Plasmacytoid dendritic cells proliferation associated with acute myeloid leukemia: phenotype profile and mutation landscape by Loria Zalmai, Pierre-Julien Viailly, Sabeha Biichle, Meyling Cheok, Lou Soret, Fanny Angelot-Delettre, Tony Petrella, Marie-Agnès Collonge-Rame, Estelle Seilles, Sandrine Geffroy, Eric Deconinck, Etienne Daguindau, Sabrina Bouyer, Elodie Dindinaud, Victor Baunin, Magali Le Gaff-Tavernier, Damien Roos-Weil, Orianne Wagner-Ballon, Véronique Salaun, Jean Feuillard, Sophie Brun, Bernard Drenou, Caroline Mayeur-Rousse, Patricia Okamba, Véronique Dorvaux, Michel Ticchioni, Johann Rose, Marie-Thérèse Rubio, Marie-Christine Jacob, Victoria Raggueneau, Claude Preudhomme, Philippe Saas, Christophe Ferrand, Olivier Adotevi, Christophe Roumier, Fabrice Jardin, Francine Garnache-Ottou, and Florian Renosi

Haematologica 2020 [Epub ahead of print]

Citation: Loria Zalmai, Pierre-Julien Viailly, Sabeha Biichle, Meyling Cheok, Lou Soret, Fanny Angelot-Delettre, Tony Petrella, Marie-Agnès Collonge-Rame, Estelle Seilles, Sandrine Geffroy, Eric Deconinck, Etienne Daguindau, Sabrina Bouyer, Elodie Dindinaud, Victor Baunin, Magali Le Gaff-Tavernier, Damien Roos-Weil, Orianne Wagner-Ballon, Véronique Salaun, Jean Feuillard, Sophie Brun, Bernard Drenou, Caroline Mayeur-Rousse, Patricia Okamba, Véronique Dorvaux, Michel Ticchioni, Johann Rose, Marie-Thérèse Rubio, Marie-Christine Jacob, Victoria Raggueneau, Claude Preudhomme, Philippe Saas, Christophe Ferrand, Olivier Adotevi, Christophe Roumier, Fabrice Jardin, Francine Garnache-Ottou, and Florian Renosi. Plasmacytoid dendritic cells proliferation associated with acute myeloid leukemia: phenotype profile and mutation landscape Haematologica. 2020; 105:xxx doi:10.3324/haematol.2020.253740

Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid dissemination of science. Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts that have completed a regular peer review and have been accepted for publication. E-publishing of this PDF file has been approved by the authors. After having E-published Ahead of Print, manuscripts will then undergo technical and English editing, typesetting, proof correction and be presented for the authors' final approval; the final version of the manuscript will then appear in print on a regular issue of the journal. All legal disclaimers that apply to the journal also pertain to this production process. Plasmacytoid dendritic cells proliferation associated with acute myeloid leukemia: phenotype profile and

mutation landscape

Loria Zalmaï1, Pierre-Julien Viailly2, Sabeha Biichle3, Meyling Cheok4, Lou Soret3, Fanny Angelot-Delettre3, Tony

Petrella5, Marie-Agnès Collonge-Rame6, Estelle Seilles3, Sandrine Geffroy4,7, Eric Deconinck8, Etienne Daguindau8,

Sabrina Bouyer9, Elodie Dindinaud9, Victor Baunin10, Magali Le Garff-Tavernier11, Damien Roos-Weil12, Orianne

Wagner-Ballon13, Véronique Salaun14,Jean Feuillard15, Sophie Brun16, Bernard Drenou17, Caroline Mayeur-Rousse18,

Patricia Okamba19, Véronique Dorvaux20, Michel Tichionni21, Johann Rose22, Marie Thérèse Rubio23, Marie Christine

Jacob24, Victoria Raggueneau25, Claude Preudhomme4,7, Philippe Saas3, Christophe Ferrand3, Olivier Adotevi3,

Christophe Roumier4,7, Fabrice Jardin2, Francine Garnache-Ottou3 and Florian Renosi3.

1Service d’hématologie biologique, Hôpital Cochin, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris, France

2INSERM U1245, Centre Henri Becquerel, Rouen, France.

3Univ. Bourgogne Franche-Comté, INSERM, EFS BFC, UMR1098, Interactions Hôte-Greffon-Tumeur/Ingénierie

Cellulaire et Génique, Besançon, France.

4INSERM U837, CHRU Lille, IRCL Laboratoire d'Hématologie, Centre de Biologie Pathologie, Lille, France.

5Department of Pathology, University of Montréal, Hôpital Maisonneuve-Rosemont, Montréal, QC, Canada

6Laboratoire de Génétique biologie, CHU Besançon, Besançon, France.

7Laboratoire d'Hématologie A, Centre de Biologie Pathologie, Boulevard du Pr Leclercq, 59037 Lille, France

8Service Hématologie, CHU Besançon, Besançon, France.

9Service d’Hématologie biologique, CHU La Milétrie, Poitiers, France.

10Laboratoire du Groupe Hospitalier de La Rochelle-Ré-Aunis, CH de La Rochelle, La Rochelle, France.

11Laboratoire d’Hématologie, Hôpital Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris, France.

12Service d’Hématologie Clinique, Sorbonne Université, Hôpital Pitié-Salpêtrière, Assistance Publique-Hôpitaux de

Paris (AP-HP), Paris, France.

13 Département d’Hématologie biologique, Hôpitaux Universitaires Henri Mondor, Assistance Publique-Hôpitaux de

Paris (AP-HP), Créteil, France.

1

14 Laboratoire d’Hématologie, CHU de Caen Normandie, Normandie Université, UNICAEN, Caen, France.

15 Laboratoire d'hématologie, CHU Dupuytren, avenue Martin Luther King, Limoges, France.

16 Laboratoire d'Hématologie et Consultations d'Hématologie Biologique, Hôpital Universitaire Carémeau, Nîmes,

France.

17 Service d’Hématologie, Groupe Hospitalier de la région Mulhouse Sud Alsace, Mulhouse, France.

18 Laboratoire d’Hématologie, CHRU Strasbourg, Hôpital de Hautepierre, 67098 Strasbourg, France.

19Laboratoire d’hématologie et auto-immunité, Hôpital de Mercy, CHR de Metz-Thionville, France.

20Service d’hématologie de l’hôpital de Mercy, CHR de Metz-Thionville, France.

21Laboratoire d’hématologie, Hôpital Pasteur, Nice, France.

22Laboratoire d’hématologie, CH du Mans, Le Mans, France.

23 Service Hématologie, CNRS UMR7365, Biopôle Université de Lorraine, CHRU Nancy, Vandœuvre-lès-Nancy,

France.

24 Laboratoire d’immunologie, CHU Grenoble, La Tronche, France.

25 Service de Biologie Médicale, Centre Hospitalier de Versailles A. Mignot, Le Chesnay, France.

Running Heads: Frequent RUNX1 mutations in acute leukemia + pDC

Contact information for correspondences:

Prof. Francine Garnache Ottou, UMR1098, Laboratoire Hématologie, EFS/B-FC, 8 rue Dr JFX Girod, 25020 Besançon,

France. Phone: Tel: +33(0)3.81.615.615, Fax: +33(0)3.81.615.617, E-mail: [email protected]

Word Count:

Abstract: 249/250

Main text: 3598/4000

Methods: 499/500

Figures: 5

Tables: 1

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Supplemental Files: 1

References: 50/50

Acknowledgments

The authors would like to thank Hugues Faucheu for helping on the design panel, Véronique Yerly-Motta for helping on the NGS platform and Fiona Ecarnot for English proofreading. This work was supported by Ligue régionale contre le Cancer (CCIRGE-BFC-2016), Fondation ARC (Aides Individuelles DOC20170505805) and

Association Laurette Fugain (ALF 2018/08).

Authorship and disclosures

FGO designed the study. FGO and FR supervised the study. ED, ED, SB, ED, VB, MLG, DRW, OWB, VS, JF,

SB, BD, CMR, PO, VD, MT, JR, MTR, MCJ, VR, ES and FGO procured patient specimens. SB performed cell sorting. TP performed anatomopathological analysis and MACR cytogenetics analysis. LZ, FR and LS performed the molecular experiments. PJV provided assistance in bioinformatics analysis. LZ and FR analyzed NGS data. FR performed statistical analysis. LZ, FR and FGO wrote the original manuscript. CP,

CR, MC, FAD, SG, FJ, CF, PS and OA revised the manuscript and provided guidance and expertise. All authors provided input and approved the final version of the manuscript. The authors declare no competing financial interests.

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ABSTRACT

Neoplasms involving plasmacytoid Dendritic Cells (pDCs) include Blastic pDC Neoplasms (BPDCN) and other pDC proliferations, where pDCs are associated with myeloid malignancies: most frequently Chronic MyeloMonocytic

Leukemia (CMML) but also Acute Myeloid Leukemia (AML), hereafter named pDC-AML. We aimed to determine the reactive or neoplastic origin of pDCs in pDC-AML, and their link with the CD34+ blasts, monocytes or conventional

DCs (cDCs) associated in the same sample, by phenotypic and molecular analyses (targeted NGS, 70 genes). We compared 15 pDC-AML at diagnosis with 21 BPDCN and 11 normal pDCs from healthy donors. CD45low CD34+ blasts were found in all cases (10-80% of medullar cells), associated with pDCs (4-36%), monocytes in 14 cases (1-10%) and cDCs (2 cases, 4.8-19%). pDCs in pDC-AML harbor a clearly different phenotype from BPDCN: CD4 + CD56- in

100% of cases, most frequently CD303+, CD304+ and CD34+; lower expression of cTCL1 and CD123 with isolated lymphoid markers (CD22/CD7/CD5) in some cases, suggesting a pre-pDC stage. In all cases, pDCs, monocytes and cDC are neoplastic since they harbor the same mutations as CD34+ blasts. RUNX1 is the most commonly mutated gene: detected in all AML with minimal differentiation (M0-AML) but not in the other cases. Despite low number of cases, the systematic association between M0-AML, RUNX1 mutations and an excess of pDC is puzzling. Further evaluation in a larger cohort is required to confirm RUNX1 mutations in pDC-AML with minimal differentiation and to investigate whether it represents a proliferation of blasts with macrophage and DC progenitor potential.

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MAIN TEXT

INTRODUCTION

Plasmacytoid Dendritic Cells (pDCs) are hematopoietic cells mainly developed from a myeloid branch including the

Macrophage DC Progenitor (MDP) with monocyte, conventional DC (cDC) and pDC differentiation potential1–4. Two types of neoplastic counterparts for pDC have been identified: the first is the well-known Blastic pDC Neoplasm

(BPDCN), initially described as CD4+ CD56+ neoplasm5–10; and the second is defined as Mature pDC Proliferation

(MPDCP) associated with a myeloid neoplasm, frequently Chronic MyeloMonocytic Leukemia (CMML), but also

MyeloDysplastic Syndrome (MDS) or Acute Myeloid Leukemia (AML), especially with monocytic differentiation 11–17.

MPDCP is not formally referenced in the WHO 2017 classification, but mentioned as a differential diagnosis of

BPDCN7,8. As for BPDCN, MPDCP occur predominantly in male patients (75%) with a median age of 69 years 18 and frequent lymph nodes or skin lesions. The mature pDC denomination refers to the morphologically mature and

CD56- phenotype (as with normal pDCs), in the absence of the blastic morphology of BPDCN8. Flow cytometry or immunohistochemistry are mandatory for BPDCN diagnosis and relatively well defined with CD4+, CD56+,

CD123+high, CD303+/-, CD304+/- cells7,19 expressing TCL1 at high levels20. Conversely, only few cases of MPDCP phenotype have been described7,14,15. In the same way, the genomic profile of MPDCP is still poorly understood, but a clonal relationship between pDC and the associated neoplasm has been demonstrated with pDCs exhibiting leukemic abnormalities such as monosomy 7, trisomy 8, del(5q), CBFB-MYH11 or internal tandem duplication of

FLT3 (FLT3-ITD) of blasts in AML16,17,21–23 and the mutational profile of monocytes in CMML24.

We collected AML harboring a heterogeneous phenotypic presentation with a population of immature blasts associated with an excess of pDCs, but also monocytes and sometimes cDCs. These cases are hereafter referred to as AML with pDC (pDC-AML). The purpose of this study was to better characterize pDC-AML by analyzing the phenotype of immature blasts and pDCs and the mutational profiles of each cell population (blasts, pDCs, monocytes, cDCs) after cell sorting, in order to determine whether they share the same profiles.

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METHODS

PATIENT SAMPLES

Primary cells are routinely referred to our center for BPDCN suspicion with cytologic and phenotypic arguments in peripheral blood (PB) or bone marrow (BM) (collection DC 2016-27 91). Some cases do not meet the criteria for a

BPDCN diagnosis, based on the WHO classification 20175,8. Especially, 15 cases were collected based on the association of CD34+ blasts of myeloid origin and pDCs (Table 1). All cases were analyzed at diagnosis, except for

N35 (Day 81 post-induction). The analysis was performed on BM aspiration (n=12) or PB (n=3). BM biopsies were rarely available, preventing from anatomopathology analysis. Fifteen normal BM aspiration performed for peripheral thrombopenia/research of metastatic infiltration, 10 PB from healthy donors and 21 previously described cases of BPDCN20 were used as controls after written informed consent. This study was approved by the

Besançon ethics committee (CPP-Est II, Besançon, France)20.

IMMUNOPHENOTYPE

Flow cytometry was performed using a FACSCanto II cytometer (BD Biosciences, San Jose, CA, USA) with DIVA 6.2 software (BD Biosciences) after cell labelling with monoclonal antibodies (Online Supplementary Table S1). The

Mean Fluorescence Intensity Ratio (MFIR) of cTCL1 was obtained by dividing the Mean Fluorescence Intensity (MFI) for cTCL1 by that of the isotype control mAb. Cells were considered positive for cTCL1 expression when MFIR was greater than 2. Isotype control was not used for MFIR of CD123 because of its high expression on pDCs; thus MFIR was calculated by dividing the MFI of CD123 on pDC by that on lymphocytes.

CELL SORTING

One to 10 million cells were sorted using an ARIA III FACS (Becton Dickinson Biosciences) after cell labelling with monoclonal antibodies (Online Supplementary Table S1) in order to select the populations of interest: T-cells

(CD45+high, CD3+), immature blasts (CD34+, CD303-), pDCs (CD123+high, CD303+), monocytes (CD14+ or CD64+,

CD123+low, CD303-) and cDC (CD34-, CD1c+, CD303-).

MOLECULAR BIOLOGY

Whole Genome Amplification was carried out using the REPLI-g® Single Cell kit (Qiagen Hilden, Germany), as recommended by the manufacturer. Next Generation Sequencing (NGS) was performed, from a HaloPlexHS Target

6

Enrichment System (Agilent Technologies Inc., Santa Clara, CA, USA) targeting 70 genes (Online Supplementary

Table S2), in paired-end, 2x150 cycles on a MiSeq platform (Illumina Inc., San Diego, CA, USA).

BIOINFORMATICS ANALYSIS

Raw data were analyzed using in-house bioinformatics pipelines (Online Supplementary Methods) with annotation of the variants via GenerateReportsTM25 yielding Variant Call Files. Finally, several filters were applied to eliminate intronic regions, synonymous mutations and polymorphisms.

STATISTICS

Statistical analyses were performed using Prism 6.0 software (GraphPad, San Diego, CA, USA). The distribution of

MFIR was studied using the D’Agostino-Pearson normality test. Quantitative data were compared using ANOVA.

Patient groups were compared using the Mann-Whitney non-parametric test for quantitative variables with a non-

Gaussian distribution, the Student t test for normally distributed variables or the Chi-square test with Yates’ continuity correction for categorical variables. Correlations between quantitative variables were investigated by linear regression analyses. All statistical tests were two-sided, with a 5% alpha risk. Results are expressed as median

[range].

Further details in the Online Supplementary Appendix.

RESULTS

PATIENTS

Fifteen patients were included, mainly elderly men with a median age of 70 years [52-87] and a sex ratio of 4:1

(Table 1). Cytological analysis of BM aspiration, associated with phenotypic data, found 11 cases of AML with minimal differentiation (M0-AML in FAB classification) with a prior history of cytopenia in one of them; one AML without maturation (AML-1); 2 acute myelomonocytic leukemia (M4-AML) with a prior history of CMML; and one

M5-AML secondary to another MyeloDysplastic Syndrome/MyeloProliferative Neoplasm (MDS/MPN) analyzed during progression under induction treatment. On BM aspiration, blast cells were observed in all cases, associated with pDCs, described as smaller with a more mature chromatin, faint basophilic cytoplasm without granulation, sometimes small vacuoles and short pseudopodia (Figure 1A-C). Lymph node involvement was reported in one patient, and skin lesions in 4 other patients (26.7%). Pathology analysis of skin lesions found myeloblast infiltration

7 without pDCs in patients N2 and N8, and pDC proliferation in patients N9 and N11. No clonal aberration was detected on karyotype for 9 cases, while 3 others had trisomy 13 (N1, N9 and N11), 2 had chromosome 7 defects

(N14, N16), and one had Y loss (N20). Two patients with normal karyotype were rearranged for KMT2A (N8, N35), and a deletion of EZH2 was detected by FISH in patient N16.

IMMUNOPHENOTYPE IDENTIFIED DIFFERENT SUBPOPULATIONS IN PATIENTS

In contrast to BPDCN, cells of interest were heterogeneous, with a significant population of CD45low CD34+ immature myeloid blasts (44%, [10-80]) without markers of pDC commitment on the one hand, and an excess of

CD4+ CD123+ HLA-DR+ cTCL1+ CD303+ pDCs (15%, [4-36]) on the other hand. Consequently, these cases more likely fit the description of pDC-AML (or MPDCP associated with AML according to the WHO classification8) where pDC were in excess, greater than 4% (median 15%) in our cohort. Indeed, a median of 0.25% of total nucleated cells was detected for the pDC contingent in 15 normal BM aspiration and 0.24% in PB, with similar ranges [0.02-0.95%] and

[0.17-0.53%], respectively. For 11/15 AML cases (73%), CD45low CD34+ immature blasts were more frequent than pDCs, whereas pDCs were preponderant in the remaining 4 cases [10-26%]. Monocytes (3% [1-10]) were also found in 14 cases, greater in acute myelomonocytic leukemia (M4-AML) than in other cases. Interestingly, cDCs were detected in patients N7 and N8 (4.8% and 19% of cells respectively) (Figure 2).

The CD45low immature blasts were CD34+ (15/15, 100% of cases), CD117+ (11/15; 73%), TdT+ (5/9; 56%), expressed myeloid markers such as CD13 (11/15, 73%) (Figure 1D,E) and/or CD33 (5/15, 33%); and less frequently CD15/65 (2 cases) or myeloperoxidase (1 case). Monocyte lineage markers were only expressed on blasts of M4/5-AML patients. T-cell markers were expressed in 2 cases (CD7 +/- CD5: patients N2 and N20) and the B-cell marker CD22 in 2 other cases (N7 and N14) which was not sufficient to meet the definition of mixed-phenotype acute leukemia, as CD3/cCD3 or CD19/cCD79a were not expressed (Figure 2). CD123 was expressed in 12 cases/15 (80%) at low level (MFIR: 15.58 [2.5-63.1], MFI: 2309 [465-8674]). In 2 cases, blasts expressed CD4, whereas CD56 was never expressed. The blastic population did not express markers strongly associated with the pDC (cTCL1, CD303 and

CD304) or cDC (CD1c, CD11c) lineages.

Associated pDCs were constantly CD123+high, CD4+/+low and CD303+ (100%), with CD304 expression in most cases

(13/15, 87%) and none of them expressed CD56. The immaturity marker CD34 was expressed in 33% of cases,

8 whereas TdT in only 1 case out of 9 tested. The CD123 expression level was significantly higher on pDCs than on the immature CD34+ blasts (MFIR: 172.9 [12.5-482.6] versus 15.58 [2.5-63.1] on CD34+ blasts, p<0.0001; MFI: 20836

[10755-45746] versus 2309 [465-8674] on CD34+ blasts, p<0.0001) (Figure 3A,B). cTCL1 was expressed in pDCs

(9/12 cases, 75%) at a statistically lower level (MFIR:5.3 [0.6-22.6]; MFI:2473 [487-20684]) than in the 21 BPDCN cases (MFIR:34 [6.0-96.0], p<0.0001; MFI:13990 [2186-125568], p=0.0006) and even than in normal pDCs

(MFIR:16.9 [3.5-45.5], p=0.0052; MFI:8106 [3389-31544], p=0.0192) (Figure 3C,D).

Interestingly, 4 patients with M0-AML expressed myeloid, B-cell or T-cell markers on both CD34 + blasts and pDCs, but with partial and lower expression on pDCs than on CD34+ blasts: CD33 and CD22 for N7, CD13 for N1, CD33 for

N36, CD5 and CD7 for N20 (Figure 2). A maturation continuum between immature blasts and pDCs was observed in some cases (Figure 1D). cDCs were identified by the expression of CD1c and CD11c without CD14 and CD64 expression in patients N7 and

N8, and were sorted for molecular studies for patient N8 only.

MUTATIONAL PROFILE

The purity of each sorted fraction is depicted in Online Supplementary Table S3. The NGS panel was informative for all cases, with a number of detected mutations ranging from 2 to 6. Fourteen genes were found to be mutated among the 70 explored (Figure 4), corresponding to transcription factors RUNX1 (11/15, 73%); epigenetic modifiers

ASXL1 (5/15, 33%), EZH2 (3/15, 20%), TET2 (4/15, 27%), DNMT3A (3/15, 20%); genes involved in splicing SRSF2

(5/15, 33%), SF3B1 (2/15, 13%), U2AF1 (1/15); RAS pathway CBL (3/15, 19%), KRAS, PTPN11 (1/15 each); cytokine signaling FLT3 (3/15, 19%) as well as other genes, such as PFH6 and WT1 (1/15 each) (Online Supplementary Table

S4). Of note, only one case was mutated for NPM1 (patient N35) and none for CEBPA. In BPDCN, 17 out of the 21 of the cohort were studied by NGS, with 0 to 5 mutations per case on 17 genes: TET2 (9/17, 53%), ASXL1 (6/17, 41%),

ZRSR2 (4/17, 24%), TP53 (3/17, 18%), IKZF1, NRAS, SRSF2, IDH1 (2/17 each, 12%), ZEB2, MET, ETV6, ATM, IKZF3,

CXCR4, NOTCH2,KRAS, JAK2 (1/17 each) (Figure 4).

Mutations were systematically found in sorted CD34+ immature blasts, pDCs, monocytes and cDCs of the same sample, and were not detected in the T-cell fraction, thus confirmed to be a non-neoplastic subpopulation (Figure

4). Variant Allele Frequencies (VAF) were quite similar between cell fractions. However, VAFs of the monocyte

9 subpopulation were lower than in blasts and pDCs in 2 cases (N13 and N36), which may indicate that this mutation is subclonal in monocytes (N13) or that there is a mixture of neoplastic and reactive non-neoplastic monocytes

(same VAF difference in all detected mutations for N36). Some mutations were subclonal in both blast and monocyte fractions: KRAS for N19, FLT3 for N20 (Figure 4). Overall, the most frequently mutated genes were the transcription factor RUNX1, splicing genes (SRSF2, SF3B1, U2AF1) and epigenetic modifiers DNMT3A and TET2.

Interestingly, RUNX1 mutations concerned all M0-AML, while none of the other cases were mutated (M4/5-AML and M1-AML). Consequently, despite low number of cases, there was a significant association between the M0-

AML subtype and RUNX1 mutations (Chi-square with Yates' correction, X2=10.32, 1df, p-value=0.0013). The majority of RUNX1 mutations detected were frameshift (n=6) or stop gain (n=3), with a biallelic invalidation in patient N13. RUNX1-mutated cases involved mainly males (M/F=8/3), with a median age of 70 years [55-87].

DISCUSSION

The WHO 2017 classification clearly recognizes BPDCN as a form of pDC neoplasm, while MPDCP is still insufficiently described and probably under-diagnosed8. MPDCP mainly concerns CMML and more rarely MDS or

AML. Our study focused on pDC-AML, using a phenotypic and mutation characterization on sorted population of 15 cases. Our gateway for the inclusion of cases is the pDC-like morphology of some blastic cells detected by cytologists in French hospitals. This point precludes us from determining the prevalence of pDC-AML among AML, due to a recruitment bias. We highlight in this study an excess of pDCs in a context of AML in order to differentiate them from BPDCN and to precise their molecular profile, not well-described so far. The clinical presentation of our cohort was close to that of BPDCN, with elderly patients and a clear male predominance, but skin lesions were less frequent (25% of cases vs 90% in BPDCN), linked to either pDC (2 cases) or myeloblast (2 cases) infiltration. The excess of pDCs in BM aspiration of pDC-AML was well over the pDC infiltrate in healthy donor BM and PB, considered to be below 1% of total nucleated cells24,26,27 and confirmed by our data. Moreover, pDCs are frequently gathered on BM smears, compatible with aggregates and in the only case of BM biopsy, clustered nodules of pDC were present, as reported by pathologists with islands of pDCs in MPDCP13,21,24. In the two cases with prior history of CMML, pDCs were not in high excess prior transformation on bone marrow smears. However, in the absence of available sample, flow cytometry assays were not assessed at the CMML stage.

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Blasts exhibited an immature phenotype with expression of CD34, CD117 and TdT, which distinguishes these cases from BPDCN. They also frequently express myeloid markers, and the expression of CD123 is definitely weaker than pDCs from the same samples or pDC blasts from BPDCN. Of note, strong pDC or cDC lineage markers (CD303,

CD304, cTCL1, ILT7 and CD1c, CD141, CD11c) were never expressed on blasts. Focusing on the 11 M0-AML cases, blasts are immature with markers of commitment to the myeloid lineage (CD13, CD33 without MPO) plus expression of lymphoid markers in some cases; this could be a sign of an original progenitor origin or oncogenic dysregulation in pDC-AML compared to other AML.

Only 4 cases of pDC-AML showed higher infiltration of pDC than blasts: 3 at diagnosis (N2, N8 and N34) and the fourth at day 81 post-induction (N35). In these 15 cases, the phenotype of pDCs is different from pDCs in healthy donors and in BPDCN. CD56, is not expressed in our 15 cases, similarly to physiological mature pDCs, only CD56+ when stimulated by Flt3L during maturation28,29. Conversely CD56 is almost systematically expressed in BPDCN5,30,31, except for rare cases7,32,33. Moreover, the pDCs in pDC-AML always expressed CD303 (100%) while 20 to 30% of

BPDCN are CD303- 6,31 and BPDCN express a lower intensity of CD303 than pDC of pDC-AML (data not shown); cTCL-

1 expression was substantially lower on pDCs of pDC-AML compared to pDCs of BPDCNs and even pDCs of healthy donors (Figure 3B). Finally, CD34 is sometimes expressed on pDCs (33% of cases), whereas CD34 is almost never expressed on BPDCN blasts. This phenotype is closer to normal pDCs in some respects (CD56-, CD303+, cTCL1+low). pDC lineage maturation is divided in 3 stages with progressive acquisition of CD303 and CD304 plus downregulation of CD34 and CD117 and gradual loss of CD13, CD33 or CD22 expression34. In our series, the expression of CD34 (6 cases) and CD13, CD33 or CD22 (3 cases) makes these pDCs closer to the intermediate stage of pDC maturation

(CD34+, CD303+, CD304+/-, CD123+). Moreover, aberrant markers of myeloid or lymphoid lineage (CD13, CD33,

CD11b, CD22, CD7, CD5) on both blasts and pDCs in the same patient support the idea of a common origin of these populations, with maturation of CD34+ blasts towards the pDC lineage by downregulation of CD34 and upregulation of CD123, CD303 and CD304. Reinforcing this hypothesis, we clearly show a maturation continuum between blasts and pDCs or monocytes (Figure 1D) with CD34 downregulation and upregulation of CD45, pDC markers (CD123,

CD304, CD303) on the pDC cells or CD14, CD64 on monocytes, as recently highlighted14,15. Another study also described similar cases of leukemia associating immature CD34+ myeloid blasts and CD34+/- CD56- pDCs, but consider these cases as an immature subgroup of BPDCN35. Altogether, a variety of teams described similar cases

11 under different denominations: MPDCP with myeloid neoplasm (MN), pDC proliferations associated with MN,

AML/MN with PDC differentiation, AML/MDS-pDCs, leukemia of ambiguous lineage, or immature Group 1 of

BPDCN14–17,22,23,35,36. There is a need to refine this poorly defined MPDCP, where the “mature” denomination should be omitted because only referring to the absence of the blastic morphology of BPDCN8.

Our data and others highlight that pDCs are present at all stages of maturation in pDC-AML, even CD34+, because they would keep their potential for maturation14,15 as already described in pDC-CMML24. In addition, pDC-AML appear to be quite similar to pPDC-CMML, as clonal monocytes are also frequently detected in our cohort (14/15

AML). In this regard, all cases of MPDCP described so far fall within our definition of pDC-AML/CMML/MDS.

Interestingly, the expression of CD123 on both blasts and pDCs opens up the potential to use new therapies targeting CD123, such as tagraxofusp, IMGN632 or CD123 CAR-T cells37–40. Crucially, these pDC-AML may respond differently from other AML or BPDCN.

Very few molecular data have been obtained to assert the clonal link between pDCs and leukemic cells in CMML and AML21–23. In our series, using sorted cell populations, we show that the same mutations are shared by blasts, pDCs, monocytes and even cDCs, thus confirming the neoplastic origin of pDCs and their shared clonal origin with blasts, monocytes and cDCs (for one tested case). Of note, no unique mutation was identified in pDCs but not in the blast compartment, or vice versa, albeit with the limitation that only 70 genes were tested. Thus, no specific mutation arising during the lineage commitment was highlighted. Blasts seem to be broadly blocked in an immature undifferentiated stage in pDC-AML compared to pDC-CMML, and maintain their ability to mature towards pDCs, monocytes or even cDCs (Figure 5A-B). The immature CD34+ blasts could be a proliferation, at the very least, of a bipotent progenitor with DC (pDC and cDC) and monocyte potential such as the MDP. The expression of CD22, CD2 and CD5 on pDCs and blasts in some cases could also suggest that they derive from a granulocyte-monocyte-lymphoid progenitor or from AXL+ SIGLEC6+ DCs, recently identified41.

The mutation status obtained does not highlight specific genes for all cases. Many of them are frequently mutated in other myeloid malignancies, involved in epigenetics (ASXL1 and TET2), splicing (SRSF2, SF3B1, U2AF1) or the RAS pathway (CBL, KRAS, PTPN11). This mutation profile is only partially similar to BPDCN, because BPDCN can also be mutated for ETV6, TP53, ZEB2, MET, ATM, IKZF3, JAK, NOTCH2 and CXCR4, plus TET2 with a high frequency (Figure

4). Cases classified as M4/5-AML were always transformations of MDS/MPN and had a similar molecular profile

12 including ASXL1, TET2, SRSF2, CBL or PTPN11 mutations, sometimes with additional mutations responsible for acute transformation (NPM1 mutation for N35). Remarkably, RUNX1 is the most frequently mutated gene in pDC-AML

(73% of cases), as already described17. Moreover, it only concerned M0-AML cases in our study and all cases of M0-

AML exhibit this mutation (100%). This point is particularly puzzling considering that this prevalence is markedly different from the described epidemiology of 20-30% of RUNX1 mutations in M0-AML42,43 and knowing that RUNX1 mutation has only been reported once in BPDCN44. Although, this study only concerns a small number of cases and the recruitment bias precludes us from determining the frequency of M0-AML and RUNX1 mutations in pDC-AML.

Our 11 PDCP-M0-AML cases were 71 years old on average, with a M/F sex ratio of 2.67, consistent with the RUNX1- mutated AML provisional entity8,42. Unfortunately, given the advanced age of patients with palliative care and low number of cases, prognostic conclusions are impossible. RUNX1 mutations were not detected in the non-neoplastic

T-cell fraction, demonstrating that these mutations are somatic. WGA prevented us to definitely obtain copy number variations, but VAF were higher than 50% for 6 patients (N8, N9, N11, N12, N16, N19) and a seventh (N13) was double mutated for RUNX1, suggesting secondary alteration of RUNX1, as already described45.

Notably, inhibition of RUNX1 is considered to increase RUNX2 and RUNX3 protein levels, following a complementary compensation mechanism that maintains the entire RUNX family at a constant level46. Then,

RUNX1 invalidation could promote a RUNX2 switch and then a pDC commitment because RUNX2 plays a crucial role in pDC diffentiation47,48. Further experiments are nevertheless required to confirm this hypothesis.

To conclude, our study identifies a group of pDC-AML requiring a differential diagnosis with BPDCN. They are characterized by an immature myeloid population (CD34+, CD117+/- CD123+low without expression of pDC markers) associated with an excess of pDCs (CD123+high, CD4+) that clearly differ from BPDCN neoplastic cells by no expression of CD56, the possible expression of CD34, higher expression of CD303 and lower expression of cTCL1.

Molecular data show that these pDCs are neoplastic and not reactive, and the mutational landscape of pDC-AML appear distinct from BPDCN, notably with frequent RUNX1 mutations. Moreover, a continuous maturation pattern suggests that these pDCs as well as monocytes could arise from the immature CD34+ blasts, potentially with MDP properties (Figure 5B). Finally, this study addresses (i) the frequency with which RUNX1-mutated AML are

13 associated with an excess of pDCs, (ii) the type of progenitors involved and (iii) its prognostic or therapeutic impact.

These questions warrant investigation in an independent and larger cohort of AML.

Note Added in Proof: during the review process of our manuscript, Xiao et al. also specifically described pDC-AML with frequent RUNX1 mutations and an original pDC transcriptional program, in approximately 5% of AML (Xiao W,

Chan A, Waarts MR, et al. Plasmacytoid dendritic cell expansion defines a distinct subset of RUNX1 mutated acute myeloid leukemia. Blood [Epub ahead of print], PMID: 32871587). Our work and this independent study argue for pDC-AML to be a distinct entity within AML, with potential therapeutic impact.

14

REFERENCES

1. Banchereau J, Briere F, Caux C, et al. Immunobiology of dendritic cells. Annu Rev Immunol. 2000;18767-811.

2. Jegalian AG, Facchetti F, Jaffe ES. Plasmacytoid dendritic cells: physiologic roles and pathologic states. Adv Anat Pathol. 2009;16(6):392-404.

3. Naik SH, Sathe P, Park H-Y, et al. Development of plasmacytoid and conventional dendritic cell subtypes from single precursor cells derived in vitro and in vivo. Nat Immunol. 2007;8(11):1217-1226.

4. Onai N, Obata-Onai A, Schmid MA, Ohteki T, Jarrossay D, Manz MG. Identification of clonogenic common Flt3+M-CSFR+ plasmacytoid and conventional dendritic cell progenitors in mouse bone marrow. Nat Immunol. 2007;8(11):1207-1216.

5. Chaperot L, Bendriss N, Manches O, et al. Identification of a leukemic counterpart of the plasmacytoid dendritic cells. Blood. 2001;97(10):3210-3217.

6. Garnache-Ottou F, Feuillard J, Ferrand C, et al. Extended diagnostic criteria for plasmacytoid dendritic cell leukaemia. Br J Haematol. 2009;145(5):624-636.

7. Facchetti F, Cigognetti M, Fisogni S, Rossi G, Lonardi S, Vermi W. Neoplasms derived from plasmacytoid dendritic cells. Mod Pathol. 2016;29(2):98-111.

8. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Revised 4th Edition. Lyon, France: International Agency for Research on Cancer; 2017.

9. Petrella T, Comeau MR, Maynadié M, et al. “Agranular CD4+ CD56+ hematodermic neoplasm” (blastic NK-cell lymphoma) originates from a population of CD56+ precursor cells related to plasmacytoid monocytes. Am J Surg Pathol. 2002;26(7):852-862.

10. Petrella T, Facchetti F. Tumoral aspects of plasmacytoid dendritic cells: what do we know in 2009? Autoimmunity. 2010;43(3):210-214.

11. Vitte F, Fabiani B, Bénet C, et al. Specific skin lesions in chronic myelomonocytic leukemia: a spectrum of myelomonocytic and dendritic cell proliferations: a study of 42 cases. Am J Surg Pathol. 2012;36(9):1302- 1316.

12. Dargent J-L, Delannoy A, Pieron P, Husson B, Debecker C, Petrella T. Cutaneous accumulation of plasmacytoid dendritic cells associated with acute myeloid leukemia: a rare condition distinct from blastic plasmacytoid dendritic cell neoplasm. J Cutan Pathol. 2011;38(11):893-898.

13. Orazi A, Chiu R, O’Malley DP, et al. Chronic myelomonocytic leukemia: The role of bone marrow biopsy immunohistology. Mod Pathol. 2006;19(12):1536-1545.

14. Hamadeh F, Awadallah A, Meyerson HJ, Beck RC. Flow Cytometry Identifies a Spectrum of Maturation in Myeloid Neoplasms Having Plasmacytoid Dendritic Cell Differentiation. Cytometry B Clin Cytom. 2020;98(1):43-51.

15. Huang Y, Wang Y, Chang Y, et al. Myeloid Neoplasms with Elevated Plasmacytoid Dendritic Cell Differentiation Reflect the Maturation Process of Dendritic Cells. Cytom A. 2020;97(1):61-69.

16. Wang P, Feng Y, Deng X, et al. Tumor-forming plasmacytoid dendritic cells in acute myelocytic leukemia: a report of three cases and literature review. Int J Clin Exp Pathol. 2017;10(7):7285-7291.

15

17. Xiao W, Goldberg AD, Famulare C, et al. Acute Myeloid Leukemia with Plasmacytoid Dendritic Cell Differentiation: Predominantly Secondary AML, Enriched for RUNX1 Mutations, Frequent Cross-Lineage Antigen Expression and Poor Prognosis. Blood. 2018;132(Supplement_1):2789.

18. Bodmer A, Menter T, Juskevicius D, et al. Sharing of a PTPN11 mutation by myelodysplastic bone marrow and a mature plasmacytoid dendritic cell proliferation provides evidence for their common myelomonocytic origin. Virchows Arch. 2017;470(4):469-473.

19. Horny HP, Feller AC, Horst HA, Lennert K. Immunocytology of plasmacytoid T cells: marker analysis indicates a unique phenotype of this enigmatic cell. Hum Pathol. 1987;18(1):28-32.

20. Angelot-Delettre F, Biichle S, Ferrand C, et al. Intracytoplasmic detection of TCL1--but not ILT7-by flow cytometry is useful for blastic plasmacytoid dendritic cell leukemia diagnosis. Cytom A. 2012;81(8):718-724.

21. Vermi W, Facchetti F, Rosati S, et al. Nodal and extranodal tumor-forming accumulation of plasmacytoid monocytes/interferon-producing cells associated with myeloid disorders. Am J Surg Pathol. 2004;28(5):585- 595.

22. Rickmann M, Krauter J, Stamer K, et al. Elevated frequencies of leukemic myeloid and plasmacytoid dendritic cells in acute myeloid leukemia with the FLT3 internal tandem duplication. Ann Hematol. 2011;90(9):1047- 1058.

23. Mohty M, Jarrossay D, Lafage-Pochitaloff M, et al. Circulating blood dendritic cells from myeloid leukemia patients display quantitative and cytogenetic abnormalities as well as functional impairment. Blood. 2001;98(13):3750-3756.

24. Lucas N, Duchmann M, Rameau P, et al. Biology and prognostic impact of clonal plasmacytoid dendritic cells in chronic myelomonocytic leukemia. Leukemia. 2019;33(10):2466-2480.

25. Viailly P-J, Mareschal S, Bertrand P, et al. GenerateReports: an Ion Torrent plugin summarizing a whole NGS experiment for clinical interpretation. 2015.

26. Wang W, Khoury JD, Miranda RN, et al. Immunophenotypic characterization of reactive and neoplastic plasmacytoid dendritic cells permits establishment of a 10-color flow cytometric panel for initial workup and residual disease evaluation of blastic plasmacytoid dendritic cell neoplasm. Haematologica. 2020 Apr 2. [Epub ahead of print].

27. Xiao W, Goldberg AD, Famulare CA, et al. Loss of plasmacytoid dendritic cell differentiation is highly predictive for post-induction measurable residual disease and inferior outcomes in acute myeloid leukemia. Haematologica. 2019;104(7):1378-1387.

28. Comeau MR, Van der Vuurst de Vries A-R, Maliszewski CR, Galibert L. CD123bright plasmacytoid predendritic cells: progenitors undergoing cell fate conversion? J Immunol. 1950 2002;169(1):75-83.

29. MacDonald KPA, Munster DJ, Clark GJ, Dzionek A, Schmitz J, Hart DNJ. Characterization of human blood dendritic cell subsets. Blood. 2002;100(13):4512-4520.

30. Feuillard J, Jacob M-C, Valensi F, et al. Clinical and biologic features of CD4(+)CD56(+) malignancies. Blood. 2002;99(5):1556-1563.

31. Garnache-Ottou F, Vidal C, Biichlé S, et al. How should we diagnose and treat blastic plasmacytoid dendritic cell neoplasm patients? Blood Adv. 2019;3(24):4238-4251.

32. Julia F, Dalle S, Duru G, et al. Blastic plasmacytoid dendritic cell neoplasms: clinico-immunohistochemical correlations in a series of 91 patients. Am J Surg Pathol. 2014;38(5):673-680. 16

33. Lamar E, Roggy A, Le Calvez G, et al. Blastic Plasmacytoid Dendritic Cell Neoplasm: Report of a Case with Atypical Cytology and Immunophenotype. J Blood Disord. 2015;2(3):1033.

34. Martín-Martín L, Almeida J, Hernández-Campo PM, Sánchez ML, Lécrevisse Q, Orfao A. Immunophenotypical, morphologic, and functional characterization of maturation-associated plasmacytoid dendritic cell subsets in normal adult human bone marrow. Transfusion. 2009;49(8):1692-1708.

35. Martín-Martín L, López A, Vidriales B, et al. Classification and clinical behavior of blastic plasmacytoid dendritic cell neoplasms according to their maturation-associated immunophenotypic profile. Oncotarget. 2015;6(22):19204-19216.

36. Tsagarakis NJ, Kentrou NA, Papadimitriou KA, et al. Acute lymphoplasmacytoid dendritic cell (DC2) leukemia: results from the Hellenic Dendritic Cell Leukemia Study Group. Leuk Res. 2010;34(4):438-446.

37. Mani R, Goswami S, Gopalakrishnan B, et al. The interleukin-3 receptor CD123 targeted SL-401 mediates potent cytotoxic activity against CD34+CD123+ cells from acute myeloid leukemia/myelodysplastic syndrome patients and healthy donors. Haematologica. 2018;103(8):1288-1297.

38. Kovtun Y, Jones GE, Adams S, et al. A CD123-targeting antibody-drug conjugate, IMGN632, designed to eradicate AML while sparing normal bone marrow cells. Blood Adv. 2018;2(8):848-858.

39. Mardiros A, Dos Santos C, McDonald T, et al. T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia. Blood. 2013;122(18):3138-3148.

40. Pemmaraju N, Lane AA, Sweet KL, et al. Tagraxofusp in Blastic Plasmacytoid Dendritic-Cell Neoplasm. N Engl J Med. 2019;380(17):1628-1637.

41. Villani A-C, Satija R, Reynolds G, et al. Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors. Science. 2017;356(6335):eaah4573.

42. Gaidzik VI, Teleanu V, Papaemmanuil E, et al. RUNX1 mutations in acute myeloid leukemia are associated with distinct clinico-pathologic and genetic features. Leukemia. 2016;30(11):2282.

43. Roumier C, Eclache V, Imbert M, et al. M0 AML, clinical and biologic features of the disease, including AML1 gene mutations: a report of 59 cases by the Groupe Français d’Hématologie Cellulaire (GFHC) and the Groupe Français de Cytogénétique Hématologique (GFCH). Blood. 2003;101(4):1277-1283.

44. Menezes J, Acquadro F, Wiseman M, et al. Exome sequencing reveals novel and recurrent mutations with clinical impact in blastic plasmacytoid dendritic cell neoplasm. Leukemia. 2014;28(4):823-829.

45. Antony-Debré I, Duployez N, Bucci M, et al. Somatic mutations associated with leukemic progression of familial platelet disorder with predisposition to acute myeloid leukemia. Leukemia. 2016;30(4):999-1002.

46. Morita K, Suzuki K, Maeda S, et al. Genetic regulation of the RUNX transcription factor family has antitumor effects. J Clin Invest. 2017;127(7):2815-2828.

47. Kubota S, Tokunaga K, Umezu T, et al. Lineage-specific RUNX2 super-enhancer activates MYC and promotes the development of blastic plasmacytoid dendritic cell neoplasm. Nat Commun. 2019;10(1):1653.

48. Chopin M, Preston SP, Lun ATL, et al. RUNX2 Mediates Plasmacytoid Dendritic Cell Egress from the Bone Marrow and Controls Viral Immunity. Cell Rep. 2016;15(4):866-878.

17

Table 1. Clinical and biological features of the cohort.

s f s ) n ) n o n 3 r o n o o y o 2 t e ti o n si g si q n b a ti s/ y e o e 1 l c a e p l l l e 1 ge m ) r ia fi ic y ra r o r p ( n u (y e r i f ( e la th la ty i n d e s si y h l a l A t t e n t a s r t u p u yo T2 n n g e a cl la a r d o d r co ie A G M c d io e e a M t O n r m m m K K C a H B o P a to a D P A c r a tr SH p W F Se xt n x I E A e F AML with no M0 N13 87 F PB mutated no no ND 46,XX[20] anomaly CD34- AML RUNX1 detected M0 N16* 59 M BM AML-MRC no no ND 46,XY,del(7)(q36)[8]/46,XY[12] ND CD34- AML AML with no M0 N2 77 M BM mutated no yes/cutaneous AML 46,XY anomaly CD34- AML RUNX1 detected AML with no M0 N20 71 M BM mutated no no ND 45,X,-Y[20] anomaly CD34- AML RUNX1 detected AML with no M0 N19 70 F BM mutated no no ND 46,XX[20] anomaly CD34+ AML RUNX1 detected AML with KMT2A(MLL) M0 yes/ N8 64 M BM rearrangement no AML 46,XY[20] rearranged CD34- AML cutaneous and mutated RUNX1 AML with no M0 N1 82 F PB mutated no no ND 47,XX,+13[13]/46,XX[7] anomaly CD34+ AML RUNX1 detected Neutropenia and Therapeutic M0 thrombocytopenia, abstention, yes/ N9$ 70 M BM AML-MRC pDCs 46,X,-Y,+13[11]/46,XY[23] ND CD34+ AML granulocytic monitoring cutaneous dysplasia£ AML with no M0 yes/ N11 68 M BM mutated no pDCs 47,XY,+13[21]/46,XY[11] anomaly CD34+ AML cutaneous RUNX1 detected AML with M0 yes/lymph N7 79 M BM mutated no ND 46,XY ND CD34- AML nodes RUNX1 AML with no M0 N12 55 M BM mutated no no ND 46,XY[30] anomaly CD34+ AML RUNX1 detected no AML without M1 N36 52 M BM no no ND 46,XY[28] anomaly CD34+ maturation AML detected Therapeutic M4 N14 85 M BM AML-MRC CMML abstention, no ND 46,XY,-7,+mar[18]/46,XY[4] ND CD34+ AML monitoring M4 N34¤ 73 M PB AML-MRC CMML Hydroxyurea no ND 46,XY[20] ND CD34- AML AML with Therapeutic mutated abstention, M5 N35 65 M BM NPM1 and MDS/MPN monitoring no ND 46,XY[20] rearranged CD34- AML KMT2A(MLL) rearrangement BM: Bone marrow; PB: Peripheral blood; AML-MRC: AML with myelodysplasia-related changes; WHO classification: World Health Organisation classification; FAB: French-American-British classification; FISH: Fluorescent In Situ Hybridization; ND: not done. $pDC nodules were described on BM biopsy. £diagnosis of SMD was not clearly affirmed before AML state. ¤Only patient N34 experienced a prior history of solid tumor with prostatic neoplasm treated by hormonotherapy. 18

Figure Legends Figure 1. Morphologies and immunophenotypes of populations of interest. A,B,C. Representative morphologic aspects of peripheral blood smears from patient N1 (Magnification x1000). A: blast cells are medium sized with a high nuclear cytoplasmic ratio, fine chromatin with proeminent nucleoli. Cytoplasm is basophilic with some rare azurophilic granulations. B. pDC are smaller with more mature chromatin. The cytoplasm is less basophilic without granulation but sometimes pseudopodia and small vacuoles under the cytoplasmic membrane. C. A blast cell (top), a pDC with pseudopodia (center) and a monocyte (bottom). D,E. Representative immunophenotype after gating on FSC-A versus FSC- H plus SSC vs FSC to select singlets, leucocytes and exclude debris (not shown). Lymphocytes in blue (CD45bright /SSCdim cells); immature blasts in black (CD34+ cells); pDCs in pink (CD123bright); monocytes in green (CD123dim, CD33bright, CD64bright); cDCs in orange (CD123+, CD33+ CD64dim). D. Patient N8: M0-AML with a continuum of phenotypic acquisition of markers (arrows) from the immature blasts: downregulation of CD13 and CD33 to pDCs or upregulation to monocytes and cDCs; downregulation of CD117 and CD34 to pDCs/monocytes/cDCs. E. Patient N12: M0-AML with Tdt+, HLA-DRbright, CD33-, cCD13+ immature blasts; CD7+ CD4+ CD56- pDCs.

Figure 2. Immunophenotypic features of pDC-AML. Positive high on 100% of cells in red (>10^4), positive on 100% of cells in orange (10^2 to 10^4), partially positive in light yellow, negative (<20%) in green, not done in grey. CD15 and CD65 are both labelled by FITC in the same tube of our panel. ¤Percentage of cells corresponds to flow cytometry, quantification on the sample used for phenotyping, possibly diluted by PB. All cases exhibited more than 20% of blasts on BM smears. $Analyses performed on sample obtained after induction of chemotherapy, 70 days after diagnosis. cCD3, CD3, CD19, cCD79a were negative for all cases and all fractions.

Figure 3. Expression of CD123 and cTCL1 on pDCs from pDC-AML. A. Comparison of Mean Fluorescence Intensity Ratio (MFIR) of CD123 between pDCs and immature CD34+ blasts in pDC-AML. B. Comparison of MFI of CD123 between pDCs and immature CD34+ blasts in pDC-AML. C. Comparison of MFIR of cTCL1 between pDCs from pDC-AML, BPDCN and non- neoplastic pDCs from healthy donors. D. Comparison of MFI of cTCL1 between pDCs from pDC-AML, BPDCN and non- neoplastic pDCs from healthy donors. P-values (unpaired Mann-Whitney test) are marked above.

Figure 4. Mutation profile of pDC-AML. Mutations detected by NGS with VAF, or Sanger sequencing (especially for ASXL1). Abnormalities in pDC-AML and BPDCN are depicted in: bright blue (monoallelic mutation); dark blue (biallelic mutation); white (absence of mutation); grey (not available). ¤Percentage of cells corresponds to flow cytometry, quantification on the sample used for phenotyping, possibly diluted by PB (all cases exhibited more than 20% of blasts on BM smears). $Analyses performed on sample obtained after induction of chemotherapy, 70 days after diagnosis. £FISH 7q36 on case N16: loss of EZH2 in 91/200 nuclei. ***VAF not available because ASXL1 c.1934dupG;p.Gly646TrpfsX12 was confirmed by Sanger sequencing.

Figure 5. Maturation model in pDC-AML. A. Representative CD45/SSC dot plot of pDC-AML, with 4 populations identified: immature CD34+ blasts in black, pDCs in pink, monocytes in green and lymphocyte in blue, with morphologies of these populations depicted above. B. The maturation model: immature blast cells are mainly proliferative without maturation, but at least part of them conserved MDP (Macrophage-DC Progenitor)-like potential of maturation leading to variable amounts of clonal pDCs, monocytes and cDCs.

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Supplemental methods

PATIENT SAMPLES: Normal BM aspiration performed for peripheral thrombopenia/research of metastatic infiltration were performed on sternal puncture. After BM examination, no qualitative or quantitative abnormalities were noticed, and cytologists concluded to physiological hematopoiesis.

CELL LABELLING: After 20 minutes of incubation, cells were washed twice with PBS buffer (Gibco Life technologies,

Paisley, UK). The percentage of positive expression of markers was determined by a threshold obtained using matched isotypic control mAbs.

CELL SORTING: 30,000 to one million cells were obtained per fraction and their purity was controlled by flow cytometry. The sorted cells were frozen in dry pellets at -20°C.

MOLECULAR BIOLOGY

After thawing the cell pellets at room temperature, the genomic DNA of each sorted fraction was extracted manually according to the manufacturer’s instructions using the QIAamp® DNA Mini kit (Qiagen, Hilden, Germany) or the

QIAamp® DNA Micro kit (Qiagen) for fractions below 150,000 cells. The DNA was eluted in 20-30 µL of elution buffer.

Concentrations ranged from 0.58-33.86 ng/µL on a Nanodrop™ND-2000 spectrophotometer (Thermo Fisher Scientific,

Wilmington, USA). After Whole Genome Amplification, the final DNA quantities obtained were about 40 µg on

Nanodrop™ND-2000.

The HaloPlexHS Target Enrichment System design included 5392 amplicons, a total sequenceable design size of 244.235 kbp and a total coverage of 98.56%. In addition to genes recurrently mutated in onco-hematology, this panel included mutated genes in BPDCN (IKZF2, IKZF3, ZEB2, HOXB9, UBE2G2, APC, MET, RB1, VHL, MLH1, RET, SHOC2, NEFH, HNF1A,

SSC5D, SF3A2, SF3BA, PAX3, GNB1, CRIPAK, MAP2K1). NPM1 and CEBPA variants were screened by Sanger sequencing with a BigDye®Terminator V3.1 Cycle Sequencing kit (Thermo Fisher Scientific) and FLT3-ITD mutations were confirmed by fragment analysis on an ABI3500 device (Applied Biosystems, Thermo Fisher Scientific). Some other variants (ASXL1, RUNX1, PFH6) detected by NGS were confirmed by Sanger sequencing on a capillary sequencer

SeqStudio Genetic Analyser (Applied Biosystems, Thermo Fisher Scientific).

BIOINFORMATICS ANALYSIS

The reads were aligned with the reference genome sequence GHCh37 (hg19) using the BWA (Burrows-Wheeler

Alignment) aligner. The calibration of the base scores (BSQR) and the realignment of the Indels were performed according to GATK Best Practices recommendations. Variant Calling was based on the VarScan algorithm. The 1

SUPPLEMENTAL DOCUMENTS

annotation of the variants was done via Generate Reports™ yielding VCF files (Variant Call Files). Finally, several filters were applied to eliminate intronic regions, synonymous mutations and polymorphisms with VAF>0.01%.

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Supplemental tables

Table S1: Antibodies used for immunophenotype or cell sorting. Expression was evaluated on the selected populations for immunophenotyping. Intracellular detection of TCL1 was performed after permeabilization with PFA 8% (Sigma-Aldrich, Saint- Louis, Missouri, USA) and lysis perm buffer : Triton X100 10% (Euromedex, Souffelweyersheim, France). After 20 minutes of incubation, the cells were washed twice with PBS buffer (Gibco Life technologies, Paisley, UK). Conjugated fluorochromes: phycoerythrin (PE), allophycocyanin (APC), allophycocyanin-H7 (APC-H7), phycoerythrin-cyanin-7 (PE-Cy7)

Antigen Conjugated fluorochrome Clone Firm Purpose CD45 Horizon V500 HI30 BD Biosciences immunophenotype CD34 Horizon V450 8G12 BD Biosciences immunophenotype CD117 APC 104D2 BD Pharmingen immunophenotype HLA-DR FITC G46-6 BD Pharmingen immunophenotype TdT FITC HT-6 Dako immunophenotype ILT7 APC 17G10.2 Affymetrix eBiosciences immunophenotype CD11c PerCP-Cy5.5 B-ly6 BD Pharmingen immunophenotype CD1c PE L161 Biolegend, Ozyme immunophenotype CD141 APC AD5-14H12 Miltenyi Biotec immunophenotype cMPO FITC MPO-7 Dako immunophenotype CD13 PE L138 BD Pharmingen immunophenotype CD33 PerCP-Cy5.5 P67.6 BD Pharmingen immunophenotype CD14 APC-H7 MφP9 BD Pharmingen immunophenotype CD64 FITC 10.1 BD Pharmingen immunophenotype CD15 FITC HI98 BD Pharmingen immunophenotype CD22 PerCP-Cy5.5 HIB22 BD Pharmingen immunophenotype CD7 Horizon V450 M-T701 BD Pharmingen immunophenotype CD5 FITC BL1a Beckman Coulter immunophenotype CD56 Horizon V450 B156 BD Biosciences immunophenotype CD4 APC-H7 SK3 BD Biosciences immunophenotype CD123 PE-Cy7 6H6 Biolegend, Ozyme immunophenotype CD303 APC AC144 Miltenyi Biotec immunophenotype CD304 PE AD5-17F6 Miltenyi Biotec immunophenotype cTCL1 PE eBio1-21 Affymetrix eBiosciences immunophenotype CD45 BV421 HI30 BD Pharmingen cell sorting CD45 Horizon V500 HI30 BD Pharmingen cell sorting CD3 Horizon V500 SK7 BD Pharmingen cell sorting CD34 APC 8G12 BD Pharmingen cell sorting CD123 PE-Cy7 6H6 Biolegend, Ozyme cell sorting CD303 FITC 201A Biolegend, Ozyme cell sorting CD304 FITC 12C2 Biolegend, Ozyme cell sorting CD1c PE L161 Biolegend, Ozyme cell sorting CD14 APC-H7 MφP9 BD Pharmingen cell sorting CD64 FITC 10.1 BD Pharmingen cell sorting

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SUPPLEMENTAL DOCUMENTS

Table S2: Amplicons included in the HaloPlexHS Target Enrichment System design for Illumina Sequencing

Target ID (gene name_exon or region) Chromosomic coordinates Size (bp) Coverage (%) APC_P_ALA1582PRO chr5:112175996-112176075 80 100 ASXL1_EX12 chr20:31022235-31027122 4888 99.71 ATM_EX1 chr11:108093559-108093913 355 100 ATM_EX10 chr11:108121428-108121799 372 100 ATM_EX11 chr11:108122564-108122758 195 100 ATM_EX12 chr11:108123544-108123639 96 90.62 ATM_EX13 chr11:108124541-108124766 226 100 ATM_EX14 chr11:108126942-108127067 126 100 ATM_EX15 chr11:108128208-108128333 126 100 ATM_EX16 chr11:108129713-108129802 90 100 ATM_EX17 chr11:108137898-108138069 172 100 ATM_EX18 chr11:108139137-108139336 200 100 ATM_EX19 chr11:108141791-108141873 83 100 ATM_EX2 chr11:108098322-108098423 102 100 ATM_EX20 chr11:108141978-108142133 156 100 ATM_EX21 chr11:108143259-108143334 76 100 ATM_EX22 chr11:108143449-108143579 131 100 ATM_EX23 chr11:108150218-108150335 118 100 ATM_EX24 chr11:108151722-108151895 174 100 ATM_EX25 chr11:108153437-108153606 170 100 ATM_EX26 chr11:108154954-108155200 247 100 ATM_EX27 chr11:108158327-108158442 116 100 ATM_EX28 chr11:108159704-108159830 127 56.69 ATM_EX29 chr11:108160329-108160528 200 100 ATM_EX3 chr11:108098503-108098615 113 100 ATM_EX30 chr11:108163346-108163520 175 100 ATM_EX31 chr11:108164040-108164204 165 100 ATM_EX32 chr11:108165654-108165786 133 100 ATM_EX33 chr11:108168014-108168109 96 89.58 ATM_EX34 chr11:108170441-108170612 172 100 ATM_EX35 chr11:108172375-108172516 142 100 ATM_EX36 chr11:108173580-108173756 177 100 ATM_EX37 chr11:108175402-108175579 178 100 ATM_EX38 chr11:108178624-108178711 88 100 ATM_EX39 chr11:108180887-108181042 156 100 ATM_EX4 chr11:108099905-108100050 146 100 ATM_EX40 chr11:108183138-108183225 88 100 ATM_EX41 chr11:108186550-108186638 89 100 ATM_EX42 chr11:108186738-108186840 103 100 ATM_EX43 chr11:108188100-108188248 149 100 ATM_EX44 chr11:108190681-108190785 105 93.33 ATM_EX45 chr11:108192028-108192147 120 100 ATM_EX46 chr11:108196037-108196271 235 100 ATM_EX47 chr11:108196785-108196952 168 100 ATM_EX48 chr11:108198372-108198485 114 100 ATM_EX49 chr11:108199748-108199965 218 100 ATM_EX5 chr11:108106397-108106561 165 100 ATM_EX50 chr11:108200941-108201148 208 100 ATM_EX51 chr11:108202171-108202284 114 100 ATM_EX52 chr11:108202606-108202764 159 100 ATM_EX53 chr11:108203489-108203627 139 100 ATM_EX54 chr11:108204613-108204695 83 100 ATM_EX55 chr11:108205696-108205836 141 100 ATM_EX56 chr11:108206572-108206688 117 100 ATM_EX57 chr11:108213949-108214098 150 100 ATM_EX58 chr11:108216470-108216635 166 100 ATM_EX59 chr11:108218006-108218092 87 0 ATM_EX6 chr11:108114680-108114845 166 100 ATM_EX60 chr11:108224493-108224607 115 100 ATM_EX61 chr11:108225538-108225601 64 100 4

SUPPLEMENTAL DOCUMENTS

ATM_EX62 chr11:108235809-108235945 137 100 ATM_EX63 chr11:108236052-108239826 3775 90.07 ATM_EX7 chr11:108115515-108115753 239 100 ATM_EX8 chr11:108117691-108117854 164 100 ATM_EX9 chr11:108119660-108119829 170 100 BCL6_AMPL1 chr3:187440236-187440399 164 100 BCL6_AMPL2 chr3:187442719-187442876 158 100 BCL6_AMPL3 chr3:187443277-187443427 151 100 BCL6_AMPL4 chr3:187444509-187444696 188 100 BCL6_AMPL5 chr3:187446138-187446342 205 100 BCL6_AMPL6 chr3:187446828-187447819 992 100 BCL6_AMPL7 chr3:187449487-187449728 242 100 BCL6_AMPL8 chr3:187451311-187451491 181 100 BCOR_P_G1529EFSX4 chrx:39914632-39914711 80 100 BIRC3_EX6 chr11:102201730-102201972 243 100 BIRC3_EX7 chr11:102206697-102206951 255 100 BIRC3_EX8 chr11:102207491-102207532 42 100 BIRC3_EX9 chr11:102207640-102210135 2496 99.6 BRAF_EX15 chr7:140453075-140453193 119 100 BTK_AMPL1 chrx:100604863-100604954 92 100 BTK_AMPL10 chrx:100613595-100613694 100 100 BTK_AMPL11 chrx:100614271-100614345 75 100 BTK_AMPL12 chrx:100615066-100615148 83 100 BTK_AMPL13 chrx:100615546-100615753 208 100 BTK_AMPL14 chrx:100617151-100617238 88 100 BTK_AMPL15 chrx:100617539-100617687 149 100 BTK_AMPL16 chrx:100624976-100625077 102 100 BTK_AMPL17 chrx:100626611-100626699 89 100 BTK_AMPL18 chrx:100629514-100629632 119 100 BTK_AMPL19 chrx:100630122-100630282 161 100 BTK_AMPL2 chrx:100608172-100608349 178 100 BTK_AMPL3 chrx:100608848-100608986 139 100 BTK_AMPL4 chrx:100609608-100609692 85 100 BTK_AMPL5 chrx:100611030-100611266 237 100 BTK_AMPL6 chrx:100611762-100611953 192 100 BTK_AMPL7 chrx:100612487-100612581 95 100 BTK_AMPL8 chrx:100613137-100613158 22 100 BTK_AMPL9 chrx:100613284-100613435 152 100 CALR_AMPL1 chr19:13049484-13049594 111 100 CALR_AMPL2 chr19:13049938-13050059 122 100 CALR_AMPL3 chr19:13050232-13050455 224 100 CALR_AMPL4 chr19:13050857-13050971 115 100 CALR_AMPL5 chr19:13051047-13051276 230 100 CALR_AMPL6 chr19:13051345-13051478 134 100 CALR_AMPL7 chr19:13051548-13051711 164 100 CALR_AMPL8 chr19:13054341-13054453 113 100 CALR_AMPL9 chr19:13054517-13054737 221 100 CARD11_EX4 chr7:2985453-2985590 138 100 CARD11_EX5 chr7:2983846-2984171 326 100 CARD11_EX6 chr7:2979383-2979562 180 100 CARD11_EX7 chr7:2978313-2978465 153 100 CARD11_EX8 chr7:2977541-2977666 126 100 CARD11_EX9 chr7:2976671-2976868 198 100 CBL_EX8 chr11:119148876-119149007 132 100 CBL_EX9 chr11:119149220-119149423 204 100 CD79A_EX5 chr19:42384934-42385439 506 100 CD79B_EX5 chr17:62006794-62006835 42 100 CDKN2A_EXON1 chr9:21974647-21975127 481 100 CDKN2A_EXON2 chr9:21970871-21971237 367 100 CDKN2A_EXON3 chr9:21967722-21968271 550 100 CEBPA_EX1 chr19:33790840-33793470 2631 100 CLLU1_EX1 chr12:92817735-92819710 1976 99.65 CLLU1_EX2 chr12:92822810-92824778 1969 100 CRIPAK_NM_175918_C_294 chr4:1388554-1388680 127 0 CRIPAK_NM_175918_C_445_-CA chr4:1388705-1388784 80 0 5

SUPPLEMENTAL DOCUMENTS

CRIPAK_NM_175918_P_X22 chr4:1388325-1388444 120 0 CSF3R_AMPL1 chr1:36931687-36931811 125 100 CSF3R_AMPL10 chr1:36937657-36937750 94 100 CSF3R_AMPL11 chr1:36937829-36938002 174 100 CSF3R_AMPL12 chr1:36938108-36938297 190 100 CSF3R_AMPL13 chr1:36939026-36939233 208 100 CSF3R_AMPL14 chr1:36939355-36939498 144 100 CSF3R_AMPL15 chr1:36940968-36941284 317 100 CSF3R_AMPL16 chr1:36945024-36945107 84 100 CSF3R_AMPL2 chr1:36931948-36932519 572 100 CSF3R_AMPL3 chr1:36932821-36932922 102 100 CSF3R_AMPL4 chr1:36933149-36933262 114 100 CSF3R_AMPL5 chr1:36933413-36933573 161 100 CSF3R_AMPL6 chr1:36933666-36933832 167 100 CSF3R_AMPL7 chr1:36934747-36934868 122 100 CSF3R_AMPL8 chr1:36935243-36935451 209 100 CSF3R_AMPL9 chr1:36937024-36937257 234 100 CXCR4_EX2 chr2:136871919-136873482 1564 100 DNMT3A_EX1 chr2:25565299-25565459 161 100 DNMT3A_EX10 chr2:25469489-25469645 157 100 DNMT3A_EX11 chr2:25469029-25469178 150 100 DNMT3A_EX12 chr2:25468889-25468933 45 100 DNMT3A_EX13 chr2:25468122-25468201 80 100 DNMT3A_EX14 chr2:25467409-25467521 113 100 DNMT3A_EX15 chr2:25467024-25467207 184 100 DNMT3A_EX16 chr2:25466767-25466851 85 100 DNMT3A_EX17 chr2:25464431-25464576 146 100 DNMT3A_EX18 chr2:25463509-25463599 91 100 DNMT3A_EX19 chr2:25463171-25463319 149 100 DNMT3A_EX2 chr2:25536782-25537030 249 100 DNMT3A_EX20 chr2:25461999-25462084 86 100 DNMT3A_EX21 chr2:25459805-25459874 70 100 DNMT3A_EX22 chr2:25458576-25458694 119 84.87 DNMT3A_EX23 chr2:25455830-25457289 1460 88.36 DNMT3A_EX3 chr2:25523008-25523112 105 100 DNMT3A_EX4 chr2:25505310-25505580 271 100 DNMT3A_EX5 chr2:25498369-25498412 44 100 DNMT3A_EX6 chr2:25497810-25497956 147 100 DNMT3A_EX7 chr2:25470906-25471121 216 100 DNMT3A_EX8 chr2:25470460-25470618 159 100 DNMT3A_EX9 chr2:25469920-25470027 108 100 ETV6_EX1 chr12:11802788-11803094 307 100 ETV6_EX2 chr12:11905384-11905513 130 100 ETV6_EX3 chr12:11992074-11992238 165 100 ETV6_EX4 chr12:12006361-12006495 135 100 ETV6_EX5 chr12:12022358-12022903 546 100 ETV6_EX6 chr12:12037379-12037521 143 100 ETV6_EX7 chr12:12038860-12038960 101 100 ETV6_EX8 chr12:12043875-12048325 4451 100 EZH2_EX1 chr7:148581256-148581441 186 100 EZH2_EX10 chr7:148514969-148515209 241 100 EZH2_EX11 chr7:148514314-148514483 170 100 EZH2_EX12 chr7:148513776-148513870 95 100 EZH2_EX13 chr7:148512598-148512638 41 63.41 EZH2_EX14 chr7:148512006-148512131 126 100 EZH2_EX15 chr7:148511051-148511229 179 100 EZH2_EX16 chr7:148508717-148508812 96 100 EZH2_EX17 chr7:148507425-148507506 82 100 EZH2_EX18 chr7:148506402-148506482 81 100 EZH2_EX19 chr7:148506163-148506247 85 100 EZH2_EX2 chr7:148544274-148544397 124 100 EZH2_EX20 chr7:148504464-148504798 335 100 EZH2_EX3 chr7:148543562-148543690 129 100 EZH2_EX4 chr7:148529726-148529842 117 100 EZH2_EX5 chr7:148526820-148526940 121 92.56 6

SUPPLEMENTAL DOCUMENTS

EZH2_EX6 chr7:148525832-148525972 141 100 EZH2_EX7 chr7:148524256-148524358 103 22.33 EZH2_EX8 chr7:148523546-148523724 179 97.21 EZH2_EX9 chr7:148516688-148516779 92 100 FBXW7_EX10 chr4:153247158-153247383 226 100 FBXW7_EX11 chr4:153245336-153245546 211 100 FBXW7_EX9 chr4:153249360-153249541 182 100 FLT3_AMPL1 chr13:28578179-28578321 143 100 FLT3_AMPL10 chr13:28602305-28602435 131 100 FLT3_AMPL11 chr13:28608014-28608138 125 100 FLT3_AMPL12 chr13:28608209-28608361 153 100 FLT3_AMPL13 chr13:28608428-28608554 127 100 FLT3_AMPL14 chr13:28609622-28609820 199 100 FLT3_AMPL15 chr13:28610062-28610190 129 100 FLT3_AMPL16 chr13:28611312-28611435 124 100 FLT3_AMPL17 chr13:28622402-28622590 189 100 FLT3_AMPL18 chr13:28623511-28623684 174 100 FLT3_AMPL19 chr13:28623762-28623921 160 100 FLT3_AMPL2 chr13:28588579-28588704 126 100 FLT3_AMPL20 chr13:28624222-28624369 148 100 FLT3_AMPL21 chr13:28626672-28626821 150 100 FLT3_AMPL22 chr13:28631474-28631609 136 100 FLT3_AMPL23 chr13:28635994-28636216 223 100 FLT3_AMPL24 chr13:28644618-28644759 142 100 FLT3_AMPL25 chr13:28674595-28674657 63 100 FLT3_AMPL3 chr13:28589284-28589403 120 100 FLT3_AMPL4 chr13:28589717-28589848 132 100 FLT3_AMPL5 chr13:28592585-28592736 152 100 FLT3_AMPL6 chr13:28597477-28597624 148 100 FLT3_AMPL7 chr13:28598988-28599090 103 100 FLT3_AMPL8 chr13:28599530-28599562 33 100 FLT3_AMPL9 chr13:28601215-28601388 174 100 GNB1_NM_002074_P_LYS57GLU chr1:1747190-1747269 80 100 GNB1_NM_002074_P_LYS89GLU chr1:1737877-1737956 80 98.75 HNF1A_NM_000545_ chr12:121432078-121432157 80 100 HNF1A_NM_000545_C_861_-G_P_X289X chr12:121432075-121432157 83 100 HOXB9_P_ARG183HIS chr17:46700428-46700507 80 100 IDH1_EX4 chr2:209113093-209113384 292 100 IDH2_EX4 chr15:90631819-90631979 161 100 IKZF1_AMPL1 chr7:50358648-50358707 60 100 IKZF1_AMPL2 chr7:50367224-50367363 140 100 IKZF1_AMPL3 chr7:50435694-50436078 385 100 IKZF1_AMPL4 chr7:50444221-50444501 281 100 IKZF1_AMPL5_P_H163Y_P_ILE164SERFS29 chr7:50450228-50450415 188 100 IKZF1_AMPL6_P_E221SFSX36 chr7:50455033-50455178 146 100 IKZF1_AMPL7 chr7:50459417-50459571 155 100 IKZF1_AMPL8_P_R502W chr7:50467606-50468335 730 100 IKZF2_P_ALA91VAL chr2:213921652-213921731 80 100 IKZF3_P_GLU318LYS chr17:37922582-37922661 80 100 IKZF3_P_THR326HISFS25 chr17:37922559-37922638 80 100 IRF8_EX1 chr16:85932774-85932829 56 100 IRF8_EX2 chr16:85936621-85936795 175 100 IRF8_EX3 chr16:85942596-85942779 184 100 IRF8_EX4 chr16:85945176-85945264 89 100 IRF8_EX5 chr16:85946737-85946842 106 100 IRF8_EX6 chr16:85948079-85948126 48 100 IRF8_EX7 chr16:85952023-85952409 387 100 IRF8_EX8 chr16:85953715-85953830 116 100 IRF8_EX9 chr16:85954712-85956211 1500 94.73 JAK2_EX12 chr9:5069925-5070052 128 100 JAK2_EX14 chr9:5073698-5073785 88 100 KIT_EX10 chr4:55593384-55593490 107 100 KIT_EX11 chr4:55593582-55593708 127 100 KIT_EX17 chr4:55599236-55599358 123 91.87 KIT_EX9 chr4:55592023-55592216 194 100 7

SUPPLEMENTAL DOCUMENTS

KRAS_EX2 chr12:25398208-25398329 122 100 KRAS_EX3 chr12:25380168-25380346 179 100 MAP2K1_EXON2 chr15:66727335-66727605 271 96.31 MAP2K1_EXON3 chr15:66729054-66729260 207 100 MET_P_ASN375SER chr7:116340223-116340302 80 100 MET_P_GLU168ASP chr7:116339603-116339682 80 100 MLH1_P_ARG385CYS chr3:37067203-37067282 80 100 MPL_EX10 chr1:43814934-43815030 97 100 MYD88_EX3 chr3:38181879-38182083 205 100 MYD88_EX4 chr3:38182248-38182339 92 100 MYD88_EX5 chr3:38182623-38184512 1890 100 NEFH_NM_021076_P_X657LYS chr22:29885559-29885638 80 65 NOTCH1_EX26 chr9:139399125-139399556 432 100 NOTCH1_EX27 chr9:139397634-139397782 149 100 NOTCH1_EX28 chr9:139396724-139396940 217 100 NOTCH1_EX34 chr9:139388885-139392010 3126 97.7 NOTCH2_EX26 chr1:120466260-120466607 348 100 NOTCH2_EX27 chr1:120465259-120465401 143 100 NOTCH2_EX28 chr1:120464859-120465069 211 100 NOTCH2_EX34 chr1:120454176-120459317 5142 99.75 NRAS_EX2 chr1:115258671-115258798 128 100 NRAS_EX3 chr1:115256421-115256599 179 100 PAX3_NM_181459_P_GLU28 chr2:223163214-223163293 80 100 PAX3_NM_181459_P_THR227ILE chr2:223096870-223096949 80 100 PHF6_P_A140T chrx:133527943-133528022 80 100 PTEN_EX5 chr10:89692770-89693008 239 100 PTEN_EX7 chr10:89717610-89717776 167 100 PTPN11_EX13 chr12:112926828-112926979 152 100 PTPN11_EX3 chr12:112888122-112888316 195 100 RB1_P_ARG358 chr13:48942646-48942725 80 100 RB1_P_ASP332GLY chr13:48941646-48941725 80 100 RET_P_SER649LEU chr10:43609955-43610034 80 100 RUNX1_EX3 chr21:36265222-36265260 39 100 RUNX1_EX4 chr21:36259140-36259393 254 100 RUNX1_EX5 chr21:36252854-36253010 157 100 RUNX1_EX6 chr21:36231771-36231875 105 100 RUNX1_EX7 chr21:36206707-36206898 192 100 RUNX1_EX8 chr21:36171598-36171759 162 100 RUNX1_EX9 chr21:36160098-36164907 4810 98.59 SETBP1_AMPL1 chr18:42281302-42281807 506 100 SETBP1_AMPL2 chr18:42449185-42449258 74 100 SETBP1_AMPL3 chr18:42456520-42456728 209 100 SETBP1_AMPL4 chr18:42529836-42533315 3480 100 SETBP1_AMPL5 chr18:42618440-42618630 191 100 SETBP1_AMPL6 chr18:42643034-42643673 640 100 SF3A2_NM_007165_P_X329X chr19:2248095-2248174 80 100 SF3B1_EX13 chr2:198267673-198267759 87 100 SF3B1_EX14 chr2:198267280-198267550 271 100 SF3B1_EX15 chr2:198266709-198266854 146 100 SF3B1_EX16 chr2:198266466-198266612 147 100 SF3B1_EX17 chr2:198266124-198266249 126 100 SF3B1_EX18 chr2:198265439-198265660 222 100 SF3B4_NM_005850_P_X383X chr1:149895522-149895601 80 100 SHOC2_P_S2G chr10:112724081-112724160 80 100 SRSF2_EX1 chr17:74732881-74733493 613 100 SSC5D_NM_001144950_P_PRO1247X chr19:56029344-56029423 80 100 SSC5D_NM_001144950_P_X1146X chr19:56029041-56029120 80 100 STAT3_EX19 chr17:40475278-40475372 95 100 STAT3_EX20 chr17:40475022-40475161 140 100 STAT3_EX21 chr17:40474303-40474512 210 100 STAT3_EX22 chr17:40469200-40469242 43 100 STAT3_EX23 chr17:40468807-40468919 113 100 STAT3_EX24 chr17:40465343-40467818 2476 95.84 TCL1A_EX1 chr14:96180284-96180533 250 100 TCL1A_EX2 chr14:96178557-96178733 177 100 8

SUPPLEMENTAL DOCUMENTS

TCL1A_EX3 chr14:96178086-96178139 54 100 TCL1A_EX4 chr14:96176304-96177218 915 100 TET2_EX1 chr4:106067842-106068136 295 100 TET2_EX10 chr4:106193721-106194075 355 100 TET2_EX11 chr4:106196205-106200960 4756 99.81 TET2_EX2 chr4:106111517-106111662 146 100 TET2_EX3 chr4:106155054-106158508 3455 98.9 TET2_EX4 chr4:106162496-106162586 91 100 TET2_EX5 chr4:106163991-106164084 94 100 TET2_EX6 chr4:106164727-106164935 209 100 TET2_EX7 chr4:106180776-106180926 151 100 TET2_EX8 chr4:106182916-106183005 90 100 TET2_EX9 chr4:106190767-106190904 138 100 TNFAIP3_EX1 chr6:138188325-138188631 307 100 TNFAIP3_EX2 chr6:138192350-138192659 310 100 TNFAIP3_EX3 chr6:138195982-138196172 191 100 TNFAIP3_EX4 chr6:138196825-138196972 148 100 TNFAIP3_EX5 chr6:138197133-138197303 171 100 TNFAIP3_EX6 chr6:138198213-138198393 181 100 TNFAIP3_EX7 chr6:138199569-138200488 920 100 TNFAIP3_EX8 chr6:138201208-138201389 182 100 TNFAIP3_EX9 chr6:138202172-138204451 2280 98.16 TP53_EX10 chr17:7573927-7574033 107 100 TP53_EX11 chr17:7571720-7573008 1289 98.37 TP53_EX2 chr17:7579839-7579940 102 100 TP53_EX3 chr17:7579700-7579721 22 100 TP53_EX4 chr17:7579312-7579590 279 100 TP53_EX5 chr17:7578371-7578554 184 100 TP53_EX6 chr17:7578177-7578289 113 100 TP53_EX7 chr17:7577499-7577608 110 100 TP53_EX8 chr17:7577019-7577155 137 100 TP53_EX9 chr17:7576853-7576926 74 100 U2AF1_AMPL1 chr21:44513202-44513369 168 100 U2AF1_AMPL10 chr21:44524425-44524512 88 100 U2AF1_AMPL11 chr21:44527551-44527614 64 100 U2AF1_AMPL2 chr21:44514571-44514683 113 100 U2AF1_AMPL3 chr21:44514755-44514908 154 100 U2AF1_AMPL4 chr21:44514765-44514898 134 100 U2AF1_AMPL5 chr21:44515538-44515656 119 100 U2AF1_AMPL6 chr21:44515794-44515863 70 100 U2AF1_AMPL7 chr21:44520553-44520639 87 100 U2AF1_AMPL8 chr21:44521466-44521552 87 100 U2AF1_AMPL9 chr21:44524415-44524522 108 100 UBE2G2_P_ASP63VAL chr21:46197231-46197310 80 100 VHL_P_PRO81SER chr3:10183733-10183812 80 100 WT1_AMPL1 chr11:32410594-32410735 142 100 WT1_AMPL10 chr11:32452066-32452095 30 100 WT1_AMPL11 chr11:32456236-32456901 666 100 WT1_AMPL2 chr11:32413508-32413620 113 100 WT1_AMPL3 chr11:32414202-32414311 110 100 WT1_AMPL4 chr11:32417793-32417963 171 100 WT1_AMPL5 chr11:32421484-32421600 117 100 WT1_AMPL6 chr11:32438026-32438096 71 100 WT1_AMPL7 chr11:32439113-32439210 98 100 WT1_AMPL8 chr11:32449492-32449614 123 100 WT1_AMPL9 chr11:32450033-32450175 143 100 ZEB2_P_ASP268ARGFS12 chr2:145161450-145161529 80 100 ZEB2_P_GLY626ARG chr2:145156839-145156918 80 100 ZEB2_P_LEU420ARG chr2:145157456-145157535 80 100 ZRSR2_AMPL1 chrx:15808609-15808669 61 100 ZRSR2_AMPL10 chrx:15838320-15838449 130 100 ZRSR2_AMPL11 chrx:15840844-15841375 532 94.92 ZRSR2_AMPL2 chrx:15809047-15809146 100 100 ZRSR2_AMPL3 chrx:15817985-15818086 102 100 ZRSR2_AMPL4 chrx:15821801-15821929 129 100 9

SUPPLEMENTAL DOCUMENTS

ZRSR2_AMPL5 chrx:15822224-15822330 107 100 ZRSR2_AMPL6 chrx:15826346-15826404 59 100 ZRSR2_AMPL7 chrx:15827313-15827451 139 100 ZRSR2_AMPL8 chrx:15833790-15834023 234 100 ZRSR2_AMPL9 chrx:15836700-15836775 76 100

10

SUPPLEMENTAL DOCUMENTS

Table S3: Purity of each fraction. ND: not done because small amounts of cells, stored for NGS analysis only.

Patient fraction purity patient fraction purity Blasts 98.5 Blasts 98.6 pDCs 96.8 pDCs 96.1 N1 N20 Monocytes 98.9 Monocytes 92.7 T-cells 99.0 T-cells 96.2 Blasts 99.1 Blasts 99.8 pDCs 98.9 pDCs 99.0 N2 N34 Monocytes 99.8 Monocytes 99.4 T-cells 99.6 T-cells 99.0 Blasts 97.8 Blasts 95.8 pDCs 98.0 pDCs ND N7 N35 Monocytes 98.3 Monocytes 99.0 T-cells 99.0 T-cells 96.8 Blasts 98.6 Blasts 98.8 pDCs 99.1 pDCs 96.2 N36 N8 Monocytes 98.9 T-cells 99.4 T-cells 98.4 T-cells 99.4 cDCs 100.0 Blasts ND

pDCs ND N9 Monocytes ND T-cells ND Blasts 99.3 pDCs 94.9 N11 Monocytes 99.4 T-cells 99.3 Blasts 99.7 pDCs 93.7 N12 Monocytes 99.5 T-cells 99.3 Blasts 98.2 pDCs 84.1 N13 Monocytes 96.0 T-cells 99.2 Blasts 96.3 pDCs 93.3 N14 Monocytes 97.9 T-cells 97.3 Blasts 97.0 pDCs 90.3 N16 Monocytes 87.0 T-cells 99.0 Blasts 98.0 pDCs 94.5 N19 Monocytes 94.6 T-cells 98.9

11

SUPPLEMENTAL DOCUMENTS

Table S4: Mutations detected by NGS.

mutated reads mutated KL_coding

Gene Transcripts Exon coordinates cDNA coordinates protein coordinates Genomic Type Sample Score Quality Frequency of Number reads total of Number Class GenerateReports InterVar dbSNP ID COSMIC tissue COSMIC ClinVar PopFreqMax ExAC_ALL SIFT polyphen2_HDIV polyphen2_HVAR LRT MutationTaster MutationAssessor FATHMM PROVEAN VEST3 MetaSVM MetaLR MCAP CADD DANN fathmm_M GERP

ASXL1 NM_015338 12 c.A2123G- p.Q708R chr20:g.3102263 nonsynony N11-Bl 1061 38.3 203 530 Uncertain Uncertain - D B B N N M T N (s=- s=0.041;rank T (s=- T s=0.004;rank s=- s=0.961;rank N s=2.94;rank= 8A>G mous SNV significance significance (s=0.036;rank (s=0.006;rank (s=0.003;rank (s=0.391;rank (s=1;rank=0.0 (s=2.25;rank= (s=1.91;rank= 0.45;rank=0. =0.025 1.007;rank=0 (s=0.058;rank =0.096 0.308;rank=0 =0.28 (s=0.322;rank 0.33 =0.437) =0.126) =0.08) =0.133) 9) 0.64) 0.233) 148) .278) =0.242) .068 =0.243) ASXL1 NM_015338 12 c.A2123G- p.Q708R chr20:g.3102263 nonsynony N11- 1044.43 58.25 113 194 Uncertain Uncertain - D B B N N M T N (s=- s=0.041;rank T (s=- T s=0.004;rank s=- s=0.961;rank N s=2.94;rank= 8A>G mous SNV mono significance significance (s=0.036;rank (s=0.006;rank (s=0.003;rank (s=0.391;rank (s=1;rank=0.0 (s=2.25;rank= (s=1.91;rank= 0.45;rank=0. =0.025 1.007;rank=0 (s=0.058;rank =0.096 0.308;rank=0 =0.28 (s=0.322;rank 0.33 =0.437) =0.126) =0.08) =0.133) 9) 0.64) 0.233) 148) .278) =0.242) .068 =0.243) ASXL1 NM_015338 12 c.A2123G- p.Q708R chr20:g.3102263 nonsynony N11- 2223.51 54.88 658 1199 Uncertain Uncertain - D B B N N M T N (s=- s=0.041;rank T (s=- T s=0.004;rank s=- s=0.961;rank N s=2.94;rank= 8A>G mous SNV pDC significance significance (s=0.036;rank (s=0.006;rank (s=0.003;rank (s=0.391;rank (s=1;rank=0.0 (s=2.25;rank= (s=1.91;rank= 0.45;rank=0. =0.025 1.007;rank=0 (s=0.058;rank =0.096 0.308;rank=0 =0.28 (s=0.322;rank 0.33 =0.437) =0.126) =0.08) =0.133) 9) 0.64) 0.233) 148) .278) =0.242) .068 =0.243) ASXL1 NM_015338 12 c.G2362T- p.E788X chr20:g.3102287 stopgain N12-BL 2122.04 48.27 474 982 Likely Uncertain - N D s=13.144;ran s=0.997;rank D s=5.17;rank= 7G>T pathogenic significance (s=0.01;rank= (s=1;rank=0.8 k=0.989 =0.813 (s=0.961;rank 0.707 0.303) 1) =0.671) ASXL1 NM_015338 12 c.G2362T- p.E788X chr20:g.3102287 stopgain N12- 1351.34 46.92 183 390 Likely Uncertain - N D s=13.144;ran s=0.997;rank D s=5.17;rank= 7G>T MONO pathogenic significance (s=0.01;rank= (s=1;rank=0.8 k=0.989 =0.813 (s=0.961;rank 0.707 0.303) 1) =0.671) ASXL1 NM_015338 12 c.G2362T- p.E788X chr20:g.3102287 stopgain N12- 2567.24 49.28 785 1593 Likely Uncertain - N D s=13.144;ran s=0.997;rank D s=5.17;rank= 7G>T PDC pathogenic significance (s=0.01;rank= (s=1;rank=0.8 k=0.989 =0.813 (s=0.961;rank 0.707 0.303) 1) =0.671) ASXL1 NM_015338 12 c.2757dupA- p.P920Tfs* chr20:g.3102327 frameshift N9-Bl 762.45 46.36 121 261 Likely rs771822198 - 0 0 1->A insertion pathogenic ASXL1 NM_015338 12 c.2757dupA- p.P920Tfs* chr20:g.3102327 frameshift N9- 34 32 93 Likely rs771822198 - 0 0 1->A insertion Mono pathogenic ASXL1 NM_015338 12 c.2757dupA- p.P920Tfs* chr20:g.3102327 frameshift N9-pDC 299.57 26.32 40 152 Likely rs771822198 - 0 0 1->A insertion pathogenic CBL NM_005188 8 c.G1211A- p.C404Y chr11:g.1191489 nonsynony N13-Bl 214.63 48.3 398 824 Likely Uncertain rs192712314 COSM34068 8(haematopoietic_and_lymphoid_ - 0.0001 0 D D D D D H D (s=- D (s=- s=0.952;rank D D s=0.511;rank s=4.917;rank s=0.99;rank= D s=5.52;rank= 91G>A mous SNV pathogenic significance tissue) (s=0;rank=0.9 (s=1;rank=0.8 (s=1;rank=0.9 (s=0;rank=0.8 (s=1;rank=0.8 (s=4.25;rank= 4.23;rank=0. 10.42;rank=0 =0.949 (s=1.065;rank (s=0.974;rank =0.953 =0.661 0.489 (s=0.99;rank= 0.821 12) 99) 71) 43) 1) 0.981) 97) .99) =0.985) =0.992) 0.9) CBL NM_005188 8 c.G1211A- p.C404Y chr11:g.1191489 nonsynony N13- 192.75 44.09 496 1125 Likely Uncertain rs192712314 COSM34068 8(haematopoietic_and_lymphoid_ - 0.0001 0 D D D D D H D (s=- D (s=- s=0.952;rank D D s=0.511;rank s=4.917;rank s=0.99;rank= D s=5.52;rank= 91G>A mous SNV Mono pathogenic significance tissue) (s=0;rank=0.9 (s=1;rank=0.8 (s=1;rank=0.9 (s=0;rank=0.8 (s=1;rank=0.8 (s=4.25;rank= 4.23;rank=0. 10.42;rank=0 =0.949 (s=1.065;rank (s=0.974;rank =0.953 =0.661 0.489 (s=0.99;rank= 0.821 12) 99) 71) 43) 1) 0.981) 97) .99) =0.985) =0.992) 0.9) CBL NM_005188 8 c.G1211A- p.C404Y chr11:g.1191489 nonsynony N13- 220.87 49.19 427 868 Likely Uncertain rs192712314 COSM34068 8(haematopoietic_and_lymphoid_ - 0.0001 0 D D D D D H D (s=- D (s=- s=0.952;rank D D s=0.511;rank s=4.917;rank s=0.99;rank= D s=5.52;rank= 91G>A mous SNV pDC pathogenic significance tissue) (s=0;rank=0.9 (s=1;rank=0.8 (s=1;rank=0.9 (s=0;rank=0.8 (s=1;rank=0.8 (s=4.25;rank= 4.23;rank=0. 10.42;rank=0 =0.949 (s=1.065;rank (s=0.974;rank =0.953 =0.661 0.489 (s=0.99;rank= 0.821 12) 99) 71) 43) 1) 0.981) 97) .99) =0.985) =0.992) 0.9) CBL NM_005188 9 c.T1253G- p.F418C chr11:g.1191492 nonsynony N8-Bl 1030.8 62.69 84 134 Likely Uncertain - 45T>G mous SNV pathogenic significance CBL NM_005188 9 c.T1253G- p.F418C chr11:g.1191492 nonsynony N8-cDC 5000 94.85 387 408 Likely Uncertain - D D D D D M D (s=- D (s=- s=0.928;rank D D s=0.447;rank s=4.731;rank s=0.986;rank D s=5.96;rank= 45T>G mous SNV pathogenic significance (s=0;rank=0.9 (s=1;rank=0.8 (s=0.999;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.455;rank 3.84;rank=0. 6.31;rank=0. =0.93 (s=1.014;rank (s=0.901;rank =0.943 =0.634 =0.431 (s=0.991;rank 0.967 12) 99) =0.916) 43) 1) =0.715) 958) 908) =0.974) =0.967) =0.917) CBL NM_005188 9 c.T1253G- p.F418C chr11:g.1191492 nonsynony N8- 2654.79 90.11 246 273 Likely Uncertain - D D D D D M D (s=- D (s=- s=0.928;rank D D s=0.447;rank s=4.731;rank s=0.986;rank D s=5.96;rank= 45T>G mous SNV mono pathogenic significance (s=0;rank=0.9 (s=1;rank=0.8 (s=0.999;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.455;rank 3.84;rank=0. 6.31;rank=0. =0.93 (s=1.014;rank (s=0.901;rank =0.943 =0.634 =0.431 (s=0.991;rank 0.967 12) 99) =0.916) 43) 1) =0.715) 958) 908) =0.974) =0.967) =0.917) CBL NM_005188 9 c.T1253G- p.F418C chr11:g.1191492 nonsynony N8-pDC 5000 98.37 301 306 Likely Uncertain - D D D D D M D (s=- D (s=- s=0.928;rank D D s=0.447;rank s=4.731;rank s=0.986;rank D s=5.96;rank= 45T>G mous SNV pathogenic significance (s=0;rank=0.9 (s=1;rank=0.8 (s=0.999;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.455;rank 3.84;rank=0. 6.31;rank=0. =0.93 (s=1.014;rank (s=0.901;rank =0.943 =0.634 =0.431 (s=0.991;rank 0.967 12) 99) =0.916) 43) 1) =0.715) 958) 908) =0.974) =0.967) =0.917) DNMT3A NM_175629 23 c.C2644T- p.R882C chr2:g.25457243 nonsynony N19-BL 630.03 42.26 251 594 Likely Uncertain rs377577594 COSM1166704 161(haematopoietic_and_lymphoi - 0.0007 0.0004 D D D D D M D (s=- D (s=- s=0.936;rank D D s=0.634;rank s=7.199;rank s=0.999;rank D s=5.72;rank= G>A mous SNV pathogenic significance COSM53042 d_tissue) (s=0;rank=0.9 (s=1;rank=0.8 (s=0.936;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.595;rank 4.28;rank=0. 7.55;rank=0. =0.93 (s=1.093;rank (s=0.951;rank =0.969 =0.945 =0.995 (s=0.991;rank 0.893 12) 99) =0.654) 43) 1) =0.761) 971) 953) =0.994) =0.984) =0.917) DNMT3A NM_175629 23 c.C2644T p.R882C chr2:g.25457243 nonsynony N19- 640.96 48.69 167 343 Likely Uncertain rs377577594 COSM1166704 161(haematopoietic_and_lymphoi - 0.0007 0.0004 D D D D D M D (s=- D (s=- s=0.936;rank D D s=0.634;rank s=7.199;rank s=0.999;rank D s=5.72;rank= G>A mous SNV mono pathogenic significance COSM53042 d_tissue) (s=0;rank=0.9 (s=1;rank=0.8 (s=0.936;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.595;rank 4.28;rank=0. 7.55;rank=0. =0.93 (s=1.093;rank (s=0.951;rank =0.969 =0.945 =0.995 (s=0.991;rank 0.893 12) 99) =0.654) 43) 1) =0.761) 971) 953) =0.994) =0.984) =0.917) DNMT3A NM_022552 23 c.C2644T- p.R882C chr2:g.25457243 nonsynony N19- 765.05 54.92 201 366 Likely Uncertain rs377577594 COSM1166704 161(haematopoietic_and_lymphoi - 0.0007 0.0004 D D D D D M D (s=- D (s=- s=0.936;rank D D s=0.634;rank s=7.199;rank s=0.999;rank D s=5.72;rank= G>A mous SNV PDC pathogenic significance COSM53042 d_tissue) (s=0;rank=0.9 (s=1;rank=0.8 (s=0.936;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.595;rank 4.28;rank=0. 7.55;rank=0. =0.93 (s=1.093;rank (s=0.951;rank =0.969 =0.945 =0.995 (s=0.991;rank 0.893 12) 99) =0.654) 43) 1) =0.761) 971) 953) =0.994) =0.984) =0.917) DNMT3A NM_022552 23 c.C2644T- p.R882C chr2:g.25457243 nonsynony N2-Bl 747.75 48.77 278 570 Likely Uncertain rs377577594 COSM1166704 161(haematopoietic_and_lymphoi - 0.0007 0.0004 D D D D D M D (s=- D (s=- s=0.936;rank D D s=0.634;rank s=7.199;rank s=0.999;rank D s=5.72;rank= G>A mous SNV pathogenic significance COSM53042 d_tissue) (s=0;rank=0.9 (s=1;rank=0.8 (s=0.936;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.595;rank 4.28;rank=0. 7.55;rank=0. =0.93 (s=1.093;rank (s=0.951;rank =0.969 =0.945 =0.995 (s=0.991;rank 0.893 12) 99) =0.654) 43) 1) =0.761) 971) 953) =0.994) =0.984) =0.917) DNMT3A NM_175629 23 c.C2644T- p.R882C chr2:g.25457243 nonsynony N2- 851.01 48.67 514 1056 Likely Uncertain rs377577594 COSM1166704 161(haematopoietic_and_lymphoi - 0.0007 0.0004 D D D D D M D (s=- D (s=- s=0.936;rank D D s=0.634;rank s=7.199;rank s=0.999;rank D s=5.72;rank= G>A mous SNV Mono pathogenic significance COSM53042 d_tissue) (s=0;rank=0.9 (s=1;rank=0.8 (s=0.936;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.595;rank 4.28;rank=0. 7.55;rank=0. =0.93 (s=1.093;rank (s=0.951;rank =0.969 =0.945 =0.995 (s=0.991;rank 0.893 12) 99) =0.654) 43) 1) =0.761) 971) 953) =0.994) =0.984) =0.917) DNMT3A NM_175629 23 c.C2644T- p.R882C chr2:g.25457243 nonsynony N2-pDC 775.42 45.41 475 1046 Likely Uncertain rs377577594 COSM1166704 161(haematopoietic_and_lymphoi - 0.0007 0.0004 D D D D D M D (s=- D (s=- s=0.936;rank D D s=0.634;rank s=7.199;rank s=0.999;rank D s=5.72;rank= G>A mous SNV pathogenic significance COSM53042 d_tissue) (s=0;rank=0.9 (s=1;rank=0.8 (s=0.936;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.595;rank 4.28;rank=0. 7.55;rank=0. =0.93 (s=1.093;rank (s=0.951;rank =0.969 =0.945 =0.995 (s=0.991;rank 0.893 12) 99) =0.654) 43) 1) =0.761) 971) 953) =0.994) =0.984) =0.917) DNMT3A NM_022552 23 c.G2645A- p.R882H chr2:g.25457242 nonsynony N36-Bl 204.63 57.14 16 28 Likely Uncertain rs147001633 COSM52944 1(breast) - 0.0009 0.0005 D P B D D M D (s=- D (s=- s=0.811;rank D D s=0.312;rank s=7.071;rank s=0.999;rank D s=5.72;rank= C>T mous SNV pathogenic significance COSM442676 410(haematopoietic_and_lymphoi (s=0.002;rank (s=0.651;rank (s=0.043;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.25;rank= 4.28;rank=0. 4.72;rank=0. =0.817 (s=0.894;rank (s=0.865;rank =0.913 =0.94 =0.997 (s=0.983;rank 0.893 d_tissue) =0.721) =0.388) =0.229) 43) 1) 0.64) 971) 802) =0.956) =0.955) =0.815) DNMT3A NM_022552 23 c.G2645A- p.R882H chr2:g.25457242 nonsynony N36- 9 22 248 Likely Uncertain rs147001633 COSM52944 1(breast) - 0.0009 0.0005 D P B D D M D (s=- D (s=- s=0.811;rank D D s=0.312;rank s=7.071;rank s=0.999;rank D s=5.72;rank= C>T mous SNV mono pathogenic significance COSM442676 410(haematopoietic_and_lymphoi (s=0.002;rank (s=0.651;rank (s=0.043;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.25;rank= 4.28;rank=0. 4.72;rank=0. =0.817 (s=0.894;rank (s=0.865;rank =0.913 =0.94 =0.997 (s=0.983;rank 0.893 d_tissue) =0.721) =0.388) =0.229) 43) 1) 0.64) 971) 802) =0.956) =0.955) =0.815) DNMT3A NM_022552 23 c.G2645A- p.R882H chr2:g.25457242 nonsynony N36- 577.67 53.3 105 197 Likely Uncertain rs147001633 COSM52944 1(breast) - 0.0009 0.0005 D P B D D M D (s=- D (s=- s=0.811;rank D D s=0.312;rank s=7.071;rank s=0.999;rank D s=5.72;rank= C>T mous SNV pDC pathogenic significance COSM442676 410(haematopoietic_and_lymphoi (s=0.002;rank (s=0.651;rank (s=0.043;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.25;rank= 4.28;rank=0. 4.72;rank=0. =0.817 (s=0.894;rank (s=0.865;rank =0.913 =0.94 =0.997 (s=0.983;rank 0.893 d_tissue) =0.721) =0.388) =0.229) 43) 1) 0.64) 971) 802) =0.956) =0.955) =0.815) EZH2 NM_0012032 8 c.C889T- p.H297Y chr7:g.14852356 nonsynony N13-Bl 562.09 36.56 215 588 Likely Uncertain - D B B D D M D (s=- D (s=- s=0.798;rank D D s=0.452;rank s=4.525;rank s=0.996;rank D s=5.28;rank= 47 4G>A mous SNV pathogenic significance (s=0.009;rank (s=0.009;rank (s=0.012;rank (s=0;rank=0.6 (s=1;rank=0.8 (s=2.82;rank= 2.71;rank=0. 5.6;rank=0.8 =0.864 (s=0.943;rank (s=0.869;rank =0.944 =0.606 =0.769 (s=0.987;rank 0.74 =0.912) =0.899) =0.916) 29) 1) 0.824) 906) 77) =0.963) =0.956) =0.862) EZH2 NM_0012032 4 c.C280T- p.Q94X chr7:g.14852980 stopgain N13-Bl 1967.64 55.65 754 1355 Likely Likely - D A s=12.367;ran s=0.998;rank D s=5.54;rank= 47 9G>A pathogenic pathogenic (s=0;rank=0.8 (s=1;rank=0.8 k=0.983 =0.912 (s=0.993;rank 0.828 43) 1) =0.938) EZH2 NM_0012032 8 c.C889T- p.H297Y chr7:g.14852356 nonsynony N13- 11.69 70 599 Likely Uncertain - D B B D D M D (s=- D (s=- s=0.798;rank D D s=0.452;rank s=4.525;rank s=0.996;rank D s=5.28;rank= 47 4G>A mous SNV Mono pathogenic significance (s=0.009;rank (s=0.009;rank (s=0.012;rank (s=0;rank=0.6 (s=1;rank=0.8 (s=2.82;rank= 2.71;rank=0. 5.6;rank=0.8 =0.864 (s=0.943;rank (s=0.869;rank =0.944 =0.606 =0.769 (s=0.987;rank 0.74 12

SUPPLEMENTAL DOCUMENTS

=0.912) =0.899) =0.916) 29) 1) 0.824) 906) 77) =0.963) =0.956) =0.862) EZH2 NM_0012032 4 c.C280T- p.Q94X chr7:g.14852980 stopgain N13- 9.99 181 1812 Likely Likely - D A s=12.367;ran s=0.998;rank D s=5.54;rank= 47 9G>A Mono pathogenic pathogenic (s=0;rank=0.8 (s=1;rank=0.8 k=0.983 =0.912 (s=0.993;rank 0.828 43) 1) =0.938) EZH2 NM_0012032 8 c.C889T- p.H297Y chr7:g.14852356 nonsynony N13- 484.22 35.34 141 399 Likely Uncertain - D B B D D M D (s=- D (s=- s=0.798;rank D D s=0.452;rank s=4.525;rank s=0.996;rank D s=5.28;rank= 47 4G>A mous SNV pDC pathogenic significance (s=0.009;rank (s=0.009;rank (s=0.012;rank (s=0;rank=0.6 (s=1;rank=0.8 (s=2.82;rank= 2.71;rank=0. 5.6;rank=0.8 =0.864 (s=0.943;rank (s=0.869;rank =0.944 =0.606 =0.769 (s=0.987;rank 0.74 =0.912) =0.899) =0.916) 29) 1) 0.824) 906) 77) =0.963) =0.956) =0.862) EZH2 NM_0012032 4 c.C280T- p.Q94X chr7:g.14852980 stopgain N13- 1681.41 53.25 508 954 Likely Likely - D A s=12.367;ran s=0.998;rank D s=5.54;rank= 47 9G>A pDC pathogenic pathogenic (s=0;rank=0.8 (s=1;rank=0.8 k=0.983 =0.912 (s=0.993;rank 0.828 43) 1) =0.938) EZH2 NM_004456 16 c.C1938A- p.Y646X chr7:g.14850872 stopgain N14-Bl 5000 100 1084 1084 Likely Likely - D A s=15.021;ran s=0.996;rank D s=5.63;rank= 6G>T pathogenic pathogenic (s=0;rank=0.8 (s=1;rank=0.8 k=0.998 =0.717 (s=0.99;rank= 0.861 43) 1) 0.894) EZH2 NM_004456 16 c.C1938A- p.Y646X chr7:g.14850872 stopgain N14- 5000 100 428 428 Likely Likely - D A s=15.021;ran s=0.996;rank D s=5.63;rank= 6G>T pDC pathogenic pathogenic (s=0;rank=0.8 (s=1;rank=0.8 k=0.998 =0.717 (s=0.99;rank= 0.861 43) 1) 0.894) EZH2 NM_004456 16 c.C1938A- p.Y646X chr7:g.14850872 stopgain N14- 5000 97.3 469 482 Likely Likely - D A s=15.021;ran s=0.996;rank D s=5.63;rank= 6G>T MONO pathogenic pathogenic (s=0;rank=0.8 (s=1;rank=0.8 k=0.998 =0.717 (s=0.99;rank= 0.861 43) 1) 0.894) EZH2 NM_0012032 2 c.C73T- p.R25X chr7:g.14854431 stopgain N34-Bl 794.95 54.49 485 890 Likely Likely COSM53003 1(haematopoietic_and_lymphoid_ - D A s=9.734;rank s=0.997;rank D s=2.63;rank= 47 8G>A pathogenic pathogenic tissue) (s=0;rank=0.8 (s=1;rank=0.8 =0.964 =0.839 (s=0.802;rank 0.303 43) 1) =0.396) EZH2 NM_0012032 2 c.C73T- p.R25X chr7:g.14854431 stopgain N34- 1027.85 70.93 344 485 Likely Likely COSM53003 1(haematopoietic_and_lymphoid_ - D A 47 8G>A mono pathogenic pathogenic tissue) (s=0;rank=0.8 (s=1;rank=0.8 43) 1) EZH2 NM_004456 2 c.C73T- p.R25X chr7:g.14854431 stopgain N34- 686.64 49.69 396 797 Likely Likely COSM53003 1(haematopoietic_and_lymphoid_ - D A s=9.734;rank s=0.997;rank D s=2.63;rank= 8G>A pDC pathogenic pathogenic tissue) (s=0;rank=0.8 (s=1;rank=0.8 =0.964 =0.839 (s=0.802;rank 0.303 43) 1) =0.396) FLT3 NM_004119 14 c.1793_1794ins p.E598delins N20-Bl 10 GGGGAAATCATA EGKSYNE TAATGA- FLT3 NM_004119 14 c.1793_1794ins p.E598delins N20- 8 GGGGAAATCATA EGKSYNE Mono TAATGA- FLT3 NM_004119 14 c.1793_1794ins p.E598delins chr13:g.2860826 nonframeshi N20- 630.11 23.50 196 834 Uncertain - GGGGAAATCATA EGKSYNE 2- ft insertion pDC (14 significance TAATGA- >TCATTATATGAT fragme TTCCCC nt analysis ) FLT3 NM_004119 14 c.1780_1781insC p.F594delins chr13:g.2860827 nonframeshi N2-Bl 1705.2 46 932 2026 Uncertain - CCCGAATGAGTA SPNEYFYVDF 5- ft insertion significance CTTCTACGTTGA >AATCAACGTAG TT- AAGTACTCATTC GGGG FLT3 NM_004119 14 c.1780_1781insC p.F594delins chr13:g.2860827 nonframeshi N2- 1640.2 42.66 1236 2897 Uncertain - CCCGAATGAGTA SPNEYFYVDF 5- ft insertion Mono significance CTTCTACGTTGA >AATCAACGTAG TT- AAGTACTCATTC GGGG FLT3 NM_004119 14 c.1780_1781insC p.F594delins chr13:g.2860827 nonframeshi N2-pDC 1642.34 42.24 1360 3220 Uncertain - CCCGAATGAGTA SPNEYFYVDF 5- ft insertion significance CTTCTACGTTGA >AATCAACGTAG TT- AAGTACTCATTC GGGG FLT3 NM_004119 20 c.2511_2511del p.I836del chr13:g.2859263 frameshift N36-Bl 669.76 38.71 48 124 Likely - CAT- 4C>ATG substitution pathogenic FLT3 NM_004119 20 c.2511_2511del p.I836del chr13:g.2859263 frameshift N36- 13 180 1358 Likely - CAT- 4C>ATG substitution Mono pathogenic FLT3 NM_004119 20 c.2511_2511del p.I836del chr13:g.2859263 frameshift N36- 2403.6 38.84 416 1071 Likely - CAT- 4C>ATG substitution pDC pathogenic KRAS NM_004985 2 c.G34T- p.G12C chr12:g.2539828 nonsynony N19-Bl 3 16 1017 Likely Likely rs121913530 COSM1140136 15(stomach) Pathogenic - RCV000013406.4 0.0001 0 D D D D D M T (s=- D (s=- s=0.905;rank D D s=0.147;rank s=7.016;rank s=0.997;rank D s=5.68;rank= 5C>A mous SNV pathogenic pathogenic COSM516 1(central_nervous_system) Pathogenic - RCV000038265.2 not (s=0.006;rank (s=1;rank=0.8 (s=0.993;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.56;rank= 1.24;rank=0. 6.99;rank=0. =0.895 (s=0.548;rank (s=0.701;rank =0.83 =0.938 =0.782 (s=0.984;rank 0.88 8(upper_aerodigestive_tract) provided,Pathogenic - =0.614) 99) =0.797) 43) 1) 0.75) 79) 972) =0.912) =0.897) =0.819) 2(oesophagus) 3(peritoneum) RCV000119791.1,RCV000013407.4 1776(lung) 18(thyroid) Pathogenic - RCV000013408.4 17(haematopoietic_and_lymphoid Pathogenic,Pathogenic - _tissue) 2(testis) 2(cervix) RCV000154401.2,RCV000013414.5 4(gastrointestinal_tract_(site_ind Pathogenic - RCV000038264.2 not eterminate)) 13(soft_tissue) provided - RCV000119790.1 34(ovary) 38(endometrium) Pathogenic - RCV000144971.3 5(skin) 11(breast) 8(small_intestine) 1(kidney) 130(pancreas) 4(liver) 15(urinary_tract) 1(adrenal_gland) 1476(large_intestine) 51(biliary_tract) 10(prostate) KRAS NM_004985 2 c.G34T- p.G12C chr12:g.2539828 nonsynony N19- 353.57 20.24 135 667 Likely Likely rs121913530 COSM1140136 15(stomach) Pathogenic - RCV000013406.4 0.0001 0 D D D D D M T (s=- D (s=- s=0.905;rank D D s=0.147;rank s=7.016;rank s=0.997;rank D s=5.68;rank= 5C>A mous SNV mono pathogenic pathogenic COSM516 1(central_nervous_system) Pathogenic - RCV000038265.2 not (s=0.006;rank (s=1;rank=0.8 (s=0.993;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.56;rank= 1.24;rank=0. 6.99;rank=0. =0.895 (s=0.548;rank (s=0.701;rank =0.83 =0.938 =0.782 (s=0.984;rank 0.88 8(upper_aerodigestive_tract) provided,Pathogenic - =0.614) 99) =0.797) 43) 1) 0.75) 79) 972) =0.912) =0.897) =0.819) 2(oesophagus) 3(peritoneum) RCV000119791.1,RCV000013407.4 1776(lung) 18(thyroid) Pathogenic - RCV000013408.4 17(haematopoietic_and_lymphoid Pathogenic,Pathogenic - _tissue) 2(testis) 2(cervix) RCV000154401.2,RCV000013414.5 4(gastrointestinal_tract_(site_ind Pathogenic - RCV000038264.2 not eterminate)) 13(soft_tissue) provided - RCV000119790.1 34(ovary) 38(endometrium) Pathogenic - RCV000144971.3 5(skin) 11(breast) 8(small_intestine) 1(kidney) 130(pancreas) 4(liver) 15(urinary_tract) 1(adrenal_gland) 1476(large_intestine) 51(biliary_tract) 10(prostate) KRAS NM_004985 2 c.G34T- p.G12C chr12:g.2539828 nonsynony N19- 2 15 792 Likely Likely rs121913530 COSM1140136 15(stomach) Pathogenic - RCV000013406.4 0.0001 0 D D D D D M T (s=- D (s=- s=0.905;rank D D s=0.147;rank s=7.016;rank s=0.997;rank D s=5.68;rank= 13

SUPPLEMENTAL DOCUMENTS

5C>A mous SNV PDC pathogenic pathogenic COSM516 1(central_nervous_system) Pathogenic - RCV000038265.2 not (s=0.006;rank (s=1;rank=0.8 (s=0.993;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.56;rank= 1.24;rank=0. 6.99;rank=0. =0.895 (s=0.548;rank (s=0.701;rank =0.83 =0.938 =0.782 (s=0.984;rank 0.88 8(upper_aerodigestive_tract) provided,Pathogenic - =0.614) 99) =0.797) 43) 1) 0.75) 79) 972) =0.912) =0.897) =0.819) 2(oesophagus) 3(peritoneum) RCV000119791.1,RCV000013407.4 1776(lung) 18(thyroid) Pathogenic - RCV000013408.4 17(haematopoietic_and_lymphoid Pathogenic,Pathogenic - _tissue) 2(testis) 2(cervix) RCV000154401.2,RCV000013414.5 4(gastrointestinal_tract_(site_ind Pathogenic - RCV000038264.2 not eterminate)) 13(soft_tissue) provided - RCV000119790.1 34(ovary) 38(endometrium) Pathogenic - RCV000144971.3 5(skin) 11(breast) 8(small_intestine) 1(kidney) 130(pancreas) 4(liver) 15(urinary_tract) 1(adrenal_gland) 1476(large_intestine) 51(biliary_tract) 10(prostate) PHF6 NM_032458 5 c.408dupT- p.H136fs chrX:g.13352797 frameshift N12-BL 1091 100 133 133 Likely - 1->T insertion pathogenic PHF6 NM_032458 5 c.408dupT- p.H136fs chrX:g.13352797 frameshift N12- 742.39 97.44 76 78 Likely - 1->T insertion PDC pathogenic PTPN11 NM_002834 13 c.A1517C- p.Q506P chr12:g.1129268 nonsynony N14-Bl 1377.65 44.09 1906 4323 Likely Uncertain rs397507548 COSM1658770 1(haematopoietic_and_lymphoid_ Pathogenic - RCV000033550.6 D P P D D H D (s=- D (s=- s=0.991;rank D D s=0.303;rank s=6.081;rank s=0.994;rank D s=5.13;rank= 97A>C mous SNV pathogenic significance tissue) 1(skin) Pathogenic - RCV000055887.1 (s=0.001;rank (s=0.869;rank (s=0.825;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=3.655;rank 2.08;rank=0. 5.51;rank=0. =0.996 (s=0.886;rank (s=0.823;rank =0.91 =0.838 =0.628 (s=0.99;rank= 0.696 Pathogenic - RCV000154371.2 =0.784) =0.458) =0.575) 43) 1) =0.944) 86) 859) =0.955) =0.94) 0.901) Pathogenic - RCV000157683.2 PTPN11 NM_002834 13 c.A1517C- p.Q506P chr12:g.1129268 nonsynony N14- 1114.28 40.82 689 1688 Likely Uncertain rs397507548 COSM1658770 1(haematopoietic_and_lymphoid_ Pathogenic - RCV000033550.6 D P P D D H D (s=- D (s=- s=0.991;rank D D s=0.303;rank s=6.081;rank s=0.994;rank D s=5.13;rank= 97A>C mous SNV MONO pathogenic significance tissue) 1(skin) Pathogenic - RCV000055887.1 (s=0.001;rank (s=0.869;rank (s=0.825;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=3.655;rank 2.08;rank=0. 5.51;rank=0. =0.996 (s=0.886;rank (s=0.823;rank =0.91 =0.838 =0.628 (s=0.99;rank= 0.696 Pathogenic - RCV000154371.2 =0.784) =0.458) =0.575) 43) 1) =0.944) 86) 859) =0.955) =0.94) 0.901) Pathogenic - RCV000157683.2 PTPN11 NM_002834 13 c.A1517C- p.Q506P chr12:g.1129268 nonsynony N14- 1155.07 42.67 643 1507 Likely Uncertain rs397507548 COSM1658770 1(haematopoietic_and_lymphoid_ Pathogenic - RCV000033550.6 D P P D D H D (s=- D (s=- s=0.991;rank D D s=0.303;rank s=6.081;rank s=0.994;rank D s=5.13;rank= 97A>C mous SNV pDC pathogenic significance tissue) 1(skin) Pathogenic - RCV000055887.1 (s=0.001;rank (s=0.869;rank (s=0.825;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=3.655;rank 2.08;rank=0. 5.51;rank=0. =0.996 (s=0.886;rank (s=0.823;rank =0.91 =0.838 =0.628 (s=0.99;rank= 0.696 Pathogenic - RCV000154371.2 =0.784) =0.458) =0.575) 43) 1) =0.944) 86) 859) =0.955) =0.94) 0.901) Pathogenic - RCV000157683.2 RUNX1 NM_0010018 2 c.411_412insCC p.G138Pfs* chr21:g.3625286 frameshift N11-Bl 373.55 62.16 23 37 Likely - 90 CC- 9->GGGG insertion pathogenic RUNX1 NM_0010018 2 c.411_412insCC p.G138Pfs* chr21:g.3625286 frameshift N11- 970.41 87.84 65 74 Likely - 90 CC- 9->GGGG insertion mono pathogenic RUNX1 NM_0010018 2 c.411_412insCC p.G138Pfs* chr21:g.3625286 frameshift N11- 2760.94 88.44 306 346 Likely - 90 CC- 9->GGGG insertion pDC pathogenic RUNX1 NM_0010018 3 c.G521A- p.R174Q chr21:g.3623178 nonsynony N12-BL 5000 100 943 943 Likely Likely rs74315450 COSM24805 1(oesophagus) 1(lung) Pathogenic - RCV000015550.25 D D D D A M D (s=- D (s=- s=0.984;rank D D s=0.521;rank s=7.634;rank s=1;rank=1 D s=5.12;rank= 90 2C>T mous SNV pathogenic pathogenic 12(haematopoietic_and_lymphoid (s=0.047;rank (s=1;rank=0.8 (s=0.987;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.91;rank= 6.47;rank=0. 3.71;rank=0. =0.987 (s=1.022;rank (s=0.988;rank =0.955 =0.954 (s=0.944;rank 0.693 _tissue) =0.413) 99) =0.807) 43) 1) 0.844) 997) 718) =0.976) =0.996) =0.609) RUNX1 NM_0010018 3 c.G521A- p.R174Q chr21:g.3623178 nonsynony N12- 5000 98.25 393 400 Likely Likely rs74315450 COSM24805 1(oesophagus) 1(lung) Pathogenic - RCV000015550.25 D D D D A M D (s=- D (s=- s=0.984;rank D D s=0.521;rank s=7.634;rank s=1;rank=1 D s=5.12;rank= 90 2C>T mous SNV MONO pathogenic pathogenic 12(haematopoietic_and_lymphoid (s=0.047;rank (s=1;rank=0.8 (s=0.987;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.91;rank= 6.47;rank=0. 3.71;rank=0. =0.987 (s=1.022;rank (s=0.988;rank =0.955 =0.954 (s=0.944;rank 0.693 _tissue) =0.413) 99) =0.807) 43) 1) 0.844) 997) 718) =0.976) =0.996) =0.609) RUNX1 NM_0010018 3 c.G521A- p.R174Q chr21:g.3623178 nonsynony N12- 5000 99.65 1129 1133 Likely Likely rs74315450 COSM24805 1(oesophagus) 1(lung) Pathogenic - RCV000015550.25 D D D D A M D (s=- D (s=- s=0.984;rank D D s=0.521;rank s=7.634;rank s=1;rank=1 D s=5.12;rank= 90 2C>T mous SNV PDC pathogenic pathogenic 12(haematopoietic_and_lymphoid (s=0.047;rank (s=1;rank=0.8 (s=0.987;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.91;rank= 6.47;rank=0. 3.71;rank=0. =0.987 (s=1.022;rank (s=0.988;rank =0.955 =0.954 (s=0.944;rank 0.693 _tissue) =0.413) 99) =0.807) 43) 1) 0.844) 997) 718) =0.976) =0.996) =0.609) RUNX1 NM_0010018 3 c.C520T- p.R174X chr21:g.3623178 stopgain N13-Bl 5000 88.73 866 976 Likely Likely COSM3356082 9(haematopoietic_and_lymphoid_ - D A s=12.982;ran s=0.998;rank D s=4.21;rank= 90 3G>A pathogenic pathogenic COSM24771 tissue) (s=0;rank=0.8 (s=1;rank=0.8 k=0.988 =0.888 (s=0.875;rank 0.488 43) 1) =0.467) RUNX1 NM_0010018 2 c.422_423insTT- p.R142X chr21:g.3625285 frameshift N13-Bl 2 12 668 Likely - 90 8->AA insertion pathogenic RUNX1 NM_0010018 3 c.C520T- p.R174X chr21:g.3623178 stopgain N13- 790.33 20.56 296 1440 Likely Likely COSM3356082 9(haematopoietic_and_lymphoid_ - D A s=12.982;ran s=0.998;rank D s=4.21;rank= 90 3G>A Mono pathogenic pathogenic COSM24771 tissue) (s=0;rank=0.8 (s=1;rank=0.8 k=0.988 =0.888 (s=0.875;rank 0.488 43) 1) =0.467) RUNX1 NM_0010018 2 c.422_423insTT- p.R142X chr21:g.3625285 frameshift N13- 5 69 1386 Likely - 90 8->AA insertion mono pathogenic RUNX1 NM_0010018 3 c.C520T- p.R174X chr21:g.3623178 stopgain N13- 1538.83 40.6 406 1000 Likely Likely COSM3356082 9(haematopoietic_and_lymphoid_ - D A s=12.982;ran s=0.998;rank D s=4.21;rank= 90 3G>A pDC pathogenic pathogenic COSM24771 tissue) (s=0;rank=0.8 (s=1;rank=0.8 k=0.988 =0.888 (s=0.875;rank 0.488 43) 1) =0.467) RUNX1 NM_0010018 2 c.422_423insTT- p.R142X chr21:g.3625285 frameshift N13- 1520.58 43.13 289 670 Likely - 90 8->AA insertion pDC pathogenic RUNX1 NM_0010018 2 c.T401C- p.L134P chr21:g.3625288 nonsynony N16-BL 5000 100 677 677 Likely Uncertain COSM444417 1(breast) - D D D D D M D (s=- D (s=- s=0.987;rank D D s=0.841;rank s=6.712;rank s=0.999;rank D s=5.31;rank= 90 0A>G mous SNV pathogenic significance (s=0;rank=0.9 (s=1;rank=0.8 (s=1;rank=0.9 (s=0;rank=0.8 (s=1;rank=0.8 (s=2.91;rank= 6.83;rank=0. 6.27;rank=0. =0.991 (s=0.961;rank (s=0.992;rank =0.988 =0.918 =0.982 (s=0.993;rank 0.75 12) 99) 71) 43) 1) 0.844) 998) 915) =0.966) =0.998) =0.936) RUNX1 NM_0010018 2 c.T401C- p.L134P chr21:g.3625288 nonsynony N16- 5000 100 400 400 Likely Uncertain COSM444417 1(breast) - D D D D D M D (s=- D (s=- s=0.987;rank D D s=0.841;rank s=6.712;rank s=0.999;rank D s=5.31;rank= 90 0A>G mous SNV MONO pathogenic significance (s=0;rank=0.9 (s=1;rank=0.8 (s=1;rank=0.9 (s=0;rank=0.8 (s=1;rank=0.8 (s=2.91;rank= 6.83;rank=0. 6.27;rank=0. =0.991 (s=0.961;rank (s=0.992;rank =0.988 =0.918 =0.982 (s=0.993;rank 0.75 12) 99) 71) 43) 1) 0.844) 998) 915) =0.966) =0.998) =0.936) RUNX1 NM_0010018 2 c.T401C- p.L134P chr21:g.3625288 nonsynony N16- 5000 100 789 789 Likely Uncertain COSM444417 1(breast) - D D D D D M D (s=- D (s=- s=0.987;rank D D s=0.841;rank s=6.712;rank s=0.999;rank D s=5.31;rank= 90 0A>G mous SNV PDC pathogenic significance (s=0;rank=0.9 (s=1;rank=0.8 (s=1;rank=0.9 (s=0;rank=0.8 (s=1;rank=0.8 (s=2.91;rank= 6.83;rank=0. 6.27;rank=0. =0.991 (s=0.961;rank (s=0.992;rank =0.988 =0.918 =0.982 (s=0.993;rank 0.75 12) 99) 71) 43) 1) 0.844) 998) 915) =0.966) =0.998) =0.936) RUNX1 NM_0010018 1 c.249_260delins p.T84Yfs* chr21:g.3625915 frameshift N19-BL 209.11 38.07 75 197 Likely - 90 TACCTTGC- 2GGGCAGGGT> insertion pathogenic GCAAGGTA RUNX1 NM_0010018 1 c.249_260delins p.T84Yfs* chr21:g.3625915 frameshift N19- 222.6 54.17 39 72 Likely - 90 TACCTTGC- 2GGGCAGGGT> insertion Mono pathogenic GCAAGGTA RUNX1 NM_0010018 1 c.249_260delins p.T84Yfs* chr21:g.3625915 frameshift N19- 146.29 29.68 46 155 Likely - 90 TACCTTGC- 2GGGCAGGGT> insertion PDC pathogenic GCAAGGTA RUNX1 NM_0010018 5 c.G755A- p.W252X chr21:g.3617172 stopgain N1-BL 1028.35 42.36 194 458 Likely Likely - D A s=13.53;rank s=0.998;rank D s=5.78;rank= 90 9C>T pathogenic pathogenic (s=0;rank=0.8 (s=1;rank=0.8 =0.992 =0.843 (s=0.982;rank 0.914 43) 1) =0.805) RUNX1 NM_0010018 5 c.G755A- p.W252X chr21:g.3617172 stopgain N1- 998.56 41.21 190 461 Likely Likely - D A s=13.53;rank s=0.998;rank D s=5.78;rank= 90 9C>T MONO pathogenic pathogenic (s=0;rank=0.8 (s=1;rank=0.8 =0.992 =0.843 (s=0.982;rank 0.914 43) 1) =0.805) RUNX1 NM_0010018 5 c.G755A- p.W252X chr21:g.3617172 stopgain N1-pDC 352.8 34.88 45 129 Likely Likely - D A s=13.53;rank s=0.998;rank D s=5.78;rank= 90 9C>T pathogenic pathogenic (s=0;rank=0.8 (s=1;rank=0.8 =0.992 =0.843 (s=0.982;rank 0.914 43) 1) =0.805) RUNX1 NM_0010018 2 c.C341A- p.S114X chr21:g.3625294 stopgain N2-Bl 1429.43 52.31 533 1019 Likely Likely COSM25125 2(haematopoietic_and_lymphoid_ - D A s=12.651;ran s=0.996;rank D s=5.31;rank= 90 0G>T pathogenic pathogenic tissue) (s=0;rank=0.8 (s=1;rank=0.8 k=0.985 =0.718 (s=0.998;rank 0.75 43) 1) =0.996) RUNX1 NM_0010018 2 c.C341A- p.S114X chr21:g.3625294 stopgain N2- 1451.51 50.61 661 1306 Likely Likely COSM25125 2(haematopoietic_and_lymphoid_ - D A s=12.651;ran s=0.996;rank D s=5.31;rank= 90 0G>T Mono pathogenic pathogenic tissue) (s=0;rank=0.8 (s=1;rank=0.8 k=0.985 =0.718 (s=0.998;rank 0.75 43) 1) =0.996) 14

SUPPLEMENTAL DOCUMENTS

RUNX1 NM_0010018 2 c.C341A- p.S114X chr21:g.3625294 stopgain N2-pDC 1714.98 53.56 1120 2091 Likely Likely COSM25125 2(haematopoietic_and_lymphoid_ - D A s=12.651;ran s=0.996;rank D s=5.31;rank= 90 0G>T pathogenic pathogenic tissue) (s=0;rank=0.8 (s=1;rank=0.8 k=0.985 =0.718 (s=0.998;rank 0.75 43) 1) =0.996) RUNX1 NM_0010018 1 c.260_260delins p.P86Sfs* chr21:g.3625915 frameshift N7-BL 297.47 43.21 261 604 Likely - 90 GCCCA- 0A>TGGGC substitution pathogenic RUNX1 NM_0010018 1 c.260_260delins p.P86Sfs* chr21:g.3625915 frameshift N7- 286.59 43.59 187 429 Likely - 90 GCCCA- 0A>TGGGC substitution mono pathogenic RUNX1 NM_0010018 1 c.260_260delins p.P86Sfs* chr21:g.3625915 frameshift N7-pDC 236.81 36.59 191 522 Likely - 90 GCCCA- 0A>TGGGC substitution pathogenic RUNX1 NM_0010018 1 c.115dupG- p.A39Gfs* chr21:g.3625929 frameshift N8-Bl 462.14 86.76 59 68 Likely - 90 4->C insertion pathogenic RUNX1 NM_0010018 1 c.115dupG- p.A39Gfs* chr21:g.3625929 frameshift N8-cDC 1302.05 99.66 296 297 Likely - 90 4->C insertion pathogenic RUNX1 NM_0010018 1 c.115dupG- p.A39Gfs* chr21:g.3625929 frameshift N8- 1208.76 100 243 243 Likely - 90 4->C insertion mono pathogenic RUNX1 NM_0010018 1 c.115dupG- p.A39Gfs* chr21:g.3625929 frameshift N8-pDC 1419.87 99.22 381 384 Likely - 90 4->C insertion pathogenic RUNX1 NM_0010018 1 c.97dupG- p.A33Pfs* chr21:g.3625931 frameshift N9-Bl 579.25 63.16 72 114 Likely - 90 2->C insertion pathogenic RUNX1 NM_0010018 1 c.97dupG- p.A33Pfs* chr21:g.3625931 frameshift N9- 325.17 42.55 40 94 Likely - 90 2->C insertion mono pathogenic RUNX1 NM_0010018 1 c.97dupG- p.A33Pfs* chr21:g.3625931 frameshift N9-pDC 870 9280 116 125 Likely - 90 2->C insertion pathogenic SF3B1 NM_012433 14 c.G1998C- p.K666N chr2:g.19826735 nonsynony N2-Bl 399.19 35.19 82 233 Likely Uncertain rs377023736 COSM132937 9(haematopoietic_and_lymphoid_ Pathogenic - RCV000203465.1 0.0001 0.0001 D D D D D H T (s=- D (s=- s=0.555;rank D D s=0.1;rank=0. s=6.136;rank s=0.999;rank D s=4.81;rank= 9C>G mous SNV pathogenic significance tissue) 1(liver) (s=0;rank=0.9 (s=1;rank=0.8 (s=0.992;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=3.725;rank 0.17;rank=0. 4.88;rank=0. =0.59 (s=0.219;rank (s=0.528;rank 772 =0.846 =0.938 (s=0.973;rank 0.613 12) 99) =0.79) 43) 1) =0.949) 656) 813) =0.862) =0.825) =0.735) SF3B1 NM_012433 14 c.G1998C- p.K666N chr2:g.19826735 nonsynony N2- 617.22 42.62 179 420 Likely Uncertain rs377023736 COSM132937 9(haematopoietic_and_lymphoid_ Pathogenic - RCV000203465.1 0.0001 0.0001 D D D D D H T (s=- D (s=- s=0.555;rank D D s=0.1;rank=0. s=6.136;rank s=0.999;rank D s=4.81;rank= 9C>G mous SNV Mono pathogenic significance tissue) 1(liver) (s=0;rank=0.9 (s=1;rank=0.8 (s=0.992;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=3.725;rank 0.17;rank=0. 4.88;rank=0. =0.59 (s=0.219;rank (s=0.528;rank 772 =0.846 =0.938 (s=0.973;rank 0.613 12) 99) =0.79) 43) 1) =0.949) 656) 813) =0.862) =0.825) =0.735) SF3B1 NM_012433 14 c.G1998C- p.K666N chr2:g.19826735 nonsynony N2-pDC 382.2 28.98 111 383 Likely Uncertain rs377023736 COSM132937 9(haematopoietic_and_lymphoid_ Pathogenic - RCV000203465.1 0.0001 0.0001 D D D D D H T (s=- D (s=- s=0.555;rank D D s=0.1;rank=0. s=6.136;rank s=0.999;rank D s=4.81;rank= 9C>G mous SNV pathogenic significance tissue) 1(liver) (s=0;rank=0.9 (s=1;rank=0.8 (s=0.992;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=3.725;rank 0.17;rank=0. 4.88;rank=0. =0.59 (s=0.219;rank (s=0.528;rank 772 =0.846 =0.938 (s=0.973;rank 0.613 12) 99) =0.79) 43) 1) =0.949) 656) 813) =0.862) =0.825) =0.735) SF3B1 NM_012433 13 c.G1774A- p.E592K chr2:g.19826770 nonsynony N7-BL 744.48 36.52 252 690 Likely Uncertain COSM132936 2(haematopoietic_and_lymphoid_ - D D D D D M D (s=- s=0.915;rank D D s=0.094;rank s=7.733;rank s=0.999;rank D s=5.7;rank=0. 5C>T mous SNV pathogenic significance tissue) (s=0.001;rank (s=1;rank=0.8 (s=0.999;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=3.325;rank 3.62;rank=0. =0.906 (s=0.569;rank (s=0.677;rank =0.763 =0.955 =0.997 (s=0.993;rank 886 =0.784) 99) =0.916) 43) 1) =0.91) 695) =0.915) =0.888) =0.945) SF3B1 NM_012433 13 c.G1774A- p.E592K chr2:g.19826770 nonsynony N7- 794.32 48.28 154 319 Likely Uncertain COSM132936 2(haematopoietic_and_lymphoid_ - D D D D D M D (s=- s=0.915;rank D D s=0.094;rank s=7.733;rank s=0.999;rank D s=5.7;rank=0. 5C>T mous SNV mono pathogenic significance tissue) (s=0.001;rank (s=1;rank=0.8 (s=0.999;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=3.325;rank 3.62;rank=0. =0.906 (s=0.569;rank (s=0.677;rank =0.763 =0.955 =0.997 (s=0.993;rank 886 =0.784) 99) =0.916) 43) 1) =0.91) 695) =0.915) =0.888) =0.945) SF3B1 NM_012433 13 c.G1774A- p.E592K chr2:g.19826770 nonsynony N7-pDC 590.09 30.54 197 645 Likely Uncertain COSM132936 2(haematopoietic_and_lymphoid_ - D D D D D M D (s=- s=0.915;rank D D s=0.094;rank s=7.733;rank s=0.999;rank D s=5.7;rank=0. 5C>T mous SNV pathogenic significance tissue) (s=0.001;rank (s=1;rank=0.8 (s=0.999;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=3.325;rank 3.62;rank=0. =0.906 (s=0.569;rank (s=0.677;rank =0.763 =0.955 =0.997 (s=0.993;rank 886 =0.784) 99) =0.916) 43) 1) =0.91) 695) =0.915) =0.888) =0.945) SRSF2 NM_003016 1 c.C284A- p.P95H chr17:g.7473295 nonsynony N11-Bl 673.3 73.49 61 83 Likely Uncertain rs751713049 COSM211029 80(haematopoietic_and_lymphoid - 0.0002 0.0001 D D D D D M T (s=- D (s=- s=0.275;rank T (s=- D s=0.981;rank s=4.52;rank= s=0.937;rank D s=2.76;rank= 9G>T mous SNV pathogenic significance COSM211505 _tissue) (s=0.001;rank (s=1;rank=0.8 (s=0.984;rank (s=0;rank=0.6 (s=1;rank=0.8 (s=2.565;rank 0.94;rank=0. 5.56;rank=0. =0.372 0.044;rank=0 (s=0.532;rank =0.999 0.605 =0.231 (s=0.901;rank 0.314 COSM211504 =0.784) 99) =0.745) 29) 1) =0.752) 753) 863) .813) =0.826) =0.507) SRSF2 NM_003016 1 c.C284A- p.P95H chr17:g.7473295 nonsynony N11- 230.44 32.35 22 68 Likely Uncertain rs751713049 COSM211029 80(haematopoietic_and_lymphoid - 0.0002 0.0001 D D D D D M T (s=- D (s=- s=0.275;rank T (s=- D s=0.981;rank s=4.52;rank= s=0.937;rank D s=2.76;rank= 9G>T mous SNV mono pathogenic significance COSM211505 _tissue) (s=0.001;rank (s=1;rank=0.8 (s=0.984;rank (s=0;rank=0.6 (s=1;rank=0.8 (s=2.565;rank 0.94;rank=0. 5.56;rank=0. =0.372 0.044;rank=0 (s=0.532;rank =0.999 0.605 =0.231 (s=0.901;rank 0.314 COSM211504 =0.784) 99) =0.745) 29) 1) =0.752) 753) 863) .813) =0.826) =0.507) SRSF2 NM_003016 1 c.C284A- p.P95H chr17:g.7473295 nonsynony N11- 729.89 43.44 139 320 Likely Uncertain rs751713049 COSM211029 80(haematopoietic_and_lymphoid - 0.0002 0.0001 D D D D D M T (s=- D (s=- s=0.275;rank T (s=- D s=0.981;rank s=4.52;rank= s=0.937;rank D s=2.76;rank= 9G>T mous SNV pDC pathogenic significance COSM211505 _tissue) (s=0.001;rank (s=1;rank=0.8 (s=0.984;rank (s=0;rank=0.6 (s=1;rank=0.8 (s=2.565;rank 0.94;rank=0. 5.56;rank=0. =0.372 0.044;rank=0 (s=0.532;rank =0.999 0.605 =0.231 (s=0.901;rank 0.314 COSM211504 =0.784) 99) =0.745) 29) 1) =0.752) 753) 863) .813) =0.826) =0.507) SRSF2 NM_003016 1 c.C284T- p.P95L chr17:g.7473295 nonsynony N16-BL 661.36 50 91 182 Likely Uncertain rs751713049 COSM146288 53(haematopoietic_and_lymphoid - 0.0001 0.0001 D P P N D M T (s=- D (s=- s=0.203;rank T (s=- T s=0.971;rank s=3.407;rank s=0.966;rank D s=2.76;rank= 9G>A mous SNV pathogenic significance COSM211506 _tissue) (s=0.001;rank (s=0.753;rank (s=0.462;rank (s=0;rank=0.6 (s=1;rank=0.8 (s=2.22;rank= 0.9;rank=0.7 5.95;rank=0. =0.249 0.484;rank=0 (s=0.363;rank =0.998 =0.468 =0.3 (s=0.89;rank= 0.314 COSM211028 =0.784) =0.414) =0.447) 29) 1) 0.631) 49) 891) .692) =0.724) 0.489) SRSF2 NM_003016 1 c.C284T- p.P95L chr17:g.7473295 nonsynony N16- 372.05 36.97 44 119 Likely Uncertain rs751713049 COSM146288 53(haematopoietic_and_lymphoid - 0.0001 0.0001 D P P N D M T (s=- D (s=- s=0.203;rank T (s=- T s=0.971;rank s=3.407;rank s=0.966;rank D s=2.76;rank= 9G>A mous SNV MONO pathogenic significance COSM211506 _tissue) (s=0.001;rank (s=0.753;rank (s=0.462;rank (s=0;rank=0.6 (s=1;rank=0.8 (s=2.22;rank= 0.9;rank=0.7 5.95;rank=0. =0.249 0.484;rank=0 (s=0.363;rank =0.998 =0.468 =0.3 (s=0.89;rank= 0.314 COSM211028 =0.784) =0.414) =0.447) 29) 1) 0.631) 49) 891) .692) =0.724) 0.489) SRSF2 NM_003016 1 c.C284T- p.P95L chr17:g.7473295 nonsynony N16- 664.33 44 110 250 Likely Uncertain rs751713049 COSM146288 53(haematopoietic_and_lymphoid - 0.0001 0.0001 D P P N D M T (s=- D (s=- s=0.203;rank T (s=- T s=0.971;rank s=3.407;rank s=0.966;rank D s=2.76;rank= 9G>A mous SNV PDC pathogenic significance COSM211506 _tissue) (s=0.001;rank (s=0.753;rank (s=0.462;rank (s=0;rank=0.6 (s=1;rank=0.8 (s=2.22;rank= 0.9;rank=0.7 5.95;rank=0. =0.249 0.484;rank=0 (s=0.363;rank =0.998 =0.468 =0.3 (s=0.89;rank= 0.314 COSM211028 =0.784) =0.414) =0.447) 29) 1) 0.631) 49) 891) .692) =0.724) 0.489) SRSF2 NM_003016 1 c.C283G- p.P95A chr17:g.7473296 nonsynony N34-Bl 730.4 38.75 112 289 Likely Uncertain COSM307352 1(haematopoietic_and_lymphoid_ - D P P D D M T (s=- D (s=- s=0.149;rank T (s=- T s=0.975;rank s=2.595;rank s=0.886;rank D s=3.85;rank= 0G>C mous SNV pathogenic significance tissue) (s=0.001;rank (s=0.889;rank (s=0.679;rank (s=0;rank=0.6 (s=1;rank=0.8 (s=2.565;rank 0.87;rank=0. 4.98;rank=0. =0.176 0.206;rank=0 (s=0.438;rank =0.999 =0.373 =0.175 (s=0.907;rank 0.434 =0.784) =0.51) =0.516) 29) 1) =0.752) 746) 821) .775) =0.777) =0.519) SRSF2 NM_003016 1 c.C283G- p.P95A chr17:g.7473296 nonsynony N34- 876.83 47.33 124 262 Likely Uncertain COSM307352 1(haematopoietic_and_lymphoid_ - D P P D D M T (s=- D (s=- s=0.149;rank T (s=- T s=0.975;rank s=2.595;rank s=0.886;rank D s=3.85;rank= 0G>C mous SNV Mono pathogenic significance tissue) (s=0.001;rank (s=0.889;rank (s=0.679;rank (s=0;rank=0.6 (s=1;rank=0.8 (s=2.565;rank 0.87;rank=0. 4.98;rank=0. =0.176 0.206;rank=0 (s=0.438;rank =0.999 =0.373 =0.175 (s=0.907;rank 0.434 =0.784) =0.51) =0.516) 29) 1) =0.752) 746) 821) .775) =0.777) =0.519) SRSF2 NM_003016 1 c.C283G- p.P95A chr17:g.7473296 nonsynony N34- 1146.61 44.48 250 562 Likely Uncertain COSM307352 1(haematopoietic_and_lymphoid_ - D P P D D M T (s=- D (s=- s=0.149;rank T (s=- T s=0.975;rank s=2.595;rank s=0.886;rank D s=3.85;rank= 0G>C mous SNV pDC pathogenic significance tissue) (s=0.001;rank (s=0.889;rank (s=0.679;rank (s=0;rank=0.6 (s=1;rank=0.8 (s=2.565;rank 0.87;rank=0. 4.98;rank=0. =0.176 0.206;rank=0 (s=0.438;rank =0.999 =0.373 =0.175 (s=0.907;rank 0.434 =0.784) =0.51) =0.516) 29) 1) =0.752) 746) 821) .775) =0.777) =0.519) SRSF2 NM_003016 1 c.C284A- p.P95H chr17:g.7473295 nonsynony N35-BL- 708.13 54.39 93 171 Likely Uncertain rs751713049 COSM211029 80(haematopoietic_and_lymphoid - 0.0002 0.0001 D D D D D M T (s=- D (s=- s=0.275;rank T (s=- D s=0.981;rank s=4.52;rank= s=0.937;rank D s=2.76;rank= 9G>T mous SNV CD34ne pathogenic significance COSM211505 _tissue) (s=0.001;rank (s=1;rank=0.8 (s=0.984;rank (s=0;rank=0.6 (s=1;rank=0.8 (s=2.565;rank 0.94;rank=0. 5.56;rank=0. =0.372 0.044;rank=0 (s=0.532;rank =0.999 0.605 =0.231 (s=0.901;rank 0.314 g COSM211504 =0.784) 99) =0.745) 29) 1) =0.752) 753) 863) .813) =0.826) =0.507) SRSF2 NM_003016 1 c.C284A- p.P95H chr17:g.7473295 nonsynony N35-BL- 300.51 25.29 44 174 Likely Uncertain rs751713049 COSM211029 80(haematopoietic_and_lymphoid - 0.0002 0.0001 D D D D D M T (s=- D (s=- s=0.275;rank T (s=- D s=0.981;rank s=4.52;rank= s=0.937;rank D s=2.76;rank= 9G>T mous SNV CD34po pathogenic significance COSM211505 _tissue) (s=0.001;rank (s=1;rank=0.8 (s=0.984;rank (s=0;rank=0.6 (s=1;rank=0.8 (s=2.565;rank 0.94;rank=0. 5.56;rank=0. =0.372 0.044;rank=0 (s=0.532;rank =0.999 0.605 =0.231 (s=0.901;rank 0.314 s COSM211504 =0.784) 99) =0.745) 29) 1) =0.752) 753) 863) .813) =0.826) =0.507) SRSF2 NM_003016 1 c.C284A- p.P95H chr17:g.7473295 nonsynony N35-Bl 284.39 29.31 34 116 Likely Uncertain rs751713049 COSM211029 80(haematopoietic_and_lymphoid - 0.0002 0.0001 D D D D D M T (s=- D (s=- s=0.275;rank T (s=- D s=0.981;rank s=4.52;rank= s=0.937;rank D s=2.76;rank= 9G>T mous SNV pathogenic significance COSM211505 _tissue) (s=0.001;rank (s=1;rank=0.8 (s=0.984;rank (s=0;rank=0.6 (s=1;rank=0.8 (s=2.565;rank 0.94;rank=0. 5.56;rank=0. =0.372 0.044;rank=0 (s=0.532;rank =0.999 0.605 =0.231 (s=0.901;rank 0.314 COSM211504 =0.784) 99) =0.745) 29) 1) =0.752) 753) 863) .813) =0.826) =0.507) SRSF2 NM_003016 1 c.C284A- p.P95H chr17:g.7473295 nonsynony N35- 818.46 60.11 107 178 Likely Uncertain rs751713049 COSM211029 80(haematopoietic_and_lymphoid - 0.0002 0.0001 D D D D D M T (s=- D (s=- s=0.275;rank T (s=- D s=0.981;rank s=4.52;rank= s=0.937;rank D s=2.76;rank= 9G>T mous SNV Mono pathogenic significance COSM211505 _tissue) (s=0.001;rank (s=1;rank=0.8 (s=0.984;rank (s=0;rank=0.6 (s=1;rank=0.8 (s=2.565;rank 0.94;rank=0. 5.56;rank=0. =0.372 0.044;rank=0 (s=0.532;rank =0.999 0.605 =0.231 (s=0.901;rank 0.314 COSM211504 =0.784) 99) =0.745) 29) 1) =0.752) 753) 863) .813) =0.826) =0.507) SRSF2 NM_003016 1 c.C284A- p.P95H chr17:g.7473295 nonsynony N35- 193.11 83.33 10 12 Likely Uncertain rs751713049 COSM211029 80(haematopoietic_and_lymphoid - 0.0002 0.0001 D D D D D M T (s=- D (s=- s=0.275;rank T (s=- D s=0.981;rank s=4.52;rank= s=0.937;rank D s=2.76;rank= 9G>T mous SNV pDC pathogenic significance COSM211505 _tissue) (s=0.001;rank (s=1;rank=0.8 (s=0.984;rank (s=0;rank=0.6 (s=1;rank=0.8 (s=2.565;rank 0.94;rank=0. 5.56;rank=0. =0.372 0.044;rank=0 (s=0.532;rank =0.999 0.605 =0.231 (s=0.901;rank 0.314 COSM211504 =0.784) 99) =0.745) 29) 1) =0.752) 753) 863) .813) =0.826) =0.507) SRSF2 NM_003016 1 c.308_308delins p.P95Rfs* chr17:g.7473293 frameshift N9-Bl 763.55 54.33 113 208 Likely - CCCCGGACTCAC 5C>GGCGGCTGT substitution pathogenic ACCACAGCCGCC GGTGTGAGTCCG - GGG SRSF2 NM_003016 1 c.308_308delins p.P95Rfs* chr17:g.7473293 frameshift N9- 27 6 22 Likely - CCCCGGACTCAC 5C>GGCGGCTGT substitution Mono pathogenic ACCACAGCCGCC GGTGTGAGTCCG - GGG SRSF2 NM_003016 1 c.308_308delins chr17:g.7473293 frameshift N9-pDC 421.1 49.43 43 87 Likely - CCCCGGACTCAC 5C>GGCGGCTGT substitution pathogenic ACCACAGCCGCC GGTGTGAGTCCG - GGG 15

SUPPLEMENTAL DOCUMENTS

TET2 NM_0011272 11 c.C4570T- p.Q1524X chr4:g.10619623 stopgain N13-Bl 1079.61 33.99 686 2018 Likely Uncertain - D s=10.896;ran s=0.997;rank D s=2.87;rank= 08 7C>T pathogenic significance (s=1;rank=0.8 k=0.971 =0.78 (s=0.907;rank 0.324 1) =0.518) TET2 NM_0011272 11 c.C4570T- p.Q1524X chr4:g.10619623 stopgain N13- 1149.25 37.01 597 1613 Likely Uncertain - D s=10.896;ran s=0.997;rank D s=2.87;rank= 08 7C>T Mono pathogenic significance (s=1;rank=0.8 k=0.971 =0.78 (s=0.907;rank 0.324 1) =0.518) TET2 NM_0011272 11 c.C4570T- p.Q1524X chr4:g.10619623 stopgain N13- 1270.25 41.92 529 1262 Likely Uncertain - D s=10.896;ran s=0.997;rank D s=2.87;rank= 08 7C>T pDC pathogenic significance (s=1;rank=0.8 k=0.971 =0.78 (s=0.907;rank 0.324 1) =0.518) TET2 NM_0011272 11 c.4679dupA- p.Y1560_S1 chr4:g.10619634 stopgain N34-Bl 2683.37 50.9 1357 2666 Likely COSM1716912 1(NS) - 08 561delinsX 5->A pathogenic TET2 NM_0011272 11 c.4679dupA- p.Y1560_S1 chr4:g.10619634 stopgain N34- 1301.13 42.86 231 539 Likely COSM1716912 1(NS) - 08 561delinsX 5->A Mono pathogenic TET2 NM_0011272 11 c.4679dupA- p.Y1560_S1 chr4:g.10619634 stopgain N34- 2110.46 47.56 681 1432 Likely COSM1716912 1(NS) - 08 561delinsX 5->A pDC pathogenic TET2 NM_0011272 3 c.C1471T- p.Q491X chr4:g.10615657 stopgain N35-BL- 2306.29 47.97 1369 2854 Likely Uncertain - U A s=6.873;rank s=0.988;rank N s=0.061;rank 08 0C>T CD34ne pathogenic significance (s=0.039;rank (s=1;rank=0.8 =0.93 =0.467 (s=0.193;rank =0.136 g =0.019) 1) =0.204) TET2 NM_0011272 11 c.G5582A- p.G1861E chr4:g.10619724 nonsynony N35-BL- 5000 49.97 879 1759 Likely Uncertain - D D D D M T D (s=- s=0.882;rank T (s=- T s=0.133;rank s=6.024;rank s=0.998;rank D s=5.33;rank= 08 9G>A mous SNV CD34ne pathogenic significance (s=0;rank=0.9 (s=1;rank=0.8 (s=1;rank=0.9 (s=1;rank=0.8 (s=2.51;rank= (s=2.42;rank= 7.25;rank=0. =0.982 0.834;rank=0 (s=0.15;rank= =0.816 =0.829 =0.885 (s=0.971;rank 0.756 g 12) 99) 71) 1) 0.734) 0.154) 943) .531) 0.477) =0.725) TET2 NM_0011272 3 c.C1471T- p.Q491X chr4:g.10615657 stopgain N35-BL- 1701.22 39.83 801 2011 Likely Uncertain - U A s=6.873;rank s=0.988;rank N s=0.061;rank 08 0C>T CD34po pathogenic significance (s=0.039;rank (s=1;rank=0.8 =0.93 =0.467 (s=0.193;rank =0.136 s =0.019) 1) =0.204) TET2 NM_0011272 11 c.G5582A- p.G1861E chr4:g.10619724 nonsynony N35-BL- 1768.45 38.5 313 813 Likely Uncertain - D D D D M T D (s=- s=0.882;rank T (s=- T s=0.133;rank s=6.024;rank s=0.998;rank D s=5.33;rank= 08 9G>A mous SNV CD34po pathogenic significance (s=0;rank=0.9 (s=1;rank=0.8 (s=1;rank=0.9 (s=1;rank=0.8 (s=2.51;rank= (s=2.42;rank= 7.25;rank=0. =0.982 0.834;rank=0 (s=0.15;rank= =0.816 =0.829 =0.885 (s=0.971;rank 0.756 s 12) 99) 71) 1) 0.734) 0.154) 943) .531) 0.477) =0.725) TET2 NM_0011272 3 c.C1471T- p.Q491X chr4:g.10615657 stopgain N35- 2480.08 48.91 1878 3840 Likely Uncertain - U A s=6.873;rank s=0.988;rank N s=0.061;rank 08 0C>T Mono pathogenic significance (s=0.039;rank (s=1;rank=0.8 =0.93 =0.467 (s=0.193;rank =0.136 =0.019) 1) =0.204) TET2 NM_0011272 11 c.G5582A- p.G1861E chr4:g.10619724 nonsynony N35- 2577.52 41.18 719 1746 Likely Uncertain - D D D D M T D (s=- s=0.882;rank T (s=- T s=0.133;rank s=6.024;rank s=0.998;rank D s=5.33;rank= 08 9G>A mous SNV Mono pathogenic significance (s=0;rank=0.9 (s=1;rank=0.8 (s=1;rank=0.9 (s=1;rank=0.8 (s=2.51;rank= (s=2.42;rank= 7.25;rank=0. =0.982 0.834;rank=0 (s=0.15;rank= =0.816 =0.829 =0.885 (s=0.971;rank 0.756 12) 99) 71) 1) 0.734) 0.154) 943) .531) 0.477) =0.725) TET2 NM_0011272 11 c.G5582A- p.G1861E chr4:g.10619724 nonsynony N35- 2 22 1416 Likely Uncertain - D D D D M T D (s=- s=0.882;rank T (s=- T s=0.133;rank s=6.024;rank s=0.998;rank D s=5.33;rank= 08 9G>A mous SNV pDC pathogenic significance (s=0;rank=0.9 (s=1;rank=0.8 (s=1;rank=0.9 (s=1;rank=0.8 (s=2.51;rank= (s=2.42;rank= 7.25;rank=0. =0.982 0.834;rank=0 (s=0.15;rank= =0.816 =0.829 =0.885 (s=0.971;rank 0.756 12) 99) 71) 1) 0.734) 0.154) 943) .531) 0.477) =0.725) TET2 NM_0011272 3 c.C1471T- p.Q491X chr4:g.10615657 stopgain N35- 180.02 20.29 14 69 Likely Uncertain - U A s=6.873;rank s=0.988;rank N s=0.061;rank 08 0C>T pDC pathogenic significance (s=0.039;rank (s=1;rank=0.8 =0.93 =0.467 (s=0.193;rank =0.136 =0.019) 1) =0.204) TET2 NM_0011272 6 c.T3731C- p.L1244P chr4:g.10616486 nonsynony N36-Bl 487.66 51.22 42 82 Likely Uncertain - D D D D M T D (s=- s=0.87;rank= T (s=- T s=0.487;rank s=5.631;rank s=0.999;rank D s=5.46;rank= 08 3T>C mous SNV pathogenic significance (s=0.001;rank (s=1;rank=0.8 (s=0.999;rank (s=1;rank=0.8 (s=3.175;rank (s=0.91;rank= 6.46;rank=0. 0.961 0.349;rank=0 (s=0.303;rank =0.95 =0.767 =0.969 (s=0.984;rank 0.799 =0.784) 99) =0.916) 1) =0.89) 0.449) 914) .736) =0.674) =0.82) TET2 NM_0011272 3 c.C1081T- p.Q361X chr4:g.10615618 stopgain N36-Bl 736.39 43.23 83 192 Likely Uncertain - N A s=11.026;ran s=0.998;rank D s=4.97;rank= 08 0C>T pathogenic significance (s=0;rank=0.0 (s=1;rank=0.8 k=0.972 =0.923 (s=0.899;rank 0.653 02) 1) =0.504) TET2 NM_0011272 7 c.T3731C- p.L1244P chr4:g.10616486 nonsynony N36- 27 129 347 Likely Uncertain - D D D D M T D (s=- s=0.87;rank= T (s=- T s=0.487;rank s=5.631;rank s=0.999;rank D s=5.46;rank= 09 3T>C mous SNV Mono pathogenic significance (s=0.001;rank (s=1;rank=0.8 (s=0.999;rank (s=1;rank=0.8 (s=3.175;rank (s=0.91;rank= 6.46;rank=0. 0.961 0.349;rank=0 (s=0.303;rank =0.95 =0.767 =0.969 (s=0.984;rank 0.799 =0.784) 99) =0.916) 1) =0.89) 0.449) 914) .736) =0.674) =0.82) TET2 NM_0011272 3 c.C1081T- p.Q361X chr4:g.10615618 stopgain N36- 9.21 201 2181 Likely Uncertain - 08 0C>T mono pathogenic significance TET2 NM_0011272 7 c.T3731C- p.L1244P chr4:g.10616486 nonsynony N36- 1433.83 43.83 508 1159 Likely Uncertain - D D D D M T D (s=- s=0.87;rank= T (s=- T s=0.487;rank s=5.631;rank s=0.999;rank D s=5.46;rank= 09 3T>C mous SNV pDC pathogenic significance (s=0.001;rank (s=1;rank=0.8 (s=0.999;rank (s=1;rank=0.8 (s=3.175;rank (s=0.91;rank= 6.46;rank=0. 0.961 0.349;rank=0 (s=0.303;rank =0.95 =0.767 =0.969 (s=0.984;rank 0.799 =0.784) 99) =0.916) 1) =0.89) 0.449) 914) .736) =0.674) =0.82) TET2 NM_0011272 3 c.C1081T- p.Q361X chr4:g.10615618 stopgain N36- 2606.61 54.62 1188 2175 Likely Uncertain - N A s=11.026;ran s=0.998;rank D s=4.97;rank= 08 0C>T pDC pathogenic significance (s=0;rank=0.0 (s=1;rank=0.8 k=0.972 =0.923 (s=0.899;rank 0.653 02) 1) =0.504) U2AF1 NM_006758 6 c.G467A- p.R156H chr21:g.4451478 nonsynony N1-BL 942.51 53.39 252 472 Likely Uncertain rs769567889 COSM1235015 1(haematopoietic_and_lymphoid_ - 0.0002 0 D P B D D M T D (s=- s=0.837;rank T (s=- T s=0.08;rank= s=6.539;rank s=0.999;rank D s=5.01;rank= 0C>T mous SNV pathogenic significance COSM1235014 tissue) 1(breast) (s=0.015;rank (s=0.798;rank (s=0.234;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.585;rank (s=0.93;rank= 4.54;rank=0. =0.824 0.306;rank=0 (s=0.249;rank 0.734 =0.9 =0.999 (s=0.981;rank 0.663 =0.555) =0.515) =0.479) 43) 1) =0.758) 0.441) 784) .748) =0.619) =0.796) U2AF1 NM_006758 6 c.G467A- p.R156H chr21:g.4451478 nonsynony N1- 815.07 48.03 220 458 Likely Uncertain rs769567889 COSM1235015 1(haematopoietic_and_lymphoid_ - 0.0002 0 D P B D D M T D (s=- s=0.837;rank T (s=- T s=0.08;rank= s=6.539;rank s=0.999;rank D s=5.01;rank= 0C>T mous SNV MONO pathogenic significance COSM1235014 tissue) 1(breast) (s=0.015;rank (s=0.798;rank (s=0.234;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.585;rank (s=0.93;rank= 4.54;rank=0. =0.824 0.306;rank=0 (s=0.249;rank 0.734 =0.9 =0.999 (s=0.981;rank 0.663 =0.555) =0.515) =0.479) 43) 1) =0.758) 0.441) 784) .748) =0.619) =0.796) U2AF1 NM_006758 6 c.G467A- p.R156H chr21:g.4451478 nonsynony N1-pDC 5000 72.06 557 773 Likely Uncertain rs769567889 COSM1235015 1(haematopoietic_and_lymphoid_ - 0.0002 0 D P B D D M T D (s=- s=0.837;rank T (s=- T s=0.08;rank= s=6.539;rank s=0.999;rank D s=5.01;rank= 0C>T mous SNV pathogenic significance COSM1235014 tissue) 1(breast) (s=0.015;rank (s=0.798;rank (s=0.234;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.585;rank (s=0.93;rank= 4.54;rank=0. =0.824 0.306;rank=0 (s=0.249;rank 0.734 =0.9 =0.999 (s=0.981;rank 0.663 =0.555) =0.515) =0.479) 43) 1) =0.758) 0.441) 784) .748) =0.619) =0.796) WT1 NM_024426 5 c.C999A- p.S333R chr11:g.3243803 nonsynony N8-cDC 392.2 39.53 187 473 Likely Uncertain - D D D U D N T N (s=- s=0.821;rank T (s=- T s=0.009;rank s=6.319;rank s=0.996;rank D s=5.33;rank= 8G>T mous SNV pathogenic significance (s=0.002;rank (s=0.996;rank (s=0.99;rank= (s=0.039;rank (s=0.961;rank (s=0.55;rank= (s=2.86;rank= 0.72;rank=0. =0.809 0.935;rank=0 (s=0.194;rank =0.246 =0.873 =0.761 (s=0.897;rank 0.756 =0.784) =0.67) 0.774) =0.242) =0.81) 0.145) 0.494) 342) .434) =0.548) =0.501) WT1 NM_024426 5 c.C999A- p.S333R chr11:g.3243803 nonsynony N8- 654.91 57.88 290 501 Likely Uncertain - D D D U D N T N (s=- s=0.821;rank T (s=- T s=0.009;rank s=6.319;rank s=0.996;rank D s=5.33;rank= 8G>T mous SNV mono pathogenic significance (s=0.002;rank (s=0.996;rank (s=0.99;rank= (s=0.039;rank (s=0.961;rank (s=0.55;rank= (s=2.86;rank= 0.72;rank=0. =0.809 0.935;rank=0 (s=0.194;rank =0.246 =0.873 =0.761 (s=0.897;rank 0.756 =0.784) =0.67) 0.774) =0.242) =0.81) 0.145) 0.494) 342) .434) =0.548) =0.501) WT1 NM_024426 5 c.C999A- p.S333R chr11:g.3243803 nonsynony N8-pDC 534.04 51.43 216 420 Likely Uncertain - D D D U D N T N (s=- s=0.821;rank T (s=- T s=0.009;rank s=6.319;rank s=0.996;rank D s=5.33;rank= 8G>T mous SNV pathogenic significance (s=0.002;rank (s=0.996;rank (s=0.99;rank= (s=0.039;rank (s=0.961;rank (s=0.55;rank= (s=2.86;rank= 0.72;rank=0. =0.809 0.935;rank=0 (s=0.194;rank =0.246 =0.873 =0.761 (s=0.897;rank 0.756 =0.784) =0.67) 0.774) =0.242) =0.81) 0.145) 0.494) 342) .434) =0.548) =0.501) ASXL1 NM_015338 12 c.C1774T p.Q592X chr20:31022289 stopgain P38 827.02 44.22 692 1565 Likely Uncertain COSM96400 3(haematopoietic_and_lymphoid_ D D s=12.011;ran s=0.998;rank D s=5.41;rank= C>T pathogenic significance tissue 1(large_intestine (s=0;rank=0.8 (s=1;rank=0.8 k=0.98 =0.887 (s=0.986;rank 0.782 43 1 =0.848 ASXL1 NM_015338 12 c.C1826T p.T609I chr20:31022341 nonsynony P4 736.82 46.68 302 647 Likely Uncertain D P P D D M T D s=0.25;rank= T T s=0.012;rank s=5.318;rank s=0.998;rank D s=5.41;rank= C>T mous SNV pathogenic significance (s=0.001;rank (s=0.931;rank (s=0.546;rank (s=0;rank=0.4 (s=0.939;rank (s=2.39;rank= (s=0.7;rank=0 (s=4.83;rank= 0.485 (s=0.744;rank (s=0.19;rank= =0.31 =0.72 =0.841 (s=0.931;rank 0.782 =0.784 =0.571 =0.503 57 =0.372 0.691 .515 0.809 =0.583 0.542 =0.571 ASXL1 NM_015338 12 c.G1924T p.G642X chr20:31022439 stopgain P11 448.29 39.56 72 182 Likely Uncertain COSM110710 1(haematopoietic_and_lymphoid_ D D s=13.06;rank s=0.993;rank D s=5.55;rank= G>T pathogenic significance tissue (s=0;rank=0.8 (s=1;rank=0.8 =0.988 =0.583 (s=0.978;rank 0.832 43 1 =0.768 ASXL1 NM_015338 12 c.C2077T p.R693X chr20:31022592 stopgain P2 317.3 50 34 68 Likely Uncertain rs373221034 COSM51388 2(large_intestine 0.0002 0.0001 N D s=13.129;ran s=0.998;rank D s=5.22;rank= C>T pathogenic significance 11(haematopoietic_and_lymphoid (s=0.007;rank (s=1;rank=0.8 k=0.989 =0.897 (s=0.948;rank 0.722 _tissue =0.317 1 =0.623 ASXL1 NM_015338 12 c.C2077T p.R693X chr20:31022592 stopgain P2 250.81 34.02 33 97 Likely Uncertain rs373221034 COSM51388 2(large_intestine 0.0002 0.0001 N D s=13.129;ran s=0.998;rank D s=5.22;rank= C>T pathogenic significance 11(haematopoietic_and_lymphoid (s=0.007;rank (s=1;rank=0.8 k=0.989 =0.897 (s=0.948;rank 0.722 _tissue =0.317 1 =0.623 ASXL1 NM_015338 12 c.G2629T p.E877X chr20:31023144 stopgain P4 623.53 46.67 147 315 Likely Uncertain N D s=10.42;rank s=0.993;rank N s=4;rank=0.4 G>T pathogenic significance (s=0.388;rank (s=1;rank=0.8 =0.968 =0.574 (s=0.428;rank 55 =0.133 1 =0.267 ASXL1 NM_015338 12 c.G4189A p.G1397S chr20:31024704 nonsynony P106 953.15 42.59 638 1498 Benign Benign rs146464648 COSM133033 3(haematopoietic_and_lymphoid_ not provided RCV000120111.1 Likely 0.0024 0.0003 P B N D L T N s=0.072;rank T T s=0.005;rank s=2.628;rank s=0.991;rank D s=4.69;rank= G>A mous SNV tissue benign RCV000353069.1 (s=0.792;rank (s=0.255;rank (s=0.007;rank (s=0.906;rank (s=1.845;rank (s=2.53;rank= (s=0.52;rank= =0.163 (s=1.006;rank (s=0.024;rank =0.128 =0.377 =0.527 (s=0.981;rank 0.584 =0.427 =0.375 =0.32 =0.363 =0.488 0.14 0.162 =0.282 =0.104 =0.792 16

SUPPLEMENTAL DOCUMENTS

ASXL1 NM_015338: 12 c.1934dupG p. chr20:g.3102244 frameshift P89 515.85 47.13 82 174 Benign rs756958159 COSM1411076 1(large_intestine 0.031 0.0016 exon12:c.193 G646WfsX1 1>G insertion 4dupG(p. 2 G646WfsX12 ATM NM_000051 29 c.T4324C p.Y1442H chr11:10816041 nonsynony P5 43.11 96 180 Likely Uncertain rs201666889 Benign RCV000115190.6 Uncertain 0.0007 0.0004 D D D D D M T D s=0.976;rank D D s=0.242;rank s=5.844;rank s=0.998;rank D s=5.36;rank= 6T>C mous SNV pathogenic significance significance RCV000168302.4 (s=0;rank=0.9 (s=1;rank=0.8 (s=0.998;rank (s=0;rank=0.8 (s=1;rank=0.5 (s=2.85;rank= (s=0.73;rank= (s=4.12;rank= =0.978 (s=0.03;rank= (s=0.507;rank =0.888 =0.8 =0.923 (s=0.99;rank= 0.765 Uncertain significance 12 99 =0.916 43 88 0.831 0.731 0.75 0.829 =0.814 0.901 RCV000212011.3 CXCR4 NM_0010085 1 c.C452T p.P151L chr2:136873058 nonsynony P2 142.59 21.92 32 146 Uncertain Uncertain T B B D D N T N s=0.456;rank T T s=0.017;rank s=3.465;rank s=0.955;rank D s=5.79;rank= 40 G>A mous SNV significance significance (s=0.18;rank= (s=0.002;rank (s=0.026;rank (s=0;rank=0.6 (s=1;rank=0.8 (s=0.185;rank (s=0.48;rank= (s=1.91;rank= =0.524 (s=0.919;rank (s=0.168;rank =0.382 =0.474 =0.265 (s=0.925;rank 0.917 0.219 =0.144 =0.195 29 1 =0.042 0.703 0.444 =0.457 =0.508 =0.556 ETV6 NM_001987 5 c.C799T p.Q267X chr12:12022693 stopgain P4 1338.24 59.95 259 432 Likely Likely D A s=13.641;ran s=0.998;rank D s=5.76;rank= C>T pathogenic pathogenic (s=0;rank=0.8 (s=1;rank=0.8 k=0.993 =0.891 (s=0.985;rank 0.907 43 1 =0.828 IDH2 NM_002168 4 c.G419A p.R140Q chr15:90631934 nonsynony P2 42.27 52 123 Likely Uncertain rs121913502 COSM41590 417(haematopoietic_and_lymphoi Pathogenic RCV000015831.26 0.0003 0.0001 D D D D A H D D s=0.98;rank= D D s=0.371;rank s=6.063;rank s=0.999;rank D s=4.75;rank= C>T mous SNV pathogenic significance d_tissue 1(NS Pathogenic RCV000292094.1 (s=0;rank=0.9 (s=1;rank=0.8 (s=0.998;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=4.16;rank= (s=2.22;rank= (s=3.81;rank= 0.982 (s=0.918;rank (s=0.852;rank =0.928 =0.835 =0.983 (s=0.983;rank 0.598 12 99 =0.875 43 1 0.977 0.871 0.718 =0.959 =0.951 =0.813 IDH2 NM_002168 4 c.G419A p.R140Q chr15:90631934 nonsynony P38 858.62 87.1 770 884 Likely Uncertain rs121913502 COSM41590 417(haematopoietic_and_lymphoi Pathogenic RCV000015831.26 0.0003 0.0001 D D D D A H D D s=0.98;rank= D D s=0.371;rank s=6.063;rank s=0.999;rank D s=4.75;rank= C>T mous SNV pathogenic significance d_tissue 1(NS Pathogenic RCV000292094.1 (s=0;rank=0.9 (s=1;rank=0.8 (s=0.998;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=4.16;rank= (s=2.22;rank= (s=3.81;rank= 0.982 (s=0.918;rank (s=0.852;rank =0.928 =0.835 =0.983 (s=0.983;rank 0.598 12 99 =0.875 43 1 0.977 0.871 0.718 =0.959 =0.951 =0.813 IKZF1 NM_0012918 3 c.G85T p.E29X chr7:50367278G stopgain P9 1156.66 46.12 392 850 Likely Likely D A s=12.295;ran s=0.998;rank D s=5.92;rank= 47 >T pathogenic pathogenic (s=0;rank=0.8 (s=1;rank=0.8 k=0.982 =0.842 (s=0.995;rank 0.955 43 1 =0.968 IKZF1 NM_006060 5 c.C445T p.Q149X chr7:50450261C stopgain P89 335.98 33.43 228 682 Likely Likely D A s=12.21;rank s=0.998;rank D s=6.08;rank= >T pathogenic pathogenic (s=0;rank=0.8 (s=1;rank=0.8 =0.981 =0.879 (s=0.988;rank 0.99 43 1 =0.871 IKZF3 NM_183229 5 c.C565A p.L189I chr17:37947696 nonsynony P5 478.39 33.92 58 171 Likely Uncertain D D D D M T N s=0.735;rank T T s=0.048;rank s=6.134;rank s=0.996;rank D s=5.49;rank= G>T mous SNV pathogenic significance (s=0.999;rank (s=0.995;rank (s=0;rank=0.5 (s=1;rank=0.8 (s=2.315;rank (s=0.6;rank=0 (s=1.82;rank= =0.743 (s=0.184;rank (s=0.401;rank =0.635 =0.846 =0.759 (s=0.987;rank 0.809 =0.899 =0.916 04 1 =0.665 .571 0.427 =0.781 =0.753 =0.858 JAK2 NM_004972 14 c.G1849T p.V617F chr9:5073770G> nonsynony P9 2124.45 56.07 2706 4826 Likely Likely rs77375493 COSM12600 1(central_nervous_system 3(lung Pathogenic,Pathogenic 0.0008 0.0007 D D D D D M D D s=0.94;rank= D D s=0.338;rank s=6.874;rank s=0.995;rank D s=5.51;rank= T mous SNV pathogenic pathogenic 33324(haematopoietic_and_lymp RCV000022629.25,RCV000015769.7 (s=0.002;rank (s=0.996;rank (s=0.934;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.085;rank (s=1.69;rank= (s=3.86;rank= 0.934 (s=0.442;rank (s=0.681;rank =0.92 =0.93 =0.697 (s=0.945;rank 0.817 hoid_tissue Pathogenic RCV000015770.7 =0.721 =0.67 =0.652 43 1 =0.579 0.83 0.724 =0.897 =0.89 =0.611 Pathogenic RCV000015771.7 other RCV000015772.68 Pathogenic RCV000022627.7 other RCV000022628.8 Pathogenic RCV000279716.1 KRAS NM_004985 3 c.A183C p.Q61H chr12:25380275 nonsynony P1 1274.84 38.59 318 824 Likely Uncertain rs17851045 COSM554 1(ovary 5(endometrium 1(breast Pathogenic,Pathogenic D B B D D M D D s=0.798;rank D D s=0.105;rank s=3.862;rank s=0.997;rank D s=5.77;rank= T>G mous SNV pathogenic significance COSM1135364 2(skin 2(small_intestine 4(liver RCV000038259.2,RCV000154530.2 (s=0.007;rank (s=0.411;rank (s=0.092;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=3.08;rank= (s=1.81;rank= (s=4.39;rank= =0.79 (s=0.264;rank (s=0.613;rank =0.781 =0.521 =0.812 (s=0.932;rank 0.91 1(kidney 35(pancreas =0.599 =0.333 =0.326 43 1 0.875 0.84 0.786 =0.87 =0.863 =0.573 1(urinary_tract 1(biliary_tract 65(large_intestine 13(lung 1(thyroid 9(haematopoietic_and_lymphoid_ tissue MET NM_000245 2 c.A1127G p.K376R chr7:116340265 nonsynony P11 2228.39 48.98 410 837 Uncertain Uncertain COSM1447453 1(large_intestine T B B N N L T N s=0.134;rank T T s=0.005;rank s=0.821;rank s=0.982;rank D s=3.67;rank= A>G mous SNV significance significance (s=0.174;rank (s=0.028;rank (s=0.018;rank (s=0.011;rank (s=0.874;rank (s=1.52;rank= (s=2.78;rank= (s=1.15;rank= =0.184 (s=1.086;rank (s=0.028;rank =0.115 =0.172 =0.389 (s=0.872;rank 0.411 =0.289 =0.184 =0.188 =0.297 =0.281 0.385 0.113 0.295 =0.063 =0.121 =0.464 NOTCH2 NM_024408 34 c.A6295G p.M2099V chr1:120459050 nonsynony P11 584.73 26.37 307 1164 Likely Likely benign rs201584590 0.001 0.0001 T B B U N N T N s=0.166;rank T T s=0.011;rank s=0.816;rank s=0.666;rank N s=0.188;rank T>C mous SNV benign (s=0.193;rank (s=0;rank=0.0 (s=0;rank=0.0 (s=0.254;rank (s=1;rank=0.0 (s=0.755;rank (s=1.45;rank= (s=1.32;rank= =0.2 (s=1.014;rank (s=0.112;rank =0.29 =0.039 =0.079 (s=0.267;rank =0.143 =0.209 26 13 =0.154 9 =0.192 0.809 0.33 =0.257 =0.402 =0.228 NRAS NM_002524 2 c.G35A p.G12D chr1:115258747 nonsynony P8 717.61 35.67 173 485 Likely Likely rs121913237 COSM564 60(skin 3(biliary_tract Pathogenic RCV000037576.2 0.0001 0 D B B U D M T D s=0.951;rank D D s=0.149;rank s=6.806;rank s=0.998;rank D s=5.58;rank= C>T mous SNV pathogenic pathogenic 49(large_intestine 1(genital_tract Pathogenic,Pathogenic (s=0.011;rank (s=0.372;rank (s=0.399;rank (s=0;rank=0.5 (s=1;rank=0.8 (s=3.105;rank (s=1.19;rank= (s=5.35;rank= =0.947 (s=0.16;rank= (s=0.565;rank =0.831 =0.925 =0.891 (s=0.911;rank 0.843 1(kidney 1(pancreas 1(ovary RCV000158986.1,RCV000203450.1 =0.555 =0.323 =0.427 59 1 =0.879 0.784 0.847 0.852 =0.842 =0.525 2(soft_tissue 4(endometrium Pathogenic,Pathogenic 2(thyroid 1(lung 3(testis RCV000380895.1,RCV000032849.7 324(haematopoietic_and_lymphoi Pathogenic RCV000144963.3 d_tissue Pathogenic RCV000158980.2 3(central_nervous_system 1(upper_aerodigestive_tract 2(NS NRAS NM_002524 2 c.G34C p.G12R chr1:115258748 nonsynony P37 678.51 29.14 250 858 Likely Likely rs121913250 COSM561 13(haematopoietic_and_lymphoid Pathogenic,Pathogenic,Pathogenic D P P U D M T D s=0.894;rank T T s=0.071;rank s=6.917;rank s=0.999;rank D s=5.58;rank= C>G mous SNV pathogenic pathogenic _tissue 1(lung 1(NS RCV000158979.1,RCV000158985.2,R (s=0.021;rank (s=0.613;rank (s=0.602;rank (s=0;rank=0.5 (s=1;rank=0.8 (s=2.275;rank (s=1.16;rank= (s=6.14;rank= =0.883 (s=0.081;rank (s=0.479;rank =0.711 =0.933 =0.986 (s=0.928;rank 0.843 1(large_intestine 1(urinary_tract CV000158978.2 Pathogenic =0.491 =0.378 =0.49 59 1 =0.649 0.781 0.901 =0.805 =0.801 =0.563 6(skin 1(salivary_gland RCV000212761.1 SRSF2 NM_003016 1 c.C284T p.P95L chr17:74732959 nonsynony P71 124.5 36.54 38 104 Likely Uncertain rs751713049 COSM146288 53(haematopoietic_and_lymphoid 0.0001 0.0001 D P P N D M T D s=0.203;rank T T s=0.971;rank s=3.407;rank s=0.966;rank D s=2.76;rank= G>A mous SNV pathogenic significance COSM211506 _tissue (s=0.001;rank (s=0.753;rank (s=0.462;rank (s=0;rank=0.6 (s=1;rank=0.8 (s=2.22;rank= (s=0.9;rank=0 (s=5.95;rank= =0.249 (s=0.484;rank (s=0.363;rank =0.998 =0.468 =0.3 (s=0.89;rank= 0.314 COSM211028 =0.784 =0.414 =0.447 29 1 0.631 .749 0.891 =0.692 =0.724 0.489 SRSF2 NM_003016 1 c.C284A p.P95H chr17:74732959 nonsynony P37 159.79 41.73 53 127 Likely Uncertain rs751713049 COSM211029 80(haematopoietic_and_lymphoid 0.0002 0.0001 D D D D D M T D s=0.275;rank T D s=0.981;rank s=4.52;rank= s=0.937;rank D s=2.76;rank= G>T mous SNV pathogenic significance COSM211505 _tissue (s=0.001;rank (s=1;rank=0.8 (s=0.984;rank (s=0;rank=0.6 (s=1;rank=0.8 (s=2.565;rank (s=0.94;rank= (s=5.56;rank= =0.372 (s=0.044;rank (s=0.532;rank =0.999 0.605 =0.231 (s=0.901;rank 0.314 COSM211504 =0.784 99 =0.745 29 1 =0.752 0.753 0.863 =0.813 =0.826 =0.507 TET2 NM_0011272 10 c.4285dupT p.E1428fsX chr4:106193822 frameshift P31 1737.4 36.88 2476 6714 Likely 08 >T insertion pathogenic

TET2 NM_0011272 3 c.C64A p.P22T chr4:106155163 nonsynony P5 202.65 23 23 100 Likely Uncertain B B U N M T N s=0.171;rank T T s=0.057;rank s=2.061;rank s=0.951;rank N s=1.68;rank= 08 C>A mous SNV pathogenic significance (s=0.03;rank= (s=0.008;rank (s=0.153;rank (s=1;rank=0.1 (s=1.995;rank (s=3.51;rank= (s=1.46;rank= =0.218 (s=1.029;rank (s=0.058;rank =0.67 =0.309 =0.257 (s=0.26;rank= 0.231 0.272 =0.287 =0.179 82 =0.543 0.35 0.634 =0.207 =0.242 0.227 TET2 NM_0011272 3 c.650delC p.V218W chr4:106155749 nonsynony P9 5000 85.9 2279 2653 Likely Uncertain D D D U D M T N s=0.796;rank T T s=0.095;rank s=4.996;rank s=0.999;rank D s=4.95;rank= 08 C> mous SNV pathogenic significance (s=0;rank=0.9 (s=1;rank=0.8 (s=0.963;rank (s=0.59;rank= (s=0.941;rank (s=2.42;rank= (s=2.66;rank= (s=1.42;rank= =0.79 (s=1.163;rank (s=0.08;rank= =0.765 =0.672 =0.955 (s=0.856;rank 0.648 12 99 =0.79 0.056 =0.373 0.702 0.126 0.35 =0.007 0.318 =0.444 TET2 NM_0011272 3 c.G2392T p.E798X chr4:106157491 stopgain P31 381.34 27.36 776 2836 Likely Uncertain COSM110792 1(haematopoietic_and_lymphoid_ N A s=10.769;ran s=0.997;rank D s=4.14;rank= 08 G>T pathogenic significance tissue (s=0;rank=0.0 (s=1;rank=0.8 k=0.97 =0.787 (s=0.64;rank= 0.477 02 1 0.32 TET2 NM_0011272 3 c.A2698T p.K900X chr4:106157797 stopgain P3 603.39 48.78 301 617 Likely Uncertain A s=10.37;rank s=0.988;rank N s=3.16;rank= 08 A>T pathogenic significance (s=1;rank=0.8 =0.967 =0.46 (s=0.311;rank 0.353 1 =0.24 TET2 NM_0011272 3 c.C2746T p.Q916X chr4:106157845 stopgain P71 1406.47 91.47 643 703 Likely Uncertain rs780710758 COSM43417 12(haematopoietic_and_lymphoid 0.0001 0 U A s=10.694;ran s=0.996;rank D s=4.93;rank= 08 C>T pathogenic significance _tissue (s=0.048;rank (s=1;rank=0.8 k=0.969 =0.769 (s=0.933;rank 0.643 =0.233 1 =0.577 TET2 NM_0011272 3 c.3140_3141del L1048QfsX chr4:106158239 frameshift P36 38.38 147 383 Likely 08 CT CT> deletion pathogenic

TET2 NM_0011272 3 c.G3404A p.C1135Y chr4:106158503 nonsynony P11 623.5 41.53 98 236 Likely Uncertain rs769422572 0.0001 0 D D D N D M T D s=0.79;rank= T T s=0.062;rank s=5.166;rank s=0.997;rank D s=4.51;rank= 08 G>A mous SNV pathogenic significance (s=0;rank=0.9 (s=0.999;rank (s=0.971;rank (s=0.245;rank (s=1;rank=0.8 (s=3.005;rank (s=1.48;rank= (s=9.85;rank= 0.824 (s=1.05;rank= (s=0.242;rank =0.685 =0.697 =0.785 (s=0.924;rank 0.544 12 =0.899 =0.818 =0.036 1 =0.862 0.317 0.988 0.145 =0.61 =0.555 17

SUPPLEMENTAL DOCUMENTS

TET2 NM_0011272 7 c.A3881G p.Y1294C chr4:106180853 nonsynony P6 1657.4 34.25 958 2797 Likely Uncertain D D D D M T D s=0.815;rank T T s=0.466;rank s=5.855;rank s=0.998;rank D s=5;rank=0.6 08 A>G mous SNV pathogenic significance (s=0;rank=0.9 (s=1;rank=0.8 (s=0.999;rank (s=1;rank=0.8 (s=2.51;rank= (s=2.43;rank= (s=8.03;rank= =0.953 (s=0.819;rank (s=0.141;rank =0.946 =0.802 =0.902 (s=0.986;rank 61 12 99 =0.916 1 0.734 0.153 0.966 =0.54 =0.461 =0.842 TET2 NM_0011272 8 c.T3986C p.L1329P chr4:106182947 nonsynony P37 1689.42 92.74 281 303 Likely Uncertain D D D D M T D s=0.888;rank T T s=0.343;rank s=6.471;rank s=0.999;rank D s=5.36;rank= 08 T>C mous SNV pathogenic significance (s=0;rank=0.9 (s=1;rank=0.8 (s=1;rank=0.9 (s=1;rank=0.8 (s=2.51;rank= (s=2.14;rank= (s=6.72;rank= =0.996 (s=0.737;rank (s=0.169;rank =0.921 =0.892 =0.992 (s=0.986;rank 0.765 12 99 71 1 0.734 0.194 0.925 =0.587 =0.509 =0.844 TP53 NM_0011261 4 c.367_368delTG p.V122DfsX chr17:7579321C frameshift P38 422.82 40.52 124 306 Likely 12 A> substitution pathogenic

TP53 NM_0012766 5 c.A503G p.H168R chr17:7578427T nonsynony P5 679.95 98.25 112 114 Likely Uncertain rs867114783 COSM3388210 1(breast 1(pancreas D P N D M D D s=0.216;rank D D s=0.479;rank s=2.461;rank s=0.975;rank D s=0.619;rank 99 >C mous SNV pathogenic significance COSM3388206 3(large_intestine 1(soft_tissue (s=0.964;rank (s=0.781;rank (s=0.025;rank (s=0.979;rank (s=3.105;rank (s=6.8;rank=0 (s=7.5;rank=0 =0.649 (s=1.055;rank (s=0.984;rank =0.948 =0.357 =0.34 (s=0.968;rank =0.167 COSM3388207 2(ovary 1(endometrium 1(lung =0.622 =0.644 =0.262 =0.395 =0.879 .998 .952 =0.982 =0.995 =0.71 COSM3388208 1(haematopoietic_and_lymphoid_ COSM3388211 tissue 1(central_nervous_system COSM43545 1(stomach 1(oesophagus COSM3388209 TP53 NM_0011261 8 c.T808A p.F270I chr17:7577130A nonsynony P8 168.47 22.22 36 162 Likely Uncertain COSM43809 1(breast 1(large_intestine D D D D D M D D s=0.874;rank D D s=0.693;rank s=6.646;rank s=0.992;rank D s=5.13;rank= 12 >T mous SNV pathogenic significance COSM1646805 2(oesophagus 2(lung (s=0.002;rank (s=1;rank=0.8 (s=0.994;rank (s=0;rank=0.8 (s=1;rank=0.8 (s=2.225;rank (s=6.8;rank=0 (s=5.2;rank=0 =0.862 (s=1.015;rank (s=0.985;rank =0.975 =0.911 =0.535 (s=0.998;rank 0.696 COSM3937598 =0.784 99 =0.807 43 1 =0.632 .998 .837 =0.974 =0.996 =0.992 COSM437484 ZEB2 NM_0011716 6 c.C801A p.F267L chr2:145158809 nonsynony P5 289.43 34.78 32 92 Likely Uncertain D D D D M T D s=0.798;rank T T s=0.099;rank s=5.688;rank s=0.998;rank D s=4.47;rank= 53 G>T mous SNV pathogenic significance (s=0.998;rank (s=0.998;rank (s=0;rank=0.8 (s=1;rank=0.5 (s=2.65;rank= (s=0.82;rank= (s=5.65;rank= =0.911 (s=0.203;rank (s=0.4;rank=0 =0.772 =0.776 =0.918 (s=0.977;rank 0.536 =0.715 =0.875 43 88 0.778 0.741 0.87 =0.776 .752 =0.763 ZRSR2 NM_005089 4 c.263delA p.K88RfsX chrX:15821871A frameshift P6 322.22 75.53 142 188 Uncertain Uncertain T B B N D L T N s=0.31;rank= T T s=0.006;rank s=2.645;rank s=0.933;rank D s=1.22;rank= > deletion significance significance (s=0.13;rank= (s=0.047;rank (s=0.024;rank (s=0.043;rank (s=0.726;rank (s=1.795;rank (s=2.03;rank= (s=1.78;rank= 0.371 (s=1.064;rank (s=0.036;rank =0.162 =0.379 =0.224 (s=0.792;rank 0.202 0.267 =0.206 =0.19 =0.238 =0.337 =0.474 0.21 0.42 =0.107 =0.155 =0.389 ZRSR2 NM_005089 7 c.C505T p.R169X chrX:15827389C stopgain P2 93.07 121 130 Likely Likely COSM1716885 1(haematopoietic_and_lymphoid_ N A s=12.662;ran s=0.998;rank D s=5.46;rank= >T pathogenic pathogenic tissue (s=0.02;rank= (s=1;rank=0.8 k=0.985 =0.919 (s=0.899;rank 0.799 0.272 1 =0.503 ZRSR2 NM_005089 8 c.C700T p.Q234X chrX:15833942C stopgain P36 119.99 52.17 12 23 Likely Likely D A s=12.985;ran s=0.998;rank D s=5.41;rank= >T pathogenic pathogenic (s=0;rank=0.8 (s=1;rank=0.8 k=0.988 =0.916 (s=0.906;rank 0.782 43 1 =0.517 ZRSR2 NM_005089 8 c.G751A p.G251R chrX:15833993G nonsynony P3 553.87 94.17 97 103 Likely Uncertain D D D D D H D s=0.938;rank D D s=0.709;rank s=6.676;rank s=0.999;rank D s=5.41;rank= >A mous SNV pathogenic significance (s=0;rank=0.9 (s=1;rank=0.8 (s=1;rank=0.9 (s=0;rank=0.8 (s=1;rank=0.8 (s=4.1;rank=0 (s=7.82;rank= =0.932 (s=0.449;rank (s=0.605;rank =0.976 =0.914 =0.996 (s=0.98;rank= 0.782 12 99 71 43 1 .974 0.96 =0.898 =0.86 0.788

18