Metabolomics and transcriptomics based multi-omics integration reveals radiation-induced altered pathway networking and underlying mechanism.

Recent advancement in integrated multi-omics has significantly contributed to many areas of the biomedical field. Radiation research has also grasped uprising omics technologies in biomarker identification to aid in triage management. Herein, we have used a combinatorial multi-omics approach based on transcriptomics together with metabolomics and lipidomics of blood from murine exposed to 1 Gy (LD) and 7.5 Gy (HD) of total-body irradiation (TBI) for a comprehensive understanding of biological processes through integrated pathways and networking. Both omics displayed demarcation of HD group from controls using multivariate analysis. Dysregulated amino acids, various PC, PE and carnitine were observed along with many dysregulated genes (Nos2, Hmgcs2, Oxct2a, etc.). Joint-Pathway Analysis and STITCH interaction showed radiation exposure resulted in changes in amino acid, carbohydrate, lipid, nucleotide, and fatty acid metabolism. Elicited immune response was also observed by Gene Ontology. BioPAN has predicted Elovl5, Elovl6 and Fads2 for fatty acid pathways, only in HD group. Collectively, the combined omics approach facilitated a better understanding of processes uncovering metabolic pathways. Presumably, this is the first in radiation metabolomics that utilized an integrated omics approach following TBI in mice. Our work showed that omics integration could be a valuable tool for better comprehending the mechanism as well as molecular interactions.

Mitochondrial gene expression signature predicts prognosis of pediatric acute myeloid leukemia patients.

Introduction: Gene expression profile of mitochondrial-related genes is not well deciphered in pediatric acute myeloid leukaemia (AML). We aimed to identify mitochondria-related differentially expressed genes (DEGs) in pediatric AML with their prognostic significance. Methods: Children with de novo AML were included prospectively between July 2016-December 2019. Transcriptomic profiling was done for a subset of samples, stratified by mtDNA copy number. Top mitochondria-related DEGs were identified and validated by real-time PCR. A prognostic gene signature risk score was formulated using DEGs independently predictive of overall survival (OS) in multivariable analysis. Predictive ability of the risk score was estimated along with external validation in The Tumor Genome Atlas (TCGA) AML dataset. Results: In 143 children with AML, twenty mitochondria-related DEGs were selected for validation, of which 16 were found to be significantly dysregulated. Upregulation of SDHC (p<0.001), CLIC1 (p=0.013) and downregulation of SLC25A29 (p<0.001) were independently predictive of inferior OS, and included for developing prognostic risk score. The risk score model was independently predictive of survival over and above ELN risk categorization (Harrell's c-index: 0.675). High-risk patients (risk score above median) had significantly inferior OS (p<0.001) and event free survival (p<0.001); they were associated with poor-risk cytogenetics (p=0.021), ELN intermediate/poor risk group (p=0.016), absence of RUNX1-RUNX1T1 (p=0.027), and not attaining remission (p=0.016). On external validation, the risk score also predicted OS (p=0.019) in TCGA dataset. Discussion: We identified and validated mitochondria-related DEGs with prognostic impact in pediatric AML and also developed a novel 3-gene based externally validated gene signature predictive of survival.

TMAO to the rescue of pathogenic protein variants.

Trimethylamine N-oxide (TMAO) is a chemical chaperone found in various organisms including humans. Various studies unveiled that it is an excellent protein-stabilizing agent, and induces folding of unstructured proteins. It is also well established that it can counteract the deleterious effects of urea, salt, and hydrostatic pressure on macromolecular integrity. There is also existence of large body of data regarding its ability to restore functional deficiency of various mutant proteins or pathogenic variants by correcting misfolding defects and inhibiting the formation of high-order toxic protein oligomers. Since an important class of human disease called "protein conformational disorders" is due to protein misfolding and/or formation of high-order oligomers, TMAO stands as a promising molecule for the therapeutic intervention of such diseases. The present review has been designed to gather a comprehensive knowledge of the TMAO's effect on the functional restoration of various mutants, identify its shortcomings and explore its potentiality as a lead molecule. Future prospects have also been suitably incorporated.

An integrative chemometric approach and correlative metabolite networking of LC-MS and 1H NMR based urine metabolomics for radiation signatures.

The increasing threat of nuclear terrorism or radiological accident has made high throughput radiation biodosimetry a requisite for the immediate response for triage. Owing to detection of subtle alterations in biological pathways before the onset of clinical conditions, metabolomics has become an important tool for studying biomarkers and the related mechanisms for radiation induced damage. Here, we have attempted to combine two detection techniques, LC-MS and 1H NMR spectroscopy, to obtain a comprehensive metabolite profile of urine at 24 h following lethal (7.5 Gy) and sub-lethal (5 Gy) irradiation in mice. Integrated data analytics using multiblock-OPLSDA (MB-OPLSDA), correlation networking and pathway analysis was used to identify metabolic disturbances associated with radiation exposure. MB-OPLSDA revealed better clustering and separation of irradiated groups compared with controls without overfitting (p-value of CV-ANOVA: 1.5 × 10-3). Metabolites identified through MB-OPLSDA, namely, taurine, creatine, citrate and 2-oxoglutarate, were found to be dose independent markers and further support and validate our earlier findings as potential radiation injury biomarkers. Integrated analysis resulted in the enhanced coverage of metabolites and better correlation networking in energy, taurine, gut flora, L-carnitine and nucleotide metabolism observed post irradiation in urine. Our study thus emphasizes the major advantage of using the two detection techniques along with integrated analysis for better detection and comprehensive understanding of disturbed metabolites in biological pathways.

Mitochondrial complex II and V activity is enhanced in pediatric acute myeloid leukemia.

BACKGROUND: Mitochondrial bioenergetic alterations are commonly observed metabolic adaptation in malignancies including acute myeloid leukemia (AML). Mitochondrial DNA alterations are well known in pediatric AML with possible prognostic significance; however, mitochondrial complex activity and its impact on disease outcome have not been previously explored. The aim of this study was to evaluate the mitochondrial complex II and complex V activity and its prognostic significance in pediatric AML patients. METHODS: Consecutive 82 de novo pediatric (≤18 years) patients with AML were included in the study along with age and sex matched controls. Bone marrow mononuclear cells were isolated from baseline bone marrow samples from all patients and controls. DNA, RNA and proteins were extracted and relative expression of mitochondrial biogenesis genes TFAM, POLG, POLRMT were estimated along with mitochondrial DNA copy number. The mitochondrial complex II and V enzymes were immunocaptured and their activity was measured by substrate specific absorbance change by kinetic ELISA. The mitochondrial complex II and V activity was compared with controls and their association with clinico-pathological features and survival outcome were analysed. Complex activity was also correlated with relative expression of biogenesis genes. RESULTS: The activity of mitochondrial complex II and V were found to be significantly enhanced (P = 0.010 and P = 0.0013 respectively) in pediatric AML patients compared to controls. The activity of mitochondrial complex II and V showed significant positive correlation with relative gene expression of mitochondrial biogenesis genes TFAM (P = 0.001 and P = 0.016 respectively) and POLG (P = 0.005 and P = 0.006 respectively). Neither of the two complex activities showed any significant association with baseline disease demographics or any clinico-pathological feature. Furthermore, the complex II and V activity did not show any impact on event free survival (P = 0.25 and P = 0.24 respectively) and overall survival (P = 0.14 and P = 0.17 respectively) in our cohort. CONCLUSION: The activity of both mitochondrial complex II and V are significantly elevated in bone marrow mononuclear cells of children with AML compared to controls. The enhanced activity may be related to upregulation of mitochondrial biogenesis genes TFAM and POLG. The enhanced activity of either of the complexes did not impact disease biology or survival outcomes in pediatric AML.

PGC1A driven enhanced mitochondrial DNA copy number predicts outcome in pediatric acute myeloid leukemia.

Mitochondrial DNA (mtDNA) copy number alterations occur in acute myeloid leukemia (AML). We evaluated regulation and biological significance of mtDNA copy number in pediatric AML patients (n = 123) by qRT-PCR, and in-vitro studies. MtDNA copy number was significantly higher (p < 0.001) and an independent predictor of aggressive disease (p = 0.006), lower event free survival (p = 0.033), and overall survival (p = 0.007). Expression of TFAM, POLG, POLRMT, MYC and ND3 were significantly upregulated. In cell lines, PGC1A inhibition decreased mtDNA copy number while MYC inhibition had no effect. PGC1A may contribute to enhanced mtDNA copy number, which predicts disease aggressiveness and inferior survival outcome.

Comparative metabolic profiles of total and partial body radiation exposure in mice using an untargeted metabolomics approach.

INTRODUCTION: A large scale population exposure to ionizing radiation during intentional or unintentional nuclear accidents undoubtedly generates a complex scenario with partial-body as well as total-body irradiated victims. A high throughput technique based rapid assessment method is an urgent necessity for stratification of exposed subjects independent of whether exposure is uniform total-body or non-homogenous partial-body. OBJECTIVE: Here, we used Nuclear Magnetic Resonance (NMR) based metabolomics approach to compare and identify candidate metabolites differentially expressed in total and partially irradiated mice model. METHODS: C57BL/6 male mice (8-10 weeks) were irradiated total-body or locally to thoracic, hind limb or abdominal regions with 10 Gy of gamma radiation. Urine samples collected at 24 h post irradiation were examined using high resolution NMR spectroscopy and the datasets were analysed using multivariate analysis. RESULTS: Multivariate and metabolic pathway analysis in urine samples collected at 24 h post-radiation exhibited segregation of all irradiated groups from controls. Metabolites associated with energy metabolism, gut flora metabolism and taurine were common to partial and total-body irradiation, thus making them potential candidates for radiation exposure. Nevertheless, a distinct metabolic pattern was observed in partial-body exposed groups with maximum changes observed in the hind limb region indicating differential tissue associated radiation sensitivity. The organ-specific changes may provide an early warning regarding the physiological system at risk after radiation injury. CONCLUSION: The study affirms potentiality of metabolite markers and comparative analysis could be an important piece of information for an integrated solution to a complex research question in terms of radiation biomarkers.

Genetic Landscape of Mitochondrial Regulatory Region in Pediatric Acute Myeloid Leukemia: Changes from Diagnosis to Relapse.

This prospective study aimed to compare the pattern of mitochondrial deoxyribonucleic acid D-loop (mt-DNA D-loop) variations in 41 paired samples of de novo pediatric acute myeloid leukemia (AML) (baseline vs. relapse) patients by Sanger's sequencing. Mean mt-DNA D-loop variation was 10.1 at baseline as compared with 9.4 per patients at relapse. In our study, 28 (68.3%) patients showed change in number of variations from baseline to relapse, 11 (26.8%) patients showed increase, 17 (41.6%) patients showed decrease, and 7 (17.1%) patients who suffered a relapse had a gain at position T489C. No statistically significant difference was observed in the mutation profile of mt-DNA D-loop region from baseline to relapse in the evaluated population of pediatric AML.

Expression of mitochondrial genes predicts survival in pediatric acute myeloid leukemia.

Deregulated mitochondrial metabolism and biogenesis have been studied in acute myeloid leukemia (AML); yet, the relevance of mitochondrial-encoded gene expression on AML outcomes is unknown. This study was conducted to assess clinical significance of expression of mitochondrial-encoded genes, namely ND3, SDHB, Cytochrome b, Cytochrome C, and ATP6, in pediatric AML. Pediatric AML patients from July 2013 to June 2016 were enrolled in this prospective study. Relative genes expression was determined using real-time PCR, and expressed as fold change. 123 AML patients were enrolled, median age 10 (range 0.7-18 years). ND3 gene expression was significantly increased in poor-risk cytogenetics (P = 0.03). In univariate analysis, high ND3 and ATP6 gene expression was significantly associated with inferior EFS (P = 0.01 and P = 0.04, respectively) and OS (P = 0.02 and P = 0.01, respectively), whereas, in multivariate analysis, ND3 gene expression emerged as the only independent prognostic factor for EFS and OS (P = 0.04 and P = 0.03). ND3 gene expression is a significant predictor of EFS and OS in pediatric AML, and should be evaluated as a potential biomarker.

Prognostic impact of mitochondrial DNA D-loop variations in pediatric acute myeloid leukemia.

The role of mitochondrial DNA (mt-DNA) changes, especially those in the regulatory D-loop region in Acute Myeloid Leukemia (AML) remains investigational. Consecutive 151 de novo pediatric AML patients, (≤18 yr) were prospectively enrolled from June 2013-August 2016, to assess the prognostic impact of mt-DNA D-loop variations (somatic/germline) on survival. For each patient, D-loop region was sequenced on baseline bone marrow and buccal swab, and mother's blood sample. In 151 AML subjects, 1490 variations were found at 237 positions; 80.9% were germline and 19.1% somatic. The mean number of variations per position was 6.3. Variations with frequency ≥6 were analyzed for their impact on survival and 4 categories were created, namely "somatic-protective", "somatic-hazardous", "germline-protective" and "germline- hazardous". Although, somatic-protective could not predict event free survival (EFS) or overall survival (OS), somatic-hazardous [(OS) HR = 2.33, p = 0.06] and germline-hazardous [(OS) HR = 2.85, p < 0.01] significantly predicted OS and EFS. Notably, the germline-protective, could significantly predict EFS (HR = 0.31, p = 0.03) and OS (HR = 0.19, p < 0.01), only when variations at ≥2 positions were present. On multivariate analysis, three positions namely 16111, 16126, 16362 and karyotype were found to be predictive of EFS. A prognostic index (PI) was developed using nomogram PI = (0.8*karyotype) + (1.0*c16111) + (0.7*t16362) + (1.2*t16126). Hazard ratio for EFS increased significantly with increasing PI reaching to a maximum of 3.3 (p < 0.01). In conclusion, the impact of mt-DNA D-loop variations on outcomes in pediatric AML depends on their nature (germline/somatic), position and mutational burden, highlighting their potential role as evolving prognostic biomarkers.

Apoptosis: A biomarker of high-risk phenotype in pediatric acute myeloid leukemia?

INTRODUCTION: Dysregulation of apoptosis has been explored in acute myeloid leukemia (AML); yet, its correlation with clinical outcomes in pediatric AML is unknown. This study was aimed to analyze percentage of apoptosis and apoptosis mediated through the intrinsic pathway with clinical outcomes in patients with pediatric AML. METHODS: This prospective study included pediatric AML patients enrolled from July 2013 to August 2016. Annexin-V (marker of total apoptosis) and caspase-9 expression (marker of intrinsic pathway) was determined in baseline bone marrow (BM) samples by flow cytometery and compared with controls (unaffected BM of solid tumors and peripheral blood [PB] of unaffected siblings). Overall survival (OS) and event-free survival (EFS) were compared using log-rank test. RESULTS: A total of 151 AML patients were enrolled, median age 10 (range: 0.7-18 years). Annexin-V expression in blast cells was significantly high in AML patients as compared to BM of subjects with solid tumors (P = 0.01) and PB of healthy subjects (P = 0.04). Caspase-9 expression in blast cells was not significantly different. Median annexin-V expression was significantly higher in patients with WBC count ≥11 000/mm3 (P = 0.02), poor-risk cytogenetics (P = 0.02), the absence of RUNX1-RUNX1T1 translocation (P = 0.004), and the absence of NPM1 mutation (P = 0.05). Patients with high annexin-V expression had significantly inferior OS (P = 0.05) in univariate analysis but not in multivariate analysis (P = 0.32). CONCLUSION: Apoptosis as a whole was found to be activated in baseline BM samples of AML patients. High apoptosis may be associated with high-risk phenotype in this disease.

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 mother's 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.

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.

High receptor tyrosine kinase (FLT3, KIT) transcript versus anti-apoptotic (BCL2) transcript ratio independently predicts inferior outcome in pediatric acute myeloid leukemia.

OBJECTIVE: In acute myeloid leukemia (AML), simultaneous expression of proliferative (FLT3, KIT) and anti-apoptotic genes (BCL2) is unknown. The aim of the study was to prospectively evaluate proliferative and anti-apoptotic gene transcripts, their interrelationship and impact on the outcome in pediatric AML patients. METHODS: We assessed proliferative and anti-apoptotic gene transcripts by Q-polymerase chain reaction (TaqMan probe) in 64 consecutive pediatric AML patients. Survival data was analyzed by Kaplan-Meier curves followed by log rank test to compare statistical significance between groups. Stepwise multivariable Cox regression method was used to evaluate independent prognostic factors. RESULTS: In univariate analysis, transcript ratio of FLT3/BCL2 and FLT3+KIT/BCL2 significantly predicted event free survival (EFS) (<0.01 and <0.01 respectively) and overall survival (OS) (<0.01 and<0.01 respectively). In stepwise Cox-regression model, high white blood cell count and high FLT3+KIT/BCL2 ratio predicted EFS (HR: 2.2 and 2.3); high hemoglobin and high FLT3+KIT/BCL2 ratio predicted OS (HR: 0.45 and 3.85). Prognostic index (PI) was calculated using the hazard coefficient of independent prognostic factors; at 57.3 months, predicted OS of patients with the highest PI of 1.8 was 8% versus 73% for the lowest PI of -0.3. The mean PI of patients who died was 1.8±0.72 versus 0.54±0.70 for those who are alive, P=0.004. CONCLUSIONS: This first study showed that individual expression of proliferative and anti-apoptotic transcripts is not as important in AML patients, rather their interrelationship and relative level probably determines the 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.

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

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.

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).

Increased coexpression of c-KIT and FLT3 receptors on myeloblasts: independent predictor of poor outcome in pediatric acute myeloid leukemia.

BACKGROUND: 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. METHODS: Using flow-cytometry, we prospectively observed in 115 AML patients that CD135, CD117, and CD135+CD117 coexpression was expressed in 95 (82%), 104 (90%), and 81 (70%) patients respectively. Median expression of CD135, CD117, and their co expression was used as cut off for high and low expression. RESULTS: FLT3 ITD (internal tandem duplication) was present in 20 (17%) patients. High coexpression did not correlate with FLT3 ITD (P = 0.432) and cytogenetics (P = 0.244). Out of 115 patients, 86 (74.7%) achieved remission. At median followup of 15.5 months, EFS and OS was 29% and 35%, respectively for the entire cohort. Patients with high coexpression of CD135 and CD117 in comparison to those with low coexpression had significantly inferior EFS (20% vs 38% P < 0.001) and OS (27% vs 44% P = 0.001). In step wise Cox regression multivariable analysis, hazard ratio for high hemoglobin, WBC count, and coexpression of CD135 and CD117 was 0.63, 1.73, and 2.46 respectively for EFS, and for OS only CD135+CD117 coexpression emerged as an independent predictor (hazard ratio 2.25). CONCLUSIONS: This is the first study to show that high coexpression of CD135+CD117 is an independent predictor of poor outcome in AML and is easily measurable by routine diagnostic flow-cytometry.

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/mm(3) 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.