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 Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 6  |  Issue : 2  |  Page : 112-118

Clinical, immunophenotype and cytogenetic profile of acute lymphoblastic leukemia in children at tertiary health care centre in India


1 Department of Pediatric Hematology Oncology, Rainbow Children's Tertiary Care Centre, Hyderabad, India
2 Department of Pediatrics, Rainbow Children's Tertiary Care Centre, Hyderabad, India
3 Professor of Pharmacology, Hyderabad, India

Date of Web Publication13-Jul-2015

Correspondence Address:
Dr. Sirisha Rani Siddaiahgari
Department of Pediatric Hematology Oncology, Rainbow Children's Tertiary Care Centre, Hyderabad - 500 034, Telangana
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0975-9727.160676

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  Abstract 

Objective: Data on acute lymphoblastic leukemia (ALL) in Indian children being limited, we analyzed the clinical, immunophenotype and cytogenetic profile of ALL in our pediatric patients. Materials and Methods: This was a prospective and retrospective observational study in which data of 103 children with ALL, aged three months to 18 years were analyzed. Results: Definite male preponderance (70.87%) was observed, 58.25% patients were aged between two to five years. Common clinical features included fever (92.33%), pallor (87.38%), hepatomegaly (85.44%) and splenomegaly (83.50). central nervous system involvement was seen in 6.80% and tumor lysis syndrome in 32.04% patients. Hemoglobin was <5 gm/dl in 43.69%, between 5-10 gm/dl in 45.63% patients; white blood cell count between 5,000-50,000/hpf seen in 69.90%, >50,000 in 20.39% patients. Platelet count was <50,000 cells/hpf in 48.54%. Blast cells in peripheral smear present in 67.96% patients of 97 in whom immunophenotyping was done, 82.44% were Pre B cell ALL. Pre T and T cell ALL was seen in 15/97 (15.46%) patients. Karyotyping revealed hyperdiploidy in 9/89, hypodiploidy in 2/89, miscellaneous changes in 35 patients. FISH analysis in 63 patients revealed Philadelphia chromosome positivity in seven (11.11%). MLL gene was positive in 12.70%. TELAML was positive in 13.64% (3/22) patients. 97.06% patients went in to remission post induction. Conclusion: Most of our patients come under standard risk by their age, WBC count, immunophenotyping and cytogentic analysis. We suggest larger multi-centric studies even in developing countries to understand the clinical profile and RT-PCR technique in cytogenic analysis for better risk stratification to help in risk adopted therapy.

Keywords: ALL, cytogenic profile, epidemiology, immunophenotyping, risk stratification, RT-PCR technique


How to cite this article:
Siddaiahgari SR, Awaghad M A, Latha M S. Clinical, immunophenotype and cytogenetic profile of acute lymphoblastic leukemia in children at tertiary health care centre in India. Muller J Med Sci Res 2015;6:112-8

How to cite this URL:
Siddaiahgari SR, Awaghad M A, Latha M S. Clinical, immunophenotype and cytogenetic profile of acute lymphoblastic leukemia in children at tertiary health care centre in India. Muller J Med Sci Res [serial online] 2015 [cited 2019 Sep 18];6:112-8. Available from: http://www.mjmsr.net/text.asp?2015/6/2/112/160676


  Introduction Top


Leukemia accounts approximately for 1/3 of cancers occurring in early childhood, with acute lymphoblastic leukemia (ALL) being the most common entity. It is biologically and clinically a distinct entity, with peak prevalence during 2-5 years of life. [1],[2] There has been insufficient and limited data available on childhood cancers in India; Chaudhuri et al. [3] have reported that 39.2% of childhood cancers in the state of West Bengal, India, are acute leukemias. Medical fraternity has witnessed an increase in the incidence in childhood leukemia throughout the world, and in India annually >10,000 cases have been reported. [4]

Studies from India have reported that ALL accounted for 60 to 85% of all childhood leukemias. [5],[6],[7] Tyagi et al. [8] reported the incidence of leukemia in Indian pediatric population as 34%, of which 25% was ALL.

Newer treatment modalities and early detection have lead the treatment success to >90% in ALL in the United Kingdom (UK) [9] and >80% in other developed countries, whereas India is lagging behind these countries in attaining this success rate. Five year survival rate has been approaching 80% or more in developed countries, [10],[11] whereas it is <40% in Morocco [12] and 50%-70% in India, [13],[14] depicting the discrepancy among the developed and developing countries.

Marwaha et al.[4] have emphasized the need for the accurate estimation of incidence and prevalence of ALL in India to estimate the true disease burden, its impact on the population and health reforms. It is important to identify the pattern and clinical presentation of disease, risk factors for prognosis, and management lacunae in ALL in Indian scenario for better risk stratification and treatment.

In spite of the existence of national registry for child-hood leukemia, challenges such as under-reporting due to lack of awareness, under-diagnosis, and insufficient infrastructure still exist, which are contributing to the paucity of accurate data, delay in recognizing the disease on time and a late referral. This can place the child in a high risk category. Hence, improved awareness among the physicians, health care providers and general public is essential. We analyzed and present here the clinical, immunophenotypic and cytogenetic profiles of children who presented with ALL at our hospital, a tertiary care centre, during a period of four years.


  Materials and Methods Top


Patients

One hundred and three (103) children of both gender, aged between 03 months and 18 years, admitted with clinical symptoms and signs of ALL, confirmed on bone marrow aspiration and biopsy, were included in the study.

Methodology

This study was done between October 2007 and December 2011. It was a retrospective and prospective observational study with duration of one year 6 months and two years 7 months, respectively. Forty one patients were studied retrospectively and 62 prospectively. Patients who received the initial treatment elsewhere or presented with relapse for the first time to our institution were excluded. Clinical, hematological and biochemical evaluation, bone marrow morphology, immunophenotyping by flow cytometry and genetic mapping by karyotyping with G banding technique, FISH analysis, and Reverse transcription polymerase chain reaction (RT-PCR) in selective cases, were performed. UKALL 2003 protocol was used for the chemotherapy as per the risk stratification. Remission status (bone marrow aspirate blast being <5%) at the end of induction period was the end point for the study.

Statistics

Data was captured on Microsoft Excel from the medical records of patients and Data processing, tabulation of descriptive statistics, calculation of inferential statistics, and graphical representations was performed primarily using the licensed Statistics/Data Analysis software Statatm version 10. (Stata Corp, College Station, TX) © 1984-2008. Descriptive analysis was used for demography and other variables. Statistical testing was performed at 0.05 level of significance. Chi square test was used for finding dependency between two variables.


  Results Top


Demography

Of 103 children, 73 (70.87%) were boys and 30 (29.13%) girls, with a mean (± SD) age of 4.36 (± 2.66) years, with a median of 4 years, minimum age was 0.4 years and maximum age was 13 years. [Figure 1] shows the age distribution of patients and [Figure 2] shows the clinical presentation.
Figure 1: Distribution of patients in the study Majority of children were in 2-5 yrs age group (58.25%), followed by ≤2 years (21.36%), between 6-10 years (16.50%) and >10 years (3.88%).

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Figure 2: Clinical presentation Fever was seen in 92.23% patients, followed by pallor (87.38%), splenomegaly (83.50%), hepatomegaly (85.44%), lymphadenopathy (66.99%), Petechial rash (59.22%), bone pains (52.434%), bleeding manifestation (46.60%), mediastinal involvement (4.85%), Icterus (4.85%), superior venacaval sign (3.88%).

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Laboratory Investigations

Seventy (67.96%) patients were diagnosed by peripheral smear further confirmation by bone marrow aspiration. Thirty three cases did not reveal any blasts in the peripheral smear but confirmed in bone marrow.

Hemoglobin (Hb) <5 gm/dl was observed in 45 (43.69%) patients; 72 (69.90%) had WBC count between 5,000-50,000 cells/hpf and 21 (20.39%) had >50,000 cells/hpf. Fifty patients (48.54%) had platelet count <50,000 cells/hpf. Bone marrow revealed L2 morphology in 72 (69.90%) patients [Table 1].

CNS penetration was found in seven (6.80%) patients, four of them had Pre B ALL, one had pre T-cell ALL and two had T-cell ALL. Tumor lysis syndrome, defined as hyperkalemia, hyperphosphatemia, hyperuricemia, and hypocalcemia, was seen in 33 (32.04%) patients.
Table 1: Laboratory investigations

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Flowcytometry

Flowcytometry, using panel of antibodies was performed for 97 patients of which 80 (82.44%) were pre B-cell ALL [Table 1]. Immunophenotype was not performed in five patients. It was inconclusive in one child due to prior treatment with steroids. Later this patient was diagnosed as biphenotypic leukemia, when developed progressive disease.

Karyotyping

Karyotyping was done for 89 patients. Normal karyotypes were seen in 43 (48.32%) patients. In two (1.12%) patients cytogenetic analysis was not possible due to lack of growth. Few important changes seen in conventional karyotyping were hyperdiploidy in nine (10.11%) patients and hypodiploidy in two cases (2.25%). Five (5.62%) children had t(9,22) in regular karyotyping and they were also found to be FISH positive.

Two patients each showed 11q deletion and 15 q deletion in regular karyotyping. Other changes observed were 13q deletion, trisomy 11, 21q deletion, t(15,21), t(1,19), t(4,14), t(7,12), t(6,21) andTEL/ AML-t(12,21) in one patient each.

FISH Analysis

FISH analysis using dual colour probes was performed for 63 patients. MLL gene was positive in 8/63 (12.70%, CI 4.25, 21.15) patients. Of 63 patients, Philadelphia chromosome was found in seven (11.11%) patients [Table 1]. FISH for TEL AML was performed in 22 patients, of which three (13.6%) showed positivity. One patient exhibited Tel AML positivity on conventional karyotyping.

Response to Treatment

Most of these patients cleared blasts by Day 7 except eight, of which five cleared blasts by day 14, whereas two T cell ALL cases who had associated high count disease took >15 days to clear blast cells from peripheral blood; one case who was Philadelphia positive took one month to clear the blast cells.

Post Induction Remission

At the end of induction, bone marrow aspiration was done which showed that all patients were in remission except one. Quantitative RT PCR was done for all Philadelphia positive case at the end of induction. All these patients had remission except one who was high risk MRD as per flowcytometry and RT PCR; Latter was considered for bone marrow transfusion (BMT). All MLL positive cases were analysed by FISH at the end of induction and none were found to be positive.

Ninety nine (99/103, 96.06%, CI 93.72, 100.40) patients had post induction remission. Two patients died during induction, one abandoned the treatment; excluding these three patients, 99/100 went into remission. One patient had progressive disease.

Risk Groups

Among 80 pre B-cell ALL cases, 71 were CD10 (common ALL antigen -CALLA) positive. WBC count 5,000-50,000 cells/hpf was seen in 52/71 CALLA positive children. WBC count >50,000 cells/hpf was seen in 10/71 patients. Thirty one CALLA positive patients had Hb <5 gms/dl. Three had CNS penetration. Of nine hyperdiploidy cases, six were CALLA positive and two were CALLA negative pre B-cell ALL; hypodiploidy was seen in two patients, both were in pre B-cell ALL.

Seven of 80 pre B-cell ALL were positive for MLL and six were positive for Philadelphia chromosome.All three patients who were TEL AML positive were in Pre B-cell ALL group.

Of 15 cases of Pre T (n = 7) and T (n = 8) cell ALL, WBC >50,000cells/hpf was seen in eight (53.33%) patients at diagnosis. Three (20%) patients showed CNS penetration. MLL and Philadelphia chromosome were positive in one each.

Post treatment complications

Febrile neutropenia was seen in 47 (48.41%) patients during induction of which two patients had febrile neutropenia related mortality.


  Discussion Top


Acute leukemia is characterized by the unrestrained clonal proliferation of hematopoietic precursor cells coupled with aberrant or arrested differentiation. Advent of newer diagnostic modalities help clinicians to identify and recognize the association between morphology, immunophenotyping, and specific cytogenetic abnormalities, leading to the development of added treatment modalities based upon specific genetic defects.

Leukemia contributes to 25-40% of all childhood leukemias in India, of which 60-80% is ALL. ALL in children is associated with male preponderance. [7],[15],[16],[17],[18] Similar observation was noted in Italian population by Foà, [19] American population by Adelman et al., [20] and in Indian pediatric population. [5],[6],[21] Our study supports this finding with definite male predominance (70.87%) which has been observed to be higher compared to other studies, which may be due to low female population trend in the country.

Peak incidence of pediatric ALL has been reported in age group 2-5 years. [1],[2] Khalid et al.[15] reported 58.7% of ALL in the age group of one to nine years while Arya et al.[16] reported mean age as 7.6 years. In India, ALL found to be more prevalent in children <5 years of age [6],[8] but Biswas et al.[5] reported the average age as 6.12 years (SD ± 2.98). Our study supports the results of the previous studies, with 58.25% of patients in 2-5 years and 16.50% in 6-10 years age group. Median age in our study was four years, similar to that observed by Idris et al.[22] in Pakistani children (4.5 years).

Usual clinical manifestations of ALL cited in the literature are fever, fatigue, pallor, and weight loss. Thrombocytopenia with platelet counts <100,000 are seen in about 75% of patients. CNS involvement is less common, with 5% of children presenting with this feature. In our study, clinical presentations were in similar lines with the previous studies, [5],[15],[23],[24] but with a higher incidence of fever (92.23%).

CNS involvement (6.80%) and enlarged mediastinum (4.85%) reported to be high in our study compared to other studies. Available data in literature on CNS involvement in ALL is 4.3% and enlarged mediastinum in 2.2% patients. [15] Hyperleukocytosis having tumor lysis syndrome was reported to be 22% of ALL cases, [25] and we have observed tumor lysis syndrome in 32.04% patients.

Study by Khalifa et al.[26] showed best remission rate with L2 morphology; children's cancer group has associated L2 with poor prognosis, [27] however, its role is controversial. Ramyar et al. noted L1 morphology predominantly in their study, [24] whereas we observed predominance of L2 cells (69.90%) in our patients, similar to the findings of other studies. [23],[26] Khalid et al.[15] reported L1 and L2 morphology in 39.1% and 37% patients, respectively. In our study, there was no difference in remission status, without much difference between both L1 and L2 morphology, similar to the observation of Khalid et al.

Previous studies [16],[24] have shown the preponderance of Pre-B ALL and our study supports this indicating that the study population was under standard group. However, higher proportion of T-Cell ALL has been mentioined in the literature [7] in India; it has been estimated that about 20-50% ALL reported are found to be of T-cell type contrast to the scenario in developed world which is 10-20% [7] and it is 15.5% in our study. Mukhopadhyay et al. have documented high rate of T cell immunophenotypes (50.4%, 252 patients) and increased heterogenecity in their patients. [28] Abitha et al. have observed that in 20 years, a change in the trend and noted decrease in T-cell ALL. [29]

Presence of CD10 determines the prognosis and also treatment response. Previous study [30] has reported CD10 positive, pre B phenotype as the predominant phenotype and it was in similar lines in our study.

Literature [1] has recorded poor prognostic features like age >10 years, WBC count >50,000/ mm 3 , mediastinal lymphadenopathy and CNS leukemia significantly more frequent in those with T-ALL. [16] We have observed these features in our study also.

Karyotyping helps to assess the genetic mapping and aids in the treatment outcome. Previous studies have reported the incidence of normal karyotype as 75% (9/12), [15] 33% (14/42), [30] 56.7% (38/67), [31] 20% (8/40), [23] whereas it was 48.32% in our study. Hyper diploid karyotype was seen in 14.2%, [21] 19%, [30] 17.5%, [23] 20.68%, [31] and 32% [17] in previous studies and it was 10.11% in our study. Distribution of standard karyotype among Indian pediatric population has been described in the literature. [32] Hypodiploidy is a considered as a poor prognostic factor. [33],[34] Higher incidence of hypodiploidy was observed in Indian pediatric population. [7] Hypodiploidy was reported to be 51.2%, [17] 37.5, [23] 10.34%, [31] where as it was low (2.25%) in our study.

Chromosomal translocations are closely associated with the prognosis of pediatric ALL cases and have been seen in 75% of pediatric ALL. [35] Mazloumi SH, et al. [21] have described translocations in 42.8% in their study population. Presence of Philadelphia chromosome is considered as a poor prognostic factor and it is usually seen in less than 5% of children with ALL. Previous studies have reported Philadelphia chromosome positivity in1.5%, [33] 2-5%, [21] 2.8%, [32] but it was higher in our study (11.11%). Complete profile of BCR-ABL positive ALL cases has been highlighted by Bhatia et al. [36] Study by Gurbaxani et al.[37] reported further higher incidence (24%, 7/24)) of Philadelphia positivity in Indian pediatric population. Sazawal et al. [38] reported Philadelphia chromosome being positive in l/35 (2.8%) and t (1; 19) in 2/35 (5.7%) whereas it was seen in 7/63 (11.11%) and in 1/89 (1.12%) patient, respectively in our study. It was positive in 23% patients in previous study [39] whereas we noticed this in 13.64% (3/22) patients. Outcome was favorable the presence of TELAML. Study Bhatia et al. [40] documents the role of multiplex RT-PCR in ALL and this has shown good positivity for TEL AML1 in pediatric ALL.

In our study, post induction remission was seen in 99 % (99/100) overall. Kumar et al. [41] have observed 100% remission, whereas Settin et al.[23] have reported 62.5% remission in Indian pediatric patients with ALL, whereas Khalid et al. [15] and Idris et al. [22] have reported 97.8% and 94% remission in Pakistani children respectively.

We observed high risk factors of cytogenetics such as hypodiploidy, MLL gene rearrangement and presence of Philadelphia chromosome in only few cases.

Most of the CD10 positive pre B-cell patients had <50,000WBC count. Hyperdiploidy is predominantly seen in CD10 positive pre B-cell ALL group. All TELAML positive patients were in pre B-cell ALL. CNS penetration was seen in 4% of pre B-cell ALL group.

More than half of T-cell ALL children have WBC count >50,000 and percentage of CNS penetration is upto 20% indicating T-cell disease being a high risk category. We observed children with one high risk factor also noticed to have other high risk factors.

Most of our patients were in the standard risk as mentioned in the literature.

This study was conducted to describe the clinical, immunophenotype and cytogenetic profild of ALL patients who were treated in our centre. Currently, these patients are being followed up for the survival rate. We performed karyotyping and FISH analysis in considerably larger number of patients; however, these tests were not performed in all patients as they were expensive and not affordable by few. Not performing flowcytometry, aberrant myeloid antigen phenotype expression and genetic mapping in all patients was the limitation of our study.


  Conclusion Top


In our study, majority of the children were in the standard risk by age, WBC count, flowcytometry and cytogenetic analysis. Association with more than one high risk factor was seen in small proportion of cases. Flowcytometry and cytogenetic analysis help in risk adopted therapy for ALL. Reverse transcription polymerase chain reaction and cytogenetic analyses helps in detecting the minimal residual disease (MRD), thereby identifying the risk group as those with high MRD are considered to be at high risk with poor treatment response and outcome. Larger studies required using RT-PCR technique in cytogenic analysis for better risk stratification even in developing countries which will help in risk adopted therapy in addition to other prognostic factors.


  Acknowledgement Top


We acknowledge all children whose data was used for analysis. We thank Dr Ramana Dandamudi (pediatric hemato-oncologist, Hyderabad), Dr Hafsa Ahmed (Hematopathologist, Rainbow children's hospital, Hyderabad), Dr Gayathri and Dr Manu Goel (Hemato-pathologists, Tapadia diagnostics, Hyderabad) for their valuable support. We thank Mr Sunoj C Varughese for his assistance in statistics. We acknowledge the support of the management of Rainbow Hospital, Hyderabad, India, where this study was conducted. We appreciate the support of paramedical staff of Rainbow children's hospital, Hyderabad during the conduct of the study.

 
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