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Related Experiment Video

Updated: May 9, 2026

Yeast As a Chassis for Developing Functional Assays to Study Human P53
14:57

Yeast As a Chassis for Developing Functional Assays to Study Human P53

Published on: August 4, 2019

Validation of a yeast functional assay for p53 mutations using clonal sequencing.

Richard Iggo1, Justine Rudewicz, Elodie Monceau

  • 1INSERM U916, Institut Bergonié Comprehensive Cancer Centre, Univ. Bordeaux, F-33000, Bordeaux, France.

The Journal of Pathology
|July 31, 2013
PubMed
Summary

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Next-generation sequencing (NGS) offers superior accuracy for detecting p53 mutations in breast tumors compared to Sanger sequencing. This improved sensitivity clarifies previous trial results and enhances future cancer research.

Area of Science:

  • Oncology
  • Molecular Biology
  • Genetics

Background:

  • A previous clinical trial (EORTC 10994/BIG 1-00) investigated p53 functional status for taxane chemotherapy selection in breast cancer patients.
  • The trial yielded negative results, prompting an investigation into potential p53 functional assay misclassification.

Purpose of the Study:

  • To re-evaluate p53 mutation detection in breast tumors using Sanger sequencing and Roche 454 next-generation sequencing (NGS).
  • To compare the reliability of different mutation detection techniques, including a p53 functional assay, Sanger sequencing, and NGS.

Main Methods:

  • Re-analysis of breast tumor biopsies using Sanger sequencing and Roche 454 NGS.
  • Direct comparison of the p53 functional assay, Sanger sequencing, and NGS on a cohort of 48 biopsies.
Keywords:
breast cancernext generation sequencingp53 yeast functional assay

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Last Updated: May 9, 2026

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Published on: August 4, 2019

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  • Analysis of pooled cDNA sequencing data for mutation detection.

    • Main Results:

    • Conventional Sanger sequencing of pooled cDNA was insensitive to low-level mutant p53.
    • NGS unequivocally identified mutations in all samples classified as mutant by the functional assay.
    • Small deletions, likely from PCR splicing, contributed to background noise in the functional assay.

    Conclusions:

    • Mutation detection techniques analyzing multiple clones, like the p53 functional assay and NGS, are more reliable than Sanger sequencing of pooled DNA.
    • The high p53 mutation rate observed in the EORTC 10994/BIG 1-00 trial is attributed to the high sensitivity of the functional assay.
    • Roche 454 NGS is a suitable method for identifying p53 mutations in previously tested tumors and for future research.