TP53 mutations are found in 30% of breast tumors and are associated with poor prognosis in distinct subtypes of breast cancer. Direct sequencing is commonly used to obtain TP53 mutation status in tumor tissue, but has limitations in detection level and is time-consuming. Methods targeting hotspots is insufficient for TP53 analysis since the mutations are widely spread along the gene1. Here we describe the development of the Ion TorrentTM next-generation semiconductor sequencing and Ion AmpliSeqTM technology (Life TechnologiesTM).

1. Assessment of TP53 Mutation Status in Breast Tumor
Tissue using the "Ion Ampliseq™ TP53 Panel"
Laxmi Silwal-Pandit1,2, Einar Rødland2, Astrid M Dalsgaard1, Inger Riise Bergheim2, Suet-Feung Chin3,4, Carlos Caldas3,4,
Anne-Lise Børresen-Dale1,2 and Anita Langerød1,2
1Department of Genetics, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Norway. 2The K.G. Jebsen Center for Breast Cancer Research, Institute for Clinical
Medicine, Faculty of Medicine, University of Oslo, Norway. 3Cancer Research UK, Cambridge Institute, UK. 4Department of Oncology, University of Cambridge, UK.
Aim
The aim was to develop and evaluate a Next Generation
Sequencing method for TP53 mutation analysis covering the huge
spectrum of mutations in the gene, and with a sequencing depth
facilitating the study of clonal and subclonal variants in breast
tumor tissue.
Material & Methods
Study design:
The verification of the panel has been performed in two phases, and
here we present the results of phase 1 and 2A (Fig.1). DNA from fresh
frozen samples (METABRIC cohort) previously characterized by Sanger
sequencing were analyzed1.
Panel design:
Non-overlapping primers (Fig. 2) were designed to provide:
• Full coverage of all coding exons and exon-intron boundaries
• Overlapping amplicons covering all exons
• Minimal off-target sequencing
• No SNPs in the last five nucleotides of primer
• Amplification of DNA extracted from FFPE material (Fig.2)
Sequencing & data analysis:
The sequencing were carried out following standard protocol for the
Ion PGM™ Sequencer. (Fig.3) Data analysis was performed on Ion
Reporter TM Software 4.0 using the TP53 panel workflow.
Results
The new panel covers TP53 coding regions comprehensively. It is
comprised of 24 primer pairs across 2 pools requiring 20 ng of DNA.
The design of short amplicons (73 to 139 bp) permits the amplification
of DNA from FFPE.
Phase 1 were carried out loading 10 samples on an Ion 318™ chip,
giving a high coverage (>500x) in 95% of the sequenced amplicons.
Average coverage uniformity was 88% for all samples and the
percentage of mapped reads on target was 97%. The overall sensitivity
was 95% (Table 1).
In Phase 2A 200 samples were analyzed loading 20 samples per 318™
chip, giving a high coverage (>500x) in 97% of sequenced amplicons.
Average coverage uniformity was 86% for all samples and the
percentage of mapped reads on target was 94%. The overall sensitivity
was 90% (Table 2). Three large deletions were not detected, whereas
two new subclonal base substitutions were found (double mutations).
Conclusion
Most mutations detected by Sanger Sequencing were also
detected by the TP53 panel, but large deletions failed to be
identified. Very few subclonal mutations were seen using ultra-
deep sequencing. The Ion Reporter facilitates data analysis for
clinical research labs with limited bioinformatic resources.
Introduction
TP53 mutations are found in 30% of breast tumors and are associated
with poor prognosis in distinct subtypes of breast cancer. Direct
sequencing is commonly used to obtain TP53 mutation status in tumor
tissue, but has limitations in detection level and is time-consuming.
Methods targeting hotspots is insufficient for TP53 analysis since the
mutations are widely spread along the gene1. Here we describe the
development of the Ion Torrent™ next-generation semiconductor
sequencing and Ion AmpliSeq™ technology (Life Technologies™).
Figure 3: Ion AmpliSeq™ Workflow
Acknowledgements
We would like to acknowledge Rosella Petraroli , Chrysanthi Ainali, and Alain
Rico at Thermo Fisher Scientific for great collaboration during the project.
Figure 2: Ion AmpliSeq ™ TP53 Panel design
International p53 Workshop, July 15-19, 2014, Stockholm
Reference: 1Silwal-Pandit et al, Clin Cancer Res; 20(13) July 1, 2014.
Figure 1: Study design
Phase 1
Analysis parameter optimization
Reproducibility study
• 30 breast tumor samples
• Analyzed in 2 labs
• Known mutation status
• Selected cohort (20 mut, 10 wt)
Phase 2A
Extended performance verification
• 200 breast tumor samples
• Known mutation status
• Unselected cohort (with regard
to mutation status)
Phase 2B
• Additional 200 tumor samples
Mutation type Mutations (no.) Miscalls (no.) Sensitivity (%)
Base substitution 10a 0 -
Deletion 6 0 -
Insertion 5 1b -
Complex (in+del) 0 0 -
TOTAL 21 8 95%
Mutation type Mutations (no.) Miscalls (no.) Sensitivity (%)
Base substitution 43 2 -
Deletion 12 3ab -
Insertion 3 0 -
Complex (ins+del) 1 1c -
TOTAL 59d 6 90%
Table 1: Sensitivity, phase 1
Table 2: Sensitivity, phase 2A
a18bp deletions close to amplicon end misaligned to reference.
b24bp deletion at amplicon end; no sequence on opposite side to allow calling.
c Complex mutation (ins+del) visible in sequence data, but not called.
d Totally 53 different type of mutations across the gene.
aOne mut previously detected by TTGE, and not by Sanger Seq, was characterized.
bComplex duplication of 18bp.