CCNE1 amplification and centrosome number abnormality in serous tubal intraepithelial carcinoma: further evidence supporting its role as a precursor of ovarian high-grade serous carcinoma

Abstract

Aberration in chromosomal structure characterizes almost all cancers and has profound biological significance in tumor development. It can be facilitated by various mechanisms including overexpression of cyclin E1 and centrosome amplification. As ovarian high-grade serous carcinoma has pronounced chromosomal instability, in this study we sought to determine whether increased copy number of CCNE1 which encodes cyclin E1 and centrosome amplification (>2 copies) occurs in its putative precursor, serous tubal intraepithelial carcinoma. We found CCNE1 copy number gain/amplification in 8 (22%) of 37 serous tubal intraepithelial carcinomas and 12 (28%) of 43 high-grade serous carcinomas. There was a correlation in CCNE1 copy number between serous tubal intraepithelial carcinoma and high-grade serous carcinoma in the same patients (P<0.001). There was no significant difference in the percentage of CCNE1 gain/amplification between serous tubal intraepithelial carcinoma and high-grade serous carcinoma (P=0.61). Centrosome amplification was recorded in only 5 (14%) of 37 serous tubal intraepithelial carcinomas, and in 10 (40%) of 25 high-grade serous carcinomas. The percentage of cells with centrosome amplification was higher in high-grade serous carcinoma than in serous tubal intraepithelial carcinoma (P<0.001). Induced expression of cyclin E1 increased the percentage of fallopian tube epithelial cells showing centrosome amplification. Our findings suggest that gain/amplification of CCNE1 copy number occurs early in tumor progression and precedes centrosome amplification. The more prevalent centrosome amplification in high-grade serous carcinoma than in serous tubal intraepithelial carcinoma supports the view that serous tubal intraepithelial carcinoma precedes the development of many high-grade serous carcinomas.

Fig. 1.

Summary of CCNE1 FISH results in ovarian high-grade serous carcinoma and serous tubal intraepithelial carcinoma. The results are classified into 5 categories and their percentages in different lesions are shown.

Fig. 2.

CCNE1 two-color FISH in a representative serous tubal intraepithelial carcinoma. The H&E of the lesion is shown at the left panel. Red signals: CCNE1 probe; green signal: CEP control probe; blue: nuclear staining with DAPI. There are many serous tubal intraepithelial carcinoma cells exhibiting CCNE1 amplification (big red dots).

Fig. 3.

Centrosome numbers as determined by immunofluorescence in high-grade serous carcinoma (HGSC), serous tubal intraepithelial carcinoma (STIC) and normal fallopian tube epithelium (FTE). A: percentage of lesions showing centrosome amplification in 33 STICs and 25 HGSCs. B: Percentage of tumor cells demonstrating centrosome amplification. All specimens containing normal fallopian tube epithelium do not have centrosome amplification. C: Percentage of tumor cells with centrosome amplification in paired STIC and corresponding HGSC. D. A positive correlation of percentage of tumor cells showing centrosome amplification between STICs and HGSCs (r²= 0.65, P< 0.005).

Fig. 4.

Detection of centrosomes using immunofluorescence in a representative serous tubal intraepithelial carcinoma. The H&E of the lesion is shown at the left panel. Red signals: γ-tubulin (stain centrosomes); green signal: α-tubulin (stains cilia); blue: nuclear staining with DAPI. There are several cells containing big red dots (i.e., centrosome amplification) in serous tubal intraepithelial carcinoma cells at the interface but not in normal fallopian tube epithelium (FTE, inset).

Fig. 5.

The effect of cyclin E1 expression on centrosome numbers on fallopian tube epithelial cells. A. Western blot analysis shows protein levels of cyclin E1 in fallopian tube epithelial cells (FT282-V) and cyclin E1 expressing cells (FT282-CE) which were transfected by a plasmid expressing CCNE1 gene. Cyclin E1 expression in other ovarian cancer cell lines is also shown. B. The percentage of cancer cells with centrosome amplification (> 2 centrosomes/tumor nucleus) in each cell line. FT282-CE cells have significantly higher percentage of cells with centrosome amplification than its parental FT282-V cells (p< 0.005).