Cytogenetics on released DNA fibers.

DNA fibers can be released from cell suspensions and frozen tissue and can be used as a template for hybridization with multiple differently labeled probes. Simultaneous hybridization with 5-10 adjacent or partly overlapping probes generates a highly specific "color barcode" for individual DNA segments. Rearrangements in this barcode can be easily detected and mapped. The resolution of DNA fiber FISH is between 2 and 500kb. In mixing experiments of cell lines with different structural abnormalities, we found a sensitivity of approximately 10%. We applied DNA fiber FISH for detection of t(11;14) in mantle cell lymphoma (MCL) and immunoglobulin (Ig) class switching in hairy cell leukemia (HCL). Using a barcode for the Ig and BCL-1 loci at 14q32 and 11q13, we detected and mapped a t(11;14) breakpoint in 35 of 36 MCL. In 5 cases complex mono-allelic rearrangements at both sides of the cyclin D1 gene were identified. In 13 HCL with phenotypic evidence of Ig class switching, including 2 cases with solely IgD, fiber FISH revealed concordant Ig class switch deletions. In most cases both alleles were affected. These results indicate that DNA fiber FISH is a very powerful method to detect and map structural DNA alterations.

Statistical methods in interphase cytogenetics: an experimental approach.

In situ hybridization (ISH) techniques on interphase cells, or interphase cytogenetics, have powerful potential clinical and biological applications, such as detection of minimal residual disease, early relapse, and the study of clonal evolution and expansion in neoplasia. Much attention has been paid to issues related to ISH data acquisition, i.e., the numbers, colors, intensities, and spatial relationships of hybridization signals. The methodology concerning data analysis, which is of prime importance for clinical applications, however, is less well investigated. We have studied the latter for the detection of small monosomic and trisomic cell populations using various mixtures of human female and male cells. With a chromosome X specific probe, the male cells stimulated monosomic subpopulations of 0, 1, 5, 10, 50, 90, 95, 99, and 100%. Analogously, when a (7 + Y) specific probe combination was used, containing a mixture of chromosome No. 7 and Y-specific DNA, the male cells simulated trisomic cell populations. Probes specific for chromosomes Nos. 1, 7, 8, and 9 were used for estimation of ISH artifacts. Three statistical tests, the Kolmogorov-Smirnov test, the multiple-proportion test, and the z'-max test, were applied to the empirical data using the control data as a reference for ISH artifacts. The Kolmogorov-Smirnov test was found to be inferior for discrimination of small monosomic or trisomic cell populations. The other two tests showed that when 400 cells were evaluated, and using selected control probes, monosomy X could be detected at a frequency of 5% aberrant cells, and trisomy 7 + Y at a frequency of 1%.(ABSTRACT TRUNCATED AT 250 WORDS)

Mosaicism of trisomy 12 in chronic lymphocytic leukemia detected by non-radioactive in situ hybridization.

Cytogenetic analysis using banding techniques of B-chronic lymphocytic leukemia (CLL) is hampered by the difficult in vitro proliferation of these tumor cells. For detection of specific cytogenetic aberrations these problems can be overcome with non-radioactive in situ hybridization (ISH). ISH may especially be applied for the detection of trisomy 12, which is the most frequent cytogenetic aberration in CLL. Sixty-seven patients with CLL, four normal controls and one lymphoblastoid B-cell line with a trisomy 12 were studied using a chromosome 12 specific probe. To determine the hybridization properties of the CLL cells, all samples were also hybridized with probes specific for chromosomes 1 and 8. All leukemias were analyzed by immunocytochemistry to determine the proportion of tumor cells. Eight cases (11%) showed a trisomy 12. After correction for the number of tumor cells, it was demonstrated that in almost all cases (7 out of 8), the aberration was present in a proportion of the tumor cells (between 30 and 72%). Except for one patient this mosaicism persisted with long-term follow-up. We conclude that the in vivo incidence of trisomy 12 in CLL is approximately 11%, and that trisomy 12 occurs in most instances in only a subpopulation of the leukemic cells. Both findings suggest that trisomy 12 in CLL is a late event.

Acquired aplastic anemia in adults. IV. Histological and CFU studies in transplanted and non-transplanted patients.

A follow-up study of 10 patients suffering from acquired aplastic anaemia, comprising methocrylate-embedded bone marrow biopsies and CFU cultures, is presented. The haematopoietic recovery patterns and changes in the inflammatory infiltration after permanent engraftment could be distinguished from those in non-transplanted patients. After anti-thymocyte globulin treatment followed by allogeneic bone marrow infusion, the recovery pattern resembled that in non-transplanted patients. The persistently low colony-forming capacity in some patients could be explained by the existence of lymphoid inhibitory cells, which suggests an immunologic auto-destructive mechanism.