AJCP / Original Article
CALR, JAK2, and MPL Mutation Profiles in Patients With
Four Different Subtypes of Myeloproliferative Neoplasms
Primary Myelofibrosis, Essential Thrombocythemia, Polycythemia
Vera, and Myeloproliferative Neoplasm, Unclassifiable
Seon Young Kim, MD, PhD,1 Kyongok Im,2 Si Nae Park,2 Jiseok Kwon,2 Jung-Ah Kim, MD,1 and
Dong Soon Lee, MD, PhD1,2
From the 1Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea; and 2Cancer Research Institute,
Seoul National University College of Medicine, Seoul, Republic of Korea.
Key Words: Calreticulin; Somatic mutation; JAK2; Myeloproliferative neoplasms
Am J Clin Pathol May 2015;143:635-644
DOI: 10.1309/AJCPUAAC16LIWZMM
ABSTRACT
Objectives: We investigated mutation profiles of
CALR, JAK2, and MPL in 199 Korean patients with
myeloproliferative neoplasms (MPNs).
Methods: In total, 199 patients with MPN (54 primary
myelofibrosis [PMF], 79 essential thrombocythemia [ET],
58 polycythemia vera [PV], and eight MPN-unclassifiable
[MPN-U]) and 4 patients with acute panmyelosis with
myelofibrosis (APMF) were retrospectively subjected to
Sanger sequencing for CALR, JAK2, and MPL.
Results: The overall frequency of CALR mutations was
12.6% (type 1 mutation, 16 patients; type 2 mutation, nine
patients): most frequent in MPN-U (37.5%), followed by
ET (17.7%) and PMF (14.8%). CALR mutations were not
found in PV or APMF. CALR and JAK2 or MPL mutations
were mutually exclusive. In PMF, the CALR mutations
were associated with lower levels of leukocytes, lower bone
marrow cellularity, and higher number of megakaryocytes.
Patients with CALR-mutated ET more frequently progressed
to the accelerated or blast phases compared with patients
with JAK2 mutations. CALR mutations were frequently
observed in the JAK2-negative MPNs, most frequently in
MPN-U.
Conclusions: The prognostic significance of CALR
mutations likely differs among the MPN subtypes.
American Society for Clinical Pathology
CME/SAM
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Upon completion of this activity you will be able to:
describe frequent molecular abnormalities in myeloproliferative
neoplasms.
describe characteristics of mutations in calreticulin gene that were
recently found in myeloproliferative neoplasms.
discuss correlation between calreticulin mutations and clinical
characteristics of patients with myeloproliferative neoplasms.
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Myeloproliferative neoplasms (MPNs) are a clonal dis-
ease of myeloid stem cells that are characterized by myeloid
cell proliferation, bone marrow (BM) fibrosis, and symp-
toms associated with the accompanying peripheral blood
cell abnormalities. The World Health Organization (WHO)
provides diagnostic criteria for the following MPN subtypes:
chronic myelogenous leukemia; BCR-ABL1positive; polycy-
themia vera (PV); essential thrombocythemia (ET); primary
myelofibrosis (PMF); chronic neutrophilic leukemia; chronic
eosinophilic leukemia, not otherwise specified; mastocyto-
sis; and MPN-unclassifiable (MPN-U).1 Common molecular
events in MPNs are the V617F mutation in the JAK2 gene,
mutations of exon 10 of the MPL gene (mainly involving
codon W515), and JAK2 mutations on exon 12, which are
included as one of the diagnostic criteria.1-4 Recently, novel
frameshift mutations in exon 9 of the calreticulin (CALR) gene
were found using next-generation sequencing in patients with
JAK2 or MPL nonmutated PMF and ET.5,6 In these studies,
Am J Clin Pathol 2015;143:635-644 635
DOI: 10.1309/AJCPUAAC16LIWZMM
Kim et al / CALR Mutations in Four MPN Subtypes
CALR mutations were detected in approximately 20% to 25%
of patients with ET and PMF and not in patients with PV.
Most CALR mutations were deletions and insertions in exon
9, which cause frameshift mutations. The type 1 (L367fs*46)
mutation, which results from a 52base pair (bp) deletion, is
found in approximately 50% of patients with CALR muta-
tions, and the type 2 (K385fs*47) mutation, which results
from a 5-bp TTGTC insertion, accounts for approximately
30% of patients with CALR mutations.5-9 In patients with ET,
CALR mutations have been associated with a lower hemoglo-
bin level, lower leukocyte count, higher platelet count, and
relatively low thrombotic risk.9-11
In this study, we investigated the mutation profiles of
CALR, JAK2, and MPL mutations in four different MPN
subtypes in Korean patients with PMF, ET, PV, and MPN-U.
In addition, we investigated the mutation profile of patients
with acute panmyelosis with myelofibrosis (APMF), which
is a very rare acute myeloid leukemia (AML) subtype char-
acterized by diffuse myelofibrosis and increased blasts of
20% or more. The BM histologic features of APMF are
sometimes indistinguishable from the acute MPN phase,
thus blurring a common distinction. We performed a correla-
tion analysis of mutation patterns with clinical, hematologic,
and cytogenetic characteristics and prognostic impacts.
Materials and Methods
Patients
A series of 199 patients who were diagnosed with MPN
and treated at Seoul National University Hospital were
included in this study. The inclusion criteria for this study
were the availability of BM samples collected at the time of
diagnosis or revisit coupled with symptom aggregation after
a follow-up period. MPNs were strictly diagnosed accord-
ing to the 2008 WHO classification criteria.1 Patients were
diagnosed with the following subtypes: 54 PMF, 79 ET, 58
PV, and eight MPN-U. Eight patients with MPN-U had pan-
myelosis without diffuse fibrosis; therefore, the prefibrotic
stage of PMF, ET, or PV could not be determined. In addi-
tion, four patients with APMF who had blast increase and
diffuse fibrosis were included in this study. The following
laboratory and clinical information was obtained for each
patient: dates of diagnosis and therapy initiation, age, sex,
ethnicity, hemoglobin levels, platelet count, conventional
G-banding cytogenetic analyses of BM cells, and the pres-
ence of splenomegaly. For patients with PMF, the Dynamic
International Prognostic Scoring Systemplus (DIPSS-plus)
risk categorizations were assessed as previously described.12
This study complied with Declaration of Helsinki. All BM
samples were collected with informed consent, and the study
636 Am J Clin Pathol 2015;143:635-644
DOI: 10.1309/AJCPUAAC16LIWZMM
was reviewed and approved by the Institutional Review
Board of Seoul National University College of Medicine.
BM Histologic Examination
Hematopathologists (S.Y.K. and D.S.L.) reviewed
Wright-Giemsastained BM smears and H&E-stained sec-
tions of the BM trephine biopsy specimens. In all patients,
immunohistochemical staining was performed for reticulin,
collagen, CD34, CD117, and CD61 using BM sections (all
from Dako, Glostrup, Denmark). BM fibrosis (MF) was
assessed according to the European consensus grading sys-
tem on a scale of MF-0 to MF-3.13
Cytogenetic Analysis
Cytogenetic studies using standard G-banding tech-
niques on heparinized BM samples were performed as part
of the diagnostic workup. At least 20 metaphases were ana-
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lyzed whenever possible. Clonal abnormalities were defined
as two or more cells with the same chromosomal gain or
structural rearrangement or at least three cells with the same
chromosome deletion. Karyotypes were recorded according
to the International System for Human Cytogenetic Nomen-
clature (ISCN) 2013.14
In addition, fluorescence in situ hybridization (FISH)
was performed in most cases (n = 158). The following com-
mercial FISH probes were used in subset of patients: a BCR/
ABL dual-color, dual-translocation probe (n = 158); the
13q14 SpectrumOrange probe (n = 66); the LSI D20S108
(20q12) probe (n = 77); the LSI EGR1 (5q31) probe (n =
27); the LSI D7S522 (7q31) probe (n = 18); the CEP 8 probe
(n = 23); and the LSI 1p32/1q25 probe (n = 21) (all from
Abbott Molecular, Des Plaines, IL). Slides were stained with
FISH probes and counterstained with DAPI, and the fluo-
rescence signals were analyzed by fluorescent microscopy
(Zeiss, Gttingen, Germany). FISH results were recorded
according to the ISCN 2013.14
DNA Extraction and Detection of
Mutations Using Sanger Sequencing
Genomic DNA was extracted from frozen BM mono-
nuclear cells of all patients. DNA was extracted using the
MagNA Pure LC DNA Isolation Kit (Roche Applied Sci-
ence, Indianapolis, IN) according to the manufacturers
instructions. DNA quality was analyzed by assessing the
260/280 absorbance ratio using an ND-1000 Spectropho-
tometer (NanoDrop Technologies, Wilmington, DE).
The following primers were used for polymerase chain
reaction (PCR) amplification of the CALR, JAK2, and MPL
genes: CALR forward, 5-CAT TCA TCC TCC AGG TCA
AG-3; CALR reverse, 5-AGG GGA ACA AAA CCA
AAA TC-3; JAK2 exon 14 forward, 5-TCC TCA GAA
CGT TGA TGG CA-3; JAK2 exon 14 reverse, 5-ATT GCT
American Society for Clinical Pathology
 AJCP / Original Article

Table 1
Clinical and Laboratory Characteristics of 199 Patients With MPN and Four Patients With APMFa
Total MPN
Variable (n = 199) PMF (n = 54) ET (n = 79) PV (n= 58) MPN-U (n = 8) APMF (n = 4)
Male/female, % male 91/108 (45.7) 32/22 (59.3) 26/53 (32.9) 28/30 (48.3) 5/3 (62.5) 2/2 (50.0)
Age, y 58.3 (8.0-83.9) 61.5 (8.0-83.3) 55.0 (19.3-83.9) 57.4 (25.8-75.9) 58.0 (31.8-73.8) 38.3 (32.7-50.4)
Hemoglobin, g/dL 13.6 (5.2-22.0) 10.4 (5.2-16.9) 13.0 (8.8-19.5) 18.2 (15.0-22.2) 13.2 (9.2-14.2) 8.1 (5.0-9.7)
Leukocytes, 109/L 9.99 (2.02-38.53) 9.56 (2.18-38.53) 8.68 (2.02-38.52) 12.74 (3.32-34.20) 11.39 (5.00-12.25) 2.46 (1.97-3.53)
Absolute neutrophils, 6.79 (1.01-35.06) 5.44 (1.01-35.06) 5.97 (1.05-34.67) 9.48 (1.73-30.44) 7.83 (3.10-9.42) 1.01 (0.41-1.87)
109/L
Platelets, 109/L 584 (10-3,519) 285 (10-1,455) 842 (483-3,519) 407 (100-1,076) 815 (20-1,879) 70 (44-106)
Circulating blasts, % 0 (0-17) 0 (0-17) 0 (0-3) 0 (0-6) 0 (0-0) 4 (0-8)
Splenomegaly 108 (54.3) 43 (79.6) 25 (31.6) 35 (60.3) 5 (62.5) 2 (50.0)
Cytogenetic abnormalities 37 (18.6) 21 (38.9) 7 (8.9) 7 (12.1) 2 (25.0) 1 (25.0)
BM blasts estimated in 1 (0-20) 1 (0-14) 0 (0-20) 1 (0-3) 0 (0-2) 12 (2-17)b
aspirates, %
BM cellularity, % 70 (25-100) 85 (25-95) 55 (30-100) 75 (35-100) 85 (55-95) 95 (85-95)
Megakaryocytes per hpf 7.5 (0-25.0) 6.5 (0-20.0) 7.5 (2.5-22.5) 6.5 (1.5-25.0) 6.5 (4.5-7.5) 5.8 (0.5-8.0)
in BMc
Reticulin fibrosis (MF 2-3) 58 (29.1) 46 (85.2) 6 (7.6) 6 (10.3) 0 4 (100)
Progression of fibrosis in 71 (35.7) 47 (87.0) 11 (13.9) 11 (19.0) 2 (25.0) 4 (100)

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subsequence disease
coursed
Progression to 21 (10.6) 12 (22.2) 6 (7.6) 3 (5.2) 0 4 (100)
accelerated or blast
phase
Thrombotic events 16 (8.0) 7 (13.0) 5 (6.3) 4 (6.9) 0 0
Major bleeding events 8 (4.0) 5 (9.3) 1 (1.3) 2 (3.5) 0 0
Allogeneic transplantation 14 (7.0) 10 (18.5) 1 (1.3) 2 (3.5) 1 (12.5) 2 (50.0)
Deceased 41 (20.6) 25 (46.3) 9 (11.4) 5 (8.6) 2 (25.0) 3 (75.0)
Follow-up, mo 55.1 (0.4-299.4) 29.2 (1.4-280.5) 58.5 (3.7-216.5) 66.6 (3.9-299.4) 12.8 (0.4-73.4) 13.9 (7.9-20.2)
APMF, acute panmyelosis with myelofibrosis; BM, bone marrow; ET, essential thrombocythemia; hpf, high-power field; MF, myelofibrosis; MPN, myeloproliferative neoplasm;
MPN-U, myeloproliferative neoplasm, unclassifiable; PMF, primary myelofibrosis; PV, polycythemia vera.
a Data are presented as the median (range) for continuous variables and the number of cases (percentage) for categorical variables unless otherwise indicated.
b All four patients with APMF had more than 20% blasts in the BM biopsy specimens (estimated by CD34 staining).
c Average megakaryocytes counted in 10 hpf (400) from bone marrow biopsy specimens.
d The number of patients who had bone marrow fibrosis (MF 2-3 at the final follow-up test).

TTC CTT TTT CAC AA-3; JAK2 exon 12 forward, 5-CTC
CTC TTT GGA GCA ATT CA-3; JAK2 exon 12 reverse,
5-CCA ATG TCA CAT GAA TGT AA-3; MPL forward,
5-TGG GCC GAA GTC TGA CCC TTT-3; and MPL
reverse, 5-ACA GAG CGA ACC AAG AAT GCC TGT-3.
The amplified 537-bp, 453-bp, 280-bp, and 212-bp frag-
ments covered CALR exons 8 and 9, the JAK2 V617F site in
exon 14, JAK2 exon 12, and exon 10 of MPL, respectively.
PCR was performed using 25 to 100 ng genomic DNA in
100 L PCR solution (10 L of 10 MG Taq-HF buffer,
0.2 mol/L of each primer, 10 L of 2 mmol/L MG dNTPs
mixture, 1 L of MG Taq-HF polymerase [Macrogen, Seoul,
Korea], and distilled water). The PCR used the following
cycle protocol: an initial 5-minute denaturation step at 94C
followed by 35 cycles of 94C for 30 seconds, 58C to 64C
for 30 seconds (depending on the primers), and 72C for
60 seconds, with a final 7-minute extension at 72C. The
PCR products were purified and sequenced using a BigDye
Terminator v3.1 cycle sequencing kit (Applied Biosystems,
Foster City, CA) and an ABI 3730 XL automatic sequencer
(Applied Biosystems) using the above-described primers.
Statistical Analysis
Fisher exact test and the 2 test were used to compare
categorical variables, and the Mann-Whitney U test was
used for continuous variables. Overall and leukemia-free
survival were estimated using the Kaplan-Meier method,
and differences between the survival curves were analyzed
using the log-rank test. Statistical analyses were performed
using SPSS version 17.0 (SPSS, Chicago, IL). P values less
than .05 were considered statistically significant.
Results
Clinical Characteristics of Enrolled Patients
The baseline characteristics of all patients are summa-
rized in Table 1. All patients were Korean, with a median
age of 58 years (range, 8-84 years). There were 91 (46%)
male and 108 female patients. Hemoglobin, leukocyte, and
platelet counts were variable among the different disease
subtypes. Twenty-one (10.6%) patients progressed to the

American Society for Clinical Pathology Am J Clin Pathol 2015;143:635-644 637

DOI: 10.1309/AJCPUAAC16LIWZMM
Kim et al / CALR Mutations in Four MPN Subtypes
accelerated or blast phases, and 71 (35.7%) patients had or
developed significant fibrosis (MF-2 or MF-3). A subset of
patients (14 [7.0%]) underwent allogeneic transplantation.
CALR and Other Mutations
In the 199 patients with MPN, CALR frameshift muta-
tions were detected in 25 (12.6%) Table 2. JAK2 V617F
mutations were detected in 134 patients (67.3%), and muta-
tions in JAK2 exon 12 were detected in two (1.0%) patients
with PV. MPL was detected in seven (3.5%) patients (six
patients with the MPL W515L mutation and one patient with
the MPL W515K mutation). Thirty-six (18.1%) patients
were triple negative for all three mutations. CALR frameshift
mutations and JAK2 or MPL mutations were mutually exclu-
sive. Among those patients without JAK2 or MPL mutations
(n = 57), the frequency of CALR frameshift mutations was
43.9%. Among the CALR frameshift mutations, 15 patients
had typical type 1 mutations (L367fs*46), and nine patients
had type 2 mutations (K385fs*47). One patient had slightly
different breakpoints and subsequent amino acid products
(L367fs*49; c.1093-1138del) with typical type 1 deletions
(c.1092_1143del). In the disease subgroups, CALR frame-
shift mutations were detected in eight (14.8%) patients with
PMF and 14 (17.7%) patients with ET. JAK2 mutations were
observed in 57.4% and 63.3% of patients with PMF and
ET, respectively. No patients with PV had a CALR frame-
shift mutation, but JAK2 mutations were found in 91.4% of
patients with PV. In all four patients with APMF, no muta-
tions in the JAK2, CALR, and MPL genes were observed.
Among the eight patients with MPN-U, three (37.5%) had
a CALR frameshift mutation, which is a significantly higher
ratio of CALR/JAK2 mutations compared with the PMF and
ET groups (3/2 vs 22/81, P = .045).
CALR Mutations and Correlation
With Clinical Characteristics
In patients with PMF, CALR frameshift mutations were
associated with lower levels of leukocytes and absolute neu-
trophils, less significant BM hypercellularity, and a higher
number of BM megakaryocytes compared with patients
with JAK2 mutations Table 3. The transfusion require-
ment decreased in patients with CALR mutations patients
compared with patients with JAK2 mutations (P = .066).
No patients progressed to accelerated or blast phase, and
no significant thrombotic events were observed in patients
with PMF who had CALR mutations, although a statistical
comparison was difficult because of the small number of
events in the JAK2-mutated groups (progression to acceler-
ated or blast phase [25.8%] and thrombotic events [16.1%]
for patients with PMF who had JAK2 mutations).
In patients with ET, patients with CALR frameshift
mutations had lower hemoglobin levels and leukocyte
638 Am J Clin Pathol 2015;143:635-644
DOI: 10.1309/AJCPUAAC16LIWZMM
and granulocyte counts and tended to have higher platelet
counts Table 4. In addition, patients with ET who had
CALR mutations more frequently progressed to acceler-
ated or blast phase disease compared with patients with a
JAK2 mutation, but the number of patients was small (n
= 3 [21.4%]; P = .032). There were slightly higher rates
of male patients (50%) among patients with ET who had
CALR mutations compared with patients with JAK2 muta-
tions (30%); however, there was no statistical significance
between these groups (P = .164). Patients with ET who had
CALR mutations had slightly higher ratios of progression to
post-ET myelofibrosis than did patients with JAK2 muta-
tions, but this difference was not statistically significant
(21.4% vs 14.0%, P = .499).
CALR Mutations and Their Correlation
With Cytogenetic Characteristics
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Cytogenetic abnormalities assessed by G-banding analy-
sis were observed in 21 (38.9%) patients with PMF and seven
(8.9%) patients with ET. Three patients with CALR mutations
had cytogenetic abnormalities. There were no significant
differences in the percentages of patients with cytogenetic
abnormalities between those with JAK2 and CALR mutations
(Tables 3 and 4; PMF, 35.5% vs 25.0%, P = .575; ET, 12.0%
vs 7.1%, P = .607). CALR frameshift mutations were not
associated with any specific cytogenetic abnormalities. Two
patients with PMF who had cytogenetic abnormalities had
46,XY,del(20)(q11.2)[20] and 47,XY,+der(1;19)(q10;p10)
[13]/46,XY[7] mutations. One patient with a del(20)(q11.2)
mutation also had deletions on 20q in 73.5% of cells by
interphase FISH analysis. One patient with ET had com-
plex abnormalities (44,XX,der(1)t(1;?3)(p36;q23),der(3;12)
(q10;q10),5,add(19)(p13.3)[11]/45,XX,der(1)
t(1;?3),del(3)(p?21p25), 5,add(19)[4]/46,XX,der(1)
t(1;?3),5,add(19)(q10),+22,mar[3]/46,XX[3]) and was
tested for cytogenetic abnormalities after progression to the
accelerated phase with 9.7% of blasts in BM. Sequential
interphase FISH analyses revealed 51.5% cells with 5q dele-
tions in the accelerated phase and 97.0% cells with 5q dele-
tions in the subsequent blast phase.
CALR Mutations and Their Prognostic Significance
When the overall and leukemia-free survivals were
compared among patients with CALR, JAK2, and MPL
mutations and triple-negative patients, no significant dif-
ferences were detected between patients with PMF and
ET Figure 1. However, in patients with PMF, those
with CALR mutations seemed to have better overall and
leukemia-free survival compared with patients with JAK2
mutations (P = .264 and P = .320). However, the small
number of patients made it difficult to reach conclusive
results (Figures 1A and 1B).
American Society for Clinical Pathology
 AJCP / Original Article

Table 2
Number of Diseases With Mutations
No. (%) of Cases
Mutation Total MPN (n = 199) PMF (n = 54) ET (n = 79) PV (n= 58) MPN-U (n = 8) APMF (n = 4)
JAK2
JAK2, V617F 134 (67.3) 31 (57.4) 50 (63.3) 51 (87.9) 2 (25.0) 0
JAK2, exon 12 2 (1.0) 0 0 2 (3.5) 0 0
CALR frameshift mutations 25 (12.6) 8 (14.8) 14 (17.7) 0 3 (37.5) 0
Type 1 mutation 16 (8.0)a 6 (11.1)a 9 (11.4) 0 1 (12.5) 0
Type 2 mutation 9 (4.5) 2 (3.7) 5 (6.3) 0 2 (25.0) 0
MPL 7 (3.5) 5 (9.3) 2 (2.5) 0 0 0
Triple negative 36 (18.1) 11 (20.4) 13 (16.5) 5 (8.6) 3 (37.5) 4 (100)
APMF, acute panmyelosis with myelofibrosis; ET, essential thrombocythemia; MPN, myeloproliferative neoplasm; MPN-U, myeloproliferative neoplasm, unclassifiable; PMF,
primary myelofibrosis; PV, polycythemia vera.
a One patient with a CALR frameshift mutation L367fs*49 (c.1093-1138del) and others with the L367fs*46 mutation.

Table 3
Clinical and Laboratory Characteristics of 54 Patients With PMF Stratified According to Mutation Profilesa

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Total PMF JAK2 Mutated CALR Mutated MPL Mutated Triple Negative P Valueb
Variable (n = 54) (n = 31) (n = 8) (n = 4) (n = 11) (JAK2 vs CALR)
Male/female, % male 32/22 (59.3) 18/13 (58.1) 6/2 (75.0) 3/1 (75.0) 5/6 (45.5) .380
Age, y 61.5 (8.0-83.3) 61.9 (43.6-83.3) 60.3 (47.1-70.1) 64.7 (57.0-71.5) 59.1 (8.0-81.9) .972
Age >65 y 22 (40.7) 13 (41.9) 3 (37.5) 2 (50.0) 4 (36.4) .820
Hemoglobin, g/dL 10.4 (5.2-16.9) 11.3 (5.2-16.9) 10.4 (7.8-12.8) 8.5 (5.7-10.9) 8.8 (7.7-12.4) .157
Leukocytes, 109/L 9.56 (2.18-38.53) 11.02 (2.26-38.53) 7.55 (2.56-15.86) 6.86 (2.18-11.79) 3.92 (2.31-15.60) .034
Absolute neutrophils, 109/L 5.44 (1.01-35.06) 8.03 (1.01-35.06) 4.42 (1.68-9.56) 4.41 (1.11-5.54) 2.27 (1.32-10.69) .018
Platelets, 109/L 285 (10-1,455) 368 (32-1,455) 548 (107-1,031) 256 (203-308) 35 (10-382) .260
Circulating blasts, % 0 (0-17) 0 (0-11) 0 (0-3) 3 (0-6) 0 (0-17) .422
DIPSS-plus risk group .637
Low 7 (13.0) 5 (16.1) 2 (25.0) 0 0
Intermediate 1 8 (14.8) 5 (16.1) 2 (25.0) 1 (25.0) 0
Intermediate 2 16 (29.6) 10 (32.3) 3 (37.5) 1 (25.0) 2 (18.2)
High 23 (42.6) 11 (35.5) 1 (12.5) 2 (50.0) 9 (81.8)
Constitutional symptoms 32 (59.3) 17 (54.8) 4 (50.0) 2 (50.0) 9 (81.8) .807
Circulating blasts 1% 17 (31.5) 9 (29.0) 2 (25.0) 3 (75.0) 3 (27.3) .821
Hemoglobin <10 g/dL 24 (44.4) 9 (29.0) 3 (37.5) 3 (75.0) 9 (81.8) .644
Transfusion requirements 30 (55.6) 15 (48.4) 1 (12.5) 3 (75.0) 11 (100.0) .066
Leukocytes >25 109/L 4 (7.4) 4 (12.9) 0 0 0 .284
Platelets <100 109/L 12 (22.2) 4 (12.9) 0 0 8 (72.7) .284
Cytogenetic abnormalities 21 (38.9) 11 (35.5) 2 (25.0) 3 (75.0) 5 (45.5) .575
Cytogenetic categories, 10 (18.5) 4 (12.9) 0 1 (25.0) 5 (45.5) .284
unfavorable
Splenomegaly 43 (79.6) 26 (83.9) 6 (75.0) 2 (50.0) 9 (81.8) .560
BM blasts 1 (0-14) 1 (0-8) 1 (0-3) 2 (0-14) 1 (0-14) .109
BM cellularity, % 85 (25-95) 85 (35-95) 70 (25-95) 60 (25-95). 85 (75-95) .016
Megakaryocytes per hpf in 6.5 (0-20.0) 6.5 (0-20.0) 10.0 (5.0-15.5) 7.8 (6.0-9.5) 6.5 (0.5-17.0) .045
BM
Reticulin fibrosis (MF 2-3) 46 (85.2) 24 (77.4) 8 (100) 4 (100) 10 (90.9) .138
Progression of fibrosis in 47 (87.0) 25 (80.7) 8 (100) 4 (100) 10 (90.9) .176
subsequent disease
course
Progression to accelerated or 12 (22.2) 8 (25.8) 0 1 (25.0) 3 (27.3) .107
blast phase
Thrombotic events 7 (13.0) 5 (16.1) 0 0 2 (18.2) .224
Major bleeding events 5 (9.3) 1 (3.2) 0 0 4 (36.4) .607
Allogeneic transplantation 10 (18.5) 4 (12.9) 0 1 (25.0) 5 (45.5) .284
Deceased 25 (46.3) 12 (38.7) 2 (25.0) 1 (25.0) 10 (90.9) .471
Follow-up, mo 29.2 (1.4-280.5) 25.9 (1.5-168.4) 79.9 (12.7-150.6) 36.3 (30.3-90.1) 15.3 (1.4-280.5) .049
BM, bone marrow; DIPSS-plus, Dynamic International Prognostic Scoring Systemplus; hpf, high-power field; MF, myelofibrosis; PMF, primary myelofibrosis.
a Data are presented as the median (range) for continuous variables and the number of cases (percentage) for categorical variables unless otherwise indicated.
b P values were calculated using the 2 test for categorical variables and the Mann-Whitney U test for continuous variables between patients with JAK2 mutations and CALR

frameshift mutations. Significant values are in boldface.

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Kim et al / CALR Mutations in Four MPN Subtypes

Table 4
Clinical and Laboratory Characteristics of 79 Patients With ET According to Their Mutation Profilesa
P Valueb
Total ET JAK2 Mutated CALR Mutated MPL Mutated Triple Negative (JAK2 vs
Variable (n = 79) (n = 50) (n = 14) (n = 2) (n = 13) CALR)
No. of males (%) 26 (32.9) 15 (30.0) 7 (50.0) 1 (50.0) 3 (23.1) .164
Age, y 55.0 (19.3-83.9) 57.3 (27.1-83.9) 56.0 (20.0-73.8) 52.1 (37.7-66.5) 38.5 (19.3-68.6) .691
Age >65 y 20 (25.3) 14 (28.0) 4 (28.6) 1 (50.0) 1 (7.7) .967
Hemoglobin, g/dL 13.0 (8.8-19.5) 13.5 (10.1-19.5) 12.2 (8.8-15.5) 12.8 (12.3-13.2) 12.6 (10.0-15.8) .033
Leukocytes, 109/L 8.68 (2.02-38.52) 9.79 (2.02-38.52) 7.29 (4.02-10.50) 7.72 (7.16-8.27) 7.30 (3.27-12.30) <.001
Absolute neutrophils, 109/L 5.97 (1.05-34.67) 6.61 (1.05-34.67) 4.11 (2.63-7.35) 4.65 (3.79-5.50) 4.75 (2.08-8.19) <.001
Platelets, 109/L 842 (483-3,519) 734 (483-2,587) 949 (490-2,450) 1,306 (1,112-1,499) 1,036 (665-3,519) .053
Circulating blasts, % 0 (0-3) 0 (0-3) 0 (0-2) 0 (0-0) 0 (0-0) .360
Splenomegaly 25 (31.7) 20 (40.0) 2 (14.3) 0 3 (23.1) .073
Cytogenetic abnormalities 7 (8.9) 6 (12.0) 1 (7.1) 0 0 .607
BM blasts, % 0 (0-20) 0 (0-20) 1 (0-11) 1 (0-2) 0 (0-3) .068
BM cellularity, % 55 (30-100) 58 (30-100) 55 (40-75) 55 (55-55) 55 (35-80) .328
Megakaryocytes per hpf in BM 7.5 (2.5-22.5) 7.5 (3.0-15.0) 7.5 (5.5-22.5) 12.5 (12.5-12.5) 8.5 (2.5-12.5) .166
Reticulin fibrosis (MF 2-3) 6 (7.6) 3 (6.0) 2 (14.3) 0 1 (7.7) .307
Progression of fibrosis in 11 (13.9) 7 (14.0) 3 (21.4) 0 1 (7.7) .499
subsequence disease course

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Progression to accelerated or 6 (7.6) 2 (4.0) 3 (21.4) 0 1 (7.7) .032
blast phase
Thrombotic events 5 (6.3) 4 (8.0) 1 (7.1) 0 0 .916
Major bleeding events 1 (1.3) 0 0 0 1 (7.7) .999
Allogeneic transplantation 1 (1.3) 1 (2.0) 0 0 0 .594
Deceased 9 (11.4) 5 (10.0) 2 (14.3) 1 (50.0) 1 (7.7) .650
Follow-up, mo 58.5 (3.7-216.5) 53.2 (3.7-199.1) 47.6 (9.5-216.5) 56.2 (55.1-57.2) 70.2 (5.1-104.9) .922
BM, bone marrow; ET, essential thrombocythemia; hpf, high-power field; MF, myelofibrosis.
a Data are presented as the median (range) for continuous variables and the number (percentage) for categorical variables unless indicated otherwise.
b P values were calculated using the 2 test for categorical variables and the Mann-Whitney U test for continuous variables in patients with JAK2 and CALR frameshift mutations.

Significant values are in boldface.

Comparison of Clinical and Laboratory Characteristics
of Type 1 and Type 2 CALR Mutations
Type 1 CALR mutations were present in six patients
with PMF and nine patients with ET, and type 2 CALR
mutations were present in two patients with PMF and
five patients with ET; therefore, there were no significant
differences between the distribution of type 1 and type 2
mutations among the MPN subtypes Table 5. Because of
the small number of patients belonging to specific MPN
subtypes with different types of CALR mutations, we
compared the clinical and laboratory characteristics of the
type 1 and type 2 CALR mutations among the total group
of patients with MPN. Patients with type 2 mutations had
slightly higher hemoglobin levels and blast counts in BM
(Table 5). Patients with type 1 mutations tended to develop
more fibrosis than did patients with type 2 mutations (56.3%
vs 22.2%, respectively, P = .099). In addition, patients with
type 2 mutations had a slightly younger median age (type
2 vs type 1, 48.8 vs 59.8 years, respectively; P = .174) and
slightly higher median platelet counts (1,081 109/L vs
763 109/L, respectively; P = .074), but these differences
were not statistically significant, likely due to the small
sample size. There were no significant differences in overall
survival between patients with type 1 and type 2 mutations
(P = .940). When the prognoses of type 1 and type 2 CALR
mutations were compared among the patients with PMF,
the patients with type 1 mutations appeared to show better
overall and leukemia-free survival rates compared with the
patients with type 2 mutations, although this difference was
not statistically significant because of the small sample size
(P = .307 and P = .341) Figure 2A and Figure 2B. Among
the ET patient group, no significant differences in overall or
leukemia-free survivals were detected between the patients
with type 1 and type 2 CALR mutations Figure 2C and
Figure 2D.
Discussion
In this study, we also observed frequent (43.9%) CALR
mutations in patients with PMF, ET, and MPN-U with-
out JAK2 and MPL mutations but not in patients with PV
without JAK2 and MPL mutations, and these findings are
consistent with previous reports.5,6,15,16 Previous studies of
FLT3 mutations among Korean patients with AML have
reported significantly lower mutation frequencies compared
with reports of other Asian and Western patients.17,18 Our
data and a previous study of Chinese patients with ET dem-
onstrated no significant difference in CALR mutation fre-
quency compared with Western studies.15 Therefore, there

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 AJCP / Original Article

A JAK2 mutated (n = 31) B JAK2 mutated (n = 31)

CALR (n = 8) CALR (n = 8)
1.0 MPL mutated (n = 4) 1.0 MPL mutated (n = 4)
Triple-negative (n = 11) Triple-negative (n = 11)

0.8 0.8

Leukemia-Free Survival
Overall Survival

0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0
0 50 100 150 200 250 300 0 50 100 150 200 250 300
Months Months

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C JAK2 mutated (n = 50) D JAK2 mutated (n = 50)
CALR (n = 14) CALR (n = 14)
MPL mutated (n = 2) MPL mutated (n = 2)
1.0 Triple-negative (n = 13) 1.0 Triple-negative (n = 13)

0.8 0.8
Leukemia-Free Survival
Overall Survival

0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0
0 50 100 150 200 250 0 50 100 150 200 250
Months Months

Figure 1 Prognosis of primary myelofibrosis (PMF; n = 54) (A, B) and essential thrombocythemia (ET; n = 79) (C, D) according
to mutation profiles. Overall survival (A) and leukemia-free survival (B) of patients with PMF according to the presence of JAK2,
CALR, and MPL mutations. Overall survival (C) and leukemia-free survival (D) of patients with ET according to the presence of
JAK2, CALR, and MPL mutations. JAK2+ vs CALR+: P = .264 (A), P = .320 (B), P = .789 (C), and P = .452 (D).

may be no significant ethnic influences in the occurrence of
CALR mutations. Interestingly, in this study, CALR muta-
tions were most frequent in patients with MPN-U (37.5%)
compared with mutations in the PMF and ET groups (14.8%
and 17.7%, respectively). We noticed that CALR mutations
were absent in patients with APMF, in whom a differential
diagnosis of MPN in blast phase was difficult. Although this
study included a small number of patients, we observed that
APMF had different pathogenetic mechanisms compared
with MPNs, although the disease presentation may be simi-
lar in many aspects.
When we compared the clinical and hematologic char-
acteristics of patients with CALR mutations and those
patients with JAK2 mutations, patients with PMF who had
CALR mutations had lower levels of leukocytosis, lower
neutrophilia levels, and higher megakaryocyte burden. A
previous study found that CALR mutations were associated
with a younger age, higher platelet count, lower thrombosis
risk, lower DIPSS-plus score, and less leukocytosis and
were less likely to be transfusion dependent.16 Our data for
patients with CALR mutations also showed a tendency of
higher platelet counts and lessened transfusion requirement,
although these differences were not statistically significant,
likely due to a small sample size. No overt tendency for a
younger age in patients with PMF who had CALR muta-
tions was evident in our study. In patients with ET, CALR
mutations were associated with lower levels of leukocytosis,
lower hemoglobin levels, and higher platelet counts, which

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Kim et al / CALR Mutations in Four MPN Subtypes

Table 5
Comparison of Clinical and Laboratory Characteristics in Patients With Type 1 and Type 2 CALR Mutationsa
Variable Total (n = 25) Type 1 (n = 16) Type 2 (n = 9) P Value
Disease classification .439
PMF 8 (32.0) 6 (37.5) 2 (22.2)
ET 14 (56.0) 9 (56.3) 5 (55.6)
MPN-U 3 (12.0) 1 (6.3) 2 (22.2)
Male/female, % male 14/11 (56.0) 9/7 (56.3) 5/4 (55.6) .973
Age, y 59.5 (20.0-73.8) 59.8 (42.8-73.8) 48.8 (20.0-69.4) .174
Hemoglobin, g/dL 12.0 (7.8-15.5) 11.7 (7.8-15.5) 12.8 (10.4-15.1) .047
Leukocytes, 109/L 7.39 (2.37-15.86) 7.37 (3.11-15.86) 7.39 (2.56-12.14) .378
Absolute neutrophils, 109/L 3.96 (1.52-9.56) 4.30 (1.68-9.56) 3.69 (1.77-7.84) .378
Platelets, 109/L 867 (107-2,450) 763 (135-2,450) 1,081 (107-1,765) .074
Circulating blasts, % 0 (0-3) 0 (0-2) 0 (0-3) 1.000
Splenomegaly 9 (36.0) 7 (43.8) 2 (22.2) .282
Cytogenetic abnormalities 3 (12.0) 2 (12.5) 1 (11.1) .918
BM blasts, % 1 (0-11) 1 (0-3) 2 (0-11) .036
BM cellularity, % 65 (25-95) 63 (25-95) 65 (40-85) .412
Megakaryocytes per hpf in BM 7.5 (5.0-22.5) 7.5 (5.0-15.5) 7.5 (6.5-22.5) .245
Reticulin fibrosis (MF 2-3) 10 (40.0) 8 (50.0) 2 (22.2) .174

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Progression of fibrosis in subsequent disease course 11 (44.0) 9 (56.3) 2 (22.2) .099
Progression to accelerated or blast phase 3 (12.0) 2 (12.5) 1 (11.1) .918
Thrombotic events 1 (4.0) 1 (6.3) 0 .444
Major bleeding events 0 0 0 1.000
Allogeneic transplantation 0 0 0 1.000
Deceased 4 (16.0) 3 (18.8) 1 (11.1) .617
Follow-up, mo 47.3 (8.5-216.5) 57.1 (8.5-216.5) 30.2 (12.7-123.1) .692
BM, bone marrow; ET, essential thrombocythemia; hpf, high-power field; MF, myelofibrosis; MPN-U, myeloproliferative neoplasm, unclassifiable; PMF, primary
myelofibrosis.
a Data are presented as the median (range) for continuous variables and the number of cases (percentage) for categorical variables unless otherwise indicated.

were compatible with previous reports examining overall
characteristics.10,11 Previous studies have also reported that
patients with CALR mutations were preferentially male and
younger in age.10,11 We also observed a preference for males
in this group; however, there were no differences in age
between the patients with CALR and JAK2 mutations. When
we investigated the prognostic relevance of CALR mutations,
patients with PMF who had CALR mutations seemed to have
a better prognosis, although this observation was difficult to
confirm because of the small sample size. Most current data
have also indicated that the CALR-mutated group may have
a favorable prognosis compared with patients with JAK2
mutations and those with triple-negative PMF.16,19 Consis-
tent with this observation, the patients with PMF who had
CALR mutations in this study had lower rates of progression
to accelerated or blast phase and a lower incidence of throm-
botic events, although the statistical significance was dif-
ficult to determine because of the small numbers of events.
In ET, the prognostic significance of a CALR mutation is
not certain. One study suggested that the CALR mutations
conferred a survival advantage in patients with ET6; how-
ever, subsequent studies have presented similar long-term
survival rates between patients with ET who had JAK2 and
CALR mutations.10,11,15,20 Previous studies have suggested a
longer thrombosis-free survival10,11,21 and similar leukemia
transformation rates20 for CALR-mutated ET. In this study,
we also did not observe significant differences in overall
survival between patients with ET who had CALR and JAK2
mutations. However, we observed three (21.4%) patients
with ET who progressed to accelerated or blast phase in
the CALR-mutated group. Because transformation to acute
leukemia in patients with ET is a rare event, this observation
is a substantially notable frequency, but prognostic signifi-
cance was not observed. The high rate of progression may be
due to the possible misclassification of patients with PMF
in early fibrotic stage to ET because of the lower leukocyte
counts, higher platelet counts, and higher megakaryocyte
burden. However, with the current diagnostic system largely
depending on morphologic and hematologic features, these
differences may be difficult to discriminate. In addition,
although the CALR-mutated group may be considered a
lower-risk group with the current observations, the risk for
leukemic transformation needs to be carefully assessed in
combination with other factors. Therefore, the prognostic
significance of CALR could be different among the MPN
categories.
Notably, CALR mutations were the most frequent in
patients with MPN-U. MPN-U is a disease entity that has
definite MPN features but fails to meet the criteria for any of
the specific MPN entities or has features that overlap two or
more of the MPN categories. Among the eight patients with
MPN-U, three showed a CALR mutation, which confounded
the discrimination of ET and early PMF. In a sense, MPN-U
has an overlapping spectrum among MPN subtypes within

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 AJCP / Original Article

A B
1.0 Type 1 CALR mutated (n = 6) 1.0 Type 1 CALR mutated (n = 6)
Type 2 CALR mutated (n = 2) Type 2 CALR mutated (n = 2)
JAK2 mutated (n = 31) JAK2 mutated (n = 31)
0.8 0.8

Leukemia-Free Survival
Overall Survival

0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0
0 50 100 150 200 0 50 100 150 200
Months Months

C D

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Type 1 CALR mutated (n = 9) Type 1 CALR mutated (n = 9)
Type 2 CALR mutated (n = 5) Type 2 CALR mutated (n = 5)
JAK2 mutated (n = 50) JAK2 mutated (n = 50)
1.0 1.0

0.8 0.8
Leukemia-Free Survival
Overall Survival

0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0
0 50 100 150 200 250 0 50 100 150 200 250
Months Months

Figure 2 Prognosis of primary myelofibrosis (PMF) (A, B) and essential thrombocythemia (ET) (C, D) according to presence of
type 1 and type 2 CALR mutations and JAK2 mutation. Overall survival (A) and leukemia-free survival (B) of patients with PMF
according to the presence of type 1 and type 2 CALR and JAK2 mutation. Overall survival (C) and leukemia-free survival (D) of
patients with ET according to the presence of type 1 and type 2 CALR and JAK2 mutations.

the WHO classification. These overlapping features resulted
in the highest frequency of CALR mutations in the MPN-U
group among the MPN categories. Classification of MPNs
based on molecular characteristics would help to reduce
ambiguity in the diagnosis of the WHO MPN subtypes.
CALR is a Ca2+-binding protein chaperone that is pri-
marily localized to the endoplasmic reticulum22 and is locat-
ed on 19p13.2. The calreticulin protein has three domains,
including an amino terminal N-domain, central proline-rich
P-domain, and carboxyl terminal C-domain.16 The func-
tion of calreticulin involves Ca2+ homeostasis, disposal of
misfolded proteins, cell adhesion, and immune responses.23
Most CALR mutations involve deletions and insertions in
the C-domain, which cause frameshift mutations and are
classified into type 1 and type 2 mutations. Previous studies
have shown higher platelet counts and lower hemoglobin
and leukocyte counts for patients with type 1 and type 2
mutations compared with patients with JAK2 mutations, and
in patients with ET, male sex and a younger age have been
associated with type 1 and type 2 variants, respectively.7 In
addition, platelet counts are higher in patients with type 2
mutations compared with those with type 1 mutations.7 In
patients with PMF, a comparison of type 2 CALR and JAK2
mutations showed more similarities than differences, and a
comparison of type 1 and type 2 CALR mutations showed
that the latter were associated with higher DIPSS-plus risk

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Kim et al / CALR Mutations in Four MPN Subtypes
scores, leukocytosis, increased peripheral blood percentages,
and decreased survival.8 Although our patient cohort was too
small for a complete analysis, the patients with type 2 CALR
mutations in our study had slightly higher platelet levels and
higher blast counts in BM and tended to be younger.
The limitations of this study are the small number
of patients, the retrospective nature of the study, and an
analysis involving a single center. An additional prospec-
tive multicenter study should be performed to allow a more
comprehensive genetic analysis and to assess the prognostic
significance in Korean patients with PMF.
In conclusion, we observed consistent mutation fre-
quencies and clinical characteristics in Korean patients with
MPN. The CALR mutation was most frequent in patients
with MPN-U. The CALR mutation was associated with pro-
gression to accelerated or blast phase in ET, but the overall
survival was superior (not statistically significant) in CALR-
mutated PMF. Therefore, the clinical significance of CALR
mutations is different among MPN subtypes and still needs
further study, specifically addressing it in the context of the
MPN disease stages.
Address reprint requests to Dr Lee: Dept of Laboratory Medicine,
Seoul National University College of Medicine, 101 Daehak-ro,
Jongno-gu, Seoul 110-744, Republic of Korea; soonlee@snu.
ac.kr.
This study was supported by grants from the Korean Health
Technology R&D Project, Ministry of Health & Welfare, Republic
of Korea (HI12C0169); Basic Science Research Program
through the National Research Foundation of Korea (NRF)
funded by the Ministry of Science, ICT and Future Planning
(NRF-2014R1A2A1A10052286); and Basic Science Research
Program through the NRF funded by the Ministry of Education
(NRF-2014R1A1A2A16049597).
References
1. Swerdlow SH, Campo E, Harris NL, et al, eds. World Health
Organization Classification of Tumours of Haematopoietic and
Lymphoid Tissues. Lyon, France: IARC Press; 2008.
2. Scott LM, Tong W, Levine RL, et al. JAK2 exon 12
mutations in polycythemia vera and idiopathic erythrocytosis.
N Engl J Med. 2007;356:459-468.
3. Pikman Y, Lee BH, Mercher T, et al. MPLW515L is a novel
somatic activating mutation in myelofibrosis with myeloid
metaplasia. PLoS Med. 2006;3:e270.
4. Jones AV, Kreil S, Zoi K, et al. Widespread occurrence of
the JAK2 V617F mutation in chronic myeloproliferative
disorders. Blood. 2005;106:2162-2168.
5. Nangalia J, Massie CE, Baxter EJ, et al. Somatic CALR
mutations in myeloproliferative neoplasms with nonmutated
JAK2. N Engl J Med. 2013;369:2391-2405.
6. Klampfl T, Gisslinger H, Harutyunyan AS, et al. Somatic
mutations of calreticulin in myeloproliferative neoplasms. N
Engl J Med. 2013;369:2379-2390.
644 Am J Clin Pathol 2015;143:635-644
DOI: 10.1309/AJCPUAAC16LIWZMM
7. Tefferi A, Wassie EA, Guglielmelli P, et al. Type 1 vs type
2 calreticulin mutations in essential thrombocythemia:
a collaborative study of 1027 patients. Am J Hematol.
2014;89:E121-E124.
8. Tefferi A, Lasho TL, Finke C, et al. Type 1 vs type 2
calreticulin mutations in primary myelofibrosis: differences in
phenotype and prognostic impact. Leukemia. 2014;28:1568-
1570.
9. Cazzola M, Kralovics R. From Janus kinase 2 to
calreticulin: the clinically relevant genomic landscape of
myeloproliferative neoplasms. Blood. 2014;123:3714-3719.
10. Rumi E, Pietra D, Ferretti V, et al. JAK2 or CALR mutation
status defines subtypes of essential thrombocythemia with
substantially different clinical course and outcomes. Blood.
2014;123:1544-1551.
11. Rotunno G, Mannarelli C, Guglielmelli P, et al. Impact
of calreticulin mutations on clinical and hematological
phenotype and outcome in essential thrombocythemia.
Blood. 2014;123:1552-1555.
12. Gangat N, Caramazza D, Vaidya R, et al. DIPSS plus: a
refined Dynamic International Prognostic Scoring System
Downloaded from http://ajcp.oxfordjournals.org/ by guest on December 5, 2016
for primary myelofibrosis that incorporates prognostic
information from karyotype, platelet count, and transfusion
status. J Clin Oncol. 2011;29:392-397.
13. Thiele J, Kvasnicka HM, Facchetti F, Franco V, van der
Walt J, Orazi A. European consensus on grading bone
marrow fibrosis and assessment of cellularity. Haematologica.
2005;90:1128-1132.
14. International Standing Committee on Human Cytogenetic
Nomenclature, Shaffer LG, McGowan-Jordan J, Schmid M.
ISCN 2013: An International System for Human Cytogenetic
Nomenclature. Unionville, CT: Karger; 2013.
15. Fu R, Xuan M, Zhou Y, et al. Analysis of calreticulin
mutations in Chinese patients with essential
thrombocythemia: clinical implications in diagnosis,
prognosis and treatment. Leukemia. 2014;28:1912-1914.
16. Tefferi A, Lasho TL, Finke CM, et al. CALR vs JAK2 vs
MPL-mutated or triple-negative myelofibrosis: clinical,
cytogenetic and molecular comparisons. Leukemia.
2014;28:1472-1477.
17. Yoo SJ, Park CJ, Jang S, Seo EJ, Lee KH, Chi HS. Inferior
prognostic outcome in acute promyelocytic leukemia with
alterations of FLT3 gene. Leuk Lymphoma. 2006;47:1788-
1793.
18. Bang SM, Ahn JY, Park J, et al. Low frequency and variability
of FLT3 mutations in Korean patients with acute myeloid
leukemia. J Korean Med Sci. 2008;23:833-837.
19. Panagiota V, Thol F, Markus B, et al. Prognostic effect of
calreticulin mutations in patients with myelofibrosis after
allogeneic hematopoietic stem cell transplantation. Leukemia.
2014;28:1552-1555.
20. Tefferi A, Wassie EA, Lasho TL, et al. Calreticulin mutations
and long-term survival in essential thrombocythemia.
Leukemia. 2014;28:2300-2303.
21. Chao MP, Gotlib J. Two faces of ET: CALR and JAK2.
Blood. 2014;123:1438-1440.
22. Eggleton P, Michalak M. Calreticulin for better or for
worse, in sickness and in health, until death do us part. Cell
Calcium. 2013;54:126-131.
23. Gold LI, Eggleton P, Sweetwyne MT, et al. Calreticulin:
non-endoplasmic reticulum functions in physiology and
disease. FASEB J. 2010;24:665-683.
American Society for Clinical Pathology