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Validation of a yeast functional assay for p53 mutations using clonal sequencing
Author(s) -
Iggo Richard,
Rudewicz Justine,
Monceau Elodie,
Sevenet Nicolas,
Bergh Jonas,
Sjoblom Tobias,
Bonnefoi Hervé
Publication year - 2013
Publication title -
the journal of pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.964
H-Index - 184
eISSN - 1096-9896
pISSN - 0022-3417
DOI - 10.1002/path.4243
Subject(s) - sanger sequencing , biology , dna sequencing , context (archaeology) , mutation , complementary dna , kras , genetics , computational biology , dna , gene , paleontology
We have previously tested biopsies from 1469 breast tumours with a p53 functional assay in the context of a prospective clinical trial ( EORTC 10994/ BIG 1–00). The goal of the trial was to determine whether p53 status could be used to select patients who would benefit from inclusion of taxanes in anthracycline‐based chemotherapy. The results of the trial were negative. To test whether this was because the functional assay misclassified the tumours, we have reanalysed two groups of biopsies by Sanger sequencing and Roche 454 next generation sequencing ( NGS ). Comparison of yeast data with pooled cDNA sequencing data in an initial cohort of 69 biopsies showed that conventional sequencing is insensitive when the mutant p53 content is low. A second cohort of 48 biopsies was used to compare directly the yeast assay with Sanger and NGS technology. The mutant sequence was difficult to detect in sequence chromatograms of pooled cDNA , whereas NGS unequivocally identified mutations in every case classified as mutant by the functional assay. The NGS data showed that small deletions, probably caused by PCR splicing, account for most of the unexplained background in the yeast assay. We conclude that mutation detection techniques that test multiple clones, such as the p53 functional assay and NGS , are more reliable than Sanger sequencing of pooled DNA ; that the high p53 mutation rate (44%) seen with the yeast assay in the EORTC 10994/ BIG 1–00 trial reflects this high sensitivity; and that NGS with Roche 454 technology could be used to identify the p53 mutations in the remaining tumours previously tested in yeast in the EORTC10994 / BIG 1–00 trial. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.