A Translocation (9;22)(p24;q11.2) In a Patient With CML

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5184-5184
Author(s):  
Daniele Costa Abreu ◽  
Ana Paula Castilho, Bachelor ◽  
Vivian Dionísio Niewiadonski, Bachelor ◽  
Mauricio Drummond ◽  
Nelson Gaburo
Keyword(s):  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Medical Information ◽  
Conflicts Of Interest ◽  
Philadelphia Chromosome ◽  
Chromosome Analysis ◽  
Chromosome 9 ◽  
Fish Analysis ◽  
Ph Chromosome

Abstract Introduction In January 2013 was received in our lab service a bone marrow sample for cytogenetic analysis. The 61 years old female patient presents an elevated white blood cell count (118,000 x10³/mm³) and clinical diagnosis as Chronic Myeloid Leukemia (CML). According the medical information the treatment began with hydroxyurea 3g daily and allopurinol 300mg daily. Methods We proceeded with cytogenetic examination of the patient’s bone marrow aspirate by conventional G-banding analysis performed on unstimulated short-term cultures (24 hrs). FISH for BCR/ABL translocation was tested using a dual fusion dual color probe. Because of the sample stability we were unable to performed RT-PCR test. Results Chromosome analysis showed the translocation (9;22)(p24;q11.2) as a sole abnormality in 100% (20/20) of analyzed metaphases. Chronic myeloid leukemia presents as a specific chromosomal abnormality the Philadelphia chromosome, t(9;22)(q34;q11) which is different from the results obtained where the region of translocation of chromosome 9 was p24 instead of the classic q34. This result suggests it is BCR/JACK2 translocation. The FISH analysis showed the presence of a complex Ph chromosome: ABL con BCRx1 (one fusion) and BCRx2;ABLx2. Conclusion The patient took imatinib without answer. She is still in clinical monitoring with persistent hyperleucocytosis and the treatment is following with hydroxyurea 500mg daily and Interferon 5000 UI three times a week. Further molecular and cytogenetic tests will be performed in a second sample to contribute with evaluation of disease progression and monitoring treatment response. Disclosures: No relevant conflicts of interest to declare.

BCR/ABL Fusion Gene On Constitutional Der(14;22) Roberstsonian Translocation in a Case of Chronic Myeloid Leukemia with Masked Philadelphia Chromosome

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4822-4822
Author(s):  
Pablo Lopez ◽  
Daniela Infante ◽  
Isabel Moro ◽  
Victoria Elizondo ◽  
Gerardo Romanelli ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Peripheral Blood ◽  
Cytogenetic Analysis ◽  
Myeloid Leukemia ◽  
Robertsonian Translocation ◽  
Philadelphia Chromosome ◽  
Chromosome 9 ◽  
Ph Chromosome ◽  
One Step

Abstract Abstract 4822 Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome (Ph) observed in more than 90% of patients with CML as a result of t(9;22)(q34;q11), leading to the formation of the BCR/ABL chimeric gene. The remaining 5–10% of CML cases exhibit a variant Ph translocation generally involving a third or even a fourth chromosome in addition to chromosome 9 and 22, potentially leading to masked Ph chromosome or reveal cryptic translocations that remains undetected under conventional cytogenetic analysis. These chromosome rearrangements can be disclosed by means of fluorescence in situhybridization (FISH) or polymerase chain reaction (PCR) procedures. A very few Ph positive CML cases were reported with constitutional robertsonian translocations, i.e. translocation between two acrocentric chromomosomes (13–15, 21–22), with breakpoints in the short arms, leading to a dicentric chromosome and thus to 45 instead of 46 chromosomes Case Report. 42 year-old woman presenting with asthenia. Physical examination: Grade 1 splenomegaly. Peripheral blood count showed: hemoglobin concentration 117g/L, platelet count: 329×109/L and white blood cell count (WBC): 199×109/L. Peripheral blood smear: myelemia exhibiting 3% of myeloid blasts. Cytogenetic analysis by G-banding performed on bone marrow metaphase cells afforded the following karyotype: 45, XX, der(14;22)(q10;q10)c?, t(9;22;11)(q34;q11;q13) [20]. The analysis of the BCR-ABLfusion gene according to standard protocols detected the presence of the b3a2 isoform. FISH studies using dual color dual fusion probes in metaphases showed a 1F2G2R signal pattern. We detect a normal ABL signal on chromosome 9 and BCR signal on chromosome 22; the fusion signal was present on the der(14;22);extra-signals BCR and ABL with reduced intensities were present on der(11) and der(9) respectively: ish der(9)(ABLdim+), der(11)(BCRdim+), der(14;22)(BCR+,ABL+) [10]. FISH analysis on interphase nuclei (n=200) presented the same signal pattern. Nuc ish (ABL, BCRx3)(BCR con ABL x1) [200]. Chromosome analysis of bone marrow cells after six months of Imatinib therapy showed the following karyotype: 45, XX, der(14;22)(q10;q10)c [20] thus demonstrating complete cytogenetic remission and that der(14;22) is a robertsonian constitutional abnormality that could be inherited and thus necessitate a familial genetic councelling to inform about the familial risk of congenital malformations and miscarriage. Discussion. To explain the formation of variant chromosome Ph translocations one-step, two-step and multi-step mechanisms have been proposed. In our case complex translocations involving four chromosomes and the participation of two acrocentric chromosomes, led to the hypothesis of the presence of a constitutional or acquired Robertsonian translocation. Karyotype analysis six months after treatment confirmed the presence of a constitutional Robertsonian translocation. According to the FISH pattern, this variant Ph chromosome was formed in one step. The occurrence of Philadelphia positive CML in a patient with a constitutional Robertsonian translocation is probably coincidental. The role of constitutional chromosomes abnormalities in hematologic malignancies is well known in Down syndrome patients and in chromosome breakage syndromes such as Fanconi anemia. In the literature, only one case of CML patients with Robertsonian t(14;22) have been described. To our knowledge this is the first report showing a Robertsonian t(14;22) in a variant Ph involving four chromosomes and exhibiting the fusion FISH signal in a derivative chromosome 14, with masked Ph. Disclosures: No relevant conflicts of interest to declare.

Variant Philadelphia Translocation with BCR/ABL Fusion Gene Site In 10q24 In a Case of Chronic Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4839-4839
Author(s):  
Rossana Bonomi ◽  
Pablo Lopez ◽  
Daniela Infante ◽  
Isabel Moro ◽  
Victoria Elizondo ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Chromosome 9 ◽  
Chromosome 22 ◽  
Fish Analysis ◽  
Signal Pattern ◽  
Ph Chromosome ◽  
Fusion Signal

Abstract Abstract 4839 Introduction. Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome (Ph) observed in more than 90% of patients with CML as a result of t(9;22)(q34;q11), leading to the formation of chimeric gene BCR/ABL encoding for proteins with abnormal tyrosine kinase activity. Cytogenetic variants of Ph chromosome can be identifed in 5 to 10% of CML patients, involving additional chromosomes other than 9 and 22. To explain the formation of variant translocations one-step, two-step and multi-step mechanisms have been proposed. Rarely, the variant Ph chromosome results from a BCR insertion on the ABL region and form a BCR/ABL fusion gene, generally mapping to 9q34, instead of the usual location at 22q11. In very few variant Ph cases, the insertion of the BCR/ABL product in a third chromosome was demonstrated. Case Report 28 year-old man, with bilateral central scotoma and gingivorragia. Physical examination: Grade 4 splenomegaly. Peripheral blood count showed hemoglobin concentration 11.5 g/dl, platelet count: 300.000/mm3, and white blood cell count 590.000/mm3. Blood smear: myelemia exhibiting 30% of myeloid blasts. Bone marrow biopsy: panmyelosis showing 20% of myeloid blasts. Cytogenetic analysis by G-banding performed in peripheral blood verified the following karyotype: 46, XY, t(9;22;10)(q34;q11;q24)[20] The analysis of the BCR-ABL fusion gene according to standard protocols detected the presence of the b3a2 isoform. Fluorescence in situ hybridization (FISH) studies using dual color dual fusion probes in metaphases showed a signal pattern 1F2G1R. The fusion signal mapped to 10q24, the red signal to 9q34, and the normal green signal to chromosome 22, while a second low intensity green signal mapped to the Ph chromosome. No signal was observed in der(9). Interphase FISH analysis in nuclei (n=200) presented the same signal pattern. Instead of using whole chromosome probes for 9 and 22, we hybridised probes used to detect DiGiorge syndrome. These probes detect gene control ARSA (spectrum green) localized at 22q13 and Tuple1 at 22q11 (spectrum orange). Two signals, green and orange were identified in normal chromosome 22. Ph chromosome showed the orange signal, whereas the green signal mapped to der(10). Discussion. The localization of the hybrid BCR/ABL gene on chromosomes other than 22q is a rare event wich can only be detected by FISH techniques. When these unusual translocation occurs, the hypothesis most often put forward is that several consecutive chromosome rearrangements have taken place. In the present case the interpretation of karyotypes, FISH data and molecular evidence lead to the following hypothesis: Insertion of the BCR sequence from chromosome 22 to chromosome 9 may have ocurred, producing a BCR/ABL fusion in der(9). The Ph chromosome detected by G-banding showed a different green fluorescence intensity in the metaphase FISH signal pattern with BCR/ABL dual color dual fusion probes, as a result of an insertion on chromosome 9. This first event was followed by the translocation between the derivative 9 and chromosome 10, being the final localization of the BCR/ABL gene in 10q24. FISH analysis using a DiGeorge syndrome probe, supports the hypothesis of a multistep mechanism underlying insertion and translocations events in the present case. The relocation of BCR/ABL fusion sequence on sites other than chromosme 22q11 represent a rare type of variant Ph translocation. At least 21 cases described in the literature, showed fusion gene BCR/ABL located at 9q24. Only 12 patients with variant Ph were reported bearing BCR/ABL on a third chromosome. All of them involved a masked Ph chromosome. To our best knowledge this is the first report showing a variant Ph chromosome detected by G-banding in a CML patient due to a BCR insertion on ABL sequences and exhibiting the fusion signal in a third chromosome. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Detection of the BCR-ABL Gene By the Real-Time PCR Method in Patients with Chronic Myeloid Leukemia in Rio Grande Do Norte, Brazil

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5432-5432
Author(s):  
Aldair Sousa Paiva ◽  
Hugo Diogenes De Oliveira Paiva ◽  
Geraldo Barroso Cavalcanti ◽  
Gioconda DR Leão ◽  
Marcos Dias Leão ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Real Time ◽  
Real Time Pcr ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Rna Extraction ◽  
Philadelphia Chromosome ◽  
Genetic Material ◽  
Chromosome 9 ◽  
The Real

Abstract Background: The Philadelphia chromosome is a cytogenetic change resulting from a reciprocal translocation of genetic material between ABL genes from chromosome 9 and BCR from chromosome 22 or t(9; 22) (q34; 11), forming the chimeric gene BCR- ABL, being associated with chronic myeloid leukemia (CML), acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML). The p190 variant is usually associated with acute forms of leukemia, including AML and ALL, whereas the p210 variant is associated with the chronic phases of CML. Due to the high sensitivity and specificity, nucleic acid amplification techniques by real-time PCR have replaced the conventional cytogenetic techniques for the identification of the Philadelphia chromosome and its p190 and p210 variants. Molecular analysis has been indicated in the initial diagnostic phase and also for the therapeutic monitoring defining the percentage of neoplastic cells present in the patients during the different phases of the treatment (Minimum Residual Disease or MRD).The aim of this study was the transcript BCR-ABL identification in patients with suspected of CML and evaluation of the gene frequency in these patients. Methods: The presence of BCR-ABL gene was investigated in blood samples from 42 patients with suspected CML. The RNA extraction was performed by phenol/chloroform method. The cDNA was submitted to PCR, using specific primers for and BCR-ABL genes by Real time PCR. Results: From all studied patients, 16 (38.10%) were negative, and 26 (59.09%) positive for one of rearrangements: p210 b3a2 and b2a2 in 18 cases (40.91%) and p190 a1a2 in 2 cases (4,76%) and double positive p120/190 in 6 cases (14,28%). We observed that the most common rearrangement was the p210 b3a2, and the molecular results were compatible with clinical and hematologic suspicion. Conclusions: The Real-timePCR, because of its specificity and sensitivity, can be considered the most used technique in routine diagnosis and investigation of MRD of CML patients. Disclosures No relevant conflicts of interest to declare.

Highly Sensitive Fluorescence In Situ Hybridization Method to Detect Double BCR/ABL Fusion and Monitor Response to Therapy in Chronic Myeloid Leukemia

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3357-3365 ◽  
Author(s):  
Gordon W. Dewald ◽  
William A. Wyatt ◽  
Amy L. Juneau ◽  
Richard O. Carlson ◽  
Alan R. Zinsmeister ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Chronic Myeloid Leukemia ◽  
Fluorescence In Situ Hybridization ◽  
Myeloid Leukemia ◽  
Philadelphia Chromosome ◽  
Response To Therapy ◽  
Chromosome 9 ◽  
Ph Chromosome ◽  
Cytogenetic Remission

Abstract We investigated a new method using fluorescence in situ hybridization and DNA probes that span the common breakpoints of t(9;22)(q34;q11.2) and that detect double BCR/ABL fusion (D-FISH) in bone marrow cells with this translocation, one on the abnormal chromosome 9 and one on the Philadelphia chromosome (Ph chromosome). D-FISH patterns were abnormal in 30 of 30 specimens with classic, simple, complex, and masked Ph chromosomes. Based on 200 nuclei from each of 30 normal specimens, the mean percentage of false-positive cells was 0.25 ± 0.39. Thirty-seven specimens from 10 patients were studied before treatment and two or more times at 4-month intervals after treatment with interferon-α2b (IFN-α2b) with or without ara-C. Based on 200 nuclei, the results of D-FISH in these specimens correlated closely with quantitative cytogenetics and accurately quantified disease within a few percent. We studied 6,000 nuclei for each of six specimens, three normal and three from patients with chronic myeloid leukemia (CML) in cytogenetic remission. The normal cutoff for 6,000 nuclei was 0.079% and patients in cytogenetic remission had residual disease ranging from 7 (0.117%) to 53 (0.883%) Ph-positive nuclei. We conclude that D-FISH can detect the Ph chromosome and its variant translocations and accurately quantify disease in CML at diagnosis and at all times after treatment, including cytogenetic remission.

Complex three-way Philadelphia translocation t(6;9;22) (p23;q34;q11.2) along with unusual t(1;4): A chronic myeloid leukemia case report

2018 ◽  
Vol 3 (2) ◽  
pp. 66
Author(s):  
M. Kumar ◽  
Bekleshwar Salona ◽  
Shiv Murti Kumar ◽  
Akshay Gore ◽  
Shivani Sharma
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
International System ◽  
Specific Region ◽  
Chromosome 9 ◽  
Fish Analysis ◽  
Standard Protocol ◽  
The Third ◽  
Ph Chromosome ◽  
Variant Ph Translocation

Chronic myeloid leukemia (CML) is a myeloproliferative disorder and genetically characterized by the presence of the Philadelphia (Ph) chromosome, resulting from a balanced reciprocal translocation between chromosome 9 and 22 at bands 9q34 and 22q11.2 [t(9;22)(q34;q11.2)]. In the formation of the Ph chromosome, the specific region of the ABL oncogene is transposed from 9q34 to the specific region of the BCR gene on chromosome 22 to form a fusion gene BCR‑ABL, which encodes a constitutively active protein, with tyrosine kinase activity. Vast majority of CML patients show classical Ph translocation, t(9;22) and 5–10% of cases are observed with variant Ph translocation. In variant Ph translocation, generally, the third chromosome involved with chromosome 9 and 22. The segment of the third chromosome usually translocates to the chromosome 9 at band 9q34. The formation of variant or complex Ph translocation is controversial topic. A wide array of additional chromosome involved in translocation with the t(9;22) has been described previously in CML patients. In this report, we present a unique and complex Ph translocation involving three chromosomes (6;9;22) in a 25-year-old female 66who was clinically diagnosed as CML. Chromosome analysis using 24 h and 48 h unstimulated culture with GTG-banding was performed according to standard protocol. A total of 20 metaphases analyzed from the bone marrow culture of the patient and karyotyping was performed on the basis of International System for Human Cytogenetic Nomenclature (2016). The FISH analysis was performed according to the standard protocol from sample for confirmation of BCR/ABL gene using dual color fusion FISH probe (Zytovision Probe). All cell’s images were captured using Olympus fluorescent microscope BX-61 equipped with a CCD camera and analyzed using Bioview FISH software.

A Study on t(9;22)(q34;q11) in Endothelial Cells in Chronic Myeloid Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4125-4125
Author(s):  
Liang Huang ◽  
Jingyi Wu ◽  
Miao Zheng ◽  
Mei Huang ◽  
Yicheng Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Chronic Myeloid Leukemia ◽  
Vascular Endothelium ◽  
Myeloid Leukemia ◽  
Magnetic Beads ◽  
Mononuclear Cells ◽  
Philadelphia Chromosome ◽  
Tissue Sections ◽  
Ph Chromosome

Abstract Chronic myeloid leukemia (CML) is a disease characterized by t(9;22)(q34;q11), the Philadelphia chromosome (Ph chromosome), and speculated to derive from the hemangioblast, which can give rise to both blood cells and endothelial cells. By using fluorescence in situ hybridization (FISH), we detected Ph chromosome in endothelial cells, which were separated with magnetic beads of CD105+, the marker for endothelial cells, from bone marrow mononuclear cells of 6 CML patients. Moreover, by using Fiction technique, a method combined immunofluorescence with FISH assay, we studied the translocation in vascular endothelium in hepatic and gastroenteric tissue sections and bone marrow trephines of another 7 CML patients. We showed that CD105+ endothelial cells of 6 CML patients with Ph chromosome were identified to harbor the same chromosome aberration as the leukemia cells, 69∼87% of bone marrow endothelial cells carried Ph chromosome. And Ph chromosome was identified in 21∼74% of microvascular endothelial cells in another 7 patients. These results confirmed that CML is derived from the hemangioblast, which can contribute to the maintenance of the blood vascular endothelium. Given specific cytotoxic T lymphocytes can be activated by the immunogenic bcr/abl peptide, we presume that the expression of bcr/abl on endothelial cells in CML patients may be one of the reasons for the incidence of chronic graft versus host disease (cGVHD) after bone marrow transplantation.

scholarly journals Highly Sensitive Fluorescence In Situ Hybridization Method to Detect Double BCR/ABL Fusion and Monitor Response to Therapy in Chronic Myeloid Leukemia

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3357-3365 ◽  
Author(s):  
Gordon W. Dewald ◽  
William A. Wyatt ◽  
Amy L. Juneau ◽  
Richard O. Carlson ◽  
Alan R. Zinsmeister ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Chronic Myeloid Leukemia ◽  
Fluorescence In Situ Hybridization ◽  
Myeloid Leukemia ◽  
Philadelphia Chromosome ◽  
Response To Therapy ◽  
Chromosome 9 ◽  
Ph Chromosome ◽  
Cytogenetic Remission

We investigated a new method using fluorescence in situ hybridization and DNA probes that span the common breakpoints of t(9;22)(q34;q11.2) and that detect double BCR/ABL fusion (D-FISH) in bone marrow cells with this translocation, one on the abnormal chromosome 9 and one on the Philadelphia chromosome (Ph chromosome). D-FISH patterns were abnormal in 30 of 30 specimens with classic, simple, complex, and masked Ph chromosomes. Based on 200 nuclei from each of 30 normal specimens, the mean percentage of false-positive cells was 0.25 ± 0.39. Thirty-seven specimens from 10 patients were studied before treatment and two or more times at 4-month intervals after treatment with interferon-α2b (IFN-α2b) with or without ara-C. Based on 200 nuclei, the results of D-FISH in these specimens correlated closely with quantitative cytogenetics and accurately quantified disease within a few percent. We studied 6,000 nuclei for each of six specimens, three normal and three from patients with chronic myeloid leukemia (CML) in cytogenetic remission. The normal cutoff for 6,000 nuclei was 0.079% and patients in cytogenetic remission had residual disease ranging from 7 (0.117%) to 53 (0.883%) Ph-positive nuclei. We conclude that D-FISH can detect the Ph chromosome and its variant translocations and accurately quantify disease in CML at diagnosis and at all times after treatment, including cytogenetic remission.

THE CASE VARIANT TRANSLOCATION T(3;9;22)(P24;Q34;Q11) IN CHRONIC MYELOID LEUKEMIA

2018 ◽  
Vol 64 (6) ◽  
pp. 810-814
Author(s):  
Kodirzhon Boboev ◽  
Yuliana Assesorova ◽  
Kh. Karimov ◽  
B. Allanazarova
Keyword(s):  
Adverse Effect ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Genetic Locus ◽  
Philadelphia Chromosome ◽  
Chromosome 9 ◽  
Banding Technique ◽  
Variant Translocation

This paper presents a case of chronic myeloid leukemia with an earlier unknown variant translocation t (3; 9; 22) (p24; q34; q11) detected by cytogenetic research using the GTG-banding technique. Despite the absence of the classical Philadelphia chromosome, the presence of chromosome 9 and 22 derivatives, as well as the BCR-ABL fusion gene, allow this translocation to be considered pathogenetic for CML. A good response of the patient to the treatment with glivec is that there is no adverse effect on the pathogenesis of the disease of an additional genetic locus (3p24) involved in complex restructuring.

Ph-Chromosome-positive chronic myeloid leukemia with associated bone marrow mastocytosis

2005 ◽  
Vol 29 (10) ◽  
pp. 1227-1232 ◽  
Author(s):  
Hermine Agis ◽  
Karl Sotlar ◽  
Peter Valent ◽  
Hans-Peter Horny
Keyword(s):  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Ph Chromosome

Targeting Chronic Myeloid Leukemia (CML) Stem Cells by BMS-214662 in Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2861-2861
Author(s):  
Cong Peng ◽  
Yiguo Hu ◽  
Francis Y. Lee ◽  
Shaoguang Li
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Cancer Cells ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Philadelphia Chromosome ◽  
Inhibitory Effect ◽  
Leukemia Stem Cells ◽  
Marrow Cells

Abstract The BCR-ABL inhibitor imatinib mesylate is the current approved treatment for Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML). While this agent is effective in the chronic phase of CML, it is less effective in advanced disease (acelerated phase or blast crisis), and resistance to imatinib is an issue at all stages of disease, particularly advanced. Resistance is mediated primarily by BCR-ABL mutations, although other mechanisms have also been implicated. Another key issue with imatinib therapy is that molecular remission in imatinib-treated CML patients is difficult to achieve, leaving patients at risk of relapse. We have previously observed that imatinib significantly prolongs survival of CML mice, but is not curative (Hu et al, Nature Genetics36[5]:453–461, 2004). We hypothesize that this can be attributed to the inability of imatinib to completely kill CML stem cells. We identified that BCR-ABL-expressing Lin-c-KIT+Sca-1+ bone marrow cells are CML stem cells in mice. We tested whether BMS-214662 (which has been shown to have an inhibitory effect on growth of non-proliferating cancer cells) (Lee et al, Proceedings of the AACR42:260s, 2001) reduces leukemia stem cell populations in CML mice. Donor bone marrow cells from C57BL/6 mice were transduced with P210BCR-ABL-IRES-GFP retrovirus, followed by transplantation into lethally irradiated C57BL/6 recipient mice. Eight days after transplantation, BMS-214662 was given orally once a day at a dose of 300 mg/kg for 7 days. Bone marrow cells from the treated CML mice were then analyzed by FACS for CML stem cells (GFP+Lin-c-Kit+Sca-1+). CML mice treated with placebo, dasatinib (a novel, oral, multi-targeted kinase inhibitor that targets BCR-ABL and SRC family kinases) 10 mg/kg, twice daily (BID), BMS-214662, or dasatinib 10 mg/kg BID in combination with BMS-214662. Numbers of leukemia stem cells per bone were significantly lower in mice treated with BMS-214662 alone, dasatinib alone, or both BMS-214662 and dasatinib, compared with placebo-treated mice. Among different treatments, the combination of BMS-214662 and dasatinib had the strongest inhibitory effect on CML stem cells. Inhibition of the leukemia stem cells by dasatinib could be due to its inhibitory effect on BCR-ABL or SRC kinases, whereas BMS-214662 must function through other mechanisms. BMS-214662 is also a farnesyl transferase inhibitor (FTI), which reduces Ras activation. However, our control experiment showed that other FTIs did not inhibit proliferation of non-proliferating cancer cells (data not shown). This suggests that BMS-214662 inhibits CML stem cells through unknown mechanisms. In summary, BMS-214662 is a potent inhibitor of CML stem cells, and combinatorial use of BMS-214662 and dasatinib may provide more durable responses, and potentially a curative therapy for CML patients. Given the proven activity of dasatinib against a spectrum of imatinib-resistant BCR-ABL mutations (O’Hare, et al. Cancer Res65[11]:4500–5, 2005; Shah et al, Science, 305:399, 2004), and the apparent activity of dasatinib against stem cells in vivo shown here, this combination could potentially suppress the emergence of resistance, further adding to the durability of response.

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