Germline GATA2 Mutations Research Articles

MDS-associated mutations in germline GATA2 mutated patients with hematologic manifestations

Germline mutation in GATA2 can lead to GATA2 deficiency characterized by a complex multi-system disorder that can present with many manifestations including variable cytopenias, bone marrow failure, myelodysplastic syndrome/acute myeloid leukemia (MDS/AML), and severe immunodeficiency. Penetrance and expressivity within families is often variable. There is a spectrum of bone marrow disease in symptomatic cytopenic patients ranging from hypocellular marrows without overt dysplasia to those with definitive MDS, AML, or chronic myelomonocytic leukemia. Relatives of probands with the same mutations may demonstrate minimal disease manifestations and normal marrows. A comprehensive clinical, hematological and genetic assessment of 25 patients with germline GATA2 mutation was performed. MDS-associated mutations were identified in symptomatic GATA2 patients both with overt MDS and in those with hypocellular/aplastic bone marrows without definitive dysplasia. Healthy relatives of probands harboring the same germline GATA2 mutations had essentially normal marrows that were overall devoid of MDS-associated mutations. The findings suggest that abnormal clonal hematopoiesis is a common event in symptomatic germline mutated GATA2 patients with MDS and also in those with hypocellular marrows without overt morphologic evidence of dysplasia, possibly indicating a pre-MDS stage warranting close monitoring for disease progression.

Prognostic Value of IPSS-R Score, Cytogenetic Aberrations, and Cytopenias on Outcome of Allogeneic Hematopoietic Stem Cell Transplant in Myelodysplastic Syndrome in GATA2 Deficiency

Sporadic or familial germline mutations in GATA2 frequently result in a bone marrow failure syndrome characterized by recurrent life-threatening infections, cytopenias, and progression to myelodysplastic syndrome (MDS), acute myelogenous leukemia (AML), or chronic myelomonocytic leukemia (CMML). Hematopoietic stem cell transplantation (HSCT) represents the only definitive therapy for GATA2 deficiency. However, the highly variable disease penetrance in different families, and in mutation positive individuals within a single family, make the timing of HSCT challenging. We assessed the utility of scoring patient characteristics according to the Revised International Prognostic Scoring System (IPSS-R) to predict survival after HSCT in a cohort of 51 patients with GATA2 deficiency who had progressed to MDS. Higher IPSS-R scores significantly correlated with reduced overall survival after transplant. In evaluating the components of the IPSS-R score for patients with GATA2 deficiency, cytopenias, including anemia, neutropenia, and thrombocytopenia, were important variables in placing these patients in higher risk IPSS-R categories. Since GATA2 deficiency patients have very low peripheral blood monocyte, B cell, and NK cell counts, we evaluated these cytopenias in conjunction with IPSS-R scoring. These cytopenias also had prognostic value for outcome after HSCT in patients with GATA2 deficiency. Cytogenetics, another component of the IPSS-R score, also placed patients with GATA2 deficiency into higher risk categories. In particular, patients harboring monosomy 7 had poorer survival outcomes than patients with normal cytogenetics or trisomy 8. Of note, all five patients with GATA2 deficiency and trisomy 1q successfully underwent HSCT. Clonal cytogenetic progression with the development of additional cytogenetic aberrations was a harbinger of myeloid progression and led to HSCT. In a cohort of four patients who underwent HSCT following progression to AML/CMML, there was only one survivor. Thus, HSCT is recommended in patients with GATA2 deficiency and intermediate or high-risk IPSS-R scores before the development of AML or CMML. DisclosuresNo relevant conflicts of interest to declare.

Monosomy 7 As the Initial Hit Followed By Sequential Acquisition of SETBP1 and ASXL1 Driver Mutations in Childhood Myelodysplastic Syndromes

Childhood myelodysplastic syndromes (MDS) account for less than 5% of pediatric hematologic malignancies and differ from their adult counterpart in terms of biology, genetics, and cure rates. Complete (-7) or partial loss (del7q) of chromosome 7 constitutes the most common cytogenetic abnormality and is associated with more advanced disease typically requiring timely hematopoietic stem cell transplantation (HSCT). Previously, we and others established a link between -7 and germline GATA2 mutations in pediatric MDS (37% of MDS/-7 cases are GATA2-deficient) as well as constitutional SAMD9/9L disorders where -7 is utilized as an escape mechanism from the growth-restrictive effect of SAMD9/9L mutations. To date, comprehensive sequencing studies have been performed in 96 children with primary MDS, as reported by Pastor et al, Leukemia 2017 and Schwartz et al, Nature Comm 2017. This work established mutations in SETBP1, ASXL1, PTPN11, RUNX1 and RAS pathway genes as common somatic drivers. However, little is known about the clonal development of -7 and the role of additional somatic mutations. The knowledge about clonal hierarchies is essential for the understanding of disease progression on molecular level and for mapping potential drug targets. The rationale for the current study was to i) define the most common somatic drivers in a large cohort of patients with childhood MDS, ii) identify clonal/subclonal mutations, iii) infer clonal architecture of monosomy 7 and track the changes over time.We studied a cohort of 576 children and adolescents with primary MDS diagnosed between 1998 and 2016 in Germany, consisting of 482 (83%) patients with refractory cytopenia of childhood (RCC) and 94 (17%) MDS with excess blasts (EB). All patients underwent deep sequencing for 30 genes relevant to pediatric MDS and additional WES was performed in 150/576 patients. Using 20 computational predictors (including CADD and REVEL), population databases and germline testing, we identified the most likely pathogenic mutations.First, we excluded germline predisposing mutations in GATA2, SAMD9/SAMD9L and RUNX1 detected in 7% (38/576), 8% (43 of 548 evaluable) and 0.7% (4/576) of patients, respectively. Then we focused on the exploration of somatic aberrations. Most common karyotype abnormalities were monosomy 7 (13%, 77/576) and trisomy 8 (3%, 17/576). A total of 104 patients carried somatic mutations, expectedly more prevalent in the MDS-EB group as compared to RCC (56%, 53/94 vs 10.6%, 51/482; p<0.0001). The most recurrent somatic hits (≥ 1% frequency within 576 cases) were in SETBP1 (4.2%), ASXL1 (3.8%), RUNX1 (3.3%), NRAS (2.9%), KRAS (1.6%), PTPN11 (1.4%) and STAG2 (1%). We next focused on the -7 karyotype as a common denominator for the mutated group. Mutations were found in 54% (43/79), and the mutational load was significantly higher in -7 vs. non-7 (1.1 vs. 0.1 mutations per patient; p<0.001). In 11 patients with -7 and concomitant SETBP1/ASXL1 driver mutations, SETBP1 surpassed ASXL1 hits (median allelic frequency: 38% vs. 24%, p<0.05), while mutations in other genes were subclonal. Notably, these clonal patterns were independent of the underlying hereditary predisposition (4/11 GATA2; 3/11 SAMD9L). To explore the clonal hierarchy in MDS/-7 we performed targeted sequencing of several hundreds of single bone marrow derived colony forming cells (CFC) in 7 patients with MDS/-7. In all cases, the -7 clone was the founding clone followed by stepwise acquisition of mutations (i.e. -7>SETBP1>ASXL1; -7>SETBP1>ASXL1>PTPN11; -7>SETBP1>ASXL1>CBL, -7>EZH2>PTPN11). Finally, we tracked clonal evolution over time in 12 cases with 2-12 available serial samples using deep sequencing complemented by serial CFC-analysis. This confirmed that SETBP1 clones are rapidly expanding, while ASXL1 subclones exhibit an unstable pattern with clonal sweeping, while additional minor clones are acquired as late events. In 2 of 11 transplanted patients who experienced relapse, the original clonal architecture reappeared after HSCT.In summary, the hierarchy of clonal evolution in pediatric MDS with -7 follows a defined pattern with -7 aberrations arising as ancestral event followed by the acquisition of somatic hits. SETBP1 mutations are the dominant driver while co-dominant ASXL1 mutations are unstable. The functional interdependence and potential pharmacologic targetability of such somatic lesions warrants further studies. DisclosuresNiemeyer:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Early Clonal Hematopoiesis Detected in Gata2 heterozygosity By Color-Barcoding of Hematopoietic Stem Cells in Zebrafish

Germline GATA2 haploinsufficiency underlies an inherited syndrome of predisposition to myeloid malignancies. The mechanism by which GATA2 mutations lead to leukemia in children and young adults remains to be determined. We generated gata2b heterozygous mutant zebrafish using CRISPR/Cas9 technology to study this process. We used a color-barcoding system in zebrafish called Zebrabow that labels each hematopoietic stem cell (HSC) born during embryogenesis with a different color. Color-barcoding was induced by a blood-specific promoter-driven Cre recombinase during development at 24 hours post fertilization when there are on average 20 HSC clones. We then examined adult hematopoiesis in wildtype and gata2b+/- zebrafish. Surprisingly, at steady state the marrow of gata2b+/- fish showed color dominance with a single color clone contributing to over 30% of granulocytes, while the myeloid output in wildtype clutchmates was polyclonal. This suggested a baseline oligoclonal state in gata2b+/- hematopoiesis. Gata2b+/- fish also had a concomitant mild myelocytopenia with 20% reduction of myeloid cells in the marrow as early as 2 months post fertilization (mpf), reminescent of monocytopenia often present in GATA2 patients. Myelocytopenia with concurrent expansion of progenitor population was more profound in gata2b-/- fish with up to 4-fold increase of progenitor to myeloid cell ratio at 3mpf. Gata2b-/- fish are adult viable, possibly due to partial compensation by a duplicated gata2a gene. Germline GATA2-associated MDS and AML often harbor additional acquired mutations in epigenetic regulators. To study the effect of somatic mutations in GATA2 heterozygosity, we induced mosaic mutagenesis in wild-type or gata2b+/-embryos by injecting 1-cell embryos with Cas9 mRNA and guide RNAs targeting zebrafish orthologs of ASXL1 and STAG2, both of which have been associated with GATA2 mutations in MDS or AML. The marrow analysis of injected gata2b+/- fish showed an increase of hematopoietic progenitor cells and worse myelocytopenia at 7 mpf compared to control injected wildtype fish (progenitor/myeloid ratio of 1.7, p < 0.05). Single color expansion was observed in over 60% of injected gata2b+/-fish as measured by Shannon index of diversity, and correlated with stag2a and stag2b frame-shift mutations with variant allele fraction of >20%. This mutations result in the predicted loss of at least 2 of the 4 total stag2 alleles. In summary, gata2b deficiency alone results in aberrant hematopoiesis with reduced HSC diversity and myelocytopenia. Mutations in stag2 lead to early enhanced clonal dominance in gata2b+/- but not wildtype fish. Our studies suggest a model in which germline GATA2 mutations lead to a state of reduced HSC diversity during development. Such a state predisposes to clonal events due to acquired mutations in epigenetic regulators resulting in clonal hematopoiesis and myeloid leukemias, establishing a link between abnormal developmental hematopoiesis and leukemia risk later in life. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2535: Single-cell transcriptome analysis on lymphocytes of GATA2 deficiency patients

Abstract GATA2 is an essential transcription factor in the proliferation and maintenance of hematopoietic stem and progenitor cells. Germline GATA2 mutations cause hematological malignancies, especially myelodysplastic syndrome and acute myeloid leukemia. Pre-leukemic phenotype of GATA2 deficient patients varies markedly. However, peripheral blood (PB) lymphopenia is characteristic for almost all the patients and encounters most prominently B and natural killer (NK) cells. We aimed to gain understanding on the effects of GATA2 deficiency in hematopoiesis by comparing expression patterns in lymphocytes in GATA2 wild type and GATA2 deficient individuals. We conducted single-cell transcriptomic analysis using 10x Genomics Chromium Single Cell 3′ Solution on PB samples from three healthy controls and three GATA2 deficient patients (p.Q328X, p.T354M, and one haploinsufficiency due to unidentified reason). Erythrocytes were lysed prior to washing, filtering and resuspending white blood cells in 0.04% BSA/PBS (1x106 cells/mL). The gel beads in emulsion were generated using Chromium Single Cell 3' v2 Reagent Kit. The sample libraries, prepared according to 10x Genomics instructions, were sequenced in Illumina HiSeq 2500. Cell Ranger v1.3 analysis pipelines (10x Genomics) were used to demultiplex and convert Chromium single cell 3' RNA-sequencing barcode; read data to FASTQ files; and generate align reads and gene-cell matrices. The data were analyzed using Seurat, an R package designed for single cell analysis. Cell type clusters were identified through known markers from Seurat's graph-based clustering. Significant gene expression differences in lymphocytes (GATA2 deficient patients vs. controls) were observed (Wilcoxon rank sum test, Bonferroni-corrected p &amp;lt;0.05). In NK cells the distinction was found e.g. in KLRB1, CD74, CCL5, STAT1, and LAG3, all linked to immune responses, and BTG1 and DDIT4, regulating cell proliferation and differentiation. Expression changes in immune system-associated genes were also noted in T cells e.g. in STAT1, IL7R, CD48, and CXCR4, as well as in NPM1, TPT1, and TXNIP, genes identified to participate in diverse cellular processes. Also, several genes encoding ribosomal proteins were differentially expressed between patients' and controls' T cells. Comparison of B cells revealed changes in e.g. GZMH, PPBP, and LCN2, which, too, play roles in the immune system. We observed significant dissimilarities in gene expression pattern in GATA2 deficient patients' vs. controls' lymphocytes. These were reflected in several genes involved in immune system and cellular processes. Connection between our findings and patients' clinical manifestations is intriguing and may have an impact in the development of future patient care. We will extend our material and include also bone marrow sample single-cell transcriptomes and healthy mutation carriers to the analysis. Citation Format: Elina A. Hirvonen, Inari Natri, Pirkko Mattila, Jenni Mattila, Esa Pitkänen, Kimmo Porkka, Outi Kilpivaara, Ulla Wartiovaara-Kautto. Single-cell transcriptome analysis on lymphocytes of GATA2 deficiency patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2535.

Natural history of GATA2 deficiency in a survey of 79 French and Belgian patients.

Heterozygous germline GATA2 mutations strongly predispose to leukemia, immunodeficiency, and/or lymphoedema. We describe a series of 79 patients (53 families) diagnosed since 2011, made up of all patients in France and Belgium, with a follow up of 2249 patients/years. Median age at first clinical symptoms was 18.6 years (range, 0-61 years). Severe infectious diseases (mycobacteria, fungus, and human papilloma virus) and hematologic malignancies were the most common first manifestations. The probability of remaining symptom-free was 8% at 40 years old. Among the 53 probands, 24 had missense mutations including 4 recurrent alleles, 21 had nonsense or frameshift mutations, 4 had a whole-gene deletion, 2 had splice defects, and 2 patients had complex mutations. There were significantly more cases of leukemia in patients with missense mutations (n=14 of 34) than in patients with nonsense or frameshift mutations (n=2 of 28). We also identify new features of the disease: acute lymphoblastic leukemia, juvenile myelomonocytic leukemia, fatal progressive multifocal leukoencephalopathy related to the JC virus, and immune/inflammatory diseases. A revised International Prognostic Scoring System (IPSS) score allowed a distinction to be made between a stable disease and hematologic transformation. Chemotherapy is of limited efficacy, and has a high toxicity with severe infectious complications. As the mortality rate is high in our cohort (up to 35% at the age of 40), hematopoietic stem cell transplantation (HSCT) remains the best choice of treatment to avoid severe infectious and/or hematologic complications. The timing of HSCT remains difficult to determine, but the earlier it is performed, the better the outcome.

Establishment of a Screening System to Identify Novel GATA-2 Transcriptional Regulators.

Hematopoietic stem cells can self-renew and differentiate into all blood cell types. The transcription factor GATA-2 is expressed in hematopoietic stem and progenitor cells and is essential for cell proliferation and differentiation. Heterozygous germline GATA2 mutations induce GATA-2 deficiency syndrome, characterized by monocytopenia, a predisposition to myelodysplasia and acute myeloid leukemia, and a profoundly reduced dendritic cell (DC) population, which is associated with increased susceptibility to viral infections. Because patients with GATA-2 deficiency syndrome could retain a wild-type copy of GATA-2, boosting residual wild-type GATA-2 activity may represent a novel therapeutic strategy for the disease. Here, we sought to establish a screening system to identify GATA-2 activators using human U937 monocytic cells as a potential model of the DC progenitor. Enforced GATA-2 expression in U937 cells induces CD205 expression, a marker of DC differentiation, indicating U937 cells as a surrogate of human primary DC progenitors. Transient luciferase reporter assays in U937 cells reveals a high promoter activity of the -0.5 kb GATA-2 hematopoietic-specific promoter (1S promoter) fused with two tandemly connected GATA-2 +9.9 kb intronic enhancers. We thus established U937-derived cell lines stably expressing tandem +9.9 kb/-0.5 kb 1S-luciferase. Importantly, forced GATA-1 expression, a repressor for GATA-2 expression, in the stable clones caused significant decreases in the luciferase activities. In conclusion, our system represents a potential tool for identifying novel regulators of GATA-2, thereby contributing to the development of novel therapeutic approaches.

Heterogeneity of GATA2-related myeloid neoplasms.

The GATA2 gene codes for a master hematopoietic transcription factor that is essential for the proliferation and maintenance of hematopoietic stem and progenitor cells. Heterozygous germline mutations in GATA2 have been initially associated with several clinical entities that are now collectively defined as GATA2 deficiency. Despite pleiotropic clinical manifestations, the high propensity for the development of myelodysplastic syndromes (MDS) constitutes the most common clinical denominator of this major MDS predisposition syndrome. The immunological phenotypes can be variable and mostly include deficiency of monocytes and/or B cells. Thus far, nearly 380 GATA2-deficient patients had been reported, with a roughly estimated prevalence of myeloid neoplasia of at least 75%. The most common abnormal karyotypes associated with GATA2-related MDS are monosomy 7, der(1;7) and trisomy 8. The overall clinical penetrance seems to be nearly complete for this transcriptopathy disorder. The high-risk MDS subtypes and karyotypes, and the underlying immunodeficiency guide decision-making toward timely stem cell transplantation.

Germline Mutations Associated with Leukemia in Childhood: New Discoveries and Emerging Phenotypes

Acute leukemia is the most common cancer of childhood. The pathogenesis of childhood leukemia is multifactorial, and most cases occur sporadically. Although the majority of genetic changes are acquired somatic mutations, recent discoveries have revealed that inherited germline mutations are present in an increasing proportion of children, predisposing them to leukemia. Recently described germline mutations in ETV6, SH2B3, and PAX5 have been associated with increased risks of acute lymphoblastic leukemia (ALL). A high incidence of germline mutations in GATA2 is seen in pediatric myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML). Susceptibility genes for adult leukemia are found at increased incidence in children with MDS/AML. Recent findings have broadened the phenotype of inherited bone marrow failure syndromes, with a decreased emphasis on phenotypic abnormalities. Newly identified germline susceptibilities to hematologic malignancies are emerging at a rapid pace with the advent of next-generation sequencing approaches. A higher incidence of germline mutations accounts for childhood leukemia more than previously thought, and affected children do not necessarily fit into previously described syndromic presentations; therefore, careful clinical consideration is required for accurate diagnosis. Furthermore, children with inherited predisposition require a multidisciplinary approach to ensure proper evaluation and treatment, including but not limited to genetic counseling for the patient and family members and close management by a hematologist and oncologist, plus evaluations by a psychologist, as well as by a stem cell transplantation specialist.

Somatic mutations in children with GATA2-associated myelodysplastic syndrome who lack other features of GATA2 deficiency

AbstractApproximately 10% of children with primary myelodysplastic syndrome (MDS) have germ line GATA2 mutations, leading to the proposal that all children with primary MDS and certain cytogenetic findings, including monosomy 7, be tested for germ line GATA2 mutations regardless of family history or other clinical features associated with GATA2 deficiency. In adults with familial GATA2-MDS, those with somatic mutations in ASXL1 experience rapid disease progression to acute myeloid leukemia (AML) and poor prognosis after stem cell transplantation; however, the prevalence of somatic mutations in primary pediatric GATA2-MDS is unclear. Here, we studied a cohort of 8 pediatric patients with MDS and lacking additional GATA2-associated clinical features or significant family history and identified heterozygous germ line GATA2 mutations in 5 patients, including 1 with a normal karyotype. For those with GATA2-MDS, we screened for somatic mutations in genes with prognostic relevance in AML/MDS, using a targeted next-generation sequencing panel. Although no somatic mutations in ASXL1 were observed, somatic mutations were found in RUNX1, SETBP1, IKZF1, and CRLF2. One subject with deleterious mutations in RUNX1, SETBP1, and IKZF1 rapidly progressed to AML with disease that was refractory to treatment. Our findings confirm the importance of GATA2 testing in primary pediatric MDS, even in the absence of other clinical features of GATA2 deficiency. Further, similar to what has been observed in adults with GATA2-MDS, somatic mutations with potential prognostic effect occur in children with MDS associated with mutations in GATA2.

Myelodysplastic and myeloproliferative disorders of childhood.

Myelodysplastic syndrome (MDS) and myeloproliferative disorders are rare in children; they are divided into low-grade MDS (refractory cytopenia of childhood [RCC]), advanced MDS (refractory anemia with excess blasts in transformation), and juvenile myelomonocytic leukemia (JMML), each with different characteristics and management strategies. Underlying genetic predisposition is recognized in an increasing number of patients. Germ line GATA2 mutation is found in 70% of adolescents with MDS and monosomy 7. It is challenging to distinguish RCC from aplastic anemia, inherited bone marrow failure, and reactive conditions. RCC is often hypoplastic and may respond to immunosuppressive therapy. In case of immunosuppressive therapy failure, hypercellular RCC, or RCC with monosomy 7, hematopoietic stem cell transplantation (HSCT) using reduced-intensity conditioning regimens is indicated. Almost all patients with refractory anemia with excess blasts are candidates for HSCT; children age 12 years or older have a higher risk of treatment-related death, and the conditioning regimens should be adjusted accordingly. Unraveling the genetics of JMML has demonstrated that JMML in patients with germ line PTPN11 and CBL mutations often regresses spontaneously, and therapy is seldom indicated. Conversely, patients with JMML and neurofibromatosis type 1, somatic PTPN11, KRAS, and most of those with NRAS mutations have a rapidly progressive disease, and early HSCT is indicated. The risk of relapse after HSCT is high, and prophylaxis for graft-versus-host disease and monitoring should be adapted to this risk.

Outcomes of Allogeneic Stem Cell Transplantation in Pediatric Patients with Myelodysplastic Syndrome and Bone Marrow Failure Due to GATA2 Haploinsufficiency: A Case Control Study

Introduction: Germline mutations in GATA2 lead to myelodysplastic syndromes (MDS) and bone marrow failure (BMF). Stem cell transplantation (SCT) remains the only curative therapy. In our previously described cohort of pediatric MDS/BMF patients with GATA2 mutation we noted a high transplant related mortality (TRM), a low relapse rate and frequent toxicities including thromboses. We hypothesized that analogous to other inherited syndromes the underlying GATA2 mutation may contribute to increased toxicity.Methods: We performed a case control study of patients treated at our center in 2000-20014 comparing pediatric MDS/BMF patients with GATA2 mutations to MDS/BMF patients without the mutation (control A) and to patients with acute leukemia (ALL or AML) (control B). Controls were randomly selected and matched for age (0-10, 11-20), stem cell source (matched related donor (MRD), matched unrelated donor (MUD), mismatched unrelated donor (mmURD) or umbilical cord blood (UCB) and conditioning regimen.We assessed if the proportion of patients who experienced a) high grade transplant related toxicity (TRT) including: infections, organ toxicities, secondary malignancies, thrombotic events; b) acute (grade II-IV) or extensive chronic GVHD (cGVHD); and, c) TRM within 100 and 365 days from transplant day, differed between the investigational and control cohorts. All pairwise comparisons of these factors were performed using a one-sided Fisher's Exact test.We reported overall survival (OS), disease free survival (DFS) and event free-survival (EFS) (events: TRM, relapse, acute GVHD, cGVHD, secondary malignancy, thrombotic events using point estimates at 5 years post-transplant (± standard error).Results: 80 patients were analyzed: 15 GATA2, 25 control A and 40 control B. The diagnoses in the GATA2 vs. the control A cohort were: RCC (n=8 vs 9), RAEB/ RAEB-T/AML-MRC (n=4 vs 11), AML (n=2 vs 0), BMF (n=0 vs 1), aplastic anemia (n=0 vs 4) and ALL (n=1 vs 0). Control B patients had AML (n=20) or ALL (n=20). Monosomy 7 was the most common cytogenetic abnormality in the GATA2 cohort [(n=9 (60%)] vs. 9 (36%) and 3 (8%) in Control A and B. The median follow-up for the study population was 3 years (range=0.14-12.1).GATA2 patients were treated with the following conditioning regimens: cyclophosphamide (CY)/total body irradiation (TBI) +/-ATG (n=11), fludarabine/CY/TBI (n=2) and busulfan/CY (n=2) and transplanted using bone marrow from a MRD (n=3), MUD (n=5) and mmURD (n=3), or UCB (n=4). GVHD prophylaxis consisted of cyclosporine A (CSA)/MTX+/- prednisone (n=9) or CSA/ mycophenolate mofetil (n=3).Thrombotic events were seen only in the GATA2 cohort (p< 0.001). For all other toxicities 10 (67%) occurred in the GATA2 group vs. 7 (28%) in control A (p=0.019), and 8 (20%) in control B (p=0.0018)]. Other comparisons were not statistically significant. Secondary malignancy was rare, but 1 (6.7%) was observed in the GATA2 cohort vs. 1 (4%) and 2 (5%) in control A or B. Severe lung disease requiring lung transplant was observed in 2 (13.3%) of the GATA2 patients and in 0 (0%) and 1 (2.5%) in control A and B, respectively. Acute and cGVHD were seen in 5 (33.3%) and 5 (33%) in the GATA2 group vs. 5 (20%) and 8 (32%) in control A and 10 (25%), 9 (22.5%) in control B. TRM within 100 and 365 days occurred in 2 (13%), 3 (20%) in the GATA2 cohort and 2 (8%), 4 (16%) in control A and 2 (5%), 7 (17.5%) in control B. TRM was the most common cause of death within 365 days in all groups. Relapse rate was not different between groups.The 5-year OS in the GATA2 cohort was 65±13% compared to 58%±10% and 45%±8% for control A and B, respectively. The 5-year EFS were 7%±6%, 31%±11% and 33%±8% for cases, A, and B, respectively. The 5-year DFS was 51%±13% in the GATA2 group, 51% ±11% in control A, and 44%±8% in control B.Conclusions: Pediatric MDS/BMF patients with GATA2 mutations had a significantly higher rate of TRT and thromboses compared to those without themutation or pre-treated leukemia patients. The 5-year EFS was lower in the GATA2 patients, but 5-year OS and DFS was comparable across the groups. Toxicities observed, especially frequent thromboses appear unique to the GATA2 cohort and warrant specific treatment considerations in the future. DisclosuresNo relevant conflicts of interest to declare.

Clinical Spectrum of Germline Mutations with Predisposition to Myeloid Neoplasms- 2016 World Health Organization Classification Update

▪Background: The 2016 revision to the World Health Organization (WHO) classification of myeloid neoplasms has specifically categorized germline mutations that are associated with myeloid clonal evolution (Arber et al. Blood 2016). This group consists of myeloid neoplasms with an isolated germline predisposition (CEBPA, DDX41), myeloid neoplasms associated with congenital thrombocytopenia (ETV6, RUNX1, ANKRD26) and germline myeloid neoplasms with multi-organ dysfunction (GATA2, chromosomal breakage disorders, telomere biology disorders etc). We carried out this study to describe the clinical spectrum of germline disorders with predisposition to myeloid neoplasms as categorized by the 2016 WHO classification revision.Methods: After Institutional Review Board (IRB) approval, the adult and pediatric bone marrow failure syndrome database (1990-2016) and the electronic medical record were queried for germline disorders involving GATA2, CEBPA, DDX41, ETV6, RUNX1, ANKRD26, Down syndrome and Noonan syndrome. Chromosomal breakage assays (Diepoxybutane/Mitomycin-C), flow-fluorescent in-situ hybridization (FISH) for telomere length assessment, Fanconi anemia complementation assays and Sanger/Next Generation sequencing (NGS) for the aforementioned germline disorders with myeloid predisposition were carried out in Clinical Laboratory Improvement Amendments (CLIA)-certified laboratories. These disorders were then classified based on the 2016 WHO classification revision.Results: 54 individuals (37 families) were included in the study. Eleven (20%) patients belonging to 5 families were identified as having germline mutations with a preexisting platelet disorder: ETV6 (n=1), ANKRD26 (n=5), RUNX1 (n=5). Forty-three (79%) patients (32 families) had inherited syndromes with multi-organ dysfunction: GATA2 (n=11, 26%), bone marrow failure syndromes (n=14, 33%) and telomere biology disorder (n=14, 33%). There was one patient with neurofibromatosis with a germline PTPN11 mutation who developed juvenile myelomonocytic leukemia, while there were three patients with Down syndrome; 2 with transient abnormal myelopoiesis and one who developed acute megakaryocytic leukemia. The clinical phenotype, prevalence and characteristics of myeloid clonal evolution and outcomes are presented in Table 1. No patients with germline CEBPA or DDX41 mutations were identified.Patients with germline platelet disorders did not have any prominent non-hematological manifestations. Erythrocytosis (20%) with long-standing thrombocytopenia (100%) was a unique feature associated with ANKRD26 mutations. Non-hematologic clues such as human papillomavirus (HPV)-driven warts, primary lymphedema (Emberger syndrome) and frequent atypical infections with monocytopenia were seen in patients with germline GATA2 mutations, and preceded myeloid clonal evolution (morphologic, cytogenetic and molecular). Notably, the age at presentation and penetrance of myeloid transformation was variable, with individuals from the same family developing symptoms during the first decade of life and others remaining asymptomatic to date (fifth decade). Somatic ASXL1 mutations were detected in all 3 (100%) patients with GATA2 mutations and in one patient with ANKRD26 mutation that developed myeloid clonal evolution. In our study myeloid clonal evolution was seen in 40% with RUNX1 mutations, 27% with GATA2 mutations, and 20% with ANKRD26 mutations. We could not calculate the same for bone marrow failure syndromes as the total number of cases seen are still being assessed. Outcomes with allogeneic stem cell transplantation were favorable in appropriately selected patients (Table 1).Conclusion: The 2016 WHO revision to the classification of myeloid neoplasms highlights the importance of recognition and molecular characterization of germline mutations (syndromic and non-syndromic) with risk for myeloid clonal evolution. While some of these disorders (GATA2, Fanconi anemia, telomere biology disorders) may have important non-hematological clues, many present with isolated thrombocytopenia (RUNX1, ETV6). The age and frequency of myeloid evolution is highly variable. Acquisition of somatic ASXL1 mutations at the time of clonal myeloid transformation highlights the role of epigenetic dysregulation in disease evolution. [Display omitted] DisclosuresKenderian:Novartis: Patents & Royalties, Research Funding.

Comprehensive Genomic Profiling for Improved Diagnosis and Therapy of Pediatric Acute Leukemias

Subsets of pediatric acute leukemias are refractory to frontline therapy or relapse with standard treatment. Pediatric leukemias are associated with distinct molecular and genomic alterations. Clinical genomic profiling has been used effectively to improve the diagnosis of adult leukemias. However, its use and utility for pediatric leukemias are not well defined. Here, we used FoundationOne Heme, a clinical grade, high-throughput, hybridization capture-based next-generation sequencing (NGS) assay for targeted sequencing of all exons of 405 genes as well as RNA sequencing of 265 genes. We analyzed the results of samples from 71 patients sequenced on the FoundationOne® Heme assay from children and young adults with acute lymphoblastic leukemia (ALL) (n= 34); early T cell precursor ALL (ETP) (n=3); acute myelogenous leukemia (AML) (n= 19); ambiguous lineage leukemia (n=3); and myelodysplastic syndrome (MDS) (n=12) who were treated at Memorial Sloan Kettering Cancer Center in the Department of Pediatrics over a 26 month period (January 2014-March2016). Age of patients ranged from 1-24 years of age (median =10 years). 42 samples were from patients who had yet to receive cytotoxic therapy (newly diagnosed) and 29 samples were from patients who had relapsed (n=24) or had refractory disease (n=5).The current average turnaround time for FoundationOne® Heme is 12-14 days. Overall 99% (70/71) of specimens were successfully profiled. We identified 230 genomic alterations, including 59.1% (136/230) missense or nonsense mutations of genes, 15.6% (36/230) copy number alterations, and 25.6% (59/230) gene fusions or rearrangements. Molecular aberrations were detected in 81% (9/11) of patients with normal karyotypes; 55% (5/9) of these were cytogenetically cryptic genomic rearrangements including NUP98-NSD1 (n=2) and 1 MLL-PTD (n=1). We identified one novel translocation involving SATB1-PDGFRB in a sample from a patient with pre B ALL. Another patient with pre B ALL was found to have a unique combination of genomic alterations including an MLL-PTD and SSBP2-JAK2. Two patients with MDS were identified as harboring germline GATA2 mutations, indicating the likely benefit of an allogeneic hematopoietic stem cell transplantation. Three patients had therapy modifications based on findings with the addition of dasatinib (ZMIZ1-ABL1), ruxolitinib (PCM1-JAK2) and trametinib (PTPN11mutation), and two patients had risk stratification altered based on MLL rearrangements which were not identifiable by conventional cytogenetics.Overall, the use of comprehensive genomic profiling led to improved accuracy of diagnosis or alteration of therapy in 10% (7/71) cases, including molecular based therapy selection (n=3), escalation of conventional chemotherapy due to high-risk features (n=2), and inclusion of stem cell transplantation (n=2). Thus, clinical genomic profiling of pediatric acute leukemia led to discovery of new pathobiology, improved accuracy of clinical diagnosis, and inclusion of targeted molecular therapies. [Display omitted] DisclosuresKobos:Janssen: Employment. He:Foundation Medicine, Inc: Employment, Equity Ownership. Roshal:BD Biosciences: Consultancy. Zhong:foundation medicine: Employment. Balasubramanian:Foundation Medicine, Inc: Employment, Equity Ownership. Nahas:Foundation medicine: Employment. Vergilio:Foundation Medicine: Employment. Ross:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment, Equity Ownership. Mughal:Foundation Medicine: Employment, Equity Ownership. Miller:Foundation Medicine: Employment, Equity Ownership. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

GATA2 regulates dendritic cell differentiation

AbstractDendritic cells (DCs) are critical immune response regulators; however, the mechanism of DC differentiation is not fully understood. Heterozygous germ line GATA2 mutations induce GATA2-deficiency syndrome, characterized by monocytopenia, a predisposition to myelodysplasia/acute myeloid leukemia, and a profoundly reduced DC population, which is associated with increased susceptibility to viral infections, impaired phagocytosis, and decreased cytokine production. To define the role of GATA2 in DC differentiation and function, we studied Gata2 conditional knockout and haploinsufficient mice. Gata2 conditional deficiency significantly reduced the DC count, whereas Gata2 haploinsufficiency did not affect this population. GATA2 was required for the in vitro generation of DCs from Lin−Sca-1+Kit+ cells, common myeloid-restricted progenitors, and common dendritic cell precursors, but not common lymphoid-restricted progenitors or granulocyte-macrophage progenitors, suggesting that GATA2 functions in the myeloid pathway of DC differentiation. Moreover, expression profiling demonstrated reduced expression of myeloid-related genes, including mafb, and increased expression of T-lymphocyte–related genes, including Gata3 and Tcf7, in Gata2-deficient DC progenitors. In addition, GATA2 was found to bind an enhancer element 190-kb downstream region of Gata3, and a reporter assay exhibited significantly reduced luciferase activity after adding this enhancer region to the Gata3 promoter, which was recovered by GATA sequence deletion within Gata3 +190. These results suggest that GATA2 plays an important role in cell-fate specification toward the myeloid vs T-lymphocyte lineage by regulating lineage-specific transcription factors in DC progenitors, thereby contributing to DC differentiation.

Prevalence, clinical characteristics, and prognosis of GATA2-related myelodysplastic syndromes in children and adolescents

AbstractGermline GATA2 mutations cause cellular deficiencies with high propensity for myeloid disease. We investigated 426 children and adolescents with primary myelodysplastic syndrome (MDS) and 82 cases with secondary MDS enrolled in 2 consecutive prospective studies of the European Working Group of MDS in Childhood (EWOG-MDS) conducted in Germany over a period of 15 years. Germline GATA2 mutations accounted for 15% of advanced and 7% of all primary MDS cases, but were absent in children with MDS secondary to therapy or acquired aplastic anemia. Mutation carriers were older at diagnosis and more likely to present with monosomy 7 and advanced disease compared with wild-type cases. For stratified analysis according to karyotype, 108 additional primary MDS patients registered with EWOG-MDS were studied. Overall, we identified 57 MDS patients with germline GATA2 mutations. GATA2 mutations were highly prevalent among patients with monosomy 7 (37%, all ages) reaching its peak in adolescence (72% of adolescents with monosomy 7). Unexpectedly, monocytosis was more frequent in GATA2-mutated patients. However, when adjusted for the selection bias from monosomy 7, mutational status had no effect on the hematologic phenotype. Finally, overall survival and outcome of hematopoietic stem cell transplantation (HSCT) were not influenced by mutational status. This study identifies GATA2 mutations as the most common germline defect predisposing to pediatric MDS with a very high prevalence in adolescents with monosomy 7. GATA2 mutations do not confer poor prognosis in childhood MDS. However, the high risk for progression to advanced disease must guide decision-making toward timely HSCT.

Acute Lymphoblastic Leukemia in a Patient with Monomac Syndrome/GATA2 Haploinsufficiency

Background: Heterozygous germline mutations in GATA2 have been described in three distinct conditions: 1) familial myelodysplastic syndrome (MDS)/ acute myeloid leukemia (AML), 2) Emberger syndrome which is characterized by lymphedema, warts and predisposition to MDS/AML, 3) MonoMac syndrome which is comprised of atypical nontuberculous mycobacterial infection, monocyte, and B and natural killer cell lymphoid deficiency. It is now recognized that these conditions represent a spectrum of hematopoietic, lymphatic and immune system disorders due to GATA2 haplosinsufficiency. MDS/AML due to GATA2 mutation shows a unique histopathology with characteristic dysplasia and is often associated with monosomy 7. Although many patients with GATA2 haploinsufficiency are initially asymptomatic the majority of patients will ultimately experience a significant complication such as severe infections due to immunodeficiency, pulmonary alveolar proteinosis (PAP), thrombotic events, bone marrow failure, MDS and progression to AML. Allogenic hematopoietic stem cell transplant (HSCT) is the only curative treatment for patients with GATA2 haploinsufficiency and those who develop MDS/AML. Here we report a unique patient who presented with with acute lymphoblastic leukemia (ALL) and was later found to have classical features of MonoMAC syndrome and GATA2 haploinsufficiency.Case Summary: A previously healthy 11 year-old girl presented with fever, cellulitis, and pancytopenia. Bone marrow biopsy and aspirate were diagnostic for B-precursor acute lymphoblastic leukemia (ALL) with associated monosomy 7 and the following karyotype: 45,XX,-7,del(9)(p13),del(10)(q24). She was treated on Dana Farber Cancer Institute (DFCI) Consortium ALL Protocol 05-001, achieving a morphological and cytogenetic remission. During induction, she developed necrotizing aspergillus pneumonia and molluscum contagiousum. Her planned course of therapy was abbreviated due to the development of restrictive lung disease associated with PAP and disseminated Mycobacterium kansasii infection. Serial off therapy bone marrow studies were obtained given poor count recovery and revealed significant morphologic dysplasia, most prominent in the megakaryocytes. These findings were reminiscent of those characteristically seen in patients with GATA2 haploinsufficiency. Her infectious complications, profound monocytopenia, PAP and bone marrow dysplasia raised concern for MonoMAC Syndrome. Sanger Sequencing of GATA2 revealed a point mutation in the regulatory enhancer region of intron 5 (c.1017+572C>T) confirming the diagnosis. More than 3 years following remission of ALL, she developed a bone marrow relapse with her initial clone. Given her diagnosis of GATA2 haploinsufficiency, HSCT was selected as consolidation therapy in second remission. She succumbed to complications of HSCT 4 months after transplantation.Conclusion: Patients with GATA2 haploinsufficiency show a heterogeneous clinical presentation and are at high risk for MDS/AML often associated with monosomy 7. The development of ALL in association with GATA2 haploinsufficiency has not been described in the literature. Hematologist and oncologists should be aware that ALL may be associated with GATA2 haploinsufficiency and should be attuned to the clinical, laboratory and histopathologic features of the MonoMAC syndrome that would prompt additional testing and potentially alter treatment regimens. As allogenic HSCT is the only definitive therapy for patients with GATA2 mutation, consideration of immediate HSCT following induction of remission should be considered in patients with ALL and GATA2 haploinsufficiency. Further, as patients with GATA2 mutations can be asymptomatic, it is imperative to screen family members for GATA2 mutations and offer genetic counselling prior to consideration as potential bone marrow donors. DisclosuresNo relevant conflicts of interest to declare.

A Targeted Next-Generation Sequencing Mutation Panel for Pediatric Acute Myeloid Leukemia and Myelodysplastic Syndrome (MDS) Detects Potential Additional Driver Mutations in Pediatric GATA2-MDS

Introduction: In adult populations, driver mutations often facilitate the progression of myelodysplastic syndrome (MDS) to acute myeloid leukemia (AML). Increased mutational burden is associated with decreased leukemia-free survival (Papaemmanuil et al, 2013); however, this phenomenon is less common in pediatric MDS. GATA2 mutations resulting in loss of function or haploinsufficiency are associated with MDS predisposition. Recent reports in adults with GATA2-MDS suggest that additional somatic driver mutations such as ASXL1, EZH2, and GATA1 contribute to rapidly progressive disease (Bodor et al, 2012; Fujiwara et al, 2014). Yet the spectrum of somatic mutations in pediatric GATA2-MDS is not well described. We applied a custom next-generation sequencing (NGS) panel inclusive of germline and acquired mutations commonly reported in pediatric AML/MDS to a subset of MDS patients with germline GATA2 mutations. Our results indicate that acquisition of somatic mutations in AML/MDS related genes may contribute to pediatric GATA2-MDS disease evolution and pathogenesis.Methods: Using Agilent SureDesign, we designed an NGS panel that targeted genes frequently mutated in pediatric AML/MDS, including 278 exonic, promoter, or intronic regions from 46 genes. Select known mutations in diagnostic specimens from a local pediatric AML/MDS cohort were used for panel validation. We applied this panel to DNA extracted from blood or marrow samples from 4 pediatric subjects diagnosed at ages 5-15 years with GATA2-MDS, and one subject age 8 with suspected GATA2-MDS. Sample NGS libraries were prepared, multiplexed, and sequenced on MiSeq flow cells in paired-end mode. Read alignment, variant calling, and annotation were performed using Agilent SureCall v3.0 software and NExtGENe software. Variants passing defined QC metric filters were reviewed for pathogenic significance. This research was performed under a local Institutional Review Board-approved protocol and in accord with the Declaration of Helsinki.Results: The subject with suspected GATA2-MDS was confirmed to have a deletion in GATA2 resulting in a frameshift. Presumed germline variants in telomerase reverse transcriptase (TERT) were found in one subject at allele frequencies consistent with heterozygosity. Four acquired mutations were found in two out of five GATA2-MDS subjects: a deletion in IKZF1 resulting in a frameshift, and mutations in both RUNX1 (n=2) and in SETBP1 (Table 1).Conclusions: Within a cohort of five GATA2-MDS subjects, we found four somatic mutations likely contributing to disease pathogenesis.Acquired mutations in SETBP1, including p.G870S, are an independent and poor prognostic indicator in adult AML (Makishima et al, 2013). Acquired mutations in RUNX1 are also common in adult MDS/AML, and both p.L56S and p.D198Gmutations have been described in adult cases of MDS progressing to AML (Pellagati et al, 2015). We also identified a frameshift mutation in IKZF1 presumed to be somatic in one subject. Large focal deletions, but not frameshifts, have been described as rare but recurrent events in pediatric AML in conjunction with monosomy 7, as with this case (de Rooij et al, 2015). Our results represent the first evidence of acquired driver mutations in pediatric GATA2-MDS, and demonstrate the utility of this comprehensive custom AML/MDS NGS panel to uncover key somatic mutational events that may be associated with rapidly progressive disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

GATA-2 Regulates Dendritic Cell Differentiation

(Background) Dendritic cells (DCs) are critical regulators of the immune response, but their differentiation mechanism remains unclear. Heterozygous germline GATA-2 mutations in humans cause MonoMAC syndrome, characterized by monocytopenia and predisposition to myelodysplasia/acute myeloid leukemia. In this syndrome, DC count decreases profoundly, with an increased susceptibility to viral infections, impaired phagocytosis, and decreased cytokine production. In the present study, we analyzed the role of GATA-2 in DC differentiation and the underlying molecular mechanisms.(Method)Gata2 haploinsufficient mice (Gata2+/−: Tsai et al. Nature 1994) and tamoxifen-inducible Gata2-knockout mice (Gata2flox/flox/ER-Cre: Charles et al. Molecular Endocrinology 2006) were used. To generate conditional Gata2 knockouts in vivo, Gata2flox/flox/ER-Cre mice were intraperitoneally injected with 1-μg tamoxifen on days 1-3 and 8-10 and evaluated on days 20-22. Isolation of splenic DCs and bone marrow (BM) precursors, including LSK (Lin- Sca1+ Kit+ cell), CMP (common myeloid-restricted progenitor), GMP (granulocyte-macrophage progenitor), CLP (common lymphoid-restricted progenitor), and CDP (common dendritic cell precursor), were separated with both MACS (Miltenyi Biotech) and BD FACSAria II (BD Biosciences). For the in vitro analysis of Gata2-knockout, BM cells were cultured with CD45.1+ BM feeder cells from SJL mice (The Jackson Laboratory) with FLT3L (200 ng/mL) and 4-hydroxytamoxifen (Sigma). For transcription profiling, SurePrint G3 mouse GE microarray (Agilent) was used, and the data was subsequently analyzed with ImmGen database (http://www.immgen.org). Promoter assay was conducted with Dual Luciferase Reporter Assay system (Promega). Quantitative chromatin immunoprecipitation (ChIP) analysis was performed using CMP fraction and erythroid-myeloid-lymphoid (EML) hematopoietic precursor cell line (ATCC) with antibodies to GATA-2 (sc-9008, Santa Cruz Biotechnology).(Results) Quantitative RT-PCR analysis showed abundant Gata2 expression in LSK and CMP fractions, with detectable expression in GMP, CLP, and CDP fractions and in vitro differentiated DCs. Although the DC count did not change in Gata2 haploinsufficient mice, it significantly and profoundly decreased in Gata2 conditional knockout mice. To examine the role of GATA-2 during DC differentiation, we knocked out Gata2 during in vitro DC differentiation, starting from LSK, CMP, GMP, CLP, and CDP fractions obtained from Gata2flox/flox/ER-Cre mice. Gata2 knockout significantly decreased CD11c+ DC counts from LSK, CMP, and CDP fractions, while those from CLP and GMP were unaffected, implying the importance of GATA-2 during DC differentiation in the pathway from LSK to CDP via CMP, not via CLP nor GMP.To elucidate the underlying molecular mechanisms, we performed expression profiling with control and Gata2 -knockout DC progenitors from CMP of Gata2flox/flox/ER-Cre mice. Gata2 knockout caused >5-fold upregulation and downregulation of 67 and 63 genes, respectively. Although genes critical for the DC differentiation, e.g., Spi1, Ikzf1, and Gfi1, were not detected among the GATA-2-regulated gene ensemble, we found significant enrichment of myeloid-related and T lymphocyte-related genes among the downregulated and upregulated gene ensembles, respectively. We focused on Gata3 upregulation (7.33-fold) as a potential key mechanism contributing to Gata2 knockout-related impaired DC differentiation. Quantitative ChIP analysis with both CMP fraction and EML cell line demonstrated obvious GATA-2 chromatin occupancy at the consensus GATA-binding motif within Gata3+190 kb, which was conserved with human. Furthermore, addition of Gata3 +190 kb region to the Gata3 promoter (~0.5 kb) significantly decreased luciferase activity, which was significantly recovered by the deletion of GATA sequence within Gata3 +190 kb, in EML cells.(Conclusion) GATA-2 seems to play an important role for cell fate specification toward myeloid versus T lymphocytes, and thus contributing to the DC differentiation. Our data offer a better understanding of the pathophysiology of MonoMAC syndrome. DisclosuresFujiwara:Chugai Pharmaceuticals. Co., Ltd.: Research Funding. Fukuhara:Gilead Sciences: Research Funding. Ishizawa:GSK: Research Funding; Takeda: Research Funding; Celgin: Speakers Bureau; Kyowa Kirin: Research Funding; Celgin: Research Funding; Janssen: Research Funding; Takeda: Speakers Bureau; Kyowa Kirin: Speakers Bureau; Pfizer: Speakers Bureau.

Development of Somatic NRAS Mutation Associated with Rapid Transition from Germline GATA2 Mutation Associated Myelodysplastic Syndrome to Acute Myeloid Leukemia

There is increasing recognition of the role of inherited germline predisposition for myeloid disorders such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The additional somatic genetic events required for development of a malignant phenotype are however poorly understood.A 25 year old woman was referred to the NHLBI hematology branch in March 2014 for a seven year history of pancytopenia. Her medical history included recurrent pneumonias, oral ulcers, severe varicella infection and arthralgias. Prior bone marrow examinations at ages 21 and 23 at outside institutions reported normocellular marrow, tri-lineage hematopoiesis and mild dyspoiesis. Cytogenetics were remarkable for trisomy 8 in 80% (aged 21) or 90% (aged 23) of metaphases.Previously unrecognized lymphedema was noted on examination. Peripheral blood counts showed WBC 2.28 K/ul [normal range: 3.98-10.04], HGB 9.9 g/dL [11.2-15.7], PLT: 67 K/ul [173-369], ALC: 0.36 K/ul [1.18-3.74] and AMC: 0.06 [0.24-0.86]. Peripheral blood flow cytometry demonstrated decreased CD3+ CD4+ (T) cells, CD19+ (B) cells and NK cells. HLA-DR15 negative. Bone marrow examination showed trilineage hematopoiesis, 50-60% cellularity, mild erythroid predominance and mildly increased, mildly atypical megakaryocytes. Blasts less than 5%. Bone marrow flow cytometry revealed severely decreased B-cells and monocytes, absent B-cell precursors, absent dendritic cells, inverted CD4:CD8 ratio, and atypical myeloid maturation pattern. Cytogenetics demonstrated stable trisomy 8 in 90% of metaphases.On the basis of this assessment the diagnosis of MDS was confirmed. Sanger sequencing revealed a GATA2 L375S mutation in the second zinc finger of known pathogenic significance.Four months later she developed increased fatigue and easy bruising with worsening thrombocytopenia (PLT: 10K/ul). Bone marrow was dramatically changed; now markedly hypercellular (90-100%) with diffuse sheets of immature cells consistent with blasts having fine chromatin, distinct or prominent nucleoli, and visible cytoplasm. Blasts were positive for CD33, CD56, CD64, CD123, and CD163; and were negative for CD34, CD14, and myeloperoxidase. Cytogenetics showed a new trisomy 20 in 65% of metaphases, in addition to previously seen trisomy 8 in 100%. A diagnosis of acute monoblastic leukemia (M5a subtype) was made.At both clinic visits bone marrow aspirate was collected on an IRB approved research sample acquisition protocol. Whole exome sequencing of 1ug DNA was performed using Agilent SureSelect v5 Exome enrichment Kits on an Illumina HiSeq 2000 with 100-bp paired-end reads (Macrogen, Rockville, MD). Data was mapped to hg19 (BWA) and processed using an in-house pipeline (Samtools/Picard/GATK/VarScan/Annovar). Mean read depth of target regions was 157 and 149. There was high correlation between both samples with the exception of a NRAS:NM_002524:exon3:c.C181A:p.Q61K mutation (57 of 180 reads) seen only in the later sample.Confirmatory ultra-deep sequencing for NRAS was performed using Illumina TruSight Myeloid Sequencing Panel on an Illumina MiSeq. No evidence of the NRAS Q61K mutation was found in the earlier March MDS bone marrow sample even when sequenced to a depth greater than 1750 reads (see figure). The mutation was confirmed in the August AML sample at a variant allele frequency of 35%. If heterozygous this would reflect a clone size of 70%, consistent with data from both cytogenetics (new trisomy 20 in 65% of metaphases) and the 76% blasts documented by bone marrow aspirate smear differential.We report here the rapid progression to AML in a patient with germline GATA2 MDS associated with development of a new trisomy 20 karyotype and a NRAS Q61K mutation. The NRAS mutation was not detectable after the patient achieved a complete remission following induction chemotherapy further supporting this association. This NRAS mutation has been implicated in the pathogenesis of multiple cancers by constitutive activation of proliferative signaling. GATA2 associated MDS is a high-risk pre-leukemic condition with the potential for rapid evolution to AML. This is the first report of acquired somatic mutations in the RAS/RTK signaling pathway in the context of germline GATA2 insufficiency associated with acute leukemic transformation. [Display omitted] DisclosuresTownsley:Novartis: Research Funding; GSK: Research Funding.