Genotyping of chimerical BCR-ABL1 RNA in chronic myeloid leukemia by integrated DNA chip.

Chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL) are associated with fusion of the BCR and ABL1 genes by chromosome translocation. The chimerical BCR-ABL1 gene encodes different fusion proteins that vary in size, depending on the breakpoint in the BCR region. Different types of fusion genes in CML and Ph(+) ALL are thought to be related to the clinical course and outcome of each patient. Currently, the genotypes are determined by PCR, followed by gel electrophoresis or DNA sequencing (among other methodologies). Our major aim was to develop a diagnostic method for CML genotyping by means of an integrated process of DNA microarray. Here, we describe a method of integrated multiplex reverse transcription, asymmetric PCR, and hybridization, all in the same reaction mixture in a chamber assembled on the surface of capture oligonucleotide probes immobilized on a glass slide. The integrated system successfully identified the four predominant types of chimerical BCR-ABL1 RNA (b3a2, b2a2, e1a2, and c3a2), which together account for 98% of CML cases. The integrated multiplex system also had a high sensitivity of detection (as little as 200 molecules of target RNA molecules). The integrated process saves time and effort, and it also the advantage of minimizing the steps needed for automated detection of different types of chimerical CML RNA.

Comprehensive therapeutic outcomes of frontline imatinib mesylate in newly diagnosed chronic phase chronic myeloid leukemia patients in Korea: feasibility assessment of current ELN recommendation.

Optimal responses during imatinib therapy are commonly defined following the European LeukemiaNet (ELN) recommendations. Achievements of these optimal responses have not, however, been comprehensively tested as response-related prognostic factors using single center data sets. We evaluated the parameters using long-term (median 63 months) outcomes from 363 chronic phase chronic myeloid leukemia patients treated with imatinib as frontline therapy at our center. Intention-to-treat analysis showed comparable rates of complete cytogenetic response (86 %), major molecular response (MMR, 54 %), and complete molecular response (MR(4.5), 8 %). Estimated overall survival, progression-free survival, and event-free survival at 7 years were 94, 88 and 84 %, respectively. Achievement of recommended optimal response at 6 months (major cytogenetic response) and 12 months (complete cytogenetic response) yielded significantly better overall, progression-free, and event-free survival. However, achievement of recommended optimal response at 18 months (MMR) provided marginal benefit only in event-free survival. Most ELN criteria were predictive of long-term outcomes, with the exception of the clinical significance of achieving MMR at 18 months. Treatment adherence in the early treatment period was one of the important independent predictors of favorable long-term outcome. Durable cytogenetic and molecular responses were maintained in a majority of patients treated with optimal dose intensity.

Long-term pattern of pleural effusion from chronic myeloid leukemia patients in second-line dasatinib therapy.

Dasatinib is a potent second-generation tyrosine kinase inhibitor approved for the treatment of chronic myeloid leukemia after imatinib failure. However, some patients treated with dasatinib experience pleural effusions (PEs). The determinants of pleural effusion in long-term dasatinib treatment (median 35 months, range 1-55) were investigated in single-center data of 65 patients enrolled in global phase 2 and phase 3 trials. Of the 65 patients, 35 (54%) developed dasatinib-induced pleural effusion (a median onset time, 20 months; range 0.2-54). The first pleural effusion developed in 15 (43%) patients within 12 months of dasatinib therapy. Disease phase (P = 0.02), dose schedule (P = 0.002) and actual daily mean dose (P = 0.0002) were significantly associated with an increased risk of pleural effusion. Twice-daily administration of dasatinib resulted in significantly more patients developing pleural effusions compared with the once-daily dosing schedule, particularly in advanced disease. In addition, a strong correlation was found between actual daily mean dose and time to onset of pleural effusions in patients treated with a daily mean dose >100 mg/day of dasatinib (P = 0.01). These data emphasize the need for dasatinib dose and schedule optimization and long-term monitoring of dasatinib-treated patients to prevent the negative clinical implications of pleural effusion.

Sensitive quantitation of minimal residual disease in chronic myeloid leukemia using nanofluidic digital polymerase chain reaction assay.

Undetectable BCR-ABL transcripts in patients with chronic myeloid leukemia (CML) should not be regarded as indicative of a cure, due to the sensitivity limit of current real-time quantitative polymerase chain reaction (RQ-PCR) technology. To demonstrate the feasibility of more sensitive approaches, 62 samples from 43 patients with CML were screened by conventional RQ-PCR, replicate RQ-PCR (rRQ-PCR), and/or nanofluidic digital PCR (dPCR). First, we confirmed the correlation of dPCR to conventional RQ-PCR using 30 patient samples with various minimal residual disease (MRD) levels. When the sensitivity limits were determined using cell line and patient sample dilutions, rRQ-PCR and dPCR with pre-amplification showed 2-3 log improvement compared to conventional RQ-PCR, and 24 of 32 PCR negative samples as assayed by conventional RQ-PCR showed detectable BCR-ABL in rRQ-PCR and/or dPCR. More important, using dPCR in conjunction with a pre-amplification step, a continuous decline in MRD level could be precisely monitored even after it became undetectable by conventional RQ-PCR. In this study, both rRQ-PCR and dPCR demonstrated successful detection of BCR-ABL transcripts not detectable in conventional RQ-PCR, and these data show the potential feasibility of highly sensitive PCR approaches for molecular monitoring and clinical relevance in future CML management by allowing further characterization of patients who achieve PCR negativity in a conventional RQ-PCR assay.

Establishment of the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL mRNA.

Serial quantitation of BCR-ABL mRNA levels is an important indicator of therapeutic response for patients with chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, but there is substantial variation in the real-time quantitative polymerase chain reaction methodologies used by different testing laboratories. To help improve the comparability of results between centers we sought to develop accredited reference reagents that are directly linked to the BCR-ABL international scale. After assessment of candidate cell lines, a reference material panel comprising 4 different dilution levels of freeze-dried preparations of K562 cells diluted in HL60 cells was prepared. After performance evaluation, the materials were assigned fixed percent BCR-ABL/control gene values according to the International Scale. A recommendation that the 4 materials be established as the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL translocation by real-time quantitative polymerase chain reaction was approved by the Expert Committee on Biological Standardization of the World Health Organization in November 2009. We consider that the development of these reagents is a significant milestone in the standardization of this clinically important test, but because they are a limited resource we suggest that their availability is restricted to manufacturers of secondary reference materials.

Chronic myeloid leukemia in the Asia-Pacific region: current practice, challenges and opportunities in the targeted therapy era.

Chronic myeloid leukemia (CML) management varies across Asia due to disparities in affluence and healthcare provision. We surveyed CML management practice at 33 hospitals in 14 countries/regions to identify treatment challenges and opportunities for harmonization. Patients were generally treated according to international guidelines; however, tyrosine kinase inhibitors (TKIs) and molecular monitoring are inaccessible to many patients not covered by national insurance or eligible for subsidized treatment. Late diagnosis and suboptimal monitoring, often due to cost and accessibility issues, are challenges. Priorities for Asia include: extending accessibility to TKIs; specialist laboratory monitoring; and enriching data to support regional CML management guidelines.

Dynamic change of T315I BCR-ABL kinase domain mutation in Korean chronic myeloid leukaemia patients during treatment with Abl tyrosine kinase inhibitors.

We analysed the dynamic change of imatinib-resistant mutations in BCR-ABL kinase domain focusing on T315I mutation during dasatinib or nilotinib therapy. Fifty-five imatinib-resistant chronic myeloid leukaemia patients (32 patients with imatinib-resistant mutations and 23 patients without mutation) in different disease phases were treated with dasatinib (median 17.3 months) or nilotinib (median 6.8 months). Among the 32 patients with baseline mutation, mutations including M244V, G250E, E255K, M351T, H396R, S417Y, E450K and E459K disappeared in 8 patients and new mutations were detected in 9 patients, all of which were T315I. Among the 23 patients without baseline mutation, 4 patients showed newly developed mutations including T315I, T315I + E459K, M244V and F359V. The T315I was the most frequently detected mutation in imatinib therapy (16%, 9 of 55) as well as in dasatinib or nilotinib therapy (24%, 11 of 44). Patients with imatinib resistant baseline mutations had a higher rate of mutation development during dasatinib or nilotinib treatment compared to patients without baseline mutations (28% vs. 17%).

Previous best responses can be re-achieved by resumption after imatinib discontinuation in patients with chronic myeloid leukemia: implication for intermittent imatinib therapy.

Although imatinib is considered as a front line therapy in patients with chronic myeloid leukemia (CML), it is still unclear whether transient imatinib discontinuation may adversely affect the outcome. This study was conducted to investigate long-term outcome after discontinuation and resumption of imatinib, and to determine whether intermittent imatinib therapy can be employed in patients with CML. Twenty six Philadelphia chromosome positive (Ph+) patients with CML discontinued imatinib when they achieved complete cytogenetic response (CCyR) or complete molecular response (CMR), and they were retreated with imatinib in case of hematologic, cytogenetic or molecular relapse. Except one patient who progressed and two patients who are in persistent molecular remission without imatinib resumption, all of 23 patients are maintaining the best response achieved after imatinib resumption with a median follow-up of 44 months. This study shows that although imatinib cannot be discontinued completely, intermittent therapy can be considered for the treatment of patients with CML in particular situations.

Analysis of Bcr-Abl kinase domain mutations in Korean chronic myeloid leukaemia patients: poor clinical outcome of P-loop and T315I mutation is disease phase dependent.

Despite durable responses to imatinib in chronic myeloid leukaemia (CML), mutations in Bcr-Abl kinase domain (KD) are known to induce imatinib resistance and cause poor clinical outcome. We characterized Bcr-Abl KD mutations in 137 Korean CML patients with imatinib resistance (n = 111) or intolerance (n = 26) using allele specific oligonucleotide polymerase chain reaction (PCR) and direct sequencing. Seventy (51%) patients harboured 81 mutations of 20 different types with increasing prevalence in advanced phase. Nine (13%) patients had multiple mutations. No mutation was found in intolerant patients. T315I was the most common mutation and P-loop was the hottest spot in Bcr-Abl KD. Patients harbouring P-loop mutation, T315I, or multiple mutations showed poor overall survival and progression free survival compared with patients harbouring other mutations. Survival analysis according to disease phase of mutation being detected and type of mutations provided correlation between P-loop or T315I mutation and poor overall survival in blast crisis, but not in accelerated phase (AP) or chronic phase (CP), indicating poor clinical outcome of particular mutations depends on disease phase. CML patients with imatinib resistance showed high rate (63%) of mutations in Bcr-Abl KD and therefore CML patients who do not respond to imatinib should be the candidates for mutation screening as molecular monitoring.

Structural modeling of V299L and E459K Bcr-Abl mutation, and sequential therapy of tyrosine kinase inhibitors for the compound mutations.

Sequential treatment with different tyrosine kinase inhibitors (TKIs) is one of the strategies for handling chronic myeloid leukemia (CML) in which dynamic change in Bcr-Abl kinase domain mutation is often an obstacle faced during TKI therapy. Here we report successful sequential therapy with different TKIs for the CML patient harboring V299L and E459K compound mutations. Molecular monitoring including quantitative analysis of BCR-ABL transcript level and mutation analysis were performed regularly for successful treatment. Additionally a drug-target complex was structurally modeled to investigate influence of amino acid substitutions on drug resistance, and to choose alternative TKI in sequential therapy, suggesting protein structural modeling can be useful approach in selecting alternative TKIs.