Evaluation of Oligonucleotide Arrays for Sequencing of the p53 Gene in DNA from Formalin-Fixed, Paraffin-Embedded Breast Cancer Specimens

Melissa Cooper, Shu Qiu Li, Tajinder Bhardwaj, Thomas E. Rohan, Rita A. Kandel

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)500-508
Number of pages9
JournalClinical Chemistry
Volume50
Issue number3
DOIs
StatePublished - Mar 2004

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p53 Genes
Oligonucleotide Array Sequence Analysis
Oligonucleotides
Paraffin
Formaldehyde
Genes
Tissue
Breast Neoplasms
Exons
DNA
Microarrays
Mutation
Polymorphism
Codon
Cohort Studies
Retrospective Studies
Processing
Neoplasms

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

Evaluation of Oligonucleotide Arrays for Sequencing of the p53 Gene in DNA from Formalin-Fixed, Paraffin-Embedded Breast Cancer Specimens. / Cooper, Melissa; Li, Shu Qiu; Bhardwaj, Tajinder; Rohan, Thomas E.; Kandel, Rita A.

In: Clinical Chemistry, Vol. 50, No. 3, 03.2004, p. 500-508.

Research output: Contribution to journalArticle

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abstract = "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{\circledR}; 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.",
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N2 - 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.

AB - 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.

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