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Role of cytogenetic profiles as prognostic factors for complete remission after induction phase in acute myeloblastic leukemia

Submitted October 19, 2021. Accepted November 18, 2021. Abstract Background Risk stratification for acute myeloid leukemia (AML) in children is a must in treatment strategy. This stratification is based on cytogenetic profiles, which are needed to determine proper management to gain better outcomes and reduce side effects of treatment. There is no such risk stratification available in Indonesia until now. Objective To evaluate the association between cytogenetic profiles of t(8,21) and inv(16) mutations with the complete response to induction phase of chemotherapy in pediatric AML. Methods A prospective study was conducted between year 2018 and 2020, involving children with AML from 4 pediatric oncology centers in Jakarta. Subjects were evaluated for cytogenetic profiles, especially t(8,21) and inv(16), as the favorable predictors for AML. Bone marrow remission was evaluated after 2 cycles of induction phase. The results were evaluated for remission rate and survival analysis. Results Karyotype data of 18 subjects were obtained. Translocation t(8;21) detected in 1 subject, and inv(16) mutation in 4 subjects. These two variables had no significant correlation with complete remission after induction phase. Nevertheless, favorable group had more tendencies to achieved remission than unfavorable group. Complete remission achieved in 61% subjects, 90% of theme had a relapse period with an average time 43 weeks. The relapse period in favorable group was shoter than in unfavorable group (34 weeks and 44 weeks, respectively). Conclusion This study shows that cytogenetic profiles of t(8;21) and inv(16) mutation can not be used as prognostic factors for complete remission after induction phase of chemotherapy in pediatric AML. [Paediatr Indones. 2021;61:343-9 ; DOI: 10.14238/pi61.6.2021.343-9].

L eukemia is the most common cancer in children, accounts for a third of all cancer types. Approximately, 15-20% of acute leukemia cases in children are acute myeloid leukemia (AML). In childhood, AML incidence increases with age, with 5 cases per 1 million in children aged 5-9 years, 9 cases per 1 million in children aged 15-19 years, and 37 cases per 100.000 in adults aged ≥ 20 years. 1 Risk-based treatment protocol is needed to improve pediatric AML outcomes. It has been applied in developed countries to decrease treatment-related death as an adverse effect of chemotherapy. Patients will be classified into low-risk or high-risk group based on clinical characteristics and cytogenetic profiles in each patient. Treatment will be given depending on the risk groups. As a result, pediatric AML outcomes has improved significantly in the past decade, with complete remission and overall survival rates 80-90% and 64-78%, respectively. 2 Unfortunately, pediatric AML outcomes in Indonesia is still poor, with complete remission rate of 32%, and overall survival in Dr. Cipto Mangunkusumo Hospital only 10% in year 2011. We do not have risk-based treatment protocol in Indonesia until now, since cytogenetic and molecular testing is not available for practical purposes.
Prognostic factors to determine the risk stratification group can be assessed mainly by cytogenetic profiles (karyotype and gene mutation). According to World Health Organization (WHO) criteria, AML with core binding factor (CBF)-associated translocation is considered favorable cytogenetic subgroup. The CBF-AML includes 2 major subtypes of favorable group, translocation t(8;21)(q22;q22), and inversion of chromosome 16 (inv(16)(p13q22)), which together account for 25% of pediatric de novo AML patients. 3 Berlin-Frankfurt-Munster (BFM) study groups and Medical Research Council (MRC) haves confirmed the overall survival (OS) rate of t(8;21) and inv(16) mutation in childhood CBF AML were 91% and 92%, respectively. 4 This study was conducted to evaluate the role of karyotype abnormalities t (8,21) and inv(16) as favorable prognostic factors of complete remission after induction phase chemotherapy in pediatric AML.

Methods
A prospective cohort study was conducted at 4 following pediatric oncology centers in Jakarta: Dr. Cipto Mangunkusumo Hospital (CMH), Gatot Soebroto Army Hospital, Dharmais Cancer Hospital, and Harapan Kita Women and Child Hospital, between year 2018 and 2020. Institutional review board approval was obtained from the CMH Board and Ethics Committee of Faculty of Medicine, Universitas Indonesia Medical School.
The inclusion criteria were the following: children aged <18 years with de novo AML who were diagnosed by morphological and leukemia immunophenotyping; underwent induction phase of chemotherapy under national AML protocol and agreed to sign the study informed consent. The exclusion criteria were: AML L3 subtype, AML in Down syndrome, or secondary AML as a result of chemotherapy (treatment related leukemia).
Consecutive sampling method was performed until number of subjects required was met or until 12 months of study period.
The national AML protocol consisted of cytarabine and daunorubicin given for 5 days. De novo AML refered to AML in patients with no clinical history of prior myelodysplastic syndrome (MDS), myeloproliferative disorder, or exposure to pontentially leukemogenic therapies or agents.
Clinical data of patients were obtained from medical records. Blood specimens from BMP required for each examination were 2 mL for morphological and immunophenotyping, 3 mL for karyotyping, and 2 mL for molecular examination. If the specimens were not sufficient, the sample would be retaken from the peripheral blood.
Subjects received the first induction phase chemotherapy, which was then followed by a second BMP within 1-4 weeks, according to patient's clinical conditions. Afterward, subjects received the second induction phase of chemotherapy and continued underwent re-examination of response to therapy within 1-4 weeks. If the 2 nd BMP result (after the first induction phase) was not in remission, then the response to therapy would be assessed on the 3 rd BMP (performed after the second induction phase). Subjects' data was collected and then processed until the 3 rd BMP was done. Response to treatment was evaluated based on morphological examination after the induction phase. Remission was defined as blasst cell < 5% in bone marrow.
The association of cytogenetic mutation and analysed using Fisher's exact test. All statistical analyses were performed using the IBM SPSS software version 20.0 (IBM Corp., Armonk, NY, USA); a P value of < 0.05 were considered indicative of statistical significance.

Results
This study was performed for 18 months, between November 2018 to March 2020. Eighteen patients were obtained as subjects for this study from 4 pediatric oncology centers. The study was discontinued due to the COVID-19 pandemic.
Of those subjects, only one had a karyotype with translocation t(8;21). Fisher's exact test showed no significant correlation between the presence of translocation t(8;21) and the achievement of postinduction remission (P=0.38) ( Table 2). Inversion of chromosome 16 was detected in 4 subjects. Bivariate analysis using Fisher's exact test showed no significant correlation between inv(16) karyotype and the incidence of remission after the induction phase (P=0.10). Nevertheless, inv(16) subjects were more likely to achieve remission than subjects who weare not inv(16) (4/4 vs. 7/14 subjects) ( Table 3).

Discussion
Approximately 70% of pediatric AML patients have cytogenetic aberrations at the time of diagnosis. The most common chromosomal abnormalities found were t(8;21) and inv(16). Translocation of t(8;21) were found variably. Incidence of t(8;21) in pediatric AML patients were 15% in Saudi Arabia, 4,5 15% in China, 6 24% in Japan, 7 26-29% in India. 8,9 A study stated that t(8;21) expression in Asian children tends to be higher compared to Europe and North America. 8 In our study t(8;21) only found in 1 of 18 subjects. This result is lower than reports from both Asian and European countries. Translocation t(8;21) as one of the favorable cytogenetic group was reported commonly found at a younger age. In our study, we found lower t(8;21) expression although most subjects were less than 10-years of age. Other factors which may influence the findings are ethnic or environmental factors that may differ from other countries. 4,6 Incidence of inv(16) in pediatric AML was reported to be less frequent than t(8;21), which was 5% in India, 8 3.5% in Japan, 7 1% in China, 6 while studies in Europe and the United States reported between 4-9%. 6,10 In our study, inv(16) mutation was found in 4 out of 18 subjects. Those findings were different compared to other reports from both Asian and European countries. This result is an important finding and needs further investigation because higher number of favorable group increases the advantage of therapy management. Age, geographical, and ethnic differences are reported to affect karyotype patterns and it is suspected that these differences are also related to exposure to certain chemicals or environmental substances. 6 Approximately 7/18 subjects in this study were in the favorable risk karyotype group. The small number of subjects with favorable karyotypes in this study can explain inferior response to induction therapy in pediatric AML patients in Indonesia. Complete remission rate in our study was 61%, which was much lower than complete remission rate reporteds in AML patients less than 15 years of age in Vietnam which was 82.6%, 11 or 92% in Saudi Arabia. 4,11 Kariotype data in Saudi Arabia with 19% higher rate of t(8;21) expression compared to this study might explain the difference of remission rate result.
The findings of t(8;21) and inv(16) in our study did not show significant relation towards induction phase therapy outcomes. Expression of inv(16) in our study was not proven to be a prognostic factor for remission to induction phase. Nevertheless, it seemed that 4 out of 4 subjects with inv(16) achieved remission while in non-inv(16) only 7 out of 14 achieved remissions ( Table 3). This showed a consistent tendency with the study hypothesis and literatures which stated that inv(16) is a good prognostic factor for AML. Our study showed that t(8;21) was not proven as a prognostic factor for remission in induction phase of AML. A meta-analysis shows the influence of ethnic and racial factors in the prognosis of AML with t(8;21) expression. 4 White ethnicity in pediatric AML have better prognosis compared to the non-whites and the effect of racial or ethnic differences is significantly seen when accompanied by KIT mutations. Genetic abnormalities finding such as chromosome 9 deletion or the addition of chromosome 4 lower the prognosis rate. None of our subjects in this study had those cytogenetic abnormality.
The complete remission rate after induction phase chemotherapy in this study was 61%. This rate was lower than other countries, both developed countries and countries with the similar level of pediatric cancer services, such as Vietnam and Pakistan. Complete remission rate in Vietnam was 82.6%, 11 Pakistan was 80%, 12 and Saudi Arabia was 86%. 5 Nevertheless, the remission rate in this study was higher than the data of pediatric AML in RSCM in year 2010 which was 32%. 13 This improvement of remission rate may be caused by protocol changes after year 2011, when the National AML protocol was started to use. Low remission rate in this study may suggest that the chemotherapy regimen that is being used in the National AML protocol is not adequately enough. The National AML protocol consists of 5 days of treatment, while the treatment protocol in other countries generally consists of 7 days of treatment, with the same drug regimen: cytarabine and daunorubicin. Based on this results, we suggest to improve the strength of existing national protocol to achieve a higher remission rate.
The survival rate in this study is also lower than other countries. The 1-year OS in this study was 47%, lower than the outcomes in other Asian countries such as China (5-year OS was 69% in year 2011) 14 and Saudi Arabia (58.8% in year 2016). 15 The outcomes of this study was worse considering that the OS for 1 year was already low and the recurrence rate was high. A previous study shows that 3-year OS of all AML patients was 22.6±5.4%, while prognosis of cytogenetic abnormality of subjects with (inv)16 was 75.0±21.7% and subject with t(8;21) was 36.0±16.1%. 5 Our study was the first study in Indonesia to examine the karyotype in children with AML. The data obtained from this study could be very useful for further management to treat AML in children in Indonesia. The limited number of study subject may explain the insignificance statistical results. In addition, our study encountered obstacles in fulfilling the sample size due to the limited numbers of new diagnosed AML patients who came to the study hospitals (Dr. Cipto Mangunkusumo Hospital/CMH, Gatot Soebroto Army Hospital, Dharmais Cancer Hospital). The COVID-19 pandemic has also affected the diagnosis confirmation in new AML patients and former patients who discontinued the treatment. The other difficulty faced in this study was technical examination of karyotypes and mutations which was complex and required several trials. The examination were carried out collectively hence re-sampling could not be done because patient had already received chemotherapy or did not survive. Another study limitation was that the laboratory could not examine samples due to the damage of the incubator and data processing computer, or the supply of reagents taking longer duration. The time discrepancy between the schedule of receiving samples and taking specimens from the subjects was also another obstacle in this study so that some patients failed to become study subjects due to the damaged samples. These factors hinder the achievement of having more study subjects.
In conclusion, this study fails to show that cytogenetic profiles of t(8;21) and inv(16) as favorable prognostic factors in pediatric AML. Nevertheless, this study finds the tendency of subjects with these chromosomal aberrancies to have better prognosis.