Gene Mutations Identified in Chemorefractory Acute Myeloid Leukemia

Abstract Introduction: Acute myeloid leukemia (AML) is a genetically heterogeneous disease. Recently, multiple recurrently mutated genes have been identified in AML and implicated in various mechanisms of leukemogenesis. However, knowledge regarding the association of gene mutations with primary or secondary resistance to chemotherapy is incomplete. Methods: We analyzed a discovery cohort of 45 patients with chemorefractory AML that were enrolled in a phase 2 clinical trial of a novel conditioning regimen prior to allogeneic stem cell transplant for patients with non-remission AML. DNA was extracted from FACS-purified leukemic cells procured from patients after failure to achieve complete remission (CR) after ≥2 cycles of induction chemotherapy after initial diagnosis ("primary refractory", N=22) or after rapid relapse (<6 months) or failure to achieve CR after ≥1 cycle of induction chemotherapy after relapse ("secondary refractory", N=23). Since TP53 mutations have been previously associated with refractory disease, we selected 29 TP53 wild-type cases from the discovery cohort and performed whole exome sequencing (WES) with a mean read depth of 72X (range 30-140). All somatically acquired gene variants identified by WES in protein-coding genes were verified by Sanger sequencing. In addition, we performed Sanger re-sequencing of 11 recurrently mutated genes in AML (TP53, RUNX1, DNMT3A, TET2, FLT3, NPM1, IDH1, IDH2, ASXL1, NRAS and KRAS) in all 45 cases. Given lack of published WES data in refractory AML, we then compared these mutation frequencies to a cohort of 151 AML patients enrolled consecutively at one center (the "university cohort") with known responses to chemotherapy or The Cancer Genome Atlas (TCGA) data which comprises de novo AML only. Results: WES of 29 TP53 wild-type refractory AML cases revealed a total of 351 confirmed somatic mutations with a median of 13 protein-coding mutations per case (range 5-22). Genes mutated in 7% (2 of 29 cases), and excluding the 11 known recurrently mutated genes listed above, that were not previously described in AML (COSMIC review) included ADAM23, CPNE7, and SIX5. We also identified mutations in NOMO3 and OAS2 in 7% (2 of 29 cases), which have been previously described in AML but at lower frequencies (1.7% [6 of 347; COSMIC] and 0.2% [1 of 347] respectively) based on review of the literature. The genes SRSF2, NOMO3 and OAS2, which were all identified in 7% (2 of 29 cases) in our discovery cohort, had no reported mutations found in the TCGA (p=0.02). Additional genes, which were found in our discovery cohort in 7% (2 of 29 cases) respectively, and were also found in the TCGA, include BCOR, FOXP1, FRYL, PHF6, STAG2, PTPN11 and SETD2. Mutational profiling of the 11 recurrently mutated genes in AML revealed a striking paucity of NPM1 mutations in primary refractory AML (range 0% [discovery cohort] - 3% [university cohort]) compared with chemosensitive AML (31% [university cohort]; p<0.001). TP53 mutations, however, were enriched in primary refractory AML (range 23% [discovery cohort] - 38% [university cohort]) compared with chemosensitive AML (4% [university cohort]; p<0.001). Of note, while FLT3 -ITD mutations were infrequently observed in primary refractory AML (range 0% [discovery cohort] - 14% [university cohort]) compared to chemosensitive AML (27% [university cohort]; p<0.01), they were highly enriched in secondary refractory AML (61% [discovery cohort] - 30% [university cohort]; p=0.03). Conclusions: 1) Whole exome sequencing of 29 TP53 wild-type refractory AML revealed recurrent mutations in ADAM23, CPNE7, NOMO3, OAS2 and SIX5. The function and prevalence of these gene mutations are not well-characterized in AML, including refractory AML and should be determined in a larger cohort of patients; 2) TP53 mutations were significantly enriched in primary refractory disease; 3) Conversely, FLT3 -ITD mutations were significantly enriched in secondary but not primary refractory disease, suggesting the frequent emergence of a chemorefractory FLT3-ITD mutated clone following treatment with conventional chemotherapy; and, 4) NPM1 mutations were significantly under-represented in primary refractory AML. While larger sequencing studies of refractory AML cases are needed, these data do not support gene mutations other than in TP53 as frequent causes of primary refractoriness to chemotherapy in AML. Disclosures Malek: Gilead Sciences: Equity Ownership; Abbvie: Equity Ownership; Janssen Pharmaceuticals: Research Funding.

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Recurrent Mutations of Multiple Components of Cohesin Complex in Myeloid Neoplasms

Blood ◽

10.1182/blood.v120.21.782.782 ◽

2012 ◽

Vol 120 (21) ◽

pp. 782-782

Author(s):

Ayana Kon ◽

Lee-Yung Shih ◽

Masashi Minamino ◽

Masashi Sanada ◽

Yuichi Shiraishi ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Gene Mutations ◽

Cohesin Complex ◽

Wild Type ◽

Myeloid Neoplasms ◽

Whole Exome ◽

Recurrent Mutations ◽

Equity Ownership

Abstract Abstract 782 Recent genetic studies have revealed a number of novel gene mutations in myeloid malignancies, unmasking an unexpected role of deregulated histone modification and DNA methylation in both acute and chronic myeloid neoplasms. However, our knowledge about the spectrum of gene mutations in myeloid neoplasms is still incomplete. In the previous study, we analyzed 29 paired tumor-normal samples with chronic myeloid neoplasms with myelodysplastic features using whole exome sequencing (Yoshida et al., Nature 2011). Although the major discovery was frequent spliceosome mutations tightly associated with myelodysplasia phenotypes, hundreds of unreported gene mutations were also identified, among which we identified recurrent mutations involving STAG2, a core cohesin component, and also two other cohesin components, including STAG1 and PDS5B. Cohesin is a multimeric protein complex conserved across species and is composed of four core subunits, i.e., SMC1, SMC3, RAD21 and STAG proteins, together with several regulatory proteins. Forming a ring-like structure, cohesin is engaged in cohesion of sister chromatids in mitosis, post-replicative DNA repair and regulation of gene expression. To investigate a possible role of cohesin mutations in myeloid leukemogenesis, an additional 534 primary specimens of various myeloid neoplasms was examined for mutations in a total of 9 components of the cohesin and related complexes, using high-throughput sequencing. Copy number alterations in cohesin loci were also interrogated by SNP arrays. In total, 58 mutations and 19 deletions were confirmed by Sanger sequencing in 73 out of 563 primary myeloid neoplasms (13%). Mutations/deletions were found in a variety of myeloid neoplasms, including AML (22/131), CMML (15/86), MDS (26/205) and CML (8/65), with much lower mutation frequencies in MPN (2/76), largely in a mutually exclusive manner. In MDS, mutations were more frequent in RCMD and RAEB (19.5%) but rare in RA, RARS, RCMD-RS and 5q- syndrome (3.4%). Cohesin mutations were significantly associated with poor prognosis in CMML, but not in MDS cases. Cohesin mutations frequently coexisted with other common mutations in myeloid neoplasms, significantly associated with spliceosome mutations. Deep sequencing of these mutant alleles was performed in 19 cases with cohesin mutations. Majority of the cohesin mutations (16/19) existed in the major tumor populations, indicating their early origin during leukemogenesis. Next, we investigated a possible impact of mutations on cohesin functions, where 17 myeloid leukemia cell lines with or without cohesin mutations were examined for expression of each cohesin component and their chromatin-bound fractions. Interestingly, the chromatin-bound fraction of one or more components of cohesin was substantially reduced in cell lines having mutated or defective cohesin components, suggesting substantial loss of cohesin-bound sites on chromatin. Finally, we examined the effect of forced expression of wild-type cohesin on cell proliferation of cohesin-defective cells. Introduction of the wild-type RAD21 and STAG2 suppressed the cell growth of RAD21- (Kasumi-1 and MOLM13) and STAG2-defective (MOLM13) cell lines, respectively, supporting a leukemogenic role of compromised cohesin functions. Less frequent mutations of cohesin components have been described in other cancers, where impaired cohesion and consequent aneuploidy were implicated in oncogenic action. However, 23 cohesin-mutated cases of our cohort had completely normal karyotypes, suggesting that cohesin-mutated cells were not clonally selected because of aneuploidy. Alternatively, a growing body of evidence suggests that cohesin regulate gene expression, arguing for the possibility that cohesin mutations might participate in leukemogenesis through deregulated gene expression. Of additional note, the number of non-silent mutations determined by our whole exome analysis was significantly higher in 6 cohesin-mutated cases compared to non-mutated cases. Since cohesin also participates in post-replicative DNA repair, this may suggest that compromised cohesin function could induce DNA hypermutability and contribute to leukemogenesis. In conclusion, we report a new class of common genetic targets in myeloid malignancies, the cohesin complex. Our findings highlight a possible role of compromised cohesin functions in myeloid leukemogenesis. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

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Whole Exome Sequencing Reveals Spectrum of Gene Mutations in Pediatric AML

Blood ◽

10.1182/blood.v120.21.124.124 ◽

2012 ◽

Vol 120 (21) ◽

pp. 124-124

Author(s):

Norio Shiba ◽

Kenichi Yoshida ◽

Yusuke Okuno ◽

Yuichi Shiraishi ◽

Yasunobu Nagata ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Conflicts Of Interest ◽

Massively Parallel Sequencing ◽

Gene Mutations ◽

Pediatric Leukemia ◽

Protein Coding ◽

Whole Exome ◽

Recurrent Mutations ◽

Pediatric Aml

Abstract Abstract 124 Background Pediatric acute myeloid leukemia (AML) comprises ∼20% of pediatric leukemia, representing one of the major therapeutic challenges in pediatric oncology with the current overall survival remains to be ∼60%. As for the molecular pathogenesis of pediatric AML, it has been well established that gene fusions generated by recurrent chromosomal translocations, including t(15;17), t(8;21), inv(16) and t(9;11), play critical roles in leukemogenesis. However, they are not sufficient for leukemogenesis, indicating apparent need of additional genetic hits, and approximately 20% of pediatric AML cases lack any detectable chromosomal abnormalities (normal karyotype AML). Currently, a number of gene mutations have been implicated in the pathogenesis of both adult and pediatric AML, including mutations of RAS, KIT and FLT3, and more recently, a new class of mutational targets have been reported in adult AML, including CEBPA, NPM1, DNMT3A, IDH1/2, TET2 and EZH2. However, mutations of the latter class of gene targets seem to be rare in pediatric AML cases, whereas other abnormalities such as a NUP98-NSD1 fusion are barely found in adult cases, indicating the discrete pathogenesis between both AML at least in their subsets. Meanwhile, the recent development of massively parallel sequencing technologies has provided a new opportunity to discover genetic changes across the entire genomes or protein-coding sequences in human cancers at a single-nucleotide level, which could be successfully applied to the genetic analysis of pediatric AML to obtain a better understanding of its pathogenesis. Methods In order to reveal a complete registry of gene mutations and other genetic lesions, we performed whole exome sequencing of paired tumor-normal specimens from 23 pediatric AML cases using Illumina HiSeq 2000. Although incapable of detecting non-coding mutations and gene rearrangements, the whole-exome approach is a well-established strategy for obtaining comprehensive spectrum of protein-coding mutations. Recurrently mutated genes were further examined for mutations in an extended cohort of 200 pediatric AML samples, using deep sequencing, in which the prevalence and relative allele frequencies of mutations were investigated. Results Whole-exome sequencing of paired tumor-normal DNA from 23 patients were analyzed with a mean coverage of more than x120, and 90 % of the target sequences were analyzed at more than x20 depth on average. A total of 237 somatic mutations or 10.3 mutations per sample were identified. Many of the recurrent mutations identified in this study involved previously reported targets in adult AML, such as FLT3, CEBPA, KIT, CBL, NRAS, WT1, MLL3, BCOR, BCORL1, EZH2, and major cohesin components including XXX and ZZZ. On the other hand, several genes were newly identified in the current study, including BRAF, CUL2 and COL4A5, which were validated for the clinical significance in an extended cohort of 200 pediatric cases. Discussion Whole exome sequencing unmasked a complexity of gene mutations in pediatric AML genomes. Our results indicated that a subset of pediatric AML represents a discrete entity that could be discriminated from the adult counterpart, in terms of the spectrum of gene mutations. Disclosures: No relevant conflicts of interest to declare.

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High Incidence of RAS Signalling Pathway Mutations in MLL–rearranged Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v120.21.539.539 ◽

2012 ◽

Vol 120 (21) ◽

pp. 539-539

Author(s):

Vera Grossmann ◽

Susanne Schnittger ◽

Alexander Kohlmann ◽

Christiane Eder ◽

Annette Fasan ◽

...

Keyword(s):

Survival Data ◽

De Novo ◽

Gene Mutations ◽

Signalling Pathway ◽

Melting Curve Analysis ◽

Prognostic Impact ◽

Tp53 Mutations ◽

Equity Ownership ◽

Ras Signalling ◽

Acute Myeloid

Abstract Abstract 539 Background: Chromosomal translocations of the MLL gene on chromosome 11q23 are associated with a unique subset of acute lymphoblastic or acute myeloid leukemias (AML). In adults, MLL rearrangements are detected in 3% of de novo AML and in 10% of therapy-related AML (t-AML) cases and are associated with poor prognosis. In addition to disease defining mutations recent high-throughput sequencing studies had shown that almost all myeloid malignancies accumulate a large number of cooperating gene mutations. Aim: Determination of somatic mutations occurring in cases harboring MLL rearrangements and investigation of the prognostic impact of molecular and additional chromosomal aberrations. Patients and Methods: We investigated a cohort of 110 adult AML (80 de novo, 30 t-AML) cases harboring an 11q23 translocation. The cohort was composed of 66 females and 44 males; median age: 55.8 years. MLL translocation partners were as follows: MLLT3 (n=46), MLLT4 (n=15), ELL (n=15); MLLT10 (n=8), others (n=26). Chromosome banding analysis data was available in all cases and survival data in 78 cases (median overall survival (OS) was 10.1 months). Patients were screened for mutations in ASXL1 (n=98), CBL (n=62), CEBPA (n=61), FLT3-ITD (n=103), FLT3-TKD (n=95), IDH1 (n=96), IDH2 (n=84), KRAS (n=107), NPM1 (n=101), NRAS (n=106), PTPN11 (n=99), RUNX1 (n=110), and TP53 (n=110) using amplicon deep-sequencing (454 Roche Life Sciences, Branford, CT), direct Sanger sequencing or melting curve analysis. Results: Overall, mutations were detected in 59/110 (53.6%) cases. We discovered that 42/110 (38.2%) MLL-translocated AML cases harbored mutations within the RAS signalling pathway (KRAS mut: 23/107; 21.5%; NRAS mut: 22/106; 20.8%; PTPN11 mut: 3/99, 3.0%) or alterations in the RAS regulating FLT3 gene (FLT3-ITD: 4/103, 3.9%, and FLT3-TKD: 10/95, 10.5%). Additional mutations were detected in the tumor suppressor gene TP53 (8/110; 7.3%), ASXL1 (6/98; 6.1%), RUNX1 (4/110; 3.6%), and IDH1 (1/96). No mutation was detected in IDH2, CBL, CEBPA, and NPM1. Most cases showed only one mutation (n=39, 66.1%), whereas 17 cases (28.8%) showed two and 3 cases (5.1%) three mutations in different genes. No difference of mutation distribution was seen between de novo and t-AML. In this cohort, no associations amongst gene mutations were observed, however, FLT3-ITD was associated with MLL-ELL (3/14 vs 1/89, P=0.008) and PTPN11 mutations with MLLT10-MLL (2/8 vs 1/91, P=0.017) alterations. In addition, KRAS mut and NRAS mut correlated with high WBC count (KRAS mut: 103.0±79 vs 59.2±67 x109/L, P=0.016; NRAS mut: 94.7±57 vs 60.4±72 x109/L, P=0.080). Further, we were interested in the prognostic impact of single gene mutations. NRAS mut and TP53 mut showed both a non-significant inferior impact on OS, i.e. OS after 2 years: 19.1% vs 46.4%, P=0.62; 0% vs 41.3%, P=0.114. Further, TP53 mutations were correlated with shorter event-free survival (EFS) (EFS after 2 years: 0% vs 20.0%, P=0.029). No associations with prognosis were observed for the remaining genes and translocation partners. In contrast, age was associated with OS and EFS (<60 years, n=59 vs ≥60 years, n=51: OS after 2 years: 51.4% vs 26.3%, P=0.003, EFS after 2 years: 28.0% vs 7.7%, P=0.004). Within the cohort of cases ≥60 years, TP53 mutations (n=5) were associated with worse EFS and OS in comparison to TP53 wild-type cases (n=45) (EFS after 2 years: 8.4% vs 0%, P= 0.006; OS after 2 years: 28.5% vs 0%, P=0.045). Of note, no correlations between mutation frequency and age were observed. We next focused on whether the number of mutations showed any impact on survival. This analysis revealed that cases with more than one mutation (n=20) showed shorter EFS (EFS after 2 years: 10.0% vs 27.3%, P=0.020). Finally, we concentrated on AML with t(9;11)(p22;q23)/MLLT3-MLL, recognized as a distinct WHO-entity. We neither detected an association of MLLT3-MLL (n=46) with OS (P=0.445) or EFS (P=0.644) in comparison to the remaining translocation partners nor a distinct gene mutation profile. However, NRAS mutations correlated with shorter OS and EFS in cases with MLLT3-MLL (after 2 years OS: 17.8% vs 48.3%, P=0.045; after 2 years EFS: 17.8% vs 35.2%, P=0.056). Conclusions: In patients with MLL-translocations a high number of secondary alterations (53.6%), predominantly in RAS pathway components (38.2%), were detected. This may have implication on novel therapeutic options in this unfavorable AML subset. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Fasan: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.

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Modulation of the Clonal Composition in Relapsed CLL: A Study Based on Targeted Deep-Sequencing of ATM, BIRC3, NOTCH1, POT1, SF3B1, SAMHD1 and TP53

Blood ◽

10.1182/blood.v126.23.1721.1721 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1721-1721

Author(s):

Sabine Jeromin ◽

Wolfgang Kern ◽

Richard Schabath ◽

Tamara Alpermann ◽

Niroshan Nadarajah ◽

...

Keyword(s):

Deep Sequencing ◽

Clonal Evolution ◽

Gene Mutations ◽

Wild Type ◽

Mutation Load ◽

Employment Equity ◽

Time Points ◽

Mutational Status ◽

Equity Ownership ◽

Time Point

Abstract Background: Relapse or refractory disease is a challenging clinical problem in the majority of chronic lymphocytic leukemia (CLL) patients. Treatment influences the clonal composition by selection and eventually induction of additional genetic abnormalities. Aim: To characterize the clonal evolution in relapsed CLL patients by deep-sequencing analysis of mutations in ATM, BIRC3, NOTCH1, POT1, SF3B1, SAMHD1 and TP53. Patients and Methods: Sequential samples of 20 relapsed CLL patients at three time-points were evaluated: A: at diagnosis (n=16) or in untreated state (n=4), B: at first relapse (n=20) and C: at second relapse (n=2). Patients were treated with diverse treatment schemes and had temporarily achieved either complete or partial remission during the course of the disease. The median time from diagnosis to first-line treatment was 13 months (1 - 169 months). All geneswere sequenced by a deep sequencing approach (Illumina, San Diego, CA). IGHV mutational status was determined by direct Sanger sequencing at time-point A. Chromosome banding analysis (CBA) and FISH data on del(17p), del(11q), trisomy 12 (+12), and del(13q) were available in 33/42 and 36/42 samples, respectively. Results: Initially, samples at first relapse were sequenced. Mutations in SF3B1 (6/20, 30%), TP53 (5/20, 25%), ATM (5/20, 25%), NOTCH1 (4/20, 20%), and SAMHD1 (3/20, 15%) were detected at high frequencies. No mutations were detected in BIRC3 and POT1. In total, 75% of cases presented with at least one mutation (Figure 1): 8 (40%) cases had one, 6 (30%) cases had two and one patient had three genes concomitantly mutated (mut). Patients were also analyzed for IGHV mutational status at diagnosis and presented with unmutated status at a frequency of 85% (17/20). Subsequently, samples from cases with mutations were analyzed at time-point A. In 12/15 (80%) cases the mutations at relapse were already detectable at time-point A with a similar load indicating presence of the main clone before and after chemotherapy. However, in 7/15 (47%) patients new gene mutations were acquired either additionally to existing mutations (n=4) or in previously wild-type cases (n=3). In 5/7 (71%) cases mutations were located in TP53. TP53 mut were the only mutations that were not detected in samples before treatment (sensitivity of 3%). Thus, TP53 mutations might have been initiated by chemotherapy or exist in a minor subclone subsequently selected by chemotherapy. Furthermore, only 4 cases had low-level mutations (3-6% mutation load) at diagnosis in either SAMHD1 or SF3B1 that eventually increased in their burden during disease course. Of note, in two patients a multibranching clonal evolution could be identified (#2, #9). For patient #2 three time-points were analyzed. At diagnosis 2 ATM mutations were detected with mutation loads of about 20%, each. In the course of the disease these mutations were lost, whereas SF3B1 mut showed a stable mutation load in all three time-points of about 40%. In contrast, mutation load of SAMHD1 increased over time from 4% to 87%. CBA was performed at diagnosis and detected independent clones with del(11q) and del(13q). Accordingly, del(11q) detected by FISH at diagnosis was lost and the percentage of cells with del(13q) increased from diagnosis to time-point C. Therefore, patient #2 shows different genetic subclones in parallel that were eradicated or selected by chemotherapy. In patient #9 two SF3B1 mutations were initially detected with the same mutation load of 10%. After treatment one mutation was lost, whereas the load of the second mutation increased indicating at least two different subclones with only one of them being sensitive to chemotherapy. This might be due to different additional aberrations. Indeed, CBA identified two clones: one with +12 alone and one in combination with del(13q). FISH revealed unchanged percentage of +12 at time-point B, whereas del(13q) positive cells were diminished. Conclusions: In 75% of relapsed CLL cases mutations in SF3B1, TP53, ATM, NOTCH1, and SAMHD1 are present at high frequencies. 80% of these mutations are already detectable before treatment initiation representing the main clone. Remarkably, TP53 mutations were the only mutations that were not detected before but only after chemotherapy. Figure 1. Distribution of gene mutations in 15 CLL cases with mutations at diagnosis or before treatment (D) and at relapse (R). Red = mutated, grey = wild-type, white = not analyzed. Figure 1. Distribution of gene mutations in 15 CLL cases with mutations at diagnosis or before treatment (D) and at relapse (R). Red = mutated, grey = wild-type, white = not analyzed. Disclosures Jeromin: MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schabath:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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The TP53 Codon72 Polymorphism Is Associated with TP53 Mutations in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v118.21.1783.1783 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1783-1783 ◽

Cited By ~ 1

Author(s):

Vera Grossmann ◽

Valentina Artusi ◽

Susanne Schnittger ◽

Frank Dicker ◽

Sabine Jeromin ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Mutation Rate ◽

Tp53 Mutation ◽

Genome Project ◽

Lymphocytic Leukemia ◽

Wild Type ◽

Employment Equity ◽

Tp53 Mutations ◽

Equity Ownership ◽

Exon 4

Abstract Abstract 1783 TP53 is one of the most important cell-cycle regulator genes and its tumor suppressor activity is fundamental in cellular responses. Mutations in TP53 are known to influence clinical outcome in diverse diseases. In particular, a relationship between TP53 mutations and a poor prognosis has been established in chronic lymphocytic leukemia (CLL), which is one of the most commonly diagnosed lymphoid malignancies in Western countries. Thus far, it has been demonstrated that TP53 mutations are associated with codon72 polymorphism in different diseases e.g. breast cancer, lung cancer, head and neck squamous cell carcinoma, and that this variant could determine cancer susceptibility. In this study, we investigated the overall TP53 mutation rate in 511 CLL and focused on the codon72 polymorphism (rs1042522) in exon 4 (transcript-ID: ENST00000269305). We initially examined the published available 1000 Genome Project results of the European cohort: from a total of 283 genomes analyzed, 137 showed an ARG/ARG genotype (48%), 124 an ARG/PRO genotype (43%) and 22 a PRO/PRO genotype (7.7%). Secondly, in order to determine a potential association between this polymorphic variant and mutations in the TP53 gene, we investigated 511 thoroughly characterized patients with CLL, all diagnosed by immunophenotyping in our laboratory. For molecular analyses, all cases were analyzed for TP53 mutations (exon 4 to exon 11) either by DHPLC and subsequent Sanger sequencing (n=210/511), or using a sensitive next-generation amplicon deep-sequencing assay (n=301/511) (454 Life Sciences, Branford, CT). We observed the occurrence of the three distinct genotypes (ARG/ARG, ARG/PRO, PRO/PRO) of codon72 in the CLL cohort and detected ARG/ARG as the most common genotype (63%), followed by ARG/PRO (31.7%), and PRO/PRO (5.3%); very similar to the distribution of the codon72 polymorphism in the 1000 Genome Project data. Moreover, mutations in TP53 were detected in 63/511 patients resulting in an overall mutation rate of 12%, which reflects the expected mutation rate in this disease. Importantly, as already demonstrated in other malignancies, we here present that also in CLL patients harboring a PRO/PRO genotype a significantly higher frequency of TP53 mutations (9/27, 33%) was observed compared to ARG/ARG (41/321, 13%, P=.037) and ARG/PRO (13/163, 8%, P=.012). With respect to the clinical outcome we confirmed a generally poor survival for the TP53 mutated cases as compared to TP53 wild-type patients (n=23 vs. 189 with clinical data available, alive at 7 years: 29.6% vs. 88.1%; P<.001). Moreover, the impact of the three distinct genotypes on outcome was analyzed. However, no correlation was detectable, neither in the cohort of TP53 mutated cases (P=.225) nor in the TP53 wild-type patients (P=.190). In summary, we demonstrated a significant association between the codon72 allelic variant and TP53 mutation rate in our CLL cohort. Patients with a PRO/PRO genotype showed a significantly higher frequency of TP53 mutations than all other genotypes. However, no prognostic impact of codon72 allelic variant was observed, neither in the TP53 wild-type nor in the TP53 mutated cohort. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Artusi:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Boeck:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment.

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Genetic Basis of Myeloid Proliferation Related to Down Syndrome

Blood ◽

10.1182/blood.v120.21.535.535 ◽

2012 ◽

Vol 120 (21) ◽

pp. 535-535

Author(s):

Kenichi Yoshida ◽

Tsutomu Toki ◽

Myoung-ja Park ◽

Yusuke Okuno ◽

Yuichi Shiraishi ◽

...

Keyword(s):

Down Syndrome ◽

Exome Sequencing ◽

High Throughput Sequencing ◽

Somatic Mutations ◽

Gene Mutations ◽

Whole Genome ◽

Discovery Cohort ◽

Whole Exome ◽

Recurrent Mutations ◽

Gata1 Mutation

Abstract Abstract 535 Background Transient abnormal myelopoiesis (TAM) represents a self-limited proliferation exclusively affecting perinatal infants with Down syndrome (DS), morphologically and immunologically characterized by immature blasts indistinguishable from acute megakaryoblastic leukemia (AMKL). Although spontaneous regression is as a rule in most cases, about 20–30% of the survived infants develop non-self-limited AMKL (DS-AMKL) 3 to 4 years after the remission. As for the molecular pathogenesis of these DS-related myeloid proliferations, it has been well established that GATA1 mutations are detected in virtually all TAM cases as well as DS-AMKL. However, it is still open to question whether a GATA1 mutation is sufficient for the development of TAM, what is the cellular origin of the subsequent AMKL, whether additional gene mutations are required for the progression to AMKL, and if so, what are their gene targets, although several genes have been reported to be mutated in occasional cases with AMKL, including JAK2/3, TP53 and FLT3. Methods To answer these questions, we identify a comprehensive spectrum of gene mutations in TAM/AMKL cases using whole genome sequencing of three trio samples sequentially obtained at initial presentation of TAM, during remission and at the subsequent relapse phase of AMKL. Whole exome sequencing was also performed for TAM (N=16) and AMKL (N=15) samples, using SureSelect (Agilent) enrichment of 50M exomes followed by high-throughput sequencing. The recurrent mutations in the discovery cohort were further screened in an extended cohort of DS-AMKL (N = 35) as well as TAM (N = 26) and other AMKL cases (N = 19) using target deep sequencing. Results TAM samples had significantly fewer numbers of somatic mutations compared to AMKL samples with the mean numbers of all mutations of 30 (1.0/Mb) and 180 (6.0/Mb) per samples in whole genome sequencing or non-silent somatic mutations of 1.73 and 5.71 per sample in whole exome sequencing in TAM and AMKL cases, respectively (p=0.001). Comprehensive detections of the full spectrum of mutations together with subsequent deep sequencing of the individual mutations allowed to reveal more complicated clonological pictures of clonal evolutions leading to AMKL. In every patient, the major AMKL clones did not represent the direct offspring from the dominant TAM clone. Instead, the direct ancestor of the AMKL clones could be back-traced to a more upstream branch-point of the evolution before the major TAM clone had appeared or, as previously reported, to an earlier founder having an independent GATA1 mutation. Intratumoral heterogeneity was evident at the time of diagnosis as the presence of major subpopulations in both TAM and AMKL populations, which were more often than not characterized by RAS pathway mutations. While GATA1 was the only recurrent mutational target in the TAM phase, 8 genes were recurrently mutated in AMKL samples in whole genome/exome sequencing, including NRAS, TP53 and other novel gene targets that had not been previously reported to be mutated in other neoplasms. The recurrent mutations found in the discovery cohort, in addition to known mutational targets in myeloid malignancies, were screened in an extended cohort of DS-associated myeloid disorders (N=61) as well as other AMKL cases, using high-throughput sequencing of SureSelect-captured and/or PCR amplified targets. Secondary mutations other than GATA1 mutations were found in 3 out of 26 TAM, 20 out of 35 DS-AMKL and 4 out of 19 other AMKL cases. Conclusion TAM is characterized by a paucity of somatic mutations and thought to be virtually caused by a GATA1 mutation in combination with constitutive trisomy 21. Subsequent AMKL evolved from a minor independent subclone acquiring additional mutations. Secondary genetic hits other than GATA1 mutations were common, where deregulated epigenetic controls as well as abnormal signaling pathway mutations play a major role. Disclosures: No relevant conflicts of interest to declare.

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Diverse and Targetable Kinase Alterations Drive Histiocytic Neoplasms

Blood ◽

10.1182/blood.v126.23.481.481 ◽

2015 ◽

Vol 126 (23) ◽

pp. 481-481

Author(s):

Benjamin Heath Durham ◽

Eli L. Diamond ◽

Julien Haroche ◽

Zhan Yao ◽

Jing Ma ◽

...

Keyword(s):

Mapk Pathway ◽

Single Agent ◽

Braf V600e ◽

Rna Seq ◽

Wild Type ◽

Employment Equity ◽

Mek Inhibitors ◽

Whole Exome ◽

Recurrent Mutations ◽

Equity Ownership

Abstract Histiocytic neoplasms are clonal, hematopoietic disorders characterized by an accumulation of abnormal monocyte-derived dendritic cells or macrophages in Langerhans Cell (LCH) and non-Langerhans (non-LCH) histiocytoses, respectively. The discovery of the BRAF V600E mutation in ~50% of patients with LCH and the non-LCH Erdheim-Chester Disease (ECD) provided the first molecular target in these patients and novel insights into the pathogenesis of these disorders. However, recurrent mutations in the majority of the ~50% of BRAF V600E-wild type patients with non-LCH are unknown. Moreover, recurrent mutations outside of the MAP kinase pathway are undefined throughout histiocytic neoplasms. To address these issues, we performed whole exome sequencing (WES) of frozen biopsies from 24 patients with LCH (n=10) or ECD (n=14) paired with peripheral blood mononuclear cells. 13/24 patients also underwent RNA sequencing (RNA-seq). All mutations in activating kinases were validated by droplet-digital PCR, while targeted-capture next-generation sequencing validated all others. Both adult (n=18; n=2 with LCH) and pediatric cases (n=9; n=8 with LCH) were included. Using combined WES/RNA-seq, activating kinase alterations were identified in 100% of patients. In LCH, 60% and 40% had BRAF V600E and MAP2K1 mutations, respectively. In non-LCH 51%, 14%, 14%, and 7% were BRAFV600E, ARAF, MAP2K1, and NRAS mutant (Fig1A). Overall, a mean of 7 non-synonymous mutations per adult patient was identified (range 1-22) compared with 5 mutations per pediatric patient (range 4-9; p =ns). Mutations affecting diverse cellular processes were found to co-exist with kinase mutations including mutations in epigenetic modifiers and the p38/MAPK pathway. In addition to kinase point mutations, RNA-seq identified recurrent, in-frame kinase fusions-a first for these disorders. All identified fusions were validated using FISH and RT-PCR. This includes novel fusions in BRAF (RNF11-BRAF and CLIP2-BRAF), as well as therapeutically important fusions in ALK (2 separate KIF5B-ALK fusions) and NTRK1 (LMNA-NTRK1;Fig1B). Expression of each fusion in Ba/F3 cells conferred cytokine-independent growth. Importantly, the BRAF fusions were found to be sensitive to MEK inhibition but resistant to vemurafenib while the ALK fusions conferred sensitivity to the ALK inhibitors crizotinib or alectinib. We next interrogated a validation cohort of 37 BRAF V600E-wild type, non-LCH, formalin-fixed, paraffin-embedded tissue samples using targeted mutational profiling for MAP2K1, ARAF, NRAS, KRAS, and PIK3CA. This revealed activating mutations in MAP2K1 (32%; n=12), NRAS (16%; n=6), KRAS (11%; n=4), PIK3CA (8%; n=3), and ARAF (3%; n=1). Three of the investigated non-LCH patients with refractory disease and progressive organ dysfunction were treated with targeted therapies based on the discovery of novel kinase alterations described above. Treatment of 2 refractory MAP2K1- mutant, non-LCH patients with MEK inhibitors (trametinib or cobimetinib) resulted in dramatic clinical improvement (Fig1C). Both patients have been maintained on MEK inhibitor single-agent therapy with a sustained clinical response for >100 days. Further evidence of effective targeted inhibition was found in a refractory ECD patient carrying an ARAF S214A mutation. This patient failed to respond to 3 lines of prior therapies and suffered near blindness due to disease infiltration in the retina and optic nerves. Given a recent report of complete response to sorafenib in a lung cancer patient with an ARAF S214C mutation, we initiated sorafenib. Within 12 weeks, there was improvement in the patientÕs eyesight and decreased infiltrative disease, coinciding with >50% decrease in mutant ARAF DNA in plasma cell-free DNA. Whole exome and transcriptome sequencing identified activating kinase mutations or translocations in all patients with the common downstream effect of activating the MAPK pathway. The preliminary, dramatic, clinical efficacy observed with use of MEK and RAF inhibitors in MAP2K1 - and ARAF-mutated, non-LCH patients further supports the central role of targeting the MAPK pathway in these tumors. The discovery of the discussed mutations and fusions in diverse kinases provides critical new insights into the genetic events central to a spectrum of adult and pediatric histiocytic neoplasms. Figure 1. Figure 1. Disclosures Off Label Use: This abstract describes use of MEK inhibitors (both tremetinib and cobimetinib) as well as sorafenib for MEK1 and ARAF mutant histiocytosis. . Stephens:Foundation Medicine, Inc.: Employment, Equity Ownership. Miller:Foundation Medicine, Inc.: Employment, Equity Ownership. Ross:Foundation Medicine Inc.: Employment. Ali:Foundation Medicine Inc.: Employment. Hyman:Chugai Pharma: Consultancy; Biotherapeutics: Consultancy; Atara: Consultancy, Honoraria.

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FLNC and MYLK2 gene mutations in a Chinese family with different phenotypes of cardiomyopathy

10.21203/rs.3.rs-26973/v1 ◽

2020 ◽

Author(s):

Xianyu Qin ◽

Ping Li ◽

Hui-Qi Qu ◽

Yichuan Liu ◽

Yu Xia ◽

...

Keyword(s):

Family Member ◽

Novel Mutation ◽

Gene Mutations ◽

Chinese Family ◽

Wild Type ◽

Human Cardiomyocytes ◽

Sarcomeric Protein ◽

Truncating Mutation ◽

Whole Exome ◽

Type Gene

Abstract Background Mutations in the sarcomeric protein filamin C (FLNC) gene have been linked to hypertrophic cardiomyopathy (HCM), in which they increase the risk of ventricular arrhythmia and sudden death. In this study, we identified a novel missense mutation of FLNC in a Chinese family with HCM and interestingly a second novel truncating mutation of MYLK2 in one family member with different phenotype. Methods We performed whole-exome sequencing in a Chinese family with HCM of unknown cause. To validate the function of a novel mutation of FLNC, we introduced the mutant and wild-type gene into AC16 cells (human cardiomyocytes), and used western blotting to analyze the expression of FLNC in subcellular fractions, and confocal microscope to observe the subcellular distribution of the protein. Results We identified a novel missense single nucleotide variant (FLNC c.G5935A [p.A1979T]) in the family, which segregates with the disease. FLNC expression levels were equivalent in both wild type and p.A1979T cardiomyocytes. However, expression of the mutant protein resulted in cytoplasmic protein aggregations, in contrast to wild type FLNC, which was distributed in the cytoplasm and did not form aggregates. Unexpectely, a second truncating mutation, NM_033118:exon8:c.G1138T:p.E380X of the MYLK2 gene, was identified in the mother of the proband with dilated cardiomyopathy, but absent in other subjects. Conclusion We identified the FLNC A1979T mutation as a novel pathogenic variant associated with HCM in a Chinese family, as well as a second causal mutation in a family member with a distinct phenotype. The possibility of more than one causal mutations in cardiomyopathy warrants clinical attention, especially for patients with atypical clinical features.

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FLNC and MYLK2 gene mutations in a Chinese family with different phenotypes of cardiomyopathy

10.1101/2020.05.10.20097519 ◽

2020 ◽

Author(s):

Xianyu Qin ◽

Ping Li ◽

Huiqi Qu ◽

Yichuan Liu ◽

Yu Xia ◽

...

Keyword(s):

Family Member ◽

Novel Mutation ◽

Gene Mutations ◽

Chinese Family ◽

Wild Type ◽

Human Cardiomyocytes ◽

Sarcomeric Protein ◽

Truncating Mutation ◽

Whole Exome ◽

Type Gene

Background: Mutations in the sarcomeric protein filamin C (FLNC) gene have been linked to hypertrophic cardiomyopathy (HCM), in which they increase the risk of ventricular arrhythmia and sudden death. In this study, we identified a novel missense mutation of FLNC in a Chinese family with HCM and interestingly a second novel truncating mutation of MYLK2 in one family member with different phenotype. Methods: We performed whole-exome sequencing in a Chinese family with HCM of unknown cause. To validate the function of a novel mutation of FLNC, we introduced the mutant and wild-type gene into AC16 cells (human cardiomyocytes), and used western blotting to analyze the expression of FLNC in subcellular fractions, and confocal microscope to observe the subcellular distribution of the protein. Results: We identified a novel missense single nucleotide variant (FLNC c.G5935A [p.A1979T]) in the family, which segregates with the disease. FLNC expression levels were equivalent in both wild type and p.A1979T cardiomyocytes. However, expression of the mutant protein resulted in cytoplasmic protein aggregations, in contrast to wild type FLNC, which was distributed in the cytoplasm and did not form aggregates. Unexpectely, a second truncating mutation, NM_033118:exon8:c.G1138T:p.E380X of the MYLK2 gene, was identified in the mother of the proband with dilated cardiomyopathy, but absent in other subjects. Conclusion: We identified the FLNC A1979T mutation as a novel pathogenic variant associated with HCM in a Chinese family, as well as a second causal mutation in a family member with a distinct phenotype. The possibility of more than one causal mutations in cardiomyopathy warrants clinical attention, especially for patients with atypical clinical features.

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A Quantitative Analysis of Subclonal and Clonal Gene Mutations Occurring Pre- and Post-Therapy in 53 Cases of Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v126.23.2909.2909 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2909-2909

Author(s):

Nisar A. Amin ◽

Erlene Kuizon Seymour ◽

Peter Ulintz ◽

Kamlai Saiya-Cork ◽

Brian Parkin ◽

...

Keyword(s):

Sequence Data ◽

Gene Mutations ◽

Post Treatment ◽

Lymphocytic Leukemia ◽

Driver Mutations ◽

Exome Capture ◽

Tp53 Mutations ◽

Equity Ownership ◽

Pre Treatment ◽

Notch1 Mutations

Abstract Introduction: The landscape of gene mutations in CLL prior to therapy is well-characterized. Comparatively less is known about gene mutations and their frequency in CLL patients that have relapsed after potent chemo-immunotherapy. Further, despite knowledge of subclonal TP53 mutations that enrich and likely drive CLL relapse in a fraction of cases, a comprehensive profile of gene mutations and their variant allele frequencies (VAFs) and clonal dynamics before and after chemo-immunotherapy in CLL is lacking. Methods: We have procured paired pre-treatment and post-treatment samples from 53 CLL cases that had relapsed after chemo-immunotherapy and purified CLL CD19+ cells and CD3+ T-cells to purity with FACS. DNA from relapsed CLL was subjected to exome capture and whole exome sequencing (WES) at a mean coverage of 72-fold (range 52-102) and sequence data analyzed using three variant callers: MuTect v.1.1.4, Strelka v.1.0.13, and VarScan2 v.2.3.7. Somatically acquired gene mutations occurring in 2 or more rCLL cases were confirmed by Sanger sequencing in relapsed CLL samples and also re-sequenced in pre-treatment samples. Genes with mutation frequencies ≥5% in rCLL underwent custom gene panel-based deep coverage re-sequencing in paired pre-treatment and post-treatment samples. Analysis of deep re-sequencing data was done using the Broad GATK HaplotypeCaller v3.3.0 in parallel with VarScan2. Selected low-level variants were measured using droplet digital PCR (ddPCR) that was adapted to detection of VAFs as low as 1/10,000. Results: In CLL relapsed from potent chemo-immunotherapy, we detected mutated TP53, NOTCH1, SF3B1, XPO1, BIRC3, MYD88, NXF1, POT1, CACNA1E, CHD2, EGR2, FAM50A, FAT3, FBXW7, MGA, SAMHD1 and ZMYM3 with frequencies ≥5%. An additional 64 genes were mutated in 2/53 rCLL cases each. We performed ultra-deep panel-based re-sequencing of the 17 genes with frequencies ≥5% in 53 paired diagnosis and relapse samples, complementing selected variants with ddPCR validation to determine VAFs. TP53 mutations constituted the most frequently enriched gene at relapse (7/53=13%) and the VAFs of all TP53 mutations substantially increased at relapse often from very minor subclones at diagnosis. Importantly, none of the clonal TP53 mutations in rCLL appeared directly induced by chemotherapy, but instead all were selected from pre-existing subclones. Similarly, subclonal mutations in SAMHD1 substantially enriched in four cases at relapse (4/53=8%) suggesting a role in resistance to chemotherapy. The majority of NOTCH1 mutations (8/13) were already fully clonal at diagnosis without further enrichment at relapse. Three (3/13) subclonal NOTCH1 mutations substantially enriched at relapse, while two (2/13) clonal NOTCH1 mutations substantially decreased. The VAFs for SF3B1 mutations similarly demonstrated three patterns: i) clonal that remained clonal (4/10), ii) clonal that substantial declined and became subclonal at relapse (4/10), and, iii) subclonal that enriched but remained subclonal (2/10) at relapse. Of the 13 remaining genes, most demonstrated no consistent enrichment or depletion or remained subclonal at relapse. Of biological interest, the genes FBXW7, MYD88, NOTCH1, NXF1, ZMYM3, XPO1, SF3B1 and POT1, were often already fully clonal in the pre-treatment samples, suggesting an early role in CLL pathogenesis rather than a later role in the development of CLL relapse. Conclusion: In this large WES study focused on gene mutations in relapsed CLL paired with analysis of subclone dynamics using deep panel re-sequencing and ddPCR, we identify the genes TP53 and likely SAMHD1 as drivers of CLL relapse in 20% of cases. Multiple other genes previously implicated as CLL drivers did not consistently enrich at relapse. Further, a subset of the mutated genes was often already fully clonal pre-treatment; these genes likely serve an important role early in CLL pathogenesis that is independent of therapy. The majority of relapsed CLL in this cohort were not associated with the recurrent clonal emergence of known CLL driver mutations and based on the gene mutations frequencies reported here, much larger rCLL cohorts would need analysis to confirm possible additional low frequency gene drivers of rCLL. Disclosures Malek: Gilead Sciences: Equity Ownership; Abbvie: Equity Ownership; Janssen Pharmaceuticals: Research Funding.

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