Chronic myeloid leukemia (CML) is a hematological malignancy driven by the BCR::ABL fusion protein. Although tyrosine kinase inhibitors such as imatinib have markedly improved clinical outcomes, treatment resistance remains a major challenge. Natural product-derived compounds may offer alternative therapeutic opportunities. This study investigated the antiproliferative and pro-apoptotic effects of newly synthesized oleanolic acid (OA) derivatives in K562 cells and evaluated their predicted interactions with the ABL kinase domain. OA derivatives were synthesized and characterized by FT-IR and NMR. Cytotoxicity was assessed using the WST-8 assay. Apoptosis, mitochondrial membrane potential, and cell-cycle distribution were analyzed by flow cytometry. Expression levels of BCL2, BAX, Caspase-3, Caspase-8, and Caspase-9 were determined by qPCR, whereas BCR::ABL protein levels were assessed by Western blotting. Boltz-2 complex prediction, molecular dynamics simulations, and MM/PBSA calculations were used to evaluate predicted binding stability, interaction patterns, and energetics. OA-Acid showed the lowest IC₅₀, whereas OACN showed the highest value . Apoptosis was highest in imatinib- and OACN-treated cells, and mitochondrial membrane potential decreased significantly in both groups. OA-Acid and MCM35 produced the greatest G0/G1 accumulation. Apoptosis-related gene expression was altered in a compound-dependent manner. OA-Acid showed the greatest decrease in BCR::ABL protein level. Computational analyses supported conformationally stable predicted interactions of OA, OA-Acid, and OACN within the ABL ATP-binding region. OA derivatives produced distinct cytotoxic, apoptotic, mitochondrial, cell-cycle, and molecular effects in K562 cells. Computational findings supported predicted interactions within the ABL kinase domain, warranting further chemical and mechanistic evaluation.

Purpose: Resveratrol is a polyphenolic compound that has been shown to stimulate fatty acid oxidation and increase the expression of genes associated with thermogenic processes. Therefore, it is important to investigate the effects of resveratrol beyond intracellular thermogenic pathways to determine side effects at the cellular level. The aim of this study was to evaluate the effect of resveratrol on intracellular R-catenin, in addition to its thermogenic effect. Materials and Methods: The effect of resveratrol on thermogenic genes such as SIRT1 gene, UCP1, PGC1 alpha, PRDM16 and R-catenin gene expression levels was analyzed by RT-PCR. The cytosolic Rcatenin level analyzed by western-blot. Results: The effect of resveratrol on thermogenic genes such as SIRT1 gene, UCP1, PGC1 alpha, PRDM16 and R-catenin gene expression levels was analyzed by RT-PCR. The cytosolic R-catenin level analyzed by western-blot. We found that the expression levels of UCP1, PPAR gamma, PRDM16 were increased by SIRT1 protein activated by resveratrol, but it had no effect on R-catenin mRNA expression. While R-catenin did not change at the mRNA level, we found that R-catenin increased at the protein level in the cell independently of the canonical Wnt pathway. Conclusion: Our results suggest that resveratrol, which is widely used as a supplement, may also act on different signaling pathways in the cell.

Wnt-signaling pathway plays a crucial role in the pathogenesis and progression of Chronic Myeloid Leukemia (CML). sFRP1 is involved in the suppression of the Wnt-signaling pathway and has been shown to be epigenetically silenced by promoter hypermethylation during CML progression. DNMT3A plays a crucial role in promoter hypermethylation and is responsible for establishing methylation patterns. We aimed to analyze the relationship between sFRP1 expression and DNMT3A, TET1, TET2 and TET3 proteins that are responsible for maintaining cellular methylation patterns; along with miRNAs miR144-3p and miR-767-5p that are known to be associated with these proteins. CML cell lines K562 and K562S which stably expresses sFRP1, were used to compare the changes in miR144-3p and miR-767-5p expression. DNMT3A, TET1, TET2 and TET3 protein levels were analyzed by Western blot. In K562S cells the expression of miR-144-3p and miR-767-5p were decreased along with DNMT3A and TET1 protein levels. On the contrary, TET2 protein was increased. Our results support other reports involving sFRP1 and methylation dynamics; as well as opening new avenues of exploration. Our data supports the conclusion that re-expression of sFRP1 protein alters the expression of factors that play important roles in the overall methylation patterns in the leukemic cell line K562.

Dishevelled (Dvl) is a scaffold protein that transmits Wnt signals to downstream effector molecules via both canonical and non-canonical Wnt signaling pathways. Deregulated activation of Dvl proteins has been reported in various solid tumors. However, it is not clear which pathway and proteins are responsible for observed aberrant activities and their relevance in disease prognosis. In addition, there is relatively limited knowledge on the role Dvl proteins may have in hematologic malignancy etiopathogenesis. In this study, we demonstrated that Dvl genes are not expressed in normal bone marrow but are expressed at different levels in the bone marrow of patients with chronic myeloid leukemia. We showed SMAD1, AHR, mTOR, BRD7 protein expressions are significantly affected by Dvl silencing and overexpression in CML cell lines. Wnt/β-catenin and Wnt/PCP signaling pathway components are effectively repressed after Dvl silencing in K562 cells, while regulator of Wnt/Ca2+ signaling showed increase in both CML cell lines. Targeting Dvl proteins increases imatinib susceptibility of the K562 and MEG-01 cell lines. In light of our data, Dvl could be a potential therapeutic target in the treatment of CML.

Amac: Kronik Miyeloid Losemi (KML), hematopoetik kok hucre (HKH) orijinli klonal miyeloproliferatif bir hastaliktir. KML’deki Bcr-Abl kimerik geni ile birlikte diger ek mutasyonlar ve epigenetik modifikasyonlar hastaligin blastik faza dogru ilerleyisi icin gereklidir. DNA metiltransferazlar (DNMTler), epigenetik olarak duzenlenmis genlerin ifade edilmesinde ve baskilanmasinda onemli rol oynayan genom metilasyonu icin anahtar proteinlerdir. Hematolojik malignitelerde DNMT3A ve diger DNA metilasyon duzenleyicilerinde mutasyonlar tanimlanmistir. R882H mutasyonu DNMT3a’da en sik gozlenen mutasyondur. Bu calismanin amaci DNMT3A R882H mutasyonunun KML hastalarindaki gorulme oranini arastirmaktir. Gerec ve Yontem: Bcr/Abl kimerik gen analizi amaciyla gonderilmis KML hastalarinin kemik iliklerinden elde edilmis olan cDNA orneklerinde, DNMT3A R882H mutasyonunu taramak icin, AciI restriksiyon enzim kesimi kullanildi. Kesim sonrasi Bcr/Abl+ ve Bcr/Abl- orneklerden random secilerek DNMT3A icin DNA dizi analizi yapildi. Elde edilen verilerin analizleri istatistiksel olarak degerlendirildi. Bulgular: 35 Bcr/Abl+ ve 60 Bcr/Abl- ornegin AciI restriksiyon enzim kesimi sonrasi bu mutasyonu tasimadiklari gozlendi. Bu orneklerden random secilerek DNA dizi analizi ile degerlendirilen orneklerde de R882H mutasyonunun olmadigi dogrulandi. Sonuc: Elde edilen sonuclarimiza gore DNMT3A-R882H mutasyonunun KML hastalarinda gozlenmedigi, Bcr/Abl+ ve Bcr/Abl- bireyler arasinda mutasyon gorulme orani acisindan herhangi bir farklilik olmadigi gozlenmistir. Hematopoetik hucrelerin proliferasyon potansiyelini artirmakta rol oynadigi bilinen DNMT3A R882H mutasyonunun KML progresyonu acisindan etkili olmadigi ortaya konmustur.

Objective: Chronic Myeloid Leukemia (CML) is a myeloproliferative disorder characterized by the chromosomal translocation t(9;22) encoding for the Bcr/Abl fusion gene. Secreted Frizzled Related Protein (sFRP) gene products are antagonists of Wnt signaling and their epigenetic silencing has been reported in different types of leukemia. While epigenetic silencing has been extensively reported for sFRP, sFRP4 is a family member less studied. We aimed to study epigenetic silencing of sFRP4 in CML patients. Patients and Methods: Epigenetics alterations in the promoter region of the sFRP4 gene were analyzed in 43 CML patients. DNA methylation of CpGs in the sFRP4 promotor region was investigated using methylation specific PCR. Conventional cytogenetic analyzes were performed directly and after 24 h short cultures from bone marrow samples of patients with a preliminary diagnosis of CML. Results: 42 out of the 43 CML patients investigated were shown to be unmethylated at the sFRP4 promoter region, while in 1 patient hemimethylation was observed. This patient was the only one in which cytogenetic progression with additional chromosomal abnormalities were identified. All others achieved major or complete cytogenetic remission. Conclusions: We observed that sFRP4 methylation is a very rare CML patients' phenomenon. Yet when observed it may implicate disease progression and therapy resistance. A large study needs to clarify the implication of methylated sFRP4 in CML progression.

Purpose: The aim of this study was to investigate the expression changes in Wnt and Frizzled genes that initiate Wnt signaling in two different Ph (+) chronic myeloid leukemia (CML) cell lines.Materials and Methods: Total RNA isolations were performed from Ph (+) CML cell lines with megakaryoblast and basophil cell types. RNA concentrations was evaluated by spectrophotometry and reverted to cDNA. Expression levels of Wnt and Frizzled gene, were analyzed b PCR and data of two cell lines were compared with each other.Results: In the analyzes, it was observed that MEG-01 cells expressed Fzd1-9 genes, while KU812 expressed Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd9 genes. It was determined that MEG-01 cells expressed Wnt2b and Wnt3 genes, while KU812 cells expressed only Wnt3 gene.Conclusion: KU812, MEG-01 cell lines differ from each other in terms of expression levels of Wnt signaling genes. It is thought that the differences between the expression levels of Wnt genes observed in the two CML Ph (+) cell lines may be caused by the differences in the cell's origin, fusion regions and cell type-specific signal transduction stimuli and pathways. Therefore, it is recommended to select more than one cell line in studies using model cell lines.

Amaç: Bu çalışmada blastik fazda farklı hücre serilerinde tutulum gösteren iki farklı Ph (+) kronik miyeloid lösemi (KML) hücre hattında, Wnt sinyal yolağı ana oyuncuları olan Wnt ve Frizzled genlerinin ifade farklılıklarının incelenmesi amaçlanmıştır. Gereç ve Yöntem: Megakaryoblast (MEG-01) ve bazofil (KU812) hücre tipine sahip Ph (+) KML hücre hatları kültüre edildikten sonra, RNA izolasyonları gerçekleştirilmiştir. RNA konsantrasyonları spektrofotometrede belirlendikten sonra, cDNA çevrimi yapılmıştır. PCR ile Wnt ve Frizzled genlerinin ifade düzeyleri analiz edilmiş, iki hücre hattının Wnt ve Frizzled ekspresyon verileri birbirleri ile karşılaştırılmıştır. Bulgular: Yapılan analizler sonrasında MEG-01 hücrelerinin Fzd1-9 genlerini, KU812’nin ise Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd9 genlerini ifade ettiği gözlenmiştir. MEG-01 hücreleri Wnt2b ve Wnt3 genlerini eksprese ederken, KU812 hücrelerinin ise sadece Wnt3’ü eksprese ettiği saptanmıştır. Sonuç: KU812 ve MEG-01 hücre hatlarının, Wnt sinyal genlerinin ifade düzeyleri açısından birbirinden farklılık gösterdiği belirlenmiştir. İki KML Ph (+) hücre hattında gözlenen Wnt sinyal genlerinin ifade düzeyleri arasındaki farklara, hücrenin orjini, füzyon bölgeleri ve hücre tipine özgü olabilecek sinyal ileti uyarı ve yolaklarındaki farklılıkların neden olabileceği düşünülmektedir. Bu nedenle model hücre hatları kullanılarak yapılan çalışmalar da birden fazla hücre hattının seçilmesi önerilmektedir.

Chronic myeloid leukemia is a clonal hematopoietic stem cell disease characterized by myeloid expansion. The hallmark of the disease is the Philadelphia chromosome, which results in the formation of the BCR-ABL oncogene, a tyrosine kinase that is involved in many signaling pathways including Wnt signaling. The latter has a unique role in chronic myeloid leukemia progression; activated signaling leads to the establishment of an additional leukemic stem cell population derived from granulocyte-macrophage progenitors. sFRP1 is an inhibitor of Wnt signaling and its expression is important for differentiation and maintenance of hematopoietic stem cells. In this study, we aimed to investigate miRNAs that target Wnt signaling by being co-induced along with the expression of sFRP1 in K562 cells. We present a detailed analysis of hsa-mir-221 -3p, hsa-mir-222-3p, hsa-mir-106b-5p, hsa-let-7f-5p, hsa-mir-182-5p, hsa-mir-191-5p, and hsa-mir-183-5p and their target genes and their involvement in Wnt signaling.

Amaç: Kronik Miyeloid Lösemi (KML), hematopoetik kök hücreden (HKH) köken alan miyeloproliferatif bir hastalıktır. MikroRNA'lar, transkripsiyon sonrası gen ekspresyonunu düzenlenleyen küçük kodlamayan RNA’lardır. miRNA’lar KML’nin progresyonunda, lösemik kök hücre büyümesi ve tirozin kinaz inhibitörü (TKİ) direncinin gelişmesinde hücre homeostazisini etkilemektedirler. Bu çalışmada KML lösemik hücresi ve KML lösemik kök hücresi (LKH) arasında değişen miRNA ekspresyon profillerinin incelenmesi amaçlanmıştır. Gereç ve yöntem: KML hücre hattı olan K562 hücrelerinden, manyetik hücre ayrımlama (MACS) yöntemi kullanılarak CD34+CD38- lösemik kök hücreleri ayrımlanmıştır. Ayrımlanan LKH’lerin saflığının %85-92 arasında olduğu akım sitometri yöntemi ile gösterilmiştir. K562 ve K562 LKH’leri arasında, gerçek zamanlı kantitatif PCR ile kanser kök hücre ilişkili 84 adet miRNA’nın ekspresyon değişimleri incelenmiştir. Bulgular: K562 ve K562 LKH’leri arasında, kök hücre ilişkili olduğu bilinen 84 adet miRNA’dan 7’sinin anlamlı düzeyde değiştiğini gözledik (P<0,05). K562 LKH’lerinde hsa-miR-29b-3p’nin ekspresyon düzeylerinde artış izlenirken; hsa-miR-320d, hsa-miR-96-5p, hsa-let-7e-5p, hsa-miR-17-5p, hsa-miR-181b-5p hsa-miR-423-5p’da azalma olduğu gözlenmiştir. Sonuç : KML lösemik hücreleri ile KML LKH’leri arasında proliferasyon, eritroid farklılaşma, kendi kendini yenileme ve apoptoz sürecinde rol alan miRNA’lar ve hedef genlerinin ekspresyonlarındaki değişim, hastalığın ilerlemesinde miRNA’larında rol oynayabileceğini göstermektedir. Bu nedenle KML LKH’lerine özgü miRNA’ların hastalığın progresyonunun ve TKI direncinin önlenmesi için yeni terapötik stratejilerin geliştirilmesinde hedef moleküller olabileceğini düşünmekteyiz.

Wnt signaling has been a topic of research for many years for its diverse and fundamental functions in physiological (such as embryogenesis, organogenesis, proliferation, tissue repair and cellular differentiation) and pathological (carcinogenesis, congenital/genetic diseases, and tissue degeneration) processes. Wnt signaling pathway aberrations are associated with both solid tumors and hematological malignancies. Unregulated Wnt signaling observed in malignancies may be due to a wide spectrum of abnormalities, from mutations in the genes of key players to epigenetic modifications of Wnt antagonists. Of these, Wnt antagonists are gaining significant attention for their potential of being targets for treatment and inhibition of Wnt signaling. In this review, we discuss and summarize the significance of Wnt signaling antagonists in the pathogenesis and treatment of hematological malignancies.

Dishevelled (Dvl) proteins are activated by Wnt pathway stimulation and have crucial roles in the regulation of β-catenin destruction complex. CYLD is a tumor suppressor and a deubiquitination enzyme. CYLD negatively regulates the Wnt/β-catenin signaling pathway by deubiquitinating Dvl proteins. Loss of function and mutations of CYLD were linked to different types of solid tumors. Loss of function in CYLD is associated with Dvl hyper ubiquitination, resulting in the transmission of Wnt signaling to downstream effectors. β-catenin upregulation is observed during disease progression in chronic myeloid leukemia (CML). Deregulated Dvl signaling may be a reason for β-catenin activation in CML; and CYLD may contribute to Dvl deregulation. First, we evaluated mRNA expression in three CML cell lines and mRNA expression of the CYLD gene was found to be present in all (K562, MEG01, KU812). Unlike solid tumors sequencing revealed no mutations in the coding sequences of the CYLD gene. DVL genes were silenced by using a pool of siRNA oligonucleotides and gene expression differences in CYLD was determined by RT-PCR and western blot. CYLD protein expression decreased after Dvl silencing. An opposite approach of overexpressing Dvl proteins resulted in upregulated CYLD expression. While previous reports have described CYLD as a regulator of DVL proteins; our data suggests the presence of a more complicated reciprocal regulatory mechanism in CML cell lines.

Chronic myeloid leukemia is a clonal myeloproliferative disorder that arises from the neoplastic transformation of the hematopoietic stem cell, in which the Wnt/β-catenin signaling pathway has been demonstrated to play an important role in disease progression. However, the role of Wnt signaling antagonists in therapy resistance and disease progression has not been fully investigated. We aimed to study the effects of Wnt/β-catenin pathway antagonists-secreted frizzled-related protein 1 and Wnt inhibitory factor 1-on resistance toward tyrosine kinase inhibitors in chronic myeloid leukemia. Response to tyrosine kinase inhibitors was analyzed in secreted frizzled-related protein 1 and Wnt inhibitory factor 1 stably transfected K562 cells. Experiments were repeated using a tetracycline-inducible expression system, confirming previous results. In addition, response to tyrosine kinase inhibitor treatment was also analyzed using the secreted frizzled-related protein 1 expressing, BCR-ABL positive MEG01 cell line, in the presence and absence of a secreted frizzled-related protein 1 inhibitor. Our data suggests that total cellular β-catenin levels decrease in the presence of secreted frizzled-related protein 1 and Wnt inhibitory factor 1, and a significant increase in cell death after tyrosine kinase inhibitor treatment is observed. On the contrary, when secreted frizzled-related protein 1 is suppressed, total β-catenin levels increase in the cell and the cells become resistant to tyrosine kinase inhibitors. We suggest that Wnt antagonists carry the potential to be exploited in designing new agents and strategies for the advanced and resistant forms of chronic myeloid leukemia.

Cancer stem cells (CSC) isolated from multiple tumor types differentiate in vivo and in vitro when cultured in serum; however, the factors responsible for their differentiation have not yet been identified. The first aim of the present study was to identify CD133high/CD44high DU145 prostate CSCs and compare their profiles with non-CSCs as bulk counterparts of the population. Subsequently, the two populations continued to be three-dimensional multicellular spheroids. Differentiation was then investigated with stem cell-related genomic characteristics. Polymerase chain reaction array analyses of cell cycle regulation, embryonic and mesenchymal cell lineage-related markers, and telomerase reverse transcriptase (TERT) and Notch signaling were performed. Immunohistochemistry of CD117, Notch1, Jagged1, Delta1, Sox2, c-Myc, Oct4, KLF4, CD90 and SSEA1 were determined in CSC and non-CSC monolayer and spheroid subcultures. Significant gene alterations were observed in the CD133high/CD44high population when cultured as a monolayer and continued as spheroid. In this group, marked gene upregulation was determined in collagen type 9 α1, Islet1 and cyclin D2. Jagged1, Delta-like 3 and Notch1 were respectively upregulated genes in the Notch signaling pathway. According to immunoreactivity, the staining density of Jagged1, Sox2, Oct4 and Klf-4 increased significantly in CSC spheroids. Isolated CSCs alter their cellular characterization over the course of time and exhibit a differentiation profile while maintaining their former surface antigens at a level of transcription or translation. The current study suggested that this differentiation process may be a mechanism responsible for the malignant process and tumor growth.

OBJECTIVE:At present, there are a limited number of facilities in Turkey that can provide reliable real-time quantitative(RQ)-PCR BCR-ABL results. The present study aimed to test a cost-effective, in-house method of BCR-ABL quantification,including verification of the method by RQ-PCR validation tests.MATERIAL AND METHODS:BCR-ABL and ABL target sequences were cloned into pJET1.2 vectors, from whichcalibrators were prepared and used as templates in RQ-PCR reactions to generate standard curves. Dilutions of K562cells (representing an in vitro simulation of BCR-ABL transcript reduction) were analyzed.RESULTS:Standard curves were generated from calibrators. These curves were then used to calculate the BCR-ABL andABL copy numbers; in which linear BCR-ABL results were obtained. Repetitive experiments showed that our methodologywas able to detect 1 BCR-ABL positive cell from amnong 1x105 cells.CONCLUSION:The method described herein is suitable for implementation with any RQ-PCR instrument and/or kit forquantify BCR-ABL transcripts.CONFLICT OF INTEREST:None declared.

Identifying gene expression differences in the Wnt signaling pathway specific to leukemic cells can be hampered by the lack of verified knowledge on the expression of WNT genes in normal blood cells. In this study we aimed to determine the expression profile of human WNT, FZD and sFRP genes in normal adult bone marrow, T and B cells; along with the hematopoietic cell lines K562, HL60, Jurkat and Namalwa. Bone marrow and peripheral blood from 16 donors were evaluated and our results were compared with the GeneNote database expression arrays. In bone marrow, only WNT3, WNT10A, FZD3, FZD7 and sFRP1 genes are constitutively expressed. Lymphocytes express WNT7A, WNT9B, FZD6 and FZD7 in addition to the genes above, but T cells differ in that they lose sFRP1 expression and gain constitutive expression of WNT16. An established “normal” profile of the Wnt genes in various blood cells will provide a fundamental basis for research investigating hematopoiesis and cellular processes during leukemic transformation.

Epigenetic silencing of sFRP genes has been shown to lead to constitutive activation of the canonical Wnt-signaling pathway. The first description of deregulated Wnt-signaling activation in a hematological malignancy was reported in chronic myeloid leukemia (CML). To investigate whether epigenetic silencing of sFRP is responsible for the observed Wnt activation in CML, we studied the methylation and mutational status of the sFRP1 promoter in 48 chronic phase CML patients. Of the 48 CML patients 41 were shown to be unmethylated, 6 patients hemi-methylated and 1 patient fully methylated at the sFRP1 promoter. Albeit observed infrequently in chronic phase CML, sFRP1 promoter methylation correlated with primary cytogenetic resistance to imatinib mesylate. sFRP1 promoter methylation may indicate a genetically more unstable form of disease resistant to therapy and provide a key biological difference in therapy resistant patients, in addition to a possible mechanism for the observed activation of canonical Wnt signaling in CML.

AIMS AND BACKGROUND Studies reporting activated Wnt signaling in all stages of chronic myeloid leukemia (CML) have demonstrated that deregulation of the pathway plays a role in the pathogenesis of this disease. Several reports have suggested mechanisms for the deregulated Wnt signaling and beta-catenin stabilization observed in CML. One possible mechanism for beta-catenin stabilization could be the acquisition of mutations at its N-terminal domain, especially in the third exon where it is marked via phosphorylation for degradation. We sought to determine whether mutations in the third exon of the beta-catenin gene are responsible for the observed Wnt activation in CML. MATERIAL AND METHODS We screened bone marrow specimens from 33 patients with CML in the chronic phase and also examined the K562 cell line for beta-catenin mutations. RESULTS None of the patients nor the K562 cell line were found to carry mutations. CONCLUSION Beta-catenin amino-terminal mutations are not observed or very rare and therefore are not the underlying mechanism of activated Wnt signaling in CML.

With the aim of determining the differential expression of WNT and FZD genes, before and after induction of apoptosis in BCR-ABL positive cells, we treated the myeloid cell line K562 and control cell line HL60 with imatinib mesylate and etoposide, and analyzed relative mRNA expression levels of WNT, FZD and sFRP genes under normal and apoptotic conditions by real-time RT-PCR. We observed marked increase in mRNA levels of FZD4, FZD5, FZD7 and WNT5b, correlating with apoptotic activity and independent of the agent or cell line used. Our results suggest the involvement of non-canonical Wnt signaling in executing programmed cell death in myeloid cell lines.

Little is understood about the basic biological mechanisms that underlie the reasons for acute transformation in chronic myeloid leukemia (CML). Progression of disease may include inactivation of one or more tumor suppressor genes (TSGs). A widely used methodology for indirectly detecting somatic inactivation of TSGs is searching loss of heterozygosity (LOH) for polymorphic loci located in or near the gene(s) of interest. We aimed to analyze DNA of chronic phase and blastic phase archive material of 15 CML patients for LOH using D1S430, D2S123, D3S1611, D11S29, D14S65, D17S520, BAT 40 markers, the dinucleotide repeat located in the ABL gene and the trinucleotide repeat located in the BCR gene (amplification of the trinucleotide in the BCR gene could not be succeeded). LOH was identified by a %50 lost of one of the alleles intensity. LOH was detected with the ABL dinucletide repeat and D2S123 marker in two patients and with the D14S65 marker in three patients. The three patients exhibiting LOH at the D14S6S locus, all proceeded through lymphoid blast crisis. The D14S65 marker is located at the 14q32 locus which contains the immunglobulin heavy chain gene and the TCL1 oncogene. 14q32 abnormalities at the molecular level, may be predictive for lymphoid blast crisis, whether or not they are detectable cytogenetically