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

12 th Congress of the European Hematology Association
A 4-year old girl, child of non-consanguineous parents, presented with
easy bruising and recurrent nosebleeds starting at the age of ambulation.
On physical examination the girl had pale skin and blond hair, pale-blue
irises with occasional outbursts of horizontal nystagmus and many
hematomas mostly on legs and arms. Both parents had normal pigmented
skin and hair. Ophthalmic examination revealed reduced visual acuity.
Laboratory investigations showed normal platelet counts and morphology,
normal prothrombin time (PT) and activated partial thromboplastin
time (APTT), normal platelet aggregation with ADP and collagen-arachidonic
acid. Bleeding time however, was prolonged (8-8-9.25
minutes) and ADP in platelets was decreased (0.9 nmol/10*8 platelets).
Platelet Function Analysis (PFA)performed at a later stage showed delayed
closure time with epinephrine (>300seconds). She was diagnosed with
Hermansky-Pudlak syndrome. Three years later, at the age of 7, our
patient presented with persisting fever, malaise and rash. The peripheral
blood showed pancytopenia with 95% blasts. Examination of the bone
marrow aspirate with morphology, immunophenotyping and cytogenetics
revealed acute myeloid leukemia (AML), FAB classification M1.
There were no good risk cytogenetics ( t(8:21), t(15:17) or inv(16) ). There
was no CNS involvement. Our patient was treated according to the
MRC-AML 12 protocol. After the first course she attained complete
remission. Two-and-a-half years later however a relapse of AML
occurred, again without CNS involvement. Treatment according to the
DCOG Relapsed AML-protocol 2000/2001 was commenced. After
achievement of a second complete remission our patient received BMT
with T-cell depleted marrow from a matched unrelated donor. Conditioning
regimen consisted ofATG, cyclophosphamide and total body irradiation
of 2?6 Gray. The transplantation procedure was uneventful. Six
weeks post-transplantation coagulation screening was repeated. Platelet
counts were normal, as were PT, APTT, platelet aggregation and PFA.
ADP in platelets has normalized to 3.1 nmol/10*8 platelets. Until one
year after BMT the patient was well without sign or symptom of a bleeding
disorder. One year after BMT a second relapse of AML occurred and
she died 3 weeks later. Conclusion. HPS is a rare disorder causing oculocutaneous
albinism and bleeding tendency. It is considered to be a defect
in intracellular vesicle formation in platelets, melanocytes and lysosomes.
There is no evidence suggesting a link between HPS and leukemia, as was
the case in our patient. We have shown that the bleeding tendency can
be successfully corrected by bone marrow transplantation, which may
be an option for those few patients with serious bleeding problems.
However, whether serious complications like lung fibrosis can be prevented
by bone marrow transplantation remains yet to be seen
0998
SIMULTANEOUS DETECTION OF FLT3, NPM1 AND WT1 MUTATIONS USING
HIGH-RESOLUTION CAPILLARY ELECTROPHORESIS
I. Kakkas, 1 K. Summers, 2 C. Fleischmann, 3 P. Virappane, 2 T.A. Lister, 2
J. Fitzgibbon2 1 Evaggelismos Hospital, ATHENS, Greece; 2 Barts and the london School of
Medicine, LONDON, United Kingdom; 3 Queen Mary University, LONDON,
United Kingdom
Background. FLT3-Internal Tandem Duplication (FLT3-ITD) and Nucleophosmin
(NPM1) mutations are the most frequent genetic alterations
in normal karyotype AML. FLT3-ITD results in inframe duplications
within the juxtamembrane region (exon 14 and 15). In contrast mutations
in NPM1 result in frameshift mutations in exon 12. Both FLT3-ITD
and NPM1 mutations are strongly associated with NK-AML prognosis
(adverse and favourable respectively). We have recently shown that the
mutations in Wilms’ Tumor-1 (WT1) cluster to exons 7 and 9 and typically
result in short regions of insertion or deletion. The change in PCR
product size makes these suitable for high throughput mutational analysis
using high-resolution Capillary Electrophoresis (CE). We have set
out therefore to establish a reliable screening method for simultaneous
detection of FLT3, NPM1 and WT1 mutation. Patients and Methods. FLT3,
NPM1 and WT1 mutation status was determined by DNA amplification
and direct sequence analysis of 83 NK-AML patients. Multiplex PCR
(QIAGEN Multiplex PCR Kit) for all 3 genes were performed using 5’ end
dye labelled reverse (R) primers [FLT3 (HEX), NPM1 (HEX), WT1 exon
7 (FAM), WT1 exon 9 (TAMRA)], followed by CE using a Genetic
Analyser. Results and Conclusions. Twenty samples were selected for
analysis based on their FLT3 (10 cases), NPM1 (10 cases) and WT1 (6 cases
- exon 7 and 2 cases - exon 9) mutation status. These included cases
with all mutation permutations and included heterozygous and
homozygous events. There was complete concordance with CE profile
and the mutation pattern obtained by direct sequencing. We can conclude
therefore that CE of fluorescently labelled PCR products provides
370 | haematologica/the hematology journal | 2007; 92(s1)
a sensitive approach for the simultaneous detection of DNA fragment
size variation up to and including 1bp difference. It is semi-quantitative,
can discriminate between low, intermediate and high mutation load and
allows the analysis of several fragments simultaneously.
0999
FINAL CLINICAL RESULTS OF A NEW INTRAVENOUS BUSULFAN DOSING STRATEGY
AS PART OF BUCY CONDITIONING REGIMEN IN CHILDREN AND ADOLESCENTS
UNDERGOING ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION (HSCT):
LOW MORTALITY AND LONG TERM DISEASE FREE SURVIVAL IN MYELOID LEUKEMIAS
H. Espérou, 1 G. Vassal, 2 B. Neven, 3 F. Mechinaud, 4 Y. Bertrand, 5
C. Galambrun, 5 ,K. Yakouben, 6 C. Paillard, 4 ,P. Bordigoni, 7 ,C. Levrault, 8
H. Zouabi, 8 G. Michel9 1 Hopital Saint Louis, PARIS; 2 Inst Gustave Roussy, VILLEJUIF, France; 3 Hop
Necker-Enfants Malades, PARIS; 4 Hopital Hotel Dieu, NANTES; 5 Hopital
Debrousse, LYON; 6 Hopital Robert Debre, PARIS; 7 Hopital d'Enfants Brabois,
VANDOEUVRE LES NANCY; 8 Institut de Recherche Pierre Fabre,
BOULOGNE; 9 Hopital La Timone, MARSEILLE, France
Background. In children and adolescents allogeneic (allo-) hematopoietic
stem cell transplantation (HSCT) is a standard therapeutic option, but
its use remains limited by the risk of transplant-related mortality (TRM).
Busulfan (Bu) is often included in conditioning regimens but under- or
overdosing may have a fatal outcome. We published that instead of an
age-based dosing a body-weight- (BW) based calculation of IVBu can
successfully target a therapeutic blood exposure without any dose adjustment
(Vassal G et al. EHA 2006). This approach was applied prospectively
in 36 patients (pts) and we report here the final clinical results. Patients
and Methods. 36 children (21 boys/15 girls); median age 7.5 y (range 0.3
to 17.2 y) and weight 27 kg (range 5.0 to 62.0). IVBu (Busilvex ® ) was given
over 2 h q6h x 16 doses according to BW: 1.0 mg/kg, 1.2 mg/kg, 1.1
mg/kg, 0.95 mg/kg, and 0.8 mg/kg for patients(pts) with 34 kg BW, respectively. Cy was administered
at 50 mg/kg qdx4. Clonazepam was used as seizures prophylaxis.
Graft-versus-host disease (GVHD) prophylaxis was given according to
local practice. Indications for HSCT were: AML (n=17: 15 CR1/2 CR2),
ALL (n=1, CR2), CML (n=3), MDS (n=1), SAA (n=1); SCD (n=7), thalassemia
(n=1), Wiskott-Aldrich Syndrome (WAS, n= 3), LAD (n=1), and
HLH (n=1). Pts received bone marrow (n=35), and PBPC (n=1) containing
6.4 ×10 6 CD34 + /kg (range 1.1-29) from matched (28/36) mismatched
(1/36) related or unrelated (7/36, 2 mismatched) donors.
Figure 1.
Results. All pts achieved sustained engraftment at day 19 (range 9-47)
for ANC > 0.5×10 9 /L, and day 30 (range 16-111) for platelets >50×10 9 /L.
Complete chimerism (>99%) was seen in 31/36, and 5/36 were mixed
chimeras but had mainly donor cells(>85%). IVBu was well tolerated: no
grade (G) IV toxicity, G III (mainly stomatitis) occurred in 14 pts. VOD
occurred in 8% (all in pts with genetic diseases) but none was severe. G
I-II and III-IV acute GVHD rates were 50% and 6%, respectively. The
median follow-up of the entire cohort is 36.7 months (range 3.9-49.1)
Two pts died of treatment-related causes (hemorrhage=1, c-GVHD=1)and