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

Cytogenetics and Molecular diagnostics II
0564
RATIONAL USE OF THE REAL-TIME QUANTITATIVE PCR PROTOCOL TO MEASURE
TELOMERE LENGTH IN BLOOD SPECIMENS
J. Ohyashiki
Tokyo Medical University, TOKYO, Japan
Background and Aims. It has been presumed that the measurement of
telomere length is troublesome and time-consuming, despite the fact
that telomere attrition is closely linked to bone marrow failure syndrome
as well as leukemia and lymphoma. We present here an automated
telomere measurement system that combines magnetic filtration and
real-time PCR. Methods. Genomic DNA was extracted from 100 µL of
whole blood by an automated magnetic bead system, then quantitative
PCR was done in a closed tube using a carefully designed pair of oligonucleotide
primers as reported by Cawthon et al. (Nucleic Acid Res 2002,
30: e47). In this assay, the quantity of telomere repeat in each experiment
sample was measured as the level of an arbitrarily chosen reference sample,
then the telomere signal was normalized to the signal from a single
copy gene to generate a relative ratio (T/S). Results. In 107 peripheral
blood specimens obtained from healthy volunteers (aged 8 to 84) and 6
leukemia cell lines, the T/S ratio was significantly associated with the
mean telomere restriction fragment (TRF) measured by traditional Southern
blot analysis [y=TRF, x=T/S, y=6.55x±2.39, R2=0.564]. The relative
telomere ratio in blood specimens obtained from healthy volunteers was
1.06±0.21 (mean ± S.D). In 87 peripheral blood specimens obtained from
various hematologic diseases, the relative telomere ratio was as follows:
chronic myeloproliferative disorders (n=34), T/S=0.93±0.41, acute
leukemia (n=31), T/S=0.77±0.75, myelodysplastic syndrome (n=23),
T/S=0.39±0.34. Of note is that the T/S ratio in myelodisplastic syndrome
was more closely linked to the length of the G-tail rather than TRF,
which included the subtelomeric region. Conclusions. Our results suggest
automated telomere measurement is suitable for high throughput of
samples. The resolution of this assay should be adequate for many genetic
and epigenetic studies. This assay will facilitate investigations of the
biology of telomeres and their roles in hemopoetic stem cell biology as
well as bone marrow failure syndrome.
0565
COOPERATING MUTATIONS OF RECEPTOR TYROSINE KINASE/RAS PATHWAY IN ADULT
CORE-BINDING FACTOR ACUTE MYELOID LEUKEMIA: DIFFERENT PATTERNS BETWEEN
AML1-ETO AND CBFB-MYH11
L.Y. Shih, C.F. Huang, C.L. Lai, T.H. Lin, J.H. Wu, M.C. Kuo, T.L. Lin
Chang Gung Memorial Hospital, TAIPEI, Taiwan
Background. Two-hit model of leukemogenesis has been proposed for
AML; class I mutations that drive proliferation and survival, and class II
mutations that block differentiation. Aims. We sought (1) to determine
the cooperating mutations of class I including receptor tyrosine kinase
(RTK)/Ras signaling pathway in adult AML patients with core-binding
factor (CBF) translocations which are class II mutations, and (2) to determine
the difference in the patterns of cooperating mutations between
AML with AML1-ETO and AML with CBFβ-MYH11. Patients and Methods.
By RT-PCR analysis, 123 adult patients were identified to have CBF
AML, 93 with AML1-ETO and 30 with CBFβ-MYH11. Bone marrow
samples at diagnosis were analyzed for FLT3-ITD, FLT3-TKD, N-ras, Kras,
c-KIT and c-FMS mutations. Mutational analysis was performed by
DNA-PCR followed by GeneScan analysis for FLT3/ITD, and by PCR-
RFLP followed by direct sequencing for FLT3-TKD, by DNA/cDNA PCR
followed by direct sequencing of all PCR products for N-ras, K-ras, c-KIT
and c-FMS. Results. Forty-six of 93 patients (49.5%) with AML1-ETO had
RTK/Ras mutations compared with 21 of 30 patients (70%) with CBFβ-
MYH11 (p=0.059). The frequencies of RTK/Ras mutations in 93 AML1-
ETO AML were 5.4% (n=5) for FLT3/ITD, 7.5% (n=7) for FLT3/TKD,
1.1% (n=1) for N-ras, 3.2% (n=3) for K-ras, 33.3% (n=31) for c-KIT, and
1.1% (n=1) for c-FMS mutation (A273V). The frequencies of RTK/Ras
mutations in 30 CBFβ-MYH11 AML were 3.3% (n=1) for FLT3/ITD,
26.7% (n=8) for FLT3/TKD, 20% (n=6) for N-ras, 20% (n=6) for c-KIT,
and none for K-ras and c-FMS mutations. All RTK/Ras mutations were
mutually exclusive except two, one of them had both N-ras and K-ras
mutations, the other harbored FLT3/TKD and c-KIT mutations. Patients
with CBFβ-MYH11 had a significantly higher frequency of FLT3/TKD
and N-ras mutations than patients with AML1-ETO (p=0.010 for
FLT3/TKD, and p=0.001 for N-ras). Taken together, c-KIT mutations
12 th Congress of the European Hematology Association
accounted for 30% in CBF AML, and there was no difference in the frequency
between AML1-ETO and CBFβ-MYH11 groups. Of the 31
patients with AML1-ETO and c-KIT mutations, 23 had mutations located
at kinase domain (exon 17), 3 in exon 8, 1 in exon 9, and 4 in exon
11. Of the 6 patients with CBFβ-MYH11 and c-KIT mutations, two each
had mutations in exons 8, 11 and 17. Patients with AML1-ETO were
more frequently associated with c-KIT mutations in exon 17 as compared
with patients with CBFβ-MYH11 (p=0.073). Conclusions. Our
results showed that occurrence of cooperating mutations of RTK/Ras
pathway are common in adult patients with CBF AML. The patterns of
mutations were different between AML1-ETO and CBFβ-MYH11
groups.
0566
COOPERATING MUTATIONS IN CHILDHOOD ACUTE MYELOID LEUKEMIA
D.C. Liang, 1 L.Y. Shih, 2 C.F Huang, 2 Y.T Chang, 1 C.L Lai, 2 H.C Liu, 1
C.P Yang3 1 Mackay Memorial Hospital, TAIPEI; 2 Chang Gung Memorial hospital,
TAIPEI; 3Chang Gung Children's Hospital, LINKOU, Taiwan
Background. There have been rare reports on the cooperating mutations
of class I mutations which drive proliferation of acute myeloid leukemia
(AML) blasts and class II mutations which block differentiation of blasts
in childhood AML. Aims. We sought to systematically study the cooperating
mutations in a large series of childhood AML. Patients and Methods.
By cytogenetics and RT-PCR analysis,166 children with newly diagnosed
AML were divided into t(8;21) (N=31), inv(16) (N=14), MLL
rearrangement (N=21), t(15;17) (N=14), Down syndrome (N=6), unfavorable
group (N=15) which included -7 or 7q-, complex karyotype and
t(6;9), and intermediate group for all other patients (N=65). Bone marrow
samples at diagnosis were examined for mutations of FLT3-ITD,
FLT3-TKD, N-Ras, K-Ras, c-KIT, c-FMS, CEBPα, and NPM1. Mutational
analysis was performed by PCR followed by GeneScan analysis for
FLT3-ITD, by PCR-RFLP followed by direct sequencing for FLT3-TKD,
and by PCR followed by direct sequencing for other genes. Results. c-KIT
mutations were more prevalent in t(8;21) than others (p