XPO1 Mutations Research Articles

Molecular and Genetic Characterization of Fit, Elderly Patients Receiving Oral Fludarabine, Oral Cyclophosphamide and Intravenous Rituximab (OFOCIR) As Initial Treatment of Chronic Lymphocytic Leukemia (CLL)

BackgroundCytogenetic characterization by fluorescence in-situ hybridization (FISH) identifies subgroups of patients with chronic lymphocytic leukemia likely to have poor responses or short remission duration following standard frontline chemoimmunotherapy. Next-generation sequencing (NGS) has identified new molecular targets associated with refractory or poorly responsive disease (eg. Notch1, SF3B1 or BIRC3) independent of cytogenetic abnormalities. We have performed genetic and molecular characterization of fit, elderly patients enrolled on the Australasian Leukemia and Lymphoma Group (ALLG) CLL5 randomized clinical trial of oral fludarabine, cyclophosphamide and rituximab (ACTRN12608000404325).MethodsPre-treatment peripheral blood and bone marrow aspirate samples were obtained from patients enrolled on a phase II randomized clinical trial investigating oral fludarabine, oral cyclophosphamide and intravenous rituximab (poFCivR) tolerance in previously untreated fit elderly patients with CLL (ALLG CLL5 study). Fitness was defined as Cumulative Illness Rating Scale (CIRS) score of ²6. Bone marrow aspirate samples were analysed for CLL-associated genomic changes with a Vysis CLL FISH probe kit (Abbott, Des Moines, IL) and ranked according to Dohner hierarchical classification. DNA was extracted from peripheral blood lymphocytes and we performed targeted exome sequencing of genes including TP53, ATM, NOTCH1, SF3B1, BIRC3, MYD88 and FBXW7 using a TruSeq Custom Amplicon Design Panel on a MiSeq DNA sequencer as per manufacturerÕs protocol (Illumina, San Diego, CA). Gene mutations were confirmed by Sanger sequencing. Data was analyzed using Illumina proprietary software, annotated using ANNOVAR software and compared to COSMIC and other genomic mutation databases.ResultsOf 116 analyzable patients enrolled on the clinical trial, 78 pts had available FISH results and 76 patients DNA sequencing. The ORR and CR for all patients on study were 96% and 56% respectively. There was no significant difference in ORR between cytogenetic risk groups (Table 1); however, only 1 of 9 patients with ATM deletion achieved CR (11%, p=0.0095). We identified 8 pts with TP53 mutations, only one patient (12.5%) achieved a CR (p=0.0084). CR rate for patients with mutations in ATM (n=9, CR 44%), NOTCH1 (n=10, CR 60%), SF3B1 (n=11, CR 91%), BIRC3 (n=2, CR 0%), XPO1 (n=6, CR 33%), myd88 (n=5, 100%) were not significantly different to patients without the respective mutations. Of 14 pts with normal FISH, 10 pts (71%) had molecular abnormalities identified by NGS (Figure 1). Median follow-up of patients is 20 months, with 91% patients alive at last follow up. At the time of analysis, there was no significant difference in progression free survival (PFS) between different FISH cytogenetic risk groups (Figure 1). Multivariable analysis identified patients with TP53 mutations (HR 4.3, p=0.04) and XPO1 mutations (HR 3.2, p=0.035) as independently associated with shorter PFS. Our analysis was limited by the small subgroups of patients with individual molecular mutations and currently relatively short follow-up of this study.ConclusionsMolecular characterization by DNA sequencing increases the yield of pre-treatment genetic alterations discovered in CLL patients. In this randomized clinical trial of elderly patients requiring first line therapy of CLL, we identified a high proportion of genomic alterations. Identification of genomic mutations may help further risk stratify CLL patients undergoing chemoimmunotherapy.Table 1NCR (n)CR (%)ORR (n)ORR (%)All patients116655611196FISH17p deletion191052178911q deletion9111889Trisomy 1215960149313q deletion3318553297No abnormal16138116100Not Done24148124100NGSAll Available7644587295mutationsTP538113787.5ATM94449100NOTCH110660990SF3B11110911091BIRC32002100XPO16233583Myd88551005100 [Display omitted] [Display omitted] DisclosuresBadoux:Roche: Honoraria. Mulligan:Roche, Abbvie: Consultancy, Honoraria. Kuss:Roche: Research Funding; Sanofi: Research Funding.

Mutational Landscape of 118 Relapsed Chronic Lymphocytic Leukemia Clinical Trial Samples; Evidence for a Multiple-Hit Profile Using Targeted Next Generation Sequencing

Background: Previous studies using next-generation sequencing (NGS) have led to the identification of a number of genes mutated frequently in CLL. Recent publications focus on the most recurrently mutated genes (TP53, SF3B1 and NOTCH1) which tend to be mutually exclusive. Large series of untreated patients have shown that these mutations have a prognostic impact. Relapse may be associated with more frequent mutational events. Further investigation of relapsed CLL genomes within a clinical trial setting using a comprehensive NGS gene panel is required.Methods: Using targeted NGS we determined the mutational spectrum of 118 refractory/relapsing CLL patients enrolled in one French and two UK prospective trials (ICLL01 from the French intergroup GCFLLC/MW-GOELAMS, NCRNCLL201, NCRNCLL202 respectively). Eighty percent of patients had an unmutated IGHV status and 21 (18%) patients carried a 17p deletion. Sequencing libraries were composed of a panel of nine recurrently mutated genes in CLL (i.e.TP53, SF3B1, ATM, NOTCH1, XPO1, SAMHD1, MED12, BIRC3 and MYD88) and run on the Illumina MiSeq instrument (Illumina Inc). On average 14.1 M reads were obtained per run of which 96.8% were identified reflecting an acceptable signal to noise ratio. Yield was 4.1 Gb and 95.9% of reads were above Q30 across 6 MiSeq runs. Data was analysed using our in-house bioinformatics pipeline consisting of a combination of two different aligners (Custom Amplicon Alignment, Illumina Inc and Stampy, Wellcome Trust centre for Human Genetics), two variant callers (GATK, Broad Institute and Platypus, Wellcome Trust centre for Human Genetics) and a stringent filtering process in order to detect SNVs and indels with a variant allele frequency down to 7%.Results: We identified a total of 196 mutations (mean=1.7/sample) in 95 (80%) patients: 138 missense mutations, 41 substitutions/indels, 12 nonsense and 5 splicing mutations. TP53, SF3B1 and ATM mutations occurred frequently in 29 (24.6%), 33 (28%) and 29 (24.6%) patients, respectively. Eighteen (15.3%) patients harbored a NOTCH1 mutation matching the range of reported frequency. Mutations in the other genes sequenced were distributed as follows: XPO1 mutations in 17 (14.4%), SAMHD1 mutations in 12 (10.2%), MED12 mutations in 10 (8.5%), BIRC3 mutations in 6 (5.1%) and MYD88 mutations in 3 (2.5%) patients. Twenty-three (20%) patients did not have any mutations present (Figure 1, cluster #1). A total of 51 (43%) patients had one gene mutated (Figure 1, cluster #2) and the remaining 44 (37%) patients had two or more genes mutated (Figure 1, clusters #3 & #4). Recurrent combinations of mutations (affecting more than 5% of patients) were found in a group of 23 (20%) patients. These combinations of mutations comprised of at least two of the following genes: TP53, SF3B1 and ATM (Figure 1, cluster #3, so called multiple-hit (MH) profile). Remarkably, mutations in these 3 genes were found significantly more frequently associated than in isolation. We then investigated the potential clinical relevance of the MH profile. This profile was associated with poorer ORR than the remaining cohort (43% vs 80%, P<.0001). None of the patients with a MH profile achieved CR compared to 24% for the remaining patients (P=.006). MH patients have also shorter median PFS of 12 months compared to 19 months in cluster #1, 23 months in cluster #2 and 18 months in cluster #4 (P=.03). Multivariate analysis for PFS including relevant factors such as 'fludarabine-refractory’ and TP53 disruption confirmed the adverse prognostic value related to the MH profile (HR=3.194 [95%CI=1.493-6.835], P=.003). Interestingly, among the TP53-disrupted patients, the MH profile retained its prognostic impact with a median PFS of 11 months for those with mut-SF3B1 and/or mut-ATM versus 22 months for those with wt-SF3B1 and wt-ATM (P=.022).Conclusion: The mutational landscape of relapsing CLL is marked by a group of patients with combined mutations of the TP53, ATM and SF3B1 genes (multiple-hit profile) and is associated with an adverse prognostic impact. In addition to TP53 and SF3B1, ATM should be sequenced at relapse to predict outcome and guide subsequent therapeutic intervention. Further studies are required to confirm these findings and to understand the subclonal distribution of these mutations. [Display omitted] DisclosuresHillmen:Pharmacyclics, Janssen, Gilead, Roche: Honoraria, Research Funding. Tournilhac:mundipharma: Honoraria, Other, Research Funding; GSK: Honoraria, Other, Research Funding; Roche: Honoraria, Other, Research Funding.

IGHV Mutation Status Does Not Add Prognostic Information In The Background Of Mutations In TP53 and SF3B1, and Deletions Of 17p and 11q, Which Are Independent Adverse Prognostic Parameters In CLL

BackgroundIn CLL, the IGHV mutation status is an established prognostic marker. However, associations between the IGHV mutation status and other genetic markers have been reported. Based on genome sequencing data, novel recurrent mutations have been identified in CLL and first data on their prognostic impact is also available. AimsIn this study we addressed the following questions in a large cohort of 934 CLL patients: 1. Associations of IGHV mutation status with cytogenetic and molecular genetic markers 2. Age dependency of IGHV mutation status, cytogenetic and molecular mutations 3. Impact on survival. Patients and MethodsIn total, in 934 CLL patients the IGHV mutation status was determined. Median age was 66.8 years (range: 29.6 - 90.5 years). Further, all patients were analyzed by DNA sequencing for mutations in TP53, SF3B1, MYD88, XPO1, NOTCH1 and FBXW7 and by FISH for TP53 deletion status as well as for del(13q), del(11q) and +12. ResultsIn the entire cohort 550/934 (58.9%) patients showed a mutated and 384 (41.1%) an unmutated IGHV status. Molecular mutations were observed in the following frequencies: NOTCH1: 12.6%, SF3B1: 11.9%, TP53: 9.2%, XPO1: 4.0%, FBXW7: 2.6%, and MYD88: 1.7%. Patients showed deletions 13q in 58.7% (13q as the sole abnormality in 45.5%), trisomy 12 in 15.6%, 11q in 12.1%, and 17p in 5.8%, respectively. The following genetic aberrations were more frequent in CLL with unmutated vs mutated IGHV status: TP53mut (14.8% vs 5.3%), SF3B1mut (19.0% vs 6.9%), XPO1mut (9.1% vs 0.4%), NOTCH1mut (24.2% vs 4.5%), del(17p) (10.7% vs 2.4%), del(11q) (24.2% vs 3.6%), +12 (21.4% vs 11.6%) (for all p<0.001). In contrast, del(13q) (45.8% vs 67.6%), del(13q) sole (26.3% vs 58.9%) (for all p<0.001), and MYD88mut (0.3% vs 2.7%, p=0.004) were less frequent in CLL with unmutated IGHV status. No association between age and IGHV status was observed. With respect to all other analyzed markers only the frequency of TP53mut and del(17p) increased significantly with age. In univariate analysis the following parameters were significantly associated with shorter overall survival (OS): IGHV unmutated (HR: 2.0), age (≤70 vs >70 yrs: HR:3.4), TP53mut (HR: 3.8), SF3B1mut (HR: 2.3), del(17p) (HR: 6.2), del(11q) (HR: 2.3) (for all p<0.001), and NOTCH1mut (HR: 1.5, p=0.05), while del(13q) sole was associated with longer OS (HR: 0.6; p=0.003). In multivariate analysis the following parameters were independently associated with shorter OS: age (≤70 vs >70 yrs: HR: 3.1; p<0.001), TP53mut (HR: 2.0; p=0.018), SF3B1mut (HR: 2.1; p=0.001), del(17p) (HR: 3.6, p<0.001), del(11q) (HR: 2.1; p=0.005), while IGHV mutation status surprisingly did not show an independent impact on OS. Next we separated the cohort according to the number of adverse prognostic markers TP53mut, SF3B1mut, del(11q), and del(17p) (group A: 0 marker (n=658), group B: 1 marker (n=197), group C: 2 or more markers with alterations (n=78)). Kaplan-Meier-analysis revealed a 5 yrs OS of 89.5% in group A, of 71.3% in group B and of 37.4% in group C (for all comparisons p<0.001). Neither within groups A, B nor within group C did the IGHV mutation status have an impact on OS. On the other hand, OS at 5 yrs differed significantly according to the group within patients with either mutated IGHV or unmutated IGHV status (mutated IGHV status: group A (n=467) 90.6%, group B (n=67) 73.7% and group C (n=16) 47.2%; A vs B: p=0.031; B vs C: p=0.022; unmutated IGHV status: group A (n=191) 86.8%, group B (n=130) 69.6% and group C (n=62) 35.8%; A vs B: p=0.006; B vs C: p<0.001). Interestingly, a significant association between a higher number of adverse prognostic markers and age was observed (p=0.019). Further we performed multivariate analysis with the number of adverse prognostic factors (TP53mut, SF3B1mut, del(11q), del(17p): 0 markers to 4 markers positive) and age per decade. Both parameters were independently associated with OS (HR: 2.3 per adverse marker positive and HR: 1.7 per decade, for both p<0.001). Conclusions1. Several adverse prognostic markers (TP53mut, SF3B1mut, NOTCH1, del(17p), del(11q)) were associated with an unmutated IGHV status. 2. However, in the background of the genetic markers TP53mut, SF3B1mut, del(11q), or del(17p) IGHV status lost its independent prognostic impact. 3. An accumulation of adverse prognostic markers worsens prognosis in CLL. Disclosures:Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Jeromin:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Extensive High-Depth Sequencing Of Longitudinal CLL Samples Identifies Frequent Mutations In MAP Kinase Signaling and Novel Mutations Activating Notch and Beta-Catenin

Recent whole-genome sequencing, whole-exome sequencing, and copy number analysis studies in small sets of patients with chronic lymphocytic leukemia (CLL) at presentation versus progression have demonstrated that clonal evolution has clinical and biologic relevance in CLL. Although the time and cost required to perform whole genome and exome sequencing are improving, challenges still exist in implementing these genomic strategies in real-time clinical practice. Here we performed DNA and RNA sequencing of an extensive panel of all genes known to be recurrently mutated in lymphoid, myeloid, and solid tumor malignancies, at high sequencing depth in patients with CLL at clinical presentation and progression. This allowed us to obtain integrated mutation, copy number, and gene fusion events in CLL and to use this data to obtain new insights into clonal evolution of CLL.Genomic DNA and total RNA was isolated from 59 CLL samples (including 29 CLL paired patients sampled at the time of initial presentation when no clinical indication for therapy was met and at a later time point of disease progression requiring therapy). The median time between samples was 2.07 years (range 0.08-4.95 years)). Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 272 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >590X for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. All clinical data as well as biomarkers previously validated for clinical use in CLL (IGHV mutational status, CD38 expression, metaphase cytogenetics and FISH) were also obtained for all samples at each time point.This targeted sequencing approach detected at least one clonal somatic mutation in 95% of CLL patients including 144 individual mutations, 3 functional rearrangements, and 1 homozygous deletion. Consistent with prior reports, mutations in NOTCH1 (25%), SF3B1 (19%), and TP53 (14%) were amongst the most commonly mutated genes in this cohort. Of note, activating mutations in KRAS (15%) and BRAF (10%) were also amongst the most commonly mutated genes targeted by somatic mutations in CLL (Figure). Taken together, mutations resulting in constitutive MAP kinase signaling occurred in 36% of all patients, second only to mutations activating Notch signaling which occurred in 40% of patients. The two most commonly mutated genes in the Notch signaling pathway were mutations in NOTCH1 followed by loss of function mutations in SPEN (7%). SPEN is a nuclear receptor transcription factor which blocks the differentiation of precursor B cells into marginal zone B cells through interactions with RBP-J. Also identified as recurrent were mutations in FAT3, an inhibitor of b-catenin signaling. In addition, sequencing identified a novel fusion event in BIRC3 fusing BIRC3 to LRRC40 as well as a truncating mutation in one patient in BRCA2 at disease progression. [Display omitted] We then performed paired mutational analysis of samples at initial sample acquisition and at disease progression requiring therapy (Figure). Mutational analysis at the time of clonal evolution identified mutations in DNMT3A, EED, IDH2, IRF4, VHL, and RB1 only at the time of disease evolution. Mutations in NOTCH1, KRAS, TP53, NRAS, and BCOR were more common at disease progression than initial presentation. In contrast, mutations in SF3B1 and XPO1 were always retained at disease progression if found at earlier disease states.These data demonstrate the utility of clinical grade, high throughput targeted sequencing in CLL to identify targetable genomic alterations, discover novel mutations not previously reported, and identify important markers of disease evolution which may represent incipient biomarkers for clinical disease progression. This includes a much more frequent occurrence of targetable mutations in the MAP kinase pathway than previously described in CLL and recurrent loss of function mutations in SPEN and FAT3 in CLL. Disclosures:Levine:Foundation Medicine, Inc: Consultancy. Heaney:Novartis: Research Funding; Sanofi-Aventis: Consultancy, Research Funding; Onconova: Research Funding; Incyte: Consultancy, Research Funding. Abdel-Wahab:Foundation Medicine, Inc: Consultancy.

SF3B1 mutations correlated to cytogenetics and mutations in NOTCH1, FBXW7, MYD88, XPO1 and TP53 in 1160 untreated CLL patients

We analyzed a large cohort of 1160 untreated CLL patients for novel genetic markers (SF3B1, NOTCH1, FBXW7, MYD88, XPO1) in the context of molecular, immunophenotypic and cytogenetic data. NOTCH1 mutations (mut) (12.3%), SF3B1mut (9.0%) and TP53mut (7.1%) were more frequent than XPO1mut (3.4%), FBXW7mut (2.5%) and MYD88mut (1.5%). SF3B1mut, NOTCH1mut, TP53mut and XPO1mut were highly correlated to unmutated, whereas MYD88mut were associated with mutated IGHV status. Associations of diverse cytogenetic aberrations and mutations emerged: (1) SF3B1mut with del(11q), (2) NOTCH1mut and FBXW7mut with trisomy 12 and nearly exclusiveness of SF3B1mut, (3) MYD88mut with del(13q) sole and low frequencies of SF3B1mut, NOTCH1mut and FBXW7mut. In patients with normal karyotype only SF3B1mut were frequent, whereas NOTCH1mut rarely occurred. An adverse prognostic impact on time to treatment (TTT) and overall survival (OS) was observed for SF3B1mut, NOTCH1mut and TP53 disruption. In multivariate analyses SF3B1mut, IGHV mutational status and del(11q) were the only independent genetic markers for TTT, whereas for OS SF3B1mut, IGHV mutational status and TP53 disruption presented with significant impact. Finally, our data suggest that analysis of gene mutations refines the risk stratification of cytogenetic prognostic subgroups and confirms data of a recently proposed model integrating molecular and cytogenetic data.

Acquisition of TP53 Mutations in Chronic Lymphocytic Leukemia (CLL) Patients During the Course of Disease Is Associated with an Unmutated IGHV Status and Mutations in XPO1 and SF3B1

Abstract 1768 Background:In chronic lymphocytic leukemia (CLL), an association between TP53 mutations (TP53mut) and a poor prognosis has been reported. Two hypotheses are discussed regarding the occurrence of TP53mut during the course of disease: 1. TP53mut are already present at first diagnosis in a small subclone, which selectively grow despite treatment, and 2. TP53mut occur de novo during disease progression. Aim:Investigation of the acquisition of TP53mut in previously TP53wt CLL cases during the course of disease. Patient and Methods:The discovery cohort included 20 cases, which were selected based on negativity for TP53mut at first diagnosis and acquisition of TP53mut during follow-up (7 female, 13 male patients; median age: 60.7 yrs, range: 41.3–76.6 yrs). 16/20 patients were treated according to various regimens (including fludarabine and rituximab). TP53 analyses of the discovery cohort were performed by DHPLC and subsequent direct Sanger sequencing and, additionally, by 454 deep-sequencing analyses (454 Life Sciences, Branford, CT) enabling a higher sensitivity. Further, a comprehensive molecular characterization included in addition to TP53 the following markers: IGHV mutation status, NOTCH1, FBXW7, SF3B1, XPO1, and MYD88. All markers had been analyzed both at initial diagnosis and at the first follow-up time point that revealed an acquired TP53mut. A second cohort (n=326) was used for comparison of the mutation frequencies of IGHV, NOTCH1, FBXW7, SF3B1, XPO1, and MYD88. Results:Next-generation sequencing confirmed 18/20 cases as wild-type at first diagnosis (median coverage: 733-fold; range: 378-1, 369; sensitivity: <2.0%). However, in 2/20 cases deep-sequencing revealed a low-level clone of TP53 aberration (4.0% and 7.0%) that had not been detected by DHPLC and Sanger sequencing. Both patients with a detectable TP53mut at first diagnosis were excluded from further analyses.New TP53mut occurred at a median of 39.0 months after first diagnosis (range: 13.7–68.4 months). The patients harbored between 1 and 4 TP53mut (mean: 1.3) with a median mutation load of 18.0% (range: 2.0–93.0%). Of note, 15/18 patients had received therapy prior to the acquisition of the TP53mut. In addition, 13/17 (76.5%; n=1 no data available) cases showed a concomitant TP53 deletion detected by fluorescence in situ hybridization. Only 1/10 of these cases showed a TP53 deletion already at first diagnosis (no data available: n=3).Mutation frequencies of the NOTCH1 PEST domain, MYD88, FBXW7, XPO1, SF3B1 and the IGHV mutation status were compared between the discovery cohort and an independent cohort of 326 newly diagnosed CLL cases. Mutation frequencies in NOTCH1 PEST domain (2/18, 11.1% vs 45/326, 13.8%), MYD88 (1/18, 5.6% vs 6/326, 1.8%), and FBXW7 (0/18, 0% vs 8/326, 2.5%) were comparable between both cohorts, while mutations in XPO1 (4/18; 22.2% vs 14/326; 4.3%, P=0.010) and in SF3B1 (4/18; 22.2% vs 27/326 8.3%, P=0.067) were observed at significantly higher frequencies in the discovery cohort. Also an unmutated IGHV status was significantly more frequent in the discovery cohort compared to the unselected comparison cohort (16/18, 88.9% vs 196/326, 60.1%; P=0.01). Of note, in one case with a MYD88 mut and another case with a SF3B1 mut, these alterations were detected at first diagnosis, but disappeared during course of disease. In contrast, an XPO1 mutation was acquired during the course of disease in parallel to the TP53mut. In the remaining cases with SF3B1 (n=3), XPO1 (n=3), and NOTCH1 (n=2), mutations were observed both at first diagnosis and follow-up time point that revealed an acquired TP53mut. Conclusions:1. A small fraction of CLL patients (2/20) harbored a subclone with TP53 mutations at first diagnosis which escaped detection by conventional laboratory assays (DHPLC and Sanger sequencing) and were only identified retrospectively by using a more sensitive next-generation sequencing assay. These subclones increased in size during the course of disease. 2. A subset of CLL patients acquired TP53mut during course of their disease. Thus, repeated TP53 mutation analyses are necessary for optimal treatment decisions. 3. An unmutated IGHV status and mutations in XPO1 and SF3B1 were more frequent in CLL patients who acquired TP53 mutations during the course of disease, suggesting that these are potential risk factors for the acquisition of TP53 mutations during disease progression. Disclosures:Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Weissmann:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Boeck:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Whole Exome Sequencing of CLL Before Second-Line Treatment with Fludarabine and Cyclophosphamide with or without Rituximab (REACH trial).

AbstractAbstract 2514Chronic lymphocytic leukemia (CLL), the most frequent leukemia in adults, still remains poorly understood. Several prognostic markers like the deletion of certain genomic regions as assayed by fluorescence in situ hybridization (FISH), the mutational status of the IGVH locus or the expression levels of ZAP70 have been identified. However, the predictive power of these markers is limited and they cannot fully explain the heterogeneity and the biology of CLL. The addition of monoclonal anti-CD20 antibody (rituximab) to chemotherapy has significantly improved progression-free survival of CLL patients even at second-line treatment (Robak et al. JCO 2010).The aim of our project was to identify new, potentially predictive markers in previously treated patients that reached complete remission after second-line treatment with FC or R-FC (Fludarabine, Cyclophosphamide and Rituximab). We performed whole exome sequencing in 25 CLL samples before second-line treatment and their corresponding disease free remission samples (peripheral blood). Disease free remission was defined as minimal residual disease levels of less than 1×10−3 as measured by the disease specific IGVH rearrangements using quantitative PCR and FISH negativity if a marker was available. The CLL samples had a median of 84% CD19 positive cells. Our CLL patients had a predominance of favorable prognostic markers, 60% being IGVH mutated, 52% with a sole 13q deletion and 16% with a trisomy 12. Only 8% had a prognostically unfavorable 11q deletion, none had a 17p deletion or more than one FISH abnormality (the following genomic regions were analyzed: 6q, 13q, 11q (ATM), trisomy 12, 17p (TP53)). The exomes of the paired CLL and disease free remission samples were captured using the Agilent Sure Select 50 Mb kit (Agilent Technologies, Santa Clara, CA, USA). Sequencing was performed with 76–80 bp paired-end reads on an Illumina IIx Genome Analyzer (Illumina, San Diego, CA, USA). The mean total sequence per exome was 6.6 Gbp, of which >80% could be aligned to the reference genome (build NCBI36/hg18). About 90% of all SureSelect exome target positions were covered ≥ 10 fold. We called single nucleotide variants (SNVs) specific for the CLL samples using VarScan (Koboldt et al Bioinformatics 2009) with custom filter settings. Annotated polymorphisms (dbSNP130) were excluded. About 80% of the SNVs that were predicted to result in a missense or nonsense mutation could be validated by Sanger sequencing. In total we detected 208 somatic missense or nonsense mutations in 198 genes in the 25 CLL exomes. Four genes were found mutated in more than one CLL sample indicating that these might be drivers for CLL.We also compared our gene list with published CLL exome and genome data to identify additional recurrently mutated genes. A total of 2756 mutated genes (harbouring non-synonymous, InDels and splice site mutations) have been described in 200 CLL patients which were predominantly analyzed at first-line treatment (Wang et al NEJM 2011, Puente et al Nature 2011 and Quesada et al Nature Genetics 2012). Within these three public datasets 369 genes were recurrent (found mutated in more than one sample). In our dataset 129 out of 198 mutated genes have not been described as mutated in these three publications. The remaining 69 genes were previously found to be mutated in CLL. Out of these 69 genes 37 can only be identified as recurrent when our data set is taken into consideration. Thus, our study increases the number of recurrently mutated genes in CLL from 369 to 406. We detected three samples with XPO1 mutations; all of which had a non-mutated IGVH status, two had a 13q deletion and one had no FISH aberration. Two patients in our cohort had SF3B1 mutations; both patients had an unmutated IGVH status; one with a 13q deletion and one with no FISH aberration. 24 out of the 25 patients had at least one recurrent mutation taking the public datasets in consideration.Our results are in agreement with other published whole exome and whole genome sequencing reports of CLL and reinforce the picture that CLL is genetically a very heterogeneous disease. In fact, almost all large scale sequencing studies of cancer genomes and exomes indicate that the number of biologically relevant mutational targets is much larger than expected. Our results also highlight the necessity to perform whole exome or whole genome sequencing on ever larger numbers of CLL samples. Disclosures:Konstandin:Roche: Research Funding. Krebs:Illumina: Honoraria. Trunzer:Roche: Employment. Weisser:Roche: Employment.

Selective inhibitors of nuclear export show that CRM1/XPO1 is a target in chronic lymphocytic leukemia

The nuclear export protein XPO1 is overexpressed in cancer, leading to the cytoplasmic mislocalization of multiple tumor suppressor proteins. Existing XPO1-targeting agents lack selectivity and have been associated with significant toxicity. Small molecule selective inhibitors of nuclear export (SINEs) were designed that specifically inhibit XPO1. Genetic experiments and X-ray structures demonstrate that SINE covalently bind to a cysteine residue in the cargo-binding groove of XPO1, thereby inhibiting nuclear export of cargo proteins. The clinical relevance of SINEs was explored in chronic lymphocytic leukemia (CLL), a disease associated with recurrent XPO1 mutations. Evidence is presented that SINEs can restore normal regulation to the majority of the dysregulated pathways in CLL both in vitro and in vivo and induce apoptosis of CLL cells with a favorable therapeutic index, with enhanced killing of genomically high-risk CLL cells that are typically unresponsive to traditional therapies. More importantly, SINE slows disease progression, and improves overall survival in the Eμ-TCL1-SCID mouse model of CLL with minimal weight loss or other toxicities. Together, these findings demonstrate that XPO1 is a valid target in CLL with minimal effects on normal cells and provide a basis for the development of SINEs in CLL and related hematologic malignancies.

NOTCH1 mutations in CLL associated with trisomy 12

AbstractTwo recent studies reported wholegenome sequencing of chronic lymphocytic leukemia (CLL) samples and found repeated mutations in the XPO1 and NOTCH1 genes. XPO1 was found mutated in 2.4% of cases, while NOTCH1 was found mutated in 12.2% or 15.1% of CLL samples. Here we report the results of sequencing of XPO1 and NOTCH1 in 186 CLL cases. Our results confirmed frequency of XPO1 mutations. However, we found only 5 NOTCH1 mutations in 127 IGVH unmutated/ZAP70+ CLL samples (4%), and one mutation was found in IGVH mutated/ZAP70− CLL for a total percentage of 1.5%. Because 4 of 6 mutated samples also showed trisomy 12, we sequenced NOTCH1 in an additional 77 cases with trisomy 12 CLLs, including 47 IGVH unmutated/ZAP70+ cases. Importantly, we found 41.9% NOTCH1 mutation frequency in aggressive trisomy 12 CLL cases. Our data suggest that activation of NOTCH1 plays a critical role in IGVH unmutated/ZAP70+ trisomy 12 CLL.

Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution. Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes. The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer.