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  • American Society of Hematology  (6)
  • Kirschner-Schwabe, Renate  (6)
  • 1
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    TP53 and KRAS Variants at Initial Diagnosis Identify an Ultra-High Risk Group of Pediatric T-Lymphoblastic Leukemia (T-ALL)
    American Society of Hematology ; 2021
    In:  Blood Vol. 138, No. Supplement 1 ( 2021-11-05), p. 1315-1315
    Details
    In: Blood, American Society of Hematology, Vol. 138, No. Supplement 1 ( 2021-11-05), p. 1315-1315
    Abstract: Introduction Patients who suffer a relapse of pediatric T-cell acute lymphoblastic leukemia (T-ALL) face a dismal prognosis. Prognostic molecular biomarkers that reliably predict the risk of relapse at the time of first diagnosis are not available. Inactivating mutations in TP53 were previously detected in approximately 10% of relapsed patients (Hof et al. J Clin Oncol. 2011) and are invariably associated with fatal outcome (Richter-Pechanska et al. Blood Cancer J. 2017). Mutations in other genes were identified to be either specific for relapse (NT5C2 and CCDC88A) or to be associated with a poor prognosis in relapse (IL7R, KRAS, NRAS, USP7, CNOT3 and MSH6) (Meyer et al. Nat Genet. 2013; Richter-Pechanska et al. Blood Cancer J. 2017). We hypothesized that subclones bearing such mutations can give rise to relapse and analyzed these 9 genes at initial diagnosis of T-ALL with targeted ultra-deep sequencing. Methods Leukemia samples collected at initial diagnosis of 81 children with T-ALL who later relapsed were analyzed. As a control group, we selected 79 children with T-ALL who remained in first remission for at least three years and were matched with regard to treatment response, treatment, age and sex. Targeted deep sequencing was performed by using the Agilent Haloplex High Sensitivity kit with unique molecular identifiers for reliable detection of mutations with very low allele frequencies (average read depth: 1,012x). Results Overall, we detected 75 mutations among 7 targeted genes in 33 / 81 relapsing and 21 / 79 non-relapsing patients. The average allele frequency (AF) of the identified mutations was 25% (0.8% - 83%; SD ± 18%). More than half of the variants (43/75) showed AFs below 30% and were thus classified as subclonal. Interestingly, 7 pathogenic TP53 mutations (subclonal: n=5, clonal: n=2) with AFs of 4.4% - 49.4% were exclusively discovered in 6 patients who experienced a relapse. While 2 of these patients received an allogeneic stem cell transplantation in first remission because of poor treatment response, the remaining 4 patients were treated by chemotherapy in the high-risk (n=1) or medium-risk (n=3) arm. None of the 79 non-relapsing control patients carried TP53 mutations. Consistent with the hypothesis of clonal evolution as a mechanism of relapse in T-ALL, Sanger Sequencing of the relapse sample of one TP53-positive patient confirmed that the subclone harboring the TP53 mutation A159D at initial diagnosis (AF 5.4%) expanded to a major clone (AF 42%) in relapse. The presence of TP53 mutations in two further TP53-positive patients in at least one available post-remission sample is also compatible with clonal selection. However, in a fourth patient the low allele frequency of the TP53 mutation at relapse indicates that the TP53 subclone persisted but did not expand during the development of relapse. In addition to TP53, we identified pathogenic KRAS mutations to be significantly enriched in relapsing patients (9 / 81) compared to non-relapsing patients (2 / 79) at the time of initial diagnosis (chi-squared test, p= 0.032; Table 1). Conclusion Subclonal and clonal mutations in TP53 and KRAS at initial diagnosis were enriched in T-ALL patients who later relapsed and identified approximately 17% of patients suffering a relapse. We thus propose that (subclonal) mutations of TP53 and KRAS may define a subgroup of high-risk T-ALL patients already at the time of first diagnosis. The identification of such mutations may complement the current risk stratification which depends on treatment response and may determine a new molecularly defined subgroup of T-ALLs that may benefit from intensified treatment strategies. Figure 1 Figure 1. Disclosures Schrappe: SigmaTau: Other: research support; Amgen: Other: research support; Servier: Honoraria; Novartis: Honoraria; JazzPharma: Honoraria; Servier: Honoraria, Other: research support; JazzPharma: Honoraria, Other: research support; SHIRE: Other: research support; Novartis: Honoraria, Other: research support. Cario: Novartis: Other: Lecture Fee. Muckenthaler: Silence Therapeutics: Research Funding. Kulozik: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BioMedX: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; bluebird bio, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Honoraria.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2021-148420
    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2021
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  • 2
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    Identification of an Ultra High-Risk and Targetable Molecular Signature in Relapsed Pediatric T-ALL
    American Society of Hematology ; 2016
    In:  Blood Vol. 128, No. 22 ( 2016-12-02), p. 1084-1084
    Details
    In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 1084-1084
    Abstract: Relapsed T-cell acute lymphoblastic leukemia (T-ALL) represents one of the major challenges of pediatric oncology as it is often resistant to treatment and fatal. In the search for genes that define critical steps of relapse and can serve as prognostic markers we compared 67 relapses (REL) and 147 samples collected at initial diagnosis (INI) by targeted sequencing of 313 leukemia-related genes. In addition to the analysis of single nucleotide variants (SNV) and small insertions and deletions (InDels), we made use of the available coverage data for profiling of copy number alterations (CNA). Of the 147 INI patients, 31 were treated according to the ALL-BFM 2000 and 116 according to the AIEOP-BFM ALL 2009 protocol. All REL patients were recruited from the ALL-REZ BFM 2002 trial. We analyzed bone marrow DNA by targeted capture of 313 genes (5964 exons) using the Haloplex Target Enrichment Kit (Agilent). We used Varscan to detect both SNV and InDels. In the absence of available remission samples, we subtracted known SNPs (dbSNP, 1000 gp) and variants present in at least one of 20 non-leukemic samples that we sequenced in parallel to the patients' samples. Only mutations with an AF 〉 10% were considered. Copy number analysis based on read-depth data was validated by MLPA analysis of 14 genes showing a sensitivity of 99% and by low coverage WGS. In total, we have SNV data on all 147 INI and 67 REL patients and CNA data on 144 and 58 patients, respectively. Altogether, we identified relapse specific genetic events in 32 of 67 RELs. Our results confirm that NT5C2 mutations are highly enriched in relapse (REL: 17/67 vs. INI: 1/147; p=0.0001). Although activation of NT5C2 was associated with the occurrence of early first relapse (p=0.02), it did not correlate with induction failure and therefore has no prognostic impact. Similarly, amplifications of chromosome 17 q11.2-24.3, a region that contains genes of the STAT and ABCA families were significantly more frequent in REL than in INI (REL 7/58 vs. INI 3/144, p=0.0068), but also had no prognostic implications. By contrast, TP53 mutations were highly predictive of a second event: all 8 patients who carried a total of 9 TP53 mutations and deletions died within 9 months after first relapse (p=0.002), whereas 17 of the other 58 (29%) survived. Inactivation of TP53 was significantly correlated with higher mutation rates in other genes compared to those with wild-type TP53 (ttest= p 〈 0.0001). TP53 alterations combined with other recurrent mutations in genes responsible for surveillance of DNA integrity (MSH6 and USP7) recognize 15 of the 49 patients with a fatal post-relapse outcome. This group appeared to be particularly resistant to induction therapy as only 2/14 (no data available for 1 pt) patients compared to 17/52 without TP53/MSH6/USP7 mutations achieved second remission and could receive stem cell transplantation (p=0.0006). Activating mutations of KRAS or NRAS thus activating the Ras/Raf/MEK/ERK pathway were found in 8 of these 49 REL patients, 6 of whom died within 3 months, indicating that these mutations cause treatment resistance (p 〈 0.05). Further, likely inactivating mutations of CNOT3 and known activating insertions of IL7R have been exclusively found in 7 REL patients who failed induction. Four of these 7 RELs also carried TP53 or RAS mutations. This combination of mutations predicts an exquisitely poor outcome and results in an ultra-high risk prognostic signature that identifies 24/49 REL patients, who failed induction treatment and died. In conclusion, targeted sequencing of relapsed T-ALL identified a molecular signature predicting an exquisitely poor outcome in 50% of relapses, who failed salvage treatment. This group of patients does not benefit from current treatment strategies thus identifying a subgroup with a dire clinical need for experimental therapy. Notably, approx. 25% REL patients who failed induction carried RAS and IL7R mutations. These patients may be considered for personalized treatment with either MEK- or JAK/STAT-inhibitors. Another 4 patients carry TP53 missense mutations and may benefit from treatment with p53 refolding compounds. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V128.22.1084.1084
    RVK:
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    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2016
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  • 3
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    Longitudinal Multilevel Omic Analysis of Pediatric T-ALL Reveals Distinct Mechanisms for Disease Progression in Type 1 and in Type 2 Relapses
    American Society of Hematology ; 2018
    In:  Blood Vol. 132, No. Supplement 1 ( 2018-11-29), p. 2826-2826
    Details
    In: Blood, American Society of Hematology, Vol. 132, No. Supplement 1 ( 2018-11-29), p. 2826-2826
    Abstract: We aimed at understanding the relapse-driving processes in pediatric T-ALL and performed an integrated longitudinal multi-level omics analysis of 13 T-ALL patients at initial diagnosis (INI) and relapse (REL). We compared the mutation (SNV/InDels) and copy number alteration (CNA) patterns as well as gene expression, methylation levels and chromatin accessibility by ATAC-Seq. Aberrant expression of T-ALL transcription factors (TAL1, TAL2,LMO2, TLX1, TLX3, NKX2.4 and NKX2.5) was preserved from initial presentation to relapse in all patients. These leukemia-driving events defined the expression patterns, methylation profiles and the chromatin accessibility landscapes. A global differential analysis of the RNA-Seq data (DESeq2, padj 〈 .05) revealed only 0.3% of the genes to be either up- or down-regulated at relapse when compared to the matched initial sample. Likewise, we detected methylation changes in only 3% of the promoters and differential chromatin accessibility in only 0.26% of the analyzed ATAC-peaks (DESeq2, padj 〈 .05). We then focused our analysis on the 2 types of relapse in pediatric T-ALL, which we have previously defined on the basis of subclonal mutation profiles (Kunz et al., 2015). These types of relapse are characterized by either clonal evolution of cells derived from the major clone at initial presentation (type 1) or emergence and evolution of a minor initial clone showing a molecular profile that is distinct from the predominant initial clone (type 2). When considering type 1 and type 2 relapses separately we identified a strong trend for type 2 relapses to acquire more mutations (p=0.0879, ttest) than type 1 relapses. Further to the known activating mutations in NT5C2 acquired at relapse by 8/13 patients no other mutations or CNAs were recurrently acquired in the relapses of this group of patients. However, mutations in proto-oncogenes or genes involved in DNA surveillance were acquired by 7/8 type 2 relapse patients in our series. Changes of CNAs also occurred more frequently in type 2 than in type 1 relapses (pval= 0.0267, ttest). This increased complexity on the genetic level was also apparent on the epigenetic level, with an increase of changes in the methylation pattern (mean difference in β value between INI and REL: type 1 - 0.00034; type2 - 0.002 (pval 〈 0.0001, chi2)), chromatin accessibility (number of differentially accessible ATAC-peaks: type 1 - 4 (0.006%) ; type 2 - 1018 (1.3%); (pval 〈 0.0001, chi2)) and on the expression level (number of differentially expressed genes: type 1 - 11; type 2 - 111, pval 〈 0.0001, chi2). When considering differences between leukemias at the time of initial diagnosis, which later develop either type 1 or type 2 relapses we found 1.016 genes to be differentially expressed (524: up; 492: down in type 1; DE-Seq2: padj 〈 0.05). Differential expression analysis revealed that genes involved in early T-cell differentiation were upregulated at initial diagnosis of type 1 in comparison to initial diagnosis of type 2, which was also reflected by more accessible chromatin surrounding their cis-regulatory regions as analyzed by ATAC-seq suggesting an early arrest of these samples during differentiation process. Altogether 1.4% of all ATAC-peaks were more accessible in type 1, whereas 0.7% of peaks were more accessible in type 2 leukemia (DE-Seq2: p 〈 0.05). Remarkably, the chromatin surrounding DNA-repair genes was more accessible in initial leukemias of type 1, which was reflected by up-regulated mRNA expression level of such genes. These data suggest that an increased propensity of DNA repair may represent an important mechanism of T-ALL to develop a type 1 relapse, an interpretation that is consistent with previous reports linking the epigenetic silencing of the methylguanine-DNA methyltransferase promoter with compromised DNA repair and longer survival in patients with glioblastoma receiving alkylating agents (Esteller et. al., 2000). In sum, the multilevel omic comparison of pediatric T-ALL that develop either a type 1 or a type 2 relapse show remarkably more complex changes of the genetic and epigenetic profiles during the transition from initial to relapsing disease. Notably, pediatric T-ALLs, who later develop a type 1 relapse display an epigenetic and transcriptomic landscape predicting an upregulation of DNA repair functions, which we suggest to potentially play a role in developing resistance to DNA damaging agents in this type of relapse. Disclosures Muckenthaler: Novartis: Research Funding. Bourquin:Amgen: Other: Travel Support. Kulozik:bluebird bio: Consultancy, Honoraria.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2018-99-117978
    RVK:
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    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2018
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  • 4
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    Targeted Deep Sequencing of Genetic Alterations Identified By Whole Exome Sequencing Reveals Clonal Evolution in Pediatric T-Lymphoblastic Leukemia
    American Society of Hematology ; 2014
    In:  Blood Vol. 124, No. 21 ( 2014-12-06), p. 491-491
    Details
    In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 491-491
    Abstract: Precursor T-cell acute lymphoblastic leukemia (T-ALL) represents one of the major challenges of pediatric oncology, because relapses are frequently refractory to treatment and fatal. The molecular understanding of progression to relapse in T-ALL is limited. We aimed at identifying patterns of clonal evolution and at describing mechanisms of relapse by comparing the genetic and epigenetic alterations in primary and in relapsed pediatric T-ALL. DNA from bone marrow of 13 patients with T-ALL at primary disease, remission and relapse was analyzed by a combination of multiplex ligation- dependent probe amplification (MLPA), Illumina 450k array, whole exome sequencing (WES) and targeted deep sequencing. Targeted deep sequencing was performed after target capture with Agilent HaloPlex. In the target capture design, all loci that showed somatic mutations in WES were included. Deep sequencing was done on all primary disease and relapse samples and on a subset of remission samples. Allele frequencies by HaloPlex were highly reproducible, corresponded well to allele frequencies of loci that were well covered in WES and were consistent after serial dilutions. Analysis of DNA methylation using the Illumina 450k array showed that methylation of relapse samples does not differ significantly from the methylation of the matching primary disease samples, with the variability between different patients being much larger than the variability within samples from the same patient. WES identified on average 10 single nucleotide variants (SNVs) and 1.8 small insertions and deletions (indels) in primary T-ALL and 23.2 SNVs and 2.6 indels in the corresponding relapse samples. Only about 30% of SNVs and indels identified in relapse were already detected in primary disease by WES, while most amplifications and deletions that had been detected by the combination of MLPA and read depth analysis of WES data were conserved from primary disease to relapse. Recurrently, we identified known and novel drivers of T-ALL (NOTCH1, FBXW7, PHF6, WT1, PTEN, NRAS, STAT5B). Targeted resequencing of mutated genes at high depth (median coverage 6233, 90% of targets covered 〉 1000x) identified rare subclonal alleles with a sensitivity in the range of 10-2 to 10-4, depending on the coverage of each individual locus. This allowed us to distinguish de novo mutations that were acquired during treatment from mutations that had already been present at initial diagnosis and were selected for in relapse. Depending on the contribution of clonal selection or de novo mutations, at least two different patterns of relapse could be identified: In a smaller proportion of leukemias, all mutations present at first diagnosis were again detected in relapse, with some additional mutations that were specific for relapse. In most leukemias, the major clone at relapse had arisen from a minor subclone at primary disease and has acquired additional mutations, indicating that clonal selection was the main contributor to the evolution of relapse. In all cases, at least one genetic alteration was detected in samples from both time points. The example of activating mutations in the nucleotidase NT5C2, which have previously been proposed to contribute to resistance against nucleoside analogues, illustrates the genetic plasticity of T-ALL: Activating NT5C2 mutations were identified in 4 out of 13 relapse samples. The only activating NT5C2 mutation that was already detected in a primary disease sample at low allele frequency was not present in the corresponding relapse sample but was replaced by another activating NT5C2 mutation. This indicates that mutations acquired during treatment may outcompete subclonal mutations that were present in the primary leukemia. In at least two relapse samples, subclonal NT5C2 mutations were detected, compatible with the notion that acquisition of resistance towards chemotherapy by mutation of NT5C2 is a late event on the way to relapse. Conclusion: The acquisition of novel genetic alterations and selection of treatment resistant subclones are main contributors to T-ALL relapse. We now aim at identifying molecular signatures that characterize treatment resistant subclones, which may be included in risk stratification algorithms of primary T-ALL. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V124.21.491.491
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2014
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  • 5
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    Pediatric T-ALLs Developing into a Type 2 Relapse Originate from Cells That Carry the Potential of Variable Maturation into Subclones with Distinct Chromatin Landscapes
    American Society of Hematology ; 2018
    In:  Blood Vol. 132, No. Supplement 1 ( 2018-11-29), p. 1545-1545
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    In: Blood, American Society of Hematology, Vol. 132, No. Supplement 1 ( 2018-11-29), p. 1545-1545
    Abstract: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that is classified according to surface marker expression. In order to reveal the cells of origin in pediatric T-ALL and to understand mechanisms of relapse we used ATAC-Seq (Assay for Transposase-Accessible Chromatin Sequencing) to compare chromatin accessibility landscapes of healthy T-cell precursors to those of T-ALL cells obtained at initial diagnosis (INI) and relapse (REL). We have FACS sorted 7 differentiation stages of normal T-cell precursors contained in the thymus of infants undergoing cardiac surgery (DN2, DN3, ISP, DPCD3-, DPCD3+, CD4+ and CD8+) and subjected these to ATAC-Seq. Unsupervised learning by principal component analysis (PCA) clustered sorted populations according to the maturation stage, demonstrating that regulatory chromatin signatures of thymocytes are highly stage-specific and re-shaped during T-cell differentiation. We next compared normal T-cell precursors at different stages of maturation to pediatric T-ALLs and found fundamental differences with 30% of open chromatin regions to be more and 28% being less accessible in T-ALL (DESeq2, padj 〈 .05), respectively. We next identified transcription factor (TF) binding sites in open chromatin regions of normal T-cell precursors and T-ALL cells by HOMER analysis. We found 76% of the accessible TF binding sites in double negative (DN2 and DN3) normal precursors to be also accessible in most (20/24) leukemias comparing to 17% of the sites accessible in the more mature (CD4+ and CD8+) stages. These data indicate that T-ALLs originate from cells with an epigenetic profile of early thymic progenitors. We then subjected the ATAC-seq data of all matched leukemia samples obtained at initial disease and at relapse to PCA. INI and REL samples derived from the same patient always clustered in close proximity and were separated according to the T-ALL driving fusion genes. A global analysis of differential accessibility revealed only 0.26% of ATAC-regions to be less- or more-accessible at relapse when compared to the matched initial samples (DESeq2, padj 〈 .05). These data indicate chromatin accessibility to be largely determined by the cell of origin and to generally remain stable during progression from initial diagnosis to relapse. We then considered the 2 types of relapse separately, which we have previously characterized on the basis of subclonal mutation profiles (Kunz et al. Haematologica, 2015). These relapse types are defined by either clonal evolution of cells derived from the predominant clone at primary disease (type 1) or emergence and evolution of a minor initial clone showing a molecular profile that differs from the major initial clone (type 2). We found that the proportion of differentially accessible ATAC-regions between INI and REL is significantly higher in type 2 (1.3%) than in type 1 (0.006%) (DESeq2, padj 〈 .05). Moreover, we trained the deconvolution algorithm CIBERSORT to recognize particular T-cell differentiation stages using ATAC-profiles of the 7 FACS-sorted healthy T-cell populations. We used regulatory chromatin landscape of non-sorted (total) thymus to assess the accuracy of deconvolution. Comparison of predicted fractions in total thymus to FACS measurements revealed highly accurate identification of the maturation stages (r2 = 0.95). CIBERSORT analysis confirmed that the profiles were largely preserved between INI and REL of each sample pair. Notably, however, while in T-ALLs that later developed into a type 1 relapse only one type of early T-cell progenitor dominated the deconvolution profile, T-ALLs that developed into a type 2 relapse showed heterogeneous profiles with contributions of progenitors at different maturation stages. In sum, these epigenomic analyses revealed that the chromatin landscape of normal T-cell precursors evolves in the course of thymic maturation and that the early maturation stages are the likely origin of T-ALL cells. Remarkably, pediatric T-ALLs that later develop a type 2 relapse consist of subclones with a variable profile of chromatin accessibility that define different stages of maturation. These data indicate that T-ALLs with the propensity to develop a type 2 relapse differ from type 1 in that they originate from early precursors that carry the potential of further development into different stages of maturation before the leukemia becomes apparent with a highly subclonal pattern. Disclosures Muckenthaler: Novartis: Research Funding. Bourquin:Amgen: Other: Travel Support. Kulozik:bluebird bio: Consultancy, Honoraria.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2018-99-118255
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    XA 33000
    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2018
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  • 6
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    Gene Panel Sequencing of Primary and Relapsed Pediatric T-ALL Shows That Relapse-Specific Mutations Are Diverse and Mostly Non-Recurrent
    American Society of Hematology ; 2015
    In:  Blood Vol. 126, No. 23 ( 2015-12-03), p. 1428-1428
    Details
    In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 1428-1428
    Abstract: Precursor T-cell acute lymphoblastic leukemia (T-ALL) remains one of the major challenges of pediatric oncology, because relapses are frequently refractory to treatment and fatal. We aimed at identifying relapse specific genetic alterations by analyzing a cohort of 147 primary T-ALL patients and of 70 relapsed T-ALL patients with targeted gene panel sequencing. In addition to the analysis of single nucleotide variants (SNVs) and small insertions and deletions (InDels), we made use of the available coverage data to characterize aberrant copy number alterations (CNA). DNA from bone marrow of these 217 pediatric T-ALL patients was analyzed by gene panel sequencing after target capture with Agilent HaloPlex. In the target capture design, exons of 324 genes were included that had been found before by whole exome sequencing to carry somatic mutations in a pilot set of relapsed T-ALL or that have been reported to be mutated in T-ALL in the literature. We did not analyze corresponding remission samples and did not discriminate between germline and somatic alterations. Only mutations with an allele frequency (AF) 〉 10% were considered and absence of the mutation in the 1000 Genomes variant catalogue was required. Copy number analysis based on read-depth data identified deletions (DEL) and amplifications (AMP). Direct comparison of CNAs by multiplex ligation-dependent probe amplification (MPLA) and gene panel sequencing was possible for 13 overlapping regions covering 14 genes in 185 samples. Recognition rate by coverage analysis was 98% (256/260) for biallelic alterations and 81% (92/114) for monoallelic alterations found by MLPA. On average, gene panel sequencing identified 6.7 mutations in initial diagnosis samples (SNVs: 5.2; InDels: 1.5) and 7.9 mutations in relapse samples (SNVs: 5.9; InDels: 2). In the group of primary leukemia and relapse samples, the average AMP/DEL per patient was 8.2 (AMP: 3.2, DEL: 5.0) and 8.8 (AMP: 4.2, DEL: 4.6), respectively. 31 genes were found to be mutated and 46 deleted/amplified in 10 or more patients (see Table 1 and 2). Table 1. Most commonly mutated genes (SNVs and InDels) Gene Total # of mutations # pts with mutation in primary T-ALL(n=147) # pts with mutation in relapsed T-ALL (n=70) NOTCH1 178 88 (60%) 38 (54%) PHF6 47 24 (16%) 16 (23%) FBXW7 42 20 (14%) 17 (24%) OBSCN 35 20 (14%) 10 (14%) DNM2 28 17 (12%) 10 (14%) PTEN 34 20 (14%) 4 (6%) XIRP2 24 19 (13%) 5 (7%) CDH23 23 11 (7%) 11 (16%) WT1 36 11 (7%) 8 (11%) NT5C2 22 1 (1%) 17 (24%) Table 2. Most common copy number alterations Amplifications Deletions Gene primary T-ALL (n=147) relapsed T-ALL (n=64) Gene primary T-ALL (n=147) relapsed T-ALL (n=64) MYB 9 (6%) 9 (15%) CDKN2A 102 (70%) 36 (59%) MYC 11 (8%) 6 (10%) CDKN2B 83 (57%) 31 (51%) NRG1 11 (8%) 3 (5%) MLLT3 28 (19%) 5 (8%) UNC5D 11 (8%) 3 (5%) PHIP 18 (12%) 6 (10%) NCOA2 11 (8%) 3 (5%) ELOVL4 18 (12%) 5 (8%) PTK2B 11 (8%) 3 (5%) MAP3K7 17 (12%) 5 (8%) FDFT1 11 (8%) 3 (5%) CASP8AP2 17 (12%) 5 (8%) ABL1 8 (5%) 3 (5%) APC 19 (13%) 3 (5%) CNOT3 8 (5%) 3 (5%) LEF1 16 (11%) 5 (8%) SMG8 3 (2%) 7 (10%) PAX5 15 (10%) 5 (8%) Potential novel mechanisms of oncogene activation are amplifications of PTK2B, a gene that has been found to be deregulated by fusion in Philadelphia-like BCP-ALL and that is potentially targetable by tyrosine kinase inhibitors, and of MYC, which has long been known to be a key player in T-ALL leukemogenesis and that is amplified in neuroblastoma and medulloblastoma. Enriched in relapse, we identified mutations in NT5C2 (p=1.4E-08), TP53 (p=0.0006) and CCDC88A (p=0.01), and amplifications of a region on chr 17q represented by the genes CLTC, ABCA5, C17orf80 and SRSF2. MLLT3 deletions were enriched in primary samples (p=0.04), consistent with the observation that MLLT3 deletions confer a lower risk of relapse in patients treated on BFM protocols. Conclusion Gene panel sequencing emerges as a suitable tool for a comprehensive genetic characterization of pediatric T-ALL. Within the group of selected genes contained in the panel, CNA were as frequent as point mutations. Only few genes were found to be specifically altered in relapse, indicating that progression to relapse may involve diverse, non-recurrent genetic alterations. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V126.23.1428.1428
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2015
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