Surender Kumar Sharawat

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.

Diagnosis of invasive fungal infections using real-time PCR assay in paediatric acute leukaemia induction.

Invasive fungal infections (IFI) lead to morbidity and mortality in neutropenic patients and in allogenic stem cell transplantation. Serum‐based fungal detection assays have limitation of specificity or sensitivity. Studies on fungal DNA detection using real‐time PCR in childhood leukaemia are lacking. The aim of this study was to develop sensitive and specific diagnostic tools for IFI in paediatric acute leukaemia patients using real‐time PCR. Of 100 randomised paediatric acute leukaemia patients receiving antifungal prophylaxis with voriconazole/amphotericin B, single peripheral whole blood sample in EDTA was used for Pan‐AC real‐time PCR assay (detects nine Candida and six Aspergillus species) in patients who failed prophylaxis due to proven, probable, possible or suspected fungal infections. PCR results were retrospectively correlated with clinical profile. Real‐time PCR test was positive in 18/29 (62%) patients who failed prophylaxis. The only patient with proven IFI (mucormycosis), real‐time PCR assay was negative. Real‐time PCR was positive in 2/4 (50%) patients with possible and 16/24 (66.6%) suspected IFI and 5/10 (50%) patients with pneumonia. By applying method A/B, sensitivity and positive predictive value could not be commented due to unproven Aspergillus or Candida infections; specificity and negative predictive values (NPV) were 41% and 100% respectively; by method C (included episodes of possible IFI as true positive), sensitivity, specificity, PPV and NPV were 50%, 36%, 11% and 81% respectively. In those with suspected IFI, 8/24 (33.3%) were PCR negative and unnecessarily received empirical antifungal therapy (EAFT). Real‐time PCR is a practical, rapid, non‐invasive screening test for excluding IFI in paediatric leukaemia. The high NPV makes real‐time PCR a promising tool to use this prior to initiating EAFT in antibiotic‐resistant febrile neutropenic patients; this would avoid toxicity, cost and hospitalisation for EAFT (ClinicalTrials.gov identifier:NCT00624143).

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.

Childhood chronic myeloid leukemia with monocytosis.

We report two cases of chronic myeloid leukemia (CML) in childhood presenting with monocytosis. History, physical examination and laboratory findings were in favor of juvenile myelomonocytic leukemia in both the cases, but reverse transcriptase polymerase chain reaction (RT-PCR) detected b2a2 and b3a2 transcript of p210 bcr-abl protein characteristic of major BCR breakpoint. Presence of monocytosis in early childhood suggests a viral infection or JMML but a possibility of CML with monocytosis needs to be considered.

Systemic mastocytosis associated with childhood acute myeloid leukemia.

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Aggressive serous epithelial ovarian cancer is potentially propagated by EpCAM + CD45 + phenotype

Epithelial ovarian carcinoma (EOC) patients often acquire resistance against common chemotherapeutic drugs like paclitaxel and cisplatin. The mechanism responsible for the same is ambiguous. We have identified a putative drug-resistant tumour cell phenotype (EpCAM+CD45+) in the ascitic fluid of EOC patients, which appears to originate from the primary tumour. These cells represent the major tumour burden and are more drug resistant compared to EpCAM+ tumour cells due to the over-expression of SIRT1, ABCA1 and BCL2 genes. We have found that the entire EpCAM+CD45+ population is highly invasive with signature mesenchymal gene expression and also consists of subpopulations of ovarian cancer stem cells (CD133+ and CD117+CD44+). Additionally, we demonstrate that the EpCAM+CD45+ tumour cells over-express major histocompatibility complex class I antigen, which enable them to evade the natural killer cell-mediated immune surveillance. Preliminary evidence obtained in OVCAR-5 cells suggests that exosomes, secreted by non-tumour cells of the ascitic fluid, play an important role in rendering drug resistance and invasive properties to the cancer cells. Identification of such aggressive tumour cells and deciphering their origin is important for designing better drug targets for EOC.

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).

Circulating T‐regulatory cells in PNET: A prospective study

Bone marrow regulatory T‐cells (Tregs) have been evaluated in patients with peripheral neuroectodermal tumor (PNET); data on peripheral blood circulating Tregs are lacking. The objective of our study was to determine baseline Tregs (both Treg frequency and absolute number) in patients with PNET and correlate with patient characteristics, and observe their change with treatment and relapse.