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

a cytogenetic hallmark for M4/M5 subtype of poor prognosis acute
myeloid leukemia (AML) characterized by blast cells erythrophagocytosis
found in both de novo and therapy-related AML cases. At the molecular
level, translocation t(8;16) fuses the MOZ gene (located on 8p11) and
CREB binding protein (Rubinstein-Taybi syndrome; CREBBP, or CBP)
gene (located on 16p13), both encoding proteins with histone acetyltransferase
activity. Recently, gene expression profiling of AML with t(8,16)
reveals a specific pattern of FLT3 and Hox genes expression. We described
here an atypical case of a secondary acute monocytic leukemia (M5) with
erythrophagocytosis and a t(8;16)(q12;p13) associated with an inv(8)
(p11q11) which shown similar gene expression signature. Materiel and
Methods. A 59-year-old man, initially diagnosed as LMNH has been treated
with ESHAP chemotherapy. 2 years later, he developed a secondary
AML with peripheral blood analysis showing anemia (Hb 9.9g/dL),
hyperleukocytosis (939×10 9 /L) and thrombocytopenia (48×10 9 /L ). Bone
marrow aspiration demonstrated hypercellular marrow with 90% blast
cells of AML-M5 type of the FAB classification, with erythrophagocytosis
feature in 5% of blast cells. According to the ISCN 2005 nomenclature,
conventional karyotype analysis at diagnosis was: 46,XY,add(3) (q11),
del(8)(q11.1),der(16)t(8;16)(q12;p13)X2,add(20)(q13)[19]/ 46,XY[1]). The
patient died four months later. The chromosomal rearrangement and the
molecular breakpoint were characterised by FISH, M-FISH and 5’ Race
molecular analyses. The expression level of Hoxa9/FLT3/MeisI transcripts
were performed by quantitative Real Time RT-PCR (RT-RQPCR) in using
specific probes for each genes and ABL control gene to normalised results.
Results. The final karyotype was: 46,XY,del(3) (q11), t(8;16)
(q11;p13),+der(16)t(8;16)(q11;p13)X2,der(20)t(X;20)(?;q13)[19]. In order
to characterize the sequences fused to CBP, we carried out 5’Race PCR
from CBP gene and PCR products were subcloned. As it was unexpected,
sequence analysis revealed an in-frame junction between exon 15
from MOZ and exon 4 from CBP genes. The MOZ/CBP fusion transcripts
were confirmed by RT-PCR performed with specific primers from
MOZ and CBP genes confirming the involvement of MOZ in this
rearrangement. As it was recently described by Camos et al. in other
t(8;16)(p11;p13), RT-RQPCR performed in this case showed an up-regulation
of the Hoxa9/FLT3/MeisI expression levels, suggesting a similar
leukaemogenesis pathway in all AML with MOZ-CBP involvement. Conclusions.
In spite of an atypical t(8;16)(q11;p13) associated with a masked
inversion inv(8)(p11q12) in a M5 AML, the molecular characterization
showed the known breakpoints in 3’ part of MOZ and in the 5’end of the
CBP genes resulting in a MOZ-CBP in frame fusion transcripts. Moreover,
as it was suggested by the Hoxa9/FLT3/MeisI overexpression in this case,
this leukaemia would correspond to the specific signature of AML with
the recurrent t(8;16)(p11;p13) previously described. These results provide
further evidence for the multiple contribution of both MOZ and CBP
genes in this particular AML.
0981
CD8+CD28- IN PERIPHERAL BLOOD OF PATIENTS WITH CUTANEUS T-CELL LYMPHOMA
(CTLC) IN DIFFERENT CLINICAL STAGES
D. Urbaniak, K. Kapelko-Slowik, J. Maj, J. Dybko, A. Jankowska,
M. Slowik, D. Wolowiec, M. Kielbinski, K. Kuliczkowski
Wroclaw Medical University, WROCLAW, Poland
Background. Patients with CTLC usually have a poor immune response.
CD8 + CD28- T cells are new types of immune suppressor cells. The study
was to analyze the level and changes of them in peripheral blood with
CTLC, their effects on immunosupression of CTLC, the influence factors
to provide reference for treatment of CTLC patients. Patients and Methods.
27 patients with CTLC were enrolled in the study: 8 women and 19
men aged 35-81, medium 57. According to Ann Arbor classification stage
I was represented by 1 patient, stage II '6 patients, stage III-5 patients,
stage IV-6 patients and stage early stage (premycoticus) was represented
by 8 patients. Peripheral blood samples were assessed from all patients
with CTLC before treatment and 8 normals. Percentage CD8 + CD28- T
cells was analyzed by flow cytometry. Results. Compared with control
group percentage of CD8 + CD28- T cells was significantly higher in
patients group p=0,005 (test U Mann Whitney). There were no significant
differences of T-cells in among patients with different clinical stages. We
revealed the correlation between CD8 + CD28- cells and advanced clinical
stage r=0,54, p=0,035 (Spearman correlation test). Conclusions. Percentage
of CD8 + CD28 – T cells is increased in peripheral blood in CTLC patients,
and correlates with advanced stage.
12 th Congress of the European Hematology Association
0982
HIGH FREQUENCY OF CMV-SPECIFIC CD4 AND CD8 IMMUNE RESPONSE AND CORRELA-
TION BETWEEN THEM IN B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA
B. Pourgheysari, 1 R. Bruton, 2 P. Moss2 1 Shahrekord Universit of Medical Sciences, SHAHREKORD, Iran; 2 University
of Birmingham, BIRMINGHAM, United Kingdom
Background. Chronic lymphocytic leukemia (CLL) is characterized by
a monoclonal proliferation of lymphocytes mainly B cells (B-CLL) in the
blood and bone marrow. Morphological and functional abnormalities of
T cells and monoclonality of them have been documented in CLL. Such
expanded cells may be specific for recognition of pathogens and
cytomegalovirus (CMV) is most likely to be involved in this phenomenon
in CLL. CMV infects the majority of the human population in their
lifetime and stimulates the generation of a vigorous CMV-specific
immune response. In addition patients who are immunosuppressed frequently
have supra-normal levels of CMV-specific T cells, which may be
a response to subclinical episodes of viral reactivation. However CMV
reactivation is an increasing concern with the use of lymphocytotoxic
antibodies used in the treatment of B-CLL and is likely to reflect inadequate
CMV-specific T cell immunity. Aims. Our study hypothesized the
relationship between expanded T cells and the CMV-specific CD4 and
CD8 immune response in B-CLL. Methods. 41 CMV seropositive and 32
CMV seronegative CLL patients were studied. The level of CMV-specific
CD4 was detected by intracellular cytokine staining and flow cytometry
and the level of CD8 immune response was detected by CMV
tetramers. Results. The frequency and absolute number of IFNγ and TNFα
positive CD4 + T cells were significantly higher in CMV positive CLL
patients compared to CMV positive age-matched control group
(p=0.0004and 0.0006 for IFNγ ;0. 003 and 0.01 for TNFα respectively) and
were higher in patients with a history of treatment compared to untreated
subjects (p=0.01 and 0.04 respectively). The level of CMV-specific
CD8 immune response was also higher in patients compared to control
group. There was a positive correlation between the level of CD4 and
CD8 immune response (p=0.009). Conclusions. These results show the
deep influence of CMV infection and the immune response to it in T-cell
abnormalities in CLL patients. This level of immune response is likely
to be required to control viral reactivation.
0983
IMMUNOPHENOTYPE OF NK AND NKT CELLS IN PATIENTS WITH CHRONIC NK
LYMPHOCYTOSIS
E. Konsta, 1 K. Psarra, 2 V. Kapsimali, 2 D. Chatzopoulou, 2 M. Mpakiri, 2
Ch. Papasteriades2 1 2 National University of Athens, ATHENS, Greece; Evaggelismos Hospital,
ATHENS, Greece
Background. Chronic natural killer cell lymphocytosis (CNKL) is a persistent
state of natural killer (NK) cell (CD3-CD16/CD56 + ) excess in the
peripheral blood that is not associated with clinical lymphoma. CNKL
is a heterogeneous but not well characterized disorder. Aims. The purpose
of this study was the definition of the phenotypic markers of
human NK- and NKT-cells in healthy individuals and patients with
CNKL. Methods. Peripheral blood lymphocytes of 20 patients with CNKL
(defined by more than 500 NK lymphocytes/µL) and 20 healthy individuals
were labeled with monoclonal antibodies CD3, CD56, CD16, CD2,
CD7, CD8, CD11a, CD57, CD11c, CD45RA, CD45RO, HLADR,
TCRÁ‰, perforin, granzyme A by direct whole blood staining and were
analyzed by four-color flow cytometry. Results. The NK cells percentage
increase was mainly due to CD16 + NK cells (p