Clinical Significance of BCR-ABL Fusion Gene Subtypes in Chronic Myelogenous and Acute Lymphoblastic Leukemias

The Philadelphia chromosome (Ph), t (9; 22) (q34; q11.2) is transcribed into a fusion gene, BCR-ABL (Awan et al., 2012; Sabir et al., 2012). This translocation is one of the most common genetic abnormalities detected in leukemia. The site of the breakpoint in the BCR gene may influence the phenotype of diseases. In the vast majority of patients, the breakpoints in the BCR gene are clustered within three well-defined regions: (I) a 55 kb sequence of the first intron, called the minor breakpoint cluster region (m-bcr); (II) a 5.8 kb region spanning exons 12-16, called the major breakpoint cluster region (M-bcr), and finally; (III) intron 19, called μ-bcr. The resultant fusion transcript (e1-a2) (m-bcr) encodes a 190 kDa chimeric protein (p190), and in cases of M-bcr, codes a 210 kDa chimeric protein (p210) and in cases of μ-bcr, a 230 kDa protein (p230) (Fausel, 2007). Large studies indicated that BCR-ABL fusion gene is a highly useful diagnostic tool that controls the effectiveness of the chronic myelogenous


Introduction
The Philadelphia chromosome (Ph), t (9; 22) (q34; q11.2) is transcribed into a fusion gene, BCR-ABL (Awan et al., 2012;Sabir et al., 2012).This translocation is one of the most common genetic abnormalities detected in leukemia.The site of the breakpoint in the BCR gene may influence the phenotype of diseases.In the vast majority of patients, the breakpoints in the BCR gene are clustered within three well-defined regions: (I) a 55 kb sequence of the first intron, called the minor breakpoint cluster region (m-bcr); (II) a 5.8 kb region spanning exons 12-16, called the major breakpoint cluster region (M-bcr), and

Clinical Significance of BCR-ABL Fusion Gene Subtypes in Chronic Myelogenous and Acute Lymphoblastic Leukemias
Yuan-Xin Ye, Juan Zhou, Yan-Hong Zhou, Yi Zhou, Xing-Bo Song, Jun Wang, Li Lin, Bin-Wu Ying*, Xiao-Jun Lu* leukemia (CML) treatment and indicates at an early stage resistance development or disease progression (Ohsaka et al., 2002;Hehlmann et al., 2007;Hunger, 2011;Ohanian et al., 2012;Sabir et al., 2012;Soheila et al., 2013).Recent research showed that BCR-ABL fusion gene can also be detected in 25-30% of adult and 2-5% of childhood cases of acute lymphoblastic leukemia (ALL), and less frequently, in acute myelogenous leukemia (AML) (Den Boer et al., 2009;Uchida et al., 2009;Yeung et al., 2012).In ALL, this genetic lesion is known to confer a very poor prognosis (Hanfstein et al., 2012), which made its detection important in planning aggressive therapies including allogeneic bone marrow transplant.For different BCR-ABL subtype, some reports indicated that M-bcr is sufficient to course CML and few CML cases with m-bcr were reported (Moorman et al., 2007;Yanada et al., 2008).In CML, the prevalence of M-bcr is higher than m-bcr (Press et al., 2007).In contrary, ALL patients showed a higher prevalence of m-bcr (Chomel et al., 2012).Although the distribution of BCR-ABL subtype have been studied in CML and ALL separately (Hanfstein et al., 2012;Marin et al., 2012), few studies have investigated the BCR-ABL subtype distribution in CML-CP, CML-BP and ALL patient simultaneity and there is still lack of data on the impact of BCR-ABL subtypes in progression of CML and ALL.It is also a matter of contention if BCR-ABL subtype may be significantly predictive for the long-term outcome of CML and ALL patients treated with imatinib (IM) first line.In this study, we focused on analyzing the distribution difference of M-bcr and m-bcr in CML-CP, CML-BP and ALL patients with bone marrow smear cytology, blood routine examination results and BCR-ABL concentration before treatment in order to make a preliminary investigation on the relationship between BCR-ABL subtype and patient feature in different disease entities.Moreover, BCR-ABL concentration after treatment for 3, 6, 9 months and 1 year were also followed-up to investigate the association between longterm outcome of CML and ALL patients treated with IM first line and BCR-ABL subtype elementarily.These data confirm and further strengthen that M-bcr and m-bcr play differently in leading to leukemia and may influence the prognosis.BCR-ABL subtype should be considered while making treatment protocols.

Study population
During June 2007 to December 2009, a total of 452 BCR-ABL positive patients diagnosed strictly according to WHO diagnostic criteria as CML-CP, CML-BP and ALL before treatment in the West China Hospital were enrolled into the present study.All patients enrolled were BCR-ABL positive and clinical data such as WBC, RBC, PLT, and primitive cell ratio for all patients were collected and assessed.80 CML-CP patients and 32 ALL patients under imatinib (IM) treatment (400mg qd for remaining life) followed-up for at least 1 year with BCR-ABL relative concentration detected after 3 months, 6 months, 9 months and 1 year were enrolled in prognosis analysis.Written Informed consents were obtained from all included individuals and this study was approved by the ethical committee of West China Hospital, Sichuan University.

RNA extraction
Bone marrow samples were collected from these patients and subjected to cytogenetic analysis and RNA isolation isolated within 24 hours using QIAamp RNA Blood Mini-Kit (QIAGEN, Hilden, Germany).

Reverse transcription (RT) and QPCR for BCR-ABL relative quantification
Reverse transcription was carried out with a cDNA Synthesis Kit (Roche Diagnostics) according to the manufacturer's instructions.The resulted cDNA was used as the template of real-time PCR reaction, which was performed in a LightCycler 2.0 real-time fluorescence PCR detection system, according to the manufacture's instruction .The concentration of BCR-ABL fusion gene in patient sample was calculated on the basis of the standard curve established with K562 cell standard samples and normalizing their amplification to glyceraldehyde-3phosphate dehydrogenase (GAPDH).The relative gene expression level was calculated on the basis of the ΔCt.

Reverse transcription (RT) PCR and electrophoresis for BCR-ABL subtyping
According to F Gabert ( 14), the ENF501 (BCR3173-3193: 5'-TCCGCTGACCATCAATAAGGA-3') was selected as the forward primer for the RT-PCR detection of M-bcr transcripts and ENF402 (BCR1727-1744: 5'-CTGGCCCAACGATGGCGA-3') was selected as the forward primer for the RT-PCR detection of m-bcr transcripts.A reverse primer, ENR 561 (ABL277-257: 5'-CACTCAGACCCT GAGGCTCAA-3') was used as reverse prime for both M-bcr and m-bcr.One-step RT-PCR was carried out with one-step RT-PCR Kit (QIAamp) according to the manufacturer's instructions.The thermal conditions consisted of reverse transcriptase at 50℃ for 30 min, Denature at 95℃ for 15min, and amplification at 95℃ 30s; 54℃ 1 min, 72℃ 1 min for 42 cycles; and extend at 72℃ for 10min.K562 cell RNA was used as M-bcr positive control and TOM-1 cell RNA as m-bcr positive control.For subtype M-bcr, a special band of 149bp was amplified and as for m-bcr, a band of 92bp was amplified.The electrophoresis condition was 150V for 30min.

Blood routine examination
Blood routine examination including red blood cell (RBC) counts, white blood cell (WBC) counts, platelet counts was examined using the fully automated hematology analyzer XE-5000™ (Sysmex, Kobe, Japan).

Bone marrow smear cytology
The bone marrow smears were stained with Wright's-Giemsa, and examined by light microscopy.Two hundred nucleated cells were classified and the percentages of all types of cells were calculated.Primitive cell ratio (%) represented the percentage of Primitive cell in total nucleated cells.

Statistical analysis
All measurements were performed in at least triplicate.Results were expressed as a range with median.SPSS software (version 15.0, SPSS Inc., USA) was used for statistical analysis.Chi-square test was used for BCR-ABL subtype distribution analysis and Kruskal Wallis test was used for quantitative data analysis.Level of statistical significance p<0.05 was used in all analyses.

Basic characteristics of studied patients
After adjustment was made for smoking, age and other general cancer-related factors, 452 patients enrolled in the current study.The average age of the patients was 39.6 years, and M/F was 270/182.All the patients were diagnosed in the light of the clinical manifestations and bone marrow cytogenetic examination report in accordance with the WHO's diagnostic criteria and were divided into three groups: group1: CML-CP patients with DOI:http://dx.doi.org/10.7314/APJCP.2014.15.22.9961Clinical Significance of BCR-ABL Fusion Gene subtypes in Chronic Myelogenous and Acute Lymphoblastic Leukemias BCR-ABL positive.(n=354); group2: CML myeloid BP patients with BCR-ABL positive.(n=26); group3: ALL patients with BCR-ABL positive.(n=72).The average age of the patients and M/F ratio in different group were shown in Table 1 and there showed no significant difference in age and M/F among CML-CP, CML-BP and ALL groups.

Association analysis of BCR-ABL subtype with BCR-ABL relative concentration after imatinib treatment
Patients with IM-treatment were followed up for at least one year and 80 CML-CP patients and 30 ALL patients were enrolled with BCR-ABL relative concentration detected after 3 months, 6 months, 9 months and 1 year.No CML-BP patients were enrolled as only 3 patients were followed long enough.80 CML-   CP patients followed-up were also divided into three groups according to the BCR-ABL subtypes.In 40 M-bcr patients, the median BCR-ABL relative concentration after 3 months, 6 months, 9 months and 1 year treatment were 1.6%, 0.05%, 0.0%, and 0.0%.In 14 m-bcr patients, the median BCR-ABL relative concentration were 5.1%, 2.41%, 1.2% and 0.3% and in 26 mixed patients, 3.2%, 0.9%, 0.6% and 0.2% respectively.Patients with m-bcr showed notable higher BCR-ABL relative concentration than M-bcr and mixed group with significantly differences and the differences enlarged with further treatment as shown in Table 4. Two patients showed m-bcr got relapse.In 32 ALL patients followed-up, 4 patients were M-bcr subtype, with median BCR-ABL relative concentration of 0.32%, 0.0%, 0.0%, 0.0% respectively.20 were m-bcr subtype with median BCR-ABL relative concentration of 1.29%, 1.02%, 0.52%, 0.98% and 8 were mixed with median BCR-ABL relative concentration of 3.26%, 0.28%, 0.1%, 0.0% respectively.Patients with m-bcr showed higher BCR-ABL relative concentration after treatment for 1 year with significant difference (p=0.012).One patient showed m-bcr got relapse.

Discussion
BCR-ABL fusion gene is one of the most commonly detected abnormal genes in leukemia.Though the BCR-ABL subtype is clearly defined, the distribution and expression levels of BCR-ABL subtypes, their relationships with clinical manifestations and prognosis in CML and ALL have not been well established yet.It has been reported that the expression of BCR-ABL is associated with the disease severity.In our previous researches (Lu et al., 2011), we have confirmed the significance of Real-time fluorescent PCR in monitoring treatment responses, and identification of relapse in CML patients.Some pervious showed (Gabert et al., 2003;Tripathi et al., 2011) that in patients who receive interferon treatment that does not induce molecular and cytogenetic remission, the increased expression of BCR-ABL suggests that the disease may CML-CP patients (n) 40 14 26 BCR-ABL relative concentration after 3 months (%) median(P5-P95) 1.6(0, 8.87) 5.1(0, 16.8) 3.2(0.0,11.5) 0.040* BCR-ABL relative concentration after 6 months (%) median(P5-P95) 0.05(0.0,7.2) 2.41(0.0,13.8) 0.9(0.0,9.0) 0.049* BCR-ABL relative concentration after 9 months (%) median(P5-P95) 0.0(0.0,4.5) 1.20(0, 8.9) 0.6 (0.0, 4.32) 0.020* BCR-ABL relative concentration after 1 year (%) median(P5-P95) 0.0(0.0,0.5)0.3(0.0,9.3) 0.2(0.0,5.76) 0.007** Replase after treatment 0 2/12 0 ALL patients(n) 4 20 8 BCR-ABL relative concentration After 3 months (%) 0.32(0.0,9.12) 1.29(0.0,7.8) 3.26(0.0,9.92) 0.59 BCR-ABL relative concentration After 6 months (%) 0(0.0, 4.62) 1.02(0.0,6.12) 0.28(0.0,6.12) 0.244 BCR-ABL relative concentration After 9 months 0(0.0, 1.72) 0.52(0.0,5.31) 0.1(0.0,5.01) 0.139 BCR-ABL relative concentration (%) After 1 year (%) 0(0.0, 0.92) 0.98(0.0,4.32)0(0.develop into an accelerated phase or a blastic phase, which is valuable for evaluating the prognosis of the patients (Cetin et al., 2012;Mu et al., 2012).In our present study, we observed significant different expression of BCR-ABL gene in CML-CP patients, CML-BP patients and ALL patients, which indicated that the expression of BCR-ABL was higher in CML-BP patients and Ph+ ALL patients than that in CML-CP patients.This result was not only consistent with other studies (Cetin et al., 2012;Ernst et al., 2012;Mu et al., 2012) but also a beneficial complement to other studies.It was reported in some early reports that m-bcr might be frequent in B lymphoid progenitor cells but uncommon in hematopoietic stem cells, explaining the rarity of m-bcr in CML but M-bcr and m-bcr per se can induce the myeloproliferative process, resulting in promotion of chronic phase CML (Bhatia et al., 2012;Gokbuget et al., 2012).These reports also showed that in patients showed a higher incidence of expression of m-bcr than M-bcr in ALL (Lee et al., 2005;Wang et al., 2011).In our study, we found distinct different distribution of M-bcr, m-bcr and mixed subtype among CML-CP, CML-BP and ALL patients.Our results confirmed that CML-CP patients have more M-bcr while ALL patients with more m-bcr, which was similar to other studies.In consistent with previous reports the occurrence of mixed subtype was higher than that reported (Li et al., 2010), this difference may result from the mutual distraction during PCR.In this study, we analyzed ages, bone marrow smear cytology results, blood routine examination results and BCR-ABL concentration before treatment in CML-CP CML-BP and ALL patients with different subtypes.In both CML-CP group and CML-BP group, patients with M-bcr showed older age with higher WBC and PLT counts.In contrast, m-bcr occurs more likely in younger CML patients, and atypical patients without markedly increased WBC counts.These results suggested that M-bcr, which encodes the p210, was associated with the typical CML with older age and markedly increased WBC counts.This finding agrees with the reports that over-expression of p210 leads to the occurrence of the CML (Killick et al., 1999).In ALL patients, our study showed that patients with M-bcr have a significantly higher WBC counts which was coincident with other reports that patients with Ph-positive ALL presented with higher WBC counts (Killick et al., 1999;Matutes et al., 2011).Overall, M-bcr occurs more frequently in typical CML patients and high-WBC ALL patients.Following the results of the IRIS multicenter trial (Druker et al., 2006), IM promptly became the standard frontline therapy of CML in CP and it is well-known that relative BCR-ABL concentration is an important prognosis factor (Mizuta et al., 2012), BCR-ABL relative concentration after treatment was chosen as an indicator of prognosis.The followed-up dates indicated that patients with M-bcr showed a lower relative BCR-ABL concentration after treatment in both CML-CP and ALL, which coincided with reports that (Al-Seraihy et al., 2009;Weinberg et al., 2010) these patients who show older ALL patients, exhibits a resistance to IM treatment more likely with a poor prognosis.These results support our predetermination that patients with M-bcr have a better prognosis than with m-bcr subtype.
No CML-BP patient was followed long enough, because most of them were under chemotherapy or allogenetic bone marrow transplantation therapy.It has been reported that the BCR-ABL protein shows an increased tyrosine kinase activity and it seems to deregulate the normal cytokine-dependent signal transduction pathways leading to the inhibition of apoptosis.From our research, patients with different BCR-ABL subtype showed clear difference in clinical feature and prognosis, which gave us a hint that different BCR-ABL subtypes may play different role in leukemogenesis.Early reports indicated that the Racfamily kinases have been implicated in BCR-ABL subtype signaling (Mulloy et al., 2010) and in the progression of CML and Ph+ ALL (Cho et al., 2005).All these prompted us a potential direction to further study the mechanism of leukemogenesis in different BCR-ABL subtypes.From the present study, we obtain some important information regarding.Firstly, we find that the expression of BCR-ABL is significantly different between CML-CP and CML-BP patients, suggesting that an abrupt increase in BCR-ABL expression may imply that the disease may start to enter an accelerated phase or a blastic crisis phase.Secondly, we confirm that BCR-ABL subtypes occur differently between the CML patients and ALL patients.Thirdly, our study suggests that patients with the m-bcr subtype may have a worse prognosis than patients with the M-bcr subtype.But there are still some limitations in our study.First, the statistical difference may be affected by the small sample sizes in CML-BP patients and ALL patients.Second, the mechanisms of M-bcr and m-bcr and their associated prognosis in different diseases need further study in a large sample of patients with a long-term follow-up.
In summary, we investigate the expression of BCR-ABL subtypes in CML-CP patients, CML-BP patients and Ph+ ALL patients, and find that M-bcr subtype and m-bcr subtype occur differently among these patients.M-bcr occurs more frequently in classical CML patients and younger ALL patients, who show a good response to imatinib.In contrast, m-bcr occurs more frequently in atypical CML patients and older ALL patients, who exhibit a resistance to imatinib treatment with a poor prognosis.