Biochemical analysis of H2O2-induced mutation spectra revealed that multiple damages were involved in the mutational process.

Reactive oxygen species (ROS) are a major threat to genomic integrity and believed to be one of the etiologies of cancers. Here we developed a cell-free system to analyze ROS-induced mutagenesis, in which DNA was exposed to H2O2 and then subjected to translesion DNA synthesis by various DNA polymerases. Then, frequencies of mutations on the DNA products were determined by using next-generation sequencing technology. The majority of observed mutations were either C>A or G>A, caused by dAMP insertion at G and C residues, respectively. These mutations showed similar spectra to COSMIC cancer mutational signature 18 and 36, which are proposed to be caused by ROS. The in vitro mutations can be produced by replicative DNA polymerases (yeast DNA polymerase δ and ε), suggesting that ordinary DNA replication is sufficient to produce them. Very little G>A mutation was observed immediately after exposure to H2O2, but the frequency was increased during the 24 h after the ROS was removed, indicating that the initial oxidation product of cytosine needs to be maturated into a mutagenic lesion. Glycosylase-sensitivities of these mutations suggest that the C>A were made on 8-oxoguanine or Fapy-guanine, and that G>A were most likely made on 5-hydroxycytosine modification.

Biochemical and photochemical mechanisms that produce different UV-induced mutation spectra.

Although UV-induced mutagenesis has been studied extensively, the precise mechanisms that convert UV-induced DNA damage into mutations remain elusive. One well-studied mechanism involves DNA polymerase (Pol) η and ζ, which produces C > T transitions during translesion synthesis (TLS) across pyrimidine dimers. We previously proposed another biochemical mechanism that involves multiple UV-irradiations with incubation in the dark in between. The incubation facilitates spontaneous deamination of cytosine in a pyrimidine dimer, and the subsequent UV irradiation induces photolyase-independent (direct) photoreversal that converts cytosine into monomeric uracil residue. In this paper, we first demonstrate that natural sunlight can induce both mutational processes in vitro. The direct photoreversal was also reproduced by monochromatic UVB at 300 nm. We also demonstrate that post-irradiation incubation in the dark is required for both mutational processes, suggesting that cytosine deamination is required for both the Pol η/ζ-dependent and the photoreversal-dependent mechanisms. Another Y-family polymerase Pol ι also mediated a mutagenic TLS on UV-damaged templates when combined with Pol ζ. The Pol ι-dependent mutations were largely independent of post-irradiation incubation, indicating that cytosine deamination was not essential for this mutational process. Sunlight-exposure also induced C > A transversions which were likely caused by oxidation of guanine residues. Finally, we constructed in vitro mutation spectra in a comparable format to cancer mutation signatures. While both Pol η-dependent and photoreversal-dependent spectra showed high similarities to a cancer signature (SBS7a), Pol ι-dependent mutation spectrum has distinct T > A/C substitutions, which are found in another cancer signature (SBS7d). The Pol ι-dependent T > A/C substitutions were resistant to T4 pyrimidine dimer glycosylase-treatment, suggesting that this mutational process is independent of cis-syn pyrimidine dimers. An updated model about multiple mechanisms of UV-induced mutagenesis is discussed.

Eleven-marker 10-color flow cytometric assessment of measurable residual disease for T-cell acute lymphoblastic leukemia using an approach of exclusion.

Measurable/minimal residual disease (MRD) status has been suggested as a powerful indicator of clinical-outcome in T-cell lymphoblastic leukemia/lymphoma (T-ALL). Multicolor flow cytometric (MFC)-based T-ALL MRD reports are limited and traditionally based on the utilization of markers-of-immaturity like TdT and CD99. Moreover, studies demonstrating the multicolor flow cytometric (MFC) approach for the assessment of T-ALL MRD are sparse. Herein, we describe an 11-marker, 10-color MFC-based T-ALL MRD method using an "approach of exclusion." METHODS: The study included 269 childhood T-ALL patients treated with a modified-MCP841 protocol. An 11-marker, 10-color MFC-based MRD was performed in bone marrow (BM) samples at the end-of-induction (EOI) and end-of-consolidation (EOC) time-points using Kaluza-version-1.3 software. RESULTS: We studied EOI-MRD in 269 and EOC-MRD in 105 childhood T-ALL patients. EOI-MRD was detectable in 125 (46.5%) samples (median, 0.3%; range, 0.0007-66.3%), and EOC-MRD was detectable in 34/105 (32.4%) samples (median, 0.055%; range, 0.0008-27.6%). Leukemia-associated immunophenotypes (LAIPs) found useful for MRD assessment were dual-negative CD4/CD8 (40.9%), dual-positive CD4/CD8 (23.3%) and only CD4 or CD8 expression (35.8%); dim/subset/dim-negative surface-CD3 (39%), dim/subset/dim-negative/negative CD5 (28.3%), dim/dim-negative/negative/heterogeneous CD45 (44.7%) and co-expression of CD5/CD56 (7.5%). EOI-MRD-positive status was found to be the most-relevant independent factor in the prediction of inferior relapse-free and overall survival. CONCLUSION: We described an 11-marker 10-color MFC-based highly sensitive MRD assay in T-ALL using an approach of exclusion. The addition of CD4 and CD8 to the pan-T-cell markers in a 10-color assay is highly useful in T-ALL MRD assessment and extends its applicability to almost all T-ALL patients.

Post-induction Measurable Residual Disease Using Multicolor Flow Cytometry Is Strongly Predictive of Inferior Clinical Outcome in the Real-Life Management of Childhood T-Cell Acute Lymphoblastic Leukemia: A Study of 256 Patients.

Background: Measurable/minimal residual disease (MRD) status is suggested as a powerful indicator of clinical-outcome in T-cell lymphoblastic leukemia/lymphoma (T-ALL). Contrary to B-cell ALL, reports on T-ALL MRD are limited and mostly based on molecular methods, mainly from developed countries. Multicolor flow cytometry (MFC)-based T-ALL studies are very few. Clinically relevant cut-off levels and ideal time-point for MRD assessment are still inconclusive. In view of lack of T-ALL MRD data from the developing world, we evaluated the prognostic value of MFC-based post-induction (PI)-MRD assessment in T-ALL in the context of standard practice. Methods: We included 256 childhood-T-ALL patients (age < 15 years) treated with a modified-MCP841 protocol, which uses high-dose cytarabine during consolidation, as a part of standard hospital practice. MRD was studied using 10-color 11-antibody MFC with any level of detectable disease being considered positive. Post-induction (PI)-MRD was available in all patients, and post-consolidation (PC) MRD was available mostly in PI-MRD-positive patients (n = 88). Results: Three years cumulative-incidence-of-relapse (3years-CIR) in PI-MRD-positive patients was inferior to negative patients (46.3% vs. 18.4%). The median relapse-free-survival (RFS), event-free-survival (EFS) and overall-survival (OS) with hazard ratio (HR) of PI-MRD-positive patients were 21.4 months vs not reached (p < 0.0001, HR-4.7), 21.6 months vs. not-reached (p = 0.0003, HR-2.01) and 37.3 months vs. not reached (p = 0.026, HR-1.64) respectively. RFS, EFS and OS of patients with PI-MRD<0.01% (n = 17) were as inferior as PI-MRD ≥ 0.01% in comparison with MRD-negative patients with HR of 4.7 (p < 0.0001), 2.45 (p = 0.0003), and 2.5 (p = 0.029), respectively. Three-years-CIR of patients with hyperleukocytosis (≥100 × 109/L) was also higher (50.5 vs. 27.6%) with inferior RFS, EFS, and OS. Among PI-MRD-positive patients, 3years-CIR, RFS, EFS, and OS of PC-MRD-positive were also inferior to that of negative patients. On multivariate analysis any-level detectable PI-MRD and hyperleukocytosis remained independently associated with inferior RFS, EFS, and OS. A combination of PI-MRD-positive status and hyperleukocytosis identified the patients with the worst clinical outcomes. Conclusion: Detectable PI-MRD using MFC was found to be the strong predictive factor of inferior clinical outcome in T-ALL patients. The combination of PI-MRD status and hyperleukocytosis provides the most influential tool for the management of T-ALL in resource constrained settings from developing world.