Recurrent Gene Mutations Research Articles

A specific missense mutation in GTF2I occurs at high frequency in thymic epithelial tumors.

We analyzed 28 thymic epithelial tumors (TETs) using next-generation sequencing and identified a missense mutation (chromosome 7 c.74146970T>A) in GTF2I at high frequency in type A thymomas, a relatively indolent subtype. In a series of 274 TETs, we detected the GTF2I mutation in 82% of type A and 74% of type AB thymomas but rarely in the aggressive subtypes, where recurrent mutations of known cancer genes have been identified. Therefore, GTF2I mutation correlated with better survival. GTF2I β and δ isoforms were expressed in TETs, and both mutant isoforms were able to stimulate cell proliferation in vitro. Thymic carcinomas carried a higher number of mutations than thymomas (average of 43.5 and 18.4, respectively). Notably, we identified recurrent mutations of known cancer genes, including TP53, CYLD, CDKN2A, BAP1 and PBRM1, in thymic carcinomas. These findings will complement the diagnostic assessment of these tumors and also facilitate development of a molecular classification and assessment of prognosis and treatment strategies.

Conserved recurrent gene mutations correlate with pathway deregulation and clinical outcomes of lung adenocarcinoma in never-smokers.

BackgroundNovel and targetable mutations are needed for improved understanding and treatment of lung cancer in never-smokers.MethodsTwenty-seven lung adenocarcinomas from never-smokers were sequenced by both exome and mRNA-seq with respective normal tissues. Somatic mutations were detected and compared with pathway deregulation, tumor phenotypes and clinical outcomes.ResultsAlthough somatic mutations in DNA or mRNA ranged from hundreds to thousands in each tumor, the overlap mutations between the two were only a few to a couple of hundreds. The number of somatic mutations from either DNA or mRNA was not significantly associated with clinical variables; however, the number of overlap mutations was associated with cancer subtype. These overlap mutants were preferentially expressed in mRNA with consistently higher allele frequency in mRNA than in DNA. Ten genes (EGFR, TP53, KRAS, RPS6KB2, ATXN2, DHX9, PTPN13, SP1, SPTAN1 and MYOF) had recurrent mutations and these mutations were highly correlated with pathway deregulation and patient survival.ConclusionsThe recurrent mutations present in both DNA and RNA are likely the driver for tumor biology, pathway deregulation and clinical outcomes. The information may be used for patient stratification and therapeutic target development.

Molecular mechanism of cholangiocarcinoma carcinogenesis.

Cholangiocarcinoma (CCA) is a highly malignant cancer of the biliary tract with a poor prognosis, which often arises from conditions causing long-term inflammation, injury, and reparative biliary epithelial cell proliferation. Several conditions are known to be major risk factors for cancer in the biliary tract or gallbladder, including primary sclerosing cholangitis, liver fluke infection, pancreaticobiliary maljunction, and chemical exposure in proof-printing workers. Abnormalities in various signaling cascades, molecules, and genetic mutations are involved in the pathogenesis of CCA. CCA is characterized by a series of highly recurrent mutations in genes, including KRAS, BRF, TP53, Smad, and p16(INK4a) . Cytokines that are affected by inflammatory environmental conditions, such as interleukin-6 (IL-6), transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), and platelet-derived growth factor (PDGF), play an important role in cancer pathogenesis. Prominent signaling pathways important in carcinogenesis include TGF-β/Smad, IL-6/STAT-3, PI3K/AKT, Wnt, RAF/MEK/MAPK, and Notch. Additionally, some microRNAs regulate targets in critical pathways of CCA development and progression. This review article provides the understanding of the genetic and epigenetic mechanism(s) of carcinogenesis in CCA, which leads to the development of new therapeutic targets for the prevention and treatment of this devastating cancer.

The mechanistic role of DNA methylation in myeloid leukemogenesis

The importance of epigenetic aberrations in the pathogenesis of leukemias has been revealed by recurrent gene mutations that highlight epigenetic pathways as well as by the clinical success of therapies like 5-azacytidine and decitabine that work through epigenetic mechanisms. However, precise mechanisms of how gene mutations lead to leukemias and how epigenetic therapies induce clinical remissions are elusive. Current scientific inquiries that take advantage of techniques that can distinguish among the various covalent cytosine modifications at single base resolution are likely to shed light on the ways epigenetic pathways drive leukemogenesis as well as how the hypomethylating drugs induce clinical remissions. The hope is that these studies will also reveal which patients are likely to respond to epigenetic therapies. Thus, the future is likely to bring a new wave of diagnostic and prognostic tools that probe the epigenomics of leukemia to help clinicians in their management of patients.

Polarity gene alterations in pure invasive micropapillary carcinomas of the breast.

IntroductionPure invasive micropapillary carcinoma (IMPC) is a special type of breast carcinoma characterised by clusters of cells presenting polarity abnormalities. The biological alterations underlying this pattern remain unknown.MethodsPangenomic analysis (n = 39), TP53 (n = 43) and PIK3CA (n = 41) sequencing in a series of IMPCs were performed. A subset of cases was also analysed with whole-exome sequencing (n = 4) and RNA sequencing (n = 6). Copy number variation profiles were compared with those of oestrogen receptors and grade-matched invasive ductal carcinomas (IDCs) of no special type.ResultsUnsupervised analysis of genomic data distinguished two IMPC subsets: one (Sawtooth/8/16) exhibited a significant increase in 16p gains (71%), and the other (Firestorm/Amplifier) was characterised by a high frequency of 8q (35%), 17q (20% to 46%) and 20q (23% to 30%) amplifications and 17p loss (74%). TP53 mutations (10%) were more frequently identified in the amplifier subset, and PIK3CA mutations (4%) were detected in both subsets. Compared to IDC, IMPC exhibited specific loss of the 6q16-q22 region (45%), which is associated with downregulation of FOXO3 and SEC63 gene expression. SEC63 and FOXO3 missense mutations were identified in one case each (2%). Whole-exome sequencing combined with RNA sequencing of IMPC allowed us to identify somatic mutations in genes involved in polarity, DNAH9 and FMN2 (8% and 2%, respectively) or ciliogenesis, BBS12 and BBS9 (2% each) or genes coding for endoplasmic reticulum protein, HSP90B1 and SPTLC3 (2% each) and cytoskeleton, UBR4 and PTPN21 (2% each), regardless of the genomic subset. The intracellular biological function of the mutated genes identified by gene ontology analysis suggests a driving role in the clinicopathological characteristics of IMPC.ConclusionIn our comprehensive molecular analysis of IMPC, we identified numerous genomic alterations without any recurrent fusion genes. Recurrent somatic mutations of genes participating in cellular polarity and shape suggest that they, together with other biological alterations (such as epigenetic modifications and stromal alterations), could contribute to the morphological pattern of IMPC. Though none of the individual abnormalities demonstrated specificity for IMPC, whether their combination in IMPC may have a cumulative effect that drives the abnormal polarity of IMPC needs to be examined further with in vitro experiments.

Double heterozygotes among breast cancer patients analyzed for BRCA1, CHEK2, ATM, NBN/NBS1, and BLM germ-line mutations.

17 double heterozygous (DH) breast cancer (BC) patients were identified upon the analysis of 5,391 affected women for recurrent Slavic mutations in BRCA1, CHEK2, NBN/NBS1, ATM, and BLM genes. Double heterozygosity was found for BRCA1 and BLM (4 patients), BRCA1 and CHEK2 (4 patients), CHEK2 and NBS1 (3 patients), BRCA1 and ATM (2 patients), CHEK2 and BLM (2 patients), CHEK2 and ATM (1 patient), and NBS1 and BLM (1 patient). DH BC patients were on average not younger than single mutation carriers and did not have an excess of bilateral BC; an additional non-breast tumor was documented in two BRCA1/BLM DH patients (ovarian cancer and lymphoplasmacytic lymphoma). Loss-of-heterozygosity (LOH) analysis of involved genes was performed in 5 tumors, and revealed a single instance of somatic loss of the wild-type allele (LOH at CHEK2 locus in BRCA1/CHEK2 double heterozygote). Distribution of mutations in patients and controls favors the hypothesis on multiplicative interaction between at least some of the analyzed genes. Other studies on double heterozygosity for BC-predisposing germ-line mutations are reviewed.

Interleukin-7 receptor mutants initiate early T cell precursor leukemia in murine thymocyte progenitors with multipotent potential

Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) exhibits lymphoid, myeloid, and stem cell features and is associated with a poor prognosis. Whole genome sequencing of human ETP-ALL cases has identified recurrent mutations in signaling, histone modification, and hematopoietic development genes but it remains to be determined which of these abnormalities are sufficient to initiate leukemia. We show that activating mutations in the interleukin-7 receptor identified in human pediatric ETP-ALL cases are sufficient to generate ETP-ALL in mice transplanted with primitive transduced thymocytes from p19(Arf-/-) mice. The cellular mechanism by which these mutant receptors induce ETP-ALL is the block of thymocyte differentiation at the double negative 2 stage at which myeloid lineage and T lymphocyte developmental potential coexist. Analyses of samples from pediatric ETP-ALL cases and our murine ETP-ALL model show uniformly high levels of LMO2 expression, very low to undetectable levels of BCL11B expression, and a relative lack of activating NOTCH1 mutations. We report that pharmacological blockade of Jak-Stat signaling with ruxolitinib has significant antileukemic activity in this ETP-ALL model. This new murine model recapitulates several important cellular and molecular features of ETP-ALL and should be useful to further define novel therapeutic approaches for this aggressive leukemia.

The Use of Molecular Genetics to Refine Prognosis in Acute Myeloid Leukemia

The discovery and application of advanced molecular techniques, such as gene and microRNA expression profiling, whole genome and exome sequencing, proteomic analysis and methylation assays, have allowed for the identification of recurrent molecular abnormalities in acute myeloid leukemia (AML) that have revolutionized our understanding of the genetic landscape of the disease. These modalities have emerged as valuable tools that permit a more comprehensive and detailed molecular characterization of AML. Many of these molecular abnormalities have been shown to predict prognosis, particularly within the context of cytogenetically normal AML. This review will discuss the major techniques and platforms that have been used to identify novel recurrent gene mutations in AML and briefly describe how these discoveries have impacted on outcome prediction.

Constitutive Activation of PKA Catalytic Subunit in Adrenal Cushing's Syndrome

BackgroundCorticotropin-independent Cushing's syndrome is caused by tumors or hyperplasia of the adrenal cortex. The molecular pathogenesis of cortisol-producing adrenal adenomas is not well understood.MethodsWe performed exome sequencing of tumor-tissue specimens from 10 patients with cortisol-producing adrenal adenomas and evaluated recurrent mutations in candidate genes in an additional 171 patients with adrenocortical tumors. We also performed genomewide copy-number analysis in 35 patients with cortisol-secreting bilateral adrenal hyperplasias. We studied the effects of these genetic defects both clinically and in vitro.ResultsExome sequencing revealed somatic mutations in PRKACA, which encodes the catalytic subunit of cyclic AMP–dependent protein kinase (protein kinase A [PKA]), in 8 of 10 adenomas (c.617A→C in 7 and c.595_596insCAC in 1). Overall, PRKACA somatic mutations were identified in 22 of 59 unilateral adenomas (37%) from patients with overt Cushing's syndrome; these mutations were not detectable in 40 patients with subclinical hypercortisolism or in 82 patients with other adrenal tumors. Among 35 patients with cortisol-producing hyperplasias, 5 (including 2 first-degree relatives) carried a germline copy-number gain (duplication) of the genomic region on chromosome 19 that includes PRKACA. In vitro studies showed impaired inhibition of both PKA catalytic subunit mutants by the PKA regulatory subunit, whereas cells from patients with germline chromosomal gains showed increased protein levels of the PKA catalytic subunit; in both instances, basal PKA activity was increased.ConclusionsGenetic alterations of the catalytic subunit of PKA were found to be associated with human disease. Germline duplications of this gene resulted in bilateral adrenal hyperplasias, whereas somatic PRKACA mutations resulted in unilateral cortisol-producing adrenal adenomas. (Funded by the European Commission Seventh Framework Program and others.)

Chromatin modifiers and the promise of epigenetic therapy in acute leukemia

Hematopoiesis is a tightly regulated process involving the control of gene expression that directs the transition from hematopoietic stem and progenitor cells to terminally differentiated blood cells. In leukemia, the processes directing self-renewal, differentiation and progenitor cell expansion are disrupted, leading to the accumulation of immature, non-functioning malignant cells. Insights into these processes have come in stages, based on technological advances in genetic analyses, bioinformatics and biological sciences. The first cytogenetic studies of leukemic cells identified chromosomal translocations that generate oncogenic fusion proteins and most commonly affect regulators of transcription. This was followed by the discovery of recurrent somatic mutations in genes encoding regulators of the signal transduction pathways that control cell proliferation and survival. Recently, studies of global changes in methylation and gene expression have led to the understanding that the output of transcriptional regulators and the proliferative signaling pathways are ultimately influenced by chromatin structure. Candidate gene, whole-genome and whole-exome sequencing studies have identified recurrent somatic mutations in genes encoding epigenetic modifiers in both acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). In contrast to the two-hit model of leukemogenesis, emerging evidence suggests that these epigenetic modifiers represent a class of mutations that are critical to the development of leukemia and affect the regulation of various other oncogenic pathways. In this review, we discuss the range of recurrent, somatic mutations in epigenetic modifiers found in leukemia and how these modifiers relate to the classical leukemogenic pathways that lead to impaired cell differentiation and aberrant self-renewal and proliferation.

Impact of Recurrent Copy Number Alterations and Cancer Gene Mutations on the Predictive Accuracy of Prognostic Models in Clear Cell Renal Cell Carcinoma

Several recently reported recurrent genomic alterations in clear cell renal cell carcinoma are linked to pathological and clinical outcomes. We determined whether any recurrent cancer gene mutations or copy number alterations identified in the TCGA (The Cancer Genome Atlas) clear cell renal cell carcinoma data set could add to the predictive accuracy of current prognostic models. In 413 patients who underwent nephrectomy/partial nephrectomy we investigated whole exome, copy number array analyses and clinical variables. We identified 65 recurrent genomic alterations based on prevalence and combined them into 35 alterations, including 12 cancer gene mutations. Genomic markers were modeled using the elastic net algorithm with preoperative variables (tumor size plus patient age) and in the postoperative setting using the externally validated Mayo Clinic SSIGN (stage, size, grade and necrosis) prognostic scoring system. These models were subjected to internal validation using bootstrap. Median followup in survivors was 45 months. Several markers correlated with adverse cancer specific survival and time to recurrence on univariate analysis. However, most of them lost significance when controlling for tumor size with or without age in the preoperative models or for SSIGN score in the postoperative setting. Adding multiple genomic markers selected by the elastic net algorithm failed to substantially add to the predictive accuracy of any preoperative or postoperative model for cancer specific survival or time to recurrence. While recurrent copy number alterations and cancer gene mutations are biologically significant, they do not appear to improve the predictive accuracy of existing models of clinical cancer specific survival or time to recurrence for clear cell renal cell carcinoma.

Prognostic factors for acute myeloid leukaemia in adults - biological significance and clinical use

Acute myeloid leukaemia (AML) is a heterogeneous disease. Prognosis of AML is influenced both by patient-specific as well as disease-specific factors. Age is the most prominent patient-specific risk factor, while chromosomal aberrations are the strongest disease-specific risk factors. For patients with cytogenetically normal AML, prognosis can be specified by mutational status of the genes NPM1, FLT3 and CEBPA. A growing number of recurrent mutations in additional genes have recently been identified, for which the prognostic effect yet has to be determined. Performance status, geriatric assessment, secondary leukaemia following myelodysplastic syndrome or cytotoxic treatment, common laboratory parameters, leukaemic stem cell frequency, bone marrow microenvironment, gene expression levels, epigenetic changes, micro-RNA's as well as kinetics and depth of response to treatment influence prognosis of AML patients. Despite the high number of established risk factors, only few predictive markers exist which can truly aid therapy decisions in patients with AML.

Molecular Diagnostics of T-Cell Lymphoproliferative Disorders

T-cell neoplasms include both mature T-cell leukemias and lymphomas and immature proliferations of precursor T cells. Molecular laboratories routinely assay suspected T-cell proliferations for evidence of clonality. In addition, some T-cell neoplasms are characterized by recurrent structural abnormalities that can be readily identified by such techniques as fluorescence in situ hybridization. New massively parallel sequencing technologies have led to the identification of numerous recurrent gene mutations in T-cell neoplasms. These findings are reviewed. As new technologies become implemented in molecular diagnostic laboratories and as targeted therapies are developed, it is anticipated that more extensive genomic characterization of T-cell neoplasms will be routinely performed in the future.

Abstract P5-10-01: A systems approach to combine genomics and emerging therapeutics to discover new treatments for TNBC

Abstract Genomic analyses of patient tumors have unearthed an overwhelming number of recurrent somatic alterations in genes that undoubtedly have dramatic effects on breast tumor biology, clinical outcomes and responses to therapeutics. In triple negative breast cancer (TNBC), there is an urgent need to translate this emerging patient genomic data into new therapeutic paradigms. To develop genotype-specific therapies, we have established an isogenic cell-line drug screen that measures the impact of gene activation on a panel of emerging, clinically relevant compounds targeting a variety of cancer pathways. We seek to uncover genotype-dependent sensitivity to drugs, synthetic lethal relationships, and validate in breast cancer cells and in vivo mouse models. We focused on emerging compounds that are already approved or in testing for human use and we expect that this work will serve as a prelude to one or more clinical trials in TNBC. RESULTS: We have developed a synthetic-lethal interaction mapping strategy to uncover the impact of gene activation on responses to a panel of emerging therapeutics. We believe that this approach can identify core synthetic lethal interactions which underlie drug sensitivity and can be used as a foundation to identify patient populations that will selectively respond to drug treatments. We have developed a high-throughput screening platform that can rapidly and accurately assess the proliferative capacity of cell lines in response to a panel of inhibitors. We have generated a panel of isogenic cell lines which closely mimic the impact of recurrent gene mutations and overexpression common in breast cancer. We have currently generated 23 cell lines expressing wild-type and/or mutant forms of these genes. To date we have screened 12 known oncogenes against our panel of 94 emerging therapeutic compounds. Using this systems approach, the resulting interaction map highlights both known and novel connections between oncogene activation and drug responses and provides a modular roadmap for the exploration of synthetic lethal relationships. Based on the coordinate activity of multiple drugs targeting the same pathway, we identified oncogenes that can drive resistance to EGFR inhibitors and PI3K/AKT inhibitors. We also recapitulated a known synthetic lethal relationship between MYC and CDK inhibition which we have previously described and we are now evaluating in a clinical trial. Therefore, our approach identified both resistance and synthetic lethal relationships that have been independently described, indicating that this platform is a good model for predicting synthetic lethality in vivo. Several novel and previously uncharacterized connections were also uncovered, including novel connections between MYC activity and several promising drugs in clinical development. Synthetic-lethal interactions discovered using this chemical-genetic systems approach and their mechanistic validation will be described in the presentation. Furthermore, the important role of patient advocacy in developing this project will be described. CONCLUSION: A novel systems biology approach that uses chemical-genetic maps of oncogenes and emerging therapeutics can define synthetic-lethal interactions and actionable therapeutics for breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-10-01.

Mutational landscape of gingivo-buccal oral squamous cell carcinoma reveals new recurrently-mutated genes and molecular subgroups

Gingivo-buccal oral squamous cell carcinoma (OSCC-GB), an anatomical and clinical subtype of head and neck squamous cell carcinoma (HNSCC), is prevalent in regions where tobacco-chewing is common. Exome sequencing (n=50) and recurrence testing (n=60) reveals that some significantly and frequently altered genes are specific to OSCC-GB (USP9X, MLL4, ARID2, UNC13C and TRPM3), while some others are shared with HNSCC (for example, TP53, FAT1, CASP8, HRAS and NOTCH1). We also find new genes with recurrent amplifications (for example, DROSHA, YAP1) or homozygous deletions (for example, DDX3X) in OSCC-GB. We find a high proportion of C>G transversions among tobacco users with high numbers of mutations. Many pathways that are enriched for genomic alterations are specific to OSCC-GB. Our work reveals molecular subtypes with distinctive mutational profiles such as patients predominantly harbouring mutations in CASP8 with or without mutations in FAT1. Mean duration of disease-free survival is significantly elevated in some molecular subgroups. These findings open new avenues for biological characterization and exploration of therapies.

Epigenetics of myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are clonal diseases of the elderly characterized by chronic cytopenias, dysplasia and a variable risk of progression to acute myeloid leukemia (AML). Aberrant methylation of tumor-suppressor gene promoters has been established for many years and recently tracked to the most immature cells of MDS, suggesting that these alterations are drivers of MDS pathogenesis. In recent years, recurrent somatic mutations in genes encoding proteins involved in DNA methylation and demethylation and in covalent histone modifications have been reported in myeloid malignancies, including MDS. Whole-genome epigenetic profiles of MDS are also emerging. In parallel with these advances in the molecular pathogenesis of MDS, clinical trials have established hypomethylating agents (HMAs) as the mainstay of therapy in the advanced forms of the disease. In this review, we summarize the current understanding of the molecular machinery involved in epigenetic regulation, discuss how epigenetic alterations arise in MDS and contribute to its pathogenesis and then discuss the mode of action of HMAs in MDS.

Mutational and Cytogenetic Analyses Of 177 CLL Patients With Trisomy 12: A Retrospective Study Of The CLL French Intergroup

BackgroundCytogenetic abnormalities are of key importance for predicting clinical course and response to therapy in patients with chronic lymphocytic leukemia (CLL). Trisomy 12 (tri12), the third most frequent chromosomal aberration in CLL patients (10-20%), is associated with an intermediate prognostic risk but represents a clinical heterogeneous entity. Recently, next generation sequencing have revealed recurrent mutations in genes that were unknown to be involved in CLL pathogenesis, including NOTCH1, MYD88, SF3B1, XPO1 and BIRC3. In patients harboring tri12, NOTCH1 mutations have been shown to be present in up to 25% of cases and to confer unfavorable outcome explaining in part the clinical heterogeneity of tri12 patients. To better understand the genetic basis and prognosis of tri12 patients, we performed a multicenter retrospective study combining extensive mutational and cytogenetic analysis. MethodsPatients carrying tri12 were identified using fluorescence in situ hybridization (FISH) and/or chromosome banding (CB). Main clinical and biological characteristics were collected and included in univariate analysis of prognostic factors, comprising age, Binet stage (A vs. B-C), splenomegaly, lymphocyte doubling time (LDT), LDH, beta2microglobulin (B2M), CD38 expression, IGHV mutational status, percentage of interphase nuclei positive (INP) for tri12, additional FISH (del13q, del11q, del17p) or chromosomal aberrations and presence of complex karyotype (> 2 CB abnormalities). Search for mutations was performed by Sanger direct sequencing for TP53 (exons 5-10), NOTCH1 (exon 34), MYD88 (exons 14-16), SF3B1 (exons 14-16) and XPO1 (exons 14-15). Primary and secondary endpoints were time to first treatment (TFT), response to therapy, time to next treatment (TNT) and overall survival (OS). ResultsThe study population comprised a total of 177 untreated patients including 112 and 75 patients with stage A and B-C CLL, respectively. The median age at diagnosis was 62 years old (range, 31-87) and 33% of patients were female. B2M was superior to 4 mg/L in 30/92 (32%) patients and LDH elevated in 65%. CD38 expression was positive (>30%) in 58% and IGHV status was unmutated in 60%. Among the whole study population, all patients were positive for tri12 by FISH and 158/165 by CB. Tri12 was associated by CB with tri19 in 21 patients (13.2%), tri18 in 12 patients (7.5%), tri3 in 1 patient (<1%), t(14;18) in 9 patients (5.7%), t(14;19) in 3 patients (2%) and del14q in 7 patients (4.5%). Complex karyotype was present in 42 patients (26%) and tri12 was the sole abnormality observed by CB in 71 patients (45%). Out of 170 patients analysed with the four probes by FISH, tri12 was the sole abnormality in 114 patients (67%) and was associated with del13q, del11q and del17p in, respectively, 44 (26%), 8 (4%) and 10 (6%) patients. The median percentage of INP for tri12 was 58% (range, 5-100). TP53, NOTCH1, MYD88 and SF3B1 mutations were tested in 113 patients and identified in, respectively, 9 (8%), 19 (17%), 1 (<1%) and 1 (<1%) patients. No mutation of XPO1 was observed. Among the stage A population of 112 patients, 64 (57%) needed treatment with a median TFT of 45 months (range, 1-170). A shorter TFT was significantly associated with a LDT inferior to one year (P=0.0001), and the presence of TP53 (P=0.04) mutation while the presence of another trisomy (P=0.03) was associated with a significantly longer TFT. By multivariate analysis, only TP53 mutation retain prognostic significance for TFT (HR=3.9, 95%CI=1.02-15.04). Among the 75 stage B-C patients, 62 were treated, 51 evaluable for response to therapy and 28 received a second line of treatment. The only prognostic variable associated with poor response to therapy was the presence of NOTCH1 mutation (P=0.01). Finally, regarding the whole tri12 population, Binet stage (A vs. B-C), splenomegaly, lymphocytosis, LDH, B2M, the percentage of INP and the IGHV unmutated status were associated with worst OS in univariate analysis. ConclusionThe most frequent molecular abnormalities observed in our tri12 cohort were TP53 and NOTCH1 mutations that occurred in, respectively, 8 and 17% of cases. NOTCH1 mutations were associated with poor response to therapy and TP53 mutations with a shorter time to first treatment. Disclosures:Leblond:Mundipharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche : Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Decision-Making At The End Of Consolidation Of Acute Myeloid Leukemia: Negative Effect Of Minimal Residual Disease Detectable With Multiparametric Flowcytometry Combined With Gene Mutations Of FLT3 On Early Relapse

▪ BackgroundAbout 70-80% of adult patients (pts) with acute myeloid leukemia (AML) achieve complete remission (CR); however, around a half of them experience a relapse. For the purpose of creating accurate decision-making process of post-consolidation strategy, stratification system using karyotype and recurrent gene mutations have been widely utilized. As is confirmed in childhood acute lymphocytic leukemia, however, the information of minimal residual disease (MRD) status would substantially improve the reliance of decision-making process of adult AML pts. Unfortunately, approximately a half of AML patients lack molecular targets suitable for MRD monitoring. The aims of this study are to evaluate the applicability of MRD detection using multiparametric flow cytometry (MPFC) and to estimate the impact of MRD measured with MPFC at the end of consolidation therapy in improving decision-making process. Patients and MethodsWe retrospectively studied 81 consecutive pts with newly diagnosed AML who received induction therapies and achieved CR in our institute between January 2007 and March 2013. Pts with acute promyelocytic leukemia were excluded. We routinely analyzed the bone marrow specimens with MPFC for the detection of leukemia-associated immunophenotypes (LAIPs) at diagnosis. Since April 2010, RT-PCR assay examined FLT3-ITD mutation in the same specimens. In pts who had traceable LAIPs, the relationships of the levels of MRD at the end of consolidation therapy with relapse free survival were analyzed. Positive MRD was defined as the detection of 0.2% and more LAIPs-positive cells with MPFC. We compared two patient groups: those with MRD at the end of consolidation (MRDp group) and those without (MRDn group). Relapse-free survival (RFS) was analyzed using the Kaplan-Meier method and the log-rank test was used for comparison between each group. A multivariate Cox regression analysis for RFS was fit to assess the effect of the followings: age at diagnosis (≥ vs. < 65 years old), the number of induction regimens required for achieving CR (≥ vs. < 2 times), cytogenetic risk groups of SWOG (unfavorable vs. favorable/intermediate). ResultsIn 57 / 81 pts, MPFC could detect LAIPs in the bone marrow specimens at diagnosis (70.4% of all subjects; 15-82 years-old; follow-up time [median] 98-2211[517] days). FLT-ITD mutations were found in 13 pts, but not in 39 pts (the remaining 5 pts were not examined). The rate of detection of LAIPs with 6-color MPFC was significantly superior to 3-color MPFC (82.1% vs. 61.0%, p<0.05). Induction chemotherapies the pts received were anthracyclin-containing regimens, such as idarubicin and cytarabin (3+7), in 52 pts (91.2%), low-dose cytarabin-based regimen in 4 pts (7.0%) and azacitidine in 1 pt. (1.8%). The MRDp and the MRDn groups were comprised of 20 and 37 pts (35.1% and 64.9%) , respectively. One-year RFS of the MRDp group was significantly inferior to the MRDn group (28.3% vs. 75.2%; log-rank p<0.0005). In the multivariable analysis using the model above, MRD positivity at the end of consolidation remains a significant predictor (HR, 2.93, 95% CI 1.16-7.45, p<0.05). In addition, the 1-year RFS in the MRDp group with FLT-ITD was significantly shorter than that in the MRDp group without FLT3-ITD (0% vs. 47.6% with positive and negative FLT3-ITD, log-rank p<0.05). In the MRDn group, however, the negative impact of FLT3-ITD was not documented (85.7% vs. 69.3% with positive and negative FLT3-ITD, log-rank p=0.954). [Display omitted] [Display omitted] ConclusionOur retrospective study confirmed that LAIPs as MRD targets were applicable to the majority of pts with AML; MRD positivity measured with LAIPs was a promising predictor for early relapses at the end of consolidation, as was previously reported. When combined with FLT-ITD status, it might become a more sensitive prognostic factor. Disclosures:Takahashi:celgene: Research Funding.

Large Conserved Domains Of Low DNA Methylation Maintained By 5-Hydroxymethycytosine and Dnmt3a

Identification of recurrent leukemia-associated mutations in genes encoding regulators of DNA methylation such as DNMT3A and TET2 have underscored the critical importance of DNA methylation in maintenance of normal physiology. To gain insight into how DNA methylation exerts the central role, we sought to determine the genome-wide pattern of DNA methylation in the normal precursors of leukemia cells: the hematopoietic stem cell (HSC), and investigate the factors that affect alterations in DNA methylation and gene expression.We performed whole genome bisulfite sequencing (WGBS) on purified murine HSCs achieving a total of 1,121M reads, resulting in a combined average of 40X coverage. Using Hidden Markov Model we identified 32,325 under-methylated regions (UMRs) with average proportion of methylation ≤ 10% and by inspecting the UMR size distribution, we discovered exceptionally large “methylation Canyons” which span highly conserved domains frequently containing transcription factors and are quite distinct from CpG islands and shores. Methylation Canyons are a distinct genomic feature that is stable, albeit with subtle differences, across cell-types and species. Canyon-associated genes showed a striking pattern of enrichment for genes involved in transcriptional regulation (318 genes, P=6.2 x 10-123), as well as genes containing a homeobox domain (111 genes, P=3.9 x 10-85). We compared Canyons with TF binding sites as identified from more than 150 ChIP-seq data sets across a variety of blood lineages (>10)19 and found that TF binding peaks for 10 HSC pluripotency TFs are significantly enriched in entirety of Canyons compared with their surrounding regions. Low DNA methylation is usually associated with active gene expression. However, half of Canyon genes associated with H3K27me3 showed low or no expression regardless of their H3K4me3 association while H3K4me3-only Canyon genes were highly expressed.Because DNMT3A is mutated in a high frequency of human leukemias24, we examined the impact of loss of Dnmt3a on Canyon size. Upon knockout of Dnmt3a, the edges of the Canyons are hotspots of differential methylation while regions inside of Canyon are relatively resistant. The methylation loss in Dnmt3a KO HSCs led Canyon edge erosion, Canyon size expansion and addition of 861 new Canyons for a total of 1787 Canyons. Canyons marked with H3K4me3 only were most likely to expand after Dnmt3a KO and the canyons marked only with H3K27me3 or with both marks were more likely to contract. This suggests Dnmt3a specifically is acting to restrain Canyon size where active histone marks (and active transcription) are already present. WGBS cannot distinguish between 5mC and 5hmC, so we determined the genome-wide distribution of 5hmC in WT and Dnmt3a KO HSCs using the cytosine-5-methylenesulphonate (CMS)-Seq method in which sodium bisulfate treatment convert 5hmC to CMS; CMS-containing DNA fragments are then immunoprecipitated using a CMS specific antiserum. Strikingly, 5hmC peaks were enriched specifically at the borders of Canyons. In particular, expanding Canyons, typically associated with highest H3K4me3 marking, were highly enriched at the edges for the 5hmC signal suggesting a model in which Tet proteins and Dnmt3a act concomitantly on Canyon borders opposing each other in alternately effacing and restoring methylation at the edges, particularly at sites of active chromatin marks. Using Oncomine data, we tested whether Canyon-associated genes were likely to be associated with hematologic malignancy development and found Canyon genes were highly enriched in seven signatures of genes over-expressed in Leukemia patients compared to normal bone marrow; in contrast, four sets of control genes were not similarly enriched. Further using TCGA data, we found that expressed canyon genes are significantly enriched for differentially expressed genes between patients with and without DNMT3A mutation (p value<0.05) Overall, 76 expressed canyon genes, including multiple HOX genes, are significantly changed in patients with DNMT3A mutation (p=0.0031). Methylation Canyons, the novel epigenetic landscape we describe may provide a mechanism for the regulation of hematopoiesis and may contribute to leukemia development. Disclosures:No relevant conflicts of interest to declare.

Prognostic Impact Of Somatic NOTCH2 Mutation In Splenic Marginal Zone Lymphoma

IntroductionNew insights in the pathogenesis of the splenic marginal zone lymphoma (SMZL) has been recently enlightened by next generation sequencing technology discovering recurrent mutations in several genes implicated in major signalling pathways. In 2 recent reports (JEM 2012), mutations in NOTCH2, a gene encoding a protein required for marginal zone B-cell development, were identified in nearly 25% of the patients, with controversial impact on clinical outcome. The aim of our analysis was to analyse the incidence and the prognostic impact of somatic NOTCH2 mutations in a large cohort of patients with SMZL homogeneously treated. MethodsA series of 105 consecutive SMZL patients requiring treatment and referred in 2 expert centers was analysed. Genomic DNA was extracted from frozen tumor samples of involved spleen or blood or bone marrow obtained at diagnosis. Tumor cell clonality was established by amplification of the rearranged IGH genes. Targeted sequencing of the NOTCH2 C-terminal coding exons 26, 27 and 34 was performed using Sanger sequencing and primers described by Kiel MJ et al (J.Exp.Med.209:9,2012). All mutations were verified in at least two independent PCR amplification and sequencing reactions. Log-rank tests were used to compare survival times -overall survival (OS) and progression-free survival (PFS)- between patients with mutated NOTCH2 and patient with wild-type NOTCH2. We controlled the effects of prognostic factors on outcome using multivariate Cox model analysis. ResultsThe median follow-up was 7 years. At diagnosis, median age was 63 years. Sex ratio was M/F 1:1.35. Nearly all patients presented with a disseminated disease and a good performance status. Forty-four percent of the patients had a high LDH level. A monoclonal component was detected in 31% of the patients. An immunological event such as haemolytic anemia, thrombocytopenia, neutropenia, coagulation disorder, and cryoglobulinemia, was observed in 27% of the patients. None of the patients was infected with hepatitis C virus. Patients were scored according to the age-adjusted International Prognostic Index (n=95), and to the IIL score system (n=76). Splenectomy was realized in 97% of the patients. Three patients were treated with R-Bendamusine without splenectomy. Adjuvant treatment after splenectomy was proposed for 24% of the patients, and included CHOP+/-R (n=16), HLX-VP16 (n=1), chlorambucil (n=4), and rituximab alone (n=1). Five and 10-year OS was estimated at 82% and 69%, respectively. Five and 10-year PFS was estimated at 61% and 46%, respectively. Age>60 was significantly associated to a shorter OS (p=0.0135).We identified NOTCH2 mutations in 10 (10%) of the patients. The detected mutations occurred only within the exon 34 (TAD and PEST domains) and represented mostly truncating or insertional events leading to frameshift mutations (8 of the 10 cases) (also 1 nonsense and 1 missense mutations). Comparison of the clinical characteristics at diagnosis between patients with or without NOTCH2 mutations did not show any significant differences. Patients with NOTCH2 mutations were characterized by a significantly worse OS (at 5 years, 32% vs 87%, p<0.0001). Presence of NOTCH2 mutations was also significantly associated with a shorter PFS (at 5-years, 16% vs 66%; p=.0008), and with a shorter time to histological transformation (at 5 years, 26% vs 6%; p=0.0051). NOTCH2 mutational status did not influence the time to first treatment. Multivariate analysis showed an independent prognostic impact of NOTCH2 mutational status on OS (RR 4.7, p=0.02). ConclusionNOTCH2 mutation was identified in 10% of the SMZL patients in our series. Our data demonstrate also, as described, than patients with NOTCH2 mutations have a significant poorer prognosis than patients without NOTCH2 mutations, with a shorter PFS and a shorter OS. NOTCH2 mutational status seems to be an independent prognostic factor. This needs to be confirmed in prospective trials in the context of new therapeutics.CA and GB equally contributed Disclosures:Coiffier:Millennium Pharmaceuticals : Consultancy.