Assessing mutant p53 in primary high-grade serous ovarian cancer using immunohistochemistry and massively parallel sequencing

Abstract

The tumour suppressor p53 is mutated in cancer, including over 96% of high-grade serous ovarian cancer (HGSOC). Mutations cause loss of wild-type p53 function due to either gain of abnormal function of mutant p53 (mutp53), or absent to low mutp53. Massively parallel sequencing (MPS) enables increased accuracy of detection of somatic variants in heterogeneous tumours. We used MPS and immunohistochemistry (IHC) to characterise HGSOCs for TP53 mutation and p53 expression. TP53 mutation was identified in 94% (68/72) of HGSOCs, 62% of which were missense. Missense mutations demonstrated high p53 by IHC, as did 35% (9/26) of non-missense mutations. Low p53 was seen by IHC in 62% of HGSOC associated with non-missense mutations. Most wild-type TP53 tumours (75%, 6/8) displayed intermediate p53 levels. The overall sensitivity of detecting a TP53 mutation based on classification as 'Low', 'Intermediate' or 'High' for p53 IHC was 99%, with a specificity of 75%. We suggest p53 IHC can be used as a surrogate marker of TP53 mutation in HGSOC; however, this will result in misclassification of a proportion of TP53 wild-type and mutant tumours. Therapeutic targeting of mutp53 will require knowledge of both TP53 mutations and mutp53 expression.

Figure 1. Flow diagram summarising inclusion and exclusion criteria from the primary tumour cohort.

Eighty-eight primary ovarian tumours underwent MPS for TP53. Two samples were excluded as paired p53 IHC was unable to be obtained. Upon pathological reassessment, 4 samples were excluded due to the identification of clear cell components. A further 6 samples were excluded as they contained <5% tumour cells based on hematoxylin and eosin staining. Of the 76 remaining samples, 72 were HGSOC and 4 were LGSOC.

Figure 2. Distribution of TP53 mutations.

Grey exons mark the un-translated regions (UTR), light blue exons denote the coding regions, while dark blue exons within the coding region correlate with the DNA binding domain. The dark red region marks the p53 response to DNA damage through apoptosis area. Intronic areas are shown by the black line joining exons. All 68 mutations identified in this study are displayed as lollypop symbols, with missense mutations above the gene marked as green circles, while all ‘other’ mutation types (nonsense, frameshift, in-frame insertion and splice site) are displayed below the gene as open red circles. Mutations occurring more than once are indicated.

Figure 3. Comparison of the ability of next generation and Sanger sequencing to detect TP53 mutations.

Top panels (a–c) display MPS data viewed using IGV software (i). Lower panels display Sanger sequencing (SS) chromatograms of mutant and wild-type reference samples viewed using Sequencher software (ii). Red arrows denote presence of mutations in SS, initially discovered by MPS. (a) Shows four mutations with frequencies of the mutant allele between 26–89% determined by MPS that were able to be detected by SS. (b) Shows five mutations with frequencies of the mutant allele between 13–29%, which were initially not detected by SS; however, with the knowledge of MPS results, were identified in the chromatograms. (c) Shows two samples with frequencies of the mutant allele between 16–18% determined by MPS that were unable to be detected at all in SS chromatograms.

Figure 4. p53 immunohistochemistry and TP53 status in HGSOC and LGSOC.

(a) Immunohistochemistry scores (‘Low’, ‘Intermediate’ or ‘High’) correlated with TP53 mutation status. (b) Representative images of p53 immunohistochemistry showing (i) ‘Low’, (ii) ‘Intermediate’ and (iii) ‘High’, scores.

Figure 5. Distribution of non-missense low and high expression TP53 mutations.

Grey exons mark the un-translated regions (UTR), light blue exons denote the coding regions, while dark blue exons within the coding region correlate with the DNA binding domain. The dark red region marks the p53 response to DNA damage through apoptosis area. Intronic areas are shown by the black line joining exons. All 26 non-missense mutations identified in our cohort are displayed along TP53. Mutations that resulted in accumulated p53 are identified with open orange circles. The mutation that resulted in ‘Intermediate’ p53 staining is identified as a yellow triangle. Mutations that resulted in loss of p53 expression are identified as closed purple circles. Mutations occurring more than once are indicated.

Figure 6. Correlation of wild-type and mutant p53 levels with TP53 mutation status (wild-type, missense and all ‘other’ mutations).

While all missense TP53 mutants resulted in ≥70% tumour cell nuclei staining positive for p53, there was considerable variation amongst p53 staining patterns for wild-type (including both HGSOC and LGSOC) and non-missense TP53 mutations. While the majority of wild-type TP53 samples clustered between 8–65% tumour cell nuclei staining positive for p53 (denoted by the broken red line), one HGSOC sample demonstrated Low staining (#695-11; 0%) and one High (#493-10; 100%). Furthermore, there were two distinct clusters of non-missense mutations, with tumour cell nuclei staining positive for p53 between 80–100% and 0–4%, that could not further be discriminated by whether the mutations were nonsense, splice site, an in-frame insertion or frameshifts. Furthermore, a single nonsense mutation displayed 55% of tumour cell nuclei staining positive for p53 (#353-09).

Figure 7. p53 protein and transcript levels in previously uncharacterized HGSOC cell lines (OV167 and OV202) and clear cell carcinoma cell line (OV207) compared to a wild-type p53 cell line (A2780).

(a) Representative Western blots of basal levels of p53 and phosphorylated p53 (Ser15) (p-p53). (b) Graphical representation of protein levels of basal p-p53 in TP53 mutant ovarian cancer cell lines relative to A2780 (N = 3, mean ± S.E.M, **P < 0.01). (c) Graphical representation of TP53 transcript levels relative to the reference gene HMBS in ovarian cancer cell lines compared to A2780 (N = 3, mean ± S.E.M, *P < 0.05). (d) TP53 mutations identified by our Fluidigm-MiSeq analysis pipeline in previously uncharacterised ovarian cancer cell lines.