation or a secondary down-regulation of CD 10 expression associated with MLL rearrangements. We performed methylation analysis of full promoter regions of the CD10 gene to investigate epigenetic mechanisms responsible for CD10 negativity. Methods: CD10-negative infant ALL with MLL/AF4, CD10-positive infant ALL with germ-line MLL, CD10-positive pre-B ALL cell line, infant AML (M5) with MLL/AF9 and pediatric AML (M2) with AML1/ETO were analyzed for VDJH status and methylation of CD10 gene promoters. Results: Three of 4 cases with infant ALL revealed complete rearrangements of VDJH gene with productive joints. Bisulfite sequencing of CD 10 type 1 and 2 promoters identified more than 84% of methylated CpG dinucleotides in all three CD10-negative infant ALL cases with MLL/AF4. The CpG dinucleotides distributed in the clusters of putative Sp 1 binding sites and functionally active regulatory regions of the promoters were fully methylated. In contrast, none or a few of the CpG dinucleotides were methylated in the CD10-positive ALL, AML (M5) with MLL/AF9 or AML (M2) with AML1/ETO. Conclusions: Structural evidence of dense methylation in the CD 10 gene promoter suggested that methylated transcription factor binding sites contribute to CD10 silencing as an epigenetic mechanism. [Table: see text] No significant financial relationships to disclose.
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Robinson, Blaine W., Meenakshi Devidas, Andrew J. Carroll, Richard C. Harvey, Nyla A. Heerema, Cheryl L. Willman, Amanda R. Hudome, et al. "Specific MLL Partner Genes in Infant Acute Lymphoblastic Leukemia (ALL) Associated with Outcome Are Linked to Age and White Blood Cell Count (WBC) at Diagnosis: A Report On the Children's Oncology Group (COG) P9407 Trial." Blood 114, no. 22 (November 20, 2009): 907. http://dx.doi.org/10.1182/blood.v114.22.907.907.
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Abstract Abstract 907 Introduction: MLL translocations are the most pervasive molecular abnormalities in infant ALL and are poor prognostic factors; however, interrelationships between specific MLL partner genes, event-free survival (EFS) and clinical covariates have been elusive due to several factors. Infant ALL is a rare disease with limited cases within MLL translocation subsets, specialized methods are needed to identify the spectrum of MLL partner genes, and most prior clinical trials defined MLL status by karyotype and analyses of only common MLL partner genes. P9407, a COG pilot study testing the utility of intensified induction therapy, developed a detailed molecular cytogenetic classifier of MLL status and partner genes to investigate relationships with EFS and correlations with clinical covariates. Methods: MLL status was defined by karyotype, FISH, Southern blot analysis, RT-PCR analysis of MLL translocations with AF4, ENL or AF9, and cDNA or genomic panhandle PCR. Kaplan-Meier plots were used to estimate EFS as a function of MLL status (rearranged, R; germline, G), MLL partner gene and, within MLL status and partner gene subsets, as a function of age at diagnosis. Fisher's exact test was used to assess correlations between MLL partner genes and age, WBC (K/ml3) or CNS status at diagnosis. Results: MLL status was determined in 210 of 221 total cases [95%; 158 R, 75%; 52 G, 25%]. Partner genes were definitively determined in 133 of the 158 MLL-R cases: AF4/78/49.4%; ENL/33/20.1%; AF9/15/9.5%; EPS15/4/2.5%; AF10/1/0.6%; ASAH3/1/0.6%; ACTN4/1/0.6%. The ACTN4 partner gene was identified in a new complex 3-way MLL/ACTN4/RYR1 rearrangement. In the other 25 MLL-R cases, RT-PCR with gene specific primers excluded AF4 (n=5) or AF4, AF9 and ENL (n=13) or MLL status was not defined beyond MLL-R or MLL-G (n=7). Analyses of 202 eligible subjects within the cohort of 210 in whom MLL status was assigned, indicated a 5-year EFS rate of 45.9±4.6%, which was worse in MLL-R than MLL-G cases (38.8±5.1% v. 66.2±9.3%, P=0.0003). By category, 5-year EFS rates were: ENL (29.0±10.0%), AF4 (34.2±7.4%), other partner genes (45.0±14.9%), AF9 (67.7±17.2%), MLL-G (66.2±9.3%) (P=0.0003). Within MLL-R, AF4 was associated with worse EFS than other partner genes including AF9 but excluding ENL (P=0.043), and ENL with worse EFS than other partner genes including AF9 but excluding AF4 (P=0.024). Also, EFS was worse when AF4 and ENL subsets were combined, compared to any other partner gene (P=0.025). Conversely, the higher EFS with AF9 than with any other partner gene became less significant when AF4 and/or ENL were excluded from the other MLL-R category. EFS in the AF9 subset was not different from that for MLL-G cases. Five-year EFS was worse in patients aged <90d than ≥90d at diagnosis within MLL-R cases overall (6.2±4.2% v. 47.4±6.5%, P<0.0001) and within the AF4 (4.6%±4.4% v. 46.6±9.5%, P<0.0001), or ENL subsets (0.0% v. 40.9±12.8%, P=0.0011), whereas age <90d did not adversely impact survival in the MLL-G cases or those with AF9 as partner gene. Compared to cases with different partner genes, cases with AF4, ENL or AF9 were not differently distributed in the <90d v. ≥90d age categories, but there were more other MLL-R cases in patients aged ≥90d at diagnosis (18% v. 3%, P=0.027). WBC was higher (≥50K) in cases with AF4 (P=0.0006) and lower (<50K) in cases with AF9 (P=0.0007). CNS status was not differently distributed by partner gene. In similar analyses of cohort 3 alone (n=136) in which treatment modifications were made for earlier toxicities, the same relationships were upheld. Conclusions: MLL translocations, young age and high WBC have correlated with poor outcome in infant ALL before, but this MLL classifier in the largest cohort of infant ALL with detailed molecular characterization reported to date, adds new elements to the impact of MLL partner genes on outcome and their connection to prognostic factors. AF4 and ENL not only negatively impact EFS, but this effect is larger in the younger infants. Regardless of age, AF9 is a favorable prognostic factor, but the other partner genes associated with better outcome occur more often in older infants. Relationships between high WBC and AF4, and low WBC and AF9 were also discovered. The newfound associations of specific MLL partner genes with age and WBC indicate how disease biology and the classical prognostic factors in this disease are integrally connected. Disclosures: No relevant conflicts of interest to declare.
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Shimada, Akira, Daisuke Tomizawa, Akitoshi Kinoshita, Kazuko Hamamoto, Ichiro Tsukimoto, Atsushi Ogawa, Takashi Taga, et al. "Heterogeneity in Infants with Acute Myeloid Leukemia: Retrospective Analysis of a Japanese Nationwide Survey." Blood 118, no. 21 (November 18, 2011): 1477. http://dx.doi.org/10.1182/blood.v118.21.1477.1477.
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Abstract 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.
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Lock, Richard B., Jennifer Richmond, Laura High, Hernan Carol, Kathryn Evans, Agnes M. Mendomo, Raushan T. Kurmasheva, Peter J. Houghton, and Malcolm A. Smith. "Effective Targeting Of The P53/MDM2 Axis In Preclinical Models Of Infant MLL-Rearranged Acute Lymphoblastic Leukemia." Blood 122, no. 21 (November 15, 2013): 71. http://dx.doi.org/10.1182/blood.v122.21.71.71.
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Abstract Introduction While the overall cure rate for the most common pediatric cancer, acute lymphoblastic leukemia (ALL) now approaches 90%, infants (<12 months) diagnosed with ALL harboring translocations in the mixed-lineage leukemia oncogene (infant MLL-ALL) experience shorter remission duration and a significantly reduced likelihood of survival (∼50%). Therefore, new treatments that can be incorporated into conventional chemotherapy regimens to extend patient remission and improve survival are urgently required. Mutations in the p53 tumor suppressor are uncommon in infant MLL-ALL, and drugs that release p53 from inhibitory mechanisms may be of therapeutic benefit. Nutlin cis-imidazole molecules selectively inhibit p53-MDM2 binding, resulting in activation of the p53 pathway in cancer cells leading to cell cycle arrest and apoptosis. The purpose of this study was to assess the efficacy of the orally available nutlin, RG7112, against patient-derived MLL-ALL xenograft models. Methods In vitro cytotoxicity was assessed by mitochondrial metabolic activity assay (Alamar blue) following 48h drug exposures. P53 protein levels and subcellular distribution were assessed by immunoblotting. Patient-derived xenografts were established from infant MLL-ALL, B-cell precursor (BCP)-ALL, or T-lineage ALL (T-ALL) bone marrow or peripheral blood (PB) biopsies in immune-deficient (NOD/SCID or NSG) mice, and their gene expression profiles generated using Illumina Human Ref-12 Expression BeadChips. Engraftment and drug responses were assessed by enumeration of the proportion of human versus mouse CD45+ cells in the PB. Mice with established disease received vehicle, RG7112 (100 mg/kg daily x 5 p.o.), a combination of vincristine (0.15 mg/kg once i.p.) dexamethasone (5 mg/kg daily x 5 i.p.) and L-asparaginase (1,000 IU/kg daily x 5 i.p.) (VXL), or RG7112 plus VXL. Anti-leukemic efficacy was assessed using an objective response measure modeled after the clinical setting, as well as the median event-free survival (EFS) of treated or control groups from treatment initiation. Therapeutic enhancement was considered to occur when the RG7112/VXL combination significantly extended mouse EFS compared with that of both of the RG7112 and VXL treated groups. Results Unsupervised hierarchical clustering of gene expression profiles revealed that the MLL-ALL (n=9), BCP-ALL (n=7) and T-ALL (n=13) xenografts clustered according to leukemia subtype. Moreover, genes previously reported to be overexpressed in MLL-ALL, including MEIS1, CCNA1, and members of the HOXA gene family, were significantly upregulated in MLL-ALL xenografts. The specificity of RG7112 was validated by cytotoxicity assays against leukemia cell lines of known p53 status; p53 wild-type cell lines (RS4;11, IC50 1.4 µM; NALM-6, IC50 3.0 µM) were markedly more sensitive than those with mutant p53 (CEM, IC50 >10 µM; JURKAT, IC50 >10 µM). The in vitro sensitivity of BCP-ALL (n=3) and infant MLL-ALL (n=4) xenografts was consistent with wild-type p53 status, with IC50s of 0.11 - 2.2 µM. Exposure of ALL xenograft cells to nutlin-3 (10 µM, 6h) caused marked p53 up-regulation and nuclear translocation. Since we had previously shown that RG7112 administered as a single agent for 14 days induced significant regressions [Complete Responses (CRs) or greater] in 7/7 infant MLL-ALL xenografts in vivo, we assessed its efficacy in a 5-day combination treatment with an induction-type regimen (VXL) against two infant MLL-ALL xenografts (MLL-5 and MLL-14). The RG7112/VXL combination caused a Partial Response in MLL-5 compared with Progressive Disease for both RG7112 and VXL. The efficacy of RG7112/VXL was even more pronounced against MLL-14, causing a Maintained CR compared with CRs for both RG7112 and VXL, which met the criteria for Therapeutic Enhancement (the median EFS of RG7112/VXL-treated mice, 65.0 days, was significantly greater, P< 0.0001, than that of RG7112, 22.2 days, and VXL, 28.5 days). Conclusions RG7112 induces significant regressions in a high proportion of infant MLL-ALL xenografts and enhances the efficacy of an induction-type regimen. The utility of targeting the p53-MDM2 axis in combination with established drugs for the clinical management of infant MLL-ALL warrants further investigation. This study was supported by NCI NO1CM42216. The authors thank Roche Pharmaceuticals, Inc., for providing RG7112. Disclosures: No relevant conflicts of interest to declare.
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Stumpel, Dominique JPM, Diana Schotte, Ellen AM Lange-Turenhout, Pauline Schneider, Lidija Seslija, Renee X. de Menezes, Victor E. Marquez, Rob Pieters, Monique L. Den Boer, and Ronald Stam. "Methylation of Specific Microrna Genes In MLL-Rearranged Infant Acute Lymphoblastic Leukemia: - Major Matters at a Micro Scale -." Blood 116, no. 21 (November 19, 2010): 2483. http://dx.doi.org/10.1182/blood.v116.21.2483.2483.
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Abstract Abstract 2483 MLL-rearranged Acute Lymphoblastic Leukemia (ALL) in infants (<1 year) represents one of the most aggressive types of childhood leukemia. In order to develop more suitable treatment strategies, a firm understanding of the biology underlying this disease is of utmost importance. MLL-rearranged ALL displays a unique gene expression profile, partly explained by erroneous histone modifications. We recently showed that t (4;11)-positive infant ALL is also characterized by pronounced promoter CpG hypermethylation. Here we investigated whether this widespread hypermethylation also affected microRNA (miRNA) expression. We performed CpG methylation analyses at 122 miRNA loci using Differential Methylation Hybridization (DMH), and miRNA expression analyses using quantitative real-time PCR on primary t (4;11)-positive infant ALL samples (n= 22) and normal pediatric bone marrows (n= 7). We identified 11 miRNAs that were markedly down-regulated in t (4;11)-positive infant ALL as a consequence of CpG hypermethylation. Seven of these miRNAs were re-activated after exposure to the de-methylating agent Zebularine. Interestingly, 5 of these miRNAs had already been associated either with the MLL gene or with leukemic MLL fusions. For one of the remaining miRNAs, i.e. miR-152, we demonstrate that high degrees of methylation strongly correlate with a poor clinical outcome. Moreover, we identified MLL and DNA methyltransferase 1 (DNMT1) as potential target genes for miR-152. Thus, genome-wide DNA methylation in MLL-rearranged infant ALL not only inactivates numerous protein-coding genes, but also affects several miRNA genes. While inhibition of methylation by Zebularine to certain extents re-activates gene expression, re-activation of miRNAs by this agent restores the suppression of associated target genes. As demethylating agents exert their functions by covalently trapping DNMT1 to the DNA, re-activation of miR-152 by Zebularine further supports demethylation by targeting DNMT1 expression. In summary, our data demonstrates an important role for genome-wide DNA methylation in suppressing miRNA expression and provides additional grounds to initiate efficacy testing of demethylating agents in MLL-rearranged ALL in vivo. Disclosures: No relevant conflicts of interest to declare.
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Tsaur, Grigory, Claus Meyer, Alexander Popov, Olga Plekhanova, Anatoly Kustanovich, Alena Volochnik, Tatyana Riger, et al. "MLL genomic DNA Breakpoints In Infant Acute Leukemia." Blood 122, no. 21 (November 15, 2013): 1350. http://dx.doi.org/10.1182/blood.v122.21.1350.1350.
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Abstract Background Infant acute leukemia is characterized by high incidence of MLL gene rearrangements. Purpose To evaluate the distribution of MLL genomic DNA breakpoints and their relation to several diagnostic parameters among infant acute leukemia. Methods 72 infants with MLL-rearranged acute lymphoblastic leukemia (ALL) (n=52), acute myeloid leukemia (AML) (n=19) and mixed phenotype acute leukemia (n=1) were included in this study based on the availability of DNA material at diagnosis. In the observed group there were 28 boys (39%) and 44 girls (61%) with median age of 4.9 mo (range 0.03-11.9). Genomic DNA breakpoint detection in MLL gene and translocation partner genes (TPG) was performed by long-distance inverse PCR (LDI-PCR). Exon-intron numbering of MLL gene was done according to I. Nilson et al, 1996. Results Majority of ALL cases (n=28; 54%) was characterized by presence of MLL-AF4 fusion gene (FG), less frequently MLL-MLLT1 (n=12; 23%), MLL-MLLT3 (n=7; 13%) and others were found (Table 1). The most common breakpoint location within MLL gene in ALL patients was intron 11, detected in 25 cases (48%). The highest variability of MLL breakpoints was found in MLL-AF4-positive patients: only 11 of 28 (39%) had breakpoints in intron 11. The most stable pattern of MLL genomic DNA breakpoints was observed in MLL-MLLT1-positive patients: 8 of 12 (67%) had breakpoints in intron 11. In AML patients two the most prevalent FGs were MLL-MLLT3 (n=7, 37%) and MLL-MLLT10 (n=5, 26%). The remaining ones are listed in Table 1. The most frequent breakpoints location was intron 8 (8 out of 19, 42%). The most stable pattern was revealed for MLL-MLLT10 FG: MLL breakpoints in 4 of 5 (80%) cases were found in intron 9 (Table 1). ALL patients who had breakpoints in intron 11 were significantly younger (median 3.0 mo, range 0.03-11.6) than all others (median 5.6 mo, range 0.7-11.9) (p=0.025) and than patients with MLL breakpoints in intron 9 (median 6.6 mo, range 3.1-11.9) (p=0.017). For AML cases we did not find any relation between age and breakpoints locations. Distribution of MLL DNA breakpoints was similar in boys and girls and did not depend on type of TPG. Genetic recombinations involving MLL gene predominantly resulted in reciprocal chromosomal translocations (n=62; 86%). Beside them, 6 (11%) insertions were identified in all MLL-MLLT10-positive cases and MLL-SEPT6-positive one. In 11 (15%) patients we found breakpoints within the regions located from 0.7 Kb to 25.4 Kb 3' of the first exon of TPGs (MLLT1 n=9; EPS15 n=1; MYO1F n=1), however fusion transcripts at cDNA level were identified and sequenced in all these cases, indicating a spliced fusion mechanism. 3-way translocations were found in 5 patients and in 1 case we found combination of insertion with interstitial deletion of chromosome 11. The list of reciprocal genes involved in these 6 cases was as follows: CEP164, DNAH6, DCPA1, MCL1 as well as non-coding regions of 2q21.2 and 2p21. We also analyzed breakpoints in TPGs. Except above mentioned spliced fusion cases, the remaining 3 breakpoints in MLLT1 as well as 3 of 4 breakpoints in EPS15 and all breakpoints in MLLT11 were within intron 1 of corresponding genes. In AF4 the major breakpoint region included intron 3 (n=19), intron 4 (n=6) and intron 5 (n=2). We also revealed 2 rare breakpoints in intron 6 and 10. In MLLT3 the most frequent breakpoint location was intron 5 (n=12), additionally 2 cases in intron 5 were identified. In MLLT10 two separate breakpoint locations were found: intron 3 (n=1) and intron 8 (n=3) in combination with intron 9 (n=1). We estimated prognostic significance of MLL breakpoint locations in 31 cases of infant ALL treated by MLL-Baby protocol. 3-year cumulative incidence of relapse was remarkably higher in patients with breakpoints in intron 11 (n=18) in comparison to patients with breakpoint localized from intron 7 to exon 11, inclusively (n=13) (0.85±0.01 and 0.57±0.02, respectively), although difference between these two groups did not achieve statistical significance (p=0.261). Median follow-up time in the observed group was 30 months (range 6–42). Conclusion In the current study we estimated clinical and prognostic significance of MLL and TPG genomic DNA breakpoints in infant acute leukemia. Our data provide additional information of molecular genetic features of MLL-rearranged infant acute leukemia. Disclosures: No relevant conflicts of interest to declare.
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Miyamura, Takako, Katsuyoshi Koh, Daisuke Tomizawa, Kanji Sugita, Koji Kato, Takashi Sato, Yoshihiro Takahashi, et al. "Nation-Wide Survey of Infant Leukemia in Japan: A Report from the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG)." Blood 112, no. 11 (November 16, 2008): 896. http://dx.doi.org/10.1182/blood.v112.11.896.896.
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Abstract Infant leukemia consists of wide spectrum of disease phenotype which includes acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and mixed lineage leukemia, and has unique biological and clinical features. However, there are few studies which have focused on its diversity from the cross-sectional view of this specific age group. We here conducted a nation-wide survey to analyze the disease distribution, characteristics, clinical courses, and outcomes of these patients in Japan. Questionnaires were sent to 170 institutions participating in the JPLSG which covers more than 95% of the hospitals in Japan, and total of 225 cases with infant leukemia and related disorders aged less than 24 months at onset who were diagnosed between 2004 and 2007 were registered. Median age at onset was 12 months, ranged from 0 to 22 months. No gender differences were observed (male, 118 cases; female, 107 cases). Thirty-nine were diagnosed as ALL with positive MLL gene rearrangements (MLL-R ALL), 74 as ALL with germline MLL (MLL-G ALL), 83 as AML, 9 as mixed lineage leukemia, and 20 as other disorders. In MLL-R ALL group, the frequency of t(4;11) was high in patients of <12 months of age (18/29 cases), whereas hyperdiploidy was detected in 20% of cases of MLL-G ALL group (12/61 cases). In AML, positive MLL gene rearrangements were detected in only 15% (13/83 cases), while t(1;22)(p13q13) or OTT-MAL was detected in 4 cases. Regardless of disease groups, early infant group of <6 months of age showed poor prognosis compared with other age groups. Particularly, all cases of ALL diagnosed at <4 weeks after birth harbored MLL gene rearrangements and either died or relapsed, indicating that more appropriate strategy is needed to improve the outcome for this specific group. Patients with MLL-G ALL less than 12 months old showed a favorable prognosis compared to those with MLL-R ALL. In AML, majority of cases were myelomonocytic/monocytic leukemia (M4/M5) or megakaryoblastic leukemia (M7), and showed favorable outcome. In conclusion, infant leukemia is still a challenging disease harboring diverse features, therefore, it is necessary to keep on-going the prospective trials, to register and follow-up these cases systematically and to standardize its treatment including supportive cares, especially for those with younger age onset.
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Schafer, Eric S., Rumen Kostadinov, Peter Murakami, Sandeep S. Negi, Maria E. Figueroa, Ari Melnick, and Patrick Brown. "Lineage, Fusion Partner and Age Differences in the Methylome of MLL-r Leukemias." Blood 120, no. 21 (November 16, 2012): 3506. http://dx.doi.org/10.1182/blood.v120.21.3506.3506.
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Abstract Abstract 3506 Background: MLL gene rearrangements (MLL-r) are seen in all ages in both acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML). The most common fusion partners are AF4, AF9 and ENL/ELL. Murine models suggest that the expression of MLL fusion proteins is necessary but not sufficient for leukemogenesis, but true cooperating lesions have been difficult to identify. Our lab and others have previously shown that MLL-r infant ALL can be defined by a unique signature characterized by genome-wide hypermethylated CpG islands leading to tumor suppressor gene silencing. In this study we aimed to discover the driving mechanism of MLL-r infant ALL epigenetic signature and whether that signature is intrinsic to the MLL-r or possibly one of the elusive cooperating leukemogenic events. Methods: We measured DNA methylation levels at roughly 225,000 CpG sites covering over 19,000 genes using the HELP (HpaII-tiny fragment Enrichment by Ligation-mediated PCR) assay to evaluate 102 leukemia samples and 12 normal controls. Gene expression using Illumina HT-12 arrays were concurrently performed for future paired expression-methylation analyses. Leukemia samples were primary diagnostic patient samples representing ages infancy to adulthood and included 60 ALL samples (36 MLL-r and 24 MLL-wt) and 42 AML samples (29 MLL-r and 13 MLL-wt). We used unsupervised hierarchical clustering and principal component analysis (PCA) to evaluate whether the methylation patterns in the 102 samples discriminate between lineage (ALL vs. AML), MLL fusion status (MLL-r vs. MLL-wt), fusion partners, and age (infant, pediatric, adult). Results: Hierarchical clustering, using the top 50% of genes that show highest variance in methylation level across all 102 samples, discriminated leukemia samples into distinct lineage specific groups (ALL vs. AML). Within the ALL cluster, there were distinct sub-clusters separating T- and B- cell disease. In addition, In B- cell disease only, MLL-r and MLL-wt formed distinct sub-clusters while no MLL-r vs MLL-wt subclustering was noticed in AML. PCA recapitulated descriptive findings. Regression analysis of the first principal component of all 102 samples showed significant linear separation of AML vs. ALL (p<0.001), but not by age or MLL-r status. However, analysis of the second principal component (diminishing the effect of lineage) showed that there was a secondary MLL-r specific signature among all samples (p<0.001). Regression analysis of the first principal component in the ALL-only samples showed significant linear separation of MLL-r vs. MLL-wt (p<0.001) and showed distinct separation by fusion partner group defined as AF4, AF9 and all other MLL-r (p<0.01). In addition, PCA showed significant distinction between infant ALL and childhood ALL (p<0.01) and trended towards significance when compared to adult disease (p=0.07). These patterns were not duplicated in AML where PCA showed no significant linear separation of MLL-r vs. MLL-wt (p=0.42) or separation by age(p>0.4). While there was no intrinsic MLL-r specific signature in AML, there was a weaker but significant clustering of MLL-r samples into two distinct groups; one consisting of AF9 samples and the other containing all other MLL-r fusion partners (p<0.03). Finally, we explored linage differences controlling for fusion partner, using false discovery rate analysis. Enumerating regions with q-values of <0.05, 29,736 probes showed differential methylation patterns when comparing infant MLL-AF9 ALL and infant MLL-AF9 AML while there were 0 probes were isolated when comparing infant MLL-AF9 AML to adult MLL-AF9 AML. Conclusions: Methylation patterns in MLL-r ALL and AML are distinct. The primary determinant of methylation signatures in MLL-r leukemia is lineage, not MLL status, even when controlling for fusion partners, suggesting that the MLL-r does not drive methylation patterns observed. However, within lineages MLL status is an important contributor to methylation patterns, but this influence is different in ALL and AML. In B-ALL, MLL-r defines a unique methylation signature, subgrouped by fusion partner and additionally, but less so, by age. In AML, MLL fusions AF9 and other MLL-r cluster together in restricted analyses but there is no intrinsic MLL-r signature. However, while lineage is the principle driver of methylation, MLL status does define a secondary significant signature irrespective of lineage. Disclosures: No relevant conflicts of interest to declare.
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Isobe, Tomoya, Kenichi Yoshida, Chika Kobayashi, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Shiro Fukuda, et al. "Genome-Wide Mutational Landscape of Infant Acute Lymphoblastic Leukemia." Blood 128, no. 22 (December 2, 2016): 4070. http://dx.doi.org/10.1182/blood.v128.22.4070.4070.
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Abstract [Background] Mixed lineage leukemia (MLL also called KMT2A) rearrangement-positive leukemia is one of the most aggressive types of leukemia. It is diagnosed predominantly in infants and typically shows a multi-lineage phenotype. Since current chemotherapy fails in more than 50% of infantile acute lymphoblastic leukemia (ALL) with MLL-rearrangement, a better understanding of biological features of the disease is important in order to develop more specific and successful treatment strategies. Despite the evident biological and clinical significance of MLL-rearrangement, it has also been shown in vitro and in vivo that MLL-rearrangement is not sufficient to induce full leukemic transformation, indicating additional hits are required for complete leukemogenesis. Recently, sequencing-based genome-wide studies have suggested collaborative involvement of RAS-PI3K pathway in infant leukemogenesis through identification of recurrent mutations affecting RAS-PI3K pathway genes, in infant ALL with or without MLL-rearrangement. However, genetic basis of infant ALL with MLL-rearrangement are not fully elucidated. [Materials and Methods] Here, we performed whole exome sequencing in leukemia samples with matched peripheral blood T lymphocyte from 46 cases of infant ALL including 3 cases with germline MLL. Our cohort included samples from the Infant Leukemia Sub-committee, Japan Children's Cancer Group. For mapping and mutation calling, we used publicly available "Genomon Pipeline". [Results] In total, 198 somatic non-silent mutations (4.3 mutations per case) were identified, including 125 missense, 4 nonsense, 8 splice site mutations, 49 frameshift indels, and 12 in-frame indels. Thirteen genes were recurrently affected, among which RAS-PI3K pathway mutations were the most frequent: KRAS (10/46 cases, 21.7%), NRAS (4/46 cases, 8.7%) and PTPN11 (3/46 cases, 6.5%). In addition, novel recurrent mutations were detected in HSP90AB1 (5/46 cases, 10.9%), MAPRE3 (2/46 cases, 4.3%) and SRCAP (2/46 cases, 4.3%), as well as known candidate driver mutations in FLT3 (7/46 cases, 15.2%), TP53 (3/46 cases, 6.5%), CHD4 (2/46 cases, 4.3%) and PAX5 (2/46 cases, 4.3%). HSP90AB1 is a member of Heat shock protein90 (Hsp90) family that encodes a group of molecular chaperones involved in stabilization and activation of multiple oncogenic proteins and pathways. Overexpression of Hsp90 is observed in ALL, chronic myeloid/lymphocytic leukemia and several non-hematological malignancies. Another recurrently mutated gene, SRCAP, encodes the core component of chromatin-remodeling Snf2-related CREBBP activator protein complex. Recurrent mutations in this gene have recently been reported in FLT3-ITD positive acute myeloid leukemia with significantly high co-occurrence of MLL3 mutations. Overall, RAS-PI3K pathway mutations were detected in 19/46 cases of infant ALL (41.3%). In our cohort, none of 3 germline MLL cases contained RAS-PI3K pathway mutations; one case had a PAX5 mutation, another had a SRCAP mutation, and the other had no candidate driver mutations. In the germline MLL case without candidate drivers, G-banding had identified a structural abnormality, t(5;15)(p15;q11.2), that is rare but reported to be predominant in infant ALL rather than other hematological/non-hematological malignancies. [Conclusion] Our results demonstrated that aberrations of cell proliferation signaling, transcription factors, as well as cell cycle/epigenetic regulators are co-operative oncogenic events in MLL-rearrangement cases. The mutation spectrum is similar to myeloid malignancies rather than lymphoid cancers. Intriguingly, we identified novel recurrent mutations in the oncogenic proteins, such as HSP90AB1, MAPRE3, and SRCAP,suggesting that these are potentially targetable by small-molecule therapy. In addition, we also illustrated the genetic differences between MLL-rearrangement and germline MLL. Thus, our mutational landscape provides a novel insight into the molecular mechanisms of infant ALL and may contribute to improving the clinical outcome of infant patients suffering from the intractable ALL. Disclosures Ogawa: Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.
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van Roon, Eddy HJ, Ronald Stam, Merel Willekes, Roland P. Kuiper, Rutger W. W. Brouwer, van IJcken F. Wilfred, and Rob Pieters. "Exome Sequencing Identifies Pxdn and DSP As Recurrently Mutated Genes in Infant ALL Patients Carrying a MLL-AF4 Translocation." Blood 124, no. 21 (December 6, 2014): 130. http://dx.doi.org/10.1182/blood.v124.21.130.130.
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Abstract Background and purpose: Acute lymphoblastic leukemia (ALL) in infants (<1 year of age) carrying MLL translocations represent a highly aggressive subtype of leukemia with an exceedingly poor prognosis. Although MLL rearrangements have been considered to be causative for this type of leukemia, recent studies using mouse models suggest that the expression of chimeric MLL fusion genes alone may not be sufficient to induce leukemia. In an attempt to identify additional mutations that synergize with MLLfusion genes in driving leukemogenesis, we performed exome-sequencing in primary infant ALL patient samples (n=13) carrying the t(4;11)-translocation, giving rise to the MLL-AF4 fusion protein. Materials and Methods: Bone marrow or peripheral blood samples from untreated infants diagnosed with t(4;11)+ ALL were collected at our laboratory as part of the international collaborative INTERFANT treatment protocols. Genomic DNA was extracted from ~5×106leukemic cells. Exome capture has been performed using the Sure Select Human All Exon v2 kit (Agilent Technologies). The samples were prepped for sequencing according to the TruSeq v3 protocol (Illumina) prior to sequencing on the Hiseq2000 with a v3 flowcell for 100bp = 7bp index (Illumina). The reads have been aligned against the human reference genome build 19 (hg19) using a BWA and NARWHAL based pipeline. Sequential filtering selected for single nucleotide variations (SNVs) in canonical splice sites, insertions/deletions (indels) providing frameshifts, indels involving regions conserved between species (phyloP >3), nonsense mutations and nonsynonymous missense mutations involving regions conserved between species. All known annotated single nucleotide polymorphisms according to dbSNP build 138 and an in-house database of 1354 sequenced exomes were excluded. Recurrent mutations were validated by Sanger sequencing on an extended infant ALL patient cohort (n=122). Results: The sequenced exomes had an average coverage depth of 102.5 reads per region or 109 per base. Exome sequencing revealed that, after filtering, t(4;11)+ infant ALL patients on average carried 71 variants (range 58-98, including rare germline variants and somatic mutations). To identify recurrent mutations we selected 23 genes in which two or more patients carried a mutation for further validation by Sanger sequencing. Although all 23 genes were successfully validated, no additional patients carrying these mutations were found. However, recurrent secondary mutations within the same functional region as the initial mutations were found in two genes: PXDN and DSP. The average incidences of the PXDN and DSP mutations amongst MLL-rearranged infant ALL patients were 32% and 20%, respectively. Interestingly, MLL-rearranged infant ALL patients carrying a PXDN mutation had a significantly (p=0.013) better prognosis compared with patients not carrying a PXDNmutation, with mean EFS rates of 6.4 vs. 3.2 years, respectively. Both gene products are involved in intracellular interactions, desmosome formation, extracellular matrix consolidation and phagocytosis and no prior associations with ALL have been made before. Conclusion: We identified 23 genes with two or more mutations in our initial exome sequencing cohort. None of these mutations were found in additional patients in our validation cohort. Despite an average of 71 variations per patient, no recurrent mutations were identified and our study provides evidence supporting previous observations that infant ALL has one of the lowest mutation rates observed in human cancer. However, we identified additional mutations within the same functional region as the initial mutation in DSP and PXDN. Interestingly, PXDN mutations show an association with a favorable prognosis in MLL-R patients. Disclosures No relevant conflicts of interest to declare.
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Pui, C. H., F. G. Behm, J. R. Downing, M. L. Hancock, S. A. Shurtleff, R. C. Ribeiro, D. R. Head, H. H. Mahmoud, J. T. Sandlund, and W. L. Furman. "11q23/MLL rearrangement confers a poor prognosis in infants with acute lymphoblastic leukemia." Journal of Clinical Oncology 12, no. 5 (May 1994): 909–15. http://dx.doi.org/10.1200/jco.1994.12.5.909.
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PURPOSE Leukemic cell characteristics were analyzed in infants less than 1 year of age with acute lymphoblastic leukemia (ALL) to determine adverse prognostic factors that might explain the poor prognosis of this group. PATIENTS AND METHODS Treatment outcomes were analyzed according to the presenting clinical and laboratory features of 30 infants treated between May 1979 and April 1993. A stepwise multivariate regression model was used to identify the most important prognostic indicator with respect to event-free survival. RESULTS Infant ALL cases were characterized by high presenting leukocyte count (median, 87 x 10(9)/L), increased frequency of CNS leukemia (50%), and blast cells with a CD10- phenotype (67%), myeloid-associated antigen expression (48%), and 11q23/MLL rearrangement (68%). The 11q23/MLL involvement was correlated with age less than 6 months, CD10- phenotype, myeloid-associated antigen expression, and high leukocyte count. Although 11q23/MLL involvement, age less than 6 months, myeloid-associated antigen expression, and female sex were each significantly associated with an inferior treatment outcome, only rearranged 11q23/MLL emerged as an independent predictor of prognosis in multivariate analysis (P = .01). Infants with this genetic abnormality had a 4.7-fold (95% confidence interval, 1.3- to 17.0-fold) increased risk in adverse events compared to other infants. CONCLUSION The 11q23/MLL involvement of blast cells identifies a major subgroup of infant ALL cases that require an innovative treatment approach. Infants who lack this genetic abnormality have an intermediate prognosis and could be treated accordingly on risk-directed protocols.
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Robinson, Blaine W., Kajia Cao, Joanne M. Hilden, Patricia A. Dinndorf, Nyla A. Heerema, Harland N. Sather, Ron McGlennen, et al. "Age Is the Strongest Determinant of Leukemia Blast Cell Gene Expression in MLL-Rearranged Infant ALL and MLL-AF4 Directs a Distinct Gene Expression Profile Related to CNS Disease." Blood 112, no. 11 (November 16, 2008): 1200. http://dx.doi.org/10.1182/blood.v112.11.1200.1200.
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Abstract In infant ALL a constellation of features (young age, hyperleukocytosis, extramedullary/CNS disease, CD10− phenotype, MLL translocation, slow early response to treatment) are associated with poor outcome. Despite the frequent MLL translocations, the biologic basis for these features is unknown. In this study, gene expression profiles of leukemia cells from diagnosis were determined in MLL-rearranged infant ALL and relationships were sought between gene expression and clinical covariates. Patients and Methods: Seventeen infants treated on COG protocol CCG 1953 were studied. Features at diagnosis were: age, 0–333 d, median 121 d; WBC count, 10.2–1,286 ×103/μL, median 229 ×103/μL; CD10−, 14, CD10+, 2, CD10 unknown, 1; CNS1 status, 11, CNS2 status, 2, CNS3 status, 4. Eight were alive and 9 were dead at survival times from 41–3384 d. MLL translocations were identified using karyotype, FISH, Southern blot and PCR. Gene expression profiling was performed with Affymetrix HG_U133 Plus2.0 arrays. Pearson’s correlation coefficients and significance levels were computed between age and log-2 transformed expression levels of each probeset. To find associations between gene expression and MLL partner genes with the age effect controlled, linear regression was run using gene expression as the dependent variable and age and partner gene (AF4 v. other) as the independent variable. Similar linear regressions were run for WBC count, CNS status (excluding CNS2) and survival status. Results: PCR identified the MLL partner genes AF4, ENL, AF9, AF10 and EPS15 in 6, 4, 2, 1 and 1 cases, respectively. In 1 case each with t(4;11)(q21;q23) or t(11;19)(q23;p13.3) partner genes were assigned based on karyotype. Another case harbored a t(6;9;11) (q21;p22;q23). Gene expression was affected more by age at diagnosis than any other factor. Remarkably, gene expression analysis by age at diagnosis as a continuous variable showed correlations of 2072 probesets, all but one of which were positive (p<0.0001). The most differentially expressed genes were related to glycosylation and signaling, adhesion, membrane and development. Gene expression analysis by partner gene with the age effect controlled yielded 118 probesets (p<0.001) that segregated cases with MLL-AF4 from cases with MLL-ENL. Many more genes were up- than down-regulated in cases with MLL-AF4. Expression of these 118 probesets in other non MLL-AF4 cases more closely resembled those in cases with MLL-ENL. CNS status significantly correlated with 208 probesets (p<0.001). Even though the partner gene was AF4 in all CNS3 status cases and there was substantial overlap, the genes associated with CNS3 status or MLL-AF4 were not entirely overlapping. The most differentially expressed genes that were correlated with CNS status and MLL-AF4 were related to macromolecule/protein localization and protein transport. With age effect controlled, few genes correlated with WBC count or outcome. Conclusions: Within infant ALL there are profound intrinsic biologic differences in leukemia cells due to age at diagnosis. MLL-AF4 translocations in infant ALL have variably been associated with more inferior outcomes than other MLL translocations. This analysis indicates that the different MLL translocations are biologically dissimilar and that cases with MLL-AF4 are distinct from cases with MLL-ENL or other partner genes. The study also shows that a specific gene expression program directs the clinical/biologic property of CNS disease, which is overlapping with the effect of AF4 involvement. The complex correlations between clinical covariates, MLL translocations and gene expression in infant ALL merit further study. It will also be important to determine if a similar gene expression profile is associated with CNS disease in other leukemia subtypes.
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Popov, Alexander, Tatiana Verzhbitskaya, Grigory Tsaur, Egor Shorikov, Liudmila Movchan, Mikhail Belevtsev, Anatoly Kustanovich, et al. "Relapse Prediction By Flow Cytometric Minimal Residual Disease Assessment in Infants with Acute Lymphoblastic Leukemia." Blood 128, no. 22 (December 2, 2016): 1731. http://dx.doi.org/10.1182/blood.v128.22.1731.1731.
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Abstract Acute lymphoblastic leukemia (ALL) in infants (less than 1 year old) is a unique tumor with distinct biological features and poor outcome even when modern treatment schemes are used. Rearrangementsof MLL-gene, located in 11q23 region, are detected in approximately 75-80% of infants with ALL and lead to treatment resistance and high relapse rate. Nevertheless patients with germline MLL also demonstrate inferior outcome compared to ALL in older children. Thus additional risk factors implementation is one of the crucial points in infant ALL management. Minimal residual disease (MRD) measurement by multicolor flow cytometry (FCM) or various PCR technics is a well-standardized method of treatment response evaluation in childhood ALL, although in infants MRD data is not so widely applied. The aim of the study was to evaluate the relapse prediction feasibility in infant ALL by FCM MRD assessment during remission induction of MLL-Baby protocol. Methods. Totally 89 infants aged from 5 days to 11 months were enrolled in the present study. In 23 cases (25.8%) MLL gene was germline (MLL-g group), 33 patients (37.1%) had MLL-AF4 fusion while in remaining 33 cases (37.1%) other types of MLL-rearrangements were found. All patients were diagnosed as B-cell precursor ALL and all were treated by well established in Russia and Belarus MLL-Baby protocol, which is specially designed for infant ALL management. MRD was measured by 6-10-color FCM in bone marrow (BM) samples obtained at day 15 and at the end of remission induction (day 36). The availability of samples at at least one of these time-points was the only criteria for study group completion. In 43 patients with known types of MLL-rearrangements MRD was also assessed by fusion gene transcript (FGT) detection in RQ-PCR after first consolidation or first high risk block (for intermediate risk and high risk groups respectively). Relapse risk was investigated by cumulative incidence of relapse (CIR) estimation. Median of follow-up was 3 years 10 months. Results. Finally at day 15 MRD was studied in 71 cases. 17 patients (23.9%) were tested MRD-negative while remaining ones displayed various levels of MRD-positivity: 8 cases (11.3%) - from 0.01% to 0.1%; 14 (19.7%) - from 0.1% to 1%; 22 (31.0%) - from 1% to 10%; 10 (14.1%) - more than 10%. Proportion of MRD-positivity was lower in MLL-g group compared to MLL-rearranged (MLL-r) patients (58.8% and 81.5% respectively, p=0.06). Prognostic impact of day 15 MRD differed due to MLL-status. In MLL-r group significant differences between MRD(+) and MRD(-) patients were observed (n=10, CIR 0.28(0.18) and n=37, CIR 0.67(0.08) respectively, p=0.025). At the same time in MLL-g group these outcome differences were not significant (n=7, CIR 0 and n=9, CIR 0.22(0.14) correspondingly, p=0.197). Interestingly, in patients carrying MLL-AF4 fusion, known to be one of the most adverse types of MLL-rearrangements, day 15 MRD-negativity predicted low relapse incidence (n=5, CIR 0 and n=16, CIR 0.68(0.12) respectively, p=0.045). Thus day 15 MRD-negativity allows to detect low-risk MLL-r infants but it is not applicable in MLL-g group. It was previously shown that any detectable level of FGT after first consolidation or first high risk block predicts very poor outcome in MLL-r cases (G. Tsaur et al, ASH-2011). In current series RQ-PCR data in patients, FCM MRD(+) at day 15, distinguished groups of intermediate and very high relapse risk (n=17, CIR 0.51(0.13) and n=18, CIR 0.84(0.09) respectively, p=0.017). At day 36 FCM MRD was assessed in 82 infants. Among them 35 (42.7%) were tested negative while remaining 47 (57.3%) were MRD(+) at various levels. Prognostic value of day 36 FCM MRD data in MLL-r group was not significant: MRD(+) patients (n=23) had CIR of 0.71(0.08) while in MRD(-) cases (n=33) CIR was 0.49(0.12), p=0.153. Conversely, in MLL-g group low end-induction MRD (less than 0.1%) lead to excellent outcome compared to patients with higher MRD (n=14, CIR 0 and n=7, CIR 0.22(0.20) respectively, p=0.010). Conclusions. Thus FCM MRD data could distinguish infants with low risk of ALL relapse, but in MLL-r and MLL-g groups different time-points are prognosticaly significant. In MLL-g patients tandem application of FCM at early time-point and RQ-PCR later could help to define groups with low, intermediate and high relapse risk. MRD data could be added to MLL-Baby protocol risk group stratification, which is currently based on type of MLL-rearrangement. Disclosures No relevant conflicts of interest to declare.
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Hilden, Joanne M., Franklin O. Smith, Joy L. Frestedt, Ronald McGlennen, William B. Howells, Poul H. B. Sorensen, Diane C. Arthur, et al. "MLL Gene Rearrangement, Cytogenetic 11q23 Abnormalities, and Expression of the NG2 Molecule in Infant Acute Myeloid Leukemia." Blood 89, no. 10 (May 15, 1997): 3801–5. http://dx.doi.org/10.1182/blood.v89.10.3801.3801_3801_3805.
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To study prognostic factors in infant acute myeloid leukemia (AML), we analyzed 44 children treated on Childrens Cancer Group protocols for MLL gene rearrangement by Southern blot, cytogenetic 11q23 abnormalities, and reactivity with monoclonal antibody 7.1. This antibody detects the human homologue of the rat NG2 chondroitin sulfate proteoglycan molecule, which has previously been reported to be expressed on human melanoma. NG2 has been found to be expressed on human leukemic blasts but not on other hematopoietic cells. In childhood AML, NG2 cell surface expression correlated with poor outcome and with some but not all 11q23 rearrangements. In childhood acute lymphoblastic leukemia, NG2 expression correlated with poor outcome and with balanced 11q23 translocations. In this study, 29 of 44 (66%) of infants with AML showed MLL rearrangement and, as expected, this group had a high incidence of French-American-British M4/M5 morphology (22/29). Of the cases tested, 35.1% (13/37) were NG2 positive. All (13/13) NG2-positive cases were rearranged at MLL, whereas only 46% (11/24) of NG2-negative cases had MLL rearrangement. NG2 expression did not correlate with poor outcome (P = .31); there was a trend towards a worse outcome with MLL rearrangement (P = .13). Thus monoclonal antibody 7.1 does not detect all cases of MLL rearrangement in infant AML.
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Andersson, Anna K., Jing Ma, Jianmin Wang, Xiang Chen, Michael Rusch, Gang Wu, John Easton, et al. "Whole Genome Sequence Analysis of 22 MLL Rearranged Infant Acute Lymphoblastic Leukemias Reveals Remarkably Few Somatic Mutations: A Report From the St Jude Children‘s Research Hospital - Washington University Pediatric Cancer Genome Project." Blood 118, no. 21 (November 18, 2011): 69. http://dx.doi.org/10.1182/blood.v118.21.69.69.
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Abstract Abstract 69 Infant (< 1 year of age) acute lymphoblastic leukemia (ALL) is a rare disease characterized by rearrangements of the Mixed Lineage Leukemia (MLL) gene at 11q23 and a poor prognosis. In an effort to determine the total complement of somatic mutations occurring in this high risk leukemia, we performed paired-end whole genome sequencing (WGS) on diagnostic leukemia blasts and matched germ line samples from 22 infants with MLL rearranged ALL using the Illumina platform. In addition, we sequenced 2 paired relapse samples. Somatic alterations, including single nucleotide variations (SNV), and structural variations (SV) including insertions, deletions, inversion, and inter- and intra-chromosomal rearrangements were detected using complementary analysis pipelines including Bambino, CREST and CONSERTING. Validation of identified somatic mutations was performed using PCR amplification of the leukemia and germ line DNA followed by Sanger or 454-based sequencing, or by array-based capture followed by Illumina-based sequencing. Analysis of the structure of MLL rearrangements at the base pair level revealed that over half had complex rearrangements that involved either three or more chromosomes, or contained at the breakpoints deletions, amplifications, insertions, or inversion of sequences. In five of the complex cases, chromosomal rearrangements were predicted to generate not only a MLL-partner gene fusion, but also novel in-frame fusions including KRAS-MLL; RAD51B-MLL / AFF1-RAD51B; MLLT10-CTNNAP3B; MLLT10-ATP5L / ATP5L-YPEL4; and CRTAM-GNL3. An analysis of the sequence surrounding the breakpoints of MLL and its partner genes suggest that the predominant mechanism of rearrangement involved non-homologous end joining. An analysis of the total number of non-silent mutations revealed infant ALL to have the lowest frequency of non-silent somatic mutations of any cancer sequenced to date. After removal of SVs and CNAs associated with the MLL rearrangements, a mean of only 2 somatic SVs and 2 SNVs affecting the coding region of annotated genes or regulatory RNAs were detected per case, with a range of non-silent mutation of between 0 and 11 per case (0–7 SV and 0–5 SNV). Despite the paucity of mutations several pathways were recurrently targeted. Mutations leading to activation of signaling through the PI3K/RAS pathway was observed in 45% of the cases with mutation of individual components including KRAS (n=4), NRAS (n=2), and non-recurrent mutations in NF1, PTPN11, PIK3R1, and the GTPase activating protein ARHGAP32 (p200Rho/GAP), which mediates cross-talk between RAS and Rho signaling. Other pathways altered include B cell differentiation, with 23% of cases containing mono-allelic deletion or gains of PAX5, 14% with deletions of the CDKN2A/B, and 2 cases with focal deletions of the non-coding RNA genes DLEU1/2. WGS of two infant ALL relapse samples and comparison with the data from their matched diagnostic samples revealed a marked increase in the number of mutations at relapse with additional SVs, SNVs, and CNAs identified. Moreover, an analysis of the allelic ratios of mutated genes revealed clonal heterogeneity at diagnosis with relapse appearing to arise from a minor diagnostic clone. Because of the exceedingly low frequency of mutations detected in infant ALL, we decided to define the frequency of non-silent SNVs in MLL rearranged leukemia occurring in older children (7–19 years of age). Exome sequencing was performed on 13 MLL leukemias (8 ALLs and 5 AMLs). This analysis revealed that non-infant pediatric MLL rearranged leukemias harbor a significantly higher number of non-silent somatic SNVs than infant ALL (mean 8/case in older patients versus 2/case in infants, p<0.001). Although the increased frequency of mutations may be a reflection of the older age, the low number of cooperating mutations in infants raises the possibility that the target cell of transformation differs between infants and older children, with the cells present during early development requiring fewer cooperating mutations to induce leukemia. In summary our analysis demonstrated an exceedingly small number of mutations required to generate infant MLL rearranged leukemia. The number of detected somatic mutations may represent the lower limit of mutations required to transform a normal human cell into cancer. Disclosures: Fioretos: Cantargia AB: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Qlucore AB: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
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Nishi, Masanori, Yujirou Sadakane, Mariko Eguchi, Minenori Eguchi-Ishimae, Hisamichi Tauchi, Sanae Kawakami, Daisuke Tomizawa, Shuki Mizutani, Kanji Sugita, and Eiichi Ishii. "Suppression of Let-7b MicroRNA and Enhanced Its Target genes in Infant Acute Lymphoblastic Leukemia with MLL Gene Rearrangements." Blood 114, no. 22 (November 20, 2009): 986. http://dx.doi.org/10.1182/blood.v114.22.986.986.
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Abstract Abstract 986 Poster Board I-8 Introduction: MicroRNAs (miRs) play an important role in leukemogenesis; specific miRs are underexpressed, whereas others are overexpressed in leukemias. In addition, to regulate the leukemogenesis, each specific miRs repress one or many target genes. These findings indicate that some specific miRs can regulate the leukemogenesis also in infant ALL. Recently, the etiology of infant leukemia has been calrified; in utero exposure to topoisomerase-II inhibitors and other factors may affect MLL gene rearrangements, which lead to induction of leukemogenesis. After birth, specific oncogenes and proliferation or differentiation factors will be disrupted via enhanced HoxA9 by the stimulation of MLL fusion genes. In this study, let-7b miR and its regulator lin28 are suppressed, followed by the enhanced expression of HoxA9 and HMGA2 oncogene in MLL-rearranged infant ALL. Materials and methods: Leukemic cells from ALL infants and ALL cell lines with and without MLL gene rearrangements were used. In microarray analysis, miR was extracted and analysis was performed by using the mirVanaTM miRNA Bioarray V2 system. Quadruplicated data were averaged, and all miRs with >2-fold different expression was considered as significant in this study. Then, TaqMan real-time PCR method was used to quantify the expression of each miR. The relative expression of each gene was calculated by setting the control normal B lymphocytes. Finally, the transfection of MLL fusion genes to HEK293 cells was performed to induce let-7b expression in vitro. Results: By the microarray analysis, 13 miRs showed >2-fold lower expression in MLL-rearranged ALLs than MLL-germline ALLs, including miR-124a, miR-153, and let-7b with >5-fold lower expression. When we focused on let-7b, expressions of let-7b, its associated genes lin28 and Dicer were significantly lower in MLL-rearranged ALLs than MLL-germline ALLs. MLL-rearranged ALLs showed high expression of HoxA9 and HMGA2, possible target genes for let-7b. The in vitro transfection of MLL fusion genes to HEK293 cells also induced the suppression of let-7b. Discussion: Our study first demonstrated that the expressions of let-7b is significantly decreased in MLL-rearranged ALL cells. Let-7 family has been reported as tumor-suppressor gene in cancer; the expression level of let-7 family increases during differentiation of cancer cells, while its target genes show a reciprocal expression pattern. However, it remains to be explored whether let-7 might be involved directly or indirectly in the leukemia-related functions. Conclusion: The interaction between HoxA9 disrupted by MLL fusion and HMGA2 oncogene regulated by specific miRs including let-7 could be involved in the pathogenesis of infant ALL with MLL gene rearrangements. These genes might become a target for therapeutic approach in the future. Disclosures: No relevant conflicts of interest to declare.
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Urtishak, Karen A., Li-San Wang, Richard Harvey, Susan R. Atlas, I.-Ming L. Chen, Blaine W. Robinson, Lea Moukarzel, et al. "Infant Acute Lymphoblastic Leukemias Are Pan-Sensitive to Obatoclax Across molecular/Cytogenetic Subtypes, Especially MLL-ENL, and gene Expression Profiles Determine Obatoclax IC50: A Report on the Children's Oncology Group (COG) P9407 Trial." Blood 116, no. 21 (November 19, 2010): 2757. http://dx.doi.org/10.1182/blood.v116.21.2757.2757.
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Abstract Abstract 2757 Introduction: The outcome of infants with acute lymphoblastic leukemia (ALL) remains poor because of the association of frequently occurring MLL translocations with drug resistance and vulnerability of the very young to treatment complications. The two most common MLL partner genes in infant ALL, AF4 (AFF1) and ENL (MLLT1), are associated with particularly poor survival. Better therapies are urgent. One candidate is obatoclax (GeminX Biotechnologies, Inc.), which targets interactions of pan-anti-apoptotic BCL-2 family proteins with BH3 proteins and is now in a Phase I trial for relapsed/refractory pediatric cancers (COG ADVL0816). Previously we showed potent single agent in vitro activity of obatoclax against MLL-rearranged infant ALL (Zhang ASH 2008). Here we evaluate correlations of obatoclax activity with MLL translocation status and gene expression profiles in a large number of cases of infant ALL to define molecular determinants of sensitivity. Methods: Bone marrow, peripheral blood or apheresis samples from the time of diagnosis in 54 infants (age 1–365 d, median 168 d; WBC count 15–1230×103/μL, median 445×103/μL) with ALL (n=52) or bilineal acute leukemia (n=2) were examined, 48 of which were from the COG P9407 trial. By molecular/cytogenetic classification, the cases were MLL-AF4+ (n= 28), MLL-ENL+ (n= 11), other MLL rearrangement positive (other MLL+) (n= 8) or MLL germline (MLL-) (n= 7). Single agent IC50 values from MTT assays after 72 h obatoclax exposures were determined in all cases (including 13 previously tested; Zhang ASH 2008) by plotting the surviving fractions. IC50s in the MLL-AF4+ group were compared to those in each of the other 3 molecular/cytogenetic groups by Wilcoxon's test. Gene expression profiling was performed on Affymetrix HG_U133 Plus2.0 arrays in 47 of the 48 COG P9407 cases. Spearman test was used to identify correlation between log2 expression levels for each probeset and IC50 values across subjects. A heatmap of significant probesets (p≤0.001) was generated by transforming expression levels to z-scores and ordering rows and columns by complete linkage hierarchical clustering. Ingenuity pathway analysis was applied to all probesets with p≤0.01 to identify pathways significantly correlated with IC50. Additional MTT assays were initiated to test sensitivity to agents targeting these pathways. Results: Even though most cases in all 4 groups were sensitive to obatoclax as indicated by IC50s within a clinically achievable range, MLL translocation status still had a significant effect on IC50. MLL-AF4+ cases were least sensitive and MLL-ENL+ cases were most sensitive to obatoclax. Respective IC50 ranges across all 54 cases were: MLL-AF4+, 26–918 nM; MLL-ENL+, 13–294 nM; other MLL+ 10–356 nM; MLL−, 31–488. Compared to MLL-AF4+, the IC50s in MLL-ENL+ cases were significantly lower (p=0.047), IC50s in other MLL+ cases were lower but the difference did not achieve significance (p=0.10), and IC50s in MLL- cases were not significantly different (p=0.64). In the 47 COG P9407 cases studied by MTT assay and gene expression profiling, 450 probesets defined a cluster of 16 cases with higher IC50s, which were predominantly MLL-AF4+ (68.7%). Ingenuity analysis identified significant correlations of the following canonical pathways with the IC50 in the same 47 cases: glycolysis/gluconeogenesis, mTOR signaling, regulation of eIF4 and p70S6K signaling, EIF2 signaling, and fructose and mannose metabolism. In preliminary analyses, cell lines with t(4;11) exhibited time and dose-dependant sensitivity to the eIF4e inhibitor ribavirin. Conclusions: In infant ALL, obatoclax has broad-spectrum activity and there is pan-sensitivity across MLL translocation subtypes and MLL− cases. Still specific MLL partner genes have a strong effect on obatoclax IC50 and there is exquisite sensitivity in MLL-ENL+ cases. This result is important because MLL-ENL is associated with particularly poor survival when conventional therapies are used. The association of differentially expressed genes in canonical cell signaling and metabolism pathways with differences in obatoclax sensitivity forms the basis to combine obatoclax with targeted agents directed at restoring these pathways to enhance responsiveness even further. Disclosures: Felix: None: Patent not licensed.
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Tomizawa, Daisuke, Katsuyoshi Koh, Jun Nagayama, Takashi Sato, Naoko Kinukawa, Keiichi Isoyama, Yoshiyuki Kosaka, et al. "Risk Stratification by MLL Gene Status and Outcome of Acute Lymphoblastic Leukemia in Infants: A Report from the Japan Infant Leukemia Study Group." Blood 108, no. 11 (November 16, 2006): 148. http://dx.doi.org/10.1182/blood.v108.11.148.148.
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Abstract Background: Acute lymphoblastic leukemia (ALL) in infants represent 2.5% to 5% of all childhood ALL, and has an inferior therapeutic outcome compared to that of older children. Among several risk factors, rearrangement in MLL gene is most closely associated with this poor outcome. This is the first series of cooperative group trials stratifying ALL in infants with MLL gene status. Procedure: Infants with ALL, age less than 12 months, were registered on two consecutive multi-center trials designated MLL96 and MLL98. All the patients were tested for MLL gene rearrangements by Southern Blot analysis and/or fluorescence in situ hybridization. According to the MLL gene status, patients with rearranged MLL gene were allocated to intensive chemotherapy followed by hematopoietic stem cell transplantation (HSCT), and those with germline MLL were treated with standard chemotherapy alone (total treatment duration was 83–85 weeks). In MLL98 study, treatment intensification and early use of HSCT were introduced compared to MLL96 study. Results: A total of 101 infants were enrolled between December 1995 and December 2002. Seventy-nine had rearranged MLL and 22 had germline MLL. Event-free survival (EFS) of all 101 infants with ALL was 52.0% at 5 years; 5-year EFS for MLL rearranged cases was 39.7%, and for MLL germline cases was 95.5% (p<0.001). Among the MLL rearranged patients, 3-year EFS improved from 34.0% in MLL96 trial (N=41) to 43.6% in MLL98 trial (N=38). Although their were no secondary malignancies among the whole enrolled patients, physical growth impairments was observed in MLL rearranged patients, while no significant late effects were observed in MLL germline patients. Conclusions: Risk stratification with MLL gene status was successful for infants with ALL. Excellent results were obtained for the germline MLL group using chemotherapy alone, and early introduction of allogeneic HSCT in first remission seemed effective for the MLL rearranged group. However, use of HSCT with less toxic conditioning regimens or development of effective chemotherapy including molecular target therapy are needed for MLL rearranged patients in order to avoid severe late effects for infants.
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Schafer, Eric, Rafael Irizarry, Sandeep Negi, Emily McIntyre, Donald Small, Maria E. Figueroa, Ari Melnick, and Patrick Brown. "Promoter hypermethylation in MLL-r infant acute lymphoblastic leukemia: biology and therapeutic targeting." Blood 115, no. 23 (June 10, 2010): 4798–809. http://dx.doi.org/10.1182/blood-2009-09-243634.
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Abstract Cooperating leukemogenic events in MLL-rearranged (MLL-r) infant acute lymphoblastic leukemia (ALL) are largely unknown. We explored the role of promoter CpG island hypermethylation in the biology and therapeutic targeting of MLL-r infant ALL. The HELP (HpaII tiny fragment enrichment by ligation-mediated polymerase chain reaction [PCR]) assay was used to examine genome-wide methylation of a cohort of MLL-r infant leukemia samples (n = 5), other common childhood ALLs (n = 5), and normals (n = 5). Unsupervised analysis showed tight clustering of samples into their known biologic groups, indicating large differences in methylation patterns. Global hypermethylation was seen in the MLL-r cohort compared with both the normals and the others, with ratios of significantly (P < .001) hypermethylated to hypomethylated CpGs of 1.7 and 2.9, respectively. A subset of 7 differentially hypermethylated genes was assayed by quantitative reverse-transcription (qRT)–PCR, confirming relative silencing in 5 of 7. In cell line treatment assays with the DNA methyltransferase inhibitor (DNMTi) decitabine, MLL-r (but not MLL wild-type cell lines) showed dose- and time-dependent cytotoxicity and re-expression of 4 of the 5 silenced genes. Methylation-specific PCR (MSP) confirmed promoter hypermethylation at baseline, and a relative decrease in methylation after treatment. DNMTi may represent a novel molecularly targeted therapy for MLL-r infant ALL.
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Kerstjens, Mark, Patricia Garrido Castro, Sandra S. Pinhanços, Pauline Schneider, Priscilla Wander, Rob Pieters, and Ronald W. Stam. "Irinotecan Induces Disease Remission in Xenograft Mouse Models of Pediatric MLL-Rearranged Acute Lymphoblastic Leukemia." Biomedicines 9, no. 7 (June 23, 2021): 711. http://dx.doi.org/10.3390/biomedicines9070711.
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Acute lymphoblastic leukemia (ALL) in infants (<1 year of age) remains one of the most aggressive types of childhood hematologic malignancy. The majority (~80%) of infant ALL cases are characterized by chromosomal translocations involving the MLL (or KMT2A) gene, which confer highly dismal prognoses on current combination chemotherapeutic regimens. Hence, more adequate therapeutic strategies are urgently needed. To expedite clinical transition of potentially effective therapeutics, we here applied a drug repurposing approach by performing in vitro drug screens of (mostly) clinically approved drugs on a variety of human ALL cell line models. Out of 3685 compounds tested, the alkaloid drug Camptothecin (CPT) and its derivatives 10-Hydroxycamtothecin (10-HCPT) and 7-Ethyl-10-hydroxycamtothecin (SN-38: the active metabolite of the drug Irinotecan) appeared most effective at very low nanomolar concentrations in all ALL cell lines, including models of MLL-rearranged ALL (n = 3). Although the observed in vitro anti-leukemic effects of Camptothecin and its derivatives certainly were not specific to MLL-rearranged ALL, we decided to further focus on this highly aggressive type of leukemia. Given that Irinotecan (the pro-drug of SN-38) has been increasingly used for the treatment of various pediatric solid tumors, we specifically chose this agent for further pre-clinical evaluation in pediatric MLL-rearranged ALL. Interestingly, shortly after engraftment, Irinotecan completely blocked leukemia expansion in mouse xenografts of a pediatric MLL-rearranged ALL cell line, as well as in two patient-derived xenograft (PDX) models of MLL-rearranged infant ALL. Also, from a more clinically relevant perspective, Irinotecan monotherapy was able to induce sustainable disease remissions in MLL-rearranged ALL xenotransplanted mice burdened with advanced leukemia. Taken together, our data demonstrate that Irinotecan exerts highly potent anti-leukemia effects against pediatric MLL-rearranged ALL, and likely against other, more favorable subtypes of childhood ALL as well.