Detection of early precursors of t(12;21) positive pediatric acute lymphoblastic leukemia during follow-up.

DNA-, RNA-, and cell-based methods provide different biologic information for determining the presence of minimal residual disease (MRD). We monitored the responses of patients with pediatric acute lymphoblastic leukemia (pALL) using DNA markers, TEL/AML1 expression, and scanning fluorescence microscopy (SFM). Using SFM, 36% of patients exhibited 1.5-3.1 log and 2.9-4.2 log higher MRD levels compared with those based on DNA and RNA markers, respectively. CD10+ ancestor cells with germline antigen receptors, but silent TEL/AML1 expression, may reside in the lymphoid stem cell compartment of treated t(12;21)-positive patients and might act as a potential source of cells for late relapses.

Automated FISH analysis using dual-fusion and break-apart probes on paraffin-embedded tissue sections.

Detecting balanced translocations using tissue sections plays an important diagnostic role in cases of hematological malignancies. Manual scoring is often problematic due to truncation and overlapping of nuclei. Reports have described automated analysis using primarily tile sampling. The aim of this study was to investigate an automated fluorescent in situ hybridization analysis method using grid sampling on tissue sections, and compare the performance of dual-fusion (DF) and break-apart (BA) probes in this setting. Ten follicular, 10 mantle cell lymphoma, and 10 translocation-negative samples were used to set the threshold of false positivity using IGH/CCND1, IGH/BCL-2 DF, and IGH BA probes. The cut-off distances of red and green signals to define fusion signals were 0.5, 1.0, and 1.2 mum for the IGH/CCND1, IGH/BCL-2 DF, and IGH BA probes, respectively. The mean false positivity of grid units was 5.3, 11.4, and 28.1%, respectively. Ten to 14 additional samples analyzed blindly and were correctly classified using each probe. Discriminating positive and negative samples using automated analysis and grid sampling was possible with each probe, although different definitions of fusion signals were required due to the different physical distances between the DNA probes. Using the DF probes resulted in lower false positivity, which was less affected by signal numbers per grid units.

Automated detection of residual leukemic cells by consecutive immunolabeling for CD10 and fluorescence in situ hybridization for ETV6/RUNX1 rearrangement in childhood acute lymphoblastic leukemia.

Among the various methods available for analyzing minimal residual disease, a new procedure for the cell-based approaches using consecutive phenotypic and genotypic analysis as revealed by immunofluorescent labeling and subsequent fluorescent in situ hybridization (FISH) has been developed. We are introducing a fluorescent microscopy-based technique by which not only cellular targets and immunological marker positivity, but also the FISH pattern was identified by automated scanning. For the latter one translocation-specific FISH pattern recognition was accomplished by using an automated scanning mode for the 3D determination of valid distances between FISH signals, to define the cutoff value for the shortest green-red spot distance differentiating positive cells from negative ones. The procedure was tested with CD10(+) acute lymphoblastic leukemia cell line harboring the t(12;21)(p13;q22) resulting in the ETV6/RUNX1 rearrangement (formerly TEL/AML1), as well as peripheral blood lymphocytes of healthy individuals. Using the combined, automated method, a sensitivity of 98.67% and a specificity of 99.97% were obtained. The mean false positivity + 2 standard deviations cutoff level (0.09%) allows detection of leukemic cells with high accuracy, even a bit below the tumor load dilution of 10(-3), a value reported to be critical in clinical decision making.

Synthesis and antibacterial activity of fused Mannich ketones.

New Mannich ketones of fused bicyclic ketones as 1-indanones and 1-tetralones were prepared using the classical acid-catalysed Mannich reaction. Known members of this family were used in comparative biological tests. Antibacterial activity of these new water-soluble compounds was reported against Pseudomonas aeruginosa, Escherichia coli, E. coli ReD31m4, Salmonella minnesota Re595, Shigella sonnei Re4350, Staphylococcus aureus, Staphylococcus saprophyticus, Micrococcus luteus and Bacillus subtilis standard strains. Human cytotoxicity of our new compounds was evaluated against HeLa cell line. Some compounds showed low cytotoxicity (56.738 nM mL(-1) for 24, 47.497 nM mL(-1) for 31 and 48.379 nM mL(-1) for 26) and proved to be efficient antibacterial agents against the Gram-positive and partly against E. coli strains. Minimum inhibitory concentrations (MIC) changed in the range of 1.56->200 microg mL(-1). The deep rough mutants showed (generally eight times) higher sensitivity toward the compounds than the smooth E. coli. Hence, the permeability of Gram-negative outer membrane can influence the MIC values of our compounds. A preliminary quantitative structure-activity relationship (QSAR) study indicated the maximum positive charge (MaxQ(+)) as the parameter that most significantly affected antibacterial activity against E. coli. In B. subtilis, the influence of a topological descriptor (first-order valence-connectivity index, XV1) was also revealed; however, other strains did not yield meaningful QSAR with the set of descriptors employed.

Crystal-storing histiocytosis associated with only one of two consecutive, but genetically unrelated B-cell lymphomas.

A localized crystal-storing histiocytosis (CSH) making the underlying marginal zone lymphoma (MZL) hardly discernible microscopically is described. Image analysis of the hyper electron dense crystals localized light microscopically in swollen histiocytic cells exhibited a major equatorial periodicity of 6.6 nm. Rarely, crystals of this type were detected within plasma cells, but were always surrounded by smooth membrane in contrast to Russell bodies. IgM/lambda restriction and VH3-21*02, DH4-17*01, JH4*02 gene usage were detected behind the lesion. Within 26 months, a genetically unrelated lymphoma of CD5-CD20-CD23-positive phenotype with a different VH1-24*01, DH2-21*02, JH2*01 heavy chain rearrangement, but with the same light chain gene usage, was identified without CSH. This might indicate that the unique condition responsible for the crystal formation is likely to rely on the sequence of the first clonally rearranged heavy chain exhibiting much higher CDRIII pI value (6.0) than the average.