Research Highlights

Mirna in Chronic Myeloid Leukemia and Imatinib Response

Evaluation of: Flamant S, Ritchie W, Guilhot J et al.: Micro-RNA response to imatinib mesylate in patients with chronic myeloid leukemia. Haematologica 95(8), 1325–1333 (2010).

miRNAs are small, endogenous, noncoding RNAs that post-transcriptionally regulate gene expression through partial base-pairing with the 3´-UTR region of target mRNAs, resulting in mRNA destabilization and translational inhibition [1]. The recent implication of miRNAs in the regulation of important biological processes altered in chronic myeloid leukemia (CML), such as the cell cycle, apoptosis and adhesion, establishes these small RNA molecules as potential players in CML pathogenesis mediated or not by BCR-ABL activity.

Using TaqMan^® (Applied Biosystems, CA, USA) low-density array system, Flamant et al. studied miRNA expression of 364 human mature miRNAs in peripheral blood mononuclear cells isolated from blood samples from 11 newly diagnosed CML patients before and within the first 2 weeks of imatinib (IM) therapy [2]. They observed a significant median fold change of more than three between day 0 and 14 for five miRNAs. These results were confirmed by retrotranscription quantitative PCR (RT-qPCR) in these patients, in 12 additional CML patients and compared with six healthy donors. After IM treatment, miR-142–3p, miR145 and miR199b-5p demonstrated decreased expression, whereas miR-150 and miR-146a demonstrated increased expression, with a trend, for all, toward a return to the normal control values. These miRNAs correlated with some clinical data, in particular miR142–3p with the Sokal risk score [2]. Their results are concordant with some of the results which appeared in two published studies [3,4], which focused on the miRNA repertoire of CML cells. Flamant observed, as Venturini did using K562 cells treated with IM, the downregulation of miR142–3p and of species produced from a common precursor (the so-called ‘miR 17–92 cluster‘). These results allowed them to validate their TaqMan^® low-density array system. However, the upregulation of miR142–3p at diagnosis was not found in CD34⁺ CML patients in Venturini‘s study [3]. Flamant also observed the upregulation of miR150, miR151 and miR10a after 14 days of IM treatment in CML patients. The miR150 and miR151 results are concordant with Agirre et al.[4], but are in partial discordance for miR10a. In fact, in Agirre‘s study, expression of miR10a did not depend on BCR-ABL activity because treatment of Mo7e-p210 cells with IM did not affect the expression of miR10a whereas in Flamant‘s study an upregulation of miR10a is observed after 14 days of IM treatment. However, some discrepancies could be due to different strategies used for miRNAs expression normalization and to types of cells used. In fact, normalization was carried out using the expression of miR-16 in Venturini‘s study, GUSB and U6 genes in Agirre‘s study, and RUN48 gene in Flamant‘s study. The independent BCR-ABL activity of miRNA was studied in vitro in Mo7e and Mo7e-p210 cells in Agirre‘s study and in vivo in peripheral blood mononuclear cells in Flamant‘s study. Finally, miR150 was found to be reduced by approximately 30-fold in both chronic phase and advanced phase patients as compared with normal cells, while the downregulation of miR-146a and the upregulation of miR142-3p and miR199-5p in chronic phase were not conserved in blast crisis phase. miR-150 targets the oncogene c-Myb, which was demonstrated to be necessary for BCR-ABL mediated transformation [5]. Recently miR-328, which profoundly affects myeloid cell differentiation has been involved in the mechanisms regulating the phenotype of CML-blast crisis progenitors [6].

Finally, Flamant‘s results provide potential new biomarkers in CML. As CML is a clonal stem cell disease, it would be interesting to confirm these results using CD34⁺ CML cells and to study their dependence on BCR-ABL activity.

Molecular Breakpoint of BCR-ABL in Chronic Myeloid Leukemia and Imatinib Response

Evaluation of: Sharma P, Kumar L, Mohanty S et al.: Response to imatinib mesylate in chronic myeloid leukaemia patients with variant BCR-ABL fusion transcripts. Ann. Haematol. 89(3), 241–247 (2010).

The BCR-ABL fusion transcript, is a molecular hallmark of the chronic myeloid leukemia (CML), which is a result of fusion of the t(9;22)(q34;q11). It encodes for a constitutively active tyrosine kinase, responsible for leukemogenesis. The breakpoints on chromosome 9 are variable over a region of 200 kb, but are always located from the 5´ end point to exon 2 of the ABL gene whereas the breakpoints on chromosome 22 can occur in three region clusters on the BCR gene. Most of them are restricted to a central region of the BCR gene called the ‘major breakpoint cluster region‘, located between BCR exons 13 and 15 (i.e., exon b2 and b4) leading to different BCR-ABL transcript types. Most of the CML patients have transcripts containing the b3a2 or b2a2 junctions. For a minority, alternative splicing induces the presence of both the b3a2 and b2a2 transcripts. So far, the clinical impact of the different transcripts remains unclear. Three main response criteria can be used to evaluate CML treatment response: complete hematological remission, complete cytogenetic response (CCyR), and major molecular response. In this article, Sharma et al., presented a cohort of 87 CML Indian patients treated with imatinib 400 mg/day in front-line therapy (n = 30) or after IFN-α and/or hydroxyurea therapy (n = 57) [1]. They analyzed the clinical impact of different transcript types on CCyR at 18 months and on the BCR-ABL/ABL expression levels. b3a2 transcript was present in 53% of patients (n = 46), b2a2 in 39% (n = 34) and both in 8% (n = 7). Patients‘ characteristics at diagnosis (age, sex, Sokal and Hasford risk, and hemoglobin (Hb), white blood cells (WBC) and platelet levels) were not different between groups. Significantly more b2a2 patients achieved CCyR compared with b3a2 patients (59 vs 28%, p = 0.04), irrespective of prior treatment, suggesting that b2a2 patients could achieve a better response to imatinib. A nonsignificant trend to a lower BCR-ABL/ABL expression was observed in b2a2 transcripts compared with b3a2 transcripts. These results are in agreement with those of Lomaia et al.[2], who reported in 27 Russian patients a shorter time to obtain CCyR in b2a2 patients and those of De Lemos et al.[3], who founded, in 22 Brazilian patients, a slight significant decrease in the mean BCR-ABL expression levels in b2a2 transcripts after 6 months of treatment, despite similar levels of transcripts at the beginning of treatment between the two groups of transcripts. Conversely, Popampalli et al.[4] did not find any difference in molecular and cytogenetic response in 90 Indian patients, Vega-Ruiz et al. reported from 480 American patients, a BCR-ABL transcript level lower at 3, 6 and 12 months for b3a2 patients than for b2a2 patients [5]. Lucas et al. reported that in 71 English patients b3a2 patients had a higher CCyR rate at 12 months than b2a2 patients and achieved CCyR faster, demonstrating a higher tyrosine kinase activity in b2a2 patients [6].

Therefore, discrepancies between studies remain in the imatinib era. This could be explained by the different response criteria to CML treatment chosen by authors, the distribution of each transcript in different cohorts, the ethnic origin and number of patients studied, and the different duration of follow up. Furthers studies are requested to know if a real impact of BCR-ABL transcripts type exists.

Role of Cyp3A4 Activity in Chronic Myeloid Leukemia and Imatinib Response

Evaluation of: Géen H, Skoglund K, Rommel F et al.: CYP3A activity influences imatinib response in patients with chronic myeloid leukaemia: a pilot study on in vivo CYP3A activity. Eur. J. Clin. Pharmacol. 66(4), 383–386 (2010).

Imatinib, a competitive inhibitor of the BCR-ABL tyrosine kinase, is the key treatment of chronic myeloid leukemia (CML). Despite the outstanding results of imatinib in the chronic phase of CML, cases of treatment failure or suboptimal response have been reportedly caused by heterogeneous molecular response. Apart from dosage errors, drug interactions or problems with noncompliance to treatment, many mechanisms of resistance are known. These mechanisms could involve the target (by mutation or amplification of BCR-ABL) or downstream pathways BCR-ABL independent, or whether the pharmacokinetics of imatinib. The oral bioavailability of imatinib is dependent on the gastrointestinal absorption and extensive first-pass metabolism. Imatinib is metabolized mainly by CYP3A4 and CYP3A5 [1]. The main metabolite in plasma, CGP74588, mostly formed by CYP3A4, represents approximately 20% of the parent drug plasma level in patients. It has a longer terminal half-life (85–95 h) than imatinib and is pharmacologically active with a potency and selectivity similar to those of imatinib [2]. Géen et al. designed this study to investigate the role of the CYP3A4 activity in the response to imatinib therapy [3]. The authors phenotyped 14 Caucasian CML patients for in vivo CYP3A4 activity using quinine as a probe drug and they correlated it with the molecular response at 12 months. An undetectable ratio of BCR-ABL:glucuronidase-β defined the complete molecular response and patients with a detectable transcript at 12 months were classified in partial molecular response. Interestingly, a higher mean CYP3A4 activity (low quinine ratio) was observed in complete molecular response patients compared with partial molecular response patients suggesting a clinical importance of the CGP74588 on efficacy of imatinib therapy. However, results may be biased by several confounding factors such as different disease phases at inclusion, different dosages of imatinib in four patients (300–600 mg), different time in the dosage of CYP3A4 activity (before, during or after imatinib therapy). Moreover in the five patients with two activity assessments (before and after 2 months of imatinib), there was no reproducibility of the measures, suggesting a variability in CYP3A4 activity. Trough imatinib plasma levels (C_min) have been reported to be associated with major molecular response (defined by the BCR-ABL level 3 log reduction relative to a standardized baseline) following standard dose of imatinib in CML with a plasma threshold of 1002 ng/ml [4]. So, it would be interesting to correlate the CYP3A4 activity with the plasma dosage of both imatinib and CGP74588 and to see if among good responders patients despite a C_min value of less than 1002 ng/l, a high CGP74588 level could explain the better outcome. Finally, the result should be confirmed in a larger and more homogenous cohort to confirm this pilot study.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.