BCL2 Negative Follicular Lymphoma with BCL6 Gene Rearrangement Show Mutations of STAT6 DNA Binding Domain

Abstract Abstract 1559 Follicular lymphoma (FL) is the most common indolent subtype of non-Hodgkin's lymphoma in the western world. The genetic hallmark of FL is the t(14;18)(q32;q21) translocation leading to the deregulation of BCL2 expression which occurs in up to 90% of the grade 1–2 FL. However, a minority of FL without BCL2 gene rearrangement harbour genetic abnormalities involving the BCL6 gene (5–15% of the cases) and show distinct pathological features. The activation of Signal Transducer and Activator of Transcription 6 (STAT-6) has been observed in Primary Mediastinal B-cell Lymphoma (PMBL) and Hodgkin Lymphoma but also in FL. Because missense mutations of STAT6 DNA binding domain have been described in PMBL, we searched for such mutations in FL. Using a PCR HRM (High Resolution Melting) assay as a screening tool and conventional Sanger sequencing in all cases with abnormal denaturation curves, we analyzed the frequency of STAT6 mutations in FL samples. We focused our screening on exon 12, which encodes part of the DNA binding domain and which had been shown to be a hotspot mutation in PMBL. A series of 40 FL lymphomas samples diagnosed at the University Hospital of Créteil (Henri Mondor) and Centre Henri Becquerel in Rouen were retrieved. These tumors showed characteristic histopathological features of FL according to the World Health Organization (WHO) classification. DNA analysis was performed on DNA extracted from fresh/frozen samples (Rouen) or FFPE (formalin-fixed and paraffin-embedded) tissues (Créteil) with standard procedures. We detected 5 (12%) mutated tumors in this series of FL. These 5 mutations were single missense mutations targeting amino acids 419–421. All mutations were observed in histological grade 1or 2 lymphomas, and no mutations were found in the 9 cases classified as 3A or 3B. Only one classical FL out of 15 cases (6%) with BCL2 rearrangement showed STAT6 mutation. Strikingly, the 4 other mutated cases showed specific features. There were 3 female and 1 male, with a mean age of 52 years, and all presented with inguinal involvement and stage III or IV Ann Arbor disease. Morphologically, these cases displayed a follicular growth pattern. The immunophenotype was CD20+, CD5-, CD10+ (3/4), BCL6+, CD23+ (3/4) but BCL2 was negative (4/4). Cytogenetically, these 4 cases were characterized by BCL6 gene rearrangement without BCL2 gene rearrangement by interphase FISH on FFPE tissue sections. Thus, STAT6 mutations were observed in 4/11 (36%) grade 1–2, and 0/6 grade 3A/3B BCL2 negative FL with BCL6 rearrangement. No case was found to be mutated in the FL group without BCL2 and BCL6 rearrangement (8 cases). In conclusion, this is the first time that mutations of STAT6 are found in FL and interestingly, they target a rare group of FL with distinct pathological and cytogenetical features. Further investigations are required to identify the mutational mechanisms involved and the oncogenic function associated with these mutations. Disclosures: No relevant conflicts of interest to declare.

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Recurrent STAT6 Mutations In Follicular Lymphoma

Blood ◽

10.1182/blood.v122.21.503.503 ◽

2013 ◽

Vol 122 (21) ◽

pp. 503-503 ◽

Cited By ~ 1

Author(s):

Sami Malek ◽

Mark S. Kaminski ◽

Hongxiu Li ◽

Peter Ouillette ◽

Sian Jones ◽

...

Keyword(s):

Dna Binding ◽

Follicular Lymphoma ◽

Massively Parallel Sequencing ◽

B Cell Lymphoma ◽

Binding Domain ◽

Read Length ◽

Single Amino Acid ◽

Primary Data ◽

Dna Binding Domain ◽

Structural Genomic

Abstract Introduction Follicular lymphoma (FL) constitutes the second most common non-Hodgkin’s lymphoma in the Western world. FL carries characteristic recurrent structural genomic aberrations. However despite recent advances, knowledge regarding the coding genome in FL is still evolving and is currently incomplete. Methods To further our understanding of the genetic basis of follicular lymphoma (FL), we used solution exon capture of sheared and processed genomic DNA isolated from FACS-sorted lymphomatous B-cells and paired CD3+ T-cells isolated from twenty three cases of FL and one case of DLBCL (which was transformed from prior FL), followed by paired-end (96-101 base pair read length per side) massively parallel sequencing. The sequence data were characterized by a mean depth of coverage of 41, and 90% of bases in the target region were covered by at least 10 reads. Bioinformatics pipelines developed by our bioinformatics core served as the primary data source to nominate candidate mutated genes in downstream data analysis. Results The bioinformatics pipeline nominated 711 distinct candidate mutations in 24 FL cases. Sanger sequence validation confirmed 39 recurrently (≥ 2 FL cases) mutated genes. Genes with confirmed mutations in ≥ 2 FL cases in the discovery cohort were subsequently selectively expanded into a combined FL validation cohort of 114 cases. In addition to frequent mutations in MLL2, CREBBP, BCL2, TNFRSF14, EZH2, OCT2, ARID1A, IRF8 and MEF2B, we here report novel mutations in STAT6 in FL. STAT6 mutations were identified in 11% (12/114) of FL and predominantly affected the DNA binding domain (DBD; comprising STAT6 amino acids 268-430). Two FL cases each carried two distinct STAT6 mutations, presumably targeting both alleles. Of interest, the majority of FL-associated STAT6 mutations affected a single amino acid codon (codon 419), resulting in the STAT6 mutants p.419D>D/G or p.419D>D/H. These FL-associated STAT6 mutations are distinct from mutations previously described in primary mediastinal B-cell lymphoma (PMBCL). Given the involvement of STAT6 in signal transduction pathways activated by multiple cell surface receptors, as well as the recently described involvement of STAT6 in antiviral innate immunity (involving an interaction between the STAT6 DBD and the protein STING), we are currently exploring functional consequences of the novel STAT6 mutations in FL and cell line models. Conclusion We report identification of somatic mutations in STAT6 in 11% of FL. These mutations predominantly affected the STAT6 DNA binding domain. We identify a novel STAT6 mutation hotspot in STAT6 codon 419 (p.419D>D/G or p.419D>D/H). Disclosures: Lebovic: Genentech: Speakers Bureau; Allos/Spectrum: Speakers Bureau; Celgene: Speakers Bureau; Onyx: Speakers Bureau.

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The Biological Impact of the Human Master Regulator p53 Can Be Altered by Mutations That Change the Spectrum and Expression of Its Target Genes

Molecular and Cellular Biology ◽

10.1128/mcb.26.6.2297-2308.2006 ◽

2006 ◽

Vol 26 (6) ◽

pp. 2297-2308 ◽

Cited By ~ 60

Author(s):

Daniel Menendez ◽

Alberto Inga ◽

Michael A. Resnick

Keyword(s):

Dna Binding ◽

Target Genes ◽

Tumor Development ◽

Human Tumor ◽

Human Cells ◽

Binding Domain ◽

Dna Binding Domain ◽

Missense Mutations ◽

The Relationship ◽

Biological Outcomes

ABSTRACT Human tumor suppressor p53 is a sequence-specific master regulatory transcription factor that targets response elements (REs) in many genes. p53 missense mutations in the DNA-binding domain are often cancer associated. As shown with systems based on the yeast Saccharomyces cerevisiae, p53 mutants can alter the spectra and intensities of transactivation from individual REs. We address directly in human cells the relationship between changes in the p53 master regulatory network and biological outcomes. Expression of integrated, tightly regulated DNA-binding domain p53 mutants resulted in many patterns of apoptosis and survival following UV or ionizing radiation, or spontaneously. These patterns reflected changes in the spectra and activities of target genes, as demonstrated for P21, MDM2, BAX, and MSH2. Thus, as originally proposed for “master genes of diversity,” p53 mutations in human cells can differentially influence target gene transactivation, resulting in a variety of biological consequences which, in turn, might be expected to influence tumor development and therapeutic efficacy.

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Only Missense Mutations Affecting the DNA Binding Domain of P53 Influence Outcomes in Patients with Breast Carcinoma

PLoS ONE ◽

10.1371/journal.pone.0055103 ◽

2013 ◽

Vol 8 (1) ◽

pp. e55103 ◽

Cited By ~ 23

Author(s):

Frédérique Végran ◽

Magali Rebucci ◽

Sandy Chevrier ◽

Muriel Cadouot ◽

Romain Boidot ◽

...

Keyword(s):

Breast Carcinoma ◽

Dna Binding ◽

Binding Domain ◽

Dna Binding Domain ◽

Missense Mutations

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Adaptive evolution of transcription factor binding affinities

10.1101/118455 ◽

2017 ◽

Author(s):

Jungeui Hong ◽

Nathan Brandt ◽

Ally Yang ◽

Tim Hughes ◽

David Gresham

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Dna Binding ◽

Adaptive Evolution ◽

Consensus Sequence ◽

Binding Domain ◽

Dna Binding Domain ◽

Missense Mutations ◽

Binding Affinities ◽

Synonymous Mutations

Understanding the molecular basis of gene expression evolution is a central problem in evolutionary biology. However, connecting changes in gene expression to increased fitness, and identifying the functional basis of those changes, remains challenging. To study adaptive evolution of gene expression in real time, we performed long term experimental evolution (LTEE) of Saccharomyces cerevisiae (budding yeast) in ammonium-limited chemostats. Following several hundred generations of continuous selection we found significant divergence of nitrogen-responsive gene expression in lineages with increased fitness. In multiple independent lineages we found repeated selection for non-synonymous mutations in the zinc finger DNA binding domain of the activating transcription factor (TF), GAT1, that operates within incoherent feedforward loops to control expression of the nitrogen catabolite repression (NCR) regulon. Missense mutations in the DNA binding domain of GAT1 reduce its binding affinity for the GATAA consensus sequence in a promoter-specific manner, resulting in increased expression of ammonium permease genes via both direct and indirect effects, thereby conferring increased fitness. We find that altered transcriptional output of the NCR regulon results in antagonistic pleiotropy in alternate environments and that the DNA binding domain of GAT1 is subject to purifying selection in natural populations. Our study shows that adaptive evolution of gene expression can entail tuning expression output by quantitative changes in TF binding affinities while maintaining the overall topology of a gene regulatory network.

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TP53 Alterations Including Missense Mutations, Aberrant Exon-Junctions and Internal Intron Retentions Are Frequent and May Contribute to MDM2 Antagonist-Resistance in B-Acute Lymphoblastic leukemia

Blood ◽

10.1182/blood.v118.21.1484.1484 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1484-1484

Author(s):

Ilaria Iacobucci ◽

Anna Ferrari ◽

Stefania Trino ◽

Annalisa Lonetti ◽

Cristina Papayannidis ◽

...

Keyword(s):

Dna Binding ◽

Board Of Directors ◽

Lymphoblastic Leukemia ◽

Binding Domain ◽

Dna Binding Domain ◽

Missense Mutations ◽

Advisory Committees ◽

Tp53 Mutations ◽

Mdm2 Antagonist ◽

Exon Junctions

Abstract Abstract 1484 MDM2, a p53-inducible phosphoprotein, binds to the N-terminus of the p53 and negatively regulates its transcriptional activity. New MDM2 antagonists, such as RO5045337 (Roche) and NSC-66811 (Merck), are now available for Phase I/II clinical development, but their activity is dependent on TP53 mutation status. Therefore, in order to efficiently treat B-progenitor acute lymphoblastic leukemia (ALL) patients with an MDM2 antagonist, we set up a sensitive assay to identify TP53 lesions. Deletions and uniparental disomy (UPD) involving TP53 were assessed on 146 DNA samples from Philadelphia-positive (Ph+)(n = 126) and Ph-negative (n = 20) ALL patients by Genome-Wide Human SNP 6.0 array (Affymetrix). No 17p UPD events were detected whereas losses were identified in 2% of cases. Mutations of TP53 were thereafter investigated in 67 samples including 60 Ph+ and 7 Ph-negative cases. Since the majority of the studies in leukemia were focused on genomic alterations and resulted in low rate of TP53 mutations, we aimed to identify RNA mutations and aberrant isoforms due to other mechanisms, such as RNA editing. To this purpose three overlapping shorter amplicons covering the entire coding cDNA sequence (GenBank accession number NM_000546.4) and the untranslated exon 1 [amplicon 1 (491 bp): exons 1–5; amplicon 2 (482 bp): exons 5–8; amplicon 3 (498 bp): exons 8–11)] and a longer amplicon (1,317 bp) starting from exon 1 and ending to exon 11 were sequenced by Sanger method. TP53 mutations were detected in only 6 cases (8.9%), suggesting that these alterations are apparently rare events in B-ALL. They included 4 missense point mutations in the DNA binding domain and in the carboxyl-terminal tetramerization and regulatory domain: C135Y (ex 5), A234T (ex 7), R290C (ex 8) and A347T (ex 10). Interestingly, in two cases we identified aberrant transcripts: 1) a TP53 isoform characterized by retention of introns 5–6–7 and predicted to encode for a truncated protein due a premature stop codon; 2) a TP53 isoform in which the DNA binding domain is lost due to an exon conjunction between the exon 4 and the 3' untraslated region (UTR)(ex4-3'UTR: 7579533–7572842, according to GRCh37/hg19). Next, in order to investigate if low rate of mutations were detectable, we also analyzed our whole transcriptome data obtained using next generation sequencing technology (Illumina/Solexa Genome Analyzer) on 3 Ph+ ALL patients. Curiously, all patients harbored clones ranging from 45% to 94% with TP53 mutations in the DNA binding and tetramerization domains: C182W (ex 5), T231A (ex 7), L330R (ex 9) in the first patient and Stop394S, D393V/H and G389Y/V (ex 11) in the second one. Moreover, in the first and third patient we detected 10 and 13 base exchanges, respectively, located in intron 6 within 7578166–7578142 region, suggesting a retention of this intron in the primary transcript and the dysfunction of the DNA-binding domain. The mechanism of intron retention (with or without mutations) was particularly intrigued since it could be a new mechanism of functional inactivation of TP53. To address this hypothesis we performed amplification of TP53 cDNA followed by single cell cloning and subsequent direct sequencing in 4 patients previously resulted wild-type by Sanger sequencing for TP53. By this approach, all patients showed cDNA alterations. In one case we identified the missense mutation S90P (ex 4) and an aberrant isoform lacking the DNA binding domain and caused by an exon-junction between exons 2 and 7 (ex2-7: 7579866–7577510). In a second patient the P190S (ex 6) and N235S (ex 7) missense mutations were detected. Moreover, an aberrant isoform lacking the DNA binding domain and characterized by an exon-junction between the first part of exon 4 and the last part of exon 7 (ex4-7: 7579581–7577532) was also identified. In the third patient the E285G (ex 8) was found associated with a 3'-UTR base exchange, which was also detected in the remaining fourth patient. In conclusion, we demonstrate for the first time that TP53 alterations at the RNA level, including missense mutations, aberrant exon junctions and internal intron retentions are highly frequent in B-ALL patients and that testing for TP53 mutations with sensitive assay based on RNA analysis is absolutely required. Supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2009, Ateneo RFO grants, PIO program, Programma di Ricerca Regione – Università 2007 – 2009. Disclosures: Soverini: Novartis: Consultancy; ARIAD: Consultancy; Bristol-Myers Squibb: Consultancy. Baccarani:Pfizer Oncology: Consultancy; Novartis: Consultancy; BMS: Consultancy; Ariad: Consultancy; Novartis: Research Funding; Pfizer Oncology: Honoraria; Novartis: Honoraria; BMS: Honoraria; Ariad: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

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Missense mutations that alter the DNA-binding domain of the MtrR protein occur frequently in rectal isolates of Neisseria gonorrhoeae that are resistant to faecal lipids

Microbiology ◽

10.1099/13500872-141-4-907 ◽

1995 ◽

Vol 141 (4) ◽

pp. 907-911 ◽

Cited By ~ 78

Author(s):

W. M. Shafer ◽

J. T. Balthazar ◽

K. E. Hagman ◽

S. A. Morse

Keyword(s):

Dna Binding ◽

Neisseria Gonorrhoeae ◽

Binding Domain ◽

Dna Binding Domain ◽

Missense Mutations

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Mutations of STAT6 DNA Binding Domain Are Characteristic of PMBL and Belong to the Genetic Signature of This Entity.

Blood ◽

10.1182/blood.v120.21.2645.2645 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2645-2645

Author(s):

Marlene Ochmann ◽

Francois Lemonnier ◽

Diane Damotte ◽

Richard Delarue ◽

Susana Ben-Neriah ◽

...

Keyword(s):

Dna Binding ◽

B Cell ◽

Copy Number ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Class Ii ◽

Biological Data ◽

Binding Domain ◽

Janus Kinase ◽

Dna Binding Domain

Abstract Abstract 2645 Primary mediastinal B-cell lymphoma (PMBL) is a separate entity of aggressive B-cell lymphoma and accounts for about 5% of aggressive lymphomas according to the World Health Organization (WHO) classification. This lymphoma affects young patients and presents as a anterior mediastinal mass consisting of large B-cells that usually express little if any surface or cytoplasmic immunoglobulin and major histocompatibility complex class I and/or class II molecules. PMBL display constitutive activation of janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway, also observed in Hodgkin lymphoma and is characterized by heterozygous missense mutations of STAT6 DNA binding domain that are not observed in diffuse large B-cell lymphoma. To determine whether these mutations could represent a diagnostic or prognostic marker useful in the clinic, we developed a PCR HRM (High Resolution Melting) assay to screen for mutations in STAT6 exon 12, which had been previously identified as a hotspot mutation. We analyzed a series of 80 patients with a mediastinal biopsy and/or histological review of PMBL, enrolled in clinical trials of the GELA (LNH-03) or the GOELAMS (075). DNAs were extracted from FFPE (formalin-fixed and paraffin-embedded) samples with QIAmp DNA mini kit (QIAGEN®) for GELA samples and fully automated Siemens Healthcare Diagnostics proprietary experimental method based on magnetic beads for GOELAMS samples. Sixteen DNAs were not analyzed due to poor amplification in PCR and four DNAs were not analyzed due to other reasons (refused protocol, inclusion at relapse, not included in the tissue microarray/TMA). All cases with abnormal denaturation curves were analyzed by Sanger sequencing. Of the remaining 60 DNA samples, we detected 20 mutated tumors in exon 12 (30%). These mutations were heterozygous single nucleotide variants, often multiple (10 cases, 8 double hit mutations and 2 triple hit mutations) and targeted mostly amino acids 417 (9 hits) and 419 (7 hits). Immunohistochemical analysis of TMA showed that the mutated tumors expressed CD23 (14/20) and MAL proteins (13/15), more frequently than non-mutated ones (10/33, p= 0.009 and 15/27, p=0.049, respectively). Interestingly, these proteins are encoded by genes belonging to the transcriptional signature of PMBL as defined by gene expression profiling in previous studies, There were no significant difference in the clinical presentation between patients with a STAT6 mutation and non-mutated cases regarding age, sex, performance status, Ann Arbor stage, LDH, IPI, treatment response, progression free survival and overall survival. However we noted an absence of elevated β2-microglobulin (1 case elevated/18) and an absence of albumin < 35 g/dl (0 case/8) in the mutated tumors compared with non-mutated tumors (11/31, p= 0.003 and 9/21, p< 0.05, respectively). Finally, we analyzed the correlations of these mutations with cytogenetic abnormalities including CIITA (Class II Transactivator) rearrangement, PD-L1/2 (Programmed death ligand) rearrangement and PD-L1/2 copy number alterations. Indeed, CIITA rearrangements have been described in 38% of PMBL and PD-L1/2 amplification were reported in 63% of PMBL. In this series of patients, CIITA rearrangements were present in 15/49 (31%) and PD-L1/2 low (3–4 copies) and high (>4 copies) level amplification observed in 33/52 (63%) of cases. It is interesting to note that STAT6-mutated cases frequently harbor CIITA rearrangement compared to STAT6-non-mutated cases (9/18 vs 6/31, p= 0.05) as well as PD-L1/2 low level copy number gains (12/19 vs 10/32, p= 0.02), suggesting that the combination of specific genomic alterations cooperate during PMBL oncogenesis. In conclusion, the frequency of this mutation suggests an in vivo selection during PMBL lymphomagenesis. The correlations with the clinico-biological data suggest that STAT6 mutations may be searched for in routine FFPE samples and may help to establish a molecular definition of PMBL that is characterized by alterations of the JAK-STAT pathway. Further investigations are required to identify the mutational mechanisms involved and the oncogenic function associated with these mutations. Disclosures: No relevant conflicts of interest to declare.

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TP53 DNA Binding Domain Mutations Predict Progression-Free Survival of Bevacizumab Therapy in Metastatic Colorectal Cancer

Cancers ◽

10.3390/cancers11081079 ◽

2019 ◽

Vol 11 (8) ◽

pp. 1079 ◽

Cited By ~ 3

Author(s):

Hung-Chih Hsu ◽

Jeng-Fu You ◽

Shu-Jen Chen ◽

Hua-Chien Chen ◽

Chien-Yuh Yeh ◽

...

Keyword(s):

Colorectal Cancer ◽

Dna Binding ◽

Metastatic Colorectal Cancer ◽

Progression Free Survival ◽

Binding Domain ◽

Dna Binding Domain ◽

Missense Mutations ◽

Treatment Benefit ◽

Free Survival ◽

Clinical Biomarkers

(1) Background: Bevacizumab-based regimens are a standard treatment for metastatic colorectal cancer (mCRC) patients, however meaningful clinical biomarkers for treatment benefit remain scarce. (2) Methods: Tumor samples from 36 mCRC patients treated with bevacizumab-based chemotherapy underwent comprehensive genomic profiling. Alterations in frequently altered genes and important signaling pathways were correlated with progression-free survival (PFS). (3) Results: Overall genetic alteration analysis of investigated genes and pathways did not identify promising new predictors of PFS. However, when considering mutation subtypes, TP53 DNA binding domain (DBD) missense mutations were associated with prolonged PFS (HR, 0.41; 95% CI, 0.13−0.65; p = 0.005). In contrast, TP53 truncating mutations were associated with short PFS (HR, 2.95; 95% CI, 1.45−27.50; p = 0.017). Importantly, neither TP53 mutation subtype was associated with overall response rate. In multivariate analysis, TP53 DBD missense mutations remained an independent PFS predictor (HR, 0.31; 95% CI, 0.13–0.77; p = 0.011). The other genetic factor independently associated with PFS were PTPRT/PTPRD deleterious alterations, which we previously identified in a screen for biomarkers of bevacizumab response. (4) Conclusions: TP53 DBD missense mutations may predict prolonged PFS in mCRC patients treated with bevacizumab-based therapy. Analyses of TP53 mutations as clinical biomarkers should take the biological impact of different mutation subtypes into consideration to improve patient stratification.

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