12th Congress of the European Hematology ... - Haematologica

0250
INACTIVATION OF SUPPRESSOR OF CYTOKINE SIGNALING-1 AND -3 (SOCS-1 AND -3)
AND SH2-CONTAINING PHOSPHATASE-1 (SHP-1) IN PHILADELPHIA-NEGATIVE CHRONIC
MYELOPROLIFERATIVE DISORDERS (CMPD)
M. Lunghi, D. Rossi, C. Deambrogi, M. Cerri, S. Rasi, S. Franceschetti,
A. Conconi, P. Riccomagno, D. Capello, G. Gaidano
Division of Hematology, NOVARA, Italy
Background. Ph-negative chronic myeloproliferative disorders (CMPD)
are a clinically overlapping group of disorders characterized by somatic
point mutations of the JAK2 gene, leading to constitutive JAK-STAT activation.
The negative regulators of cytokine signaling SOCS-1, SOCS-3
and SHP-1 have a crucial function in the down-regulation of JAK-STAT
activation in response to cytokines. SHP-1, SOCS-1 and SOCS-3 may be
silenced by aberrant DNA methylation and/or mutation in human malignancies.
Aims. To test epigenetic and genetic inactivation of SOCS-1,
SOCS-3 and SHP-1 in CMPD and acute myeloid leukaemia (AML).
Methods. The study was based on: i) 112 CMPD, including 43 essential
thrombocythemia (ET), 28 polycythemia vera (PV), 24 idiopathic
myelofibrosis in pre-fibrotic phase (MF), 11 atypical chronic myeloid
leukemia (ACML), and 6 chronic myelomonocytic leukemia (CMML);
ii) 20 AML post-CMPD, including 10 AML from ET, 5 AML from PV and
5 AML from MF. For comparison, 20 normal bone marrow samples were
also investigated. All cases were analysed for SOCS-1, SOCS-3 and SHP-
1 aberrant methylation by methylation-specific PCR and for JAK2V617F
mutation status by allele specific PCR. SOCS-1 and SOCS-3 mRNA levels
were detected by real-time RT-PCR. Mutation of SOCS-1 and SOCS-
3 were tested by DNA direct sequencing. Results. SOCS-3 aberrant
methylation occurred with high frequency both in CMPD (46/112, 41%)
and in AML post-CMPD (10/17; 59%), and was distributed throughout
the different WHO categories: 20/43 (46%) ET, 13/28 (46%) PV, 5/24
(21%) MF, 5/11 (45%) ACML and 3/6 (50%) CMML. Methylation of
SOCS-1 and SHP-1 occurred with lower frequency both in CMPD
(14/112, 12.5% for SOCS-1; 8/112, 7% for SHP-1) and in AML post-
CMPD (3/20, 15% for SOCS-1; 1/20, 5% for SHP-1). In particular, SOCS-
1 methylation was detected in 5/43 (12%) ET, 5/28 (18%) PV, 3/24
(12.5%) MF, 1/11 (9%) ACML, 0/6 CMML. SHP-1 methylation was
observed in 4/43 (9%) ET and 4/28 (14%) PV, while it was absent in MF,
ACML and CMML. All normal bone marrow samples (n=20) scored
negative for SOCS-1, SOCS-3 and SHP-1 methylation. JAK2V617F mutation
was detected in 66/112 (59%) Ph-CMPD, including 24/43 (56%)
ET, 23/28 (82%) PV, 19/24 (79%) MF, and in 5/20 (25%) AML post-
CMPD. SOCS-3, SOCS-1 and SHP-1 methylation occurred in both
JAK2V617F-positive (26/66, 39% for SOCS-3; 11/66, 17% for SOCS-1,
6/66, 9% for SHP-1) and JAK2V617F-negative CMPD (12/46, 26% for
SOCS-3; 2/46, 4% for SOCS-1, 2/46, 4% for SHP-1). This pattern of
SOCS-3, SOCS-1 and SHP-1 methylation was conserved also when the
analysis was restricted to PV, ET and MF each as a single group and after
stratification for JAK2V617F mutation. By combining the results of SHP-
1, SOCS-1 and SOCS-3 methylation status, 29/66 (44%) JAK2V617F
mutated cases carried SHP-1 and/or SOCS-1 and/or SOCS-3 methylation
as opposed to 29/46 (43%) germline cases. Similar results were obtained
in JAK2V617F-positive and JAK2V617F-negative AML post-CMPD. To
verify the correlation between aberrant methylation and gene expression,
we analyzed SOCS-3 and SOCS-1 mRNA levels by real-time RT-
PCR. SOCS-3 mRNA levels were significantly higher in unmethylated
samples (n=10) compared to methylated samples (n=6; p=0.005) and to
normal bone marrow (n=10; p=0.03). Similar results were obtained for
SOCS-1. SOCS-1 and SOCS-3 missence mutations were detected in
2/104 (2%) and 1/93 (1%) CMPD, respectively. Conclusions. i) Inactivation
by aberrant methylation of SOCS-3, SOCS-1 and SHP-1 is involved
in the pathogenesis of CMPD, is selectively associated with neoplastic
hemopoiesis and correlates with reduced gene expression; ii) methylation
of SOCS-3, SOCS-1 and SHP-1 occurs in both JAK2V617F positive
and negative cases; iii) the methylation rate of SOCS-3, SOCS-1, and
SHP-1 is similar in CMPD and in AML post-CMPD, suggesting that
SOCS-3, SOCS-1 and SHP-1 silencing is not involved in leukemic transformation;
iv) SOCS-1 and SOCS-3 mutations are rarely involved in
CMPD.
12 th Congress of the European Hematology Association
0251
CHARACTERIZATION OF DIFFERENTIALLY EXPRESSED MICRORNAS IN GRANULOCYTES
FROM PRIMARY MYELOFIBROSIS
P. Guglielmelli, 1 A. Pancrazzi, 1 C. Bogani, 1 L. Tozzi, 1 R. Zini, 2 A. Bosi, 1
R. Manfredini, 2 A.M. Vannucchi1 1 2 Florence University, FLORENCE; University of Modena and Reggio Emilia,
MODENA, Italy
Background. Despite advances in defining diagnostic and prognostic criteria
in patients with Primary Myelofibrosis (PMF), and the recent
description of Val617Phe mutation in JAK2 exon12 and of MPL W515K/L
mutation, the molecular defect(s) associated with the development of
PMF remain still largely to be defined. Comparative transcriptome
microarray analysis has allowed us to evidentiate a complex pattern of
aberrantly regulated genes in PMF. The underlying mechanism is still
poorly understood, but microRNAs might be supposed to play a role in
abnormal gene regulation. AIMS. As an approach to identify possibly
aberrantly regulated microRNAs in PMF, we used a Human Panel including
150 individual miRNA assays. We performed a comprehensive transcriptome
comparative microRNA analysis of normal and PMF granulocytes.
METHODS. To this purpose, we prepared four pools, each comprising
three subjects, of granulocytes from PMF subjects, two from
JAK2V617F wild-type (WT) and two from homozygote patients; two
pools from blood donors were used as controls. The method uses stemlooped
primers for reverse transcription (RT) of the miRNA, followed by
quantitative real-time PCR. The cDNA was analysed with the aid of a
TaqMan MicroRNA Assay Human Panel Kit (Applied Biosystem).
Results. Ninety six differentially expressed microRNAs were identified;
87 were decreased and 9 were increased. In order to validate these data,
we have selected 7 miRNAs which were extremely aberrantly regulated
in PMF and we performed a Real-time PCR (RT-PCR) using single
TaqMan MicroRNA Assay (Applied Biosystem); these were carried out
in an indipendent cohort of normal controls and patients with PMF, polycythemia
vera (PV), essential thrombocythemia (ET) and idiopathic erytrocytosis
(IE). Three of these gene (miR-150, miR-95 and miR-183)
allowed to discriminate patients with PMF from both healthy subjects,
IE and others chronic myeloproliferative disorders. We found no difference
in expression profile between WT and JAK2V617F homozygote
patients. With the aim to confirm the biological effects of these microR-
NAs, we have combined miRNA expression data with previously
described transcriptosome of CD34 + PMF using microarray analysis
(Guglielmelli et al, Stem Cells, 2007:165-73). After identified a number
of predicted miRNA 'Targets, we have measured gene expression levels
in granulocytes by RT-PCR. We found that MYB, MYCN, LEPR and
PRAME were significantly increased in PMF compared with healthy
controls as expected for the low levels of miR-150 and miR-183; on the
other hand, both DTR and FOSB, and their putative regulatory miR-95
and miR-31, were found concurrently decreased. Conclusions. Recent
studies have implicated miRNAs in a number of fundamental cell
processes and in hematopoiesis. These data show an unique expression
profiling of PMF patients as compared to normal controls, with an
observed overall miRNA downregulation, as reported in other cancer
cells. Moreover, 3 miRNAs were identified as a predictive signature of
PMF as opposed to other myeloproliferative disorders. Finally, we
showed correlation between some microRNAs and abnormally regulated
putative target genes, suggesting their possible role in disease pathogenesis.
haematologica/the hematology journal | 2007; 92(s1) | 91