Chronic Myeloid Leukemia, Version 1.2019, NCCN Clinical Practice Guidelines in Oncology.

Chronic myeloid leukemia (CML) is defined by the presence of Philadelphia chromosome (Ph), resulting from a reciprocal translocation between chromosomes 9 and 22 [t(9;22] that gives rise to a BCR-ABL1 fusion gene. CML occurs in 3 different phases (chronic, accelerated, and blast phase) and is usually diagnosed in the chronic phase. Tyrosine kinase inhibitor (TKI) therapy is a highly effective first-line treatment option for all patients with newly diagnosed chronic phase CML (CP-CML). The selection TKI therapy should be based on the risk score, toxicity profile of TKI, patient's age, ability to tolerate therapy, and the presence of comorbid conditions. This manuscript discusses the recommendations outlined in the NCCN Guidelines for the diagnosis and management of patients with CP-CML.

The story of tyrosine kinase inhibitors discontinuation in clinical practice.

Chronic myeloid leukemia is the first example of successful clinical use of tyrosine kinase inhibitors (TKIs). Blockade of oncogenic BCR-ABL1 tyrosine kinase activity by imatinib translated into an impressive demonstration of clinical and biological control of the disease. Historically, the primary objective of TKIs use was to achieve long-term survival. Today this is a reality for the majority of patients. The rapid development of BCR-ABL1 quantification by RT-qPCR has facilitated the monitoring of residual disease. Molecular response has emerged as a new therapeutic objective, opening the door to TKIs discontinuation. The first prospective stop study in patients in deep molecular response was initiated in 2004, with today nearly 15 years of follow-up. The pioneering observations of this study followed by numerous stop-studies have led to a new therapeutic goal of treatment-free remission. Five cases of patients who discontinued TKIs are reviewed, illustrating the current issues and challenges surrounding this approach.

Discontinuation of tyrosine kinase inhibitors in chronic myeloid leukemia: Recommendations for clinical practice from the French Chronic Myeloid Leukemia Study Group.

BACKGROUND: The ultimate goal of chronic myeloid leukemia management in the tyrosine kinase inhibitor (TKI) era for patients who obtain deep molecular responses is maintaining a durable off-treatment response after treatment discontinuation; this situation is called treatment-free remission (TFR). Knowledge accumulated during the last 10 years justifies moving TFR strategies from research to clinical practice. METHODS: Twenty experts from the French Chronic Myeloid Leukemia Study Group (France Intergroupe des Leucémies Myéloïdes Chroniques), including 17 hematologists, 2 molecular biologists, and 1 cytogeneticist, critically reviewed published data with the goal of developing evidence-based recommendations for TKI discontinuation in clinical practice. RESULTS: Clinically relevant questions were addressed, including the selection of candidate patients (with known prognostic factors for outcomes taken into account), detailed monitoring procedures during the treatment-free phase, a definition of relapse requiring therapy resumption, and monitoring after treatment reintroduction. CONCLUSIONS: This work presents consensus statements with the aim of guiding physicians and biologists by means of pragmatic recommendations for safe TKI discontinuation in daily practice. Cancer 2018;124:2956-63. © 2018 American Cancer Society.

Bis-three-way junction nanostructure and DNA machineries for ultrasensitive and specific detection of BCR/ABL fusion gene by chemiluminescence imaging.

A novel G-quadruplex DNAzyme-driven chemiluminescence (CL) imaging method has been developed for ultrasensitive and specific detection of BCR/ABL fusion gene based on bis-three-way junction (bis-3WJ) nanostructure and cascade DNA machineries. Bis-3WJ probes are designed logically to recognize BCR/ABL fusion gene, which forms the stable bis-3WJ nanostructure for the activation of polymerase/nicking enzyme machineries in cascade, resulting in synthesis of DNAzyme subunits. These DNAzyme subunits can form integrated DNAzyme by self-assembly to catalyze CL substrate, thus providing an amplified signal for the sensing events or outputs for AND logic operation. The imaging method achieved ultrasensitive detection of BCR/ABL fusion gene with a low detection limit down to 23 fM. And this method exhibited wide linear ranges over seven orders of magnitude and excellent discrimination ability toward target. In addition, an acceptable recovery was obtained in complex matrix. It is notable that this biosensing strategy possesses merits of homogenous, isothermal and label-free assay system. Therefore, these merits endow the developed imaging method with a potential tool for CML diagnosis.

Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques.

Chronic Myeloid Leukemia (CML) is characterized by a balanced translocation juxtaposing the Abelson (ABL) and breakpoint cluster region (BCR) genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR) defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i) CML can be properly diagnosed at onset, (ii) follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1) when BCR-ABL1IS transcripts are between 1-10%, and (iii) rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients.

Incidence of Bcr-Abl kinase domain mutations in imatinib refractory chronic myeloid leukemia patients from South India.

Mutations in the Bcr-Abl kinase domain (KD) are a major cause for acquired resistance to imatinib (IM) treatment and have been associated with progression and poor prognosis in chronic myeloid leukemia patients. The present study includes 63 patients resistant to standard imatinib dose of 400 mg according to ELN guidelines. Direct sequencing method is used for mutational analysis. The present study revealed 15 exonic mutations in 46.03 % of patients; among them, seven cases (24.13 %) had multiple mutations. Mutations were found to be higher in sokal high- (45.0 %) and intermediate- (68.42 %) compared to low-risk (29.16 %) group. Mutations were observed in 38.09 % of patients with EUTOS (European Treatment and Outcome Study) high risk and in 50.0 % with low risk. The frequency of mutations was 50.0 % in advanced phase, 47.36 % in late chronic-phase, and 43.33 % in chronic-phase patients. 42.10 % of patients with primary resistance and 52.0 % with secondary resistance had mutations. P-loop and T315I mutations were associated with poor survival in advanced phase patients (85.71 %) (P = 0.03). No significant variation was observed with Bcr-Abl transcript levels between the patients with the presence or absence of mutations (P = 0.73). Bcr-Abl levels were found to be significantly elevated in P-loop and T315I mutation carriers (P = 0.001) and also in T315I mutation-positive patients (P = 0.01). P-loop mutations and T315I are frequent in advanced phases and strongly associated with poor prognosis and survival. Hence, the identification of mutations in IM-resistant CML patients will help in treatment optimization with 2nd- or 3rd-generation tyrosine kinase inhibitors (TKIs).

Prognostic value of regulatory T cells in newly diagnosed chronic myeloid leukemia patients.

BACKGROUND: Chronic myeloid leukemia (CML) is a clonal disease, characterized by a reciprocal t(9, 22) that results in a chimeric BCR/ABL fusion gene. Regulatory T cells (Tregs) constitute the main cell population that enables cancer cells to evade immune surveillance. OBJECTIVE: The purpose of our study was to investigate the level of Tregs in newly diagnosed CML patients and to correlate it with the patients' clinical, laboratory and molecular data. We also aimed to assess the effect of treatment using tyrosine kinase inhibitor (TKI) on Treg levels. METHODS: Tregs were characterized and quantified by flow cytometry in 63 newly diagnosed CML patients and 40 healthy controls. TKI was used in 45 patients with chronic phase CML, and the response to therapy was correlated with baseline Treg levels. RESULTS: The percentages of Tregs were significantly increased in CML patients compared to the controls. Treg numbers were significantly lower in patients with chronic phase CML versus the accelerated and blast phases, and were significantly lower in patients with complete molecular remission (CMR) compared to those patients without CMR. CONCLUSION: Tregs may play a role in the maintenance of CML. Moreover, the decrease of their levels in patients with CMR suggests that Tregs might have a clinical value in evaluating the effects of therapy.

[A multicenter comparison study on the quantitative detection of bcr-abl (P210) transcript levels in China].

OBJECTIVE: To investigate the comparability of bcr-abl (P210) transcript levels detected in different hospitals. METHODS: Ten hospitals in China took part in the four times of sample exchange and comparisons from April, 2010 to August, 2011. The exchange samples were prepared by Peking University People's Hospital. Firstly, the BCR-ABL (P210)(+) cells from a newly diagnosed chronic myeloid leukemia patient were 10-fold serially diluted by BCR-ABL (P210)(-) cells and they covered 4 magnitudes. Then, TRIzol reagents were thoroughly mixed with cells in each tube. Every 12 samples (three samples per magnitude) were sent to the other 9 hospitals. The cell number of each sample was 8×10(6). The detection of bcr-abl transcript levels by real-time quantitative PCR were performed in every hospital according to their own protocols. Conversion factors (CF) were calculated using regression equation. RESULTS: Differences in bcr-abl transcript levels did exist among results of 10 hospitals in each comparison. In general, the results of the most of hospitals were in line with the dilutions of cells. CF of every hospital fluctuated. Three hospitals had relatively stable CF, and their ranges were 2.8 - 5.2, 1.2 - 2.8 and 2.2 - 6.8, respectively; two hospitals had unstable CF with ranges 0.76 - 7.0 and 2.1 - 18.7; three hospitals couldn't be calculated CF one or two times because of the significant deviation of the results from the actually bcr-abl transcript levels, and their ranges of CF which could be calculated were 1.9 - 19.2, 3.6 - 7.6 and 0.18 - 14.7; One hospital only had two CF (3.3 and 5.0) because of the replacement of an important reagent during the period of comparisons. CONCLUSIONS: Comparability of bcr-abl (P210) transcript levels between different hospitals could be achieved through CF which acquired by sample exchange and comparison. The stable and reliable detection system is the premise to acquire correct CF.

A multiple reaction monitoring (MRM) method to detect Bcr-Abl kinase activity in CML using a peptide biosensor.

The protein kinase Bcr-Abl plays a major role in the pathogenesis of chronic myelogenous leukemia (CML), and is the target of the breakthrough drug imatinib (Gleevec™). While most patients respond well to imatinib, approximately 30% never achieve remission or develop resistance within 1-5 years of starting imatinib treatment. Evidence from clinical studies suggests that achieving at least 50% inhibition of a patient's Bcr-Abl kinase activity (relative to their level at diagnosis) is associated with improved patient outcomes, including reduced occurrence of resistance and longer maintenance of remission. Accordingly, sensitive assays for detecting Bcr-Abl kinase activity compatible with small amounts of patient material are desirable as potential companion diagnostics for imatinib. Here we report the detection of Bcr-Abl activity and inhibition by imatinib in the human CML cell line K562 using a cell-penetrating peptide biosensor and multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer. MRM enabled reproducible, selective detection of the peptide biosensor at fmol levels from aliquots of cell lysate equivalent to ~15,000 cells. This degree of sensitivity will facilitate the miniaturization of the entire assay procedure down to cell numbers approaching 15,000, making it practical for translational applications in patient cells in which the limited amount of available patient material often presents a major challenge.

Detection of a rare BCR-ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS).

Hypothesis directed proteomics offers higher throughput over global analyses. We show that immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) in H929 multiple myeloma (MM) cancer cells led to the discovery of a rare and unexpected BCR-ABL fusion, informing a therapeutic intervention using imatinib (Gleevec). BCR-ABL is the driving mutation in chronic myeloid leukemia (CML) and is uncommon to other cancers. Three different IP-MS experiments central to cell signaling pathways were sufficient to discover a BCR-ABL fusion in H929 cells: phosphotyrosine (pY) peptide IP, p85 regulatory subunit of phosphoinositide-3-kinase (PI3K) IP, and the GRB2 adaptor IP. The pY peptides inform tyrosine kinase activity, p85 IP informs the activating adaptors and receptor tyrosine kinases (RTKs) involved in AKT activation and GRB2 IP identifies RTKs and adaptors leading to ERK activation. Integration of the bait-prey data from the three separate experiments identified the BCR-ABL protein complex, which was confirmed by biochemistry, cytogenetic methods, and DNA sequencing revealed the e14a2 fusion transcript. The tyrosine phosphatase SHP2 and the GAB2 adaptor protein, important for MAPK signaling, were common to all three IP-MS experiments. The comparative treatment of tyrosine kinase inhibitor (TKI) drugs revealed only imatinib, the standard of care in CML, was inhibitory to BCR-ABL leading to down-regulation of pERK and pS6K and inhibiting cell proliferation. These data suggest a model for directed proteomics from patient tumor samples for selecting the appropriate TKI drug(s) based on IP and LC-MS/MS. The data also suggest that MM patients, in addition to CML patients, may benefit from BCR-ABL diagnostic screening.

Purification of TAT-CC-HA protein under native condition, and its transduction analysis and biological effects on BCR-ABL positive cells.

BCR-ABL oncoprotein is the cause of chronic myeloid leukemia. The homologous oligomerization of BCR-ABL protein mediated by BCR coiled-coil (CC) domain plays an important role in ABL kinase activation. The HIV-1 TAT peptide has been used extensively for the introduction of proteins into cells. We recombinated a TAT-CC-HA protein to interrupt the homologous oligomerization of BCR-ABL. The expression conditions for TAT-CC-HA were optimized. The TAT-CC-HA fusion protein was purified with Ni+-NTA resin. TAT-CC-HA fusion protein was added into the cultures of Ba/F3-p210, 32D-p210, K562, KU812, Ba/F3, 32D, and HL-60 cells. It was found that TAT-CC-HA could transduce into these cells. It was confirmed that TAT-CC-HA fusion protein was internalized by Ba/F3-p210, K562, and Ba/F3 cells and located in the cytoplasm observed by confocal laser scanning fluorescence microscope. The transduction of TAT-CC-HA fusion protein into K562 cells was in a dose-dependent and time-dependent manner. The result of coimmunoprecipitation assay indicated that TAT-CC-HA could interact with BCR-ABL in K562 cells. The effects of TAT-CC-HA fusion protein on cell growth and apoptosis were detected by MTT test and flow cytometry. Our findings suggested that TAT-CC-HA fusion protein could specifically inhibit the growth of BCR-ABL positive cells, and specifically induce apoptosis of BCR-ABL positive cells, while not affect the growth and apoptosis of BCR-ABL negative cells.

Cloning, expression, purification and functional characterization of the oligomerization domain of Bcr-Abl oncoprotein fused to the cytoplasmic transduction peptide.

Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require accurate cellular localization for function. Cytoplasmic transduction peptide (CTP) is a newly designed transduction peptide that carries molecules across the cell membrane with a preference to localize in the cytoplasmic compartment and is, therefore, applicable for cytoplasmic targeting. The Bcr-Abl fusion protein, playing major causative role in chronic myeloid leukemia (CML), is a cytoplasmic oncoprotein that contains an N-terminus oligomerization domain (OD) mediating homodimerization of Bcr-Abl proteins, and an intact OD in Bcr-Abl is required both for the activation of its transforming activity and tyrosine kinase. Therefore, disrupting Bcr-Abl oligomerization represents a potential therapeutic strategy for inhibiting Bcr-Abl oncogenicity. In this study, we explored the possible homodimerization-disrupting and tyrosine kinase inhibiting effect of the transduction of OD in Bcr-Abl positive K562 cells. By expressing in Escherichia coli a CTP-OD-HA fusion protein followed by Ni+-NTA affinity purification, immunoblot identification and enterokinase cleavage, we showed that the CTP-OD-HA protein was structurally and functionally active in that it potently transduced and primarily localized into the cytoplasmic compartment, heterodimerized with Bcr-Abl, and potently inhibited the phospho-tyrosine pathways of Bcr-Abl oncoprotein at a low concentration of 4 microM. These results delineate strategies for the expression and purification of therapeutic molecules for intracytoplasmic protein based therapeutics and the CTP-OD-HA-mediated killing strategy could be explored as a promising anti-leukemia agent or an adjuvant to the conventional therapeutic modalities in chronic myeloid leukemia, such as in vitro purging.

[Real-time PCR quantification of bcr/abl chimera and WT1 genes in chronic myeloid leukemia].

Quantification of mRNAs deriving from malignant cells is useful for estimating leukemic states. In this study, we have developed RT-PCR methods using real-time PCR detection system, a LightCycler, for quantification of bcr/abl chimerical genes in peripheral blood and bone marrow of chronic myeloid leukemia patients. Total amounts of RNA extracted were corrected using beta-actin gene as an internal standard. The coefficients of variation of intra-assay variation and inter-assay variation for each gene were within a range of 1.7-26.0% which showed more precise quantification than the competitive PCR method. The coefficients of variation of assay are within a range of 7.7-27.6% in the case of using three samples of normal subjects from blood collecting to quantification of bcr gene. Bcr/abl and WT1 genes could be measured from 10(2) to 10(8) copies and 10 to 10(5) copies with linearity, respectively. Using real-time PCR detection with LightCycler system, 2 x 10(3) K562 cells among 2 x 10(6) total cells demonstrated the bcr/abl gene, while 2 x 10(1) K562 cells among 2 x 10(6) total cells could be detected using the nested PCR method. In tests of seven clinical samples, five samples demonstrated bcr/abl and WT1 genes, while those in two other patients after bone marrow transplantation and a normal subject could not detected. This result suggests that our quantitative method reflect the clinical stages of CML patients.

Polarized distribution of Bcr-Abl in migrating myeloid cells and co-localization of Bcr-Abl and its target proteins.

Bcr-Abl plays a critical role in the pathogenesis of Philadelphia chromosome-positive leukemia. Although a large number of substrates and interacting proteins of Bcr-Abl have been identified, it remains unclear whether Bcr-Abl assembles multi-protein complexes and if it does where these complexes are within cells. We have investigated the localization of Bcr-Abl in 32D myeloid cells attached to the extracellular matrix. We have found that Bcr-Abl displays a polarized distribution, colocalizing with a subset of filamentous actin at trailing portions of migrating 32D cells, and localizes on the cortical F-actin and on vesicle-like structures in resting 32D cells. Deletion of the actin binding domain of Bcr-Abl (Bcr-AbI-AD) dramatically enhances the localization of Bcr-Abl on the vesicle-like structures. These distinct localization patterns of Bcr-Abl and Bcr-Abl-AD enabled us to examine the localization of Bcr-Abl substrate and interacting proteins in relation to Bcr-Abl. We found that a subset of biochemically defined target proteins of Bcr-Abl redistributed and co-localized with Bcr-Abl on F-actin and on vesicle-like structures. The co-localization of signaling proteins with Bcr-Abl at its sites of localization supports the idea that Bcr-Abl forms a multi-protein signaling complex, while the polarized distribution and vesicle-like localization of Bcr-Abl may play a role in leukemogenesis.

A new method for the detection of bcr/abl sequences amplified by polymerase chain reaction (PCR) following bone marrow transplantation.

Disease recurrence is a major problem in patients receiving bone marrow transplantation for chronic myeloid leukemia. Residual malignant cells are the most likely source of recurrence. Detection of minimal residual disease early during therapy may provide an additional prognostic value and help in identifying patients who are at high risk of relapse. PCR followed by hybridization is the most sensitive method to investigate the persistence of leukemic cells. However, more reproducible methods suitable to standardization and quantification are required in clinical practice. In this study, we describe a novel PCR assay combined with immunological and colorimetric detection of the bcr-rearrangement. Residual bcr/abl rearranged cells were observed in 7 patients. Our results show that the assay is equally sensitive as RT-PCR, more versatile in terms of standardization and easily adaptable as a diagnostic test.

P210 Bcr-Abl interacts with the interleukin-3 beta c subunit and constitutively activates Jak2.

Chronic myelogenous leukemia is a neoplasm of pluripotent hematopoietic cells. Cytokines such as interleukin-3 and granulocyte-macrophage colony-stimulating factor regulate the growth and differentiation of hematopoietic precursors. These cytokines activate two distinct signals to the nucleus. One signal is through the Ras pathway, and the second involves activation of Jak2. We demonstrated that Bcr-Abl co-immunoprecipitates with and constitutively phosphorylates the common beta c chain of the interleukin-3 (IL-3) and granulocyte-macrophage-macrophage colony-stimulating factor (GM-CSF) receptors. Our data show that formation of this complex leads to the constitutive activation of Jak2. Previously, it has been demonstrated that Bcr-Abl interacts with Grb2 and Shc, which in turn activates the Ras pathway. Thus, Bcr-Abl can activate signalling through both pathways in a factor-independent fashion.

[Fluorescent in-situ hybridization technique (FISH) in the diagnosis of Philadelphia translocation in chronic myeloid leukemia]. / Application de la technique d'hybridation in situ fluorescente (FISH) au diagnostic de la translocation de Philadelphie dans la leucémie myéloïde chronique.

The Philadelphia chromosome (Ph) resulting from translocation t(9;22)(q34;q11) is observed in more than 90% of patients with chronic myeloid leukemia (CML). Its molecular consequence is the genesis of a fusion gene BCR-ABL between the 5' sequences of the BCR gene (chromosome 22) and the 3' end of the ABL gene (chromosome 9). Fluorescence in situ hybridization (FISH) using specific DNA probes provides a useful tool for the detection of t(9;22) and BCR-ABL rearrangement. We report our results using the FISH technique for t(9;22) assessment in the hematopoietic cells of patients with Ph-positive CML. The DNA libraries pBS 9 and pBS 22 containing multiple sequences derived from chromosomes 9 and 22 have been used to identify t(9;22) in metaphase cells. The cos bcr-51 and cos abl-18 probes that hybridize to unique sequences specific to the BCR and ABL genes have the ability to detect the BCR-ABL rearrangement in metaphase cells as well as in interphase nuclei. FISH is a sensitive and specific technique that represents a valuable complement to conventional cytogenetics. The BCR-ABL rearrangement can be detected in metaphase spreads of insufficient quality or from interphase nuclei in the case of terminally differentiated cells or of cells which do not divide in vitro. When the efficiency of hybridization and detection is good, a large number of cells can be analyzed. This is of major significance in assessment of response to treatment and definition of a cytogenetic remission. However, interphase cytogenetics may be difficult due to variations in signal resolution and background level. The FISH technique can also be used to detect the BCR-ABL rearrangement in cases of Ph negative BCR-ABL positive CML.