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

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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Impact of Eltrombopag on Expansion of Clones with Somatic Mutations in Refractory Aplastic Anemia

Blood ◽

10.1182/blood.v126.23.300.300 ◽

2015 ◽

Vol 126 (23) ◽

pp. 300-300 ◽

Cited By ~ 1

Author(s):

Bhumika J. Patel ◽

Bartlomiej Przychodzen ◽

Michael J. Clemente ◽

Cassandra M. Hirsch ◽

Tomas Radivoyevitch ◽

...

Keyword(s):

Aplastic Anemia ◽

Board Of Directors ◽

Deep Sequencing ◽

Somatic Mutations ◽

Clonal Evolution ◽

Clonal Expansion ◽

Quantitative Detection ◽

Sequencing Analysis ◽

Advisory Committees ◽

Before And After

Abstract Despite documented success of immunosuppressive therapy (IST) in the treatment of aplastic anemia (AA), a significant minority of patients remain refractory, most responses are incomplete, and allogeneic stem cell transplantation is not available for older patients or those with significant comorbidities. Until introduction of the cMpl agonist eltrombopag, anabolic steroids were the most commonly used salvage drugs. At least theoretically, engaging growth factor receptors with eltrombopag has the potential to promote the evolution or expansion of mutant clones and thereby increase the rate of progression to secondary MDS, a feared complication of AA occurring in 10-20% of patients. Recently we and others reported detection of clonogenic somatic mutations typical of MDS in patients with AA and PNH. Subsequent study demonstrated that mutations characteristic of sMDS can be found in some patients at presentation of AA and may constitute risk for subsequent progression to MDS. As the risk of MDS evolution was a prominent concern when filgrastim was more widely used in management of AA, now similar questions have been raised regarding use of eltrombopag, be it as salvage therapy or to complement IST. Recently, one of our primary refractory patients receiving eltrombopag progressed to AML. This clinical observation led to investigation of the impact of eltrombopag on evolution and clonal expansion using deep sequencing of a cohort of patients with AA. DNA from bone marrow cells was sequenced before and after initiation of eltrombopag to evaluate clonal expansion or evolution using a targeted multi-amplicon deep sequencing panel of the top 60 most commonly mutated genes in MDS. Among 208 AA patients treated at Cleveland Clinic, we identified 13 patients (median age 68 yrs.) who were treated with eltrombopag for IST-refractory AA; median duration of treatment was 85 wks. The overall response rate, defined as sustained improvement in blood counts and transfusion independence after 12 weeks of therapy, was 46% (6/13), while 38% (5/13) of patients showed stable disease with intermittent transfusions (one of whom underwent HSCT). Among the two non-responders, one patient developed a PNH clone and another progressed to AML (see below). Expansion of PNH granulocytes after eltrombopag treatment was observed in 2 patients. Two patients had chromosomal abnormalities at initial diagnosis, one with t (10; 18) in 2 metaphases, and one with an extranumeral Y chromosome. Use of next generation sequencing (NGS) allows for the quantitative detection of clonal events. We hypothesized that serial analysis by NGS before and after eltrombopag therapy may provide clues as to potential effects of this drug on clonal evolution. Sequencing analysis before eltrombopag treatment revealed the presence of a sole clonal mutational event in 3/13 cases, including CEBPA, EZH2, and BCOR. In the patient with a CEBPA mutation, the mutation persisted during treatment with minimal clonal expansion evidenced by a change in VAF from 53% to 65%. In the second patient, NGS results revealed the initial presence of an EZH2 mutation. A post eltrombopag sample clearly identified acquisition of additional clonal events in genes highly associated with advanced disease and clonal evolution (RUNX1 and U2AF1), as well as slight expansion of a persistent EZH2 clone from 2 to 8%. The third patient harbored a BCOR mutation which expanded markedly, increasing from 8% to 21%, and was accompanied by a hematological response. Sequencing results after eltrombopag treatment revealed the acquisition of new somatic mutations in 5/13 (38%) cases: 2 new CEBPA mutations, 1 new BCOR mutation, and, as discussed, one case with an initial EZH2 mutation in which RUNX1 and U2AF1 mutations were later discovered. In the 5th patient, evolution to AML was observed and accompanied by a large DNMT3A and U2AF1 clone that was absent on initial evaluation. In conclusion, we did observe occasional expansion of clones with potentially leukemogenic mutations during treatment with eltrombopag. At our institution a case control study of patients with refractory aplastic anemia without treatment with eltrombopag is ongoing; ideally a prospective trial would be needed to confirm results. Our results suggest that the initial detection of certain somatic mutations (CBL, SETBP1 and RUNX1) associated with post-AA MDS may contraindicate use of eltrombopag in AA. 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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Blinatumomab Associated Seizure Risk in Patients with Down Syndrome and B-Lymphoblastic Leukemia: An Interim Report from Children's Oncology Group (COG) Study AALL1731

Blood ◽

10.1182/blood-2021-148967 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2304-2304

Author(s):

Amanda M. Li ◽

Karen R Rabin ◽

John Kairalla ◽

Cindy Wang ◽

Meenakshi Devidas ◽

...

Keyword(s):

Down Syndrome ◽

Board Of Directors ◽

De Novo ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Early Report ◽

Advisory Committees ◽

Neurologic Recovery ◽

Increased Risk ◽

Pre Treatment

Abstract INTRODUCTION Children with Down Syndrome (DS) and B-lymphoblastic leukemia (B-ALL) are at an increased risk of both relapse and treatment-related mortality, compared to those without DS. On COG study AALL1731 for de novo B-ALL, patients with DS and higher risk features (DS-High) are non-randomly treated with a regimen replacing intensive elements of conventional chemotherapy with three 28-day cycles of blinatumomab, with the combined goals of reducing toxicity and enhancing anti-leukemic efficacy. The DS-High group includes all NCI high risk (HR) patients; NCI standard risk (SR) patients with end-induction minimal residual disease positivity (>0.01%), unfavorable cytogenetics, CNS3 status, steroid pre-treatment, neutral cytogenetics with CNS2 status, or testicular disease. Neurotoxicity is a known risk of blinatumomab, with an incidence of 4% in block 1 and 1% in block 2 among pediatric patients with relapsed ALL (Brown et al, JAMA 2021). However, the specific risk in patients with DS has not been described to date. Here, we provide an early report of increased seizure incidence associated with blinatumomab in older DS-High patients enrolled on AALL1731 to date. METHODS We reviewed seizure incidence among patients with DS enrolled on AALL1731 from June 2019 to June 2021 who had proceeded to receive blinatumomab. Blinatumomab was administered at a dose of 15 mcg/m 2/day, using dexamethasone pre-medication in cycle 1. Infusions were interrupted for seizures, with resumption at 5 mcg/m 2/d permitted following full resolution for grade 1-3 seizures. RESULTS Among DS NCI HR patients, 8 of 47 (17%) had a seizure during blinatumomab infusion (Table 1). All 8 seizures occurred in patients over 10 years old. Six of the 8 seizures occurred in the first cycle of blinatumomab, most in the first 3 days of the infusion. Four had concomitant fever or cytokine release syndrome. Seizures were grade 2 (n=2) or grade 3 (n=6), and all resolved with full neurologic recovery. Of the 8 patients, 5 elected to resume blinatumomab; no further seizures occurred in these patients. There was no indication of increased seizure risk among NCI SR DS-High patients (1 seizure among 11 patients), or among DS or non-DS patients receiving blinatumomab on other study strata (0 of 7 DS SR-Avg; 1 of 146 non-DS SR-Avg; and 2 of 120 non-DS SR-High). CONCLUSIONS The incidence of seizures associated with blinatumomab in DS-ALL patients older than 10 years appears higher than previously reported in children without DS. The majority of seizures occurred within the first 3 days, all fully resolved with no sequelae, and no patient who resumed blinatumomab infusion at a lower rate experienced further seizures. Seizure prophylaxis may be advisable in DS patients while receiving blinatumomab, particularly those >10 years of age. Further follow-up and a larger sample size are needed to confirm incidence and identify risk factors predisposing DS patients to neurologic toxicity. Figure 1 Figure 1. Disclosures Li: Novartis Canada: Membership on an entity's Board of Directors or advisory committees. Raetz: Pfizer: Research Funding; Celgene: Other: DSMB member. Loh: MediSix therapeutics: Membership on an entity's Board of Directors or advisory committees. Gupta: Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Rau: Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; AbbVie Pharmaceuticals: Other: Spouse is employee and stock holder; Servier Pharmaceuticals: Consultancy. OffLabel Disclosure: This trial includes the use of blinatumomab in combination with chemotherapy for treatment of de novo B-lymphoblastic leukemia.

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Distinct Features of Chip-Derived and De Novo MDS

Blood ◽

10.1182/blood-2018-99-117763 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2572-2572

Author(s):

Yasunobu Nagata ◽

Hideki Makishima ◽

Cassandra M. Hirsch ◽

Hassan Awada ◽

Abhinav Goyal ◽

...

Keyword(s):

Board Of Directors ◽

Somatic Mutations ◽

De Novo ◽

Meta Analysis ◽

Genetic Alterations ◽

Low Risk ◽

Alternative Methods ◽

Molecular Characteristics ◽

Study Cohort ◽

Advisory Committees

Abstract Subclinical clonal expansions, referred to as clonal hematopoiesis of indeterminate potential (CHIP), are present in blood of otherwise healthy individuals and their frequency increases with age. While many CHIP-associated mutations are present in MDS, only a small proportion of asymptomatic individuals with CHIP progress to MDS. We assumed that: i) a proportion of CHIP mutations will eventually serve as ancestral hits that manifest as MDS upon acquisitions of additional genetic alterations, and ii) MDS from antecedent CHIP may be a MDS disease subtype that is distinct from de novo MDS characterized by more penetrant primary hits. Separating ancestral vs. secondary hits in MDS patients and comparing by meta-analyses their frequencies to those in CHIP may enable molecular and clinical characterizations of CHIP-related MDS. Our study cohort consisted of 1,809 clinically annotated MDS patients. Deep targeted NGS was conducted for a panel of the 36 most frequently mutated "myeloid" genes, which revealed 3,971 somatic mutations in MDS patients after removing SNPs and errors. To discriminate between dominant and subsequent rising secondary mutations, we used a stringent binominal distribution algorithm to define VAFs confidence intervals via loci read counts. Sample maximal VAF mutations were defined as "dominant" and those with overlapping VAF 95% CI were defined as "co-dominant"; mutations with lower non-overlapping 95% CIs were "secondary". These definitions are consistent with those of other methods such as Pyclone, with 95% concordance (1,253/1,317). They yield 1,474 (36%) dominant and 1,372 (35%) secondary mutations. We compared a meta-analysis of frequently mutated genes in 1,693 healthy CHIP individuals with somatic mutations from the CHIP meta-analysis to dominant mutations in MDS patients. Mutations in DNMT3A, TET2, ASXL1, and JAK2 were more frequent in CHIP than MDS [e.g.,DNMT3A; 52% (888/1,693) vs. 6% (110/1,809), p<.001]. Hence MDS patients with dominant mutations in these 4 genes were defined as CHIP-derived MDS (C-MDS). Other dominant mutations such as U2AF1, RUNX1 and STAG2 which were not identified in individuals with CHIP were deemed not CHIP-derived MDS (NC-MDS). And in between, mutations in TP53, SF3B1 and SRSF2, were identified in both cohorts [e.g., TP53; 4% (71/1,693) vs. 5% (97/1,809), p=.11], and such cases will be denoted as C/NC-MDS patients. We set out to compare clinical, molecular and demographic features of C- vs. NC-MDS. There were 459 (25%) C-MDS and 498 (28%) NC-MDS cases out of 1809 MDS patients. 95% of patients with C-MDS (437/459) had at least one TET2 (51%, 234/459), DNMT3A (24%, 110/459) or ASXL1 (20%, 93/459) dominant mutation. The top 5 dominant mutations in patients with de novo MDS were U2AF1 (15%, 75/498), ZRSR2 (10%, 52/498), RUNX1 (9%, 47/498), STAG2 (9%, 43/498), and EZH2 (8%, 40/498). 52% (257/498) of patients with de novo MDS had at least one of these mutations. Focusing on secondary mutations, patients with C-MDS had a significantly higher frequency of secondary TET2 and ZRSR2 mutations than those with de novo [e.g., TET2, 17% (77/459) vs. 8% (41/498), p<.001]. In contrast, secondary ASXL1 mutations were more frequent in NC-MDS [7% (31/459) vs. 18% (88/498), p<.001]. C-MDS cases were older and had more low risk subtypes than NC-MDS cases [(average Age) 72 vs. 69, p<.001, (low risk subtypes) 56% (256/459) vs. 45% (222/498), p<.001]. Patients with C-MDS had better prognosis than those with NC-MDS [hazard ratio .75 (.63-.91), p=.003]. Prospective sequencing of serial samples of CHIP to development of MDS may be needed to fully reveal the landscape of C-MDS. This may not be practical, as over a million healthy donors would need to be followed for years to obtain adequate numbers of C-MDS cases. If our hypothesis that there exist clinical and molecular characteristics of C-MDS is correct, alternative methods that begin with MDS cases could then be employed. Regardless, detecting and monitoring CHIP is warranted, as this will lead to biomarkers that predict risks and thus preventative interventions. Disclosures Nazha: MEI: Consultancy. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; 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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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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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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Somatic Mutations in Therapy-Related Myeloid Neoplasms Are Influenced By Therapeutic Modality and Clonal Hematopoiesis of Indeterminate Potential

Blood ◽

10.1182/blood-2018-99-119950 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3084-3084 ◽

Cited By ~ 1

Author(s):

Teodora Kuzmanovic ◽

Bhumika J. Patel ◽

Srinivasa Reddy Sanikommu ◽

Yasunobu Nagata ◽

Hassan Awada ◽

...

Keyword(s):

Board Of Directors ◽

Somatic Mutations ◽

De Novo ◽

Germ Line ◽

Molecular Signature ◽

Therapeutic Modality ◽

Advisory Committees ◽

Therapeutic Success ◽

Clonal Hematopoiesis ◽

Iatrogenic Complications

Abstract Treatment-associated myeloid neoplasias (tMN) are serious iatrogenic complications of cytotoxic therapies applied to primary malignancies (PM). With increased therapeutic success rates and prolonged survival of cancer patients, tMN may become more prevalent. Although tMN diagnosis is trivial to the extent that previous therapies are known, tMN may represent coincidental primary MDS/AML not causally linked to chemotherapy (ctx) or radiation (rtx). Other seemingly tMN cases may carry germ line predisposition responsible for co-occurrence of 2 neoplastic processes, and finally others are truly treatment-related MN. With the recognition of clonal hematopoiesis of indeterminate potential (CHIP), it is also likely the preexisting CHIP would be either selected for, eliminated by cancer therapies, or that ctx or rtx lead to emergence of CHIP. tMN may also manifest without antecedent CHIP, and thus could be either CHIP-derived or de novo. Some of the problems in assigning somatic mutational pattern to tMN may be alleviated by application of proper control groups which include sMN (MN after PM treated only surgically). In our cohort of 1058 patients, we identified 109 cases of such sMN, 266 tMN with a history of rtx or ctx for PM, and 683 of primary MN (pMN), having no PM. Of these 65 sMN, 145 tMN, and 683 pMN were sequenced by NGS. Using these three patient groups, we sought somatic mutations that distinguish them. tMN presented as more aggressive disease: diagnosed older vs. pMN/sMN (68 years, p<.001) , shorter latency from PM to MN vs. sMN (8.7 vs. 10.5 years, p=.085), complex cytogenetics vs. pMN, sMN (p=1.4x10-5, p=2.7x10-4) including chromosome 7 (p=1.7x10-7, p=8.4x10-5) and 5 abnormalities (p=.044, p=.09), 50% tMN were advanced MDS/AML vs. 35% pMN, 42% sMN (p=.00016, p=.25). The most common mutations in all 3 groups were TET2, DNMT3A, ASXL1 and SRSF2. Mutations in SF3B1 and JAK2 were less common in tMN vs. pMN, sMN (p=.058, p=.014; p=.011, p=.327) while those in TP53, KIT, EZH2,WT1 were most frequently mutated in tMN vs. pMN/sMN (OR 2.6, p=.002, OR 6, p=.011, OR 1.9, p=.083, OR 3.2, p=.08). Mutations in ETV6 and EZH2 were only found in rtx vs. ctx-treated tMN cases (p=.046, p=.004). TP53 mutations were associated with ctx (OR 7.2, p=0.062), and when combined with cases which received both ctx and rtx vs. rtx alone, TP53 mutation was 9.3x was common (p=.014). In tMN TP53 and EZH2-mutated cases, a higher proportion of transversions was observed vs. those found in pMN (p=.055, p= 0.052). The domains mutated in TP53 tMN vs. pMN/sMN cases were similar, while EZH2-mutated tMN were enriched for hits in domain 2 (p=0.0604). This suggests that the type of treatment utilized influences the molecular signature of tMN in terms of frequency of mutations as well as types of mutations found. A meta-analysis of 9 CHIP studies was performed to pool overall frequencies of MN-related genes. These frequencies were compared to those of ancestral events (determined by recapitulation of clonal hierarchy via variant allele frequency and zygosity) in our cohort. This yielded 3 categories of mutations: those that are CHIP-derived (frequency in CHIP> ancestral frequency in MN), from de novo MN (mutations in gene not seen in CHIP), or those found in both CHIP and MN, termed mix-derived. CHIP-derived hits were TET2, DNMT3A, JAK2 while STAG2, EZH2, APC, MLL, WT1 were de novo hits. Using this categorization scheme tMN break down as 19% CHIP-derived, 38% de novo, and 24% mix-derived. CHIP-derived tMN were, on average, 6 years older than de novo tMN (p=.019). This also held true for the age of PM diagnosis, where CHIP-derived cases were, on average, 10 years older than de novo tMN (p=.0175), suggesting that age of PM may be correlated with acquisition of CHIP in tMN. CHIP-derived vs. de novo tMN latencies did not differ. The molecular signatures of tMN are influenced by therapies utilized for PM as well as CHIP. Targeted sequencing for germ line predisposition genes is under way for these patients to further characterize their molecular profiles. Disclosures Nazha: MEI: Consultancy. Gerds:Apexx Oncology: Consultancy; Incyte: Consultancy; Celgene: Consultancy; CTI Biopharma: Consultancy. Carraway:Jazz: Speakers Bureau; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; FibroGen: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Agios: Consultancy, Speakers Bureau. 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; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy.

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Mutations in Genes Associated with Familial Predisposition to Myeloid Neoplasms: Their Frequency and Associations with Pretreatment Characteristics in Adult Patients (Pts) with Presumably Sporadic De Novo Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood-2018-99-120085 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1478-1478

Author(s):

Ann-Kathrin Eisfeld ◽

Jessica Kohlschmidt ◽

Krzysztof Mrózek ◽

Alice S. Mims ◽

Christopher J. Walker ◽

...

Keyword(s):

Survival Rate ◽

Family History ◽

Board Of Directors ◽

Somatic Mutations ◽

De Novo ◽

Risk Group ◽

Germline Mutations ◽

World Health ◽

Advisory Committees ◽

Myeloid Neoplasms

Abstract The effects of germline variants in the development of myeloid neoplasms, including AML, were largely neglected for decades. However, several myeloid neoplasms with germline predisposition have been recently recognized as separate entities in the 2016 revision to the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia. In addition to genes whose mutations are associated with bone marrow failure syndromes, and are long-known contributors to Mendelian disorders that have myelodysplastic syndromes/AML as the main clinical feature (e.g., germline CEBPA, GATA2 and RUNX1 mutations), 3 more genes were included: ANKRD26, DDX41 and SRP72. Mutations in these genes were described in few families, and are thus considered to be very rare. However, it is possible that their frequency might be underestimated, because the associated phenotypes are often vague and family history not routinely considered. To establish the frequency of ANKRD26, DDX41 and SRP72 mutations, and to characterize molecular and clinical features associated with these mutations, we determined mutational status of 83 cancer- and leukemia-associated genes using 2 targeted sequencing panels in diagnostic samples of 1,021 clinically well-characterized adult pts with de novo AML AML treated on trials conducted by the Alliance for Clinical Trials in Oncology. Mutations in the 3 familial genes were found in 46 pts (4.5%), specifically, mutations in ANKRD26 in 15, DDX41 in 17 and SRP72 in 19 pts. Three pts had mutations in either 2 or all 3 genes. Mutations occurred at varying variant allele fractions (VAFs, median: 0.47; range: 0.10-0.98), with 76% of mutations observed with VAFs >35%. Mutations were found throughout the genes. Pts harboring mutations in any of the 3 genes were predominantly younger (median age, 54 years; range, 19-77), 65% of them were male, and 48% belonged to the 2017 European LeukemiaNet (ELN) favorable genetic risk group. The co-mutation profiles partially differed among the genes. NPM1 mutations were the most frequent co-mutations, occurring in 47%, 41%, and 42% of pts with mutations in ANKRD26, DDX41, and SRP72, respectively. However, ANKRD26-mutated pts frequently harbored FLT3-ITD and mutations in DNMT3A, IDH2 and SRSF2 genes (each detected in 20% of pts). DDX41-mutated pts commonly had mutations in NRAS (18%), SMARCA2 (12%) and TP53 (12%). SRP72-mutated pts often had mutations in TET2 (26%), CEBPA (23%) and IDH1 (21%). With the exception of a higher complete remission rate in ANKRD26-mutated pts (93% compared with 73% for DDX41- and 81% for SRP72-mutated pts), the clinical outcomes were very similar. Considering all 3 genes combined, the median 3-year disease-free survival rate of 25% and median 3-year overall survival rate of 44% resembled those of pts belonging to the ELN intermediate risk group. We next tested whether the variants detected in our cohort of pts with presumably sporadic AML were of germline or somatic origin. We performed Sanger sequencing on germline material (buccal swab or remission samples) of 28 pts who had mutations detected at VAF>35% and material available. Germline origin was determined for the mutations detected in 24 of 28 pts tested (86%; ANKRD26, 9/10 tested pts; SRP72, 9/11 pts; DDX41, 8/10 pts). Of the detected germline changes, 7/9 ANKRD26 mutations, 6/10 DDX41 mutations and 5/9 SRP72 mutations were predicted to have deleterious effects on the respective proteins via Polyphen. The 1 pt with mutations in all 3 genes were found to be somatic mutations. Additional genes whose germline mutations are known to occur in families, such as GATA2, CEBPA and RUNX1, were sequenced for somatic mutations in our pt cohort, but not yet tested for potential germline origin in our analysis. Thus, it is likely that the frequency of familial AML mutations is even higher in our cohort. To our knowledge, this is the first study that tested the frequency of 3 leukemia-predisposing gene mutations in a large cohort of adults with presumably sporadic AML. The relatively high number of germline mutations in these 3 genes highlights the importance of testing for germline mutations. Thus, inclusion of those genes in diagnostic sequencing panels should be considered, and critical consideration of obtained family history should be strongly encouraged for providers taking care of pts with myeloid malignancies. Support: U10CA180821, U10CA180882, U10CA180861, U24CA196171 Disclosures Mims: Novartis: Consultancy; Abbvie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Powell:Rafael Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Kolitz:Magellan Health: Consultancy, Honoraria.

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

Blood ◽

10.1182/blood.v126.23.1645.1645 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1645-1645 ◽

Cited By ~ 1

Author(s):

Aziz Nazha ◽

Manja Meggendorfer ◽

Niroshan Nadarajah ◽

Kassy E Kneen ◽

Tomas Radivoyevitch ◽

...

Keyword(s):

Board Of Directors ◽

Somatic Mutations ◽

Catalytic Domain ◽

Germ Line ◽

Hot Spot ◽

Prognostic Impact ◽

Nucleotide Polymorphisms ◽

Advisory Committees ◽

Myeloid Neoplasms ◽

Increased Risk

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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Clonal Events of Aplastic Anemia Related to the Evolution to Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v128.22.4290.4290 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4290-4290 ◽

Cited By ~ 1

Author(s):

Eiju Negoro ◽

Michael Clemente ◽

Naoko Hosono ◽

Aziz Nazha ◽

Wenyi Shen ◽

...

Keyword(s):

Board Of Directors ◽

Somatic Mutations ◽

De Novo ◽

Low Frequency ◽

Gene Mutations ◽

Progression Free Survival ◽

Similar Proportion ◽

Advisory Committees ◽

Cross Sectional ◽

Patients At Risk

Abstract Somatic mutations constitute clonal markers now amenable to monitoring by deep NGS. While transient and low frequency clones have been described in AA, their pathophysiologic link to the overtly clonal complication of AA, secondary MDS following AA (sMDS), has not been established. Clarification of this relationship may provide clues as to the genesis of sMDS. Identification of predictive markers for AA patients at risk for this complication is necessary. The etiology of sMDS within AA may include either expansion of a preexisting clone or truly late clonogenic events. In both instances, progression may result in clonal escape. To address these questions we studied 258 AA and 60 PNH patients, identifying 35 patients (11%) who evolved to sMDS. Cytogenetic analysis showed abnormal karyotype in 76% cases; 5% had complex karyotype and -7/del(7q) was present in 67% of cases. The presence of a PNH clone was detected in a similar proportion of cases that transformed to sMDS vs. those that did not (P=.76). For comparison, we have also analyzed primary de novo cases of MDS (pMDS) with (N=19) and without (N=161) -7/del(7q). In contrast to sMDS, -7/del(7q) was present in 12% of cases of pMDS. Using WES on 8 cases and a 60 gene targeted panel on 15 cases, confirmed mutational events and chromosomal aberrations were found in 21/23 patients with sMDS; 18/23 cases of sMDS had at least 1 confirmed somatic mutation. By comparison of mutational profiles ASXL1, RUNX1, PIGA, SETBP1, and CBL were most common in sMDS (26.1%, 21.7%, 18%, 13% and 13%, respectively). Because sMDS 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 appeared to be more frequent in sMDS vs. pMDS (27% vs. 0%, P=.03). In contrast, TP53 was more common in pMDS (7% vs. 32%, P=.1). Similarly, there were several other distinctive differences between all sMDS and pMDS irrespective of cytogenetics: mutations in U2AF1 were common in pMDS, mutations in RUNX1 appeared to be more frequent in sMDS vs. pMDS (22% vs. 5.5%, P=.02). Mutations in PIGA gene constituted a marker for sMDS derivation from AA. To discern a possible biological relationship we have also compared mutational profiles of hypocellular pMDS and sMDS, but no significant differences aside from PIGA prevalence were found. If sMDS is derived from mutations present at the AA stage, one would expect overlaps in the mutational spectrum of AA before and after evolution to MDS. DNMT3A, BCOR/BCORL1, and PDGFR family mutations were found at higher frequency in AA while ASXL1, RUNX1, SETBP1, PIGA, and CBL were higher in sMDS. Thus, cross-sectional analysis suggests that most of the clonal events occurring during the course of AA do not initiate sMDS. To further examine these findings we performed serial sequencing analyses: in 7 patients with sMDS WES was performed and clonal architecture was analyzed. We then queried whether mutations present in MDS were detected in archival samples at presentation using deep targeted NGS (depth 5-10x104 rds. In 4/7 cases the alterations appeared to be ancestral events for sMDS evolution. When an additional 68 AA cases were studied by deep NGS, somatic mutations were present in 31% of AA patients at presentation. Patients with clonal events at presentation tended toward worse progression free survival compared to patients without mutations (P=.1). 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 gene mutations (n=21) before immunosuppressive therapies (IST) had worseoverall survival compared to patients without these mutations (n=47; P=.009). To assess the potential impact of IST, we also investigated a subset of 37 patients (25 responders/ 12 refractory) following IST. Clonal somatic events were identified in 42 of them, but there was no association between the response to IST and somatic mutations at presentation (P=.7). Our results demonstrate that while subclonal mutations indicative of oligoclonal hematopoiesis are frequent in AA, the presence of specific permissive ancestral somatic events at the outset of AA predisposes patients to sMDS, a feature that has diagnostic and prognostic implications. 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. Maciejewski:Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau.

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Long-Term Experience with Large Granular Lymphocytic Leukemia Evolving after Solid Organ and Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood-2019-130167 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1226-1226

Author(s):

Hassan Awada ◽

Reda Z. Mahfouz ◽

Jibran Durrani ◽

Ashwin Kishtagari ◽

Deepa Jagadeesh ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Viral Infections ◽

De Novo ◽

Post Transplantation ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Post Transplant ◽

Stat3 Mutations

T-cell large granular lymphocyte leukemia (T-LGLL) is a clonal proliferation of cytotoxic T lymphocytes (CTL). T-LGLL mainly manifest in elderly and is associated with autoimmune diseases including rheumatoid arthritis (RA), B cell dyscrasias, non-hematologic cancers and immunodeficiency (e.g., hypogammaglobulinemia). LGL manifestations often resemble reactive immune processes leading to the dilemmas that LGLs act like CTL expansion during viral infections (for example EBV associated infectious mononucleosis). While studying a cohort of 246 adult patients with T-LGLL seen at Cleveland Clinic over the past 10 years, we encountered 15 cases of overt T-LGLL following transplantation of solid organs (SOT; n=8) and hematopoietic stem cell transplantation (HSCT; n=7). Although early studies reported on the occurrence of LGL post-transplant, these studies focused on the analysis of oligoclonality skewed reactive CTL responses rather than frank T-LGLL. We aimed to characterize post-transplantation T-LGLL in SOT and HSCT simultaneously and compare them to a control group of 231 de novo T-LGLL (cases with no history of SOT or HSCT). To characterize an unambiguous "WHO-defined T-LGLL" we applied stringent and uniform criteria. All cases were diagnosed if 3 out of 4 criteria were fulfilled, including: 1) LGL count >500/µL in blood for more than 6 months; 2) abnormal CTLs expressing CD3, CD8 and CD57 by flow cytometry; 3) preferential usage of a TCR Vβ family by flow cytometry; 4) TCR gene rearrangement by PCR. In addition, targeted deep sequencing for STAT3 mutations was performed and charts of bone marrow biopsies were reviewed to exclude other possible conditions. Diagnosis was made 0.2-27 yrs post-transplantation (median: 4 yrs). At the time of T-LGLL diagnosis, relative lymphocytosis (15-91%), T lymphocytosis (49-99%) and elevated absolute LGL counts (>500 /µL; 93%) were also seen. Post-transplantation T-LGLL were significantly younger than de novo T-LGLL, (median age: 48 vs. 61 yr; P<.0001). Sixty% of post-transplantation T-LGLL patients were males. Fifteen% of patients had more cytogenetic abnormalities compared to de novo T-LGLL, had a lower absolute LGL count (median: 4.5 vs. 8.5 k/µL) and had less frequent neutropenia, thrombocytopenia and anemia (27 vs. 43%, 33 vs. 35% and 20% vs. 55%; P=.01). TCR Vb analysis identified clonal expansion of ≥1 of the Vb proteins in 60% (n=9) of the patients; the remaining 40% (n=6) of the cases had either a clonal process involving a Vb protein not tested in the panel (20%; n=3) or no clear expansion (20%; n=3). Signs of rejection were observed in 20% (n=3/15) and GvHD in 13% (n=2/15) of the patients. Post-transplantation, 27% of cases presented with neutropenia (absolute neutrophil count <1.5 x109/L; n=4), 33% with thrombocytopenia (platelet count <150 x109/L; n=5) and 25% with anemia (hemoglobin <10 g/dL; n=3). T-LGLL evolved in 10 patients (67%; 10/15) despite IST including cyclosporine (n=5), tacrolimus (n=4), mycophenolate mofetil (n=5), cyclophosphamide (n=1), anti-thymocyte globulin (n=1), and corticosteroids (n=6). Lymphadenopathy and splenomegaly were seen in 13% (n=2) and 33% (n=5) of the patients. Other conditions observed were MGUS (20%; n=3) and RA (7%; n=1). Conventional cytogenetic showed normal karyotype in 89% (n=11, tested individuals 13/15). Somatic STAT3 mutations were identified in 2 patients. Sixty% of cases (n=9) were seropositive for EBV when tested at different time points after transplant. Similarly, 53% (n=8) were seropositive for CMV, of which, 5 were positive post-transplantation and 3 pre-/post-transplantation. The complexity of T-LGLL expansion post-transplantation might be due to several mechanisms including active viral infections, latent oncogenic viral reactivation and graft allo-antigenic stimulation. However, in our cohort graft rejection or GvHD was encountered in a few patients (2 allo-HSCT recipients). Autoimmune conditions were present in 50% of SOT recipients (n=4/ 8, including RA, ulcerative colitis, systemic lupus erythematosus). Some of our patients also had low immunoglobulin levels. Overt EBV (post-transplant lymphoproliferative disorder) and CMV reactivation was diagnosed in only 27% (4/15) of the patients. In sum we report the long term follow up of a cohort of T-LGLL and emphasize the expansion of T-LGLL post-transplant highlighting the difficulty in assigning one unique origin of LGLL. Disclosures Hill: Genentech: Consultancy, Research Funding; Takeda: Research Funding; Celegene: Consultancy, Honoraria, Research Funding; Kite: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Consultancy, Honoraria; Amgen: Research Funding; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; TG therapeutics: Research Funding; AstraZeneca: Consultancy, Honoraria. Majhail:Atara Bio: Consultancy; Mallinckrodt: Honoraria; Nkarta: Consultancy; Anthem, Inc.: Consultancy; Incyte: Consultancy. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

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