Spectrum Of Genetic Alterations In Acquired Aplastic Anemia

Abstract Acquired aplastic anemia (AA) is a prototype of idiopathic bone marrow failure, which is caused by immune-mediated destruction of hematopoietic progenitors. However, its natural course could be more complicated than expected for a simple immune-mediated disorder, as in the development of apparently acquired (somatic) clonal disorders such as paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML), during its course. Although these evidences suggest a pathogeneic link between these disorders, the clonal architecture in AA has not been fully explored. In order to genetically define the origin of clonal hematopoiesis in patients with acquired AA, we sought gene mutations, by targeted deep sequencing of peripheral blood DNA from 192 Japanese patients with AA for mutations, using a panel of 51 genes including common mutational targets in myeloid malignancies using a SureSelect custom kit. An extended cohort of 293 American AA patients was further analyzed for targeted sequencing of granulocyte-derived DNA; for these cases, multiple sequential specimens with germline controls and complete clinical follow-up data were available. Exome sequencing was also performed for selected cases. In the Japanese cohort, about 40% were severe or very severe diseases with an excellent response to immunosuppressive therapies (IST). In total, 43 somatic mutations were detected in 18% of the cases with the mean allelic burden of 18%. Mutations were most frequent in DNMT3A (3.6%), followed by ZRSR2 (3.1%), ASXL1 (2.6%), BCOR (2.0%) and more biased to nonsense (25.6%), frameshift (14.0%), splice site changes (7.0%) and non-frameshift indel (11.6%), indicating driver roles of these mutations in many cases. Mutations were associated with older age (p=0.014) and a better response to IST (p=0.040). We next examined an extended cohort of 293 AA cases from the United States, for which samples were collected at 6 months after treatment in the patients of severe or very severe disease. Except for 12 cases, CD3(+) cells were available and used to confirm the somatic origin of mutations by comparison with CD3 cells representing germline sequence. All patients had received IST, with overall response about 65%. As of the date of submission, data analysis was completed for 86 of the 293 cases, in whom we confirmed somatic mutations detected in BM samples from 45 cases (53%) 6 months after IST, with the mean number of mutations and the mean allelic burden were 1.08 and 15.3%, respectively. Similar to the finding in the Japanese cohort, BCOR (13.8%), DNMT3A (11.5%), and ASXL1 (10.3%) were most frequently mutated. PIGA (6.9%) and CSMD1 (4.6%) were also mutated in 6.9% and 4.6%, respectively. Again, mutations were associated with older age. Although there was no significant difference in response to IST (p=0.133) between mutation (+) and (-) cases, responders showed significantly higher numbers of mutations compared with non-responders (p=0.033). Evolution to MDS/AML occurred in 12 out of the 45 cases with mutations, while 13 out of the 62 cases without mutations developed MDS/AML. Therefore, candidate genes associated with some but not most evolution events. We further performed whole exome sequencing in 6 cases, for whom sequential samples were available: in 5 of 6 cases, somatic mutations were detected and the mean number of mutations was 9. There was evidence over time of clonal selection with or without progression to MDS or AML. Small clones of cells containing RUNX1 and U2AF1-mutated clones present in the initial specimen showed expansion in size with progression to MDS (0.003 to 0.46 and 0.013 to 0.097, respectively). In conclusion, mutations in common target genes in myeloid malignancies can drive clonal evolution during the course of AA. However, overall there was no correlation between the presence of mutations and clinical evolution to MDS/AML, as many patients with evidence of clones containing mutations remained stable. Clonal expansion and the appearance and disappearance of clones occurred in some cases without clinical changes. The marrow failure environment may favor selection of mutant clones. In addition, other genetic/epigenetic alterations, including chromosomal instability induced by telomere shortening (accompanying abstract by Dumitriu and Feng) provide a mechanism of oncogenesis. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:Aplastic anemia&MDS International Foundation: Research Funding; NIH: Research Funding.

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Somatic Mutations in Schinzel-Giedion Syndrome Gene SETBP1 Determine Progression in Myeloid Malignancies

Blood ◽

10.1182/blood.v120.21.2.2 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2-2 ◽

Cited By ~ 1

Author(s):

Hideki Makishima ◽

Kenichi Yoshida ◽

Nhu Nguyen ◽

Masashi Sanada ◽

Yusuke Okuno ◽

...

Keyword(s):

Research Funding ◽

Somatic Mutations ◽

Bone Marrow Cells ◽

Germ Line ◽

Cancer Susceptibility ◽

Gain Of Function ◽

Myeloid Malignancies ◽

Runx1 Expression ◽

Myeloid Progenitors

Abstract Abstract 2 MDS and other chronic myeloid malignancies such as MDS/MPN are characterized by a frequent progression to secondary AML (sAML), a likely multistep process of acquisition of genetic abnormalities. Genes involved in congenital genetic cancer susceptibility syndromes are often targets of somatic mutations in various tumors. For instance, germ-line mutations of SETBP1 are associated with Schinzel-Giedion syndrome (SGS), which is characterized by skeletal malformations, mental retardation and frequent neuroepithelial tumors. While SETBP1 overexpression in myeloid malignancies links to poor prognosis, somatic mutations of SETBP1 were not previously identified in leukemias. When we performed whole exome sequencing of 20 cases with myeloid malignancies, in addition to detecting previously described lesions, such as TET2, CBL and ASXL1, we identified a somatic SETBP1 mutation (D868N) in 2 cases with RAEB. Analysis of DNA from CD3+ cells from these patients confirmed its somatic nature. Sanger sequencing was applied to all coding exons in an additional 48 cases, leading to detection of 2 additional somatic mutations (G870S and I871T) in 2 patients with CMML and sAML, respectively. These findings prompted us to further expand our screening cohort: targeted SETBP1 sequencing was performed in a total of 734 patients (283 with MDS, 106 with sAML, 167 with MDS/MPN, 138 MPN and 146 with primary AML): 52 mutations were detected in 52 patients (7.1%); D868N, G870S and I871T alterations were more frequently observed (N=27, N=16 and N=5, respectively), while D868Y, S869N, D880E and D880N were less prevalent. These mutations, of which 92% (48 out of 52) were identical to those in the SGS germ line, were detected in 15% with CMML (24/156), 15% with sAML (16/106) and 7% CML blast phase (2/28). Clinically, mutant cases were associated with higher age (p=.014), deletion of chromosome 7q (p=.0005) and shorter median survival (28 vs. 13 months, p<.0001). As shown in the analysis of 11 paired samples of progressing MDS patients, all SETBP1 mutations were acquired during leukemic evolution. In addition to mutations, SETBP1 overexpression can be found in 12% and 26% of cases of MDS and sAML, respectively, a finding linking higher activity of SETBP1 to leukemic progression. To directly test whether SETBP1 mutations represent gain-of-function, we performed retroviral transduction of murine Setbp1 engineered with two of the somatic mutations, D868N and I871T, and evaluated the ability of the mutants to immortalize normal murine myeloid progenitors. With a low viral titer of 1 x105 cfu, both Setbp1 mutants caused efficient immortalization of myeloid progenitors, similar to overexpressed WT Setbp1. In addition, cells immortalized with mutant Setbp1 proliferated faster than cells with WT Setbp1. These data suggest that mutations of SETBP1 in our study represent gain-of-function in leukemias. The in vitro immortalization effect of overexpressed WT Setbp1 was associated with and dependent on Hoxa9 and Hoxa10 overexpression. We performed quantitative RT-PCR and western blot experiments to evaluate expression of these genes in our mutant cases. Relative HOXA9 and HOXA10 mRNA expression values were higher in all mutant cases (N=7) than median of those in WT cases (N=4). Also, both HOXA9 and HOXA10 proteins were detected in all cases with SETBP1 mutations, suggesting that HOXA9 and HOXA10 induction is consistently associated with SETBP1 mutations similar to observations in forced expression of WT Setbp1. Moreover, in agreement with findings in primary cells showing that SETBP1 mutations or high SETBP1 expression share a common genetic association with RUNX1 mutations, Runx1 expression was reduced after in vitro immortalization of normal bone marrow cells by forced Setbp1 overexpression and two Runx1 promoter sequences were amplified after ChIP performed with antibody specific for exogenous Setbp1 protein. Moreover, Setbp1 shRNA knockdown resulted in enhanced Runx1 transcription consistent with the negative regulation of this gene by Setbp1. These results indicate that SETBP1 is associated with decreased activity of RUNX1 due to hypomorphic mutations or by direct down-modulation WT RUNX1 expression bypassing the need for mutations. In sum, somatic recurrent SETBP1 mutations are lead to gain of function and are associated with molecular pathogenesis of myeloid leukemic transformation of various primary myeloid subentities. Disclosures: Makishima: Scott Hamilton CARES Initiative: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

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Whole Exome Sequencing to Predict Response to Hypomethylating Agents in MDS

Blood ◽

10.1182/blood.v120.21.1698.1698 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1698-1698 ◽

Cited By ~ 1

Author(s):

Holleh D Husseinzadeh ◽

Edward P Evans ◽

Kenichi Yoshida ◽

Hideki Makishima ◽

Andres Jerez ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Research Funding ◽

Somatic Mutations ◽

Prolonged Exposure ◽

Mutational Analysis ◽

Outcome Analysis ◽

Individual Treatment ◽

Hypomethylating Agents ◽

Whole Exome

Abstract Abstract 1698 Hypomethylating agents decitabine and azacitidine are standard treatments for myelodysplastic syndromes (MDS). In their use, one hopes to rectify cytopenias and prolong survival by retarding further disease progression. However, individual treatment responses vary from complete remission (CR) to complete refractoriness. In general, at least 4 cycles of therapy are administered prior to assessing response. Thus, patients may have prolonged exposure to ineffective therapy, suffering toxicities without clinical benefit, while alternative and potentially more effective treatments are delayed. Currently, there are no reliable phenotypic or mutational markers for predicting response to hypomethylating agents. Once whole exome sequencing (WES) became available for more routine analysis, we theorized that somatic mutational patterns may help identify patients who would most benefit from these drugs, thereby maximizing response rate by rational patient selection. To pursue this hypothesis, we screened a cohort of 168 patients with MDS who received either azacitidine or decitabine for the presence of somatic mutations. Only those who received sufficient therapy, i.e., completed at least 4 cycles, were selected for outcome analysis. Targeted Sanger sequencing, including a panel of up to 19 genes frequently affected by somatic mutations was performed. For a representative subset of 26 patients (this subset is expanding) of whom there were 15 responders and 11 non-responders, mutational analysis was performed by WES to select target genes for further analysis. WES utilizes paired DNA (tumor vs. CD3+ lymphocytes) to produce raw sequence reads aligned using Burrows-Wheeler Aligner (BWA). Variants are detected using the Broad Institute's Best Practice Variant Detection GATK toolkit. Median age was 68 years (range, 55–85), 50% were female, and MDS subtypes were as follows: RA/RCUD/RARS 13%, RCMD 16%, RAEB-1/2 20%, MDS/MPN & CMML-1/2 31%, and sAML 20%. Response was assessed using IWG 2006 criteria at 4 and 7 months after therapy initiation. Overall response was 48%; rate of CR (including marrow/cytogenetic CR) was 28%, any HI 20%, SD 22%, and no response 29%. The cohort was then dichotomized into “responders” and “non-responders,” with responders classified as those achieving CR or any HI. Baseline patient characteristics were similar between both groups, including average age at treatment initiation, disease subtypes, proportion of abnormal/complex karyotypes, and presence of common cytogenetic aberrations. Overall, the most frequently mutated genes include TET2/IDH1/IDH2, SRSF2, ASXL1, SF3B1, RUNX1, EZH2/EED/SUZ12, SETBP1, CBL, and PPIAF2. The highest rate of refractoriness was noted in mutants of TET2/IDH1/IDH2 (67%), SF3B1 (67%), U2AF1/2 (67%). We also identified several genes whose mutants were few but associated exclusively with refractory disease (100%), including KIT, ZRSR2, PRPF8, LUC7L2. We next applied a recursive partitioning algorithm to construct a decision tree for identifying the most pivotal mutations associated with response: we found mutant CBL and PPFIA2 to be strongly associated with response, whereas mutant U2AF1/2, SF3B1 and PRPF8 were strongly associated with refractoriness. Our final approach was to dichotomize the cohort by the presence/absence of each mutation/group of mutations, and response within mutant vs. wild type cases was compared. Among refractory cases, TET2/IDH1/IDH2 (26%) and SF3B1 (17%) were most frequently mutated; among responders, mutations in RUNX1 (19% vs. 4%]), CBL (14% vs. 0%), SRSF2 (23% vs. 9%), and SETBP1 (18% vs. 4%) were most frequent. When multiple genes were combined in “either-or” fashion, mutation in TET2, SF3B1, PRPF8, or LUCL71 was significantly associated with refractoriness (52%, p=.0287), whereas mutations of RUNX1, CBL, SRSF2, SETBP1, or PPFIA2 mutation was significantly associated with response (86%, p=.0001). Mutational patterns appear to predict response to standard hypomethylating agents. Identification of the most predictive genes could guide development of molecular maker-based selection of patients for hypomethylating agent therapy, but will require ongoing analysis and additional prospective testing for validation. Disclosures: Advani: Genzyme: Honoraria, Research Funding; Immunomedics: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

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Strengthening the Evidence of Relevant Somatic Mutations of Individual Patients By Pairing Sequencing Deduced Allelic Burden, Leukemic Blast Percent and Cluster Analysis

Blood ◽

10.1182/blood.v126.23.2589.2589 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2589-2589

Author(s):

Marcus Celik Hansen ◽

Sadudee Chotirat ◽

Chirayu Udomsakdi Auewarakul ◽

Peter Hokland

Keyword(s):

Cluster Analysis ◽

Exome Sequencing ◽

Somatic Mutations ◽

Signal To Noise Ratio ◽

Tumor Burden ◽

Clinical Settings ◽

Leukemic Blast ◽

Signal To Noise ◽

Cluster 2 ◽

Allelic Burden

Abstract Introduction Exome sequencing can detect somatic mutations at an unprecedented scale. However, high false-positive rates due to multiple technical contributors affecting signal-to-noise ratio is an unsolved inherent problem. Furthermore, the current literature does not consistently assess or report the allelic burden of detected and scrutinized mutations, potentially leading to focus on irrelevant mutations. We hypothesized that this issue can be addressed by a structured usage of allele frequencies in combination with detected mutations. Employing improved techniques to assess the molecular signature shortly after diagnosis could also result in improved risk stratification. Here, we present proof-of-principle that identification of relevant mutations, and extent of clonality, can be accomplished by pairing hypothetical allelic burden deduced from sequencing reads to leukemic burden in individual samples. Methods 7 samples were used for whole exome sequencing derived from bone marrow aspirated from two different patients. Of these, 4 were leukemia samples (diagnostic and two relapse samples of one T-cell/myeloid mixed-phenotype leukemia and a diagnostic from AML, M0), reflecting different clinical situations, and 3 samples served as controls (keratinocytes, and fibroblasts in the first, remission in the second) to include different clinical situations. Raw data processing, mutations analysis and downstream analysis of sequencing data was performed as described earlier (Hansen & al., MethodsX 2015, Hansen & al., Br J Haematol. 2015), following GATK Best Practice workflow and MuTect default parameters. Cluster analysis was utilized to mathematically divide somatic SNVs into read frequency clusters on the basis of squared Euclidean distance, thus enabling the retrieval of mean allelic burden and expected allele frequencies juxtaposed with flow cytometry-derived leukemic blast percent. The initial condition of the classification model was default set to accommodate separation of background (cluster 1: noise or low read), heterozygous mutations (cluster 2), and an outlier bin for possible homozygous mutations (cluster 3). Results and discussion In order to reference the leukemic burden of a given sample we employed data from immunophenotyping as a surrogate marker for malignancy. Without filtering of detected mutations by exome sequencing the partly stochastic nature of the signal is evident (fig. 1A). An exponential-like continuum of non-rejected mutations is found when these are sorted according to frequencies (black points), making it difficult to deduce any clonal nature of the malignancy, and to evaluate the validity of possibly relevant mutations found. Using a minimum depth of coverage threshold of 30 the signal-to-noise ratio is increased (fig. 1B), and, subsequently, two clones were resolved at diagnosis (fig. 1C) by cluster analysis. This resolution decreases at lower tumor burden showing expected sensitivity towards lower allelic reads (fig. 1D), although driver mutations of the persistent clone could be detected. At second relapse only one distinct sAML phenotyoe clone and, now homozygous, CDKN2 AR80* could be detected (fig. 1E) with the mutations of secondary clone present at diagnosis (cluster 2) lost due to selective pressure from treatment, i.e. FLT3D835Y. The observations are backed by another diagnostic sample from a patient with AML with NRAS and BCOR mutations (fig. 1F), also with a distinct clone. Here, too much emphasis could easily be given to NRASG12D if the frequency had not been assessed. Conclusion We have addressed the pertinent question regarding false positive observations arising from deep sequencing or emphasizing mutations found in the low allele frequency fractions. From this dataset we have, despite the low number of samples, accomplished to suggest a formalized approach for single sample mutational analysis. As a consequence, we can now show that malignant clones with high tumor burden can be resolved semi-spatially by sequencing, generally applicable to a wide range of clinical settings. We conclude that this approach is amenable in single patient situations. While further studies are needed to ultimately test the applicability of this approach in the clinical settings, the perspectives to this observation become evident as sequencing depth and cost continue to develop in an inversely correlated manner. Disclosures No relevant conflicts of interest to declare.

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Exome Sequencing of Aggressive Natural Killer Cell Leukemia and Drug Profiling Highlight Candidate Driver Pathways in Malignant Natural Killer Cells

Blood ◽

10.1182/blood.v126.23.700.700 ◽

2015 ◽

Vol 126 (23) ◽

pp. 700-700

Author(s):

Olli Dufva ◽

Tiina Kelkka ◽

Shady Awad ◽

Nodoka Sekiguchi ◽

Heikki Kuusanmäki ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Exome Sequencing ◽

Cell Lines ◽

Hierarchical Clustering ◽

Research Funding ◽

Drug Sensitivity ◽

Somatic Mutations ◽

Nk Cell ◽

Negative Regulator

Abstract Background Natural killer (NK) cell malignancies are rare lymphoid neoplasms characterized by aggressive clinical behavior and poor treatment outcomes. Clinically they are classified as extranodal NK/T-cell lymphoma, nasal type (NKTCL) and aggressive NK cell leukemia (ANKL). Both subtypes are almost invariably associated with Epstein-Barr virus (EBV). Recently, genomic studies in NKTCL have identified recurrent somatic mutations in JAK-STAT pathway molecules STAT3 and STAT5b as well as in the RNA helicase gene DDX3X in addition to previously detected chromosomal aberrations. Here, we identified somatic mutations in 4 cases of ANKL in order to understand whether these entities share common alterations at the molecular level. To further establish common patterns of deregulated oncogenic signaling pathways operating in malignant NK cells, we performed drug sensitivity profiling using NK cell lines representing ANKL, NKTCL and other malignant NK cell proliferations. We aimed to identify sensitivities to agents that selectively target components of pathways required for survival of malignant NK cells in an unbiased manner. Methods Exome sequencing was performed on peripheral blood or bone marrow of ANKL patients using the NK cell negative fraction or other healthy tissue as control. Profiling of drug responses was performed with a high-throughput drug sensitivity and resistance testing (DSRT) platform comprising 461 approved and investigational oncology drugs. The NK cell lines KAI3, KHYG-1, NKL, NK-YS, NK-92, SNK-6 and YT and IL-2-stimulated and resting NK cells from healthy donors were used as sample material. All drugs were tested on a 384-well format in 5 different concentrations over a 10,000-fold concentration range for 72 h and cell viability was measured. A Drug Sensitivity Score (DSS) was calculated for each drug using normalized dose response curve values. Results The ANKL patients displayed mutations in genes reported as recurrently mutated in NKTCL, such as FAS, TP53, NRAS, STAT3 and DDX3X. Additionally, novel alterations in genes previously implicated in the pathogenesis of NKTCL were detected. These included an inactivating mutation in INPP5D (SHIP), a negative regulator of the PI3K/mTOR pathway and a missense mutation in PTPRK, a negative regulator of STAT3 activation. Interestingly, the total number of nonsilent somatic mutations in 3 out of 4 ANKL patients (97, 82 and 45) was remarkably high compared to other hematological malignancies analyzed in our variant calling pipeline. Analysis of drug sensitivities in NK cell lines showed a close correlation between all cell lines and a markedly higher correlation with those of IL-2 stimulated than resting healthy NK cells, suggesting that malignant NK cells may share a common drug response pattern. Furthermore, in an unsupervised hierarchical clustering the NK cell lines formed a distinct group from other leukemia cell lines tested (Fig. A). Among pathway-selective compounds (namely, kinase inhibitors and rapalogs), the drugs most selective for malignant NK cells fell into two major categories: PI3K/mTOR inhibitors (e.g. temsirolimus, buparlisib) and inhibitors of aurora and polo-like kinases such as rigosertib and GSK-461364 (Fig. B). JAK inhibitors (e.g. ruxolitinib, gandotinib) and CDK inhibitors (e.g. dinaciclib) showed strong efficacy in both malignant NK cells and IL-2 activated healthy NK cells. Conclusions Our exome sequencing results suggest that candidate driver alterations affecting similar signaling pathways underlie the pathogenesis of ANKL as has been reported in NKTCL. Drug sensitivity profiling highlights the PI3K/mTOR pathway as a potential major driver of malignant NK cell proliferation, whereas JAK-STAT signaling appears to be essential in both healthy and malignant NK cells. Components of these pathways harbored mutations in our small cohort of ANKL patients and have been shown to be deregulated by mutations or other mechanisms in previous studies, underlining their importance as putative drivers. The systematic large-scale characterization of drug responses also identified these pathways as potential targets for novel therapy strategies in NK cell malignancies. Figure 1. (A) Unsupervised hierarchical clustering based on drug sensitivity scores (DSS) of NK, AML, CML and T-ALL cell lines. (B) Scatter plot comparing DSS of malignant NK cell lines (average) and healthy IL-2 stimulated NK cells. Figure 1. (A) Unsupervised hierarchical clustering based on drug sensitivity scores (DSS) of NK, AML, CML and T-ALL cell lines. (B) Scatter plot comparing DSS of malignant NK cell lines (average) and healthy IL-2 stimulated NK cells. Disclosures Mustjoki: Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.

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Molecular Spectrum of CSF3R variants Correlate with Specific Myeloid Malignancies and Secondary Mutations

Blood ◽

10.1182/blood-2018-99-116554 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4389-4389

Author(s):

Vera Adema ◽

Cassandra M. Hirsch ◽

Bartlomiej Przychodzen ◽

Yasunobu Nagata ◽

Tomas Radivoyevitch ◽

...

Keyword(s):

Amino Acid ◽

Board Of Directors ◽

Research Funding ◽

Cytoplasmic Domain ◽

Tier 2 ◽

Juxtamembrane Domain ◽

Advisory Committees ◽

Myeloid Malignancies ◽

Tier 1 ◽

Allelic Burden

Abstract Different CSF3R mutations (CSF3RMT) result in aberrant G-CSF signaling pathways and are linked to a wide range of myeloid disorders. Loss-of-function mutations in its extracellular domain cause severe congenital neutropenia (SCN). Activating mutations in the juxtamembrane region have been associated with a variety of myeloid malignancies. Truncating mutations in the cytoplasmic domain are associated with SCN cases that progress to MDS or AML. In this study, we evaluate the extent to which different CSF3RMT associate with disease onset, progression to leukemia and neutrophil counts in patients (pts) diagnosed with myeloid malignancies. We identified CSF3RMT cases in a cohort of 1400 pts [median age 71 years (yrs)]. We analyzed somatic and germline mutational patterns, and cross-sectional correlation with other gene mutations in CSF3RMT. A stringent algorithm based on conserved amino acid residues and alterations of protein features was used to predict the pathogenic significance of CSF3RMT. We identified 44 CSF3RMT: 33 germline (CSF3RGL) and 11 somatic (CSF3RS) variants. Most CSF3RGL were found in pts (median age 63 yrs) with MDS or related conditions (87% of all mutant cases), conversely these mutations were present in 5% (n= 22/424) of MDS, 3% (n= 7/244) MDS/MPN and <1% (n= 3/392) of AML and in 1 out of 3 pts with aCML tested. Mutations were mostly missense and located between the cytoplasmic (58%: M696T, R698C (isoform III), D732N, P733T, S744F, Y752*, E808K), and extracellular (42%: C131Y, E149Q, A208V, Q216H, D320N, E405K, S413L, Y562H) domains. No mutations were detected in the juxtamembrane domain. Variants were grouped in Tier-1 (61%: C131Y, E149Q, A208V, Q216H, D320N, E405K, S413L, Y562H Y752*, E808K) and Tier-2 (variants with uncertain significance, 39%: S413L, M696T, R689C, D732N, P733T, S744F). E808K and R698C were the most common amino acid changes in Tier-1 (53%) and Tier-2 (44%), respectively. A total of 4/7 pts with E808K progressed to AML (but none with R698C), supporting previous observations that E808K (or E785K) represents a pathogenic variant predisposing to leukemia. A total of 46% (n=14) of pts with CSF3RGL had neutropenia [median 0.9x109/L (0.02-1.22x109/L)] at the time of sampling. Two pts diagnosed with a prior cancer manifested sustained neutropenia before the diagnosis of MDS and MDS/MPN. G-CSF was administered in 21% of pts. Alterations in -7/7q- were common (21%). Some pts also harbored other somatic mutations in NF1 (15%), DNMT3A (12%), SETBP1 (12%), or U2AF1 (12%). Of note, 1 patient carried mutations in WAS and GATA2 and another carried a mutation in VPS45, which have been previously associated with SCN/MDS. The patient with aCML harbored also a CSF3RS (T615A). Overall combined allelic burden in pts cohort was 2% vs. 1.6% expected allelic burden in control populations for the same variants (P=.02). CSF3R S were found in 11 pts (median age 71 yrs) with AML or MDS related conditions (73% of all mutant cases), conversely these mutations were present in 1.4% (n= 6/424) of AML, <1% in MDS (n= 2/244) and MDS/MPN (n= 1/392) and in 2/3 pts with aCML tested. Mutations were missense in 63% of pts, T618I being most recurrent (n=5/11; 45%). Frameshifts accounted for 36% of the mutations and were localized in the cytoplasmic domain (Q741*, Q749*, Y752*, Q768*). All mutations were heterozygous. At the time of sampling 3/11 pts had leukocytosis and 3/11 had neutropenia. Mutations were distributed between the juxtamembrane domain (55%) and the cytoplasmic domain (45%). Mutations in the extracellular domain were not detected. Pts with sAML mostly carried mutations in the juxtamembrane domain (67%), those with MDS carried only in cytoplasmic domain, and those with MDS/MPN or aCML carried mutations in both the juxtamembrane and extracellular domains. There was one somatic and one RUNX1GL mutation. The cytogenetic abnormalities -7/7q- were detected in 18% (2/11) of cases. Interestingly, T618I was found solely in pts with sAML. Focusing on associations between CSF3RMT and mutations in the class III receptor tyrosine kinases CSF1R, FLT3, and KIT we identified only FLT3 to be co-mutated with CSF3RMT. All 3 pts (2 CSF3RGL and 1 CSF3RS) with such co-mutations evolved to AML. In sum, we found that CSF3RGL do not commonly co-occur with CSF3RS, suggesting that the neutropenia observed at the sampling time most likely is causative of undetected GL variants and/or is representative of a long unrecognized disease. Disclosures Nazha: MEI: Consultancy. Carraway:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Speakers Bureau; Jazz: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Speakers Bureau; FibroGen: Consultancy. Santini:Otsuka: Consultancy; AbbVie: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Amgen: Membership on an entity's Board of Directors or advisory committees. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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Distinctive Genetic Features of Plasma Cells in POEMS Syndrome

Blood ◽

10.1182/blood.v128.22.4404.4404 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4404-4404

Author(s):

Yuhei Nagao ◽

Naoya Mimura ◽

June Takeda ◽

Motohiko Oshima ◽

Kenichi Yoshida ◽

...

Keyword(s):

Rna Sequencing ◽

Copy Number ◽

Monoclonal Gammopathy ◽

Research Funding ◽

Somatic Mutations ◽

Poems Syndrome ◽

M Protein ◽

Target Sequencing ◽

The Mean ◽

Genetic Profiles

Abstract Introduction: Polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome is a rare paraneoplastic disease due to an underlying monoclonal plasma cell (PC) dyscrasia. Despite of dynamic symptoms associated with highly elevated VEGF, monoclonal PCs are thought to be quite small, and pathogenic significance of these PCs remains undetermined. In this study, we performed whole exome sequencing (WES), target sequencing, and RNA sequencing of PCs in patients with POEMS syndrome in order to define its genetic profiles. Methods: Patients diagnosed with POEMS syndrome at Chiba University Hospital from July 2014 to June 2016 were enrolled. DNA was extracted from either PCs which were isolated from patients' bone marrow (BM) using CD138 MACS (Miltenyi) or buccal cells as controls. WES and target sequencing were performed using HiSeq2500 (Illumina) and MiSeq (Illumina), respectively. The data of WES and target sequencing were analyzed by Empirical Bayesian mutation Calling (EBCall). Copy number was analyzed using the data of WES. RNA sequencing of PCs isolated by MACS and FACS sorting was conducted using HiSeq 1500 (Illumina). PCs from some patients diagnosed with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) were also collected as controls for RNA sequencing. Results: Twenty POEMS patients (M:F 12:8, mean age 42.6, range 16-78; 15 newly diagnosed, 5 refractory or relapsed cases) were included in this study. Regarding the types of M protein, 55% (11/20) were IgA-λ, 25% (5/20) were IgG-λ, and each individual case of the following; IgA-λ+IgG-λ, BJP-λ, IgG-κ, and Castleman's variant with no M protein. The mean serum VEGF was 6,471 pg/ml (range 1,190-13,800), and the mean PCs percentage in the BM was 4.4% (range 0.8-10.5). WES was performed in 15 cases; a total of 359 somatic mutations in 334 genes were revealed in 93.3% of cases (14/15) with a mean number of 23.9 (range 0-119) in each. All these mutated genes were significantly enriched in several pathways related to cell adhesion. Importantly, frequently mutated genes in MM such as NRAS, KRAS, and TP53 were not identified. Among all mutations, 1.7% were frameshift insertions, 2.0% were frameshift deletions, 4.2% were stop gains, 0.8% were non-frameshift deletions, 60.2% were other non-synonymous single nucleotide variants (SNVs), 29.5% were synonymous SNVs, and 1.7% were splicing mutations which were within 2-bp of a splicing junction. Copy-number variations were detected in 33.3% of cases (5/10) including -13 (2 cases), +1q (2 cases), and hyperdiploidy (2 cases). To carry out target sequencing in all 20 cases, we defined 51 target genes which included recurrently mutated genes from our WES data, frequently mutated genes in hematopoietic and lymphoid tissues according to the database (COSMIC), and 15 frequently mutated genes in MM (NRAS, KRAS, TP53, BRAF, CDKN2C, FGFR3, BIRC3, DIS3, CYLD, KDM6A, LRP1B, FAM46C, COL6A3, DNAH5, and KRT6A). A total of 60 somatic mutations were revealed in 65% of cases (13/20), and 9 new somatic mutations were found in the cases in which WES was also performed. Ten recurrently mutated genes were identified; KLHL6 in 20% of cases (4/20), each of LTB, RYR1 in 15% of cases (3/20), and each of EHD1, EML4, HEPHL1, HIPK1, PCDH10, USH2A, and ZNF645 in 10% of cases (2/20). Among frequently mutated genes in MM, only 3 genes (FAM46C, LRP1B, and DNAH5) were mutated, each in a single case. We finally conducted RNA sequencing of the FACS-sorted PCs in 5 POEMS patients compared to 5 MGUS and 4 MM patients. Upregulated genes were significantly enriched in some gene sets, gene ontology terms, and pathways related to immune response and cell adhesion, whereas downregulated genes were related to tumorigenesis. Of note, VEGF was not significantly upregulated in POEMS patients. Principal component analysis distinguished the 3 disease groups of patients with marginal overlaps between POEMS and MGUS, and also MGUS and MM. Conclusions: Our data clearly demonstrate that the genetic profiles of PCs in POEMS syndrome are distinct from those in MM and MGUS. Notably, PCs may not be the main source of extremely elevated VEGF in POMES syndrome. On-going further investigation will help clarify the molecular pathogenesis of POEMS syndrome. Disclosures Ogawa: Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

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Inhibition of Notch Signaling in T Cells Prevents Immune-Mediated Bone Marrow Failure.

Blood ◽

10.1182/blood.v114.22.180.180 ◽

2009 ◽

Vol 114 (22) ◽

pp. 180-180

Author(s):

Gloria T Shan ◽

Ivy Tran ◽

Ashley R Sandy ◽

Ann Friedman ◽

Yi Zhang ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Notch Signaling ◽

Aplastic Anemia ◽

Research Funding ◽

Bone Marrow Failure ◽

Comprehensive Cancer Center ◽

Cancer Center ◽

Immune Mediated

Abstract Abstract 180 Aplastic anemia is a severe bone marrow disorder characterized by the loss of hematopoietic stem cells (HSC). HSC destruction is thought to be T cell-mediated in a majority of patients with aplastic anemia. Global immunosuppression and HSC transplantation can induce disease remission, but these treatments are not effective in all patients and can promote life-threatening complications. Thus, novel immunomodulatory approaches are needed in this disorder. Notch is a conserved cell-cell communication pathway that can regulate T cell differentiation and function with context-dependent effects. To study the role of Notch signaling in pathogenic T cells causing immune-mediated bone marrow failure, we inhibited canonical Notch signaling in mature T cells through conditional expression of the pan-Notch inhibitor DNMAML (ROSA-DNMAMLf × Cd4-Cre mice). We used two complementary mouse models of immune-mediated bone marrow failure that mimic features of aplastic anemia: administration of C57BL/6 (B6) T cells into sublethally irradiated (500 rads) minor histocompatibility antigen mismatched BALB/b recipients (Chen et al., J Immunol 2007; 178:4159), or infusion of B6 lymphocytes into unirradiated MHC-mismatched B6×DBA F1 recipients. In contrast to control B6 T cells which led to lethal bone marrow failure in virtually all recipients, DNMAML-expressing Notch-deprived T cells were profoundly deficient at inducing HSC loss in both disease models, leading to markedly improved long-term survival (>90%). Notch-deficient T cells showed a modest decrease in overall expansion within secondary lymphoid organs, but their accumulation in the target bone marrow was preserved. Upon restimulation with anti-CD3 and anti-CD28 antibodies, DNMAML T cells had decreased production of IL-2 and interferon gamma. Activated CD4+ and CD8+ DNMAML T cells had reduced interferon gamma, granzyme B, and perforin transcripts despite preserved induction of the master transcription factors Tb×21 (encoding T-bet) and Eomes. In vivo infusion of CFSE-labeled host-type target cells revealed a decreased cytotoxicity in DNMAML as compared to control B6 T cell recipients. These observations point to a novel spectrum and mechanism of Notch action in mature T cells. Since we have shown recently that canonical Notch signaling is dispensable for the maintenance of adult HSCs (Maillard et al., Cell Stem Cell 2008, 2:356), our findings suggest that Notch inhibition could represent a novel therapeutic modality to target the T cell response and reverse immune-mediated HSC destruction in aplastic anemia. Disclosures: Shan: American Society of Hematology: Research Funding. Zhang:University of Michigan Comprehensive Cancer Center: Research Funding; Damon Runyon Cancer Research Foundation: Research Funding. Maillard:Damon Runyon Cancer Research Foundation: Research Funding; American Society of Hematology: Research Funding; University of Michigan Comprehensive Cancer Center: Research Funding.

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Karyotypic and Genetic Abnormalities Associated with Clonal Evolution in Paroxysmal Nocturnal Hemoglobinuria.

Blood ◽

10.1182/blood.v120.21.2371.2371 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2371-2371

Author(s):

Hideki Makishima ◽

Kenichi Yoshida ◽

Michael J. Clemente ◽

Masashi Sanada ◽

Yasunobu Nagata ◽

...

Keyword(s):

Stem Cell ◽

Research Funding ◽

Somatic Mutations ◽

Chromosomal Abnormalities ◽

Clonal Evolution ◽

Clonal Expansion ◽

Novel Mutations ◽

Jak2 Mutation ◽

Myeloid Malignancies ◽

Whole Exome

Abstract Abstract 2371 PNH is a clonal stem cell disease. While nonmalignant, PNH shows certain similarities to MDS and other neoplasms affecting hematopoietic stem and progenitor cells, including persistence of an aberrant clone, clonal expansion, and phenotypic abnormalities. In a small proportion of patients, subtle chromosomal abnormalities can be found and cases of otherwise classical PNH due to microdeletions involving the PIG-A locus have been described, illustrating similarities to other malignant conditions. PIG-A gene mutations lead to defective biosynthesis of GPI anchors and are responsible for the PNH phenotype. Similarly, phenotypic features of stem cells affected by PIG-A mutations are believed to be responsible for the extrinsic growth advantage and clonal expansion in the context of immune mediated suppression of hematopoiesis. While this scenario is plausible, there are also observations suggesting that intrinsic factors may be also involved. For instance, PNH persists after successful immunosuppression, often for many years, suggesting activation of stem cell maintenance genes. Furthermore, PNH clones can also be encountered (albeit at a very low frequency) in healthy individuals, and PNH can present in a pure form without aplastic anemia. Such extrinsic factors may include additional, secondary genetic events such as somatic mutations. Supporting this theory, clonal rearrangement of chromosome 12, which leads to overexpression of the transcription factor HMGA2 gene, were found in cells with the PIG-A mutation from 2 PNH cases. Also, we recently reported 3 PNH cases with JAK2 V617F mutation, who presented with a MPN phenotype and thrombosis. We theorized that study of clonal architecture in PNH will reveal clues as to the pathogenesis of clonal evolution of the PNH stem cell. We applied next generation whole exome sequencing to detect somatic mutations in PNH cases (N=6). The subsequent validation set included 45 PNH cases. PNH and non-PNH cells were sorted using magnetic beads. DNA from both fractions was analyzed by whole exome sequencing and results of the non-PNH cells were subtracted from the results of the PNH clone. We found biallelic PIG-A mutations in 2 female cases and a single mutation in each male case. In an index female case with thrombosis, a novel somatic heterozygous mutation of NTNG1 (P24S) was detected, while the patient was negative for the JAK2 mutation. Allelic frequency with the NTNG1 mutation (53/160 sequence reads (33%)) was larger than that with a concomitant heterozygous PIG-A mutation (intron 5 splice donor site G<A) (78/333 reads (23%)). In this case, the size of the other heterozygous PIG-A mutation (G68E) was less (31/194 (16%)) than the other PNH clone. These findings suggest that there are 2 different PNH clones in one case and that the NTNG1 mutation might be acquired before PIG-A gene was mutated. Moreover, NTNG1 encodes a GPI-anchored cell membrane protein and the mutation (P24S) was located in the predicted signal peptide. All together, 3 novel mutations were discovered, including MAGEC1 (C747Y) and BRPF1 (N797S) mutations. Of note, BRPF1 mutations have been also reported in AML. Interestingly, BRPF1 encodes a component of MOZ/MORF complex, positively regulating the transcription of RUNX1. To screen pathogenic karyotypic lesions in PNH clonal expansions, we combined metaphase cytogenetics and single nucleotide polymorphism arrays. We detected 14 somatic chromosomal abnormalities in 13 out of 26 PNH cases (50%). Of note is that a microdeletion on 2q13 resulted in the loss of an apoptosis-inducing gene BCL2L11, suggesting a contribution to growth advantage. Somatic UPD lesions strongly suggest the presence of homozygous mutations, for example the SET nuclear oncogene, which is located in UPD9q32qter was observed in another PNH case. Overall, the discovery of these novel mutations, as well the previously described JAK2 mutation, indicates that the pathophysiology of PNH clonal evolution partially overlaps that of other myeloid malignancies. In sum, various novel somatic karyotypic abnormalities and mutations are frequently detected in PNH clones using technology with comprehensive and high resolution. Some of these aberrations play a similar role in the clonal evolution of myeloid malignancies. These results suggest new therapeutic strategies similar to those for other myeloid malignancies should be considered in PNH cases with addition mutations. Disclosures: Makishima: Scott Hamilton CARES Initiative: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

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Identifying Immunogenetic Features That Distinguish Immune Mediated, Immunosuppression-Sensitive Disease in Aplastic Anemia (AA)

Blood ◽

10.1182/blood.v120.21.3481.3481 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3481-3481

Author(s):

Manuel G. Afable ◽

Theresa Guo ◽

Bartlomiej P Przychodzen ◽

Lisa Rybicki ◽

Michael J. Clemente ◽

...

Keyword(s):

Stem Cell ◽

Aplastic Anemia ◽

Internal Control ◽

Research Funding ◽

Univariate Analysis ◽

Significant Proportion ◽

Minor Allele ◽

Cell Transplant ◽

Hematopoietic Stem ◽

Immune Mediated

Abstract Abstract 3481 A significant proportion of patients with idiopathic AA respond to immunosuppressive therapy (IST), suggestive an ex juvantibus autoimmune pathogenesis. Refractory cases may be due to either insufficient intensity of IST or exhaustion of stem cell reserves. Similarly, there may be a non-immunological component of the disease that cannot be distinguished clinically to date. Multiple trials of various ISTs often delay potentially curative therapy with bone marrow transplantation (BMT); hence, identification of this subtype is of great importance. Immune-mediated AA may have measurable immunogenetic determinants, such as HLA genes, that modify susceptibility to, character of, or intensity of autoimmune reactions. We hypothesize that genetic polymorphisms may exist in immune cytokines, cytokine receptors, and immune regulatory genes that may be markers of immune-mediated disease. We applied a custom cancer chip (Illumina) containing 211,155 probes for mostly non-synonymous single-nucleotide polymorphisms (SNPs) to perform focused bioanalysis on 32 immunogenetic polymorphisms. Our cohort included 152 patients; AA (N=91), AA/PNH (N=38), and PNH (N=23). Results were compared to internal control and previously published results with comparable populations. Median age was 45 years (5–80 years); 92% (N=109) had normal cytogenetics; 67% (N=96) had IST with an overall response rate (ORR) of 65%; 11% (N=17) underwent hematopoietic stem cell transplant; 43% (N=49/114) were positive for HLA DR15. The following genotypes had higher incidence in our AA cohort vs. controls: IL1A C(1202)T (rs1800794) CC 60% vs. 35% (p=<.001), IL4 –33 T>C (rs2070874) CC 68% vs. 29% (p=<.001), IL4 –590 C>T (rs2243250) CC 95% vs. 74% (p=<.001), IL2 T-330-G (rs2069762) TT 47% vs. 37% (p=.04), IL6 NT565A>G (rs1800797) AA 17% vs. 8% (p=.006), IL10 –1082G>A (rs1800896) AA 39% vs. 20% (p=<.001), TNF -308 G>A (rs1800629) GG 95% vs. 71% (p=<.001) and IL10 –592 A>C (rs1800872) AA 16% vs. 6% (p=.005). Among AA subtypes, we compared the frequency of each genotype: IL10 –592 A>C (rs1800872) was more frequent in AA vs. PNH (p=.003); both homozygous genotypes, CC and TT of TGF b1 codon 10 +869 C/T (rs1800470) were increased in AA vs. AA/PNH (p=.007). Frequency of CC genotype of CR2 C>T (rs2802221) was increased in AA vs. PNH (p=.003). Differences were also present in CR2 (S663P exon 11, rs4308977) and CR2 (rs6667140) in AA vs. AA/PNH (p=.001), AA vs. PNH (p=.031), and in AA vs. AA/PNH (p=.002). Next, we identified prognostic factors for response via univariate analysis which showed a trend towards higher ORR in AA/PNH vs. AA (88% vs. 59%, p=.06), as previously described. However, neither karyotype nor presence of HLA DR15 affected ORR. Interestingly, there was a trend towards higher ORR in patients with the following genotypes: IL4 –33 T>C CC vs. CT vs. TT (80% vs. 53% vs. 50%, respectively), p=.08; IL4 –590 C>T CC vs. CT vs. TT (69% vs. 33.3% vs. 0, respectively), p=.06. We analyzed (logistic regression) 3 variables (presence of PNH, IL4 –33 T>C SNP, and IL4 –590 C>T SNP) identified in univariate analysis (p<.15) and found that polymorphism in IL4 –33 T>C particularly the CC genotype is prognostic for response (p=.038) while IL4 –590 C>T polymorphism showed a trend towards significance (p=.09). We also employed unbiased screening of non-synonymous SNPs in 116 cases and confirmed in an additional 120. 3 SNPs were outstanding based on differential frequency between patients and controls. There was a difference between dominant/minor (C/T) alleles rs13050238 (CRYZL1) between patients and controls [213/9 (95%, dominant allele) vs. 3747/25 (99%), p<1×108] suggesting carriers of at least one copy of the T allele (CT/TT) are at higher risk of developing AA. Similarly, the minor allele G (A/G) in rs6451268 (RANBP3L) has protective potential [194/30 (87%) vs. 3721/207 (95%), p<1×107]. Minor allele T (C/T) in rs911178 (ZKSCAN3) may also be a protective allele [193/33 (85.53%) vs. 3641/287 (92.69%), p=<1×105]. This gene may regulate expression of genes favoring tumor progression in colorectal cancer. In summary, the T allele of IL1A 1202, IL4 –33, IL4–590; G allele of IL2 –330, IL6 NT565, IL10 –1082; A allele of TNF -308 and C allele of IL10 –592 may be protective against development of AA. SNPs in IL4 –33 T>C and IL4 –590 C>T may be predictive of response to IST and may be considered along with the presence of PNH as favorable prognostic factor. Disclosures: Maciejewski: NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

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Novel Recurrent Mutations in the Ras-Like GTP-Binding Gene Rit1 in Myeloid Malignancies

Blood ◽

10.1182/blood.v120.21.558.558 ◽

2012 ◽

Vol 120 (21) ◽

pp. 558-558

Author(s):

Inées Góomez-Seguíi ◽

Hideki Makishima ◽

Andres Jerez ◽

Kenichi Yoshida ◽

Bartlomiej P Przychodzen ◽

...

Keyword(s):

Research Funding ◽

Somatic Mutations ◽

Small Gtpases ◽

Refractory Anemia ◽

Myeloid Malignancies ◽

Gtp Binding ◽

Myeloid Neoplasms ◽

Relative Ratio ◽

Recurrent Mutations ◽

Ras Family

Abstract Abstract 558 In addition to chromosomal and epigenetic abnormalities, somatic mutations constitute key pathogenic lesions in myeloid neoplasms. Individual somatic mutations or various combinations may be both valuable prognostic markers and targets for new rational therapies. Among them, RAS family genes are ubiquitous oncogenes associated with various cancers. Recurrent canonical mutations in the nucleotide binding domains in NRAS and KRAS result in constitutively activated proteins. In myeloid neoplasms, RAS mutations convey a poor prognosis and are often found in acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) and, rarely, myeloproliferative neoplasms (MPN). We applied whole exome sequencing to paired germline vs. leukemia samples in 65 cases of MDS, 36 MDS/MPN and 32 sAML. We focused our study on the RAS protein superfamily of small GTPases and identified mutations in 3% and 6% of KRAS and NRAS, respectively. Most significantly, we identified somatic recurrent mutations in the F82 residue of Ras-like without CAAX1 (RIT1) gene in 2 patients with chronic myelomonocytic leukemia (CMML) and secondary AML (sAML), respectively. We confirmed the somatic nature of both mutations in sorted CD3+ cells from each patient (pt). RIT1 gene encodes a member of Ras-related GTPases, involved in the p38 MAPK and AKT signaling pathway that mediates cellular survival in response to stress. RIT1 gene amplification has been found in 26% of hepatocellular carcinoma. However, neither amplification nor mutations of this gene have been reported in myeloid malignancies. We thus focused this line of experimentation on this somatic mutation. To establish clinical associations we further studied a cohort of 322 patients with various myeloid malignancies by Sanger sequencing and detected somatic RIT1 mutations in an additional 6 (2%) cases. All mutations were located in exon 5, in the 81 and 82 residues, which encode the switch II domain of this protein, an effector region very close to the GTP-binding site G3, and which is highly conserved among species. Among the 8 mutant cases, 5 (63%) pts had CMML, resulting in a higher frequency of mutations in this subcohort of pts (5 out of 57 CMML, 9%). The other 3 mutations were found in one primary (p)AML (M5b subtype) (1 out of 58 pAML, 2%) and two high-grade MDS, one refractory anemia with excess blasts (RAEB)-2 and one sAML(RAEB-T in the FAB-classification) (2 out of 80, 2.5%). RIT1 mutations were heterozygous in all cases except for one case with trisomy 1 and duplication of the mutant allele. In the cases of WES, we estimated an allelic frequency of ∼35%, consistent with the presence of a heterozygous mutation in ∼70% of sample cells. Because of the large size of the clone and serial samples showing RIT1 mutation since the time of initial diagnosis, it is likely that RIT1 may be of ancestral origin. As RAS-family gene amplifications have been described in cancer, we also studied the presence of amplifications of the RIT1 locus (1q22) by SNP-A. We found 10 cases characterized by a gain involving the RIT1 region (1q21.1-q44): 4 (40%) cases had a diagnosis of CMML, 4 (40%) had myelofibrosis, whereas the remaining patients had MDS (one RAEB-1 and a RA). Quantitative RT-PCR showed RIT1 overexpression in mutants and in patients with 1q amplification (median normalized relative ratio 0,51 and 0,40, respectively) compared to patients with wild type RIT1 and no amplification in 1q (median normalized relative ratio 0,15; P=.039). We theorized that activating RIT1 mutations may constitute a suitable therapeutic target. Because AKT inhibitors can block AKT phosphorylation and therefore reverse the antiapoptotic effect of mutant RIT1, we tested whether AKT inhibitor V (Triciribine) can selectively abrogate the growth of primary cells with RIT1 mutation. In in vitro suspension cultures, a 65% of reduction proliferation was observed with significant effects even at 0.1μM concentrations. In sum, somatic recurrent RIT1 mutations are novel lesions involved in the molecular pathogenesis of myeloid cancers, presumably early in the development of the disease. Moreover, amplifications of RIT1 also lead to overexpression of this Ras-like GTP-ase. Specifically, these abnormalities appear to be more frequent in patients with CMML, but also can be found in other types of MDS. Disclosures: Makishima: Scott Hamilton CARES Initiative: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

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