GATA2 Mutations Are Frequent in Intermediate-Risk Karyotype AML with Biallelic CEBPA Mutations and Are Associated with Favorable Prognosis

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LETTERS TO THE EDITOR GATA2 mutations are frequent in intermediate-risk karyotype AML with biallelic CEBPA mutations and are associated with favorable prognosis

Leukemia (2013) 27, 482–485; doi:10.1038/leu.2012.174 review board before its initiation. GATA2 mutation analysis was performed on either bone marrow or peripheral blood samples. Sanger sequencing of GATA2 exons 4 and 5 was performed The GATA binding protein 2 (GATA2) belongs to the GATA family following RT–PCR using the following primers: GATA2_Ex4-F: of transcription factors, which contain zinc fingers in their DNA 50-GGTTAAGCAGGCCCCCGTGTC-30, GATA2_Ex4-R: 50-ATTAACCGCC binding domain. The human GATA2 gene, located on 3q21, is AGCTCCTGCC-30, GATA2_Ex5-F: 50-CAGCCTGCTGACGCTGCCTT-30, encoded by six exons and contains a conserved DNA-binding GATA2_Ex5-R: 50-GCCTCTTGCCTGGCAGCACA-30. Mutation status of domain composed of two multifunctional zinc finger domains other genes was available as follows: NPM1: n ¼ 212, FLT3-ITD: (ZFDs). GATA2 is an indispensable transcription factor for hematopoiesis as it maintains the proliferative progenitor-cell phenotype. Table 1. Demographics and clinical and molecular characteristics of Its downregulation is necessary for differentiation and thus GATA2 AML patients according to GATA2 mutations status expression is tightly regulated by several transcription factors such as NOTCH1, PU.1 and EVI1 as well as by the cytokines IL-1 Characteristics GATA2wt GATA2mut P and TNFa.1 As GATA2 has been shown to have a key role in (N ¼ 191) (N ¼ 21) controlling the proliferation and differentiation of hematopoietic cells, defects arising from mutations in this gene may contribute No. of %Of No. of %Of patients total patients total to hematopoietic disorders, including leukemia. So far, there are few studies analyzing the role of GATA2 Age (years) in hematological malignancies. Zhang et al.2 analyzed genetic Median 60 54 n.s. Range 16–85 23–83 alterations of transcription factors in 85 cases of BC-CML (chronic myeloid leukemia in blast crisis) and, among others, identified Gender Female 92 43.4 15 7.1 0.064 two mutations in GATA2: a p.Leu359Val substitution within the Male 99 46.7 6 2.8 C-terminal ZFD of GATA2 was found in eight cases with myelomo- FAB subtype (n ¼ 203) n.s. noblastic features, whereas an in-frame deletion of six amino acids M0 2 100.0 0 0.0 (D341–346) spanning the C-terminal border of the C-terminal ZFD M1 78 38.4 12 5.9 was detected in one patient demonstrating myeloid BC with M2 73 36.0 8 3.9 M4 19 100.0 0 0.0 eosinophilia. Further studies indicated that GATA2 Leu359Val not M5 6 100.0 0 0.0 only increased transactivation activity of GATA2 but also enhanced M6 4 89.7 1 10.3 its inhibitory effects on the activity of PU.1, a major transcription Type of disease (n ¼ 212) n.s. factor for myeloid cell differentiation, via aberrant protein–protein de-novo AML 184 86.8 20 9.4 2 t-AML 2 1.0 1 4.8 interaction. Furthermore, heterogeneous GATA2 mutations s-AML 5 2.4 0 0.0 (GATA2mut) have been described in families with predisposition to myelodysplastic syndrome (MDS) and acute myeloid leukemia Hemoglobin, g/dl (n ¼ 159) n.s. 3,4 Median 9.4 9.3 (AML). GATA2mut in these diseases are presumed to Range 4.0–14.4 7.1–12.1 differentially affect binding of other proteins, leading to variable WBC count, Â 109/L (n ¼ 163) n.s. consequences on target gene regulation and cell fate determi- median 38.1 48.1 nation. Interestingly, two mutations were repeatedly found in Range 1.0–249.5 6.0–182.0 different syndromes associated with AML and MDS: p.Thr354Met 9 3 Platelet count, x10 /L (n ¼ 163) n.s. and p.Thr355del. Recently, GATA2mut have also been described Median 87 56 in a single study with a frequency of 40.6% in a cohort of 32 CN- Range 5–950 9–225 AML (cytogenetically normal AML) patients with biallelic CEBPA Bone marrow blasts, % (n ¼ 187) n.s. gene mutations.5,6 All GATA2mut were missense mutations and Median 62 61 were located in the N-terminal ZFD. Range 4–98 22–90 On the basis of the previously mentioned study,5,6 we first Correlating mutations k evaluated GATA2mut in intermediate-risk karyotype AML with monoCEBPA (n ¼ 22/212) 22 10.4 0 0.0 0.041 biCEBPA (n ¼ 98/212) 80 37.7 18 8.5 o0.001 m biallelic CEBPA (biCEBPA) mutations for frequency, association with NPM1 (n ¼ 45/212) 43 20.3 2 0.9 n.s. other mutations and impact on outcome. We therefore analyzed FLT3-ITD (n ¼ 45/212) 45 21.4 0 0.0 0.009 k FLT3-TKD (n ¼ 4/210) 4 1.9 0 0.0 n.s. 98 such cases for GATA2mut by direct Sanger Sequencing of exons MLL-PTD (n ¼ 14/211) 14 6.6 0 0.0 n.s. 4 and 5, which contain the two highly conserved N-and C-terminal RUNX1 (n ¼ 23/211) 22 10.4 1 0.5 n.s. ASXL1 (n ¼ 31/212) 30 14.2 1 0.5 n.s. ZFDs of GATA2. Additionally, we screened 22 intermediate-risk WT1 (n ¼ 19/211) 17 8.1 2 0.9 n.s. karyotype AML cases with monoallelic CEBPA (monoCEBPA) mutations and 92 cases with CEBPA wild-type (CEBPAwt) for Abbreviations: AML, acute myeloid leukemia; biCEBPA, biallelic CEBPA; FAB, GATA2mut. The study design adhered to the tenets of the French-American-British; monoCEBPA, monoallelic CEBPA; WBC, white blood cell. Significant values are written in bold. Declaration of Helsinki and was approved by our institutional

Accepted article preview online 3 July 2012; advance online publication, 20 July 2012 Letters to the editor 483 n ¼ 212, CEBPA: n ¼ 212, MLL-PTD: n ¼ 211, RUNX1: n ¼ 211, ASXL1: count or cytogenetics (Table 1). In 103 cases, immunophenotyping n ¼ 212, FLT3-TKD: n ¼ 210, WT1: n ¼ 211. In all, 156 cases (73.6%) data were available. Cases with GATA2mut (n ¼ 10) had a were CN-AML and 56 (26.4%) had intermediate-risk aberrant higher expression of CD133 (52±29% vs 29±27%, P ¼ 0.015), cytogenetics according to Medical Research Council criteria.7 CD34 (67±30% vs 42±31%, P ¼ 0.018) and HLA-DR (59±28% vs Female/male ratio was 107/105 and age ranged from 15.7 to 38±24%, P ¼ 0.017) as well as lower expression of CD11b 84.9 years (median: 59.7) (Table 1). (19±14% vs 37±24%, P ¼ 0.003) and CD36 (10±5% vs Overall, in 21/212 patients (9.9%) GATA2mut were detected. 23±15%, Po0.001) and thus had a more immature phenotype However, as the total cohort was not unselected the frequency as compared with GATA2wt. With regard to cytomorphology, we was calculated separately for the biCEBPA (n ¼ 18/98, 18.3%) and observed a preponderance of AML M1 (n ¼ 12/21) and AML M2 the CEBPAwt cohort (n ¼ 3/92, 3.3%). All mutations were point (n ¼ 8/21) subtypes in GATA2mut cases. In addition, GATA2mut mutations. In detail, most mutations (n ¼ 14, 66.7%) were located were strongly associated with biCEBPA mutations (n ¼ 18/98 in the N-terminal ZFD (aa 294–344). Five patients harbored a 18.3% vs 3/92, 3.3% in CEBPAwt, Po0.001), whereas GATA2mut mutation in the C-terminal ZFD (aa 349–398). One patient had two were mutually exclusive of monoCEBPA mutations (0/22; mutations in the N-terminal ZFD and one patient had each one P ¼ 0.041) and of FLT3-ITD (0/45; P ¼ 0.009) (Figure 1b). Further- mutation in both the N-and C-terminal ZFDs, respectively more, we observed GATA2mut in three patients without biCEBPA (Figure 1a; Table 2). Of note, no patient harbored the mutations mutations. Two of these patients had an additional NPM1 reported in BC-CML or familial AML-MDS, suggesting a different mutation and the third patient harbored a mutation in RUNX1. oncogenic mechanism in these diseases. All mutations were As GATA2 has been described to be an MDS/AML predisposing analyzed by PolyPhen prediction (http://genetics.bwh.harvard. gene and germline mutations were reported,3,4 we analyzed edu/pph2/) and were identiﬁed as probably damaging indicating remission samples of 10/21 GATA2mut cases. In all 10 cases the a loss of GATA2 function. This result is in contrast to GATA2mut GATA2mut detected at initial diagnosis were not detectable in found in BC-CML that have been described as gain of function remission and thus was somatic and not germline (Supplementary mutations resulting in enhanced DNA binding and co-activator Table 1). In four GATA2mut patients, sample material was available recruitment and furthermore have been shown to result in at diagnosis, remission and also at relapse. Two patients carried repression of the myeloid master regulator PU.1. additional biCEBPA mutations at diagnosis, one patient showed an GATA2mut tended to be more frequent in females than in males additional NPM1 mutation and one patient showed additional (n ¼ 15/107, 14.0% vs 6/105, 5.7%, P ¼ 0.064). There was no mutations in NPM1, WT1 and NRAS. Only the patient with the association with age, leukocyte count, hemoglobin level, platelet additional NPM1 mutation showed the same GATA2 mutation at

294 Exon 4 344 349Exon 5 398

ZFD 1 ZFD 2 His GIn lle His Pro Ser Val Arg Lvs GIn GIn Leu Asp Phe 361 379 317 321 308 317 318 321 354 362 329 307 320 321 As Arg Leu Ala Arg Thr Leu Arg Gly Arg Leu Asn Asn Leu

wild-type mutated CEBPA biallelic CEBPA monoallelic no data available

120 GATA2 mut 18.4% GATA2 wt 100

60 4.4%

40 3.2% 4.3% 10.5% Total case numbers 20

0 monoCEBPA biCEBPA FLT3-ITD NPM1 MLL-PTD RUNX1 WT1 FLT3 -TKD ASXL1 (0/22) (18/98) (0/45) (2/45) (0/14) (1/23) (2/19) (0/4) (1/31) p=0.041 p<0.001 p=0.009 Case Numbers (GATA2 mut/positive cases) Figure 1. (a) Distribution of GATA2 mutations within the N- and C-terminal ZFDs detected within the total cohort of 212 patients. Numbers in circles below the indicated mutations indicate the frequency of the respective mutations. If no number is given, then the mutation was observed once. (b) Alignment of gene mutations for 212 patients. Each column represents one of the 212 analyzed samples. Mutations in the nine investigated genes are shown by colored bars. A red bar indicates a mutation; a gray bar, no mutation; whereas a white bar indicates that no data were available. (c) Concomitant events of GATA2 with other mutations are also shown as a bar chart. The gray part represents GATA2wt, the red one GATA2mut within the analyzed subcohorts. GATA2mut frequencies and signiﬁcances (P-values) are denoted; numbers of mutated/analyzed cases of the subcohorts are given in parenthesis below the bars.

& 2013 Macmillan Publishers Limited Leukemia (2013) 482 – 516 Letters to the editor 484 Table 2. GATA2 mutations detected within the total cohort of 212 patients

GATA2 exon Mutation Allele Amino-acid Case numbers with PolyPhen predictiona type change change mutations (%)

4 Missense c.1252G4T p.Arg307Leu 1/21 (4.8) Probably damaging Missense c.1255G4C p.Arg308Pro 1/21 (4.8) Probably damaging Missense c.1293C4G p.Leu321Val 1/21 (4.8) Probably damaging Missense c.1282A4G p.Asn317Ser 1/21 (4.8) Probably damaging Missense c.1282A4T p.Asn317lle 1/21 (4.8) Probably damaging Missense c.1285C4T p.Ala318Val 3/21 (14.3) Probably damaging Missense c.1291G4A p.Gly320Asp 1/21 (4.8) Probably damaging Missense c.1294T4G p.Leu321Arg 1/21 (4.8) Probably damaging Missense c.1293C4T p.Leu321Phe 5/21 (23.8) Probably damaging Missense c.1294T4A p.Leu321His 1/21 (4.8) Probably damaging Missense c.1321G4A p.Arg329Gln 1/21 (4.8) Probably damaging 5 Missense c.1391C4A p.Thr354Lys 1/21 (4.8) Probably damaging Missense c.1414G4A p.Arg361His 1/21 (14.3) Probably damaging Missense c.1417G4A p.Arg362Gln 3/21 (9.5) Probably damaging Missense c.1468T4A p.Leu379Gln 1/21 (4.8) Probably damaging ahttp://genetics.bwh.harvard.edu/pph2.

GATA2 mutated

GATA2 wild-type

p=0.001 p=0.025

GATA2/ biCEBPA mutated

GATA2 wild-type/ biCEBPA mutated GATA2/ biCEBPA mutated

GATA2 wild-type/ biCEBPA mutated

p=0.058 p=0.280

Figure 2. Kaplan Meier survival analysis of 194 intermediate-risk AML patients. (a) OS within the total cohort 194 patients. Data are shown for GATA2 mutated patients (n ¼ 20) and GATA2 wild-type patients (n ¼ 174; alive at 2 years: 100.0% vs 53.1%). (b) EFS within the total cohort 194 patients. Data are shown for GATA2 mutated patients (n ¼ 20) and GATA2 wild-type patients (n ¼ 174; alive at 2 years: 63.8% vs 35.7%). (c)OS within 80 patients with biCEBPA mutations. Data are shown for biCEBPA mutated/GATA2 mutated patients (n ¼ 17) and biCEBPA mutated/ GATA2 wild-type patients (n ¼ 63; alive at 2 years: 100.0% vs 76.1%). (d) EFS within 80 patients with biCEBPA mutations. Data are shown for biCEBPA-mutated/GATA2-mutated patients (n ¼ 17) and biCEBPA-mutated/GATA2-wild-type patients (n ¼ 63; alive at 2 years: 63.8% vs 49.2%).

both diagnosis and ﬁrst relapse. In the other three patients, the (2y-OS) (100% vs 53.1%, P ¼ 0.001) and 2-year event free survival GATA2mut were absent at relapse (Supplementary Table 2). (EFS) (63.8% vs 35.7%, P ¼ 0.025) compared with GATA2wt cases This strongly suggests GATA2mut as secondary events. (Figures 2a and b). In univariable analysis, GATA2mut were Clinical follow-up data were available in 194/212 patients and in associated with better EFS (P ¼ 0.03) and OS (P ¼ 0.041). However, 20/21 patients with GATA2mut. With regard to prognosis, patients in multivariable analysis GATA2mut lost their impact on EFS and with GATA2mut had signiﬁcantly better 2-year overall survival OS (Supplementary Table 3). Because of the high coincidence

Leukemia (2013) 482 – 516 & 2013 Macmillan Publishers Limited Letters to the editor 485 of GATA2 and biCEPBA mutations, the prognostic impact of is greatly appreciated. In addition, we are grateful for the data management support GATA2mut in dependence on biCEBPA mutations was analyzed. performed by Tamara Alpermann. Patients with biCEBPA mutations and additional GATA2mut (n ¼ 17) had a better 2-year OS compared with patients with A Fasan1, C Eder1, C Haferlach1, V Grossmann1, A Kohlmann1, biCEBPA mutations and GATA2wt (n ¼ 63) (100% vs 76.1% F Dicker1, W Kern1, T Haferlach1 and S Schnittger1 P ¼ 0.058) (Figure 2c). Interestingly, GATA2 had been shown to 1MLL Munich Leukemia Laboratory, Munich, Germany interact with CEPBA by forming protein complexes and this E-mail: [email protected] interaction is critical for the suppression of adipocyte differentiation.8 Thus, the coincidence of mutations in both genes could impair interaction and provoke a differentiation advantage REFERENCES of these cells resulting in the favorable prognosis of patients harboring both GATA2mut and biCEBPA mutations. 1 Vicente C, Conchillo A, Garcia-Sanchez MA, Odero MD. The role of the GATA2 transcription factor in normal and malignant hematopoiesis. Crit Rev Oncol Hematol In conclusion, we confirmed a strong association of GATA2mut 2012; 82:1–17. with biCEBPA mutations in a large set of intermediate-risk AML 2 Zhang SJ, Ma LY, Huang QH, Li G, Gu BW, Gao XD et al. Gain-of-function patients. Furthermore, we did not observe GATA2mut in patients mutation of GATA-2 in acute myeloid transformation of chronic myeloid leukemia. with monoCEPBA mutations underlining the different biology of Proc Natl Acad Sci USA 2008; 105: 2076–2081. AML with biallelic vs monoCEPBA mutated AML. For the first time, 3 Hahn CN, Chong CE, Carmichael CL, Wilkins EJ, Brautigan PJ, Li XC et al. this study also provides data on the frequency of GATA2mut Heritable GATA2 mutations associated with familial myelodysplastic syndrome and in CEPBAwt AML, in which a low frequency of only 3.3% was acute myeloid leukemia. Nat Genet 2011; 43: 1012–1017. detected. Analyses performed at relapse demonstrated that 4 Ostergaard P, Simpson MA, Connell FC, Steward CG, Brice G, Woollard WJ et al. GATA2mut are secondary events. Furthermore, GATA2mut are Mutations in GATA2 cause primary lymphedema associated with a predisposition to acute myeloid leukemia (Emberger syndrome). Nat Genet 2011; 43: associated with female sex and favorable impact on survival. 929–931. GATA2 thus seems to be a promising new marker to identify 5 Dufour A, Konstandin N, Ksienzyk B, Zellmeier E, Benthaus T, Yaghmaie M et al. patients with even more favorable prognosis in the subgroup of High frequency of GATA2 mutations in cytogenetically normal acute myeloid patients with the prognostically favorable biCEBPA mutated AML. leukemia with biallelic CEBPA mutations identified by exome sequencing. ASH Abstract 2011; 72. 6 Greif PA, Dufour A, Konstandin NP, Ksienzyk B, Zellmeier E, Tizazu B et al. CONFLICT OF INTEREST GATA2 zinc finger 1 mutations associated with biallelic CEBPA mutations define a CH, WK, TH and SuS are equity owners of and AF, CE, VG, AK and FD are employed by unique genetic entity of acute myeloid leukemia. Blood 2012; e-pub ahead of print the MLL Munich Leukemia Laboratory. 30 May 2012. 7 Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison G et al. The importance of diagnostic cytogenetics on outcome in AML: analysis ACKNOWLEDGEMENTS of 1,612 patients entered into the MRC AML 10 trial. The Medical Research We thank all clinicians for sending samples to our laboratory for diagnostic purposes, Council Adult and Children’s Leukaemia Working Parties. Blood 1998; 92: and for providing clinical information and follow-up data. In addition, we would like 2322–2333. to thank all co-workers at the MLL Munich Leukemia Laboratory for approaching 8 Tong Q, Tsai J, Tan G, Dalgin G, Hotamisligil GS. Interaction between GATA and the together many aspects in the field of leukemia diagnostics and research. Especially C/EBP family of transcription factors is critical in GATA-mediated suppression the technical assistance of Madlen Ulke, who performed Sanger Sequencing analyses, of adipocyte differentiation. Mol Cell Biol 2005; 25: 706–715.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

Glucocorticoid sensitivity of T-cell lymphoblastic leukemia/ lymphoma is associated with glucocorticoid receptor-mediated inhibition of Notch1 expression

Leukemia (2013) 27, 485–488; doi:10.1038/leu.2012.192 (T-ALL)-associated genetic lesion has been initially reported to result in deletion of Notch1 ectodomain and constitutive activa- Notch signaling can regulate a wide variety of cellular processes, tion of its intracellular region.5 Further studies have shown that including apoptosis1 differentiation,2 and drug resistance.3 At activating mutations in the NOTCH1 gene are present in over 50% the molecular level, Notch signaling has been demonstrated to of human T-ALL cases, making NOTCH1 the most prominent onco- function as a transcriptional activator.4 Receptor-ligand interaction gene specifically involved in the pathogenesis of this disease.5–7 renders Notch susceptible to cleavage by ADAM-type metallo- In T-ALL, activation of Notch signaling has been implicated in the proteases at site S2, which creates a short-lived intermediate, regulation of multiple cellular pathways, including those resulting which is in turn cleaved by g-secretase within the transmembrane in apoptosis,8 tissue infiltration9 and glucocorticoid resistance.10 domain at site S3. This frees the intracellular domain of Notch The link between Notch and glucocorticoid resistance is of parti- (Notch-IC), which translocates to the nucleus to form a trans- cular interest, as glucocorticoids were among the first drug classes criptional activation complex with the DNA-binding factor CSL. used in the treatment of patients with T-cell leukemia and are That the Notch signaling pathway has a crucial role in cancer is still the essential components of treatment (Inaba and Pui in11 and firmly established as a rare T-cell acute lymphoblastic leukemia references therein). They seem to exert their cytotoxic effects

Accepted article preview online 13 July 2012; advance online publication, 31 July 2012

& 2013 Macmillan Publishers Limited Leukemia (2013) 482 – 516