Evaluation of oligonucleotide arrays for sequencing of the p53 gene in DNA from formalin-fixed, paraffin-embedded breast cancer specimens.
BACKGROUND
Routine tissue processing has generated banks of paraffin-embedded tissue that could be used in retrospective cohort studies to study the molecular changes that occur during cancer development. The purpose of this study was to determine whether a p53 microarray could be used to sequence the p53 gene in DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues.


METHODS
DNA was extracted from 70 FFPE breast cancer tissue specimens. p53 was sequenced with an oligonucleotide microarray (p53 GeneChip; Affymetrix), and the results were compared with the results obtained from direct sequencing.


RESULTS
DNA was extracted from 62 of 70 cases. We identified 26 mutations in 24 of the 62 cases by the p53 GeneChip. No polymorphisms were detected, and exon 4 could not be evaluated in 20 cases. There were 43 genetic alterations detected by direct sequencing in 35 of the 62 cases. These consisted of 26 polymorphisms and 17 mutations in exons or splice sites. Fifteen mutations were identified by both methods. Direct sequencing detected significantly more gene alterations (43 of 54) in DNA extracted from FFPE tissue than the p53 GeneChip (26 of 54; P = 0.018). However, if the changes in exon 4 were eliminated from this comparison, the p53 GeneChip detected 26 of 27 mutations compared with direct sequencing, which identified 16 of 27 mutations. (P = 0.016).


CONCLUSIONS
A combination of oligonucleotide microarray and direct sequencing may be necessary to accurately identify p53 gene alterations in FFPE breast cancer. The p53 GeneChip cannot be used to detect exon 4 polymorphisms (codon 72) in FFPE breast cancer tissue.
Cooper
Melissa
M
Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada.
Li
Shu-Qiu
SQ
Bhardwaj
Tajinder
T
Rohan
Thomas
T
Kandel
Rita A
RA
D001943:Breast Neoplasms / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics*; D005260:Female; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D010232:Paraffin; D011110:Polymorphism, Genetic; D016610:Tissue Embedding; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D004273:DNA, Neoplasm; D005404:Fixatives; D016159:Tumor Suppressor Protein p53; D005557:Formaldehyde; D010232:Paraffin