Panagiota, Thol et al.
Prognostic effect of calreticulin mutations after HSCT
Prognostic effect of calreticulin mutations in patients with myelofibrosis after
allogeneic hematopoietic stem cell transplantation
Victoria Panagiota1*, Felicitas Thol1*, Birgit Markus2, Boris Fehse3, Haefaa Alchalby3,
Anita Badbaran3, Ulrich Lehmann4, Christian Koenecke1, Rabia Shahswar1, Anuhar
Chaturvedi1,
Michael
Stadler1,
Matthias
Eder1,
Gudrun
Göhring5,
Michael
Koenigsmann6, Arnold Kloos1, Arne Trummer1, Thomas Schroeder7, Guido Kobbe7,
Christian Thiede8, Uwe Platzbecker8, Brigitte Schlegelberger5, Hans-Heinrich Kreipe4,
Arnold Ganser1, Nicolaus Kröger3& and Michael Heuser1&
Supplementary data
Supplementary methods
Cytogenetic and molecular analysis
Cytogenetic analysis was performed by G- and fluorescence R-banding. Cytogenetic
risk was defined following criteria by Hussein et al. (1). Molecular analyses were
performed at Hannover Medical School, as described before (2). In brief, mononuclear
cells from bone marrow or peripheral blood were enriched by Ficoll density gradient
centrifugation and stored in liquid nitrogen until further use. Genomic DNA was extracted
from frozen samples using the All Prep DNA/RNA Kit (Qiagen, Hilden, Germany),
according to the manufacturer’s recommendations. CALR was amplified using the
following primers: CALR forward 5’-CTGGTCCTGGTCCTGATGTC-3’ and CALR
reverse
5’-CCAAATCCGAACCAGCCTG-3’.
The
PCR
fragments
were
directly
sequenced and analyzed using Mutation Surveyor software (SoftGenetics, State
College, PA). All mutations were confirmed in an independent experiment.
Statistical analysis
Outcome analysis was performed for CALR mutated or wildtype patients with
myelofibrosis who underwent alloHSCT, as only this patient subset had a significant
proportion of CALR mutated patients. Pair wise comparisons were performed by
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Panagiota, Thol et al.
Prognostic effect of calreticulin mutations after HSCT
Kolmogorov-Smirnov or Student’s t-test for continuous variables and by two-sided chisquared tests for categorical variables, and are provided for exploratory purposes.
Overall survival (OS) endpoints, measured from the date of alloHSCT, were death
(failure) and alive at last follow-up (censored). Cumulative incidence of relapse (CIR)
endpoints, measured from the date of alloHSCT, were relapse (failure) and alive in CR
at last follow-up (censored). Non-relapse mortality (NRM) endpoints, measured from the
date of alloHSCT, were death from non-relapse causes (failure), and alive in CR at last
follow-up (censored), while patients with relapse were censored at the date of relapse.
The Kaplan-Meier method and log-rank test were used to estimate the distribution of OS,
CIR and NRM, and to compare differences between survival curves. The Gray test was
used to compare CIR and NRM as competing risks (3). For multivariate analysis, a Cox
proportional hazards model was constructed for OS and NRM adjusting for potential
confounding covariates (4). The following variables were considered by univariate
analysis for OS and NRM: Age above vs. below median age, primary vs. secondary
myelofibrosis, dynamic international prognostic scoring system (DIPSS, low vs.
intermediate-1 vs. intermediate-2 vs. high) (5), cytogenetic risk according to Hussein et
al. (low vs. intermediate-1 vs. intermediate-2 vs. high) (1), JAK2 mutation status, MPL
mutation status, donor sex, donor type (related vs. unrelated), HLA match (matched vs.
mismatched), and CMV serostatus of recipient and donor (recipient and donor negative
vs. recipient negative/donor positive or recipient positive/donor negative or recipient
positive/donor positive). Variables with P ≤0.1 in univariate analysis were included in the
model. To provide quantitative information on the relevance of results, 95% confidence
intervals (CIs) of hazard ratios (HR) were computed. The two-sided level of significance
was set at P <.05. The statistical analyses were performed with the statistical software
package SPSS 21.0 (IBM Corporation, Armonk, NY) and the R software package.
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Prognostic effect of calreticulin mutations after HSCT
Supplementary Tables
Supplementary Table S1. CALR mutations in patients with primary or secondary
myelofibrosis. Sequence numbering is in accordance to the DNA coding sequence of
Ensembl transcript ENST00000316448 and the protein sequence of ENSEMBL protein
ENSP00000320866.
Patient
Nucleotide change
Amino acid change
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
c.1120_1128delAAGAAACinsTACGTA
c.1099_1150del
c.1103_1148del
c.1122delG
c.1139_1140insTC
c.1099_1150del
c.1099_1150del
c.1099_1150del
c.1099_1150del
c.1151-1154delACAAinsTATGTC
c.1099_1150del
c.1099_1150del
c.1102_1135del
c.1099_1150del
c.1154_1155_insTTGTC
c.1099_1150del
c.1099_1150del
c.1154_1155_insATGTC
c.1099_1150del
c.1099_1150del
c.1099_1150del
c.1099_1150del
c.1099_1150del
c.1099_1150del
c.1099_1150del
c.1099_1150del
c.1099_1150del
c.1154_1155insTTGTC
p.K374fs
p.L367fs
p.K368fs
p.K374fs
p.E380fs
p.L367fs
p.L367fs
p.L367fs
p.L367fs
p.D384fs
p.L367fs
p.L367fs
p.K386fs
p.L367fs
p.K385fs
p.L367fs
p.L367fs
p.E386fs
p.L367fs
p.L367fs
p.L367fs
p.L367fs
p.L367fs
p.L367fs
p.L367fs
p.L367fs
p.L367fs
p.K385fs
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Panagiota, Thol et al.
Prognostic effect of calreticulin mutations after HSCT
Supplementary Table S2. Multivariate analysis for OS and NRM in myelofibrosis
patients with mutated (n=28) or unmutated CALR (n=105).
Overall survival
Age
Above vs below median
Donor sex
Female vs male
CALR mutation status
Mutated vs. unmutated
HR
95% CI
P
3.0
1.56-5.77
.001
1.92
1.05-3.51
.033
0.41
0.15-1.16
.094
Non-relapse mortality
Age
Above vs below median
CALR mutation status
Mutated vs. unmutated
Donor sex
Female vs male
HR
95% CI
P
3.36
1.55-7.24
.002
0.22
0.05-0.93
.039
1.46
0.73-2.93
.28
NOTE: Hazard ratios greater than or less than 1 indicate an increased or decreased
risk, respectively, of an event for the first category listed.
Abbreviations: HR, hazard ratio; CI, confidence interval.
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Prognostic effect of calreticulin mutations after HSCT
Supplementary Figures
Supplementary Figure S1. Prognostic effect of donor match on OS, CIR and NRM.
(A) OS according to donor match (MRD, matched related donor; MUD, matched
unrelated donor; MMUD, mismatched unrelated donor) (B) CIR according to donor
match (C) NRM according to donor match.
B
A
C
MRD (n=29)
MUD (n=61)
MMUD (n=43)
MRD (n=29)
MUD (n=61)
MMUD (n=43)
Supplementary Figure S2. Overall survival of patients with primary or secondary
myelofibrosis after alloHSCT.
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Panagiota, Thol et al.
Prognostic effect of calreticulin mutations after HSCT
Supplementary Figure S3. Overall survival of patients with myelofibrosis according to
(A) age above or below the median age and (B) female vs male donor sex.
A
B
Younger (below median age, n=67)
Older (above median age, n=66)
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Panagiota, Thol et al.
Prognostic effect of calreticulin mutations after HSCT
Supplementary Figure S4. Cumulative incidence of non-relapse mortality
according to age and donor sex. (A) Cumulative incidence of non-relapse mortality in
myelofibrosis patients with age above or below the median age. (B) Cumulative
incidence of non-relapse mortality in myelofibrosis patients according to female vs male
donor sex.
B
A
Older (above median age, n=66)
Younger (below median age, n=67)
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Panagiota, Thol et al.
Prognostic effect of calreticulin mutations after HSCT
Supplementary Figure S5. Prognostic effect of CMV serostatus of donor and
recipient on NRM and OS.
(A) Non-relapse mortality in patients where donor and recipient had negative CMV
serostatus compared to patients where at least one of donor or recipient had positive
CMV serostatus. (B) OS in patients where donor and recipient had negative CMV
serostatus compared to patients where at least one of donor or recipient had positive
CMV serostatus.
A
B
CMV: patient negative,
donor negative (n=33)
CMV: all other
Combinations (n=100)
CMV: patient negative,
donor negative (n=33)
CMV: all other
Combinations (n=100)
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Panagiota, Thol et al.
Prognostic effect of calreticulin mutations after HSCT
Supplementary Figure S6. Prognostic effect of acute GvHD on NRM and OS.
(A) Non-relapse mortality in patients with and without acute GvHD. (B) OS in patients
with and without acute GvHD.
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Panagiota, Thol et al.
Prognostic effect of calreticulin mutations after HSCT
Supplementary Figure S7. Prognostic effect of chronic GvHD on NRM and OS.
(A) Non-relapse mortality in patients with and without chronic GvHD. (B) OS in patients
with and without chronic GvHD.
B
A
with cGvHD, n=66
no cGvHD, n=50
no cGvHD, n=50
with cGvHD, n=66
Supplementary References
1.
Hussein K, Pardanani AD, Van Dyke DL, Hanson CA, Tefferi A. International
Prognostic Scoring System-independent cytogenetic risk categorization in
primary myelofibrosis. Blood 2010 Jan 21; 115(3): 496-499.
2.
Thol F, Kade S, Schlarmann C, Löffeld P, Morgan M, Krauter J, et al. Frequency
and prognostic impact of mutations in SRSF2, U2AF1, and ZRSR2 in patients
with myelodysplastic syndromes Blood 2012; 119(15): 3578-3584.
3.
Gray RJ. A class of K-sample tests for comparing the cumulative incidence of a
competing risk. Annals of Statistics 1988; 16: 1141-1154.
4.
COX DR. Regression models and life tables. J R Stat Soc B 1972 1972///; 34:
187-202.
5.
Passamonti F, Cervantes F, Vannucchi AM, Morra E, Rumi E, Pereira A, et al. A
dynamic prognostic model to predict survival in primary myelofibrosis: a study by
the IWG-MRT (International Working Group for Myeloproliferative Neoplasms
Research and Treatment). Blood 2010 Mar 4; 115(9): 1703-1708.
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