Littérature scientifique sur le sujet « T-cell acute lymphoblastic leukemia/lymphoma, gene-expression profiling, genome-wide copy number analysis, microRNA »

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.

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.

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.

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.

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.

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.

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.

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 &gt; 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 &gt; 15% (n = 647) identified by TPS encompassing especially all mutations in genes relevant for WHO diagnosis and prognostication. 26/171 mutations with a VAF &lt; 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.

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.

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.

ABSTRACT In the first part of our work we focused our attention on the biological question about the differences between two pathologies: T-cell lymphoblastic leukemia and T-cell lymphoblastic lymphoma. These two diseases share many features such as immunophenotypic features, lymphoblast morphology and clinical characteristics and are differentially diagnosed only on the base of bone marrow involvement. We tried to understand whether T-cell leukemia and lymphoma are a unique pathology with a different manifestation or whether they are two different diseases. The results obtained by gene expression profiling revealed an intrinsic difference in the expression of 78 genes between T-ALL and T-LBL. In particular since these genes belong to the angiogenesis and the chemotactic response we supposed that the two malignancies have different ability to respond to several cyto- and chemokines and that T-LBL need to modulate transcription to promote angiogenesis as well as to deal with hypoxic conditions. Also by analysis of copy number we were able to identify some abnormalities that seemed to be specific for each group regardless the limited data set of patients. Although this work provides additional elements in the characterization of these two pathologies, many studies have yet to be done, especially on the comprehension of the different capability of cells to migrate and invade the bone marrow compartment. The complete understanding of the molecular characteristics of T-LBL and T-ALL represents the driving element toward the design of fully successful therapeutic approaches. The second part of the study focused on the genetic characterization of two different groups of T-ALL patients on the basis of MRD response. With the aim to find biological correlates with the outcome for HR and nonHR patients, we performed many analyses, starting from copy number analysis to microRNAs expression profiling. Furthermore, we tried to integrate all the data in order to delineate common characteristics for each group of patients. First of all, the study of copy number revealed the presence of multiple abnormalities in all patients: we found known and unknown lesions, and in some cases we were able to associate them with HR or nonHR patients. The improvement of copy number results was obtained by the study of translocations. Also in this case we found one or more translocation in the majority of patients and we identified that the most recurrent were SIL-TAL1 and TLX3 translocations. Moreover, we tested the Notch1 mutations and, as expected, about 60% of patients were mutated for Notch1, with a tendency for Notch1 mutations to be more frequent in the nonHR group. The second step was the analysis of gene and microRNAs expression. The initial unsupervised analysis between HR and nonHR group failed to distinguish the two groups; but the successively supervised analysis revealed the distribution of MR patients in an equal manner between HR and SR patients. Thus an unsupervised analysis without MR patients showed a specific pattern of expression for each group (SR and HR). The GSEA analysis performed highlighted the enrichment of two specific pathways: the mir-215/192 pathway and the methylation pathway. The results obtained by the expression profile of about 700 microRNAs in the HR and nonHR group and those achieved by the combined analysis of GEP and microRNAs suggested miR-215 and miR-107 as the most differentially expressed and provided some possible target genes of these microRNAs. Moreover, to delineate specific pattern of expression not driven by MRD but by other alterations, we used the data derived from copy number, translocation and mutational analyses to supervise genetic subgroups. Significant results were obtained for Nocth1 mutated vs non-mutated, TLX3 translocated vs non-translocated, PTEN and LEF1 deleted vs non-deleted. We also tried to integrate all the data provided by both genomic and trasncriptomic analyses to understand whether the distribution of MR and the different signature of HR and SR were correlated with specific gene lesions. SIL-TAL1 fusion gene and the deletion of LEF1 and PTEN seemed to be specific for the HR group while the TLX3-translocation seemed to be peculiar for the nonHR group of patients. In conclusion HR and nonHR patients seemed to show some peculiar lesions and patterns of expression that could be justify the different response to therapy. In summary, several high throughput methodologies have been applied to the selected subgroup of patients to study the biological correlates of the different response to therapy. By this work we tried to provide a better characterization of T-ALL and to give a way of interpretation for the different outcome of T-ALL patients.