Purpose Gemtuzumab ozogamicin (GO), a CD33-targeted immunoconjugate, is a re-emerging therapy for acute myeloid leukemia (AML). CD33 single nucleotide polymorphism rs12459419 C>T in the splice enhancer region regulates the expression of an alternatively spliced CD33 isoform lacking exon2 (D2-CD33), thus eliminating the CD33 IgV domain, which is the antibody-binding site for GO, as well as diagnostic immunophenotypic panels. We aimed to determine the impact of the genotype of this splicing polymorphism in patients with AML treated with GO-containing chemotherapy. Patients and Methods CD33 splicing single nucleotide polymorphism was evaluated in newly diagnosed patients with AML randomly assigned to receive standard five-course chemotherapy alone (No-GO arm, n = 408) or chemotherapy with the addition of two doses of GO once during induction and once during intensification (GO arm, n = 408) as per the Children's Oncology Group AAML0531 trial. Results The rs12459419 genotype was CC in 415 patients (51%), CT in 316 patients (39%), and TT in 85 patients (10%), with a minor allele frequency of 30%. The T allele was significantly associated with higher levels of D2-CD33 transcript ( P < 1.0E) and with lower diagnostic leukemic cell surface CD33 intensity ( P < 1.0E). Patients with the CC genotype had significantly lower relapse risk in the GO arm than in the No-GO arm (26% v 49%; P < .001). However, in patients with the CT or TT genotype, exposure to GO did not influence relapse risk (39% v 40%; P = .85). Disease-free survival was higher in patients with the CC genotype in the GO arm than in the No-GO arm (65% v 46%, respectively; P = .004), but this benefit of GO addition was not seen in patients with the CT or TT genotype. Conclusion Our results suggest that patients with the CC genotype for rs12459419 have a substantial response to GO, making this a potential biomarker for the selection of patients with a likelihood of significant response to GO.
• Earlier use of high-dose cytarabine during induction II therapy improves EFS for ML-DS patients.• MRD assessment by flow cytometry after induction I is a new prognostic variable for ML-DS.Patients with myeloid leukemia of Down syndrome (ML-DS) have favorable event-free survival (EFS), but experience significant treatment-related morbidity and mortality. ML-DS blast cells ex vivo have increased sensitivity to cytarabine (araC) and daunorubicin, suggesting that optimizing drug dosing may improve outcomes while reducing toxicity. The Children's Oncology Group (COG) AAML0431 trial consisted of 4 cycles of induction and 2 cycles of intensification therapy based on the treatment schema of the previous COG A2971 trial with several modifications. High-dose araC (HD-araC) was used in the second induction cycle instead of the intensification cycle, and 1 of 4 daunorubicincontaining induction cycles was eliminated. For 204 eligible patients, 5-year EFS was 89.9% and overall survival (OS) was 93.0%. The 5-year OS for 17 patients with refractory/ relapsed leukemia was 34.3%. We determined the clinical significance of minimal residual disease (MRD) levels as measured by flow cytometry on day 28 of induction I. MRD measurements, available for 146 of the 204 patients, were highly predictive of treatment outcome; 5-year disease-free survival for MRD-negative patients (n 5 125) was 92.7% vs 76.2% for MRD-positive patients (n 5 21) (log-rank P 5 .011). Our results indicated that earlier use of HD-araC led to better EFS and OS in AAML0431 than in past COG studies. A 25% reduction in the cumulative daunorubicin dose did not impact outcome. MRD, identified as a new prognostic factor for ML-DS patients, can be used for risk stratification in future clinical trials. This trial was registered at www.clinicaltrials.gov as #NCT00369317. (Blood. 2017;129(25):3304-3313)
Purpose The Children's Oncology Group AAML0631 trial for newly diagnosed pediatric acute promyelocytic leukemia (APL) was a phase III historically controlled trial to determine the survival of patients receiving arsenic trioxide (ATO) consolidation and reduced doses of anthracyclines. Patients and Methods Patients age 2 to 21 years with de novo APL confirmed by PML-RARα polymerase chain reaction were stratified as standard risk (SR) or high risk (HR) on the basis of diagnostic WBC count. All patients received all-trans retinoic acid (ATRA) during induction, each consolidation course, and maintenance. All patients received two cycles of ATO therapy during consolidation 1, an additional two (SR) or three (HR) consolidation courses that included high-dose cytarabine and anthracycline, and maintenance therapy comprising ATRA, oral methotrexate, and mercaptopurine. Results One hundred one patients (66 SR and 35 HR) were evaluable for outcome. The 3-year overall survival was 94%, and event-free survival (EFS) was 91%. For SR and HR patients with APL, the overall survival was 98% versus 86% ( P = .003), and EFS was 95% versus 83% ( P = .03), respectively. The EFS for SR patients in AAML0631 was noninferior to that of patients in the AIDA 0493 historical control, which used a significantly higher anthracycline dose and did not include ATO consolidation. Relapse risk for patients in AAML0631 from end consolidation 1 (after ATO treatment) was only 4% at 3 years and did not differ significantly between SR and HR patients. Conclusion ATO consolidation cycles were well tolerated in pediatric patients with APL and allowed significant reduction in cumulative anthracycline doses while maintaining excellent survival and a low relapse risk for both SR and HR patients with APL.
Purpose: KIT mutations (KIT þ) are common in core binding factor (CBF) AML and have been associated with varying prognostic significance. We sought to define the functional and clinical significance of distinct KIT mutations in CBF pediatric AML. Experimental Design: Following transfection of exon 17 (E17) and exon 8 (E8) mutations into HEK293 and Ba/F3 cells, KIT phosphorylation, cytokine-independent growth, and response to tyrosine kinase inhibitors (TKI) were evaluated. Clinical outcomes of patients treated on COG AAML0531 (NCT01407757), a phase III study of gemtuzumab ozogamicin (GO), were analyzed according to mutation status [KIT þ vs. wild-type KIT (KIT À)] and mutation location (E8 vs. E17). Results: KIT mutations were detected in 63 of 205 patients (31%); 22 (35%) involved only E8, 32 (51%) only E17, 6 (10%) both exons, and 3 (5%) alternative exons. Functional studies demonstrated that E17, but not E8, mutations result in aberrant KIT phosphorylation and growth. TKI exposure significantly affected growth of E17, but not E8, transfected cells. Patients with KIT þ CBF AML had overall survival similar to those with KIT À (78% vs. 81%, P ¼ 0.905) but higher relapse rates (RR ¼ 43% vs. 21%; P ¼ 0.005). E17 KIT þ outcomes were inferior to KIT À patients [disease-free survival (DFS), 51% vs. 73%, P ¼ 0.027; RR ¼ 21% vs. 46%, P ¼ 0.007)], although gemtuzumab ozogamicin abrogated this negative prognostic impact. E8 mutations lacked significant prognostic effect, and GO failed to significantly improve outcome. Conclusions: E17 mutations affect prognosis in CBF AML, as well as response to GO and TKIs; thus, clinical trials using both agents should be considered for KIT þ patients.
for subsequent relapses, we usually treat patients who have relapsed episodes of TTP with rituximab. The values and preferences of patients and physicians are essential for these treatment decisions.
Glycine-N methyltransferase (GNMT) is a potential tumor suppressor that is commonly inactivated in human hepatoma. We systematically investigated how GNMT regulates methyl group kinetics and global DNA methylation. HepG2 cells (GNMT inactive, GNMT-) and cells transfected with GNMT expressed vector (GNMT+) were cultured in low (10 μmol/L), adequate (100 μmol/L), or high (500 μmol/L) l-methionine, each with 2.27 μmol/L folate. Transmethylation kinetics were studied using stable isotopic tracers and GC-MS. Methylation status was determined by S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) levels, SAM:SAH ratio, DNA methyltransferase (DNMT) activity, and methylated cytidine levels in DNA. Compared with GNMT- cells, GNMT+ cells had lower homocysteine and greater cysteine concentrations. GNMT expression increased methionine clearance by inducing homocysteine transsulfuration and remethylation metabolic fluxes when cells were cultured in high or adequate l-methionine. In contrast, homocysteine remethylation flux was lower in GNMT+ cells than in GNMT- cells and homocysteine transsulfuration fluxes did not differ when cells were cultured in low methionine, suggesting that normal GNMT function helps to conserve methyl groups. Furthermore, GNMT expression decreased SAM and increased SAH levels and reduced DNMT activity in high or adequate, but not low, methionine cultures. In low methionine cultures, restoring GNMT in HepG2 cells did not lead to sarcosine synthesis, which would waste methyl groups. Methylated cytidine levels were significantly lower in GNMT- cells than in GNMT+ cells. In conclusion, we have shown that GNMT affects transmethylation kinetics and SAM synthesis and facilitates the conservation of methyl groups by limiting homocysteine remethylation fluxes.
Glycine N-methyltransferase (GNMT) is a major hepatic enzyme that converts S-adenosylmethionine to S-adenosylhomocysteine while generating sarcosine from glycine, hence it can regulate mediating methyl group availability in mammalian cells. GNMT is also a major hepatic folate binding protein that binds to, and, subsequently, may be inhibited by 5-methyltetrafolate. GNMT is commonly diminished in human hepatoma; yet its role in cellular folate metabolism, in tumorigenesis and antifolate therapies, is not understood completely. In the present study, we investigated the impacts of GNMT expression on cell growth, folate status, methylfolate-dependent reactions and antifolate cytotoxicity. GNMT-diminished hepatoma cell lines transfected with GNMT were cultured under folate abundance or restriction. Folate-dependent homocysteine remethylation fluxes were investigated using stable isotopic tracers and gas chromatography/mass spectrometry. Folate status was compared between wild-type (WT), GNMT transgenic (GNMT tg ) and GNMT knockout (GNMT ko ) mice. In the cell model, GNMT expression increased folate concentration, induced folate-dependent homocysteine remethylation, and reduced antifolate methotrexate cytotoxicity. In the mouse models, GNMT tg had increased hepatic folate significantly, whereas GNMT ko had reduced folate. Liver folate levels correlated well with GNMT expressions (r = 0.53, P = 0.002); and methionine synthase expression was reduced significantly in GNMT ko , demonstrating impaired methylfolate-dependent metabolism by GNMT deletion. In conclusion, we demonstrated novel findings that restoring GNMT assists methylfolate-dependent reactions and ameliorates the consequences of folate depletion. GNMT expression in vivo improves folate retention and bioavailability in the liver. Studies on how GNMT expression impacts the distribution of different folate cofactors and the regulation of specific folate dependent reactions are underway.
Glycine N-methyltransferase (GNMT) is a folate binding protein commonly diminished in human hepatoma yet its role in tumor development remains to be established. GNMT binds to methylfolate but is also inhibited by it; how such interactions affect human carcinogenesis is unclear. We postulated that GNMT plays a role in folate-dependent methyl group homeostasis and helps maintain genome integrity by promoting nucleotide biosynthesis and DNA repair. To test the hypothesis, GNMT was over-expressed in GNMT-null cell lines cultured in conditions of folate abundance or restriction. The partitioning of folate dependent 1-carbon groups was investigated using stable isotopic tracers and GC/MS. DNA damage was assessed as uracil content in cell models, as well as in Gnmt wildtype (Gnmt 1/1 ), heterozygote (Gnmt) and knockout (Gnmt 2/2 ) mice under folate deplete, replete, or supplementation conditions. Our study demonstrated that GMMT 1) supports methylene-folate dependent pyrimidine synthesis; 2) supports formylfolate dependent purine syntheses; 3) minimizes uracil incorporation into DNA when cells and animals were exposed to folate depletion; 4) translocates into nuclei during prolonged folate depletion.In conclusion, loss of GNMT impairs nucleotide biosynthesis. Over-expression of GNMT enhances nucleotide biosynthesis and improves DNA integrity by reducing uracil misincorporation in DNA both in vitro and in vivo. To our best knowledge, the role of GNMT in folate dependent 1-carbon transfer in nucleotide biosynthesis has never been investigated. The present study gives new insights into the underlying mechanism by which GNMT can participate in tumor prevention/suppression in humans.
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