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

12 th Congress of the European Hematology Association
cooperating mutations were only found in 2 patients: FLT3-TKD + K-
Ras in one with t(11;19) and c-KIT + N-Ras in another with inv(16) , all
others with two cooperating mutations occurred in the intermediate
group: FLT3-ITD + NPM1 in 5, FLT3-ITD + CEBPα in 4, FLT3-TKD + K-
Ras, FLT3-TKD + CEBPα, and N-Ras + K-Ras in one each. Conclusions.
In childhood AML, the core-binding factor leukemias are more frequently
associated with c-KIT mutations. AML with inv(16) has more N-Ras
mutations. Patients with intermediate cytogenetic risk group have more
FLT3-ITD, CEBPα, and NPM1 mutations.
0567
DETECTION OF PATIENT-SPECIFIC BCR-ABL GENOMIC DNA IN CML PATIENTS WITH NO
DETECTABLE BCR-ABL BY QUANTITATIVE REVERSE TRANSCRIPTASE PCR
D. Ross, 1 P.A. Bartley, 2 A.A. Morley, 2 J.F. Seymour, 3 S. Branford, 1
T.P. Hughes1 1 IMVS, ADELAIDE; 2 Flinders University & Medical Centre, ADELAIDE;
3 Australasian Leukaemia & Lymphoma Group, MELBOURNE, Australia
Background. With prolonged imatinib therapy for CML an increasing
proportion of patients have persistently undetectable BCR-ABL using
real-time reverse transcriptase quantitative PCR (RQ-PCR), referred to
as complete molecular remission (CMR). However, most patients in CMR
have residual disease, and imatinib withdrawal may result in early
relapse. More sensitive molecular methods are required to identify
patients at higher risk of relapse and to quantify residual disease so that
the efficacy of therapy given after attaining CMR can be assessed. Aims.
To determine if a patient-specific DNA PCR assay can detect residual disease
below the level of CMR as measured by RQ-PCR. Methods. The
patient-specific DNA PCR assay was developed after determining the
genomic BCR-ABL breakpoint in each patient. DNA was extracted from
cells frozen at presentation. The BCR-ABL breakpoint was identified
using long template PCR with a forward primer in BCR and reverse
primer in ABL. The sequence flanking the breakpoint was amplified and
characterised. A sensitive DNA PCR assay with patient-specific primers
and probe was then used for detection of BCR-ABL in follow-up samples.
First, the breakpoint region was amplified using TaqGold DNA
polymerase, then the breakpoint amplicon was detected in real-time
PCR using nested primers and a TaqMan probe spanning the breakpoint.
Each PCR was performed in two independent experiments. There
were two negative controls in each assay; no template, and DNA from
another CML patient. DNA PCR results were compared with prior RQ-
PCR Results. RNA was reverse transcribed with random hexamer primers
and Superscript II. BCR-ABL cDNA was quantified using RQ-PCR with
TaqMan probes on the ABI Prism 7000. Results. We report on two DNA
PCR assays developed for the first patients enrolled in the Australasian
Leukaemia and Lymphoma Group study of imatinib withdrawal in
patients with CMR for ≥2 years. To assess specificity each DNA assay
was tested on 5 other CML patients. No non-specific amplification was
detected. The limit of detection for serial dilutions of the breakpoint
amplicon from presentation samples was similar for both patients suggesting
comparable sensitivity. Both patients had received imatinib treatment
for ≥3 years with no detectable BCR-ABL by RQ-PCR for 2 years
prior to cessation of imatinib (baseline). Patient #1 had molecular relapse
by RQ-PCR 3 months after imatinib cessation. Retrospective analysis by
DNA PCR detected BCR-ABL in this patient’s samples collected immediately
prior to imatinib cessation and 6 months earlier. In contrast, for
patient #2 the available DNA samples at baseline and 14 months earlier
were negative by DNA PCR. This patient remains in CMR 6+ months
after imatinib cessation. Conclusions. A major limitation of sensitive RQ-
PCR is the potential for BCR-ABL contamination from other patient
samples. Patient-specific DNA detection should overcome this problem.
This DNA PCR assay for BCR-ABL is more sensitive than RQ-PCR in
the two patients analysed to date. In these cases DNA PCR results correlated
with remission/ relapse status after cessation of imatinib therapy.
We aim to develop a quantitative DNA PCR assay which might
enable more precise enumeration of residual CML cells.
212 | haematologica/the hematology journal | 2007; 92(s1)
0568
SENSITIVE DETECTION OF C-KIT POINT MUTATIONS BY D-HPLC AND QUANTITATIVE PCR
IN PERIPHERAL BLOOD AND BONE MARROW SAMPLES FROM PATIENTS WITH SYS-
TEMIC MASTOCYTOSIS
P. Erben, S. Lauber, T. Ernst, M.C. Mueller, T. Schenk, J. Kruth, G.
Metzgeroth, P. Paschka, R. Hehlmann, A. Reiter, A. Hochhaus
Universitätsklinikum Mannheim, MANNHEIM, Germany
The majority of patients (pts) with systemic mastocytosis (SM) is
characterized by the presence of the transforming mutation D816V of
the c-kit gene, resulting in a factor-independent activation of the receptor
tyrosine kinase KIT. The mutation is regarded the causative event for
the pathogenesis and a potential target for therapeutic intervention with
novel tyrosine kinase inhibitors like dasatinib, nilotinib (AMN107) and
midostaurin (PKC412). However, the sensitivity of screening procedures
for mutations are compromised by a minor proportion of malignant
cells in the bone marrow (BM) sample. We therefore established and
compared sensitive strategies for the detection of c-kit mutations in BM
and peripheral blood (PB) samples on mRNA level: (i) D-HPLC (denaturing-high
performance liquid chromatography) combined with direct
sequencing in case of a positive D-HPLC signal and (ii) a LightCyclerTM
based quantitative PCR assay for the D816V mutation amplifying the ckit
wildtype (wt) and the mutated allelen. Levels of the D816V mutation
are expressed as ratios D816V C-KIT/wt C-KIT. The different techniques
have been established using serial dilutions of c-kit D816V positive
HMC-1 cells in a background of NB4 cells harboring wildtype c-kit
and pts mRNA in control mRNA. D-HPLC and the quantitative PCR
were optimized for the detection of the D816V mutation down to 0.1-
0.5% fraction of the mutated clone (cell line and RNA dilution). No false
positive results were observed in 20 different control samples obtained
from healthy donors PB samples. In comparison, the detection limit for
D816V mutations by conventional sequencing was 10 to 15%. These
techniques were applied to BM (n=122) and PB (n=65) samples from
102 pts (55 m, 47 f) meeting the WHO criteria for SM. Median age was
51 yrs (range, 23-81). At diagnosis, D-HPLC was positive in 89% of the
BM samples (110/123) and the quantitative PCR in 89% of the BM samples
(59/66) whereas conventional sequencing revealed the D816V mutation
in 73% of the BM samples (90/123). Two patients showed the
D816H and one patient the D816L mutation. The analysis of PB samples
revealed D-HPLC positivity in 48% of the samples (31/65) with a
consecutive detection of the D816V mutation by direct sequencing alone
in 40% of pts (26/65). D816V mutation was detected by quantitative
PCR in 58% of pts (14/24). The mutant proportion of the D816V mutation
analysed in the positive samples by quantitative PCR in BM samples
was in median 22% (range, 0.5-67%) and in PB samples 22% (range,
2.8-91%). In conclusion, i. D-HPLC is a reliable and sensitive method for
the screening of c-kit mutations in SM which is superior to conventional
direct sequencing. ii. The quantitative PCR assay for the most common
mutation D816V offers easy handling but overlooks rare mutations
and is suitable for the surveillance of pts with SM during therapy
with novel tyrosine kinase inhibitors. iii. Employing these sensitive techniques,
the D816V mutation could be detected in BM as well as in PB
samples, but the reliability for a positive result of the mutation was
higher in BM.
0569
A NOVEL DENATURING-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (D-HPLC)-
BASED METHOD FOR KIT MUTATION SCREENING OF PATIENTS WITH SYSTEMIC
MASTOCYTOSIS
S. Colarossi, 1 S. Soverini, 1 A. Gnani, 1 M. Rondoni, 1 S. Gatto, 2
S. Merante, 2 E. Gottardi, 3 D. CIlloni, 3 P. Musto, 2 M. Tiribelli, 2
R. Zanotti, 2 G. Martinelli, 1 M. Baccarani1 1 Dept. of Hematology/Oncology Seràgnoli, BOLOGNA; 2 Hematology,
ALESSANDRIA; 3 Dept of Clinical and Biological Science, ORBASSANO
(TURIN), Italy
Background. Systemic mastocytosis (SM) is a clonal disorder characterized
by anormal growth and accumulation of mast cells (MC) in various
tissues. Kit is a tyrosine kinase transmembrane receptor on the surface
of MC. The presence of an enzimatic site (ES) mutation (e.g.,
D816V) renders Kit resistant to inhibition by imatinib, whereas Kit with
juxtamembrane (JM) mutations (e.g., V560G) remains sensitive to imatinib.
Sensitive methods are required to assure appropriate therapeutic
management of SM patients as the proportion of malignant cells in the
available tissues which carry the mutation is small. Aims. Our aims were: