TET 2 Alterations in Myeloid Malignancies, Impact on Clinical Characteristics, Outcome, and Disease Predisposition

Abstract TET2 mutations are the most common somatic genetic lesions in myeloid neoplasms. TET2 mutant clones have been found also in healthy individuals, increase with age, and convey an increased risk for myeloid clonal diseases. The TET2 gene is very polymorphic, with hundreds of single nucleotide polymorphisms (SNPs) of unknown clinical impact, but with some variants that may be pathogenically important. Similarly, somatic mutations affect all portions of the gene, and can be missense or truncating, homo-, hemi- and heterozygous. While the majority of TET2 mutations are ancestral, they can also be subclonal, implicating the clonal architecture in the consequences of TET2 lesions. This diversity may be hampering establishment of the clear prognostic impact of TET2 mutations. Taking advantage of a large cohort of patients (pts, N=4985 including 1616 MDS, 871 MDS/MPN, 1782 pAML, 304 sAML, 333 MPN, and 79 therapy-related MDS/AML/MDS-MPN) analyzed by targeted deep sequencing for TET2 and other common myeloid lesions, we examined the distribution and impact of TET2 mutations. DNA sequencing of all coding exons of TET2 and 61 other genes representing the most common somatic mutations in myeloid neoplasms. Nonsynonymous alterations not present in SNP database (dbSNP) were annotated as somatic mutations or SNPs if present in myeloid and T cells whenever available. Nonsynonymous alterations not in dbSNP or ExAC databases and not confirmed to be somatic were excluded. Overall, TET2 somatic mutations (TET2mut) were present in 920 pts (18%); 38% of MDS/MPN, 19% pAML, 16% MPN, 16% sAML, 12% MDS, and 13% of therapy related MDS/AML/MDS-MPN. Mutations included 16% missense, 33% frameshift deletions, 18% frameshift insertions, and 33% nonsense. TET2mut pts were older than those with TET2 wild type (TET2wt, 72 vs. 67 yrs, p<.001), had a higher presenting WBC (6 vs. 4 x103 /uL, p <.001), and lower blast % (3 vs. 7%, p =.03). Similar findings were observed in each myeloid subtype. Overall, median OS for TET2mut pts was similar to TET2wt (12 vs. 17 mo, p =.20). Median OS was similar in TET2mut pts compared to TET2wt in pts with MDS (23 vs. 23 mo, p =.77), MDS/MPN (15 vs. 21 mo, p=.1), pAML (9 vs. 14 mo, p =.77), sAML (6 vs. 9 mo, p =.07), and MPN (30 vs. 35 mo, p =.66). Neither the type of mutation (mis-, nonsense vs. truncating) nor location (catalytic domain vs. other) impacted the OS. Using variant allelic frequencies (VAF), we established a clonal hierarchy in individual cases; 24% of TET2 mutations were ancestral, 17% subclonal, and 59% codominant. TET2 mutations were ancestral in 23% of MDS samples, 29% of MDS/MPN, 25% of pAML, and 19% of sAML. Whether the mutation was ancestral or subclonal did not impact OS. The presence of TET2mut was associated with different mutations in each myeloid subtype. In MDS, TET2mut were associated with APC (p<.001), ASXL1 (p<.001), BCOR (p<.001), BCORL1 (p<.001), ETV6 (p<.04), SUZ12 (p<.001), RAD21 (p<.02), NF1 (p<.001), KDM6A (p<.001), ZRSR2 (p<.001), and U2AF1 (p=.02), in MDS/MPD correlated with ASXL1 (p<.04), NRAS (p<.02), and SRSF2 (p<.05), in pAML with JAK2 (p<.001), RUNX1 (p =.05), and CBL (p=.05), and in sAML with RUNX1 (p<.001), ASXL1 (p<.001), BCORL1 (p=.01), SUZ12 (p=.02), STAG2 (p=.05), and JAK2 (p<.001). When we next focused on germ line variants, we identified 2518 SNPs of TET2. All recurring SNPs were ranked according to the difference in their frequencies between pts and healthy controls. A large number of these SNPs were more common in our pts compared to controls, among them we identified 2 SNPs (both located in the dioxygenase domain) with a significantly higher frequency: SNP1 (OR 10.6, p<.0001), and SNP2 (OR 6.7, p=.02). We further investigated whether these SNPs were mutually exclusive or increased the risk for acquisition of somatic TET2 mutations; 91% of cases with SNP1 and 67% with SNP2 also acquired somatic mutations in TET2. In silico and crystallographic analyses showed that SNP1 is adjacent to the iron binding site (7th beta stand in the jelly roll motif) and is predicted to change the orientation of a-KG binding and thereby to be hypomorphic. SNP2 is located in a hot spot area known to be targeted by 3 recurrent somatic mutations. In conclusion, both somatic mutations as well as germ line variants affect TET2 in myeloid neoplasia. The interaction between clonal mutations and germ line lesions may lead to gain of function and thus a growth advantage. These mechanisms are currently being explored. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Sekeres:TetraLogic: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees.

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Pathogenic Relevance of Germ Line TET2 Alterations

Blood ◽

10.1182/blood.v128.22.3160.3160 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3160-3160 ◽

Cited By ~ 1

Author(s):

Cassandra M. Hirsch ◽

Aziz Nazha ◽

Kassy E Kneen ◽

Manja Meggendorfer ◽

Bartlomiej P Przychodzen ◽

...

Keyword(s):

Somatic Mutations ◽

Catalytic Domain ◽

Rare Variants ◽

Germ Line ◽

Bone Marrow Failure ◽

Genetic Syndromes ◽

Advisory Committees ◽

Myeloid Neoplasm ◽

Myeloid Neoplasms ◽

Biallelic Mutations

Abstract Several familial mutations predisposing to the development of leukemia have been identified, mostly in association with childhood or adolescent presentations of myeloid neoplasms or bone marrow failure states. However, many of these genetic syndromes have variable penetrance and latency. Thus, late presentations of familial diseases are possible as with DDX41 mutations associated with adult myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Growing evidence exists to support the notion that germ line (GL) alterations may play a greater than previously known role in otherwise typical spontaneous adult myeloid neoplasms. These alterations may include known predisposition leukemia genes (i.e.,. ETV6, RUNX1, CEBPa, and DDX41), heterozygous alterations known to generate recessive disorders, or new unidentified genes. The search for such predisposition genes is difficult but we hypothesized that genes frequently damaged by somatic mutations may also be affected by GL alterations, a similar principle as seen for CEBPa or TP53. Further, that a collection of rare variants (MAF<1%) within a gene could be associated with risk of MDS according to the allelic burden (AB) theory. To develop proof-of-principle of this theory we focused on the gene TET2. In comparisons of the AB of rare TET2 GL variants of patients vs. ethnically-matched controls (selected according to ethnic indicator SNPs), MDS patients examined by whole exome sequencing (WES, N=368) had more rare coding-sequence variants than controls (p<.001). On average, patients had 3 TET2 rare variants, while controls had only 1.5 variants. This suggests that the burden of TET2 rare variants could increase risks of myeloid neoplasms. This finding is in agreement with several other observations; 1.) TET2 is frequently mutated and extremely polymorphic; 2.) it is often associated with biallelic mutations, of which some may be a combination of GL and somatic lesions; and 3.) GL alterations in TET2 may predispose to acquisition of additional lesions. We analyzed a cohort of 4159 patients with myeloid neoplasms using WES and targeted deep next generation sequencing (NGS). Extensive scoring algorithms, along with mutational databases were used to detect GL variants. Serial tumor samples were examined to discriminate GL from somatic defects. We identified 2518 TET2 SNPs in 1475 patients. Multiple SNPs were seen in the same patient, 28% of patients had 2 SNPs, 11% had 3 and 6% had 4 or more SNPs. Of those 52% were found in proximity to the catalytic domain, and 10% were homozygous. TET2 SNPs irrespective of their population frequency were found in 21% of MDS, 27% of MDS/MPN, 31% of MPN, and 33% of AML patients. We focused our analysis on the top 10 canonical SNPs with the highest odds ratio (OR) when comparing frequencies in our population to that of healthy controls derived from the ExAC base of 60,000 individuals. The SNPs with the highest OR was present in 2% of patients (p.Val1718Leu) vs. 0.5% of controls. Combinations of SNPs (biallelic configuration) were then investigated; SNPs p.Leu1721Trp and p.Pro363Leu co-occurred in 8% of patients, and p.Val218Met, p.Leu34Phe and p.His1778Arg co-occurred in 3%. We also queried whether GL TET2 SNPs create a haplotype (i.e., increased the risk for acquisition of somatic TET2 mutations) or are mutually exclusive. Overall, 32% of cases with any TET2 SNP also acquired a TET2 somatic mutation vs the WT form for these (68%). Conversely, among the somatic mutant carriers, for example SNP p.Met1701Ile, SNP p.Tyr864His and SNP p.Gly355Asp had an OR vs. controls of 1.1. In particular, 55% of all TET2 mutants had SNP p.Leu1721Trp in a biallelic fashion vs. 46% of somatic WT cases. This suggests a predisposition haplotype. We also examined the relationship between unrelated somatic mutations and "top OR" TET2 SNPs. No correlation was seen between SNPs and other somatic mutations. In sum, several GL TET2 variants or their combination may constitute complex, likely low penetrance predisposing factors for myeloid neoplasm that also interact with somatic lesions and lead to cancer decades later. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Maciejewski:Celgene: Consultancy, Honoraria, Speakers Bureau; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau.

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Landscape of Subclonal Mutations in Myelodysplastic Syndromes (MDS) Allows for a Novel Hierarchy of Clonal Advantage By Combining Germline and Somatic Mutations

Blood ◽

10.1182/blood.v128.22.957.957 ◽

2016 ◽

Vol 128 (22) ◽

pp. 957-957

Author(s):

Yasunobu Nagata ◽

Hideki Makishima ◽

Tomas Radivoyevitch ◽

Cassandra M. Hirsch ◽

Bartlomiej P Przychodzen ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Somatic Mutations ◽

Dna Analysis ◽

Germ Line ◽

Wide Spectrum ◽

Median Number ◽

Prognostic Impact ◽

Advisory Committees ◽

Disease Manifestation

Abstract Targeted and unbiased next generation sequencing (NGS) has contributed to a better understanding of the molecular pathogenesis of myeloid neoplasms, including MDS. Discovery efforts have identified novel classes of mutated genes, while deep NGS approaches have yielded a better appreciation of clonal hierarchy, inter-case variability and intra-tumor heterogeneity. MDS is a disease continuum characterized by a wide spectrum of often overlapping lesions that determine phenotype, while also serving as initiation and progression events. In addition to somatic lesions, germ line (GL) alterations can serve as bona fidenon-clonal ancestral events that play an underappreciated role in MDS pathogenesis. While some of these lesions are associated with childhood familial leukemia syndromes, others are unknown, and are likely characterized by a low/variable penetrance and delayed disease manifestation. To delineate clonal dynamics in MDS, we sequenced whole exomes of 262 cases with primary MDS and related disorders. For validation and confirmation we also deep sequenced a cohort of 1,686 additional cases with a various type of myeloid malignancies. An extensive bioanalytic pipeline and confirmatory sequencing, including GL DNA analysis, was used to discriminate somatic vs. GL lesions and exclude sequencing artifacts. Initially we focused on driver somatic events in significantly mutated genes. All somatic mutations were subjected to clonal hierarchy analysis using variant allele frequencies (VAFs). In selected cases (n = 180), serial analyses were performed. Using VAF rankings of each event, a position within the clonal hierarchy was assigned; while each patient has a single dominant clone, some may have a founding chromosomal abnormality and others may have VAFs too close to distinguish, i.e. have co-dominant events. In general, multiple subclonal events are detected in each patient. For the purpose of this analysis we distinguished between 2 types of ancestral events: 1) driver non-clonal mutations (e.g., GL TP53, RUNX1, ETV6) and 2) predisposition non-clonal events (FA genes, telomerase genes, BRCA1/2). The latter do not influence the clonal architecture. Based on average sequencing depth, 5,474 somatic mutations were identified: 241 (92%) were clonal dominant and 234 (89%) were sub-clonal (secondary) events. The median number of mutations in subclonal events per case was 13. The number of mutations in subclonal events was higher than that in events that were clonal dominant (4,881 vs 593). No genes were mutated in a purely dominant fashion and some genes were almost entirely subclonal, e.g., RAS and FLT3. For each dominant event, there is a frequent secondary lesion, e.g., dominant TET2 mutations are followed by subclonal second TET2 events, SRSF2 and ASXL1 lesions. Thus, novel relationships between dominant and subclone events were found, indicating the presence of invariant functional interactions among different mutations in MDS pathogenesis. In a confirmatory cohort studied by NGS targeted to a selected panel of significantly mutated genes, the number of subclonal events increased due to greater coverage and thus sensitivity. The spectrum of dominant events, however, should not differ as they are inherently associated with a high clonal burden. For examples, TP53 clonal mutations frequently co-occur with TP53 subclonal mutations (12%, p=.004), but are exclusive of STAG2 subclonal mutations. EZH2 clonal and ASXL1 secondary mutations also co-occur. Classifications of clonal and secondary events may have prognostic and diagnostic implications. We identified a spectrum of novel predisposition and non-clonal driver variants by comparing to ethnically weighted control populations. Eight mutations (3%, 8/262 cases) in 3 genes (DDX41, TP53, and ELANE) were identified as driver non-clonal mutations because identical mutations were reported in familial leukemia syndromes, while 16 mutations (6%) in 3 genes (CSF3R, BRCA1, and RPL5) were identified as non-clonal predisposition events. Detailed understanding of such clonal dynamics and complexity of clonal hierarchical complexity may have clinical significance, both for somatic mutations and for germline events. Increasing clonal burden of extracted genes associated with predictive prognostic impact should be prospectively validated in a more uniform and larger cohort of MDS cases. Disclosures Makishima: The Yasuda Medical Foundation: Research Funding. Mukherjee:Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding. Sole:Celgene: Membership on an entity's Board of Directors or advisory committees. Carraway:Celgene: Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Ogawa:Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding. Maciejewski:Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Speakers Bureau.

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NFE2 Mutations Impact AML Transformation and Overall Survival in Patients with Myeloproliferative Neoplasms (MPN)

Blood ◽

10.1182/blood-2020-136177 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 36-36

Author(s):

Clemence Marcault ◽

Lin-Pierre Zhao ◽

Rafael Daltro De Oliveira ◽

Juliette Soret ◽

Nicolas Gauthier ◽

...

Keyword(s):

Overall Survival ◽

Board Of Directors ◽

Somatic Mutations ◽

Response To Therapy ◽

Age At Diagnosis ◽

Prognostic Impact ◽

Leukemic Transformation ◽

Advisory Committees ◽

Increased Risk

Introduction: MPN are a heterogeneous group of chronic hematological malignancies often resulting from a combination of a driver gene mutation (JAK2, MPL or CALR) and a variety of somatic mutations harboring diverse prognosis values. A subset of MPN patients carry somatic mutations in the hematopoietic transcription factor NFE2 (nuclear factor erythroid 2) resulting in a functionally enhanced truncated form of NFE2 (Jutzi JS et al., JEM, 2013). Moreover, epigenetically induced overexpression of NFE2 has recently been reported in the majority of MPN patients (Peeken JC et al., Blood, 2018). In transgenic murine models, NFE2 overexpression results in an MPN phenotype (thrombocytosis, leukocytosis, EPO-independent colony formation, characteristic bone marrow histology and expansion of stem and progenitor compartments) and has recently been shown to predispose to the acquisition of additional genetic abnormalities and subsequent leukemic transformation (Kaufmann KB et al., JEM, 2012) (Jutzi JS et al., Blood, 2019). However, clinical impact of NFE2 mutations in MPN patients remains unknown. The aim of this study was to evaluate the phenotypic characteristics and prognostic impact of NFE2 somatic mutations in a large mono-centric cohort of MPN patients. Methods: A total of 1243 consecutive patients were diagnosed with MPN according to WHO criteria and followed in our hospital between January 2011 and May 2020. This study included 707 of them in whom a next-generation sequencing (NGS) molecular analysis targeting 36 myeloid genes was performed at diagnosis and/or during follow-up. Clinical and biological characteristics at time of diagnosis and follow-up were collected from medical charts and electronic medical records. Statistical analyses were performed using the STATA software (STATA 15.0 Corporation, College Station, TX). Results: In our cohort, 411 patients presented with polycythemia vera (PV), 577 with essential thrombocythemia (ET), 184 with primary or pre-fibrotic myelofibrosis (PMF), 59 with unclassified MPN and 12 with MDS/MPN. Median age at diagnosis was 51 years [40-63]. 73.1% patients had a JAK2V617F mutation, 14.1% a CALR mutation and 3.3% a MPL mutation. Overall, 64 (9.05%) patients harbored a NFE2 mutation with a variant allelic frequency (VAF) ≥ 0.5% and 36 had a VAF ≥ 5%, the latest were considered as NFE2 mutated for the rest of the study as VAF <5% may refer to a minor clone without clinical relevance. NFE2 mutations were present in 7.3%, 5.3% and 3.6% of PV, PMF and ET patients respectively. No significant association between the presence of NFE2 mutation and clinical/molecular MPN characteristics (driver mutation, constitutional symptoms, splenomegaly, blood counts, cytogenetic and other molecular features) was observed using a logistic regression association model. Median follow-up was 103.8 months, IQR [47.2; 175.6]. In terms of response to therapy, 52.8% of patients achieved complete response, complete hematological response or clinical improvement in NFE2 mutated vs 61.7% in non-mutated patients (p= 0.026). Interestingly, presence of a NFE2 mutation (HR 9.92, 95%CI[3.21; 30.64], p< 0.001), age at diagnosis (HR 1.09, 95%CI[1.05; 1.12], p< 0.001), PMF subtype (HR 6.92, 95%CI[2.81; 17.06], p < 0.001) and high-risk mutations (ASXL1, EZH2, SRSF2, IDH1/2 and U2AF1) (HR 2.45, 95%CI[1.14; 5.28], p=0.021) were independently associated with AML/MDS transformation free survival (TFS) in a COX regression multivariate analysis (Figure A). Presence of a NFE2 mutation was also independently associated with overall survival (OS) (HR 9.37, 95%CI [4.18; 21.03], p<0.001) (Figure B). Median TFS were 216.1 months and not reached, while median OS were 144.2 months and not reached for NFE2 mutated and non-mutated patients, respectively. No difference was observed in terms of thrombo-hemorrhagic events (HR 0.73; 95%CI [0.10; 5.21], p=0.752) and secondary myelofibrosis free survivals (HR 0.67; 95%CI [0.09; 4.87], p=0.693). Conclusion: In this retrospective study we show that presence of NFE2 mutations with a VAF ≥5% is independently associated with an increased risk of leukemic transformation and shorter overall survival. These findings are in line with recently reported animal models and suggest that NFE2 mutations screening should be routinely performed in MPN patients. Disclosures Rea: Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Kiladjian:AOP Orphan: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Benajiba:Gilead Foundation: Research Funding.

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Myelodysplastic Syndrome (MDS)-Determining Clonal Events at Presentation of Aplastic Anemia (AA)

Blood ◽

10.1182/blood.v126.23.1652.1652 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1652-1652

Author(s):

Eiju Negoro ◽

Naoko Hosono ◽

Wenyi Shen ◽

Tetsuichi Yoshizato ◽

Bhumika J. Patel ◽

...

Keyword(s):

Aplastic Anemia ◽

Board Of Directors ◽

Somatic Mutations ◽

De Novo ◽

Initial Presentation ◽

Progression Rate ◽

Advisory Committees ◽

Increased Risk ◽

Slow Expansion ◽

Secondary Mds

Abstract Historically, the evolution rate of aplastic anemia (AA) to MDS approaches 15% in 10 yrs; thus, AA can be considered a major predisposition factor for secondary MDS (sMDS). The likely etiology includes expansion of a preexisting clone or truly late clonogenic events. In both instances, progression can be a result of a clonal escape, but confirmation of the presence of mutant cells at presentation would indicate that initial autoimmune processes may represent a tumor surveillance reaction. We studied 326 patients with AA and 47 patients with paroxysmal nocturnal hemoglobinuria (PNH) and identified 36 cases (progression rate: 11% in median follow up of 6 years) that evolved to MDS or AML (median time to progression: 3.2 yrs.; transplanted patients were not censored). Cytogenetic analysis upon progression showed abnormal karyotype in 83% of cases; 7% had complex karyotype and -7/del(7q) was present in 62% of cases. The presence of a PNH clone was detected in 17% of cases that transformed to sMDS vs. 35% in non-progressors (P=.1). For comparison, we have also analyzed primary de novo cases of MDS (pMDS) with (N=94) and without (N=557) -7/del(7q). In contrast to sMDS, -7/del(7q) was present in 14.4% of cases in pMDS. Because sMDS following AA or PNH included a high proportion of patients with -7/del(7q), we compared sMDS with -7/del(7q) to pMDS with -7/del(7q) for coexisting mutational events. Mutations in RUNX1, CBL, SETBP1 and ASXL1 appeared to be more frequent in sMDS vs. pMDS (28.6% vs. 2.1%, 21.4% vs. 2.1%, 21.4% vs. 5.3%, 21.4% vs. 10.6%, P=.003, P=.02, P=.07, P=.37, respectively). In contrast, TP53 and DMT3A were more common in pMDS (7.1% for sMDS vs. 17%, 0% for sMDS vs. 8.5%, P=.69, P=.59). Similarly, there were several other distinctive differences between all sMDS and pMDS irrespective of the cytogenetics: mutations in SF3B1, SRSF2, NPM1, DNMT3A were common in primary AML but entirely absent from cases after AA; mutations in RUNX1 and SETBP1 appeared to be more frequent in sMDS vs. pMDS (26.3% vs. 8.3%, 21.1% vs. 3.2%, 15.8% vs. 3.9%, P=.03, P=.005, respectively). Whole exome NGS was performed after progression, with confirmed somatic mutations subsequently tracked back by targeted deep NGS applied to serial samples starting at initial presentation. Confirmed mutational events and chromosomal aberrations were found in 19/36 patients with sMDS; 17/19 cases of sMDS had at least 1 confirmed somatic mutation. Remarkably, in retrospective analysis in 6/7 cases studied serially, at least one of the identified mutations was detectable at presentation when deep targeted sequencing (depth 5,000~20,000 reads) was performed. In 5 of these cases the alterations appeared to be ancestral events for sMDS evolution. When anadditional 77 AA or PNH cases were studied by deep sequencing, somatic mutations were present in 48% of AA patients at presentation. Detection of clonal events at presentation was associated with an increased risk of subsequent MDS evolution (14/37 mutant cases vs. 3/40 nonclonal cases evolved, P=.002). Mutations found at both initial presentation and upon evolution were suggestive of a slow expansion of previously cryptic clones (ASXL1, CUX1, TET2, CBL, RUNX1, and SETBP1). Patients with these genes (n=18) had worseoverall survival compared to patients without these mutations (P=.03). To assess the potential impact of immunosuppressive therapies (IST), we also investigated a subset (out of 77) of 53 patients (39 responders and 14 refractory cases) following IST. Clonal somatic events were identified in 27 of them, but there was no association between the response to IST and somatic mutations at presentation. Our results demonstrate that while subclonal mutations indicative of oligoclonal hematopoiesis are frequent in AA, the presence of permissive ancestral somatic events at the outset of AA predisposes patients to sMDS, a feature that had diagnostic and prognostic implications. Disclosures Sekeres: Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees.

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Overall Survival in Patients with JAK2 and ASXL1 Positive Myeloproliferative Neoplasms

Blood ◽

10.1182/blood-2019-128309 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5383-5383

Author(s):

Murtadha Al-Khabori ◽

Shoaib Al-Zadjali ◽

Iman Al Noumani ◽

Khalil Al Farsi ◽

Salam Al-Kindi ◽

...

Keyword(s):

Overall Survival ◽

Board Of Directors ◽

Myeloproliferative Neoplasms ◽

Prognostic Significance ◽

Jak2 V617f ◽

World Health ◽

Prognostic Impact ◽

Advisory Committees ◽

Myeloid Neoplasms ◽

The Impact

Objectives: Mutations in additional sex combs-like transcriptional regulator 1 (ASXL1) have been previously described in myeloid neoplasms (21% in non-Myeloproliferative [MPN; Tefferi A, Leukemia, 2010) and have been associated with a more aggressive disease [Rocquain J et al, BMC Cacer, 2010]. They can also be found in patients with JAK2 positive MPN [Abdel-Wahab O et al, Cancer Research, 2010). Disruption of ASXL1 gene leads to MPN phenotype in zebrafish model (Gjini E, Dis Model Mech, 2019). The co-expression and the prognostic significance of ASXL1 in patients with JAK2 positive MPN are not yet fully defined. We therefore planned to define the prognostic impact of ASXL1 mutations on the Overall Survival (OS) of patients with JAK2 positive MPN. Methods: We included patients with JAK2 V617F positive MPN diagnosed according to the World Health Organization 2016 criteria and treated at the three largest hematology centers in Oman. The entire coding region of ASXL1 gene was sequenced using Next Generation Sequencing (NGS; Ion PGM Sequencer; Thermo Fisher Scientific®). The library was constructed and the templates were prepared using the PGM tool and the variants were annotated using the ClinVar database and the prediction from the Scale-Invariant Feature Transform (SIFT) and or Polymorphism Phenotyping (Polyphen) algorithms. The NGS analysis was done on the frozen diagnostic bone marrow samples. The survival probability was estimated using Kaplan-Meier estimator and Cox regression was used to assess the impact of predictors on the OS outcome. An alpha threshold of 0.05 was used. The R program (version 3.1.2) was used for all statistical analyses. Results: A total of 58 patients with JAK2 V617F positive MPN were included. All of these patients were found to have mutated ASXL1 using the NGS (ASXL1 p.Leu815Pro was found in all patients). The median age of this cohort was 62 years (InterQuartile Range [IQR]: 44 - 70) and female to male ratio was 25:33. The median hemoglobin, hematocrit, white blood cell count and platelet count was 14.7 g/dL, 58%, 11.5 x109/L and 518 x109/L respectively. Out of the 58 patients included, 28 had polycythemia vera, 20 had essential thrombocythemia, 8 had myelofibrosis and 2 had MPN-Unclassified. The median time from diagnosis to last follow up or death was 13 months (IQR: 3-39). During this period, 5 patients died. The probability of OS at 3 years was 88%. The median OS was not reached. In the univariable analysis, age was a statistically significant predictor of OS (p = 0.0355) but not gender (p = 0.434) and MPN subtype (p = 0.7). In the multivariable analysis model of the previous three factors, age remained statistically significant (Hazard ratio = 1.13, p = 0.041). Conclusions: ASXL1 is mutated in high proportion of patients with JAK2 positive MPN. Despite the negative impact of ASXL1 in patients with non-MPN myeloid neoplasms, the patients with combined positivity of JAK2 and ASXL1 in this study had a very good OS probability. Age was a predictor of OS in the univariable and multivariable models. We recommend the development and the assessment of ASXL1 inhibitors as therapeutic strategies in patients with MPN. Disclosures Al-Khabori: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; NovoNardisk: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Shire (Takeda): Membership on an entity's Board of Directors or advisory committees; SOBI: Honoraria; AstraZeneca: Honoraria; Abbvie: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

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Impact of Non-JAK2 Molecular Mutations and Cryptic SNP Lesions in Myelofibrosis Patients Treated with Ruxolitinib

Blood ◽

10.1182/blood.v124.21.3194.3194 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3194-3194

Author(s):

Jing Ai ◽

Valeria Visconte ◽

Ali Tabarroki ◽

Ahmad Zarzour ◽

Christopher Gerace ◽

...

Keyword(s):

Board Of Directors ◽

Dose Escalation ◽

Somatic Mutations ◽

Similar Response ◽

Prognostic Impact ◽

Severe Thrombocytopenia ◽

Spleen Size ◽

Advisory Committees ◽

Myeloid Neoplasm

Abstract The identification of the JAK2V617F mutation in myeloproliferative neoplasms (MPN) paved the way for the pivotal studies that led to the FDA approval of a JAK1/2 inhibitor, ruxolitinib (rux) in patients (pts) with myelofibrosis (MF). Improvement in splenomegaly and debilitating disease-related symptoms were the primary clinical responses observed with rux. Although JAK2 mutational status did not impact response/survival in MF pts, cytogenetics had an impact on prognosis. In a related myeloid neoplasm specifically myelodysplastic syndromes, molecular mutations (TET2/DNMT3A) predict for better therapeutic response to DNA methyltransferase inhibitors. We hypothesized that somatic mutations and single nucleotide polymorphism array (SNP-A) lesions are frequent in MF pts treated with rux and may affect their clinical outcomes. To further investigate the predictive and prognostic impact of SNP-A lesions and somatic mutations in MF pts in the rux era, we studied 54 MF pts who received at least 12 weeks of rux therapy (tx) using a modified dose escalation approach (Tabarroki A et al. 55th ASH; Abstract 1586). Clinical (total symptom score [TSS], spleen size), cytogenetic (metaphase cytogenetics [MC], SNP-A), hematologic and survival data were collected before and 12-weeks post rux tx. Categorical data were analyzed using X2 test. A p-value of <.05 was considered statistically significant. Sanger sequencing for genes relevant to myeloid neoplasm pathophysiology like TET2, CBL, LNK, DNMT3A, TP53, SF3B1, U2AF1, SRSF2, ASXL1, EZH2, JAK2, CALR, and IDH1/2 was performed. The median age of the cohort was 66 yrs (41-89); male/female: 28/26. The median follow-up time after initiation of tx was 17 months. The median overall follow-up of the cohort from the time of diagnosis was 35 months. Using DIPSS-plus, pts were stratified as high (24, 44%), int-2 (22, 41%) and int-1 (8, 15%) risk groups. Baseline median WBC=9.4k/μL, Hgb=10.2g/dL, PLT=212k/μl, TSS=20, and median palpable spleen size=13cm. Post-tx median WBC=9.9k/μL, Hgb=10.1g/dL, PLT=150k/μL, TSS=4, and spleen size=6cm. MC identified cytogenetic abnormalities in 24/54 (44.4%) pts. The most frequent chromosomal defects included del(20), +8, and +9. Serial MC was available for 20 pts and no cytogenetic evolution was identified. SNP-A data were available for 29 pts, of which 28 pts had SNP-A lesions. The most commonly involved chromosomes were 9 (15.1%), 20 (14.1%), and 14 (8.5%). Compared to MC analysis, additional SNP-A lesions were found in 66% of pts. Of note 39% of the pts had normal karyotypes but with pathologic SNP-A lesions; another 27% had pathologic SNP-A lesions besides the abnormal MC. Serial SNP-A analysis was available in 10 pts who while on rux tx did not develop any additional/new SNP-A lesion. There was no difference in spleen response rates or TSS between those who carried SNP-A lesions versus those who did not. Molecular analysis was possible for 34 pts. The most frequent somatic mutations observed involved JAK2 (70.6%), ASXL1 (24%), CALR (24%), SRSF2 (15%), and U2AF1 (9%). Pts with int-2 and high DIPSS plus scores were more likely to carry at least 1 mutation in any gene compared to pts with int-1 scores (int-2 vs int-1, p=.05; high vs int-1, p=.06). After a median follow-up of 35 months from diagnosis, 95% of the pts were still alive. 3 pts died from disease progression: 1 had a sole SRSF2 mutation, 1 had an SRSF2 plus CALR mutation, and 1 had a TET2 plus TP53 mutation. SRSF2 mutant pts had more severe thrombocytopenia pre-rux tx (91 vs. 203k/μL; p=.04). ASXL1 mutant pts had increased spleen sizes pre-rux (21 vs. 15cm, p=.06), but had similar response post-rux (10 vs. 8cm, p=0.6) compared to the wild-type. SRSR2 mutant pts had higher DIPSS-plus score (4.4 vs. 3; p=.05). Our study showed that MF pts treated with a rux modified dose escalation approach resulted in meaningful clinical and splenic responses regardless of molecular mutation status. Frequently found cryptic SNP-A lesions in MF pts may explain their poorer outcomes compared to pts with other MPNs. The fact that pts did not acquire additional/new SNP-A lesions during rux tx may be one of the mechanisms of improved survival in these pts. ASXL1, CALR, SRSF2 and U2AF1 were the most frequent non-JAK molecular mutations in MF pts treated with rux and were more frequent in high risk pts. Further studies are necessary to elucidate the clinical/ biological effects of these mutations in MF pts treated with rux. Disclosures Tiu: Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees.

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Impact and Function of Somatic PHF6 Mutations in Myeloid Neoplasms

Blood ◽

10.1182/blood.v124.21.3581.3581 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3581-3581

Author(s):

Brittney Dienes ◽

Bartlomiej P Przychodzen ◽

Michael Clemente ◽

Wenyi Shen ◽

Chantana Polprasert ◽

...

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Board Of Directors ◽

Somatic Mutations ◽

Suppressor Gene ◽

Advisory Committees ◽

Nonsense Mutations ◽

Dysmorphic Features ◽

Normal Bone ◽

Myeloid Neoplasms

Abstract Borjeson−Forssman−Lehmann syndrome (BFLS), a hereditary X-linked disorder characterized by mental retardation, truncal obesity, gynecomastia, hypogonadism and other dysmorphic features, is known to be caused by germline (GL) mutations of plant homeo domain finger protein 6 (PHF6). PHF6 is a highly conserved 41kDa protein showing ubiquitous expression in a variety of tissues, including bone marrow, CD34+ cells and blood leukocytes. Human PHF6 is located on chrXq26.2. Recently, rare somatic nonsense mutations and deletions have been detected in patients with T-ALL and AML and found in some T-ALL cell lines. Patients with BFLS with PHF6 mutations have been reported to develop leukemia, suggesting PHF6 mutations may predispose cancer. Although the actual function and molecular pathogenesis is unknown, PHF6 has been suggested to be a tumor suppressor gene involved in the control of myeloid development. In an index case of a young adult female patient with proliferative CMML with dysmorphic features, we have identified remarkable GL mosaicism for PHF6 mutation (p.K44fs), confirmed by deep sequencing of marrow, CD3+ cells and skin tissue. Subsequently, we screened patients with myeloid neoplasms by targeted multi-amplicon sequencing to determine the prevalence and distribution of PHF6 gene alterations. Sequencing results from 1072 cases were analyzed (728 by targeted deep sequencing and 344 by whole exome sequencing). In total, we identified 21 cases with PHF6 mutations, 13 of which were frameshift or nonsense mutations. Previously, PHF6 have been included in screening panels by Haferlach et al., (Leukemia 2014) and Papaemmanuil et al., (Blood 2013) and somatic mutations were found in 24/944 and 21/738 cases of MDS, respectively. These results along with ours suggest that PHF6 mutations are common driver events. The somatic nature of these defects was confirmed by analysis of non-clonal CD3+ lymphocytes, thus, PHF6 mutations occur at a frequency of 2.0% and are most frequently observed among patients with secondary AML (33%, P=.0021). Gender distribution showed a strong male predominance (76%), likely due to the location of PHF6 on chrX and indicating that retention of a single copy of PHF6 may be protective. SNP-array karyotyping showed that deletions of Xq, involving the PHF6 locus (Xq26), were present in about 1.2% of myeloid neoplasms and affect only female patients. As a family, plant homeo domain (PHD) finger genes are affected by mutations associated with various cancers. JARID1A, PHF23, NSD1 and NSD3 were described to serve as fusion partners with the NUP98 in a subset of AML cases. The most frequent chromosomal aberration observed in conjunction with PHF6 mutations was trisomy-8 (P=.08). The most commonly associated somatic mutations were in RUNX1 (N=7; P=.001), U2AF1 (N=5), ASXL1 (N=5), IDH1 (N=4), and DNMT3A (N=4). Interestingly, 6/7 cases with concomitant PHF6 and RUNX1 mutations showed a poor prognosis AML. Subsequent analysis of clonal architecture using variant allelic frequency calculations and serial samples for these cases suggested that PHF6 may function as a founder driver gene while RUNX1 mutations are acquired as secondary events. Recent studies proposed that PHF6 deficiency leads to impaired cell proliferation, cell cycle arrest at G2/M phase and an increase of DNA damage. To examine DNA damage and quantify double stranded breaks (DSBs) in primary cells from PHF6-mutants, those with wild-type (WT) PHF6 and normal bone marrow we used a flow cytometric anti-γH2AX assay, following induction of DNA damage with Camptothecin. As judged by greater percentages of anti-γH2AX labeled cells, DSBs were more common in mutant cases consistent with more DNA damage present in PHF6 mutant compared to WT MDS and normal bone marrow cells. In conclusion, our results indicate that PHF6 mutations are generally present in more aggressive types of myeloid neoplasms, frequently associated with RUNX1 mutations. Our functional in vitro studies along with recently published reports suggest an association of PHF6 deficiency with genomic instability and thereby provide a basis for a mutator phenotype conveyed by ancestral lesions, consistent with its role as a tumor suppressor gene. Disclosures Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen Corp: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim Corp: Membership on an entity's Board of Directors or advisory committees.

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Mutation Profiling of Therapy-Related Myeloid Neoplasms Using Next-Generation Sequencing Demonstrates Distinct Profiles from De Novo Disease

Blood ◽

10.1182/blood.v124.21.4611.4611 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4611-4611

Author(s):

Alan H. Shih ◽

Franck Rapaport ◽

Stephen S. Chung ◽

Emily K Dolezal ◽

Sean Hobson ◽

...

Keyword(s):

Next Generation Sequencing ◽

Board Of Directors ◽

Tp53 Mutation ◽

Somatic Mutations ◽

De Novo ◽

Next Generation ◽

Advisory Committees ◽

Myeloid Neoplasms ◽

Mutation Profile ◽

Generation Sequencing

Abstract Therapy-related Myeloid Neoplasms (tMN) comprise a poor risk subset of myelodysplastic syndromes and acute myelogenous leukemia, are increasing in incidence, and represent a serious complication following treatment for primary malignancies. In our previous study of 11 genes in 38 tMN patient samples, the data suggested that the mutational spectrum of tMN was distinct from de novo myeloid malignancies. To confirm this finding and to refine the tMN mutation profile, we investigated the mutation profile in samples from 88 patients and 28 genes using Sanger and next-generation sequencing approaches. We performed amplification using RainDance microfluidic PCR, followed by HiSeq next-generation sequencing. Mutations were identified using a modified pipeline for SNP calling employing variant detection software programs. Our study cohort included 88 patients, 71 of whom had complete clinical data for analyses. Patients had a history of epithelial and hematologic malignancies (³2 malignancies n=11; breast n=9; colorectal n=5; head and neck n=4; genital-urinary n=6; lung n=1; lymphoma n=25; melanoma n=2; ovarian n=1; sarcoma n=2; other, n=5). Treatment of primary cancers included chemotherapy alone (n=27), radiation alone (n=8), autologous stem cell transplant (n=11), or chemotherapy plus radiation (n=25). The median latency time between primary malignancy treatment and tMN diagnosis was 5.7yrs (range, 0.7 - 30.9 yrs). Median age at tMN diagnosis was 64yrs (range, 26 - 85 yrs). International Prognostic Scoring System (IPSS) risk group for MDS at tMN diagnosis were Low risk (n=8), Int-1 (n=11), Int-2 (n=30), High risk (n=9). We identified somatic mutations in 56 of 88 (64%) patients (83 patients were evaluated by next-generation sequencing and 5 by Sanger sequencing only). Mutations in TP53 were most common and were detected in 27/88 patients (30.7%), followed by mutations in TET2 in 12/88 (13.6%), DNMT3A in 9/88 (10.2%), NRAS in 8/83 (9.6%), KRAS in 5/83 (6.0%), and KIT in 5/83 (6.0%). Gene mutations detected at lower frequencies included those in ASXL1 in 5/88 (5.7%), RUNX1 in 2/83 (2.4%), EZH2 in 1/88 (1.1%), and SF3B1 in 1/88 (1.1%). Of the 58 patients with complete sequencing and FISH data, 4 patients exhibited biallelic somatic TP53 mutations and 3 patients had TP53 mutation combined with del 17p TP53 loss, demonstrating that 7 of 58 evaluable patients (12.1%) experienced biallelic loss of TP53. We also identified biallelic mutations in TET2 and DNMT3A in 2 separate patients. 25 patients had 2 or more concurrent somatic mutations. The highest number of co-occurring mutations in one patient was 5 mutations; 12 patients had 2 somatic mutations. The most common co-occurrence was TP53 and TET2, which was observed in 5 patients. All 5 ASXL1 mutations co-occurred with additional mutations. By analyzing variant allele frequencies (VAFs) in patients with multiple mutations, we observed that some tMN patients harbored multiple clones with distinct VAFs. This observation was also supported by the co-occurrence of typical class I driver mutations in the same patient, (e.g. KRAS 6% and NRAS 21% VAF; NRAS 9% and KIT 34%; NRAS 26% and KIT 9% in individual patients). The allele frequency data also suggested that ASXL1 is likely an early occurring mutation as the VAF was higher than for other co-occurring mutations (mean VAF ASXL1 50%, other co-occurring genes 23.5%, p<.05 t-test). Because of previous reports on the prognostic significance of point mutations in myeloid malignancies (e.g. TP53 in MDS and TET2 in AML), we tested the impact of individual mutations on prognosis. TP53 mutation or loss was associated with worse prognosis in tMN (OS 17.6 vs 25.2 mos, n=72, p<.11 log-rank test) (Fig A). TET2 mutation and KRAS or NRAS mutations did not predict for a difference in prognosis, although analysis was limited by cohort size. TP53 mutation was also associated with del 5q / monosomy 5 (p<.0001, Chi-square test, n=51). Our data reveal that tMNs display distinct mutation profile compared to de novo disease (Fig B). TP53 mutations and loss are the most common abnormalities and predict for adverse outcome. Epigenetic modifier mutations also occur in tMNs and can serve as disease-initiating mutations. Collectively our results demonstrate that characterizing these mutation profiles can enhance our understanding of disease mechanisms in tMNs and may guide the development of future therapies for these difficult to treat disorders. Figure 1 Figure 1. Disclosures Sekeres: Celgene Corp.: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees.

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Clinical and Biological Implications of CUX1 Mutations in Myeloid Neoplasms

Blood ◽

10.1182/blood.v128.22.3156.3156 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3156-3156

Author(s):

Mai Aly ◽

Naoko Hosono ◽

Przychodzen Bartlomiej ◽

Hideki Makishima ◽

Nagata Yasunobu ◽

...

Keyword(s):

Board Of Directors ◽

Lower Risk ◽

Somatic Mutations ◽

Myeloproliferative Neoplasms ◽

Loss Of Function ◽

Wild Type ◽

Advisory Committees ◽

Molecular Features ◽

Myeloid Neoplasms ◽

Normal Cytogenetics

Abstract Recurrent somatic mutations of CUX1 are described in myeloid neoplasms. CUX1 is located at chromosome 7q22.1; -7/del(7q) involving CUX1 locus are common abnormalities in myelodysplastic syndromes (MDS). Mutations and loss of heterozygosity involving CUX1 have been also described in breast, lung and uterine cancers. Preliminary functional studies, lack of a mutational hotspot and coincidental deletions suggest loss of function/hypomorphic consequences of these molecular defects. CUX1 (p200), contains 4 evolutionarily conserved DNA-binding domains, including 3 CUT repeats and a CUT homeodomain. Functionally, CUX1 regulates many genes involved in DNA replication and chromosome segregation. Cell-based assays have established a role for CUX1 in the control of cell-cycle progression, cell motility, and invasion .The objective of this study is to assess the molecular context and clinical significance of CUX1 mutations and deletions in myeloid neoplasms. We analyzed a subset of 1478 patients [24% lower-risk MDS, 17% higher-risk MDS, 22% primary (p)AML, 14% secondary AML, 14% MDS/myeloproliferative neoplasms (MPN) and 9% MPN] for the presence of CUX1 mutations and deletions. No CUX1 mutations were found in core binding factor AML. We correlated the presence of these lesions with clinical parameters, cytogenetic abnormalities, and molecular features including clonal architecture and associated somatic mutations. Copy number variation and their boundaries were analyzed by Single Nucleotide Polymorphism (SNP) arrays and mutations by multiamplicon deep sequencing utilizing a panel targeting 60 most commonly mutated genes in myeloid neoplasms. In total cohort 4 % of patients had CUX1 mutations and 6% had locus deletions (affecting ch 7q commonly deleted region: 7q22.1) including 90% of del (7q) cases. Expression of CUX1 is significantly lower in AML with -7/del(7q) compared to AML with normal cytogenetics (p<.00001) and also in MDS with -7/del(7q) compared tohealthy controls (p=.004). Additionally, decreased expression of CUX1 was found in 15% of MDS and 8% of AML patients without -7/del(7q) or related mutations. Cases with lower expression had worse OS compared to patients with higher expression (p=.002). In terms of configuration, most mutations were heterozygous, 5% of mutations were hemizygous and 4% were homozygous (due to UPD). Among 75 somatic CUX1mutations; 72% were missense, 20% where frame shift and 8% where non sense. CUX1 mutations were associated with either lower-risk MDS (p=.0001) and pAML (p=.04) while deletions involving the CUX1 locus were significantly related to higher-risk MDS (p=.05). Heterozygous CUX1 mutations were more commonly associated with normal cytogenetics (p=.01). Patients with -7/del(7q) frequently represented del(5q) (p=.04) and thrombocytopenia (p=.001). The OS of patients with CUX1 mutations was shorter (p=.04) as was that of patients with CUX1/deletions (p=.02) when compared to wild type. We subsequently studied the molecular background of CUX1 alterations. CUX1 mutations (vs. wild type) were associated with TET2 (31% vs. 14%, p=.006), ASXL1 (29% vs. 9%, p=.0005), BCOR (28% vs. 8%, p=.0004), and cohesion mutations (26%, vs. 5%, p=.0005), while NPM1 mutations showed the reverse relationship (1% vs. 7%, p=.03). RAS and CUX1 mutations were mutually exclusive (0% vs. 6%, p=.03). When we analyzed clonal hierarchy in the context of CUX1 mutations; dominant CUX1 mutations (24%; mean VAF=49%); were accomplished by ASXL1 (21%) and SRSF2 (14%) mutations which were the most common secondary events in this context. Phenotypically, dominant CUX1 mutations were associated with MDS/MPN (42%) and MDS (33%). 14% of CUX1 mutant cases did not harbor any other alterations and were not associated with a discernable phenotype. Secondary CUX1 lesions (62%; mean VAF=22%) were found in the context of dominant TET2 mutations (16%). The pathomorphologic context of secondary CUX1 mutation did not differ from that of primary lesions. AML seemed to be underrepresented (p=.006) and MPN overrepresented (p=.019) among dominant CUX1 mutant cases. In conclusion, CUX1 lesions including locus deletions with haploinsuffciency, mutations and a fraction of cases with decreased CUX1 expression can be encountered in MDS and related neoplasms, chiefly AML. CUX1 dysfunction is associated with poor survival likely due to its distinct molecular background. Disclosures Makishima: The Yasuda Medical Foundation: Research Funding. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

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Clinical Impacts of Germline DDX41 Mutations on Myeloid Neoplasms

Blood ◽

10.1182/blood-2020-140174 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 38-40

Author(s):

Hideki Makishima ◽

Yasuhito Nannya ◽

June Takeda ◽

Yukihide Momozawa ◽

Ryunosuke Saiki ◽

...

Keyword(s):

Board Of Directors ◽

Somatic Mutation ◽

Ethnic Diversity ◽

Research Funding ◽

Somatic Mutations ◽

Healthy Individuals ◽

Advisory Committees ◽

Missense Variants ◽

Myeloid Neoplasms ◽

Sporadic Cases

DDX41 was identified as a causative gene for late-onset familial myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). While DDX41 is thought to be one of the most frequent targets of germline mutations responsible for sporadic cases with AML/MDS and other myeloid neoplasms, the entire spectrum of pathogenic DDX41 variants and their effect size therein are still to be elucidated, and so was the clinical picture of DDX41-mutated myeloid neoplasms. In this study, through an international collaboration, we investigated DDX41 variants in a total of 5,609 sporadic cases with different myeloid neoplasms from different ethnicities, using next generation sequencing. Mutations in the major driver genes commonly mutated in AML/MDS were also examined. Frequencies of germline DDX41 variants were compared between sporadic cases with myeloid neoplasms and healthy individuals (n=13,906). We also characterized genetic/clinical features of DDX41-mutated myeloid neoplasms. We identified a total of 208 (3.6%) patients with DDX41 variants, of whom approximately 50% had both germline and somatic mutations, whereas 37% and 13% had either germline or somatic mutations alone, respectively. Somatic mutations were found in 58% of patients with germline mutation, which was significantly higher than those without (0.21%) (P<0.0001). No somatic mutation was identified in healthy individuals. Among 174 germline variants, truncating and missense mutations were found in 93 and 81 cases, respectively, whereas only 1.9% of somatic mutations were truncating (P<0.0001). Among 21 cases with somatic mutations alone, 4 had multiple somatic mutations and an additional 4 had loss-of-heterozygosity of the DDX41 locus (5q35.3), including 3 with uniparental disomy and 1 with deletion. Thus, 8 out of 21 cases with somatic mutation alone were suspected to have biallelic DDX41 mutations. Germline DDX41 variants showed a conspicuous ethnic diversity; the most frequent germline variants were A500fs in Japan, D140fs in USA, Q41* in Germany, G218D in Italy, M1I in Sweden, S21fs in Thailand. The M1I variant was also seen in other European countries, but not in Japan or Thailand, while no A500fs mutation was found in Europe. Among the Japanese population, significant enrichment in myeloid neoplasms was observed not only for truncating variants, such as A500fs (odds ratio (OR)=12.1) and E7X (OR=11.0) but also for missense variants, including Y259C (OR 14.3) and E256K (OR 7.81), frequently accompanied by a somatic DDX41 mutation (Figure 1). Patients with germline and/or somatic DDX41 variants were significantly older than those without (P=0.00076) and more prevalent in male than female (OR=3.14; P<0.0001). DDX41 variants were significantly more frequent in MDS (4.7%) and AML (2.9%), compared with other myeloid neoplasms (0.58%). Among AML, mutations were more frequent in AML with myelodysplasia-related changes (P<0.00001). Patients with MDS having both germline and somatic mutations were more likely to classified in refractory anemia with excess blasts (RAEB), compared with those with germline or somatic alone (P=0.029). DDX41 variants were significantly associated with lower WBC and granulocyte counts. Most frequently co-occurring mutations included those in ASXL1, SRSF2, TET2, CUX1, and DNMT3A, of which only CUX1 mutations were statistically significant. Overall, no difference was observed in overall survival (OS) between DDX41-mutated and unmutated cases. However, among RAEB cases, DDX41 variants were associated with a significantly longer OS (P=0.0039). In summary, the majority of DDX41-mutated cases had a germline variant, although a minority had somatic mutations alone. Pathogenic DDX41 alleles have a large ethnic diversity, where not only truncating variants but also missense variants are associated with an increased risk of the development of myeloid neoplasms. Disclosures Kanda: Chugai Pharma: Honoraria, Research Funding; Merck Sharp & Dohme: Honoraria; Mundipharma: Honoraria; Ono Pharmaceutical: Honoraria; Nippon Shinyaku: Honoraria, Research Funding; Takeda Pharmaceuticals: Honoraria; Alexion Pharmaceuticals: Honoraria; Shire: Honoraria; Mochida Pharmaceutical: Honoraria; Daiichi Sankyo: Honoraria; Shionogi: Research Funding; Meiji Seika Kaisha: Honoraria; Sanofi: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Janssen: Honoraria; Pfizer: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Novartis: Honoraria; Kyowa Kirin: Honoraria, Research Funding; Astellas Pharma: Honoraria, Research Funding. Miyazaki:NIPPON SHINYAKU CO.,LTD.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria; Novartis Pharma KK: Honoraria; Astellas Pharma Inc.: Honoraria; Otsuka Pharmaceutical: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Celgene: Honoraria. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria. Ogawa:Otsuka Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai Co., Ltd.: Research Funding.

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