Cohort Of Chronic Myeloid Leukemia Patients Research Articles

Single Cell Analysis of Genes Involved in Asymmetric Hematopoietic Stem Cell (HSC) Division in Chronic Myeloid Leukemia (CML) : Evidence of the Regulation of Tetraspanin CD63 during Leukemia Progression

Tyrosine kinase inhibitor (TKI) therapies have profoundly changed the natural history and survival of CML patients. However, the majority of the patients remain on long-lasting therapies and switch one TKI to another, either because of intolerance or resistance. All TKI cessation trials showed approximately 50% of leukemia recurrence rates even after several years of TKI therapies, suggesting the persistence of residual leukemic stem cells in patients in deep molecular response (DMR). The resistance and persistence of CML HSC are related to intrinsic (BCR-ABL independent survival) or extrinsic (leukemic niche ) mechanisms some of which have been extensively studied. However, there has been no data with regard to the role of asymmetric cell division pathways in the persistence and progression of CML stem cell clone. Indeed, During asymmetric division process of hematopoietic stem cells, some proteins are differently segregated between daughter HSCs such as endosome associated tetraspanin proteins (CD53, CD63) and mitochondria-associated fission regulator Dynamin related protein-1(DRP1). Similarly, it has been shown that asymmetric organelle inheritance can influence HSC decisions towards differentiation versus self-renewal. Interestingly, the tetraspanin CD63 is known to be involved in the quiescence of human and murine HSC via activation of TGF-beta pathway, CD63 hi cells exhibiting increased self-renewal potential after the asymmetric division with reduced differentiation potential. Materials and Methods We have analyzed the transcriptome performed in a cohort of CML patients in chronic phase (CP) and blast phase (BP) (GSE4170) as well as in a validation cohort of RNA-sequencing (GSE100026). Similarly, single cell transcriptome analysis of Lin- CD34+ CD38- CML progenitors (GSE76312) were included in the study evaluating the expression of genes involved in asymmetric cell division. We have then generated an asymmetric hematopoietic stem cell risk score using a panel of genes involved in the asymmetric division of HSCs and correlated these results with the transcriptome of the CML cohorts above. A pseudotime analysis was performed to evaluate single cell trajectories of the genes identified. Results To determine the potential role of asymmetric cell division markers in the progression of CML, we have analyzed the expression of several of these genes during CML progression in two independent transcriptome cohorts. The regulation concordance between the two distinct cohorts was principally observed on asymmetric molecules which were found to be down-regulated during blast crisis as compared to chronic phase. The regulation concordance between the two distinct cohorts was principally observed on asymmetric molecules down regulated during blast crisis as compared to chronic phase suggesting less of asymmetric properties during advanced phase of the disease. The down regulation in patients in blast crisis was confirmed at single cell level for the expression of SELL, CD63, NUMB, HK2 and LAMP2. CD63 expression has also been found to be of interest in CML chronic phase. Indeed, single cell neural network and deep learning in CP samples showed that the CD63 expression was the best asymmetric gene marker predicting a poor prognosis and this appeared to be induced by opposite regulation of HK2 and NUMB. In patients in CP-CML, the single cell trajectory reconstitution revealed the major regulatory role of CD63 highlighting a trajectory cluster implicating HSPB1, PIM2, ANXA5, LAMTOR1, CFL1, CD52, RAD52, MEIS1, PDIA3, all genes known to be involved also in hematopoietic malignancies such as myelodysplasia, AML and ALL. Conclusion We show here for the first time the identification of the expression of genes involved in the asymmetric division of CML HSC. Among these genes we identified the tetraspanin CD63 as being down-regulated during CML progression. The expression of the genes involved in the asymmetric HSC division should require further studies in CML, either to predict persistence of CML or progressive disease. Figure 1 : Single cell transcriptome of Lin- CD34+ CD38- CML progenitors in CP links CD63 expression to progressive disease. A/ Piechart of counts for chronic phase cells analyzed according to prognosis; B/ pseudotime transformation of single cell transcriptome according patient prognosis; C/ Pseudotime expression heatmap; D/ Pseudotime expression of CD63 in CP.

ENOX2 NADH Oxidase: A BCR-ABL1-dependent Cell Surface and Secreted Redox Protein in Chronic Myeloid Leukemia (CML).

Chronic myeloid leukemia (CML) is a disease caused by the acquisition of BCR-ABL1 fusion in a hematopoietic stem cell. In this study, we focused on the oncofoetal ENOX2 protein as a potential secretable biomarker in CML. We used cell culture, Western blot, quantitative RT-PCR, ELISA, transcriptome analyses and bioinformatics technics to investigate ENOX2 mRNA and protein expression. Western-blot analyses on UT-7 and TET-inducible Ba/F3 cell lines demonstrated the up-regulation of ENOX2 protein. BCR-ABL1 has been found to induce ENOX2 overexpression in a kinase-dependent manner. We confirmed increased ENOX2 mRNA expression on a cohort of CML patients at diagnosis. In a series of CML patients, ELISA assays showed a highly significant increase of ENOX2 protein levels in the plasma of patients with CML compared to controls. Transcriptomic dataset reanalyzes confirmed ENOX2 mRNA overexpression in the chronic phase of the disease. Bioinformatic analyses identified several genes whose mRNA expression was positively correlated to ENOX2 in a BCR-ABL1 context. Some of them encode proteins involved in cellular functions compatible with the growth deregulation observed in CML. Our results highlight the upregulation of a secreted Redox protein in a BCR-ABL1-dependent manner in CML. Our data suggest that ENOX2, through its transcriptional program, plays a significant role in BCR-ABL1 leukemogenesis.

MANAGEMENT OF PATIENTS WITH CHRONIC MYELOID LEUKEMIA – AN ACTUAL ISSUE OF ONCOLOGY AND PUBLIC HEALTH

Objectives: The aims of the study were to identify and evaluate the epidemiological aspects and management options in chronic myeloid leukemia (CML). Methods: We performed the analytical, cohort and prospective-retrospective study of 134 patients with CML, who were treated and followed up in the Institute of Oncology from Moldova between 2005–2021. The quantitative reverse transcription polymerase chain reaction (RT-PCR) was accomplished in order to determine the expression of the BCR-ABL p210 and p190 transcripts. Tyrosine kinase inhibitors (TKIs) were used as a front-line therapy in the newly diagnosed CML patients and in the cases of resistance to non-TKIs chemotherapy and interferon-?. Results: The diagnosis of CML was established in chronic phase in 122 (91,04 ± 2,32%) patients, in the accelerated and acute phases – in 12 (8,96 ± 2,03%). BCR-ABL p210 transcript range was 21.84–100% IS. Under the first-line TKIs therapy, the complete molecular response was obtained in 24.3% of cases. No significant differences were registered in the rate of complete hematologic response (92.3% vs 92.8%) and complete molecular response (23.8% vs 24.7%) under the TKIs medication between the elderly patients and the whole cohort of CML patients. The overall one- and 5-year survival in elderly patients treated with TKI was 97.6 and 79%, being comparable with the respective parameters in the totality of CML patients (98.5% and 87%, correspondingly). Conclusions: The treatment with TKIs remains a curative option of choice for CML patients regardless of the age, gender and CML phase.

Between a Rock and a Hard Place: Paraproteinemia Associated with Chronic Myeloid Leukemia

Introduction: Though originating from different cell origins, several reports have described patients with the coexistence of chronic myeloid leukemia (CML) and paraproteinemias, mostly in patients treated with tyrosine kinase inhibitors (TKIs).Objective: To determine whether the prevalence of paraproteinemia in a large cohort of chronic phase (CP-) CML patients is higher than expected in the general population.Methods: We conducted a cross-sectional study among out-patient CP-CML patients treated in a tertiary medical center. Between February 2019 and May 2021 consecutive CP-CML patients who consented to the study, performed the following tests: quantitative immunoglobulin (Ig) levels, serum-free light chains (sFLC), serum protein electrophoresis (SPEP), and if abnormal also serum protein immunofixation (IFIX). The primary outcome was the prevalence of paraproteinemia among this cohort of CML patients. We conducted further analyses to evaluate whether various patient, disease and treatment-related variables are predictors of paraproteinemia within our cohort.Results: One hundred patients, 50% males, with a median age of 63.5 (range 26-91.6) years were recruited for the study. The median time from CML diagnosis to enrollment was 6.3 (range 0.1-23.5) years. Cohort's characteristics are depicted in table 1.Monoclonal (M-) protein was detected on SPEP and IFIX in eight patients (8%), whose median age was 66.8 (range 46-74) years. Among them, two patients had an abnormal sFLC, elevated IgG or IgA levels, on a bone marrow biopsy over 10% monoclonal plasma cells were present, and in the absence of myeloma defining events they were diagnosed with IgG-kappa and IgA-kappa smoldering multiple myeloma (SMM), respectively. The other six patients had normal sFLC ratio and characteristics compatible with low-risk monoclonal gammopathy of undetermined significance (MGUS). Six patients with a detectable M-protein were treated with a TKI [imatinib (n=2), dasatinib (n=3), bosutinib (n=1)] and two were in treatment-free remission (TFR). All continue observation for their paraproteinemia.None of the patients in our cohort was diagnosed with active MM. The presence of anemia was predictive of paraproteinemia, albeit with borderline statistical significance in both univariate (p=0.053) and multivariate analysis (p=0.069). Apart from anemia, no predictors for paraproteinemia were found among various other patient and disease related variables.Conclusions: This is the largest study describing the prevalence of paraproteinemia among CML patients. We found the prevalence of MGUS in CP-CML patients (6%) to be higher than in the general population above the age of 50 (3.2%), and a non-negligible prevalence of SMM (2%). Based on these data, we recommend considering screening for paraproteinemia coexistence in CML patients. Further studies and extended follow-up are needed to elucidate the true clinical significance of our findings. [Display omitted] DisclosuresLeader: Bayer: Honoraria; Leo Pharma: Honoraria; Novartis: Honoraria; Pfizer: Consultancy, Honoraria; Sanofi: Honoraria.

CML-102: Is Paraproteinemia More Prevalent in Patients with Chronic Myeloid Leukemia?

Context: Though originating from different cell origins, several reports have described patients with the coexistence of chronic myeloid leukemia (CML) and paraproteinemias, mostly in patients treated with tyrosine kinase inhibitors (TKIs). Objective: Determine whether the prevalence of paraproteinemia in a large cohort of chronic phase (CP-) CML patients is higher than in the general population. Methods: This is a cross-sectional study among ambulatory CP-CML patients treated in a tertiary medical center. Between February 2019 and May 2021, consecutive CP-CML patients who consented to the study performed the following tests: serum protein electrophoresis & immunofixation (SPEP, IFIX), serum-free light chains (sFLC), and quantitative immunoglobulin (Ig) levels. Primary outcome was the prevalence of paraproteinemia among this cohort of CML patients. Secondary outcomes evaluated the correlations between paraproteinemia and various patient, disease, and treatment-related variables. Results: 100 patients, half males, with a median age of 63.5 years (range 26-91.6) were recruited for the study. The median time from CML diagnosis to enrollment was 6.3 years (range 0.1-23.5). Monoclonal (M-) protein was detected on SPEP and IFIX in 8 patients (8%), whose median age was 66.8 years (range 46-74). Among them, two patients also had an abnormal sFLC and elevated IgG or IgA levels and were diagnosed with IgG-kappa and IgA-kappa smoldering multiple myeloma (SMM), respectively. The other six patients had normal sFLCs and characteristics compatible with low-risk monoclonal gammopathy of undetermined significance (MGUS). Six patients with a detectable M-protein were treated with a TKI [imatinib (n=2), dasatinib (n=3), bosutinib (n=1)] and two were in treatment-free remission (TFR). All continue observation for their newly diagnosed paraproteinemia. None of the patients in our cohort was diagnosed with active MM. Conclusions: This is the largest study describing the prevalence of paraproteinemia among CML patients. We found the prevalence of MGUS in CP-CML patients (6%) to be higher than in the general population above the age of 50 (3.2%) and a non-negligible prevalence of SMM (2%). Based on these data, we recommend considering screening for paraproteinemia coexistence in CML patients. Further studies and extended follow-up are needed to elucidate the true clinical significance of our findings.

Характеристика мутацій кіназного домену гена BCR/ABL1 у пацієнтів з хронічною мієлоїдною лейкемією, первинно резистентних до терапії іматинібом

The chronic myeloid leukemia (CML) development is associated with the formation of the BCR/ABL1 fusion gene and the BCR/ABL1 protein with increased tyrosine kinase activity. Despite the high efficiency of targeted therapy, up to 30% of patients do not respond on such therapy i.e. are primary resistant. The presence of BCR/ABL1 kinase domain mutations is considered to be one of the reasons of tyrosin kinase inhibitors resistance. To evaluate the frequency of BCR/ABL1 kinase domain mutations in Ukrainian cohort of CML patients with primary resistance to imatinib therapy, we retrospectively studied BCR/ABL1 kinase domain mutations in peripheral blood of 107 CML patients. The nucleotide sequence was determined by direct sequencing by Sanger. Mutations were reported in 45 of 107 (41.7%) CML patients. Two mutations at a time were revealed in 8 patients. So a total of 53 mutations were found out. Among them 49 were missense-mutations and 4 - deletions of different regions of the BCR/ABL1 kinase domain gene. The missense-mutations F359I/V (12 patients), T315I (8 patients) and G250E (6 patients) were most common. By localization, the mutations majority (23 of 53) was in the P-loop, 10 mutations - in the contact site, 13 mutations - in the catalytic domain and 6 – in the A-loop. Of the detected mutations, 26 (49%) resulted in a disruption of the hydrogen bond between BCR/ABL1-tyrosine kinase and imatinib. Significant reduction in overall survival was found in patients with BCR/ABL1 kinase domain mutations compared with patients with wild-type of BCR/ABL1 gene (p=0.018). The estimated 3-year overall survival was 83.4% (95% CI: 77.0%-89.8%) and 94.3% (95% CI: 91.0%-97.3%), respectively. Therefore, mutations of the BCR/ABL1 kinase domain are one of the mechanisms of primary resistance in CML patients on imatinib therapy. The occurrence of BCR/ABL1 gene mutations impairs the prognosis of imatinib therapy response.

ETS1 Expression Is Upregulated in Chronic Myeloid Leukemia (CML) and the ETS1 Transcriptional Program Correlates with Disease Progression Towards Blast Crisis

Chronic myeloid leukemia is a clonal myeloproliferative neoplasm characterized by the occurrence of the Ph1 chromosome in a primitive hematopoietic stem cell with a major amplification of myeloid compartment. Despite the major progress obtained by the use of tyrosine kinase inhibitors (TKI), resistances to these drugs occur with progression towards blast crisis (BC) during which there is an increase of BCR-ABL expression. To evaluate the potential targets of BCR-ABL in this context, a gene profiling analysis of the UT7-11 cell line overexpressing BCR-ABL fusion protein was performed using a whole transcriptome microarray. One of the highly upregulated genes was ETS1 with a fold change of +35.86. ETS1 proto-oncogene, the founder member of the ETS-domain family of transcription factors such as TEL and FLI1, is the human homolog of the avian erythroblastosis virus E26. They play a crucial role in stem cell biology, tumorigenesis and ETS1 has been shown to be involved in the regulation of granulocytic differentiation. Its role in CML pathophysiology has not been studied so far. We first showed by Western blots that ETS1 protein was highly increased in UT7-11 cells as compared to parental UT7 cells. Using a DOX-Inducible BCR-ABL system, we have shown that ETS1 expression was downregulated upon inhibition of BCR-ABL expression. ETS1 expression was a tyrosine kinase dependent event as its expression was reduced in the presence of TKI. To determine if ETS1 expression is upregulated in primary leukemic cells, we have analyzed leukemic cells of CML patients at diagnosis (n= 40) as compared to healthy controls (n= 30) showing an increase of ETS1 mRNA expression in primary CML cells in a highly significant manner (p < 0.0007). We then analyzed ETS1 targets using chip-sequencing in K562 cells, performed on HG19 human genome allowing to predict 3209 proximal promoters (-3000 upstream pb, + 50pb around Transcription Starting Sites) which represents a promoter enrichment of +33.68 (p-value=4.9E-324) as compared to the distribution of peaks in the whole genome. Chromosomes 19, 16, 17, 11, 12 and 1 were found over-represented with ETS1 bound events in Chip-sequencing. Integration of BCR-ABL transcriptome with ETS1 promoters identified by Chip-Sequencing led to the identification of 130 ETS1-targets activated by BCR-ABL, allowing reclassification of BCR-ABL transfected samples with transcriptome data by unsupervised classification. ETS1 transcriptional program regulated by BCR-ABL analysis performed on CD34+ from CML patients during progression of the disease (GSE47927) allowed discrimination of the different states of the progression (from the chronic phase to accelerated phase and BC p-value=1.63E-33). This analysis was also found significant by Gene Set Enrichment Analysis ETS1_BC specific geneset (NES=+1.52, p-value<0.0001). Among this ETS1 program specific of BC, we identified 2 targets that are discriminant for CD34+ cells of CML patients without cytogenetic response under Imatinib therapy. These two genes were Dynamin 3 (DNM3) and LIMS1/ PINCH1. Dynamin 3 is a member of the motor proteins implicated in cell motility and cytokinesis and it is implicated in megakaryocyte development whereas LIMS1 / PINCH1 is essentially involved in cell migration and adhesion. In order to validate ETS1 and its target expressions in a novel cohort of CML patients, we have analyzed the expression of DNM3 and PINCH1 in whole blood samples in CML patients ( n= 60 ) as compared to healthy controls (n= 32). We have found that, similar to ETS1, DNM3 and LIMS1 / PINCH1 mRNA were highly upregulated in primary CML and this increase was highly significant. In this new cohort of patients, DNM3 transcript levels were inversely correlated to WBC count (r=-0.32, p-value=0.011) and especially in male gender (r=-0.43, p-value=0.012). The expression of LIMS1 was inversely correlated with the age at diagnosis and the major molecular response at 12 months. Thus, our results show for the first time the major upregulation of ETS1 transcriptional program in CML. ETS1 transcriptional program has been found to correlate with the gene expression pattern involved in the progression of the disease in the transcriptomic analysis of CML CD34+ cells. The novel druggable targets that we identified in the BCR-ABL-activated transcriptional program is currently under investigation to determine their use in patients with resistance to targeted therapies. DisclosuresNo relevant conflicts of interest to declare.

Pattern of chronic myeloid leukemia in the imatinib era in a Sub-Saharan African setting

Chronic myeloid leukemia (CML) is an orphan disease in Africa because of the inaccessibility to specific treatment and the high cost of diagnosis and monitoring patients. The aim of this study was to report CML treatment response in a developing country in the tyrosine kinase inhibitor era. We conducted a longitudinal study of our cohort of CML patients. Socio-demographic, diagnosis, therapeutic, and treatment response parameters were studied. Sokal score, disease phase at diagnosis, delay from diagnosis to treatment, and treatment response were analyzed for their impact on survival. Fifty-five patients with a diagnosis of CML and who received treatment with imatinib for a minimum of 3months were included in this study. Median follow-up was 170 patient-years. The sex ratio (M/F) was 1.62 and median age at diagnosis was 42years. At diagnosis, 85.5% of the patients were in chronic phase (CP), 12.7% in accelerated phase (AP), and 1.8% in blast crisis (BC). Sokal risk score distribution was as follows: low risk 29.8%, intermediate risk 38.3%, and high risk 31.9%. Median time from first symptoms to first medical visit was 6.2months and median time from first medical visit to cytogenetic and or molecular confirmation was 12.4months. Mean delay time from first medical visit to imatinib initiation was 12.5months (95% CI 6.3-18.7). The complete hematologic response (CHR) at 3months, the major cytogenetic response (MCR) at 12months, and the major molecular response (MMR) at 24months were respectively 82.4, 75, and 25%. The 2-year overall survival rate was 81%. Advanced phase at the diagnosis, discontinuation of imatinib therapy over 15% of the time, lack of CHR at 3months, lack of MCR at 12months, and progression of the disease during imatinib therapy were associated with a risk of death (p ≤ 0.05). Our data confirm the improved prognosis of CML treated with imatinib in the setting of a developing country. However, response rates are lower than in developed countries, and additional efforts should be made to facilitate early diagnosis and improve access to TKI, treatment compliance, and regular molecular monitoring of patients.

Is Adherence to Imatinib Mesylate Treatment Among Patients with Chronic Myeloid Leukemia Associated with Better Clinical Outcomes in Qatar?

BACKGROUNDDespite the revolutionary success of introducing tyrosine kinase inhibitors (TKIs), such as imatinib mesylate (IM), for treating chronic myeloid leukemia (CML), a substantial proportion of patients’ treatments fail.AIMThis study investigates the correlation between patient adherence and failure of TKIs’ treatment in a follow-up study.METHODSThis is a follow-up study of a new cohort of CML patients. Adherence to IM is assessed using the Medication Event Monitoring System (MEMS 6 TrackCap, AARDEX Ltd). The 9-item Morisky Medication Adherence Scale, medication possession ratio (MPR) calculation, and the electronic medical records are used for identifying potential factors that influence adherence. Clinical outcomes are assessed according to the European Leukemia Net 2013 guidelines via reverse transcriptase quantitative polymerase chain reaction measurement of the level of BCR-ABL1 transcripts in peripheral blood. Response is classified at the hematological, cytogenetic, and molecular levels into optimal, suboptimal, or failure.RESULTSA total of 36 CML patients (5 citizens and 31 noncitizen residents) consented to participate in the study. The overall mean MEMS score was 89. Of the 36 patients, 22 (61%) were classified as adherent (mean: 95) and 14 (39%) were classified as nonadherent (mean: 80.2). Adherent patients were significantly more likely to obtain optimal response (95%) compared to the nonadherent group (14.3%; P < 0.0001). The rate of poor adherence was as high as 39% using MEMS, which correlates with 37% treatment failure rate. The survey results show that 97% of patients increased the IM dose by themselves when they felt unwell and 31% of them took the missing IM dose when they remembered. Other factors known to influence adherence show that half of patients developed one or more side effects, 65% of patients experienced lack of funds, 13% of patients declared unavailability of the drug in the NCCCR pharmacy, and 72% of patients believed that IM would cure the disease. The MPR results reveal that 16% of patients had poor access to treatment through the hospital pharmacy.DISCUSSION AND CONCLUSIONThis is the first prospective study to evaluate CML patients’ adherence and response to IM in Qatar. The high rate of treatment failure observed in Qatar is explained by poor adherence. An economic factor (unaffordable drug prices) is one of the main causes of nonadherence and efforts should be made locally to improve access to medication for cancer diseases. Other risk factors associated with poor adherence could be improved by close monitoring and dose adjustment. Monitoring risk factors for poor adherence and patient education that include direct communication between the health-care teams, doctors, nurses, pharmacists, and patients are essential components for maximizing the benefits of TKI therapy and could rectify this problem. The preliminary results show that patients’ response to treatment may be directly linked to patients’ adherence to treatment. However, further in-depth and specific analysis may be necessary in a larger cohort.

Transient presence of clonal chromosomal aberrations in Ph-negative cells in patients with chronic myeloid leukemia remaining in deep molecular response on tyrosine kinase inhibitor treatment

Advancements in treatment of chronic myeloid leukemia (CML) turned this formerly fatal neoplasm into a manageable chronic condition. Therapy with tyrosine kinase inhibitors (TKIs) often leads to significant reduction of disease burden, known as the deep molecular response (DMR). Herein, we decided to analyze the cohort of CML patients treated in our center with TKIs, who obtain and retain DMR for a period longer than 24 months. The aim of the study was to evaluate the frequency of clonal cytogenetic aberrations in Philadelphia-negative (Ph-) cells in patients with DMR during TKI treatment. The analyzed data was obtained during routine molecular and cytogenetic treatment monitoring, using G-banded trypsin and Giemsa stain (GTG) karyotyping and reverse transcription quantitative PCR. We noticed that approximately 50% of patients (28 of 55) in DMR had, at some follow-up point, transient changes in the karyotype of their Ph- bone marrow cells. In 9.1% of cases (5 of 55), the presence of the same aberrations was observed at different time points. The most frequently appearing aberrations were monosomies of chromosomes 19, 20, 21, and Y. Statistical analysis suggests that the occurrence of such abnormalities in CML patients correlates with the TKI treatment time.

Studying the impact of presence of point mutation, insertion mutation and additional chromosomal abnormalities in chronic myeloid leukemia patients treated with imatinib mesylate in the State of Qatar

Background: Imatinib is failing as a first line treatment in more than 40% of chronic myeloid leukemia (CML) patients in Qatar. We thus investigated ABL1 kinase domain mutations and additional chromosomal abnormalities (ACAs) as underlying mechanisms to explain this high rate of treatment failure. Methods: Between November 2006 and December 2011, all CML patients in Qatar were studied for BCR-ABL1 kinase domain mutations and ACAs. Total RNA was extracted and cDNA was produced via reverse transcriptase polymerase chain reaction (RT-PCR). PCR was used with special precautions to avoid amplification of wild type ABL1; the ABL1 kinase domain was then screened for mutations by direct DNA sequencing technology to detect the emergence of mutant clone. Cytogenetic analysis of bone marrow (BM) metaphases and fluorescence in situ hybridization (FISH) of peripheral blood (PB) and BM interphases were performed according to standard protocols. European Leukemia Net (ELN) response criteria were employed to identify the failing cases. Results: 26 out of 33 CML patients were eligible for the study, 22 CP and 4 AP. 14 failed Imatinib treatment, 2 had BCR-ABL1 kinase domain mutations; one patient had the G1739A mutation which leads to the exchange of glutamic acid at position 459 to lysine (E459K) (rs1064156) in the c-terminal loop while the other patient had a unique insertion of three nucleotides (AAG) at position 1432 which adds an amino acid Lysine to position 356 of the catalytic domain and a complex karyotyping at diagnosis, 6 had additional chromosomal abnormalities as an underlying mechanism of resistance, 4 patients had no identifiable cause of resistance and 2 patients were intolerant to treatment. There was a significant difference in median overall survival between patients with Ph chromosome only and patients with ACAs. Conclusions: In this study as we continued observing CML patients for nearly 5 years, the Imatinib failure reached as high as 54%. The resistance rate observed in Qatar is still higher than that reported by the IRIS study which is as high as 35%. In our cohort of CML patients, point mutation and unique tri-nucleotide insertions were identified. However, these mutations could explain only 14% of treatment failure. Additional chromosomal abnormalities were the most common cause of Imatinib failure in our patients' cohort and were documented in 50% of cases. 14% of patients stopped IM due to intolerance; and the mechanisms of resistance remained unknown in 28% of patients. Other causes such as patients' adherence to Imatitinb is being prospectively investigated.

Clonal chromosomal abnormalities in CD34+/CD38− hematopoietic cells from cytogenetically normal chronic myeloid leukemia patients with a complete cytogenetic response to tyrosine kinase inhibitors

Letter to the Editor Clonal chromosomal abnormalities in Ph-negative cells (CCA/Ph-) have been identified in 3-15% of chronic myeloid leukemia (CML) patients with a partial or complete cytogenetic response (CCyR) to imatinib(1). Trisomy 8 and deletions of chromosome 7 [either monosomy 7 or del 7(q)] account for the majority of cases, although a range of other karyotypic changes were seen at lower frequency. A recent study reported CCA/Ph- in patients on dasatinib, indicating that the phenomenon is not limited to imatinib therapy. In some patients, CCA/Ph is associated with a myelodysplastic syndrome (MDS), and progression to acute myeloid leukemia has been reported(1, 2). A single center report suggested inferior survival in newly diagnosed patients who develop CCA/Ph-(3). However, a larger study found that CCA/Ph- does not adversely affect the prognosis of patients with a major cytogenetic response (MCyR) to imatinib, indicating that this is a mostly benign condition(4). The reported incidence of CCA/Ph- is based on metaphase karyotyping, which is limited by the small number of cells analyzed and by the fact that only cells are assayed that can be induced to divide within the culture period. We thus hypothesized that conventional karyotyoping may underestimate the incidence of CCA/Ph- and decided to screen CD34+/CD38− cells from a cohort of CML patients with a CCyR to tyrosine kinase inhibitor (TKI) therapy for abnormalities of chromosomes 7 and 8. This primitive cell compartment is known to be enriched for hematopoietic progenitor and stem cells(5). We find CCA/Ph- in CD34+/CD38− cells from 4/19 patients, suggesting CCA/Ph- is more common than previously appreciated.

Economic impact of mortality risk reduction with first-line imatinib (IM) versus interferon-α (IFN) in patients newly diagnosed with chronic phase (CP) chronic myeloid leukemia (CML) under the Brazilian public healthcare system perspective over a 5-year period

17068 Background: Patients receiving imatinib had a significant survival advantage compared with patients receiving IFN therapy (Roy et al. Blood 2006; Kantarjian et al. Blood 2006). Although reimbursed as second line therapy for CP CML patients who did not respond to INF-a, IM was not considered for public reimbursement as first line treatment in Brazil based on drug costs. An economic evaluation considering the mortality risk reduction with first line IM versus INF was performed under the Brazilian Public Healthcare System perspective, comparing the costs to avoid one death of a chronic phase CML patient over a 5-year period. Methods: Risk of death in 5 years was defined by survival rates in the 60 month follow-up of IRIS for IM (Rim= 10%) (Druker et al. JCO 2006), MD Anderson cohort of CML patients treated with INF-a for INF-a (RINF-a= 38%) (Cortes et al. American J. Med 1996) and a population based-survey in Norway for the natural course of the disease (Rnon-treatment= 67%). IRIS INF group survival rate was not used due to high crossover to IM in the trial. Risk of death in the natural course of the disease was the basis for the absolute risk reduction (ARR) calculation (ARRim= 57%; ARRINF-a= 29%). Number need to treat (NNT) was obtained by the inverse of ARR value for each first line treatment (NNT =1/ARR). Costs were estimated based on the Brazilian public healthcare reimbursement payment (APAC-SUS) for chronic phase CML treatment. A base case economic model of 100 patients for each treatment option was constructed focused on drug costs and adverse events (AE) from the IRIS study for both groups. Clinical guidelines from two public hematology centers were used to estimate AE treatment costs. Costs were discounted at a 6% annual rate. Results: The treatment costs and NNTs for a 5-year period are US$ 62,135 and 3.45 for INF-a, and US$ 93,397 and 1.75 for IM, respectively. The costs to avoid one death over a 5-year period are US$ 214,368 for INF-a and US$ 163,445 for IM. Conclusions: The costs for avoiding one death of a chronic phase CML patient over a 5-year period is US$ 50,923 higher with first line INF-a than it would be with first line imatinib. [Table: see text]