Study and Survey of BCR-ABL Transcript Types in Chronic Myeloid Leukemia (CML) Young Egyptian Patients

INTRODUCTION

Chronic myeloid leukaemia (CML) is a myelo-proliferative disorder with the cytogenetic hallmark of Philadelphia chromosome, characterized by a reciprocal translocation t(9;22)(q34;q11). This translocation results when the Abelson (ABL) gene from chromosome 9 merges with the breakpoint cluster region (BCR) gene on chromosome 22, resulting in a BCR-ABL fusion gene on 22q11 that encodes for uncontrolled tyrosine kinase activity. (Nagrani et al.,2011), (Kaaren et al.,2009). 

BCR-ABL gene codes for a protein tyrosine kinase (PTK) that is the cause of leukemic transformation of hematopoietic stem cells (Quintas-Cardama and Cortes 2009).

 Breakpoints in chromosome 22 are located in major breakpoint cluster region (M-BCR), leading to origin to two transcripts, e14a2 (B3A2) or e13a2 (B2A2) coding for PTK of slightly different length (P210) (Melo 1996). Much more rarely, the breakpoint is located outside the M-BCR, either in the minor BCR (mBCR), leading to a fusion gene (e1a2) that codes for a similar PTK (P190) which is more common in Philadelphia-positive (ph+) acute lymphoblastic Leukaemia, or in the micro BCR (µBCR) leading to a fusion gene (e19a2) coding for another PTK (P230) that is the molecular marker of a neutrophilic variant of CML. (Quintas-Cardama and Cortes 2009).

 Other, rare transcripts have also been reported. Therefore, more than 90% of CML are B3A2 or B2A2. Alternative splicing or mispricing of the primary transcript can lead to a contemporary expression of both transcripts (B3A2/B2A2), and sometimes also to a detectable co-expression of the e1a2 transcript (P190) (Melo J 1996).

Both B3A2 and B2A2 code for a protein (P210) with a tyrosine kinase (TK) function, but with a slightly different length (25 amino acids). It is not known if their activity is identical. It has been reported that they may affect the characteristics of the disease and the risk (Pfirrmann, et al. 2017). 

It has not been found that they affect survival, but it has been reported that they affect the rate, the speed, and the depth of the molecular response to tyrosine kinase inhibitors (TKIs) (Jain, et al. 2016). Moreover, it has been reported that the two may have a different immunogenicity, an important difference that may affect the probability of achieving a deep or a complete molecular remission (Tarafdar, et al. 2017).

The incidence of B3A2 and B2A2 has been reported in some studies (Hanfstein, et al.2014), but not in the majority of the studies of treatment with TKIs. It is not known if the incidence may be affected by ethnic, geographic, gender, and age variables. The incidence and the country, region, gender and age distribution of the rare types are not known. Therefore, the true incidence of BCR-ABL transcript types is unknown. The definition of the incidence of the transcript type is a necessary prerequisite to any other study of the biologic and clinical value of the transcript type. (Hanfstein, et al.2014).

        The disease is characterized by three phases, namely chronic phase (CP), accelerated phase (AP) and blast crisis (BC). According to the European Leukaemia Net (ELN), the criteria for BC CML are percentage of blasts plus promyelocytes in peripheral blood or bone marrow >20%, progressive splenomegaly, thrombocytopenia (<100 X 103/µL) unrelated to therapy, and karyotypic evolution (Baccarani et al., 2013).

              Little is known about burden and epidemiological information concerning CML in Egypt. There is recent interest to observe incidence and mortality because of advent of new diagnostic and treatment policies for CML. (Azzazi and Mattar, 2013). 

The incidence of Ph+ CML increases with age: the reported median age at diagnosis is more than 60 years in epidemiologic registries and of approximately 50 to 55 years in clinical trials. Following the most widely accepted recommendations that set at 65 years the boundary between young and old persons, a relevant proportion of CML patients at diagnosis are “elderly.” (Gugliotta et al., 2011). 

The first Arab Leukaemia Network (ALN) report demonstrated that age-specific rates for CML in Egypt and Arab nations are lower by at least two decades compared to western populations (highest in age group 30-35 years). (Azzazi and Mattar, 2013).

AIM OF THE WORK

The primary objective is to assess the incidence of different BCR-ABL transcript types, among young Egyptian CML patients in different centers in Egypt to assess the impact of ethnic and demographic variables on disease outcome, particularly according to gender and age. 

And also to assess the relationships of different transcript types with the disease characteristics and response to treatment type.

Secondary objectives are:

a) To assess the incidence of transcript types by gender (male, female) and by age (by decades, from 18 to 50 years)

b) To assess the incidence of transcript types in different CML treating centres. 

c) To assess the frequency of transcript types according to the source of the data (Clinical Centres, Diagnostic Laboratories, National or Regional or Multi-institutional Registries, academic studies). 

PATIENTS AND METHODS

    Type of Study: Epidemiologic, non-interventional, retrospective, multi-centric study.

    Study Setting : The data was collected from the already existing CML databases of Clinical Centers, Diagnostic Laboratories, National, Regional, or Multi-institutional Study Groups, as well as from academic investigator-sponsored studies.

The data consists only of the number of cases with any given transcript type (B3A2, with B2A2, with B3A2/B2A2, and with other, rare, transcript types), either overall, or, whenever and wherever possible, depending on the database, according to gender (males, females) and age (by decades, from 18 years to < 50 years). Date of birth, date of diagnosis, clinical or biological baseline data, treatment data, and response and outcome data are also collected. The anonymity is ensured.

The definition of the transcript type was as reported in each original database. It is acknowledged that there are no internationally shared definitions or recommendations for distinguishing B3A2 from the mixed type B3A2/B2A2. Therefore, these two groups were analyzed both separately and together.

The collection of data from different sources is necessary to limit the bias that is intrinsic to any different source (the data from diagnostic laboratories, from Institutional databases or from clinical trial databases may refer to patients registered or enrolled with different criteria). Overlapping was avoided by calculating the incidence by source.

The study is multi central. The invitation to join the study and to contribute has been sent on to the investigators and the institutions that have shown interest in CML.

Data was collected from several sources:

a) The databases of Ain Shams University CML clinic and Hematology and Bone Marrow units.

b) The databases of Laboratories testing BCR-ABL for diagnostic purposes.

C) Other contributing medical centers.

 Data will be stored at department of Hematology and Bone Marrow Transplantation in Ain Shams University Hospital.

Study Period: Patients were evaluated for two years after the approval by the Ethical Committee of Ain Shams University faculty of medicine.

Study Population: All cases of newly diagnosed young Ph+ BCR-ABL+ CML already registered in already existing databases, between 2000 and 2019. The collection and the analysis of the data were restricted to the patients who were newly diagnosed, in order to avoid that the data of the same patient can be registered more than once. 

For the purposes of this study, "newly diagnosed" means ≤ 12 months from the first diagnosis, either treated or untreated.

Selection criteria for cases: 

Inclusion Criteria: 

Age more than or equal to 18 years old and less than 50 years old.

Newly diagnosed Ph+ BCR-ABL+ CML 

No co-morbidities to interfere with treatment.

Signed informed consent.

   Exclusion Criteria: 

Patients previously diagnosed as CML.

Patients with any immunological disease e.g.: SLE.

Previously treated patients.

      Selection criteria for controls : No controls are required      

Sampling Method: simple random sample 

Sample Size: 93 newly diagnosed Ph+ BCR-ABL+ CML patients. 

Ethical Considerations: No special consideration is required as it is non interventional study.  

Study Tools: Polymerase chain reaction (PCR) on peripheral blood sample from newly diagnosed Ph+ BCR-ABL+ CML patients.

Statistical Analysis: Statistics is descriptive: distribution, mean, SD, median, quartile, etc, calculations and comparisons are made by basic statistical methods (chi squared and chi squared variants).

Statistical Package: All statistical analysis was carried out using Statistical package for Social Science(SPSS).

RESULTS

Table (1): Demographic data of patients

Demographic Data

Study group (n=93)

Age

Mean ± SD

Range

35.35 ± 8.609

18-48

Males

Females

N (%)

N (%)

51 (54.8%)

42 (45.2%)

BCR-ABL transcript type

b2-a2

N (%)

36 (38.7%)

b3-a2

N (%)

45 (48.4%)

Both

N (%)

12 (12.9%)

Sokal score

High

N (%)

3 (3%)

Intermediate

N (%)

60 (65%)

Low

N (%)

30 (32%)

Hasford score

Low

N (%)

48 (52%)

Intermediate

N (%)

45 (48%)

High

N (%)

0 (0%)

Table (2): Null hypothesis test summary

Fig (1): Comparison between initial PCR levels in the 3 groups.

Fig (2): Comparison between PCR levels in the 3 groups at 3 months.

Table (4): Statistical significance of differences between PCR levels in the 3 groups at 3 months.

DISCUSSION

CML has a worldwide incidence of 1-1.5 cases per 100,000 inhabitants. CML constitutes 15-20% of all leukemias. The median age at diagnosis is 40-60 years, and although it is rare below 20 years, all age groups can be affected, CML has a slight male predominance (Fletcher et al., 2011). 

CML incidence rates in western countries vary from 0.6 to 2 cases per 100,000 inhabitants (Azzazi and Mattar, 2013). 

Highest rates were reported from Switzerland, USA, Italy, Australia, Germany, and UK. Lower rates were reported for Netherlands, Sweden, China, and India. An estimated 24,090 deaths are expected to occur in 2014 in USA. Death rates for leukemia have been declining for the past several decades; from 2006 to 2010, rates decreased by 0.8% per year among males and by 1.3% per year among females. (Nagrani et al., 2011).

              Little is known about burden and epidemiological information concerning CML in Egypt. There is recent interest to observe incidence and mortality because of advent of new diagnostic and treatment policies for CML. (Azzazi and Mattar, 2013). 

     In patients with Ph-positive CML the prediction of resistance to targeted therapy with tyrosine kinase inhibitors may help timely identification of those who would benefit from alternative therapies. Based on clinical response to Imatinib, criteria have been established to define failure or suboptimal response in early chronic phase CML (Verma et al., 2010).

Achievement of a cytogenetic response at 3–6 months under treatment with a second generation tyrosine kinase inhibitor was highly predictive for a major cytogenetic response at 12 months and was associated with increased progression-free and overall survival. However, these criteria are time-dependent and require observation for several months (Fabarius et al., 2011).

In the present study, we reported data of 93 young Egyptian patients with newly-diagnosed chronic phase CML.

They were 42 female patients and 51 male patients (45% and 55% respectively). 

Mean age was 35.35 years (ranged between 18 and 50 years).

Patients were divided into 3 groups according to BCR-ABL transcript type. 36 patients had b2-a2, 45 patients had b3-a2 and 12 patients had both. (38.7%, 48.4% and 12.9% respectively).

           Risk stratification according to Sokal score was done. 30 patients were of low risk, 60 patients were of intermediate risk and 3 patients were of high risk (32%, 65% and 3% respectively).

Mean Sokal score in patients with b2-a2 was 0.79, in patients with b3-a2 was 0.88 and in patients with both was 0.83.

           Risk stratification according to Hasford score was done. 48 patients were of low risk, 45 patients were of intermediate risk and there were no high risk patients (52%, 48% and 0% respectively).

Mean Hasford score in patients with b2-a2 was 652.9, in patients with b3-a2 was 831.9 and in patients with both was 914.4.

 All patients had low EUTOS risk score. 

Mean EUTOS score in patients with b2-a2 was 41.9, in patients with b3-a2 was 54.7 and in patients with both was 53.5.

  All patients presented with splenomegaly, the mean size of spleen was 18 cm at diagnosis. 

All patients presented with leukocytosis, mean basophilic count was (3×103/µL), mean eosinophilic count was (6.35×103/µL), mean platelet count was (364.4×10 3/µL).

Patients’ BCR-ABL levels were measured by PCR at 5 time points:

Initially, at 3 months, at 6 months, at 9 months and at 12 months.

      Patients with b2-a2 had higher PCR levels than the other 2 groups initially and at 3 months. The difference was statistically significant.

           Initially, PCR was higher in patients with b2-a2 compared to patients with b3-a2 and to patients with both (p=0.001 and p=0.011 respectively).

           At 3 months, PCR was higher in patients with b2-a2 compared to patients with b3-a2 (p=0.018).

           All other differences were not statistically significant.

CONCLUSION

Chronic myeloid leukaemia (CML) is a myeloproliferative disorder characterized by the presence of Philadelphia chromosome.

The disease has three phases, chronic phase (CP), accelerated phase (AP) and blastic crisis (BC). 

CML has a worldwide incidence of 1-1.5 cases per 100,000 inhabitants. 

    BCR-ABL gene codes for a protein tyrosine kinase (PTK) that is the recognized cause of the leukemic transformation of hematopoietic stem cells (Quintas-Cardama and Cortes 2009). 

   This hybrid gene is generated by a reciprocal translocation between chromosome 9 (ABL) and chromosome 22 (BCR), to a fusion gene that is located on chromosome 22 (Philadelphia chromosome). In CML, the breakpoints in chromosome 22 are located in major breakpoint cluster region (M-BCR), leading to origin to two transcripts, e14a2 (B3A2) or e13a2 (B2A2) coding for PTK of slightly different length (P210) (Melo 1996). Much more rarely, the breakpoint is located outside the M-BCR, either in the minor BCR (mBCR), leading to a fusion gene (e1a2) that codes for a similar PTK (P190), the latter is more common in Ph+ acute lymphoblastic leukemia, or in the so called micro BCR (µBCR) leading to a fusion gene (e19a2) coding for another PTK (P230) that is the molecular marker of a neutrophilic variant of CML. (Quintas-Cardama and Cortes 2009). Other, rare transcripts have also been reported. Therefore, more than 90% of CML are B3A2 or B2A2. Alternative splicing or mispricing of the primary transcript can lead to a contemporary expression of both transcripts (B3A2/B2A2), and sometimes also to a detectable co-expression of the e1a2 transcript (P190) (Melo 1996).

Both B3A2 and B2A2 code for a protein (P210) with a TK function, but with a slightly different length (25 amino acids). It is not known if their activity is identical. It has been reported that they may affect the characteristics of the disease and the risk (Pfirrmann et al. 2017). It has not been found that they affect survival, but it has been reported that they affect the rate, the speed, and the depth of the molecular response to TKIs (Jain et al. 2016). Moreover, it has been reported that the two P210 may have a different immunogenicity, an important difference that may affect the probability of achieving a deep or a complete molecular remission ( Tarafdar et al. 2017).

The incidence of B3A2 and B2A2 has been reported in some studies ( Hanfstein et al.2014), but not in the majority of the studies of treatment with TKIs. It is not known if the incidence may be affected by ethnic, geographic, gender, and age variables. The incidence and the country, region, gender and age distribution of the so-called rare types are not known. Therefore, the true incidence of BCR-ABL transcript types is unknown. The definition of the incidence of the transcript type is a necessary prerequisite to any other study of the biologic and clinical value of the transcript type.

 The results of the treatment of chronic phase (CP) Ph+ BCR-ABL1+ CML with TKIs are excellent, survival being very close to the survival of non-leukemic individuals (Baccarani et al. 2013). 

Therefore, there is little room for an improvement of survival. However, there is large room for an improvement of treatment-free remission (TFR), that is the major goal of current treatment (Baccarani et al. 2013).. 

The transcript type is important because it may affect the probability of achieving the deep molecular response that is required to achieve TFR (Baccarani et al. 2013).