Comparison of ERCC1/XPF genetic variation, mRNA and protein levels in women with advanced stage ovarian cancer treated with intraperitoneal platinum

Abstract

Objective: Approximately 20% of patients receiving platinum-based chemotherapy for epithelial ovarian cancer (EOC) are refractory or develop early recurrence. Identifying these patients early could reduce treatment-associated morbidity and allow quicker transfer to more effective therapies. Much attention has focused on ERCC1 as a potential predictor of response to therapy because of its essential role in the repair of platinum-induced DNA damage. The purpose of this study was to accurately measure protein levels of ERCC1 and its essential binding partner XPF from patients with EOC treated with platinum-based therapy and determine if protein levels correlate with mRNA levels, patient genotypes or clinical outcomes.

Methods: ERCC1 and XPF mRNA and protein levels were measured in frozen EOC specimens from 41 patients receiving intraperitoneal platinum-based chemotherapy using reverse transcription polymerase chain reaction and western blots. Genotypes of common nucleotide polymorphisms were also analyzed. Patient outcomes included progression free (PFS) and overall survival (OS).

Results: Expression of ERCC1 and XPF were tightly correlated with one another at both the mRNA and protein level. However, the mRNA and protein levels of ERCC1 were not positively correlated. Likewise, none of the SNPs analyzed correlated with ERCC1 or XPF protein levels. There was an inverse correlation between mRNA levels and patient outcomes.

Conclusion: Neither genotype nor mRNA levels are predictive of protein expression. Despite this, low ERCC1 mRNA significantly correlated with improved PFS and OS.

Fig. 1

ERCC1 and XPF mRNA expression in EOC. (A) Graph showing the levels of ERCC1 mRNA in individual ovarian tumors on the primary vertical axis and XPF mRNA levels on secondary vertical axis. Tumors on the horizontal axis are organized by increasing levels of ERCC1 mRNA. (B) Scatter plot showing the ERCC1 mRNA level vs. the XPF mRNA level in the same tumor. The correlation between mRNA levels was highly significant (r=0.83, p<0.001).

Fig. 2

ERCC1 and XPF protein expression in EOC. (A) Representative immunoblot of protein extracts from tumors to measure ERCC1 and XPF protein levels. Actin was used as a loading control. The asterisks indicate unrelated cross-reacting bands. (B) Graph showing the corrected immunoblot-band intensities for both ERCC1 (white bars) and XPF (grey bars) in each of the EOCs analyzed. The solid black line represents the ratio of band intensities of ERCC1 to XPF. Tumors on the horizontal axis were organized by increasing levels of XPF protein. (C) Scatter plot of corrected XPF protein levels vs. ERCC1 protein levels in individual tumors. The correlation between ERCC1 and XPF protein levels was highly significant (r=0.85, p<0.001).

Fig. 3

Comparison of ERCC1 and XPF mRNA and protein levels. (A) Scatter plot of ERCC1 protein vs. mRNA for individual tumors. There was a significant negative correlation between mRNA and protein levels (r=−0.41, p=0.048); (B) Scatter plot of XPF protein vs. mRNA for individual tumors. There was no correlation between the two; (C) Graph of ERCC1 protein level (bars; primary vertical axis) for individual tumors and ERCC1 mRNA (grey line; secondary vertical axis). Tumors were organized by increasing levels of ERCC1 mRNA; (D) Graph of XPF protein level (bars; primary vertical axis) for individual tumors and XPF mRNA (grey line; secondary vertical axis). Tumors were organized by increasing levels of XPF mRNA.

Fig. 4

Comparison of EOC patient outcomes and ERCC1 or XPF mRNA expression. ERCC1 mRNA expression was categorized as high, medium or low and its impact on (A) PFS and (B) OS was graphed in Kaplan-Meier plots. Similarly, the impact of XPF mRNA expression on (C) PFS and (D) OS were charted.