Analysis of changes in DNA sequence copy number by comparative genomic hybridization in archival paraffin-embedded tumor samples.
Analysis of previously unknown genetic aberrations in solid tumors has become possible through the use of comparative genomic hybridization (CGH), which is based on competitive binding of tumor and control DNA to normal metaphase chromosomes. CGH allows detection of DNA sequence copy number changes (deletions, gains, and amplifications) on a genome-wide scale in a single hybridization. We describe here an improved CGH technique, which enables reliable detection of copy number changes in archival formalin-fixed paraffin-embedded tumor samples. The technique includes a modified DNA extraction protocol, which produces high molecular weight DNA which is necessary for high quality CGH. The DNA extraction includes a 3-day digestion with proteinase K, which remarkably improves the yield of high molecular weight DNA. Labeling of the test DNA with a directly fluorescein-conjugated nucleotide (instead of biotin labeling) improved significantly the quality of hybridization. Using the paraffin-block technique, we could analyze 70 to 90% of paraffin blocks, including very old samples as well as samples taken at autopsy. CGH from paraffin blocks was highly concordant (95%) with analyses done from matched freshly frozen tumor samples (n = 5 sample pairs; kappa coefficient = 0.83). The method described here has wide applicability in tumor pathology, allowing large retrospective prognostic studies of genetic aberrations as well as studies on genetic pathogenesis of solid tumors, inasmuch as premalignant lesions and primary and metastatic tumors can be analyzed by using archival paraffin-embedded samples.
Isola
J
J
Department of Laboratory Medicine, University of California, San Francisco.
DeVries
S
S
Chu
L
L
Ghazvini
S
S
Waldman
F
F
D002874:Chromosome Mapping / Q000379:methods*; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics*; D018628:Gene Dosage; D005821:Genetic Techniques; D006801:Humans; D008722:Methods; D009369:Neoplasms / Q000235:genetics*; D016612:Paraffin Embedding
D004273:DNA, Neoplasm