A novel multiplexing, polymerase chain reaction-based assay for the analysis of chromosome 18q status in colorectal cancer.
Chromosome 18q allelic loss has been reported to have prognostic significance in stage II colorectal carcinoma. We have developed a fluorescent multiplex polymerase chain reaction assay to analyze five microsatellite markers (D18S55, D18S58, D18S61, D18S64, and D18S69) for allelic loss at the long arm of chromosome 18. Amplicon detection and evaluation was accomplished by capillary electrophoresis using an ABI 310 genetic analyzer. Robustness of the assay when performed on DNA extracted from formalin-fixed, paraffin-embedded tissue sections was confirmed by analyzing its repeatability and reproducibility. Allelic loss was assessed in 61 stage II colorectal tumors and was detected in 58% (31 of 53) of tumors not showing instability. As part of the study, results of 207 previous polymerase chain reaction/polyacrylamide-based assays were re-evaluated by two independent observers to determine the degree of concordance of visual evaluation. In the case of stage II colorectal tumors, when electropherogram results were compared with those obtained from visual evaluation of the same markers after polyacrylamide gel electrophoresis, discrepancies between observers were detected in 16.4% of determinations. In conclusion, we have developed a robust and reliable assay for multiplexed loss of heterozygosity determination that improves assessment of chromosome 18q allelic loss in colorectal tumors processed as routine formalin-fixed, paraffin-embedded specimens.
Erill
Nadina
N
Biopat, Universitat de Barcelona, Spain.
Colomer
Anna
A
Calvo
Miquel
M
Vidal
August
A
Román
Ruth
R
Verdú
Montse
M
Cordón-Cardó
Carlos
C
Puig
Xavier
X
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002887:Chromosomes, Human, Pair 18 / Q000235:genetics*; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D019656:Loss of Heterozygosity / Q000235:genetics*; D008297:Male; D018895:Microsatellite Repeats / Q000235:genetics; D008875:Middle Aged; D009367:Neoplasm Staging; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results