Imatinib-treated Patients Research Articles

Imatinib Resistance and/or Intolerance in Clinical Practice in Europe: The “Unmet Needs in CML and Ph+ALL” (UNIC) Study.

Imatinib is widely used for the treatment of chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL), and is associated with improved response rates in these patients. However, the management of patients who show resistance or intolerance to imatinib has been identified as an unmet need. The UNIC study aimed to quantify this unmet need. UNIC is a cross-sectional study, with retrospective chart review of patients currently treated for CML or Ph+ALL in Austria, Belgium, France, Italy, Netherlands, Spain, Sweden and UK. Patients were recruited September 2006-March 2007. The study was designed to estimate the proportion ofi.patients ever treated with imatinib andii.imatinib-treated patients who have experienced imatinib resistance and/or intolerance (primary objectives).A registry was collected of potentially-eligible patients - those aged ≥18 years and treated for CML/Ph+ALL at the participating centers (academic, non-academic, private clinic or other). Case Report Forms (CRFs) were completed for patients who met the inclusion criteria, until the recruitment target was reached. Data were collected at the most recent visit and retrospectively through clinical chart review. Of the 4139 patients in the registry, CRFs were completed for 1716 and analyzable for 1599 (CML, n=1551; Ph+ALL, n=48). Characteristics of the included population were representative of those of the registry. Of the included CML patients, 98% were in chronic phase and 2% were in advanced phases. In total, 1493 (96%) CML and 46 (96%) Ph+ALL patients had received imatinib. By the last observation, 48% of all imatinib-treated patients needed a change in imatinib dose, and >20% discontinued imatinib therapy (CML, 21%; Ph+ALL, 37%). A patient was defined as imatinib resistant if reported as such by the physician in the medical chart, and intolerant to imatinib (or other concurrent treatment) if toxicity led to a change in imatinib use, as reported in the medical chart. Imatinib resistance and treatment intolerance reported during the study are shown in the table. Seven percent of CML and Ph+ALL patients discontinued imatinib due to toxicity.Number (%) of patients [95% confidence interval] according to the physician assessmentCML (N=1493)Ph+ALL (N=46)Resistant to imatinib241 (16.1) [14.3; 18.1]6 (13.0) [4.9; 26.3]Intolerant to treatment583 (39.0) [36.6; 41.6]17 (37.0) [23.2; 52.5]Resistant to imatinib and/or intolerant to treatment688 (46.1) [43.5; 48.6]20 (43.5) [28.9; 58.9]Resistant to imatinib and intolerant to treatment136 (9.1) [7.7; 10.7]3 (6.5) [1.4; 17.9]This is the largest European observational study of CML/Ph+ALL patients to date. As expected, nearly all patients were exposed to imatinib therapy. Nearly half of all imatinib-treated CML and Ph+ALL patients experienced resistance to imatinib and/or intolerance to treatment including imatinib.

Platelet-Derived Growth Factor Receptor Inhibition and Chemotherapy for Castration-Resistant Prostate Cancer with Bone Metastases

To further assess preclinical and early clinical evidence that imatinib mesylate, a platelet-derived growth factor receptor (PDGFR) inhibitor, modulates taxane activity in prostate cancer and bone metastases, a randomized study was conducted. Men with progressive castration-resistant prostate cancer with bone metastases (n = 144) were planned for equal randomization to i.v. 30 mg/m(2) docetaxel on days 1, 8, 15, and 22 every 42 days with 600 mg imatinib daily or placebo, for an improvement in median progression-free survival from 4.5 to 7.5 months (two-sided alpha = 0.05 and beta = 0.20). Secondary end points included differential toxicity and bone turnover markers, tumor phosphorylated PDGFR (p-PDGFR) expression, and modulation of p-PDGFR in peripheral blood leukocytes. Accrual was halted early because of adverse gastrointestinal events. Among 116 evaluable men (57 docetaxel + imatinib; 59 docetaxel + placebo), respective median times to progression were 4.2 months (95% confidence interval, 3.1-7.5) and 4.2 months (95% confidence interval, 3.0-6.8; P = 0.58, log-rank test). Excess grade 3 toxicities (n = 23) in the docetaxel + imatinib group were principally fatigue and gastrointestinal. Tumor p-PDGFR expression was observed in 12 of 14 (86%) evaluable bone specimens. In peripheral blood leukocytes, p-PDGFR reduction was more likely in docetaxel + imatinib-treated patients compared with docetaxel + placebo (P < 0.0001), as were reductions in urine N-telopeptides (P = 0.004) but not serum bone-specific alkaline phosphatase (P = 0.099). These clinical and translational results question the value of PDGFR inhibition with taxane chemotherapy in prostate cancer bone metastases and are at variance with the preclinical studies. This discordance requires explanation.

Imatinib Mesylate in Chronic Myeloid Leukemia

Chronic myeloid leukemia has become a paradigm for the discovery of target therapeutic approaches in the field of onco-hematology. Recognition of the tyrosine kinase activity of the p210Bcr-Abl oncoprotein led to the development of compounds targeting against BCR-ABL and then controlling the leukemic proliferation. Imatinib mesylate, one of the first tyrosine kinase inhibitors developed, was found effective and safe. According to five-years experience with this drug, it is recommended that the golden standard for initial treatment of newly diagnosis chronic myeloid leukemia patients should be 400 mg Imatinib daily. In this brief review, we discuss the current tools for the effective management of chronic myeloid leukemia with Imatinib, providing the updated results of IRIS and RIGHT clinical trials and then the suggestions how Imatinib-treated patients should be monitored.

Spectrum and prognostication of KIT and PDGFRA mutation in gastrointestinal stromal tumors

Aims Recent studies reported various mutation rates in gastrointestinal stromal tumors (GISTs) and inconsistent prognostic value of mutation in GIST patients. Our purpose was to analyze the frequency and spectrum of KIT and PDFGRA in a large series study and to determine if the presence of mutation and mutation type serve as prognostic factors in GIST patients. Methods A total of 134 GISTs were subjected to mutation analysis of KIT (exons 9, 11, 13, and 17) and PDGFRA (exons 10, 12, 14, and 18). Clinicopathologic characteristics and survivals were correlated to KIT mutation. Results Approximately 69% of GISTs had KIT/ PDGFRA mutation in Taiwanese GIST patients, with 99% of mutations occurred in KIT and 1% occurred in PDGFRA. Mutation rate was significantly increased in GISTs with mitotic counts >5 per 50 high power fields ( χ 2 test, p = 0.045). However, KIT mutations, regardless of the location (exons 9 versus 11) and type (missense, insertion, and deletion, including deletion specifically involving codons 557 and 558) of mutation, were not significantly associated with poor progression-free survivals. Comparing the overall survival in imatinib-treated patients, there was no significant difference between patients with exon 11 mutation and those with exon 9 mutation ( p = 0.473). Conclusions GISTs were commonly associated with KIT mutation, but rarely associated with PDGFRA mutation in Taiwan. The presence of KIT mutation and mutation type was not significant prognostic factors in GIST patients without imatinib treatment, suggesting that there is no need to stratify GIST patients by mutation status in clinical trials of targeted therapy.

Choi criteria are superior to Response Evaluation Criteria in Solid Tumours (RECIST) for evaluating GI stromal tumours in imatinib-treated patients,

Choi criteria are superior to Response Evaluation Criteria in Solid Tumours (RECIST) for evaluating GI stromal tumours in imatinib-treated patients,

Growth Factor Stimulation Reduces Residual Quiescent Chronic Myelogenous Leukemia Progenitors Remaining after Imatinib Treatment

The BCR/ABL tyrosine kinase inhibitor imatinib mesylate is highly effective in the treatment of chronic myelogenous leukemia (CML) but fails to eliminate all leukemia cells. Residual leukemia stem and progenitor cells persist in imatinib-responsive patients and may be a potential source of relapse. Previous studies indicate that imatinib preferentially targets dividing cells, and nondividing progenitor cells are resistant to imatinib-mediated apoptosis. We investigated whether growth factor stimulation of progenitor proliferation could reduce the number of residual nondividing cells remaining after imatinib treatment. CML and normal CD34(+) cells were labeled with 5-(and 6-)-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track cell division and cultured in low or high concentrations of growth factor to determine effects of growth factor stimulation on nondividing cells. High growth factor concentrations significantly enhanced CML proliferation with or without imatinib treatment and significantly reduced the number of viable, nondividing CFSE bright cells remaining after imatinib exposure. Stimulation with high growth factor before imatinib treatment further reduced the number of residual nondividing CML CD34(+) cells. Importantly, clinically achievable concentrations of granulocyte macrophage colony-stimulating factor alone or in combination with granulocyte colony-stimulating factor also significantly reduced nondividing CML CD34(+) cells. These results support the potential efficacy of growth factor stimulation in reducing the residual leukemia progenitor population in imatinib-treated patients.

Early detection of response to imatinib therapy for gastrointestinal stromal tumor by using 18F-FDG-positron emission tomography and computed tomography imaging

A 41-year old female with metastatic gastrointestinal stromal tumor was referred to 18F-FDG-positron emission tomography and computed tomography (PET/CT) scan before and after one-month treatment with imatinib (Glivec, Gleevec, Novartis, Basel, Switzerland), a tyrosine kinase inhibitor (400 mg/d). Metabolic response was evaluated before and after one month of therapy. The decrease of the maximum standardised uptake value (SUV) was 79% (from 9.8 to 2.1). Positron emission tomography demonstrated complete metabolic response after one-month of imatinib treatment. Additionally, the previous lesion was compared with the coronal computerized tomographic image. There was no difference in the size of the tumor before and after therapy according to CT images. However, metabolic activity was inhibited. 18F-FDG-PET is a valuable method for the detection of response to one-month imatinib treatment in patients with gastrointestinal stromal tumors.

Achieving a Major Molecular Response at the Time of a Complete Cytogenetic Response (CCgR) Predicts a Better Duration of CCgR in Imatinib-Treated Chronic Myeloid Leukemia Patients

Achieving a Major Molecular Response at the Time of a Complete Cytogenetic Response (CCgR) Predicts a Better Duration of CCgR in Imatinib-Treated Chronic Myeloid Leukemia Patients

Contribution of ABL Kinase Domain Mutations to Imatinib Resistance in Different Subsets of Philadelphia-Positive Patients: By the GIMEMA Working Party on Chronic Myeloid Leukemia

ABL kinase domain mutations have been implicated in the resistance to the BCR-ABL inhibitor imatinib mesylate of Philadelphia-positive (Ph+) leukemia patients. Using denaturing high-performance liquid chromatography and sequencing, we screened for ABL kinase domain mutations in 370 Ph+ patients with evidence of hematologic or cytogenetic resistance to imatinib. Mutations were found in 127 of 297 (43%) evaluable patients. Mutations were found in 27% of chronic-phase patients (14% treated with imatinib frontline; 31% treated with imatinib post-IFN failure), 52% of accelerated-phase patients, 75% of myeloid blast crisis patients, and 83% of lymphoid blast crisis/Ph+ acute lymphoblastic leukemia (ALL) patients. Mutations were associated in 30% of patients with primary resistance (44% hematologic and 28% cytogenetic) and in 57% of patients with acquired resistance (23% patients who lost cytogenetic response; 55% patients who lost hematologic response; and 87% patients who progressed to accelerated phase/blast crisis). P-loop and T315I mutations were particularly frequent in advanced-phase chronic myeloid leukemia and Ph+ ALL patients, and often accompanied progression from chronic phase to accelerated phase/blast crisis. We conclude that (a) amino acid substitutions at seven residues (M244V, G250E, Y253F/H, E255K/V, T315I, M351T, and F359V) account for 85% of all resistance-associated mutations; (b) the search for mutations is important both in case of imatinib failure and in case of loss of response at the hematologic or cytogenetic level; (c) advanced-phase chronic myeloid leukemia and Ph+ ALL patients have a higher likelihood of developing imatinib-resistant mutations; and (d) the presence of either P-loop or T315I mutations in imatinib-treated patients should warn the clinician to reconsider the therapeutic strategy.

Increasing Frequency and Marked Stability of Complete Molecular Response Is Observed in Imatinib-Treated CML Patients with Long-Term Follow Up.

The degree of reduction of BCR-ABL in imatinib-treated patients with chronic phase CML is an important indicator of prognosis. The IRIS trial established that with first-line therapy patients with a major molecular response (MMR, 3 log reduction from a standardized baseline value for untreated patients) have a significantly more favorable progression free survival. Although 40% achieved a MMR by 12 months, very few had undetectable BCR-ABL according to strict PCR sensitivity criteria. We measured peripheral blood BCR-ABL levels by quantitative PCR at 3 to 6 month intervals in 155 patients with chronic phase CML enrolled in clinical trials of imatinib for up to 6 years. We aimed to (i) determine if BCR-ABL levels continued to decline over time, and (ii) evaluate the stability and significance of undetectable BCR-ABL. The patients included the Australasian subset of IRIS trial patients treated with 400mg of imatinib; 29 first-line patients evaluated for a median of 69 months (25th to 75th percentile range (pr) 58–72) and 24 second-line patients for a median of 54 months of imatinib (pr 38–60). 102 de-novo patients enrolled in the TIDEL trial of 600mg imatinib were evaluated for a median of 39 months (pr 30–42). Complete molecular response (CMR) was defined as undetectable BCR-ABL at a PCR sensitivity of at least 4.5 logs below the standardized baseline value confirmed on subsequent analysis after at least 3 months. The BCR control transcript level determined sensitivity and was dependent on RNA quality and reverse transcription efficiency. Of note CMR may not indicate eradication of leukemic cells, rather a reduction of BCR-ABL below the detection limit. CMR occurred in 34 patients who had 178 analyses after achieving CMR (median 4 tests per patient) and a median follow up of 15 months (pr 9–24). Very low level BCR-ABL was detected in 3 patients, the remaining 31 had undetectable BCR-ABL on every subsequent assay. Of the IRIS trial patients treated with first-line imatinib, 41% achieved a CMR by 69 months, a frequency significantly higher than occurred in these patients at 24 months (7%, p=0.006). The rate of CMR appeared to increase substantially beyond the 3 year time point (7%, 24% and 34% at 3, 4 and 5 years). 75 patients achieved MMR but not CMR and were followed for a median of 24 months after achieving MMR (pr 17–33). Six of 75 patients (8%) lost MMR as defined by >2-fold rise in BCR-ABL and loss of MMR on 2 consecutive analyses. The median fold rise was 18-fold (4 to 1900-fold), of whom 1 went on to blast crisis. Four of the 6 patients had BCR-ABL mutations detected at the time of the rise and 1 of the remaining patients had duplicate Ph. MMR was lost in these 6 patients within 18 months of its achievement. The overall rate of CMR and MMR (including patients with CMR) did not differ significantly between the 3 treatment groups at the 3 year time point (CMR 7%, 8% and 18%; MMR 66%, 71% and 70% for first-line 400mg, second-line 400mg and first-line 600mg respectively). In conclusion at a median follow-up of 5.75 years of 400mg first-line imatinib, CMR was achieved in 41% of patients. Importantly, of all patients who achieved a CMR in this study using strict criteria to define the sensitivity of analysis, none have lost MMR and 91% have maintained CMR. The slow acquistion and marked stability of CMR favour the notion that the leukemic stem cell pool is steadily declining with prolonged exposure to imatinib.

A Half-Log Increase in Quantitative RT-PCR for BCR-ABL (RQ-PCR) as a Trigger for Kinase Domain Mutation Analysis.

RQ-PCR for BCR-ABL is standard of care for monitoring patients with chronic myeloid leukemia on Abl kinase inhibitor therapy, and mutations in the kinase domain (KD) of BCR-ABL are the most frequent mechanism of acquired drug resistance. From a practical point of view, it is important to establish an RQ-PCR trigger for mutation analysis that is sufficiently sensitive, while avoiding a high rate of false positive results. In one study, a single 2-fold (0.3 log) rise in BCR-ABL transcript levels predicted mutations in 61% (1). However, the results were not confirmed in a similar study(2). We therefore analyzed the correlation between a slightly more conservative 0.5 log rise in RQ-PCR levels and mutation detection in an unselected cohort of imatinib-treated CML patients. A database search identified 273 samples (from 150 patients) with a half-log or greater RQ-PCR rise. Mutation analysis (by direct DNA sequencing) has been completed on 54 samples. BCR-ABL levels were higher in 44 samples with successful amplification and sequencing compared to 10 without sufficiently good sequencing quality [median log-drop 0.31 (range −1.2 − 2.4) vs. 2.2 (range 0.17 – 3.2), p<0.0001]. KD mutations were detected in 11 samples (25%) and included L248V, G250E, Y253H, E282G, E292V, T315I (n=2), F359V+H396R, V379I, F382L, F482S). A similar mutation rate was detected by using a trigger of a 1.0-log increase of RQ-PCR levels [4 mutations out of 19 eligible samples (21%)], but with this higher trigger level, 7 patients with mutations would be excluded from detection. We then analyzed the risk of relapse according to mutation detection. The 44 samples with a >0.5 log RQ-PCR increase were from 34 different patients. Eleven of these 34 patients had cytogenetic or hematologic progression at the time of the mutation analysis and 7 never achieved even a partial cytogenetic response (<65%). Of 16 remaining eligible patients, 9 subsequently relapsed at a median of 5.8 (range 2.6–17) months after mutation detection. Freedom from progression was shorter in patients with a half-log RQ-PCR increase compared to patients without (34 months from imatinib start vs. median survival not reached, p = 0.019).Conclusion: In our hands, a half-log increase of BCR-ABL levels is associated with mutation detection in 25% of samples, yields a significantly increased risk of subsequent relapse, and may be used as a practical trigger for kinase domain sequencing. Analysis of additional samples is ongoing to determine the ideal trigger level that optimizes specificity and sensitivity.

Monitoring minimal residual disease and controlling drug resistance in chronic myeloid leukaemia patients in treatment with imatinib as a guide to clinical management

Imatinib mesylate, binding to the inactive conformation of Bcr-Abl tyrosine kinase and suppressing the Ph chromosome positive clone, has revolutionized the treatment of chronic myeloid leukaemia (CML) patients. Given the high rates of clinical and cytogenetic remission achieved, the molecular monitoring of BCR-ABL transcript levels by RT-qPCR has become always more important to assess minimal residual disease. Recently, recommendations for harmonizing current methodologies for detecting and measuring BCR-ABL transcripts in CML patients have been suggested. Studies of imatinib-treated patients have determined that the BCR-ABL levels measured early in therapy may predict durable cytogenetic remission and in turn prolonged progression free-survival or acquisition of resistance. The major mechanism of imatinib resistance is clonal expansion of leukaemia cells with mutations in the Bcr-Abl fusion tyrosine kinase. The early reduction of such mutations may allow timely treatment intervention to prevent or overcome resistance. We review current trends in the management of chronic myeloid leukaemia patients undergoing treatment with tyrosine kinase inhibitors.

Evaluation of the Function of Human Invariant NKT Cells from Cancer Patients Using α-Galactosylceramide-Loaded Murine Dendritic Cells

NKT cells play a role in immunological regulation of certain diseases, and their frequency and/or function may be related to disease prognosis. However, it is often difficult to evaluate NKT cell function in patients with malignancies due to reduced numbers of NKT cells as well as the dysfunction of the APCs used as stimulators. We found that NKT cell function could not be evaluated by conventional ELISPOT assays, confirming the impaired function of APCs in chronic myelogenous leukemia (CML)-chronic phase patients. To overcome this problem, we have established a sensitive assay using murine dendritic cells to evaluate the function of small numbers of human NKT cells independent of autologous APCs. We found that imatinib-treated CML-chronic phase patients showing a complete cytogenetic response had NKT cells capable of producing IFN-gamma, whereas NKT cells from patients who were only partially responsive to imatinib treatment did not produce IFN-gamma. Functional NKT cells found in imatinib-treated, CML-complete cytogenetic response patients may offer the promise of effective immunotherapy with ex vivo-generated alpha-galactosylceramide-pulsed dendritic cells. This new approach should be available for evaluating the functions of NKT cells and APCs in cancer patients.

Compliance and persistency with imatinib

6038 Background: Imatinib is an oral therapy with efficacy in chronic myelogenous leukemia (CML) and gastrointestinal stromal tumors (GIST). Optimal dosing and adherence to treatment is critical to achieve the best clinical outcomes. This study examined compliance and persistency with imatinib and identified the clinical and patient characteristics related to compliance. Methods: Claims data from a US health plan were used to identify imatinib-treated patients from 6/1/01–3/31/04 who had continuous pharmacy and medical benefits in the 3 months prior and 12 months following initiation of imatinib therapy, and a diagnosis of CML or GIST (ICD-9-CM 205.1, 205.10, or 205.11 for CML; 159.0, 159.8, or 159.9 for GIST). Compliance was defined by medication possession ratio (MPR = total days supply of imatinib in the first year divided by 365). Persistency was defined as failure to refill imatinib within 30 days from the run-out date of the prior prescription. Multivariate analyses were used to identify key factors associated with compliance. Results: Total 878 imatinib-treated patients were identified of whom 413 had at least 15 months’ continuous eligibility. Sixty-nine percent (n = 286) were diagnosed with CML, 8% (n = 34) with GIST, and 23% (n = 93) with neither. Results are presented for CML and GIST patients. The average age was 51 and 58% were males. The average starting daily dose was 424 mg, with 80% (n = 255) initiating on 400 mg daily. The mean MPR was 76%. Overall, 28% patients discontinued imatinib for at least 30 consecutive days during the 1-year follow up period. Multivariate analyses indicated MPR improved with age until age 51 and then deteriorated (p &lt; 0.001) but at a diminishing rate, decreased as the number of medications increased (p &lt; 0.001), and was lower in women (p = 0.005) and patients with more cancer complications (p &lt; 0.001). In addition, women were more likely to discontinue than men (OR = 2.08; p = 0.003). Conclusions: Compliance to imatinib was about 75% with 30% of patients interrupting therapy for at least 30 consecutive days in the first year. It has been found that interruption of imatinib therapy may lead to rapid tumor progression in GIST (PASCO05 Le Cesne #9031). Not having patients take the correct doses on a regular basis may lead to sub therapeutical clinical outcomes. [Table: see text]

Achieving a Major Molecular Response at the Time of a Complete Cytogenetic Response (CCgR) Predicts a Better Duration of CCgR in Imatinib-Treated Chronic Myeloid Leukemia Patients

Most patients with chronic-phase chronic myeloid leukemia (CML) who receive imatinib achieve a complete cytogenetic remission (CCgR) and low levels of BCR-ABL transcripts. CCgR is durable in the majority of patients but relapse occurs in a subset. To determine the potential of quantitative reverse transcription-PCR of BCR-ABL to predict cytogenetic relapse, we serially monitored residual disease in 97 CML patients with an imatinib-induced CCgR. Patients with late chronic phase CML after IFN-alpha failure were treated with imatinib (400 mg daily). During the imatinib median follow-up time of 36 months (range, 12-54 months), disease monitoring occurred by cytogenetics and quantitative PCR. Twenty percent of patients experienced cytogenetic relapse at a median of 18 months after CCgR and a median of 24 months after starting imatinib. None of the possible prognostic factors studied in univariate and multivariate analyses seemed to predict for loss of cytogenetic response but the reduction of BCR-ABL transcript levels at the time of CCgR is an important prognostic factor. In our study, we showed not only that achieving a major molecular remission at 12 months is predictive of a durable cytogenetic remission but also that patients who achieved a major molecular remission (expressed both as the BCR-ABL/beta2 microglobulin ratio % <0.0005 and as a 3-log reduction from median baseline value) already at the time of first achieving a CCgR have significantly longer cytogenetic remission durations than those without this magnitude of molecular response (P < 0.05).

Limited clinical value of regular bone marrow cytogenetic analysis in imatinib-treated chronic phase CML patients monitored by RQ-PCR for BCR-ABL

Real-time quantitative polymerase chain reaction (PCR) for BCR-ABL mRNA in the peripheral blood (RQ-PCR) provides an accurate and reliable measure of response to therapy in chronic myeloid leukaemia (CML). We wanted to determine in what circumstances additional clinically relevant information was provided by simultaneous cytogenetic analysis in RQ-PCR monitored patients receiving imatinib treatment. We analysed 828 simultaneous RQ-PCR and bone marrow cytogenetic analyses from 183 patients with chronic phase CML with a median follow-up of 20 months. Cytogenetic progression was defined as Philadelphia (Ph)-positive clonal evolution, loss of complete cytogenetic response or an increase of > or = 20% Ph-positive cells. Cytogenetic progression occurred in 24/183 (13%) patients. At the time of cytogenetic progression, none of the 24 patients had a major molecular response (MMR; > or = 3-log reduction in BCR-ABL from standardised baseline). There were 320 RQ-PCR results from 95 patients indicating MMR. No abnormality was detected in any of the corresponding cytogenetic analyses. A policy of regular RQ-PCR monitoring with cytogenetic analysis targetted only to patients who have not achieved, or have lost MMR would represent a rational approach to monitoring and spare most patients the discomfort of multiple marrow aspirates. This approach depends upon availability of an accurate, reproducible RQ-PCR assay with ongoing quality assurance.

Clinical usefulness of transcription-mediated amplification and hybridization protection assay in imatinib-treated chronic myelogenous leukemia patients

Clinical usefulness of transcription-mediated amplification and hybridization protection assay in imatinib-treated chronic myelogenous leukemia patients

Lymphoid Foci in Bone Marrow of Patients with Chronic Myeloid Leukaemia Treated with Imatinib.

Imatinib treatment of CML is successful but long term. In spite of reported inhibition of T lymphocyte proliferation and activation, we noticed multiple lymphoid foci occurring in BM trephines from CML patients on Imatinib. Method: Patients with chronic phase CML were monitored with BM biopsies (generally 3 monthly) for 1 to 4 years on Imatinib (34 patients) and for 1.5 to 12 years on Interferon (22 patients). Three hundred and thirty-one trephine biopsies were assessed for the occurrence of lymphoid foci. Selected trephines with foci on recut sections were examined by immunocytochemistry with CD3 and CD20, and available aspirates were tested by flow cytometry and PCR for evidence of monoclonality. Results: Lymphoid foci in bone marrow trephines of patients with CML chronic phase occurred more frequently in Imatinib-treated patients (28/34 patients; 82%) than in Interferon-treated patients (9/22 patients; 41%) (p =0.001). Of 175 trephine biopsies from Imatinib-treated patients 68 (39%) contained lymphoid foci, compared to 18 of 156 (12%) trephines from Interferon-treated patients (p= 0.001). In 13/34 Imatinib-treated patients the BM trephine was positive on only one occasion, with 15/34 having multiple positive biopsies. In comparison BM trephine biopsies from random patients with various diagnoses have been described to have lymphoid foci in 3% of cases (Thiele et al, J Clin Pathology 1999,52,294). The majority of foci were interstitial but there were occasional paratrabecular foci. There was no difference between the incidence of foci in Imatinib-treated patients who had previously been treated with Interferon (11/14) and those who had Imatinib as first-line therapy (17/20). Lymphocyte numbers in BM aspirates were above normal in 32/175 aspirates and not coincident with the presence of foci in the corresponding trephines. Foci in the trephines examined by immunocytochemistry showed that the foci were a mixture of CD3 and CD20 positive cells. Flow cytometric immunophenotyping of available BM aspirates of patients with lymphoid foci showed no monoclonality, apart from 1 patient who developed monoclonal Non-Hodgkins lymphoma confined to the bone marrow during treatment with Imatinib. Only 2 patients had foci present at diagnosis of CML and one of these Imatinib-treated patients was the one who later developed NHL. Response to treatment as assessed by cytogenetic response could not be correlated with the development of lymphoid foci in the BM during treatment, as all Imatinib treated patients achieved complete cytogenetic remission. However patients with multiple positive trephine biopsies showed a trend (p=0.095) towards better molecular response as measured by a greater than 4 log reduction in BCR/ABL (8/14) than those with no lymphoid foci (1/6). Conclusion: Lymphoid foci occur frequently in the BM trephines of Imatinib- treated chronic phase CML patients; more commonly than in Interferon-treated CML patients. Both these patient groups have a higher number of lymphoid foci than is reported in random BM biopsies. This finding seems in contradiction to in vitro findings of Inatimib suppression of lymphocytic activation and proliferation. The occurrence of multiple foci suggests that the BM trephines and aspirates should continue to be monitored in Imatinib-treated patients. This early data also suggests that there may be a correlation of molecular response to Imatinib with the development of multiple lymphoid foci in the bone marrow.

ATP Binding Domain Mutants of BCR-ABL in Patients with Chronic Myeloid Leukemia Treated with Imatinib.

Objective:Imatinib (also called STI571 or Gleevec), a competitive inhibitor at the ATP binding site of BCR-ABL, has been shown remarkable clinical activity in patients with chronic myeloid leukemia (CML). However, a significant proportion of Imatinib-treated CML patients with advanced stage disease develop resistance. In this study, we try to detect ATP binding domain mutants of BCR-ABL in Imatinib-treated CML patients and explore the possibility that ATP binding domain mutants might confer resistance to imatinib.Patients and Methods:We analyzed bone marrow samples from 16 Imatinib-treated CML patients, including 6 chronic phase, 2 accelerated phase (AP) and 8 blast crisis (BC) patients. 8 patients were sensitive to imatinib and 8 patients resistant. A polymerase chain reaction strategy was used to amplify and sequence the ATP binding domain of BCR-ABL.Results:Point mutations were found in the ATP binding domain of BCR-ABL in 3 of 16 patients. In 2 of 8 imatinib-resistance patients, a single nucleotide change was detected and resulted in a threonine to isoleucine substitution at position 315(Thr315Ile) of ABL kinase. This point mutation has proved to interfere with drug binding and cause resistance to imatinib. In addition, we found a mutation (Lys357Arg) remain sensitive to imatinib.Discussion:Chronic myeloid leukemia (CML) is a hematopoietic disorder characterized by the malignant expansion of bone marrow stem cells. Its malignant clonal marker is Philadelphia chromosome (Ph) which harbors the BCR-ABL fusion gene. The latter encodes a chimeric BCR-ABL protein, identified as having a central role in the pathogenesis of CML. As a competitor for ATP binding of BCR-ABL, Imatinib selectively induces apoptosis and blocks proliferation in BCR-ABL positive cells and has been shown to have high activity in CML. Clinical studies demonstrate that Many Imatinib-treated CML patients with advanced stage disease respond initially but then relapse. Till now, the resistant mechanisms of imatinib have not all been known. In this study, we find that ATP binding domain mutants of BCR-ABL is one of the potential mechanisms of resistance to imatinib, but not all mutants associate with imatinib resistance and some mutants remain sensitive to imatinib.

Targeting Chronic Myeloid Leukemia (CML) Stem Cells by BMS-214662 in Mice.

The BCR-ABL inhibitor imatinib mesylate is the current approved treatment for Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML). While this agent is effective in the chronic phase of CML, it is less effective in advanced disease (acelerated phase or blast crisis), and resistance to imatinib is an issue at all stages of disease, particularly advanced. Resistance is mediated primarily by BCR-ABL mutations, although other mechanisms have also been implicated. Another key issue with imatinib therapy is that molecular remission in imatinib-treated CML patients is difficult to achieve, leaving patients at risk of relapse. We have previously observed that imatinib significantly prolongs survival of CML mice, but is not curative (Hu et al, Nature Genetics 36[5]:453–461, 2004). We hypothesize that this can be attributed to the inability of imatinib to completely kill CML stem cells. We identified that BCR-ABL-expressing Lin-c-KIT+Sca-1+ bone marrow cells are CML stem cells in mice. We tested whether BMS-214662 (which has been shown to have an inhibitory effect on growth of non-proliferating cancer cells) (Lee et al, Proceedings of the AACR 42:260s, 2001) reduces leukemia stem cell populations in CML mice. Donor bone marrow cells from C57BL/6 mice were transduced with P210BCR-ABL-IRES-GFP retrovirus, followed by transplantation into lethally irradiated C57BL/6 recipient mice. Eight days after transplantation, BMS-214662 was given orally once a day at a dose of 300 mg/kg for 7 days. Bone marrow cells from the treated CML mice were then analyzed by FACS for CML stem cells (GFP+Lin-c-Kit+Sca-1+). CML mice treated with placebo, dasatinib (a novel, oral, multi-targeted kinase inhibitor that targets BCR-ABL and SRC family kinases) 10 mg/kg, twice daily (BID), BMS-214662, or dasatinib 10 mg/kg BID in combination with BMS-214662. Numbers of leukemia stem cells per bone were significantly lower in mice treated with BMS-214662 alone, dasatinib alone, or both BMS-214662 and dasatinib, compared with placebo-treated mice. Among different treatments, the combination of BMS-214662 and dasatinib had the strongest inhibitory effect on CML stem cells. Inhibition of the leukemia stem cells by dasatinib could be due to its inhibitory effect on BCR-ABL or SRC kinases, whereas BMS-214662 must function through other mechanisms. BMS-214662 is also a farnesyl transferase inhibitor (FTI), which reduces Ras activation. However, our control experiment showed that other FTIs did not inhibit proliferation of non-proliferating cancer cells (data not shown). This suggests that BMS-214662 inhibits CML stem cells through unknown mechanisms. In summary, BMS-214662 is a potent inhibitor of CML stem cells, and combinatorial use of BMS-214662 and dasatinib may provide more durable responses, and potentially a curative therapy for CML patients. Given the proven activity of dasatinib against a spectrum of imatinib-resistant BCR-ABL mutations (O'Hare, et al. Cancer Res 65[11]:4500–5, 2005; Shah et al, Science , 305:399, 2004), and the apparent activity of dasatinib against stem cells in vivo shown here, this combination could potentially suppress the emergence of resistance, further adding to the durability of response.