Daria Pašalić
Department of Medical Chemistry, Biochemistry and Clinical Chemistry
Zagreb University School of Medicine
Šalata ul 2.
10 000 Zagreb, Croatia
Phone +385 (1) 4590 205; +385 (1) 4566 940
E-mail: dariapasalic [at] gmail [dot] com

Useful links

P08-1 (Oral presentation)

Horvat I, Radić Antolic M, Zadro R. PO8-1: T315I mutation detection in patients resistant to imatinib mesylate therapy. Biochemia Medica 2009;19(Suppl 1):S136-S137.
Clinical Institute for Laboratory Diagnostics, Zagreb Clinical Hospital Centre, Zagreb, Croatia
Corresponding author:ivanahorvat13 [at] gmail [dot] com
Introduction: Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the presence of BCR-ABL fusion gene and consequently bcr-abl fusion protein. Although the discovery of tyrosine kinase inhibitor (TKI), imatinib mesylate, improved the treatment of CML patients, a proportion of patients develop resistance to drug resulting in increased bcr-abl level. One of possible reasons for resistance are mutations in ABL kinase domain. Some mutations can be overcome by increasing the drug dose or by using the new generation of TKI. The only mutation resistant to currently available TKI is T315I. The aim of this study was to detect if the presence of T315I is the cause for increase or constantly high bcr-abl level.
Materials and methods: Twenty-four CML patients with inadequate response to therapy were included in the study: 10 patients with suboptimal response, 8 patients with failure in response to therapy and 6 patients with > 1 log increase of bcr-abl level over time. ASO-PCR for T315I detection was performed according to Kang et al (Haematologica, 2006; 91:659). Positive control was DNA sample from a patient with confirmed T315I mutation.
Results: T315I was detected in 4/24 patients (17%). All patients with mutation had high bcr-abl levels (12.7-38.1%; 24.9-26.1%; 12.5-30.4% according to International Scale and permanently positive qualitative PCR for the last patient). Mean bcr-abl level before the mutation request for all 4 patients was 23.1% and the patients did not respond to the increase of therapeutic dose.
Conclusion: ASO-PCR enables, with sensitivity of 1/1000, detection T315I which is one of important prognostic factors for disease course as the treatment of choice for mutation carriers is bone marrow transplantation.
P08-2 (Oral presentation)
Čuković-ČavkaS1, BožinaN2, Ladić A1, Krznarić Ž1, Sertić J2, Vucelić B1. PO8-2: Genetic polymorphism of thiopurine methyltransferase in Croatian inflammatory bowel disease patients. Biochemia Medica 2009;19(Suppl 1):S137-S138.
1Division of Gastroenterology, Zagreb University Hospital Centre, Zagreb, Croatia
2Clinical Institute for Laboratory Diagnostics, Zagreb Clinical Hospital Centre, Zagreb, Croatia
Corresponding author:nbozina [at] kbc-zagreb [dot] hr
Introduction: Thiopurines are important drugs used in the treatment of steroid-dependent and refractory inflammatory bowel disease. They exhibit their activity once they are metabolized via methylation by thiopurine methyltransferase (TPMT) enzyme. TPMT displays a range of activities due to genetic polymorphism meaning that frequency of certain TPMT alleles determines interindividual variations. Therefore the likelihood of adverse effect and interindividual differences in therapeutic efficacy may relate to TPMT genotype. It was established in some studies that TPMT testing should be introduced prior to the initiation of thiopurines in therapy. Regarding to different frequences of polymorphic TPMT alleles reported from different populations (in Caucasians 11%) the aim of this study was to evaluate the frequency of the most common TPMT variants in our inflammatory bowel disease patients (IBD).
Patients and methods: The allelic variants of the TPMT gene (*2, *3A,*3C) were analyzed by polymerase chain reaction-based assays (PCR-RFLP and Real-time PCR) in 326 adult IBD patients.
Results: Among the patients enrolled in the study, 9 patients were heterozygous for a variant TPMT allele (2.7%), while the rest had a wild-type allele. Frequencies of deficient alleles TPMT*2, TPMT*3A, and TPMT*3C were 0.6%, 0.9% and 1.2% respectively.
Conclusion: TPMT*3C was the most frequently ocurring nonfunctional TPMT allele in Croatian IBD population. Regarding to relatively lower frequency of TPMT polymorphisms we need further investigation in cohort of healthy controls prior definite conclusion about benefit of pharmacogenetically guided dosing in our population.
P08-3 (Oral presentation)
Wagner J1, Dorner S2, Stipoljev F3, Lauc G1, Liehr T4, Brečević L5. PO8-3: Partial monosomy 4q and partial trisomy 13q: phenotype and molecular mapping of the breakpoints. Biochemia Medica 2009;19(Suppl 1):S138-S139.
1School of Medicine Osijek, Osijek, Croatia
2Osijek University Hospital, Osijek, Croatia
3Sveti Duh General Hospital, Zagreb, Croatia
4Institute for human genetics and anthropology, Jena, Germany
5Croatian Institute for Brain Research, Zagreb, Croatia
Corresponding author:jwagner [at] mefos [dot] hr
Introduction: Phenotype in patients with segmental aneuploidy often vary in their clinical manifestation depending on the size of the chromosomal region involved. Deletions of chromosome regions 4q31, 4q32, and 4q33-4qter lead to a distinctive malformation syndrome (deletion 4q syndrome) of facial dysmorphism, cardiac and limb defects and developmental delay. More distal 4q deletions involving bands q34-q35 have been found in patients presenting with less characteristic features and less severe mental retardation. Partial trisomy 13q (13q14-qter) has also been shown to cause a characteristic phenotype associated with severe mental deficiency resembling that of trisomy 13. Although del4q and dup13q syndromes vary in their phenotypes, they have also several features in common. Herein presented are the clinical and molecular findings in a 1-year-old female with a karyotype 46,XX,der(4)t(4;13)(q34.1;q14.3)mat, whose phenotype exhibits the features of the two distinctive syndromes.
Methods: The chromosome breakpoints and the size of segments involved in the rearrangement were determined by FISH applying chromosome 4 and 13 specific molecular banding (MCB), subtelomeric probes for 13qter and 4qter, and BACs mapped in 13q21.1 and 13q14.3, respectively.
Results: According to the labeling scheme for all 169 array-mapped MCB libraries (Weise et al., 2008) it was estimated that the duplication encompassed 73.95 Mb of 13q, and the deletion of chromosome 4 14.6 Mb. The report of further cases with very good clinical and molecular characterization will add to a more precise description of the deletion 4q syndrome and will pinpoint the critical region on chromosome 13 responsible for trisomy 13.
P08-4 (Oral presentation)
Franić Šimić I.PO8-4: Early detection of genomic changes with I – FISH in patients with atipical cells in urine. Biochemia Medica 2009;19(Suppl 1):S139.
Zagreb Clinical Hospital Centre, Zagreb, Croatia
Corresponding author:ivafranic [at] yahoo [dot] com
Introduction: Bladder cancer is one of the most prominent cancers in men. About 90,000 cases are newly diagnosed in Europe and 260,000 worldwide. With highly sensitive, specific and non invasive tehniques of molecular biology, like I-FISH, it is possible to detect genomic changes i urine cells and in that way differenciate malignant and benignant cells.
Materials and methods: UroVysion test detects aneuploidy of chromosomes 3, 7, 17 and loss of thew 9p21 locus via fluorescence in situ hybridization (FISH). A total of 270 urine specimens were analysed.
Results: 113 (42%) speciments werw pozitiv. In 24 (21%) samples we found numerical and structural changes, in 83 (74%) only numerical changes and in 6 (5%) only structural changes.
Conclusion: I-FISH allows to detect genomic changes in bladder cancer cells and to differenciate between malignant and benignant cells.
Kardum Paro MM1, Šiftar Z1, Vrhovac R2, Jakšić B2. PO8-5: FLT3 gene internal tandem duplication (FLT3/ITD) in acute myeloid leukemia (AML). Biochemia Medica 2009;19(Suppl 1):S140.
1Institute of Clinical Chemistry, Merkur University Hospital, Zagreb, Croatia
2Merkur University Hospital, Zagreb, Croatia
Corresponding author:mariana [dot] kardum [at] zg [dot] htnet [dot] hr
Introduction: The FLT3 gene encodes a tyrosine kinase receptor that regulates proliferation and differentiation of hematopoietic stem cells. Mutation of the FLT3 receptor, either by internal tandem duplication of the juxtamembrane domain (ITD) or by point mutation of the aspartic acid 835 of the kinase domain (KD), causes constant activation of the FLT3 receptor. Although FLT3/ITD is the most common mutation in adult de novo AML, they are variable in length and therefore associated with a poor AML prognosis.
Aim: To indicate the FLT3/ITD presence by reverse-transcriptase-polymerase chain reaction (RT-PCR) (Noguera NI et al.) in adult de novo AML patients.
Materials and methods: From 20 samples (14 bone marrows and 6 peripheral bloods) of adult de novo AML patients (11 men and 9 woman, median age of 62 years; range 36– 82 years) mononuclear cells were isolated. RT-PCR according to Noguera NI et al. with specific forward and reverse FLT3 primers on exons 14 and 15 and electrophoresis on agarose gel were used for indicating the FLT3/ITD presence. Negative cases (FLT3/WT) had one expected product of 130 base pairs (bp), whereas all positive cases had additional amplicon longer than the expected product of 130 bp.
Results: Negative cases (FLT3/WT) had only one expected product of 130 bp. All positive cases appeared on agarose gel as one additional amplicon longer than the expected product of 130 bp. 20% (4/20) of adult de novo AML patients were FLT3/ITD+ (3/14 bone marrows; 21% and 1/6 peripheral blood; 17%).
Conclusion: RT-PCR and electrophoresis on agarose gel is adequate as a screening method for FLT3/ITD presence in adult de novo AML patients. In the future their variability in length should be investigated because of AML prognosis.
Čipak A1, Nikolac N1, Šimundić AM1, Reljić A2, Justinić D2, Vrkić N1. PO8-6: Insulin receptor (IR) and insulin receptor substrate 1 (IRS-1) polymorphisms and prostate cancer. Biochemia Medica 2009;19(Suppl 1):S141.
1University Department of Chemistry, Sestre Milosrdnice University Hospital, Zagreb, Croatia
2Department of Urology, Sestre milosrdnice University Hospital, Zagreb, Croatia
Corresponding author:andrea [dot] cipak [at] gmail [dot] com
Background: In recent years, a few genes in the insulin signalling pathway have been associated with prostate cancer (PC) susceptibility. Two such gene candidates are the insulin receptor (IR) and the insulin receptor substrate 1 (IRS-1) genes. Both, the IR and IRS-1 are involved in appropriate cell proliferation, tissue development and growth. The aim of this study was to examine the association of IR 1058 C/T and IRS-1 G972R polymorphisms with PC. We also aimed to examine possible association with cancer severity assessed by Gleason score.
Materials and methods: We have studied 119 patients with PC and 61 patients with benign prostate hyperplasia (BPH) as a control group. Patients with PC were divided into subgroups of higher (Gleason score 7-9) and lower (Gleason score 4-6) malignancies. The genotyping of two polymorphisms (IR 1058 C/T and IRS-1 G972R) was performed by the PCR-RFLP method.
Results: There was no difference in genotype (P = 0.794) nor allelic (P = 0.621) frequency of the IRS-1 G972R polymorphism between PC and BPH patient groups. However, there was a significant difference in IR 1058 C/T polymorphism genotype and allelic distribution. Carriers of the polymorphic allele (C/T+T/T) were more frequent in a group of BPH, than in the PC group (54.10% vs. 37.82%); P = 0.040; OR (95% CI) = 0.52 (0.28-0.96). No difference was found in IRS-1 and IR polymorphism distribution in subgroups according to Gleason score.
Conclusion: IR 1058 C/T polymorphism seems to be associated with prostate cancer, whereas IRS-1 G972R is not. Neither polymorphism is associated with the degree of prostate cancer malignancy.
Marušić Vrsalović M1, Livun A1, Pejša V2, Romić Ž1, Kušec R1,2. PO8-7: BCR-ABL oncogene mutations in imatinib treated chronic myeloid leukemia. Biochemia Medica 2009;19(Suppl 1):S142.
1Department for Laboratory Diagnostics, Dubrava University hospital, Zagreb, Croatia
2Department for Internal Medicine, Dubrava University hospital, Zagreb, Croatia
Corresponding author:rkusec [at] irb [dot] hr
Introduction: Targeting the tyrosine kinase activity of BCR-ABL represents promising therapeutic strategy in chronic myeloid leukemia (CML). Despite remarkable efficiency of the tyrosine kinase inhibitor STI571 (imatinb), resistance in some patients has been observed.
Patients and methods: Out of 34 imatinib treated CML patients at University Hospital Dubrava, five of them were showing consistently high levels of BCR-ABL transcripts monitored by real-time PCR (TaqMan technology). We screened those patients for Thr315Ile, Phe311Leu and Met351Thr point mutations using allele-specific oligonucleotide (ASO)-DNA-PCR (with sensitivity of 1/10,000 cells).
Results: Thr315Ile mutations was not detected. All patients showed Met351Thr mutation and in two of them it was present prior to imatinib therapy. One patient acquired Phe311Leu mutation during imatinib therapy.
Conclusion: Increased proportion of mutated sequences during treatment detected by ASO-PCR suggests clonal evolution of mutated cells. Our data imply that in CML patients treated with imatinib, ABL mutations are not restricted to the accelerated phase of the disease, and that, in some cases, mutations occur in chronic phase of the disease and prior to STI571 therapy.
Horvat I1, Radić Antolic M1, Sertić D2, Müller M3, Zadro R1. PO8-8: Standardization of molecular monitoring of bcr-abl fusion transcript according to International Scale. Biochemia Medica 2009;19(Suppl 1):S143.
1Clinical Institute for Laboratory Diagnostics, Zagreb Clinical Hospital Centre, Zagreb, Croatia
2Department of Hematology, Zagreb Clinical Hospital Centre, Zagreb, Croatia
Corresponding author:ivanahorvat13 [at] gmail [dot] com
Introduction: The most frequent method for monitoring therapy in chronic myeloid leukemia patients was qualitative PCR. Development of quantitative real-time PCR enables nowadays relative bcr-abl quantification. The result depends on the probe and housekeeping gene selection as well as in the standards used. For this reason the subproject “Molecular Monitoring“ within the European Leukemia Net intends to standardize the results obtained in different laboratories.
Materials and methods:The aim of the study was to prepare 30 RNA samples that homogenously cover a range of > 3 logs between 0.001 and 10% bcr-abl, to perform the quantification by the method routinely used in laboratory and to send the same samples to the reference laboratory of the Mannheim University Hospital in order to obtain conversion factor for expression of results according to International Scale (IS). The procedure started with the reception of 4 lysates with different dilutions of bcr-abl positive WBC in healthy donors WBC from reference laboratory.
Results: Quantification of bcr-abl was performed by the method routinely used in laboratory and the results sent to reference laboratory revealed the preliminary conversion factor of 0.7830. Next, 30 RNA samples were prepared in 6 aliquots, 2 x 107 WBC each. Bcr-abl quantification was performed in 3 aliquots. Other 3 aliquots together with the obtained results were sent to the reference laboratory where the same procedure was repeated. Comparison of results from both centers revealed the final conversion factor of 0.3911 that generates the results according to IS.
Conclusion: Standardization enables the expression of results according to IS and comparability of results within institutions. Monitoring of dynamics of the change in bcr-abl fusion transcript quantity enables proper disease monitoring and decision in treatment options.