|Year : 2021 | Volume
| Issue : 4 | Page : 214-219
Acute lymphoblastic leukemia in Indian children at a tertiary care center: A multiparametric study with prognostic implications
Shweta Jha1, Dinesh Kumar2
1 Assistant Professor, Anatomy, Netaji Subhas Medical College and Hospital, Bihta, Patna, Bihar, India
2 Director Professor, Anatomy, Maulana Azad Medical College, New Delhi, India
|Date of Submission||23-Apr-2021|
|Date of Decision||20-Aug-2021|
|Date of Acceptance||18-Sep-2021|
|Date of Web Publication||28-Oct-2021|
Assistant Professor, Anatomy, Netaji Subhas Medical College and Hospital, Bihta, Patna, Bihar
Source of Support: None, Conflict of Interest: None
Background: Leukemia is a varied group of hematological malignancies due to uncontrolled proliferation of blast cells. Among childhood leukemias, acute lymphoblastic leukemia (ALL) comprise 70%–80% of all childhood leukemias in India. The current study aims to report the various prognostic markers of disease severity. Methodology: Bone marrow and peripheral blood samples from 20 patients of ALL were subjected to cytogenetic and flow cytometric analysis after recording clinical history and laboratory findings. Patients were classified according to immunophenotyping markers.: For risk stratification, patients were divided into two subgroups B ALL and T ALL. The age group of majority of patients was 1–9 years (90%) with 5% each belonging to <1 and >9 years. Male: female ratio was 1.1:1. Results: Hepatosplenomegaly, lymphadenopathy, and mediastinal involvement was found in 45%, 40%, and 5% of patients, respectively. Hemoglobin levels <5 g/dl and >5 g/dl were seen in 50% of patients in each range. White blood cell counts >50,000 were seen in 3 (15%) of patients. Cytogenetic analysis revealed hypodiploid karyotype for majority (64%) of cases, normal karyotype in 28% and hyperdiploidy in rest (7%). Structural aberrations like t (21;4), del (5p), dic (5) were found all in B ALL subgroup. Patients were stratified into high and standard risk groups based on good and prognostic factors. Conclusions: This study reinforces the significance of immunophenotyping cytogenetics, clinical presentation as a prognostic tool, and their significance in risk stratification.
Keywords: Acute lymphoblastic leukemia, cytogenetics, flow cytometry, immunophenotyping
|How to cite this article:|
Jha S, Kumar D. Acute lymphoblastic leukemia in Indian children at a tertiary care center: A multiparametric study with prognostic implications. Natl J Clin Anat 2021;10:214-9
|How to cite this URL:|
Jha S, Kumar D. Acute lymphoblastic leukemia in Indian children at a tertiary care center: A multiparametric study with prognostic implications. Natl J Clin Anat [serial online] 2021 [cited 2021 Dec 8];10:214-9. Available from: http://www.njca.info/text.asp?2021/10/4/214/329497
| Introduction|| |
Leukemia is a diverse group of hematologic neoplasm that results from partial or total change of blast cells. Leukemia is the most common childhood cancer in India with frequency varying between 25% and 40%. Out of these, 60%–85% of all leukemias reported are acute lymphoblastic leukemia (ALL). The frequency of T ALL is higher in India (20%–50%) as compared to western nations (10%–20%). ALL is the most common malignancy in childhood, especially in white males under the age of 14 years. The medical examination, flow cytometry, immunophenotyping, morphology, cytochemistry, molecular, and cytogenetic study are helpful in diagnosis and classification of ALL. The cytogenetics evaluation helps in recognition chromosomal aberrations, classification with staging, and prognosis of it. Diagnosis and accurate classification of acute leukemia is made possible due to distinctive patterns of cluster of differentiation surface antigen expression. This plays a pivotal role in giving appropriate treatment. The authors have identified the existing gap of information regarding prognostic markers in pediatric ALL in Indian scenario.
The current study was conducted to evaluate clinical features, laboratory findings, immunophenotyping, and cytogenetic features of pediatric ALL patients. The correlation among these features is examined for severity and risk assessment.
| Materials and Methods|| |
This was cross-sectional observational study.
The study was performed in the Department of Anatomy, Pathology and Paediatrics of Maulana Azad Medical College, Delhi, between November 2011 and April 2013 after obtaining Ethical Clearance from Institutional Ethics Committee (No. F.11/IEC/MAMC/10).
Pediatric patients recruited from pediatrics ward of Lok Nayak Hospitals, Delhi.
Selection of patients
Patients in pediatric age group (≤12 years) who were hematologically confirmed cases of ALL (blast cells >25% in bone marrow aspirate) were selected for this study.
Age >12 years. Associated malignancies were ruled out by the presence of blast cells >25% in bone marrow and presence B ALL (cluster of differentiation [CD19], CD22, CD79) CD markers and T ALL (CD3) by immunophenotyping and clinical features.
During bone marrow aspiration, to prevent microbial contamination of sample was aspirated by using sterile instruments, gloves, and gown and cleaning the aspiration site by antiseptics (e.g. Povidone-iodine) and transferring it using sterile closed vacutainers. Contamination of cell culture lines was prevented using laminar airflow.
0.6 ml bone marrow was aspirated from iliac crest and 3 ml of peripheral blood sample under septic precautions mentioned above, in a heparinized vacutainer. Clinical history was taken, and informed consent was obtained from parents. Samples were sent for culture and peripheral blood smear examination.
0.6 ml of aspirate was added to 15 ml of RPMI culture medium in three separate tubes for immediate, 24 and 48 h culture method. The culture tubes were placed in CO2 incubator for 24 and 48 h.
0.05 ml of colchicine was added to the incubated culture tubes at 22 and 46 h respectively to arrest mitotic division in metaphase. After 2 h, tubes were centrifuged at 1000 RPM. Postdiscarding the supernatant, 5 ml of hypotonic solution (prepared by addition of 0.56 g KCL in 100 ml of distilled water) was added to the pellet. Incubation was done for 45 min followed by centrifugation at 1000 rpm. 5 ml of fixative (methanol: glacial acetic acid 3:1) was added to pellet after discarding the supernatant. The solution was centrifuged 1000 rpm for 10 min. The addition of fixative and centrifugation was repeated till pellet turned white. Cell suspension (formed by addition of minimal media and buffer to pellet) was dropped from height of 30 cm on a chilled glass slide at an angle of 30°. Well-banded metaphase spreads were observed using a binocular microscope and the position of metaphase spreads was recorded. Metaphase spreads were captured using a camera and the image was transferred to an image analyzer. Karyotypes were analyzed according to the International System of Cytogenetic Nomenclature 2009.
Immunophenotyping data obtained by routine flow cytometry analysis, clinical and hematological parameters such as age, gender, cerebrospinal fluid cytology (presence of blast cells), hemoglobin levels, white blood cell (WBC) levels, platelet count were noted for each patient.
Data were recorded on Microsoft excel. Data processing and application of inferential statistics were done using statistical software Stata version 14.0 (State Corp LP, TX). Descriptive statistics was used to study demography and other variables. Categorical data were expressed as frequency and percentage. Quantitative data were expressed as mean ± standard deviation (SD).
| Results|| |
To stratify patients according to prognostic factors, various clinical, hematological, immunophenotyping, and cytogenetic features at diagnosis were recorded. B ALL is defined by the presence of CD markers for B lineage precursors CD10, CD19, and HLADR. T ALL by expression of markers for T lineage-like CD3 predominantly. Nineteen (95%) patients in this study were found to be in B ALL subgroup and 1 (5%) in T ALL subgroup [Table 1] and [Table 2].
|Table 1: Presenting clinical characteristics of 20 pediatric acute lymphoblastic leukemia patients (B acute lymphoblastic leukemia and T acute lymphoblastic leukemia)|
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|Table 2: Antigen expression in immunophenotyping in 20 cases of acute lymphoblastic leukemia|
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The most common age group in this study was 1–9 years with 90% of patients lying in this age group. The age of patients in this study averaged 4.2 years with SD of 2.6. The other subgroups were one patient each in <1 year and one patient more than 9 years (5% in each respectively). This patient, who was more than 9 years of age, was also the only T ALL case [Table 1] and [Figure 1].
Male: female ratio was 1.1:1 in B ALL subgroup there was male preponderance (1.3:1), while the T ALL patient was female [Table 1] and [Figure 1].
The most common symptoms were fever (92%), hepatosplenomegaly (45%), and lymphadenopathy (40%). Mediastinal widening was seen in patient with T ALL but, the same was absent in B ALL. None of our patients had central nervous system (CNS) involvement at presentation [Table 1] and [Figure 2], [Figure 3].
|Figure 2: The comparative frequency of presenting clinical symptoms seen in present study|
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|Figure 3: The comparative frequency of clinical signs seen in present study|
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Blast cells in bone marrow at diagnosis were in range of 25%–60%, 60%–90%, and >90% in 35%, 45% and 20% patients, respectively [Table 3].
The mean hemoglobin level was 5.5 ± 1.86 g/dl. Hemoglobin levels of <5 g/dl were seen in 50%. At the initial presentation, the patient with T ALL had higher hemoglobin levels (7.4 g/dl) than the patient with B ALL (5.4 ± 1.8). Average WBC count for all patients at presentation was 28.83 ± 51.72 with patient with T ALL having very high counts of 164,000/hpf. Three patients (6%) were found to have WBC counts >50,000 out of which two belonged to B-ALL category and one T ALL. The mean platelet count was 41.1 ± 44.09 with 10 (50%) patients having severe thrombocytopenia (20.0 × 109 cells/L) [Table 4]. Pancytopenia was seen in 8 (40%) of patients [Table 3] and [Table 4].
|Table 4: Baseline laboratory features of 20 children with acute lymphoblastic leukemia|
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Karyotypic analysis could be done in 14 cases. [Table 5] shows the frequency of various karyotypes both numerical and structural categorized according to immunophenotypes. According to modal number chromosomes, all cases showed some numerical abnormality showing mixture of normal and abnormal cell lines. These phenomena are seen due to clonal nature of disease. Ploidy status of the patient is a strong prognostic factor in ALL with hypodiploidy indicating poor prognosis and hyperdiploidy indicating good prognosis. Normal karyotype was seen in 4 (28%) of cases, hypodiploidy in 9 (64%), and hyperdiploidy in 1 (7%) cases. Structural defects seen in this study were deletions (5p), translocations like t (21;4), t (13p), and dicentric (dic 5) chromosome [Table 5].
|Table 5: Karyotyping (numerical and structural) findings in 20 cases with acute lymphoblastic leukemia|
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CD10 positive B ALL (pre-B ALL) were seen in 17 patients and CD10 negative B ALL (pro B ALL) were seen in 2 patients [Table 2] and [Table 4]. T ALL case was identified by the presence of dedicated marker for Thymic line precursor marker CD3 seen in 1 patient [Table 2].
| Discussion|| |
Varied clinical and biological features describe pediatric ALL. Five-year overall survival rate (OS) of ALL has drastically improved (89% worldwide and 45%–81% in India) due to a better understanding of pathophysiology and risk-stratified tailored therapy given to patients. Prognostic factors are utilized to determine an individual patient's risk assessment of relapse. Treatment is then adjusted by patient stratification into different therapeutic risk groups.,
The improvement in the OS and cure rates of Indian patients is encouraging. However, the results are still inferior to the West. This could be due to Indian patients having more risk factors with bad prognosis. This study was an attempt to study various factors that affect outcome in North Indian pediatric population and stratify patients into risk groups that require different treatment approaches according to severity.
Age at presentation is a strong prognostic factor in ALL., Age <1 and more than 9 years are poor prognostic indicators., This is because infancy is associated with other unfavorable prognostic factors such as high presenting WBC count, CD10 negative B-ALL immunophenotype, and presence of MLL rearrangements. Age more than 9 years is known to have higher relapse rates, and remission rates are poor for this age group. In contrast, age 1–9 years is considered to have good prognosis compared to other two subgroups. In the present study, the majority of patients belonged to 1–9 years of age group (90%) with one patient each in below 1 year and above 9 years of age group. The only patient with age more than 9 years had other unfavorable prognostic factors like T ALL immunophenotype, hypodiploidy, WBC count >50 × 109 cells/L making it a high risk case prognostically [Figure 4]. The mean age of patients in the present study was 4.2 with SD of 2.6. This is in accordance to previous data which suggest peak incidence of ALL being reported in the age group of 2–5 years. In a previous study mean age of 7.6 years was reported which is higher than what was seen in this study. The findings of this study are similar to those observed in a study on Pakistani pediatric population (mean age group 4.5 years) and another Indian study in Telangana region [Table 1] and [Figure 1].
|Figure 4: Risk stratification algorithm classifying patients in present study into standard risk and high risk groups on the basis of prognostic factors found|
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In this study, there was preponderance of male patients which is in accordance with previous literature. [1, 8, 11, 13, 14] Prognostically, females have been found to have superior survival rates as compared to males.
B ALL cases in our study were 95% and T cell was 5%. T ALL frequency (5%) in our study was lesser as compared to findings reported by Indian (20%–50%) and western authors (10%–20%).,,, One study on south Indian population reported the prevalence of T ALL as 15.5% which is higher than our study. The finding is similar to study on the Brazilian population which showed 93% of B-lineage ALL and 7% of T lineage. T ALL is a poor prognostic indicator., In our study, there was one case of T ALL. This patient had other unfavorable prognostic features like age >9 years, hypodiploidy, severe leucocytosis (>105 cells/μL of blood), hepatosplenomegaly, lymphadynopathy, and mediastinal involvement. The patient was stratified in high-risk category [Figure 4] needing more intensive treatment [Table 1] and [Table 2].
CD10-positive pre-B ALL has a better outcome than CD negative pro B cases.,, In this study, CD10 positive pre-B ALL was seen in 17 cases (85%). In a study on Brazilian population, 90% of patients had B ALL immunophenotype out of which CD10 positive cases were 65% [Table 2] and [Table 4].
According to modal number of chromosomes, ploidy status predicted survival, even when age, initial leukocyte count were not considered. Pretreatment cytogenetics is one of the most important prognostic indicators in ALL. Hypodiploidy is a known bad prognostic factor, while hyperdiploidy is good prognostic factor. This is probably due to more number of translocations associated with hypodiploid karyotype and favorable prognostic factors like age between 1 and 9 years, a low WBC count, B ALL immunophenotype associated with hyperdiploid status.
In 14 out of 20 cases, karyotyping results could be obtained [Table 5]. Out of these, 4 (28%) patients had normal karyotype, 9 (65%) were hypodiploid and 1 (7%) was hyperdiploid. Patient with T ALL immunophenotype showed hypodiploid status. Our results were dissimilar from other studies where the percentage of patients with normal karyotype was 78%, 48%. The incidence of hypodiploidy (65%) in our study was higher than other studies 51.2%, 10.3%, 2.25%, and 9%.,,, In contrast, hyperdiploid (7%) frequency was less as compared to other studies (10.11%, 20.68%). Ploidy status shows regional variations; hence, it is important to classify patients in each geographical region according to ploidy.
The four major translocations observed in B ALL in childhood were t (12;21), t (1;19), t (9;22)/BCR-ABL and t (4;11)/MLL gene and those associated with T ALL were t (8;14)(MYC gene). These translocations are used to define clinicopathological entities that are used for risk stratification. In the present study, translocation in B ALL was found to be t (21;4) and one case involving translocation of short arm of chromosome 13. No case of BCR-ABL fusion was found in this study, probably due to use of routine karyotypic analysis and nonavailability of FISH technique. Translocations are known to have bad prognostic value particularly in B ALL where such rearrangements create fusion genes with transforming properties.
The significance of hematological parameters lies in the fact that it has been found WBC more than 50,000 increases death risk by about 22 times. Leukocyte count in the above range was seen in 3 (6%) of our patients, indicating poor prognosis [Figure 4]. This is lower as compared to other Indian studies, (23.7% and 20.39%). Platelet count <50,000/hpf was seen 85% of patients which is higher than other studies (68% and 48.54%), Hb levels ofHb<5g/dl was seen in 50% of our patient as compared to study in South Indian population where it was 43.69%.
The most common clinical features in this study were hepatosplenomegaly (45%) and lymphadenopathy (40%). Mediastinal widening which is an established bad prognostic indicator was there in 1 (5%), this is similar to incidence seen by another Indian study [Table 1] and [Figure 3]. This patient who was also the only case of T ALL. CNS involvement at presentation, a poor prognostic indicator seen in other studies (6.8% and 2.1%), was not seen in our study [Table 1].
[Table 6] summarizes favorable prognostic factors as age 1–9 years, WBC count at <50,000/hpf, B-cell immunophenotype, CD10 positivity, hyperdiploidy >50 chromosomes. Unfavorable prognostic factors are Age <1 and >9 years, WBC 50,000/hpf, T ALL, CD10 negativity and hypodiploidy.,,,
|Table 6: Favorable and unfavorable prognostic factors predicting outcome in acute lymphoblastic leukemia|
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A risk assessment algorithm was created on the basis above-mentioned prognostic factors found in our study [Figure 4]. High risk patients were identified by the presence of any one of the following features: age <1 or >9 years, WBC count >50,000, immunophenotype: T ALL, CD10 (−) B ALL, Cytogenetics: Hypodiploidy, (35%) [Figure 4]. Rest were classified as standard-risk patients (65%). According to national comprehensive cancer network guidelines for pediatric ALL, patients in standard-risk groups should be treated with induction, consolidation, and maintenance therapy. Cases of infant ALL (one in the present study) should be treated with Interfant-based regimen. T ALL patients (one in our study) need systemic chemotherapy.
| Conclusions|| |
In our study, majority of patients were classified under standard risk (65%) when stratified according to age at presentation, immunophenotyping, WBC count, and cytogenetic findings. More than one risk factor was seen in one patient who was >9 years, T ALL patient with hypodiploid karyotype and WBC count >100,000. There was one case of Infant ALL which in itself is a major risk factor. T ALL frequency in the present study was less (5%) as compared to other studies. Hypodiploidy (64%) in our study was higher than other studies. Due to the variable genetic makeup of different population groups, prognostic variables significant in one may not be important in another; hence, it is important to risk-stratify patients at all centers to tailor therapy, reduce relapse risk, improve treatment outcome, and minimize toxic deaths.
The limitations of this study were limited sample size, nonavailability of FISH technique for genetic evaluation. OS and response to induction therapy could not be assessed.
Portions of this manuscript were published as apart of article titled “Cytogenetic Pattern Profiling in cases of acute lymphoblastic leukemia in pediatric age group. Journal of Anatomical Society of India. 2017;66(1): 48-53). Shweta Jha, Dinesh Kumar, J M Kaul, Tejinder Singh, A P Dubey. et al. (Copyright Elsevier).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]