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  • 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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    Mutational Analysis of Mir-29 Family Members in Chronic Lymphocytic Leukemia
    American Society of Hematology ; 2011
    In:  Blood Vol. 118, No. 21 ( 2011-11-18), p. 1770-1770
    Details
    In: Blood, American Society of Hematology, Vol. 118, No. 21 ( 2011-11-18), p. 1770-1770
    Abstract: Abstract 1770 Background: MicroRNAs (miRNAs) are small non-coding RNA molecules regulating gene expression by inhibition of mRNA translation and/or stability. Numerous miRNAs were described as tumor suppressor genes or oncogenes. In this respect, miR-29 family belongs to the most important miRNAs involved in CLL biology. Three different isoforms of the miR-29 (miR-29a-2, miR-29b-2 and miR-29c) were shown to be down-regulated in aggressive CLL (Calin et al., 2004; Mraz et al., 2009). Moreover, miR-29 was suggested to target the expression of MCL1, CDK6 and TCL1, a critical oncogene in aggressive CLL. Recently, the generation of transgenic mice over-expressing miR-29 in B-cells demonstrated its direct role in CLL pathogenesis. Additionally, mutations in miR-29c and miR-29b-2 have been also detected in CLL patients (Calin et al., 2005); however, their impact and frequency have yet to be elucidated. Aims: In this study, we searched for novel sequence variations in three members of the miR-29 family (miR-29a, miR-29b-2 and miR-29c) using re-sequencing microarray and Sanger sequencing. Methods: The pre-miRNAs (29a, 29b-2, 29c) and 107 other pre-miRNAs were analyzed by a custom re-sequencing microarray (Affymetrix, 50K) in 98 high-risk CLL patients (81.6% of patients had unmutated IgVH; 37.8% had a mutation in the TP53 gene). To cover the whole genomic area of pre-miRNAs, ∼20 nucleotides from both 5' and 3' ends of pri-miRNAs were also analysed. MiRNAs were amplified from genomic DNA isolated from peripheral blood cells (CLL lymphocytes or mononuclear cells) using long range PCR. Amplicons were pooled, fragmented and co-hybridized on the array according to the manufacturer's protocol. Sanger sequencing was used to confirm the presence of the mutations detected by microarray. The genomic regions (455-bp, 940-bp) of pri-miR-29c and pri-miR-29b-2, respectively, were further analyzed by Sanger sequencing in another 192 CLL patients (63.5% with unmutated IgVH; 21.4% with TP53 gene mutation). Results: Using resequencing microarray and confirmatory Sanger sequencing, one heterozygous variation (A/G +22 in 3') was found in the pri-miR-29a in one of 98 CLL patients. Screening of pri-miR-29c (455-bp genomic area) detected two substitutions in 4 of 192 CLL patients. In particular, the known G/A substitution (-31 in 5') was found as heterozygous in two patients. Additionally, novel heterozygous T/A substitution (+137 in 3') was found in two other patients. Screening of 940-bp genomic region of the pri-miR-29b-2 detected insertion (+A) (+107 in 3') in 22 of 192 patients. However, the known G/A substitution (+212 in 3') was not detected in our cohort. Furthermore, 2 novel heterozygous variations in pri-miR-29b-2 (C/G −169 in 5' and A/G − 256 in 5') were found in 5 and 2 patients, respectively. According to our results, pri-miR-29b-2 seems to be the most frequently mutated member of the miR-29 family, since three types of variations were detected in 29 of 192 CLL patients (15.1%). Two substitutions were found in pri-miR-29c in 4 of 192 CLL patients (2.1%) and only one substitution was detected in pri-miR-29a in one of 98 CLL patients (1%). Conclusion: We herein confirm that both known mutations and novel sequence variations are present in three members of the miR-29 family which is already implicated in CLL pathogenesis or progression. Altogether, we have found 6 types of variations in 34 of 290 analyzed CLL patients (11.7%). The insertion (+A) (+107 in 3') in pri-miR-29b-2 was the most common variation detected in our cohort of CLL patients; however, its impact on CLL pathogenesis has to be elucidated. Supported by the grants IGA-MZ-CR NT11218-6/2010, NS10439-3/2009, NS9858-3/2009, MPO-CR-FR-TI2/254 and MSMT-CR-MSM0021622430. Disclosures: Mayer: Roche: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Astellas: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Novartis: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Fresenius Medical Care: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Pfizer: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Genzyme: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; GSK: Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Amgen:.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V118.21.1770.1770
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2011
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  • 3
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    Whole Exome Sequencing In Relapsed Pediatric T-ALL: Progression Into Relapse Is Characterized By An Increased Number Of Somatic Mutations and a Conservation Of Mutations In Leukemogenic Driver Genes
    American Society of Hematology ; 2013
    In:  Blood Vol. 122, No. 21 ( 2013-11-15), p. 228-228
    Details
    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 228-228
    Abstract: Acute precursor T-lymphoblastic leukemia (T-ALL) remains a serious challenge in pediatric oncology, because relapses carry a particularly poor prognosis with high rates of induction failure and death despite generally excellent treatment responses of the initial disease. It is critical, therefore, to understand the molecular evolution of pediatric T-ALL and to elucidate the mechanisms leading to T-ALL relapse and to understand the differences in treatment response between the two phases of the disease. We have thus subjected DNA from bone marrow samples obtained at the time of initial diagnosis, remission and relapse of 14 patients to whole exome sequencing (WES). Eleven patients suffered from early relapse (duration of remission 6-19 months) and 3 patients from late relapse (duration of remission 29-46 months).The Agilent SureSelect Target Enrichment Kit was used to capture human exons for deep sequencing. The captured fragments were sequenced as 100 bp paired reads using an Illumina HiSeq2000 sequencing instrument. All sequenced DNA reads were preprocessed using Trimmomatic (Lohse et al., Nucl. Acids Res., 2012) to clip adapter contaminations and to trim reads for low quality bases. The remaining reads greater than 36bp were mapped to build hg19 of the human reference genome with Stampy (Lunter & Goodson, Genome Res. 2011), using default parameters. Following such preprocessing, the number of mapped reads was 〉 95% for all samples. Single-nucleotide variants (SNVs) were called using SAMtools mpileup (Li et al., Bioinformatics, 2009). The number of exonic SNVs varied between 23,741 and 31,418 per sample. To facilitate a fast classification and identification of candidate driver mutations, all identified coding SNVs were comprehensively annotated using the ANNOVAR framework (Wang et al., Nat. Rev. Genet., 2010). To identify possible somatic driver mutations, candidate SNVs were filtered for non-synonymous, stopgain or stoploss SNVs, requiring an SNV quality greater or equal to 50, and requiring absence of segmental duplications. Leukemia-specific mutations were identified by filtering against the corresponding remission sample and validated by Sanger sequencing of the genomic DNA following PCR amplification. We identified on average 9.3 somatic single nucleotide variants (SNV) and 0.6 insertions and deletions (indels) per patient sample at the time of initial diagnosis and 21.7 SNVs and 0.3 indels in relapse. On average, 6.3 SNVs were detected both at the time of initial diagnosis and in relapse. These SNVs were thus defined as leukemia specific. Further to SNVs, we have also estimated the frequency of copy number variations (CNV) at low resolution. Apart from the deletions resulting from T-cell receptor rearrangement, we identified on average for each patient 0.7 copy number gains and 2.2 copy number losses at the time of initial diagnosis and 0.5 copy number gains and 2.4 copy number losses in relapse. We detected 24/27 copy number alterations both in initial diagnosis and in relapse. The most common CNV detected was the CDKN2A/B deletion on chromosome 9p. Nine genes were recurrently mutated in 2 or more patients thus indicating the functional leukemogenic potential of these SNVs in T-ALL. These recurrent mutations included known oncogenes (Notch1), tumor suppressor genes (FBXW7, PHF6, WT1) and genes conferring drug resistance (NT5C2). In several patients one gene (such as Notch 1, PHF6, WT1) carried different mutations either at the time of initial diagnosis and or in relapse, indicating that the major leukemic clone had been eradicated by primary treatment, but that a minor clone had persisted and expanded during relapse. The types of mutations did not differ significantly between mutations that were either already present at diagnosis or those that were newly acquired in relapse, indicating that the treatment did not cause specific genomic damage. We will further characterize the clonal evolution of T-ALL into relapse by targeted re-sequencing at high depth of genes with either relapse specific or initial-disease specific mutations. In conclusion, T-ALL relapse differs from primary disease by a higher number of leukemogenic SNVs without gross genomic instability resulting in large CNVs. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V122.21.228.228
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    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
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  • 4
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    Whole-Exome Sequencing Links CARD11 Inactivation with SCID
    American Society of Hematology ; 2012
    In:  Blood Vol. 120, No. 21 ( 2012-11-16), p. 258-258
    Details
    In: Blood, American Society of Hematology, Vol. 120, No. 21 ( 2012-11-16), p. 258-258
    Abstract: Abstract 258 Primary immunodeficiencies represent model diseases for the mechanistic understanding of the human innate and the adaptive immune response and are per se clinically highly relevant, because in SCID patients infections by opportunistic pathogens are typically life-threatening early in life. We identified an infant of consanguineous parents suffering from a novel form of SCID, who presented with a life-threatening Pneumocystis jirovecii pneumonia. This entity was characterized by agammaglobulinemia and profoundly deficient T-cell function despite quantitatively normal T- and B-lymphocytes. Lymphocyte proliferation was strongly inhibited after stimulation of PBMCs with T-cell mitogens such as PHA, Con A, or anti-CD3 monoclonal antibody. The expression of several T-cell response associated cytokines upon stimulation with PMA/ionomycin was dramatically reduced in comparison to normal controls. By contrast, proliferation induced by the classical B-cell mitogen PWM was almost comparable to healthy controls. Immunophenotyping revealed a predominantly naïve phenotype (CD45RA+ CCR7+) in CD4+ and CD8+ T-lymphocytes, whereas central memory T-lymphocytes (CD45RA− CCR7+) were nearly absent. B-lymphocytes from peripheral blood were mainly naïve B-cells (CD27−) with a uniformly immature transitional B-lymphocyte phenotype (CD24++, CD38++). Patient B-lymphocytes retained the ability to proliferate and differentiate in response to BCR-independent stimuli, while their response to BCR activation was defective. Our findings thus revealed a combined defect of TCR-mediated T-lymphocyte functions and BCR-mediated B-lymphocyte functions but did not enable us to link the immunological phenotype with one of the known molecularly defined categories of SCID. Diagnostic whole-exome sequencing and systematic variant categorization revealed a single pathogenic homozygous nonsense mutation of the caspase recruitment domain 11 (CARD11) gene. CARD11 is a scaffold protein that is known to be required for the assembly and activation of the NF-kB complex. In reconstitution assays we demonstrated that the patient derived truncated CARD11 protein is defective in antigen receptor signaling and NF-kB activation. Several lines of evidence substantiate the involvement of the identified CARD11 mutation in the new form of SCID that we report here. First, PCR and Sanger re-sequencing validated the truncating CARD11 mutation to be homozygous in the patient and heterozygous in the parents, in agreement with the recessive transmission of the mutation through the healthy consanguineous parents. Second, CARD11 is a scaffold protein required for TCR- and BCR-induced NF-kB activation as well as lymphocyte activation and proliferation, which is specifically expressed in hematopoietic cells, consistent with a causative role of CARD11 mutations in the context of an immune disorder. Third, the GUK domain of CARD11, which is missing in the mutated form of CARD11 due to truncation, was previously reported to be necessary for NF-kB activation by PMA/ionomycin treatment, further supporting the presumed damaging nature of the homozygous CARD11 mutation observed in the female patient reported here. Finally, the immunological findings in this patient are compatible with the phenotype of a previously described Card11 −/− k.o. mouse, which shows a selective defect in NF-κB activation leading to diminished antigen receptor or PKC mediated proliferation and defective cytokine production in T-cells and B-cells. Thus, we have identified an inactivating CARD11 mutation linking defective NF-kB signaling with a novel cause of autosomal recessive SCID, which must be considered in the diagnostic assessment of patients with suspected SCID but with quantitatively normal T-cells. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V120.21.258.258
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2012
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  • 5
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    Constitutional PIGA mutations cause a novel subtype of hemochromatosis in patients with neurologic dysfunction
    American Society of Hematology ; 2022
    In:  Blood Vol. 139, No. 9 ( 2022-03-03), p. 1418-1422
    Details
    In: Blood, American Society of Hematology, Vol. 139, No. 9 ( 2022-03-03), p. 1418-1422
    Abstract: Muckenthaler et al describe a novel form of hemochromatosis caused by a constitutional PIGA mutation in 3 children with associated neurologic dysfunction. Hemochromatosis results from decreased hepcidin, which is regulated by HFE, hemojuvelin (HJV), and transferrin receptor 2. HJV is a glycosylphosphatidylinositol-linked protein, so PIGA mutation leads to decreased HJV expression. Interestingly, none of the children had evidence of paroxysmal nocturnal hemoglobinuria. The cause of the novel association with central nervous system manifestations remains to be elucidated.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.2021013519
    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2022
    detail.hit.zdb_id: 1468538-3
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  • 6
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    Validation of the 2022 European Leukemianet Genetic Risk Stratification of Acute Myeloid Leukemia
    American Society of Hematology ; 2022
    In:  Blood Vol. 140, No. Supplement 1 ( 2022-11-15), p. 3408-3409
    Details
    In: Blood, American Society of Hematology, Vol. 140, No. Supplement 1 ( 2022-11-15), p. 3408-3409
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2022-167022
    RVK:
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    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2022
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  • 7
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    Whole Exome Sequencing Identifies Novel Lyst-Missense Mutations In Incomplete Childhood Chediak-Higashi-Syndrome Presenting As Hemphagocytic Lymphohistiocytosis (HLH)
    American Society of Hematology ; 2013
    In:  Blood Vol. 122, No. 21 ( 2013-11-15), p. 3479-3479
    Details
    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 3479-3479
    Abstract: Chediak Higashi Syndrome (CHS) is caused by defective membrane targeting of components of the lysosome, which results from inactivation of the lysosomal trafficking regulator LYST. Clinically, CHS is typically characterized by partial albinism, susceptibility to infection, lymphoproliferation with acceleration to HLH. The immunodeficiency can be cured by allogeneic stem cell transplantation (HSCT), but transplanted patients can develop picture resembling spinocerebellar degeneration in early adult life. Depending on the type of mutation, CHS can vary from a most severe childhood form with null-mutations to milder adult onset forms with hypomorphic mutations. We report on a previously healthy boy, who presented at the age of 3 years with life threatening features of HLH but no clinical features of CHS. The patient was treated with HSCT from an unrelated HLA-identical stem cell donor 4 years ago and is developing normally since. Analysis of HLH candidate genes did not result in the identification of the genetic cause at that time. At the time of the next pregnancy whole exome sequencing of DNA that had been obtained before HSCT was performed to enable specific genetic counseling. The Agilent SureSelect Target Enrichment Kit was used and the captured fragments were sequenced as 100 bp paired reads using an Illumina HiSeq2000 sequencing instrument. All sequenced DNA reads were preprocessed using Trimmomatic (Lohse et al. 2012) to clip adapter contaminations and to trim reads for low quality bases. The remaining reads greater than 36bp were mapped to build hg19 of the human reference genome with Stampy (Lunter & Goodson, 2011), using default parameters. Following such preprocessing, the number of mapped reads was 〉 95% for all samples. Single-nucleotide variants (SNVs) were called using SAMtools mpileup (Li et al. 2009). The number of exonic SNVs varied between 23,741 and 31,418 per sample. To facilitate a fast classification and identification of candidate driver mutations, all identified coding SNVs were comprehensively annotated using the ANNOVAR framework (Wang et al., Nat. Rev. Genet., 2010). To identify possible pathogenic mutations, candidate SNVs were filtered for nonsynonymous, stopgain or stoploss SNVs, requiring an SNV quality greater or equal to 100, and requiring absence of segmental duplications. Only SNVs that were not contained in dbSNP were considered for further analysis. No homozygous and 122 heterozygous SNVs meeting those requirements were identified. Only one gene, LYST, was affected by two different SNVs and was selected for further analysis because of its known relationship to HLH. Sanger sequencing confirmed the compound heterozygous genotype for the two novel LYST missense mutations Q3057K and R3785H in the patient and the heterozygous genotype for one of these mutations in the parents. We then specifically searched for typical features of CHS in the pre-HSCT diagnostic material. The typical large lysosomal granules in blood cells could not be identified. By contrast, light microscopy of the patient’s hair showed a silvery aspect and chunky dyspigmentation in the medulla. Little granular melanin was detected in the hair cortex. Electron microscopy revealed an uneven distribution of pigment and giant melanosomes in some keratinocytes, compatible with a partial albinism. We thus conclude that this patient suffers from an incomplete albeit immunologically most severe Chediak-Higashi syndrome, which led to an early accelerated phase resembling primary HLH. This report highlights the diagnostic power of whole exome sequencing, which enables an unbiased mutation analysis and the identification of unexpected causes of genetic diseases with atypical phenotypes. At the same time, this case also highlights some of the ethical challenges associated with diagnostic genomic analyses: While a specific and clinically validated diagnosis enabled specific genetic counseling, the family now has to face the unexpected uncertainty about the neurologic prognosis of incomplete Chediak-Higashi-syndrome, which may possibly progress into untreatable neurodegeneration during early adulthood despite successful allogeneic stem cell transplantation. Apart from adding to the knowledge of the genetic and phenotypic complexity of CHS, this patient also underlines the necessity of careful counseling before diagnostic genomic analyses are offered to patients and their families. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V122.21.3479.3479
    RVK:
    XA 33000
    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
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  • 8
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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
    Details
    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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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2018
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  • 9
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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
    detail.hit.zdb_id: 1468538-3
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  • 10
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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:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2018
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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