Age, JAK2V617F and SF3B1 mutations are the main predicting factors for survival in refractory anaemia with ring sideroblasts and marked thrombocytosis

Abstract

Refractory anaemia with ring sideroblasts (RARS) and marked thrombocytosis (RARS-T) is a provisional entity in the World Health Organisation 2008 classification and has previously been shown to have a high proportion of JAK2V617F (Janus Kinase 2) and SF3B1 (Splicing Factor 3B subunit 1) mutations. The purpose of the present study was to analyse the frequency of SF3B1 mutations in a large cohort of 111 patients with RARS-T and 33 patients with RARS and to explore the prognostic impact of SF3B1 mutational status on RARS-T. The frequency of SF3B1 mutations in RARS-T (96/111, 86.5%) and RARS (28/33, 84.8%) was similar. In RARS-T, median survival was better in SF3B1-mutated patients than in SF3B1-non-mutated patients (6.9 and 3.3 years, respectively, P=0.003). RARS can be differentiated from RARS-T by the frequency of JAK2V617F (0% vs 48.6%). In RARS-T patients, SF3B1 (P=0.021) and JAK2 mutations (P=0.016) were independent factors for a better prognosis. Altogether, our results confirm that RARS-T is an independent entity that should be recognised by the next World Health Organisation classification. The assessment of SF3B1 mutations is of prognostic interest in RARS-T patients. Younger age, JAK2V617F and SF3B1 mutations are the main predicting factors for survival in RARS-T.

Full text

ORIGINAL ARTICLE
Age, JAK2
V617F
and SF3B1 mutations are the main predicting
factors for survival in refractory anaemia with ring sideroblasts
and marked thrombocytosis
J Brose´us
1
, T Alpermann
2
, M Wulfert
3
, L Florensa Brichs
4,10
, S Jeromin
2
, E Lippert
5
, M Rozman
6,10
, F Lifermann
7
, V Grossmann
2
,
T Haferlach
2
, U Germing
3
, E Lun˜o
8,10
, F Girodon
9
and S Schnittger
2
for the MPN and MPNr-EuroNet (COST Action BM0902)
Refractory anaemia with ring sideroblasts (RARS) and marked thrombocytosis (RARS-T) is a provisional entity in the World Health
Organisation 2008 classification and has previously been shown to have a high proportion of JAK2
V617F
(Janus Kinase 2) and SF3B1
(Splicing Factor 3B subunit 1) mutations. The purpose of the present study was to analyse the frequency of SF3B1 mutations in a
large cohort of 111 patients with RARS-T and 33 patients with RARS and to explore the prognostic impact of SF3B1 mutational
status on RARS-T. The frequency of SF3B1 mutations in RARS-T (96/111, 86.5%) and RARS (28/33, 84.8%) was similar. In RARS-T,
median survival was better in SF3B1-mutated patients than in SF3B1-non-mutated patients (6.9 and 3.3 years, respectively,
P¼0.003). RARS can be differentiated from RARS-T by the frequency of JAK2
V617F
(0% vs 48.6%). In RARS-T patients, SF3B1
(P¼0.021) and JAK2 mutations (P¼0.016) were independent factors for a better prognosis. Altogether, our results confirm that
RARS-T is an independent entity that should be recognised by the next World Health Organisation classification. The assessment of
SF3B1 mutations is of prognostic interest in RARS-T patients. Younger age, JAK2
V617F
and SF3B1 mutations are the main predicting
factors for survival in RARS-T.
Leukemia (2013) 27, 1826–1831; doi:10.1038/leu.2013.120
Keywords: refractory anaemia with ring sideroblasts and marked thrombocytosis; SF3B1; prognostic impact; survival
INTRODUCTION
Refractory anaemia with ring sideroblasts (RARS) and marked
thrombocytosis (RARS-T) has been proposed in the World Health
Organisation 2001 classification of tumours of haematopoietic
and lymphoid tissues and retained as a provisional entity in the
classification of 2008.
1
RARS-T is classified in the myelodysplastic/
myeloproliferative (MDS/MPN) disorders group, because it
presents with the dysplastic features of RARS
2
and the
myeloproliferative features of essential thrombocythemia (ET).
3
RARS-T is characterised by a high rate of JAK2
V617F
(Janus Kinase 2)
mutations
4–15
and/or the presence of the mutation MPL
W515L/R
(MyeloProliferative Leukemia).
16,17
The classification of RARS-T as an
entity that is independent from RARS or ET is currently a matter of
debate. Several specialists favour the hypothesis that RARS-T is a
form of ET with Z15% of ring sideroblasts in the bone marrow
18
while others think that RARS-T develops from RARS with
secondary thrombocytosis accompanying the acquisition of the
JAK2
V617F
mutation.
19
Recent publication from our group in a European retrospective
multicentre collaborative study demonstrated that RARS-T was
independent from RARS and ET from a clinical and biological as
well as prognostic point of view.
20
Our results have recently been strengthened by the discovery of
the association between myelodysplastic syndromes and muta-
tions involving components of the RNA splicing machinery,
including U2AF35 (U2 small nuclear RNA Auxiliary Factor 35), ZRSR2
(Zinc finger CCCH type,RNA-binding motif and Serine/aRginine
rich 2), SRSF2 (Serine/aRginine-rich Splicing Factor 2) and SF3B1
(Splicing Factor 3B subunit 1). SF3B1 mutations (SF3B1
mut
) are found
in about 20% of total MDS and correlate strongly with the
presence of Z15% of ring sideroblasts (MDS-RS; 64–82.6% in
RARS, 57–76% in refractory cytopenia with multilineage dysplasia
and ring sideroblasts (RCMD-RS) and 66.7–72% in RARS-T).
21–27
On
the other hand, mutations of SF3B1 are found at a lower frequency
in MDS with o15% ring sideroblasts, which confirms the
specificity of SF3B1
mut
in MDS-RS. SF3B1 mutations are rare in
myeloproliferative neoplasms and particularly in ET (0–3%).
22,28
The high frequency of SF3B1 mutations suggests that these
mutations have a pathophysiological role in these diseases,
probably through perturbations of RNA splicing. The link
between SF3B1-mutated status and ring sideroblasts has been
confirmed in a recent experimental study on murine models.
29
About one quarter of MDS-RS are SF3B1
wt
and somatic mutations
of SRSF2 or ZRSR2 have been described in about 7% of MDS-RS,
21
1
Haematology Laboratory, University Hospital, Nancy, France;
2
MLL Munich Leukemia Laboratory, Munich, Germany;
3
Department of Haematology, Oncology and Clinical
Immunology, Heinrich-Heine-Universita
¨t, Du¨ sseldorf, Germany;
4
Laboratorio Citologia Hematolo
`gica, Servicio Patologia, Hospital del Mar, Barcelona, Spain;
5
Haematology
Laboratory, University Hospital, Bordeaux, France;
6
Unidad de Hematopatologı
´a, Departamento de Patologı
´a, Hospital Clı
´nic, IDIBAPS, Barcelona, Spain;
7
Department of Internal
Medicine, Hospital of Dax, Dax, France;
8
Servicio de Hematologı
´a, Servicio de Salud del Principado de Asturias, Oviedo, Spain and
9
Haematology Laboratory, University Hospital,
Dijon, France. Correspondence: Professor F Girodon, Haematology Laboratory, University Hospital, Plateau technique de biologie, 2 rue Ange
´lique Ducoudray, Dijon, cedex 21079,
France or Dr S Schnittger, MLL Munich Leukemia Laboratory, Munich, Germany.
E-mail: francois.girodon@chu-dijon.fr or susanne.schnittger@mll.com
10
On behalf of the Spanish Group of Hematological Cytology (GECH).
Received 20 March 2013; accepted 10 April 2013; accepted article preview online 18 April 2013; advance online publication, 14 May 2013
Leukemia (2013) 27, 1826– 1831
&
2013 Macmillan Publishers Limited All rights reserved 0887-6924/13
www.nature.com/leu

which suggests that other mutant genes may have a role in the
appearance of ring sideroblasts. Furthermore, a recent study
showed that RARS-T presented with a particular genetic pattern
with a high frequency of JAK2
V617F
and SF3B1 mutations,
confirming the classification of RARS-T in the category of
myelodysplastic/myeloproliferative neoplasms.
30
Finally, as precedent studies have been performed on little
RARS-T cohorts, the prognostic impact of SF3B1
mut
status remains
controversial,
22,24,25,27,31
and there is a need for a study on a larger
cohort. Our purpose was to analyse the frequency of SF3B1
mutations in a large cohort of 111 RARS-T and to explore the
prognostic impact of SF3B1 mutations in this disorder.
PATIENTS AND METHODS
Patient selection
According to the World Health Organisation 2008 classification, patients
were diagnosed with RARS-T if they fulfilled the following criteria: (i)
anaemia (haemoglobin level o125 g/l for females and o135 g/l for males)
with erythroid dysplasia and Z15% ring sideroblasts; (ii) thrombocytosis of
Z450 10
9
platelets/l; (iii) o5% blast cells in the bone marrow; (iv) the
presence of large atypical megakaryocytes similar to those observed in
BCR-ABL1-negative myeloproliferative neoplasms; (v) no secondary cause
of ring sideroblasts; and (vi) no karyotype abnormalities, such as del(5q),
t(3;3)(q21;q26) or inv(3)(q21q26).
1
To obtain a comprehensive data set of this rare entity, samples from
seven European centres in three European countries were collected. The
total cohort comprised 111 cases of RARS-T and 33 cases of RARS.
Data record
For each patient, demographic (gender, age at diagnosis, date of death or
last follow-up) and biological data (blood cell count, bone marrow
exploration, ring sideroblasts, karyotype, molecular explorations) were
collected.
The SF3B1 mutations were analysed with a sensitive next-generation
amplicon deep-sequencing assay (454 Life Sciences, Branford, CT, USA)
with a median coverage of 500 reads. This approach was able to detect
mutations with a sensitivity o1%.
The JAK2
V617F
mutation was analysed by allele-specific real-time PCR to
estimate allele burden according to methods published by Lippert et al.
32
and Schnittger et al.
33
with a sensitivity of 1%. JAK2
exon12
analysis was
performed according to the method of Schnittger et al.,
34
and the MPL
W515
mutations were analysed by high-resolution melting curve analyses
followed by Sanger sequencing if positive, as previously published by
Schnittger et al.
35
Statistical analyses
Standardised overall survival was estimated by the Kaplan–Meier method
and based on the time from diagnosis to death or last contact. Survival
curves for the different groups were compared using the log rank test.
Multivariate analysis was performed using Cox’s proportional hazards
model.
Approval for the study was obtained from the ethics committee of each
institution, and the procedures were carried out in accordance with the
Helsinki Declaration of 1975, as revised in 2000.
RESULTS
Demographic and biological features
A total of 144 cases (111 RARS-T and 33 RARS including 72 males
and 72 females) were recorded in the study. Median age at
diagnosis was 73.9 years (range: 44.4–96.1 years). The median
follow-up was 37.5 and 55.2 months for the RARS-T and RARS
cohort, respectively (Table 1). Survival data were available in 130
(97 RARS-T and 33 RARS) of the 144 patients.
Frequencies and characterisation of mutations
A karyotype was available in 128 cases. One hundred and ten
(85.9%) patients carried a normal karyotype, whereas 18 (14.1%)
patients showed aberrant karyotypes, which was equally
distributed between RARS-T and RARS patients. Even if the IPSS
(International Prognostic Scoring System) score can only be
applied to MDS de novo, we calculated it to check if we had a
homogeneous group of patients. Most of the patients of the total
cohort (133 out of 144) had an IPSS score of 0.
ASF3B1
mut
was noted in 124 out of the 144 patients (86.1%).
A total of 127 SF3B1 mutations were detected in these 124
patients (28 RARS and 96 RARS-T). Three RARS-T cases carried two
Table 1. Demographic and biological characteristics of RARS-T and
RARS patients
RARS-T patients RARS patients
n111 33
Male (%) 46.8 60.6
Age at diagnosis (years)
Median 74.3 71.1
Range 44.4–92.1 48.4–96.1
20–50 years, n(%) 4 (3.6) 2 (6.1)
50–70 years, n(%) 36 (32.5) 9 (27.2)
470 years, n(%) 71 (63.9) 22 (66.7)
Available survival data (n)97 33
Median follow-up (years) 3.1 4.6
WBC ( 10
9
/l)
Median 7.6 5.2
Range 2.1–60.0 1.6–17.3
Hb (g/l)
Median 96.5 91.0
Range 51.0–131.0 69.0–128.0
Platelets ( 10
9
/l)
Median 646 314
Range 452–1500 61–444
450–600, n(%) 54 (48.6)
4600, n(%) 57 (51.4)
Ring sideroblasts (%)
Median 52 40
Range 16–97 19–85
SF3B1 mutations (%) 86.5 84.8
Tested (n) 111 33
Mutated (n)9628
p.Lys700Glu 51 16
p.Lys666Glu/Thr/Asp/Asn 16 2
p.His662Asp/Gln 11 2
p.Glu622Asp 7 2
p.Arg625Gys/Leu/Gly 5 3
p.Thr663Pro 2 2
p.Met784_Lys785delinsIle 1 0
p.Asp781Gly 0 1
Two different mutations 3 0
JAK2
V617F
mutations (%) 48.6 0
Tested (n) 111 33
MPL mutations (%) 1 0
Tested (n) 102 27
IPSS
0 104 29
0.5 5 3
121
Abbreviations: Hb, haemoglobin; IPSS, International Prognostic Scoring
System; JAK2, Janus Kinase 2; MPL, MyeloProliferative Leukaemia; RARS,
refractory anaemia with ring sideroblasts; RARS-T, refractory anaemia with
ring sideroblasts and marked thrombocytosis; SF3B1, Splicing Factor 3B
subunit 1; WBC, white blood cells.
Impact of SF3B1 mutations on the prognosis of RARS-T
J Brose
´us et al
1827
&2013 Macmillan Publishers Limited Leukemia (2013) 1826 – 1831

different mutations. With the exception of one p.Arg549Cys in
exon 12 and two in exon 16, all mutations were located in exons
14 and 15. All but one del/ins mutations (p.Met784_Lys785del/
insIle) were missense mutations. In detail, the most frequent
mutation was p.Lys700Glu (68/127 53.5%), followed by p.Lys666-
Glu/Thr/Asp/Asn mutations (n¼18, 14.2%), p.His662Asp/Gln
(n¼13, 10.2%), p.Arg625Cys/Leu (n¼10, 7.9%), p.Glu622Asp
(n¼9, 7.1%) and p.Thr663Pro (n¼4, 3.1%). Five further mutations
were detected in single cases only. Frequencies and positions of
mutations are illustrated in Table 1 and Figure 1. Median
mutation/wildtype load was 40% (range: 15–78%). Small sub-
clones with SF3B1
mut
were not detected.
Frequency of mutations in RARS and RARS-T
The frequency of SF3B1 mutations in RARS-T (96/111, 86.5%) was
similar to that in RARS (28/33, 84.8%). By contrast, both entities
differed by the presence of the JAK2
V617F
mutation, which was
detected in 54/144 (37.5%) in the total cohort but in 54/111
(48.6%) in RARS-T compared with none (0/33) in RARS (Po0.001).
Among the RARS-T SF3B1
mut
, 46/96 (47.9%) harboured a JAK2
V617F
mutation. JAK2
V617F
allele burden was very heterogeneous with a
median of 49% (range: 1–100%). No JAK2
exon12
mutation (111
tested) was observed, whereas only one case with the MPL
W515L
mutation was noted in a RARS-T (102 tested; Table 1 and Figure 2).
Biological association
The presence of SF3B1
mut
was analysed with respect to age, sex,
white blood cell count, haemoglobin levels, platelet counts, blast
counts, percentage of ring sideroblasts, karyotype and JAK2
V617F
allele burden. In RARS-T, SF3B1 mutations were more frequent in
females (56/59, 94.9%) than in males (40/52, 76.9%) (P¼0.010),
and mean ring sideroblast counts were higher in SF3B1
mut
than in
SF3B1
wt
(55% vs 38%) (P¼0.007). No further correlations were
detected for these parameters.
Impact of mutations on outcome
The difference in survival between RARS-T and RARS was not
statistically significant (median survival 10.7 vs 6.2 years,
respectively, P40.05). On the other hand, in the total cohort,
patients with SF3B1
mut
had longer survival than those with
Figure 1. Positions and characterisation of mutations in the HEAT domains 1–6 (exons 12–16) of the SF3B1 gene. Missense mutations are
indicated in green and the rare ins/del mutation in yellow. HEAT, Huntingtin, elongation factor 3, protein phosphatase 2A, Tor1; PPP1R8,
protein phosphatase 1, regulatory subunit 8; aa, amino acid.
Figure 2. Distribution of SF3B1,JAK2
V617F
and MPL
W515L
mutations in RARS-T and RARS.
Impact of SF3B1 mutations on the prognosis of RARS-T
J Brose
´us et al
1828
Leukemia (2013) 1826 – 1831 &2013 Macmillan Publishers Limited

SF3B1
wt
(8.0 vs 1.8 years, respectively, Po0.001; Figure 3a). When
restricted to RARS-T (85 SF3B1
mut
and 12 SF3B1
wt
), median overall
survival was 6.9 years in SF3B1
mut
vs 3.3 years in SF3B1
wt
(P¼0.003; Figure 3b). In RARS, survival was 10.7 years in the
SF3B1
mut
(n¼28) and 1.8 years in SF3B1
wt
patients (n¼5;
P¼0.001; Figure 3c). In RARS-T patients, the survival rates at 2, 4
and 6 years within the JAK2
V617F
sub-cohort were 94.9, 84.3 and
67.4%, respectively, while within the JAK2
wt
sub-cohort, they were
79.7, 69.6 and 32.2%, respectively. JAK2
V617F
(n¼50) was then
associated with a more favourable outcome compared with
JAK2
wt
(n¼47; P¼0.019; Figure 3d).
Cox regression analysis
In the total cohort including RARS-T and RARS cases in univariate
analysis, age (P¼0.020), ring sideroblast count (P¼0.008) and
SF3B1 mutational status (Po0.001) were prognostically significant,
but SF3B1 mutational status was the only independent prognostic
factor (P¼0.001) in multivariable analysis.
In the RARS-T cohort in univariate analysis, age (P¼0.038), ring
sideroblast count (P¼0.014), JAK2
V617F
(P¼0.024) and SF3B1
mut
(P¼0.006) were prognostically significant but only age (P¼0.044),
JAK2
V617F
(P¼0.016) and SF3B1
mut
(P¼0.021) were independent
prognostic parameters in multivariable analysis. Survival was
better in patients with age r80 years, JAK2
V617F
and SF3B1
mutations. Taking into account these three prognostic factors, a
model for survival in RARS-T patients was constructed in which
each risk factor (that is, age 480 years, SF3B1
wt
and JAK2
wt
) was
worth 1 point and defined two groups of risk: high risk for patients
with a score of 2 or 3 (n¼13) and low risk when the score is 0 or 1
(n¼84). Median survival in the high-risk group was 1.6 vs 8.0 years
in the low-risk group (Po0.001; corresponding survival rates at 2,
4 and 6 years are 31.4, 31.4 and 0% for high-risk patients vs 95.8,
84.4 and 62.8% for low-risk patients; Figure 4).
DISCUSSION
Up to now, SF3B1 mutations have been studied in small series of
RARS-T patients. In this study, we provide data on SF3B1 mutations
in a large cohort of RARS-T patients, which is, to the best of our
knowledge, the largest published to date.
SF3B1 mutations were observed with a high frequency in both
RARS-T and RARS patients (86.5% and 84.8%, respectively). These
proportions are slightly higher than those already published, as
SF3B1 mutations have been found in 64–82.6% of RARS
21–24
and
66.7–72% in RARS-T.
23,24
The slightly higher frequency of SF3B1
mutations in the current study may be due to the larger cohort of
RARS-T patients than in other studies. There may also be
differences in methodology, for example, direct sequencing vs
non-sequencing-based screening strategies.
However, RARS differs from RARS-T in that there are no
JAK2
V617F
mutations in RARS,
4,14
whereas there is a high
frequency in RARS-T.
4–13,15,19
We recently showed that RARS-T
differed from RARS and ET from a clinical, biological and
SF3B1mut
(n=113, median 8.0 yrs)
p<0.001
SF3B1wt
(n=17, median 1.8 yrs)
1
2
4
5
11
17
1010
12
14
2028
4556
7588
113
Patients at risk
1
1
2
3
712
668
9
12
15
31
4156
66
85
Patients at risk
SF3B1mut
(n=85, median 6.9 yrs)
SF3B1wt
(n=12, median 3.3 yrs)
p=0.003
1
2
24
5
444
58
1314
15
19
22
28
Patients at risk
SF3B1mut
(n=28, median 10.7 yrs)
SF3B1wt
(n=5, median 1.8 yrs)
p=0.001
334
5
791822
28
3647
3
34
4
5
7
14
21
3137
50
Patients at risk
JAK2wt
(n=47, median 5.4 yrs;
6-yr survival rate 32.2%)
p=0.019
JAK2V617F
(n=50, median 20.4 yrs;
6-yr survival rate 67.4%)
ab
cd
Figure 3. Kaplan–Meier analysis for survival since diagnosis according to SF3B1 and JAK2
V617F
mutation status. SF3B1 in (a) total cohort, (b)
RARS-T, (c) RARS and (d)JAK2
V617F
in RARS-T.
Impact of SF3B1 mutations on the prognosis of RARS-T
J Brose
´us et al
1829
&2013 Macmillan Publishers Limited Leukemia (2013) 1826 – 1831

prognostic point of view, suggesting that RARS-T could be
considered as a unique entity.
20
The results of our current study
showing the presence of both SF3B1 and JAK2
V617F
mutations in a
high proportion of RARS-T confirm that RARS-T is a unique entity.
Indeed, RARS-T was associated with high rates of SF3B1 mutations
(86.5%) and JAK2
V617F
mutations while ET patients have a low
frequency of SF3B1 mutations, and in RARS, no JAK2
V617F
mutations were detected.
A minority of RARS-T patients (13.5%) presented without SF3B1
mutations. This could be due to another mutation affecting
components of the RNA splicing machinery as mutations of SRSF2,
U2AF35 and ZRSR2 have already been described in 12.4, 7.3 and
3.1% of MDS, respectively,
36
or due to mutations of proteins
associated with SF3B1 (SF3B4 and SF3B14).
These results are in line with our hypothesis that RARS-T is an
independent entity characterised by a particular molecular
combination associating mutations that give a myeloproliferative
advantage (JAK2
V617F
,MPL
W515R/L
or other unknown mutations)
and mutations of components of the splicing machinery
responsible for myelodysplastic features (SF3B1 in most cases,
and possibly SRSF2,U2AF35 or ZRSF2 in the remaining cases).
Conflicting results on the prognostic impact of SF3B1 mutations
in MDS have been reported as several studies noted a good
prognostic impact in MDS, whereas others hypothesised that SF3B1
mutations were associated with good-prognosis MDS subgroups
but lost their prognostic impact in RARS and in RCMD-RS.
27
In our large cohort of RARS-T patients, SF3B1 mutations were
associated with female sex, higher ring sideroblast counts and a
longer overall survival than in SF3B1
wt
patients. In a multivariable
analysis, age 480 years at diagnosis, SF3B1
wt
as well as JAK2
wt
were independent factors of a worse prognosis. Based on these
three independent parameters, a prognostic score for RARS-T
patients was created to define two risk groups: high risk when
there were two or three risk factors, low when there was only one
or no risk factor. Median survival was 1.6 vs 8.0 years in the high-
and low-risk group, respectively, underlying the relevance of such
score in RARS-T patients.
Exploring SF3B1 mutations in MDS associated with ring
sideroblasts is of interest from a prognostic point of view,
particularly as specific treatment will be available.
37,38
Also,
allele-specific PCR have been designed, and these could be
useful for monitoring minimal residual disease in MDS.
39
In summary, this study confirms that RARS-T should be
considered an independent entity. In RARS-T patients, age o80
years at diagnosis, SF3B1 and JAK2 mutations are independent
factors for better survival and may be used to stratify patients.
CONFLICT OF INTEREST
SS and TH declare part ownership of the MLL Munich Leukemia Laboratory. TA, SJ
and VG are employed by the MLL Munich Leukemia Laboratory. All the other authors
declare no conflict of interest.
ACKNOWLEDGEMENTS
We thank the Medical Doctors from the Haematology Department and Laboratory of
the University Hospital of Dijon, the Spanish Group of Hematological Cytology
(GECH), as well as Philip Bastable for revising the manuscript. EL and FL are grateful to
the Tumor Bank of the CHU of Bordeaux. This work was supported by grants from the
association ‘Tulipes contre le cancer’ (Cha
ˆlon s/Sao
ˆne, Burgundy, France) and from
FEHH (Spain) and 2009 SGR 541 (Generalitat de Catalunya).
REFERENCES
1 Vardiman JW, Bennett JM, Bain BJ, Baumann I, Thiele J, Orazi A. Myelodysplastic/
myeloproliferative neoplasms, unclassifiable. In: Swerdlow SH, Campo E, Lee
Harris N, Jaffe ES, Pileri SA, Stein H et al. (eds) WHO Classification of Tumours
of Haematopoietic and Lymphoid Tissue. 4th edn. IARC: Lyon, France, 2008,
pp 85–86.
2 Hasserjian RP, Gatterman N, Bennett JM, Brunning RD, Thiele J. Refractory anemia
with ringed sideroblasts. In: Swerdlow SH, Campo E, Lee Harris N, Jaffe ES, Pileri
SA, Stein H et al. (eds) WHO Classification of Tumours of Haematopoietic and
Lymphoid Tissue. 4th edn. IARC: Lyon, France, 2008, pp 96–97.
3 Thiele J, Kvasnicka HM, Orazi A, Tefferi A, Gisslinger H. Essential thrombo-
cythaemia. In: Swerdlow SH, Campo E, Lee Harris N, Jaffe ES, Pileri SA, Stein H et al.
(eds) WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue.4th
edn. IARC: Lyon, France, 2008, pp 48–50.
4 Ceesay MM, Lea NC, Ingram W, Westwood NB, Gaken J, Mohamedali A et al. The
JAK2 V617F mutation is rare in RARS but common in RARS-T. Leukemia 2006; 20:
2060–2061.
5 Boissinot M, Garand R, Hamidou M, Hermouet S. The JAK2-V617F mutation and
essential thrombocythemia features in a subset of patients with refractory anemia
with ring sideroblasts (RARS). Blood 2006; 108: 1781–1782.
6 Flach J, Dicker F, Schnittger S, Kohlmann A, Haferlach T, Haferlach C. Mutations of
JAK2 and TET2, but not CBL are detectable in a high portion of patients with
refractory anemia with ring sideroblasts and thrombocytosis. Haematologica
2010; 95: 518–519.
7 Gattermann N, Billiet J, Kronenwett R, Zipperer E, Germing U, Nollet F et al. High
frequency of the JAK2 V617F mutation in patients with thrombocytosis (platelet
count4600 109/L) and ringed sideroblasts more than 15% considered as MDS/
MPD, unclassifiable. Blood 2007; 109: 1334–1335.
8 Hellstrom-Lindberg E, Cazzola M. The role of JAK2 mutations in RARS and other
MDS. Hematology Am Soc Hematol Educ Program 2008; 52–59.
9 Raya JM, Arenillas L, Domingo A, Bellosillo B, Gutierrez G, Luno E et al. Refractory
anemia with ringed sideroblasts associated with thrombocytosis: comparative
analysis of marked with non-marked thrombocytosis, and relationship with JAK2
V617F mutational status. Int J Hematol 2008; 88: 387–395.
10 Remacha AF, Nomdedeu JF, Puget G, Estivill C, Sarda MP, Canals C et al. Occur-
rence of the JAK2 V617F mutation in the WHO provisional entity: myelodysplastic/
myeloproliferative disease, unclassifiable-refractory anemia with ringed
sideroblasts associated with marked thrombocytosis. Haematologica 2006; 91:
719–720.
11 Renneville A, Quesnel B, Charpentier A, Terriou L, Crinquette A, Lai JL et al. High
occurrence of JAK2 V617 mutation in refractory anemia with ringed sideroblasts
associated with marked thrombocytosis. Leukemia 2006; 20: 2067–2070.
12 Schmitt-Graeff AH, Teo SS, Olschewski M, Schaub F, Haxelmans S, Kirn A et al.
JAK2V617F mutation status identifies subtypes of refractory anemia with ringed
sideroblasts associated with marked thrombocytosis. Haematologica 2008; 93:
34–40.
1112713
High risk
668912153142576684
Lowrisk
Patients at risk
Low risk group
(n=84, median 8.0 yrs;
6-yr survival rate 62.8%)
High risk group
(n=13, median 1.6 yrs;
6-yr survival rate 0%)
p<0.001
Figure 4. Kaplan–Meier analysis for survival according to two risk
groups. High risk comprising patients with at least two of the
following risk factors: age 480 years, SF3B1wt, JAK2wt; and low risk:
one or less of the risk factors.
Impact of SF3B1 mutations on the prognosis of RARS-T
J Brose
´us et al
1830
Leukemia (2013) 1826 – 1831 &2013 Macmillan Publishers Limited

13 Szpurka H, Tiu R, Murugesan G, Aboudola S, Hsi ED, Theil KS et al. Refractory
anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T),
another myeloproliferative condition characterized by JAK2 V617F mutation.
Blood 2006; 108: 2173–2181.
14 Steensma DP, Tefferi A. JAK2 V617F and ringed sideroblasts: not necessarily
RARS-T. Blood 2008; 111: 1748.
15 Wang SA, Hasserjian RP, Loew JM, Sechman EV, Jones D, Hao S et al. Refractory
anemia with ringed sideroblasts associated with marked thrombocytosis harbors
JAK2 mutation and shows overlapping myeloproliferative and myelodysplastic
features. Leukemia 2006; 20: 1641–1644.
16 Pardanani AD, Levine RL, Lasho T, Pikman Y, Mesa RA, Wadleigh M et al. MPL515
mutations in myeloproliferative and other myeloid disorders: a study of 1182
patients. Blood 2006; 108: 3472–3476.
17 Schnittger S, Bacher U, Haferlach C, Dengler R, Krober A, Kern W et al. Detection of
an MPLW515 mutation in a case with features of both essential thrombocyt hemia
and refractory anemia with ringed sideroblasts and thrombocytosis. Leukemia
2008; 22: 453–455.
18 Wardrop D, Steensma DP. Is refractory anaemia with ring sideroblasts and
thrombocytosis (RARS-T) a necessary or useful diagnostic category? Br J Haematol
2009; 144: 809–817.
19 Malcovati L, Della Porta MG, Pietra D, Boveri E, Pellagatti A, Galli A et al. Molecular
and clinical features of refractory anemia with ringed sideroblasts associated with
marked thrombocytosis. Blood 2009; 114: 3538–3545.
20 Broseus J, Florensa L, Zipperer E, Schnittger S, Malcovati L, Richebourg S et al.
Clinical features and course of refractory anemia with ring sideroblasts associated
with marked thrombocytosis. Haematologica 2012; 97: 1036–1041.
21 Yoshida K, Sanada M, Shiraishi Y, Nowak D, Nagata Y, Yamamoto R et al. Frequent
pathway mutations of splicing machinery in myelodysplasia. Nature 2011; 478:
64–69.
22 Papaemmanuil E, Cazzola M, Boultwood J, Malcovati L, Vyas P, Bowen D et al.
Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts. N Engl J Med
2011; 365: 1384–1395.
23 Visconte V, Makishima H, Jankowska A, Szpurka H, Traina F, Jerez A et al. SF3B1, a
splicing factor is frequently mutated in refractory anemia with ring sideroblasts.
Leukemia 2012; 26: 542–545.
24 Malcovati L, Papaemmanuil E, Bowen DT, Boultwood J, Della Porta MG, Pascutto C
et al. Clinical significance of SF3B1 mutations in myelodysplastic syndromes
and myelodysplastic/myeloproliferative neoplasms. Blood 2011; 118: 6239–6246.
25 Damm F, Thol F, Kosmider O, Kade S, Loffeld P, Dreyfus F et al. SF3B1 mutations in
myelodysplastic syndromes: clinical associations and prognostic implications.
Leukemia 2012; 26: 1137–1140.
26 Damm F, Kosmider O, Gelsi-Boyer V, Renneville A, Carbuccia N, Hidalgo-Curtis C et al.
Mutations affecting mRNA splicing define distinct clinical phenotypes and
correlate with patient outcome in myelodysplastic syndromes. Blood 2012; 119:
3211–3218.
27 Patnaik MM, Lasho TL, Hodnefield JM, Knudson RA, Ketterling RP, Garcia-Manero
Get al. SF3B1 mutations are prevalent in myelodysplastic syndromes with ring
sideroblasts but do not hold independent prognostic value. Blood 2012; 119:
569–572.
28 Visconte V, Makishima H, Maciejewski JP, Tiu RV. Emerging roles of the
spliceosomal machinery in myelodysplastic syndromes and other hematological
disorders. Leukemia 2012; 26: 2447–2454.
29 Visconte V, Rogers HJ, Singh J, Barnard J, Bupathi M, Traina F et al. SF3B1
haploinsufficiency leads to formation of ring sideroblasts in myelodysplastic
syndromes. Blood 2012; 120: 3173–3186.
30 Jeromin S, Haferlach T, Grossmann V, Alpermann T, Kowarsch A, Haferlach C et al.
High frequencies of SF3B1 and JAK2 mutations in refractory anemia with ring
sideroblasts associated with marked thrombocytosis strengthen the assignment
to the category of myelodysplastic/myeloproliferative neoplasms. Haematologica
2013; 98:15–17.
31 Cazzola M, Rossi M, Malcovati L. Biologic and clinical significance of somatic
mutations of SF3B1 in myeloid and lymphoid neoplasms. Blood 2013; 121:
260–269.
32 Lippert E, Boissinot M, Kralovics R, Girodon F, Dobo I, Praloran V et al. The JAK2-
V617F mutation is frequently present at diagnosis in patients with essential
thrombocythemia and polycythemia vera. Blood 2006; 108: 1865–1867.
33 Schnittger S, Bacher U, Kern W, Schroder M, Haferlach T, Schoch C. Report on two
novel nucleotide exchanges in the JAK2 pseudokinase domain: D620E and E627E.
Leukemia 2006; 20: 2195–2197.
34 Schnittger S, Bacher U, Haferlach C, Geer T, Muller P, Mittermuller J et al. Detection
of JAK2 exon 12 mutations in 15 patients with JAK2V617F negative polycythemia
vera. Haematologica 2009; 94: 414–418.
35 Schnittger S, Bacher U, Haferlach C, Beelen D, Bojko P, Burkle D et al.
Characterization of 35 new cases with four different MPLW515 mutations and
essential thrombocytosis or primary myelofibrosis. Haematologica 2009; 94:141–144.
36 Thol F, Kade S, Schlarmann C, Loffeld 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: 3578–3584.
37 Rymond B. Targeting the spliceosome. Nat Chem Biol. 2007; 3: 533–535.
38 Webb TR, Joyner AS, Potter PM. The development and application of small
molecule modulators of SF3b as therapeutic agents for cancer. Drug Discov Today
2013; 18:43–49.
39 Matsuda K, Ishida F, Ito T, Nakazawa H, Miura S, Taira C et al. Spliceosome-related
gene mutations in myelodysplastic syndrome can be used as stable markers
for monitoring minimal residual disease during follow-up. Leuk Res 2012; 36:
1393–1397.
Impact of SF3B1 mutations on the prognosis of RARS-T
J Brose
´us et al
1831
&2013 Macmillan Publishers Limited Leukemia (2013) 1826 – 1831