Comparative FISH analysis of numerical chromosome 7 abnormalities in 5-μm and 15-μm paraffin-embedded tissue sections from prostatic carcinoma

 Interphase fluorescence in situ hybridization (FISH) was performed on 15-μm-thick paraffin sections from prostatic carcinomas using a chromosome 7-specific α-satellite DNA probe. A confocal laser scanning microscope (CLSM) was used for optical sectioning of the thick sections and reconstruction of 3D images. The number of FISH signals was determined by a gallery of optical sections evaluating only complete nuclei. To investiate the influence of section thickness and truncation and nuclei on scoring results, we compared the FISH data from 15-μm sections with signal counts obtained from 5-μm sections. The latter were evaluated by conventional fluorescence microscopy in the same tumor regions previously defined and marked on the slides. After statistical analysis of spot frequencies in tumor and non-tumorous cells (χ² test), we transferred the signal frequencies into a cytogenetic classification (−7, +7, polysomy 7). Based on this classification, most cases showed more than one chromosome 7 aberration type. Trisomy 7 (+7) became apparent in 15-μm-thick sections in all 19 tumors, polysomy 7 (>3 spots) in 18/19 cases, and monosomy 7 (−7) in 13/19 cases. In 5-μm sections, however, trisomy 7 and polysomy 7 were found in only 7/19 and 13/19 cases, respectively, and monosomy 7 in 7/19 cases. When comparing the classification results of tumor cells of the same tumor regions originating either from 5-μm or 15-μm sections, the following discrepancies were noted: in 15-μm sections exclusively, in 12/19 tumors, trisomy 7 was found; in 6/19 cases, polysomy 7; in 8/19 cases, monosomy 7. The high proportion of cases with tumor nuclei expressing only one hybridization signal of chromosome 7 in 15-μm sections could be confirmed as monosomy 7 in five selected cases by double-hybridization using centromere-specific probes for chromosomes 7 and 12. These results demonstrate that numerical chromosome 7 aberrations are more frequently observed in thick (15-μm) paraffin-embedded tissue sections by evaluating only complete nuclei. The use of routine sections (5-μm) for interphase cytogenetic analyses is compromised by a remarkable underestimation of the real chromosome copy numbers. Accepted: 7 November 1996     

Comparison of triple helical COMBO-FISH and standard FISH by means of quantitative microscopic image analysis of abl/bcr positions in cell nuclei

In this study, a novel DNA fluorescence labelling technique, called triple helical COMBO-FISH (Combinatorial Oligo Fluorescence In Situ Hybridisation), was compared to the standard FISH (Fluorescence In Situ Hybridisation by means of commercially available probe kits) by quantitative evaluation of the nuclear position of the hybridisation signals of the Abelson murine leukaemia (abl) region and the breakpoint cluster region (bcr) in 3D-conserved cell nuclei of lymphocytes and CML blood cells. Two sets of 31 homopyrimidine oligonucleotides each, corresponding to co-localising sequences in the abl region of chromosome 9 and in the bcr region of chromosome 22 were synthesised. Probe types and sizes (in bases) as well as the binding mechanisms of both FISH techniques were completely different. In accordance to established findings that cell type specific radial positioning of chromosomes and sub-chromosomal elements is evolutionarily conserved, no significant difference was found between the two FISH techniques for the radial localisation of the barycentre of the analysed genomic loci. Thermal denaturation and hypotonic treatment of cell nuclei subjected to standard FISH, however, led to different absolute radii and volumes of the cell nuclei, in comparison to the quantities determined for the triple helical COMBO-FISH technique; the chromatin appears to shrink in laterally enlarged, flat nuclei. Consequently, the absolute distances of the homologous labelled sites shifted to greater values. For precise quantitative microscopic analysis of genomic loci, fluorescence labelling procedures are recommended that well maintain the native chromatin topology. Triple helical COMBO-FISH may offer such an approach.     

Comparison of triple helical COMBO-FISH and standard FISH by means of quantitative microscopic image analysis of abl/bcr positions in cell nuclei

In this study, a novel DNA fluorescence labelling technique, called triple helical COMBO-FISH (Combinatorial Oligo Fluorescence In Situ Hybridisation), was compared to the standard FISH (Fluorescence In Situ Hybridisation by means of commercially available probe kits) by quantitative evaluation of the nuclear position of the hybridisation signals of the Abelson murine leukaemia (abl) region and the breakpoint cluster region (bcr) in 3D-conserved cell nuclei of lymphocytes and CML blood cells. Two sets of 31 homopyrimidine oligonucleotides each, corresponding to co-localising sequences in the abl region of chromosome 9 and in the bcr region of chromosome 22 were synthesised. Probe types and sizes (in bases) as well as the binding mechanisms of both FISH techniques were completely different. In accordance to established findings that cell type specific radial positioning of chromosomes and sub-chromosomal elements is evolutionarily conserved, no significant difference was found between the two FISH techniques for the radial localisation of the barycentre of the analysed genomic loci. Thermal denaturation and hypotonic treatment of cell nuclei subjected to standard FISH, however, led to different absolute radii and volumes of the cell nuclei, in comparison to the quantities determined for the triple helical COMBO-FISH technique; the chromatin appears to shrink in laterally enlarged, flat nuclei. Consequently, the absolute distances of the homologous labelled sites shifted to greater values. For precise quantitative microscopic analysis of genomic loci, fluorescence labelling procedures are recommended that well maintain the native chromatin topology. Triple helical COMBO-FISH may offer such an approach.     

High-level detection of gene amplification and chromosome aneuploidy in extracted nuclei from paraffin-embedded tissue of human cancer using FISH: a new approach for retrospective studies

A novel application of fluorescence in situ hybridization (FISH) to isolated nuclei is described. The method detects gene amplification and chromosome aneuploidy in extracted nuclei from paraffin-embedded tissue of human cancer with greater sensitivity and specificity than existing FISH methods. In this study, the method is applied to signal detection of the HER-2/neu (c-erbB-2) gene, whose amplification is one of the most common genetic alterations associated with human breast cancer. Nuclei were extracted and isolated from formalin fixed, paraffin embedded tissue of 43 different carcinomas (breast, ovary, endometrium, gastrointestinal stromal tumor and malignant mesothelioma). FISH was performed both on sections and extracted nuclei of each tissue using chromosome enumeration probes (CEP) for the centromeric regions of chromosomes 8 and 17, and a locus specific identifier (LSI) for the HER-2/neu oncogene. Differences between ploidy calculated in sections and extracted nuclei were seen in 3 breast carcinomas and 1 gastrointestinal stromal tumor (GIST). Furthermore, 1 breast cancer, previously considered to be borderline for HER-2/neu gene amplification turned out to be clearly amplified. Nuclei extraction and isolation bypass all the problems related to signal interpretation in tissue sections, and the adoption of this new technique, which improves the signal quality in several neoplastic samples, is suggested.     

In situ hybridization: Alkaline phosphatase visualization of biotinylated probes in cryostat and paraffin sections

Summary Alkaline phosphatase immunochemical systems were evaluated for use in the demonstration ofin situ hybridized biotin-labelled probes in frozen and fixed sections of tonsil. Three probes were used: total genomic DNA, pHY2.1, a human repetitive sequence which hybridizes to a 2.12 KB sequence on the Y chromosome (2000 repeats) and a 2.0 KB sequence on the autosomes (100–200 repeats), and human papilloma virus type II. Indirect, three- and five-stage detection methods were compared on cryostat sections. The indirect method involved the application of a streptavidin, biotinylated alkaline phosphatase sequence. The three-stage procedure comprised a mouse monoclonal anti-biotin, rabbit anti-(mouse immunoglobulin), mouse APAAP system. In the five-stage method the indirect and three-stage reagents were sequentially applied. Alkaline phosphatase was demonstrated using a Fast Red naphthol-capture method.The total genomic DNA probe was used initially to investigate hybridization conditions including the optimum temperature of denaturation, which was found to be higher than previously reported. The five-stage detection method gave the most sensitive results for the Y sequence probe, with intense demonstration of the Y body in male nuclei and autosomal sequences in female nuclei. This method was then applied to fixed tissue sections and gave Y body signals on Bouin's and Carnoy's fixed tissue. On the other hand tissue fixed using formalin-based solutions required proteolytic digestion as a pretreatment to hybridization for a Y body signal. The application of this methodology to viral diagnosis in routine fixed anogenital tissue and cytological preparations was also demonstrated.     

High-resolution cDNA microarray CGH mapping of genomic imbalances in osteosarcoma using formalin-fixed paraffin-embedded tissue

Formalin-fixed paraffin embedded (FFPE) tumor tissue provides an opportunity to perform retrospective genomic studies of tumors in which chromosomal imbalances are strongly associated with oncogenesis. The application of comparative genomic hybridization (CGH) has led to the rapid accumulation of cytogenetic information on osteosarcoma (OS); however, the limited resolving power of metaphase CGH does not permit precise mapping of imbalances. Array CGH allows quantitative detection and more precise delineation of copy number aberrations in tumors. Unfortunately the high cost and lower density of BACs on available commercial arrays has limited the ability to comprehensively profile copy number changes in tumors such as OS that are recurrently subject to genomic imbalance. In this study a cDNA/EST microarray including 18,980 human cDNAs (which represent all 22 pairs of autosomal chromosomes and chromosome X) was used for CGH analysis of eight OS FFPE. Chromosomes 1, 12, 17, and X harbored the most imbalances. Gain/amplification of X was observed in 4/8 OS, and in keeping with other recent genomic analyses of OS, gain/amplification of 17p11.2 was often accompanied by a distal deletion in the region of the p53 gene. Gain/amplification of the X chromosome was verified using interphase FISH carried out on a subset of OS FFPE sections and OS tissue arrays.     

Variations in alphoid DNA sequences escape detection of aneuploidy at interphase by FISH technique.

The advent of a new staining technique, termed fluorescence in situ hybridization (FISH), allows the rapid identification of the genomic constitution of an individual with aneuploidy even in interphase nuclei through the use of a series of chromosome-specific DNA probes, an approach termed "interphase cytogenetics." However, alphoid DNA sequences of every centromere are polymorphic (heteromorphic), and the number of targeted sequences may be below the detection level of a specific DNA probe, thus escaping detection and resulting in the imprecise identification of the chromosomal constitution at interphase. The limitations associated with the FISH technique have dire consequences which are emphasized here with an example in which the presence of an additional chromosome 21 in two siblings born consecutively with trisomy 21 (Down syndrome) was not detected by "interphase cytogenetics." The copy number of alphoid DNA sequences of one of the paternal chromosomes 21 was low and resulted in discordance between domain numbers at interphase and actual chromosome numbers at metaphase in both children. This is an isolated incident that could have led to a misdiagnosis if FISH were the only test employed. Although the advantages of this technology are undeniably enormous, the present finding has made it apparent that precise standards and reliability of the procedure must be established prior to its routine application.     

Fluorescence in situ hybridization on vibratome sections of plant tissues

This protocol describes the application of fluorescence in situ hybridization (FISH) to three-dimensionally (3D) preserved tissue sections derived from intact plant structures such as roots or florets. The method is based on the combination of vibratome sectioning with confocal microscopy. The protocol provides an excellent tool to investigate chromosome organization in plant nuclei in all cell types and has been used on tissues of both monocot and dicot plant species. The visualization of 3D well-preserved tissues means that cell types can be confidently identified. For example, meiocytes can be clearly identified at all stages of meiosis and can be imaged in the context of their surrounding maternal tissue. FISH can be used to localize centromeres, telomeres, repetitive regions as well as unique regions, and total genomic DNAs can be used as probes to visualize chromosomes or chromosome segments. The method can be adapted to RNA FISH and can be combined with immunofluorescence labeling. Once the desired plant material is sectioned, which depends on the number of samples, the protocol that we present here can be carried out within 3 d.     

Improved correction of quantitative nuclear DNA (ploidy) measurements in tissue sections

OBJECTIVE: To evaluate, using a computer model, the advantages for ploidy measurements of selecting only center-containing sections of nuclei in ultrathin, very thick or relatively thick tissue sections. STUDY DESIGN: The computed corpuscle sectioning program was run on a personal computer. Its synthetic data were corrected by a variety of correction algorithms. RESULTS: When only center-containing sections of nuclei were selected in ultrathin sections, spherical nuclei could be corrected perfectly, and mildly prolate ellipsoidal nuclei with a selection bias in favor of elliptical nuclear section profiles could be corrected with high fidelity. Ultrathin sections most faithfully represented the true height of the peak of highest ploidy and showed better peak discrimination than other choices of section thickness, but small sample size, wavy sections, markedly inhomogeneous intranuclear DNA distribution and oblate ellipsoidal nuclei represented significant limitations of this approach. As nuclear prolation increased, peak definition worsened, and the peak of highest ploidy was falsely shortened. Results were unaffected by errors in the estimation of section thickness when an internal diploid standard was used. The effect of variable internuclear DNA concentration in mildly or moderately prolate ellipsoidal nuclei was nil. The choice of correction algorithm was unimportant, except that the reference curve method was better able to analyze oblate ellipsoidal nuclei, wavy sections and nuclei with inhomogeneous intranuclear DNA, and provided superior insight into nuclear and section parameters. Thick and very thick sections did not require correction and, unlike ultrathin sections, were immune to markedly inhomogeneous intranuclear DNA distribution, to nonspherical nuclear shape and to focal variation in section thickness (waviness), but (in relatively thick more than in very thick sections) the height of the peak of highest ploidy was falsely shortened, often markedly, and peak definition was worse. CONCLUSION: Choice of section thickness and selection of only center-containing nuclear sections for analysis with a bias in favor of elliptical nuclear section profiles in ultrathin sections are very important for optimal results; the choice of correction algorithm is less important.     

Fluoreszenz-in-situ-Hybridisierung in der humangenetischen Diagnostik

The field of genetic diagnostics incorporates a variety of methods that complement each other. Therefore, the development of new methods calls for a review of the advantages and limitations of established and new technologies. Fluorescence in situ hybridization (FISH) is routinely applied in genetics. Custom-designed and commercially available probes allow for nearly unlimited and targeted visualization of genomic DNA using either metaphase spreads, interphase nuclei, tissue sections, or living cells. FISH applications are particularly important for the detection of structural rearrangements such as microdeletions, translocations, inversions, and insertions, as well as for identification of marker chromosomes, characterization of chromosome breakpoints, and prenatal aneuploidy testing. Furthermore, the analysis of genetic heterogeneity, including mosaicism, is accomplished by evaluating single cells. FISH may also be combined with fluorescent antibodies against cell surface markers and correlated to specific morphologic features of cells and tissues.     

Defects in lamin B1 expression or processing affect interphase chromosome position and gene expression

Radial organization of nuclei with peripheral gene-poor chromosomes and central gene-rich chromosomes is common and could depend on the nuclear boundary as a scaffold or position marker. To test this, we studied the role of the ubiquitous nuclear envelope (NE) component lamin B1 in NE stability, chromosome territory position, and gene expression. The stability of the lamin B1 lamina is dependent on lamin endoproteolysis (by Rce1) but not carboxymethylation (by Icmt), whereas lamin C lamina stability is not affected by the loss of full-length lamin B1 or its processing. Comparison of wild-type murine fibroblasts with fibroblasts lacking full-length lamin B1, or defective in CAAX processing, identified genes that depend on a stable processed lamin B1 lamina for normal expression. We also demonstrate that the position of mouse chromosome 18 but not 19 is dependent on such a stable nuclear lamina. The results implicate processed lamin B1 in the control of gene expression as well as chromosome position.     

Influence of section thickness, mean nuclear diameter and nuclear crowding on DNA ploidy in histologic sections of melanocytic skin lesions

OBJECTIVE: To determine the influence of section thickness, nuclear diameter (MND) and area percentage of nuclei (a measure of nuclear crowding) on histologic DNA ploidy assessed by image cytometry (ICM) of primary melanocytic skin neoplasms (MSNs). STUDY DESIGN: Initially a feasibility study was performed to determine if comparable DNA ploidy histograms could be obtained from cell disaggregates and tissue sections. Following this, DNA ICM was performed on Feulgen-stained tissue sections (4, 6, 8 and 10 microns thick) from 30 primary MSNs (20 benign, 10 malignant) with nuclear diameters from 5.6 to 8.6 microns. Area percentage of nuclei was assessed in all cases at all section thicknesses. RESULTS: The feasibility study produced comparable results for cytocentrifuge and tissue section preparations. For sectioned MSNs, DNA ploidy histograms from 4-micron sections had a higher coefficient of variation of the 2c peak than those from 6-, 8- and 10-micron sections. Ten-micrometer sections had marked overlapping of nuclei, and only small numbers of cells could be measured, giving inadequate results. MND and area percentage of nuclei did not have an important influence on the results. CONCLUSION: Adequate DNA ploidy profiles can be obtained by DNA ICM on 6- and 8-micron-thick histologic sections of MSNs, provided that a strict measurement protocol is followed.     

Adjusting interphase FISH results in epithelial tissue sections to whole cell complement

OBJECTIVE: To derive an equation to compensate for the discrepancies between whole cell preparations and tissue sections for more accurate enumeration of fluorescence in situ hybridization (FISH) signals per cell. STUDY DESIGN: Mean centromere signal counts in touch preparations and corresponding 4-6-micron sections of paraffin-embedded tissue were calculated. Mean widest nuclear diameters were also determined from the tissue sections. The observed data were analyzed to define the volumetric relationships between tissue sections and whole cell preparations. RESULTS: Analysis of results from six lung specimens yielded an equation that approximates whole versus sectioned nuclear volume and permits accurate quantification of mean FISH signal count in histologic sections, as follows: [formula: see text].     

Laser capture microdissection MALDI for direct analysis of archival tissue

MALDI mass spectra were obtained from cancer cells isolated by laser capture microdissection (LCM) of archived tissue. Frozen human lung tissue from adenocarcenoma and squamous cell carcenoma cases were cut into 5 to 15 microm thick sections, stained with hematoxylin and dehydrated. Cancer cells were isolated by LCM, mixed with matrix solution, and deposited on a MALDI target for mass spectrometric analysis. For comparison with LCM isolated cells, tissue sections were placed directly on the MALDI target without microdissection. Tissue sections frozen in optimal cutting temperature (OCT) solution and cut into 8 microm thick sections gave the best performance with direct MALDI analysis. Between 15 and 20 peaks were observed in the mass region between 1,000 and 4,000 Da, and roughly half of these peaks were common to either squamous cells or adenocarcenoma. Additional peaks were observed in the non-LCM mass spectra and these may result from biomolecules in the healthy tissue. When compared to fresh tissue, both LCM and non-LCM archived tissue produced fewer peaks, possibly due to degradation of the biomolecules in the archived tissue.     

Cytoplasmic intermediate filaments are stably associated with nuclear matrices and potentially modulate their DNA-binding function

The tight association of cytoplasmic intermediate filaments (cIFs) with the nucleus and the isolation of crosslinkage products of vimentin with genomic DNA fragments, including nuclear matrix attachment regions (MARs) from proliferating fibroblasts, point to a participation of cIFs in nuclear activities. To test the possibility that cIFs are complementary nuclear matrix elements, the nuclei of a series of cultured cells were subjected to the Li-diiodosalicylate (LIS) extraction protocol developed for the preparation of nuclear matrices and analyzed by immunofluorescence microscopy and immunoblotting with antibodies directed against lamin B and cIF proteins. When nuclei released from hypotonically swollen L929 suspension cells in the presence of digitonin or Triton X-100 were exposed to such strong shearing forces that a considerable number were totally disrupted, a thin, discontinuous layer of vimentin IFs remained tenaciously adhering to still intact nuclei, in apparent coalignment with the nuclear lamina. Even in broken nuclei, the distribution of vimentin followed that of lamin B in areas where the lamina still appeared intact. The same retention of vimentin together with desmin and glial IFs was observed on the nuclei isolated from differentiating C2C12 myoblast and U333 glioma cells, respectively. Nuclei from epithelial cells shed their residual perinuclear IF layers as coherent cytoskeletal ghosts, except for small fractions of vimentin and cytokeratin IFs, which remained in a dot-to cap-like arrangement on the nuclear surface, in apparent codistribution with lamin B. LIS extraction did not bring about a reduction in the cIF protein contents of such nuclei upon their transformation into nuclear matrices. Moreover, in whole mount preparations of mouse embryo fibroblasts, DNA/chromatin emerging from nuclei during LIS extraction mechanically and chemically cleaned the nuclear surface and perinuclear area from loosely anchored cytoplasmic material with the production of broad, IF-free annular spaces, but left substantial fractions of the vimentin IFs in tight association with the nuclear surface. Accordingly, double-immunogold electron microscopy of fixed and permeabilized fibroblasts disclosed a close neighborhood of vimentin IFs and lamin B, with a minimal distance between the nanogold particles of ca. 30 nm. These data indicate an extremely solid interconnection of cIFs with structural elements of the nuclear matrix, and make them, together with their susceptibility to crosslinkage to MARs and other genomic DNA sequences under native conditions, complementary or even integral constituents of the karyoskeleton.     

Lamin A/C and polymeric actin in genome organization

In this work, we have studied the structural and functional linkage between lamin A/C, nuclear actin, and organization of chromosome territories (CTs) in mammary carcinoma MCF-7 cells. Selective down-regulation of lamin A/C expression led to disruption of the lamin A/C perinuclear layer and disorganization of lamin-bound emerin complexes at the inner nuclear membrane. The silencing of lamin A/C expression resulted in a decrease in the volume and surface area of chromosome territories, especially in chromosomes with high heterochromatin content. Inhibition of actin polymerization led to relaxation of the structure of chromosome territories, and an increase in the volumes and surface areas of the chromosome territories of human chromosomes 1, 2 and 13. The results show an important role of polymeric actin in the organization of the nuclei and the chromosome territories.