Analysis of Genomic Landscape of Large Granular Lymphocyte Leukemia Reveals Etiologic Insights

Introduction: Large Granular Lymphocyte (LGL) leukemia is a rare lymphoproliferative disorder characterized by clonal expansion of either CD3+ cytotoxic T cells expressing T cell receptor (TCR) alpha beta or CD3- natural killer (NK) cells. A less frequent CD3+ T cell subtype expresses TCR gamma delta (GD). Here, we present the molecular landscape of known LGL types (T, NK, GD) from analysis of the largest patient cohort assembled to date. An integrative analysis of genomic datasets from all LGL subtypes is necessary to more precisely define the shared and unique etiology of this rare disorder. Methods: We collected paired saliva and PBMC samples and related clinical information from 116 LGL leukemia patients after informed consent. The assembled cohort consisted of 93 T-LGL, 11 NK-LGL, and 12 GD T-LGL patients. Genomic analyses were performed on the leukemic (PBMC) and germline (saliva) samples after whole exome sequencing (WES) and transcriptome sequencing (RNAseq, PBMC only). Results: Somatic mutations were detected in the previously described potential drivers STAT3 (n=56), TNFAIP3 (n=9), and PIK3R1 (n=4). We also identified somatic mutations in CDH8 (n=3) and CCL22 (n=4), which we postulate as putative drivers based on mutational clustering. CDH8 was mutated in all three LGL subtypes, but CCL22 somatic mutations were only observed in NK-LGL patients. We observed that STAT3 and CCL22 together account for 64% (7/11) of NK-LGL cases. STAT3 is the most recurrently mutated gene in LGL leukemia, yet concurrent molecular and clinical features are incompletely defined. Interestingly, patients with STAT3 mutations have a higher mutational burden (P=0.0006) compared to those with wild-type (WT) STAT3. This effect is independent of the age of the patient, which correlates with the mutational burden (R=0.26, P=0.0039) and agrees with the finding that the dominant mutational signatures in this cohort exhibit clock-like properties. We also observed that patients with STAT3 mutations are enriched (P=0.0273) for additional mutations in chromatin modifier enzymes such as KMT2D, TET2, DNMT3A, and SETD1B (Figure 1). We found that ~10% of the samples exhibit broad somatic copy-number aberrations, and a patient with somatic mutations in STAT3 and KMT2D displayed high-level microsatellite instability. STAT3 mutations were also significantly associated with increased expression of genes involved in apoptosis, complement activation, and interferon cytokine signaling compared to STAT3 WT (FDR < 0.05). Early-onset LGL patients with age 51 years or less (n=28), as defined by the bottom quartile of the cohort, displayed no differential enrichment of the somatic driver genes. Interestingly, the age of the patient was significantly associated with absolute neutrophil counts (ANC) (P = 0.0068), with younger patients exhibiting lower neutrophil counts, even after adjusting for the presence of STAT3 mutation, as it is associated with lower ANC. As neutropenia is a hallmark feature of LGL leukemia and often a trigger for initiating therapy, the association of young age with lower neutrophil counts and lower somatic mutational burden suggests other mechanisms may be involved. Focusing on the germline variants, we found that 17 of the patients (14.6%) had at least one pathogenic or likely pathogenic germline variant with known oncogenic association as annotated using CharGer. 5 patients had pathogenic mutations in known tumor suppressors including FANCC (n=1), BRCA1 (n=1), PALB2 (n=1), MUTYH (n=1), and SDHA (n=1), while 1 patient had pathogenic mutations in ALK, a known oncogene. Conclusions: We report on the genomic analyses done on whole exome and RNA-seq data from the largest cohort assembled for LGL leukemia to date. We show that the presence of STAT3 mutation is significantly associated with an increase in mutation burden and additional somatic mutations in chromatin modifiers, hinting at potential pathogenic mechanisms within STAT3 mutated patients. By combining LGL subtypes in our analysis, we were able to identify CDH8 as a putative driver that is present in T, NK, and GD subtypes. Additionally, we report CCL22 mutations specific to NK-LGL leukemia, however did not detect any subtype specific mutations in GD T-LGL. We found that about 15% of the patients carry at least one pathogenic germline variant with known oncogenic associations. These findings highlight emerging etiologic insights into this rare disorder. Disclosures Feith: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees. Loughran:Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees.

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Patterns of Clonal Evolution Assessed By Whole Exome Sequencing during Progression from MDS to AML Are Related to Therapy

Blood ◽

10.1182/blood.v128.22.4309.4309 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4309-4309

Author(s):

María Abáigar ◽

Jesús M Hernández-Sánchez ◽

David Tamborero ◽

Marta Martín-Izquierdo ◽

María Díez-Campelo ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Clonal Evolution ◽

Clonal Expansion ◽

Driver Mutations ◽

Advisory Committees ◽

Disease Evolution ◽

Mutational Burden ◽

Whole Exome ◽

Leukemic Phase

Abstract Introduction: Myelodysplastic syndromes (MDS) are hematological disorders at high risk of progression to acute myeloid leukemia (AML). Although, next-generation sequencing has increased our understanding of the pathogenesis of these disorders, the dynamics of these changes and clonal evolution during progression have just begun to be understood. This study aimed to identify the genetic abnormalities and study the clonal evolution during the progression from MDS to AML. Methods: A combination of whole exome (WES) and targeted-deep sequencing was performed on 40 serial samples (20 MDS/CMML patients evolving to AML) collected at two time-points: at diagnosis (disease presentation) and at AML transformation (disease evolution). Patients were divided in two different groups: those who received no disease modifying treatment before they transformed into AML (n=13), and those treated with lenalidomide (Lena, n=2) and azacytidine (AZA, n=5) and then progressed. Initially, WES was performed on the whole cohort at the MDS stage and at the leukemic phase (after AML progression). Driver mutations were identified, after variant calling by a standardized bioinformatics pipeline, by using the novel tool "Cancer Genome Interpreter" (https://www.cancergenomeinterpreter.org). Secondly, to validate WES results, 30 paired samples of the initial cohort were analyzed with a custom capture enrichment panel of 117 genes, previously related to myeloid neoplasms. Results: A total of 121 mutations in 70 different genes were identified at the AML stage, with mostly all of them (120 mutations) already present at the MDS stage. Only 5 mutations were only detected at the MDS phase and disappeared during progression (JAK2, KRAS, RUNX1, WT1, PARN). These results suggested that the majority of the molecular lesions occurring in MDS were already present at initial presentation of the disease, at clonal or subclonal levels, and were retained during AML evolution. To study the dynamics of these mutations during the evolution from MDS/CMML to AML, we compared the variant allele frequencies (VAFs) detected at the AML stage to that at the MDS stage in each patient. We identified different dynamics: mutations that were initially present but increased (clonal expansion; STAG2) or decreased (clonal reduction; TP53) during clinical course; mutations that were newly acquired (BCOR) or disappearing (JAK2, KRAS) over time; and mutations that remained stable (SRSF2, SF3B1) during the evolution of the disease. It should be noted that mutational burden of STAG2 were found frequently increased (3/4 patients), with clonal sizes increasing more than three times at the AML transformation (26>80%, 12>93%, 23>86%). Similarly, in 4/8 patients with TET2 mutations, their VAFs were double increased (22>42%, 15>61%, 50>96%, 17>100%), in 2/8 were decreased (60>37%, 51>31%), while in the remaining 2 stayed stable (53>48%, 47>48%) at the AML stage. On the other hand, mutations in SRSF2 (n=3/4), IDH2 (n=2/3), ASXL1 (n=2/3), and SF3B1 (n=3/3) showed no changes during progression to AML. This could be explained somehow because, in leukemic phase, disappearing clones could be suppressed by the clonal expansion of other clones with other mutations. Furthermore we analyzed clonal dynamics in patients who received treatment with Lena or AZA and after that evolved to AML, and compared to non-treated patients. We observed that disappearing clones, initially present at diagnosis, were more frequent in the "evolved after AZA" group vs. non-treated (80% vs. 38%). By contrast, increasing mutations were similar between "evolved after AZA" and non-treated patients (60% vs. 61%). These mutations involved KRAS, DNMT1, SMC3, TP53 and TET2among others. Therefore AZA treatment could remove some mutated clones. However, eventual transformation to AML would occur through persistent clones that acquire a growth advantage and expand during the course of the disease. By contrast, lenalidomide did not reduce the mutational burden in the two patients studied. Conclusions: Our study showed that the progression to AML could be explained by different mutational processes, as well as by the occurrence of unique and complex changes in the clonal architecture of the disease during the evolution. Mutations in STAG2, a gene of the cohesin complex, could play an important role in the progression of the disease. [FP7/2007-2013] nº306242-NGS-PTL; BIO/SA52/14; FEHH 2015-16 (MA) Disclosures Del Cañizo: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen-Cilag: Membership on an entity's Board of Directors or advisory committees, Research Funding; Arry: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Recurrent Missense Mutations in the STAT3 Gene in LGL Leukemia Provide Insights to Pathogenetic Mechanisms and Suggest Potential Diagnostic and Therapeutic Applications

Blood ◽

10.1182/blood.v118.21.936.936 ◽

2011 ◽

Vol 118 (21) ◽

pp. 936-936

Author(s):

Hanna Koskela ◽

Samuli Eldfors ◽

Henrikki Almusa ◽

Emma Andersson ◽

Pekka Ellonen ◽

...

Keyword(s):

T Cell ◽

Exome Sequencing ◽

Missense Mutation ◽

Whole Exome Sequencing ◽

Somatic Mutations ◽

Index Patient ◽

Missense Mutations ◽

Whole Exome ◽

Stat3 Mutations ◽

Lgl Leukemia

Abstract Abstract 936 BACKGROUND: T-cell large granular lymphocyte (LGL) leukemia is an uncommon lymphoproliferative disorder characterized in most cases by expansion of mature, clonal CD3+CD8+ cytotoxic T lymphocytes (CTLs). The pathogenesis of LGL-leukemia is unknown, and leukemic cells closely resemble normal terminally differentiated effector memory CTLs. While resistance to apoptotic pathways (Fas/Fas ligand, sphingolipid) and activation of survival signaling pathways (Ras) have been implicated in LGL leukemia, the underlying genetic defects have not yet been elucidated. We aimed to identify somatic mutations in LGL leukemia by whole exome sequencing of leukemic and matched healthy control cells. METHODS: Our index patient is a 70 year-old male with untreated CD8+ LGL leukemia diagnosed in 2009 with a clonal rearrangement in the T-cell receptor (TCR) delta and gamma gene. He has been asymptomatic with grade 2 neutropenia and an absolute lymphocyte count of 4–15 ×109/L. The patient had one large predominant T-cell clone: 94% of CD8+ cells consisted of a single Vβ16 clone, as assessed by flow cytometry. No clonal expansions were observed in the CD4+ fraction. DNA was extracted from FACS-sorted CD8+ (leukemic) and CD4+ (control) cells and sequenced by exome capture using an Agilent SureSelect All exon 50 MB capture kit and the Illumina GAII sequencing platform. Candidate somatic mutations were identified with a bioinformatics pipeline consisting of BWA for sequence alignment, Samtools for alignment filtering and Varscan for somatic mutation calling. Mutations were manually reviewed in IGV for alignment artifacts and validated by capillary sequencing. DNA samples from 8 additional untreated LGL-leukemia patients were used for further screening of confirmed somatic mutations by capillary sequencing. From six of these patients DNA was extracted from CD8 sorted cells and from two patients from whole blood. RESULTS: Whole exome sequencing of CD8+ leukemic DNA from the index patient identified a missense mutation in the STAT3 gene (D661V), which was subsequently confirmed by capillary sequencing. As STAT3 signaling has been associated with LGL leukemia pathogenesis previously, we next designed primers for the secondary screening of the six exomes of STAT3 SH2 region from the remaining patients. Another recurrent somatic missense mutation (STAT3 Y640F) was identified in two additional patients. Thus, three out of nine LGL patients (33%) showed evidence of mutations in the STAT3 SH2 region. Both missense mutations found (D661V and Y640F) were located in the area of the SH2 domain known to mediate STAT3 protein dimerization and activation. The Y640F mutation alters a conserved tyrosine residue leading to a hyperactivating STAT protein (Scarzello et al. Mol Biol Cell, 2007) and was recently found in a human inflammatory hepatocellular adenoma causing cytokine-independent tyrosine phosphorylation and activation as well as cytokine-dependent hyperactivation of STAT3 (Pitali et al., J Exp Med, 2011). The D661V mutation has not been described previously. CONCLUSIONS: Our data imply for the first time that STAT3 is a common mutational target in LGL leukemia, revealing insights to the molecular pathogenesis of this rare disease. Known structural and functional data on STAT biology imply that the mutations are leading to STAT3 hyperactivation and could also confer ligand-independent signaling. While confirmatory data from a larger series of patients are necessary, our results pinpoint STAT3 mutations and aberrations in the STAT3 pathway as key pathogenetic events in true clonal LGL leukemia. Detection of STAT3 mutations could therefore be applied in the diagnostic assessment, disease stratification and therapeutic monitoring of LGL patients. Disclosures: Koskela: Novartis: Honoraria. Kuittinen:Roche: Consultancy. Porkka:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Mustjoki:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria.

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Whole-Exome Sequencing In Myelodysplastic Syndromes With 5q- and Normal Karyotype

Blood ◽

10.1182/blood.v122.21.1551.1551 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1551-1551

Author(s):

Vera Adema ◽

Mar Mallo ◽

Leonor Arenillas ◽

María Díez-Campelo ◽

Elisa Luño ◽

...

Keyword(s):

Bone Marrow ◽

Exome Sequencing ◽

Board Of Directors ◽

Whole Exome Sequencing ◽

Research Funding ◽

Somatic Mutations ◽

Normal Karyotype ◽

Advisory Committees ◽

Whole Exome ◽

5Q Deletion

Abstract Introduction Myelodysplastic Syndromes (MDS) are a heterogeneous group of clonal myeloid stem cells disorders with high prevalence in the elderly characterized by inefficient hematopoiesis, peripheral blood (PB) cytopenias, and an increased risk of transformation to acute myeloid leukemia (AML). The karyotype is the clinical parameter with the strongest prognostic impact according the IPSS-R (Greenberg et al., 2012). The most frequent cytogenetic alteration is the chromosome 5q deletion (del[5q]) which as a single anomaly, confers a good prognosis and predicts an excellent response to lenalidomide. Whether other genetic abnormalities routinely cooperate with del(5q) is not known. Whole-exome sequencing (WES) is a powerful tool to identify somatic mutations in protein coding genes that might cooperate with del(5q). In order to better understand the genetic basis of MDS with del(5q), we performed whole-exome sequencing (assessing 334,378 exons) of tumor-normal paired samples from 21 MDS patients. Herein we describe the preliminary findings. The analysis is ongoing and the complete results will be presented in the meeting. Methods A total of 21 patients with MDS (16 with del(5q) as a sole abnormality, 3 with del(5q) and additional alterations and 2 with normal karyotype) were included in our study. We examined a total of 25 tumor samples (21 diagnostic bone marrow (BM) samples with matched CD3+ cells as a controls, additional BM samples from 3 patients during lenalidomide treatment and 1 bone marrow sample from a del(5q) patient after AML progression). DNA was extracted from BM samples and from isolated peripheral blood CD3+ cells (magnetic-activated cell sorting (MACS), MiltenyiBiotec GmbH, Germany). The purity of CD3+ cells was assessed by FC 500 flow cytometer (Beckman Coulter, Hialeah, Fl, USA). Only DNA that fulfilled quality controls required by WES were submitted. For each diagnostic sample, we performed Conventional G-banding cytogenetics and fluorescence in situ hybridization (FISH, to confirm or dismiss 5q deletions). Whole-exome targeted capture was carried out on 3 μg of genomic DNA, using the SureSelect Human Exome Kit 51Mb version 4 (Agilent Technologies, Inc., Santa Clara, CA, USA). The captured and amplified exome library was sequenced with 100 bp paired-end reads on an Illumina HiSeq2000. Whole-exome sequencing data were analyzed using an in-house bioinformatics pipeline as previously reported. Somatic mutations identified as alterations present in tumor but not in the matched CD3+ sample were validated by Sanger sequencing. Results In our preliminary analysis of WES from 12 patients (10 patients with 5q- and 2 patients with normal karyotype), a total of 249 non-silent somatic variant candidates were identified, of which 146 were confirmed as somatic mutations. Recurrent mutations were observed in three genes (ASXL1, NBPF10 and SF3B1) in 3 different patients. Seven genes (HRNR, JAK2, POTEG, MUC5B, PHLDA, TTN, ZNF717) were mutated in two patients. Mutations in several genes known to be mutated in MDS (ASXL1, JAK2, RUNX1, SF3B1, SRSF2 and TET2) were also identified. Patients with the 5q deletion had an average of 11 mutations whereas patients with normal karyotype had a higher mean (14.5). Mutated genes identified in both groups were HRNR, JAK2, MUC5B, NBPF10 and SF3B1. No mutations in TP53 were detected in this subset. Pathway analysis of the complete list of somatically mutated genes will be carried out once all 21 patients are analyzed. The four in-treatment samples will be examined from their matched diagnostic samples. Conclusions Whole-exome sequencing of largely del(5q) MDS patient samples identified both known and previously unreported somatic mutations. Analysis of additional samples will allow a more complete description of the genes and pathways that may cooperate with del(5q) in the development and progression of MDS. Acknowledgments Financial support: This work has been supported (in part) by a grant from Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (PI 11/02010); by Red Temática de Investigación Cooperativa en Cáncer (RTICC, FEDER) (RD07/0020/2004; RD12/0036/0044); Acción COST BM0801: European Genomics and Epigenomics Study on MDS and AML; Sociedad Española de Hematología y Hemoterapia (SEHH) and MDS Celgene. Footnotes Rafael Bejar and Francesc Sole contributed equally. Disclosures: Díez-Campelo: Novartis and Celgene: Honoraria, Research Funding. Cañizo:Celgene Jansen-Cilag Arry Novartis: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Sanchez:Celgene: Honoraria, Research Funding. Bejar:Genoptix: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Solé:Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

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Targeted Sequencing Reveals a Relationship Between Mutational Burden and Clinical Phenotype in MPNs

Blood ◽

10.1182/blood.v126.23.4061.4061 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4061-4061

Author(s):

Bartlomiej Getta ◽

Franck Rapaport ◽

Sean Devlin ◽

Chen Zhao ◽

Kristina Marie Knapp ◽

...

Keyword(s):

Board Of Directors ◽

Somatic Mutations ◽

Clinical Phenotype ◽

Disease Phenotype ◽

Advisory Committees ◽

Mutational Burden ◽

Normal Cytogenetics ◽

Serial Samples ◽

Over Time ◽

Stat Pathway

Abstract The Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) Essential Thrombocytosis (ET), Polycythemia Vera (PV), and Myelofibrosis (MF) are characterized by mutations, which drive JAK-STAT pathway activation. Several studies have demonstrated the presence of recurrent somatic mutations outside of the JAK-STAT pathway, which accumulate over time, and may impact disease phenotype and outcome. We sought to determine the influence of somatic mutations on clinical phenotype and prognosis. We sequenced a total of 30 genes recurrently mutated in myeloid malignancies in a cohort of 162 MPN patients (pts) using a next generation sequencing platform. The cohort included 49 pts with ET, 26 PV, 38 Primary Myelofibrosis (MF), 11 Post ET MF, 14 Post PV MF, 12 with leukemic transformations of MPN (LT), 7 with MPN-unclassified (MPN-U) and 5 others. Median age was 59 years and 79 were men. A Total of 288 gene mutations were identified with the most commonly mutated genes being JAK2 (n=121, 74%), TET2 (n=31, 19%), DNMT3A (n=18, 11%), ASXL1 (n=16, 10%), IDH2 (n=10, 6%), RAS (n=12, 7%), TYK2 (n=8, 5%) and TP53 (n=7, 4%). We did not find any mutations in NPM1, CBL, SRSF2 and no FLT3 -ITD. CALR was not assessed in 20 pts and these were excluded from mutation number analysis. Importantly, we identified a relationship between the absolute number of mutations found per pt, disease phenotype, and age (table 1). Pts with/without prior chemotherapy or radiotherapy exposure did not have a difference in mutation number (1.5 vs. 1.9). Cases of ET or PV with fibrotic transformation had more mutations in ASXL1, RAS, EZH2, PHF6 and MPL than pre fibrotic ET or PV suggesting these may be relevant in disease progression and development of fibrosis. Mutations in TET2, RAS and PHF6 were more frequent in cases with LT compared to those with chronic phase MPN. Pts over 40 were more likely to have mutations in TET2 (p=0.026) and JAK2 (p=0.019) and ASXL1 mutations were more common in pts with abnormal cytogenetics than in those with normal cytogenetics (p=0.003). Thrombotic events, which are an important cause of morbidity in MPN patients, negatively correlated with mutations in ASXL1 (p=0.044). Prognosis as measured by DIPPS and DIPSS-Plus scores appeared to correlate with the average number of mutations found in MF patients (table 2). We examined several cases for which serial samples were available, and noted the acquisition of new mutational events despite ongoing therapy. We noted that the most commonly acquired mutations occurred in epigenetic modifying (DNMT3A, TET, IDH, ASXL1) and in growth signaling pathway (RAS, CBL) genes. These occurred despite active therapy and often without an overt change in clinical phenotype. Further details of these serial samples will be presented. We conclude that the number and spectrum of somatic mutations correlate with disease phenotype of MPN. Younger pts have fewer mutations, as do pts with normal cytogenetics. JAK2 and TET2 mutations were more common in older pts. We show that a subset of pts acquire mutations in epigenetic modifiers and in genes involved in growth signaling pathways during disease course, and that mutations in TET2, RAS and PHF6 were enriched at the time of leukemic transformation. Taken together, these results indicate that mutations outside the JAK-STAT pathway influence disease phenotype, and that the acquisition of mutations over time may predict for disease progression. Serial evaluation of mutational burden over time therefore warrants exploration in the clinical setting. Table 1. Average number of mutations appeared to correlate with disease phenotype, age and abnormal cytogenetics. Average Number of Mutations N Mean (SD) P-value Age < 40 years 13 1.4 (0.9) 0.026 Age > 40 years 12 2 (1) No Thrombosis 113 2 (1) 0.712 Thrombosis 28 1.9 (1) Normal Cytogenetics 64 1.8 (0.9) 0.016 Abnormal Cytogenetics 40 2.3 (1.2) ET/PV/PMF 99 1.8 (0.8) 0.029 LT 10 3 (1.5) ET/PV 66 1.6 (0.7) 0.01 Post ET/PV MF 22 2.3 (1.1) ET 44 1.6 (0.7) < 0.001 PV 22 1.5 (0.9) PMF 33 2.2 (0.9) Post ET/PV MF 22 2.3 (1.1) LT 10 3 (1.5) Table 2. Disease prognostic scores in MF appear to correlate with the average number of mutations found per patient. Risk category N Average number mutations DIPSS Low 8 1.5 Intermediate-1 19 2.4 Intermediate-2 9 1.8 High 1 5 DIPSS-Plus Low 6 1.5 Intermediate-1 12 2 Intermediate-2 14 2.2 High 1 5 Figure 1. Comutation map of genomic alterations. Each hash mark on x-axis represents an individual patient. Figure 1. Comutation map of genomic alterations. Each hash mark on x-axis represents an individual patient. Disclosures Levine: CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy.

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MLL/AF9 Expression Causes Leukemia in Zebrafish

Blood ◽

10.1182/blood-2021-154125 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4330-4330

Author(s):

Maryam Saberi ◽

Omid Delfi ◽

Dona Wathsala Madola ◽

Peter J. Browett ◽

Purvi M Kakadia ◽

...

Keyword(s):

Acute Leukemia ◽

Exome Sequencing ◽

Board Of Directors ◽

Whole Exome Sequencing ◽

Somatic Mutations ◽

Transgenic Fish ◽

Advisory Committees ◽

Whole Exome ◽

Transgenic Embryos ◽

Marrow Cells

Abstract Background Acute Leukaemia (AL) is a genetically heterogeneous disorder caused by somatic mutations and acquired chromosomal translocations. Translocations can lead to the formation of fusion genes such as the MLL/AF9 fusion, which results from the t(9;11)(p22;q23). A better understanding the molecular pathophysiology of AML, of the mechanisms of treatment resistance, disease relapse can be achieved by developing animal models. The MLL/AF9 fusion is frequently used to model AML in mice. However, to date, no MLL/AF9 leukemia models in zebrafish have been reported. Aim Our aim was to establish a transgenic zebrafish leukemia model using the human MLL/AF9 fusion gene. Methods To generate transgenic fish, two constructs (pTol2-Runx1+23: MLL-AF9-IRES-EGFP-cmlc-GFP and pTol2-Runx1+23: MLL-AF9-IRES-mCherry-cmlc-GFP) were injected together with Tol2 transposase mRNA into one-cell stage zebrafish embryos. We used the murine Runx1+23 enhancer to direct MLL/AF9 expression to hematopoietic stem cells and EGFP or mCherry as fluorescent markers. We selected transgenic embryos 24 hours post-fertilization based on the heart marker expression (cmlc). Results 29% (100 of 340 embryos) of the transgenics reached adulthood (6 weeks). After 6 to 24 months, 77% (77) of them developed signs of sickness. They became less active with protruding eyes and hump formation on the nose. Some started bleeding from the gills and/or showed tumor formation around the abdomen and head. Sick fish were euthanized and dissected. The autopsies showed pale and dysmorphic kidneys, pale and enlarged spleens, and in some cases white granular spots on the spleen. Histological sections revealed increased kidney, spleen, and liver cellularity with massive cellular infiltration of cells in these organs. In flow cytometry, kidney marrow cells from the transgenic fish showed a different forward scatter (FSC) and side scatter (SSC) profile compared to that of the normal zebrafish kidney marrow cells. The transgenic F 0 zebrafish showed increased numbers of lymphoid cells (12 fish), precursor cells (9 fish), or myeloid cells (6 fish). Peripheral blood smears showed many intermediate-sized mononuclear cells with an increased nuclear-cytoplasmic ratio, with nuclei containing dipsersed chromatin and inconspicuous nucleoli resembling blasts. There were occasional myelocytes and no mature granulocytes. These data are consistent with the development of acute leukemia in our MLL/AF9 transgenic fish. Whole-mount in situ hybridization (WISH) was performed on F 1 embryos. RNA probes for early hematopoietic markers (gata1, scl, runx1, ikaros, cmyb, mpx, and lyz) were hybridized to 24 and 48 hpf F1 transgenic embryos. There were expression changes of these markers compared to age-matched wild-type larvae, including low expression of gata1, scl, cmyb and high expression of lyz, mpx and ikaros in the caudal hematopoietic organ. We also performed transplantation experiments with the kidney marrow cells from diseased fish to test whether the disease was transplantable. The disease was serially transplantable into secondary and tertiary recipient fish. Transplanted fish had a significantly shorter latency to disease development of only 2 to 6 weeks. The morphological evidence and the serial transplantability of the disease proves that we have in fact succeeded in establishing an MLL/AF9-driven acute leukemia model in zebrafish. The long latency and incomplete penetrance observed in our F 0 MA9 zebrafish, along with a shorter latency in the transplanted fish, suggests that additional somatic mutations are required for leukemogenesis in this model. We performed whole exome sequencing to find cooperating somatic mutations and RNASeq to identify differentially expressed genes in MA9 leukemia fish. Whole exome sequencing on six samples identified putative somatic mutations in genes such as Stat5, Cyp2j20, Ms4a17a.3, Tapbp.1 and Herc5.3, which have been reported to be mutated in human cancer. RNA-seq analysis on seven samples showed 67 differentially expressed genes with a q value < 0.05 (e.g., cxcl32b.1, myof1, ctdsp2, egr3, il2rb) and nine enriched pathways with a P-value of < 0.054 (e.g.: KRAS, TP53, MEK) in our transgenic leukemic MLL/AF9 fish. Conclusion Our MLL/AF9 zebrafish acute leukemia model will be a helpful tool to understand leukemia biology and enable testing of new therapeutic strategies. Disclosures Browett: AbbVie: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees.

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Systematic STAT3 Mutation Testing Identifies Patients with Unsuspected T-Cell Large Granular Lymphocytic Leukemia

Blood ◽

10.1182/blood.v128.22.919.919 ◽

2016 ◽

Vol 128 (22) ◽

pp. 919-919

Author(s):

Elizabeth A. Morgan ◽

Mark N. Lee ◽

Daniel J. DeAngelo ◽

David P. Steensma ◽

Richard M. Stone ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Hematologic Malignancies ◽

Sh2 Domain ◽

Lymphocytic Leukemia ◽

Advisory Committees ◽

Stat3 Mutation ◽

Stat3 Mutations

Abstract The initial clinical presentation of T-cell large granular lymphocytic leukemia (T-LGL) and myelodysplastic syndromes (MDS) can be similar, each characterized by unexplained peripheral cytopenias. However, these diseases are pathobiologically distinct and associated with stark differences in prognosis and therapy. T-LGL is a clonal lymphoid disorder defined by phenotypically abnormal cytotoxic T cells and an indolent clinical course, while MDS is a clonal disorder of hematopoietic stem cells defined by ineffective hematopoiesis, morphologic dysplasia, and an elevated risk of acute leukemia. Despite these differences, distinction between T-LGL and MDS can be challenging and misdiagnosis can significantly delay initiation of appropriate therapy. The recent identification of STAT3 mutations in LGL may facilitate this distinction: activating STAT3 mutations occur in 40-70% of T-LGL cases, primarily within the SH2 domain, but have not been reported in patients with MDS without concomitant T-LGL. STAT3 is included within our clinical next generation sequencing (NGS) panel, which is used to evaluate patients with known or suspected hematologic malignancies, primarily acute myeloid leukemia, MDS and myeloproliferative neoplasms, as well as various lymphocytic leukemias. We report the frequency and type of STAT3 mutations within our patient population and assess the impact of this information on diagnosis. Between 1/1/2015 and 6/30/2016, 3414 samples (primarily peripheral blood (PB) or bone marrow (BM)) from 2530 unique patients evaluated at Brigham and Women's Hospital and/or Dana-Farber Cancer Institute underwent clinical NGS with a custom, 95-gene, amplicon-based panel (PMID: 27339098). Exons 2-17 and 21-23 of the STAT3 gene were analyzed in each sample using reference transcript 1 (NM_139276). We identified 40 patients with 40 candidate STAT3 mutations (Figure 1). Based on domain localization, variant allele fraction, and population allele frequency, we classified these sequence variants as somatic SH2 domain mutations (n = 21), somatic non-SH2 domain mutations of unknown significance (n = 5) or germline variants (n = 14). Of the 21 patients with somatic SH2 domain mutations, 9 carried a prior diagnosis of T-LGL and 8 were concurrently diagnosed with T-LGL by conventional diagnostic criteria (clonal aberrant T cells in the setting of neutropenia, anemia, or lymphocytosis). The final 4 patients with STAT3 SH2 domain mutations were unexpected diagnoses of T-LGL. These 4 patients were initially referred from outside institutions for MDS based on the reported presence of unilineage erythroid dysplasia (n=2), unquantified ring sideroblasts (n=1), or pancytopenia with unspecified marrow findings (n=1). In 3 of these cases, the STAT3 mutation discovery prompted T-cell flow cytometric analysis of peripheral blood, which revealed an aberrant immunophenotype, and T-cell receptor gamma gene rearrangement studies, which were clonal; these tests are pending in the 4th case. BM evaluation was performed in 12 of 21 patients, including the 4 with suspected MDS; in all cases, the findings did not meet diagnostic criteria for MDS by expert hematopathology review and all showed a normal karyotype. Five additional cases demonstrated somatic non-SH2 domain STAT3 mutations of unknown pathobiologic significance: 3 myeloid neoplasms, 1 chronic lymphocytic leukemia, and 1 autoimmune hemolytic anemia. Additional non-STAT3 mutations were also frequently identified in tumors other than T-LGL. Our experience demonstrates that STAT3 sequencing is a critical component of the evaluation of unexplained cytopenias, and identification of a mutation can clarify ambiguous phenotypes thus averting the consequences of misdiagnosis or diagnostic delay. Notably, several series have reported that MDS and T-LGL can infrequently occur concurrently and thus identification of a STAT3 mutation and a clonal T-LGL population does not exclude the possibility of concomitant MDS. In our cohort, however, no T-LGL patient with a STAT3 mutation demonstrated pathologic evidence of MDS and the majority (19 of 21) showed no other myeloid-associated somatic mutations. In addition, 5 cases in our cohort had likely somatic, non-SH2 domain STAT3 mutations in the context of disparate clinical scenarios, suggesting that these mutations may have a pathogenic role in other hematologic malignancies, a subject of future study. Disclosures DeAngelo: Ariad: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Amgen: Consultancy; Baxter: Consultancy; Incyte: Consultancy; Celgene: Consultancy. Stone:Celator: Consultancy; Jansen: Consultancy; ONO: Consultancy; Agios: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Consultancy; Amgen: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy; Merck: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Sunesis Pharmaceuticals: Consultancy; Xenetic Biosciences: Consultancy. Lindsley:MedImmune: Research Funding; Takeda Pharmaceuticals: Consultancy.

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Molecular Analysis Of Circulating Tumor Cells Identifies Mutations That Are Distinct From Those Present In The Bone Marrow Of Patients With Multiple Myeloma

Blood ◽

10.1182/blood.v122.21.533.533 ◽

2013 ◽

Vol 122 (21) ◽

pp. 533-533

Author(s):

Yuji Mishima ◽

Jens Lohr ◽

Yu-Tzu Tai ◽

Ludmila Flores ◽

Yosra Aljawai ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Board Of Directors ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Research Funding ◽

Somatic Mutations ◽

Advisory Committees ◽

Whole Exome ◽

Equity Ownership

Abstract Background Clonal evolution involves simultaneous evolution of multiple co-existant subclones. Recent studies have suggested that clonal heterogeneity is critical during the progression of Multiple Myeloma (MM). Circulating tumor cells (CTCs) have been identified in many patients with solid tumors and hematological malignancies. Recent studies have suggested that CTCs can be identified in patients with Multiple Myeloma. The aims of this study were to identify the phenotypic characteristics of CTCs in patients with Mutliple Myeloma at different stages of the disease, to determine whether somatic mutations present in the bone marrow clones are also identified in CTCs or whether specific subclones are more prone to enter the systemic circulation. These subclones may have a higher likelihood of inducing dissemination into extramedullary sites and potential for drug resistance. Methods We analyzed the peripheral blood samples of 466 patients diagnosed with Multiple Myeloma at different stages of progression. Two plasma cell leukemia patients were included in the study. Freshly collected peripheral blood was processed to obtain white blood cell fractions. The cells were stained with eight antibodies including CD19, CD38, CD138, CD45, CD56, CD28, CD44, and CD183 and CTCs were purified by gating on CD19-/CD38+/CD138+ cells. Among them, ten of the CTC samples were selected to analyze somatic mutation using whole exome sequencing. Briefly, 1µg of genomic DNA was extracted form sorted cells followed by shearing, end repair, and ligation to barcoded adaptors. The DNA was size-selected, subjected to exonic hybrid capture and sequenced on Illumina HiSeq flow cells with an average depth of coverage of 100x. Results Of the 466 samples analyzed, the number of CTCs identified ranged from 0.01% to 61% of total WBC count. CTCs were detected in 61.4% of all samples analyzed. CTCs were detected in 64.5% of relapsed MM, 63.4% of newly diagnosed MM, 24.0% of smoldering MM, and 25.0% of MGUS patients. Significant differences of the surface markers including CD45, CD28, CD56, and CD44 were not observed in the different stages of MM disease progression. For further characterization of CTCs, we performed whole exome sequencing of CTCs in 10 MM samples, of which 5 had sequencing of their matched tumor cells collected from BM as well as matched normal germline cells to examine whether circulating tumor cells possess any distinctive somatic mutations. The sequence analysis revealed that both CTCS and marrow restricted tumor cells have substantial numbers of protein-coding mutations. CTCs and bone marrow cells shared 5-38% similar mutations, while interestingly the rest of the mutations were exclusively present in either the CTCs or bone marrow samples. We identified a total of 347 somatic mutations, which included 199 CTC specific mutations. Several known driver mutations were observed, i.e. BRaf V600E mutation present in the CTC samples but not in the matching bone marrow samples in one patient. Twelve of these CTC mutations were shared at least in two patients including ZNF721, NBPH10, F5, and PRDM15. Conclusion These data suggest subclonal out growth of CTCs from one of the parent clones with acquisition of additional mutation over time outside of the bone marrow microenvironment. Further validation of the unique mutations in CTCs may provide mechanistic insight into myeloma cell dissemination, and so potentially inform treatment strategies. Disclosures: Tai: Onyx: Consultancy. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership. Munshi:Celgene Corporation: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Millennium: The Takeda Oncology Company: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Onyx Pharmaceuticals Inc: Membership on an entity’s Board of Directors or advisory committees. Ghobrial:Noxxon: Research Funding; BMS: Advisory board, Advisory board Other, Research Funding; Onyx: Advisoryboard Other; Sanofi: Research Funding.

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Next-Generation Sequencing Analysis of Clonal Hierarchy and Dynamics in T-Large Granular Lymphocyte Leukemia Suggests Emergence of STAT3 Clones within Pre-Existing Dominant T-Cell Repertoire Responses Otherwise Silenced in Normal Individuals

Blood ◽

10.1182/blood.v128.22.2731.2731 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2731-2731

Author(s):

Peter W Chomczynski ◽

Michael J. Clemente ◽

Srinivasa Reddy Sanikommu ◽

Alek d Nielsen ◽

Cassandra M. Hirsch ◽

...

Keyword(s):

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Cell Repertoire ◽

Advisory Committees ◽

Normal Individuals ◽

Stat3 Mutation ◽

Serial Samples ◽

Stat3 Mutations ◽

Over Time

Abstract T-large granular lymphocyte leukemia (T-LGLL) is a clonal lymphoproliferative disorder of cytotoxic T-cells (CTL) that is associated with cytopenias, predominantly neutropenia and reticulocytopenic anemia. From a scientific point of view, T-LGLL provides a natural model to study the dynamics of CTL responses; the heterogeneity of the disorder allows for examining the diversity of CTL responses in both autoimmune disorders and putatively chronic reactive conditions. A proportion of patients may have an extreme reactive process that mimics an indolent neoplastic lymphoproliferation. NGS and deep T-cell repertoire (TCR) sequencing provide insight into the clonal dynamics at work in T-LGLL patients. A large proportion of T-LGLL patients present with a bona-fide low-grade leukemia; this notion is supported by the discovery of recurrent somatic STAT3 mutations in some patients. STAT3 clonal burden represents an excellent marker that can be serially monitored along with clinical milestones to ultimately gain a more comprehensive understanding of disease etiology and natural history. We collected a cohort of 183 LGLL patients and screened them via deep NGS for mutation status of STAT3. In 36% of patients, 4 distinct somatic mutations (Y640F, N647I, D661V, D661Y) were identified in the SH2 domain of STAT3. In patients with wildtype STAT3, no somatic mutation was implicated in clonal expansion except for a small minority with STAT5 mutations present. We performed a longitudinal analysis of 20 representative STAT3-mutated T-LGLL patients with up to 10-year follow-up and an average of 7 analyzed blood samples per case. All serial samples were deep-sequenced to detect and determine the VAF of the known STAT3 mutations. Overall, STAT3 mutation VAF had a significant, inverse relationship to both hemoglobin and absolute neutrophil count (ANC) (both p<=0.001). In 7/11 cases harboring the Y640F mutation, chemotherapy led to remission accompanied by a decrease in VAF; 3 were asymptomatic and received no treatment. In patients with D661V or D661Y, 6/9 achieved remission with treatment. Only 1/3 cases with N647I entered remission. This longitudinal cohort can be sub-categorized into distinct patterns of clonal dynamics: 1) emerging STAT3 mutation in 20% of patients with a decrease in ANC as VAF of STAT3 clones expand; 2) an opposite trend in 40% of patients where VAF decreased due to therapeutic manipulations; 3) stable VAF in 20% of patients with little change over time; 4) codominant or dominant/secondary STAT3 mutations with distinct subclonal burden in 20% of patients. We performed deep TCR NGS on a representative subset of 9 patients to explore how STAT3 mutations correlated with T-cell clonal expansions. The data were processed by an extensive bioanalytic pipeline to quantify the relative abundance of each CDR3 rearrangement within a patient's TCR. Our cohort had an average of over 53,000 CDR3 templates per sample and was compared with 587 healthy controls. Our results demonstrate multiple patterns of clonal dynamics over the course of T-LGLL. Within each case, the immunodominant clones in serial samples were identified and correlated with STAT3 VAF burden over time. When patients were in remission, both STAT3 VAF and clonality were typically low. Interestingly, functional remission occurred in 2 cases despite increases in both clonality and STAT3 VAF. In 5/9 cases, the T-LGLL process involved 1 STAT3 mutation and 1 corresponding pathogenic clonotype displaying similar dynamics over time. In patients with 2 mutations, multiple high-frequency clonotypes were observed. Most significantly, comparison of STAT3 VAF and the dominant clonotype(s) revealed that STAT3 mutation can arise within a pre-existing clonal expansion that may harbor 2 branching mutations in extreme cases. Identification of CDR3 rearrangement sequences allowed for analysis of the distribution of clonotypes among patients and controls. The pathogenic clonotypes found in T-LGLL patients were detected in a high proportion of controls but at extremely low frequencies. This suggests that these potentially autoimmune clones exist in normal individuals but are effectively suppressed. No pathogenic clonotypes were shared among disease patients. In sum, analysis of clonal dynamics suggests that STAT3 mutations can occur in the context of pre-existing oligoclonal responses and involve otherwise low-frequency clonal specificities. Disclosures Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium/Takeda: 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. Mustjoki:Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Research Funding.

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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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Association of MHC Class I Chain-Related Gene a (MICA) Polymorphisms with Allogeneic Hematopoietic Cell Transplantation Outcomes in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-112745 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2075-2075

Author(s):

Sagar S. Patel ◽

Betty K. Hamilton ◽

Lisa Rybicki ◽

Dawn Thomas ◽

Arden Emrick ◽

...

Keyword(s):

Board Of Directors ◽

Binding Affinity ◽

Myeloid Leukemia ◽

Research Funding ◽

Somatic Mutations ◽

Hematopoietic Cell Transplantation ◽

Advisory Committees ◽

Post Transplant ◽

Transplant Outcomes ◽

Nkg2d Receptor

Abstract Background MHC class I chain-related gene A (MICA) is a polymorphic ligand of the natural killer (NKG2D) receptor on immune effector cells. The activating NKG2D receptor controls immune responses by regulating NK cells, NKT cells and γδ-T cells. Dimorphisms at sequence position 129 of the MICA gene confers varying levels of binding affinity to NKG2D receptor. MICA previously has been associated with post-allogeneic hematopoietic cell transplantation (alloHCT) outcomes including graft-versus-host-disease (GvHD), infection, and relapse. However, it is unclear how MICA interacts with cytogenetic and somatic mutations in regards to these outcomes in acute myeloid leukemia (AML). Methods We conducted a single center, retrospective analysis of adult AML patients in first or second complete remission (CR1, CR2), who underwent T-cell replete matched related or unrelated donor alloHCT. Analysis was limited to those who had MICA data available for donors and recipients. In addition to cytogenetic risk group stratification by European LeukemiaNet criteria (Döhner H, et al, Blood 2016), a subset of patients had a 36-gene somatic mutation panel assessed prior to alloHCT by next-generation sequencing. Dimorphisms at the MICA-129 position have previously been categorized as weaker (valine/valine: V/V), heterozygous (methionine/valine: M/V), or stronger (methionine/methionine: M/M) receptor binding affinity. Fine and Gray or Cox regression was used to identify the association of MICA and outcomes with results as hazard ratios (HR) and 95% confidence intervals (CI). Results From 2000 - 2017, 131 AML patients were identified meeting inclusion criteria. Median age at transplant was 54 years (18-74), with 98% Caucasian. Disease status at transplant included 78% CR1 and 22% CR2. Cytogenetic risk stratification showed 13% of patients as favorable, 56% as intermediate, and 31% as adverse-risk. The five most common somatic mutations were FLT3 (15%), NPM1 (14%), DNMT3A (11%), TET2 (7%), and NRAS (6%). 60% of patients had a related donor. A myeloablative transplant was performed in 84% of patients and 53% had a bone marrow graft source. The most common conditioning regimen used was busulfan/cyclophosphamide (52%). 12% of patients were MICA mismatched with their donor. The distribution of donor MICA-129 polymorphisms were 41% V/V, 53% M/V, and 6% M/M. In univariable analysis, donor-recipient MICA mismatch tended to be associated with a lower risk of infection (HR 0.49, CI 0.23-1.02, P=0.06) and grade 2-4 acute GvHD (HR 0.25, CI 0.06-1.04, P=0.06) but was not associated with other post-transplant outcomes. In multivariable analysis, donor MICA-129 V/V was associated with a higher risk of non-relapse mortality (NRM) (HR 2.02, CI 1.01-4.05, P=0.047) (Figure 1) along with increasing patient age at transplant (HR 1.46, CI 1.10-1.93, p=0.008) and the presence of a TET2 mutation (HR 6.00, CI 1.77-20.3, P=0.004). There were no differences between the V/V and the M/V+M/M cohorts regarding somatic mutational status, cytogenetics and other pre-transplant characteristics and post-transplant outcomes. With a median follow-up of 65 months for both cohorts, 45% vs. 49% of patients remain alive, respectively. The most common causes of death between the V/V and the M/V+M/M cohorts was relapse (38% vs. 62%) and infection (31% vs. 8%), respectively. Conclusion While previous studies have demonstrated associations of somatic mutations and cytogenetics with survival outcomes after alloHCT for AML, we observed mutations in TET2 and the V/V donor MICA-129 polymorphism to be independently prognostic for NRM. Mechanistic studies may be considered to assess for possible interactions of TET2 mutations with NK cell alloreactivity. The weaker binding affinity to the NKG2D receptor by the V/V phenotype may diminish immune responses against pathogens that subsequently contribute to higher NRM. These observations may have implications for enhancing patient risk stratification prior to transplant and optimizing donor selection. Future investigation with larger cohorts interrogating pre-transplant AML somatic mutations with MICA polymorphisms on post-transplant outcomes may further elucidate which subsets of patients may benefit most from transplant. Disclosures Nazha: MEI: Consultancy. Mukherjee:Pfizer: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Projects in Knowledge: Honoraria; BioPharm Communications: Consultancy; Bristol Myers Squib: Honoraria, Speakers Bureau; Takeda Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; LEK Consulting: Consultancy, Honoraria; Aplastic Anemia & MDS International Foundation in Joint Partnership with Cleveland Clinic Taussig Cancer Institute: Honoraria. Advani:Amgen: Research Funding; Pfizer: Honoraria, Research Funding; Glycomimetics: Consultancy; Novartis: Consultancy. Carraway:Novartis: Speakers Bureau; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: 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; Agios: Consultancy, Speakers Bureau. Gerds:Apexx Oncology: Consultancy; Celgene: Consultancy; Incyte: Consultancy; CTI Biopharma: Consultancy. Sekeres:Celgene: 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; Opsona: Membership on an entity's Board of Directors or advisory committees. Maciejewski: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; Apellis Pharmaceuticals: Consultancy. Majhail:Incyte: Honoraria; Anthem, Inc.: Consultancy; Atara: Honoraria.

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