Katy Knight

A population study of imatinib in chronic myeloid leukaemia demonstrates lower efficacy than in clinical trials

A population study of imatinib in chronic myeloid leukaemia demonstrates lower efficacy than in clinical trials

Clinical evaluation of BCR-ABL peptide immunisation in chronic myeloid leukaemia: results of the EPIC study

Peptides from the e14a2 BCR-ABL junction will elicit T-cell responses in vitro. Here, 19 imatinib treated CML patients in first chronic phase were vaccinated with BCR-ABL peptides spanning the e14a2 fusion junction, some of which were linked to the pan DR epitope PADRE to augment CD4+ T cell help. Six vaccinations were given over 9 weeks, together with sargramostim. All patients developed mild local reactions. T cell responses to PADRE were seen in all patients. Fourteen of 19 patients developed T cell responses to BCR-ABL peptides. The development of an anti-BCR-ABL T cell response correlated with a subsequent fall in BCR-ABL transcripts. No molecular benefit was seen in the 5 patients not in major cytogenetic response (MCR) at baseline. However, of the 14 patients in MCR at baseline, 13 developed at least 1 log fall in BCR-ABL transcripts, though this occurred several months after completing vaccination, consistent with an effect at a primitive CML stem cell level. Vaccination may improve the fall in BCR-ABL transcripts in patients who have received imatinib for more than 12 months. BCR-ABL peptide vaccination may improve control of CML, especially in patients responding well to imatinib. Randomised trials are required to address this further.

Chronic myeloid leukemia patients with the e13a2 BCR-ABL fusion transcript have inferior responses to imatinib compared to patients with the e14a2 transcript

The clinical significance of the type of BCR-ABL transcript in newly diagnosed patients with chronic myeloid leukemia treated with imatinib remains uncertain. The findings of this study suggest that the BCR-ABL fusion transcript type may have an impact on cytogenetic response to imatinib in patients with chronic myeloid leukemia. Background Chronic myeloid leukemia is characterized by a reciprocal translocation between chromosomes 9 and 22, creating the fusion gene BCR-ABL. The clinical significance of the type of BCR-ABL transcript in newly diagnosed patients in chronic phase treated with imatinib 400 mg from initial diagnosis remains unknown. Design and Methods We analyzed the clinical outcome of 78 newly diagnosed chronic phase patients, aged 16 or over, treated with imatinib 400 mg. Of these, 71 expressed either e13a2 or e14a2 transcripts. BCR-ABL transcripts were assayed by quantitative real-time polymerase chain reaction. Results After 12 months of treatment, 54% of the e14a2 patients had achieved a complete cytogenetic response, compared to 25% of the e13a2 patients (p=0.01). Kaplan-Meier analysis of the time to achieve complete cytogenetic response revealed that e14a2 patients had more rapid response rates, compared to e13a2 patients (p=0.006). e14a2 patients had a higher event-free survival rate in the first 12 months of treatment, although overall survival did not differ significantly between the patients with the two types of transcript. Human organic cation transporter protein 1 mRNA levels did not differ between the patients with the two types of transcript. The pre-treatment pCrKL/CrKL ratio (a surrogate marker of BCR-ABL tyrosine kinase activity) was higher in patients with e13a2 transcripts than in those with e14a2 (p=0.017). Conclusions Patients expressing the e14a2 transcript type have a higher rate and more rapid complete cytogenetic responses than e13a2-expressing patients, which may be due to higher BCR-ABL tyrosine kinase activity. Knowledge of the transcript type may yield additional prognostic information, although this requires testing on larger datasets.

Simultaneous determination of nilotinib, imatinib and its main metabolite (CGP-74588) in human plasma by ultra-violet high performance liquid chromatography

We describe a high performance liquid chromatography (HPLC) method that separates two of the currently licenced tyrosine kinase inhibitors (TKIs); nilotinib (AMN107, Tasigna) and imatinib (STI571, Glivec), together with its main metabolite, CGP-74588, from human plasma. After solid phase extraction the drug mix was separated through a Gemini C6-phenyl column (150 mm x 4.6mm, i.d.; 5 microm) (Phenomenex), UK) under isocratic mobile phase conditions of methanol:50mM ammonium acetate (pH 8) (65:35, v/v) with ultra-violet (UV) detection at 260 nm wavelength. For all compounds the intra-day coefficient of variation and bias were <3% and <5% respectively; and inter-day were <4% and <9%. This simple and novel method may be used to quantify levels of TKIs when used alone or in combination with drug treatments for clinical samples.

A prospective evaluation of cardiac function in patients with chronic myeloid leukaemia treated with imatinib

In vitro studies have suggested that imatinib may be toxic to cardiac myocytes. Though retrospective studies have not shown clinical heart failure, these did not look for subtle cardiac damage. We have carried out a prospective cardiac assessment in 59 chronic myeloid leukaemia (CML) patients treated with imatinib for a median of 3.4 years, using echocardiography and MUGA scanning, with the latter repeated after a further year. We report no evidence of myocardial deterioration, either at baseline or over 12 months of imatinib treatment. Imatinib cardiotoxicity is not an important clinical consideration for CML patients or their advisors.

BCR-ABL1 tyrosine kinase activity at diagnosis, as determined via the pCrkL/CrkL ratio, is predictive of clinical outcome in chronic myeloid leukaemia.

Imatinib has become first-line therapy in chronic myeloid leukaemia (CML), but recent studies suggest that more than one-third of patients fail imatinib over 2–3 years. (de Lavallade et al, 2008; Lucas et al, 2008) It would be clinically helpful to prospectively identify patients unlikely to achieve a complete cytogenetic response (CCRe) following imatinib treatment. Prognostic scoring methods, such as the Sokal score, activity of the imatinib uptake transporter, hOCT1 (human organic cation transporter 1) (Wang et al, 2008) and BCR-ABL1 transcript type (Lucas et al, 2009) have been shown to correlate with clinical outcome. However, none of these parameters are sufficiently powerful to reliably prospectively predict patients destined to fare poorly. The phosphorylation status of CrkL has been identified as a surrogate marker of BCR-ABL1 tyrosine kinase activity (White et al, 2005), because BCR-ABL1 tyrosine kinase activity cannot be reliability detected in CML patient samples (Patel et al, 2007). White et al (2005) showed, by Western blotting, that changes in the level of CrkL phosphorylation after 1 month of imatinib treatment correlated with clinical outcome. In this prospective study we developed a fluorescenceactivated cell sorting (FACS) protocol to investigate the predictive value of measuring pCrkL, CrkL and the pCrkL/ CrkL ratio (Lucas et al, 2009) in fresh peripheral blood from 20 untreated chronic phase CML patients prior to commencing either imatinib or nilotinib treatment. For CrkL phosphorylation determination, cells were resuspended in 500 ll of 2% paraformaldehyde (VWR, Lutterworth, UK) fixed for 10 min at 37 C and chilled on ice for 1 min. Cells were harvested by centrifugation (770 g, 3 min), 500 ll 90% methanol (Fisher Scientific, Loughborough, UK) added, vortexed briefly and incubated on ice for 30 min. Cells were then washed (throughout with 1 ml of incubation buffer containing phosphate-buffered saline and 0Æ5% bovine serum albumin), harvested, and resuspended in 25 ll incubation buffer and incubated at room temperature for 10 min. Antibodies (pCrkL antibody Cat# 3181 lot 3 and 4, Cell Signalling Technology, Danvers, MA, USA; CrkL Cat#sc-319, Santa Cruz Biotechnology, Santa Cruz, CA, USA; control anti-normal-rabbit immunoglobulins Cat#AB-105-C, R&D Systems, Abingdon, UK) were added to a final concentration of 28 lg/ml, vortexed and incubated at room temperature for 40 min then washed twice, resuspended in fluorescein-labelled goat-anti-rabbit second layer antibody Alexa Fluor 488 (10 lg/ml; Invitrogen, Paisley, UK), and incubated at room temperature in the dark for 30 min. Twice washed cells were analysed using flow cytometry (FACScalibur; Becton Dickinson, Oxford, UK), with cellquest pro software (Becton Dickinson) for data analysis. The levels of pCrkL and CrkL present in the sample were determined as the geometric mean fluorescence intensity (MFI) minus the MFI value of the control sample. The pCrKL/CrKL ratio of a sample was determined via:

Naturally occurring CD4+ CD25+ FOXP3+ T-regulatory cells are increased in chronic myeloid leukemia patients not in complete cytogenetic remission and can be immunosuppressive

OBJECTIVE Clinical presentation of chronic myeloid leukemia (CML) requires not only the deregulated tyrosine kinase BCR-ABL, but also the failure of an immune response against BCR-ABL-expressing cells. T-cell responses against BCR-ABL and other antigens are well-described, but their relevance to the in vivo control of CML is unclear. The suppressive role of naturally occurring T regulatory (T-reg) cells in antitumor immunity is well-established, although little is known about their role in modulating the T-cell response to BCR-ABL. MATERIALS AND METHODS Naturally occurring T-reg cells were characterized and quantified by flow cytometry in 39 CML patients and 10 healthy donors. Their function was studied by observing their effect on responses to purified protein derivative, a recall antigen, and on the response of an autologous T-cell line recognizing BCR-ABL. RESULTS T-reg cells were CD4(+), CD25(+), FOXP3(+), CD127(low), and CD62L(high). T-reg numbers in patients in complete cytogenetic remission were significantly lower than in patients not in complete cytogenetic remission (p < 0.01). T-reg cell depletion using anti-CD25 selection enhanced proliferative responses to purified protein derivative. Furthermore, the interferon-γ and/or granzyme-B production of effector cells specific for viral peptides or a BCR-ABL HLA-A3-restricted peptide was inhibited when autologous T-reg cells were present. CONCLUSIONS Taken together, these data suggest a role for T-reg cells in limiting immune responses in CML patients and this may include immune responses to BCR-ABL. The increased frequency of T-reg cells in patients with high levels of BCR-ABL transcripts indicates that an immune mechanism may be important in the control of CML.

BCR-ABL peptide vaccination in healthy subjects: immunological responses are equivalent to those in chronic myeloid leukaemia patients.

We and others have reported that vaccination of chronic myeloid leukaemia (CML) patients with e14a2 BCR-ABL junctional peptides can elicit moderate but transient T cell responses. To determine whether CML patients may be tolerised to BCR-ABL, here we used the same schedule to vaccinate 5 healthy subjects. Although IFN-γ and granzyme-B production, and proliferative responses to the vaccine peptides were detected in all 5 cases, responses were statistically similar to CML patients. CML patients are therefore not appreciably tolerised to BCR-ABL, and junctional peptides may only be moderately immunogenic, underlining the importance of antigen immunogenicity when designing vaccination strategies.

A population study showing that the advent of second generation tyrosine kinase inhibitors has improved progression-free survival in chronic myeloid leukaemia

BACKGROUND Population based data suggest the proportion of patients failing imatinib in chronic myeloid leukaemia (CML) is higher than the reported one-third of patients in clinical trials. Clinical trials have demonstrated second generation tyrosine kinase inhibitors (TKI) dasatinib and nilotinib can restore complete cytogenetic remission (CCR) and major molecular response (MMR) to many patients failing imatinib, but their impact in the general population is not clear. DESIGN AND METHODS We report CML outcome in a population of 2.3 million people in a geographically contiguous area of North West England and North Wales. RESULTS Between 2003 and 2009, 192 new CML cases were diagnosed, of whom 184 were in chronic phase and 160 started on imatinib. The maximal CCR rate was 65% at 24 months and the maximal MMR rate was 50% at 36 months. Patients diagnosed since second generation TKI became available for imatinib failure had a more rapid cumulative CCR and MMR rate and a significantly improved progression free survival (p=0.022) than those diagnosed before this time. CONCLUSION The study indicates that second generation TKI have improved CML outcome in the general population.