title: Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization.
abstract: Chromogenic (CISH) and fluorescent (FISH) in situ hybridization have emerged as reliable techniques to identify amplifications and chromosomal translocations. CISH provides a spatial distribution of gene copy number changes in tumour tissue and allows a direct correlation between copy number changes and the morphological features of neoplastic cells. However, the limited number of commercially available gene probes has hindered the use of this technique. We have devised a protocol to generate probes for CISH that can be applied to formalin-fixed, paraffin-embedded tissue sections (FFPETS). Bacterial artificial chromosomes (BACs) containing fragments of human DNA which map to specific genomic regions of interest are amplified with phi29 polymerase and random primer labelled with biotin. The genomic location of these can be readily confirmed by BAC end pair sequencing and FISH mapping on normal lymphocyte metaphase spreads. To demonstrate the reliability of the probes generated with this protocol, four strategies were employed: (i) probes mapping to cyclin D1 (CCND1) were generated and their performance was compared with that of a commercially available probe for the same gene in a series of 10 FFPETS of breast cancer samples of which five harboured CCND1 amplification; (ii) probes targeting cyclin-dependent kinase 4 were used to validate an amplification identified by microarray-based comparative genomic hybridization (aCGH) in a pleomorphic adenoma; (iii) probes targeting fibroblast growth factor receptor 1 and CCND1 were used to validate amplifications mapping to these regions, as defined by aCGH, in an invasive lobular breast carcinoma with FISH and CISH; and (iv) gene-specific probes for ETV6 and NTRK3 were used to demonstrate the presence of t(12;15)(p12;q25) translocation in a case of breast secretory carcinoma with dual colour FISH. In summary, this protocol enables the generation of probes mapping to any gene of interest that can be applied to FFPETS, allowing correlation of morphological features with gene copy number.
authors.affiliation: The Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, London, UK.
authors.forename: Maryou B K
authors.initials: MB
authors.lastname: Lambros
authors.forename: Pete T
authors.initials: PT
authors.lastname: Simpson
authors.forename: Chris
authors.initials: C
authors.lastname: Jones
authors.forename: Rachael
authors.initials: R
authors.lastname: Natrajan
authors.forename: Charlotte
authors.initials: C
authors.lastname: Westbury
authors.forename: Dawn
authors.initials: D
authors.lastname: Steele
authors.forename: Kay
authors.initials: K
authors.lastname: Savage
authors.forename: Alan
authors.initials: A
authors.lastname: Mackay
authors.forename: Fernando C
authors.initials: FC
authors.lastname: Schmitt
authors.forename: Alan
authors.initials: A
authors.lastname: Ashworth
authors.forename: Jorge S
authors.initials: JS
authors.lastname: Reis-Filho
mesh_terms: D008949:Adenoma, Pleomorphic / Q000235:genetics / Q000473:pathology; D018070:Biological Specimen Banks; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology; D018270:Carcinoma, Ductal, Breast / Q000235:genetics / Q000473:pathology; D018275:Carcinoma, Lobular / Q000235:genetics / Q000473:pathology; D002863:Chromogenic Compounds; D022202:Chromosomes, Artificial, Bacterial; D015342:DNA Probes / Q000096:biosynthesis*; D005260:Female; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D016612:Paraffin Embedding; D010336:Pathology / Q000379:methods*; D012468:Salivary Gland Neoplasms / Q000235:genetics / Q000473:pathology; D012680:Sensitivity and Specificity; D016707:Tissue Fixation; D014178:Translocation, Genetic
chemical_list: D002863:Chromogenic Compounds; D015342:DNA Probes