Exp Mol Pathol  2001 Jun;70(3):195-200 

Rapid, efficient genotyping of clinical tumor samples by laser-capture
microdissection/PCR/SSCP.

Dillon D, Zheng K, Costa J.

Department of Pathology, Yale University School of Medicine, New Haven,
Connecticut 06520, USA.

Background: 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. Copyright 2001 Academic Press.

PMID: 11417998 [PubMed - indexed for MEDLINE]