Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Granulocyte Colony-Stimulating Factor/administration & dosage , Hematopoietic Stem Cell Mobilization , Heterocyclic Compounds/administration & dosage , Leukemia, Myeloid, Acute/therapy , Stem Cell Transplantation , Adolescent , Adult , Aged , Autografts , Benzylamines , Cyclams , Cytarabine/administration & dosage , Etoposide/administration & dosage , Female , Humans , Male , Middle Aged , Mitoxantrone/administration & dosageABSTRACT
Traditional response criteria in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are based on bone marrow morphology and may not accurately reflect clonal tumor burden in patients treated with non-cytotoxic chemotherapy. We used next-generation sequencing of serial bone marrow samples to monitor MDS and AML tumor burden during treatment with epigenetic therapy (decitabine and panobinostat). Serial bone marrow samples (and skin as a source of normal DNA) from 25 MDS and AML patients were sequenced (exome or 285 gene panel). We observed that responders, including those in complete remission (CR), can have persistent measurable tumor burden (that is, mutations) for at least 1 year without disease progression. Using an ultrasensitive sequencing approach, we detected extremely rare mutations (equivalent to 1 heterozygous mutant cell in 2000 non-mutant cells) months to years before their expansion at disease relapse. While patients can live with persistent clonal hematopoiesis in a CR or stable disease, ultimately we find evidence that expansion of a rare subclone occurs at relapse or progression. Here we demonstrate that sequencing of serial samples provides an alternative measure of tumor burden in MDS or AML patients and augments traditional response criteria that rely on bone marrow blast percentage.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Clonal Evolution/genetics , Epigenesis, Genetic/drug effects , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Myelodysplastic Syndromes/drug therapy , Myelodysplastic Syndromes/genetics , Aged , Aged, 80 and over , Bone Marrow/pathology , Exome , Female , Genes, p53 , High-Throughput Nucleotide Sequencing , Histone Deacetylase Inhibitors/administration & dosage , Humans , Leukemia, Myeloid, Acute/diagnosis , Male , Middle Aged , Mutation , Myelodysplastic Syndromes/diagnosis , Polymorphism, Single Nucleotide , Remission Induction , Treatment Outcome , Tumor BurdenABSTRACT
The complex chromosomal aberrations found in therapy-related acute myeloid leukemia (t-AML) suggest that the DNA double-strand break (DSB) response may be altered. In this study we examined the DNA DSB response of primary bone marrow cells from t-AML patients and performed next-generation sequencing of 37 canonical homologous recombination (HR) and non-homologous end-joining (NHEJ) DNA repair genes, and a subset of DNA damage response genes using tumor and paired normal DNA obtained from t-AML patients. Our results suggest that the majority of t-AML patients (11 of 15) have tumor-cell intrinsic, functional dysregulation of their DSB response. Distinct patterns of abnormal DNA damage response in myeloblasts correlated with acquired genetic alterations in TP53 and the presence of inferred chromothripsis. Furthermore, the presence of trisomy 8 in tumor cells was associated with persistently elevated levels of DSBs. Although tumor-acquired point mutations or small indels in canonical HR and NHEJ genes do not appear to be a dominant means by which t-AML leukemogenesis occurs, our functional studies suggest that an abnormal response to DNA damage is a common finding in t-AML.
Subject(s)
Biomarkers, Tumor/genetics , Bone Marrow/pathology , DNA Breaks, Double-Stranded , DNA Damage/genetics , Granulocyte Precursor Cells/pathology , Leukemia, Myeloid, Acute/pathology , Leukemia, Myeloid, Acute/therapy , Animals , Chromosomes, Human, Pair 8/genetics , Comet Assay , Comparative Genomic Hybridization , DNA Repair/genetics , Flow Cytometry , Gene Expression Profiling , Histones/metabolism , Humans , Leukemia, Myeloid, Acute/genetics , Mice , Mice, Inbred C57BL , Mutation/genetics , Oligonucleotide Array Sequence Analysis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Suppressor Protein p53/geneticsABSTRACT
The fiber optic biosensor performs fluoroimmunoassays at the surface of multimode optical fibers. The effectiveness of protein A, an immunoglobulin binding protein, for antibody immobilization on the surface of these fiber probes has been investigated. No difference was observed in the binding of fluorescently-labeled goat-IgG by rabbit anti-goat IgG regardless of whether the capture antibody was bound to the probe surface via protein A or covalently attached. However, in a sandwich immunoassay for the F1 antigen of Yersinia pestis, probes with rabbit anti-plague IgG bound to the surface via protein A generated twice the signal as probes with the antibody covalently attached. Assay regeneration was also examined with protein A probes since antibody-antigen complexes have been successfully eluted from protein A under low pH conditions. Protein A probes coated with rabbit anti-goat IgG obtained nearly identical signal levels at 500 and 5000 ng/ml of Cy5.5 goat IgG five consecutive times following regeneration with glycine-HCl, 2% acetic acid, pH 2.5.
