Evidence supporting a role for the immune checkpoint protein B7-H3 in NK cell-mediated cytotoxicity against AML.

We observed that the immune checkpoint protein B7-H3 is overexpressed in acute myeloid leukemia (AML) patients with poor treatment outcomes. Inhibition of B7-H3 expression or blocking of its activity using a novel monoclonal antibody (T-1A5) in AML cells significantly enhanced natural killer (NK) cell-mediated cytotoxicity in AML cells in vitro and in vivo. Moreover, a human-mouse chimera of this antibody (ChT-1A5) induced antibody-dependent cell-mediated cytotoxicity (ADCC) in B7-H3+ primary AML cells, but not in normal hematopoietic cells, suggesting the specify of this antibody for AML cells. Epitope mapping studies identified that both T-1A5 and ChT-1A5 antibodies bind to the FG-loop region of B7-H3, which is known to regulate the immunosuppressive function of B7-H3. Furthermore, treatment with ChT-1A5 in combination with human NK cells significantly prolonged survival in AML patient-derived xenograft (PDX) models. Our results suggest that the ChT-1A5 antibody can inhibit the immunosuppressive function of B7-H3 protein as well as induce ADCC in B7-H3+ AML.

TP-0184 inhibits FLT3/ACVR1 to overcome FLT3 inhibitor resistance and hinder AML growth synergistically with venetoclax.

We identified activin A receptor type I (ACVR1), a member of the TGF-ß superfamily, as a factor favoring acute myeloid leukemia (AML) growth and a new potential therapeutic target. ACVR1 is overexpressed in FLT3-mutated AML and inhibition of ACVR1 expression sensitized AML cells to FLT3 inhibitors. We developed a novel ACVR1 inhibitor, TP-0184, which selectively caused growth arrest in FLT3-mutated AML cell lines. Molecular docking and in vitro kinase assays revealed that TP-0184 binds to both ACVR1 and FLT3 with high affinity and inhibits FLT3/ACVR1 downstream signaling. Treatment with TP-0184 or in combination with BCL2 inhibitor, venetoclax dramatically inhibited leukemia growth in FLT3-mutated AML cell lines and patient-derived xenograft models in a dose-dependent manner. These findings suggest that ACVR1 is a novel biomarker and plays a role in AML resistance to FLT3 inhibitors and that FLT3/ACVR1 dual inhibitor TP-0184 is a novel potential therapeutic tool for AML with FLT3 mutations.

Evaluation of Vascular Endothelial Growth Factor (VEGF) and Thrombospondin-1 as Biomarkers of Metronomic Chemotherapy in Progressive Pediatric Solid Malignancies.

OBJECTIVE: We compared Vascular Endothelial Growth factor (VEGF) and Thrombospondin-1 between patients with progressive paediatric malignancies randomized to metronomic chemotherapy versus placebo to determine their role as biomarker. METHODS: In this double-blinded, placebo-controlled randomized study of 108 progressive pediatric malignancies, serum VEGF and thrombospondin-1 levels were evaluated using ELISA at baseline, A2 (week-9 or earlier if progressed) and A3 (week-18 or earlier if progressed). RESULTS: Mean VEGF and thrombospondin-1 at baseline, A2 and A3 and the change from baseline to A2 were not different between two groups. In metronomic arm, responders (those completing 3 cycles) had significantly lower mean (SD) baseline VEGF levels [659.7(362.1) vs 1143.9 (622.0) µg/mL] (P=0.002) and significant decrease in thrombospondin-1 from baseline to A2 [-4.43(8.0) µg/mL vs 1.7(11.3) µg/mL] (P=0.04), as compared to non-responders. Similar changes were not observed in responders on placebo arm. No consistent trend of these biomarkers was observed. CONCLUSIONS: VEGF and thrombospondin-1 are not reliable biomarkers for response to metronomic chemotherapy.

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.

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.

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.

Cytogenetic Profiles of 472 Indian Children with Acute Myeloid Leukemia.

OBJECTIVE: To analyze the cytogenetic abnormalities of a large cohort of consecutive pediatric Acute Myeloid Leukemia (AML) patients, treated on a uniform protocol. DESIGN: Review of case records. SETTING: Pediatric Cancer Center of tertiary care hospital between June 2003 and June 2016. PARTICIPANTS: 617 consecutive de novo pediatric AML patients were screened and 472 patients were found eligible. Eligibility criteria included non M3 patients, successful cytogenetic profile and availability of complete records. MAIN OUTCOME MEASURE: Cytogenetic profile. RESULTS: Gum-hypertropy, chloromas and rate of complete remission were significantly different between European Leukemia Network classification (ELN) cytogenetic risk groups (P<0.01). t (8;21) (141, 29.8%), loss of Y chromosome (61,12.9%) and trisomy 8 (39, 8.3%) were the most common abnormalities. Among the chromosomal gains, trisomy 8 and trisomy 21 (both P<0.01) were significantly different among the three ELN risk groups. Among the chromosome losses, monosomy 5, 7 (both P<0.01) and 9 (P=0.03), loss of X and loss of Y (both P<0.01) were statistically different amongst three cytogenetic risk groups. Event-free survival (P<0.01) and overall survival (P<0.01) were found to be significantly different among the three risk groups. CONCLUSIONS: The higher frequency of t (8; 21) and its association with chloroma in Indian pediatric patients is different from other studies around the world.

Myeloid Sarcoma Predicts Superior Outcome in Pediatric AML; Can Cytogenetics Solve the Puzzle?

BACKGROUND: The purpose of our study was to evaluate the clinical, cytogenetic, and molecular features, and survival outcomes in patients with acute myeloid leukemia (AML) with myeloid sarcoma (MS) and compare them with patients with AML without MS. PATIENTS AND METHODS: This was a retrospective analysis of de novo pediatric AML patients with or without MS diagnosed at our cancer center between June 2003 and June 2016. RESULTS: MS was present in 121 of 570 (21.2%), the most frequent site being the orbit. Patients with MS had a younger median age (6 years vs. 10 years) and presented with higher hemoglobin and platelet but lower white blood cell count compared with patients without MS. Further, t (8; 21) (P < .01), loss of Y chromosome (P < .01), and deletion 9q (P = .03) were significantly higher in patients with AML with MS. Event-free survival (EFS; P = .003) and overall survival (OS; P = .001) were better among patients with AML with MS (median EFS 21.0 months and median OS 37.1 months) compared with those with AML without MS (median EFS 11.2 months and median OS 16.2 months). The t (8; 21) was significantly associated with MS (odds ratio, 3.92). In a comparison of the 4 groups divided according to the presence or absence of MS and t (8; 21), the subgroup of patients having MS without concomitant t (8; 21) was the only group to have a significantly better OS (hazard ratio, 0.53; 95% confidence interval, 0.34-0.82; P = .005). CONCLUSION: Although t (8; 21) was more frequently associated with MS, it did not appear to be the reason for better outcome.

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.