Manorama Bhargava

Concurrent methylation of multiple genes in childhood ALL: Correlation with phenotype and molecular subgroup

Multiple genes have been shown to be independently hypermethylated in lymphoid malignancies. We report here on the extent of concurrent methylation of E-cadherin, Dap-kinase, O6MGMT, p73, p16, p15 and p14 in 129 pediatric ALL cases. While most of these genes demonstrated methylation in a proportion of cases, O6MGMT, p16 and p14 were infrequently methylated (11, 7 and 3%, respectively). Methylation of at least one gene was found in the vast majority (83%) of cases. To determine the extent and concordance of methylation we calculated a methylation index (MI=number of methylated genes/number of studied genes) for each sample. The average MI was 0.28, corresponding to 2/7 methylated genes. MI was correlated with standard prognostic factors, including immunophenotype, age, sex, WBC and presence of specific translocations (TEL-AML1, BCR-ABL, E2A-PBX1 or MLL-AF4). We determined that children ⩾10 years old and children presenting with high WBC (⩾50 × 109/l) both associated with a higher MI (P<0.01 and <0.05, respectively). T-ALLs demonstrated a lower MI (median=0.17) than precursor B ALLs (median=0.28). Among the different molecular subgroups, MLL-ALLs had the highest MI (mean=0.35), while ALLs carrying the t(1;19) had the lowest MI (mean=0.07). The most common epigenetic lesion in childhood ALL was methylation of E-cadherin (72%) independent of the molecular subtype or other clinicopathological factors.

Frequencies of the major subgroups of precursor B-cell acute lymphoblastic leukemia in Indian children differ from the west

Frequencies of the major subgroups of precursor B-cell acute lymphoblastic leukemia in Indian children differ from the West

Relationship of Maternal Serum Ferritin with Foetal Serum Ferritin, Birth Weight and Gestation

Haemoglobin and ferritin estimations employing the micro-ELISA technique were done in 308 random selected mothers in labour and their newborns. The values of haemoglobin and serum ferritin as well as birth weight and gestation of babies born to iron depleted, and mildly and moderately anaemic mothers were no different from those of newborns of non-anaemic women. However, the values of serum ferritin per se in all these newborns were much lower than what are generally reported from the western countries. Babies born to severely anaemic women, on the other hand, showed elevated levels of haemoglobin and serum ferritin, and lower birth weights and gestation. Thus, mild to moderate iron deficiency in the mother does contribute to lower iron reserves in the foetus, if not frank iron depletion, and severe iron deficiency anaemia to lower birth weight and gestation.

Effect of Maternal Anaemia and Iron Depletion on Foetal Iron Stores, Birthweight and Gestation

India is considered to have the highest prevalence of nutritional anaemia in women. Between 6040% of pregnant women have been found to be anaemic, mainly due to iron deficiency (1). There are also reports on the development of iron deficiency anaemia in Indian infants already at 6 months of age (2). In view of this, it was considered pertinent to study the effect not only of anaemia but also of iron depletion in the mother on fetal iron stores. The influence of these factors on birth weight and gestation was also determined.

Pattern of relapse in childhood ALL: challenges and lessons from a uniform treatment protocol.

This retrospective analysis of 254 children less than 15 years of age treated with MCP-841 protocol from June 1992 to June 2002 was undertaken to identify the pattern of relapse and determine management lacunae. Two hundred twenty-three (87.8%) children achieved a complete remission of whom 40 (17.9%) relapsed. The mean age of relapsed patients was 6.5 years. The male/female ratio was 9:1. There were 23 (57.5%) isolated bone marrow (BM), 7 (17.5%) isolated central nervous system (CNS), 2 (5%) isolated testicular, 5 (12.5%) BM+testes and 1 each of BM+CNS, CNS+testes, and isolated bone relapses. Twenty-seven children (67.5%) relapsed on-therapy whereas 13 (32.5%) relapsed posttherapy. All 9 CNS relapses occurred on-therapy whereas 5/8 (62.5%) of testicular relapses occurred posttherapy. Lymphadenopathy was the only significant predictor for relapse. High-risk features such as age less than 1 year and greater than 10 years (P=0.047) and white cell count greater than 50.0×109/L (P=0.044) were significantly more frequent in patients with early on-therapy relapse than in patients with off-therapy relapse. The overall survival in the entire study cohort was 67±3.5%. Modest survival outcome, relapse while on chemotherapy and the higher incidence of CNS and testicular relapse indicate the need for reappraisal of our treatment protocol. There is a need of identifying risk factors and high-risk groups in our set of patients and risk-stratified intensification of chemotherapy in them.

Pattern of immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements in childhood acute lymphoblastic leukemia in India.

In 120 cases of acute lymphoblastic leukemia (median age 8 years), IgH chain gene was rearranged in 99% B-Cell Precursor (BCP) ALLs and 13% T-ALLs. One or the other TCR locus was rearranged not only in all T-ALLs, but also in 87% of BCP-ALLs. TCR-beta rearrangement in BCP-ALL was associated with a higher mean age at presentation (8.7 vs. 6.2 years, P=0.008), lower mean platelet counts (61.2x10(9)/l vs. 103.7x10(9)/l, P=0.003) and a poorer DFS (% cummulative survival 0 vs. 88.9+/-10.5, P=0.004). TCR-gamma rearrangement in T-ALL was associated with a higher mean WBC count (186.3x10(9)/l vs. 63. 4x10(9)/l, P=0.002). Also, the pattern of rearrangement of these genes appeared to be different from the West; viz. TCR-beta rearrangement in a higher proportion of BCP-ALLs (58%, 95% confidence intervals 45-69%), invariable deletion of Cgamma1 and only monoallelic rearrangement for TCR-delta locus. This repertoire of gene rearrangement may have a bearing on the poor treatment outcome reported previously from our geographic region.

Immunological subtypes of acute lymphoblastic leukemia in North India

Pretreatment immunologic marker analysis in 152 adult and childhood patients of ALL and ALL/lymphoma employing multiple monoclonal antibodies and hetero-antisera revealed three major subgroups, i.e. T-ALL (37.7%), N-ALL (33.1%) and C-ALL (21.5%). The early age peak was absent, males predominated in all the subgroups and T-ALL had increased incidence of thymic mass. Leucocyte counts of 50,000 X 10(6)/l were equally frequent in the three groups. T-ALL showed marked heterogeneity by showing a variety of markers such as T-helper/inducer, T-suppressor/cytotoxic, p-24, Ia and CALLA. These results show a high prevalence of unfavourable prognostic factors in ALL in our geographic region which might be related to socioeconomic and/or environmental factors.

β-Thalassemia mutations in subjects with borderline HbA2 values: a pilot study in North India

Abstract Background: Interpreting hemoglobin high performance liquid chromatograms with borderline HbA2 values is often problematic, especially in antenatal cases if the partner is a known thalassemia trait. Methods: We tested for underlying β-thalassemia mutations in 25 subjects with borderline HbA2 values (between 3.0%–4.0%). Amplification refractory mutation system (ARMS-PCR) was used to detect the five common Indian β-thalassemia mutations: (IVS-I-5 (G>C), IVS-I-1 (G>T), codons 8/9 (+G), codons 41/42 (-TTCT) and 619 bp deletion). β-Globin gene sequencing was performed if no mutation was detected. Results: A β-globin gene defect was identified in 8 (32%) of the 25 cases with HbA2 levels ranging from 3.5%–3.9%. ARMS-PCR revealed IVS-I-5 (G>C) in three, 619 bp deletion in two and codons 41/42 (-TTCT) in one case. Two cases had CAP +1 (A>C) mutation on gene sequencing. IVS-I-1 (G>T) and codons 8/9 (+G) were not found in this small cohort. Conclusions: Mutation analysis should be offered to all at-risk couples with borderline HbA2, especially those with values between 3.5% and 4.0% and microcytic hypochromic indices. Significant mutations different from those in other ethnic populations were seen in this small institution-based study.

Fetal liver infusion in aplastic anaemia

Forty patients with severe aplastic anaemia received an intravenous infusion of 0.004 to 11.1 x 10(8) (median: 8 x 10(8) hematopoietic cells prepared from the fetal livers of 8-32 week old abortuses. Five patients, who died within 15 days of fetal liver infusion, are excluded from analysis. Twenty-two of the 35 evaluable patients (62%) responded favourably. Six of the 7 patients with good response were alive after 9 to 44 months (median: m = 20); one died 106 months after fetal liver infusion due to renal lithiasis. Four of the 7 with moderate response were alive after 9 to 31 months; 3 died within 16 months. Of 8 patients with minimal response, one was lost to follow-up and the others died in 3.4 to 10 months (m = 6). Median survival of responders was 15.7 months. Bone marrow cellularity became normal in 12 patients following fetal liver infusion. In seven patients, there was a relapse; 6 regained a normal bone marrow cellularity after a second or third fetal liver infusion. These data strongly suggest a role of fetal liver infusion in inducing bone marrow recovery. Of 13 non-responders, 4 were lost to follow-up and 9 died within 20 days-4.3 months (m = 1.6). Fetal liver infusion appears to be an effective therapy in patients with severe aplastic anaemia.

Expression of genes implicated in multidrug resistance in acute lymphoblastic leukemia in India.

Abstract In order to investigate the phenomenon of multidrug resistance as a possible mechanism for poor response to treatment in patients with acute lymphoblastic leukemia (ALL) from India, a series of 32 cases of de novo untreated ALLs were analyzed by a cDNA-PCR approach to estimate the relative mRNA levels of the MDR-associated genes encoding MDR1, MRP, GSTπ, and GSTμ. The expression of β2 microglobulin served as an internal standard. Quantifiable transcripts were observed in 20 patients for MRP, in 5 for MDR1, in 24 for GSTπ, and in 19 for GSTμ. The values ranged from undetectable to 132% of the control A549 cell line for MRP, undetectable to 49% of the HL60/DNR control cell line for MDR1, undetectable to 268% of A549 control cell line for GSTπ, and undetectable to 247% of A549 control cell line for GSTμ mRNA. Increased MRP levels were associated with increased GSTπ and GSTμ levels (p<0.01 for both), and increased levels of MDR1 were associated with increased GSTπ levels (p<0.05). The present observations showed no correlation between the MDR1 and MRP values with treatment outcome, in terms of either achieving a complete remission or predilection to early relapse. In view of some recent studies that envisage MRP as an energy-dependent pump involved in the efflux of GSH conjugates, the simultaneous up-regulation of transcription of all these genes might well be part of an integrated detoxification response that has been switched on after exposure to an environmental stress.