Claire M. Lucas

Assessment of BCR-ABL1 Transcript Levels at 3 Months Is the Only Requirement for Predicting Outcome for Patients With Chronic Myeloid Leukemia Treated With Tyrosine Kinase Inhibitors

PURPOSE We studied BCR-ABL1 transcript levels in patients with chronic myeloid leukemia in chronic phase (CML-CP) at 3, 6, and 12 months after starting imatinib to identify molecular milestones that would predict for overall survival (OS) and other outcomes more reliably than serial marrow cytogenetics. PATIENTS AND METHODS We analyzed 282 patients with CML-CP who received imatinib 400 mg/d as first-line therapy followed by dasatinib or nilotinib if treatment with imatinib failed. We used a receiver operating characteristic curve to identify the cutoffs in transcript levels at 3, 6, and 12 months that would best predict patient outcome. We validated our findings in an independent cohort of 95 patients treated elsewhere. RESULTS Patients with transcript levels of more than 9.84% (n = 68) at 3 months had significantly lower 8-year probabilities of OS (56.9% v 93.3%; P < .001), progression-free survival, cumulative incidence of complete cytogenetic response, and complete molecular response than those with higher transcript levels. Similarly, transcript levels of more than 1.67% (n = 87) at 6 months and more than 0.53% (n = 93) at 12 months identified high-risk patients. However, transcript levels at 3 months were the most strongly predictive for the various outcomes. When we compared OS for the groups defined molecularly at 6 and 12 months with the usual cytogenetic milestones, categorization by transcript numbers was the only independent predictor for OS (relative risk, 0.207; P < .001 and relative risk, 0.158; P < .001, respectively). CONCLUSION A single measurement of BCR-ABL1 transcripts performed at 3 months is the best way to identify patients destined to fare poorly, thereby allowing early clinical intervention.

Effective dasatinib uptake may occur without human organic cation transporter 1 (hOCT1): implications for the treatment of imatinib-resistant chronic myeloid leukemia

We have previously shown that imatinib uptake into chronic myeloid leukemia (CML) cells is dependent on human organic cation transporter 1 (hOCT1; SLC22A1), and that low hOCT1 expression is an important determinant of clinical outcome to imatinib treatment. We hypothesized that dasatinib might be transported differently than imatinib, possibly accounting for its favorable effects in imatinib-resistant patients. (14)C-dasatinib uptake was greater in KCL22-transfected cells with pcDNA3-hOCT1 plasmid (high hOCT1-expressing cells) than in control cells (P = .02). However, hOCT inhibitors did not decrease dasatinib uptake into either control or primary cells, in contrast to their block on imatinib uptake. Dasa-tinib decreased the level of phosphorylated CrkL to 49.9% in control and 40.3% in high hOCT1-expressing cells. Dasa-tinib efflux was investigated in confluent ABCB1-transfected MDCKII cell monolayers. Both dasatinib and imatinib were transported from the basal to the apical layer, indicating that they were transported by ABCB1, which was confirmed using the ABCB1 inhibitor PSC833 (P = .001 and P < .001, respectively). Compared with imatinib, dasatinib achieved superior intracellular levels and BCR-ABL suppression even in cells with low or blocked hOCT1. Efflux of dasatinib and imatinib appear similar via ABCB1. Dasatinib may therefore offer an advantage over imatinib in patients with low hOCT1 expression.

Cancerous inhibitor of PP2A (CIP2A) at diagnosis of chronic myeloid leukemia is a critical determinant of disease progression

Prospective identification of patients whose chronic myeloid leukemia (CML) will progress to blast crisis is currently not possible. PP2A is a phosphatase and tumor suppressor that regulates cell proliferation, differentiation, and survival. Cancerous inhibitor of PP2A (CIP2A) is a recently described inhibitor of PP2A in breast and gastric cancer. The aim of this study was to investigate whether CIP2A played a role in CML and whether PP2A or its inhibitor proteins CIP2A or SET could predict clinical outcome. At the time of diagnosis of CML, patients who will later progress to blast crisis have significantly higher levels of CIP2A protein (P < .0001) than patients who do not progress, suggesting that PP2A is functionally inactive. We show that the potential mechanism for disease progression is via altered phosphorylation of the oncogene c-Myc. Knockdown of CIP2A results in increased PP2A activity, decreased c-Myc levels, and a decrease in BCR-ABL1 tyrosine kinase activity. We demonstrate that CIP2A levels at diagnosis can consistently predict patients who will progress to blast crisis. The data show that CIP2A is biologically and clinically important in CML and may be a novel therapeutic target.

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

Nilotinib concentration in cell lines and primary CD34(+) chronic myeloid leukemia cells is not mediated by active uptake or efflux by major drug transporters.

Imatinib mesylate and nilotinib are highly effective at eradicating the majority of chronic myeloid leukemia (CML) cells; however, neither agent induces apoptosis of primitive CML CD34+ cells. One possible explanation is that CD34+ cells do not accumulate sufficient intracellular drug levels because of either inadequate active uptake or increased efflux. To determine the interaction of nilotinib with major clinically implicated drug transporters, we analyzed their interactions with MDR1 (ABCB1), MRP1 (ABCC1), ABCG2 (BCRP) and human organic cation transporter (hOCT)1 in CML cell lines and primitive (CD34+) primary CML cells. Nilotinib is neither dependent on active import by hOCT1, nor effluxed through the ATP-binding cassette transporters analyzed. Indeed, we found nilotinib to be an inhibitor of hOCT1, MDR1 and ABCG2. The efflux transporters MDR1, MRP1 and ABCG2 are expressed on CML CD34+ cells at 13.5, 108 and 291% of control, respectively, although hOCT1 expression was absent; however, inhibition of efflux transporter activity did not potentiate the effect of nilotinib on apoptosis, Bcr–Abl inhibition or CML CD34+ cell proliferation. Therefore, we have found no evidence for either active uptake of nilotinib through hOCT1 or efflux through MDR1, MRP1 or ABCG2, and it is therefore unlikely that these transporters will have any effect on the clinical response to this drug.

Combining BCR-ABL1 transcript levels at 3 and 6 months in chronic myeloid leukemia: implications for early intervention strategies

Several groups have shown that that the BCR-ABL1 transcript level measured at 3 or 6 months after starting treatment with tyrosine kinase inhibitors strongly predicts clinical outcomes for patients with chronic myeloid leukemia. In this work, we asked whether the prognostic value of the 3-month transcript level could be improved by combining the 3- and 6-month results. We classified patients treated with imatinib and patients treated with dasatinib according to their transcript levels at 3 months and 6 months. The patients who met the 3-month landmark but failed the 6-month one had outcomes identical to those of patients who met both landmarks, whereas the patients who failed the first landmark but met the second one had prognoses similar to those who failed both landmarks. In summary, early intervention strategies can be based robustly just on the transcript level at 3 months. This trial was registered at www.clinicaltrials.gov as # NCT01460693.

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.

Second generation tyrosine kinase inhibitors prevent disease progression in high-risk (high CIP2A) chronic myeloid leukaemia patients

High cancerous inhibitor of PP2A (CIP2A) protein levels at diagnosis of chronic myeloid leukaemia (CML) are predictive of disease progression in imatinib-treated patients. It is not known whether this is true in patients treated with second generation tyrosine kinase inhibitors (2G TKI) from diagnosis, and whether 2G TKIs modulate the CIP2A pathway. Here, we show that patients with high diagnostic CIP2A levels who receive a 2G TKI do not progress, unlike those treated with imatinib (P=<0.0001). 2G TKIs induce more potent suppression of CIP2A and c-Myc than imatinib. The transcription factor E2F1 is elevated in high CIP2A patients and following 1 month of in vivo treatment 2G TKIs suppress E2F1 and reduce CIP2A; these effects are not seen with imatinib. Silencing of CIP2A, c-Myc or E2F1 in K562 cells or CML CD34+ cells reactivates PP2A leading to BCR-ABL suppression. CIP2A increases proliferation and this is only reduced by 2G TKIs. Patients with high CIP2A levels should be offered 2G TKI treatment in preference to imatinib. 2G TKIs disrupt the CIP2A/c-Myc/E2F1 positive feedback loop, leading to lower disease progression risk. The data supports the view that CIP2A inhibits PP2Ac, stabilising E2F1, creating a CIP2A/c-Myc/E2F1 positive feedback loop, which imatinib cannot overcome.

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:

Low leukotriene B4 receptor 1 leads to ALOX5 downregulation at diagnosis of chronic myeloid leukemia

ALOX5 is implicated in chronic myeloid leukemia development in mouse leukemic stem cells, but its importance in human chronic myeloid leukemia is unknown. Functional ALOX5 was assessed using an LTB4 ELISA and ALOX5, and LTB4R1 mRNA expression was determined via a TaqMan gene expression assay. LTB4R1 and 5-LOX protein levels were assessed by cell surface flow cytometry analysis. At diagnosis ALOX5 was below normal in both blood and CD34+ stem cells in all patients. On treatment initiation, ALOX5 levels increased in all patients except those who were destined to progress subsequently to blast crisis. LTB4 levels were increased despite low ALOX5 expression, suggesting that the arachidonic acid pathway is functioning normally up to the point of LTB4 production. However, the LTB4 receptor (BLT1) protein in newly diagnosed patients was significantly lower than after a period of treatment (P<0.0001). The low level of LTB4R1 at diagnosis explains the downregulation of ALOX5. In the absence of LTB4R1, the arachidonic acid pathway intermediates (5-HEPTE and LTA4) negatively regulate ALOX5. ALOX5 regulation is aberrant in chronic myeloid leukemia patients and may not be important for the development of the disease. Our data suggest caution when extrapolating mouse model data into human chronic myeloid leukemia.