Prognosis of patients with chronic myeloid leukemia presenting in advanced phase is defined mainly by blast count, but also by age, chromosomal aberrations and hemoglobin.

Chronic myeloid leukemia (CML) is usually diagnosed in chronic phase, yet there is a small percentage of patients that is diagnosed in accelerated phase or blast crisis. Due to this rarity, little is known about the prognosis of these patients. Our aim was to identify prognostic factors for this cohort. We identified 283 patients in the EUTOS population-based and out-study registries that were diagnosed in advanced phase. Nearly all patients were treated with tyrosine kinase inhibitors. Median survival in this heterogeneous cohort was 8.2 years. When comparing patients with more than 30% blasts to those with 20-29% blasts, the hazard ratio (HR) was 1.32 (95%-confidence interval (CI): [0.7-2.6]). Patients with 20-29% blasts had a significantly higher risk than patients with less than 20% blasts (HR: 2.24, 95%-CI: [1.2-4.0], P = .008). We found that the blast count was the most important prognostic factor; however, age, hemoglobin, basophils and other chromosomal aberrations should be considered as well. The ELTS score was able to define two groups (high risk vs non-high risk) with an HR of 3.01 (95%-CI: [1.81-5.00], P < .001). Regarding the contrasting definitions of blast crisis, our data clearly supported the 20% cut-off over the 30% cut-off in this cohort. Based on our results, we conclude that a one-phase rather than a two-phase categorization of de novo advanced phase CML patients is appropriate.

Research on epigenetic mechanism of SFRP2 in advanced chronic myeloid leukemia.

Secreted frizzled-related protein 2 (SFRP2) has been reported to act as a tumor suppressors. This study aims to detect the biological role of SFRP2 in advanced chronic myeloid leukemia (CML). In this study we examined bone marrow samples from 45 CML patients and 10 healthy donors. K562 and KCL22 cells were cultured and treated with demethylation drug and histone deacetylase inhibitor (HDACi). KCL22 and K562 cells were transfected with lentiviral vector (LV)-SFRP2, LV-control. The cells were then subjected to proliferation and apoptosis assays, real time polymerase chain reaction (PCR), Methylation-specific PCR (MSP), Western blotting, co-immunoprecipitation (CoIP) and Chromatin immunoprecipitation (ChIP), We found that SFRP2 was down-regulated in the accelerated and blast phase of CML, whereas, the levels of WNT1, WNT3 and WNT5A were up-regulated in the accelerated and blast phase of CML. Overexpression SFRP2 inhibited proliferation, promoted apoptosis and activated the WNT pathway. CoIP-MS results showed that SFRP2 interacted with WNT1 and WNT5A. ChIP-seq result indicated that the promoter of H3K4me3 and H3K27me3 were able to interact with SFRP2. In conclusion, our findings demonstrated the SFRP2 act as a potential therapeutic target for advanced CML. Furthermore, our results support the use of demethylation drugs and HDACi as a potential CML treatment strategy.

Life after ponatinib failure: outcomes of chronic and accelerated phase CML patients who discontinued ponatinib in the salvage setting.

Ponatinib is a pan-tyrosine kinase inhibitor (TKI) with efficacy in multirefractory CML patients who have failed other TKIs. Despite excellent response rates, resistance or intolerance may develop. We conducted a retrospective review of the outcome of patients with chronic (CP) and accelerated (AP) phase CML refractory to prior TKI who discontinued ponatinib for resistance or intolerance. Nineteen CP patients, discontinued due to resistance (n = 13), toxicity (n = 5) and to pursue stem cell transplantation (n = 1). At discontinuation, 14 were still in CP, three had progressed to AP and two to blast phase (BP). Three CP patients improved their cytogenetic response (CyR) to complete CyR (CCyR), two after SCT and one on omacetaxine. None of the 12 patients, without a major cytogenetic response at ponatinib discontinuation, including all patients treated with subsequent TKIs, responded to therapy. Seventeen AP patients, stopped ponatinib due to resistance (n = 15) or intolerance (n = 2). At discontinuation, 14 were still in AP and three had progressed to BP. Four patients were treated with SCT and one achieved major molecular response. None of the 12 patients treated with non-SCT approaches responded to subsequent therapy. Median survival for all patients was 16.6 months after ponatinib discontinuation (31, 9 and 13 months for patients in CP, AP and BP, respectively). Median survival was 60 months for patients who discontinued ponatinib for toxicity and 11 months for those who discontinued for resistance. Long-term outcome of patients with ponatinib failure are poor with estimated one-year OS and EFS rates of 54% and 40%, respectively. New treatment options are required for this subset of patients.

Constitutional t(8;22)(q24;q11.2) that mimics the variant Burkitt-type translocation in Philadelphia chromosome-positive chronic myeloid leukemia.

Constitutional translocations that coincide with t(9;22)(q34;q11.2) may lead to unnecessary treatments in chronic myeloid leukemia (CML) patients, as, under the standard criteria, a diagnosis of CML with additional chromosomal abnormalities indicates an accelerated phase (AP). In the present report, a 47-year-old male had pain in the right foot due to gout. Peripheral blood examination showed leukocytosis with left shift. Bone marrow aspiration revealed myeloid hyperplasia with megakaryocytosis. RT-PCR revealed the major BCR-ABL fusion transcript, and CML in the chronic phase was diagnosed, followed by nilotinib treatment. Although WBC counts decreased immediately, G-banding analysis showed 46,XY,t(8;22)(q24;q11.2),t(9;22)(q34;q11.2) [20]. The t(8;22)(q24;q11.2) translocation is known to be recurrent in Burkitt's lymphoma. The diagnosis was changed to CML in AP, leading to B-lymphoid crisis. Unexpectedly, the karyotype was 46,XY,t(8;22)(q24;q11.2) [20] in hematological complete remission, even after 3 months. Fluorescence in situ hybridization on metaphase spreads revealed the MYC signal on the der(22)t(8;22), indicating that the 8q24 breakpoint was centromeric to MYC at 8q24.21. G-banding analysis of phytohemagglutinin-stimulated peripheral blood T-lymphocytes also indicated 46,XY,t(8;22)(q24.1;q11.2). We conclude that the t(8;22) is constitutional in this patient. As the tumor suppressor gene TRC8/RNF139 is disrupted by constitutional t(8;22)(q24.13;q11.21) in dysgerminoma, it may be associated with the onset of CML.

BCL11A expression in acute phase chronic myeloid leukemia.

Chronic myeloid leukemia (CML) has chronic and acute phases. In chronic phase myeloid differentiation is preserved whereas in acute phase myeloid differentiation is blocked. Acute phase CML resembles acute myeloid leukemia (AML). Chronic phase CML is caused by BCR-ABL1. What additional mutation(s) cause transition to acute phase is unknown and may differ in different persons with CML. BCL11A encodes a transcription factor and is aberrantly-expressed in several haematological and solid neoplasms. We analyzed BCL11A mRNA levels in subjects with chronic and acute phase CML. BCL11A transcript levels were increased in subjects with CML in acute phase compared with those in normals and in subjects in chronic phase including some subjects studied in both phases. BCL11A mRNA levels were correlated with percent bone marrow blasts and significantly higher in lymphoid versus myeloid blast crisis. Differentiation of K562 with butyric acid, a CML cell line, decreased BCL11A mRNA levels. Cytology and flow cytometry analyses showed that ectopic expression of BCL11A in K562 cells blocked differentiation. These data suggest BCL11A may operate in transformation of CML from chronic to acute phase in some persons.

Sensitive Replicate Real-Time Quantitative PCR of BCR-ABL Shows Deep Molecular Responses in Long-Term Post-Allogeneic Stem Cell Transplantation Chronic Myeloid Leukemia Patients.

Real-time quantitative PCR (RT-qPCR) is commonly used for follow-up of chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors, but its current sensitivity does not allow detection of very low BCR-ABL levels. Therefore RT-qPCR negativity is not synonymous with complete molecular response. Replicate RT-qPCR had shown increased sensitivity in tyrosine kinase inhibitor-treated patients and was, therefore, used here to evaluate whether RT-qPCR-negative post-allogeneic stem cell transplantation (SCT) patients harbor detectable disease. Samples from 12 patients were tested at 2 time points using 82 replicates of BCR-ABL RT-qPCR. One patient (38 months after SCT) had detectable transcripts at baseline and none at the follow-up test, done at a median of 107 months after SCT. This suggests cure from CML in the majority of allogeneic SCT patients who have no transcripts detectable by replicate RT-qPCR for BCR-ABL.

A phase I study of danusertib (PHA-739358) in adult patients with accelerated or blastic phase chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia resistant or intolerant to imatinib and/or other second generation c-ABL therapy.

Danusertib is a pan-aurora kinase inhibitor with potent activity against Abl kinase including the gatekeeper T315I mutant. A phase 1 dose escalation study of danusertib was conducted in patients with accelerated or blastic phase chronic myeloid leukemia or Philadelphia chromosome-positive acute lymphoblastic leukemia. Two dosing schedules were studied: schedule A, in which danusertib was given by 3-hour intravenous infusion daily for 7 consecutive days (days 1-7) in a 14-day cycle, and schedule B, in which the danusertib was given by 3-hour intravenous infusion daily for 14 consecutive days (days 1-14) in a 21-day cycle. A total of 37 patients were treated, 29 with schedule A and eight with schedule B. The recommended phase 2 dose for schedule A was 180 mg/m(2). Enrollment to schedule B was stopped early because of logistical problems with the frequency of infusions. Febrile neutropenia and mucositis were dose-limiting toxicities in schedule A. Four patients with T315I ABL kinase mutation, all treated with schedule A, responded. Danusertib has an acceptable toxicity profile and is active in patients with Bcr-Abl-associated advanced hematologic malignancies. This study was registered with the European Clinical Trails Data Base (EudraCT number 2007-004070-18).

Phase distribution of chronic myeloid leukemia in Bangladesh.

BACKGROUND: Here, we report the phase distribution of chronic myeloid leukemia (CML), defined based on the World Health Organization criteria, among 63 patients in Bangladesh. All patients were diagnosed based on complete blood count, bone marrow examination including bone marrow aspiration and reverse-transcriptase polymerase chain reaction (RT-PCR). Out of 63 patients, 42 were male and 21 were female. The mean age of the subjects was 37.4 years, with an age range of 17-60 years. The majority of patients (86%) were classified in the chronic phase (CP), 7 (11%) in the accelerated phase (AP) and two (3%) in blast crisis (BC). The most frequent patient age ranges were 21-30 years for CP, 41-50 years for AP and 41-50 years for BC. RESULTS: The Philadelphia chromosome was detected in 48 patients by RT-PCR. The mean total leukocyte counts, platelet counts, hemoglobin levels and marrow blast frequencies were 101 × 10(9)/L, 409 × 10(9)/L, 12.2 g/dl and 2.8% for CP; 121 × 10(9)/L, 418 × 10(9)/L, 8.7 g/dl and 15% for AP and 311 × 10(9)/L, 396 × 10(9)/L, 9.2 g/dl and 26% for BC, respectively. CONCLUSION: This study concluded that most CML patients in Bangladesh are from a younger age group (31-40 years). In addition, males were more commonly affected, although females were afflicted with this disease at a younger age.

Discovery of somatic mutations in the progression of chronic myeloid leukemia by whole-exome sequencing.

We performed whole-exome sequencing in samples representing accelerated phase (AP) and blastic crisis (BC) in a subject with chronic myeloid leukemia (CML). A total of 12.74 Gb clean data were generated, achieving a mean depth coverage of 64.45 and 69.53 for AP and BC samples, respectively, of the target region. A total of 148 somatic variants were detected, including 76 insertions and deletions (indels), 64 single-nucleotide variations (SNV), and 8 structural variations (SV). On the basis of annotation and functional prediction analysis, we identified 3 SNVs and 6 SVs that showed a potential association with CML progression. Among the genes that harbor the identified variants, GATA2 has previously been reported to play important roles in the progression from AP to BC in CML. Identification of these genes will allow us to gain a better understanding of the pathological mechanism of CML and represents a critical advance toward new molecular diagnostic tests for the development of potential therapies for CML.

Tyrosine kinase inhibitors as initial therapy for patients with chronic myeloid leukemia in accelerated phase.

BACKGROUND: Accelerated phase CML most frequently represents a progression state in CML. However, some patients present with AP features at the time of diagnosis. There is limited information on the outcome of these patients who received TKIs as initial therapy. PATIENTS AND METHODS: We analyzed the outcome of 51 consecutive patients with CML who presented with features of AP at the time of diagnosis, including blasts ≥ 15% (n = 6), basophils ≥ 20% (n = 22), platelets < 100 × 10(9)/L (n = 3), cytogenetic clonal evolution (n = 17), or more than 1 feature (n = 3). Patients received initial therapy with imatinib (n = 30), dasatinib (n = 5), or nilotinib (n = 16). RESULTS: The rate of complete cytogenetic response for patients treated with imatinib was 80%, and with dasatinib or nilotinib was 90%. Major molecular response (MMR) (Breakpoint Cluster Region (BCR)-Abelson (ABL)/ABL ≤ 0.1%, International Scale [IS]) was achieved in 69% of patients including complete molecular response (BCR-ABL/ABL ≤ 0.0032% IS) in 49%. MMR rates for patients treated with imatinib were 63%, and with 2GTKIs, 76%. Overall survival at 36 months was 87% with imatinib and 95% with 2GTKIs. CONCLUSION: TKIs should be considered standard initial therapy for patients with AP at the time of diagnosis.

Identification of copy number alterations by array comparative genomic hybridization in patients with late chronic or accelerated phase chronic myeloid leukemia treated with imatinib mesylate.

The outcome of treating chronic myeloid leukemia (CML) with imatinib mesylate (IM) is inferior when therapy is commenced in late chronic or accelerated phase as compared to early chronic phase. This may be attributed to additional genomic alterations that accumulate during disease progression. We sought to identify such lesions in patients showing suboptimal response to IM by performing array-CGH analysis on 39 sequential samples from 15 CML patients. Seventy-four cumulative copy number alterations (CNAs) consisting of 35 losses and 39 gains were identified. Alterations flanking the ABL1 and BCR genes on chromosomes 9 and 22, respectively, were the most common identified lesions with 5 patients losing variable portions of 9q34.11 proximal to ABL1. Losses involving 1p36, 5q31, 17q25, Y and gains of 3q21, 8q24, 22q11, Xp11 were among other recurrent lesions identified. Aberrations were also observed in individual patients, involving regions containing known leukemia-associated genes; CDKN2A/2B, IKZF1, RB1, TLX1, AFF4. CML patients in late stages of their disease, harbor pre-existing and evolving sub-microscopic CNAs that may influence disease progression and IM response.

Hematopoietic stem cell transplantation for T315I-mutated chronic myelogenous leukemia.

BACKGROUND: The T315I mutation of BCR/ABL gene is known to produce complete resistance of chronic myelogenous leukemia (CML) to all currently available BCR/ABL inhibitors. The data suggesting poor median survival of these patients may indicate that they should be primary candidates for allogeneic stem cell transplantation (alloSCT). However, evidence on efficiency of this treatment modality in CML with T315I mutation is lacking. CASE REPORT: A 25-year-old patient was diagnosed with Philadelphia chromosome positive CML in accelerated phase. As he did not have an HLA-identical sibling or fully-matched unrelated bone marrow donor, treatment with low dose tyrosine kinase inhibitor - imatinib was initiated. Despite satisfactory hematological remission, he failed to achieve complete cytogenetic remission within the first year of treatment. Moreover, despite escalation of imatinib dosage, the disease relapsed after further 3 months of treatment. Molecular studies revealed T315I mutation of BCR/ABL gene. He responded poorly to interferon alpha (IFN-alpha) and we decided to perform alloSCT from a partially mismatched (8/10 HLA allele match) unrelated donor. The course of transplantation was complicated by staphylococcal sepsis, grade I skin acute GvHD and limited chronic skin GVHD. However, the goal of alloSCT was achieved and the patient remains in complete molecular remission at week +68 post-transplantation. CONCLUSIONS: The clinical course of this case supports the idea that allogeneic hematopoietic transplantation is a viable treatment option for patients with CML bearing T315I mutation.

Molecular monitoring and mutation analysis of patients with advanced phase CML and Ph+ ALL receiving dasatinib.

As a result of the excellent responses achieved in chronic phase chronic myeloid leukemia since the introduction of imatinib, sensitive techniques such as reverse transcriptase real-time PCR are warranted to monitor patients receiving tyrosine kinase inhibitors (TKI). Our objective was to determine the value of molecular monitoring Ph-positive leukemias under dasatinib treatment. We used real-time PCR and ABL1 kinase domain sequencing on sequential samples from 11 patients with Philadelphia-positive leukemias who received dasatinib. We were able to detect pre-existing mutations in the kinase domain of BCR-ABL1 in four patients, particularly in patients with high BCR-ABL1 transcript levels. Most mutations disappeared with dasatinib, however, in five patients a clone with T315I appeared during dasatinib treatment. We conclude that sensitive molecular monitoring with real-time PCR for BCR-ABL1 transcripts and mutation screening of the ABL1 kinase domain of patients with Philadelphia-positive leukemias are valuable for patient management, however, mutation findings should be interpreted with caution, as mutant clones not always behave in vivo as predicted by in vitro assays.

The growth factor independence-1 (Gfi1) is overexpressed in chronic myelogenous leukemia.

The activation of ABL tyrosine kinase in BCR/ABL-positive chronic myelogenous leukemia (CML) leads to a diversity of biological changes related to the pathogenesis of the disease. In CML patients, we determined the expression of growth factor independence-1 (Gfi1), a transcription repressor with weak oncogenic activity. Our data demonstrated that the Gfi1 mRNA level in both the mononuclear cells and purified CD34(+) cells from CML were significantly higher as measured by quantitative real-time PCR. Using flow cytometry and Western blot, we also showed that the Gfi1 protein content was increased in CML CD34(+) cells. The expression of Gfi1 was correlated with BCR/ABL significantly. Gfi1 may be implicated in the pathogenesis of CML and can serve as a potential target for the management of the disease.

Presentating phases of chronic myeloid leukaemia.

OBJECTIVE: To determine the frequency of three phases of chronic myeloid leukaemia at first presentation. STUDY DESIGN: Case series. PLACE AND DURATION OF STUDY: Department of Oncology, Combined Military Hospital (CMH), Rawalpindi, from June 2006 to December 2007. METHODOLOGY: Forty-five patients of either gender with Chronic Myeloid Leukaemia (CML) at their first presentation in outpatient department were included in the study by consecutive sampling technique. All patients were diagnosed on blood complete picture and bone marrow examination including aspiration, trephine and cytogenetics at Armed Forces Institute of Pathology (AFIP). Each phase was defined on the basis of World Health Organization (WHO) criteria. RESULTS: Out of 45, there were 31 (68.9%) male and 14 (31.1%) female patients. The mean age of presentation was 37.9 years. The pattern of presentation revealed 35 (77.8%) in Chronic Phase (CP), 7 (15.5%) in Accelerated Phase (AP) and 3 (6.7%) in Blast Crisis (BC). Philadelphia chromosome was detected in 39 (86.7%) cases on culture method. Splenomegaly was observed in 37 (82.2%) patients. The mean total leukocyte count, platelet count, haemoglobin and marrow blast were 214.3 x 10(9)/L, 551.4 x 10(9)/L, 9.94 g/dl and 9.3% respectively. CONCLUSION: CML presented at a younger age in the chronic phase.

Phase 3 study of dasatinib 140 mg once daily versus 70 mg twice daily in patients with chronic myeloid leukemia in accelerated phase resistant or intolerant to imatinib: 15-month median follow-up.

Dasatinib is the most potent BCR-ABL inhibitor, with 325-fold higher potency than imatinib against unmutated BCR-ABL in vitro. Studies have demonstrated the benefits of dasatinib 70 mg twice daily in patients with accelerated-phase chronic myeloid leukemia intolerant or resistant to imatinib. A phase 3 study compared the efficacy and safety of dasatinib 140 mg once daily with the current twice-daily regimen. Here, results from the subgroup with accelerated-phase chronic myeloid leukemia (n = 317) with a median follow-up of 15 months (treatment duration, 0.03-31.15 months) are reported. Among patients randomized to once-daily (n = 158) or twice-daily (n = 159) treatment, rates of major hematologic and cytogenetic responses were comparable (major hematologic response, 66% vs 68%; major cytogenetic response, 39% vs 43%, respectively). Estimated progression-free survival rates at 24 months were 51% and 55%, whereas overall survival rates were 63% versus 72%. Once-daily treatment was associated with an improved safety profile. In particular, significantly fewer patients in the once-daily group experienced a pleural effusion (all grades, 20% vs 39% P < .001). These results demonstrate that dasatinib 140 mg once daily has similar efficacy to dasatinib 70 mg twice daily but with an improved safety profile. This trial is registered at www.clinicaltrials.gov as #CA180-035.

[Multiplex fluorescence in situ hybridization for detecting complex chromosomal aberrations in chronic myeloid leukemia in blast crisis].

OBJECTIVE: To investigate the value of multiplex fluorescence in situ hybridization (M-FISH) for the detection of complex chromosomal abnormalities (CCA) of chronic myeloid leukemia in blast crisis (CML-BC). METHODS: M-FISH was used to study 26 cases of CML-BC with CCA assayed by conventional cytogenetics (CC). RESULTS: Sixty-nine kinds of structural rearrangements were detected by M-FISH besides typical t (9;22) translocation, among them only 10 were balanced ones and 59 unbalanced ones including 1 insertion, 6 deletions, 52 translocations and derivative chromosomes. In addition, 23 numerical abnormalities were detected. All chromosomes were involved in CML-BC, and chromosomes 17, 2, 8, 16 involvements were the most frequent. M-FISH failed to find out the abnormal clone in 1 case, discovered CCA clones that were missed CC in 6 cases. Clarified 16 kinds of aberrations which could not be identified CC and corrected 5 aberrations made wrong description by CC. Thirty-five kinds of translocations were found by M-FISH which were missed by CC. The aberrations of der (9) t (16; 6; 9; 22) and der (18) t (16; 18; 19) we found were reported in the literature for the first time. CONCLUSIONS: M-FISH can refine CCA in CML-BC, find out or correct the missed or misidentified abnormalities by CC. The frequent secondary chromosomal abnormalities in CML-BC with CCA are different from that in CML.

Overexpression of cyclin D1 in accelerated-phase chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a bi- or triphasic disease. Molecular markers distinct for the phase evolution would be clinically helpful. For signaling transformation and proliferation activities in CML, Bcr-Abl is the pivotal protein. As downstream signals of Bcr-Abl , RAS, PI3-K, and Stat 5 may lead to cell cycle progression mediated by increased expression of cyclin Ds. We analyzed copy numbers of bcr-abl and cyclin D1 transcripts by reverse transcription (RT) and competitive PCR titration in bone marrow cells of 20 patients with CML, 10 in chronic phase (CP) and the other 10 in accelerated phase (AP). The level of bcr-abl transcripts in the AP was not significantly higher than that in the CP; in contrast, the level of cyclin D1 transcripts in the AP was significantly higher than that in the CP (p <0.001). Cyclin D1 RNA expression in the CP of CML was also found to have clinical relevance to time to AP transformation. The median time to AP transformation for the CP patients with cyclin D1 transcripts of 1.50 x 10(4)/microg RNA was significantly shorter than that for those with cyclin D1 transcripts < 1.50 x 10(4)/microg RNA (15 vs. 67 months, p=0.0354) although confirmation to conduct in a larger patient group is required. These results suggest that the expression level of cyclin D1 RNA in bone marrow cells is predictive of the phase evolution in CML and may be helpful in treatment decision-making.