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

0545
STABILIZED BONE MARROW IS NOT SUPERIOR TO PERIPHERAL BLOOD FOR MOLECU-
LAR ANALYSIS OF CML PATIENTS WITH RESISTANCE TO TYROSINE KINASE INHIBITORS
T. Ernst, M.C. Müller, P. Erben, N. Härtel, C. Walz, R. Hehlmann,
A. Hochhaus
Universitätsklinikum Mannheim, MANNHEIM, Germany
Background. Recommendations for treatment surveillance of CML
patients on tyrosine kinase inhibitors include systematic molecular monitoring
by accurate quantification of BCR-ABL transcripts. Emergence of
BCR-ABL kinase domain mutations is considered the major cause of
resistance. Inadequate response or loss of response to therapy requires
an early and accurate measurement of BCR-ABL transcripts and BCR-
ABL kinase mutations indicating the need to change the therapeutic
strategy. The sensitivity of the respective assays clearly depends on quality
and quantity of RNA derived from peripheral blood (PB) and bone
marrow (BM) leukocytes. Bedside RNA preservation systems (e.g. PAXgene)
have been developed in order to maintain RNA stability during
shipment of the sample from the clinical site to the laboratory. Aims.
RNA stabilization systems have been tested for PB but not BM cells in
previous studies. Therefore, we sought to investigate the applicability of
stabilization systems for BM samples and to compare the performance
of the system for quantification of BCR-ABL transcripts and detection
of BCR-ABL kinase domain mutations in both tissues. Methods. Simultaneously
stabilized PB and BM samples (PAXgene system, 2.5 mL each)
were obtained from n=49 imatinib resistant CML patients in chronic
phase to compare RNA yield and purity, quantitative results for housekeeping
genes (total ABL and β-glucuronidase, GUS) by RT-PCR, ratios
BCR-ABL/ABL and BCR-ABL/GUS, and BCR-ABL mutations analyzed
by sensitive denaturing high-performance liquid chromatography (D-
HLPC) and by direct sequencing. Results. RNA yield was significantly
higher in BM (median 9.9 µg RNA/mL BM) than in PB (median 3.8 µg
RNA/mL blood, p=0.0013). Using 10 µg RNA for the generation of 40 µL
cDNA by reverse transcription, the number of housekeeping gene transcripts
indicating sample quality and sensitivity of RT-PCR was comparable
between PB and BM (median ABL copies/2 µL cDNA 13,750 vs
26,230, p=0.52; median GUS copies/2 µL cDNA 38,830 vs 59,560,
p=0.22, respectively). Further, ratios BCR-ABL/ABL (PB vs BM, median
47% vs 57%, p=0.07) and ratios BCR-ABL/GUS (PB vs BM, median 27%
vs 23%, p=0.33) were not significantly different. BCR-ABL kinase
domain mutations were detected in 26 of 49 (53%) patients, and in 27
of 49 (55%) patients using PB or BM, respectively. In one patient the
D276G mutation was only detectable in PB (20% mutated alleles) but
not in BM cells. However, in three patients mutations were only found
in BM but not in PB: Mutation H396R (n=1, 20% mutation fraction) and
the silent mutation E499E (n=2, mutation fraction 30% and 40%, respectively).
In general, the relative fraction of the mutated clone was significantly
higher in PB than in BM samples (median 70% vs 50%,
p=0.0023). Summary and conclusions. Optimum sample quality is a crucial
requirement for molecular monitoring of CML patients on therapy. Sensitivities
of the assays depend on a sufficient amount of non-degraded
RNA in the sample after transit to the laboratory. We conclude that BM
is suitable for stabilization with RNA preservation systems but is not
superior to PB for quantification of BCR-ABL transcripts and mutation
analysis in CML patients.
12 th Congress of the European Hematology Association
Chronic myeloid leukemia - Clinical
0546
PLEURAL AND PULMONARY EVENTS IN PATIENTS TREATED WITH DASATINIB FOR
CHRONIC MYELOID LEUKEMIA IN CHRONIC PHASE
PH. Rousselot, 1 A. Bergeron, 2 D. Réa, 2 V. Levy, 2 C. Picard, 2
V. Meignin, 2 J. Tamburini, 2 H. Bruzzoni, 2 F. Calvo,2 A. Tazi, 2
P. Rousselot1 1 Hôpital de Versailles, LE CHESNAY; 2 Hôpital Saint-Louis, PARIS, France
Background. Dasatinib is a novel multi-targeted kinase inhibitor of
BCR-ABL and SRC family kinases indicated for patients with chronic
myeloid leukemia (CML) or Ph + acute lymphoblastic leukemia resistant
to or intolerant of imatinib. Aims. To describe the characteristics and
management of pleural and pulmonary events observed in association
with dasatinib 70 mg b.i.d therapy. Methods. This was a single-center case
series of 40 patients with chronic-phase (CP) CML resistant to or intolerant
of imatinib, who received treatment with dasatinib in clinical trials.
Results. Nine of these 40 patients (22.5%) developed pleural effusions
and/or pulmonary manifestations. Clinical symptoms included
dyspnea, cough, and chest pain; extra-thoracic symptoms (fever, myalgia,
arthralgia, or paresthesiae) were present in 3 cases. High-resolution
CT scans identified pleural effusions in 6 patients, and lung abnormalities
were present in 8. All pleural effusions were exudative and contained
lymphocytes. Three patients underwent pleural biopsy: no abnormalities
were identified in one patient, while a lymphocytic infiltration
and a myelocytic infiltration were observed in the other 2 cases. BAL
revealed lymphocytic alveolitis in 4 patients with lung abnormalities
and neutrophilic alveolitis in a further case. Treatment with dasatinib
was interrupted in all but one case (this patient was administered steroid
therapy instead) and 2 patients received antibiotics empirically; diuretics
were not routinely given. Resolution of these lung manifestations
was evident for all 9 patients. Re-introduction of dasatinib at a reduced
40-mg b.i.d. dose was successfully achieved in 3 of 4 patients without
any recurrence; the fourth patient developed a pleural effusion. Summary
and conclusions. Pleural effusions and pleuro-pulmonary manifestations
observed in association with dasatinib 70 mg b.i.d therapy resolve upon
treatment interruption. Dose reduction appears to allow resumption of
dasatinib therapy.
0547
A COMBINATION OF DAUNORUBICIN, CYTARABINE AND IMATINIB MESYLATE FOR
PATIENTS WITH DE NOVO PHILADELPHIA POSITIVE ACUTE MYELOBLASTIC LEUKEMIA
AND MYELOID BLAST CRISIS CML : RESULTS OF THE AFR01 DOSE ESCALATING STUDY
PH. Rousselot, 1 B. Deau, 1 F. Nicolini, 2 J. Guilhot, 3 F. Huguet, 4 B. Rio, 5
A. Guerci, 6 L. Legros, 7 C. Pautas, 8 D. Réa, 9 E. Raffoux, 9 C. Berthou, 10
D. Guyotat, 11 P. Cony-Makhoul, 12 M. Gardembas, 13 S. Castaigne, 1
M. Michallet, 2 F.X. Mahon, 14 F. Guilhot, 3 P. Rousselot1 1 Hôpital de Versailles, LE CHESNAY; 2 CHU Lyon, LYON; 3 CHU La Miléterie,
POITIERS; 4 CHU Purpan, TOULOUSE; 5 Hôpital Hôtel-Dieu, PARIS; 6 CHU
Nancy, NANCY; 7CHU Nice, NICE; 8 Hôpital Mondor, CRÉTEIL; 9 Hôpital
Saint-Louis, PARIS; 10 Hôpital Morvan, BREST; 11 Hôpital Nord, SAINT ETI-
ENNE; 12 Hôpital d'Annecy, ANNECY; 13 CHU Angers, ANGERS; 14 Hôpital
Haut-Lévêque, BORDEAUX, France
Background. Only 15% of patients (pts) with chronic myelogenous
leukaemia in myeloid blast crisis (MBC CML) and treated with imatinib
mesylate (IM) achieve a complete haematological remission (CHR) and
28% return to chronic phase (CP). For pts in CHR, the median duration
of response is only 10 months and overall survival is around 7 months.
We conducted a dose escalating study to assess the safety and the efficacy
of IM associated with chemotherapy. Patients and methods. Pts ≥ 18
years with MBC CML and de novo Philadelphia positive AML not previously
treated with tyrosine kinase inhibitors (TKI) were eligible. In the
first part of the study a fixed dose of IM (600 mg/d) was administered
combined with increasing dosages of daunorubicin (cohort 1: no
daunorubicin, cohorts 2, 3 : 15 and 30 mg/m 2 /day of daunorubicin IV day
1 to day 3 respectively) and cytarabine (Ara-C) 100 mg/m 2 /d for 7 days.
G-CSF was administered from day 9 until recovery. Responding pts
received a second identical course. Hematopoietic stem cell transplantation
(HSCT) was then offered to all eligible pts. Intermediate dose Ara-
C and IM maintenance was proposed for the others. Pts included in the
second part of the study were treated with IM 600 mg/d in combination
haematologica/the hematology journal | 2007; 92(s1) | 203