Anton K. Raap

In situ hybridization as a tool to study numerical chromosome aberrations in solid bladder tumors

SummaryMethods for single- and double-target in situ hybridization (ISH) to, cells isolated from solid transitional cell carcinomas (TCCs) of the urinary bladder are described. Single cell suspensions were prepared from solid tumors of the urinary bladder by mechanical disaggregation and fixed in 70% ethanol. Using two DNA probes specific for the centromeres of chromosomes #1 and #18, ISH procedures were optimized for these samples. Human lymphocytes and cells from the T24 bladder tumor cell line were used as controls. In lymphocyte nuclei and metaphase chromosome spreads, ISH showed two major spots for each of the probes. About 80% of the nuclei from T24 cells showed three spots for both the chromosome #1 and #18 specific probes. When nuclei from TCCs were analyzed, often the number of spots for chromosome #1, and to a lesser extent for chromosome #18, differed from the number expected on basis of flow cytometric ploidy measurements. The double target-ISH method in all cases allowed the correlation of numerical aberrations for chromosomes #1 and #18 in one and the same cell. By such analyses a profound heterogeneity in chromosome number was detected in most tumors. In order to optimize the reproductbility of the method and the interpretation of the ISH-signals, criteria for their analysis have been determined. This procedure can now be applied on a routine basis to solid tumor specimens.

Up-converting phosphor reporters for nucleic acid microarrays.

An important application of robotically spotted DNA microarrays is the monitoring of RNA expression levels. A clear limitation of this technology is the relatively large amount of RNA that is required per hybridization as a result of low hybridization efficiency and limiting detection sensitivity provided by conventional fluorescent reporters. We have used a recently introduced luminescent reporter technology, called UPT (up-converting phosphor technology). Down-converting phosphors have been applied before to detect nucleic acids on filters using time-resolved fluorometry. The unique feature of the phosphor particles (size 0.4 μm) used here is that they emit visible light when illuminated with infrared (IR) light (980 nm) as a result of a phenomenon called up-conversion. Because neither support material of microarrays nor biomolecules possess up-conversion properties, an enhanced image contrast is expected when these nonfading phosphor particles are applied to detect nucleic acid hybrids on microarrays. Comparison of the UPT reporter to cyanin 5 (Cy5) in a low-complexity model system showed a two order of maginitude linear relationship between phosphor luminescence and target concentration and resulted in an excellent correlation between the two reporter systems for variable target concentrations (R2 = 0.95). However, UPT proved to be superior in sensitivity, even though a wide-field microscope equipped with a xenon lamp was used. This higher sensitivity was demonstrated by complementary DNA (cDNA) microarray hybridizations using cDNAs for housekeeping genes as probes and complex cDNA as target. These results suggest that a UPT reporter technology in combination with a dedicated IR laser array-scanner holds significant promise for various microarray applications.

In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes.

Methods are needed to study single molecules to reveal variability, interactions and mechanisms that may go undetected at the level of populations of molecules. We describe here an integrated series of reaction steps that allow individual nucleic acid molecules to be detected with excellent specificity. Oligonucleotide probes are circularized after hybridization to target sequences that have been prepared so that localized amplification reactions can be initiated from the target molecules. The process results in strong, discrete detection signals anchored to the target molecules. We use the method to observe the distribution, within and among human cells, of individual normal and mutant mitochondrial genomes that differ at a single nucleotide position.

New strategy for multi-colour fluorescence in situ hybridisation: COBRA: COmbined Binary RAtio labelling

Multicolour in situ hybridisation (MFISH) is increasingly applied to karyotyping and detection of chromosomal abnormalities. So far 27 colour analyses have been described using fluorescently labelled chromosome painting probes in a so-called combinatorial approach. In this paper a new strategy is presented to use efficiently the currently available number of spectrally separated fluorophores in order to increase the multiplicity of MFISH. We introduce the principle of COBRA (COmbined Binary RAtio labelling), which is based on the simultaneous use of combinatorial labelling and ratio labelling. Human chromosome painting in 24 colours is accomplished using four fluorophores only. Three fluorophores are used pair wise for ratio labelling of a set of 12 chromosome painting probes. The second set of 12 probes is labelled identically but is also given a binary label (fourth fluorophore). The COBRA method is demonstrated on normal human chromosomes and on a lymphoma (JVM) cell line, using probes enzymatically labelled with fluorescein, lissamine and cy5 as primary fluorophores, and diethylaminocoumarin (DEAC), a blue dye, as combinatorial fourth label to demonstrate incorporated digoxigenin. In addition, the principle was tested using chemical labelling. The first set of 12 painting probes was therefore labelled by ULS (Universal Linkage System), using DEAC, cy3 and cy5 as primary labels, and the second set was labelled similarly, but also contained a digoxigenin-ULS label, which was indirectly stained with fluorescein. Subsequently, a mathematical analysis is presented and methods are indicated for achieving an MFISH multiplicity of 48, 96 or even higher using existing technology.

Detection of chromosome aberrations in interphase tumor nuclei by nonradioactive in situ hybridization

In a blind study, chromosome aberrations in tumor cells were analyzed by conventional cytogenetic techniques (G banding) and nonradioactive in situ hybridization with chromosome-specific probes. The material was obtained directly from patients with hematologic diseases and from colon tumor derived cell lines. The cytogenetic data obtained with G banding were in accord with those obtained by in situ hybridization to metaphase chromosomes. Most importantly, in situ hybridization to interphase nuclei gave reliable results and even allowed detection of cell subpopulations that were not detected by analyzing metaphase chromosomes. Furthermore, in retrospect, even structural aberrations could be detected in interphase nuclei; abnormal cells with either an i(1q) or a translocation der(1)t(1;7) could be identified. Our results show that the application of in situ hybridization in combination with routine cytogenetic techniques offers significant advantages for cytogenetic analysis of solid tumors and hematologic malignancies.

Bi-color detection of two target DNAs by non-radioactive in situ hybridization

SummaryA non-radioactive in situ hybridization technique is described which allows the simultaneous detection of different DNA sequences. To demonstrate the feasibility of the proccdure, metaphases and interphase nuclei of a human-mouse somatic cell hybrid were simultaneously hybridized with mercurated total human DNA and a biotinylated mouse satellite DNA probe. After the hybridization, the probes were detected immunocytochemically using two different and independent affinity systems. By this approach we visualized the two DNA target sequences in metaphase chromosomes and in interphase nuclei with FITC and TRITC fluorescence, or blue (alkaline phosphatase) and brown (peroxidase) precipitated enzyme products. This method not only allows detection of intact chromosomes but also the visualization of rearrangements between parts of human and mouse chromosomes. Furthermore, the technique demonstrates the high topological resolution of nonradioactive in situ hybridizations.

The Glial and Mesenchymal Elements of Gliosarcomas Share Similar Genetic Alterations

The cellular origin of the sarcomatous component of gliosarcomas is controversial. It is not clear if the sarcoma arises in transition from the glial cells that comprise the gliomatous component or independently arises from non-neoplastic mesenchymal cells of the tumor stroma. Using comparative genomic hybridization (CGH) along with cytogenetic analysis, fluorescence in situ hybridization (FISH) analysis, and polymerase chain reaction (PCR) analysis of microsatellite allelic imbalance, we have evaluated the genetic alterations in the gliomatous and sarcomatous components of five gliosarcomas. The glial element was grade 4 fibrillary astrocytoma (glioblastoma multiforme) in all five tumors. The sarcoma elements were fibroblastic without osseous, chondroid, or angiosarcomatous differentiation. Gain of chromosome 7, loss of chromosome 10, deletions of the chromosome 9 p-arm, and alterations of chromosome 3 were frequently observed, demonstrating that gliosarcomas can be genetically classified as belonging to the spectrum of glioblastomas. Furthermore, the sarcomatous and gliomatous portions of each gliosarcoma investigated were similar with respect to both the presence and absence of specific genetic alterations. This observation supports the hypothesis that the sarcomatous component of a gliosarcoma either arises from the same common precursor cell as the gliomatous portion, or it arises from the gliomatous portion itself.

Somatic pairing of chromosome 1 centromeres in interphase nuclei of human cerebellum

SummaryInterphase nuclei isolated from paraffin-embedded tissue of four normal brains were hybridized with biotinated repetitive DNA probes specific for the (peri) centromeric regions of chromosomes 1 and 7. Hybridization results were visualized with a peroxidase-DAB system after which the number of specific signals per nucleus was counted using bright field microscopy. Using the probe specific for chromosome 7 (p7t1), both the cerebral and the cerebellar samples showed 2 spots in 82% and 83%, respectively, of the nuclei. In situ hybridization with the chromosome 1 probe (pUC1. 77) showed two spots in 69% of the cerebral nuclei. In cerebellar samples, hybridization with pUC1.77 resulted in only one large spot per nucleus in 82% of the cells. The average spot size in nuclei with one signal was about 1.6 times as large as that in nuclei with two signals. These observations suggest that the single large spot in the cerebellar cells is not the result of monosomy of chromosome 1 but that it reflects somatic pairing of the two chromosome 1 centromeres. Based on the size and the fraction of nuclei with one large spot, the small granular neuron is the most likely candidate. The difference between cerebral and cerebellar samples indicates that this somatic pairing of chromosome 1 is a cell-type-dependent phenomenon.

Array-Based Comparative Genomic Hybridization Analysis Reveals Recurrent Chromosomal Alterations and Prognostic Parameters in Primary Cutaneous Large B-Cell Lymphoma

PURPOSE To evaluate the clinical relevance of genomic aberrations in primary cutaneous large B-cell lymphoma (PCLBCL). PATIENTS AND METHODS Skin biopsy samples of 31 patients with a PCLBCL classified as either primary cutaneous follicle center lymphoma (PCFCL; n = 19) or PCLBCL, leg type (n = 12), according to the WHO-European Organisation for Research and Treatment of Cancer (EORTC) classification, were investigated using array-based comparative genomic hybridization, fluorescence in situ hybridization (FISH), and examination of promoter hypermethylation. RESULTS The most recurrent alterations in PCFCL were high-level DNA amplifications at 2p16.1 (63%) and deletion of chromosome 14q32.33 (68%). FISH analysis confirmed c-REL amplification in patients with gains at 2p16.1. In PCLBCL, leg type, most prominent aberrations were a high-level DNA amplification of 18q21.31-q21.33 (67%), including the BCL-2 and MALT1 genes as confirmed by FISH, and deletions of a small region within 9p21.3 containing the CDKN2A, CDKN2B, and NSG-x genes. Homozygous deletion of 9p21.3 was detected in five of 12 patients with PCLBCL, leg type, but in zero of 19 patients with PCFCL. Complete methylation of the promoter region of the CDKN2A gene was demonstrated in one PCLBCL, leg type, patient with hemizygous deletion, in one patient without deletion, but in zero of 19 patients with PCFCL. Seven of seven PCLBCL, leg type, patients with deletion of 9p21.3 and/or complete methylation of CDKN2A died as a result of their lymphoma. CONCLUSION Our results demonstrate prominent differences in chromosomal alterations between PCFCL and PCLBCL, leg type, that support their classification as separate entities within the WHO-EORTC scheme. Inactivation of CDKN2A by either deletion or methylation of its promoter could be an important prognostic parameter for the group of PCLBCL, leg type.

Sensitivity of various visualization methods for peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry

SummaryVarious chromogen protocols for visualizing peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry were compared with respect to their sensitivity. They were tested on tissue sections of human skeletal muscle and in an antigen spot test using antibodies against slow skeletal muscle myosin. The chromogens included 3-amino-9-ethylcarbazole (AEC), 3, 3′-diaminobenzidine (DAB),p-phenylenediamine-pyrocatechol (PPD-PC) and 4-chloro-1-naphthol (CN) in peroxidase histochemistry, and 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium salt (BCIP-NBT), BCIP-tetra nitro blue tetrazolium salt (TNBT) and various combinations of substituted naphthol phosphate-diazonium salt in alkaline phosphatase histochemistry. DAB, CN, and PPD-PC were also employed with imidazole and DAB in addition to Co2+ and Ni2+ ions. The results indicate that DAB-imidazole and DAB-Co2+ and Ni2+ ions are the most sensitive chromogen protocols for visualizing peroxidase activity. Although no large differences were found between the various chromogen protocols for visualizing alkaline phosphatase activity, the protocol BCIP-TNBT is especially recommended. Furthermore, the various chromogen protocols were evaluated as to stability of chromogen solutions and final precipitates, background staining, localization properties, and enhancement of enzyme activity.