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Introduction: Ponatinib exhibits a high inhibition potency on wild-type and most mutated forms of the BCR:ABL1 kinase, but also a significant cardiovascular toxicity. Improving the efficacy/safety ratio should allow patients to safely draw benefit from the drug., Areas Covered: Based on pharmacological findings and international guidelines on chronic myeloid leukemia and cardiovascular risk management, as well as on the most recent data collected in real-life studies and in a randomized phase II trial, we propose a decision-tree of dose selection of the drug., Expert Opinion: We distinguish (1) highly resistant patients according to poor previous response to second generation tyrosine kinase inhibitors (complete hematologic response or less) or to mutational status (T315I, E255V, alone or within compound mutations), requiring a starting daily dose of 45 mg, reduced to 15 or 30 mg according to the patient's profile, preferentially upon major molecular achievement (3-log reduction or MR3, BCR:ABL1 ≤ 0.1% IS ); (2) less-resistant patients justifying an initial dose of 30 mg, reduced to 15 mg upon MR2 (BCR:ABL1 ≤ 1% IS ) or preferentially MR3 in patients with a favorable safety profile; (3) intolerant patients to be treated by 15 mg.

Introduction: Chronic myeloid leukemia is now a highly treatable leukemia due to the availability of multiple tyrosine kinase inhibitors (TKIs) inhibiting the BCR-ABL1 oncogene. Some patients with CML can display resistance or intolerance to multiple TKIs, oftentimes due to the presence of mutations in BCR-ABL1, such as T315I, which limits effective treatment options. Ponatinib is a third-generation, rationally-designed TKI with clinically meaningful activity in this difficult-to-treat population. Ponatinib is associated with an increased risk of arterial occlusive events (AOEs) which has required a reexamination of its dosing in order to limit the risk of these events., Areas Covered: This review will provide an overview of the mechanism of action of ponatinib and the safety and efficacy data from clinical trials in chronic myeloid leukemia., Expert Opinion: Ponatinib is a potent pan-BCR-ABL1 TKI with substantial activity in patients with more resistant or advanced CML. Its efficacy needs to be balanced with the increased risk of vascular events, which seems to be at least partially diminished by the implementation of mitigation strategies aimed at modifying cardiovascular risk factors and adaptive dosing of the drug.

We report the long-term outcome of 139 patients treated with imatinib in late chronic phase after IFN failure. Median follow-up was 16.6 years and the estimated 18-year OS was 64.8%. 18-year EFS and PFS were 69% and 64.4%, respectively. Fifty (36%) patients stopped imatinib, 72% received a second line. b2a2 transcript was associated with a significantly inferior 18-year OS ( p  = 0.008), FFS ( p  = 0.036), PFS ( p  = 0.013) compared to the b3a2 type, whilst the type of transcript did not influence the time to response achievement. Failure to achieve MMR at 12 months significantly reduced the chance of reaching a DMR ( p  = 0.001). Imatinib discontinuation after achieving a sustained deep molecular response was attempted in 14 patients; 12 (86%) are still in treatment-free remission (TFR) at the last follow-up. Our experience confirms the long-term efficacy of imatinib after IFNα failure in real-life setting and documents the possibility of attempting a TFR in this subset of patients.

To evaluate long-term real-life results of dasatinib therapy among chronic phase chronic myeloid leukemia patients resistant or intolerant to imatinib, we retrospectively analyzed data of 118 patients treated in centers participating in the database INFINITY. With median follow-up of 37 months, estimated 5-year cumulative incidences of complete cytogenetic and major molecular responses were 78% and 68%, respectively. The estimated 5-year probability of overall survival (OS) and event-free survival (EFS) were 86% and 83%, respectively. Both OS and EFS were significantly improved among patients with BCR-ABL1 transcript level ≤10% at 3 months. Dasatinib toxicity was tolerable however persistent in almost half our patients, even after years of therapy. Pleural effusion occurred in 29% of patients and was responsible for 30% of dasatinib discontinuations. Our results confirmed very good efficacy and acceptable toxicity of dasatinib in second line setting and support the evidence and importance of high-quality real-life CML patient management.

Treatment-free remission (TFR) in patients with chronic myeloid leukemia in chronic phase (CML-CP) is considered a feasible option, especially with the ability of second-generation tyrosine kinase inhibitors to induce higher rates of sustained deep molecular response (DMR). DASFREE is an open-label, single-arm, multicenter phase II trial assessing TFR after dasatinib discontinuation in patients with CML-CP ( N  = 84). At 2 years, TFR was 46% in all patients. Multivariate analyses revealed statistically significant associations between 2-year TFR and duration of prior dasatinib (≥median; p = .0051), line of therapy (first line; p = .0138), and age (>65 years; p = .0012). No disease transformation occurred, and the most common adverse events experienced off treatment were musculoskeletal (observed in 30 patients); however, dasatinib withdrawal events were reported in nine patients (11%) by the investigator. Overall, these findings support the feasibility of discontinuing dasatinib for patients with CML-CP in sustained DMR in the first line and beyond.

Forty-three chromosomal abnormalities in Philadelphia-negative metaphases (Ph-CAs) appeared in 35 of 432 patients in chronic phase chronic myeloid leukemia (CP-CML) undergoing tyrosine kinase inhibitor (TKI) treatments. These CAs were mostly common in trisomy-8 (16 cases), trisomy-Y (five cases), and monosomy-7 (five cases). Furthermore, Ph- CAs were significantly associated with higher platelet count (494 × 10 9 /L vs. 326 × 10 9 /L, p  = .006), and higher incidence of true clonal evolution in Ph-positive metaphase (22.9% vs. 9.1%, p  = .017). Additionally, patients with Ph- CAs had worse rates of complete cytogenetic remission (76% vs. 86%, p  = .0091), major molecular remission (55% vs. 76%, p  = .001), progression-free survival (47% vs. 86%, p  < .001), but a similar overall survival rates compared to those in patients without Ph- CAs. In conclusion, Ph- CAs may predict worse response to TKI therapies and survival in patients with CP-CML, thus requiring close cytogenetic monitoring.

Chronic myeloid leukemia (CML) is a stem cell derived malignant disorder result of translocation t(9;22)(q34;q11) called Philadelphia chromosome (Ph + ). microRNAS (miRNAs) are involved in several biological processes, altering the progression of various pathologies, including CML. This study evaluated whether circulating miRNAs display differential expression profiles in peripheral blood of CML-Chronic Phase (CML-CP) patients newly diagnosed in comparison with CML-CP treated with imatinib. We obtained peripheral blood samples from CML-CP Ph + patients divided among group 1 (untreated newly diagnosed) and group 2 (treated with imatinib). A pool of total leukocytes from healthy donors was considered as control group. Expression analyses were performed for 768 miRNAs by RT-qPCR array. Bioinformatic tools were used to identify significant pathways and interaction networks. We found 80 deregulated miRNAs between the groups and, according to bioinformatic analysis, they are involved in different pathways, including molecular mechanisms of cancer. The study allows better understanding of disease molecular behavior, and it is useful for possible monitoring CML treatment and prognostic biomarkers identification.

Imatinib has been successful in the management of chronic myeloid leukemia (CML) but some patients experience adverse reactions or develop resistance to its use. The roles of some polymorphisms in genes encoding enzymes critical for the biotransformation of imatinib have been previously examined. This study, hence, evaluated some other unstudied functionally significant polymorphisms in CYP1A2, CYP2C8, CYP2C9, and CYP3A5. Trough imatinib blood levels and genotypes were determined in 42 CML patients by an HPLC-UV technique and a Sequenom iPLEX assay, respectively. Statistical analysis of the influence of genetic polymorphisms on standardized trough level detected no significant relationship. However, higher trough levels were observed in two homozygous carriers of CYP2C8*2 while diminished imatinib levels were seen in two homozygous carriers of CYP3A5*7. The study findings suggest that polymorphisms in drug metabolizing enzymes may be significant for imatinib therapy only in instances where all copies of the relevant studied genes are functionally impaired.

Tyrosine kinase inhibitor (TKI) therapy is the current treatment of choice for patients with chronic phase chronic myeloid leukemia (CML) leading to rapid and durable hematological as well as molecular responses. However, emergence of resistance to TKIs has been the major obstacle to treatment success on long term. In this regard kinase domain mutations are the most common mechanism of therapy failure. In this study, we analyzed peripheral blood samples from 17 CML patients who had developed resistance to various TKIs by using next-generation sequencing parallel to Sanger sequencing. BCR-ABL1 kinase domain mutations have been found in 59% of the cohort. Our results demonstrate that next-generation sequencing results in a higher mutational detection rate than reported with conventional sequencing methodology. Furthermore, it showed the clonal diversity more accurately.

Patients diagnosed with chronic myeloid leukemia (CML) and treated with BCR-ABL1 tyrosine kinase inhibitors (TKIs) have long life spans. Selection of an appropriate first-line therapy can be difficult as both the unique characteristics of each TKI and patient need to be taken into account to find the optimal match. Patient characteristics include comorbidities, concomitant medications, lifestyle, risk factors, BCR-ABL1 transcript type (e.g. b2a2 or b3a2) and additional chromosomal abnormalities. Just as patients differ, side effects, drug-drug interactions, administration plans, dosing schedules and treatment-related expenses across TKIs also vary. Alignment of these characteristics with the appropriate TKI is key to successfully initiating CML treatment. Continued success relies on communication between the patient and the healthcare team, adherence and optimization of therapy once it is initiated. In this review, we discuss these factors, in addition to TKI efficacy and safety, the cost of therapy, the future of treating CML and treatment-free remission.

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.

We conducted this study to identify the factors for predicting poor outcomes in chronic myeloid leukemia patients who failed to achieve a 3-month early molecular response (EMR). Of the 413 newly diagnosed, chronic phase, chronic myeloid leukemia patients receiving imatinib (IM), 120 (29.1%) failed to achieve a 3-month EMR. With a median follow-up of 67.0 months, 39 patients continued IM treatment with at least complete cytogenetic response (CCyR), and 81 patients permanently discontinued IM treatment. The cumulative incidence rates of CCyR and major molecular response (MMR) by 3 years were 90.1 ± 3.9% and 53.7 ± 7.3%, respectively. After adjusting for potential factors, multivariate analyses showed that a transcript type of e13a2, compared with e14a2, and a larger spleen size were independent factors for failure of overall MMR. The predictive factors outlined in this study may provide valuable information for high-risk patients who would benefit from early decision-making regarding therapy change.

Multiple BCR-ABL tyrosine kinase inhibitors (TKIs) are available for the treatment of chronic myeloid leukemia in chronic phase (CML-CP), and several baseline and on-treatment predictive factors have been identified that can be used to help guide TKI selection for individual patients. In particular, early molecular response (EMR; BCR-ABL ≤10% on the International Scale at 3 months) has become an accepted benchmark for evaluating whether patients with CML-CP are responding optimally to frontline TKI therapy. Failure to achieve EMR is considered an inadequate initial response according to the National Comprehensive Cancer Network guidelines and a warning response according to the European LeukemiaNet recommendations. Here we review data supporting the importance of achieving EMR for improving patients' long-term outcomes and discuss key considerations for selecting a frontline TKI in light of these data. Because a higher proportion of patients achieve EMR with second-generation TKIs such as nilotinib and dasatinib than with imatinib, these TKIs may be preferable for many patients, particularly those with known negative prognostic factors at baseline. We also discuss other considerations for frontline TKI choice, including toxicities, cost-effectiveness, and the emerging goals of deep molecular response and treatment-free remission.

The protein tyrosine phosphatase SHP-1 dephosphorylates BCR-ABL1, thereby serving as a potential control mechanism of BCR-ABL1 kinase activity. Pathways regulating SHP-1 expression, which could be exploited in the therapeutics of TKI-resistant chronic myeloid leukemia (CML), remain unknown. Moreover, the questions of whether there is any kind of SHP-1 deregulation in CML, contributing to disease initiation or evolution, as well as the question of prognostic significance of SHP-1, have not been definitively answered. This study shows moderately lower SHP-1 mRNA expression in chronic phase CML patients in comparison to healthy individuals and no change in SHP-1 mRNA levels after successful TKI treatment. Mutational analysis of the aminoterminal and phosphatase domains of SHP-1 in patients did not reveal genetic lesions. This study also found no correlation of SHP-1 expression at diagnosis with response to treatment, although a trend for lower SHP-1 expression was noted in the very small non-responders' group of the 3-month therapeutic milestone.

A better understanding of events triggering chronic myeloid leukemia progression is critical for optimized clinical management of chronic myeloid leukemia (CML). We sought to validate that increased expression of Musashi 2 (MSI2), a post-transcription regulator, is associated with progression and prognosis. Screening of 152 patients with CML showed that MSI2 was significantly decreased among patients with CML in chronic phase (CP) at diagnosis (p < 0.0001), but found no significant difference between the normal control group and treated patients with CML in CP. Moreover MSI2 was significantly increased (p < 0.0001) in patients with advance disease (AD) CML. Furthermore, our human hematopoietic cell line data imply that MSI2 and BCR-ABL1 mRNA expression are correlated. However, these data cast a doubt on earlier reports that MSI2 effects HES1 expression via NUMB-NOTCH signaling.

Background: Genetic variations in membrane transporters may contribute to imatinib mesylate (IM) resistance in chronic myeloid leukemia (CML). Objective To investigate the relationship between SLCO1B3, SLCO1A2, and ABCA3 polymorphisms and IM response in CML patients., Methods: Patients in chronic phase CML (N = 118) were studied. All patients were treated with a standard dose of IM (400 mg/day) and classified into one of the two groups according to their responses. Major molecular response (MMR) and complete molecular response (CMR) were evaluated. Criteria for response failure were established according to European LeukemiaNet (2009). Analysis of the SLCO1B3 c.334T > G (rs4149117) and c.699G > A (rs7311358), SLCO1A2 c.516A > C (rs11568563) and c.-62-361G > A (rs3764043), and ABCA3 c.1755C > G (rs323043) and c.4548-191C > A (rs150929) polymorphisms was carried out by real-time polymerase chain reaction., Results: SLCO1A2 and ABCA3 polymorphisms have similar frequencies between responders and non-responders. SLCO1B3 699GG and 344TT genotypes were more frequent in the responder group (63.8%) than in the non-responder group (44.7%, P = 0.042). Furthermore, carriers of 699GA/AA and 334TG/GG genotypes presented a higher probability of not responding to the standard dose of IM (odds ratio: 2.17; 95% confidence interval: 1.02-4.64, P = 0.04). Poor CMR for ABCA3 4548-91C > A was observed in patients with the CC/CA genotype when compared to AA carriers in the responder group (P = 0.014)., Conclusions: SLCO1B3 699GG and 344TT genotypes are associated with non-response to IM, while ABCA3 4548-91 CC/CA genotypes are related to poor CMR in CML patients treated with standard-dose imatinib.

Abstract With improved outcome for patients with chronic myeloid leukemia (CML) treated with tyrosine kinase inhibitors (TKIs), treatment discontinuation has become increasingly attractive to patients. We analyzed the outcomes of patients who chose to discontinue TKI therapy regardless of their ongoing response. Thirty-five patients with chronic phase CML discontinued TKI in complete cytogenetic response. Of them, 51% discontinued due to adverse effects, 23% due to long complete molecular response (CMR) (> 5 years), 9% due to pregnancy and 17% due to financial problems. After TKI discontinuation, patients were followed for a median of 16 months. Among 27 patients (77%) who discontinued TKIs in CMR, 11 (41%) had a molecular relapse after a median of 3.5 months. In univariate analysis we observed that patients with ≥ 64 months of CMR before TKI discontinuation had superior cumulative proportions of sustained CMR and major molecular response (MMR) at 12 months after discontinuation: 88.9% vs. 45.5% (p = 0.02) and 100% vs. 75% (p = 0.05), respectively. Patients treated with high dose imatinib or second generation TKIs had a higher cumulative proportion of sustained MMR at 12 months after discontinuation than patients treated with standard dose imatinib: 100% vs. 72.2% (p = 0.03), respectively. Of the five patients who stopped TKI in MR(4.5) (molecular response of 4.5-log reduction) one lost cytogenetic response. All three patients who discontinued TKIs in MMR lost cytogenetic response; one progressed to accelerated phase. Thirteen patients (37%) restarted TKIs after loss of response: 11 improved their response, and for two it is too early to assess. Treatment discontinuation can lead to sustained CMR in some patients, but risk of relapse is higher if patients discontinue TKIs when not in CMR.

Asian patients with chronic myeloid leukemia (CML) tend to have different characteristics compared with patients from other regions, including younger age and smaller body size. The phase 3, open-label, randomized DASISION trial (NCT00481247), comparing dasatinib 100 mg once daily (QD) (n = 259) with imatinib 400 mg QD (n = 260) in newly diagnosed chronic phase CML (CML-CP), included a sizeable East Asian population (n = 60: dasatinib; n = 48: imatinib). In East Asian patients, dasatinib showed favorable 24-month rates of major molecular response (68% vs. 50% for imatinib) and complete cytogenetic response (92% vs. 88%), and more patients achieved BCR-ABL1 transcript levels ≤ 10% at 3 months with dasatinib (91% vs. 69%), similar to the overall population. Relative to non-East Asian patients, the incidence of rash, fluid-related events and grade 3/4 neutropenia and thrombocytopenia appeared to be higher in East Asians, regardless of treatment. Pharmacokinetic analysis revealed statistically non-significant increased dasatinib exposure among East Asian patients. Results support the use of dasatinib 100 mg QD as first-line CML treatment in both East Asian and non-East Asian patients.

Dasatinib has demonstrated efficacy in patients with chronic-phase chronic myeloid leukemia (CML) who had resistance or intolerance to imatinib. However, some patients also develop resistance or intolerance to dasatinib. To identify potential molecular pathways involved in primary resistance to dasatinib in CML, we analyzed gene expression profiles of mononuclear cells of 7 imatinib-resistant patients, collected before and after 1-year dasatinib treatment. Large-scale gene expression was measured with Agilent microarrays covering protein-coding genes and long (>200 nt) noncoding RNAs (lncRNAs). Sets of genes and lncRNAs significantly differentially expressed (>1.5 fold-change; q value ≤10%) were identified. Ingenuity Pathway Analysis pointed to a number of functions, canonical pathways and gene networks that were significantly enriched with differentially expressed genes. In addition to protein-coding genes, lncRNAs have been recently implicated in pathways leading to tumorigenesis. Our data point to new possible regulatory elements involved in dasatinib resistance in CML.

The mitochondrial respiratory chain (MRC) consists of protein complexes I, II, III, IV and V that support oxidative phosphorylation (OXPHOS), which depends on electron transport to generate adenosine triphosphate (ATP). Electron "leakage" from the MRC generates reactive oxygen species (ROS). Chronic myeloid leukemia in chronic phase (CML-CP) stem cells (LSCs) produce high levels of mitochondrial ROS, causing oxidative DNA damage, resulting in genomic instability, generating imatinib-resistant BCR-ABL1 kinase mutants and additional chromosomal aberrations. Using global mRNA microarray analysis combined with Ingenuity Pathway Analysis we found that LSCs display enhanced expression of genes encoding MRC complexes I, II, IV and V. However, expression of genes encoding complex III was deregulated. Treatment with imatinib did not correct the aberrant levels of MRC genes. Therefore we postulate that abnormal expression of MRC genes may facilitate electron "leakage" to promote the production of ROS and accumulation of genomic instability in LSCs in imatinib-naive and imatinib-treated patients.

To investigate the correlation of trough imatinib mesylate (IM) levels with cytogenetic or molecular responses, we measured trough IM levels in patients with chronic myeloid leukemia, chronic phase (CML-CP), at 6 months of treatment with a standard dose of IM. Eighty-seven newly diagnosed patients with CML-CP were prospectively enrolled. Seventy-eight patients (89.7%) showed an optimal response (complete or partial cytogenetic response) at 6 months. Trough IM levels were 1378 ± 725 ng/mL. When categorized into two groups, there was a statistically significant difference in numbers of patients with optimal and suboptimal responses at 6 months (group with  <1000: 80.6% vs. 19.4%;  ≥ 1000: 94.6% vs. 5.4%; p = 0.032), and in numbers of patients with early major molecular response (early-MMR) and without MMR at 6 months (group with  <1000: 3.2% vs. 96.8%;  ≥ 1000: 21.4% vs. 78.6%; p = 0.047). In conclusion, the incidence of optimal cytogenetic response or early-MMR in patients with CML-CP treated with IM for 6 months was significantly higher in those with a trough level of  ≥ 1000 compared with those with a level of <1000. Dose escalation of IM can be one option in patients with CML showing suboptimal response or resistance to the standard dose of IM, especially with low trough plasma IM levels (<1000 ng/mL).

Acquired resistance to imatinib in the advanced phase of chronic myeloid leukemia (CML) has been associated with mutations in the kinase domain (KD) of BCR-ABL. On the contrary, the prognostic implication of KD mutations in early chronic phase (CP) patients at diagnosis before imatinib-based therapy has not yet been established. We have reviewed the status of mutations in 43 patients with early CP-CML on the samples collected at diagnosis. Mutations were identified by direct sequencing (DS) with BidDye Terminator v 1.1. cycle sequencing kit and analyzed with a 3130 ABI capillary electrophoresis system. Eight out 13 (61.5%) high Sokal risk patients showed the following mutations: Y253C, S265R, E255K, F359Y, N374S, E255V, E255V, E255V. Three patients progressed during imatinib and second-line inhibitors and died of blastic phase CML at 23, 33, and 69 months. Another patient with intermediate Sokal risk showed D363G mutation at diagnosis, progressed under imatinib, was allografted and he is now alive in major molecular remission (MMR). No low-risk patient carried KD mutation at diagnosis. In conclusion, KD mutations conferring high-level imatinib resistance are present in patients with de novo CML and in some of them lead to disease progression.

Chronic myelogenous leukemia (CML) is genetically characterized by the reciprocal translocation of chromosome 9 and 22. Around 5-8% of CML develop complex variant Ph translocations involving one or more chromosomal regions besides 9 and 22. Chromosome 3 is not frequently involved in complex translocations in CML. We report in this study a case of CML displaying a t(3;9;22) 3-way translocation. A review of the literature appears to indicate that CML patients with this translocation tend to have an aggressive course and poor outcome. Additional 3-way chromosome translocations associated with CML are also reviewed.

Chronic myeloid leukemia (CML) is characterized by the formation of the Philadelphia chromosome and oncogenic signaling by the resulting Bcr-Abl fusion protein. Understanding the molecular basis of CML has led to the development of highly effective targeted therapies that block Bcr-Abl tyrosine kinase activity. Imatinib, the current first-line therapy for CML, induces durable treatment responses in most patients. However, patients may develop imatinib resistance, which is often due to BCR-ABL mutations. With the availability of second generation tyrosine kinase inhibitors, an effective therapeutic option other than stem cell transplantation is available following imatinib failure. Randomized trial data suggest that dasatinib treatment is superior to imatinib dose escalation in patients with imatinib resistance. Nilotinib, a recently approved analogue of imatinib, has also demonstrated encouraging treatment responses in patients with imatinib-resistant CML. Other agents (including bosutinib and INNO-406) are in clinical development. With the potential availability of multiple treatment options for patients with CML, it may be possible to tailor treatment according to individual patient or disease characteristics, for example, BCR-ABL mutations. Future CML treatment may involve combination strategies. Overall, targeted agents have significantly improved the prognosis of patients diagnosed with CML.

Using IgH DNA fingerprinting we have previously demonstrated clonal immunoglobulin heavy chain (IgH) gene rearrangements during chronic phase (CP) chronic myeloid leukemia (CML) in patients destined to develop lymphoid blast crisis (L-BC). In view of this we decided to follow a cohort of CP CML patients to determine the frequency with which abnormal IgH fingerprints are found and their relationship, if any, to treatment regimen. Thirty three, initially CP, CML patients were studied on 111 occasions over a 16 month period using consensus PCR amplification of the third complementarity determining region (CDR3) of the IgH gene and high resolution polyacrylamide gel electrophoresis (IgH DNA fingerprinting). Of these 33 patients, thirteen received interferon-alpha (IFN) containing regimens and 15 non-IFN containing regimens throughout the study period. Five patients received variable therapy. During the period of observation 7 patients experienced disease progression: 5 accelerated phase, I L-BC and I myeloid blast crisis (M-BC). Abnormal IgH fingerprints were seen in 29 of the 111 (26%) specimens analysed. The 28 patients who received uniform therapy (IFN or non-IFN) over the 16 months were classified as "normal" (n = 18, normal pattern on all occasions) or "abnormal" (n = 10, abnormal on 1 or more occasions). Analysis by patient group (normal vs abnormal) showed that fingerprint abnormalities were associated with an elevated peripheral blood lymphocyte count (p = 0.0001) but not with changes in the total white cell count. Comparison of the IFN vs. non-IFN groups showed the former all had normal patterns whereas 10 of 15 non-IFN therapy patients were abnormal (p = 0.00023). The peripheral blood lymphocyte counts in the normal vs abnormal patients within the non-IFN group were not significantly different. The patient who developed L-BC demonstrated a persistent IgH fingerprint pattern abnormality from 7 months prior to the diagnosis of L-BC. The M-BC patient had a normal pattern at all times. We conclude that: (1) abnormal IgH fingerprints are found in a significant number of CP CML patients; (2) in this cohort the use of IFN was associated with normal CP CML IgH fingerprints, and (3) detection of abnormal IgH fingerprints may be highly predictive for the lineage of impending blast crisis.

Antisense oligonucleotides were used to determine the role of the BCR-ABL gene in the proliferation of chronic myeloid leukaemia (CML) clonogenic cells. Peripheral blood Philadelphia chromosome positive cells were obtained from eight CML patients at diagnosis (chronic phase = 7; accelerated phase = 1). Mononuclear cells were incubated with synthetic antisense 18-mer oligonucleotides complementary to the two different junctions b2a2 or b3a2. The type of junction (b2a2 or b3a2) was previously determined by RT-PCR techniques. Cells incubated for 12 to 14 hours with or without sense oligonucleotides served as controls. After incubation with oligonucleotides, the cell DNA synthesis was analysed by flow cytometry using the BrdUrd/DNA method and the cell plating efficiency in methylcellulose was determined. In six of the seven patients in chronic phase, there was a significant inhibition of CFU-GM production which was only 68.4 +/- 19%; (p < .01) of that found in controls. The S phase index, which depends upon the percentage of S phase cells as well as the fluorescence intensity, was 48 +/- 29% (p < .01) of the control values for the seven patients in chronic phase. Interestingly, for the only CML patient in accelerated phase, antisense oligomers had no inhibitory effect on either the production of CFU-GM or the number of S phase cells. In improving the specificity of oligomers, it might be useful for gene-targeted anti-leukemic therapy and/or bone marrow purging.

To determine the role of the mutated RAS oncogene during development into the blast phase, we sequentially analysed RAS oncogene mutations in the bone marrow of 27 patients with chronic myeloid leukemia (CML). DNA from CML patients in chronic and blast phases and nude mouse tumor DNA formed by a tumorigenicity assay (in vivo selection assay) were subjected to the polymerase chain reaction (PCR) and oligonucleotide hybridization. In addition, one patient in the chronic phase and five in the blast phase were also analysed. PCR analysis of DNA from the leukemic patients revealed that 3.6% (1 of 28) and 15.6% (5 of 32) of the patients in the chronic and blast phases, respectively, had RAS mutations. N- or K-RAS oncogene mutations were found mostly in the blast phase (4 of the 5 patients with the RAS oncogene mutation). Of the 5 patients with the RAS oncogene mutation, three developed myeloblastoma, a myeloblast cell tumor, in the blast phase. None of the 28 patients without the RAS mutation developed myeloblastoma. These results suggest that the RAS oncogene mutation occurred in the late stage of the disease and contributed to transformation to the blast phase in some CML patients. The findings also indicate an association between the presence of the RAS mutation and the formation of myeloblastoma.

We studied 36 DNA samples of 18 patients affected with chronic myeloid leukemia (CML) for the presence of mutations in the first exon of the BCR gene was divided into four regions amplified by polymerase chain reaction (PCR). By single strand conformation polymorphism analysis (SSCP) and direct sequencing of amplified fragments, we found different banding profiles in 9 out of 18 patients in the PCR fragment spanning nucleotide 506-826. In one patient, sequence analysis revealed the presence of a point mutation at nucleotide 669 (A-T; Gln-Leu). No difference was found between DNA samples collected during the chronic phase and the blastic transformation. No different mobility shifts of single stranded PCR products were found in the other amplified fragments. The activation of BCR-ABL involves direct interaction between BCR first exon sequences and the tyrosine kinase regulatory domains of ABL. In the first BCR exon, and around the mutated sequences two SH-2-binding sites, are retained. These domains are essential for BCR-ABL-mediated transformation. Our results demonstrate the presence of point mutation in this regulatory region, which may suggest a role for the altered BCR sequence in activation of the BCR-ABL oncogene.

A model for the pathogenesis of chronic myeloid leukaemia (CML) is proposed. It relies on a comparison between normal steady-state and regenerating haemopoiesis and suggests that chronic phase CML stem cells have a finite capacity for self-renewal. According to the model, metamorphosis of the disease occurs once the potential for chronic phase cell production has been exhausted. The model considers also the generation of leukocytosis in the chronic phase and the origin of the terminal phase. Comparison with normal regenerating haemopoiesis allows discrimination between features of CML that are fundamentally abnormal and those which are normally associated with regeneration.

Forty-eight patients with chronic myeloid leukemia (CML) in chronic phase (CP) were treated by autologous stem cells transplantation (ASCT) and alpha Interferon (IFN) with three approaches: 1) ASCT at diagnosis followed by IFN, 2) ASCT post IFN with cells collected after an interval from IFN discontinuance, followed by IFN, 3) ASCT in patients selected by cytoconversion obtained with IFN, performed soon after IFN discontinuance. Following ASCT, a major karyotype response (more than 65% Ph1 negative cells, MKR) was observed at least once in 40%, 53% and 83% of patients from the three groups, respectively. At last follow-ups (median 39, 40 and 21 months, respectively) 19%, 13% and 67% of patients still present a MKR with 2 patients from group 1 and 4 patients from group 3 being 100% Ph' negative. Projected survival from diagnosis is 77% at 52 months for patients from group 1 and 47% at 75 months for patients from group 2. Present data indicate that 1) IFN can stabilize results obtained with ASCT, 2) ASCT can potentiate responses to IFN, 3) combined ASCT and IFN can improve survival. Longer follow-up of patients and randomized studies are required to define the real impact on disease outcome by these treatment approaches.

The Philadelphia chromosome (t9;22)(q34;q11) is a characteristic abnormality in chronic myeloid leukemia. In greater than 95% of the cases, the breakpoint occurs with the M-bcr region of the BCR gene on chromosome 22. Several studies have attempted to correlate the location of the breakpoint within the M-bcr with a clinical parameter. The majority of studies have examined the relationship between the site of breakpoint and the median chronic phase duration (CPD). Some studies have reported a correlation, with 5' breakpoint patients who have a longer median CPD than patients with a 3' breakpoint. However, other groups have reported that no correlation exists. Furthermore, data from some of the latter groups have suggested that a correlation may exist with the lineage of blast crisis which developed and 3' breakpoint patients had a higher than expected number of lymphoid blast crisis. A correlation between high platelet counts at diagnosis and patients with a 3' breakpoint or those who expressed a b3-a2 BCR-ABL mRNA has also been described. No consistent conclusion from any of these studies can be drawn. This may due, in part, to some degree of patient and sample selection, although environmental, genetic or life-style factors may also contribute.

This is an interim report of the Roferon A/ABMT protocol by ICSG on CML aimed at investigating the feasibility and potential of combining treatment with alpha-IFN with ABMT in Ph+ CML. Of 675 Ph+ CML patients recruited between January 1989 and January 1991 by 44 Italian institutions, 398 were 55 or less years old and eligible for the protocol. Of 132 patients who completed IFN treatment 118 had evaluable karyotype; of these only 48 showed > 25% Ph--metaphases and were eligible for BM harvest. In 24 patients BM was collected and 13 were submitted to ABMT. The major causes of drop out from the protocol were shift to allogeneic BMT, accelerated blastic phase, patient refusal and logistic problems. Data on hematologic reconstitution are presently available in 11 patients: Neutrophils were > 0.5 x 10(9)/l in 23 days (median), (range 16-40 days); platelets reached 50 x 10(9)/l in 28 days (median), (range 25-100 days). One patient had a very delayed BM take and was rescued with autologous peripheral blood stem cells collected at diagnosis.

While much is known about CML at both the clinical and molecular biological levels, the precise relationship between the disease at these two levels is unclear. The appearance of the fusion gene bcr-abl and disorders in the regulation of the myc gene, and perhaps other oncogenes which code for nucleoproteins, appear to play integral roles in the genesis of the chronic and blastic phases of the disease. The resistance of this disease to cytotoxic therapy appears to reflect both "classical" drug resistance and the ability of those cells which survive cytotoxic therapy to rapidly replace the killed cells thereby offsetting the effects of chemotherapy ("regrowth resistance"). The clinical evolution of the disease is compatible with two fundamentally different processes: one compatible with a deterministic chaotic model and the other involves two basically independent linear phenomena which overlap and intersect as the blastic phase appears and replaces the chronic phase.

The mRNA encoding the chimeric BCR/ABL oncogene, which is transcribed from the Philadelphia chromosome in human chronic myelogenous leukemia, has a 5' noncoding sequence greater than 500 bases in length which is highly GC rich and contains a short open reading frame. This untranslated sequence has a dramatic inhibitory effect upon translational efficiency in vitro. However, when BCR/ABL message is expressed in certain cell types such as the NIH 3T3 cell line, the 5' noncoding region has little inhibitory effect on translational efficiency.