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This chapter describes the various methods derived from the protocol of standard fluorescent in situ hybridization (FISH) that are used in human, animal, plant, and microbial studies. These powerful techniques allow us to detect and physically map on interphase nuclei, chromatin fibers, or metaphase chromosomes probes derived from single-copy genes to repetitive DNA sequences. Other variants of the technique enable the co-localization of genes and the overall comparison of the genome among individuals of the same species or of different taxa. A further variant detects and localizes bacteria on tissues and cells. Overall, this offers a remarkable multiplicity of possible applications ranging from strict physical mapping, to clinical and evolutionary studies, making it a powerful and informative complement to other molecular, functional, or genomic approaches.

Childhood leukemia is a common pediatric cancer in the developed world, the disease is biologically diverse and there is much discussion as to its causal mechanisms. Acute lymphoblastic leukemia (ALL) is the most common subtype and infants with ALL have a greatly increased risk of treatment failure. There are molecular and biological properties of leukemic cells that determine treatment outcome; these can usually be attributed to distinct genetic abnormalities that alter the normal proliferative and survival signals of hematopoietic cells. Experimental evidence for the existence of leukemic stem cells (LSC) has been obtained, and it is presumed that these cells arise from mutations in normal hematopoetic stem cells or progenitor cells, and they are difficult to eradicate. LSC seem to be surprisingly different from their normal counterparts and therefore are obvious new targets for drug therapy. Therapeutic concepts using monoclonal antibodies have substantially improved response rates in patients with malignant lymphomas and are currently being evaluated in other types of cancer.

Although the histologic distinction between pheochromocytomas and head and neck paragangliomas is clear, little is known about the genetic differences between them. To date, various sets of genes have been found to be involved in inherited susceptibility to developing both tumor types, but the genes involved in sporadic pathogenesis are still unknown. To define new candidate regions, we performed CGH analysis on 29 pheochromocytomas and on 24 paragangliomas mainly of head and neck origin (20 of 24), which allowed us to differentiate between the two tumor types. Loss of 3q was significantly more frequent in pheochromocytomas, and loss of 1q appeared only in paragangliomas. We also found gain of 11q13 to be a significantly frequent alteration in malignant cases of both types. In addition, recurrent loss of 8p22-23 was found in 62% of pheochromocytomas (including all malignant cases) versus in 33% of paragangliomas, suggesting that this region contains candidate genes involved in the pathogenesis of this abnormality. Using FISH analysis on tissue microarrays, we confirmed genomic deletion of this region in 55% of pheochromocytomas compared to 12% of paragangliomas. Loss of 8p22-23 appears to be an important event in the sporadic development of these tumors, and additional molecular studies are necessary to identify candidate genes in this chromosomal region.

Molecular cytogenetic techniques enabled us to clarify numerical and structural alterations previously detected by conventional cytogenetic techniques in 37 patients who had myelodysplastic syndromes with complex karyotypes. Using high-resolution comparative genomic hybridization (HR-CGH), we found the most recurrent alterations to be deletion of 5q (70%), 18q (35%), 7q (32%), 11q (30%), and 20q (24%), gain of 11q (35%) and 8q (24%), and trisomy of chromosome 8 (19%). Furthermore, in 35% of the patients, 20 amplifications were identified. These amplifications were shown by FISH to involve some genes previously described as amplified in hematological malignancies, such as ERBB2, MLL, and RUNX1. In addition, two other genes, BCL6 and BCL2, which are classically related to apoptosis and non-Hodgkin lymphoma, were shown for the first time to be involved in amplification. Genomic alterations involving different subtelomeric regions with losses in 4p16, 5p15.3, 6q27, 18p11.3, and 18q23 and gains in 1p36.3 and 19p13.3 were detected by HR-CGH. Array CGH analysis of the subtelomeric regions in some samples was able to confirm a number of these alterations and found some additional alterations not detected by conventional CGH.

Comparative genomic hybridization (CGH) has been widely used to detect copy number alterations in cancer and to identify regions containing candidate tumor-responsible genes; however, gene expression changes have been described only in highly amplified regions (amplicons). To study the overall impact of slight copy number changes on gene expression, we analyzed 16 T-cell lymphomas by using CGH and a custom-designed cDNA microarray containing 7,657 genes and expressed sequence tags related to tumorigenesis. We evaluated mean gene expression and variability within CGH-altered regions and explored the relationship between the effects of the gene and its position within these regions. Minimally overlapping CGH candidate areas (6q25, 13q21-q22, and 19q13.1) revealed a weak relationship between altered genomic content and gene expression. However, some candidate genes showed modified expression within these regions in the majority of tumors; these candidate genes were evaluated and confirmed in another independent series of 23 T-cell lymphomas by use of the same cDNA microarray and by FISH on a tissue microarray. When all the CGH regions detected for each tumor were considered, we found a significant increase or decrease in the mean expression of the genes contained in gained or lost regions, respectively. In addition, we found that the expression of a gene was dependent not only on its position within an altered region but also on its own mechanism of regulation: genes in the same altered region responded very differently to the gain or loss of genetic material. Supplementary material for this article can be found on the Genes, Chromosomes, and Cancer website at http://www.interscience.wiley.com/jpages/1045-2257/suppmat/index.html., (Copyright 2004 Wiley-Liss, Inc.)

N-Bromosuccinimide (NBS) is a known protein reagent able to modify amino acids and proteins, resulting in oxidation of tryptophan, tyrosine and histidine residues, as well as sulfhydryl, alcohol and phenol groups. These properties make NBS a suitable reagent to selectively block certain amino acid residues in biochemistry, and also permit the histochemical detection of proteins by oxidative deamination followed by the Schiff reaction. In this paper we show that, under ultraviolet excitation, NBS selectively reveals the cytoplasmic granules of mammalian eosinophils and chicken heterophils, rendering considerable white--blue fluorescence, in a remarkable fluorogenic reaction which rapidly increases at the beginning of the observation. This emission slightly decays afterwards and then remains almost stable still yielding a high level of emission after 10min of continuous excitation. Possible mechanisms underlying these results are discussed and we propose NBS as a very suitable fluorogenic reagent for the microscopical detection and analysis of proteins.