Rapid, efficient genotyping of clinical tumor samples by laser-capture microdissection/PCR/SSCP.
Mutation analysis is becoming increasingly important in clinical practice, since sporadic mutations in tumors often correlate with prognosis and/or therapeutic response. However, the labor-intensive nature of the molecular analyses has limited the routine clinical use of tumor genotyping. Laser-capture microdissection (LCM) allows procurement of relatively pure tumor cell populations. We have investigated the possibility that the use of laser-capture microdissection would allow elimination of time-consuming intermediate steps in tumor genotyping. Design. Archival formalin-fixed, paraffin-embedded tissues from seven cases of colorectal adenocarcinoma were laser- and hand-microdissected and subsequently evaluated by PCR/SSCP/sequencing for Ki-ras exon 1 and p53 exons 5, 7, and 8. Results. Mutations in Ki-ras exon 1 and/or p53 exons 5 and 7 were detected in five of the seven samples. In the hand-microdissected samples, confident identification of mutations was possible in several cases only after band excision, DNA elution, reamplification, and verification of mutant enrichment by a second SSCP analysis prior to sequencing. In the laser-microdissected samples, confident mutation identification was possible in all cases with direct sequencing of the original PCR product, reducing the time required for molecular analysis to 3 days. Conclusion. Using laser-capture microdissection, mutant signals are strong enough to sequence directly from original PCR products. With rapid, efficient genotyping by LCM/PCR/SSCP, results can be incorporated directly into the surgical pathology report.
Dillon
D
D
Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Zheng
K
K
Costa
J
J
D001483:Base Sequence; D003062:Codon / Q000235:genetics; D003110:Colonic Neoplasms / Q000235:genetics* / Q000473:pathology; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology*; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D004210:Dissection / Q000379:methods; D005091:Exons; D016158:Genes, p53; D011905:Genes, ras; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D012004:Rectal Neoplasms / Q000235:genetics* / Q000473:pathology
D003062:Codon; D004273:DNA, Neoplasm