Radhika Bakhshi

Mitochondrial D-loop variations in paediatric acute myeloid leukaemia: a potential prognostic marker.

The D‐Loop region of mitochondrial DNA (mtDNA) is the regulatory region for its replication and transcription. There are two hypervariable regions (HV‐I, HV‐II) and the rate of mutation in these regions is 100‐ to 200‐fold that of nuclear DNA. In the current study, the entire D‐loop region of mtDNA was amplified in two overlapping polymerase chain reaction fragments and variations were evaluated in 44 paediatric acute myeloid leukaemia (AML) patients by direct DNA sequencing methods. Median age of the patients was 8·5 years (1–18 years) and the male:female ratio was 3·8:1. A total of 222 variations were observed at 118 positions in the D‐Loop of 35/44 (79·5%) AML patients. The most common variations were T→C (24·6%) and C→T (21·4%) followed by A→G (15·8%). There was no significant difference in the event‐free survival (EFS) of patients with or without any variations (P = 0·40). Three variations in HV‐I, namely 16126T→C (P = 0·05), 16224T→C (P < 0·01) and 16311T→C (P < 0·001), were significantly associated with inferior EFS. In conclusion, this is the largest study to show a high frequency of mtDNA variations in paediatric AML and their potential relevance as a prognostic marker in this disease.

Increased coexpression of c‐KIT and FLT3 receptors on myeloblasts: Independent predictor of poor outcome in pediatric acute myeloid leukemia

Significance of mutations in FLT3 and c‐KIT genes in AML has been well established, but role of their coexpression has not been evaluated. The aim of this study was to evaluate clinical significance of FLT3 (CD135) and c‐KIT (CD117) coexpression on myeloblasts in AML.

BAX/BCL2 RMFI ratio predicts better induction response in pediatric patients with acute myeloid leukemia

BAX/BCL2 ratio in pediatric AML has not been evaluated. In this first prospective study, we evaluated BAX/BCL2 transcript and RMFI ratio in 64 patients using real‐time PCR (TaqMan Probe chemistry) and flow‐cytometry, respectively. There was no correlation of BAX/BCL2 transcript ratio with RMFI ratio (R = −0.05; P = 0.715). Patients with WBC count >50,000/mm3 had lower BAX/BCL2 RMFI ratio (P = 0.043), whereas no difference in ratio was observed among patients of different cytogenetics subgroups (P = 0.786). Higher BAX/BCL2 RMFI ratio was associated positively with CR rate (P = 0.03), but this study was unable to show that it translated into improved EFS or OS. Pediatr Blood Cancer 2013;60E63‐E66.

Genetics and management of retinoblastoma

Retinoblastoma is the most common intraocular tumor in childhood. In majority of early stage retinoblastoma, the eyeballs as well as vision can be preserved with chemotherapy and local intraocular therapy with laser or photocoagulation. However, more than half the patients in India and other developing nations present in advanced stage of the disease. This article reviews the genetics, clinical approach, and treatment options for retinoblastoma focussing on advances in chemotherapy for intraocular retinoblastoma (chemoreduction), as well as improvement in survival in advanced retinoblastoma with surgery, chemotherapy, radiotherapy, and bone marrow transplantation.

High fms-like tyrosine kinase-3 (FLT3) receptor surface expression predicts poor outcome in FLT3 internal tandem duplication (ITD) negative patients in adult acute myeloid leukaemia: A prospective pilot study from India

Background & objectives: Mutations in fms-like tyrosine kinase 3 (FLT3) receptor have significant role in assessing outcome in patients with acute myeloid leukaemia (AML). Data for FLT3 surface expression in relation to FLT3 internal tandem duplication (ITD) status and outcome are not available from India. The objective of the current study was to investigate adult patients with AML for FLT3 expression and FLT3 ITD mutation, and their association with long-term outcome. Methods: Total 51 consecutive de novo AML patients aged 18-60 yr were enrolled in the study. FLT3 ITD was detected by polymerase chain reaction (PCR); flowcytometry and qPCR (Taqman probe chemistry) were used for assessment of FLT3 protein and transcript, respectively. Kaplan Meier curves were obtained for survival analysis followed by log rank test. Results: FLT3 ITD was present in eight (16%) patients. Complete remission was achieved in 33 (64.6%) patients. At 57.3 months, event free survival (EFS) was 26.9±6.3 per cent, disease free survival (DFS) 52.0±9.2 per cent, and overall survival event (OS) 34.5±7.4 per cent. FLT3 surface expression was positive (>20%) by flow-cytometry in 38 (88%) of the 51 patients. FLT3 surface expression and transcripts were not associated with FLT3 ITD status. FLT3 expression was significantly associated with inferior EFS (P=0.026) and OS (P=0.018) in those who were negative for FLT3 ITD. Interpretation & conclusions: This study evaluated FLT3 ITD mutation along with FLT3 expression in AML patients, and associated with survival. Negative impact of FLT3 surface expression on survival was observed in AML patients who were FLT3 ITD negative.

FLT3-ITD mutation in relation to FLT3 expression in pediatric AML: a prospective study from India.

There is lack of data with regard to FLT3 expression in FLT3-ITD positive pediatric AML patients. Further, FLT3-ITD has not been systematically analyzed for outcome from Indian subcontinent. Amongst 64 consecutive pediatric AML patients, FLT3-ITD was present in 12 (19%) patients. All patients with FLT3-ITD achieved CR; those with FLT3-ITD mutation had inferior DFS (P = .029). FLT3 expression by flow-cytometry was observed in all FLT3-ITD positive patients, whereas 40/52 (77%) FLT3-ITD negative patients expressed FLT3 (P = .06). FLT3 expression in 12 FLT3-ITD positive patients was unable to show an association between FLT3 expression and outcome. In FLT3-ITD negative patients, higher surface expression of FLT3 significantly predicted poor EFS (P = .001) and OS (P = .007).

Pattern of mitochondrial D-loop variations and their relation with mitochondrial encoded genes in pediatric acute myeloid leukemia

Role of mitochondrial DNA variations, particularly in D loop region, remains investigational in acute myeloid leukaemia (AML). Consecutive 151 pediatric AML patients were prospectively enrolled from June 2013 to August 2016, for evaluating pattern of variations in mitochondrial D-loop region and to determine their association, if any, with expression of mitochondrial-encoded genes. For each patient, D-loop region was sequenced on baseline bone marrow, buccal swab and mothers blood sample. Real time PCR was used for relative gene expression of four mitochondrial DNA encoded genes viz. Nicotinamide-adenine-dineucleotide-dehydrogenase subunit 3 (ND3), Cytochrome-B (Cyt-B), Cytochrome c oxidase-I (COX1) and ATP-synthetase F0 subunit-6 (ATP6). Total 1490 variations were found at 237 positions in D-Loop; 1206 (80.9%) were germline and 284 (19.1%) were somatic. Positions 73-263 were identified as a probable hotspot region. G bases appeared to be most stable nucleotide (least number of single base substitutions) whereas T appeared to be most susceptible to variations with germline T-C being the commonest. Gene expression of Cyt-B was found to be significantly higher for any variation (somatic or germline) at positions 16,192 and 16,327 while it was significantly lower for variations at positions 16,051 and 207. Any variation at positions 152, 207 and 513 significantly decreased COX1 expression while those at positions 16,051 and 152 attenuated ATP6 expression. This first study evaluated type and overall pattern of D-loop variations in AML, and also showed that some of these variations in D loop region might have an effect on the mitochondrial-encoded genes which is new and valuable information in AML genomics.

Quantitative assessment of BAX transcript and flow cytometric expression in acute myeloid leukemia: a prospective study.

Abstract Objective Quantitative assessment of BAX transcripts and protein in acute myeloid leukemia (AML). Methods We quantitatively evaluated BAX gene transcripts by real-time polymerase chain reaction (TaqMan probe chemistry) and protein expression by flow cytometry. Results Consecutive 112 AML patients with a median age of 16 (1–59) years were recruited in the study. By flow cytometry, the percentage expression was in linear correlation with relative median fluorescent intensity (RMFI; R = 0.4425; P < 0.001). However, there was no linear relationship between the transcript copies of the BAX with its RMFI (R = −0.0559; P = 0.586). The expression of the BAX at both protein and transcript level was significantly higher in AML patients as compared with normal control. RMFI of the BAX were higher in the cohort with lower white blood cell count (P = 0.029). None of the other baseline characteristics correlated with either the BAX transcript or the RMFI. BAX expression did not correlate with complete remission rate, event free, disease free, and overall survival. Conclusion BAX gene expression in AML was evaluated first time with two different methods but did not correlate with the survival outcome

Relative Receptor Tyrosine Kinases and Anti-Apoptotic Transcripts Hold Potential for Predicting Inferior Outcome in Adult Acute Myeloid Leukemia: A Prospective Pilot Study

INTRODUCTION Acute myeloid leukemia is characterized by accumulation of immature cells because of imbalance between proliferation and apoptosis. In AML, simultaneous expression of proliferative (FLT-3, c-KIT) and antiapoptotic genes (BCL-2), are unknown. PATIENTS AND METHODS We prospectively assessed proliferative and antiapoptotic gene transcripts using Taqman probe chemistry in 48 adult AML patients. A stepwise Cox regression model was applied for independent prognostic factors. RESULTS Thirty-two of 48 (75%) patients achieved complete remission. At follow-up ranging from 0.5 to 57.3 months, event-free survival (EFS) was 26.9 ± 6.3% (range, 15.5%-39.6%) and OS 34.5 ± 7.46% (range, 20.5%-48.9%). High white blood cell count correlated with an inferior complete remission rate (P = .021). Cytogenetics and FLT-3 internal tandem duplication did not predict EFS or OS. The transcripts of FLT-3, c-KIT, and BCL-2 showed a significant linear association with each other in Pearson correlation (FLT-3 vs. c-KIT: R = 0.8234; P < .001; c-KIT vs. BCL-2: R = 0.3377; P = .01; FLT-3 vs. BCL-2: R = 0.3815; P = .007). In a validation cohort (Microarray Data Set GSE1159) of adult AML patients, the global gene expression profile depicted a similar interrelationship. Patients with a greater platelet count were associated with increased transcript levels of BCL-2 (P = .034). In univariate analysis, a high transcript level of FLT-3 and high transcript ratio of FLT-3/BCL-2 and FLT-3 and c-KIT/BCL-2 significantly predicted OS (P = .043, .028, and .028, respectively). In a stepwise Cox regression model, high FLT-3 and c-KIT/BCL-2 ratio predicted OS (HR, 2.29). CONCLUSION To our knowledge, this is the first study that evaluated proliferative and antiapoptotic transcripts simultaneously, and results have shown that it is the relative levels of these transcripts that determine outcome in AML patients rather than their expression in isolation.

BAX/BCL2RMFI ratio predicts better induction response in pediatric patients with acute myeloid leukemia: Clinical Significance ofBAX/BCL2Expression in Pediatric AML

BAX/BCL2 ratio in pediatric AML has not been evaluated. In this first prospective study, we evaluated BAX/BCL2 transcript and RMFI ratio in 64 patients using real‐time PCR (TaqMan Probe chemistry) and flow‐cytometry, respectively. There was no correlation of BAX/BCL2 transcript ratio with RMFI ratio (R = −0.05; P = 0.715). Patients with WBC count >50,000/mm3 had lower BAX/BCL2 RMFI ratio (P = 0.043), whereas no difference in ratio was observed among patients of different cytogenetics subgroups (P = 0.786). Higher BAX/BCL2 RMFI ratio was associated positively with CR rate (P = 0.03), but this study was unable to show that it translated into improved EFS or OS. Pediatr Blood Cancer 2013;60E63‐E66.