Abstract
Aim: The purpose of the present study was to investigate the possible association between Excision repair cross-complementing group 1 (ERCC1) score and platinum resistance in first-line chemotherapy for ovarian cancer. Patients and Methods: ERCC1 Expression was determined using immunohisto-chemistry in 68 patients with platinum-responding tumor and 30 with platinum-resistant tumors. The primary end-point of this study was the association between the expression of ERCC1 protein with resistance to standard platinum-based chemotherapy in primary ovarian cancer. Results: In pairwise comparisons, the overall survival (OS) for patients with ovarian cancer, who were non-responders to platinum-based chemotherapy with low or intermediate H-score for ERCC1 was better than that of non-responders with high H-score for ERCC1 [median OS=21 (16.8-25.2 months) and 28 (14.6-41.4 months) vs. 15 months (6.2-23.8 months), p-value=0.048, and p-value=0.017, respectively]. Conclusion: There were no significant differences in the progression-free survival between those with low, intermediate and high H-score for ERCC1. There is no statistically significant relationship between ERCC1 score and response to platinum-based chemotherapy in patients with primary ovarian cancer.
The majority of patients with epithelial ovarian cancer have advanced-stage disease at the time of diagnosis. Primary cytoreductive surgery followed by platinum-based chemotherapy is considered to be a standard treatment procedure for patients with advanced ovarian cancer (1). Platinum compounds induce their cytotoxic effect by binding to DNA molecules in the form of a platinum DNA adduct. Although the mechanism of platinum resistance in vivo is not clearly understood, laboratory studies on cancer cell lines suggest that nucleotide excision repair (NER) is the main mechanism responsible for this resistance by increased platinum DNA adduct removal. The NER pathway is a network of many proteins forming the DNA repair system. The excision repair cross complementing-group 1 (ERCC1) gene has the leading role in the NER pathway because of its damage recognition and excision ability (2).
ERCC1 is one subunit of the DNA repair endonuclease ERCC1- xeroderma pigmentosum group F (XPF). The enzyme was discovered for its essential role in NER (3). ERCC1-XPF is also essential for the repair of DNA interstrand crosslinks (ICL) (4, 5), highly cytotoxic lesions induced by bifunctional genotoxins such as cisplatin (6). ERCC1-XPF is the only enzyme required for both NER and interstrand crosslinks (ICL) repair and, therefore, removal of all types of platinum-induced DNA damage (7).
Two single nucleotide polymorphisms (SNPs) of the ERCC1 gene, codon 118 C/T and C8092A, have been reported to be functional, but their influence on platinum resistance and survival is not yet clear (8).
Studies evaluating ERCC1 expression in relationship to patient survival have shown a correlation of its increased expression with reduced survival in lung, colon, pancreatic, gastric, bladder, and esophageal cancer (9-16).
Although platinum-based chemotherapy remains the cornerstone for treatment for ovarian cancer, most patients will experience relapse and will further develop platinum resistance. Platinum resistance is frequently defined as progression during platinum-based chemotherapy or recurrence within six months of completed platinum-based chemotherapy. Pre-clinical and clinical data have suggested a potential use of ERCC1 as a molecular predictor of clinical resistance to platinum-based chemotherapy. ERCC1 is a key in DNA repair mechanisms in tumor cells damaged by treatment with platinum agents (17). Since successful first-line chemotherapy following optimal primary cytoreductive surgery is crucial for a favorable outcome in ovarian cancer, many prognostic and predictive markers have been investigated to individualize the treatment, maximize therapeutic effect, and minimize treatment toxicity. In the present retrospective study, we investigated the possible association between ERCC1 score (immunohistochemical expression of ERCC1 protein) and platinum resistance in first-line chemotherapy for patients with ovarian cancer.
Materials and Methods
A total of 200 patients diagnosed with epithelial ovarian cancer (EOC) in the last three years at Charité University Hospital, Virchow campus were identified. The collective being studied consisted of 100 patients with platinum-sensitive and 100 with platinum-resistant ovarian cancer.
Platinum resistance was defined according to Gynecologic Cancer InterGroup (GCIG) criteria as relapse within six months after platinum-based chemotherapy.
Immunohistrochemistry for ERCC1 was carried out for only 68 platinum-sensitive and 30 platinum-resistant cases. In other cases, the analysis of ERCC1 was not possible because of insufficient pathological specimen or lack of documentation. The final collective of this study consisted of 98 patients. All samples were provided by the ovarian cancer tumor bank (www.toc-network.de) at the Charité medical university.
Immunohistochemistry for ERCC1. Tumor specimens from 98 patients were evaluated for ERCC1 expression. Rabbit monoclonal antibody against ERCC1 (clone EPR7277, dilution 1:50, Abcam, Cambridge, UK) was used to detect the ERCC1 expression in this study.
ERCC1 measurement was performed on a Ventana BENCHMARK® XT Instrument using UltraView DAB kit (Ventana Medical Systems, Tucson, USA). Briefly, for epitope retrieval, slides were exposed to heat EDTA, then endogenous peroxidase activity was blocked by incubation with 3% H2O2 (ERCC1: 30 min EDTA and 4 min H2O2. Primary antibody incubation was carried out for 32 min at 37°C. Immunoreaction was revealed by secondary-antibody incubation for 8 min with 3’-3’-diaminobenzidine as the chromogen and Mayer‘s hematoxylin as the counterstain. Positive and negative controls were run in parallel.
Microscopic analyses. An experienced investigator who was unaware of clinical data evaluated ERCC1 staining under a light microscope. She recorded whether and what percentage of the tumor cells expressed ERCC1. Nuclear immunoreactivity of samples was scored as positive for ERCC1. The staining intensity was graded on a scale of 0-3: 0, no staining; 1, mild staining; 2, moderate staining; 3, strong staining.
The percentage of positive tumor cells was calculated for each specimen, and a proportion score was assigned: 0 if 0%, 0.1 if 1-9%, 0.5 if 10-49%, and 1 if 50% or more. This proportion score was multiplied by the staining intensity of nuclei to obtain an overall score, a final semiquantitative H-score. A score of 3 was assessed as high expression, a score of between 1.5 and 2.5 as intermediate, and a score between 0 and 1 as low expression.
Statistical method. The primary end-point of this study was to investigate the association between the immunohistochemical expression of ERCC1 protein with response to standard chemotherapy combination of carboplatin and paclitaxel in patients with primary ovarian cancer.
The secondary end-points were progression-free survival (PFS) and overall survival (OS). Recurrence was defined as any cinically or histologically confirmed disease recurrence or progression. Death from disease was defined as any complications associated with cancer as well as strict disease mortality. PFS was measured from the date of diagnosis to the date of relapse, progression, death, or last follow up; OS was measured from the date of diagnosis to the date of death or date of last follow-up. Kaplan–Meier event-free survival curves were used to estimate 2-year rates, and the log-rank (Mantel-Cox) test was used to compare the curves for statistical significance.
Results
A total of 200 patients with ovarian cancer in two subgroups (100 patients who were responders to platinum-based chemotherapy and 100 non-responders) were reviewed for this analysis. The final cohort consists of 68 responders and 30 non-responders.
The median age for this cohort was 60 (range=18-79) years. The predominant histological type was serous papillary carcinoma in 92.9% of patients. Overall, 80.6% of patients had stage III disease [according to the International Federation of Obstetricians and Gynaecologists (FIGO) classification system (18)] and 95.9% of them had low-grade disease at the time of primary diagnosis. Optimal debulking was achieved in about three quarters of patients; in 20.4% of patients a residual tumor from 1-10 mm was left. Table I presents the clinical data and demographic information for these patients. All patients were treated with six chemotherapy cycles of carboplatin and paclitaxel with or without bevacizumab.
Overall, 97% out of the 98 cases of primary ovarian cancer showed expression of ERCC1 protein in the immunohistochemical study. The patients were classified into three groups according to the cumulative H-score of ERCC1 expression: Low H-score: 31 patients (31.6%); intermediate H-score: 45 patients (45.9%); and high H-score: 22 patients (22.4%).
Figure 1 shows representative examples of tissues with different scores of ERCC1 expression.
A low H-score for expression of ERCC1 was found in 27.9% of platinum-sensitive cases and in 40% of platinum non-sensitive cases. The rate of intermediate H-score for expression of ERCC1 was comparable in both groups of patients (45.6% vs. 46.7%, respectively); whereas 26.5% of platinum-sensitive patients had a high H-score for expression of ERCC1 in comparison with 13.3% of platinum non-sensitive cases [(OR)=2.34, 95%Confidence Interval (95%CI)=0.65-9.18].
The median OS in the platinum responder group was 65 months (95%CI=62.24-67.76 months), which decreased dramatically in the platinum non-responder group to 21 months (95%CI=16.53-25.47 months, p<0.001). The median OS in the platinum responder group varied according to the H-score for ERCC1 with 59, 76, and 65 months for low, intermediate and high H-score, respectively. In the platinum non-responder group the median OS declined from 21 and 28 months in patients with low and intermediate H-score for ERCC1, respectively, to only 15 months in patients with a high H-score for ERCC1. In pairwise comparisons, was the overall survival for ovarian cancer patients, who were non-responder to platinum-based chemotherapy better with low or intermediate H-score for ERCC1 versus non-responders with high H-score for ERCC1 [median OS=21 (16.8-25.2 months) and 28 (14.6-41.4 months) vs. 15 months (6.2-23.8 months), p-value=0.048, and p-value=0.017, respectively]. Figure 2 shows OS in responders and non-responders according to the different H-score for ERCC1 expression.
The median PFS in platinum responders was 31 months (95%CI=23.32-38.68 months) in comparison with that for platinum non-responders 9 months (95%CI=8.14-9.86 months). There were no significant differences in PFS between the low, intermediate and high H-score of ERCC1 in each group of patients. Figure 3 shows PFS in responders and non-responders according to the different H-score for ERCC1 expression.
Discussion
A number of studies have examined the association between ERCC1 expression (17, 19-22) and clinical outcomes including response to platinum-based therapy, PFS and OS in patients with EOC (23). The outcomes of these studies were discriminative, ranging from increased rate of platinum resistance (24), worse PFS and overall OS (25-27), similar PFS (25-28), to similar OS (25-28).
In our current study, we were unable to confirm any statistically significant association between ERCC1 expression and resistance to platinum-based chemotherapy. PFS and OS did not significantly differ between the different scores for ERCC1 expression. We only noted that the OS in the non-responder group was better in those with low or intermediate H-score for ERCC1 expression in comparison with non-responders with a high H-score.
Stadlmann et al. analyzed 80 samples of ovarian cancer utilizing 8F-1 antibody for immunohistochemy and found low overall ERCC1 expression (20.3%) and no association between protein expression and platinum responsiveness (p=0.21) (21), which correlates with our results.
In a study of Steffensen et al., immunohistochemy with the 8F-1 antibody against ERCC1 was used to examine 100 tumor specimens: 45% of specimens were positive, which was associated with a significantly poorer response to platinum-based chemotherapy but not with a worse OS (20). Lin et al. corroborated these findings by demonstrating that low ERCC1 protein expression was significantly associated with drug sensitivity in 63 patients (29).
Patients' characteristics.
In a further published study of Steffensen and co-workers, ERCC1 protein overexpression was found in 13.9% of tumors. Platinum resistance was found in 75% of tumors with positive ERCC1 protein expression compared with 27% among the patients with negative tumor staining for ERCC1 (p=0.0013). These findings translated into a significant difference in PFS in both univariate (p=0.0012) and multivariate (p=0.006) analyses. They concluded that their data suggest a positive association between positive ERCC1 protein expression and clinical resistance to platinum-based chemotherapy (8).
The presence of platinum DNA adducts, but not ERCC1 mRNA expression, in peripheral blood leukocytes was associated with better survival, but was not an independent predictor of clinical outcome in advanced EOC in a study by Darcy et al. (30). Weberpals et al. provided evidence that patients with low levels of mRNA expression of tumoral ERCC1 had improved OS (22).
In a study of Rubatt et al., in which patients who participated in GOG-172 and GOG-182 trials and provided tumor samples for translational research were included, 27% of tumors were ERCC1-positive. ERCC1 expression was not associated with clinical characteristics or platinum-responsiveness. Women with ERCC1-positive versus-negative tumors had similar median PFS (17.9 months versus 17.5 months, respectively, p=0.59), median OS (52 months versus 47 months, respectively, p=0.30), risk of disease progression [adjusted hazard ratio (HR)=0.90, 95%CI=0.71-1.15, p=0.41), and risk of death (adjusted HR=0.81, 95% CI=0.61-1.07, p=0.14). ERCC1 expression, as measured by immunohistrochemistry, was not associated with single nucleotide polymorphisms (SNPs), in codon 118 and C8092A, of the ERCC1 gene (7).
Imunohistochemical staining for (ERCC1), clone EPR7277, magnification 20×10. a: No staining, score 0; b: mild staining, score 1; c: moderate staining, score 2; d: strong staining, score 3.
The investigation of the relationship between ERCC1 protein expression and platinum resistance, PFS and OS in ovarian cancer, as study end-points, produced conflicting results. This led to increasing concern on studying genotypes of ERCC1.
Krivak et al. successfully evaluated SNPs in codon 118 of ERCC1 in 278 out of 280 of patients with advanced ovarian, peritoneal or tubal cancer, who were treated with carboplatin plus paclitaxel-based combination chemotherapy. They demonstrated that patients with CT or TT genotype compared with the CC genotype at codon 118 had better OS and similar PFS. They hypothesized that the improved survival observed in patients with the T-carrying genotypes may be attributable, at least, in part to enhanced platinum sensitivity resulting from lower levels of ERCC1 transcription, leading to less efficient repair of platinum-induced DNA adducts. Their conclusion was that the polymorphism in codon 118 in the DNA repair gene ERCC1 was an independent predictor for better survival in such patients treated with intravenous carboplatin- and paclitaxel-based chemotherapy. The relationship between the C8092A polymorphisms in ERCC1 and survival was modest, with an effect size that was not always statistically significant (23).
Smith et al. demonstrated that codon 118 genotype was not associated with PFS or OS in a cohort of 178 patients with stage I-IV ovarian cancer who were treated with platinum-based chemotherapy without or in combination with paclitaxel (26). Kim et al. concluded the same after studying 118 epithelial ovarian cancer cases treated with different regimes of platinum-based chemotherapy (27).
Kang et al. studied 60 patients with epithelial ovarian cancer treated with platinum-based chemotherapy after primary cytoreductive surgery, and demonstrated that women with the CT/TT versus CC genotypes in codon 118 in ERCC1 had similar OS but they did not examine the association between the codon 118 polymorphism and PFS (24).
OS in responders and non-responders according to the different H-score for ERCC1 expression.
PFS in responders and non-responders according to the different H-score for ERCC1 expression.
Krivak et al. hypothesized that differences in the minor allelic frequencies in codon 118 in ERCC1, country, type and number of agents included in first-line chemotherapy, stage of disease, clinical end-points, sample sizes, study design as well as clinical management and follow-up schedule, likely contribute, at least in part, to the disparities observed in these various studies (23).
From our study we conclude that there is no statistically significant relationship between the ERCC1 H-score and the response to platinum-based chemotherapy in patients with primary ovarian cancer. A high H-score for ERCC1 in non-responders was a predictive of poor survival. Further larger and multi-centric studies are needed in order to validate these results.
- Received October 13, 2013.
- Revision received November 18, 2013.
- Accepted November 21, 2013.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
