Journal articles on the topic 'T-cell acute lymphoblastic leukemia/lymphoma, gene-expression profiling, genome-wide copy number analysis, microRNA'
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1
Mullighan, Charles G., Xiaoping Su, Jing Ma, Wenjian Yang, Mary V. Relling, William L. Carroll, Gregory Reaman, et al. "Genome-Wide Profiling of High-Risk Pediatric Acute Lymphoblastic Leukemia (ALL): The ALL Pilot Project for the Therapeutically Applicable Research To Generate Effective Treatments (TARGET) Initiative." Blood 110, no. 11 (November 16, 2007): 229. http://dx.doi.org/10.1182/blood.v110.11.229.229.
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Abstract Recent studies have identified a high frequency of recurrent acquired DNA copy number abnormalities in pediatric ALL, most commonly involving genes regulating lymphoid development (Nature2007;446:758). The majority of B-progenitor ALL cases in this study harbored recurring chromosomal abnormalities such as hyperdiploidy and recurring translocations, many of which are associated with favorable outcome. Data from large cohorts of poor risk B-ALL have not been reported. Here we report genomic profiling data from a cohort of 221 pediatric ALL cases treated on the Children’s Oncology Group P9906 study from 2000–2003 with an augmented BFM regimen (N Engl J Med1998;338:1663). The study targeted poor prognosis ALL cases, using age/WBC criteria designed to identify a subset of patients with NCI high risk ALL that historically had a very poor outcome. Cases with favorable (trisomy 4 + 10; ETV6-RUNX1) or unfavorable (BCR-ABL1 or hypodiploid) genetic features were excluded. The cohort included 25 TCF3-PBX1 and 19 MLL-rearranged cases. Profiling of DNA copy number abnormalities was performed using Affymetrix 250k Sty and Nsp arrays, reference normalization, dChipSNP, and circular binary segmentation. Germline SNP array data was available for 210 cases. The most frequent abnormalities were deletions of CDKN2A in 101 cases (45.7%), the B-lineage transcription factors PAX5 (N=68, 30.5%) and IKZF1 (Ikaros, N=32, 14.5%), ETV6 (N=29, 13.1%), RB1 (N=25, 11.3%), BTG1 (N=23, 10.4%), the tumor suppressor candidate TSC22D1 (N=20, 9%), the microRNA cluster at 13q14 (N=19, 8.6%), and deletion (N=3) or amplification (N=20, 4 focal, 16 broad) of the retinoic acid pathway gene CCDC26. Other recurring copy number abnormalities with relevance to leukemogenesis included deletions of BTLA, EBF, IL3RA, ERG, TOX, RAG1/2, KRAS, NRAS, NR3C2 and the adenosine deaminase pathway genes ADAR and ADARB2. An unexpected finding was focal deletion involving DMD (dystrophin) at Xp21.1 in 15 (6.8%) cases. Copy number abnormalities were uncommon in MLL rearranged leukemias, suggesting that fewer secondary mutations are needed for leukemogenesis in this genetic subtype of ALL. Clustering of copy number data using non-negative matrix factorization identified 11 clusters of cases, including clusters driven by isochromosome 7q, deletions of 9p, gains of 1q, multiple whole chromosomal gains, and intrachromosomal amplification of chromosome 21 (iamp21). These findings confirm the high frequency of deletions involving genes regulating B cell development and cell cycle such as PAX5 and CDKN2A in both standard and poor risk B-ALL. Furthermore ETV6 (TEL) deletions are shown to be common in B-ALL cases lacking the ETV6-RUNX1 translocation. The identification of novel recurring abnormalities (IL3RA, KRAS, NRAS) emphasizes the importance of high resolution copy number analysis in leukemia. These data are being integrated with gene expression data to select genes for resequencing as part of the TARGET project.
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Mussolin, Lara, Katia Basso, Antonella Lettieri, Manisha Brahmachary, Giovanni Cazzaniga, Andrea Califano, Wei-Keat Lim, Giuseppe Basso, Andrea Biondi, and Angelo Rosolen. "Genomic and Transcriptomic Analyses Revealed Differences and Similarities Between T-Lbl and T-ALL ." Blood 114, no. 22 (November 20, 2009): 2943. http://dx.doi.org/10.1182/blood.v114.22.2943.2943.
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Abstract Abstract 2943 Poster Board II-919 Introduction: T-cell acute lymphoblastic leukemia (T-ALL) and lymphoma (T-LBL) share common morphologic and immunophenotypic features and are treated with similar therapeutic approaches. Nonetheless, they show distinct clinical presentations suggesting that they may represent two different biological entities. In order to gain insights into the biological characteristics of these T-cell malignancies we applied genomic and transcriptomic approaches on residual diagnostic specimens from pediatric patients affected by T-ALL or T-LBL. Patients and methods: Genome-wide gene expression profiling (HG-U133Plus2.0, Affymetrix) was performed for 20 patients affected by T-LBL and 10 patients affected by T-ALL. In order to control for normal cells contamination present in the nodal biopsies (T-LBL) and bone marrow aspirates (T-ALL), we used gene expression profiles of B-cell lymphoblastic lymphoma and common (CD10+) lymphoblastic leukemia, respectively. Results: Genes differentially expressed in nodal versus bone marrow-derived samples were subtracted from the T-LBL versus T-ALL signature allowing the identification of a subset of genes able to discriminate the two T-cell malignancies regardless of their localization. This gene signature includes genes involved in chemotactic response, cell adhesion and angiogenesis which may play a role in the different tumor cell localization. Indeed, genes involved in promoting angiogenesis, invasion and nodal localization were up-regulated in T-LBL. Copy number analysis was performed using single nucleotide polymorphism (SNP) arrays (Human Mapping 100K arrays, Affymetrix) on a subset of the samples (9 T-ALL and 9 T-LBL) analyzed by gene expression profiling. This analysis detected approximately 200 genetic loci recurrently affected by copy number alterations in T-ALL and/or T-LBL. The most common aberration was the 9p21.3 deletion which includes CDKN2A/B. Consistent with previous reports amplifications involving MYB were identified in two cases of T-ALL. Although most aberrations were commonly found in both entities several were recurrently detected in T-LBL but not in T-ALL and vice versa. Conclusion: Taken together these results suggest that T-LBL and T-ALL share a large fraction of their biological features; nevertheless each malignancy displays also a unique pattern of genetic lesions and specific gene expression signatures, which may contribute to the understanding of the distinct evolution of these T-cell malignancies. Disclosures: No relevant conflicts of interest to declare.
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Flotho, Christian, Susana C. Raimondi, and James R. Downing. "Identification of Aberrantly Regulated Genes through Comparison of Genome-Wide Expression Profiles and Karyotype in Hyperdiploid >50 Chromosomes Pediatric Acute Lymphoblastic Leukemia." Blood 104, no. 11 (November 16, 2004): 420. http://dx.doi.org/10.1182/blood.v104.11.420.420.
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Abstract We have demonstrated that expression profiling of leukemic blasts can accurately identify the known prognostic subtypes of ALL, including T-ALL, E2A-PBX1, TEL-AML1, MLL rearrangements, BCR-ABL, and hyperdiploid >50 chromosomes (HD>50). Interestingly, almost 70% of the genes that defined HD>50 ALL localized to chromosome 21 or X. To further explore the relationship between gene expression and chromosome dosage, we compared the expression profiles obtained using the Affymetrix U133A&B microarrays of 17 HD>50 ALLs to 78 diploid or pseudodiploid ALLs. Our analysis demonstrated that the average expression level for all genes on a chromosome could be used to predict chromosome copy numbers. Specifically, the copy number for each chromosome calculated by gene expression profiling predicted the numerical chromosomal abnormalities detected by standard cytogenetics. For chromosomes that were trisomic in HD>50 ALL, the mean chromosome-specific gene expression level was increased approximately 1.5-fold compared to that observed in diploid or pseudodiploid ALL cases. Similarly, for chromosome 21 and X, the mean chromosome-specific gene expression levels were increased approximately 2-fold, consistent with a duplication of the active X chromosome and tetrasomy of chromosome 21, a finding verified by standard cytogenetics in >90% of the HD>50 cases. These finding indicate that the aberrant gene expression levels seen in HD>50 ALL primarily reflect gene dosages. Importantly, we did not observe any clustering of aberrantly expressed genes across the duplicated chromosomes, making regional gain or loss of genomic material unlikely. Paradoxically, however, a more detailed analysis revealed a small but statistically significant number of genes on the trisomic/tetrasomic chromosomes whose expression levels were markedly reduced when compared to that seen in diploid or pseudodiploid leukemic samples. Using the Statistical Analysis of Microarrays (SAM) algorithm we identified 20 genes whose expression was reduced >2-fold despite having an increase in copy number. Interestingly, included within this group are several known tumor suppressors, including AKAP12, which is specifically silenced by methylation in fos-transformed cells, and IGF2R and IGFBP7, negative regulators of insulin-like growth factor signaling. In addition to the silencing of a small subset of genes, we also identified 21 genes on these chromosomes whose expression levels were markedly higher (>3-fold) than would be predicted solely based on copy number. Although the mechanism responsible for their increased expression remains unknown, included in this group are four genes involved in signal transduction (IL3RA, IL13RA1, SNX9, and GASP) and a novel cytokine, C17, whose expression is normally limited to CD34+ hematopoietic progenitors. Taken together, these data suggest that aberrant growth in HD>50 ALL is in part driven by increased expression of a large number of genes secondary to chromosome duplications, coupled with a further enhanced expression of a limited number of growth promoting genes, and the specific silencing of a small subset of negative growth regulatory genes. Understanding the mechanisms responsible for the non-dosage related changes in gene expression should provide important insights into the pathology of HD>50 ALL.
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Figueroa, Maria E., Shann-Ching Chen, Anna K. Andersson, Wei Liu, Cheng Cheng, Letha A. Phillips, Yushan Li, James R. Downing, Ari M. Melnick, and Charles Mullighan. "Integrated Genetic and Epigenetic Analysis of Childhood Acute Lymphoblastic Leukemia Reveals a Synergistic Role for Structural and Epigenetic Lesions In Determining Disease Phenotype." Blood 116, no. 21 (November 19, 2010): 537. http://dx.doi.org/10.1182/blood.v116.21.537.537.
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Abstract Abstract 537 Acute lymphoblastic leukemia (ALL), the commonest childhood malignancy, is characterized by recurring gross and submicroscopic structural genetic alterations that contribute to leukemogenesis. Disordered epigenetic regulation is a hallmark of many tumors, and while analysis of DNA methylation of limited numbers of genes or ALL samples suggests epigenetic alterations may also be important, a large-scale integrative genome-wide analysis evaluating DNA methylation in ALL has not been performed. Here, we report an integrated epigenomic, transcriptional and genetic analysis of 167 childhood ALL cases, comprising B-progenitor ALL with hyperdiploidy (N=26), ETV6-RUNX1 (N=27), TCF3-PBX1 (N=9), BCR-ABL1 (N=19), rearrangement of MLL (MLLr) (N=20), rearrangement of CRLF2 (N=11, CRLF2r), deletion of ERG (N=11), miscellaneous or normal karyotype (N=14), and T-lineage ALL (N=30), including 4 MLLr cases and 8 cases with early T-cell precursor immunophenotype. Genome-wide profiling of structural DNA alterations was performed for all cases using Affymetrix 500K and SNP 6.0 arrays. Affymetrix U133A gene expression profiling data was available for 154 cases. Genome-wide methylation profiling was performed using the HELP microarray assay, which measures methylation at approximately 50,000 CpGs distributed among 22,722 Refseq promoters. Methylation data was compared to that of normal pro-B (CD34+CD19+sIg-), pre-B (CD34-CD19+sIg-) and mature B (CD34-CD19+sIg+) cells FACS-sorted from bone marrow of 6 healthy individuals. Unsupervised hierarchical clustering of the top 4043 most variable methylation probesets identified 9 B-ALL clusters with significant correlation to specific genetic lesions including ETV6-RUNX1, MLLr, BCR-ABL1, CRLF2r, TCF3-PBX1 and ERG deletion. T-ALLs and hyperdiploid B-ALLs also defined specific DNA methylation clusters. Supervised analysis including limma and ANOVA identified distinct DNA methylation signatures for each subtype. Notably, the strength of these signatures was subtype dependent, with more differentially methylated genes observed in ALL cases with genetic alterations targeting transcriptional regulators (e.g. ETV6-RUNX1 and MLLr) and fewer genes in cases with alterations deregulating cytokine receptor signaling (e.g. CRLF2r). Aberrant DNA methylation affected specific and distinct biological processes in the various leukemia subtypes implicating epigenetic regulation of these pathways in the pathogenesis of these different forms of ALL (e.g. TGFB and TNF in ERG deleted leukemias; telomere and centriole regulation in BCR-ABL1 ALL). Aberrantly methylated genes were also enriched for binding sites of known or suspected oncogenic transcription factors that might represent cooperative influences in establishing the phenotype of the various B-ALL subtypes. Most importantly, an integrated analysis of methylation and gene expression of these ALL subtypes demonstrated striking inversely correlated expression of the corresponding gene transcripts. The methylation signatures of each subtype exhibited only partial overlap with those of normal B cells, indicating that the signatures do not simply reflect stage of lymphoid maturation. In a separate approach, we discovered that 81 genes showed consistent aberrant methylation across all ALL subtypes, including the tumor suppressor PDZD2, HOXA5, HOXA6 and MSH2. Inverse correlation with expression was confirmed in 66% of these genes. These data suggest the existence of a common epigenetic pathway underlying the malignant transformation of lymphoid precursor cells. Integrative genetic and epigenetic analysis revealed hypermethylation of genes on trisomic chromosomes that do not show increased expression, suggesting that epigenetic silencing may control genes within amplified regions and explain why only selected genes are overexpressed. Finally, analysis of individual genes targeted by recurring copy number alterations in ALL revealed a subset of genes also targeted by abnormal methylation, with corresponding changes in gene expression (e.g. ERG, GAB1), suggesting that such genes are inactivated far more frequently than suggested by genetic analyses alone. Collectively, the data support a key role of epigenetic gene regulation in the pathogenesis of ALL, and point towards a scenario where genetic and epigenetic lesions cooperatively determine disease phenotype. Disclosures: No relevant conflicts of interest to declare.
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Willman, Cheryl L., Huining Kang, Richard C. Harvey, I.-Ming Chen, Charles Mullighan, James R. Downing, Mary V. Relling, et al. "Use of Genomic Technologies to Identify Novel Genetic Abnormalities and Therapeutic Targets In Acute Lymphoblastic Leukemia." Blood 112, no. 11 (November 16, 2008): sci—8—sci—8. http://dx.doi.org/10.1182/blood.v112.11.sci-8.sci-8.
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Abstract With the progressive intensification of chemotherapy, the majority of children with ALL now achieve long-term survival. In parallel, a number of molecular subtypes of ALL have been identified that are associated with treatment outcomes, which are either excellent (TEL-AML1 or trisomy of chromosomes 4, 10, and 17), intermediate (MLL rearrangements or E2a-PBX), or very poor (BCR-ABL or hypodiploidy). Yet, the underlying genetic abnormalities in the majority of children with ALL, such as those with “high-risk” disease who remain resistant to current therapies, remain to be discovered. Supported by the NCI SPECS and TARGET Initiatives, the Children’s Oncology Group (COG), and The Leukemia & Lymphoma Society, we are using comprehensive genomic technologies (i.e., expression profiling, genome-wide analyses of DNA copy number abnormalities [CNAs] and germline polymorphisms, and direct gene sequencing) to develop molecular classifiers for outcome prediction that can be used to discover novel underlying genetic abnormalities and therapeutic targets in ALL. Our work has focused on a cohort of 220 children with “high-risk” ALL registered to COG Trial 9906. Using supervised learning methods on gene expression profiles, molecular classifiers predictive of relapse-free survival (RFS) and minimal residual disease (MRD) at end-induction have been developed. A 38-gene molecular risk classifier predictive of RFS (MRC-RFS) can distinguish two groups of high-risk ALL patients with different relapse risks: low (4 yr RFS: 81%, n=109) vs. high (4 yr RFS: 50%, n=98) (P< 0.0001). In multivariate analysis, the best predictor combines MRC-RFS and end-induction flow MRD, classifying children into low- (87% RFS), intermediate- (62% RFS), or high-risk (29% RFS) groups (P<0.0001). A 21-gene molecular classifier predictive of MRD can effectively substitute for end-induction MRD, yielding a combined classifier that similarly distinguishes three risk groups at pre-treatment (low: 82% RFS; intermediate: 63% RFS; and high: 45% RFS) (P< 0.0001). This combined molecular classifier was further validated on an independent cohort of 84 children with high-risk ALL registered to COG Trial 1961 (P = 0.006). Using unsupervised clustering methods, 8 distinct cluster groups based on gene expression were identified, 6 of which were entirely novel. Two of the novel clusters were associated with strikingly different outcomes (95% 4-year RFS vs 20% 4-year EFS). Novel underlying genetic abnormalities and genes that may represent novel therapeutic targets have been identified in each of these clusters. Interestingly, children of Hispanic ethnicity were disproportionately represented in the poorest outcome clusters. CNAs were revealed in genes regulating B lymphoid development in 50.2% of cases (PAX5 in 30.7% and IKZF1 in 24.9%). In addition, recurring CNAs were detected in a number of other genes known to play roles in transformation, including CDKN2A/B, RB1, BTG1, IL3RA, NRAS, KRAS, NR3C2, and ERG. CNAs in IKZF1, EBF, and BTLA were strongly associated with the poorest outcome clusters defined by gene expression profiling. Deletion of IKZF1 was particularly associated with negative outcome (p=0.002). These ongoing studies demonstrate that molecular classifiers can be used to distinguish distinct prognostic groups within high-risk ALL, significantly improving risk classification schemes and the ability to prospective identify children who will respond to or fail current therapies. These classifiers are now being integrated into the design of COG clinical trials. The discovery of novel cluster groups and underlying genetic abnormalities is being exploited to develop new therapeutic targets for this disease.
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Clappier, Emmanuelle, Wendy Cuccuini, Anna Kalota, Antoine Crinquette, Jean-Michel Cayuela, Willem A. Dik, Anton W. Langerak, et al. "A New Subtype of T-Cell Acute Leukemia in Very Young Children Is Defined by a Translocation Targeting the C-MYB Oncogene, and a Specific Gene Expression Signature." Blood 110, no. 11 (November 16, 2007): 982. http://dx.doi.org/10.1182/blood.v110.11.982.982.
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Abstract Cytogenetics and expression studies have pointed out an increasing number of oncogenes in T-cell acute lymphoblastic leukemias (T-ALL), demonstrating the complexity of T-ALL oncogenesis and the requirement for a network of cooperative genomic events. Here, we identified two types of genomic alterations involving the C-MYB locus at 6q23 in human T-ALL, both associated with transcriptional deregulation. First, using a TCRB-based FISH screening, we found a new reciprocal translocation, t(6;7)(q23;q34), that juxtaposed the TCRB and C-MYB loci (n=6 cases). Breakpoints were fully characterized at the molecular level in 3 cases. Second, a genome wide copy-number analysis by array-CGH (customized 4K BAC/PAC arrays) identified short cryptic duplications which always include the C-MYB gene (MYBdup, n=11 out of 80 primary pediatric and adult T-ALL, 14%). Somatic origin of the genomic gain was demonstrated using paired leukemic and remission samples. The minimal region of gain was mapped to 230 Kb using 244K oligonucleotide a-CGH, and fiber-FISH demonstrated tandem duplication. Interestingly, these genomic events are reminiscent of the frequent myb retroviral insertions in murine leukemia. Expression analysis by RQ-PCR and microarrays data showed stronger C-MYB expression in the MYB-rearranged cases compared to other T-ALLs and normal controls (thymus, BM, and PBL). Moreover, allele-specific approaches showed a dramatically skewed allele expression in the TCRB-MYB cases, suggesting that the translocation-driven deregulated expression overcomes a cellular attempt to downregulate C-MYB. Considering that the C-MYB transcription factor has been involved at several key steps throughout normal thymic differentiation, these data strongly suggest that deregulation of C-MYB due to genomic events is oncogenic in T-ALL. Integrated analysis of clinical, genomic, and large-scale gene expression data showed that the MYB translocation defines a new T-ALL subtype. Strikingly, 5 out of 6 patients with the translocation had a very young age for T-cell leukemia (1.1, 1.3, 1.8, 2.5, and 2.9 year-old; median 2.2 versus 9.4 year-old for 355 pediatric T-ALL in the FRALLE 93/2000 pediatric trials, p<10−4). Gene expression profiling showed that these cases cluster in a new branch distinct from other subtypes and are characterized by a specific cell cycle/mitosis signature. By contrast, the MYBdup alteration was associated to previously defined T-ALL subtypes (TAL, TLX1/HOX11, TLX3/HOX11L2, MLL, CALM-AF10, and immature cases), suggesting an additional oncogenic event. NOTCH1 activating mutations and p16/CDKN2A/ARF deletions were found in both the TCRB-MYB and MYBdup cases, according to multi-step T-cell oncogenesis. This work is the first clear demonstration of the recurrent involvement of the C-MYB locus in a human neoplasia. Moreover the t(6;7) translocation defines a new T-ALL subtype associated with very young age for T-cell leukemia and a distinct biology.
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Schmitz, Maike, Paulina Mirkowska, Petra Breithaupt, Barbara Meissner, Gunnar Cario, Andre Schrauder, Felix Niggli, et al. "Leukemia-Initiating Cells Are Frequent in Very High Risk Childhood Acute Lymphoblastic Leukemia and Give Rise to Relatively Stable Phenotypes in Immunodeficient Mice." Blood 114, no. 22 (November 20, 2009): 86. http://dx.doi.org/10.1182/blood.v114.22.86.86.
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Abstract Abstract 86 High-risk acute lymphoblastic leukemia (ALL) has been proposed to arise from a limited immature leukemia-initiating cell (LIC) compartment that may confer treatment resistance. Recent reports challenge this view. Using syngeneic leukemia mouse models, higher frequencies of LIC were detected. Moreover, sorted ALL subpopulations with different degree of lineage maturation could reconstitute the same leukemia phenotype effectively in ALL xenograft models. We have established a xenotransplantation model of primary B-cell precursor ALL cells in immunodeficient NSG mice (NOD/SCID strain with additional IL-2 receptor common gamma chain deletion), starting from carefully selected very high-risk (VHR) and standard-risk (SR) patients as defined by minimal residual disease (MRD)-based risk stratification criteria on the ALL-BFM-2000 treatment protocol. Median time to engraftment in NSG mice was similar for 11 VHR and 8 SR patients with 8 (range 6–18) weeks for VHR patients and 12 (range 4–32) weeks for SR patients. The comparison of the immunophenotype of ALL cells at diagnosis and after up to three passages in NSG mice revealed a concordance of 90% using 8 cell surface markers that are most commonly included in the diagnostic flow cytometry panels, with no differences between passage rounds. Discordant observations included loss of CD22 expression in 6/19 cases and low levels of CD13 co-expression in 4/19 cases in xenografted samples. To get insight into the genetic stability upon serial passages of ALL cells in NSG mice, we performed Affymetrix Genome-Wide Human SNP Array 6.0 analyses and transcriptional profiling to compare copy number alterations (CNAs) in diagnostic ALL samples and after serial transplantation in NSG mice. Three VHR ALL cases having data from two passages in NSG available were included in a pilot analysis, comparing leukemic DNA to the patients' constitutional DNA. A small number of focal CNAs (>100-<1000 Kb) were detected, most of them previously reported in larger studies. In all cases, CNAs corresponding to expected immunoglobulin and/or T-cell receptor gene rearrangements were seen. In one case, 5 out of 12 CNAs and 2 copy-neutral losses of heterozygosity were maintained after two passages in mice. In a second case with high hyperdiploidy, 15 out of 17 CNAs were stable. In both samples, small deletions in 9p21.3 were maintained. A third case with a t(17;19) translocation lost all 3 CNAs present at diagnosis, but acquired a small deletion in 9p21.3. Between 1 and 6 CNAs were acquired after two passages in mice. An analysis of copy number ratios suggested that selection of subclones had occured. To characterize the LIC compartment of ALL cells in VHR ALL we performed limiting dilution experiments by orthotopic intrafemoral xenotransplantation of primary ALL cells in NSG mice. Hundred thousand unsorted ALL cells generated leukemia in NSG mice in 5/5 cases, and 100 cells were sufficient for engraftment (in 4/5 cases) without conditioning, despite the xenograft barrier. Secondary transplantations demonstrated conserved self-renewal properties. Collectively, our data indicate that this xenograft model of treatment-resistant ALL is remarkably stable with successive passages in mice. This approach will constitute a powerful model to scrutinize clonal evolution of cytogenetic lesions in resistant leukemia and to evaluate new therapeutic options in the fraction of patients that is expected to be recruited in future phase I/II studies. Furthermore, our data indicate that a large proportion of the ALL cell population can recapitulate the leukemia phenotype and thus retain LIC properties. These data also support the use of unsorted populations for therapeutic modeling in NSG mice. Disclosures: No relevant conflicts of interest to declare.
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Haferlach, Claudia, Wencke Walter, Manja Meggendorfer, Anna Stengel, Constance Baer, Stephan Hutter, Niroshan Nadarajah, Heiko Müller, Wolfgang Kern, and Torsten Haferlach. "Benchmarking of Whole Genome Sequencing (WGS) and Whole Transcriptome Sequencing (WTS) As Diagnostic Tools for the Genetic Characterization of Acute Myeloid Leukemia (AML) and Acute Lymphoblastic Leukemia (ALL) in Adults." Blood 138, Supplement 1 (November 5, 2021): 273. http://dx.doi.org/10.1182/blood-2021-150260.
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Abstract Background: In AML and ALL the application of WHO classification and ELN guidelines requires a combination of cytogenetics and targeted sequencing for specific mutations to determine the diagnostic and prognostic subgroup. WGS and WTS have emerged as comprehensive techniques that allow the simultaneous analysis and identification of all genetic alterations in a single approach with possible turnaround times of 1 week. Aim: Evaluate the accuracy of WGS and WTS in providing all relevant genetic information in a clinical setting. Patients and Methods: The cohort comprised 738 AML, 293 BCP-ALL and 124 T-ALL. The diagnosis was established following WHO guidelines. WGS (100x, 2x151bp) and WTS (50 Mio reads, 2x101bp) were performed on a NovaSeq instrument. Variants were called with Strelka2, Manta and GATK using a tumor w/o normal pipeline, fusions with Arriba, STAR-Fusion and Manta. Results: The combination of WGS and WTS detected all chromosomal and molecular abnormalities in the AML and ALL cohorts relevant for disease stratification and prognostication as identified by chromosome banding analysis (CBA) and targeted panel sequencing (TPS). A very high concordance between CBA and WGS was revealed for the detection of balanced structural variants (SV) with the added benefit of WGS to also detect cytogenetically cryptic rearrangements (i.e.: ETV6-MN1, NUP98-KDM5A), which all were confirmed either by FISH or RT-PCR. Fusion calling by WTS identified 96% of the WHO subtype defining rearrangements and detected 20 additional fusion transcripts relevant for disease stratification (e.g. EP300-ZNF384, TCF3-HLF) including 9 fusion transcripts that led to prognostic reassignment or could serve as a potential treatment target. Breakpoints of unbalanced SV can occur in repetitive sequences of the genome, hampering the detection by WGS. However, adding copy number alteration (CNA) calls to the analyses allows also reliable identification of unbalanced SV. WGS outperformed CBA in cases with insufficient in vitro proliferation due to suboptimal pre-analytics (i.e. longer transport time) and identified 36 chromosomal aberrations in 12 cases with CBA not evaluable. WGS's independence of in vitro cell proliferation was most impactful in ALL: 40 T-ALL cases showed a normal karyotype according to CBA. WGS detected SVs in 16 (40%) and CNAs in 20 (50%) of these cases, confirming the normal karyotype for only 9 samples. In the BCP-ALL cohort, CNV analysis identified 29 low hypodiploid and 16 high hyperdiploid karyotypes, 6 of which were missed by CBA. Due to the higher resolution and unrestricted, genome-wide assessment, WGS detected relevant gene deletions (RB1, ERG, PAX5, CDKN2A, IKZF1, ETV6, BTG1) in 59% of ALL cases, providing additional diagnostic and prognostic information. In the AML cohort CBA and WGS detected 795 CNA concordantly. In addition WGS called 54 CNA with size 1-5 MB (below the detection limit of CBA), i.e. 3 BCOR deletions in inv(3)(q21q26) cases and 67 CNA with size > 5 MB, which were missed by CBA. 35 CNA were missed by WGS due to small clone sizes (median 6% as determined by FISH). WGS detected copy neutral loss of heterozygosity (CN-LOH) in AML most frequently on 21q (n=17), 4q (n=15), 13q (n=15), 11q (n=13) and in T-ALL on 9p (n=19), mostly encompassing CDKN2A/B deletions. Expression profiling provided additional diagnostic information for 57 ALL cases (41 BCR-ABL1-like, 16 DUX4 rearranged) that can only insufficiently be obtained by WGS or CBA. WGS reliably detected all gene mutations with a VAF > 15% (n = 647) identified by TPS encompassing especially all mutations in genes relevant for WHO diagnosis and prognostication. 26/171 mutations with a VAF < 15% were missed by WGS. Evaluation of WGS data for 121 genes recurrently mutated in hematologic neoplasms revealed an additional 2 mutations per sample on average (range: 0-9) which might qualify as targets for therapy. Conclusions: WGS and WTS provide all necessary genetic information to accurately determine the diagnostic and prognostic subgroup according to WHO and ELN guidelines in AML and ALL. Compared to today's gold standards, these novel methods provide a comprehensive genome wide characterization with higher resolution that directly identifies genes of impact, offering the basis for targeted treatment selection and monitoring of residual disease. Both can be implemented with automated analysis pipelines, consequently reducing time and error rates. Figure 1 Figure 1. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.
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Mullighan, Charles G., Ryan Morin, Jinghui Zhang, Martin Hirst, Yongjun Zhao, Chunhua Yan, Richard Finney, et al. "Next Generation Transcriptomic Resequencing Identifies Novel Genetic Alterations in High-Risk (HR) Childhood Acute Lymphoblastic Leukemia (ALL): A Report From the Children's Oncology Group (COG) HR ALL TARGET Project." Blood 114, no. 22 (November 20, 2009): 704. http://dx.doi.org/10.1182/blood.v114.22.704.704.
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Abstract Abstract 704 Relapsed ALL is a leading cause of childhood cancer death, and the biologic factors responsible for relapse are poorly understood, particularly in cases lacking sentinel chromosomal alterations. Recent studies from the Children's Oncology Group high risk ALL TARGET (Therapeutically Applicable Research to Generate Effective Targets) project that used genome-wide profiling of DNA copy number alterations and candidate gene resequencing have identified novel biomarkers of relapse (IKZF1 alteration) and therapeutic targets (JAK mutation). As a complementary approach to identify novel genomic alterations, we used second generation sequencing technology to sequence the tumor transcriptome of three cases from the COG P9906 high risk (HR) B-precursor ALL trial. The selected cases had previously been profiled by high resolution SNP and gene expression arrays and candidate gene resequencing, and lacked known sentinel chromosomal rearrangements. Each case bore features previously associated with poor treatment outcome: a gene expression profile (GEP) similar to that of BCR-ABL1 positive ALL (all cases), deletion or mutation of IZKF1 (two cases), and JAK mutation (JAK2 R867Q, one case). cDNA libraries were generated from poly-A enriched RNA and 36-50 base paired-end sequencing performed using the Illumina Genome Analyzer. Sequence alignment, variant detection and fusion transcript identification were performed using custom scripts and multiple published reference alignment and de-novo assembly algorithms. A total of 115-127 million total and 93-97 million mapped, unique reads were obtained per case. The average depth of coverage of Refseq exons ranged from 25- to 39-fold. A minimum of 5 putative fusion transcripts were identified per case, some of which were known from prior transcriptome sequencing to be recurring false positives. However, a novel transcript with an in-frame fusion of exon 9 of the striatin gene STRN3 to exon 18 of JAK2 (STRN3-JAK2) was identified in one case, and confirmed by RT-PCR and direct Sanger sequencing. Fusion of NUP214 to ABL1 was identified in a second case and also confirmed by direct sequencing. The NUP214-ABL1 rearrangement has previously only been identified in T-lineage ALL. In this case, the translocation was accompanied by amplification of the NUP214-ABL1 region at 9q. RT-PCR screening of an additional 60 high-risk ALL cases with GEP data suggestive of kinase alteration identified an additional two cases with NUP214-ABL1 fusion, each of which was accompanied by NUP214-ABL1 amplification. These two novel fusion transcripts are predicted to result in aberrant kinase signaling, and are candidates for novel therapeutic intervention. Both occurred in ALLs with a BCR-ABL1-like GEP that lacked known JAK mutations, suggesting that additional novel activating kinase mutations can be discovered via detailed sequence analysis of the 50% of BCR-ABL1-like ALLs that do not have JAK mutations. Aberrant splice variants and truncated isoforms arising from DNA copy number alterations, including internal deletions of PAX5 and truncating deletions of BTG1 were also identified using the transcriptome sequencing data. In addition, these data identified over 400 candidate non-synonymous single nucleotide and insertion/deletion variations in each patient. Known mutations involving PAX5, IKZF1 and JAK2 were robustly identified. Whole genome sequencing of matched normal DNA is underway to remove germline variation from the list of putative variants, and transcriptomic sequencing of additional cases of HR childhood ALL are being performed. Together, these data indicate that transcriptomic sequencing is a powerful method to identify novel genetic alterations in ALL, and may be used to identify novel targets for therapeutic intervention. Disclosures: No relevant conflicts of interest to declare.
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van der Sligte, Naomi E., Frank JG Scherpen, Arja ter Elst, Victor Guryev, Frank N. van Leeuwen, and Evelina SJM de Bont. "Effect of IKZF1 Deletions on Signal Transduction Pathways in Philadelphia Chromosome-Negative B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL)." Blood 124, no. 21 (December 6, 2014): 3770. http://dx.doi.org/10.1182/blood.v124.21.3770.3770.
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Abstract Overall survival rates for children with Acute Lymphoblastic Leukemia (ALL), the most common type of leukemia in children, are approaching 90% (Mullighan 2013). In the past 5 years, genome wide approaches, studying DNA copy number alterations in ALL, have increased the list of risk stratifications and included IKZF1 deletions to the list of unfavorable prognostic factors. IKZF1 deletions can be identified in approximately 70% of the Philadelphia chromosome-positive (Ph+) and in 15% of the Philadelphia chromosome-negative (Ph-) children with ALL and are associated with an increased risk on relapse and a decreased overall survival (Mullighan 2009, Kuiper 2010, van der Veer 2013). IKZF1 deletions observed in B-cell precursor ALL (BCP-ALL) are typically mono-allelic, resulting in the expression of a dominant-negative isoform (Mullighan 2008). A unique gene expression signature was revealed in IKZF1 deleted BCP-ALL patients, characterized by the downregulation of genes regulating B-cell lineage development and DNA repair upon DNA damage response genes and upregulation of cell cycle/apoptosis genes, JAK/STAT signaling and stem cell self-renewal (Iacobucci 2012). At the level of signal transduction, western blot analysis showed that IKZF1 deletions resulted in B cell receptor (BCR) signaling defects and upregulation of phospho-STAT5 in 2 and 4 Ph+ ALL patients, respectively (Trageser 2009, Iacobucci 2012). However, effects of IKZF1 deletions on signaling pathways in Ph-ALL have not been extensively studied. Pediatric Ph- BCP-ALL patients (N=46) were screened for IKZF1 deletions by multiplex ligation-dependent probe amplification analysis. A total of 15 patients carried an IKZF1 deletion. We performed a kinase activity profile (IKZF1 deleted N=15, IKZF1 wild type N=31) as well as a human phospho-proteome array (IKZF1 deleted N=11, IKZF1 wild type N=17) to elucidate active signal transduction pathways. Kinase activity profiling is a potent high throughput technique using peptides of 11 amino acids in length representing known human phosphorylation sites. In the obtained kinase activity profiles we studied differences in peptide phosphorylation intensities. 37 peptides were differentially expressed between IKZF1 deleted and wild type pediatric Ph- BCP-ALL patients (P ≤ 0.05, Figure 1). From these 37 peptides we first examined peptides derived from proteins involved in the BCR signaling and STAT5. On the kinome array, peptides derived from Src_Y352, CBL_Y371, SYK_Y526, PLCg2_Y753, PLCg2_Y1217, STAT5a_S780, STAT5a_Y694, and STAT5b_Y679 were present but showed no differences in phosphorylation intensities between IKZF1 deleted and IKZF1 wild type Ph- BCP-ALL samples. Neither could we detect differences in phosphorylation intensities of Fyn_Y420, Lyn_Y397, Src_Y419, STAT5a_Y694, STAT5b_Y699, and STAT5a/b_Y694/Y699 using human phospho-proteome arrays, confirming the kinome profiling results. We did, however observe a distinct kinome profile upon hierarchical clustering of 46 BCP-ALL primary samples, based on the 37 peptides identified by t-test (Figure 1). IKZF1 deleted cases showed high phosphorylation of 14 peptides including peptides derived from Akt1_Y326 and Cav1_Y14 (Figure 1). Loss of IKZF1 has been associated with glucocorticoid resistance. Since Akt inhibition reverses glucocorticoid resistance in T cell ALL (Piovan, 2013) and Caveolin 1 is involved in focal adhesion and chemoresistance (Faggi, 2014) we hypothesize that Akt and Caveolin 1 inhibition might convert glucocorticoid resistance in Ph-IKZF1 deleted pediatric BCP-ALL, which requires further investigation. Together, we conclude that kinome profiling revealed a distinct peptide phosphorylation pattern for IKZF1 deleted pediatric Ph- BCP-ALL including novel therapeutic targets. Disclosures No relevant conflicts of interest to declare.
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Gruber, Tanja A., Jing Ma, Sanne Noort, Yu Liu, Michael P. Walsh, Stephanie Nance, Yanling Liu, et al. "Integrative Analysis of Pediatric Acute Leukemia Identifies Immature Subtypes That Span a T Lineage and Myeloid Continuum with Distinct Prognoses." Blood 134, Supplement_1 (November 13, 2019): 918. http://dx.doi.org/10.1182/blood-2019-127411.
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Acute myeloid leukemia (AML) comprises a heterogeneous group of malignancies that are linked by the presence of blasts displaying morphologic and immunophenotypic features of myeloid cell differentiation. With the development of genome-wide gene expression profiling (GEP), array-base comparative genomic hybridization methodologies, and next generation sequencing technologies, the field has gained a greater understanding of the molecular features of this malignancy. Several pathologic lesions have been found to have prognostic implications contributing to a continuous refinement of risk stratification in the context of modern therapy. Recently, the Children's Oncology Group (COG)-National Cancer Institute (NCI) TARGET AML initiative molecularly characterized 993 pediatric AML cases including 197 specimens that underwent comprehensive whole genome sequencing. Of these, 94 carried one of three oncogenic fusions known to be strong drivers of leukemogenesis: RUNX1-RUNX1T1, CBFB-MYH11 and KMT2A rearrangements (KMT2Ar). Among all the alterations detected only ten occurred in more than 5% of subjects, all of which had been previously described. This suggested that low-frequency molecular subsets may exist that require larger cohorts to fully elucidate. To address this limitation, we selected 122 pediatric AML specimens that lacked RUNX1-RUNX1T1, CBFB-MYH11 and KMT2Ar by clinical testing for whole genome (WGS), exome (WES) and RNA (RNAseq) sequencing to enrich for cases that carry low-frequency events. GEP coupled with somatic mutation calls and outcome data were utilized to identify distinct molecular subtypes with prognostic implications. Structural variations, copy number alterations, single nucleotide variations and indels were determined by our established pipelines, as well as an evaluation for regulatory rearrangements driving oncogene overexpression through enhancer hijacking. In addition to known AML somatic mutations and rearrangements in genes such as CEBPA, GATA2, NPM1, WT1, FLT3, NRAS, KRAS, ETV6, Cohesin, NUP98 and KAT6A, we identified rare novel events in known oncogenic drivers. These include a GATA2-ITD as well as the repositioning of a distal MYC enhancer to ectopically activate either the MECOM or BCL11B loci. Interestingly, several AML cases carrying loss of function mutations in polycomb repressive complex 2 (PRC2) genes were found to resemble an early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) GEP by gene set enrichment analysis. ETP-ALL exhibits aberrant expression of stem cell and myeloid markers and has been shown to have a GEP consistent with transformation of a stem cell progenitor. Further, mixed phenotype acute leukemias (MPAL) with T and myeloid lineage characteristics have been previously suggested to be in this spectrum of immature leukemias. We therefore hypothesized that these PRC2-mutated AML cases represented the myeloid end of this continuum. To provide global transcriptional context to these ETP-like AMLs and evaluate a comprehensive cohort encompassing a range of pediatric myeloid malignancies, we integrated results from previously published AML, MPAL, acute megakaryoblastic Leukemia (AMKL), and ETP-ALL datasets that had RNAseq and either WES or WGS available for a total of 436 cases. t-SNE visualization using a 381 gene list derived from the top 100 most variably expressed transcripts within each cohort revealed a clear molecular classifier identifying groups that had consistent mutational compositions and disease outcomes but were agnostic of immunophenotype. This approach allowed the distinction of 63 ETP-like cases comprising a mixture of AML, MPAL, and ETP-ALL leukemias which fell into two subgroups distinguished by FLT3-ITD and PRC2 alterations. Irrespective of treatment approach, FLT3-ITD positive ETP-like leukemias enjoyed a favorable outcome whereas those with PRC2 mutations had a poor prognosis. Our data support a refined classification of pediatric myeloid malignancies based on molecular determinants that can be used for risk stratification in therapeutic trials. Disclosures Gruber: Bristol-Myers Squibb: Consultancy. Rubnitz:AbbVie: Research Funding. Reinhardt:Jazz: Other: Participation in Advisory Boards, Research Funding; CSL Behring: Research Funding; Novartis: Other: Participation in Advisory Boards; Roche: Research Funding. Locatelli:bluebird bio: Consultancy; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zwaan:Roche: Consultancy; Servier: Consultancy; Daiichi Sankyo: Consultancy; Novartis: Consultancy; Sanofi: Consultancy; Pfizer: Consultancy, Research Funding; BMS: Research Funding; Celgene: Consultancy, Research Funding; Jazz pharmaceuticals: Other: Travel support; Janssen: Consultancy; Incyte: Consultancy.
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Miles, Rodney R., Jonathan Downie, Mona S. Jahromi, Deepa Joshi, Vladimir Rodic, Markus Muschen, Jun J. Yang, et al. "VPREB1 Deletions Occur Independent of Lambda-Light Chain Rearrangement and Predict Worse Outcome In Pediatric Acute Lymphoblastic Leukemia (ALL)." Blood 116, no. 21 (November 19, 2010): 273. http://dx.doi.org/10.1182/blood.v116.21.273.273.
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Abstract Abstract 273 VPREB1 deletions occur in 1/3 of pediatric ALL (pALL) patients, but have been considered a normal consequence of λ light chain rearrangement due to VPREB1's location among λ chain variable region genes. Recently, VPREB1 deletions were reported in high risk pALL samples with BCR-ABL1-like gene expression (Den Boer 2009) and VPREB1 under-expression was found in a 38-gene signature associated with relapse (Kang 2010). We characterized VPREB1 deletions to determine if they are part of normal λ light chain rearrangement or represent a bona fide copy number alteration (CNA) that predicts worse outcome in pALL. We first used the genome-wide Molecular Inversion Probe (MIP) 330K Cancer Panel (Affymetrix) to identify VPREB1 deletions in a cohort of 52 pALL patients from the University of Utah (n=47 Precursor B-cell (Pre-B), 4 Precursor T-cell (Pre-T), 1 relapse Pre-B ALL). We found focal VPREB1 deletions in 25% (n=12/48) of Pre-B ALL and no (n=0/4) Pre-T ALL patients. We also identified a distinct and often homozygous deletion (14 kb in length) located ≂f 78 kb upstream of VPREB1 that could represent a potential enhancer region; this possible enhancer deletion was seen in 16.7% (n=8/48) of Pre-B and none of the Pre-T ALL patients. 50% of Pre-B ALL samples had at least one deletion of B-cell developmental gene (SPI1, IKZF1, BCL11A, TCF3, EBF1, PAX5, FOXP1, LEF1, VPREB1, BLNK), and of these, half (n=12/24) had deletion only of VPREB1 or its possible enhancer. We next sorted blood leukocytes from 10 healthy volunteers for quantitative PCR (qPCR) analysis on κ V-J junction (deletion=attempted κ rearrangement), λ V-J junction (deletion=attempted λ rearrangement), LOC96610 and PRAME (distal to VPREB1, among λ-light chain variable genes), VPREB1, and VPREB1 enhancer (proximal to VPREB1, among λ-light chain variable genes). As predicted for normal light chain rearrangement, we observed no deletions in monocytes; 100% of κ-sorted B-cells had κ V-J deletions without lambda deletions; and 100% of λ-sorted B-cells had both κ V-J and λ V-J deletions. We also analyzed 10 mature B-cell (Burkitt) lymphoma samples and observed VPREB1 deletions only in lambda-expressing samples with both κ and λ V-J deletions, and deletions always extended contiguously downstream to λ chain V-J region. We re-analyzed 30 Pre-B ALL samples (Utah cohort) and 11 Pre-B ALL cell lines by qPCR. Results validated MIP CNA patterns and differed from normal sorted B-cell and mature B-cell (Burkitt) results, displaying several types of rearrangements: 1) No light chain rearrangements with no VPREB1/enhancer deletion (n=5/30 [17%] clinical samples and 2/11 [18%] Pre-B cell lines), 2) Normal light chain (κ or κ/λ) rearrangement with no VPREB1/enhancer deletion (n=5/30 [17%] samples and 6/11 [55%] cell lines), 3) Normal light chain (κ or κ/λ) rearrangements with contiguous VPREB1/enhancer deletion (n=1/30 [3%] samples and no cell lines), 4) λ-light chain rearrangement with non-contiguous VPREB1/enhancer deletion (n=13/30 [43%] samples and 2/11 [18%] cell lines), and 5) No λ light chain rearrangement and focal VPREB1/enhancer deletion (n=6/30 [20%] samples and 1/11 [9%] cell lines). In the Utah Pre-B ALL cohort (n=47), VPREB1/enhancer deletions were significantly associated (Fisher's Exact Test, 2-tailed) with Hispanic ethnicity (p= 0.013), relapse (p= 0.027), death (p=0.026), and Day 14 M2 Marrow (5-25% blasts; p=0.038). Finally, we examined the publicly available St. Jude Children's Research Hospital ALL dataset (NEJM 351:533) and found VPREB1 under-expression associated with higher LC50 (resistance) for DNR (p=0.04) and VCR (p=0.04), but not PRED or ASP. In summary, we have shown VPREB1 deletions occur outside the context of normal light chain rearrangement. Unlike physiologic λ rearrangements, VPREB1 deletions do not always extend to the λ V-J junction in pALL. We also identified a focal, homozygous deletion just proximal to VPREB1 that may affect a possible enhancer region for this B-cell developmental gene. VPREB1/enhancer deletions were the only B-cell developmental gene lost in 25% of our cohort, perhaps defining a new Pre-B ALL subtype. Finally, deletion of VPREB1 (and its possible enhancer) predict worse clinical outcome and VPREB1 down-regulation correlates with in vitro drug resistance. Further studies of VPREB1's function in pediatric ALL may improve our understanding of leukemogenesis and could further refine clinical risk stratification. Disclosures: No relevant conflicts of interest to declare.
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Hauer, Julia, Charles G. Mullighan, Estelle Morillon, Gary P. Wang, Julie Bruneau, Nicole Brousse, Arndt Borkhardt, et al. "Leukemia Prone B-Precursor Population in a p19ARF-/-RAG1-/- Mouse Model." Blood 114, no. 22 (November 20, 2009): 3971. http://dx.doi.org/10.1182/blood.v114.22.3971.3971.
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Abstract Abstract 3971 Poster Board III-907 Aberrant V(D)J-recombination may contribute to the oncogenic transformation of B-lymphoid precursor cells, suggesting that ongoing aberrant activity of recombinase activating gene (RAG) during lymphoid development is one of the potentially dangerous factors. Unexpectedly, deletions of RAG1 have been observed in a subset of cases of human B-progenitor acute lymphoblastic leukemia. Thus, the role of RAG1-mediated recombination in the pathogenesis of B-cell progenitor ALL remains unclear. The setting of RAG1-deficient severe combined immunodeficiency provides a unique opportunity to address this question. RAG1-deficient patients harbour an atypical CD34+CD19+ B-lymphoid precursor population in the bone marrow that is not present in healthy individuals. The role of these cells in the development of leukaemia has so far not been reported. To analyze the role of RAG1 deficiency in the pathogenesis of leukaemia, we analyzed mice that lacked expression of p19ARF (and were thus tumour prone) and RAG1. p19ARF-/- (A) mice develop predominantly solid tumours, and in a minority of cases, T-lineage leukemia/lymphoma. p19ARF-/-RAG1-/- (AR) mice spontaneously developed B-precursor leukaemia (CD19+IgM-) at an incidence of 30% within 52 weeks of follow up, indicating that the simultaneous loss of both p19ARF and RAG1 promotes the development of B-lineage leukemia. Array CGH analysis of the resulting leukemia using customized microarrays incorporating genome wide coverage and dense tiling of genes involved in B-cell development identified recurring aneuploidy of chromosomes 1, 5 and 14, but a notable absence of RAG1-mediated DNA copy number alterations that are observed in RAG-sufficient mouse models for ALL. In support of this, we identified a leukemia-prone Sca1+CD19+ precursor cell population in the bone marrow of AR mice, which was not present in A mice. Transplantation of AR haematopoietic precursor cells (Sca1+) into irradiated wild type recipients accelerated leukaemia incidence to 100% in contrast to transplantation of AR Sca1+CD19- population, which resulted in a decreased incidence of B-precursor cell leukaemia. The leukaemia prone AR Sca1+CD19+ population showed a high BrdU uptake with an abnormal cell cycle progression and seems to be committed to the B cell lineage, as colony forming assays demonstrated. Co-culture of the AR Sca1+CD19+ population with OP9delta1 cells failed to differentiate these cells into T-precursor cells, however ongoing proliferation of B-lineage cells was observed. Further characterisation of the RAG1-/- lymphoid precursor compartment revealed an equivalent Sca1+CD19+ population that was not leukaemia prone, and expressed p19ARF. This suggests that p19ARF is an important factor implied in tumour suppression in this B-precursor compartment. Here we show for the first time in a mouse model that RAG1 loss of function in combination with inactivation of p19ARF contributes to leucemogenesis via accumulation of a leukaemia prone B-precursor cell population in the bone marrow. Disclosures: No relevant conflicts of interest to declare.
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Zhang, Jinghui, Charles G. Mullighan, Richard C. Harvey, Kenneth E. Buetow, William L. Carroll, I.-Ming Chen, Meenakshi Devidas, et al. "Mutations in the RAS Signaling, B-Cell Development, TP53/RB1, and JAK Signaling Pathways Are Common in High Risk B-Precursor Childhood Acute Lymphoblastic Leukemia (ALL): A Report From the Children's Oncology Group (COG) High-Risk (HR) ALL TARGET Project." Blood 114, no. 22 (November 20, 2009): 85. http://dx.doi.org/10.1182/blood.v114.22.85.85.
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Abstract Abstract 85 Despite the overall favorable treatment outcome for childhood ALL, about 20% of patients still experience relapse. Moreover, many ALL cases lack sentinel chromosomal alterations, and the genetic factors contributing to leukemogenesis in these cases are poorly understood. Genome-wide profiling of DNA copy number alterations (CNA) coupled with focused candidate gene resequencing has identified novel genetic alterations that contribute to leukemogenesis and are associated with treatment outcome in ALL. However, large-scale analysis of somatic sequence mutations in ALL has not yet been performed. As part of the COG HR-ALL TARGET (Therapeutically Applicable Research to Generate Effective Targets) Project, 125 genes (selected based on recurrent CNAs, gene expression profiles and known cancer genes) were sequenced in 187 HR B-precursor ALL patients enrolled in COG P9906. The entire coding region and UTRs of each gene were sequenced and more than 98% of the targeted nucleotides have high quality sequence coverage. We found that somatic mutations are frequent in genes that encode for proteins involved in signal transduction, B-cell development and p53/RB signaling. A notable finding was the presence of somatic mutations resulting in constitutive activation of RAS signaling in at least 39% of the cohort. Seventy-three cases have at least one mutation in NRAS (30), KRAS (28), PTPN11 (9), FLT3 (7) and NF1 (6), including 7 patients with multiple mutations (KRAS and NRAS (3), FLT3 and NF1 (1), PTPN11 and FLT3 (1), PTPN11 and FLT3 (1), PTPN11 and NRAS (1), PTPN11 and KRAS (1)). There was no association between RAS pathway mutations and event-free survival or cumulative incidence of relapse in this HR patient cohort. Notably, RAS pathway mutations were uncommon in ALL cases with TCF3-PBX1 (1/22 cases) or MLL translocations (2/18 cases), but occurred frequently in cases lacking known sentinel cytogenetic lesions (68/145 cases, 47%, p<0.0001). Sequence mutations that are known or predicted to impair normal B-cell development were observed in at least 14% of the cohort (PAX5 (21), IKZF1 (7)), with confirmatory germline sequencing underway to clarify whether lymphoblast sequence alterations present in other B-cell development genes including BLK, ETV6, IKZF3, TCF3, RAG1, and BCL11A are somatic or germline. Sequence mutations disrupting TP53/RB1 signaling ((TP53 (10), RB1 (4), CDKN2A (4)) occurred in 10% of cases. Activating sequence mutations in JAK family members (JAK2 (16) and JAK1 (3)) were present in 10% of the cases. Of the 103 cases (55%) with at least one sequence mutation in these four (RAS signaling, B-cell development, p53/RB, and JAK) pathways, 30 have somatic mutations in multiple pathways. When both CNAs and sequence mutations are considered, 94% of the cases have lesions in at least one of the pathways and 31% of the cases have somatic alterations in all three of the B-cell development, RAS, and p53/RB1 signaling pathways, suggesting that aberrations in multiple pathways may be central to development of HR-ALL. Our results contrast sharply with known genetic alterations in T-cell ALL, where RAS pathway alterations are relatively uncommon (<10% of cases). PTEN somatic mutations and deletions occur in approximately 25% of cases of T-cell ALL, but we found no PTEN alterations in this cohort, suggesting differential activation of RAS and PI-3K signaling pathways in T-cell and HR B-precursor ALL. Validation of putative deleterious mutations in other sequenced genes (e.g., TBL1XR1, BLK, and CREBBP) is ongoing. These results from the TARGET COG HR-ALL Pilot Project confirm and extend prior knowledge of genetics of this subtype of ALL and point the way to new potential therapeutic strategies for this patient population. Disclosures: No relevant conflicts of interest to declare.
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Iacobucci, Ilaria, Anna Ferrari, Annalisa Lonetti, Cristina Papayannidis, Viviana Guadagnuolo, Emanuela Ottaviani, Mariachiara Abbenante, et al. "The Inactivation of the Tumor Suppressor Genes CDKN2A/ARF by Genomic Deletions Frequently Occurs and Worsens Prognosis In Adult BCR-ABL1 Positive Acute Lymphoblastic Leukemia (ALL) Patients." Blood 116, no. 21 (November 19, 2010): 3136. http://dx.doi.org/10.1182/blood.v116.21.3136.3136.
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Abstract Abstract 3136 Deletions of the 9p21 chromosomal region are frequent events for the development of a variety of cancers, including solid tumors and hematological malignancies, such as childhood ALL. This region contains the 40-kb region encoding the p16INK4a/CDKN2A (cyclin-dependent kinase inhibitor 2a) tumor suppressor gene and two other related genes, p14/ARF and p15INK4b/CDKN2B, all of which encode critical factors for the regulation of cell cycle and/or apoptosis. In order to explore the frequency and size of deletions affecting the 9p21 locus in adult BCR-ABL1-positive ALL, to determine the main mechanism of inactivation and to investigate the influence on the prognosis, 112 patients were analyzed: 82 (73%) de novo cases, 11 (10%) unpaired relapse cases, 19 (17%) diagnosis-relapse matched samples. The median age was 53 years (range: 18–76) and the median blast percentage was 90% (range, 18–99). Affymetrix single nucleotide polymorphism (SNP) arrays (GeneChip® Human Mapping 250K NspI and Genome-Wide Human SNP 6.0) were used to identify at a high resolution copy number changes on 9p21. PCR amplification and mutation screening of each exon by cloning and subsequent sequencing were also performed. Moreover, gene-expression profiling was performed (Affymetrix Human Exon 1ST Array) to draw a specific signature associated with deletions. At diagnosis, CDKN2A/ARF and CDKN2B genomic alterations were identified in 29% and 24% of patients, respectively. Deletions were monoallelic in the majority of cases (72%) with a median of 1,012 kb in size (range, 2.8–31,319 kb). In some of them, 43%, the minimal overlapping region of the lost area spanned only the CDKN2A/ARF/CDKN2B gene locus, but more often (57%) the loss was considerable larger and extended sometimes over the entire short arm eliminating a large number of genes. In contrast, cases with bi-allelic inactivation were 28% with the majority of deletions (75%) limited to CDKN2A/ARF/CDKN2B genes. FISH analysis was performed using three different BAC clones, but since they overlooked microdeletions we only appreciated a mild fluorescent signal reduction. In order to assess whether 9p21 loss is responsible for progression, the genomic status of this locus was assessed at the time of relapse and an almost significant increase in the detection rate of CDKN2A/ARF loss (47%) compared to diagnosis (p = 0.06) was found. Functionally, deletions led to a strong down-regulation at the transcript level of CDKN2A/ARF (p= 0.0005), as demonstrated by Fluidigm Dynamic Array real-time qPCR assay (Fluidigm Corporation, South San Francisco, CA) which enables to perform TaqMan nano-reactions at high sensitivity. Finally, the mutation screening performed on each exon of ARF, CDKN2A and CDKN2B genes showed that the 9p21 locus is rarely affected by point mutations with only the identification of the missense D146N in the CDKN2A exon 2. Furthermore, 2 mutations were found in the 5′UTR of CDKN2A [UTR A 21965011 (33%), UTR G/A 21965011 (47%), UTR C/T 21964875 (3%)] and a variation, known as SNP was found in the exon 2 (rs3731249). Finally, 2 SNPs were found in the 3′ UTR region of CDKN2A/ARF (rs11515 and rs3088440). The role of the mutations identified in the UTR is not yet defined, but the comparison with the germline material from saliva samples showed that these mutations were inherited. Since the prognostic relevance of CDKN2A/ARF deletions is still controversial in literature, we addressed this issue in our study cohort, finding out that deletions in this locus are significantly associated with poor outcome both in terms of disease free-survival (p=0.003) and cumulative incidence of relapse (p= 0.004). In conclusion, the inactivation of the tumor suppressor genes CDKN2A/ARF is a frequent event in Ph+ ALL. The main mechanism of inactivation is represented by genomic deletions, whereas missense mutations are rare. Deletions are frequently acquired at the leukemia progression and work as a poor prognostic marker, impairing disease free-survival and cumulative incidence of relapse. Novel treatment strategies targeting the ARF-MDM2-p53 pathway may be effective in this subset of patients and in vitro studies are ongoing to confirm this hypothesis. Supported by: European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Disclosures: Baccarani: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.