The status of epidermal growth factor receptor in borderline ovarian tumours

Abstract

The majority of borderline ovarian tumours (BOTs) behave in a benign fashion, but some may show aggressive behavior. The reason behind this has not been elucidated. The epidermal growth factor receptor (EGFR) is known to contribute to cell survival signals as well as metastatic potential of some tumours. EGFR expression and gene status have not been thoroughly investigated in BOTs as it has in ovarian carcinomas. In this study we explore protein expression as well as gene mutations and amplifications of EGFR in BOTs in comparison to a subset of other epithelial ovarian tumours. We studied 85 tumours, including 61 BOTs, 10 low grade serous carcinomas (LGSCs), 9 high grade serous carcinomas (HGSCs) and 5 benign epithelial tumours. EGFR protein expression was studied using immunohistochemistry. Mutations were investigated by Sanger sequencing exons 18-21 of the tyrosine kinase domain of EGFR. Cases with comparatively higher protein expression were examined for gene amplification by chromogenic in situ hybridization. We also studied the tumours for KRAS and BRAF mutations. Immunohistochemistry results revealed both cytoplasmic and nuclear EGFR expression with variable degrees between tumours. The level of nuclear localization was relatively higher in BOTs and LGSCs as compared to HGSCs or benign tumours. The degree of nuclear expression of BOTs showed no significant difference from that in LGSCs (mean ranks 36.48, 33.05, respectively, p=0.625), but was significantly higher than in HGSCs (mean ranks: 38.88, 12.61 respectively, p< 0.001) and benign tumours (mean ranks: 35.18, 13.00 respectively, p= 0.010). Cytoplasmic expression level was higher in LGSCs. No EGFR gene mutations or amplification were identified, yet different polymorphisms were detected. Five different types of point mutations in the KRAS gene and the V600E BRAF mutation were detected exclusively in BOTs and LGSCs. Our study reports for the first time nuclear localization of EGFR in BOTs. The nuclear localization similarities between BOTs and LGSCs and not HGSCs support the hypothesis suggesting evolution of LGSCs from BOTs. We also confirm that EGFR mutations and amplifications are not molecular events in the pathogenesis of BOTs.

Keywords: EGFR; borderline ovarian tumours.

Figure 1. Expression of EGFR by immunohistochemistry in ovarian tumours

A, B. a case of BOT showing moderate nuclear expression; [A: magnification x10; B: magnification; x20]. C, D. a BOT case showing nuclear expression of variable intensity [C: magnification x20; D: magnification x40]. E. a BOT case showing strong nuclear and moderate cytoplasmic expression [magnification x20]. F. a BOT case with negative nuclear staining and weak cytoplasmic expression [magnification x20].

Figure 2. Representative chromatograms of mutations/SNPs revealed from sequences analysis of EGFR by Sanger sequencing

Red arrows point to the site of mutations/SNPs: A. rs1050171, c.2361 G> A, exon 20. B. rs17290559, c.2508 C>T, exon 21. C. rs41420046, c.2487 G>, exon 21. D. COSM26129 c.2572 C>T (p.L858L), exon 21. These molecular variations were detected only in exons 20 and 21 among BOTs and LGSC.

Figure 3. Representative images for the results of Chromogenic In situ hybridization (CISH) using EGFR specific probe

A. a case of LGSC and B. a case of BOT showing no EGFR gene amplification/aneuploidy [magnification x40]. Six or more brown dots in the nucleus in more than 50% of tumour cells indicate amplification of the EGFR gene, while 3-5 are considered aneuploidy.