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A thesis of the Master of Philosophy degree

Molecular and cytogenetic features and their clinical implications of Korean Philadelphia chromosome-negative

myeloproliferative neoplasm patients reclassified by 2016 WHO criteria

2016 년 WHO 진단기준에 따라

재분류한 한국인 필라델피아

염색체 음성 골수증식종양 환자의

분자 및 세포 유전학적 특성과

이에 따른 임상적 의의

February 2020

Department of MedicineLaboratory Medicine Major

Seoul National University College of MedicineJiwon Yun

의학석사 학위논문

2016년 WHO 진단기준에 따라

재분류한 한국인 필라델피아

염색체 음성 골수증식종양 환자의

분자 및 세포 유전학적 특성과

이에 따른 임상적 의의

Molecular and cytogenetic features and their clinical implications of Korean Philadelphia chromosome-negative

myeloproliferative neoplasm patients reclassified by 2016 WHO criteria

2020년 2월

서울대학교 대학원의학과 검사의학전공

윤 지 원

i

Abstract

Introduction: The 2016 World Health Organization (WHO)

myeloproliferative neoplasm (MPN) diagnostic criteria announced

updated criteria of prefibrotic primary myelofibrosis (prefibrotic PMF)

whose diagnosis is difficult in differentiating with essential

thrombocythemia (ET). Myeloproliferative neoplasm, unclassifiable

(MPN-U) shows findings that do not meet all the criteria for any

specific disorder. Herein, the first goal is to reclassify the diagnosis of

Philadelphia chromosome-negative MPN by from 2008 to 2016

classification in focus on MPN-U. The second one is to identify useful

markers to discriminate between ET and prefibrotic PMF. The last one

is to broaden the understanding of characteristics of Korean MPN

patients using the clinical, cytogenetic, telomere lengths, molecular,

and BM histologic features.

Methods: In 53 MPN and 6 AML evolved from MPN patients

diagnosed in Seoul National University Hospital between 2005 and

2014, G-banding, fluorescence in situ hybridization (FISH), targeted

capture sequencing for 88 hematopoiesis-related genes, and telomere

length (TL) measurement were performed. Survival analysis was

performed including the assessment of progression. Additionally, 99

MPN cases diagnosed in the hospital between 2017 and 2019 were

analyzed to find out CD34 positive megakaryocytes.

Results: By applying 2016 WHO criteria, 38.5% and 15.4% of MPN-U

were reclassified to prefibrotic PMF and overt PMF, respectively. In

ii

survival analysis, the updated criteria showed a better stratification of

MPN diagnosis. In our study, the mutated genes found in prefibrotic

PMF but not in ET were CSF3R, DNMT3A, SF3B1, and SRSF2, which

are potential candidate markers for the differential diagnosis between

ET and prefibrotic PMF. The genomic profile of Korean MPN was

similar to that of the previous study. We found novel MPL mutations

(MPL D128N, D261Y) in a PMF patient. Although telomere length of

ET was not shorter than that of normal control, that of prefibrotic PMF

was (P = 0.0635), suggesting telomere length as a potential marker for

differentiating two diseases. 44.4% of MPN cases showed CD34-

positive megakaryocytes but CD34 positive percentages in total

megakaryocytes are not different between ET and prefibrotic PMF.

Additional ASXL1 mutation was related to lower hemoglobin

concentration in PMF patients.

Conclusion: By revealing the reallocation of MPN-U into prefibrotic

PMF and overt PMF according to 2016 WHO criteria, we found that

the updated criteria provide a precise diagnosis of MPN. Genomic

study and telomere length analysis can help the discrimination of ET

and prefibrotic PMF. Our overall analysis provides a wider

understanding of the clinical, cytogenetic, telomere lengths, molecular,

and BM histologic findings of Korean MPN patients.

----------------------------------------------------------------------------------------------

Keywords: Myeloproliferative neoplasm; WHO classification;

Myeloproliferative neoplasm, unclassifiable; essential thrombocythemia;

early/prefibrotic primary myelofibrosis; telomere length; multigene

sequencing; CD34 positive megakaryocyte

Student Number: 2018 – 25644

iii

CONTENTS

Abstract ...................................................................................................... i

Contents....................................................................................................iii

List of tables and figures.......................................................................... iv

List of abbreviations ................................................................................vi

Introduction .............................................................................................. 1

Material and Methods............................................................................... 6

Results ..................................................................................................... 18

Discussion ................................................................................................ 55

References ............................................................................................... 62

Abstract in Korean.................................................................................. 65

iv

LIST OF TABLES AND FIGURES

Figure 1 Summary of the present study design 6

Table 1. Gene panel for targeted sequencing 14

Figure 2. Variant call strategy 17

Table 2. Baseline characteristics of the 59 MPN patients 19

Table 3. Classification of MPN subtypes according to 2008 and 2016

WHO criteria 20

Figure 3. Transitions of the diagnosis in patients with ET, MPN-U or

PMF between WHO classification 2008 and 2016 22

Figure 4. Cytogenetic features detected by conventional G-banding

and/or fluorescent in situ hybridization (FISH) in 52 patients with

BCR/ABL1-negative MPN patients 23

Table 4. Cytogenetic analysis by G-banding and/or FISH in 53 MPN

and 6 AML evolved from MPN patients 24

Figure 5. Mutation profile of 53 Korean MPN patients 29

Figure 6. Distribution of mutations of 53 MPN and 6 AML evolved

from MPN patients using 88 genes panel 30

Table 5. Number of mutated genes per patient according to the MPN

subtypes 33

Table 6. Comparison of the variant allele frequency of ASXL1, CALR,

and TET2 in ET, prefibrotic PMF, and overt PMF 35

Table 7. Frequency of canonical mutated and triple-negative Korean

MPN patients 39

Figure 7. Distribution of CALR mutation and its subtypes in 53 MPN

v

and 6 AML evolved from MPN patients 40

Figure 8. Telomere length (telomere/centromere ratio) according to

the MPN subtype, AML evolved from MPN, and normal adult

control 42

Figure 9. Kaplan-Meier curves according to the MPN subtypes and

AML evolved from MPN (a) overall survival rate (b) progression-free

survival rate 45

Figure 10. Comparison of the survival curves between WHO

classification 2008 and 2016 46

Figure 11. Correlogram of mutated genes in (A) 53 MPN patients. (B)

33 PMF patients 47

Figure 12. (A) CD34-positive megakaryocytes positive rates (B)

CD34-positive percentage of total megakaryocytes according to MPN

subtypes 53

Table 8. Specific mutations and their clinical relevance 54

vi

LIST OF ABBREVIATIONS

MPN; Myeloproliferative neoplasm

PMF; Primary myelofibrosis

MPN-U; Myeloproliferative neoplasm, unclassifiable

ET; Essential thrombocythemia

AML; Acute myeloid leukemia

JAK2; Janus kinase 2

MPL; Myeloproliferative leukemia virus oncogene

CALR; Calreticulin

VAF; variant allele frequency

1

Introduction

The first description of the entity prefibrotic PMF(primary

myelofibrosis) was published in 1999 (1). 2001 World Health

Organization (WHO) myeloid classification described clinical and

pathologic findings of pre-fibrotic stage of PMF. 2008 WHO

classification also defined diagnostic criteria of PMF including pre-

fibrotic stage. 2016 WHO classification separated diagnostic criteria of

prefibrotic PMF from overt PMF. Therefore, the diagnostic criteria for

prefibrotic PMF have altered over time, although the fundamental

histopathological features have not, and the minor criteria have been

changed.

2016 WHO Classification of Tumors of Hematopoietic and

Lymphoid Tissues have two major changes compared to the 2008 WHO

classification in the diagnosis of primary myelofibrosis. It defines the

diagnostic criteria of prefibrotic PMF. Prefibrotic PMF is characterized

by milder fibrosis (MF-0~1) than overt PMF and the absence of

leukoerythroblastic reaction. Leukoerythroblastic reaction is one of

several minor criteria used to diagnose overt PMF. Additionally, 2016

WHO includes the major clonal CALR marker and suggests that

2

mutations of JAK2 V617F, CALR, MPL, ASXL1, EZH2, TET2,

IDH1/IDH2, SRSF2, and SF3B1 can help determine clonality (2).

Following the introduction of prefibrotic PMF, the differential

diagnosis between prefibrotic PMF and ET has become a difficult

problem, although histologic differences are suggested. Patients with

prefibrotic PMF exhibit a more frequent evolution to overt

myelofibrosis and acute leukemia and inferior overall survival

compared with those with ET (3). Patients with overt PMF showed

inferior OS compared with those with prefibrotic PMF. Overall,

survival was progressively shortened depending on the fibrosis grade in

patients with PMF (4). Altogether, these results support prefibrotic

PMF as a distinct clinicopathologic entity. Consequently, it has become

important to distinguish prefibrotic PMF from ET or overt PMF in bone

marrow examination.

Myeloproliferative neoplasm, unclassifiable (MPN-U) may be

diagnosed when patients meet the general criteria to classify their

conditions as MPNs, but do not meet all the criteria for any specific

disorder, or they may exhibit characteristics from more than one

category. MPN-U may represent very early stages or very late

transformational stages of a specific MPN, such as PV, ET, or PMF.

Regarding the heterogeneity of MPN-U, Gianelli et al. grouped MPN-

3

U cases into three morphological clusters by multiple correspondence

analysis—ET-like, PMF-like, and PV-like (5).

Iurlo et al. applied the 2016 revised diagnostic criteria to

previously published series of MPN-U by WHO 2008 criteria, to see

whether the use of the modified criteria would allow a more precise

MPN subtype diagnosis by decreasing the number of cases classified as

MPN-U. In their study, by means of the revised WHO 2016 diagnostic

criteria, MPN-U was reduced by about 30% (6).

As an example of the impact of mutations on the prognosis of

MPN, CALR mutations have been associated with favorable and ASXL1

mutations with unfavorable survival in PMF, independent of DIPSS-

plus risk category (7). CALR+ASXL1− patients showed the longest

survival, at a median of 10.4 vs 2.3 years in CALR–ASXL1+ patients vs

5.8 years in CALR+ASXL1+ or CALR−ASXL1− patients. Based on these

observations, 2 new prognostic models that incorporate mutational

status have been devised and presented at the 2014 American Society of

Hematology annual meeting. The first was referred to as MIPSS70+

Version 2.0 (Mutation and Karyotype-Enhanced International

Prognostic Scoring System for Primary Myelofibrosis), which included

high molecular risk (HMR) mutations (ASXL1, EZH2, SRSF2, IDH1,

IDH2, and U2AF1), and one favorable mutation (CALR type 1/like) (8).

4

The second was GIPSS (Genetically Inspired Prognostic Scoring

System for Primary Myelofibrosis), which identified that absence of

type 1/like CALR mutation and presence of ASXL1, SRSF2, or

U2AF1Q157 mutation, as inter-independent predictors of inferior

survival (9).

Telomere attrition and telomerase activity have been reported in

various hematologic neoplasms (10, 11). Regarding MPN, the telomere

length is reduced, and telomere activity is upregulated in Philadelphia

chromosome-negative MPN (12). A reduced telomere length in

Philadelphia chromosome-negative MPN was associated with a worse

prognosis. However, no study has investigated the telomere length

among MPN subtypes.

CD34-positive megakaryocytes were reported in various bone

marrow disorders such as myelodysplastic syndromes (13). Tang G. et

al. reported that high-level CD34 expression in megakaryocytes

independently predicted an adverse outcome in patients with

myelodysplastic syndromes (14). There is no study about CD34-

positive megakaryocytes in MPN and their subgroups.

We aimed to reclassify myeloproliferative neoplasm by WHO

2016 and to track the revised diagnosis of MPN-U. We also focused on

the differential markers between ET and prefibrotic PMF. This study

5

provides the integrative analysis of the clinical, cytogenetic, telomere

lengths, molecular features, and BM histologic findings of Korean

MPN patients.

6

Material and Methods

Summary of the present study design

Patients

Figure 1. Summary of the present study design

7

Fifty-three patients and six patients who were diagnosed with

MPN and AML evolved from MPN, respectively between 2005 and

2014 at Seoul National University Hospital were included in this study.

MPNs were diagnosed according to 2008, and 2016 WHO

classification criteria (2). Unless otherwise stated, the diagnosis was

based on the WHO classification 2016 version.

Bone marrow histological examination

Hematopathologists reviewed Wright-Giemsa-stained BM

smears and hematoxylin and eosin (H&E)-stained sections of the BM

trephine biopsies. In all patients, immunohistochemical (IHC) staining

was performed for reticulin and collagen using BM sections (all from

Dako, Glostrup, Denmark). In some patients, CD34, CD117, and CD61

IHC staining were performed (from Dako, Glostrup, Denmark). BM

fibrosis (MF) was assessed according to the European consensus

grading system on a scale of MF-0 to MF-3 (15).

We analyzed a total of 99 MPN cases diagnosed between 2017

and 2019 to find CD34-positive megakaryocytes using CD34 stained

BM biopsy section. They include 16 PV, 25 ET, 26 prefibrotic PMF, 19

overt PMF, 8 PVMF, and 5 ETMF cases.

8

G-banding & fluorescent in situ hybridization (FISH)

Chromosome analysis was performed using the conventional

G-banding technique. The heparinized BM samples were collected, and

white blood cells (WBCs) were sorted by centrifugation and cultured in

RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA)

at 37℃, in 5% CO2 for 24 hours. Colcemid treatment was performed to

inhibit mitosis. Each specimen in the medium was centrifuged, and the

upper layer was decanted. Next, KCl was added at 37℃ for 20 minutes.

For fixation, 1 mL of Carnoy’s solution was used. After preparation of

the slide, Leishman’s G-banding staining was performed according to

the standard protocol. At least 20 metaphase cells per patient were

analyzed using Metafer 4 software (MetaSystems, Altlussheim, FRG).

The karyotype designation was based on the principles of the

International System for Human Cytogenetic Nomenclature (ISCN

2013).

Interphase FISH analysis was performed on mononuclear cells

of BM aspirates to detect common cytogenetic abnormalities related to

MPN using BCR/ABL1 (Vysis Inc., Downers Grove, IL, USA), 13q

deletion (Vysis Inc., Downers Grove, IL, USA), trisomy 8 (Vysis Inc.,

Downers Grove, IL, USA), and 20q deletion (Vysis Inc., Downers

9

Grove, IL, USA). The FISH slides with BM cells were fixed with

methanol: acetic acid (3:1), treated with 2× sodium saline citrate (SSC)

for 30 minutes at 37°C and dehydrated with 70%, 85%, and 100%

ethanol for 3 minutes each. Next, 10 μL of the probe mixture solution

was placed onto the slides, which were codenatured at 75°C for 3

minutes. Thereafter, the slides were hybridized overnight at 39°C in a

humidified chamber. After hybridization, the slides were warmed in a

solution containing 0.4% SSC and 0.3% nonylphenol polyethylene

glycol (NP-40) at 73°C for 2 minutes. Subsequently, the chromosomal

DNA was counterstained with 6.6 μL of 4,6-diamidino-2-phenylindole

dihydrochloride. The fluorescent signals were analyzed using a

fluorescence microscope (Zeiss, Germany). At least 200 cells in each

specimen were assessed. The FISH results were recorded according to

ISCN 2013. The normal cut-off values for the deletion, amplification,

or translocation of chromosomal regions were based on the means (±

three standard deviations), and the binomial distribution function of 20

negative controls was analyzed.

Telomere quantitative fluorescence in situ hybridization

Telomere quantitative FISH was performed using the telomere

10

peptide nucleic acid (PNA) FISH kit (Dako Cytomation Denmark A/S,

Glostrup, Denmark). A Cy3-PNA probe was hybridized to telomeres,

and a fluorescein isothiocyanate (FITC)-labeled PNA probe (Dako) was

hybridized to the centromere of the chromosome. After mixing one

microliter of the centromere probe and ten microliters of the telomere

probe, quantitative FISH was performed according to the

manufacturer’s instructions. A Zeiss Axioplan 2 imaging microscope

(Carl Zeiss MicroImaging GmbH, München, Germany) was used to

visualize metaphase and interphase quantitative FISH. The ISIS-

Telomere module (MetaSystems GmbH, Altlussheim, Germany) was

used for analysis (16). The software calculates the telomere to

centromere (T/C) fluorescence intensity ratio, which is a measure of the

telomere length (TL), for each individual chromosome arm within each

metaphase and interphase nucleus (16). The T/C ratio multiplied by 100

was used as the TL. At least 30 interphase nuclei were scanned for each

sample, and at least two metaphase cells were karyotyped and captured

for TL measurements.

DNA extraction

Genomic DNA was extracted from frozen BM mononuclear

11

cells of all patients. All the patients did not have matched germline

samples sequenced. DNA was extracted using the MagNA Pure LC

DNA Isolation Kit (Roche Applied Science, Indianapolis, IN, USA)

according to the manufacturer’s instructions. The DNA quality was

analyzed by assessing the 260/280 absorbance ratio using an ND-1000

Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).

Multigene sequencing & variant calling strategy

Fifty-nine BM specimens of MPN patients were analyzed by

multigene targeted next-generation sequencing (NGS). An in-house

gene panel comprising 88 known hematopoiesis and other cancer-

related genes was used (Table 1). The algorithm to search for probable

somatic variants is shown in Fig 2.

Sequence quality control (QC) was performed using FastqQC

0.11.2 and was mapped to the human reference genome sequence NCBI

b37 using BWA-MEM 0.7.12. Potential PCR duplicates were removed

using Picard MarkDuplicates (http://broadinstitute.github.io/picard).

The BAM files were realigned using the Genome Analysis Toolkit

(GATK) 3.3 IndelRealigner, and base quality scores were recalibrated

using the GATK base quality recalibration tools. For germline variant

12

calling, GATK’s HaplotypeCaller was used. To filter out low-quality

variants, variants with either a depth <10 or a variant allele fraction <1%

were removed. Next, we removed common single-nucleotide

polymorphisms (SNPs) by removing variants that were annotated as

having a minor allele frequency in either of the following databases:

dbSNP 132, ≥ 0.005 allele frequency in 1000 Genomes (2012/04), ≥

0.005 allele frequency in ESP 6500, or ≥ 0.002 allele frequency in the

in-house Korean SNP database (n=917). We rescued candidate

mutations registered in the COSMIC V60 database. Annotation of the

variants was performed using ANNOVAR. The functional effects of the

missense variants were predicted using 3 types of in silico tools:

Sorting Tolerant From Intolerant (SIFT), PolyPhen 2 HDIV, and ‐

Combined Annotation Dependent Depletion (CADD).

Statistical analysis

Comparisons of the quantitative variables between two groups

of patients were carried out by Mann–Whitney test. The Kruskal–

Wallis test was applied when comparing more than two groups.

Estimates of the overall survival (OS) and progression-free survival

(PFS) were performed using the Kaplan–Meier method, and differences

13

among survival curves were analyzed using the log-rank test. Analysis

of the data was carried out using SPSS statistical analysis software

(SPSS 23.0 Inc, Chicago, IL, USA), and GraphPad Prism (GraphPad

Prism version 7.00 for Windows, GraphPad Software, La Jolla

California, USA), and R-project software. P values less than 0.05 were

considered statistically significant.

Ethics

This study complied with the Declaration of Helsinki. All BM

samples were collected with informed consent, and the study was

reviewed and approved by the Institutional Review Board of Seoul

National University College of Medicine (IRB No. 1311-091-535).

14

Table 1. Gene panel for targeted sequencing

Genes NCBI Id. Position Pathway/Ontology

ASXL1 171023 20q11.1 Chromatin modification

ATM 472 11q22.3 DNA repair

ATRX 546 Xq21.1 Chromatin modification

BARD1 580 2q35 DNA repair

BCOR 54880 Xp11.14 Transcription

BIRC3 330 11q22.2 Receptor/Kinases

BRAF 673 7q34 RAS pathway

BRCC3 79184 Xq28 DNA repair

BRD2 6046 6p21.3 Transcription

BRD4 23476 19p13.1 Other

CALR 811 19p13.13 Other

CARD6 84674 5p13.1 Other

CBL 867 11q23.3 RAS pathway

CCND1 595 11q13.3 Cell cycle

CDKN2A 1029 9p21 Cell cycle

CEBPA 1050 19q13.1 Transcription

CHD2 1106 15q26.1 Other

CSF1R 1436 5q32 Receptor/Kinases

CSF3R 1441 1p34.3 Receptor/Kinases

DAP3 7818 1q22 Other

DDX3X 1654 Xp11.4 Other

DIS3 22894 13q22.1 Other

DNMT3A 1788 2p23 DNA methylation

EEF1E1 9521 6p24.3 Other

EGR2 1959 10q21.3 Transcription

ETV6 2120 12p13.2 Transcription

EZH2 2146 7q35-36 Chromatin modification

FAM46C 54855 1p12 Other

FAT4 79633 4q28.1 Other

15

FBXW7 55294 4q31.3 Receptor/Kinases

FLT3 2322 13q12 Receptor/Kinases

GATA1 2623 Xp11.23 Transcription

GATA2 2624 3q21.3 Transcription

HIST1H1E 3008 6p22.2 Other

IDH1 3417 2q33.3 DNA methylation

IDH2 3418 15q26.1 DNA methylation

IKZF1 10320 7p13 Transcription

ITPKB 3707 1q42.12 Signaling

JAK2 3717 9p24 Receptor/Kinases

KIAA0355 9710 19q13.11 Other

KIT 3815 4q12 Receptor/Kinases

KLHL6 89857 3q27.1 Other

KRAS 3845 12p12.1 RAS pathway

LAMB4 22798 7q31.1 Other

LRP1B 53353 2q21.2 Other

MAPK1 5594 22q11.22 Signal/Kinase

MED12 9968 Xq13.1 Other

MPL 4352 1p34.2 Receptor/Kinases

MYD88 4615 3p22.2 Signaling

NF1 4763 17q11.2 RAS pathway

NFKBIE 4794 6p21.1 Other

NOTCH1 4851 9q34.3 Receptor/Kinases

NPM1 4869 5q35 Transcription

NRAS 4893 1p13.2 RAS pathway

PHF6 84295 Xq26.2 Transcription

PLEKHG5 57449 1p36.31 Other

POLG 5428 15q25 Other

POT1 25913 7q31.33 Other

PRKD3 23683 2p22.2 Signaling

PRPF40B 25766 12q13.12 Splicing

PTEN 5728 10q23.3 Other

16

PTPN11 5781 12q24.1 RAS pathway

RAD21 5885 8q24.11 Cohesin

RB1 5925 13q14 Cell cycle

RIPK1 8737 6p25.2 Other

RUNX1 861 21q22.3 Transcription

SAMHD1 25939 20q11.23 Other

SCRIB 23513 8q24.3 Other

SETBP1 26040 18q12.3 Other

SF1 7536 11q13.1 Splicing

SF3A1 10291 22q12.2 Splicing

SF3B1 23451 2q33.1 Splicing

SH2B3 10019 12q24.12 Signaling

SMARCA2 6595 9p24.3 Other

SMC1A 8243 Xp11.22 Cohesin

SMC3 9126 10q25.2 Cohesin

SRSF2 6427 17q25.1 Splicing

STAG2 10735 Xq25 Cohesin

TCF12 6938 15q21.3 Transcription

TET2 54790 4q24 DNA methylation

TGM7 116179 15q15.2 Other

TP53 7157 17p13.1 Transcription

U2AF1 7307 21q22.3 Splicing

U2AF2 11338 19q13.42 Splicing

WT1 7490 11p13 Transcription

XPO1 7514 2p15 Other

ZMYM3 9203 Xq13.1 Other

ZRSR2 8233 Xp22.1 Splicing

17

Figure 2. Variant call strategy

18

Results

Patient demographics

We included 59 consecutive MPN patients (29 males and 30

females; median age: 66 years; age range: 19–84 years). The median

follow-up period from the diagnosis was 54 months. The baseline

clinical parameters at diagnosis, canonical mutation, and prognostic

scoring of MPN are described in Table 2.

A total of 53 MPNs were retrospectively reclassified according

to 2008 and 2016 WHO classification criteria. Except for MPN-U, ET,

prefibrotic PMF, overt PMF, post-PV MF, post-ET MF patients by 2008

WHO criteria were not transitioned to other MPN subtypes in 2016

criteria (Table 3). According to WHO 2016 criteria, essential

thrombocythemia (n=4), prefibrotic primary myelofibrosis (n=8), overt

primary myelofibrosis (n=25), myeloproliferative neoplasm,

unclassifiable (n=6), post-ET myelofibrosis (n=5), and post-PV

myelofibrosis (n=5) were included. Additionally, 6 AML patients were

evolved from ET (n=4), PV (n=1), and PMF (n=1).

19

Table 2. Baseline characteristics of the 59 MPN patients

PV ET PMF MPNU

Total (N) 6 13 34 6

Age (median, range) 58 (49-72) 57 (28-73) 61 (42-84) 58 (19-70)

Female sex 3 (50.0%) 10 (76.9%) 16 (47.1%) 1 (16.7%)

F/U months (median, range) 141 (54-269) 102 (52-233) 66 (7-210) 64 (17-277)

Laboratory finding at initial diagnosis (mean ± SD)

Hemoglobin (g/dL) 17.9 ± 2.6 13.3 ± 2.2 11.2 ± 3.1 10.7 ± 2.9

Platelet (103/μl) 417.7 ± 185.7 1127.6 ± 498.1 499.9 ± 331.2 442.8 ± 349.6

WBC (103/μl) 14.2 ± 5.3 12.4 ± 5.7 11.6 ± 7.7 11.1 ± 6.1

Presence of thrombotic event 2 0 1 0

Secondary transformation (N, %)

Total 6 9 6 1

AML 2 5 5 0

sMF 4 4 NA 0

Lymphoid crisis 0 0 1 0

Canonical driver mutation

JAK2 V617F 5 6 20 1

JAK2 exon12 1 0 0 0

CALR 0 4 5 4

MPL 0 0 3 0

Triple negative 0 3 6 1

DIPSS plus risk group

Low NA NA 7 NA

Intermediate-1 NA NA 8 NA

Intermediate-2 NA NA 8 NA

High NA NA 10 NA

MIPSS70 risk group

Low NA NA 5 NA

Intermediate NA NA 16 NA

High NA NA 12 NA

MIPSS70-plus version 2.0 risk group

Very low NA NA 0 NA

Low NA NA 11 NA

Intermediate NA NA 9 NA

High NA NA 9 NA

Very high NA NA 4 NA

20

Abbreviation: PV, polycythemia vera; ET, essential thrombocythemia; PMF, primary myelofibrosis; SD, standard deviation; WBC, white blood count; NA, not applicable; DIPSS, Dynamic International Prognostic Scoring System; MIPSS70, Mutation-Enhanced International Prognostic Score System for Transplantation-Age Patients With Primary Myelofibrosis; AML,

acute myeloid leukemia; sMF, secondary myelofibrosis.

DIPSS plus, MIPSS70, MIPSS70-plus version 2.0 scoring were performed in 33 PMF patients.

Table 3. Classification of MPN subtypes according to

2008 and 2016 WHO criteria

WHO classification version

MPN type 2008 2016

ET 4 4

Prefibrotic PMF 3 8

Post-PV MF 5 5

Post-ET MF 5 5

Overt PMF 23 25

MPN-U 13 6

21

Reclassification of MPN-U according to different WHO

classification versions

WHO 2008 criteria required at least two minor criteria to

diagnose prefibrotic/overt PMF; leukoerythroblastosis, increase in

serum lactate dehydrogenase level, anemia, and splenomegaly.

Accordingly, some MPNs that has PMF-like feature but do not satisfy

the minor criteria were diagnosed as MPN-U in WHO 2008 criteria.

Because WHO 2016 criteria required at least one minor criterion to

diagnose pre-fibrotic/overt PMF, the majority of MPN-U at WHO 2008

reclassified. Seven of 13 (53.8%) MPN-U in WHO 2008 moved to

other MPN subtypes in WHO 2016, specifically, 5 patients (38.5%) to

prefibrotic PMF and 2 patients (15.4%) to overt PMF. Six patients

(46.2%) remained as MPN-U (Fig. 3).

22

Cytogenetic features

Cytogenetic aberrations by G-banding and/or FISH were

observed in 21/52 (28.8%) MPN patients (Fig. 4, Table 4). Fifteen

patients by G-banding and 6 patients by FISH showed aberrations

(either 13q deletion, trisomy 8, 20q deletion, or ABL 3 copies due to

trisomy 9). The most common abnormalities by G banding were

structural abnormalities (11/15 patients, 73.3%), followed by combined

Figure 3. Transitions of the diagnosis in patients with ET, MPN-U or PMF between WHO classification 2008 and 2016

23

abnormality (3/15 patients, 20.0%), and abnormality (2/15 patients,

13.3%). The most frequent molecular cytogenetic change by FISH was

20q deletion (3/6, 50.0%), followed by 13q deletion (1/6, 16.7%),

trisomy 8 (1/6, 16.7%), and trisomy 9 (1/6, 16.7%).

Figure 4. Cytogenetic features detected by conventional G-banding and/or fluorescent in situ hybridization (FISH) in 52 patients with BCR/ABL1-negative MPN patients

24

Table 4. Cytogenetic analysis by G-banding and/or FISH in 53 MPN and 6 AML evolved from MPN

patients

Patient Subtype Karyotype

FISH abnormality, %

BCR/ABL1 Rearr.

20q12 del

13q14 del

trisomy 8

P259 ET 46,XX[20] 0.0 NT NT NT

P261 ET 46,XY[20] 0.0 NT NT NT

P266 ET 46,XY,1qh+[21] (normal variant) 0.0 0.0 0.0 NT

P269 ET 46,XY[20] NT NT NT NT

P208 ETAML44~47,XX,add(3)(p25),add(7)(q?32),add(9)(q?22),add(?15)(q22),-16,add(19)(q13.1),+1~7mar,inc[cp11]/46,XX[4]

NT 0 NT 0.0

P209 ETAML 44,XX,der(1)t(1;?3)(p36;q23),der(3;12)(q10;q10),-5,add(19)(p13.3),inc[20] NT NT NT NT

P251 ETAML46,XX,+1,der(1;15)(q10;q10)[11]/46,XX,der(20)t(1;20)(q12;p13)[3]/46,XX,+1,der(1;14)(q10;q10)[2]/46,XX[4]

0.0 0.0 NT 0.0

P201 ETMF43,XX,der(5)t(5;21)(q?31;q11.2),-7,-12,der(17)t(12;17)(p11.2;q13),-18,-21,+mar1[7]/44,XX,add(?4)(q10),der(5)t(5;21),-7,-21[5]/44,sl,+mar2[4]/44,sl,+mar3[3]/44,sdl1,del(11)(q?14q?23)[1]

NT NT NT NT

P213 ETMF 46,XX[1] NT NT NT NT

P220 ETMF 46,XX[19] 0 0 0 NT

P223 ETMF NT 0.0 0.0 0.0 NT

25

P249 ETMF 46,XX,t(10;12)(q24;q24.1)[13] NT NT NT NT

P205 MPNU 46,XY[18] 0.0 0.0 0.0 NT

P218 MPNU 46,XY[21] 0.0 0.0 0.0 NT

P225 MPNU 46,XY[20] 0.0 NT NT NT

P233 MPNU 46,XY[19] 0.0 0.0 0.0 NT

P258 MPNU 46,XY,inv(12)(q15q24.1)[20] 0.0 0.0 NT 0.0

P268 MPNU 46,XX[3] 0.0 NT NT NT

P203 PMF NT 0.0 NT NT NT

P207 PMF 46,XX,del(20)(q11.2)[10] 0.0 57.5 0.0 NT

P211 PMF 46,XX.del(20)(q11.2)[10]/46,XX[11] 0.0 26.0 0.0 NT

P215 PMF 46,XX[17] 0.0 0.0 0.0 NT

P217 PMF Insufficient mitosis 0.0 0.0 0.0 NT

P219 PMF 46,XY[20] 0.0 0.0 NT 0.0

P222 PMF 46,XX,dup(1)(q21q32)[20] 0.0 NT NT NT

P224 PMF 46,XY[20] NT NT NT NT

P230 PMF 46,XY,del(9)(q22q32),add(12)(q24),del(?13)(q?14)[cp6] 0.0 0.0 53.5 NT

P231 PMF 46,XY,del(13)(q12q14)[18]/46,XY[2] NT NT NT NT

P235 PMF 46,XX,t(7;8)(q32;p12)[19]/46,XX[1] 0.0 NT NT NT

P236 PMF 46,XY,inv(9)(p11q13)[20] NT NT NT NT

26

P237 PMF 47,XY,+9[7]/46,XY[13] 0.0 NT NT NT

P240 PMF 46,XY[12] 0.0 0.0 NT 0.0

P242 PMF 46,XX[18] 0.0 NT NT NT

P243 PMF 92,XXYY[6]/46,XY[14]1

0.0 NT NT NT

P244 PMF 47,XY,+8[9]/48,idem,+mar[5]/46,XY[1] 0.0 0.0 0.0 90.5

P247 PMF 46,XX[6] 0.0 NT NT NT

P252 PMF 46,XY[20] NT 0.0 NT 0.0

P254 PMF 46,XY[12] NT NT NT NT

P255 PMF 46,XX[20] 0.0 NT NT NT

P256 PMF 46,XX[20] 0.0 NT NT NT

P270 PMF 46,XX[20] NT NT NT NT

P274 PMF 46,XY,del(20)(q11.2)[20] NT 54.5 NT NT

P319 PMF 46,XX[20] NT NT NT NT

P227 PMFAML 46,X,del(Y)(q12),inv(9)(p11q13)[16]/46,XY,inv(9)[8] NT 0.0 NT 0.0

P202 prePMF 46,XX[20] 0.0 NT NT NT

P221 prePMF 46,XY[20] 0.0 NT NT NT

P239 prePMF 46,XX,9qh+[20] (normal variant) 0.0 NT NT NT

P246 prePMF 46,XX[20] NT NT NT NT

P250 prePMF 46,XX[20] 0.0 NT NT NT

27

P264 prePMF 46,XY[20] 0.0 NT NT NT

P271 prePMF 46,XY[22] 0.0 NT NT NT

P210 PVAML44~60,XX,-X[4],+1[4],+2[4],del(5)(q13)[9],-7[3],der(7)t(7;16)(q32;q24)[6],+8[5],+9[3],+13[3],+17[3],+19[3],der(19)t(19;?)(p10;?)[2],+21[3][cp16]/46,XX[1]

0.0 0.0 0.0 NT

P206 PVMF43,XX,add(3)(p13),add(5)(q13),-7,der(15;22)(q10;q10),der(17)t(9;17)(p13;p11.2),-18,-18,+mar[7]/44,sl,+7,-9,+add(9)(q10)x2,-14,+15,-der(15;22),+18,-mar[4]/45,sdl,+add(9)[2]/46,XX[1]

NT NT NT NT

P214 PVMF 46,XY[20] 0.0 NT NT NT

P229 PVMF 46,XY,del(9)(q?32)[7]/45,XY,der(3;16)(p10;q?10),der(6)t(3;6)(q?21;p?23),-16[2]/46,XY[11] 0.0 NT NT NT

P238 PVMF NT 0.0 0.0 0.0 NT

P248 PVMF 46,XY[7] 0.0 NT NT NT

Abbreviation: ET, essential thrombocythemia; ETAML, acute myeloid leukemia evolved from ET; ETMF, post-ET

myelofibrosis; MPNU, myeloproliferative neoplasm, unclassifiable; overt PMF, primary myelofibrosis, overt fibrotic stage; PMFAML, acute myeloid leukemia evolved from PMF; prePMF, primary myelofibrosis, prefibrotic/early stage; PV, polycythemia vera; PVAML, acute myeloid leukemia evolved from PV; PVMF, post-PV myelofibrosis, Rearr., rearrangement; NT, not tested

1 92,XXYY karyotype is estimated to be observed in the mitosis process, which is not abnormal karyotype.

28

Mutational profile and number of mutated genes in Korean

MPN patients

Fig. 5 shows the frequency of probable somatic mutation in 53

MPN patients. The most commonly mutated gene was JAK2 (23.6%,

29 cases), followed by ASXL1 (16.3%, 20 cases), CALR (9.8%, 12

cases), TET2 (8.1%, 10 cases), and SF3B1 (4.9%, 6 cases). Mutations

were classified according to type (missense, nonsense, affecting a

splice site, or others). All types of JAK2 mutations were missense

(V617F) except for one inframe deletion case on exon 12. The inframe

deletion is N542-E543del (COSMIC ID: COSM24440). The types of

ASXL1 were nonsense or frameshift. All CALR mutations were

frameshift. Fig 6. shows the distribution of mutations in 53 MPN and 6

AML evolved from MPN patients using 88 multigene panels.

The number of mutated genes per patient is as follows: ET,

1.50 ± 1.29; prefibrotic PMF, 2.25 ± 1.28; overt PMF, 2.00 ± 1.13 (P =

0.6672, Kruskal-Wallis test) (see Table 5).

29

Figure 5. Mutation profile of 53 Korean MPN patients

30

P V P MF

ASXL1 1 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 1 0 1 0 1 0 0 0 0 0 0 1 0 0 2 2 0 1 0 1 0 1 0 1 1 0 0 0 1 2 1 0 0 0 0 1 0 0 0 0

ATM 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ATRX 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

BARD1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

BCOR 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

BIRC3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

BRAF 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

BRCC3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

BRD2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

BRD4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0

CALR 1 1 1 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 1 0 1 0 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0

CARD6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

CBL 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

CCND1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

CDKN2A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

CEBPA 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

CHD2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

CSF1R 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

CSF3R 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

DAP3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

DDX3X 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

DIS3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0

DNMT3A 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0

EEF1E1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

EGR2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ETV6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

EZH2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1

FAM46C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

FAT4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

FBXW7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

FLT3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

GATA1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0

GATA2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

HIST1H1E 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

IDH1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

AML evolved fromET prefibrotic PMF overt PMF MPNU ETMF PVMF

ET

31

P V P MF

IDH2 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

IKZF1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

ITPKB 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

JAK2 0 0 0 0 1 1 1 0 1 1 1 0 1 0 0 1 0 0 1 1 1 0 1 1 0 1 0 1 0 1 1 1 0 0 0 0 1 0 1 0 0 0 0 1 1 1 1 0 1 1 1 1 1 0 0 1 1 1 1

KIAA0355 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

KIT 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

KLHL6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

KRAS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0

LAMB4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0

LRP1B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0

MAPK1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

MED12 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

MPL 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 3 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

MYD88 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

NF1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

NFKBIE 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

NOTCH1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

NPM1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

NRAS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

PHF6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

PLEKHG5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

POLG 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

POT1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

PRKD3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

PRPF40B 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

PTEN 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

PTPN11 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

RAD21 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

RB1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

RIPK1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

RUNX1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1

SAMHD1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

SCRIB 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

SETBP1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

SF1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

PVMFAML evolved from

ETET prefibrotic PMF overt PMF MPNU ETMF

32

P V P MF

SF3A1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

SF3B1 0 0 0 0 0 0 0 1 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

SH2B3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

SMARCA2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

SMC1A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

SMC3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

SRSF2 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

STAG2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

TCF12 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

TET2 2 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 1 0 0 0 0 0 0 0 0

TGM7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

TP53 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 1 0 0 1 0 2 1 0

U2AF1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

U2AF2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

WT1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

XPO1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ZMYM3 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ZRSR2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

PVMFAML evolved from

ETET prefibrotic PMF overt PMF MPNU ETMF

Figure 6. Distribution of mutations of 53 MPN and 6 AML evolved from MPN patients 88 genes panel

33

Table 5. Number of mutated genes per patient according

to the MPN subtypes

MPN type Number of mutated genesMean ± SD

ET (n=4) 1.50 ± 1.29

Prefibrotic PMF (n=8) 2.25 ± 1.28

Overt PMF (n=25)

2.31 ± 1.30

2.04 ± 1.21

Post-PV MF (n=5) 2.80 ± 0.843.00 ± 1.33

Post-ET MF (n=5) 3.20 ± 1.79

AML evolved from MPN (n=6) 2.17 ± 1.72

MPN-U (n=6) 2.33 ± 1.63

34

Comparison of the mutation profiles and variant allele

frequency (VAF) among ET, prefibrotic PMF, and overt

PMF

In this study, ET harbors ASXL1, CALR, TET2 mutation, while

prefibrotic PMF harbors CSF3R, DNMT3A, JAK2, SF3B1, SRSF2 in

addition to mutated genes in ET. Overt PMF shows mutations of ATM,

BCOR, EZH2, IDH2, KRAS, LRP1B, MPL, NFKBIE, NOTCH1, NPM1,

POLG, SH2B3, ZMYM3, ZRSR2 in addition to those in prefibrotic PMF.

We compared the VAF of ASXL1, CALR, and TET2 mutations

among ET, prefibrotic PMF, and overt PMF, which are commonly

observed mutations in the 3 diseases (Table 6). The VAFs (mean ± SD)

of ASXL1 in ET, prefibrotic PMF, and overt PMF were 0.21± NA,

0.38± NA, and 0.22 ± 0.15, respectively (P = 0.8077). The VAFs (mean

± SD) of CALR in ET, prefibrotic PMF, and overt PMF were 0.16 ±

0.03, 0.37 ± 0.16, and 0.24 ± 0.10, respectively (P = 0.2479). The VAFs

(mean ± SD) of TET2 in ET, prefibrotic PMF, and overt PMF were 0.29

± 0.15, 0.33 ± 0.11, and 0.33 ± 0.13, respectively (P = 0.9714).

35

Table 6. Comparison of the variant allele frequency of ASXL1, CALR, and TET2 in ET, prefibrotic PMF,

and overt PMF

1. ASXL1

MPN type Pt ID Exon nucleotide change amino acid change variant type VAFMean ± SD

of VAFET P266 14 c.1926del p.Gly645Valfs*58 frameshift 0.21 0.21 ± NAprefibrotic PMF P241 14 c.2421del p.Pro808Leufs*10 frameshift 0.38 0.38 ± NA

overt PMF

P207 14 c.2926C>T p.Gln976* nonsense 0.18

0.22 ± 0.15

P211 14 c.2077C>T p.Arg693* nonsense 0.27P215 14 c.1934dup p.Gly646Trpfs*12 frameshift 0.06P215 14 c.2757dup p.Pro920Thrfs*4 frameshift 0.34P222 14 c.1934dup p.Gly646Trpfs*12 frameshift 0.07P223 14 c.2455G>T p.Gly819* nonsense 0.43P240 14 c.1934dup p.Gly646Trpfs*12 frameshift 0.13P240 14 c.2122C>T p.Gln708* nonsense 0.18P244 14 c.1720del p.Ile574Phefs*129 frameshift 0.39P252 14 c.1934dup p.Gly646Trpfs*12 frameshift 0.01P319 14 c.2530del p.Thr844Profs*23 frameshift 0.38

2. CALR

MPN type Pt ID Exon nucleotide change amino acid change variant type VAFMean ± SD

of VAF

ETP259 9 c.1092_1143del p.Leu367Thrfs*45 frameshift 0.13

0.16 ± 0.03P266 9 c.1092_1143del p.Leu367Thrfs*45 frameshift 0.19P269 9 c.1092_1143del p.Leu367Thrfs*45 frameshift 0.16

prefibrotic PMFP250 9

c.1154_1155insTTGTC

p.Lys385Asnfs*46 frameshift 0.260.37 ± .0.16

P271 9c.1154_1155insTTG

TCp.Lys385Asnfs*46 frameshift 0.48

overt PMF P215 9 c.1092_1143del p.Leu367Thrfs*45 frameshift 0.27 0.24 ± 0.10

36

P219 9 c.1092_1143del p.Leu367Thrfs*45 frameshift 0.31P247 9 c.1092_1143del p.Leu367Thrfs*45 frameshift 0.13

3. TET2

MPN type Pt ID Exon nucleotide change amino acid change variant type VAFMean ± SD

of VAF

ETP259 8 c.4035dup p.Asn1346* nonsense 0.34

0.29 ± 0.15P266 6 c.3628_3629del p.Leu1210Valfs*12 frameshift 0.41P266 8 c.4011T>G p.Tyr1337* nonsense 0.13

prefibrotic PMFP202 6 c.3689dup p.Leu1231Profs*12 frameshift 0.40

0.33 ± 0.11P221 11 c.5734C>T p.His1912Tyr missense 0.25

overt PMFP231 5 c.3578G>A p.Cys1193Tyr missense 0.42

0.33 ± 0.13P256 6 c.3797A>C p.Asn1266Thr missense 0.24

4. P-value between MPN subtypes

• ASXL1: Mann Whitney test between 2 subtypes cannot be performed because it requires at least two values in each group. Kruskal-Wallis

test was used among 3 subtypes. • CALR, TET2: Mann Whitney test was used between 2 subtypes. Kruskal-Wallis test was used among 3 subtypes.

ASXL1 CALR TET2

ET vs prefibrotic PMF NA 0.2594 > 0.9999

ET vs overt PMF NA 0.7300 0.8000

prefibrotic PMF vs overt PMF NA > 0.9999 > 0.9999

ET vs prefibrotic PMF vs overt PMF 0.8077 0.2479 0.9714

Abbreviation: ET, essential thrombocythemia; PMF, primary myelofibrosis; NA, not applicable

37

Canonical mutations and triple-negative Korean MPN

patients

The frequencies of JAK2 V617F, JAK2 exon 12, MPL, and

CALR mutation in Korean MPN patients were 54.2%, 1.7%, 5.1%, and

22.0%, respectively. The frequency of triple-negative MPN was 16.9%

in this study. The frequencies of JAK2, MPL, CALR mutated, and

triple-negative Korean PMF (pre & overt) patients were 57.6%, 9.1%,

15.1%, and 18.2%, respectively (Table 7).

Distribution of CALR mutation

CALR mutation was observed in 13 MPN patients (Fig. 7) and

CALR, JAK2, and MPL mutations were mutually exclusive. Eleven

patients (84.6%) harbored type 1 and the remainder (15.3%) harbored

type 2 mutation. Patients with type-1 CALR mutation were MPN-U

(4/11 patients, 36.4%), ET (3/11 patients, 27.2%), overt PMF (3/11

patients, 27.2%), and AML evolved from ET (1/11 patients, 9.1%). All

patients with type 2 mutation were patients with prefibrotic PMF, while

all ET patients with CALR mutation harbored type 1 mutation

(P=0.1000, Fisher’s exact test). Of the 5 CALR-mutated PMF patients,

38

3 patients (60%) harbored type 1 mutation and 2 patients (40%)

harbored type 2 mutation.

39

Table 7. Frequency of canonical mutated and triple-negative Korean MPN patients

MPN subtype JAK2 V617F JAK2 exon 12 MPL exon 10 CALR exon 9 Nonmutated

JAK2/MPL/CALR (%)Total

ET 0 (0.0%) 0 (0.0%) 0 (0.0%) 3 (75.0%) 1 (25.0%) 4

PMF 19 (57.6%) 0 (0.0%) 3 (9.1%) 5 (15.1%) 6 (18.2%) 33

PVMF, PVAML 5 (83.3%) 1 (16.7%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 6

ET, ETMF, ETAML 6 (46.2%) 0 (0.0%) 0 (0.0%) 4 (30.8%) 3 (23.1%) 13

PMF, PMFAML 20 (58.8%) 0 (0.0%) 3 (8.8%) 5 (14.7%) 6 (17.6%) 34

MPNU 1 (16.7%) 0 (0.0%) 0 (0.0%) 4 (66.7%) 1 (16.7%) 6

All patients 32 (54.2%) 1 (1.7%) 3 (5.1%) 13 (22.0%) 10 (16.9%) 59

40

Figure 7. Distribution of CALR mutation and its subtypes in 53 MPN and 6 AML evolved from MPN patients

41

Telomere length analysis in Korean MPN patients

We measured the telomere length (T/C ratio) of bone marrow-

nucleated cells in 29 normal adults, 40 MPN patients, and 4 AML

evolved from MPN patients. Fig. 8 shows the distribution of mean

telomere lengths in normal adults and MPN subtypes. Except for ET,

the mean telomere lengths of prefibrotic PMF, overt PMF, post-ET MF,

post-PV MF, and MPN-U were significantly shorter than those of

normal adults (all P < 0.05). The mean telomere lengths of ET,

prefibrotic PMF, and overt PMF were 14.5 ± 1.4, 10.1 ± 3.1, and 9.3 ±

3.7, respectively (P=0.0471, Kruskal-Wallis test). The TLs of ET were

longer than those of overt PMF (P = 0.0422). However, no significant

difference was found in the TLs between ET and prefibrotic PMF (P =

0.1795) and between prefibrotic PMF and overt PMF (P > 0.9999).

42

NCBM

(n=2

9)

ET (n=4

)

pre P

MF (n

=5)

overt P

MF (n

=20)

ETMF (n

=3)

PVMF (n

=3)

MPN

U (n

=5)

AM

L from

MPN

(n=4

)

Mean

telo

mere

len

gth

(T

/L r

ati

o)

Figure 8. Telomere length (telomere/centromere ratio) according to the MPN subtype, AML evolved from MPN, and normal adult control

Abbreviation: ET, essential thrombocythemia; ETMF, post-ET myelofibrosis; MPNU, myeloproliferative neoplasm, unclassifiable; overt PMF, primary myelofibrosis, overt fibrotic stage; prePMF, primary myelofibrosis, prefibrotic/early stage; PV, polycythemia vera; PVMF, post-PV myelofibrosis, NCBM, normal control bone marrow; AML, acute myeloid leukemia; MPN, myeloproliferative neoplasm

43

Survival analysis

The overall survival and progression-free survival according to

the MPN subtype are as follows: ET, undefined for both; prefibrotic

PMF, undefined for both; overt PMF, 62 months and 54 months,

respectively; post-ET myelofibrosis, 65 months and 58 months,

respectively; post-PV myelofibrosis, 26 months for both; MPN-U, 72

months and 64 months, respectively (see Kaplan-Meier curves in Fig.

9). ET and prefibrotic PMF showed a better OS than other MPN

subtypes in both overall survival and progression-free survival

(P=0.0225 and P=0.0146, respectively). Post-ET myelofibrosis and

post-PV myelofibrosis showed no significant difference in the overall

survival and progression-free survival (P=0.4805 and P=0.2651,

respectively). Additionally, the overall survival and progression-free

survival of AML evolved from MPN are 1 month for both. The median

survival according to the initial MPN subtypes are as follows: 1 month,

1 month, and 6 months in ET, PV, and PMF, respectively (P=0.6037).

All the patients with AML evolved from PV and PMF died within 5

years after the diagnosis, whereas one fourth of AML evolved from ET

were alive at that time.

Fig. 10 shows the comparison of survival curves between

44

WHO classification 2001~2008 and 2016. In WHO classification

2001~2008, the overall survival and progression-free survival were as

follows: ET, undefined for both; MPN-U, 91 months for both; PMF, 62

months and 50 months, respectively. In WHO classification 2016, the

overall survival and progression-free survival were as follows: ET,

undefined for both; MPN-U, 72 months and 64 months, respectively;

prefibrotic PMF, undefined for both; overt PMF, 62 months and 54

months, respectively.

45

0 50 100 150 2000

50

100

Months

Perc

ent su

rviv

al

ET (n=4)

AML from MPN (n=6)

ETMF (n=5)

MPNU (n=6)

prefibrotic PMF (n=8)

overt PMF (n=25)

PVMF (n=5)

P = 0.0002

0 50 100 150 2000

50

100

Months

ET (n=4)

AML from MPN (n=6)

ETMF (n=5)

MPNU (n=6)

prefibrotic PMF (n=8)

overt PMF (n=25)

PVMF (n=5)

P < 0.0001

(A)

(B)

Figure 9. Kaplan-Meier curves according to the MPN subtypes and AML evolved from MPN (a) overall survival rate (b) progression-free survival rate

Abbreviation: ET, essential thrombocythemia; ETMF, post-ET myelofibrosis; MPNU, myeloproliferative neoplasm, unclassifiable; overt PMF, primary myelofibrosis, overt fibrotic stage; prePMF, primary myelofibrosis, prefibrotic/early stage; PV, polycythemia vera; PVMF, post-PV myelofibrosis; AML, acute myeloid leukemia; MPN, myeloproliferative neoplasm

46

Figure 10. Comparison of the survival curves between WHO classification 2008 and 2016

Overall survival: (A) WHO classification 2008 (B) WHO classification 2016. Progression-free survival: (C) WHO classification 2008 (D) WHO classification 2016.

Abbreviation: ET, essential thrombocythemia; MPNU, myeloproliferative neoplasm, unclassifiable; overt PMF, primary myelofibrosis, overt fibrotic stage; prePMF, primary myelofibrosis, prefibrotic/early stage

47

Favorable and adverse mutated genes in 53 Korean MPN

patients

Before identifying favorable and adverse variables of genes in

Korean MPN patients, we plotted a correlogram of mutated genes in

the 53 MPN and 33 PMF patients. Of 88 genes, 56 and 66 genes were

never mutated in 53 MPN and 33 PMF patients, respectively. We

analyzed the remaining 31 and 21 genes to find correlations with each

other. Fig. 11 shows the correlogram of mutated genes in 53 MPN and

33 PMF patients. In 53 MPN patients, U2AF1 and ZMYM3 gene are

strongly correlated with each other (constant of coefficients = 1). Also,

ATM and BCOR, ASXL1 and EZH2, BRD4 and TP53, CSF3R and MPL,

LAMB4 and TP53 are moderately correlated with each other (all

constant of coefficients ≥ 0.5). Randomly excluding ZMYM3, ATM,

EZH2, BRD4, and CSF3R, we assigned 26 genes as factors in the

multivariable-Cox regression and did not consider the MPN subtype as

a covariable. In the 53 MPN patients, there are no significant adverse or

favorable mutated genes. According to the correlogram of 33 PMF

patients, there are so many correlations between genes, which is not

appropriate to make the Cox proportional hazards model. Especially,

POLG and ZMYM3 gene are strongly correlated with each other

48

Figure 11. Correlogram of mutated genes in (A) 53 MPN patients (B) 33 PMF patients

(constant of coefficients = 1).

(A)

(B)

49

Factors influencing survival in MPN patients: Univariate

analysis in Korean MPN patients

l Number of mutated genes in prefibrotic and overt PMF

We compared the survival rates according to the number of

mutated genes in prefibrotic and overt PMF by the 2016 WHO

classification. In the 88-gene panel, the number of mutated genes in

PMF patients ranged from 0 to 4. Each group name indicated the

number of mutated genes. The overall survival and progression-free

survival according to the subgroups were as follows: Group 0: 62

months and 39 months, respectively; Group 1: undefined for both;

Group 2: 130 months for both; Group 3: 24 months and 11 months,

respectively; Group 4: 27 months for both (P = 0.0029 for overall

survival, P = 0.0059 for progression-free survival, log-rank test).

l JAK2/MPL/CALR mutated and triple-negative status in

prefibrotic and overt PMF

We compared the survival rates according to the mutation

status (JAK2 V617F/MPL exon 10 mutation/CALR exon 9

mutation/nonmutated JAK2/MPL/CALR) for prefibrotic and overt PMF

patients. There is no JAK2 exon 12 mutated PMF in this study. The

overall survival and progression-free survival according to the

50

subgroup were as follows: JAK2 V617F mutated: 69 months for both;

MPL exon 10 mutated: undefined for both; CALR exon 9 mutated:

undefined for both; triple-negative: 58 months and 44 months,

respectively (P = 0.2055 for overall survival, P = 0.1499 for

progression-free survival, log-rank test).

l Presence of splenomegaly in 53 MPN patients

Splenomegaly status of 53 MPN patients was assessed by

radiological findings. On univariate analysis, patients with

splenomegaly had shorter overall survival and progression-free survival

compared with patients without splenomegaly: overall survival:

49 versus 112 months, respectively, P = 0.0172, log-rank test;

progression-free survival: 44 versus 101 months, respectively, P =

0.0122, log-rank test.

l Bone marrow fibrosis (MF) grading in 53 MPN patients

MF grading of 53 MPN patients was evaluated by reticulin and

collagen staining in bone marrow biopsy sections. The overall survival

and progression-free survival according to the subgroups were as

follows: MF-0: undefined for both; MF-1: undefined for both; MF-2:

112 months and 101 months, respectively; MF-3: 42 months and 40

months, respectively (P = 0.0286 for overall survival, P = 0.0154 for

51

progression-free survival, log-rank test). When regrouping as MF-0~1

and MF-2~3, the overall survival and progression-free survival were as

follows: MF-0~1, undefined for both; MF-2~3, 58 months and 50

months, respectively (P = 0.0129 for overall survival, P = 0.0041 for

progression-free survival, log-rank test).

l Telomere lengths in Korean MPN patients

The telomere lengths (T/C ratio) were analyzed in 40 MPN

patients. We found the patients whose mean telomere lengths were the

same or less than 6.933 showed an inferior overall survival than those

whose mean telomere length was more than 6.933 (P = 0.0054).

Presence of CD34-positive megakaryocytes in MPN

Of 99 MPN cases, 44 cases (44.4%) showed CD34-positive

megakaryocytes. CD34-positive megakaryocytes positive rates

according to MPN subtypes are as follows: PV (50.0%), ET (48.0%),

prefibrotic PMF (57.7%), overt PMF (26.3%), post-PV myelofibrosis

(25.0%), and post-ET myelofibrosis (40.0%). CD34-positive

percentage of total megakaryocytes according to MPN subtypes are as

follows (mean ± SD): PV (4.23 ± 10.41), ET (2.21 ± 3.00), prefibrotic

52

PMF (15.96 ± 27.72), overt PMF (1.65 ± 4.57), post-PV myelofibrosis

(1.13 ± 2.10), and post-ET myelofibrosis (8.82 ± 15.24) (P=0.1527)

(Fig. 12).

Specific mutations and their clinical relevance

Among PMF patients, the presence of splicing gene mutations

was not associated with a difference in hemoglobin concentration,

white blood cell counts, and platelet counts at diagnosis. On the other

hand, in PMF patients, those with ASXL1 mutations were related to

lower hemoglobin concentration at diagnosis compared to those

without (8.67 ± 1.30 g/dL versus 11.77 ± 3.39 g/dL, P=0.0106) (Table

8).

53

Figure 12. (A) CD34-positive megakaryocytes positive rates (B) CD34-positive percentage of total megakaryocytes according to MPN subtypes

Abbreviation: ET, essential thrombocythemia; ETMF, post-ET myelofibrosis; PMF, primary myelofibrosis, PV, polycythemia vera; PVMF, post-PV myelofibrosis

(A)

(B)

54

Table 8. Specific mutations and their clinical relevance

(A) Additional splicing gene mutation acquisition in primary

myelofibrosis is not associated with a difference in hemoglobin

concentration, white blood cell counts, and platelet counts at diagnosis.

(B) Additional ASXL1 mutation in primary myelofibrosis is associated

with lower hemoglobin concentration at diagnosis. Data presented as

mean (±standard deviation). Hb, hemoglobin; Plt, platelet; WBC, white

blood count.

(A)

Splicing mutation No splicing mutation

P

Hb (g/dL) 9.8 (3.7) 11.3 (3.1) 0.1656

Plt (103/μl) 462.8 (356.3) 494.1 (344.4) 0.7575

WBC (103/μl) 11.8 (4.2) 11.9 (8.0) 0.9181

(B)

ASXL1 mutated No ASXL1 P

Hb (g/dL) 8.7 (1.3) 11.8 (3.4) 0.0106

Plt (103/μl) 312.2 (104.0) 551.8 (377.5) 0.2544

WBC (103/μl) 9.6 (3.9) 12.7 (8.0) 0.2898

55

Discussion

By applying the 2016 WHO criteria, 53.8% of MPN-U by

2008 WHO criteria were reclassified to prefibrotic PMF (38.5%) and

overt PMF (15.4%). There is no case reclassified to PV or ET. 46.2%

of MPN-U remained as MPN-U. 53.8% of MPN-U by 2008 WHO

criteria had PMF-like features but did not meet the diagnostic minor

criteria of PMF. Among 7 MPN-U patients who were reclassified to

PMF by 2016 WHO criteria, 4 had the only leukocytosis; 1 had only

splenomegaly; 1 had only anemia; the other had leukocytosis, left-

shifted neutrophil, and splenomegaly. In 7 patients, 5 patients

harbored canonical mutation such as JAK2 or CALR but the others

did not carry canonical mutation: one did not harbor any somatic

mutation and the other harbored POLG and ZMYM3 mutations.

Iurlo et al. reduced 30% of MPN-U by applying the 2016

WHO criteria in their study, but in our study 53.8% of MPN-U were

reallocated. One large-series study reported that, in patients

previously classified as ET, the diagnosis of ET was confirmed in

891 patients (81%) and was reallocated to prefibrotic PMF in 180

56

(16%) cases (3). Another study revealed that early/prefibrotic PMF

accounted for 14% of patients who were previously diagnosed with

ET (17). In the present study, by applying the 2016 WHO criteria,

patients previously classified as ET and PMF were not allocated to

other MPN subtypes.

WHO 2016 added prefibrotic PMF to the MPN subtypes, and

the diagnostic criteria of WHO 2016 included additional molecular

markers such as MPL and CALR. However, JAK2, MPL, and CALR

are commonly included in the diagnostic criteria of both ET and

prefibrotic PMF. Reflecting the mutational profiles of ET, prefibrotic

PMF, and overt PMF, the mutation profiles become more diverse

according to disease severity. Gene mutations found in prefibrotic

PMF but not in ET are potential candidate markers for the differential

diagnosis between ET and prefibrotic PMF. In this study, CSF3R,

DNMT3A, SF3B1, and SRSF2 mutations were detected in prefibrotic

and overt PMF but not in ET. The results of the present study can be

helpful in the molecular discrimination between ET and prefibrotic

PMF. On the other hand, ASXL1, CALR, and TET2 genes are

commonly mutated in ET, prefibrotic PMF, and overt PMF but the

VAFs of the genes are not significantly different among these

diseases.

57

The genomic profile of MPN in Koreans was similar to those

reported in Grinfeld et al.’s study (18). Thirty-one genes were

mutated in 53 patients, 2.25 ± 1.31 mutated genes per patient were

observed in our study, while 33 genes were mutated in 2053 patients

in Grinfeld et al.’s study. The frequent mutations were JAK2, ASXL1,

CALR, TET2, and SF3B1 in the present study, and JAK2, CALR,

TET2, ASXL1, and DNMT3A in Grinfeld et al.’s study. Herein, we

found a novel MPL mutation (MPL D128N, D261Y) in a patient with

overt PMF. The patients also harbor MPL W515L, which is a

canonical exon 10 missense mutation. MPL D128N and D261Y are

exon 3 and exon 5 missense mutations, respectively. The frequency

of triple-negative MPNs was 16.9% in Korean MPN. Remarkably,

the frequencies of nonmutated JAK2/MPL/CALR in prefibrotic and

overt PMF patients were higher than those in other reports: 7% in

Italy (19), 8.6% in Italy and Spain (20), 17% in China (21), and 18.2%

in Korea.

Of specific interest, CALR mutations were all type 2 in

prefibrotic PMF and all type 1 in ET, suggesting prefibrotic PMF

and ET are separate diseases. As previously known, type 1-like

mutations were mainly associated with a myelofibrosis phenotype

and type 2-like CALR mutations were preferentially associated with

58

an essential thrombocythemia phenotype, findings that were not

consistent with our results (19). The overall frequency of CALR

mutation among MPN was 20.0% (13/59 patients) with type 1

predominance (84.6%). Compared with Chinese (52.0%), Spanish

& Italian (72.0%), and Italian reports (65.0%), the proportion of

type 1 CALR mutation was rather high in Korean MPN (84.6%)

(19-21).

Regarding telomere length analysis, the telomere lengths of

MPN were shorter than those of the normal control, a finding that is

consistent with previous study findings (12). There is a tendency that

the mean TL of ET was the longest, followed by that of prefibrotic

PMF and overt PMF, suggesting that a shorter TL correlated with

disease progression. Spanoudakisa et al. found that a reduced

telomere length is an adverse prognostic factor in Philadelphia-

negative MPN patients, in which patients with TL < 27% showed

poorer survival (12). Similarly, our study showed that TL < 6.933

indicated an adverse prognosis.

In survival analysis, compared with the WHO classification

2008 version, the 2016 version showed more precise stratification of

MPN patients (P-value: 0.2714 versus 0.0424 in overall survival rate,

59

0.2497 versus 0.0355 in progression-free survival rate). Prefibrotic

PMF showed a better prognosis than overt PMF and MPN-U and a

worse prognosis than ET. This result is consistent with previous study

findings (22). The introduction of a new entity called prefibrotic

PMF has contributed to the accurate stratification and prediction of

the prognosis of MPN. When comparing the survival rates according

to the number of mutated genes for PMF, we found that the rate did

not decrease in proportion to the number of mutations. Interestingly,

the survival rates of Group 0 (mutated genes are none) and Group 4

(mutated genes are four) were similar because it is assumed that

Group 0 contains triple-negative PMF patients, which are known to

have a worse prognosis.

In CD34-positive megakaryocytes analysis, we found that

nearly half of MPN cases showed CD34-positive megakaryocytes

whose expression was variable – from very weak to moderate.

CD34-positive megakaryocytes were observed in more than 40% of

PV, ET, and prefibrotic PMF, whereas in less than 40% of overt PMF,

post-PV myelofibrosis, and post-ET myelofibrosis. On the other hand,

severe fibrosis interfered with discriminating CD34–positive

megakaryocytes, which infers that CD34-positive megakaryocytes

60

could be underestimated in more fibrotic diseases such as overt PMF,

post-PV myelofibrosis, and post-ET myelofibrosis. Of interest,

prefibrotic PMF showed a wide range of CD34 positive percentage of

total megakaryocytes and in some cases, most megakaryocytes

expressed CD34 antigen. CD34-positive percentage of total

megakaryocytes was not significantly higher in prefibrotic PMF

(15.96 ± 27.72 versus 2.21 ± 3.00; P=0.1661) when compared with ET

but significantly higher when compared with overt PMF (15.96 ±

27.72 versus 1.65 ± 4.57; P=0.0136). Therefore, it is not appropriate to

use CD34-positive megakaryocytes as a discriminating marker

between ET and prefibrotic PMF. Further comparison of CD34

expression in megakaryocytes with normal control and

myelodysplastic syndrome is needed.

Among PMF patients, splicing gene mutations were not

associated with the hematologic parameters at diagnosis, but ASXL1

mutation was related to lower hemoglobin concentration, which is

known to be an adverse prognostic marker of PMF.

In conclusion, our results support the existence of prefibrotic

PMF by revealing the reallocation of MPN-U into prefibrotic PMF

and overt PMF but not to ET. The results of the present study suggest

61

that WHO 2016 criteria successfully differentiated between the early

and far advanced stages of PMF. By applying the WHO 2016 criteria

of MPN, MPN-U was markedly decreased. Although the

morphologic distinction between ET and prefibrotic PMF is harsh,

multigene sequencing in the present study demonstrated a

discriminated pattern between prefibrotic PMF and ET. Additionally,

the telomere lengths can be a potential marker of differentiating

prefibrotic PMF and ET. Our findings contribute to a better

understanding of the clinical, cytogenetic, telomere lengths, molecular

features, and BM histologic characteristics of Korean MPN patients.

62

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초 록

서론: 2016년 WHO 골수증식종양 진단 기준은 업데이트된 전

섬유화 단계 원발성골수섬유증 진단 기준을 제시하였고, 이 질

환의 진단은 본태성혈소판 증가증과 감별하기가 어렵다. 미분

류 골수증식종양은 어느 한 가지의 골수증식종양 세부질환의

모든 진단기준을 충족하지 않는 특성을 보인다. 여기서 우리의

첫번째 목표는 WHO 2008년에서 2016년 진단기준으로 가면서

필라델피아 염색체 음성 골수증식종양을 재분류하되, 미분류

골수증식종양에 초점을 맞추었다. 두번째 목표는 본태성혈소판

증가증과 전섬유화 단계 원발성골수섬유증을 구별할 수 있는

유용한 마커를 찾는 것이다. 마지막 목표는 임상적, 세포유전

학적, 텔로미어 길이, 분자유전학적, 골수 조직학적 특성을 통

해 한국인 골수증식종양 환자의 특징에 대한 이해를 넓히는

것이다.

방법: 2005 년부터 2014 년까지 서울대학교 병원에서 골수증식

종양으로 진단된 53 명의 환자 및 골수증식종양에서 급성골수

성백혈병으로 이환된 6 명의 환자를 대상으로, 핵형 검사(G-

banding), 형광제자리부합법(FISH), 88 개의 조혈 관련 유전자

패널로 구성된 타겟 시퀀싱, 텔로미어 길이 분석을 시행하였다.

질병 진행 평가가 포함된 생존 분석도 진행하였다. 뿐만 아니

라, CD34 양성 거대핵세포를 관찰하기 위해 이 병원에서 2017

66

년부터 2019 년까지 진단된 99 개의 골수증식종양 케이스도 분

석하였다.

결과: 2016 년 WHO 진단기준을 적용했을 때, 미분류 골수증식

종양의 38.5%와 15.4%는 각각 전섬유화 단계 및 섬유화 단계

원발성 골수섬유증으로 재분류되었다. 생존 분석에서는 업데이

트된 진단기준이 골수증식종양 진단을 좀 더 잘 계층화함을

알 수 있었다. 우리 연구 결과에서 전섬유화 단계 골수섬유증

에서 발견되나 본태성 혈소판증가증에서 발견되지 않는 돌연

변이를 가진 유전자는 CSF3R, DNMT3A, SF3B1, SRSF2 이며, 이

들은 두 질환을 감별하기 위한 잠재적 후보 마커로 생각된다.

한국인 골수증식종양의 유전적 프로파일은 이전 연구에서와

비슷했다. 우리는 새로운 MPL 돌연변이 (MPL D128N, D261Y)

를 한 명의 골수섬유증 환자에서 발견하였다. 본태성 혈소판의

텔로미어 길이는 정상에 비해 짧아져 있지 않았으나, 전섬유화

단계 골수섬유증의 텔로미어 길이는 정상에 비해 짧았다

(P=0.0635). 이는 텔로미어 길이가 두 질환을 감별하는데 잠재

적 마커로 이용될 수 있음을 시사한다. 골수증식종양의 44.4%

에서 CD34 양성 거대핵세포가 관찰되었으나 본태성 혈소판증

가증과 전섬유화 단계 골수섬유증에서의 CD34 양성 거대핵세

포 분율은 차이가 없었다. 추가적인 ASXL1 돌연변이는 골수

섬유증 환자에서 낮은 헤모글로빈 농도와 연관이 있었다.

결론: 2016년 WHO 진단기준에 따라 미분류 골수증식종양이

전섬유화 단계 및 섬유화 단계 골수섬유증으로 재분류되는 것

67

을 밝히면서, 우리는 업데이트된 진단기준이 골수증식종양의

정확한 진단을 제공함을 알게 되었다. 분자 유전학적 검사와

텔로미어 길이 분석은 전섬유화 단계 골수섬유증과 본태성 혈

소판증가증의 감별에 도움이 될 수 있다. 우리의 종합적 분석

은 한국인 골수증식종양 환자의 임상적, 세포유전학적, 텔로미

어 길이, 분자유전학적, 골수 조직학적 특성에 대한 폭넓은 이

해를 제공한다.

----------------------------------------------------------------------------------------------

주요어: 골수증식종양; WHO 진단기준; 미분류 골수증식종양;

본태성 혈소판증가증; 전섬유화 단계 원발성 골수섬유증, 텔로미어

길이, 다유전자 시퀀싱, CD34 양성 거대핵세포

학번: 2018 - 25644