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1
Ying, Hsia-Yuan, Yanwen Jiang, Ana Ortega-Molina, Huimin Geng, Dylan McNally, Ling Wang, Ashley Doane, et al. "Crebbp Mutations Disrupt Dynamic Enhancer Acetylation in B-Cells, Enabling HDAC3 to Drive Lymphomagenesis." Blood 128, no. 22 (December 2, 2016): 735. http://dx.doi.org/10.1182/blood.v128.22.735.735.
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Abstract Somatic mutations in CREBBP occur frequently in germinal center derived lymphomas such as DLBCL and FL. However whether or how these mutations might contribute to lymphomagenesis is still largely unknown. Most CREBBP mutations are predicted to result in loss of function since they target the histone acetyltransferase (HAT) domain or give rise to premature stop codon prior to the HAT domain. Here, we show that Crebbp shRNA knockdown (KD) accelerated lymphomagenesis in VavP-Bcl2 transgenic mice, a model that recapitulates human GC-derived lymphomas. The median time to lymphoma onset in VavP-Bcl2/CrebbpKD mice was 114 days, significantly shorter than control VavP-Bcl2/GFP mice (193 days, p=0.04). Histopathology revealed that VavP-Bcl2/CrebbpKD lymphomas were more aggressive and widely disseminated than VavP-Bcl2/GFP lymphomas. CREBBP can regulate gene enhancer function through H3K27 acetylation. ChIP-seq in VavP-Bcl2/CrebbpKD lymphoma cells revealed significant reduction of H3K27ac peaks compared to control lymphoma cells (N = 1717, Kolmogorov-Smirnov test, p<2.2E-16). Loss of H3K27ac was markedly skewed towards enhancers. We observed similar loss of enhancer H3K27ac in human DLBCL cells after CREBBP shRNA KD. Enhancer H3K27ac loss was significantly associated with repression of nearby genes in both murine (FDR q=0.044) and human lymphoma cells (FDR q=0). RNA-seq performed in three independent FL or DLBCL patient cohorts revealed a characteristic CREBBP mutant gene expression signature featuring prominent transcriptional repression (p=1.32E-14, p=0.001, and p=0.0002 respectively). Notably, the human patient CREBBP mutant signature was highly enriched in murine and human cell line CREBBP shRNA profiles (FDR=0, GSEA), indicating that CREBBP KD signature was highly similar to CREBBP mutant signature in humans. Functional analysis of the CREBBP mutant/KD signature showed significant enrichment of GC exit pathways including genes induced by CD40, IRF4 and plasma cell differentiation; as well as immune response processes including antigen processing and presentation, such as MHC class II genes (BH-adjusted p<0.05). To better understand mechanism we performed an integrative analysis of CREBBP signatures against databases of B-cell transcription factor and epigenome profiles. This analysis yielded significant enrichment (BH-adjusted p<0.05) for i) enhancers bound by the BCL6 transcriptional repressor and its SMRT/HDAC3 corepressor complex, ii) enhancers that are normally deacetylated in GC B-cells, and iii) genes induced by BCL6 siRNA. This is notable because in normal GCs BCL6 represses enhancers by recruiting SMRT/HDAC3 complexes to deacetylate H3K27. Hence our data suggest that CREBBP is a counteracting HAT to BCL6/SMRT/HDAC3. Indeed, conditional knockout of Hdac3 in GC B-cells in mice resulted in impaired GC formation and a transcriptional signature featuring upregulation of the same genes that are repressed by CREBBP KD (GSEA FDR=0). Moreover, CREBBP KD in DLBCL cells resulted in H3K27ac loss at BCL6/SMRT/HDAC3 regulated enhancers, including those nearby CDKN1A, NFATC1, FOXP1, and MHC II genes, such as HLA-DQA1 and HLA-DRB5. CREBBP KD also resulted in silencing of these genes. Since we show HDAC3 is the opposing HDAC to CREBBP then we reasoned that CREBBP mutant DLBCLs might be especially dependent on HDAC3. Indeed we observed that HDAC3 shRNA resulted in profound suppression of CREBBP mutant DLBCL cells in vitro and in vivo (DLBCL xenografts in mice, p=0.005), whereas CREBBP WT cell lines were barely affected by HDAC3 KD. The opposing effects of BCL6/SMRT/HDAC3 and CREBBP on MHC class II could have implications for immune surveillance. Accordingly CREBBP KD induced significant loss of cell surface HLA-DR molecules (p<0.05), and these cells exhibited up to 90% less capability to stimulate T-cell response in allogeneic mixed lymphocyte reaction experiments. The loss of MHC class II molecules and T-cell response was rescued when CREBBP loss of function cells were exposed to a specific HDAC3 inhibitor. In summary, CREBBP mutations drive lymphomagenesis by enabling unopposed suppression of enhancers by BCL6/SMRT/HDAC3 complexes, resulting in a repressive transcriptional programming that disrupts GC exit and evades immune surveillance. HDAC3 targeted therapy may rescue these effects and serve as a precision approach for CREBBP mutant lymphomas. Disclosures Scott: Celgene: Consultancy; Roche: Honoraria; Janssen: Consultancy; BC Cancer Agency: Patents & Royalties: Inventor on a patent licensed to NanoString Technologies. Tam:Millennium Pharmaceuticals, Inc.: Consultancy. Melnick:Janssen: Research Funding.
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Schroers-Martin, Joseph G., Joanne Soo, Gabriel Brisou, Florian Scherer, David M. Kurtz, Brian Sworder, Michael S. Khodadoust, et al. "Recurrent Crebbp Mutations in Follicular Lymphoma Appear Localized to the Committed B-Cell Lineage." Blood 136, Supplement 1 (November 5, 2020): 30–31. http://dx.doi.org/10.1182/blood-2020-142761.
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Background: Follicular lymphoma (FL) is genetically characterized by translocations involving the BCL2 locus on chromosome 18q21. However, up to 70% of healthy individuals also carry detectable t(14;18)-positive cells, suggesting BCL2 translocation is critical but not sufficient for FL development. Chromatin modifying genes (CMGs) including KMT2D, CREBBP, EZH2, and EP300 are almost ubiquitously mutated in FL. We previously reported the direct characterization by ultra-deep sequencing of pre-diagnostic blood and tissue specimens from 19 subjects who ultimately developed FL. CREBBP lysine acetyltransferase (KAT) domain mutations were the most commonly observed precursor lesions, detected in blood a median of 7.5 years before diagnosis in patients developing FL (8/19, 42%) but not in healthy adults with or without detected BCL2 translocations (0/13, p=0.01 and 0/20, p<0.001, respectively) (Schroers-Martin et al, ASH Annual Meeting 2017). While the BCL2 translocation is thought to occur in pre-B-cell precursors in the bone marrow, the high prevalence of CMG mutations raises the possibility that, analogous to myeloid age-related clonal hematopoiesis, such early lesions could occur in hematopoietic stem cells (HSCs) rather than in the committed B-cell lineage. Methods: To address this question of mutational hierarchy, we studied blood, bone marrow aspirate, and lymph node samples from 6 patients with CREBBP or KMT2D mutations in their FL tumor. We analyzed flow-sorted purified hematopoietic cell populations by deep sequencing, including isotype-specific mature B-cell, mature T-cell, and precursor populations encompassing HSCs and common lymphoid progenitors (CLPs). Patients additionally underwent routine clinical sequencing of tumor biopsy and marrow specimens. Results: Bulk sequencing of a diagnostic bone marrow aspirate from patient FL002 revealed a CREBBP mutation concordant with FL tumor biopsy. To ascertain the population bearing this mutation, we sorted to high purity viable marrow aspirate cells (Fig. A). The CREBBP mutation was confirmed in the mature B-cell compartment at an AF of 40.3% but was not detected in other cell populations. To validate this finding, a similar sorting strategy was employed on viable bone marrow aspirate or peripheral blood samples from another 4 FL patients bearing CREBBP mutations (Fig B). In each case, CREBBP was absent from the CD34+ precursor population. In patient CIML004 a characteristic KMT2D stop mutation was likewise absent in precursors. An atypical case sheds additional light on the localization of early FL mutations. Patient LYM267 was diagnosed with Grade 1-2 FL bearing CREBBP and NRAS mutations. Eight years into a prolonged remission after chemoimmunotherapy, he developed cutaneous and gingival myeloid sarcoma without radiographic or histopathological evidence of FL recurrence. While the CREBBP mutation was not detected in myeloid sarcoma or bone marrow biopsies, concordant NRAS mutations and clonal VDJ rearrangements were seen in all 3 compartments (Fig. C). This unusual clonal lineage favors the occurrence of the CREBBP mutation later than the branch point between morphologically distinct lymphoid and myeloid tumors, likely in the committed B-cell lineage after pre-BCR rearrangements (Fig D). Conclusions: HSCs are believed to be the cell of origin in several lymphoid leukemias, and mouse models have demonstrated lymphoma development with induced CREBBP lesions in HSCs (Horton et al Nat Cell Bio 2017). However, in sorted hematopoietic cell populations from marrow and peripheral blood, we observed CREBBP mutations in B-cell lineages but never in CD34+/CD20- precursor populations or paired lymphoid/myeloid disease. Our data therefore are not in support of HSCs as a precursor reservoir in FL. Given that cells harboring the t(14;18) translocation in healthy individuals appear derived from the germinal center, recurrent mutations in CREBBP are likely to occur after the pre-B-cell stage. Figure Disclosures Kurtz: Genentech: Consultancy; Foresight Diagnostics: Other: Ownership; Roche: Consultancy. Khodadoust:Kyowa Kirin: Consultancy; Seattle Genetics: Consultancy. Nadel:Innate Pharma: Research Funding; Institut Roche: Research Funding. Diehn:RefleXion: Consultancy; Varian Medical Systems: Research Funding; Illumina: Research Funding; Roche: Consultancy; BioNTech: Consultancy; AstraZeneca: Consultancy. Roulland:Celgene/BMS: Research Funding; Roche: Honoraria. Alizadeh:Pharmacyclics: Consultancy; Genentech: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Chugai: Consultancy; Gilead: Consultancy; Pfizer: Research Funding; Roche: Consultancy.
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Tokunaga, Kenji, Shunichiro Yamaguchi, Eisaku Iwanaga, Tomoko Nanri, Taizo Shimomura, Hitoshi Suzushima, Mitsuya Hiroaki, and Norio Asou. "Crebbp HAT Domain Mutations Are Frequently Detected in Adult Acute Lymphoblastic Leukemia." Blood 120, no. 21 (November 16, 2012): 1419. http://dx.doi.org/10.1182/blood.v120.21.1419.1419.
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Abstract Abstract 1419 Aims: Molecular pathogenesis of acute lymphoblastic leukemia (ALL) has largely been verified in pediatric patients and the identification of genetic alterations have contributed to stratifying therapeutic applications. In adult patients with ALL, cytogenetic and genetic abnormalities have not sufficiently been elucidated and therapeutic improvement has been hindered. CREB binding protein (CREBBP) is a transcriptional coactivator that interacts with a diverse range of transcription factors and regulates transcription by histone acetylation in hematopoiesis. Mutations of the CREBBP gene are recently found in approximately 2–4% of pediatric patients with ALL. Especially in relapsed cases, the mutations prevail (18–63%) and are possible markers for prediction of relapse in pediatric ALL. In adult patients with ALL, the clinical significance of CREBBP mutations remains to be determined. Here we examined adult ALL patients in an attempt to determine the incidence, clinical characteristics and prognostic impact of the CREBBP mutations. Methods: We investigated 71 adult patients with newly diagnosed ALL treated with JALSG protocols between 1986 and 2010. Age ranged from 15 to 86 years, with a median of 54 years. CREBBP mutations are dominantly identified in histone acetyltransferase (HAT) domain. HAT domain in the CREBBP gene was amplified with RT-PCR using RNA isolated from the peripheral blood or bone marrow mononuclear cells at diagnosis and was subjected to direct sequencing. We compared clinical profiles between patients with and without CREBBPHAT domain mutations. This study was approved by the Institutional Review Boards and informed consent was obtained from each patient according to guidelines based on the revised Declaration of Helsinki. Results: CREBBP HAT domain mutations were detected in 8 of 71 (11.3%) patients: one nonsense mutation, five insertion mutations with frameshifts, and five missense mutations. Two patients harbored biallelic mutations. The mutations at diagnosis in adult patients were seen more frequently than those in pediatric patients ever reported. Such mutations were not completely identical to those detected in pediatric ALL, but were seen in the region within the HAT domain, indicating that such mutations are loss-of-function mutations. The mutations were found in both B-cell (6/53: 11.3%) and T-cell (1/9: 11.1%) ALL, and distributed in patients harboring IKZF1 alterations (3/31: 9.7%) or the BCR-ABL fusion gene (2/19: 10.5%). There were no statistical difference in age, sex, leukocyte, platelet counts and complete remission rate between patients with and without the CREBBP HAT domain mutations. Patients with the mutations had a trend with worse cumulative incidence of relapse (P=0.4637), relapse-free survival (P=0.4195) and OS (P=0.2349) compared to patients lacking the mutations, but statistical significance was not detected in this small cohort. Conclusions: CREBBP HAT domain mutations at diagnosis in adult ALL are found more frequently than in pediatric ALL. This may be one of the mechanisms that adult ALL has been associated with poor OS compared with pediatric ALL. In this study, CREBBP HAT domain mutations were observed in various subtypes of ALL: both B-cell and T-cell ALL, and both Philadelphia chromosome positive and negative ALL. In pediatric ALL, CREBBP mutations were frequently seen in relapsed patients but not in previously untreated patients. These observations suggest that CREBBP mutations play an important role in an additional late event(s) leading to the development and progression of ALL. Our study implies the possibility that mutations of the CREBBP gene are associated with the pathogenesis and prognostic marker of adult ALL and represent specific epigenetic modifiers in adult ALL, serving as potential therapeutic targets. Disclosures: No relevant conflicts of interest to declare.
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Idoia, García-Ramírez, Shashank Shrishrimal, Ines Gonzalez-Herrero, Alberto Martín-Lorenzo, Guillermo rodriguez-Hernandez, Romain Duval, Dalia Moore, et al. "CREBBP Loss Cooperates with BCL2 Over-Expression to Promote Lymphoma in Mice." Blood 128, no. 22 (December 2, 2016): 458. http://dx.doi.org/10.1182/blood.v128.22.458.458.
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Abstract Follicular lymphoma (FL) is genetically characterized by translocations of the BCL2 oncogene that are found in ~90% of patients, and mutations of chromatin modifying genes that are found in up to 96% of patients. The latter include inactivating mutations of KMT2D and CREBBP, and activating mutations of EZH2, among others. However, CREBBP has yet to be investigated using this approach. We recently defined the evolutionary hierarchy of somatic mutations in FL and found that CREBBP mutations were most frequently acquired as early events during disease evolution and were maintained throughout disease progression and transformation. Recent studies, using transgenic mouse models, have shown that inactivation of KMT2D and introduction of the activating EZH2 mutation results in perturbed B-cell development and lymphomagenesis. Here, we extended upon these observations by performing targeted next generation sequencing of an additional cohort of tumors allowing the identification of the spectrum of CREBBP mutations across 200 FLs. This identified CREBBP mutations in 55% of tumors, and found that 31% of these mutations reside within the lysine acetyltransferase domain. Furthermore, 30% of mutations altered a single amino acid, arginine 1408, to either a cysteine or histidine residue. We performed a sensitive in vitro acetyltransferase assay for these point mutants and show that they result in >90% loss of catalytic activity. As our results show that CREBBP mutations result in a loss of function, we modeled these events in mice by floxing one or both alleles of Crebbp and crossing with the Mb1-cre strain. This yielded mice that deleted Crebbp specifically in B-cells. We additionally crossed these mice with the EµBcl2 strain that over-expresses Bcl2 in B-cells. Inactivation of Crebbp in B-cells was associated with deficits in B-cell development, with significantly reduced numbers of total B-cells that were contributed to by reductions in multiple B-cell subsets. These deficits were partially rescued by the EµBcl2 transgene. After 14-21 months, some mice became ill and necropsy revealed lymphadenopathy and splenomegaly as a result of B-cell lymphoma. We noted increased penetrance and decreased latency of lymphoma with one vs two alleles of Crebbp deleted, and with absence vs presence of the EµBcl2 transgene (Figure 1). We investigated the molecular etiology of these tumors by isolating splenic B-cells from these mice and performing transcriptome profiling and epigenetic profiling for the histone H3 lysine 18 acetylation (H3K18Ac) mark that is catalyzed by Crebbp. Transcriptional profiling identified a signature of 335 genes with increased expression and 370 genes with decreased expression, including an incremental increase in Myc expression when one or both alleles of Crebbp were deleted, respectively. Surprisingly, changes in transcript abundance were not associated with changes in H3K18Ac in the proximal regulatory regions of those genes. Regions of significantly altered H3K18Ac were instead localized primarily to intragenic regions. Analysis of the DNA sequences in these regions identified a significant enrichment of motifs that contained Myc consensus sequences, and these were present in >60% of regions with altered H3K18Ac. In addition, ChIP-seq data from the ENCODE database showed a strong level of Myc binding to the center of these regions with altered H3K18Ac. Together, our results demonstrate that inactivating mutations of Crebbp may have a role in altering B-cell development. The significant induction of Myc expression that was associated with Crebbp deletion, and epigenetic changes in regions that are bound by Myc, suggest that Crebbp inactivation may have a role in the induction of Myc expression and activity. This may be important with respect to transformation of FL, which may proceed via induction of MYC. However, our results also demonstrate some important discrepancies between the role of CREBBP mutations in human FL, and the role of Crebbp deletion in murine models. Disclosures Lunning: Celgene: Consultancy; Spectrum: Consultancy; TG Therapeutics: Consultancy; Gilead: Consultancy; Genentech: Consultancy; Juno: Consultancy; Bristol-Myer-Squibb: Consultancy; AbbVie: Consultancy; Pharmacyclics: Consultancy.
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Chafai Elalaoui, Siham, Wiam Smaili, Julien Van-Gils, Patricia Fergelot, Ilham Ratbi, Mariam Tajir, Benoit Arveiler, Didier Lacombe, and Abdelaziz Sefiani. "Clinical description and mutational profile of a Moroccan series of patients with Rubinstein Taybi syndrome." African Health Sciences 21, no. 2 (August 2, 2021): 960–67. http://dx.doi.org/10.4314/ahs.v21i2.58.
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Background: Rubinstein-Taybi syndrome (RSTS; OMIM 180849) is a rare autosomal dominant developmental disorder with an estimated prevalence of one case per 125,000 live births. RSTS is characterized by typical face, broad thumbs and halluces, short stature, and intellectual disability. Facial dysmorphy is characteristic with microcephaly, low frontal hairline, arched eyebrows, long eyelashes, convex profile of nose, narrow palate, and micrognathia. RSTS is mainly due to mutations or microdeletions of the CREBBP gene (about 60%) and more rarely of the EP300 gene (8%).
Objective: Clinical description and identification of mutations of patients with Rubinstein Taybi syndrome.
Methods: PCR and direct sequencing of CREBBP gene.
Results: We report here, the clinical and molecular data of a series of six Moroccan patients with a phenotype of RSTS. The molecular study of the major gene CREBBP (by Sanger Sequencing followed by CGH array, if sequence normal) revealed point mutations in five patients. For the sixth patient, CGH array revealed a microdeletion carrying the CREBBP gene. Through this work, we emphasize the importance of clinical expertise in the diagnosis, management and genetic counseling in Rubinstein Taybi syndrome.
Keywords: Rubinstein Taybi syndrome; CREBBP gene; mutation; Moroccan.
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Green, Michael R., Shingo Kihira, Chih Long Liu, Ramesh V. Nair, Raheleh Salari, Andrew J. Gentles, Jonathan Irish, et al. "Mutations in early follicular lymphoma progenitors are associated with suppressed antigen presentation." Proceedings of the National Academy of Sciences 112, no. 10 (February 23, 2015): E1116—E1125. http://dx.doi.org/10.1073/pnas.1501199112.
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Follicular lymphoma (FL) is incurable with conventional therapies and has a clinical course typified by multiple relapses after therapy. These tumors are genetically characterized by B-cell leukemia/lymphoma 2 (BCL2) translocation and mutation of genes involved in chromatin modification. By analyzing purified tumor cells, we identified additional novel recurrently mutated genes and confirmed mutations of one or more chromatin modifier genes within 96% of FL tumors and two or more in 76% of tumors. We defined the hierarchy of somatic mutations arising during tumor evolution by analyzing the phylogenetic relationship of somatic mutations across the coding genomes of 59 sequentially acquired biopsies from 22 patients. Among all somatically mutated genes, CREBBP mutations were most significantly enriched within the earliest inferable progenitor. These mutations were associated with a signature of decreased antigen presentation characterized by reduced transcript and protein abundance of MHC class II on tumor B cells, in line with the role of CREBBP in promoting class II transactivator (CIITA)-dependent transcriptional activation of these genes. CREBBP mutant B cells stimulated less proliferation of T cells in vitro compared with wild-type B cells from the same tumor. Transcriptional signatures of tumor-infiltrating T cells were indicative of reduced proliferation, and this corresponded to decreased frequencies of tumor-infiltrating CD4 helper T cells and CD8 memory cytotoxic T cells. These observations therefore implicate CREBBP mutation as an early event in FL evolution that contributes to immune evasion via decreased antigen presentation.
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Meyer, Stefanie, Sofija Vlasevska, Laura Garcia Ibanez, Claudio Scuoppo, Riccardo Dalla-Favera, and Laura Pasqualucci. "Targeting Histone Acetyltransferase Gene Inactivation in Diffuse Large B Cell Lymphoma." Blood 132, Supplement 1 (November 29, 2018): 671. http://dx.doi.org/10.1182/blood-2018-99-117542.
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Abstract Diffuse Large B-cell Lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma, accounting for ~30% of de-novo diagnoses and also arising as a frequent clinical evolution of indolent lymphomas. Although curable in a substantial fraction of cases, one third of patients do not achieve durable remissions, highlighting the need for novel, targeted therapies. Over the past decade, we and others have identified the CREBBP acetyltransferase and, less frequently, its paralogue EP300 as highly recurrent targets of inactivating somatic mutations/deletions in DLBCL and follicular lymphoma (FL) (30% and 60% of patients, respectively), indicating a prominent role in the pathogenesis of these tumors (Pasqualucci et al., Nature 2011). In most cases, mutations are heterozygous and the residual wildtype (WT) allele is expressed, suggesting a haploinsufficient tumor suppressor role. Indeed, germinal center (GC)-specific loss of Crebbp perturbs the expression of genes that are relevant to the normal biology of this structure, i.e. the lymphoma cell of origin, and cooperates with BCL2 deregulation to increase the incidence of tumors recapitulating the features of the human disease (Zhang et al., Cancer Discovery 2017). Intringuingly, while CREBBP binds to virtually all GC-specific superenhancers, no detrimental effects were observed upon its deletion in mice, suggesting the existence of compensatory mechanisms. Consistent with this hypothesis, inactivation of CREBBP and EP300 rarely coexist in human DLBCL and FL, suggesting that cells require a certain amount of acetyltransferase activity. To investigate whether EP300 compensates for CREBBP loss in the GC, we analyzed the GC responses in compound mouse models engineered to specifically delete these two genes (alone and in combination) upon SRBC immunization and induction of a Cγ1-driven Cre-recombinase. While CrebbpKOmice showed a mild increase in GC formation, as reported, loss of Ep300 led to ~40% reduction in the percentage of GC cells (mean: 1.8% vs 3.1% in WT littermates; p<0.05), documenting that these two enzymes play non-entirely overlapping roles in this population. Importantly, GC formation was completely abrogated in CrebbpKOEp300KO mice and dramatically impaired in CrebbpHETEp300KO mice, as compared to both WT and single EP300KO mice. These data suggest that GC B cells require a minimum amount of acetyltransferase activity, and reveal a potential therapeutically exploitable dependency of Crebbp-mutated GC B cells on Ep300. In order to probe if a similar dependency exists in neoplastic GC B cells, we used an inducible CRISPR/Cas9 system to delete EP300 (or a control non-genic region) in 4 DLBCL cell lines representative of the various CREBBP genotypes found in DLBCL, and monitored cell proliferation and survival in competition assays over 12 days. Compared to CREBBPWT, CREBBP heterozygous and homozygous mutant cells were significantly counter-selected from the total population following doxycycline induced EP300 deletion (~30% at day 7). Moreover, no EP300-edited clones were recovered from the CREBBP mutant lines in single cell plating assays, compared to CREBBP WT (p<0.01). Thus, DLBCL cells remain addicted to the residual EP300 aceyltransferase activity, supporting the existence of a therapeutic window for EP300 inhibitors. To explore this concept further, we generated isogenic DLBCL clones carrying WT or defective CREBBP alleles (n=4 each), and performed drug-sensitivity assays with 2 novel small molecule inhibitors that specifically target the CREBBP/EP300 HAT or BRD domain. While, at higher doses, both inhibitors interfered with cell growth in all clones, CREBBPKO cells were significantly more sensitive than their isogenic WT pairsat low nanomolar ranges (IC50: 60nM vs 300nM). Importantly, we were able to design an in vitro protocol that was toxic to CREBBPKO cells but tolerated by CREBBPWT cells, providing a proof of concept for therapeutically targeting these molecules. In conclusion, we show that CREBBP and EP300 have differential roles in normal GC B cell development and that CREBBP mutated cells are addicted to the residual EP300 activity. This dependency is maintained in DLBCL cells, providing the basis for the potential application of acetyl transferase inhibition into the clinical settings. Disclosures No relevant conflicts of interest to declare.
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Mullighan, Charles, Jinghui Zhang, Lawryn H. Kasper, Stephanie Lerach, Debbie Payne-Turner, Letha A. Phillips, J. Racquel Collins-Underwood, et al. "CREBBP Mutations In Relapsed Acute Lymphoblastic Leukemia." Blood 116, no. 21 (November 19, 2010): 413. http://dx.doi.org/10.1182/blood.v116.21.413.413.
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Abstract Abstract 413 Relapsed acute lymphoblastic leukemia (ALL) is a leading cause of death due to disease in young people, but the biologic determinants of treatment failure remain poorly understood. To identify novel sequence mutations contributing to relapsed in ALL, we resequenced 300 genes in matched diagnosis and relapse samples from 23 patients with ALL. The cohort included B-progenitor ALL with high hyperdiploidy (N=3), TCF3-PBX1 (N=1), ETV6-RUNX1 (N=3), rearrangement of MLL (N=3), BCR-ABL1 (N=3), and low hyperdiploid, pseudodiploid, or miscellaneous karyotypes (N=10). This identified 52 somatic non-synonymous mutations in 32 genes, many of which were novel, including mutations in the transcriptional coactivators CREBBP and NCOR1, the transcription factors ERG, SPI1, TCF4 and TCF7L2, components of the Ras signalling pathway, histone genes, genes involved in histone modification (CREBBP and CTCF), and genes previously shown to be targets of recurring DNA copy number alteration in ALL. Analysis of an extended cohort of 63 diagnosis-relapse cases and 200 acute leukaemia cases that did not relapse found that 19% of relapse cases had sequence or deletion mutations of CREBBP, which encodes the transcriptional coactivator and histone acetyltransferase (HAT) CREB-binding protein (CBP). The mutations were either present at diagnosis, acquired at relapse, or duplicated to homozygosity at the time of relapse. Moreover, several mutations acquired at relapse were detected in subclones at diagnosis, suggesting that the mutations confer a selective advantage and promote resistance to therapy. The mutations either resulted in truncated alleles or deleterious substitutions in highly conserved residues of the HAT domain. To examine the functional consequences of the mutations, we introduced wild type or mutant Crebbp alleles into Cbp/Ep300flox/flox murine embryonic fibroblasts, (dKO MEFs), and examined histone acetylation, expression of CREBBP target genes, and cellular proliferation. The HAT domain mutations resulted in impaired acetylation of the key Crebbp substrate, H3K18, and resulted in impaired transcriptional regulation of multiple CREBBP targets and pathways, including cAMP, dsRNA and dexamethasone responsive genes. The latter observation suggests that CREBBP mutations may directly result in resistance to corticosteroid therapy, which is a hallmark of high risk ALL. Together, these data these results extend the landscape of genetic alterations in leukemia, and identify mutations targeting transcriptional and epigenetic regulation as a mechanism of resistance in ALL. Disclosures: Pui: EUSA Pharma: Honoraria; Enzon: Honoraria; Sanofi-Aventis: Honoraria.
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Tokunaga, Kenji, Shunichro Yamaguchi, Taizo Shimomura, Hitoshi Suzushima, Yutaka Okuno, Hiroaki Mitsuya, and Norio Asou. "Accumulation Of Gene Alterations Of TP53, Crebbp and IKZF1 Is a Prognostic Factor In Adult Acute Lymphoblastic Leukemia." Blood 122, no. 21 (November 15, 2013): 1386. http://dx.doi.org/10.1182/blood.v122.21.1386.1386.
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Abstract Aims Mutations of the genes associating with cell differentiation or proliferation are recognized as factors of tumorigenesis or prognosis in hematological malignancies. In pediatric acute lymphoblastic leukemia (ALL), alterations of IKZF1 (a factor of lymphocyte differentiation), TP53 (a cell cycle regulator) and CREBBP (a histone modifier) are found as possible prognostic markers for stratification of treatments. On the other hand, in adult ALL, clinical significance of such alterations remains to be determined. In the present work, we examined whether the mutations in those genes affected the incidence and prognosis in adult ALL patients. Methods We investigated 87 adult patients with newly diagnosed ALL treated with JALSG protocols between 1986 and 2011. Age ranged from 15 to 86 years, with a median of 51 years. We obtained cDNA and genomic DNA from the peripheral blood or bone marrow mononuclear cells at diagnosis. CREBBP mutations are dominantly identified in the histone acetyltransferase (HAT) domain. HAT domain in the CREBBP gene was amplified by PCR using cDNA and was subjected to direct sequencing. Additionally other histone modifiers, EZH2, EED, and UTX, were sequenced as the same as in CREBBP. TP53 exons 5 – 8 and 10, in which mutations were commonly reported, were sequenced using genomic DNA. We amplified IKZF1 using RT-PCR for detecting aberrant dominant negative isoforms: Ik6 and Ik10. Genomic deletions of IKZF1 were assessed with RQ-PCR or genomic DNA PCR. We compared clinical profiles between patients with and without such gene mutations. The present study was approved by the Institutional Review Boards and informed consent was obtained from each patient according to guidelines based on the revised Declaration of Helsinki. Results In 87 adult patients with ALL, alterations of CREBBP, EED, TP53 and IKZF1 were detected in 7 (9.5%), 3 (4.8%), 6 (6.9%) and 42 (50%), respectively. None of EZH2 and UTX mutation was found. The alterations of CREBBP and IKZF1 at diagnosis in adult patients were more frequent than those in pediatric patients ever reported. Some gene mutations were not found frequently. Each gene mutation per se did not significantly affect prognosis. We tried to predict the prognosis by scoring gene mutations and chromosomal abnormalities. Philadelphia chromosome (Ph) has great impact to prognosis of patients with ALL. We scored the number of mutated genes and Ph for each patient. As the score was higher, adult patients with ALL had poorer relapse-free survival (P=0.0439) and OS (P=0.4819), but statistical significance was not detected in this small cohort. Conclusions and Discussion Single gene mutations, such as IKZF1, can predict the prognosis in pediatric ALL. In adult ALL, however, only few gene mutations are reported to be promising prognostic factors which have impacts to treatment outcomes. Scoring system may be a useful method for predicting prognosis and stratifying treatment in adult ALL. Our study implies the possibility that a variety and heterogeneity of genetic alterations in adult ALL are associated with the pathogenesis for treatment resistance and prognostic marker of adult ALL. Disclosures: No relevant conflicts of interest to declare.
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Dixon, Zach, Julie A. E. Irving, and Lindsay Nicholson. "Crebbp K nockdown Does Not Impact on Glucocorticoid Induced Apoptosis in Childhood Acute Lymphoblastic Leukemia." Blood 126, no. 23 (December 3, 2015): 1429. http://dx.doi.org/10.1182/blood.v126.23.1429.1429.
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Abstract Childhood acute lymphoblastic leukemia (ALL) is the most common childhood cancer and, despite a cure rate approaching 90%, relapse is a significant cause of death in young people. Recently it has been shown that inactivating mutations in the histone acetyltransferase, CREB binding protein (CREBBP or CBP) are frequently seen at relapse in childhood ALL, with enrichment in high hyperdiploid and hypodiploid cases. Mutations are usually heterozygous, suggesting haploinsufficiency, and are often acquired at relapse, implying a role in drug resistance. Since glucocorticoid (GC) response genes are known targets of CREBBP and, given the pivotal role of GCs in ALL therapy, it has been postulated that CREBBP mutations confer GC chemoresistance. CREBBP is a multifunctional protein, playing a role in cAMP dependent signalling, acetylation mediated activation of p53 and inactivation of BCL6 and a range of DNA damage repair pathways including base excision repair (BER) and direct DNA damage repair. To assess the role of CREBBP haploinsufficiency in ALL, RNAi techniques were used to create isogenic CREBBP knockdown models of ALL. CREBBP knockdown was carried out using small hairpin RNA (shRNA) transduction (termed shCBP cells) or small interfering RNA (siRNA) transfection (termed siCBP cells) in the PreB 697 B-cell precursor cell line (t(1;19)) and the hypodiploid MHH-CALL-2 cell line, as well as high hyperdiploid primagraft ALL cells. Knockdown of at least 50% of control was confirmed at both mRNA and protein level. The functional impact of CREBBP knockdown in cells was determined by analysis of known CREBBP target residues; acetyl H3K18 and H3K27, and transcription of cAMP dependent genes (CXCR4, MKNK2, DUSP5, DUSP10 and RGS16). To assess the impact of CREBBP knockdown on response to GCs, cells were treated with dexamethasone and expression of the classic glucocorticoid receptor (GR) targets; GILZ and FKBP51, was assessed by quantitative reverse transcriptase PCR (QRT-PCR). Alamar blue cell viability assays were used to determine the sensitivity of each CREBBPknockdown model to dexamethasone compared to isogenic controls. Three out of four cell models displayed a reduction in H3K18 or H3K27 acetylation compared to isogenic control, indicating a relevant functional impact of CREBBP knockdown. Cell lines showed a trend towards reduced induction of some of the selected cAMP dependent targets but statistical significance was not achieved (p values >0.2). Gene expression profiling and Ingenuity Pathway Analysis of PreB 697 shCBP cells compared to isogenic control predicted that upstream transcription of NR3C1, the gene encoding the GR, would be affected in CREBBP knockdown cells. However, while induction of GILZ and FKBP51 in PreB 697 shCBP cells in response to GC was significantly impaired in knockdown compared to control cells (GILZ p=0.009, FKBP51 p=0.03), they were no more resistant to dexamethasone (p=0.9). This was mirrored in siCBP cell lines and primagraft cells, where a significant impairment in basal expression of GILZ and/or FKBP51 was seen in some lines (GILZ reduction; p=0.03 PreB 697 shCBP, p=0.02 PreB 697 siCBP, FKBP51 reduction; p=0.01 primagraft siCBP cells) but no significant impairment in the transcriptional induction of these genes in response to GC compared to isogenic control was observed (p values >0.5). Importantly, no decreased sensitivity to dexamethasone was seen in any model after CREBBP knockdown (p values >0.1). CREBBP knockdown in ALL cells had no significant effect on the induction of cAMP dependent genes, had a variable effect on GR target expression, but consistently showed no impact on GC sensitivity, regardless of cytogenetic context. These data show that the acquisition of CREBBP mutations at relapse in childhood ALL is not mediated through GC resistance and suggest that other CREBBP associated mechanisms, such as DNA damage repair, may influence drug response. Understanding the role of CREBBP in carcinogenesis and drug resistance is crucial as it is implicated as a tumour suppressor in a growing number of cancers, making it a potential multi-tumour target for novel therapies. Disclosures No relevant conflicts of interest to declare.
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Huntly, Brian J. P., Sarah Jayne Horton, George Giotopoulos, Haiyang Yun, Shabana Vohra, Olivia Sheppard, Rachael Bashford-Rogers, et al. "Early Loss of CREBBP Confers Malignant Stem Cell Properties on Lymphoid Progenitors." Blood 128, no. 22 (December 2, 2016): 460. http://dx.doi.org/10.1182/blood.v128.22.460.460.
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Abstract Loss-of-function mutations of the cyclic-AMP response element binding protein, binding protein (CREBBP) gene have recently been described at high frequencies across a spectrum of lymphoid malignancies, particularly follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). The multiple effects of this epigenetic regulator on developmental and homeostatic processes have been extensively studied, however, exactly how CREBBP functions as a tumor suppressor and the reasons for its particular predilection for suppression of lymphoid tumors remains unclear. In addition, for many mature lymphoid malignancies, the existence of cancer stem cells is unproven and their provenance and the initial target cell for transformation a source of ongoing debate. Here we use multiple mouse strains to model loss of Crebbp in different lymphoid compartments to address these questions. We demonstrate that early loss of Crebbp in hematopoietic stem and progenitor cells (HSPC), through disruption by the Mx1-Cre recombinase, leads to increased development of hematological malignancies, particularly of the B-lymphoid lineage that mimic features of human lymphomas. Theses B-cell malignancies are of long latency and are preceded by significant alterations in the proliferation, self-renewal and differentiation of lymphoid progenitors, allowing hyperproliferative lymphoid progenitors whose differentiation is blocked to accumulate. Using an aberrant surface phenotype that resembles the eventual tumor, we identify a pre-malignant population in the peripheral blood of animals that is often evident many months before any disease characteristics. We demonstrate pre-malignant stem cell characteristics for this population in functional experiments, where it generates high level reconstitution of peripheral blood in transplant recipients, but only gives rise to disease in these animals after a long latency. We also utilize this unique cellular population in longitudinal genome scale analyses (clonality, RNA-Seq, ChIP-Seq and exome sequencing) to document the mechanisms of malignant evolution. Linking the increased rate of mutation we describe to Crebbp loss, we also demonstrate increased DNA damage and an altered DNA-damage response in premalignant lymphoid progenitors. Importantly, using a Cd19-Cre recombinase that excises only within committed lymphoid cells, we are able to demonstrate that when Crebbp is lost at a later stage of lymphoid development, the marked cellular abnormalities described above are completely lost and the development of tumors is no different from normal (Figure, below). Taken together, these findings define the developmental stage-specific tumor suppressor functions of Crebbp and shed light on the cellular origins and subsequent evolution of lymphoid malignancies. In addition, the altered response to DNA damage that we demonstrate upon loss of Crebbp, allied to the increased exposure to physiological DNA-damage during lymphoid ontogeny offers an explanation for the high incidence of CREBBP mutations in mature lymphoid malignancies. Figure Left panel, Kaplan Meier graph for Mx1-Cre Crebbp mice with loss of Crebbp in the HSPC compartment demonstrates significantly shorter survival vs WT littermates with intact expression of Crebbp. In contrast, when Crebbp is excised in a later lymphoid compartment through Cd19-Cre mediated recombination, right panel, no difference in survival is noted from WT littermate controls. Figure. Left panel, Kaplan Meier graph for Mx1-Cre Crebbp mice with loss of Crebbp in the HSPC compartment demonstrates significantly shorter survival vs WT littermates with intact expression of Crebbp. In contrast, when Crebbp is excised in a later lymphoid compartment through Cd19-Cre mediated recombination, right panel, no difference in survival is noted from WT littermate controls. Disclosures Huntly: Novartis: Speakers Bureau; BMS: Speakers Bureau; Ariad: Speakers Bureau; Pfizer: Speakers Bureau.
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Ji, Mengmeng. "Histone-Modifying Gene Mutations Are Related to Tumor Progression and Response to Histone Deacetylase Inhibitors in Patients with Peripheral T-Cell Lymphoma Not Otherwise Specified." Blood 128, no. 22 (December 2, 2016): 4110. http://dx.doi.org/10.1182/blood.v128.22.4110.4110.
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Abstract Objective Histone modifications are critically involved in pathogenesis of peripheral T-cell lymphoma not otherwise specified (PTCL-NOS).Histonedeacetylaseinhibitors (HDACIs) exhibited potential anti-lymphoma activity through restoring normal chromatin modification patterns. Here we detected histone-modifying gene mutations in PTCL-NOS and correlated them with tumor progression and therapeutic response to different HDACIs. Methods Mutations of important lymphoma-associated histone-modifying genes (MLL2, TET2, CREBBP, EP300, SETD2 and EZH2) were detected by targeted sequencing in tumor samples of 105 newly diagnosed patients with PTCL-NOS and compared with clinical parameters. Moreover, T-lymphoma cell lines bearing representative mutations were treated with four clinically available HDACIs (valproic acid, VPA;suberoylanilidehydroxamic acid, SAHA;Romidepsin,Romi; andChidamide,Chid). Results A total of 65 histone-modifying gene mutations were identified in 39 of 105 PTCL patients (Figure 1A). As illustrated in Figure 1B, somatic mutations in MLL2 (encoding H3K4-specific methyltransferase) were most frequently observed (20.0%), followed by mutations of TET2 (13.3%), EP300 and CREBBP (encoding H3K18 acetyltransferase, 7.6% and 6.7%, respectively), and SETD2 (encoding H3K36 methyltransferase, 7.6%). No EZH2 mutation was detected. Clinically, mutation-positive PTCL patients presented with shorteroverall survival than patients without mutations (P=0.0038, Figure 1C). Based on the crystal structure of each gene (Figure 1D), most mutations were located on SET domain of MLL2 (p.5397-5513), HAT domain of EP300 (p.1287-1663) and SET domain of SETD2 (p.1550-1667), which could lead to loss of H3K18ac, H3K4me3 and H3K36me3 expression, respectively. Meanwhile, Jurkatcells were treated with VPA, SAHA, Romiand Chidfor 48h and the results showed that all the HDACIs increased the expression of EP300/CREBBP, as compared to the untreated group. Interestingly, VPA and SAHA specifically upregulated SETD2 expression, while Romi and Chid upregulated MLL2 expression (Figure 2A). No obvious change of TET2 expression was found during HDACI treatment. Thereafter,Jurkat cells were transfected with MLL2 mutant (p.V5389M), EP300 mutant (p.H1377R) and SETD2 mutant (p.R1598_). Compared with the WT, mutant reduced the level of H3K18ac, H3K4me3 and H3K36me3 expression, respectively. All the HDACIs restored H3K18 hypo-acetylation induced by EP300 mutant. VPA and SAHA restored H3K36me3 expression inhibited by SETD2 mutant, whileRomi andChid restored H3K4me3 expression inhibited by MLL2 mutant (Figure 2B-2D). Conclusion Our study provided the mutational spectrum of histone-modifying genes in PTCL-NOS. HDACIs targeted differently histone H3 acetylation or methylation modulated by the mutations, suggestive their distinct therapeutic efficiency in PTCL-NOS. Figure. Figure. Figure. Figure. Disclosures No relevant conflicts of interest to declare.
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Mosquera Orgueira, Adrián, Roi Ferreiro Ferro, José Ángel Díaz Arias, Carlos Aliste Santos, Beatriz Antelo Rodríguez, Laura Bao Pérez, Natalia Alonso Vence, et al. "Detection of new drivers of frequent B-cell lymphoid neoplasms using an integrated analysis of whole genomes." PLOS ONE 16, no. 5 (May 4, 2021): e0248886. http://dx.doi.org/10.1371/journal.pone.0248886.
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B-cell lymphoproliferative disorders exhibit a diverse spectrum of diagnostic entities with heterogeneous behaviour. Multiple efforts have focused on the determination of the genomic drivers of B-cell lymphoma subtypes. In the meantime, the aggregation of diverse tumors in pan-cancer genomic studies has become a useful tool to detect new driver genes, while enabling the comparison of mutational patterns across tumors. Here we present an integrated analysis of 354 B-cell lymphoid disorders. 112 recurrently mutated genes were discovered, of which KMT2D, CREBBP, IGLL5 and BCL2 were the most frequent, and 31 genes were putative new drivers. Mutations in CREBBP, TNFRSF14 and KMT2D predominated in follicular lymphoma, whereas those in BTG2, HTA-A and PIM1 were more frequent in diffuse large B-cell lymphoma. Additionally, we discovered 31 significantly mutated protein networks, reinforcing the role of genes such as CREBBP, EEF1A1, STAT6, GNA13 and TP53, but also pointing towards a myriad of infrequent players in lymphomagenesis. Finally, we report aberrant expression of oncogenes and tumor suppressors associated with novel noncoding mutations (DTX1 and S1PR2), and new recurrent copy number aberrations affecting immune check-point regulators (CD83, PVR) and B-cell specific genes (TNFRSF13C). Our analysis expands the number of mutational drivers of B-cell lymphoid neoplasms, and identifies several differential somatic events between disease subtypes.
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Sokol, Ethan, Smruthy Sivakumar, Brennan Decker, Jeffrey Ross, and Priti Hegde. "Abstract P5-13-02: Serially biopsied BRCA1/2 mutant breast tumors frequently acquire alterations in BRCA1, BRCA2, and CREBBP." Cancer Research 82, no. 4_Supplement (February 15, 2022): P5–13–02—P5–13–02. http://dx.doi.org/10.1158/1538-7445.sabcs21-p5-13-02.
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Abstract Introduction Tumors with alterations in BRCA1 or BRCA2 (BRCAm) are sensitive to poly ADP ribose polymerase inhibitors (PARPi), with significant benefit in ovary, breast, prostate, and pancreatic cancers. Despite strong early responses on PARPi, many patients eventually exhibit relapse. Previous literature has identified BRCA1/2 reversion as a common class of acquired alteration in the resistance setting; however, additional resistance pathways have not been well characterized. Here, we examined 100 BRCAm patients who were profiled with serial biopsy during clinical care to identify possible therapy resistance alterations. Methods Comprehensive genomic profiling (CGP) was carried out in a Clinical Laboratory Improvement Amendments (CLIA)-certified, CAP (College of American Pathologists)-accredited laboratory (Foundation Medicine Inc., Cambridge, MA, USA). Tissue biopsy CGP was performed on FFPE blocks examining at least 324 genes for all classes of alterations (FoundationOne® and FoundationOne® CDx). Liquid biopsy CGP was performed examining at least 62 genes (FoundationACT®, FoundationOne®Liquid).One hundred breast cancer patients with baseline BRCAm were serially profiled with CGP testing during routine clinical care (n=50 tissue then liquid; n=50 tissue for both tests). As a comparator, we examined 1,294 patients without a baseline BRCAm (BRCAwt) profiled with serial biopsy (n = 585 tissue then liquid; n=709 tissue for both tests). Co-occurrence analyses (Fisher’s exact) were run on the full research dataset, including 12,198 breast-biopsied (local) and 16,586 metastatic-biopsied samples. All classes of genomic alterations were included in the analysis. Results Of the serially biopsied samples, 7.2% (100/1,394) had baseline BRCAm. In patients with baseline BRCAm, acquired alterations were frequently observed in BRCA1/2 (17%), ESR1 (16%), TP53 (15%), MYC (12%), CREBBP (10%), RB1 (9%), PIK3CA (9%), and NF1 (5%). To understand if any of these are specific to the BRCAm population, we compared the milieu of acquired alterations to a serially biopsied BRCAwt cohort (n=1,294). While many of the alterations were common and shared across both cohorts, likely due to endocrine therapy use, acquired alterations in BRCA1, BRCA2, and CREBBP were specifically enriched in the BRCAm population (all p<0.005). Acquired BRCA1/2 mutations were predicted reversion mutations and spanned a range of mechanisms, including large deletion/rearrangement events that remove the exon of the truncal mutation, point mutations that change a nonsense BRCA1/2 to a missense event, upstream indels that revert the frame of the truncal BRCA1/2 event, and splice site mutations that lead to a skipping of the truncal BRCA1/2. Of note, a majority of cases with a reversion involved a rearrangement event (9/17; 53%). For patients with a baseline BRCA1/2 short variant mutation, there was a trend towards a higher rate of reversion in BRCA2 v BRCA1 (23.1% v 18.5%, p>0.05). Acquired BRCAm were never observed in cases with baseline deletion of BRCA1/2. The high rate of acquired CREBBP alterations was specific to the BRCAm population (10% BRCAm v 1% BRCAwt; p = 0.0013), suggesting a possible role for CREBBP in platinum or PARPi resistance. Consistent with this, BRCAm and CREBBP significantly co-occur in the metastatic setting (Odds ratio, OR = 1.6; p = 0.016) but not in the local setting (OR = 1.1; p = 0.68). Conclusions Analysis of serially biopsied BRCAm breast cases revealed frequent acquisition of BRCA1/2 reversion mutations and CREBBP alterations that are not frequently observed in BRCAwt samples. Additional studies are warranted to investigate the possible role of CREBBP in PARPi therapy resistance. Citation Format: Ethan Sokol, Smruthy Sivakumar, Brennan Decker, Jeffrey Ross, Priti Hegde. Serially biopsied BRCA1/2 mutant breast tumors frequently acquire alterations in BRCA1, BRCA2, and CREBBP [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-02.
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Lamble, Adam J., Robert B. Gerbing, Jenny L. Smith, Rhonda E. Ries, Edward A. Kolb, Todd A. Alonzo, and Soheil Meshinchi. "Crebbp Alterations Are Associated with a Poor Prognosis in De Novo AML." Blood 138, Supplement 1 (November 5, 2021): 3451. http://dx.doi.org/10.1182/blood-2021-154052.
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Abstract Introduction: The translocation, t(8;16)(p11;p13), results in the fusion between KAT6A and CREBBP and has been associated with a poor prognosis in both pediatric and adult acute myeloid leukemia (AML). This lesion has therefore been re-classified as high risk on the active Phase 3 Children's Oncology Group (COG) trial for de novo AML, AAML1831 (NCT04293562). Less is known about the prognostic significance of CREBBP sequence variants. Methods: CREBBP variant status was determined in patients with AML enrolled on 4 successive COG trials for de novo pediatric AML (NCT00003790, NCT00070174, NCT01407757, NCT01371981). Fusions involving CREBBP were prospectively obtained via conventional cytogenetics and retrospectively confirmed via RNAseq. Insertions and deletions (indels) leading to frameshift mutations and single nucleotide variants (SNVs) were retrospectively interrogated via next generation sequencing. Results: Of 2216 patients (age: 0-29.8 years), 55 (2.5%) patients had an alteration involving CREBBP. Sixteen (29%) of these were a fusion involving CREBBP (CREBBP/fus), with KAT6A being the most common translocation partner (n=15) and the remaining translocation involving ANK1. The remaining 39 patients (71%) had a CREBBP mutation (CREBBP/mut), including 19 with an indel (CREBBP/indel) leading to a frameshift mutation and 20 with a SNV (CREBBP/SNV). We compared clinical and biologic characteristics between the three cohorts. CREBBP/fus patients were significantly younger than CREBBP/indel and CREBBP/SNV patients (median ages of 2.6 vs. 7.8 vs. 11.9 years; p=0.027). There was a higher prevalence of t(8;21)/RUNX1-RUNX1T1 in CREBBP/indel patients compared to CREBBP/SNV patients (42.1% vs. 5%; p=0.008). In contrast, CREBBP/SNV patients were more likely to be associated with a normal karyotype (40% vs. 5.3%; p=0.02). There was a similar prevalence of co-occurring high-risk lesions in CREBBP/indel (n=5; CBFA2T3-GLIS2, KMT2A-AFF1, KMT2A-MLLT4, MLLT10-PICALM, NUP98-HOXA9) and CREBBP/SNV (n=7; DEK-NUP214, ETV6-FOXO1, FUS-ERG, NUP98-NSD1, ETV6-MNX1, FLT3-ITDx2) patients. There was otherwise no difference between presenting WBC count, FLT3-ITD, NPM1, CEBPA, remission rates or MRD status after Induction 1 therapy. Patients with any CREBBP alteration had a significantly worse 5-year event free survival (EFS) compared to patients without (25.9% vs. 45.2%; p=0.002) and this inferior EFS overlaps with contemporarily defined high-risk patients (Figure 1a). Evaluation of outcomes based on type of alteration demonstrated a similar 5-year EFS of 33.3% and 23.1% between CREBBP/fus and CREBBP/mut patients, respectively (Figure 1b; p=0.832). This poor EFS was maintained in the CREBBP/indel patients with a co-occurring t(8;21) (n=8, 5-year EFS 12.5%). When patients with co-occurring high-risk lesions were excluded from analysis, the remaining CREBBP/mut (n=27) patients maintained their poor EFS (29.6%). Despite their poor EFS, CREBBP/mut patients had an analogous overall survival (OS) to non-CREBBP patients (57.4% vs. 62.3%; p=0.499, Figure 1c), demonstrating that these patients could be successfully salvaged following relapse. In contrast, all patients with CREBBP/fus that relapsed subsequently died from their disease (OS 33.3%). Conclusions: In a large study of CREBBP alterations in pediatric patients with de novo AML, we show that these patients have a dismal EFS, regardless of alteration type. Further, despite enrichment of t(8;21), the favorable prognosis typically conferred by this alteration was abrogated by the co-occurrence of CREBBP/indel. Similarly, by excluding patients with co-occurring high-risk lesions from analysis, we show that these poor outcomes persist in a cohort of patients that would otherwise be considered low risk. Translocations between CREBBP and KAT6A in patients over 90 days of age are considered high risk on the active COG phase 3 trial. Given the inferior EFS and high salvage rates associated with other CREBBP alterations, intensification of upfront treatment, including hematopoietic stem cell transplant, should be considered in this population. The authors would like to acknowledge Astellas Pharma Global Development, Inc. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Al-Qattan, Mohammad M., Zuhair A. Rahbeeni, Zuhair N. Al-Hassnan, Abdulaziz Jarman, Atif Rafique, Nehal Mahabbat, and Faris A. S. Alsufayan. "Chromosome 16p13.3 Contiguous Gene Deletion Syndrome including the SLX4, DNASE1, TRAP1, and CREBBP Genes Presenting as a Relatively Mild Rubinstein–Taybi Syndrome Phenotype: A Case Report of a Saudi Boy." Case Reports in Genetics 2020 (January 9, 2020): 1–5. http://dx.doi.org/10.1155/2020/6143050.
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The classic Rubinstein–Taybi syndrome Type 1 (RSTS1, OMIM 180849) is caused by heterozygous mutations or deletions of the CREBBP gene. Herein, we describe the case of a Saudi boy with chromosome 16p13.3 contiguous gene deletion syndrome (OMIM 610543) including the SLX4, DNASE1, TRAP1, and CREBBP genes, but presenting with a relatively mild RSTS1 syndrome phenotype. Compared with previously reported cases with severe phenotypes associated with 16p13.3 contiguous gene deletions, our patient had partial deletion of the CREBBP gene (with a preserved 5′ region), which might explain his relatively mild phenotype.
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Sima, Aurora, Roxana Elena Smădeanu, Anca Angela Simionescu, Florina Nedelea, Andreea-Maria Vlad, and Cristina Becheanu. "Menke–Hennekam Syndrome: A Literature Review and a New Case Report." Children 9, no. 5 (May 22, 2022): 759. http://dx.doi.org/10.3390/children9050759.
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Background: Menke–Hennekam syndrome (MHS) is a rare and recently described syndrome consecutive to the variants in exon 30 or 31 in CREBBP (CREB-binding protein gene). The CREB-binding protein (CREBBP) and EP300 genes are two commonly expressed genes whose products possess acetyltransferase activity for histones and various other proteins. Mutations that affect these two genes are known to cause Rubinstein–Taybi syndrome (RTS); however, with the application of whole exome sequencing (WES) there were reports of variants that affect specific regions of exon 30 or 31 of these two genes but without the specific phenotype of RTS. Material and Methods: A review of the available literature was conducted, aimed at underscoring the difficulties in diagnosing MHS based on phenotype particularities. Results: Five applicable studies were identified by searching PubMed, Web of Science, and Scopus databases for publications up to November 2021 using the key terms “Menke–Hennekam syndrome” and “CREBBP”. Conclusions: In this paper, we present a new case and highlight the importance of exome sequencing to identify different mutations of exons 30 and 31 of the CREBBP gene involved in MHS, and we make formal recommendations based on our literature review.
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Gotti, Giacomo, Maneka Puligandla, Kristen E. Stevenson, Brenton G. Mar, Barbara L. Asselin, Uma Athale, Luis A. Clavell, et al. "Frequency and Prognostic Significance of Recurrent Gene Mutations in Pediatric B-ALL: Report from the DFCI ALL Consortium." Blood 136, Supplement 1 (November 5, 2020): 15–16. http://dx.doi.org/10.1182/blood-2020-136190.
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The application of next-generation sequencing (NGS) approaches to leukemia markedly expanded our understanding of the molecular landscape of pediatric acute lymphoblastic leukemia (ALL), but NGS integration into clinical care and therapeutic decision making is still limited. The prognostic impact of discovered mutations in uniformly treated patients with newly diagnosed B-ALL is not well characterized, and there is no consensus regarding the clinical significance of many of these findings. We investigated the frequency of mutations affecting common molecular pathways in pediatric B-ALL that are potentially druggable with targeted therapies, to define the prognostic role of these mutations, as measured by end-induction minimal residual disease (MRD) and event-free survival (EFS). We analyzed 159 patients (median age 6 years, range 1-18) with newly diagnosed Ph-negative B-ALL treated between 2005-2015 according to the Dana-Farber Cancer Institute (DFCI) ALL Consortium Protocols 05-001 and 11-001. Diagnostic leukemia samples were sequenced using a validated clinical NGS panel. We focused on mutations affecting the following pathways: Ras (NRAS, KRAS, PTPN11, NF1 and BRAF), cell cycle regulation (CCND3, CDKN2A/B and RB1), PI3K signaling (PTEN, PIK3CA, PIK3R1, PIK3C2B, MTOR, TSC1 and TSC2), Polycomb repressive complex 2 (PRC2) (EED, EZH2 and SUZ12), and JAK/STAT signaling (CRLF2, JAK2, JAK3, MPL, SH2B3 and SOCS1). Additionally, we analyzed mutations affecting CREBBP, FLT3, PAX5, SETD2 and TP53. Mutations occurring in the ExAC database at a frequency greater than 0.01% were excluded. A Fisher exact test and Wilcoxon rank sum test were used for categorical and continuous variables. High end-induction MRD, defined as > 10-3, was assessed by an Ig-TCR PCR assay. EFS was estimated using Kaplan and Meier method and tested between groups using a log-rank test. Induction death/failure, relapse or death were considered as events. Overall, 108 of the 159 patients (68%) carried at least one mutation in the studied genes. Most common mutations were in the Ras pathway (47%), CREBBP (9%), JAK pathway (8%), FLT3 (8%), PI3K pathway (5%), PAX5 (4%), SETD2 (4%), TP53 (4%), cell cycle regulation (3%) and PRC2 complex (3%). We investigated the distribution of mutations among common cytogenetic groups: ETV6-RUNX1, high hyperdiploidy (HHD) (51-65 chromosomes), hypodiploidy (<45 chromosomes), KMT2A-rearranged, TCF3-PBX1, and intra-amplification chromosome 21 (iAMP21). There was a strong association between TP53 and hypodiploidy (43% vs 2%; p=0.001). Ras pathway and FLT3 mutations were enriched in HHD (67% vs 39%; p=0.004) and (17% vs 4%; p=0.016), respectively. FLT3 mutations were mutually exclusive with ETV6-RUNX1 (12% vs. 0%, p=0.038), and Ras pathway mutations were rare in this subgroup (p=0.018). PAX5 mutations were enriched in children >10 years of age (p=0.035). There were no associations with sex. Ras pathway mutations were associated with high end-induction MRD (68% vs 42%; p=0.045), and this association was stronger for clonal (variant allele frequency (VAF) >25%) mutations (n=33) (63% vs 19%; p=0.0002). Focusing on HHD, CREBBP mutations frequently co-occurred with clonal Ras pathway mutations (83% vs 17%; p=0.032). Among HHD patients with evaluable MRD (n=33), 7 patients had high MRD, and 3 of these had CREBBP mutations (p=0.052). The analyzed cohort was enriched for higher risk ALL disease (Table 1). The 5-year EFS was 80%±3% among these 159 patients. Overall, no EFS difference was observed based on Ras pathway (p=0.35). Among HHD ALL, the presence of CREBBP or clonal Ras pathway mutations was significantly associated with inferior 5-yr EFS (50%±20% vs 97%±3%; p=0.0006, Figure 1) and (70%±13% vs 100%; p=0.007, Figure 2), respectively. In conclusion, our findings provide insight into the prognostic significance of the most common mutations in pediatric HHD B-ALL in a uniformly treated cohort of patients as part of the DFCI ALL Consortium. The presence of CREBBP and clonal Ras pathway mutations may be associated with upfront chemotherapy resistance as demonstrated by high end-induction MRD. Further analysis of Ras pathway mutations segregated by VAF is warranted. Future trials may integrate these findings into risk stratification of HHD ALL. With prospective continued clinical use of NGS assays, we will further clarify the role of mutations and their contribution to disease outcomes in B-ALL. Disclosures Mar: Blueprint Medicines Corporation: Current Employment, Current equity holder in publicly-traded company. Stegmaier:Novartis: Research Funding; Auron Therapeutics: Consultancy. Silverman:Takeda: Other: advisory board; Servier: Other: advisory board; Syndax: Other: advisory board.
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Los-de Vries, G. Tjitske, Wendy B. C. Stevens, Erik van Dijk, Carole Langois-Jacques, Andrew J. Clear, Phylicia Stathi, Margaretha G. M. Roemer, et al. "Genomic and microenvironmental landscape of stage I follicular lymphoma, compared with stage III/IV." Blood Advances 6, no. 18 (September 26, 2022): 5482–93. http://dx.doi.org/10.1182/bloodadvances.2022008355.
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Abstract Although the genomic and immune microenvironmental landscape of follicular lymphoma (FL) has been extensively investigated, little is known about the potential biological differences between stage I and stage III/IV disease. Using next-generation sequencing and immunohistochemistry, 82 FL nodal stage I cases were analyzed and compared with 139 FL stage III/IV nodal cases. Many similarities in mutations, chromosomal copy number aberrations, and microenvironmental cell populations were detected. However, there were also significant differences in microenvironmental and genomic features. CD8+ T cells (P = .02) and STAT6 mutations (false discovery rate [FDR] <0.001) were more frequent in stage I FL. In contrast, programmed cell death protein 1–positive T cells, CD68+/CD163+ macrophages (P < .001), BCL2 translocation (BCL2trl+) (P < .0001), and KMT2D (FDR = 0.003) and CREBBP (FDR = 0.04) mutations were found more frequently in stage III/IV FL. Using clustering, we identified 3 clusters within stage I, and 2 clusters within stage III/IV. The BLC2trl+ stage I cluster was comparable to the BCL2trl+ cluster in stage III/IV. The two BCL2trl– stage I clusters were unique for stage I. One was enriched for CREBBP (95%) and STAT6 (64%) mutations, without BLC6 translocation (BCL6trl), whereas the BCL2trl– stage III/IV cluster contained BCL6trl (64%) with fewer CREBBP (45%) and STAT6 (9%) mutations. The other BCL2trl– stage I cluster was relatively heterogeneous with more copy number aberrations and linker histone mutations. This exploratory study shows that stage I FL is genetically heterogeneous with different underlying oncogenic pathways. Stage I FL BCL2trl– is likely STAT6 driven, whereas BCL2trl– stage III/IV appears to be more BCL6trl driven.
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Pasqualucci, Laura, David Dominguez-Sola, Annalisa Chiarenza, Giulia Fabbri, Adina Grunn, Vladimir Trifonov, Lawryn H. Kasper, et al. "Genome-Wide Analysis Reveals Frequent Inactivating Mutations of Acetyltransferase Genes In B-Cell Lymphoma." Blood 116, no. 21 (November 19, 2010): 474. http://dx.doi.org/10.1182/blood.v116.21.474.474.
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Abstract Abstract 474 B cell non-Hodgkin lymphoma (B-NHL) comprises a variety of biologically and clinically distinct diseases whose pathogenesis is associated with largely unique genetic lesions affecting oncogenes and tumor suppressor genes. The identification of the complete set of genes and cellular pathways that are altered in the lymphoma cell is critical for a full understanding of the genesis and maintenance of these malignancies. Using genome-wide copy number analysis and high throughput whole exome sequencing, we have recently found that the two most common types of B-NHL, follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL), are associated with frequent structural alterations inactivating the genes that encode CREBBP and, in a smaller fraction of cases, EP300, two highly related histone and non-histone acetyltranferases (HAT) that act as transcriptional coactivators in multiple signaling pathways. Overall, ~29% (n=39/134) of DLBCL and 32% (n=15/47) of FL samples display genomic deletions and/or somatic point mutations that remove or inactivate the HAT domain of CREBBP. In 19 additional cases (15 DLBCL and 4 FL), mutations and deletions were found in the EP300 gene, also predicting its functional loss. With few exceptions, structural alterations of CREBBP and EP300 were mutually exclusive, suggesting that inactivation of these loci is at least in part functionally equivalent. Thus, in total ~36% of all DLBCL (n=49/134) and at least 40% of FL cases (n=19/47) display genetic aberrations targeting acetyltransferase genes. Interestingly, while these lesions were observed in both DLBCL phenotypic subtypes, their frequency appears to be significantly higher in germinal center B cell type DLBCL, where they account for ~46% of the cases (n=30/65) as compared to 27% in activated B cell type DLBCL (n=11/69; p<0.01). CREBBP and EP300 structural alterations commonly affect a single allele, suggesting that reduction in HAT dosage is important for lymphomagenesis. While the functional consequences of these lesions are likely to be broad on gene transcription, we demonstrate specific defects in the acetylation-mediated inactivation of the BCL6 onco-protein and activation of the p53 tumor suppressor, both of which contribute to transformation. These results identify CREBBP/EP300 alterations as a major pathogenetic mechanism shared by common forms of B-NHL, and have direct implications for the development of therapeutic regimens targeted to acetylation/deacetylation mechanisms. Disclosures: No relevant conflicts of interest to declare.
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Di Fede, Elisabetta, Valentina Massa, Bartolomeo Augello, Gabriella Squeo, Emanuela Scarano, Anna Maria Perri, Rita Fischetto, et al. "Expanding the phenotype associated to KMT2A variants: overlapping clinical signs between Wiedemann–Steiner and Rubinstein–Taybi syndromes." European Journal of Human Genetics 29, no. 1 (July 8, 2020): 88–98. http://dx.doi.org/10.1038/s41431-020-0679-8.
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AbstractLysine-specific methyltransferase 2A (KMT2A) is responsible for methylation of histone H3 (K4H3me) and contributes to chromatin remodeling, acting as “writer” of the epigenetic machinery. Mutations in KMT2A were first reported in Wiedemann–Steiner syndrome (WDSTS). More recently, KMT2A variants have been described in probands with a specific clinical diagnosis comprised in the so-called chromatinopathies. Such conditions, including WDSTS, are a group of overlapping disorders caused by mutations in genes coding for the epigenetic machinery. Among them, Rubinstein–Taybi syndrome (RSTS) is mainly caused by heterozygous pathogenic variants in CREBBP or EP300. In this work, we used next generation sequencing (either by custom-made panel or by whole exome) to identify alternative causative genes in individuals with a RSTS-like phenotype negative to CREBBP and EP300 mutational screening. In six patients we identified different novel unreported variants in KMT2A gene. The identified variants are de novo in at least four out of six tested individuals and all of them display some typical RSTS phenotypic features but also WDSTS specific signs. This study reinforces the concept that germline variants affecting the epigenetic machinery lead to a shared molecular effect (alteration of the chromatin state) determining superimposable clinical conditions.
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Mullighan, Charles G., Jinghui Zhang, Lawryn H. Kasper, Stephanie Lerach, Debbie Payne-Turner, Letha A. Phillips, Sue L. Heatley, et al. "CREBBP mutations in relapsed acute lymphoblastic leukaemia." Nature 471, no. 7337 (March 2011): 235–39. http://dx.doi.org/10.1038/nature09727.
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Loeffler, Markus, Markus Kreuz, Andrea Haake, Dirk Hasenclever, Heiko Trautmann, Christian Arnold, Karsten Winter, et al. "Integrated Somatic Mutation and DNA Methylation Analysis Reveal Genomic and Epigenomic Co-Evolution In Follicular Lymphomas." Blood 122, no. 21 (November 15, 2013): 502. http://dx.doi.org/10.1182/blood.v122.21.502.502.
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Abstract Background Follicular lymphoma (FL) is a B-cell lymphoma whose cytogenetic hallmark is the translocation t(14;18)(q32;q21) which juxtaposes the BCL2 oncogene to the immunoglobulin heavy chain locus (IGHV). FLs maintain key features of normal germinal center reactions, such as ongoing somatic hypermutation (SHM) of IGHV genes and selection for a functional B-cell receptor. SHM is mediated by activation-induced cytidine deaminase (AID) leading to single nucleotide exchange in IGHV genes and to a much lesser extent in non-IG genes. It was our objective to investigate the clonal evolution of t(14,18) FL from primary to relapse tumors simultaneously on several genetic and epigenetic levels. Methods We studied paired primary and relapsed tumors from 33 patients with t(14;18)-positive FL (76 samples: 25 pairs; 6 trios and 2 quadruples). In a core set of 19 patients we performed Sanger and next generation sequencing of the clonal VHDHJH rearrangements of the IGHV locus. We performed deep sequencing of 9 genes (BCL2, BCL6, MYC, RHOH, PAX5, IRF4, C2TA, REL and PIM1) targeted by aberrant SHM (aSHM) in lymphoma for 69 FL samples including the complete core set. We furthermore analyzed mutations in the coding regions of 10 candidate driver genes of lymphomagenesis (BCL2, MLL2, CREBBP, TNFRSF14, EZH2, EP300, MEF2B, BCL6, MYC, TP53) by deep sequencing. In addition to genetic analyses, evolutionary patterns of DNA-methylation were addressed by Illumina 27k arrays. Results We found strong evidence for ongoing selection against replacement mutations in the IGHV genes both in complementarity determining regions and framework regions, consistent with ongoing dependence of FL on a functional B-cell receptor and stimulation by antigens. Using mean normalized Hamming distance as a quantitative measure for the evolutionary divergence of paired samples (IGHV-divergence) and analyzing phylogenetic trees we classified the patterns of evolution into 3 categories: “No Evolution” (shared IGHV sequences in primary and relapse tumors), “Sequential Evolution” (relapse sequences emerge out of primary ones), “Divergent Evolution” (sequences of primary and relapse sample appear disjoint). We observed a mutation frequency of 62.0 per 100 kb in aSHM targets. These mutations were strongly enriched at the WRCY/RGYW target motifs characteristic for the SHM/AID machinery (OR=3.4; p<0.001). The most frequently altered locus was BCL2 affected in 68 of 69 samples (with 315.3 mutations per 100 kb) likely due to the recombination of the translocated allele into the IGH locus. We detected 197 single nucleotide variants (SNV) affecting the candidate driver genes (4.4 mutations per 100 kb) of which 145 were protein-changing. The genes most frequently affected were CREBBP (52 SNVs in 43/69 samples) and MLL2 (54 in 39/69). High incidence of CREBBP mutations and the high mutated allele frequencies of these mutations suggest that CREBBP deregulation is an early event in FL lymphomagenesis. We defined the aSHM-divergence as the proportion of observed aSHM mutations not present in both samples and found a significant correlation between the IGHV-divergence and the aSHM-divergence in the non-IG aSHM targets (r=0.724 [0.40-0.88]). We calculated a measure for divergence in DNA-methylation within patients by the proportion of all paired CpGs showing a methylation difference of at least 25%. DNA-methylation-divergence was found to correlate with IGHV-divergence (r=0.516 [0.24-0.72]) indicating that evolutionary divergence also affects the level of DNA-methylation. Conclusion Our observations demonstrate correlated evolutionary changes on all genomic and epigenomic levels in FL. This process most likely originates from an ongoing germinal center reaction with a functional BCR driving FL cells, sustaining a continuously active AID-machinery leading to addition of IGHV-mutations and to aberrant mutations in non-IG aSHM-target sites. The drifts in DNA-methylation patterns might be a consequence of additional mutations found in histone modifying genes such as CREBBP. The individual pattern of tumor evolution is likely to impact prognosis and clinical decision making which needs to be investigated in larger series. (Acknowledgment: BMBF/PTJ 0315452) Disclosures: No relevant conflicts of interest to declare.
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Nann, Dominik, Joan Enric Ramis-Zaldivar, Inga Müller, Blanca Gonzalez-Farre, Janine Schmidt, Caoimhe Egan, Julia Salmeron-Villalobos, et al. "Follicular lymphoma t(14;18)-negative is genetically a heterogeneous disease." Blood Advances 4, no. 22 (November 19, 2020): 5652–65. http://dx.doi.org/10.1182/bloodadvances.2020002944.
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Abstract Fifty-five cases of t(14;18)− follicular lymphoma (FL) were genetically characterized by targeted sequencing and copy number (CN) arrays. t(14;18)− FL predominated in women (M/F 1:2); patients often presented during early clinical stages (71%), and had excellent prognoses. Overall, t(14;18)− FL displayed CN alterations (CNAs) and gene mutations carried by conventional t(14;18)+ FL (cFL), but with different frequencies. The most frequently mutated gene was STAT6 (57%) followed by CREBBP (49%), TNFRSF14 (39%), and KMT2D (27%). t(14;18)− FL showed significantly more STAT6 mutations and lacked MYD88, NOTCH2, MEF2B, and MAP2K1 mutations compared with cFL, nodal marginal zone lymphoma (NMZL), and pediatric-type FL (PTFL). We identified 2 molecular clusters. Cluster A was characterized by TNFRSF14 mutations/1p36 alterations (96%) and frequent mutations in epigenetic regulators, with recurrent loss of 6q21-24 sharing many features with cFL. Cluster B showed few genetic alterations; however, a subgroup with STAT6 mutations concurrent with CREBBP mutations/16p alterations without TNFRSF14 and EZH2 mutations was noted (65%). These 2 molecular clusters did not distinguish cases by inguinal localization, growth pattern, or presence of STAT6 mutations. BCL6 rearrangements were demonstrated in 10 of 45 (22%) cases and did not cluster together. Cases with predominantly inguinal presentation (20 of 50; 40%) had a higher frequency of diffuse growth pattern, STAT6 mutations, CD23 expression, and a lower number of CNAs, in comparison with noninguinal cases (5.1 vs 9.1 alterations per case; P < .05). STAT6 mutations showed a positive correlation with CD23 expression (P < .001). In summary, t(14;18)− FL is genetically a heterogeneous disorder with features that differ from cFL, NMZL, and PTFL.
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Schroers-Martin, Joseph G., Joanne Soo, Gabriel Brisou, Florian Scherer, David M. Kurtz, Brian Sworder, Michael S. Khodadoust, et al. "Tumor-Confirmed Follicular Lymphoma Mutations Are Detectable in Peripheral Blood Years Prior to Clinical Diagnosis." Blood 138, Supplement 1 (November 5, 2021): 709. http://dx.doi.org/10.1182/blood-2021-151058.
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Abstract Background: Mutations in chromatin modifying genes (CMGs) including KMT2D, CREBBP, EZH2, and EP300 have been inferred as early events in follicular lymphoma (FL) by truncal status in mature tumors and persistence between diagnosis and relapse. We previously reported frequent detection of CREBBP lysine acetyltransferase (KAT) domain mutations in pre-diagnostic blood and tissue specimens from individuals later developing FL (Schroers-Martin et al, ASH Annual Meeting 2017). However, the limited availability of paired tumor biopsies has precluded confirmation of concordance between precursor lesions and subsequent clinical malignancy. Methods: The American Cancer Society (ACS) Cancer Prevention Study-II (CPS-II) LifeLink cohort collected screening blood or saliva samples from over 100,000 cancer-free American participants between 1998 and 2002. To evaluate detection of tumor-confirmed variants in pre-diagnostic specimens, we identified 29 FL patients with available FFPE tumor biopsy and screening sample (Fig A). The median age at screening was 71 years (range 56-83) with a median time to FL diagnosis of 56 months (TTD, range 6-139). Tumor biopsies were sequenced utilizing hybrid capture sequencing for commonly mutated lymphoma genes. DNA extracted from pre-diagnostic blood or saliva cell pellet specimens was sequenced utilizing error-corrected CAPP-Seq (Newman et al Nat Biotech 2016) to a median depth of 5204x. We sequenced to similar depths peripheral blood DNA from control cohorts of individuals with detectable t(14;18) but no subsequent lymphoma diagnosis (n=14) and healthy individuals without detectable t(14;18) by PCR (n=20). Results: Coding mutations were identified from all tumors with a mutational distribution similar to prior FL sequencing studies. Tumor-derived variants were detected in 7 of 29 paired pre-diagnostic specimens (24%) at a median TTD of 44 months (range 11-112 months, Fig B). The statistical significance of detection was assessed using a previously described approach based on Monte Carlo sampling (Newman et al Nat Biotech 2016) and the error distribution of affected loci in control cohorts. While an outlier case contained concordant TNFRSF14, FOXO1, and STAT6 mutations 90 months pre-diagnosis at an elevated allelic fraction (AF) of 1.8%, the mean AF of other detected precursor variants was 0.091%. Individuals with detected variants were not older (Fig C) nor significantly closer to clinical diagnosis (Fig D). The most frequently detected lesions were CREBBP (6/15 cases, 40%) and BCL2 (3/13, 23%) with one case demonstrating a fuller mutational profile including FOXO1 and ARID1A at 44 months before diagnosis. All detected precursor CREBBP variants localized to the KAT domain, reflecting prior observations in pre-diagnostic samples without confirmatory biopsy (Fig E). Of note, saliva cell pellets may contain 30% or more hematopoietic DNA (Kaur et al Chimerism 2012) and we detected tumor-confirmed variants in both saliva and blood screening specimens (Fig F) with no significant difference in AF (Fig G). In an illustrative independent case with available imaging, a patient undergoing radical prostatectomy was found to have involvement of a pelvic lymph node with in situ follicular neoplasia (ISFN). Staging PET/CT showed no evidence of FL (Fig H) and he was followed expectantly for 4 years without emergent disease. Eight years after surgery he presented with inguinal swelling and bilateral FDG-avid adenopathy on PET/CT. Excisional biopsy confirmed low grade FL and sequencing for M7-FLIPI revealed a CREBBP KAT domain variant. Retrospective sequencing of serial peripheral blood specimens from his initial surveillance showed detectable CREBBP R1446C at the earliest collected time point (AF range 0.019-0.046%) rising to AF 0.082% after clinical diagnosis. Conclusions: Precursor FL mutations are detectable in peripheral blood and saliva years prior to clinical diagnosis with a spectrum of variants enriched in CREBBP and BCL2 and concordant with subsequent FL tumors. Such lesions may assist in stratifying individuals at elevated risk of clinical malignancy, including after identification of pathologic precursors such as ISFN. Figure 1 Figure 1. Disclosures Kurtz: Foresight Diagnostics: Consultancy, Current holder of stock options in a privately-held company; Roche: Consultancy; Genentech: Consultancy. Khodadoust: Alexion, AstraZeneca Rare Disease: Other: Study investigator; CRISPR Therapeutics, Nutcracker Therapeutics: Research Funding; Myeloid Therapeutics: Membership on an entity's Board of Directors or advisory committees. Diehn: Roche: Consultancy; AstraZeneca: Consultancy; RefleXion: Consultancy; BioNTech: Consultancy; Varian Medical Systems: Research Funding; Illumina: Research Funding; CiberMed: Current holder of stock options in a privately-held company, Patents & Royalties; Foresight Diagnostics: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Roulland: BMS: Research Funding. Alizadeh: Bristol Myers Squibb: Research Funding; Gilead: Consultancy; CAPP Medical: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Celgene: Consultancy, Research Funding; Roche: Consultancy, Honoraria; Janssen Oncology: Honoraria; Foresight Diagnostics: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Cibermed: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Forty Seven: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.
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Ding, Ling-wen, Qiao-Yang Sun, Anand Mayakonda, Kar-Tong Tan, Wenwen Chien, De-Chen Lin, Yan-Yi Jiang, et al. "Mutational Profiling of Acute Lymphoblastic Leukemia with Testicular Relapse." Blood 128, no. 22 (December 2, 2016): 2809. http://dx.doi.org/10.1182/blood.v128.22.2809.2809.
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Abstract Relapse acute lymphoblastic leukemia (ALL) is the leading cause of childhood cancer deaths. Although relapse usually occurs in the bone marrow (medullary), extramedullary relapse occasionally occurs. Currently, the clonal origin and evolution of extramedullary relapse remain elusive. We selected two pediatric B-ALL patients who experienced testicular ALL relapse and interrogated their leukemic cells (diagnosis, remission, bone marrow relapse and testicular relapse) with whole exome sequencing. Case D483 (5.6 years old at diagnosis of ALL) developed bone marrow and testicular relapse 5 years after diagnosis of B-ALL. At diagnosis he was treated as an intermediate risk with hyperdiploid-ALL with the absence of any well-known ALL fusion-oncogene. Mutations of KRAS (G12D) and CREBBP (S1436C) were found in the founding leukemic clone at diagnosis and persisted in the bone marrow and testis at relapse). Mutation of CREBBP has been frequently found in ALL (particularly in hyperdiploid subtype) and is correlated with increased incidence of relapsed ALL. A MEF2B mutation (R17Q) was found in the bone marrow and testicular relapse sample. Missense mutation of this gene is frequently found in diffuse large B cell lymphoma (DLBCL); this protein regulates the expression of the proto-oncogene BCL6 and contributes to malignant transformation. Second child, case D727 (1.3 years old at diagnosis) harbored a MLL-AF9 fusion and was assigned as a high risk-ALL at diagnosis. Two NT5C2 mutations occurred at relapse, being present at different VAF in bone marrow and testicle: missense mutation R367Q was present with a VAF of 33.5% in bone marrow and 4.5% in testicle; while D407V was present with a VAF of 6.5% in bone marrow and 35.5% in the testicular relapse. NT5C2 encodes a 5'-nucleotidase involved in purine metabolism. The missense mutations (R367Q and D407V) identified here, have been reported as recurrent mutational hotspots of NT5C2 in relapse ALL and have been functionally validated. These mutations increase the 5'-IMP nucleotidase activity of NT5C2 protein leading to resistance to 6-mercaptopurine, a drug that was a component of the treatment regime of this patient. To understand the evolutionary trajectories of these two ALL cases, we analyzed clonal evolution based on their sequencing data. In patient D483, the relapse leukemia was directly evolved from the diagnosis leukemia clone: all of the mutations at diagnosis were persisted at relapse, and four mutated genes (MEF2B, KCNG1, AIM1, OTUD5) were acquired at both bone marrow and testicular relapse with different variant allele frequency (VAF). In patient D727, however, a faction of mutations present at diagnosis were subsequently lost at relapse, suggesting that relapsed leukemia arose from an ancestral subclone that developed before the overt leukemia at diagnosis. The mutational pattern and VAF cluster analysis results suggest that relapse in the patients' testicle represents an independently subclones from the relapse in their bone marrows. Taken together, our sequencing results suggest that relapse of patient D483 was directly evolved from the diagnosis leukemic clone; while the relapse leukemia cells (both bone marrow and testicle) of patient D727 was likely derived from a common ancestral clone, and the testicular relapse arose independently from the bone marrow relapse leukemia. Disclosures Lill: Sanofi: Speakers Bureau; California Cord Blood Services: Consultancy; Kite: Research Funding.
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Gan, Meifu, Zaixian Tai, Yijian Yu, Chao Zhang, and Juan Xu. "Next-generation sequencing shows the genomic features of ovarian clear cell cancer and compares the genetic architectures of high-grade serous ovarian cancer and clear cell carcinoma in ovarian and endometrial tissues." PeerJ 11 (January 26, 2023): e14653. http://dx.doi.org/10.7717/peerj.14653.
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Ovarian clear cell carcinoma (OCCC) is a special histological type of epithelial ovarian cancer (EOC) that is not derived from epithelial cells of the ovarian or fallopian tube as the most common type of ovarian cancer, high-grade serous ovarian carcinoma (HGSOC), but is closely related to endometriosis and similar to endometrial clear cell carcinoma (ECCC) at morphologic and phenotypic features. However, limited data was shown in OCCC genomic features and compared with that in OCCC, HGSOC and ECCC. Herein, we utilized next-generation sequencing analysis of a panel of 1,021 genes to profile the mutational alterations in 34 OCCC and compared them to those from HGSOC (402 cases) and ECCC (30 cases). In result, the ARID1A and PIK3CA are high-frequency mutations of OCCC. Clonal architectures showed that all the mutations of genes occur in the later stage in the OCCC progress, whereas KRAS mutation is the earlier event compared with mutation of ARID1A or PIK3CA, which usually occurs in a group of ARID1A or PIK3CA mutations. The mutation frequency of main driver genes is similar between OCCC and ECCC, while TP53 is the main mutation in HGSOC and ECCC. Shared mutational signatures between OCCC and ECCC tissues with commonly observed a C>T change indicated a common carcinogens-exposed between these two carcinomas, but HGSOC and ECCC have common and distinct mutational signatures across cohorts respectively. In addition, we identified some novel CNV gains in NF1, ASXL1, TCF7L2, CREBBP and LRP1B and loss in ATM, FANCM, RB1 and FLT in OCCC. Our study offered a new perspective for OCCC tumorigenesis from two organs, the ovary and uterus, at genomic architectures and revealed novel CNV events for helping to provide theoretical support for OCCC treatment.
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Pasqualucci, Laura. "Molecular Dissection of Diffuse Large B-Cell Lymphoma." Blood 124, no. 21 (December 6, 2014): SCI—14—SCI—14. http://dx.doi.org/10.1182/blood.v124.21.sci-14.sci-14.
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Diffuse large B-cell lymphoma (DLBCL), the most common form of human lymphoma, is an aggressive malignancy comprising multiple phenotypically and genetically distinct subtypes, approximately 40% of which are incurable. These tumors may arise de novo or from the transformation of more indolent lymphomas, as observed in 30-40% of follicular lymphoma (FL) and 5-12% of chronic lymphocytic leukemia cases. Over the last decade, the introduction of next-generation sequencing technologies combined with genome-wide copy number analysis has allowed a comprehensive definition of the genetic lesions that are associated with the pathogenesis of these malignancies, leading to the identification of several previously unappreciated targets1-3. These lesions, in turn, uncovered dysregulated cellular pathways that represent potential targets for improved diagnosis and therapy. Among the most common genetic alterations found in both de novo DLBCL and transformed FL (tFL) are those targeting histone/chromatin modifiers; in particular, loss-of-function mutations in the genes encoding for the H3K4 methyltransferase MLL2 and the acetyltransferases CREBBP/EP300, together with gain-of-function mutations of the EZH2 H3K27 methyltransferase are observed in over 50% of DLBCL and 90% of tFL patients, suggesting a major role for these enzymes in altering gene expression during malignant transformation. Interestingly, sequential analysis of tumor samples isolated at FL diagnosis and at evolution to DLBCL indicates that inactivating mutations of CREBBP and MLL2 represent early events acquired during the initial expansion of a common ancestral clone4. Disruption of epigenetic modifiers by genetic alterations may thus contribute to malignant transformation by shaping the epigenetic landscape of the cancer cell as well as by perturbing specific biological programs. In line with this hypothesis, we have shown that mutations of CREBBP/EP300 disrupt the balance between acetylation-mediated activation of the p53 tumor suppressor and inactivation of the BCL6 proto-oncogene5. The lecture will cover recent advances in our understanding of the genetic basis of this disease, with emphasis on the role of epigenetic regulators in normal germinal center development and lymphomagenesis, as revealed by in vitro and in vivo studies. References: 1. Pasqualucci L, Trifonov V, Fabbri G, et al. Analysis of the coding genome of diffuse large B-cell lymphoma. Nat Genet. 2011; 43: 830-837. 2. Morin RD, Mendez-Lago M, Mungall AJ, et al. Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma. Nature. 2011; 476: 298-303. 3. Lohr JG, Stojanov P, Lawrence MS, et al. Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing. Proc Natl Acad Sci U S A. 2012; 109: 3879-3884. 4. Pasqualucci L, Khiabanian H, Fangazio M, et al., Genetics of follicular lymphoma transformation. Cell Rep. 2014; 6:130-140. 5. Pasqualucci L, Dominguez-Sola D, Trifonov V, et al. Inactivating mutations of acetyltransferase genes in B-cell lymphoma. Nature. 2011; 471: 189-195. Disclosures No relevant conflicts of interest to declare.
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Dutto, Ilaria, Claudia Scalera, Micol Tillhon, Giulio Ticli, Gianluca Passaniti, Ornella Cazzalini, Monica Savio, et al. "Mutations in CREBBP and EP300 genes affect DNA repair of oxidative damage in Rubinstein-Taybi syndrome cells." Carcinogenesis 41, no. 3 (August 29, 2019): 257–66. http://dx.doi.org/10.1093/carcin/bgz149.
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Abstract Rubinstein-Taybi syndrome (RSTS) is an autosomal-dominant disorder characterized by intellectual disability, skeletal abnormalities, growth deficiency and an increased risk of tumors. RSTS is predominantly caused by mutations in CREBBP or EP300 genes encoding for CBP and p300 proteins, two lysine acetyl-transferases (KAT) playing a key role in transcription, cell proliferation and DNA repair. However, the efficiency of these processes in RSTS cells is still largely unknown. Here, we have investigated whether pathways involved in the maintenance of genome stability are affected in lymphoblastoid cell lines (LCLs) obtained from RSTS patients with mutations in CREBBP or in EP300 genes. We report that RSTS LCLs with mutations affecting CBP or p300 protein levels or KAT activity, are more sensitive to oxidative DNA damage and exhibit defective base excision repair (BER). We have found reduced OGG1 DNA glycosylase activity in RSTS compared to control cell extracts, and concomitant lower OGG1 acetylation levels, thereby impairing the initiation of the BER process. In addition, we report reduced acetylation of other BER factors, such as DNA polymerase β and Proliferating Cell Nuclear Antigen (PCNA), together with acetylation of histone H3. We also show that complementation of CBP or p300 partially reversed RSTS cell sensitivity to DNA damage. These results disclose a mechanism of defective DNA repair as a source of genome instability in RSTS cells.
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Huang, Yao-hui, and Weili Zhao. "Somatic Mutations of Epigenetic Regulator Genes in Diffuse Large B-Cell Lymphoma." Blood 128, no. 22 (December 2, 2016): 1756. http://dx.doi.org/10.1182/blood.v128.22.1756.1756.
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Abstract Background. Diffuse large B-cell lymphoma (DLBCL) is one of the most aggressive types of B-cell lymphoma with high heterogeneity, accounting for 30-40% of newly diagnosed non-Hodgkin lymphoma (NHL), and dysfunction of epigenetic regulation has been found as a common and important feature of B cell lymphomas. To identify epigenetic associated genes mutations in DLBCL, including KMT2D, CREBBP, EP300, EZH2 and MEF2B, we sequenced tumour DNA from 226 Chinese DLBCL cases by applying next generation sequencing technology (NGS). A total of 679 consecutive Chinese patients with previously untreated DLBCL at our institution from December 2006 and January 2016 were enrolled in this study, and we assessed the predictive value of clinical and mutational pattern of epigenetic associated genes in a large single-institution cohort of these patients. Methods. Genomic DNA was extracted from 226 subjects with DLBCL formalin-fixed paraffin-embedded tumor tissue, using a QIAamp DNA FFPE Tissue Kit (Qiagen). Specific primers, producing amplicons about 200 bp at the coding regions of the genes of interest , were designed at the UCSC website (http://genome.ucsc.edu/cgi-bin/hgGateway ). Microfluidic PCR reactions ran in a 48 ¡Á 48 Access array system (Fluidigm) with FastStart High Fidelity PCR system (Roche) and high-throughput DNA sequencing was performed on Illumina Genome Analyzer IIx (GAIIx) and HiSeq2000 systems, according to the manufacturer's instructions. SAMtools version 0.1.19 was used to generate chromosomal coordinate-sorted bam files and to remove PCR duplications. Sequences for epigenetic associated genes were obtained from the UCSC Human Genome database, using the corresponding mRNA accession number as a reference, and those containing splice-site, nonsense or coding-region indel mutations, were selected for Gene Ontology analysis. All of the results were also confirmed by Sanger sequencing. Baseline characteristics of patients were analysed using two-sided c2 test. Overall survival (OS) was estimated using the Kaplan-Meier method and compared by log-rank test. Univariate hazard estimates were generated with unadjusted Cox proportional hazards models. Covariates demonstrating significance with P<0.100 on univariate analysis were included in the multivariate model. Statistically significance was defined as P<0.05. All statistical analyses were carried out using Statistical Package for the Social Sciences (SPSS) 20.0 software (SPSS Inc., Chicago, IL, USA). Results. Overall, 105 of 226 Chinese DLBCL cases were identified to have at least one mutation in epigenetic regulator genes. Somatic mutations in KMT2D were most frequently observed (24.3%), followed by CREBBP, EP300, EZH2 and MEF2B (15.5%,10.6%,4.4% and 2.2%, respectively)(Figure1,A,B). Association of mutated genes according to the conceptual classification. Circos plot of mutated genes according to the function is shown, and overlap mutations between epigenetic regulator genes mutations were frequently observed (Figure1, C). Clinically, mutation-positive DLBCL patients presented shorter OS than patients those without mutations (P=0.0286, Figure 1,D) among 226 DLBCL cases. A total of 679 Chinese DLBCL cases were enrolled in univariate analysis, and R-IPI, Complete Remission (CR), epigenetic related mutations were significant prognostic factors for OS. In further multivariate analysis, R-IPI (RR=2.72,95%CI=1.619-4.567,P<0.000), CR (RR=0.129,95%CI=0.076-0.219,P<0.000), epigenetic related mutations (RR=1.605,95%CI=1.007-2.557,P=0.046) are independent prognostic factor for OS. Conclusion. Our study provided the mutational spectrum of epigenetic regulator genes in DLBCL, and the relationships between mutations and clinic suggested some therapeutic efficiency. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Jin, Ying, Yamei Chen, Huarong Tang, Qian Li, Pansong Li, Xiao Hu, Yan-Fang Guan, et al. "Clinical potential of ctDNA-based TMB in small cell lung cancer recieving chemoradiotherapy." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): 3536. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.3536.
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3536 Background: Small cell lung cancer (SCLC) is an aggressive tumor with poor prognosis. Chemotherapy and / or radiotherapy is the main choice of SCLC treatment. Circulating tumor DNA (ctDNA) has received substantial attention in recent years owing to the potential of patient stratification and monitoring. Here, we assessed the value of prediction and prognosis using ctDNA in SCLC. Methods: SCLC patients (pts) with limited-stage disease (LD) receiving chemoradiotherapy and extensive-stage disease (ED) receiving chemotherapy were enrolled. Baseline plasma samples were collected for NGS using a 1021-gene-panel. Mutational features and blood-based tumor mutation burden (bTMB) were analyzed using ctDNA. pyClone software was used to cluster the mutations. The mutations in the cluster with the highest cancer cell fraction (CCF) were defined as clonal mutations. Progression-free survival (PFS) was followed. Results: 58 SCLC pts (35 LD and 23 ED) and 58 plasma samples were enrolled. Smoking pts accounted for 84% (49/58). In all samples, recurrent genes were TP53 (86%), RB1 (57%), LRP1B (34%), CREBBP (26%), and MLL3 (22%). The median of bTMB and clone count were 7.9 [0-26] and 7 [0-25]. Significant higher bTMB and clone count were observed in ED pts compared with LD (Mann Whitney test, p = 0.019 and p = 0.041, respectively). Mutated CREBBP (10/23 ED versus 5/35 LD) was enriched in ED (Fisher exact test, p = 0.017 and OR = 0.223). Mutations in NOTCH signaling pathway were enriched in ED (l6/23 ED versus 13/35 LD, p = 0.031, OR = 0.265). In LD group, there were trend toward prolonged PFS in pts with higher bTMB(p = 0.065), and pts with higher clonal bTMB (cbTMB) exhibited significant longer PFS (p = 0.016, HR 0.37, 95% CI [0.12-1.11]). Patients with alteration in PIK3CA showed shorter PFS than wild type (p < 0.001, HR 0.11, 95% CI [0-2.86]). There were no significant difference in median PFS in LD stage pts with any detectable pathway alterations. Whereas, LD pts whose ctDNA contained RTK-RAS signaling pathway alterations exhibited shorter PFS than pts without those alterations (p = 0.135). In ED pts, NOTCH1 gene wild type displayed longer PFS than mutant type (p = 0.036, HR 0.38, 95% CI [0.1-1.53]). There were no difference in PFS between pts with higher and lower bTMB and cbTMB. Conclusions: ctDNA can characterize the mutational feature of SCLC. There are differences in the molecular characteristics between ED and LD pts. Clonal bTMB is a potential prognostic biomarker for LD SCLC chemoradiotherapy. The prognostic marker of ED chemotherapy is different from LD.
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Plaksa, I. L., M. R. Savchuk, N. V. Shved, N. A. Savelov, D. N. Khmelkova, А. A. Isaev, and R. V. Deev. "Mutation profile of the tall cell variant of papillary thyroid carcinoma: analysis of 5 cases using wide-panel next-generation sequencing." Head and Neck Tumors (HNT) 11, no. 1 (April 24, 2021): 78–85. http://dx.doi.org/10.17650/2222-1468-2021-11-1-78-85.
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The study objective is to analyze the mutation profile of the tall cell variant (TCV) of papillary thyroid carcinoma (PTC).Materials and methods. The main inclusion criteria according to the WHO classification (2017) was PTC composed of at least 30 % of tall cells. Genetic examination was conducted using the FoundationOne CDx assay (USA) with median depth of coverage of >500x. This study included 5 patients (1 man and 4 women) with a mean age of 52.6 years (range: 48-56 years). The tumor size varied between 0.4 x 0.5 cm and 11.0 x 9.0 cm. All patients have undergone surgical treatment: hemithyroidectomy for patient No. 1 with a small tumor (pT1b); thyroidectomy for patient No. 2 (pT3b); extensive thyroidectomy with the removal of paratracheal tissue for patients No. 3, 4, and 5 (No. 3 - pT3bN0; No. 4 - pT3bN1b; No. 5 - pT3bN1b). Three out of the five patients also had adenomatous goiter. The mean follow-up time was 3.4 to 5.2 years.Results. Tumors in all patients were characterized by low mutational load (0 to 4 mutations per 1 million nucleotides (megabase)) and no microsatellite instability. All study participants were found to have p.V600E mutation in the BRAF gene; two patients had c.-124C>T mutation in the promoter region of the TERT gene. All patients carried mutations with unknown clinical significance: p.V562I in the EPHB1 gene (in 2 patients); mutations in the genes AR, CREBBP, EP300, ERCC4, FLT1, IKBKE, JAK2, MAF, MLL2, MST1R, MYC, MYCL1, NTRK2, TSC2 (each mutation registered in one patient). One individual with the largest tumor and the most aggressive disease was found to have amplifications of the BTG2, MAP3K1, SMAD2, and TBX3 genes.Conclusion. In 5 patients analyzed in this study, the mutation profile of TCV PTC was characterized by low mutational load, no microsatellite instability, and presence of p.V600E mutation in the BRAF gene in all cases. Some patients also had c.-124C>T mutation in the TERT gene and p.V562I mutation in the EPHB1 gene.
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Hilton, Laura K., Kostiantyn Dreval, Shaghayegh Soudi, Susana Ben-Neriah, Manuela Cruz, Brett Collinge, Krysta M. Coyle, et al. "Constrained FL: A Genetically Distinct Subgroup of Follicular Lymphoma with Low Rates of Somatic Hypermutation and a Reduced Propensity for Histologic Transformation." Blood 138, Supplement 1 (November 5, 2021): 807. http://dx.doi.org/10.1182/blood-2021-153445.
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Abstract Introduction: Follicular lymphoma (FL) is an indolent disease that undergoes histological transformation (HT) to aggressive diffuse large B-cell lymphoma (DLBCL) in 8-15% of patients. FLs frequently share genetic features with DLBCL, especially those of the germinal center B-cell-like (GCB) cell-of-origin (COO) and the EZB/C3 genetic subgroup, and approximately 80% of transformed FL (tFL) are classified as GCB. Our current understanding of the genetics of FL and tFL is based on a variety of studies, most of which have sequenced tumors in small case numbers or using targeted approaches such that the potential role of non-coding mutations and aberrant somatic hypermutation (aSHM) in predicting HT have not previously been fully explored. Methods: Whole genome sequencing (WGS) data from 212 FL (including 24 from patients that subsequently underwent HT) and 241 de novo DLBCL were analyzed. Simple somatic mutations (SSMs) were called using an ensemble of somatic variant callers, while structural variants (SVs) were called with Manta and copy number variants (CNVs) with Battenberg and GISTIC. Fluorescence in situ hybridization with break-apart probes (BA-FISH) was used to identify MYC, BCL2, and BCL6 translocations, and with IGH /BCL2 dual-fusion probes (DF-FISH) for a subset of FLs. To compare the genetics of FL and DLBCL, 83 significantly mutated genes (SMGs) were identified with dNdS, MutSigCV, HotMaps, and OncoDriveFML, and non-silent mutations were tabulated for their presence in each genome. For 38 hypermutated regions, we used a region-specific threshold to binarize the data to aSHM/no aSHM. Recurrent missense mutations in FOXO1, MYD88L265P, CREBBP lysine acetyltransferase (KAT) domain, EZH2Y646, MEF2B, and STAT6 were tabulated separately from other mutations in these genes. Using only the FL tumors from patients with no evidence of subsequent transformation and all available de novo DLBCLs, we trained a random forest classifier to separate these two entities using this set of 129 features, including MYC and BCL6 translocations. To validate this classifier, we fit a linear model to the number of FL votes from each discovery case, utilizing the 65 features (including 19 aSHM features) that were adequately sequenced in a validation cohort of 127 tFL. Statistical tests were corrected for multiple comparisons where necessary. Results: This large cohort of FL and DLBCL genomes has enabled the curation of an extensive list of novel and established FL driver genes and the identification of distinguishing genetic features among SMGs and CNVs. Loci that are significantly enriched for mutations in FL vs. DLBCL include the CREBBP KAT domain (OR 11.5, P < 0.0001), RRAGC (OR 9.61, P < 0.001), and ATP6V1B2 (OR 11.17, P < 0.001). Deletions of ARID1A (OR 4.74, P < 0.1), PTEN (OR 3.65, P < 0.01), and TNFRSF14 (OR 3.31, P < 0.01) were among the CNVs significantly enriched in FL. Out of 156 FLs, 24 (15%) were negative for BCL2 translocations by BA-FISH, but 4 (17%) of these had BCL2 translocations detected from WGS data. All 4 of these cryptic events were confirmed using IGH /BCL2 DF-FISH. Using a threshold of 0.7, the linear model separated discovery FL cases into a more DLBCL-like subgroup, termed dFL (n = 107), and a genetically homogeneous subgroup enriched for the FL-associated features, which we describe as constrained FL (cFL, n = 105). This separation is supported by more mutations in dFL vs cFL across several aSHM loci, including the transcription start sites for BCL6, BCL7A, DTX1, and ZFP36L1 (Figure 1), consistent with reduced exposure to the germinal center reaction in cFL. Within the targeted capture validation cohort of tFL, 30 (24%) tumors were classified as cFL and 97 (76%) as dFL. The tFL cohort was significantly depleted for mutations in the CREBBP KAT domain (OR 0.59, P < 0.05), and were significantly less frequently classified as cFL (OR 0.30, P < 0.0001) compared to the discovery FLs. Conclusions: The distinction between cFL and dFL is strongly driven by CREBBP KAT domain mutations and different rates of aSHM genome wide. Given the known early clonal nature of CREBBP mutations in FL and its role in regulating germinal center cycling, we speculate that CREBBP KAT mutations may limit the exposure of FL to the dark zone, reducing the opportunity for aSHM and creating an evolutionary constraint that may limit the opportunity for HT. This classification may serve as a useful biomarker to identify FLs at higher risk of HT. Figure 1 Figure 1. Disclosures Coyle: Allakos, Inc.: Consultancy. Grande: Sage Bionetworks: Current Employment. Slack: Seagen: Consultancy, Honoraria. Steidl: Curis Inc.: Consultancy; Trillium Therapeutics: Research Funding; Epizyme: Research Funding; Bayer: Consultancy; Seattle Genetics: Consultancy; AbbVie: Consultancy; Bristol-Myers Squibb: Research Funding. Scott: Janssen: Consultancy, Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy; NanoString Technologies: Patents & Royalties: Patent describing measuring the proliferation signature in MCL using gene expression profiling.; Incyte: Consultancy; Rich/Genentech: Research Funding; Celgene: Consultancy; BC Cancer: Patents & Royalties: Patent describing assigning DLBCL COO by gene expression profiling--licensed to NanoString Technologies. Patent describing measuring the proliferation signature in MCL using gene expression profiling. . Morin: Epizyme: Patents & Royalties; Foundation for Burkitt Lymphoma Research: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy.
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Yoshida, Kenichi, Norio Shiba, Yuichi Shiraishi, Akira Shimada, Kiminori Terui, Motohiro Kato, Yusuke Okuno, et al. "Whole Exome Sequencing Reveals Clonal Evolution Pattern and Driver Mutations Of Relapsed Pediatric AML." Blood 122, no. 21 (November 15, 2013): 1410. http://dx.doi.org/10.1182/blood.v122.21.1410.1410.
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Abstract Background Pediatric acute myeloid leukemia (AML) comprises ∼20% of pediatric leukemia, representing one of the major therapeutic challenges in pediatric oncology. Nearly 40% of patients still relapse after present first-line therapies and once the relapse occurs, the long-term survival rates decrease, ranging from 21% to 34%. As for the pathogenesis of AML relapse, the recent development of massively parallel sequencing technologies has provided a new opportunity to investigate comprehensive genetic alterations that are involved in tumor recurrence of adult AML. However, little is known about the molecular details of relapsed pediatric AML. Methods In order to reveal the clonal origin and the major mutational events in relapsed pediatric AML, we performed whole exome sequencing of 4 trio samples from diagnostic, relapsed and complete remission phases using Illumina HiSeq 2000. Copy number abnormalities were also detected using whole exome sequencing. Subsequently, deep sequencing of identified mutations was performed to evaluate intra-tumor heterogeneity and the clonolocal history of relapsed clones. Results Whole-exome sequencing of 12 samples from 4 patients were analyzed with a mean coverage of more than x100, and 95 % of the targeted sequences were analyzed at more than x20 depth on average. A total of 98 somatic mutations were identified, where mean number of non-silent mutations was higher at relapsed phase than at the time of diagnosis (14.0/case vs 10.5/case) (p=0.270). Assessment of clonality using variant allele frequencies of individual mutations suggested that some mutations were subclonal mutations, consisting of intra-tumor heterogeneity both at the time of diagnosis and at relapse. In all 4 patients, relapsed AML evolved from one of the subclones at the initial phase, which was accompanied by many additional mutations including common driver mutations that were absent or existed only with lower allele frequency in the diagnostic samples, indicating a multistep process of leukemia recurrence. Forty-six out of the 98 mutations were specific either at the time of diagnosis (n = 16) or at relapse (n = 30). Relapse-specific mutations and copy number changes were found in several genes including known drivers such as NRAS and CREBBP. These mutations were further investigated in an extended cohort of relapsed pediatric AML samples using targeted sequencing to evaluate their prevalence. In some cases, AML relapse may accompany a dynamic clonal change. For example, some bona fide driver mutations, such as KRAS mutations, that were predominant at the time of diagnosis disappeared in relapsed samples. Discussion Whole exome sequencing unmasked clonal structure of primary and relapsed pediatric AML, which helped to understand the underlying mechanism of relapse in pediatric AML. Our results suggested that pediatric AML has intra-tumor heterogeneity and subclonal mutations such as NRAS and CREBBP occurring in one of the subclones could drive the AML relapse. Disclosures: No relevant conflicts of interest to declare.
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Zhang, Hui, Maoxiang Qian, Shirley, Kow Yin Kham, Shuguang Liu, Chuang Jiang, Xujie Zhao, Yi Lu, et al. "Whole Transcriptome Sequencing Identified a Distinct Subtype of Acute Lymphoblastic Leukemia with Abnormalities of CREBBP and EP300." Blood 128, no. 22 (December 2, 2016): 3912. http://dx.doi.org/10.1182/blood.v128.22.3912.3912.
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Abstract While acute lymphoblastic leukemia (ALL) is a prototype of cancer that can be cured by chemotherapy alone, current ALL treatment regimens rely primarily on conventional cytotoxic agents with significant acute and long-term side effects. Better understanding of genomic landscape of ALL is critical for developing molecularly targeted therapy and implementing genomics-based precision medicine in this cancer. In particularly, sentinel chromosomal translocations are common in ALL and often involve key transcription factors important for hematopoiesis. Epigenetic regulator genes are also frequently targeted by somatic genomic alterations such as sequence mutations (e.g., CREBBP) and gene fusions (e.g., MLL, EP300). To comprehensively define transcriptomic abnormalities in childhood ALL, we performed RNA-seq of an unselected cohort of 231 children enrolled on the MaSpore frontline ALL protocols in Singapore or Malaysia. In total, we identified 58 putatively functional and predominant fusion genes in 125 patients (54.1%), the majority of which have not been reported previously. In particular, we described a distinct ALL subtype with a characteristic gene expression signature driven by chromosomal rearrangements of the ZNF384 gene with different partners (i.e., histone acetyl-transferases EP300 and CREBBP, TAF15, and TCF3). In 9 of 11 ALL cases with ZNF384 rearrangements, the breakpoint in this gene was invariably between exon 2 and exon 3, resulting in deletion of the 5'-UTR and then in-frame fusion of the entire ZNF384 coding sequence with the partner genes. The top two most significantly up-regulated genes in the ZNF384-rearranged group were CLCF1 and BTLA, whose expression levels were 15.5- and 15.0-fold higher than in ALL cases with wildtype ZNF384, respectively. In fact, ZNF384 binding was identified within the CLCF1 and BTLA loci (particularly the promoter regions) by chromatin immunoprecipitation sequencing in B lymphoblasoid cells. Using luciferase transcription driven by CLCF1 promoter in HEK293T cells as a model system, we observed significantly greater transcription activity with EP300-ZNF384 fusion compared to cells expressing wildtype ZNF384, suggesting that this chimeric gene resulted in gain of ZNF384 function. Similar results were obtained with luciferase transcription assay driven by the BTLA promoter. In human ALL cells, CLCF1 and BTLA promoter activities were consistently and significantly higher in ZNF384-rearranged ALL than in ALL cell line with wildtype ZNF384. To examine the effects of ZNF384 fusion on hematopoietic stem and progenitor cell (HSPCs) function, we also evaluated colony forming potential of HSPC in vitro upon ectopic expression of ZNF384 fusions. While there was marked suppression of colonies from myeloid and erythoid lineages, expression of EP300-ZNF384 or CREBBP-ZNF384 significantly stimulated preB cell colony formation. However, neither EP300- nor CREBBP-ZNF384 fusion was able to transform mouse hematopoietic precursor cell Ba/f3 in vitro, but instead increased the transforming potential of other oncogenic mutations (NRASG12D). EP300-ZNF384 and CREBBP-ZNF384 fusion proteins lacked the histone acetyltransferase (HAT) domain, and showed only 25% and 10% of HAT activity of full-length EP300 and CREBBP, respectively, with dominant-negative effects. Also, expression of EP300-ZNF384 led to significant decrease in global H3 acetylation in Ba/f3 cells in vitro. Finally, in NRASG12D-transformed Ba/f3 cells, co-expression of EP300-ZNF384 or CREBBP-ZNF384 substantially potentiated cytotoxic effects of histone deacetylase inhibitor vorinostat. Similarly, in a panel of human ALL cell lines, ZNF384-rearrangement was also associated with increased sensitivity to vorinostat, suggesting that some ZNF384-rearranged ALL may benefit from therapeutic agents targeting histone acetylation regulation. In conclusion, our results indicate that gene fusion is the major class of genomic abnormalities in childhood ALL and chromosomal rearrangements involving EP300 and CREBBP may cause global epigenetic deregulation in ALL with potentials for therapeutic targeting. Disclosures No relevant conflicts of interest to declare.
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Sun, Qiao-Yang, Ling-wen Ding, Kar-Tong Tan, Wenwen Chien, Anand Mayakonda, Allen Eng Juh Yeoh, Norihiko Kawamata, et al. "Mutational Landscape of Pediatric Acute Lymphoblastic Leukemia." Blood 128, no. 22 (December 2, 2016): 452. http://dx.doi.org/10.1182/blood.v128.22.452.452.
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Abstract Pediatric ALL is the most common childhood tumor and the leading cause of childhood cancer deaths. To gain a better understanding of the landscape of somatic mutations in ALL, we performed whole exome and targeted sequencing of 240 pediatric B-ALL patients with their matched remission samples. The significantly mutated genes fall into several common categories: RAS/receptor tyrosine kinases, epigenetic regulators, transcription factors involved in lineage commitment and p53/cell cycle pathway. RAS/receptor tyrosine kinases: the most frequently mutated genes were members of RAS signaling (NRAS, KRAS, FLT3, PTPN11). Besides the well know hotspot mutations [G12D/V/C (NRAS 13 cases, KRAS 13 cases), G13D (NRAS 14 cases, KRAS 11 cases) and Q61H/L/R/K (NRAS 15 cases, KRAS 1 case)], novel mutational sites were also identified for KRAS: A146T/P (3 cases), K117N/T (4 cases) and V14I (1 case). High frequency missense mutations of PTPN11 clustered in SH2 domain (included the canonical hotspot A72T (5 cases) and E76K/V (4 cases)) and tyrosine-phosphatase catalytic domain (G503R/V). For FLT3, well-appreciated activating hotspot mutations in the kinase domain (D835Y/Y842C) and several novel recurrent mutationswere identified. Epigenetic regulators: hotspot mutations were identified in histone H3K36 methyltransferase WHSC1. Mutation E1099K located in the SET domain, was identified in 10 patients as well as two of the 5 ALL cell lines that we sequenced (RS4;11, SEM). Stable silencing of E1099K mutant WHSC1 in RS4;11 cells by either lentiviral shRNA or CRISPR guide RNA (sgRNA) markedly reduced clonogenic growth both in vitro and in vivo, underscoring the critical role of WHSC1 in lymphoid malignancies. Two highly-related histone/non-histone acetyltransferases, CREBBP and EP300, were also prominently mutated in our cohort. Mutations of CREBBP predominantly occurred in the acetyltransferase domain, particularly in the hotspot R1446C/H. Mutations of chromatin remodeling genes (ARID1A and ARID2) have been identified in a number of cases. Silencing of ARID1A in ALL cell lines by lentiviral shRNA resulted in upregulation of the pro-growth regulator c-MYC, while forced expression of ARID1A reduced c-MYC luciferase reporter activity. In addition, silencing of ARID1A by either shRNA or CRISPR-sgRNA resulted in enhanced clonogenic growth, suggesting that ARID1A may be involved in the c-MYC pathway and modulates the ALL cell proliferation. Mutations of epigenetic regulators were also found in the polycomb complex (EZH2, EED, SUZ12), chromatin/nucleosome structure modifying proteins (CHD2, CHD3, CHD4), TET family proteins [TET1 (2 cases), TET2 (5 cases)] and histone modification proteins (HDAC1, SIRT1, BCOR, BRD8, lysine demethylase PHF2/KDM6A, histone acetyltransferase KAT6B). Transcription factors and p53/cell cycle pathway: a number of alterations of transcription factors essential for hematopoietic and lymphoid differentiation were noted including the lineage regulator PAX5 (5 missense, 3 indels) and ETV6 (6 cases, 3 were frameshift indel and 1 was a splice-site mutations). In addition, mutations were also found in other lineage transcription factors (IKZF2, IKZF3, EBF1), WT1 (6 cases, including 3 indels and 1 stop-gain mutations), RUNX family member [RUNX2 (7 cases), RUNX1 (1 case)], ERG1 (3 cases), GATA1/3 (1 case each) and CTCF. Somatic mutations of genes involved in the p53 pathway occurred in 18 patients, including TP53, ATM and the kinases that regulate p53 activities (HIPK1, HIPK2). Germline TP53 pathogenic variants were found in these 2 patients. Taken together, we extensively interrogated the mutational landscape of a large cohort of pediatric ALL samples by exome and targeted resequencing. This study provides a detailed mutational portrait of pediatric ALL and gives new insights into the molecular pathogenesis of this disease. Disclosures Kantarjian: Amgen: Research Funding; ARIAD: Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer Inc: Research Funding; Delta-Fly Pharma: Research Funding; Novartis: Research Funding. Ogawa:Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding.
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Ennishi, Daisuke, Christoffer Hoffer, Hennady Shulha, Anja Mottok, Pedro Farinha, Fong Chun Chan, Barbara Meissner, et al. "Clinical Significance of Genetic Aberrations in Diffuse Large B Cell Lymphoma." Blood 124, no. 21 (December 6, 2014): 703. http://dx.doi.org/10.1182/blood.v124.21.703.703.
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Abstract Background: Although R-CHOP has significantly improved outcome in diffuse large B-cell lymphoma (DLBCL), 40% of patients still experience relapsed/refractory disease. Further investigation into the genomic architecture of DLBCL is needed to determine the biological correlates that underlie treatment failure. Recent studies using next-generation sequencing strategies have described the landscape of recurrent mutations in DLBCL. However, with the exception of TP53 and FOXO1, little is known about the clinical relevance of recurrent mutations and importantly, the interactions of these genetic alterations in DLBCL. Moreover, an integrated analysis of copy number alterations and recurrent mutations annotated across cell-of-origin (COO) distinctions for a large cohort of DLBCL cases who have received uniform therapy is lacking. The present study examined the frequency and clinical impact of recurrent genetic aberrations of DLBCL using high-resolution technologies in a large population-based DLBCL cohort. Methods: We analyzed 348 newly diagnosed DLBCL cases that were uniformly treated with R-CHOP at the BC Cancer Agency (Vancouver) with available DNA extracted from fresh frozen biopsy material (tumor content >30%). Matched germ line DNA was available for 67 patients. Comprehensive clinical annotation was available through the BCCA Lymphoid Cancer Database. Targeted re-sequencing of the coding exons of 56 genes was performed using a Truseq Custom Amplicon assay. Gene selection was based on mutational frequencies that have been previously described in DLBCL mutational landscape publications. High-resolution copy number analyses were performed using Affymetrix SNP 6.0 arrays. Tissue microarrays were constructed using duplicate 0.6mm cores from 332 cases, and breakapart FISH assays for MYC, BCL2 and BCL6 and IHC staining for MYC, BCL2 and cell of origin proteins were performed. COO classification was available in 331 cases, according to gene expression profiling by the Lymph2Cx assay using the NanoString platform (Scott, Blood 2014;123) in 299 patients as well as Hans algorithm (Hans, Blood 2004;103) in 32 cases with low tumor content. 194 cases were assigned to GCB subtype, 107 cases, ABC/non-GCB and 30 were unclassifiable. Results: In the mutation analysis, we identified 2,757 SNVs and 245 small indels. The mean depth of coverage was 634. Recurrent mutation frequencies varied between 0 and 58, with a mean of 8.25 per case. 98% of cases harbored at least one mutation and 95% of cases multiple mutations. 10 mutated genes were detected significantly more frequently in the GCB subtype including CREBBP, GNA13, EZH2, TNFRSF14, IRF8,STAT3, BCL2, SGK1, MEF2B and CD83, and 4 mutated genes, MYD88, CD79B, PRDM1 and PIM1, in the ABC subtype. In the copy number analysis, 45 significant amplification peaks and 57 deletion peaks were revealed by the GISTIC algorithm. As previously reported, 9p21.3, including CDKN2A,were more frequently detected in the ABC subtypes. With a median follow up of 6.5 years for living patients, the 5 y disease specific survival (DSS) and time to progression (TTP) of all patients were 72% and 64%, respectively. The clinical cohort was representative of registry data from BC based on a comparison of patient characteristics and survival outcomes with 1,194 control DLBCL R-CHOP patients. The ABC subtype was significantly associated with an inferior DSS and TTP (both p<0.0001). In univariate analyses we identified several gene mutations and copy number aberrations significantly associated with survival in all patients. Of these genes, MYD88 and TP53 mutations were associated with significantly inferior TTP in the ABC subtype (p=0.04) and GCB subtype (p=0.002), respectively, while TMEM30A, CREBBP, PIM1 and BTG1 mutations were associated with prognosis in DLBCL. Our analyses confirm the poor prognosis conferred by TP53 mutations in DLBCL and, importantly, identified several novel genetic alterations associated with survival stratified by COO distinctions. Conclusions: Our approach using next generation sequencing and high resolution SNP array provides an accurate estimation of frequency and clinical significance of recurrent genetic alterations of DLBCL in a uniformly R-CHOP-treated large population-based cohort of patients. Disclosures No relevant conflicts of interest to declare.
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Van‐Gils, Julien, Sophie Naudion, Jérôme Toutain, Gwenaelle Lancelot, Tania Attié‐Bitach, Sophie Blesson, Bénédicte Demeer, et al. "Fetal phenotype of Rubinstein‐Taybi syndrome caused by CREBBP mutations." Clinical Genetics 95, no. 3 (January 11, 2019): 420–26. http://dx.doi.org/10.1111/cge.13493.
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Maitre, Elsa, Cécile Tomowiak, Benjamin Lebecque, Fontanet Bijou, Khaled Benabed, Dina Naguib, Pauline Kerneves, et al. "Deciphering Genetic Alterations of Hairy Cell Leukemia and Hairy Cell Leukemia-like Disorders in 98 Patients." Cancers 14, no. 8 (April 10, 2022): 1904. http://dx.doi.org/10.3390/cancers14081904.
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Hairy cell leukemia (cHCL) patients have, in most cases, a specific clinical and biological presentation with splenomegaly, anemia, leukopenia, neutropenia, monocytopenia and/or thrombocytopenia, identification of hairy cells that express CD103, CD123, CD25, CD11c and identification of the V600E mutation in the B-Raf proto-oncogene (BRAF) in 90% of cases. Monocytopenia is absent in vHCL and SDRPL patients and the abnormal cells do not express CD25 or CD123 and do not present the BRAFV600E mutation. Ten percent of cHCL patients are BRAFWT and the distinction between cHCL and HCL-like disorders including the variant form of HCL (vHCL) and splenic diffuse red pulp lymphoma (SDRPL) can be challenging. We performed deep sequencing in a large cohort of 84 cHCL and 16 HCL-like disorders to improve insights into the pathogenesis of the diseases. BRAF mutations were detected in 76/82 patients of cHCL (93%) and additional mutations were identified in Krüppel-like Factor 2 (KLF2) in 19 patients (23%) or CDKN1B in 6 patients (7.5%). Some KLF2 genetic alterations were localized on the cytidine deaminase (AID) consensus motif, suggesting AID-induced mutations. When analyzing sequential samples, a clonal evolution was identified in half of the cHCL patients (6/12 pts). Among the 16 patients with HCL-like disorders, we observed an enrichment of MAP2K1 mutations in vHCL/SDRPL (3/5 pts) and genes involved in the epigenetic regulation (KDM6A, EZH2, CREBBP, ARID1A) (3/5 pts). Furthermore, MAP2K1 mutations were associated with a bad prognosis and a shorter time to next treatment (TTNT) and progression-free survival (PFS), independently of the HCL classification.
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Vardarajan, Badri N., Giuseppe Tosto, Roger Lefort, Lei Yu, David A. Bennett, Philip L. De Jager, Sandra Barral, et al. "Ultra-rare mutations in SRCAP segregate in Caribbean Hispanic families with Alzheimer disease." Neurology Genetics 3, no. 5 (August 24, 2017): e178. http://dx.doi.org/10.1212/nxg.0000000000000178.
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Objective:To identify rare coding variants segregating with late-onset Alzheimer disease (LOAD) in Caribbean Hispanic families.Methods:Whole-exome sequencing (WES) was completed in 110 individuals from 31 Caribbean Hispanic families without APOE ε4 homozygous carriers. Rare coding mutations segregating in families were subsequently genotyped in additional families and in an independent cohort of Caribbean Hispanic patients and controls. SRCAP messenger RNA (mRNA) expression was assessed in whole blood from mutation carriers with LOAD, noncarriers with LOAD, and healthy elderly controls, and also from autopsied brains in 2 clinical neuropathologic cohort studies of aging and dementia.Results:Ten ultra-rare missense mutations in the Snf2-related CREBBP, activator protein (SRCAP), were found in 12 unrelated families. Compared with the frequency in Caribbean Hispanic controls and the Latino population in the Exome Aggregation Consortium, the frequency of SRCAP mutations among Caribbean Hispanic patients with LOAD was significantly enriched (p = 1.19e-16). mRNA expression of SRCAP in whole blood was significantly lower in mutation carriers with LOAD, while the expression in whole blood and in the brain was significantly higher in nonmutation carriers with LOAD. Brain expression also correlated with clinical and neuropathologic endophenotypes.Conclusions:WES in Caribbean Hispanic families with LOAD revealed ultra-rare missense mutations in SRCAP, a gene expressed in the brain and mutated in Floating-Harbor syndrome. SRCAP is a potent coactivator of the CREB-binding protein and a regulator of DNA damage response involving ATP-dependent chromatin remodeling. We hypothesize that increased expression in LOAD suggests a compensatory mechanism altered in mutation carriers.
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Antic, Zeljko, Jiangyan Yu, Simon V. van Reijmersdal, Renske Kuiper, Edwin Sonneveld, Frank N. van Leeuwen, Peter M. Hoogerbrugge, Esmé Waanders, and Roland P. Kuiper. "Genomic Architecture and Clonal Dynamics of Early Relapsed BCP-ALL." Blood 128, no. 22 (December 2, 2016): 4072. http://dx.doi.org/10.1182/blood.v128.22.4072.4072.
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Abstract Relapse represents the most common cause of therapy failure in B-cell precursor ALL acute lymphoblastic leukemia (BCP-ALL), and is caused by selective outgrowth of therapy-resistant leukemic cells. Two-third of BCP-ALL relapses present after treatment, i.e. after two years. These relapses may originate from leukemic (sub)clones that remained in a quiescent state during treatment or that could not be reached by the chemotherapeutics. Relapses that occur during treatment are different in that they display clonal outgrowth in the presence of chemotherapeutics, and these patients have poorer outcomes. The aim of this study is to explore the genomic abnormalities in leukemia with early relapse, and investigate the clonal dynamics of relapses that arise during treatment. We included 17 BCP-ALL cases which relapse during treatment (<2yrs) according to DCOG protocols ALL9, ALL10 or ALL11. Median remission time was 1.08 yrs (range 0.48-1.95). Whole exome sequencing was performed on DNA isolated at time of first diagnosis, complete remission and relapse from bone marrow or peripheral blood, with an average read depth on target of 108x. After mapping of the reads, variants were called using HaplotypeCaller. In total, we identified 1771 somatic mutations in 1562 genes. Per case, a median of 21 mutations were detected at diagnosis (range 10-630) and 31 at relapse (range 10-652). A hypermutation profile was observed in one diagnosis-relapse pair, and two additional relapses. All cases harbored mutations shared between diagnosis and relapse, which were mostly part of the major clone at both time points. However, the fraction of shared mutations varied considerably between cases, ranging from <10% in 3 cases to >80% in 4 cases. Based on the clonal dynamics, 3 distinct groups were recognized. Group I includes two cases, with relapses within 6 months, in which the (sub)clonal mutation spectrum between diagnosis and relapse was identical. Group II (n=10) presented with a relapse closely resembling the major clone at diagnosis. Mostly, these relapses acquired new mutations and they often branched off from the major clone already before the time of diagnosis. Finally, Group III (n=5) consists of cases in which the relapse originates from a minor subclone at diagnosis that hardly resembled the major clone, suggesting a clonal switch during treatment. Next, we analyzed the genes with mutations that were predicted to be damaging (truncating and non-synonymous conserved missense variants). We performed pathway analysis for these genes and identified RAS pathway genes to be frequently mutated among shared mutations, while mutations in genes involved in epigenetic regulation, chromatin condensation and regulation of transcription were acquired. In total, 7 of the genes with (predicted) pathogenic mutations in relapse were affected in at least two cases, including known genes like KRAS, CREBBP, and WHSC1 (NSD2). CREBBP mutations were never part of the major clone at diagnosis and were present in cases with numerical chromosomal aberrations. Both cases with hotspot E1099K mutation in WHSC1 were t(1;19) translocation-positive. Recent studies showed that somatic mutational signatures, composed of the six substitution subtypes in a 3-nucleotide context, expose specific biological processes underlying tumor development, including defects in genomic maintenance and repair. Currently 30 mutational signatures have been described [http://cancer.sanger.ac.uk/cosmic/signatures]. Despite the low number of mutations in most samples, we identified at least 5 of these signatures, including the most common signature 1, a signature associated with aberrant AID/APOBEC activity (signature 2), and three signatures associated with mismatch repair deficiency (6, 15, 26). Most cases carried multiple signatures, but signature 2 was very prominent is one relapse and one diagnosis-relapse pair. Most signatures were preserved from diagnosis to relapse suggesting that the same mutational processes remained active. Taken together, our results show considerable heterogeneity in the group of children with early relapse of BCP-ALL. Two cases with the shortest remission times relapsed without notifiable somatic changes, whereas most other early relapses appeared to arise from minor or newly appearing subclones. These findings demonstrate the strong clonal selection that occurs during treatment in cases with very early relapse. Disclosures No relevant conflicts of interest to declare.
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Fitzgibbon, Jude. "Genomic Discovery, Prognosis, and Target Therapy Development." Blood 128, no. 22 (December 2, 2016): SCI—6—SCI—6. http://dx.doi.org/10.1182/blood.v128.22.sci-6.sci-6.
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Abstract For over three decades now, the t(14;18)(q32.3;q21.3) has been synonymous with the indolent B cell malignancy, follicular lymphoma (FL). Through the widespread uptake of next generation sequencing, we recognize that these tumors acquire many additional genetic aberrations, targeting several distinct signaling pathways (HVEM, mTORC1, NOTCH, NF-κB and JAK-STAT) all of which fall onto a backbone of epimutations that arise in nearly all FL tumors and preferentially target the histone methyl (KMT2D) and acetyl (CREBBP) transferase machinery. We are still in the early stages of understanding how these mutations impact on the biology of FL, their weight of contribution to FL initiation, progression and transformation and indeed whether we can harness this new knowledge to improve on existing treatment. At the very least, we now realize that the acquisition of these mutations and onset of FL may occur several years earlier from the study of rare examples of clonally-related tumors developing post bone marrow transplantation in both donor and recipient. This has helped formulate the notion of an uncharacterized common progenitor pool of B cells (CPC) that are capable of seeding each new genetically distinct episode of disease and providing an explanation for the relapsing remitting nature of FL. We can also now infer the nature of the apparent early driver mutations that seemingly propagate the disease in all FL clones, including the CPC, by using a gene's mutation load or clonal composition as a surrogate marker. It is apparent that many epimutations represent early events and appear to alter normal B cell programming by favoring sets of genes that promote proliferation and survival while switching off anti-tumor mechanisms, with CREBBP-mutated FLs for example showing a decreased antigen presentation gene signature. The prospect of following a precision medicine approach informed by a patient's mutation composition should be encouraged given the frequency of activating EZH2 mutations, an underappreciated role of the NF-κB pathway and mutations in the mTORC1 pathway, that are unique to FL, together with the availability of targeted therapeutic agents. There is of course a need to focus efforts on the 20% of patients that succumb to early disease relapse, and the emergence of a new clinical-genetic risk model (m7-FLIPI), as an outcome predictor offers a promising first step in developing a robust risk stratification approach. We do however need to embrace these new opportunities and embed biomarker discovery and validation studies as part of all FL trial programs. Indeed, a case can be made for utilizing material from specific retrospective studies, where updated mutation analysis may demonstrate a potential novel predictive and therapeutic biomarker (e.g. RRAGC mutations/mTOR inhibitors). Altogether, while the genetic portrait of FL is still far from complete, we are now much closer to delivering a final canvas and with it the prospect of changing the landscape of future FL treatment. Disclosures Fitzgibbon: Epizyme: Research Funding; Gilead: Honoraria; Janssen: Honoraria; Celgene: Honoraria.
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Rose-Zerilli, Matthew JJ, Marina Parry, Viktor Ljungstrom, Jane Gibson, Jun Wang, Renata Walewska, Helen Parker, et al. "Deep-Sequencing Reveals the Molecular Landscape of Splenic Marginal Zone Lymphoma: Biological and Clinical Implications." Blood 124, no. 21 (December 6, 2014): 76. http://dx.doi.org/10.1182/blood.v124.21.76.76.
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Abstract Whilst the spectrum and clinical significance of gene mutations and immunogenetic features in common mature B-cell malignancies is well established, their incidence, biological and clinical importance in splenic marginal zone lymphoma (SMZL) remains uncertain. Accordingly we screened 175 well-characterised SMZL cases for mutations in 768 genes (Haloplex Target Enrichment System) with a known or postulated role in B-cell physiology, B-cell malignancies in general and SMZL pathophysiology in particular. After sequencing (HiSeq 2000) we achieved a mean depth across our gene panel of 297-fold (range 128-702x), with more than 85% of all bases covered at >50-fold. After comprehensive filtering, 1,374 single nucleotide variants and insertions/deletions were identified. 221 genes were recurrently mutated at a gene frequency of 2-16% [n=2-28]. Sanger sequencing confirmed 86/86 selected variants in our recurrent genes, and showed 99% concordance between our Haloplex and Sanger sequencing of NOTCH2 exon 34, which was performed in all patients. Comprehensive validation of both germ-line Haloplex [n=18 patients] and Sanger sequencing established the sensitivity and specificity of our approach, and confirmed the biological importance and somatic origin of the genes described herein. To identify biologically relevant genes, we employed MutSigCV analysis, an algorithm that identifies significantly mutated genes by accounting for background mutation rate, DNA replication time and the gene size. 18 mutated genes were identified with TP53 [n=28], KLF2 [n=21], MYD88 [n=12], NOTCH2 [n=17], TNFAIP3 [n=13] and CCND3 [n=15] being the most significant; genes that encode components of pathways important in the regulation and differentiation of mature B-cells were also identified, including CREBBP [n=9], MAP3K6 [n=5], KDM2B [n=7], SETD1B [n=6], TRAF3 [n=9], ARID1A [n=10], BIRC3 [n=3], BCL10 [n=5], BTG1 [n=3], ATM [n=10], NFKBIE [n=4] and DDI1 [n=4]. Then, we searched for significant pairwise gene correlations and mutually exclusive relationships between our mutated genes demonstrating the following: (1) independent events, such as MYD88, where a mutation is invariably observed as an isolated event; (2) cancer drivers that have a similar proportion of co-occurring and mutually-exclusive relationships, such as NOTCH2, TP53, TNFAIP3 and CREBBP, and (3) genes such as KLF2, CCND3 and ARID1A that have proportionally more co-occurring relationships, thus suggesting a synergistic function to promote tumorigenesis. Finally, we studied clonal evolution, by differentiating between early, clonal events, and later, subclonal mutations (ABSOLUTE algorithm), and we were able to classify clonal or subclonal mutation in 6/18 of our MutSigCV genes. Paradigmatically, we observed that all the CREBBPmutations were fully clonal. Amongst our most novel findings was KLF2, or Krüppel-like factor 2, mutations that were distributed across the entire protein, with a cluster in the C2H2 domain and were all somatically acquired. All mutations tested were clonal, significantly associated with del(7q) (P=0.001), IGHV1-2*04 gene usage (P<0.001) and other gene mutations including NOTCH2 (all P<0.001). Together, these observations suggest that the potential cell survival advantage provided by an early KLF2 mutation allows the acquisition of additional functionally synergistic gene mutations to promote tumourigenesis. Genes associated with reduced time to first treatment (TTFT) included KLF2 (HR 1.93, 95%CI 1.16-3.32, p=0.01), and NOTCH2 (HR 2.13, 95%CI1.26-3.58, p=0.003). TP53 mutations were associated with shorter event-free (EFS) and overall survival (OS) (HR 2.17, 95%CI 1-4.74, p=0.05 and HR 2.16, 95%CI 1.05-4.42, p=0.032, respectively). Using multivariate Cox proportional hazard analysis, which included base-line clinical varaibles, both NOTCH2 (HR 2.12, 95%CI 1.02-4.4, p=0.044) and 100% germline IGHV gene identify (HR 2.19, 95%CI 1.05-4.55, p=0.036) were independent risk factors for TTFT. Furthermore, the presence of TP53 mutation was an independent risk factor for OS (HR 2.16, 95%CI 1.05-4.43, p=0.032). In summary, we have defined the mutational landscape in a large cohort of patients with SMZL and identified novel recurrent mutations, especially involving KLF2. Importantly we show for the first time, that gene mutations and immunogenetic features have independant prognostic significance. Disclosures Anagnostopoulos: Gilead Sciences: Research Funding. Fazi:Rhizen Pharmaceuticals SA: Research Funding. Ghia:Merck: Consultancy; GSK, Roche Italia: Consultancy; Gilead, Pharmacyclics, Boehringer Ingelheim, Celgene, Roche Italia: Membership on an entity's Board of Directors or advisory committees.
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О.Р., Исмагилова,, Адян, Т.А., Бескоровайная, Т.С., and Поляков, А.В. "Molecular-genetic analysis of Rubinstein-Taybi syndrome in Russia." Nauchno-prakticheskii zhurnal «Medicinskaia genetika, no. 9 (September 30, 2022): 48–51. http://dx.doi.org/10.25557/2073-7998.2022.09.48-51.
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Синдром Рубинштейна-Тейби (СРТ) - редкая наследственная патология, характеризующаяся умственной отсталостью и задержкой физического развития в сочетании с определённым комплексом внешних проявлений и аномалиями различных органов и систем. Диагностический алгоритм нацелен на поиск мутаций в двух основных генах: CREBBP и EP300, обнаруживаемых примерно в 60% клинически определённых случаев СРТ. В настоящей работе представлены результаты молекулярно-генетического анализа выборки российских пациентов с СРТ. Несмотря на то, что около 40% пациентов не получают подтверждения клинического диагноза, существуют перспективы для более глубокого понимания патогенеза заболевания и усовершенствования принципов диагностики. Rubinstein-Taybi syndrome (RTS) - rare hereditary disorder characterized by intellectual disability and growth retardation in conjunction with specific craniofacial and skeletal features and a wide range of multiple congenital anomalies. To date, mutations in two genes: CREBBP and EP300 can be discovered in about 60% of clinically identified patients with RTS. We herein report the result of molecular analysis in a cohort of Russian RTS patients. Despite the fact that we cannot confirm the diagnosis of RTS in 40% of causes, there are possibility for a better understanding of causative molecular mechanisms and improving the diagnostic process.
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Ko, Jaemin, Jason H. Pomerantz, Hazel Perry, Joseph T. Shieh, Anne M. Slavotinek, Snehlata Oberoi, and Ophir D. Klein. "Case Report of Floating-Harbor Syndrome With Bilateral Cleft Lip." Cleft Palate-Craniofacial Journal 57, no. 1 (June 27, 2019): 132–36. http://dx.doi.org/10.1177/1055665619858257.
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Floating-Harbor syndrome (FHS) is a rare genetic disorder caused by heterozygous mutations in the Snf2-related CREBBP activator protein ( SRCAP) gene. The syndrome is characterized by proportional short stature, delayed bone maturation, delayed speech development, and facial dysmorphism. Submucous cleft palate and cleft lip have been reported in FHS, but to our knowledge orofacial clefting in this condition has not been assessed in detail. Here, we report on a case of bilateral cleft lip in a patient with FHS confirmed by exome sequencing.
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Donahue, Timothy F., Aditya Bagrodia, François Audenet, Mark T. A. Donoghue, Eugene K. Cha, John P. Sfakianos, Dahlia Sperling, et al. "Genomic Characterization of Upper-Tract Urothelial Carcinoma in Patients With Lynch Syndrome." JCO Precision Oncology, no. 2 (November 2018): 1–13. http://dx.doi.org/10.1200/po.17.00143.
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Purpose Patients with Lynch syndrome (LS) have a significantly increased risk of developing upper-tract urothelial carcinoma (UTUC). Here, we sought to identify differences in the patterns of mutational changes in LS-associated versus sporadic UTUCs. Patients and Methods We performed targeted sequencing of 17 UTUCs from patients with documented LS-associated germline mutations (LS-UTUCs) using the Memorial Sloan Kettering Integrated Molecular Profiling of Actionable Cancer Targets targeted exon capture assay and compared the results with those from a recently characterized cohort of 82 patients with sporadic UTUC. Results Patients with LS-UTUC were significantly younger, had had less exposure to tobacco, and more often presented with a ureteral primary site compared with patients with sporadic UTUC. The median number of mutations per tumor was significantly greater in LS-UTUC tumors than in tumors from the sporadic cohort (58; interquartile range [IQR], 47-101 v 6; IQR, 4-10; P < .001), as was the MSIsensor score (median, 25.1; IQR, 17.9-31.2 v 0.03; IQR, 0-0.44; P < .001). Differences in the genetic landscape were observed between sporadic and LS-associated tumors. Alterations in KMT2D, CREBBP, or ARID1A or in DNA damage response and repair genes were present at a significantly higher frequency in LS-UTUC. CIC, NOTCH1, NOTCH3, RB1, and CDKN1B alterations were almost exclusive to LS-UTUC. Although FGFR3 mutations were identified in both cohorts, the R248C hotspot mutation was highly enriched in LS-UTUC. Conclusion LS- and sporadic UTUCs have overlapping but distinct genetic signatures. LS-UTUC is associated with hypermutation and a significantly higher prevalence of FGFR3 R248C mutation. Prospective molecular characterization of patients to identify those with LS-UTUC may help guide treatment.
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Green, Michael R. "Chromatin modifying gene mutations in follicular lymphoma." Blood 131, no. 6 (February 8, 2018): 595–604. http://dx.doi.org/10.1182/blood-2017-08-737361.
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Abstract Follicular lymphoma (FL) is an indolent malignancy of germinal center B cells. Although the overall survival of FL patients has recently improved with the introduction of novel therapies, there is significant heterogeneity in patient outcome and a need for rationally designed therapeutic strategies that target disease biology. Next-generation sequencing studies have identified chromatin modifying gene (CMG) mutations as a hallmark of FL, highlighting epigenetic modifiers as an attractive therapeutic target in this disease. Understanding the complex roles of these mutations will be central to identifying and adaptively targeting associated vulnerabilities. Recent studies have provided insight into the functional consequences of the most frequently mutated CMGs (KMT2D, CREBBP, and EZH2) and point to a role for these events in modifying normal B-cell differentiation programs and impeding germinal center exit. However, the majority of FL tumors serially acquire multiple CMG mutations, suggesting that there is a level of cross talk or cooperation between these events that has not yet been defined. Here, I review the current state of knowledge on CMG mutations in FL, discuss their potential as therapeutic targets, and offer my perspective on unexplored areas that should be considered in the future.
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Melchardt, Thomas, Clemens Hufnagl, Oliver Weigert, David M. Weinstock, Nadja Kopp, Lukas Weiss, Daniel Neureiter, et al. "Clonal Evolution in Relapsed or Refractory Diffuse Large B Cell Lymphoma." Blood 124, no. 21 (December 6, 2014): 77. http://dx.doi.org/10.1182/blood.v124.21.77.77.
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Abstract Background: Despite modern chemoimmunotherapy more than a third of patients with diffuse large B cell lymphoma (DLBCL) will experience relapse or refractory disease. Multiple mutations associated with the biology of DLBCL have recently been identified by next-generation sequencing in primary tumor samples, but little is known about their prognostic role. Furthermore, histological comparison of the primary tumor and relapsed disease is often not available in clinical practice due to the lack of centralized assessment and complicated by the difficulties to perform exome-wide sequencing in formalin fixed tissues. Therefore, the role of certain mutations and their mechanisms in clonal evolution during relapse is unknown and the rising of chemo-resistant DLBCL subclones has not been described in the literature so far. Methods: We identified all patients with available histologically confirmed relapsed or refractory DLBCL in our single center cohort of 346 patients with aggressive lymphoma treated at our tertiary cancer center in Salzburg, Austria. Primary formalin fixed paraffin embedded tumor sample, sample of refractory or relapsed disease and matched germ line were available for targeted next generation sequencing in 27 patients. A targeted exon capture and next-generation sequencing of all coding exons of 104 selected genes known to be frequently mutated in lymphoma were performed on a HiSeq 2500. Results: Sequencing was successful in 96.8% of all samples resulting in 25 patients with sequencing of the primary tumor and 24 patients with available pairs of primary lymphoma and histologically confirmed relapse. In these 24 patients two relapse samples were available in 10 patients and three relapse samples in one patient. Non-synonymous mutations were present in 74 of the 104 genes tested. Individual tumor samples showed between 0 and 29 non-synonymous mutations (median: 10). Less than six non-synonymous mutations in the primary tumor were associated with a better median OS than more mutations (28 versus 15 months p=0.031). We also compared the frequency of mutated genes in our cohort consisting of high risk patients defined by actual relapse with the literature containing patients with extensive sequencing but only little clinical data and clinical follow-up. Common mutated genes such as CARD11, CD58, CD79B, CREBBP, EZH2, BTG1 or B2M showed no difference in frequency to our patient cohort indicating no or only small driver function in resistant or refractory disease. Nevertheless, mutations previously reported to be at low frequency in DLBCL were significantly more often observed in our primary samples (NOTCH1, MYC, RB1, FAT2, ATM, SMARCA4, BCL7A) and relapsed samples (TP53, MCL1, ATM, FAT2, MYC, RB1, SMARCA4) of high risk patients when compared to the literature. We also observed the gain and the loss of several mutations between first diagnosis and histologically confirmed relapse. Overall, we observed an increase of the amount of non-synonymous mutations at first relapse in 12, no change in 6 and a decrease in 6 paired cases. A completely stable pattern of non-synonymous mutations was detected in 4 cases, but in the majority of cases relevant dynamic was observed: e.g.: gain of non-synonymous mutations in the p53 gene was seen in 3 patients (5 mutations), in the TNFRSF-14 gene in 2 patients (3 mutations), in the RB1 gene in 1 patient (1 mutation), in the NOTCH2 gene in 3 patients (4 mutations) and in the MYD88 gene in 1 patient (1 mutation) or loss of non-synonymous mutations in the CREBBP gene in 3 patients (3 mutations) or in the CRAD11 gene in 2 patients (2 mutations). Monitoring of subclones during disease was also possible using the allelic fraction over time e.g.: showing an increase of the NOTCH1 mutation burden in 2 biopsies after first diagnosis. Discussion: To the best of our knowledge clonal evolution detected by next generation sequencing has not been reported in DLBCL so far. We demonstrate the feasibility of such an approach from fixed tissue samples and using a curated set of target genes. In analogy to other lymphoid malignancies we can show the increase of allelic burden of certain mutations over time and the loss or gain of several others. While this approach is limited by the bias introduced by the selection of genes in the gene set, we feel that deep sequencing of selected mutations will provide further insights into subclone dynamics, which may be responsible for clonal evolution. Disclosures No relevant conflicts of interest to declare.
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Isharwal, Sumit, Francois Audenet, Eugene J. Pietzak, Eugene K. Cha, Gopa Iyer, Ahmet Zehir, Barry S. Taylor, et al. "Comparison of genomic alterations in bladder urothelial tumors with and without telomerase reverse transcriptase promoter mutation using a next-generation sequencing assay." Journal of Clinical Oncology 35, no. 6_suppl (February 20, 2017): 310. http://dx.doi.org/10.1200/jco.2017.35.6_suppl.310.
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310 Background: Telomerase reverse transcriptase (TERT) is the most frequently altered gene in urothelial cancer (UC), detected across all grades and stages of disease. We sought to characterize TERT alterations within a prospective cohort of UC treated at our institute and compare the frequency of genomic alterations in TERT promoter mutant vs wild-type UC specimens. Methods: Patients diagnosed with bladder urothelial tumors were enrolled onto an institutional review board approved prospective sequencing protocol. Tumor and matched germline DNA were analyzed for somatic point mutations, truncations, copy number alterations, and insertions/deletions using the MSK-IMPACT NGS assay that detects alterations in all exons and select introns of 410 oncogenes and tumor suppressor genes as well as the entire TERT promoter region. Results: 329 UC were sequenced of which 236 (71.7%) harbored TERT mutations, the majority being promoter region hotspots (chr5: 1295228 G > A [81%] and chr5:1295250 G > A [16%]). Patients with TERT promoter mutations were significantly older than those without (69.01±10.70 years vs. 65.44±11.78 years, p = 0.0317). UC with TERT promoter mutations had significantly higher mutation count [median 10 (range: 2-76) vs median 5 (range: 0-119)] as well as copy number alterations [median 0.11 (range: 0-0.68) vs median 0.047 (range: 0-0.65)]. Between UC with and without TERT promoter mutations, there was a very significant difference in mutation frequencies of ARID1A(33% vs 11%), PIK3CA(28% vs 13%), FGFR3(32% vs 16%), CREBBP(19% vs 4%), CDKN1A(17% vs 2%), ERBB2(26% vs 9%), ERCC2(15% vs 3%), TSC1(11% vs 0%), MT2C(17% vs 9%) (all with p < 0.005). Conclusions: TERT is the most frequently altered gene in bladder cancer with the majority of TERT alterations comprised of two hotspot mutations. UC with TERT promoter mutations tends to occur in older patients and is associated with overall higher mutation count and copy number alterations. A number of genes are differentially mutated in UC with and without TERT promoter mutations and may suggest a link between TERT promoter mutations and distinct mutations profiles in UC.
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Zhou, Xiaolong Alan, Jingyi Yang, Kimberly G. Ringbloom, Maria Estela Martinez-Escala, Kristen E. Stevenson, Alexander T. Wenzel, Damiano Fantini, et al. "Genomic landscape of cutaneous follicular lymphomas reveals 2 subgroups with clinically predictive molecular features." Blood Advances 5, no. 3 (February 1, 2021): 649–61. http://dx.doi.org/10.1182/bloodadvances.2020002469.
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Abstract Primary cutaneous follicle center lymphomas (PCFCLs) are indolent B-cell lymphomas that predominantly remain skin restricted and manageable with skin-directed therapy. Conversely, secondary cutaneous involvement by usual systemic follicular lymphoma (secondary cutaneous follicular lymphoma [SCFL]) has a worse prognosis and often necessitates systemic therapy. Unfortunately, no histopathologic or genetic features reliably differentiate PCFCL from SCFL at diagnosis. Imaging may miss low-burden internal disease in some cases of SCFLs, leading to misclassification as PCFCL. Whereas usual systemic FL is well characterized genetically, the genomic landscapes of PCFCL and SCFL are unknown. Herein, we analyzed clinicopathologic and immunophenotypic data from 30 cases of PCFCL and 10 of SCFL and performed whole-exome sequencing on 18 specimens of PCFCL and 6 of SCFL. During a median follow-up of 7 years, 26 (87%) of the PCFCLs remained skin restricted. In the remaining 4 cases, systemic disease developed within 3 years of diagnosis. Although the SCFLs universally expressed BCL2 and had BCL2 rearrangements, 73% of the PCFCLs lacked BCL2 expression, and only 8% of skin-restricted PCFCLs had BCL2 rearrangements. SCFLs showed low proliferation fractions, whereas 75% of PCFCLs had proliferation fractions >30%. Of the SCFLs, 67% had characteristic loss-of-function CREBBP or KMT2D mutations vs none in skin-restricted PCFCL. Both SCFL and skin-restricted PCFCL showed frequent TNFRSF14 loss-of-function mutations and copy number loss at chromosome 1p36. These data together establish PCFCL as a unique entity with biological features distinct from usual systemic FL and SCFL. We propose 3 criteria based on BCL2 rearrangement, chromatin-modifying gene mutations (CREBBP, KMT2D, EZH2, and EP300), and proliferation index to classify cutaneous FL specimens based on the likelihood of concurrent or future systemic spread.