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. 2019 Oct 1;8(10):1186.
doi: 10.3390/cells8101186.

Clonal Evolution of TP53 c.375+1G>A Mutation in Pre- And Post- Neo-Adjuvant Chemotherapy (NACT) Tumor Samples in High-Grade Serous Ovarian Cancer (HGSOC)

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Free PMC article
Case Reports

Clonal Evolution of TP53 c.375+1G>A Mutation in Pre- And Post- Neo-Adjuvant Chemotherapy (NACT) Tumor Samples in High-Grade Serous Ovarian Cancer (HGSOC)

Marica Garziera et al. Cells. .
Free PMC article

Abstract

Carboplatin/paclitaxel is the reference regimen in the treatment of advanced high-grade serous ovarian cancer (HGSOC) in neo-adjuvant chemotherapy (NACT) before interval debulking surgery (IDS). To identify new genetic markers of platinum-resistance, next-generation sequencing (NGS) analysis of 26 cancer-genes was performed on paired matched pre- and post-NACT tumor and blood samples in a patient with stage IV HGSOC treated with NACT-IDS, showing platinum-refractory/resistance and poor prognosis. Only the TP53 c.375+1G>A somatic mutation was identified in both tumor samples. This variant, associated with aberrant splicing, was in trans configuration with the 72Arg allele of the known germline polymorphism TP53 c.215C>G (p. Pro72Arg). In the post-NACT tumor sample we observed the complete expansion of the TP53 c.375+1G>A driver mutant clone with somatic loss of the treatment-sensitive 72Arg allele. NGS results were confirmed with Sanger method and immunostaining for p53, BRCA1, p16, WT1, and Ki-67 markers were evaluated. This study showed that (i) the splice mutation in TP53 was present as an early driver mutation at diagnosis; (ii) the mutational profile was shared in pre- and post-NACT tumor samples; (iii) the complete expansion of a single dominant mutant clone through loss of heterozygosity (LOH) had occurred, suggesting a possible mechanism of platinum-resistance in HGSOC under the pressure of NACT.

Keywords: HGSOC; NACT; NGS; TP53; chemoresistance.

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Graphical representation of CA-125 levels across the disease/treatment course of the patient diagnosed with HGSOC. The two time points, at D-LPS (pre-NACT) and at IDS (post-NACT), in which blood and ovarian tumor tissue samples were collected, are highlighted. CA-125: cancer antigen 125; D-LPS: diagnostic laparoscopy; NACT: neo-adjuvant chemotherapy; IDS: interval debulking surgery; HGSOC: high-grade serous ovarian cancer.
Figure 2
Figure 2
Graphic representation of VAF observed for SNVs in TP53 identified by NGS in blood (reference) and tumor tissue samples, collected at pre-therapy (pre-NACT) at the D-LPS and post-therapy (post-NACT) at the IDS, from a patient with HGSOC. (a) Bar-graph shows VAF decrease in pre- (27.71%) and post-NACT (4.21%) tumor samples, indicating prevalence of the WT “C” allele (blue bar) compared to the Alt “G” allele (yellow bar) at IDS for the TP53 c.215C>G (p.P72R) SNP, present in heterozygosis (57.31%) in the blood reference sample; (b) Bar-graph shows VAF increase in pre- (50.13%) and post-NACT (94.33%) tumor samples highlighting prevalence of the Alt “A” allele (red bar) compared to the WT “G” allele (green bar) in the HGSOC tissue sample collected at IDS of TP53 c.375+1G>A mutation, absent in the blood reference sample (only WT “G” allele, green bar). VAF: variant allele frequency; NACT: neo-adjuvant chemotherapy; HGSOC: high-grade serous ovarian cancer; D-LPS: diagnostic laparoscopy; IDS: interval debulking surgery; Alt: alternative; WT: wild-type.
Figure 3
Figure 3
Confirmatory Sanger electropherograms of TP53 variants identified by NGS in a patient with HGSOC, in tumor samples collected at D-LPS (pre-NACT) and at IDS (post-NACT), and in reference (germline) blood sample. (a) Sequencing result in the reference blood sample showing polymorphism TP53 c.215C>G in heterozygosis (blue box) and the WT “G” allele at the TP53 position c.375+1G>G (black box); (b) Sequencing result in the tumor sample collected at the D-LPS (pre-NACT) of the polymorphism TP53 c.215C>G (increased peak for WT “C” allele, blue box) and mutation TP53 c.375+1G>A (heterozygosis, red box), in trans configuration; (c) Sequencing result in the tumor sample collected at the IDS (post-NACT) of the polymorphism TP53 c.215C>G indicating homozygosis of the WT “C” allele or loss of the Alt “G” allele (blue box) and mutation TP53 c.375+1G>A indicating homozygosis of the Alt “A” mutated allele or loss of the WT “G” allele (red box), in trans configuration. HGSOC: high-grade serous ovarian cancer; D-LPS: diagnostic laparoscopy; IDS: interval debulking surgery; NACT: neo-adjuvant chemotherapy; SNP: single nucleotide polymorphism; NGS: next-generation sequencing; WT: wild-type.
Figure 4
Figure 4
Representative model of double TP53 SNVs in trans configuration identified in the pre-NACT/chemo-naïve HGSOC sample at D-LPS, with clone expansion and somatic LOH in the post-NACT tumor sample collected at IDS. In normal cells (Blood) at the germline level, only the TP53 SNP c.215C>G was detected; in the chemo-naïve/untreated (Tumor pre-NACT) sample, the splice mutation TP53 c.375+1G>A was present with the minor Alt allele in trans to the WT allele of polymorphism TP53 c.215C>G; in the post-therapy/treated (Tumor post-NACT) sample, the complete clone expansion with somatic LOH was observed with loss of the minor Alt allele for TP53 c.215C>G SNP and loss of the WT allele for TP53 c.375+1G>A mutation. Alt allele of the mutation TP53 c.375+1G>A is represented in red, the reference WT in green; Alt allele of the polymorphism TP53 c.215C>G is colored in orange, the reference WT allele in blue; the minor Alt alleles are in lower-case letters, the WT alleles are in upper-case letters. VAF: variant allele frequency; NACT: neo-adjuvant chemotherapy; HGSOC: high-grade serous ovarian cancer; D-LPS: diagnostic laparoscopy; IDS: interval debulking surgery; Alt: alternative; WT: wild-type; LOH: loss of heterozygosity.
Figure 5
Figure 5
Hematoxilin and eosin (H&E, 5×, 10× and 20× magnification) of HGSOC sections collected at D-LPS (pre-NACT) and at IDS (post-NACT) and immunohistochemical staining for p53 (10×, 20× and 40× magnification). (ac) H&E of bouin-fixed, paraffin-embedded tumor tissue collected at D-LPS showing the serous ovarian carcinoma architecture of HGSOC case with somatic mutation in TP53 (a: 5×; b: 10×; c: 20×); (df) Absent (-) nuclear p53 expression on tumor tissue collected at D-LPS of HGSOC case with somatic mutation in TP53 (d: 10×; e: 20×; f: 40×); (gi) H&E of formalin-fixed, paraffin-embedded tumor tissue collected at IDS showing the serous ovarian carcinoma architecture of HGSOC case with somatic mutation in TP53 (g: 5×; h: 10×; i: 20×); (ln) Absent (-) nuclear p53 expression on tumor tissue collected at IDS of HGSOC case with somatic mutation in TP53 (l: 10×; m: 20×; n: 40×). No cytoplasmic staining was observed. NACT: neo-adjuvant chemotherapy; HGSOC: high-grade serous ovarian cancer; D-LPS: diagnostic laparoscopy; IDS: interval debulking surgery.
Figure 6
Figure 6
Immunohistochemical staining for BRCA1, p16, WT1, and Ki-67 of HGSOC sections collected at D-LPS from a patient diagnosed of HGSOC, before NACT (20× and 40× magnification). (a,b) Positive nuclear staining for BRCA1 observed in ~95% of tumor cells (a: 20×, b: 40×); (c,d) Diffuse nuclear and cytoplasmic staining of p16 exhibited by ~95% of tumor cells (c: 20×, d: 40×); (e,f) Nuclear and cytoplasmic staining for WT1 observed in ~70% of tumor cells (e: 20×, f: 40×);(g,h) Diffuse nuclear staining of Ki-67 observed in ~95% of tumor cells (g: 20×, h: 40×). NACT: neo-adjuvant chemotherapy; HGSOC: high-grade serous ovarian cancer; D-LPS: diagnostic laparoscopy; BRCA1: BRCA1 DNA repair associated; WT1: Wilms tumor 1.

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References

    1. Ferlay J., Colombet M., Soerjomataram I., Mathers C., Parkin D.M., Piñeros M., Znaor A., Bray F. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int. J. Cancer. 2019;144:1941–1953. doi: 10.1002/ijc.31937. - DOI - PubMed
    1. Wiedemeyer W.R., Beach J.A., Karlan B.Y. Reversing Platinum Resistance in High-Grade Serous Ovarian Carcinoma: Targeting BRCA and the Homologous Recombination System. Front. Oncol. 2014;4:34. doi: 10.3389/fonc.2014.00034. - DOI - PMC - PubMed
    1. Siegel R.L., Miller K.D., Jemal A. Cancer statistics, 2015. CA Cancer J. Clin. 2015;65:5–29. doi: 10.3322/caac.21254. - DOI - PubMed
    1. Associazione Italiana di Oncologia Medica (AIOM) Linee Guida Tumori dell’Ovaio 2018. AIOM; Italy: 2018. [(accessed on 12 August 2019)]. Available online: http://www.aiom.it/linee-guida-aiom-2018-tumori-dellovaio/
    1. Winter W.E., Maxwell G.L., Tian C., Carlson J.W., Ozols R.F., Rose P.G., Markman M., Armstrong D.K., Muggia F., McGuire W.P., et al. Prognostic factors for stage III epithelial ovarian cancer: A Gynecologic Oncology Group Study. J. Clin. Oncol. 2007;25:3621–3627. doi: 10.1200/JCO.2006.10.2517. - DOI - PubMed

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