Chronic Myelogenous Leukemia Patients Research Articles

Novel combined Ato-C treatment synergistically suppresses proliferation of Bcr-Abl-positive leukemic cells in vitro and in vivo

Chronic myelogenous leukemia (CML) accounts for 15–20% of all leukemias affecting adults. Despite recent advances in the development of specific Bcr-Abl tyrosine kinase inhibitors (TKIs), some CML patients suffer from relapse due to TKI resistance. Here, we assessed the efficacy of a novel combinatorial arsenic trioxide (ATO) and cisplatin (CDDP) treatment (Ato-C) in human Bcr-Abl-positive leukemic cells. Combination index analyses revealed that a synergistic interaction of ATO and CDDP elicits a wide range of effects in K562, KU-812, MEG-A2, and KCL-22 cells. Notably, Ato-C synergistically enhanced apoptosis and decreased the survival of both acquired TKI-resistant CML cells and the cells expressing mutant Bcr-AblT315I. In addition, Ato-C dramatically decreased the phosphorylation level of forkhead transcription factor FOXO1/3a and STAT5 as well as c-Myc protein level. Interestingly, results of gene set enrichment analysis showed that Ato-C significantly downregulates the expression of MYC- and/or E2F1-target genes. Furthermore, Ato-C significantly suppressed the proliferation of MEG-A2-derived tumor when compared with that following monotherapy in vivo. Collectively, these results suggest that combined Ato-C treatment could be a promising alternative to the current therapeutic regime in CML.

Alkynylnicotinamide-Based Compounds as ABL1 Inhibitors with Potent Activities against Drug-Resistant CML Harboring ABL1(T315I) Mutant Kinase.

The introduction of imatinib into the clinical scene revolutionized the treatment of chronic myelogenous leukemia (CML). The overall eight-year survival rate for CML has increased from about 6 % in the 1970s to over 90 % in the imatinib era. However, about 20 % of CML patients harbor primary or acquired resistance to tyrosine kinase inhibitors. ABL1 point mutations in the BCR-ABL1 fusion protein, such as ABL1(T315I), typically emerge after prolonged kinase inhibitor treatment. Ponatinib (AP24534) is currently the only approved CML drug that is active against the ABL1(T315I) mutation. However, ponatinib has severe cardiovascular toxicities; hence, there have been efforts to find safer CML drugs that work against ABL1 secondary mutations. We reveal that isoquinoline- or naphthyridine-based compounds, such as HSN431, HSN576, HSN459, and HSN608 potently inhibit the enzymatic activities of ABL1, ABL1(T315I), and ABL1(E255K). These compounds inhibit the proliferation of ABL1-driven CML cell lines, K652 and KCL22 as well as the drug-resistant cell line, KCL22-IR, which harbors the secondary mutated ABL1(T315I) kinase.

Coexistence of JAK2 or CALR mutation is a rare but clinically important event in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors.

There are 7 designated conditions under the category of myeloproliferative neoplasms (MPN), including chronic myelogenous leukemia (CML) and classical MPN, that is, polycythemia vera (PV), essential thrombocythaemia (ET), and primary myelofibrosis (PMF). Recently, reports about Philadelphia and JAK2 V617F-positive MPN cases have been described in literature. The coexistence of different molecular defects may change the clinical and laboratory manifestation of MPN and may result in an inappropriate interpretation of the response to treatment with tyrosine kinase inhibitors in CML patients. The morphological, cytogenetic, and molecular genetic data from a retrospective analysis of 592 adult patients aged 18-86years diagnosed with CML were analyzed. In 5 CML patients, the presence of JAK2 V617F or CALR mutation was confirmed. Three of 4 TKI-treated patients did not reach complete hematologic response due to the persistence of thrombocytosis and/or splenomegaly. In all of them (in 3 with JAK2 V617F mutation and 1 with CALR mutation), thrombocytosis was present at the time when complete cytogenetic response was documented. In 3 out of 4 reported CML patients, thrombocytosis and/or splenomegaly were still present even at the time when deep molecular response was reached. In our opinion, a detailed evaluation and appropriate interpretation of clinical and laboratory data in such a category of patients seem to be extremely important, especially when a decision about the TKI change due to therapy failure is considered.

Divergent roles for antigenic drive in the aetiology of primary versus dasatinib-associated CD8+ TCR-V\u03b2+ expansions

CD8+ T-cell expansions are the primary manifestation of T-cell large granular lymphocytic leukemia (T-LGLL), which is frequently accompanied by neutropenia and rheumatoid arthritis, and also occur as a secondary phenomenon in leukemia patients treated with dasatinib, notably in association with various drug-induced side-effects. However, the mechanisms that underlie the genesis and maintenance of expanded CD8+ T-cell receptor (TCR)-Vβ+ populations in these patient groups have yet to be fully defined. In this study, we performed a comprehensive phenotypic and clonotypic assessment of expanded (TCR-Vβ+) and residual (TCR-Vβ−) CD8+ T-cell populations in T-LGLL and dasatinib-treated chronic myelogenous leukemia (CML) patients. The dominant CD8+ TCR-Vβ+ expansions in T-LGLL patients were largely monoclonal and highly differentiated, whereas the dominant CD8+ TCR-Vβ+ expansions in dasatinib-treated CML patients were oligoclonal or polyclonal, and displayed a broad range of memory phenotypes. These contrasting features suggest divergent roles for antigenic drive in the immunopathogenesis of primary versus dasatinib-associated CD8+ TCR-Vβ+ expansions.

Discriminating myelodysplastic syndrome and other myeloid malignancies from non-clonal disorders by multiparametric analysis of automated cell data

BackgroundWe investigated the usefulness of novel complete blood count (CBC) data for discriminating myeloid malignancies from non-clonal CBC abnormalities. MethodsData were obtained during routine CBC tests of 119 samples from 37 myelodysplastic syndrome (MDS) patients, 92 samples from 45 myeloproliferative neoplasm (MPN) patients, and 15 samples from 11 chronic myelogenous leukemia (CML) patients using a DxH800 (Beckman Coulter). Data obtained from patients with hypocellular bone marrow and from those with other non-clonal diseases with CBC abnormalities were included in the comparisons. ResultsFor cell population data of neutrophils, the means of median, upper median, lower median, and low angle light scatters were significantly lower in MDS patients than in patients without hematological malignancies. Low hemoglobin density (LHD) did not significantly differ between the MDS and non-clonal cytopenia patients, but it was significantly higher in the MPN and CML patients. We selected 13 parameters and scored the MDS diagnosis using cut-off values obtained from receiver operating characteristic (ROC) curve analysis. Using a score > 9, MDS was distinguished from non-clonal cytopenia with a sensitivity of 92.4% and a specificity of 85.4%. ConclusionsMultiparametric analyses of new automated parameters are useful for discriminating MDS from non-clonal cytopenia.

How is the relationship between TWIST-1 and BCR-ABL1 gene expressions in chronic myeloid leukaemia patients?

Background: The activation and increased expression of BCR-ABL1 lead to malignant chronic myelogenous leukaemia (CML) cells, as well as the resistance to antitumour agents and apoptosis inducers. Moreover, TWIST-1 protein is a prognostic factor of leukemogenesis, and its level is raised in CML patients with cytogenetic resistance to imatinib. So, there is a likely relationship between BCR-ABL1 and TWIST-1 genes.Objective: The aim of the study was to assess the relationship between TWIST-1 and BCR-ABL1 expressions.Methods: Peripheral blood samples were obtained from 44 CML patients under treatment and also from ten healthy subjects as normal controls. The expression of TWIST-1 and BCR-ABL1 genes was measured using real-time PCR, and ABL1 was used as the reference gene. The gene expression was evaluated by REST software.Results: The expression levels of TWIST-1 and BCR-ABL1 genes in CML patients was changed 40.23 ± 177.75-fold and 6 ± 18-fold, respectively.Discussion: No significant relationship was observed between the expressions of TWIST-1 and BCR-ABL1 genes. All patients with TWIST-1 expression levels ≥100-fold had failure of response to treatment.Conclusion: The probability of the relationship between BCR-ABL1 and TWIST-1 is still debatable, and the average of TWIST-1 expression has been higher in patients without response to treatment. Definitive conclusion needs further investigations.

Prognostic Implications of Derivative Chromosome 9 Deletions in Patients with Advanced-Stage Chronic Myelogenous Leukemia.

Elucidation of cryptic BCR/ABL1 gene rearrangement is exceptionally important in the clinical diagnosis and prognosis of chronic myelogenous leukemia (CML). Previous reports indicated an adverse prognostic effect of atypical BCR/ABL1 gene rearrangements with submicroscopic ABL1-BCR deletions on derivative chromosome 9 [der(9)] in CML patients. Dual color dual fusion locus-specific BCR/ABL1 fluorescent in situ hybridization (FISH) analysis together with G-banding using trypsin and Giemsa (GTG banding) was performed in 489 patients at different stages of CML to investigate the spectrum of BCR/ABL1 gene rearrangements. Among the study group analyzed, a significantly higher frequency of BCR/ABL1 gene rearrangements that is consistent with der(9) deletion were observed in the blast crisis (BC) phase at 41.67%, followed by the accelerated phase (AP) at 36.84%, the imatinib mesylate (IM)-resistant chronic phase (CP) at 23.08%, and the lowest incidence was found in de novo CP at 16.61%. 1R1G1F (1 red, 1 green, 1 fusion) with concurrent loss of ABL1-BCR fusion gene on der(9) chromosome was the major signal pattern identified in each group. The results from the current study show that this unusual BCR/ABL1 gene rearrangement is one of the steering forces toward disease progression in CML. Patients in AP/BC of CML with der(9) deletion showed poor response to IM therapy; however, patients with der(9) deletion in the early phases of CML responded well to IM treatment. Therefore, the establishment of an atypical FISH signal pattern in CML is of paramount important because it is associated with adverse clinical prognostic implications in advanced stages of the disease.

Role of ZNF224 in c-Myc repression and imatinib responsiveness in chronic myeloid leukemia.

The transcription factor ZNF224 plays a key proapoptotic role in chronic myelogenous leukemia (CML), by modulating Wilms Tumor protein 1 (WT1) dependent apoptotic genes transcription. Recently, we demonstrated that Bcr-Abl signaling represses ZNF224 expression in Bcr-Abl positive CML cell lines and in CML patients. Interestingly, Imatinib and second-generation tyrosine kinase inhibitors specifically increase ZNF224 expression.On the other hand, Bcr-Abl positively modulates, via JAK2 activation, the expression of the c-Myc oncogene, which is required for Bcr-Abl oncogenic transformation in CML. Consequently, JAK2 inhibitors represent promising molecular therapeutic tools in CML.In this work, we demonstrate that ZNF224 is a novel transcriptional repressor of c-Myc in CML. We also show that ZNF224 induction by Imatinib and AG490, a specific JAK2 inhibitor, is responsible for the transcriptional repression of c-MYC, thus highlighting the crucial role of the ZNF224/c-Myc axis in Imatinib responsiveness.Interestingly, we also report that ZNF224 is induced by AG490 in Imatinib-resistant CML cells, leading to c-Myc repression and apoptosis induction. These findings suggest that the development of molecular tools able to induce ZNF224 expression could provide promising means to bypass Imatinib resistance in CML.

Homozygous HLA-C1 is Associated with Reduced Risk of Relapse after HLA-Matched Transplantation in Patients with Myeloid Leukemia

Natural killer (NK) cells assume graft-versus-leukemia alloreactivity after hematopoietic stem cell transplantation (HSCT) through their inhibitory killer cell immunoglobulin-like receptors (KIRs). KIR2D family members recognize HLA-C alleles with Asn80 (HLA-C1) or Lys80 (HLA-C2). The predominance of HLA-C1 over HLA-C2 and the frequent presence of KIR2DL1 are characteristic of Japanese people. We compared clinical outcomes among homozygous HLA-C1 (HLA-C1/C1) patients and heterozygous HLA-C1/C2 patients who underwent HLA-matched HSCT for hematologic malignancies by assessing the data of 10,638 patients from the Japanese national registry. HLA-C1/C1 recipients had a lower rate of relapse than HLA-C1/C2 recipients after transplantation for acute myelogenous leukemia (AML) (hazard ratio [HR], .79; P = .006) and chronic myelogenous leukemia (CML) (HR, .48; P = .025), but not for acute lymphoblastic leukemia (HR, 1.36), lymphoma (HR, .97), or low-grade myelodysplastic syndrome (HR, 1.40). We then grouped AML and CML patients together and divided them into several subgroups. Advantages of HLA-C1/C1 recipients over HLA-C1/C2 recipients regarding relapse were observed irrespective of donor relation (related: HR, .79, P = .069; unrelated: HR, .77, P = .022), preparative regimen (myeloablative: HR, .79, P = .014; reduced intensity: HR, .73, P = .084), and occurrence of acute graft-versus-host disease (yes: HR, .70, P = .122; no, HR .71, P = .026) or cytomegalovirus reactivation (reactivated: HR .67,P = .054; nonreactivated: HR .71, P = .033); however, these advantages were not observed in recipients with a delay in achieving complete chimerism (HR, 1.06). The advantage of decreasing relapse and extending relapse-free survival of C1/1 over C1/2 KIR-ligand status was most pronounced in T cell-depleted HSCT (HR, .27; P < .001 and HR, .30; P = .002, respectively) and in children age <15 years (HR, .29; P < .001 and HR .31; P < .001, respectively). Our findings represent an important mechanism responsible for the immunity against HLA-C2–negative myeloid leukemia cells after HLA-matched transplantation.

Metabolomics of Chronic Myelogenous Leukemia Based on GC-MS

To investigate the metabolism characteristics, to search for potential biomarkers associated with disease and to explore related metabolic pathways by analyzing the plasma metabolic profile of patients with chronic myelogenous leukemia (CML) through metabolomies. Twenty-six newly diagnosed CML patients in the First Affilated Hospital of Soochow University from February 2015 to April 2015, 26 allogeneic hematopoietic stem cell donors as healthy controls and 26 patients treated with tyrosine kinase inhibitors (TKI) to obtain the best efficacy as post-treatment controls were enrolled in this study. All the metabolites of plasma were extracted by Gas Chromatography-Mass Spectrometer(GC-MS) to collect metabolic fingerprint. Multivariate pattern recognition analysis and t test were combined to screen out the metabolic biomarkers at different time points. The receiver operating characteristic curve analysis was used to evaluate the clinical efficacy of metabolites, and the metabolic pathway analysis was performed. Significantly different metabolite expression mode was found seen between CML and healthy control groups. Six changed metabolites in CML were confirmed by multivariate and variate statistical analyses. Compared with the healthy controls, the levels of tetradecanoic acid and glycerol were decreased, the lactic acid, myo-inositol, d-galactose and glycine in CML patients also increased (all VIP>1,P<0.05, AUC>0.7). The plasma metabolites in CML patients after treatment with tyrosine kinase inhibitors (TKI) showed a recovery trend toward to normal levels. The plasma metabolic pathways of CML were mainly related with galactose, pyruvate, glycerolipid, inositol phosphate and glycine, serine and threonine metabolism (all impact value>0.10). Significant changes in plasma metabolite levels were found in CML patients. Metabolomics combined with multivariate pattern recognition analysis may be a new tool to assist diagnosis.

Chk1 inhibitors overcome imatinib resistance in chronic myeloid leukemia cells

Drug resistance to tyrosine kinase inhibitors (TKIs) is currently a clinical problem of chronic myelogenous leukemia (CML). Bcr-Abl protein depletion is considered as a way to overcome drug resistance to TKIs. In our study, Chk1 inhibitors, AZD7762 and MK-8776, had strong antitumor effects on CML cell line KBM5 and imatinib-resistant form KBM5T315I. Moreover, Chk1 inhibitors showed a strong cytotoxic effect on leukemia cells from primary CML and imatinib-resistance CML patients, but low cytotoxic effect on normal human mononuclear cells. Then, we found that Chk1 inhibitors induced apoptosis and increased DNA damage in CML cell lines with the degradation of the Bcr-Abl protein. Using the proteasome inhibitor and an immunoprecipitation assay, we found that Chk1 inhibitors triggered the degradation of Bcr-Abl through ubiquitination, which is depending on E3 ubiquitin ligase CHIP. At last, MK-8776 showed a significant tumor-suppressive effect of KBM5T315I cell in xenograft tumor models. Taking together, these findings suggest that targeting Chk1 may overcome TKIs resistance for the treatment of CML.

Emodin reverses leukemia multidrug resistance by competitive inhibition and downregulation of P-glycoprotein.

Development of multidrug resistance (MDR) is a continuous clinical challenge partially due to the overexpression of P-glycoprotein (P-gp) for chronic myelogenous leukemia (CML) patients. Herein, we evaluated the inhibitory potency of emodin, a natural anthraquinone derivative isolated from Rheum palmatum L, on P-gp in P-gp positive K562/ADM cells. Competition experiments combined with molecular docking analysis were utilized to investigate the binding modes between emodin and binding sites of P-gp. Emodin reversed adriamycin resistance in K562/ADM cells accompanied with the decrease of P-gp protein expression, further increasing the uptake of rhodamine123 in both K562/ADM and Caco-2 cells, indicating the inhibition of P-gp efflux function. Moreover, when incubated with emodin under different conditions where P-gp was inhibited, K562/ADM cells displayed increasing intracellular uptake of emodin, suggesting that emodin may be the potential substrate of P-gp. Importantly, rhodamine 123 could increase the Kintrinsic (Ki) value of emodin linearly, whereas, verapamil could not, implying that emodin competitively bound to the R site of P-gp and noncompetition existed between emodin and verapamil at the M site, in a good accordance with the results of molecular docking that emodin bound to the R site of P-gp with higher affinity. Based on our results, we suggest that emodin might be used to modulate P-gp function and expression.

Relapse and cytogenetic evolution in myeloid neoplasms.

Based on the current WHO Classification of Myeloid Neoplasms, cytogenetic findings play a central role in the diagnostic classification of the myeloid malignancies. Cytogenetic abnormalities detected at primary diagnosis may change over time. Karyotype changes can be characterized as cytogenetic evolution, cytogenetic regression or a combination of both. While the exact mechanism of cytogenetic evolution is not completely understood, the process of cytogenetic evolution is not random, but follows different, and often disease-specific patterns during progression and relapse of myeloid neoplasms. Important lessons were learned from the cytogenetic evolution pathways observed over the course of chronic myelogenous leukemia (CML), progressing through chronic phase into accelerated phase and blast crisis. Cytogenetic evolution pathways of CML are divided into major and minor route abnormalities. The major route changes include an extra Ph chromosome (+Ph) trisomy 8 (+8) and the occurrence of an i(17q). The six most common minor route abnormalities include -7, -17, +17, +21 and -Y and one structural change, t(3;21). Recently an increased number of CML cases with karyotype abnormalities in Ph-negative cells have been reported in patients treated with imatinib. These abnormalities include trisomy 8, abnormalities of chromosome 7, and chromosome 20. The significance of the Ph-negative karyotype changes in subsequent development of myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) is unclear. Nevertheless, the occurrence of clonal abnormalities in Ph-negative cells underlines the importance of conventional cytogenetic studies in monitoring of CML patients. In AML, karyotype changes commonly occur between diagnosis and relapse status post chemotherapy. Karyotype evolution seems more common in patients who had unfavorable aberrations at diagnosis as compared to patients with favorable or intermediate abnormalities. Karyotype evolution results in shortened remission duration as compared to cases without cytogenetic change. Recent studies on cytogenetic evolution at relapse after allogeneic hematopoietic cell transplantation (allo-HCT) were similar to the data observed in chemotherapy-treated AML. Serial bone marrow evaluations after allo-HCT offer insights into the dynamics of karyotype evolution, notably, they demonstrated that a detection of karyotype abnormalities is usually followed by a relapse within the next 90 days. As a contrast, karyotype abnormalities were not observed in patients who do not relapse in the next 3 months. CGE at relapse was associated with significantly decreased postrelapse and post-transplantation survival compared with the non-CGE group. Very few data exist regarding a potential association between the dose or certain types of chemotherapy and cytogenetic evolution. Based on the results of a single study conducted recently, no specific chemotherapy regimen emerged to predispose for cytogenetic evolution. Further studies are necessary to evaluate the impact of the altered bone marrow environment and immunosuppression on karyotype stability.

Motivation of Chinese patients with chronic myelogenous leukemia who have stopped the tyrosine kinase inhibitor

Objective To analyze the motivation of Chinese patients with chronic myelogenous leukemia (CML) who have stopped the tyrosine kinase inhibitor (TKI). Methods Forty-seven CML patients who have stopped TKI provided informed consent prior to their participation in the study. These patients were divided into relapse and non-relapse group at the endpoint of the observation. None of the patients received any CML-associated therapies after TKI cessation. The reasons of withdrawal were analyzed statistically. Results The reasons for cessation included patient's request due to cost (59.57%, 28/47), patient's plan to getting pregnant (8.52%, 4/47), side-effect of TKI (23.40%,11/47) and other reasons (8.52%, 4/47). At the endpoint of observation, 23 patients suffered molecular relapse. Among them, 15 cases (65.22%) were due to cost; 1 case (4.35%) was due to getting pregnant, 5 cases (21.74%) were due to side-effect and 2 cases (8.69%) were due to other reasons. There was more frequency relapse in the group of insufficient cost. Conclusion The motivation of Chinese CML patients who have stopped TKI might show impact on the outcome, and the motivation is mainly related with history of drug reduction and withdrawal. Key words: Leukemia, myelogenous, chronic; Treatment-free remission; Withdrawal

Treatment-Free Remission: a New Therapeutic Goal in Chronic Myelogenous Leukemia.

Chronic myelogenous leukemia (CML) is a chronic myeloproliferative neoplasm characterized by the presence of Philadelphia chromosome [t(9:22)] leading to the presence of pathognomonic fusion gene product, BCR-ABL1. This leads to constitutive activation of ABL1 kinase. CML was a difficult-to-treat illness until the advent of small molecule tyrosine kinase inhibitor (TKI), imatinib which revolutionized therapy of CML. Since then, multiple second- and third-generation TKIs have been formulated which have proven effective and has led to marked improvement in survival. In this article, we review currently available data on possibility of holding TKI therapy in patients in deep remission [treatment-free remission (TFR)] and safety of this approach. As CML treatment has become more effective, new questions have emerged, most important being whether the treatment with TKIs can ever be stopped. This is especially relevant in patient experiencing side effects from therapy or who may be subject to increased health risks due to treatment. There is now evidence that some CML patients who have achieved stable deep molecular response can safely stop TKI. Furthermore, patients can safely re-establish remission after restarting their TKI therapy in the situation of relapse. CML is highly treatable disease, but the treatment has untoward physical and socioeconomic consequences. The idea of TFR is hence attractive. There is a growing body of evidence that some CML patients who have achieved stable deep molecular response can safely stop TKI.

Prostaglandin E1 and Its Analog Misoprostol Inhibit Human CML Stem Cell Self-Renewal via EP4 Receptor Activation and Repression of AP-1

Effective treatment of chronic myelogenous leukemia (CML) largely depends on the eradication of CML leukemic stem cells (LSCs). We recently showed that CML LSCs depend on Tcf1 and Lef1 factors for self-renewal. Using a connectivity map, we identified prostaglandin E1 (PGE1) as a small molecule that partly elicited the gene expression changes in LSCs caused by Tcf1/Lef1 deficiency. Although it has little impact on normal hematopoiesis, we found that PGE1 treatment impaired the persistence and activity of LSCs in a pre-clinical murine CML model and a xenograft model of transplanted CML patient CD34+ stem/progenitor cells. Mechanistically, PGE1 acted on the EP4 receptor and repressed Fosb andFos AP-1 factors in a β-catenin-independent manner. Misoprostol, an FDA-approved EP4 agonist, conferred similar protection against CML. These findings suggest that activation of this PGE1-EP4 pathway specifically targets CML LSCs and that the combination of PGE1/misoprostol with conventional tyrosine-kinase inhibitors could provide effective therapy for CML.

How to detect the rare BCR-ABL (e14a3) transcript: A case report and literature review.

The Philadelphia (Ph; BCR-ABL) chromosome originates from a translocation event between chromosomes 9 and 22, and results in the BCR-ABL fusion gene. In chronic myelogenous leukemia (CML), the BCR-ABL gene is mainly coded for by a major breakpoint cluster region (M-bcr, e13a2 and e14a2). However, in some patients, BCR-ABL genes are encoded by a minor (m)-bcr, e1a2, and a micro (µ)-bcr region, e19a2. These transcripts revealed a different clinical course. The present study described a CML patient whose cytogenetics and FISH analyses of bone marrow revealed a karyotype of 46, XY t(9,22) (q34;q11), while the commercial kits of quantitative PCR (qPCR) failed to detect the BCR-ABL fusion gene. Further multiplex Reverse transcription-PCR (RT-PCR) and sequencing analyses identified a rare e14a3 (b3a3) fusion transcript.

Impact of Imatinib on the Fertility of Male Patients with Chronic Myelogenous Leukaemia in the Chronic Phase.

Imatinib is a first-line tyrosine kinase inhibitor for treating chronic myelogenous leukaemia (CML) and has greatly improved the prognosis of this disease. An increasing number of CML patients of reproductive age are diagnosed each year, and the impact of imatinib on fertility is a major concern. Providing useful advice to these patients regarding the choice of their therapeutic treatment is very important. This study examined the impact of imatinib on the fertility of male patients with CML in the chronic phase. We performed a study of 48 adult male CML patients in the chronic phase (CML-CP), 50 healthy control subjects, and 10 male patients with infertility. Imatinib levels in semen and plasma were measured using high-performance liquid chromatography/mass spectrometry. We examined the effects of imatinib on sperm parameters and the male reproductive system using a computer-assisted sperm assay and ultrasound, respectively. We analysed sex hormone levels in the sera of CML-CP patients using an enzyme-linked immunosorbent assay. Imatinib levels in semen were comparable to plasma levels in CML-CP patients. CML-CP patients treated with imatinib exhibited reduced sperm density, counts, survival rates, and activity. Ultrasound demonstrated that the shape and size of the testis and epididymis in CML-CP patients undergoing imatinib treatment were normal. However, 19 of these patients exhibited a hydrocele in their tunica vaginalis, with a large dark area of effusion (0.7-2.9cm in width). Sex hormone levels in the sera of the CML-CP patients were normal. These results suggest that imatinib crosses the blood-testis barrier and reduces sperm density, sperm count, survival rates, and activity in CML-CP patients. However, imatinib did not affect the structure of reproductive organs or sex hormone levels.

Expression of SHP-1 mRNA in Patients with Myelogenous Leukemia and Its Clinical Significance

To investigate the expression and clinical significance of SHP-1 mRNA in patients with myelogenous leukemia. The SYBR Green-based qRT-PCR was used to assess SHP-1 mRNA levels in 54 patients with chronic myelogenous leukemia (CML), 30 cases of de novo acute myelogenous leukemia (AML) and 10 persons without malignancy as controls. The relative expression levels of SHP-1 mRNA in control group (CG), chronic phase CML (CP-CML) group, advanced phase of CML (including accelerated phase CML and blastic phase CML) group and AML group were 1.15±0.62, 4.96±1.76, 2.60±0.90 and 0.45±0.20, respectively. The expression of SHP-1 mRNA in patients with CML significantly increased in comparison with that in CG(P<0.05). Meanwhile, the expression of SHP-1 mRNA in CP-CML group very significantly increased as compared with that in advanced stage of CML group(P<0.0001). The expression of SHP-1 mRNA in AML group significantly decreased as compared with that in CG group(P=0.0442). In CP-CML group, statistical analysis showed that SHP-1 mRNA expression at baseline in optimal responders (5.712±0.4476) was significantly higher than that in the suboptimal or failed responders (4.044±0.3701)(P=0.0090). Meanwhile, the SHP-1 mRNA expression in AML patients was higher than that in CR group (0.4984±0.05164) and non-CR group (0.3537±0.02388)(P=0.0017). The SHP-1 mRNA levels in CML patients are higher than that in AML patients, and probably correlats with disease progression of CML. The mRNA expression level of SHP-1 may be a molecular marker to predict early response to inatinib treatment in CP-CML and AML.

Specific Monoclonal Antibody Against Bcr/Abl Out-of-Frame Alternative Proteins as Diagnostic Tool in Chronic Myelogenous Leukemia Patients.

More recently, alternative splicing of specific genes are investigated for their therapeutic potential. In particular, we reported the existence of BCR-ABL alternative splicing isoforms, in about 80% of Philadelphia-positive patients, which lead to the expression of aberrant proteins. These fusion proteins are characterized by an orphan initial and correct Bcr portion attached to a 112 amino acid sequence, arising from the impairment in the reading frame (reading of ABL exon 4 and 5). We demonstrated that these Abl-out-of-frame (OOF) isoforms could have an immunological role with therapeutic implications. The aim of this study was to characterize a new monoclonal antibody (MAb) specific for Abl-OOF protein portion, for diagnostic use, to detect this biomarker in Philadelphia chromosome-positive chronic myelogenous leukemia (CML) patients and to generate novel approaches in the immunotherapy. 5F11G11 MAb recognizes the OOF protein portion of the native full-length Bcr/Abl-OOF protein expressed in cells transiently transfected, as demonstrated by immunoprecipitation and immunofluorescence. In addition, we demonstrate the MAb's ability to recognize the alternative hybrid Bcr/Abl fusion protein expressed in leukemic cells from CML patients, to support the possible use of 5F11G11 MAb as a diagnostic tool to select patients with Philadelphia chromosome-positive CML that could be eligible for an immunotherapeutic approach with this new antigen.