BCR-ABL1 transcript types showed distinct laboratory characteristics in patients with chronic myeloid leukemia

Abstract

In chronic myeloid leukemia (CML) two main types of messenger RNA (e14a2 and e13a2) can be produced by BCR-ABL1 gene rearrangement. Due to conflicting results, the clinical value of these transcripts remains controversial. The aim of this study was to identify associations of e14a2 and e13a2 transcripts with laboratory variables and also the response to treatment. This study included 203 adult patients with CML treated with Imatinib as first-line drug in a reference hematology center in Northeast Brazil. Clinical and laboratory data were obtained after informed consent. Samples were collected for RNA extraction and analyzed by reverse transcription-polymerase chain reaction (PCR), according to the international protocol BIOMED-1. The LeukemiaNet 2013 criteria were used to establish the molecular response. The frequency distribution of the BCR-ABL1 transcripts was e14a2 (64%), e13a2 (34%), and double positives (2%). The results showed a statistically significant association of the e14a2 transcript type with thrombocytosis (P = 0.0005) and the e13a2 with higher leukocyte count (P = 0.0491). In a subgroup of 44 patients, the molecular response to treatment with Imatinib was assessed by quantitative PCR at 3 months (BCR-ABL1 ≤ 10%), 6 months (BCR-ABL1 ≤ 1%), or 12 months (BCR-ABL1 ≤ 0.1%). Although patients with the transcript e14a2 showed higher frequency of good responses than patients with the transcript e13a2, this difference was not statistically significant. In agreement with published data, our results showed association of the BCR-ABL1 transcript e14a2 with thrombocytosis and the BCR-ABL1 transcript e13a2 with higher leukocytosis in patients with chronic myeloid leukemia.

Introduction

The influence of the genomic breakpoint location and its association with specific clinical-biological characteristics and response to therapy remains controversial (De Braekeleer, 2016). In this regard, some authors found no significant association but others suggested a possible prognostic value of the transcript types in relation to better response to treatment with Imatinib (de Lemos et al., 2005; Lucas et al., 2009; Sharma et al., 2010).

Although various breakpoints within the BCR and ABL genes have been described, more than 95% of patients with chronic myeloid leukemia (CML) produce an mRNA in which either the BCR exon 13 (e13) or BCR exon 14 (e14) is fused to the ABL exon 2 (a2), yielding fusion forms e13a2 and e14a2, respectively (Apperley, 2015). The e13a2 and e14a2 transcript types produce a 210-kDa fusion protein (p210), an abnormal tyrosine kinase known to be critical for the clinical and pathologic features of CML (van Dongen et al., 1999). The production of fusion proteins increases the diversity of protein-protein binding domains associated with tyrosine kinase activity (Hai et al., 2014). Agents that block the tyrosine kinase activity, such as Imatinib, have been successfully used for treatment (Hughes et al., 2003).

The identification of prognostic molecular markers may be important to characterize the response of CML patients subjected to current treatments. The aim of this study was to detect the different transcript types of p210 BCR-ABL1 gene rearrangement in patients with CML and identify possible associations with variables of prognostic importance.

Material and Methods

The study included 203 patients over 18 years old of both genders diagnosed with CML from January 2009 to May 2015 at Hemope Hospital in Recife, Brazil. It was approved by the Institutional Research Ethics Committee (No. 13/2013) with clinical and laboratory data obtained from patients’ records and database. Peripheral blood samples were obtained for molecular analysis after informed consent and Ethics Committee approval.

The identification of the transcript p210 BCR-ABL1 gene rearrangement was performed by reverse transcription-polymerase chain reaction (RT-PCR) according to the international BIOMED-1 protocol (van Dongen et al., 1999). The LeukemiaNet 2013 criteria were used to establish the molecular response (Baccarani et al., 2013). Statistical analysis was done using the Stata 12.0 software. The Student t-test and the Mann-Whitney U-test were used and P ≤ 0.05 was considered statistically significant.

Results

The male:female ratio was 1.4:1 and the median age at diagnosis was 48 years old, range between 18 and 92 years. The majority of patients were in chronic phase of the disease (95%). At diagnosis, the blood count showed leukocytosis above 50 x 10⁹/L in 89% of cases, anemia in 72%, and thrombocytosis in 40%. The BCR-ABL1 rearrangement was found in all samples with the following distribution of the transcripts: e14a2 (N = 130; 64%), e13a2 (N = 68; 34%), and both types (N = 5; 2%). The comparison between variables at diagnosis and the two main transcript types is shown in Table 1.

Table 1: Clinical and laboratory data of 203 adult patients with chronic myeloid leukemia according to BCR-ABL1 transcript types.

Discussion

The clinical value of the transcript types in CML remains controversial (Table 2). Meissner et al. (1999), Cruz et al. (2014), and Al-Achkar et al. (2016) found no significant association between the transcript type and various clinical and laboratory parameters. However, the e14a2 transcript has been associated with high platelet count (Inokuchi et al., 1991; Perego et al., 2000; Rosas-Cabral et al., 2003; Bennour et al., 2013) and age at diagnosis (Bennour et al., 2013).

Table 2: Main published studies about the association between p210 BCR-ABL1 transcript types and laboratory data or prognosis in patients with chronic myeloid leukemia.

Regarding the main variables at diagnosis, results in our patient group are similar to those already published (Barboza et al., 2000; Bennour et al., 2013; Smith et al., 2014). In agreement with published data, our study showed a statistically significant association between the e14a2 transcript and high platelet count (Bennour et al., 2013), but also between the e13a2 transcript with high leukocyte count (Hanfstein et al., 2014). The statistical analysis for the e13a2/e14a2 was not possible, due to the small number of patients with this transcript (N = 5).

Some authors have found a better molecular response with the e14a2 transcript (Hanfstein et al., 2014; Lin et al., 2016; Jain et al., 2016) or e13a2 (de Lemos et al., 2005; Sharma et al., 2010). Lin et al. (2016) showed that males with e13a2 were a less favorable group in their response to imatinib treatment. In our study, it was not possible to demonstrate association between the transcript type and molecular response.

The identification of the type of transcript is important not only for minimal residual disease monitoring as different transcript types seem to represent distinct biological entities (Hanfstein et al., 2014), but also because the transcript type could be used to select treatment regimen for patients with CML (Jain et al., 2016).

Conclusion

Our data show an association between the BCR-ABL1 transcript type e14a2 with thrombocytosis and the BCR-ABL1 e13a2 with leukocytosis in patients with chronic myeloid leukemia.

Acknowledgment

The authors thank Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco - FACEPE (#BIC-1248-4.01/14) for funding the study, and Prof. Ulisses Montarroyos for statistical support.