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

12 th Congress of the European Hematology Association
without PRF mutation showed normal PRF expression by flow cytometry;
2 patients showed A91V heterozygous polymorphism with
reduced and absent PRF expression, respectively; 1 patient showed heterozygous
G1070A mutation; genetic analysis could not be performed
in 3 patients. Summary and Conclusions. In this series of patients the occurrence
of HLH in adults was higher than expected and it was associated
with a high incidence of lymphoma, also of B cell lineage, and of Herpes
virus DNA detection. Impaired CTL appears to be, at least in some
patients, independent from PRF mutations, raising the question of
whether and how these genetic events can be related, in acquired HLH
of adults, to the deficiency of the lytic activity of PRF.
0286
THE PROGNOSTIC SIGNIFICANCE OF CELL CYCLE REGULATORY GENES EXPRESSION IN
DIFFUSE LARGE B-CELL LYMPHOMAS
F. Kontsioti, 1 P.V. Pappa, 1 R.D. Rontogianni, 2 P.S. Papageorgiou, 1
E.C. Economopoulou, 1 T.P. Tsirigotis, 1 K.E. Kavada, 1
G.V. Giannopoulou, 1 K.I. Kaitsa, 1 G.K. Girkas, 1 P.E. Papageorgiou, 1
D.J. Dervenoulas,1 E.T. Economopoulos1 1 2 Attikon University General Hospital, ATHENS; Evangelismos Hospital,
ATHENS, Greece
Background. Diffuse large B-cell lymphoma (DLBCL) is an aggressive
malignancy of mature B cells with heterogeneous prognosis. Biological
markers have been used in an attempt to improve the discriminative
capacity of the International Prognostic Index (IPI). Abnormalities of cell
cycle constitute a marker of clonal expansion and may contribute to disease
progression; thus the investigation of cell cycle regulatory genes
may be of prognostic relevance. Aims. The aim of the present study was
the investigation of gene expression levels of cell cycle regulatory genes
in DLBCL and their relation to prognosis. Methods. The analysis included
45 newly diagnosed cases of diffuse large B-cell lymphomas and 15
cases of non-neoplastic lymphadenopathies used as controls. The IPI was
available for all lymphoma cases. RNA was extracted from fresh frozen
tissues and analyzed by RNase Protection Assay (RPA) with Riboquant
kit (BD Biosciences). Two sets of probes were used that included cyclins,
cyclin dependent kinases and their inhibitors. In more detail h-CC1 set
of probes detected mRNA for the genes cdk-1,cdk-2,cdk-3,cdk-
4,p16,p27,p21,PISSLRE and h-CYC1 set of probes detected mRNA for the
genes of cyclins A,B,C,D1,D2,D3 and A1.The intensity of each band was
compared to that of the housekeeping genes GAPDH and L32 using the
Image Master Software(Pharmacia). Moreover, immunohistochemistry
was performed on paraffin-embedded tissue sections in order to verify
the expression of germinal center proteins, bcl6 and CD10. Results. The
expression levels of the cell cycle regulatory genes did not correlate with
any of the clinical parameters nor the prognostic markers. 85% of patients
that didn’t have complete remission to first line therapy, expressed the
cyclinA1 (p=0,03). 92% of the patients with low IPI (0,1) expressed
PISSLRE, while only 56% of the patients with high IPI (2,3,4,5),expressed
this gene. Moreover, cdk2 and cdk4 expression levels were significantly
higher in cases expressing bcl-6 protein (p=0,013, p=0,004 respectively).
The mean expression level of cyclin B was higher in cases with positive
expression of protein CD10 (p=0,007). All patients with negative expression
of CD10 had a lower expression level of cyclinA1 compared to normal
RNA pool (p=0,037). Moreover, patients expressing cyclinA1 had
significantly shorter disease free survival (p=0,03), while the expression
of PISSLRE was associated with significantly better overall survival
(p=0,0329). Summary and Conclusions. Taking into consideration the heterogeneity
of clinical behaviour of DLBCL, the cell cycle regulatory genes
may serve as biologic markers with prognostic significance. Our study
has shown that the expression level of cyclinA1 is associated with significantly
lower response to first line therapy and lower disease free survival.
Moreover the expression of PISSLRE seems to be associated with
low risk disease and higher overall survival, which emphasizes the potential
role of this gene as a tumour suppressor.
0287
Z-GUGGULSTERONE DOWNREGULATES SURVIVIN AND INDUCES CELL DEATH IN LARGE
B CELL LYMPHOMA CELLS IN VITRO
M. Angelopoulou, K. Lilakos, V. Salpeas, S. Sachanas,
T.P. Vassilakopoulos, M.P. Siakantaris, M.N. Dimopoulou,
P. Korkolopoulou, P. Panayiotidis, G.A. Pangalis
University of Athens, ATHENS, Greece
Background. Survivin is a member of the Inhibitor of Apoptosis Pro-
104 | haematologica/the hematology journal | 2007; 92(s1)
teins and has recently gained attention as a possible therapeutic target
in neoplasia, due to its dual role both as an antiapoptotic protein and as
a cell cycle regulator. It is overexpressed in malignant cells and confers
resistance to chemotherapy and other stimuli triggering apoptosis. Survivin
as well as other antiapoptotic proteins are under NF-κB control. Z-
Guggulsterone (Z-GGS) is a plant sterol, which has been used in inflammatory
conditions, is a potent NF-κB suppressor and might affect Survivin.
Aims. To evaluate Survivin expression by two large B cell lymphoma
cell lines and study the effect of Z-GGS on Srvivin expression and
cell viability. Methods. DB and HT cell lines were treated with increasing
concentrations (10 µM, 20 µM and 30 µM) of Z-GGS, for 24, 48 and
72 hours. Survivin expression was tested by flow Cytometry and quantitative
real time PCR using the Universal Probe Library hydrolysis
probes and expressed as Survivin/abl ratio. Cell viability was assessed
by the MTT assay. Results. Both cell lines were positive for Survivin at
baseline by flow cytometry (66% of total cells for DB and 95% for HT).
Treatment of DB cells with 10, 20 and 30̪ Z-GGS resulted in a 44%,
49% and 68% reduction of Survivin expression at 24 hours, respectively,
whereas the effect on HT was less prominent with a 10% reduction
at 24 hours with 30 µM Z-GGS. Survivin transcripts decreased as well,
with the maximum effect observed at 72 hours with 3 µM Z-GGS for
both cell lines: Survivin/abl was 0.009 for untreated cells vs 0.0008 with
30 µM Z-GGS for DB cells and 0.0135 vs 0.0005 for HT cells. Linearity
was observed for increasing concentrations of Z-GGS at 72 hours. Cell
viability was practically unaffected at any time point with 10 and 20 µM
Z-GGS for both cell lines, whereas 30 µM Z-GGS resulted in a 63% and
78% cell death at 48 and 72 hours respectively for DB cells and 67% and
83% for HT cells. Conclusions. The steroid Z-GGS downregulates Survivin
expression in B-lymphoma cells in vitro and induces cell death at
30 µM concentration. Further experiments will clarify its possible role
in the treatment of B-cell malignanciies
0288
CD5 POSITIVE SPLENIC MARGINAL ZONE LYMPHOMA : CLINICAL, CYTOLOGICAL,
IMMUNOLOGICAL, MOLECULAR AND CYTOGENETICAL FEATURES OF A SERIES OF 35
CASES
M. Baseggio, F. Petinataud, E. Callet-Bauchu, F. Berger,
A. Traverse-Glehen, C. Thieblemont, D. Morel, G. Salles, P. Felman
Centre Hospitalier Lyon-Sud, PIERR-BENITE, France
Splenic marginal zone B-cell lymphoma (SMZL) has been recently
recognized owing to clinical, cyto-histological, immunological, cytogenetical
characteristics. The immunological profile, usually CD5, CD10,
CD23, CD43, cyclinD1 negative B-cells, is however in lack of specific
markers which could be useful in doubtful cases. For instance, the report
of CD5 positive cases without histological control is still a matter of
debate, and in fact the differential diagnosis with B-CLL or MCL is sometimes
difficult. Nevertheless CD5 postivity has been reported in some
histologically typical MZL cases and proposed as a marker for a more
aggressive and disseminated disease. Here we described a series of 35
CD5 positive SMZL histologically proven in 26 cases and without
t(11;14) detected by FISH, that were compared to a series of 44 typical
CD5 negative SMZL. From a clinical, cytological and immunological
point of view, the CD5 positive SMZL cases presented many similarities
with the group of CD5 negative SMZL. The main differences consisted
in a higher lymphocytosis at diagnosis (4,95 G/L in CD5 positive
group and 1,60G/L in CD5 negative group, respectively -p=0.003-) and
a more frequent IgM isotype of the monoclonal component (75% vs
53%). Interestingly, all CD5 positive cases in peripheral blood were CD5
positive on splenic specimen studied by flow cytometry and no CD5
negative B-cells were detected in spleen compartment. The tumoral
mass assessed by spleen weight seemed similar between both
groups.These results suggest that CD5 expression gives B-cells a higher
propensity to recirculate from spleen. A good correlation was
observed with the CD5 expression detected by immunohistochemistry.
The few CD5 negative cases on sections corresponded in fact with a
faint CD5 expression by FCM. Karyotypic changes were similar in both
groups and included the chromosomal abnormalities previously
described in SMZL. However, some differences were observed : trisomy
18 was more frequent (29% vs 11%) and the 7q deletion was less frequent
(23% vs 36%) in the CD5 positive group than in the CD5 negative
group, respectively. The frequency of 17p deletion, leading to an
alteration of P53 and a more aggressive clinical course was similar in
both groups. Most cases were mutated in the CD5 positive group (82%)
in contrast to the CD5 negative group (55%). The IgVH analysis showed
a VH4 usage gene in 4 cases (23%) of the 17 available CD5 positive cases,
whereas in the CD5 negative group, an underrepresentation of VH4