2008 Barcelona - European Society of Human Genetics

Cancer genetics
ity of the malignant clone in CML . We appreciate that all new cases
might bring new insights, with a special emphasis on prognostic impact
of additional chromosomal changes in imatinib therapy era .
Financial support: CNCSIS (Project 60GR/2007), CEEX (Project
111/2006) .
Acknowledgments: The authors thank Mrs . Marioara Cristea for technical
assistance .
P04.090
complex karyotype and cryptic t(9;22) translocation at the onset
of chronic myeloid leukemia - case report
A. Lungeanu1 , A. Arghir1 , G. Cardos1 , S. Chirieac1 , M. Ciochinaru2 ;
1 2 ”Victor Babes” National Institute, Bucharest, Romania, ”Dr. Carol Davila” Clinical
Hospital, Bucharest, Romania.
Among patients with chronic myeloid leukemia (CML) positive for Philadelphia
chromosome, 5-8% show variant translocations in which at
least a third chromosome in addition to 9q34 and 22q11 is involved .
We report an apparently Ph negative CML patient with unusual complex
karyotype showing a typical fusion transcript detected by revers
transcription PCR (RT-PCR) and BCR/ABL fusion gene localized in a
complex rearranged chromosome identified by FISH.
Cell cultures from bone marrow and peripheral blood, and GTG banding
were performed for karyotype investigation . Dual fusion BCR/ABL
probe, chromosome painting (WCP 9 and WCP 22 ) and locus specific
BAC (1p36 .33 and 17p11 .2) probes for metaphase FISH analysis were
used . RT-PCR for BCR/ABL fusion transcripts detection was applied .
Bone marrow cytogenetic analysis showed a complex rearrangement
consisting of chromosome 1 deletion, 17p addition, telomere-to-telomere
fusion of chromosomes 21 and 22, but no standard Philadelphia
translocation . Constitutional aberrations were ruled out by peripheral
blood karyotyping . RT-PCR detected BCR/ABL fusion transcript . FISH
analysis showed two fusion signals for hybrid gene, and confirmed
the complexity of rearrangements involving chromosomes 1, 9, 17, 21
and 22 .
The FISH techniques allowed us the identification of chromosome rearrangements
that could not otherwise be detected by conventional
banding procedures .
The frequency, formation mechanisms and clinical significance of such
rare type of clonal rearrangements need to be investigated .
Financial support: CNCSIS (Project 60GR/2007), CEEX (Project
111/2006) .
Acknowledgments: The authors thank Prof . Dr . Jean-Michel Dupont
and Mrs . Dominique Blancho for kindly providing BAC probes and Mrs .
Marioara Cristea for technical assistance .
P04.091
major causes of imatinib resistance in chronic myelogenous
leukemia: BcR-ABL kinase domain mutations and clonal
evolution
N. Meggyesi 1 , A. Bors 1 , A. Szilvási 1 , S. Lueff 2 , Á. Bátai 2 , E. Ádám 2 , A. Kozma 2 ,
G. Halm 2 , S. Nahajevszky 2 , B. Kapás 2 , Z. Csukly 2 , N. Lovas 2 , P. Reményi 2 , T.
Masszi 2 , A. Tordai 1 , H. Andrikovics 1 ;
1 Hungarian National Blood Transfusion Service, Budapest, Hungary, 2 Dept. of
Hematology and Stem Cell Transplantation, St. Istvan and St. Laszlo Hospital,
Budapest, Hungary.
Imatinib mesylate, a targeted BCR-ABL tyrosine kinase inhibitor (TKI)
has become the standard drug for the treatment of chronic myelogenous
leukemia (CML) . Mutations in the kinase domain (KD) of BCR-
ABL and nonrandom, karyotypic abnormalities (referred to as clonal
evolution) contribute to imatinib-resistance . Imatinib-resistance may
be defined as primary or secondary, hematologic or cytogenetic and
it occurs in 10-15% of patients with CML . The aim of our study was to
screen for BCR-ABL KD mutations and additional chromosomal abnormalities
(ACA) in 35 Hungarian patients (33 CML and 2 Philadelphia
chromosome positive ALL) with imatinib-resistance . Point mutations
in the entire tyrosine kinase domain (aa 230-490) were detected by
Sanger-sequencing after two separate, two step semi-nested PCR .
The presence of T315I, M244V, Y253H, M351T, F359V, L384M mutations
were confirmed by PCR-RFLP. Clonal alterations were assessed
by standard cytogenetic techniques . Overall, twelve different mutations
(aa . exchange M244V, G250E, Y253H, E255V, E279K, D276G, T315I,
M351T, F359I/V, V379E, L384M, E460K) were identified in 21 patients.
M244V was the most frequent mutation (4 patients) . T315I occurred in
2 ALL and one CML patients . In three cases, double mutations, and in
8 patients, extra Philadelphia chromosome were detected . ACA was
present overall in 13 patients . KD mutations were found in 75% (9/12)
in hematologic, and 43% (9/21) in cytogenetic TKI resistance . In summary,
BCR-ABL KD mutations and clonal evolution are common in
imatinib-resistant CML patients . Early detection of emerging mutant
clones may guide therapeutic decisions, because the degree of imatinib-resistance
varies among different mutants .
P04.092
Mutation profile of BCR-ABL kinase domain in imatinib primary
and secondary resistant chronic myeloid leukemia cases
S. Kutsev, M. Velchenko, S. Mordanov;
Rostov State Medical University, Rostov-on-Don, Russian Federation.
Chronic myeloid leukemia (CML) is hematopoietic stem cell disorder
characterized by balanced translocation t(9;22) . Fusion gene BCR-
ABL appeared in the result of t(9;22) gives rise to Abl tyrosine kinase
activation with followed leukomogenic effects . Bcr-Abl selective tyrosine
kinase inhibitor imatinib mesylate revolutionized CML therapy and
allow achieve complete cytogenetic remission (CCR) in most cases
of chronic phase of CML . Nevertheless refractoriness (primary resistance)
or relapse of initial response (secondary resistance) are observed
in over 30% cases of CML . More than 40 different point mutations
of BCR-ABL decrease sensitivity to imatinib . To study BCR-ABL
mutations profile in imatinib resistant cases of CML we have studied
BCR-ABL kinase domain mutations in 27 patients (middle age 43 y .o .
from 21 to 60) with chronic phase of Ph+ CML that did not achieve
any cytogenetic response (95-100% Ph+ BM cells) after 1 year imatinib
therapy 400 mg daily (n=22) or loose cytogenetic response (n=5) .
Mutation status was studied by direct sequencing of BCR-ABL cDNA
samples . BCR-ABL kinase domain mutations were founded in 7 patients
(25,9%). The mutational spectrum included five missense mutations:
M244V, L248V, Y253N, M351T, T315I . Five primary resistant
cases were characterized by L248V mutation (3 cases), T315I (1) and
M244V+M351T (1) . Two patients with secondary resistance shown
L248M (1) and Y253N (1) mutations . In conclusion, mutation analysis
of primary refractoriness or relapse of initial response CML patients is
very useful tool for changing of CML strategy therapy include imatinib
dose escalation, new generation of BCR-ABL inhibitors, combination
therapy and BMT .
P04.093
Differential expression pattern of ITPA gene in cmL patients
B. Hassannia 1 , M. Behmanesh 1 , M. Akbari 2,3 , Y. Nakabeppu 4 ;
1 Department of Genetics, Faculty of Basic sciences, Tarbiat Modares University,
Tehran, Islamic Republic of Iran, 2 Department of Medical Genetics, Faculty
of Medical Sciences, Tarbiat Modares University, Tehran, Islamic Republic of
Iran, 3 3Tehran Medical Genetics Laboratory, No 98, Taleghani Street, Tehran,
Islamic Republic of Iran, 4 Division of Neurofunctional Genomics, Department of
Immunobiology and Neuroscience, Medical Institute of, Fukuoka, Japan.
Genetic material in nucleus or mitochondria is the most significant
intracellular target of damages that cells exposure every day . One
of the most important of these damages is oxidative deamination of
DNA and free nucleotides in the cell pool . Incorporation of deaminated
nucleotides such as inosine triphosphate (ITP, dITP) into DNA or RNA
can increase the frequency of base substitution mutation . It has been
suggested that presence and accumulation of these rough nucleotides
can lead to genetic and chromosomal instability which is the perquisite
of different types of diseases or cancer . The evidences demonstrate
the role of inosine triphosphate pyrophosphates (ITPase) encoded by
ITPA gene, in protecting the cells by omitting the rough deaminated
purines nucleotides of the cell pool . Chronic myelogenous leukemia
(CML) is a type of cancer which is mainly characterized by the presence
of Philadelphia chromosome . There are some reports about existence
of several structural and numerical chromosome abnormalities in
addition to Philadelphia chromosome in these patients . The objective
of this study is to compare ITPA gene expression in CML patients versus
normal samples to examine the possible dysfunction of ITPA gene
activity as an important predisposing factor for genetic instability .
Our results revealed different expression pattern of ITPA gene expression
between two groups .