# Acquired TET2 mutation in one patient with familial platelet disorder with predisposition to AML led to the development of pre-leukaemic clone resulting in T2-ALL and AML-M0

Vladimir T. Manchev, Hind Bouzid, Iléana Antony-Debré, Betty Leite, Guillaume Meurice, Nathalie Droin, Thomas Prebet, Régis T. Costello, William Vainchenker, Isabelle Plo, et al.

► To cite this version:

Vladimir T. Manchev, Hind Bouzid, Iléana Antony-Debré, Betty Leite, Guillaume Meurice, et al. Acquired TET2 mutation in one patient with familial platelet disorder with predisposition to AML led to the development of pre-leukaemic clone resulting in T2-ALL and AML-M0. Journal of Cellular and Molecular Medicine, 2017, 21 (6), pp.1237 - 1242. 10.1111/jcmm.13051. hal-01537098

HAL Id: hal-01537098

https://hal.sorbonne-universite.fr/hal-01537098

Submitted on 12 Jun 2017

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Acquired TET2 mutation in one patient with familial platelet disorder with predisposition to AML led to the development of pre-leukaemic clone resulting in T2-ALL and AML-M0

Vladimir T. Manchev<sup>a, b, #</sup>, Hind Bouzid<sup>a, b, #</sup>, Iléana Antony-Debré<sup>a</sup>, Betty Leite<sup>c</sup>, Guillaume Meurice<sup>d</sup>, Nathalie Droin<sup>a, c</sup>, Thomas Prebet<sup>e, f</sup>, Régis T. Costello<sup>g</sup>, William Vainchenker<sup>a</sup>, Isabelle Plo<sup>a</sup>, M'boyba Diop<sup>a, d</sup>, Elizabeth Macintyre<sup>h</sup>, Vahid Asnafi<sup>h</sup>, Rémi Favier<sup>a, i</sup>, Véronique Baccini<sup>j</sup>, Hana Raslova<sup>a, *</sup>

Received: September 19, 2016; Accepted: October 30, 2016

## Abstract

Familial platelet disorder with predisposition to acute myeloid leukaemia (FPD/AML) is characterized by germline *RUNX1* mutations. thrombocytopenia, platelet dysfunction and a risk of developing acute myeloid and in rare cases lymphoid T leukaemia. Here, we focus on a case of a man with a familial history of *RUNX1*<sup>R174Q</sup> mutation who developed at the age of 42 years a T2-ALL and, 2 years after remission, an AML-M0. Both AML-M0 and T2-ALL blast populations demonstrated a loss of 1p36.32-23 and 17q11.2 regions as well as other small deletions, clonal rearrangements of both *TCRγ* and *TCRδ* and a presence of 18 variants at a frequency of more than 40%. Additional variants were identified only in T2-ALL or in AML-M0 evoking the existence of a common original clone, which gave rise to subclonal populations. Next generation sequencing (NGS) performed on peripheral blood-derived CD34<sup>+</sup> cells 5 years prior to T2-ALL development revealed only the missense *TET2*<sup>P1962T</sup> mutation at a frequency of 1%, which increases to more than 40% in fully transformed leukaemic T2-ALL and AML-M0 clones. This result suggests that *TET2*<sup>P1962T</sup> mutation in association with germline *RUNX1*<sup>R174Q</sup> mutation leads to amplification of a haematopoietic clone susceptible to acquire other transforming alterations.

**Keywords:** FPD/AML • predisposition to leukaemia • *RUNX1* • *TET2* • AML-M0 • T2-ALL

## Introduction

Acquired genetic alterations in *RUNX1* are frequently associated with numerous myeloid malignancies, especially acute myeloid leukaemia (AML) (40% of FAB M0 immature AMLs [1]) and more rarely with T

<footnote><sup>a</sup> INSERM UMR 1170, Gustave Roussy, Université Paris-Saclay, Equipe Labellisée par la Ligue Nationale Contre le Cancer, Villejuif, France</footnote>

<footnote><sup>b</sup> Université Paris Diderot, Paris, France</footnote>

<footnote><sup>c</sup> Gustave Roussy, Université Paris-Saclay, Genomic Platform UMS AMMICA, Villejuif, France</footnote>

<footnote><sup>d</sup> Gustave Roussy, Université Paris-Saclay, Bioinformatic Core Facility UMS AMMICA, Villejuif, France</footnote>

<footnote><sup>e</sup> Faculté de Médecine, Aix-Marseille Université, Marseille, France</footnote>

<footnote><sup>f</sup> Département d'Hématologie, Institut Paoli-Calmettes, Marseille, France</footnote>

<footnote><sup>g</sup> Assistance Publique-Hôpitaux de Marseille, Hôpital de La Conception, Service d'Hématologie et Thérapie Cellulaire, Faculté de Médecine, Aix-Marseille Université, INSERM UMR 1090 TAGC, Marseille, France</footnote>

<footnote><sup>h</sup> Hematology and INSERM U1151, Institut Necker-Enfants Malades, Université Sorbonne Paris Cité, Descartes and Assistance Publique-Hôpitaux de Paris, Paris, France</footnote>

<footnote><sup>i</sup> Assistance Publique-Hôpitaux de Paris, Hôpital Trousseau, Service d'Hématologie biologique, Paris, France</footnote>

<footnote><sup>j</sup> Assistance Publique-Hôpitaux de Marseille, Hôpital Nord, Laboratoire d'Hématologie, Faculté de Médecine, Aix-Marseille Université, INSERM UMR_S 1062, Marseille, France</footnote>

<footnote>*Correspondence to: Hana RASLOVA.
E-mail: hana.raslova@gustaveroussy.fr</footnote>

<footnote><sup>#</sup>These authors equally contributed.</footnote>
<table><caption>**Table 1** Gene mutations common to both T2-ALL and AML-M0</caption><thead><tr><th>Gene, chr</th><th>Protein</th><th>Variant frequency in T2-ALL (%)</th><th>Variant frequency in AML-M0 (%)</th></tr></thead><tbody><tr><td>PHF6, chrX</td><td>S247Y</td><td>63</td><td>96</td></tr><tr><td>IRS4, chrX</td><td>G1154V</td><td>49</td><td>92</td></tr><tr><td>HCFC1, chrX</td><td>R1982H</td><td>55</td><td>89</td></tr><tr><td>CXCR4, chr2</td><td>V116Afs*55</td><td>36</td><td>80</td></tr><tr><td>EZH2, chr7</td><td>S669N</td><td>34</td><td>44</td></tr><tr><td>EPHA10, chr1</td><td>R268H</td><td>30</td><td>29†</td></tr><tr><td>CTNND2, chr5</td><td>R82*</td><td>28</td><td>48</td></tr><tr><td>NRF1, chr7</td><td>R206W</td><td>27</td><td>50</td></tr><tr><td>ASXL1, chr20</td><td>R693*</td><td>27</td><td>52</td></tr><tr><td>PTPN13, chr4</td><td>R1838Q</td><td>25</td><td>47</td></tr><tr><td>PPP2R2B, chr5</td><td>K363Q</td><td>25</td><td>51</td></tr><tr><td>JAK1, chr1</td><td>R879H</td><td>24</td><td>54</td></tr><tr><td>JAK3, chr19</td><td>R657Q</td><td>21</td><td>55</td></tr><tr><td>TET2, chr4</td><td>P1962T</td><td>23</td><td>47</td></tr><tr><td>MSRB2, chr10</td><td>T59I</td><td>23</td><td>42</td></tr><tr><td>PCNL2, chr1</td><td>R1169W</td><td>21</td><td>52†</td></tr><tr><td>MYO1D, chr17</td><td>R560Q</td><td>21</td><td>48</td></tr><tr><td><strong>NOTCH1, chr9</strong></td><td><strong>K1607delinsNLQ</strong></td><td><strong>29</strong></td><td><strong>97</strong></td></tr><tr><td>OPRD1, chr1</td><td>I279N</td><td>18</td><td>36</td></tr><tr><td>RRP7A, chr22</td><td>F241del</td><td>11</td><td>20</td></tr></tbody></table>

*stop codon, †possibly damaging, in bold: mutation present at heterozygous state in T2-ALL and at homozygous state in AML-M0.

cell acute lymphoblastic leukaemia (T-ALL) (25% of early thymic immature T-ALL (ETP-ALL) [2]).

Germline RUNX1 mutations are found in FPD/AML characterized by thrombocytopaenia, and a 35% life-time risk of developing myelodysplastic syndrome (MDS) and/or AML. [3] T-ALL development has also been reported in rare cases [4, 5]. In AML, contrary to T-ALL, the leukaemic transformation is almost always associated with a somatic RUNX1 mutation on the second allele [4, 6, 7]. Acquired mutations in CDC25C and GATA2 have also been identified at a high frequency but only in a Japanese cohort [4, 7–9], suggesting that environmental and/or ethnic factors may play an important role in leukaemia transformation. Somatic mutations in FLT3, PHF6, KIT, KRAS, RAD21, BCOR, BCORL1, CBL, CEBPA, MPL, TP53, WT1, SRSF2, DNMT3A, TET2 and ASXL1 were described in patients who developed AML/MDS and mutations in PHF6, WT1, NOTCH1, FLT3 and ASXL1 in patients with T-ALL [4, 5, 7, 9].

<table><caption>**Table 2** Genes mutated only in T2-ALL</caption><thead><tr><th>Gene, chr</th><th>Protein</th><th>Variant frequency in T2-ALL (%)</th></tr></thead><tbody><tr><td>NOTCH1-bis, chr9</td><td>K1488N</td><td>20</td></tr><tr><td>PRB4, chr12</td><td>G164R</td><td>20</td></tr><tr><td>ITGAX, chr16</td><td>D810Y</td><td>19</td></tr><tr><td>AQP5, chr12</td><td>L74M</td><td>14†</td></tr><tr><td>GRIN3B, chr19</td><td>L824M</td><td>14</td></tr><tr><td>CUBN, chr10</td><td>A2914S</td><td>13</td></tr><tr><td>OBSCN, chr1</td><td>A8630E</td><td>12</td></tr><tr><td>SCARF2, chr22</td><td>R147S</td><td>12</td></tr><tr><td>DDX3X, chrX</td><td>G11W</td><td>12</td></tr><tr><td>PDZD2, chr5</td><td>L34I</td><td>11</td></tr><tr><td>MCM3, chr6</td><td>A461D</td><td>11</td></tr><tr><td>WNT3, chr17</td><td>R60S</td><td>11</td></tr><tr><td>ZIK1, chr19</td><td>A364delinsVLYF*</td><td>11</td></tr><tr><td>BCOR, chrX</td><td>L1203Sfs*33</td><td>11</td></tr></tbody></table>

*stop codon, †possibly damaging, in bold: mutation present at heterozygous state in T2-ALL and at homozygous state in AML-M0.

# Materials and methods

## Patient samples

Biologic samples of one FPD/AML pedigree were collected between 2006 and 2013 after informed consent, in accordance with the Declaration of Helsinki. Genomic DNA was extracted from fibroblasts, CD34⁺ cells, peripheral blood mononuclear and bone marrow total cells using kit Qiagen (Les Ulis, France).

## CGH arrays

Comparative genomic hybridization (CGH) arrays were performed on human CGH 2x400K (G4448A) (Agilent, Les Ulis, France) by hybridization of sample versus normal-matched commercial reference. CGH data and protocols have been submitted to ArrayExpress at the EBI with the accession number E-MTAB-4623.

## Exome capture and sequencing (v4+UTR, 70 Mb)

Library preparation, capture, sequencing and variant detection have been carried out by IntegraGen. Exons were captured from blood DNA using Agilent SureSelect Human All Exon v5–70 Mb kit and sequenced on IlluminaHiSEQ 2000 instrument as previously described [10]. WES
<table><caption>**Table 3** Genes mutated only in AML-M0</caption><thead><tr><td>Gene, chr</td><td>Protein</td><td>Variant frequency in AML-M0 (%)</td></tr></thead><tbody><tr><td>SUDS3, chr12</td><td>R325H</td><td>50</td></tr><tr><td>SAP130, chr2</td><td>G803-G804insPTQN</td><td>36</td></tr><tr><td>GGT1, chr22</td><td>R107C</td><td>30</td></tr><tr><td>LDB2, chr4</td><td>K355N</td><td>27</td></tr><tr><td>ENPP2, chr8</td><td>V785M</td><td>26</td></tr><tr><td>ATP8B1, chr18</td><td>R941G, G940V</td><td>23, 23</td></tr><tr><td>LOC441155, chr6</td><td>A81T</td><td>22</td></tr><tr><td>PVRIG, chr7</td><td>C62F</td><td>19</td></tr><tr><td>MMEL1, chr1</td><td>P743H</td><td>18</td></tr><tr><td>TMEM181, chr6</td><td>E111*</td><td>18</td></tr><tr><td>RBFOX3, chr17</td><td>E263*</td><td>18</td></tr><tr><td>GCGR, chr17</td><td>E362*</td><td>18</td></tr><tr><td>CENPC, chr4</td><td>E302*</td><td>17</td></tr><tr><td>PHACTR2, chr6</td><td>G201C</td><td>17</td></tr><tr><td>MUC20, chr3</td><td>T447M</td><td>15</td></tr><tr><td>FAM71F2, chr7</td><td>P198H</td><td>15</td></tr><tr><td>WDR60, chr7</td><td>G1009C</td><td>15</td></tr><tr><td>CSH2, chr17</td><td>V27I</td><td>15</td></tr><tr><td>BAG6, chr6</td><td>P252T</td><td>14</td></tr><tr><td>BCLAF1, chr6</td><td>S38Yfs*38</td><td>14</td></tr><tr><td>MYO9B, chr19</td><td>A1659S</td><td>14</td></tr><tr><td>SLC17A7, chr19</td><td>R314S</td><td>14</td></tr><tr><td>WDR52, chr3</td><td>E1818*</td><td>12</td></tr><tr><td>SOX7, chr8</td><td>P209T</td><td>12</td></tr><tr><td>C19orf26, chr19</td><td>G349W</td><td>12</td></tr><tr><td>MXRA5, chrX</td><td>E937*</td><td>12</td></tr><tr><td>SOBP, chr6</td><td>D569Y</td><td>11</td></tr><tr><td>HSPA12B, chr20</td><td>R650S</td><td>11</td></tr><tr><td>SUV39H1, chrX</td><td>C41*</td><td>11</td></tr></tbody></table>

data and protocols have been submitted to ArrayExpress at the European Bioinformatics Institute (EBI) with the accession number E-MTAB-4679. Variants present in the internal control and in public databases at

a frequency of >1% were excluded, and only non-synonymous mutations predicted by PolyPhen2 to be probably or possibly deleterious were analysed.

## Targeted NGS

The mutated regions were amplified by PCR using primers listed in Table S1. PCR products were end-repaired, extended with an 'A' base on the 3' end, ligated with indexed paired-end adaptors (NEXTflex, Bioo Scientific, Saint Marcel, France) using the Bravo Platform (Agilent) and amplified by PCR for four cycles. Amplicon libraries were sequenced in an IlluminaMiSeq flow cell using the onboard cluster method, as paired-end sequencing (2 × 250 bp reads) (Illumina, San Diego, CA, USA).

## Results

We focus on a man with a familial history of RUNX1<sup>R174Q</sup> mutation, previously referenced as All-1 [11, 12], who developed at the age of 42 years an EGIL (European Group of Immunological Characterization of Leukemia) T2-ALL with a loss of the 1p36 and 17q12 regions and the ASXL1<sup>R693*</sup> mutation in the leukaemic clone. The patient was pre-treated with corticosteroids and then treated according to the French GRAALL2003 protocol based on a polychemotherapy [13]. As 15 days later the blasts were still present in the bone marrow, a second induction course with idarubicin and cytarabine was performed. The patient achieved complete remission [5].

Two years later, he was admitted to the hospital for relapse with 87% blasts in peripheral blood. The clinical course was aggressive and the patient died 1 month after hospitalization. The blasts exhibited basophilic cytoplasm and a lack of azurophilic granules (Figure S1A) and were characterized by immature marker expression (CD34<sup>+</sup>, CD13<sup>+</sup>, HLA-DR<sup>+</sup>, CD33<sup>low</sup>, CD7<sup>+</sup> and CD56<sup>low</sup>) and absence of cytoplasmic and surface expression of CD3 and cyMPO (Figure S1B, C). This phenotype was initially described by Suzuki as myeloid/NK-cell precursor leukaemia [14] and represents a distinct subtype of AML-M0 [15]. In contrast to previously described cases, we detected low expression of TdT, CD10, CD19, cyCD79a and CD5 (Figure S1B, C).

To investigate whether initial (T2-ALL) and relapsed (AML-M0) leukaemic blasts originated from the same clone, we performed first aCGH on both blast populations. In both samples, aCGH revealed loss of 1p36.32-23 and of 17q11.2 and other small deletions (Table S2). One additional deletion on chromosome 11 and five duplicated regions on chromosome 14 were found exclusively in T2-ALL (Table S2). In the AML-M0 blasts, the loss of the 1p36.32-23 region was associated with two large amplifications at the same break point (1p36.33-32 and 1p36.23-1p31.2) surrounding the deleted region (Figure S2). Other additional deletions and duplications were identified (Table S2). These results suggested that T2-ALL and AML-M0 blasts originated from the same clone, but harboured also specific features acquired during disease progression. Furthermore, identical clonal rearrangements of TCRγ (Vγ9-Jγ1.1) and TCRδ (Dδ2-Jδ1 and Dδ2-Jδ3) (Figure S3 and data not shown) were identified in both T2-ALL and AML-M0 blasts strongly supporting their clonal affiliation.

<footnote>*stop codon.</footnote>
<figure><img src="image_2.png" /><figcaption>**Fig. 1** Clonal evolution in FPD/AML patient carrying germline *RUNX1*<sup>R174Q</sup> mutation. The clonal evolution with the different mutations occurring at various stages of the disease (thrombocytopenia, T2-ALL, AML-M0) is represented. Frequency of the variants is listed in the Tables 1-3. The other deletions and amplifications are listed in Table S2.</figcaption></figure>

Finally, analysis of WES revealed 34 variants in the T2-ALL and 49 in the AML-M0. Interestingly, 20 variants were common between these two cell populations (Table 1). Taking into consideration that T2-ALL blasts represented about 58% of the sequenced DNA and AML-M0 blasts 87%, 18 genes were found to be mutated in the original clone at a frequency of more than 40% (or about 100% for X-linked genes) (Table 1), suggesting that they were all present as heterozygous mutations. The results were confirmed by NGS (Table S3). Alterations in genes frequently associated with myeloid and/or lymphoid malig- nancies, such as *PHF6*, *EZH2*, *ASXL1*, *JAK1*, *JAK3*, *TET2* and *NOTCH1*, were found. We also identified nine probably deleterious (*CXCR4*, *IRS4*, *HCFC1*, *CTNND2*, *NRF1*, *PTPN13*, *PPP2R2B*, *MSRB2* and *MYO1D*) and two possibly deleterious *de novo* variants (*EPHA10*, *PCNXL2*) in genes that were also found with a frequency of >40% but had not previously been causally linked to AML/MDS. A second heterozygous *NOTCH1*<sup>K1488N</sup> mutation occurred only in the T2-ALL (Table 2), while the initial *NOTCH1*<sup>K1607delinsNAK</sup> mutation became homozygous in AML-M0 (Table 1). PRB4 mutated with an approxi- mate 40% frequency only in T2-ALL could be a passenger mutation. In the AML-M0, only one variant was present at 50% but absent in T2-ALL, *SUDS3*<sup>R325H</sup> (Table 3). SUDS3 is a subunit of the histone

deacetylase-dependent SIN3A corepressor complex interacting with *RUNX1* and its genetic alterations could lead to a deregulation of *RUNX1* transcriptional program. No acquired *RUNX1* mutation was found in T2-ALL and AML-M0.

To examine whether some of the potentially deleterious variants accumulated before leukaemia development, we screened 20 variants found with a high penetrance in both T2-ALL and AML-M0 clones by NGS on CD34⁺ cells obtained from the patient’s peripheral blood 5 years prior to T2-ALL development. The only somatic mutation identified was a TET2<sup>P1962T</sup> at a frequency of 1% (Table S3) reaching more than 40% at leukaemic stages. These results suggest that a missense TET2<sup>P1962T</sup> mutation in association with germline *RUNX1*<sup>R174Q</sup> mutation led to amplification of an haematopoietic clone susceptible to acquire other transforming alterations (Fig. 1).

## Discussion

Here, we describe a unique case of one FPD/AML patient who devel- oped first one T2-ALL and less than 2 years later an AML-M0. We show that these two leukaemic clones originate from the common
subclone harbouring alterations in genes frequently mutated in both types of leukaemia. We cannot exclude that AML-M0 was induced by the chemotherapy used for T2-ALL treatment, as it involved anthracyclines and cyclophosphamide known to generate genomic instability. Interestingly, one of the variant TET2<sup>P1962T</sup> was present at a frequency of 1% in haematopoietic progenitors of this patient 5 years prior to T2-ALL development. A missense TET2 mutation at the same position (P1962L) has been described in AML [16] and T-lymphoblastic lymphoma [17], and the role of TET2 in the clonal haematopoiesis preceding transformation in FPD/AML has been recently reported in another case of FPD/AML [18]. However, this is the first report of a TET2<sup>P1962T</sup> mutation leading to the amplification of a leukaemic clone.

Our results suggest the following model of transformation: the
germline *RUNX1* mutation induces both alteration in haematopoiesis
(more particularly increasing cycling) and a genetic instability [11].
The acquisition of a somatic *TET2* mutation enhances self-renewal of
haematopoietic stem/progenitor cells [17, 19, 20], leading to a clonal
haematopoiesis. This is not a clonal haematopoiesis of undetermined
potential (CHIP) in this germline context, but a true pre-leukaemic
state responsible for the emergence of progressively divergent
haematological malignancies.

In conclusion, this report presents a FPD/AML patient with a germ-
line *RUNX1* mutation and an acquired *TET2* mutation leading to clonal
haematopoiesis. During a five-year follow-up, the haematopoietic clone
acquired other genetic alterations including mutations in 18 genes. These
subclonal alterations led first to T2-ALL development, and later to a T/
Myeloid phenotype with an AML-M0 development (Fig. 1).

Clonal haematopoiesis in asymptomatic *RUNX1* carriers under the
age of 50 years was already reported [9] suggesting together with
our results that the identification of clonal haematopoiesis before leu-
kaemia development in FPD/AML patients could serve as a marker of
pre-leukaemic state. This finding might be helpful in patient care,
especially if bone marrow transplantation is considered.

Acknowledgements

This work was supported by French grants from the Agence Nationale de la Recherche (ANR-jeunes chercheurs, HR 2010), the ARC (projet libre, IP 2012). M.V.T. was supported by a doctoral fellowship from Société Francais d'Hématologie (SFH). I.A.D. was supported by a postdoctoral fel-

lowship from ARC. The WES was cofunded by French Foundation for Rare Diseases. B.H. was supported by a doctoral fellowship from Ligue National Contre le Cancer. WES has been carried out by IntegraGen (France).

Conflict of interest disclosure

The authors declare no competing financial interests. The online ver-
sion of the article contains a data supplement.

Author contribution

MVT, BH, ADI, AB, MG, DN and DMK performed genetic analysis and analysed mutational data. MVT, BH, VW, PI, ME, AV, FR, BV and RH conceptualized the idea, designed the research and analysed data. FR, PT, CTR and BV provided samples and data. MVT, BH, ADI, ME, AV, BV and RH wrote the manuscript, which was approved by all the authors.

**Supporting information**

Additional Supporting Information may be found online in the
supporting information tab for this article:

**Figure S1** Morphology and phenotype of AML-M0 blasts.

**Figure S2** Comparative genomic hybridization array on T2-ALL and AML-M0 blast populations.

**Figure S3** TCRδ and TCRγ rearrangement analysis.

**Table S1** List of primers used for NGS.

**Table S2** Deletions and insertions found in AML-M0 btasts and/or T2-ALL blasts by CGH array.

**Table S3** Variants found in AML-M0 blasts, T2-ALL blasts and CD34+ cells by NGS.

References

1. Ichikawa M, Yoshimi A, Nakagawa M, et al. A role for RUNX1 in hematopoiesis and myeloid leukemia. *Int J Hematol*. 2013; 97: 726-34.

2. **Grossmann V, Kern W, Harbich S, et al.** Prognostic relevance of RUNX1 mutations in T-cell acute lymphoblastic leukemia. *Haematologica*. 2011; 96: 1874-7.

3. Owen CJ, Toze CL, Koochin A, et al. Five new pedigrees with inherited RUNX1 mutations causing familial platelet disorder with

propensity to myeloid malignancy. *Blood*. 2008; 112: 4639-45.

4. Antony-Debre I, Duployez N, Bucci M, et al. Somatic mutations associated with leukemic progression of familial platelet disorder with predisposition to acute myeloid leukemia. *Leukemia*. 2015; 30: 999-1002.

5. Prebet T, Carbuccia N, Raslova H, et al. Concomitant germ-line RUNX1 and acquired ASXL1 mutations in a T-cell acute

lymphoblastic leukemia. *Eur J Haematol*. 2013; 91: 277-9.

6. Preudhomme C, Renneville A, Bourdon V, et al. High frequency of RUNX1 biallelic alteration in acute myeloid leukemia secondary to familial platelet disorder. *Blood*. 2009; 113: 5583-7.

7. **Haslam K, Langabeer SE, Hayat A, et al.** Targeted next-generation sequencing of familial platelet disorder with predisposition to acute
myeloid leukaemia. *Br J Haematol.* 2015; 175: 161-3.

8. **Yoshimi A, Toya T, Kawazu M, et al.** Recurrent CDC25C mutations drive malignant transformation in FPD/AML. *Nat Commun.* 2014; 5: 4770.

9. **Churpek JE, Pyrtel K, Kanchi KL, et al.** Genomic analysis of germ line and somatic variants in familial myelodysplasia/acute myeloid leukemia. *Blood.* 2015; 126: 2484-90.

10. **Gnirke A, Melnikov A, Maguire J, et al.** Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. *Nat Biotechnol.* 2009; 27: 182-9.

11. **Antony-Debre I, Manchev VT, Balayn N, et al.** Level of RUNX1 activity is critical for leukemic predisposition but not for thrombocytopenia. *Blood.* 2015; 125: 930-40.

12. **Bluteau D, Gilles L, Hilpert M, et al.** Down-regulation of the RUNX1-target gene

NR4A3 contributes to hematopoiesis deregulation in familial platelet disorder/acute myelogenous leukemia. *Blood.* 2011; 118: 6310-20.

13. **Huguet F, Leguay T, Raffoux E, et al.** Pediatric-inspire therapy in adults with Philadelphia chromosome-negative acute lymphoblastic leukemia: the GRAALL-2003 study. *J Clin Oncol.* 2009; 27: 911-8.

14. **Suzuki R, Yamamoto K, Seto M, et al.** CD7+ and CD56+ myeloid/natural killer cell precursor acute leukemia: a distinct hematolymphoid disease entity. *Blood.* 1997; 90: 2417-28.

15. **Oshimi K.** Progress in understanding and managing natural killer-cell malignancies. *Br J Haematol.* 2007; 139: 532-44.

16. **Weissmann S, Alpermann T, Grossmann V, et al.** Landscape of TET2 mutations in acute myeloid leukemia. *Leukemia.* 2012; 26: 934-42.

17. **Quivoron C, Couronne L, Della Valle V, et al.** TET2 inactivation results in pleiotropic hematopoietic abnormalities in mouse and is a recurrent event during human lymphomagenesis. *Cancer Cell.* 2011; 20: 25-38.

18. **Sakurai M, Kasahara H, Yoshida K, et al.** Genetic basis of myeloid transformation in familial platelet disorder/acute myeloid leukemia patients with haploinsufficient RUNX1 allele. *Blood Cancer J.* 2016; 6: e392.

19. **Kunimoto H, Fukuchi Y, Sakurai M, et al.** Tet2 disruption leads to enhanced self-renewal and altered differentiation of fetal liver hematopoietic stem cells. *Sci Rep.* 2012; 2: 273.

20. **Moran-Crusio K, Reavie L, Shih A, et al.** Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation. *Cancer Cell.* 2011; 20: 11-24.