CALR exon 9 mutations are somatically acquired events in familial cases
of essential thrombocythemia or primary myelofibrosis
Elisa Rumi,1,2Ashot S. Harutyunyan,3Daniela Pietra,1Jelena D. Milosevic,3Ilaria C. Casetti,2Marta Bellini,2
Nicole C. C. Them,3Chiara Cavalloni,1Virginia V. Ferretti,1Chiara Milanesi,1Tiina Berg,3Emanuela Sant’Antonio,2
Emanuela Boveri,4Cristiana Pascutto,1Cesare Astori,1Robert Kralovics,3,5and Mario Cazzola,1,2on behalf of the
Associazione Italiana per la Ricerca sul Cancro Gruppo Italiano Malattie Mieloproliferative Investigators
1Department of Hematology Oncology, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Policlinico San Matteo, Pavia, Italy;
2Department of Molecular Medicine, University of Pavia, Pavia, Italy;3CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences,
Vienna, Austria;4Anatomic Pathology Section, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; and5Department of Internal Medicine I, Division of
Hematology and Blood Coagulation, Medical University of Vienna, Vienna, Austria
• Somatic indels of CALR exon
9 are present in about 20% to
25% of sporadic patients with
essential thrombocythemia or
• These mutations are found
also in familial cases of
essential thrombocythemia or
primary myelofibrosis as
somatically acquired events.
Somatic mutations in the calreticulin (CALR) gene were recently discovered in patients
with sporadic essential thrombocythemia (ET) and primary myelofibrosis (PMF) lacking
JAK2 and MPL mutations. We studied CALR mutation status in familial cases of
myeloproliferativeneoplasm. In a cohortof 127 patients,CALR indels wereidentifiedin 6
of 55 (11%) subjects with ET and in 6 of 20 (30%) with PMF, whereas 52 cases of
polycythemia vera had nonmutated CALR. All CALR mutations were somatic, found in
granulocytes but not in T lymphocytes. Patients with CALR-mutated ET showed a higher
disease progression (P 5 .047) compared with those with JAK2 (V617F). In conclusion, a
significant proportion of familial ET and PMF nonmutated for JAK2 carry a somatic
mutation of CALR. (Blood. 2014;123(15):2416-2419)
Myeloproliferative neoplasms (MPNs), including polycythemia
vera (PV), essential thrombocythemia (ET), and primary myelofi-
brosis (PMF), have in most instances sporadic occurrence.1
However, familial clustering of MPN has been reported by us and
others.2,3Common mutations involved in the pathogenesis of MPN
also in familial cases,3-5although rare cases of germline MPL, CBL,
and TET2 mutations have been reported.6-8The JAK2 GGCC
haplotype confers susceptibility to MPN, but does not explain
Two recent articles demonstrated that most of the patients with
sporadic ET or PMF not associated with JAK2 or MPL alterations
carry somatic mutations of calreticulin (CALR).9,10The clinical
course ofsporadic CALR-mutatedpatientsismoreindolent thanthat
of JAK2-mutated patients.9
The role of CALR mutations in familial MPN remains to be
clarified. In this study, we aimed to investigate the frequency of
CALR mutations in familial MPN, their germline or somatic oc-
currence, and their correlation with clinical phenotype.
Patients and methods
This study was approved by the Ethics Committee of Fondazione Istituto di
Ricovero e Cura a Carattere Scientifico Policlinico San Matteo, Pavia, Italy.
The procedures followed were in accordance with the Helsinki Declaration,
and samples were obtained with patients’ written informed consent.
We identified in our database a total of 154 consecutive patients with
familial MPN diagnosed and followed from 1970 to 2013 at the Department
Scientifico Policlinico San Matteo, Pavia. DNA was available in 127 cases
belonging to 78 families, which were included into the study. Patients were
defined as familial cases if 2 or more individuals within the same pedigree
use at the time of the first observation, as previously described.11
JAK2 (V617F) mutation status was assessed in granulocyte DNA as
JAK2 exon 12 mutations, MPL exon 10 mutations, and CALR exon 9
pedigrees with all affected members negative for JAK2, MPL, and CALR were
evaluated for clonality using X-chromosome–inactivation pattern (XCIP).12
Submitted January 17, 2014; accepted February 14, 2014. Prepublished
online as Blood First Edition paper, February 19, 2014; DOI 10.1182/blood-
E.R. and A.S.H. contributed equally to this paper.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
© 2014 by The American Society of Hematology
2416BLOOD, 10 APRIL 2014 x VOLUME 123, NUMBER 15
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Overall survival was estimated using the Kaplan-Meier product limit
method, and survival curves were compared by the log-rank test. The cu-
mulative incidence of disease transformation and that of thrombotic events
were estimated with a competing risk approach, considering death from all
causes as a competing event.17The comparison of cumulative incidence
curves in different groups of patients was carried out using the Pepe-Mori
test.18All P values were considered statistically significant when smaller
than .05 (2-tailed). Statistical analyses were performed using Stata12.1
(StataCorp LP, College Station, TX) software.
Results and discussion
Of 127 patients, 94 carried JAK2 mutations (91 JAK2 V617F and 3
any mutations. We did not find any MPL mutation. The distribution
of mutations according to diagnosis was significantly different
PMF, as reported in Figure 1A.
All CALR mutations were demonstrated to be somatically ac-
familial MPN patients are listed in Table 1. As reported previously
for sporadic MPN,9the type 1 (n 5 4) and type 2 (n 5 6) CALR
mutations were predominant, with the remaining 2 patients carrying
type 27 and a novel type (which we numbered continuously as type
41) CALR mutation. The novel type 41 is a complex mutation,
consisting of 2 separate deletion events of 1 bp and 7 bp, as reported
in Figure 1B; this mutation creates the longest mutant CALR
protein described so far.
The clinical phenotype within the familial cluster was
homogenous in 41 (53%) of 78 families (PV in 20 families, ET
in 16 families, PMF in 5 families), whereas the remaining 37
families (47%) exhibited mixed phenotypes. In 45 of 78 families,
DNA was available for all affected members; the somatic
mutational and diagnosis pattern are reported in supplemental
phenotype (ET) and coexistence of JAK2 and CALR mutations
(supplemental Figure 1).
Figure 1. Distribution of mutations according to
diagnosis and description of the novel CALR
mutation. (A) JAK2 mutations were present in 42 of
52 (80.8%) patients with PV, 40 of 55 (72.7%) patients
with ET, and 12 of 20 (60%) patients with PMF. CALR
mutations were identified in 6 of 55 (10.9%) patients
with ET and 6 of 20 (30%) patients with PMF. The
remaining 10 patients (19.2%) with PV, 9 patients
(16.4%) with ET, and 2 patients (10%) with PMF did
not carry any mutation. (B) The novel type 41 is
a complex mutation, consisting of 2 separate deletion
events of 1 bp and 7 bp. These 2 deletions were shown
to be on the same allele by polymerase chain reaction
product subcloning and sequencing, as described
previously.9The first deletion introduces a frameshift
to alternative frame, which is common to all CALR exon
9 mutations, whereas the second deletion causes
a frameshift to the third alternative frame at the end
of exon 9. Because the next stop codon comes later
in the third frame, this mutation creates the longest
CALR protein described so far (20 amino acids longer
than the wild type). Each vertical bar represents
an amino acid: blue bars indicate negatively charged
amino acids, red bars indicate positively charged
amino acids, white bars indicate uncharged amino
acids, and black bars indicate stop codons. Horizontal
black bars below the frames denote the amino acid
stretch encoded by the respective frame. The arrows
indicate the location of the 2 deletions. 39 UTR, 39
untranslated region of the gene.
Table 1. Somatic CALR mutations found in familial MPN patients
Patient ID Sample IDDiagnosis CALR mutation typeCALR mutationMutation burden, %
S47A9SM MPC09_213PMF1 c.1092_1143del27
S29A12SM MPC12_115 ET1 c.1092_1143del48
S48A7SMMPC07_162 ET2 c.1154_1155insTTGTC24
S106A7SM MPC07_395PMF2 c.1154_1155insTTGTC44
S20A10SM MPC10_100ET2 c.1154_1155insTTGTC27
S113A10SM MPC10_288ET2 c.1154_1155insTTGTC40
S157A10SM MPC10_594PMF2 c.1154_1155insTTGTC47
S100A11SMMPC11_627 ET2 c.1154_1155insTTGTC 47
S167A6SM 906ET 27 c.1123_1125delinsTGTTT50
S17A99SM f5p3PMF41 [c.1143del;1214_1220del]49
BLOOD, 10 APRIL 2014 x VOLUME 123, NUMBER 15FAMILIAL MYELOPROLIFERATIVE NEOPLASMS 2417
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Interestingly, there were 3 families with all patients diagnosed
with PV and nonmutated JAK2 and 3 families with all patients diag-
nosed with ET and nonmutated JAK2, MPL, and CALR (supple-
mental Table 2). Twelve of 14 samples from these 6 families were
analyzed by Affymetrix SNP 6.0 arrays: 10 had normal karyotype
and 2 had aberrations of the Y chromosome, which can be also
related to aging. Five female patients were evaluated for XCIP: 2
XCIP. Affected members of 1 of our families diagnosed with ET
were recently found to carry a germline JAK2 mutation,16as also
shown by other investigators.19,20Overall, absence of somatic
mutations, normal karyotype, and polyclonal hematopoiesis in
suggest that these patients are very likely to be cases of hereditary
thrombocytosis or erythrocytosis.21,22
We did not find significant differences in terms of clinical pheno-
type (age, sex, leukocyte count, hemoglobin, platelet count, ery-
thropoietin, splenomegaly, thrombosis) at diagnosis according to
of patients (supplemental Tables 3 and 4). The only significant
difference (P 5 .017) was a higher platelet count in CALR-mutated
ET patients (median 1222 3 109/L, range 563-1536) in comparison
with JAK2-mutated ET patients (median 657 3 109/L, range 456-
1500), confirming what was previously observed in ET sporadic
The whole cohort was observed for a median follow-up from
17 (13.4%) patients had thrombotic events and 11 (8.7%) patients
developed disease progression (7 secondary myelofibrosis and 4
progression at 10 years according to diagnosis and mutational status
are reported in supplemental Table 5.
The molecular status (CALR-mutated vs JAK2-mutated vs non-
with ET and PV, whereas it did in patients with PMF (P 5 .001). In
detail, in PMF, the median overall survival was 1.3 years in patients
with nonmutated CALR/JAK2/MPL and not reached in CALR-
cases.24In ET, CALR-mutated patients showed a lower incidence of
0% vs 6.6%, P 5 .047) in comparison with JAK2-mutated patients
(supplemental Figure 2A-B), although the low number of patients
does not allow any firm conclusion. The lower rate of thrombosis in
In conclusion, in familial MPN, CALR mutations are somatically
acquired and are associated with ET or PMF phenotype, as in
sporadic MPN. In addition, in familial ET, CALR mutations are
associated with a lower risk of thrombosis with respect to JAK2
(V617F), as observed in sporadic ET.
The authors thank Riccardo Soldinger for editorial assistance.
Studies performed at the Department of Hematology Oncology,
Fondazione IRCCS Policlinico San Matteo, and Department of
Molecular Medicine, University of Pavia, were supported by grants
from the Associazione Italiana per la Ricerca sul Cancro (AIRC),
Fondo per gli investimenti della ricerca di base (FIRB, project no.
RBAP11CZLK), Ministero dell’Istruzione, dell’Universit` a e della
Ricerca (MIUR, PRIN 2010-2011) (M.C.), and from the Italian
Ministry of Health (GR-2010-2312855) (E.R.). In particular, M.C.
Clinical Oncology 5 per mille” AIRC Gruppo Italiano Malattie
Mieloproliferative (project #1005). The studies performed in
Vienna were supported by funding from the Austrian Science Fund
(FWF23257-B12, FWF4702-B20) (R.K.).
Contribution: E.R., A.S.H., R.K., and M.C. conceived this study,
C.C., E.S., and C.A. collected clinical data; A.S.H., D.P., J.D.M.,
and C.P. did statistical analyses; and E.B. studied bone marrow
Conflict-of-interest disclosure: R.K. reports having a pending
patent application on the use of calreticulin gene mutations for the
declare no competing financial interests.
Correspondence: Elisa Rumi, Department of Hematology
Oncology, Fondazione IRCCS Policlinico San Matteo, Viale Golgi
19, 27100 Pavia, Italy; e-mail: email@example.com; and
Mario Cazzola, Department of Hematology Oncology, Fondazione
IRCCS Policlinico San Matteo, Viale Golgi 19, 27100 Pavia, Italy;
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BLOOD, 10 APRIL 2014 x VOLUME 123, NUMBER 15FAMILIAL MYELOPROLIFERATIVE NEOPLASMS2419
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online February 19, 2014
2014 123: 2416-2419
Sant'Antonio, Emanuela Boveri, Cristiana Pascutto, Cesare Astori, Robert Kralovics and Mario
Nicole C. C. Them, Chiara Cavalloni, Virginia V. Ferretti, Chiara Milanesi, Tiina Berg, Emanuela
Elisa Rumi, Ashot S. Harutyunyan, Daniela Pietra, Jelena D. Milosevic, Ilaria C. Casetti, Marta Bellini,
cases of essential thrombocythemia or primary myelofibrosis
exon 9 mutations are somatically acquired events in familial
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