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

0385
CYTOPLASMIC MUTATED NUCLEOPHOSMIN DEFINES THE MOLECULAR STATUS OF A
SIGNIFICANT FRACTION OF MYELOID SARCOMAS
A. Liso, 1 D. Lenze, 2 R. Hasserjian, 3 S. Coupland, 2 D. Jaehne, 2
C. Soupir, 3 M.P. Martelli, 1 N. Bolli, 1 F. Bacci, 1 V. Pettirossi, 1
A. Santucci, 1 M.F. Martelli, 1 S. Pileri, 1 H. Stein, 2 B. Falini 1
1 Hematology, FOGGIA, Italy; 2 Charitè-Universitatsmedizin, BERLIN, Germany;
3 Massachusetts General Hospital, BOSTON, USA
Detection of genetic lesions is critical for classification, prognostic
stratification, and monitoring of minimal residual disease of acute
myeloid leukaemia (AML). Information on genetic lesions associated
with myeloid sarcoma (MS), a tumor mass consisting of myeloblasts or
immature myeloid cells at an extra-medullary site, is still limited. This
is mainly due to the fact that fresh cells are usually not available for
cytogenetic and/or molecular studies, since the diagnosis of MS is frequently
unexpected and/or the size of the sample is small, such as a skin
punch biopsy. Since MS is usually treated in the same way as AML, the
frequent lack of available cytogenetics in MS, represents a significant
disadvantage, and the availability of techniques applicable to paraffin
samples to detect specific genetic lesions would be of great help both for
diagnostic and prognostic purposes. Aberrant cytoplasmic expression of
nucleophosmin (NPM), a multifunctional shuttling protein usually located
in the nucleus, is a key property of a large subgroup of AML exhibiting
distinguishing biological and clinical features, that we termed NPMc +
(cytoplasmic-positive) AML (Falini B. et al., NEJM 352:254-266, 2005;
Falini B. et al., 109:874-885, 2007). In NPMc+ AML, leukemic cells harbour
NPM1 gene mutations generating NPM leukemic mutants that are
exported at high rate from the nucleus and accumulate in the cytoplasm
(Falini B. et al., Blood 107:4514-4523, 2006). Aberrant cytoplasmic NPM
expression and/or NPM1 mutations have been investigated in AML.
However, there is no information on the role of NPM in MS, a tumor that
may develop de novo (preceding bone marrow involvement), present
concurrently with AML, or represent manifestation of AML relapse or
blastic transformation of a pre-existing chronic myeloproliferative disorder.
Since NPM1 mutations cause aberrant cytoplasmic dislocation of
NPM that is fully predictable by immunohistochemistry (Falini B. et al.,
Blood 108:1999-2005, 2006), we used anti-NPM monoclonal antibodies
recognizing both wild-type and mutated NPM to detect cytoplasmic
NPM in paraffin-embedded samples from 181 MS retrieved from the
archives of four large Institutions. Reactivity was evaluable in 173/181
cases: 146 (85.0%) with nucleus-restricted NPM (predictive of unmutated
NPM1) and 26 (15.0%) with aberrant cytoplasmic NPM (predictive
of NPM1 mutations). The presence or absence of NPM1 mutations was
confirmed in paraffin sections by immunostaining with specific antibodies
against the NPM mutants and, in a subset of cases, also by a polymerase
chain reaction (PCR) assay of NPM1 sequence we developed for
application on paraffin-embedded formalin-fixed samples. NPMc+ MS
showed the same distinctive features as NPMc + AML, including frequent
M4 or M5 morphology, CD34-negativity, association with normal karyotype,
and no previous history of myelodysplastic or chronic myeloproliferative
disorders. Thus, immunohistochemistry can serve as a surrogate
of molecular studies for detecting NPM1 mutations in MS. This
study identifies NPM1 mutations as the most frequent genetic lesion so
far reported in MS, accounting for 15% of cases. Our genetic findings
have clear implications for the upcoming WHO classification of myeloid
neoplasms. Clinical prospective studies aimed to assess the prognostic
value of NPM1 mutations (in combination with FLT3-ITD) in MS are also
warranted.
12 th Congress of the European Hematology Association
Platelets, vascular biology and
transfusion medicine
0386
A PLACEBO CONTROLLED, CROSS-OVER STUDY OF CITRATE EFFECTS ON BONE
METABOLISM IN HEALTH VOLUNTEERS
Y. Chen, C. Bieglmayer, H. Heinzl, P. Hoecker, M. Dettke
AKH Vienna, VIENNA, Austria
Background. Citrate is the anticoagulants of choice when performing
apheresis donations. Less is known about the possible effects of citrateinduced
hypocalcemia on bone turnover. Aims. Aim of the study was to
investigate the possible effects of citrate on biochemical markers of bone
metabolism. Methods. A placebo controlled, cross-over trial was performed
in 10 male volunteers. Volunteers received two standardized
infusion (80 min.) containing either citrate (1.5 mg/kg/min) or saline
solution, separated by a wash-out period of 2 weeks. At each study time
point blood and urinary samples were collected before, during, and after
completion of the infusions. Samples were analysed for short- and longterm
markers of bone turnover, PTH and electrolytes. Results. Application
of citrate led to a profound decrease in serum level of iCa, potassium
and phosphate. Decrease of iCa was inversely related to the increase
in iPTH and the urinary excretion of Ca. Infusion of citrate resulted in a
time-dependent steadily increased of serum levels of the bone formation
marker osteocalcin (OC) and the bone resorption marker C-telopeptide
of type 1 collagen (CTX). Peak levels of both bone markers were reached
at the end of the citrate infusion (compared to base +34% for OC and
+57% for CTX, respectively). Both bone parameters showed a positive
correlation to the increase of PTH, as determined at the end of the citrate
infusion. In addition, changes of OC but not CTX were related to
the variations in serum iCa. Alterations in both bone markers were still
detectable 90 min after completion of the citrate infusion. In contrast, no
alterations were observed in the serum levels of the long-term bone
markers bone specific AP, tartrate-resistant acid phosphatase 5b, osteoprotegerin
or RANKL. Conclusions. Infusion of citrate equal to the dose
used in voluntary platelet apheresis donation results in profound alterations
of biochemical markers of bone turnover. The pattern of variations
in the serum levels of the bone markers OC and CTX resembled to those
of an increased bone resorption.
0387
A SMALL THROMBOPOIETIC MOLECULE NIP-004 IS EFFECTIVE FOR THE TREATMENT OF
THROMBOCYTOPENIA INDUCED BY INTERFERON-ALPHA
A. Yamane, 1 T. Nakamura, 2 M. Ito, 3 Y. Ohnishi, 3 Y. Ikeda, 1
Y. Miyakawa1 1 School of Medicine, Keio University, TOKYO; 2 Nissan Chemical Industries,
LTD., SAITAMA; 3 CIEA, KANAGAWA, Japan
Background. Human interferon-α (IFN) is useful to treat chronic hepatitis
C. However, doctors often reluctantly reduce the dose of IFN, or discontinue
therapy, because of IFN-induced thrombocytopenia. Although
IFN is believed to directly inhibit megakaryopoiesis, its mechanisms are
not clearly understood. Aims. In this study, we evaluated the efficacy of
a small non-peptidyl thrombopoietic molecule, NIP-004, for the treatment
of IFN-induced thrombocytopenia and studied the inhibitory
mechanisms of IFN for megakaryopoiesis. Methods. In vivo assay: After
2.4 Gy-irradiation, 1×10 5 of human umbilical cord blood-derived CD34 +
cells was intravenously injected into immunodeficient NOD/Shi-scid/IL-
2Rγ-null (NOG) mice. Pegylated-recombinant human interferon-α2b
(PEG-Intron ® ) and NIP-004 were subcutaneously administered into the
mice. The number of platelets and human megakaryocyte ploidy were
obtained by a flow cytometer. in vitro assay: The colony formation of
human megakaryocytes (CFU-MK) assay was performed using human
bone marrow (BM)-derived CD34-positive cells in collagen based semisolid
culture, and the proplatelet (PPT) formation assay was performed
using human BM-derived CD34-positive cells in serum-free liquid culture,
with the various combination of c-Mpl activator (thrombopoietin
or NIP-004) and IFN. Results. When IFN at 30 microgram/kg was administered
three times a week for 3 weeks into NOG mice transplanted
with human CD34 + cells, the number of human platelets was significantly
reduced by 40% compared to the control mice. Seven weeks treatment
with IFN resulted in 70% reduction of human platelets
[27.7±6.8×10 6 /mL (control), 8.8±1.8×10 6 /mL (IFN), n=5, p