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

12 th Congress of the European Hematology Association
od of approximately 27 days, WBC also showed oscillations with a period
of about 27 days. After change to anagrelide, oscillations promptly
ceased. Patient 2 is also a female (53 years at diagnosis). During therapy
with HU platelets (nadir 68 000 /µL, zenith 1 690 000 /µL) and WBCs
oscillated with a period of approximately 27 days. She still remains
under therapy with HU. Patient 3 is a 73-year-old woman who was
treated with HU for 4 months during which she showed marked oscillations
(nadir 71 000 /µL, zenith 1 145 000 µL) with an approximate
period of 29 days which ceased under change to anagrelide. Patient 4
(female, 52 years at diagnosis) exhibited oscillations of platelet count
(nadir 104 000 /µL, zenith 1 420 000 µL) with a period of approximately
30 days. Patient 5 (male, 62 years) showed under with HU for 10
months marked oscillations (nadir 46 000 /µL, zenith 1 122 000 /µL)
with a period of approximately 30 days. The oscillations disappeared
with switching therapy to busulphane. None of the five patients experienced
thromboembolic events under HU therapy. Summary. We have
identified 5 patients with marked oscillations under HU therapy. The
mean period was 28.2 days. No thromboembolic complications over an
accumulated observation period of 55 patient months have been
observed. Since it is unclear in which percentage of HU treated patients
such oscillations do occur and whether unrecognized oscillations may
account for dosing difficulties, systematic analyses of larger patient
cohorts are required.
1080
PRENATAL GENOTYPING OF FETAL RHD IN MATERNAL PLASMA FROM RHD
NEGATIVE WOMEN
A. Stamna, 1 B. Zournatzi, 1 A. Manitsa, 2 N. Vavatsi-Christaki1 1 Medicine Scool, AUTH, THESSALONIKI; 2 Thalassamia Prevention
Unit,Hippokration, THESSALONIKI, Greece
The Rhesus (Rh) system is one of the most important and complex
blood group systems in humans. The Rh antigens are encoded by two
highly homologous, closely linked, genes on the short arm of chromosome
Anti-D antibody was once the most common cause of haemolytic
disease of the fetus (HDN). In most cases, the anti-D of an RhD negative
mother results from immunization by transplacental haemorrhage
during a previous birth of an RhD positive baby. With the introduction
of the Rh immunoglobulin prophylaxis program, there has been a dramatic
drop in mortality due to Rh HDN. The purpose of this study was
to confirm the accuracy of a non- invasive prenatal determination of the
RHD typing and determination of the fetal DNA in the maternal plasma
from RhD negative women with the use of real time PCR. We studied
27 RhD negative pregnant women while 6 positive subjects were
used as controls and the results were compared with serologic RhD typing
of the newborns. DNA was extracted from maternal plasma using
QIAamp DNA Blood Mini Kit (Qiagen) and then analyzed for the RhD
gene with a Real-Time PCR and Taqman method. Amplification was
carried out in a Light-Cycler instrument (Roche Biochemicals). Among
27 RhD negative women, 7 were in their second trimester of pregnancy
and 20 were in their third trimester. Eighteen fetuses were RhD positive,
and 9 were RhD negative. Fetal D status was predicted with 100
percent accuracy from maternal plasma. The discovery of fetal DNA in
maternal plasma has opened up an approach for noninvasive prenatal
diagnosis. Molecular genetics of Rh blood group system has become a
reality in practical transfusion medicine the last years. Pregnant women
carrying RhD negative fetuses that can be specifically detected by RhD
genotyping may be excluded from receiving unnecessary Rh Ig prophylaxis.
1081
METHYLATION STATUS OF P57KIP2 AND P16INK4 IN PATIENTS WITH PLASMA
CELL NEOPLASMS
E. Hatzimichael, 1 A. Dasoula, 2 L. Benetatos, 1 A. Vassou, 1 I. Georgiou, 3
M. Syrrou, 2 K.L. Bourantas1 1 University Hospital of Ioannina, IOANNINA; 2 Laboratory of General Biology,
IOANNINA; 3 Laboratory of Genetics, IOANNINA, Greece
Background. Growing evidence has implicated silencing of tumor suppressor
genes (TSG) by aberrant methylation in the molecular pathogenesis
of several human cancers. The p57KIP2 and the p16INK4 are cyclindependent
kinases (CDKIs) that have been implicated in tumorigenesis
as TSG. Although the cytogenetic and molecular abnormalities underlying
plasma cell (PC) neoplasms are becoming better understood the role
of CDKIs methylation, if any, is largely unknown. Aims. In this study we
398 | haematologica/the hematology journal | 2007; 92(s1)
wished to estimate the frequency of p57KIP2 and p16INK4a methylation
in patients with PC neoplasms, to see whether there is any implication
in the pathogenesis of the disease and to find any possible correlation
with clinical and laboraratory variables. Methods. The methylation-specific
polymerase chain reaction (MSP) with primers for methylated and
unmethylated alleles of the p57KIP2 and p16INK4a gene was employed
to study prospectively bone marrow samples from 22 patients with multiple
myeloma (MM) and 2 patients with Waldenström’s macroglobulinemia
(WM). Age range was 47-84 years, median 68 years. All samples
were taken at diagnosis, except for one patient that sample was taken
when progression to plasma cell leukemia (PCL) occurred. Genomic DNA
was extracted using the QIAmp DNA mini kit of Qiagen. Bone marrow
DNA from 11 individuals with leukopenia or thrombocytopenia that
were proved to have no haematological malignancy served as negative
controls. Human male genomic DNA universally methylated for all genes
(Intergen Company, Purchase, NY) was used in all experiments as positive
control for methylated alleles. Results. MM patients were classified
using the Durie and Salmon criteria; 3 patients had smoldering myeloma,
1 patient stage IA disease, 9 patients IIA, 5 patients IIIA and 4 patients
IIIB. Classical cytogenetic analysis was available in 9/23 patients and was
normal in all but one patient that had a 45,X,-Y karyotype. Patients
younger than 60 years requiring treatment received VAD chemotherapy
and high dose melphalan with PBSC rescue, whereas patients older than
60 years received oral melphalan and methylprednisolone. Four patients
(3 MM and 1 PCL) died due to refractory disease and the remaining 20
patients are on regular follow up. In all experiments there was a strong
visual band for the positive control. All patient samples were found to be
completely negative for methylation of the p57KIP2 gene whereas 4
patients with MM (14%; one patient with smoldering myeloma, 2
patients with stage III disease and 1 patient with PCL) and 1 patient with
WM were found to be methylated for the p16INK4 gene. 2/3 MM
patients and 1/1 PCL patient with p16INK4 methylation died due to disease
progression or refractory disease. Conclusions. P57KIP2 methylation
has not been studied before and it seems that it is not a frequent event
in this group of patients. The prevalence of p16INK4 methylation found
falls within the range of 10% to 51% previously reported in the literature.
P16INK4 methylation might be associated with advanced disease.
Further studies are needed to confirm the above mentioned results.
1082
EBV RELATED TRANSFORMATION EVENTS IN CLL
H.A. Sayala, C. Patalappa, S.J. O’Connor, A.S. Jack, P. Hillmen,
R.G. Owen
HMDS, Leeds General Infirmary, LEEDS, United Kingdom
Less than 5% of patients with CLL undergo histological transformation
to diffuse large B cell lymphoma (DLBL) while transformation to
Classical Hodgkins lymphoma (CHL) is also recognised. It has been
assumed that such events reflect genetic changes in the CLL clone but
there is now emerging evidence to suggest that at least some transformation
events may be Epstein Barr virus (EBV) related neoplasms. In this
analysis we have reviewed the clinical and laboratory features of 15
CLL patients with biopsy proven histological transformation to DLBL
and 2 patients who developed CHL. Histological sections were assessed
for the expression of a wide range of immunophenotypic markers as
well as EBV latent membrane protein 1 (LMP-1). Of the 15 patients
developing DLBL 5 appeared on phenotypic grounds to be related to the
underlying CLL clone. In the remaining patients the tumour cells
appeared phenotypically distinct as they lacked CD5 and CD23 and
demonstrated expression of germinal centre markers in some instances.
LMP1 positivity was demonstrable in 5 patients with DLBL (1 apparently
related and 4 unrelated to the underlying CLL). Of the 2 patients who
developed CHL 1 was associated with EBV and lacked CD20 expression.
All the patients with EBV+ tumours were heavily pretreated (median prior
therapies 4, range 1-5) while the median time from original diagnosis
to histological transformation was 74.5 months (range 17-114). Previous
therapies included chlorambucil (n= 4), fludarabine monotherapy
(n=3), FC (n=2), FCR (n=1), high dose methyl prednisolone (n= 1) and
alemtuzumab (n=2). 2 patients received only one line of therapy for CLL
and 1 of theses was heavily immunosuppressed with steroids and azathioprine
for autoimmune neutropenia. 4 patients presented with nodal
disease and 2 patients presented with extranodal disease (bone marrow
and liver). The outcome of the EBV associated tumors in these patients
was death in 2 patients (opportunistic infection and complications of
intensive chemotherapy), remission with intensive combination
chemotherapy in 2 patients 1 of whom died 10 months later of progressive
CLL, and spontaneous remission of nodal disease in 1 patient. 1