Sequential Multilocus Fluorescence In Situ Hybridization Can Detect Complex Patterns of Increased Gene Dosage at the Single Cell Level in Tissue Sections

Abstract

A lthough some impressive applications of multicolor fluorescence in situ hybridization (M-FISH) have been demonstrated on cytogenetic preparations, there are no reports of studies that carry over these advanced multitarget techniques to histological sections of tumor tissues in molecular pathology. Despite recent advances in protocols, M-FISH with a simultaneous multicolor painting tool does not seem to be a feasible approach in histological sections, mainly because of the inherent problem of the third dimension, which leads to complex overlays of both fluorescence signals and nuclei of tumor cells. To overcome these technical limitations, we introduce here an innovative and robust method for the detection of multiple targets by interphase FISH in formalin-fixed and paraffinembedded tissue, called sequential multilocus fluorescence in situ hybridization (SML-FISH).Each single tissue section (10 m) obtained from two different paraffin blocks of formalin-fixed tissue from a surgical resection specimen of primary adenocarcinoma of distal esophagus (Union Internationale Contre le Cancer [UICC] Classification: pT1, pN0, pM0; Graduation: G2) was mounted on a glass slide. Tissue pretreatment was done by heating the slides for 20 minutes in a microwave oven (750 W); additionally, pronase E (0.05%) digestion for 10 minutes was performed. Denaturation was performed as described elsewhere (Werner et al, 1999). The following commercially available fluorescent-labeled, locus-specific (LSI), and associated centromere DNA probes (CEP) (Vysis, Inc., Downers Grove, Illinois) were sequentially applied according to manufacturers' instructions in the listed order: LSI D7S486 (c-met), CEP 7, LSI c-myc, CEP 8, LSI Cyclin D1, CEP 11, LSI Her-2/neu, CEP 17, LSI 20q13.2, and CEP 20. After hybridization of the first probe (LSI D7S486), the slides were placed in ϫ2 SSC, 0.1% NP-40 (pH 7.4) at 73°C for 2 minutes followed by embedding with an antifade solution (Vysis, Inc.) containing 4',6-diamidino-2-phenylindole (DAPI) as nuclear counterstaining. Threedimensional volume data sets for the corresponding fluorescence channel (DNA probe and nuclear counterstaining) were generated using a confocal laser scanning microscope (CLSM, Zeiss LSM 510; Carl Zeiss, Jena, Germany). For these purposes each area was scanned in high resolution mode (lens PNF 40ϫ/ 1.3 Oil; scaling X ϭ Y ϭ 0.22 m, Z ϭ 0.5 m) resulting in an image stack of DNA probes and nuclear counterstaining of each tumor area. After image aquisition, the first DNA probe (LSI D7S486) was washed out by heating the section in ϫ2 SSC at 73°C for 25 minutes, followed by denaturation at 73°C for 5 minutes in 70% formamide/ϫ2 SSC. After deletion of the DNA probe which followed after the laser scanning procedure before the next hybridization, the section was evaluated to ensure that there were no DNA probespecific fluorescent signals left. Then, the procedure (hybridization, nuclear counterstaining, image aquisition of the same tissue areas, and deletion of the probe) was repeated for each of ...

Human archival tissues provide a valuable source for the analysis of spatial genome organization

“…The tissues analyzed in the present study were fixed in a buffered 4% formaldehyde solution, which has been recommended particularly for 3D preservation . To enable sufficient DNA probe penetration during in situ hybridization in formalinfixed tissue sections, we pretreated the sections with heat and pronase E to provide a feasible compromise between optimal penetration of probe DNA into the cell nucleus, little nonspecific fluorescence background, and structural preservation of the genome microarchitecture (Walch et al 2001). An important factor for systematic investigations of intraindividual and interindividual changes in the nuclear architecture is the reliability of 3D-FISH experiments to assess the 3D positions of chromosome territories down to the level of chromatin domains of about 1 Mb, as well as other nuclear structures.…”

Aurora Kinase A Messenger RNA Overexpression Is Correlated with Tumor Progression and Shortened Survival in Head and Neck Squamous Cell Carcinoma

“…Tissue pretreatment and fluorescence in situ hybridization were done with a-satellite repeat sequence DNA probes for chromosomes 9, 15, and 20 (Chrombios GmbH, Raubling, Germany) based on a protocol previously published by our group (22,29). The selection of chromosomes was based on a recent publication describing chromosomal instability in oral squamous cell carcinoma (30).…”

Significance of HER2 Low-Level Copy Gain in Barrett's Cancer: Implications for Fluorescence In situ Hybridization Testing in Tissues

“…Fluorescence in situ hybridization A commercially available assay with fluorescence-labeled locusspecific DNA probes for HER2 and chromosome-17 centromeric a-satellite (Chrombios) was hybridized on the specimen according to a protocol described previously (21,22). The hybridization specificity of the probes was tested in metaphase spreads and lymphocytes served as internal controls in tumor tissue samples.…”