Subject(s)
Chromosome Deletion , Chromosomes, Human, Pair 5/genetics , Leukemia, Myeloid, Acute/genetics , Myeloproliferative Disorders/genetics , Aged , Cytogenetic Analysis , Female , Humans , In Situ Hybridization, Fluorescence , Leukemia, Myeloid, Acute/pathology , Leukemia, Myeloid, Acute/surgery , Male , Middle Aged , Myeloproliferative Disorders/pathology , Myeloproliferative Disorders/surgery , Oligonucleotide Array Sequence Analysis , PrognosisABSTRACT
Development of highly sensitive quantitative assays for cytomegalovirus (CMV) DNA detection is crucial for identification of immunodeficient patients at high risk of CMV disease. We designed 2 internally controlled competitive quantitative assays, enzyme-linked immunosorbent assay (ELISA)-based and real-time polymerase chain reaction (PCR) tests, using amplification of the same segment of the CMV genome. The aim of this study was to compare sensitivity, specificity, and laboratory performance characteristics of these assays. In both assays, a 159-bp segment of UL83 gene was amplified. External and internal controls were constructed by cloning the amplification product and heterogenous DNA segment flanked by target sequences for CMV-derived primers into bacterial plasmids, respectively. Real-time PCR was performed on LightCycler (Roche Diagnostics, Mannheim, Germany), and amplicons were detected using fluorescence resonance energy transfer probes. Alternatively, PCR products were labeled by digoxigenin, hybridized to immobilized probes, and detected by ELISA. The assays were tested on genomic DNA isolated from laboratory strains of CMV, QCMD control panel, and CMV DNA-positive peripheral blood DNA samples from hematopoietic stem cell transplant recipients, previously characterized by pp65 antigenemia and qualitative nested PCR. Real-time and ELISA-based PCR assays showed a linear course of 1-10(8) and 10-10(5) copies of CMV DNA per reaction, respectively. When compared with ELISA-based PCR, real-time PCR showed superiority in inter- and intra-assay reproducibility. Both assays were highly specific in detecting CMV DNA. No difference in amplification efficiency of internal or external standards and wild-type CMV DNA was found. The assays exhibited 83% concordance in CMV DNA detection from clinical samples, all discrepant samples having low CMV DNA copy numbers. There was a good correlation between viral DNA loads measured by the 2 assays. Statistically significant correlation was observed between the numbers of CMV DNA copies and pp65-positive leukocytes in the samples tested. Both variants of competitive PCR are adequately sensitive to be used for CMV DNA quantitation in clinical samples. LightCycler PCR, having superior performance characteristics and being less time-consuming, seems to be more suitable for routine diagnosis.
Subject(s)
Cytomegalovirus Infections/diagnosis , Cytomegalovirus/isolation & purification , DNA, Viral/blood , Enzyme-Linked Immunosorbent Assay/methods , Hematopoietic Stem Cell Transplantation , Polymerase Chain Reaction/methods , Cytomegalovirus/genetics , Humans , Phosphoproteins/blood , Phosphoproteins/immunology , Polymerase Chain Reaction/instrumentation , Sensitivity and Specificity , Viral Matrix Proteins/blood , Viral Matrix Proteins/immunologyABSTRACT
Telomere length was evaluated by terminal repeat fragment method (TRF) in 50 patients with myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) arising from MDS and in 21 patients with untreated primary AML to ascertain, whether telomere erosion was associated with progression of MDS towards overt leukemia. Heterogeneity of TRF among MDS FAB subgroups (P=0.004) originated from its shortening in increased number of patients during progression of the disease. Chromosomal aberrations were present in 32% MDS patients with more eroded telomeres (P=0.022), nevertheless a difference between mean TRF in the subgroups with normal and abnormal karyotype diminished during progression of MDS. A negative correlation between individual TRF and IPSS value (P=0.039) showed that telomere dynamics might serve as a useful prognostic factor for assessment of an individual MDS patient's risk and for decision of an optimal treatment strategy.
Subject(s)
Biomarkers, Tumor/genetics , Genomic Instability , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/genetics , Myelodysplastic Syndromes/diagnosis , Myelodysplastic Syndromes/genetics , Telomere/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Child , Chromosome Aberrations , Disease Progression , Humans , Karyotyping , Middle Aged , Prognosis , Risk FactorsABSTRACT
The replication error (RER+) phenotype, characterized by microsatellite instability (MSI) has been recently related to mutations of genes involved in DNA mismatch repair pathway. These genetic alterations were first described in hereditary non polyposis colorectal cancer (HNPCC). We examined 44 patients with hematological malignancies (27 AML, 9 MDS, 2 CML-BP and 6 T-ALL) for evidence of MSI. Twenty seven percent of our patients showed differences for only one marker. In four cases (9.1%) MSI was observed in multiple markers and these cases were described as RER+ phenotype. Presented data suggest that this phenomenon may play a role in at least a subset of patients with hematological malignancies.
