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. 2018 May 18;19(5):1510.
doi: 10.3390/ijms19051510.

Identification of Novel Somatic TP53 Mutations in Patients With High-Grade Serous Ovarian Cancer (HGSOC) Using Next-Generation Sequencing (NGS)

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Case Reports

Identification of Novel Somatic TP53 Mutations in Patients With High-Grade Serous Ovarian Cancer (HGSOC) Using Next-Generation Sequencing (NGS)

Marica Garziera et al. Int J Mol Sci. .
Free PMC article

Abstract

Somatic mutations in TP53 are a hallmark of high-grade serous ovarian cancer (HGSOC), although their prognostic and predictive value as markers is not well defined. Next-generation sequencing (NGS) can identify novel mutations with high sensitivity, that may be repurposed as potential druggable anti-cancer targets and aid in therapeutic decisions. Here, a commercial NGS cancer panel comprising 26 genes, including TP53, was used to identify new genetic markers of platinum resistance and patient prognosis in a retrospective set of patients diagnosed with epithelial ovarian cancer. Six novel TP53 somatic mutations in untreated tumors from six distinct patients diagnosed with HGSOC were identified: TP53 c.728_739delTGGGCGGCATGA (p.Met243_Met247del, in-frame insertion or deletion (INDEL); TP53 c.795_809delGGGACGGAACAGCTT (p.Gly266_Phe270del, in-frame INDEL); TP53 c.826_827GC>AT (p.Ala276Ile, missense); TP53 c.1022insT (p.Arg342Profs*5, frameshift INDEL); TP53 c.1180delT (p.Ter394Aspfs*28, frameshift INDEL); and TP53 c.573insT (p.Gln192Serfs*17, frameshift INDEL). Novel TP53 variants were validated by classical sequencing methods and their impact on protein expression in tumors explored by immunohistochemistry. Further insights into the potential functional effect of the mutations were obtained by different in silico approaches, bioinformatics tools, and structural modeling. This discovery of previously unreported TP53 somatic mutations provides an opportunity to translate NGS technology into personalized medicine and identify new potential targets for therapeutic applications.

Keywords: TP53 gene mutations; high-grade serous ovarian cancer (HGSOC); next-generation sequencing (NGS).

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Distribution of novel somatic mutations detected by NGS along the human TP53 gene in HGSOC. UTR: untranslated region; CDS: coding sequence; DBD: DNA binding domain; OD: oligomerization domain; INDEL: insertion/deletion; Ala: alanine; Ile: isoleucine; Arg: arginine; Pro: proline; Ter: termination; Asp: aspartic acid; Gln: glutamine; Ser: serine; fs: frameshift.
Figure 2
Figure 2
Sanger sequencing validation of novel TP53 somatic mutations discovered in HGSOC by NGS. The chromatograms show the sequences of novel TP53 mutations relative to genomic DNA from tumor tissue samples (Mutant) and the wt sequence related to genomic DNA isolated from PBMCs (Reference) from the matched patient. The position of nucleotide substitution is indicated by a red arrow. (a) Case ID 305, novel heterozygous somatic mutation in exon 7 (in-frame INDEL). The 12 deleted nucleotides are highlighted in the reference chromatogram; (b) Case ID 519, novel heterozygous somatic mutation in exon 8 (in-frame INDEL). The 15 deleted nucleotides deleted are highlighted in the Reference chromatogram; (c) Case ID 627, novel heterozygous somatic mutation in exon 7 (missense mutation); (d) Case ID 738, novel heterozygous somatic mutation in exon 10 (frameshift INDEL); (e) Case ID 751, novel heterozygous somatic mutation in exon 11 (frameshift INDEL); (f) Case ID 761, novel heterozygous somatic mutation in exon 6 (frameshift INDEL). del: deletion; ins: insertion.
Figure 2
Figure 2
Sanger sequencing validation of novel TP53 somatic mutations discovered in HGSOC by NGS. The chromatograms show the sequences of novel TP53 mutations relative to genomic DNA from tumor tissue samples (Mutant) and the wt sequence related to genomic DNA isolated from PBMCs (Reference) from the matched patient. The position of nucleotide substitution is indicated by a red arrow. (a) Case ID 305, novel heterozygous somatic mutation in exon 7 (in-frame INDEL). The 12 deleted nucleotides are highlighted in the reference chromatogram; (b) Case ID 519, novel heterozygous somatic mutation in exon 8 (in-frame INDEL). The 15 deleted nucleotides deleted are highlighted in the Reference chromatogram; (c) Case ID 627, novel heterozygous somatic mutation in exon 7 (missense mutation); (d) Case ID 738, novel heterozygous somatic mutation in exon 10 (frameshift INDEL); (e) Case ID 751, novel heterozygous somatic mutation in exon 11 (frameshift INDEL); (f) Case ID 761, novel heterozygous somatic mutation in exon 6 (frameshift INDEL). del: deletion; ins: insertion.
Figure 3
Figure 3
p53 immunohistochemistry of HGSOC samples carrying a novel TP53 somatic mutation (20× and 40× magnification). (a,b) Case ID 305 (in-frame INDEL mutation), overexpression of nuclear p53 (~80% of positive tumor cells) with high intensity (++); (c,d) Case ID 519 (in-frame INDEL mutation): overexpression of nuclear p53 (~80% positive tumor cells) with high intensity (++); (e,f) Case ID 627 (missense mutation), strong overexpression of nuclear p53 (~100% positive tumor cells) with very high intensity (+++); (g,h) Case ID 738 (frameshift INDEL mutation), underexpression of nuclear p53 equivalent to p53 (−) (<5% of tumor cells with weak staining (−/+)); (i,l) Case ID 751 (frameshift INDEL mutation), moderate nuclear p53 overexpression (~60% positive tumor cells) with ~30% with moderate (+) and ~30% high (++) intensity; (m,n) Case ID 761 (frameshift INDEL mutation), completely absent nuclear p53 expression (−). No cytoplasmic p53 staining was observed. del: deletion; Met: methionine; Gly: glycine; Phe: phenylalanine; Ala: alanine; Ile: isoleucine; Arg: arginine; Pro: proline; Ter: termination; Asp: aspartic acid; Gln: glutamine; Ser: serine; fs: frameshift.

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