Relationship of Disease Parameters with Post-Induction MRD in Children with Acute Lymphoblastic Leukaemia
Abstract
Background: Minimal Residual Disease (MRD) following chemotherapy is one of the strongest independent prognostic markers for Acute Lymphoblastic Leukaemia (ALL). Patients with detectable MRD have an increased risk of relapse. There is significant relationship of MRD with other prognostic factors. But this relationship has not been fully evaluated in paediatric patients with ALL. So, the aim of this study is to determine the relationship of initial total count of WBC with MRD at the end of induction. Methodology: This prospective cross-sectional study was conducted in the Department of Paediatric Haematology & Oncology, Dhaka Medical College Hospital (DMCH), from May 2022 to April 2023. A total of thirty children diagnosed with ALL of both sexes between 1 and 19 years who received induction chemotherapy were included in this study. MRD was done at D29 of induction from bone marrow by reverse transcription polymerase chain reaction (RT-PCR) method. Results: This study found that 33.3% patient developed positive MRD. The study showed that significant positive correlation between the initial total count of WBC and MRD level. The analysis also showed a significant relationship between high-risk group, patients receiving regimen B and M3 category bone marrow with MRD (p<0.001). Conclusion: There is significant positive correlation between the initial total count of WBC and MRD level. There is also a significant association with post induction MRD positivity with high-risk group, use of regimen B and patients with M3 marrow.
Keywords: Acute Lymphoblastic Leukaemia, End of induction, Initial total count of WBC, MRD
Introduction
Acute Lymphoblastic Leukaemia (ALL) is the commonest paediatric malignancy and accounts for most common cause of cancer related death in paediatric age group. In 1960 survival rate was 10-20%. At present, approximately 75-85% of children with ALL can be cured through the application of risk-oriented treatment protocols. This improvement of survival is due to stratify the patients according to risk groups on the basis of prognostic factors.1-3 Minimal Residual Disease (MRD) following treatment is the most important prognostic factors for ALL. In many protocols, MRD is measured at different time points such as during induction, after induction and early consolidation.4
MRD is defined as the presence of leukaemic blasts after treatment below the levels of morphologic detection within the bone marrow and more rarely in peripheral blood circulation.5-7 The aim of MRD monitoring is to determine treatment response and risk of relapse. It is also used to modify the treatment of ALL.8 MRD measurement is based on the detection of tumour cells beyond the morphological margin after chemotherapy of haematopoietic stem cell transplantation. Here, identification and enumeration of rare subpopulations within the bone marrow were done leading to post-therapeutic monitoring of disease. In polymerase chain reaction (PCR) leukaemia specific DNA sequences are amplified. Multicolour flow cytometry can detect 1 in 10,000 leukaemic cells whereas PCR can detect 1 in 1,000,000 cells.9-11 Although MRD is the one of the important prognostic factors, its relationship with other prognostic variables have not been fully explored in childhood ALL.12,13 However, it is not well established whether MRD is an independent risk factors or associated with other risk factors.14
In a retrospective, cross sectional study Meraj et al. found that post consolidation MRD has an important relationship with National Cancer Institute (NCI) risk groups such as age and CNS status.15 However, relationship of initial total count of White Blood Cell (WBC) with MRD at the end of induction in children with ALL has not been studied separately in Bangladesh.
So, the aim of this study is to identify the relationship of initial total count of WBC with MRD at the end of induction in children with ALL.
Methods
Study design and subjects: This was a prospective cross-sectional study conducted from May 2022 to April 2023 in the Department of Paediatric Haematology and Oncology, Dhaka Medical College Hospital (DMCH), Dhaka. A total of 30 children diagnosed with ALL of both sexes less than 19 years of age who received induction phase of chemotherapy were included in this study. Children aged less than 1 year or ≥19 years were excluded from this study. All the information regarding the study was collected in a structured questionnaire after taking informed written consent.
Study procedure and parameters: Demographic data regarding age, sex, socio-economic status along with clinical presentations were collected. The diagnosis of ALL was made on the basis of complete blood count and peripheral blood film examination, bone marrow study (morphology and immunophenotyping). Cytospin of CSF is used to identify CNS status. Other tests such as serum electrolytes, uric acid, inorganic phosphate, calcium were done to detect tumour lysis syndrome. All the investigations except immunophenotyping were done in laboratory of Department of Paediatric Haematology and Oncology, Department of Clinical Pathology, DMCH. Immunophenotyping was done from Department of Paediatric Haematology and Oncology, BSMMU. All patients of ALL were treated with modified UKALL 2003 protocol, regimen ‘A’ or ‘B’ according to risk stratification. In addition to this treatment, general supportive care measures were administered to all patients. All patients were monitored regularly from the start of chemotherapy up to 35 days of induction of remission.
Detection of MRD: MRD was checked at D29 of induction from bone marrow by reverse transcriptase polymerase chain reaction method from Department of Paediatric Haematology and Oncology, BSMMU. There are no definitive guidelines on which risk stratification of MRD can be done. However, based on huge number of studies, B-ALL MRD can be roughly categorized in the following categories such as MRD <0.01% negative, MRD > 0.01% and < 0.1% intermediate, MRD >0.1% positive.16-18 Children with ALL can be stratify into two risk group including standard risk group and high risk group. Standard risk group includes 1 to 9 years of age, girl, initial WBC count <500000/cumm of blood, B cell ALL, no CNS involvement, post-induction day 29 MRD <0.01%. High risk group includes age <1 year or >10 years, boy, initial WBC count >500000/cumm of blood, T cell ALL, CNS involvement, post-induction day 29 MRD >0.01%. Bone marrow remission status in ALL can be classified in three category M1 bone marrow <5%, M2 bone marrow 5-25%, M3 bone marrow >25%.11
Statistical analysis: Statistical analyses were performed using IBM SPSS Statistics 26. Data were expressed as frequency, percentage p-value was reached from Chi-square test. p<0.05 is considered level of significant. Pearson coefficient test was done to see the correlation between initial TC of WBC with MRD level.
Ethics statement and consent of the participants: The study was approved by the Institutional Review Board of Dhaka Medical College Hospital (DMCH).
Results
Patient’s characteristics in details: During the study period, 30 patients received induction chemotherapy. Among them positive MRD was found in 10 patients. Figure 1 showed 33.3% of patient had positive MRD, 3.4% patient had intermediate MRD and 63.3% patient had negative MRD. Table I summarizes the different characteristics of the patients.
Figure 1. MRD status in post induction ALL patient
As it was evident from Table I, age, sex, CNS status, immunophenotyping did not exhibit statistically significant associations with MRD (p>0.05). In this study, the analysis showed a statistically significant relationship among the risk groups, patient receiving regimen B with positive MRD (p<0.001), indicating that high-risk patients were more prone to develop positive MRD status. There is a significant association between bone marrow status at day 29 and MRD status, with patients in M3 category having a higher proportion of positive MRD compared to those in M1 and M2 categories (p<0.001).
Relationship between Initial TC of WBC & end of induction MRD: Table II shows that the initial WBC count was less than 50×109/L, all patients had negative MRD after treatment except 2 patients - 1 patient had intermediate MRD, and 1 patient had positive MRD. On the other hand, when the initial WBC count was more than 50×09/L, the majority patient had positive MRD. Analysis indicates that this difference is highly significant (p<0.001).
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The Figure 2 shows the significant positive correlation between the initial total count of WBC and Minimal Residual Disease (MRD) levels in ALL patients (r=0.722, p<0.001).
Figure 2. Pearson coefficient test. Scatter diagram showing the correlation between initial TC of WBC with MRD level among ALL patients (n=30)
Discussion
There are several well-established prognostic variables in children with ALL such as age, initial total count of white blood cell (WBC), immunophenotyping and cytogenetic characteristics of the leukaemic blasts and individual response to therapy. In case of ALL response to therapy measurement is the backbone of therapy. MRD is the gold standard for response measurement in children with ALL.19-21 Among prognostic factors of ALL, post induction and consolidation MRD have been recognised as the most important. In a retrospective, cross sectional study, Meraj et al. found that post induction MRD positive in 37% of patients and post consolidation MRD positive in 44% of patients.21 In a study conducted by Children's Oncology Group found that 28.6% of patients end induction MRD positivity.13 In our study we found that end induction MRD positive in 33.3% of patients.
Different characteristics of patients such as age, sex, initial total count of WBC, immunophenotyping, CNS status, prophase response, risk group and regimen are related to post induction MRD.13 Dario Campana found that infants and children >10 years of age, high risk group, presence of BCR-ABL had significant association with positive MRD.22 Current study found that there was no significant association between age of the patients and end of induction MRD.
T-ALL has considered poor outcome than those with Pre-B ALL.23,24 We found no association between immunophenotyping and MRD positivity.
Our findings are similar to literature, and we found no significant variation in post induction bone marrow MRD level when comparing the CNS status of the patients - indicates that presence of CNS disease at diagnosis does not indicate chemo resistant leukemia.14
Children with M1 or M2 marrows are less likely to be MRD positive at D29 in comparison to M3 marrow.13 Jovanovska et al. found that children with M2 or M3 marrow on D15 were more prone to develop MRD positivity at D29.25 We also found that there is a significant association between MRD positive at day 29 with patients in M3 category bone marrow.
Poor response to prophase is strongly associated with MRD positivity and risk of relapse.13 However, in our study prophase response was not seen.
Meraj et al. found in their retrospective cross-sectional study that post induction MRD positivity was more common in high-risk group and patients getting regimen B.21 Similar findings were observed in our study. Meraj et al. also found that MRD positivity was more common in initial high total count of WBC, high risk group and patient getting regimen B.21,22 In our study, post induction MRD positivity was more common in high-risk group and patient getting regimen B.
This study found that rate of MRD positivity was 33.3% amongst the paediatric patients with ALL.
Conclusion
This study found that significant positive correlation between the initial total count of WBC and Minimal Residual Disease (MRD) levels at the end of induction. The study also found that post induction MRD positivity has a significant association with high-risk group, use of regimen B and patients with M3 marrow.
Limitations
Were small sample size and single centre study. So, we would recommend multicentre research to evaluate association of these features with post induction MRD.