Excision Repair Cross-complementation Group 1 Expression Levels Research Articles

Expression of astrocyte-elevated gene-1 indicates prognostic value of fluoropyrimidine-based adjuvant chemotherapy in resectable stage III colorectal cancer.

It is unclear which prognostic factor such as pathological features and gene mutation are majorly relevant for stage III disease and whether they aid in determining patients who will be benefit from postoperative adjuvant chemotherapy. The expression of astrocyte-elevated gene-1 (AEG-1), thymidylate synthase (TS), excision repair cross-complementation group 1 (ERCC1), epidermal growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF) was examined to investigate their role in adjuvant chemotherapy for patients with resectable stage III colorectal cancer (CRC). A significant positive correlation was observed between AEG-1, TS, ERCC1, EGFR, and VEGF gene expression levels in CRC cell lines, and low AEG-1 and TS expression were highly sensitive to 5-fluorouracil treatment. Our results showed that AEG-1 expression was high in T4 and caused CRC recurrence or metastasis. Patients with T4, high AEG-1, TS and VEGF expression had a significantly short disease-free survival and overall survival. In multivariate Cox regression analysis, high AEG-1 expression could be an independent prognostic factor indicating poor survival in patients with resectable stage III CRC treated with adjuvant chemotherapy. In conclusion, AEG-1 expression and tumor grade are potential prognostic factors for recurrence and survival in patients with stage III CRC receiving adjuvant fluoropyrimidine-based chemotherapy.

Abstract 353: Expression of astrocyte-elevated gene-1 is associated with prognostic value of adjuvant chemotherapy in resectable stage III colorectal cancer

Abstract Background: Administration of postoperative adjuvant chemotherapy has reduced the risk of tumor recurrence and improved survival for patients with resected colorectal cancer (CRC). Regarding the prognostic factors, including pathologic features and gene mutation have major relevance for stage III disease, it is not well known whether they might help select patients who would fare favorably when received adjuvant chemotherapy. OBJECTIVE: Expression of astrocyte-elevated gene-1 (AEG-1), thymidylate synthase (TS), excision repair cross-complementation group 1 (ERCC1), epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF) were needed to investigate the role involved in adjuvant combination therapy for patients with resectable stage III colon cancer. Methods: AEG-1, TS, ERCC1, EGFR and VEGF were investigated by CRC cell lines and intra-tumor immunohistochemistry stain. Furthermore, this study collected 29 patients with stage III colorectal cancer who underwent surgical resection from 2011 to 2016 and received postoperative adjuvant chemotherapy. The culmination of clinical data registration included surgical procedures, tumor size, number of lymph node metastases, and types of chemotherapy drugs. Result: A significant positive correlation between AEG-1, TS, ERCC1, EGFR, and VEGF gene expression levels were revealed in CRC cell lines. Cell lines with lower AEG-1 and TS expression had higher sensitivity to 5-FU treatment. In clinical studies, we found that higher AEG-1 expression was noted in T4 and also caused CRC recurrence or metastasis. Patients with T4 and stage IIIC CRC had a significantly shorter DFS (T4 v.s. T1-3, DFS, months, median: 4.1 ± 1.2 v.s. non-reach [NR], p < 0.001; IIIC v.s. IIIA or IIIB, DFS, months, median: 10.6 ± 5.3 v.s. NR, p = 0.037; respectively). Patients with higher AEG-1 and TS expression had a shorter DFS (AEG-1, high v.s. low, months, median: 12.8 ± 2.4 v.s. NR, p = 0.003; TS, high v.s. low, months, median: 13.8 ± 2.3 v.s. NR, p = 0.012; respectively). In multivariate Cox Regression analysis, advanced tumor invasion (T4) and high AEG-1 expression could be an independent prognostic factor to cause poor survival in resectable stage III CRC patients treated with adjuvant chemotherapy. Conclusion: Expressions of AEG-1 and tumor grade as a candidate prognostic factor of recurrence in stage III colorectal cancer patients receiving adjuvant fluoropyrimidine-based chemotherapy. A prospective cohort study should be guaranteed to validate these factors to be predictive markers of adjuvant chemotherapy benefit for stage III CRC. Citation Format: Chung-Yu Chen, Peng-Sheng Lai, Ying-Yin Chen, Long-Wei Lin. Expression of astrocyte-elevated gene-1 is associated with prognostic value of adjuvant chemotherapy in resectable stage III colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 353.

Predictive Value of Excision Repair Cross-Complementation Group 1 in the Response to Platinum-Based Chemotherapy in Esophageal Cancer: A Meta-Analysis

Introduction: Platinum is widely used in the treatment of esophageal cancer. In clinical practice, it is significant to distinguish patients who respond to platinum from those who do not. Excision repair cross-complementation group 1 (ERCC1) is thought to be the key in the resistance to platinum. However, whether it is related to the platinum-based chemotherapy response on real esophageal cancer patients is controversial. We conducted this meta-analysis to explore the association between ERCC1 polymorphisms, its expression levels and platinum-based chemotherapy response, and identify the most sensitive genotypes. Methods: The study was carried out according to the Cochrane handbook for systemic reviews of intervention. The study protocol has been registered on PROSPERO. Results: Three studies were included in the analysis of C8092A polymorphisms, 5 in the C118T, and another 6 in ERCC1 expression levels. In C118T polymorphisms, compared to wild genotype, patients with mutant genotypes had a significantly higher response rate. As for C8092A polymorphisms, the mutant genotypes also presented a better response than the wild genotype. The pooled analysis indicated a significantly higher response rate in patients with a low expression of ERCC1. Conclusions: ERCC1 is a valuable biomarker for platinum-based chemotherapy in esophageal cancer. Patients with ERCC1 mutations or low-level ERCC1 expression are more sensitive to platinum-based chemotherapy.

EGFR exon 19-deletion aberrantly regulate ERCC1 expression that may partly impaired DNA damage repair ability in non-small cell lung cancer.

BackgroundEpidermal growth factor receptor (EGFR) activating mutations are usually associated with DNA damage repair (DDR) deficiency. However, the precise mechanism has remained elusive. In this study, we aimed to investigate whether EGFR exon 19 deletion mutation downstream signals contributed to DDR deficiency by downregulation of excision repair cross‐complementation group‐1 (ERCC1), a key factor in DDR, expression and function.MethodsWe first measured cell survival, DNA damage (γ‐H2AX foci formation) and damage repair (ERCC1 and RAD51 foci formation) ability in response to DNA cross‐linking drug in EGFR exon 19 deletion and EGFR wild‐type cells separately. We then investigated the involvement of EGFR downstream signals in regulating ERCC1 expression and function in EGFR exon 19 deletion cells as compared with EGFR wild‐type ones.ResultsWe observed increased γ‐H2AX, but impaired ERCC1 and RAD51 nuclear foci formation in EGFR exon 19 deletion cells as compared with EGFR wild‐type ones treated with DNA cross‐linker. In addition, we identified that inhibition of EGFR exon 19 deletion signals increased ERCC1 expression, whereas blocked wild‐type EGFR signals decreased ERCC1 expression, on both mRNA and protein levels. Furthermore, EGFR exon 19 deletion downstream signals not only inhibited ERCC1 expression but also influenced ERCC1 foci formation in response to DNA cross‐linker.ConclusionOur findings indicated that the aberrant EGFR exon 19 deletion signals were not only associated with decreased expression of ERCC1 but were also involved in impaired ERCC1 recruitment in response to DNA cross‐link damage, thereby providing us with more evidence for exploring the mechanism of DDR deficiency in EGFR mutant NSCLC.

The age-related expression decline of ERCC1 and XPF for forensic age estimation: A preliminary study

The age-related capacity decline of DNA damage repair in human peripheral blood has been demonstrated. Excision repair cross-complementation group1 (ERCC1) and Xeroderma pigmentosum complementation group F (XPF) were rate-limiting enzyme in nucleotide excision repair (NER) which was known as the most important DNA damage repair system. Consequently, we hypothesized that the expression and/or activity of ERCC1 and XPF may be associated with age. However, little was known about the quantitative relationship of ERCC1 and XPF expression levels with age. The aim of the present study was to analyze the correlation of ERCC1 and XPF expression levels with age by detecting the ERCC1 and XPF mRNA levels in peripheral blood mononuclear cells(PBMCs) and protein levels in plasma in healthy ethnic Han Chinese individuals, and finally find new molecular markers for forensic age estimation by establishing the mathematical model between ERCC1 and XPF expression levels and age. The results showed that the ERCC1 and XPF mRNA relative expression levels in PBMCs declined in an age-dependent manner (r=-0.578/-0.844, respectively, P<0.01). The formula for age estimation based on the ERCC1 and XPF mRNA relative expression levels decline in PBMCs were Y=3.3E-5x2-0.0261x+1.9175 (R2=0.3244, P<0.01) and Y=0.0003x2-0.0459x+2.0439 (R2=0.729, P<0.01), respectively. There were no significant differences of the ERCC1 or XPF protein expression levels in plasma between age groups (P>0.05). Furthermore, there were no significant differences of the ERCC1 or XPF mRNA and/or protein expression levels between males and females(P>0.05). It suggested that the ERCC1 and XPF mRNA expression levels could be considered as valuable additional tool in individual age estimation, especially in cases where traditional morphologic method was inefficient or absent in forensic practice.

Evaluating the Prognostic Value of ERCC1 and Thymidylate Synthase Expression and the Epidermal Growth Factor Receptor Mutation Status in Adenocarcinoma Non-Small-Cell Lung Cancer.

The present study evaluated the prognostic value of the epidermal growth factor receptor (EGFR) mutation status, and excision repair cross-complementation group 1 (ERCC1) and thymidylate synthase (TS) expression following intercalated tyrosine kinase inhibitor (TKI) therapy and platinum- and pemetrexed-based chemotherapies (subsequent second-line treatment) for patients with adenocarcinoma non-small-cell lung cancer (AC-NSCLC). In total, 131 patients with AC-NSCLC were enrolled. The EGFR mutation status and ERCC1 and TS expression were evaluated through direct DNA sequencing and immunohistochemical analyses, respectively. The EGFR mutation status and ERCC1 and TS expression were the significant predictors of clinical outcomes. The EGFR mutation status was the main outcome predictor for overall survival (OS) benefits in the overall population. Further exploratory ERCC1 and TS expression analyses were conducted to provide additional insights. Low TS expression was predictive of improved OS of patients with negative EGFR-mutated advanced AC-NSCLC, whereas high ERCC1 expression resulted in poor OS in patients with positive EGFR-mutated advanced AC-NSCLC. TS and ERCC1 expression levels were effective prognostic factors for negative and positive EGFR-mutated AC-NSCLC, respectively. In conclusion, the present results indicate that the EGFR mutation status and TS and ERCC1 expression can be used as the predictors of OS after subsequent second-line treatments for AC-NSCLC.

Analysis of ERCC1, BRCA1, RRM1 and TUBB3 as predictors of prognosis in patients with non-small cell lung cancer who received cisplatin-based adjuvant chemotherapy: A prospective study.

Although adjuvant platinum-based chemotherapy has been demonstrated to improve survival in patients with completely resected non-small cell lung cancer (NSCLC), individualized approaches to therapy are urgently required to improve the treatment efficacy and reduce unnecessary toxicity. It was hypothesized in the present study that the protein levels of excision repair cross-complementation group 1 (ERCC1), breast cancer 1 (BRCA1), ribonucleotide reductase M1 (RRM1) and class III β-tubulin (TUBB3) may influence the therapeutic effect of adjuvant cisplatin-based chemotherapy. The expression of ERCC1, BRCA1, RRM1 and TUBB3 in tissues obtained from 84 patients with NSCLC was analyzed in the present non-interventional study by immunohistochemistry prior to adjuvant chemotherapy. All patients received adjuvant cisplatin-based chemotherapy. The primary endpoint in the present study was disease free survival (DFS). Out of the 84 tumors, the expression of ERCC1, BRCA1, RRM1 and TUBB3 was identified in 46 (55%), 11 (13%), 73 (87%) and 76 (90%) tissues, respectively. A beneficial response to adjuvant cisplatin-based chemotherapy in DFS was associated with the absence of the expression of ERCC1 [hazard ratio (HR), 2.166; 95% confidence interval (CI), 1.049-4.474; P=0.037] and BRCA1 (HR, 2.419; 95% CI, 1.127-5.193; P=0.023), but not with the expression status of RRM1 (HR, 0.568; 95% CI, 0.234-1.379; P=0.212) or TUBB3 (HR, 1.874; 95% CI, 0.448-7.842; P=0.39). In addition, patients lacking the expression of ERCC1 and BRCA1 benefited more from adjuvant cisplatin-based chemotherapy compared with patients that expressed either ERCC1 or BRCA1 (HR, 3.102; 95% CI, 1.343-7.163; P=0.008). The expression of ERCC1 and BRCA1 was significantly associated with the DFS time in patients with NSCLC treated with adjuvant cisplatin-based chemotherapy, respectively. The combination of the ERCC1 and BRCA1 expression levels may be a promising prognostic prediction for adjuvant cisplatin-based chemotherapy.

Predictive Role of ERCC1 Expression in Head and Neck Squamous Cell Carcinoma Patients Treated with Surgery and Adjuvant Cisplatin-Based Chemoradiation

Objective: ERCC1 (excision repair cross-complementation group 1) expression predicts survival in patients with locally advanced head and neck squamous cell carcinoma (HNSCC) treated with chemoradiation. In order to evaluate the predictive role in the adjuvant setting, we investigated ERCC1 expression in radically resected HNSCC patients who underwent surgery and cisplatin chemoradiation. Methods: ERCC1 expression levels were determined by immunohistochemistry in primary tumor tissues from 48 patients with stage III-IV cancers. The median follow-up was 38.5 months (range: 5-121). Results: High ERCC1 expression was observed in 36 (75%) patients. Univariate analysis showed that patients with high levels of ERCC1 had significantly worse disease-free survival and overall survival (OS) than patients with low levels (HR = 7.15; 95% CI, 1.68-30.35; p = 0.008 and HR = 9.90; 95% CI, 1.33-73.96; p = 0.025, respectively). In the multivariate analysis, high ERCC1 expression (HR = 7.36; 95% CI, 1.72-31.4; p = 0.007) together with high-risk category (HR = 2.69; 95% CI, 1.01-7.18; p = 0.048) were the best predictors for relapse. High ERCC1 expression was the only unfavorable independent determinant for OS (HR = 9.53; 95% CI, 1.27-71.35; p = 0.028). Conclusions: This investigation suggests that ERCC1 expression might be useful to predict prognosis in radically resected HNSCC patients treated with surgery and chemoradiation.

ERCC1 expression levels predict the outcome of platinum-based chemotherapies in advanced bladder cancer

The objective of this study was to provide a precise evaluation of whether expression levels of excision repair cross-complementation group 1 (ERCC1) are associated with objective response, overall survival (OS), and median survival in patients with advanced bladder cancer treated with platinum-based chemotherapy. Systematic computerized searches of the electronic databases PubMed, EMBASE, Ovid, ASCO, and CNKI were performed and a meta-analysis was carried out to evaluate the correlation between ERCC1 expression levels and objective response rate, OS, or progression-free survival in patients with advanced bladder cancer receiving platinum-based chemotherapy. References within the articles identified were also searched manually. STATA package version 11.0 was used for the comprehensive quantitative analyses. A total of six studies involving 356 patients, of which ERCC1 expression was high/positive in 138 (38.8%) and low/negative in 218 (61.2%), were included in the meta-analysis. The median age of the patients was 63.7 years. The objective response rate favored patients with ERCC1 low/negative expression after platinum-based chemotherapy, but showed no significant difference [odds ratio 0.86, 95% confidence interval (CI) 0.36-2.06, P=0.734]. The median OS time and the median progression-free survival time were significantly prolonged when ERCC1 low/negative expression was compared with ERCC1 high/positive expression (hazard ratio 0.69, 95% CI 0.54-0.89, P=0.004, and hazard ratio 0.76, 95% CI 0.66-0.89, P=0.000, respectively). In conclusion, low/negative expression of ERCC1 was associated with higher objective response, median progression-free survival, and median OS in patients with advanced bladder cancer treated with platinum-based chemotherapy. ERCC1 may be a suitable marker of prognosis and sensitivity to platinum-based chemotherapy in patients with advanced bladder cancer. Larger studies and further clinical trials are warranted to confirm these findings.

The Relationship Between ERCC1 Expression and Clinical Outcome in Patients With FIGO Stage I to Stage II Uterine Cervical Adenocarcinoma

Several studies have suggested that excision repair cross-complementation group 1 (ERCC1), a protein involved in nucleotide excision repair, is associated with resistance to platinum agent-based chemotherapy or chemoradiotherapy with platinum agents in various types of cancer. Herein we evaluated ERCC1 protein expression in uterine cervical adenocarcinoma and the relationship between this expression, clinicopathological factors, and clinical outcome, particularly in patients receiving adjuvant cisplatin-based chemotherapy or chemoradiotherapy with cisplatin. Thirty-six patients with International Federation of Gynecology and Obstetrics (FIGO) stage IB to stage IIB cervical adenocarcinoma who underwent radical hysterectomy were evaluated. Excision repair cross-complementation group 1 protein expression was examined by immunohistochemistry in tumor tissues. The relationship between ERCC1 expression levels and clinicopathological factors (age, FIGO stage, histological grade, tumor size, vascular invasion, cervical stromal invasion, and lymph node metastases) and prognosis was evaluated. No significant differences between ERCC1 expression levels and clinicopathological factors were observed. The patients in the ERCC1 high-expression group (n = 7) experienced significantly worse disease-free survival than the patients in the ERCC1 low-expression group (n = 29; P = 0.005). Among the 25 patients who received cisplatin-based chemotherapy or chemoradiotherapy with cisplatin, those with high ERCC1 expression (n = 5) also experienced significantly worse disease-free survival than those with low ERCC1 expression (n = 20; P = 0.002). Moreover, univariate and multivariate analyses revealed that high ERCC1 expression was an independent prognostic factor in patients receiving cisplatin-based chemotherapy or chemoradiotherapy with cisplatin. This is the first analysis of the association between ERCC1 expression and clinical outcomes in patients with uterine cervical adenocarcinoma. High ERCC1 protein expression was revealed to be associated with worse disease-free survival in the patients who received adjuvant cisplatin-based chemotherapy or chemoradiotherapy with cisplatin and was shown to be an independent prognostic factor. Further evaluation with a larger number of patients is required to confirm these preliminary observations.

Molecular Characteristics of ERCC1-Negative versus ERCC1-Positive Tumors in Resected NSCLC

Excision repair cross-complementation group 1 (ERCC1) is a protein involved in repair of DNA platinum adducts and stalled DNA replication forks. We and others have previously shown the influence of ERCC1 expression upon survival rates and benefit of cisplatin-based chemotherapy in patients with resected non-small-cell lung cancer (NSCLC). However, little is known about the molecular characteristics of ERCC1-positive and ERCC1-negative tumors. We took advantage of a cohort of 91 patients with resected NSCLC, for which we had matched frozen and paraffin-embedded samples to explore the comparative molecular portraits of ERCC1-positive and ERCC1-negative tumors of NSCLC. We carried out a global molecular analysis including assessment of ERCC1 expression levels by using both immunohistochemistry (IHC) and quantitative reverse transcriptase PCR (qRT-PCR), genomic instability, global gene and miRNA expression, and sequencing of selected key genes involved in lung carcinogenesis. ERCC1 protein and mRNA expression were significantly correlated. However, we observed several cases with clear discrepancies. We noted that ERCC1-negative tumors had a higher rate of genomic abnormalities versus ERCC1-positive tumors. ERCC1-positive tumors seemed to share a common DNA damage response (DDR) phenotype with the overexpression of seven genes linked to DDR. The miRNA expression analysis identified miR-375 as significantly underexpressed in ERCC1-positive tumors. Our data show inconsistencies in ERCC1 expression between IHC and qRT-PCR readouts. Furthermore, ERCC1 status is not linked to specific mutational patterns or frequencies. Finally, ERCC1-negative tumors have a high rate of genomic aberrations that could consequently influence prognosis in patients with resected NSCLC.

The NER proteins are differentially expressed in ever smokers and in never smokers with lung adenocarcinoma

The expression levels of excision repair cross-complementation group 1 (ERCC1), replication protein A (RPA) and xeroderma pigmentosum group F (XPF) nucleotide excision repair proteins may be important in the response to platin-based therapy in lung cancer patients. It is not known whether ERCC1, RPA and XPF expression levels differ between ever smokers (ES) and never smokers (NS). ERCC1, RPA and XPF expression levels were immunohistochemically evaluated in 125 patients with resected lung adenocarcinoma (AC) and carefully reviewed smoking status. ERCC1 was correlated with XPF (P = 0.001), but not with RPA (P = 0.11). In the univariate analysis, ERCC1 and XPF levels were higher in NS compared with ES (P = 0.004 and P = 0.003, respectively). In the multivariate analysis, the smoking status was predictive of the ERCC1 level [odds ratio (OR) 2.5, 95% confidence interval (CI) 1.03-6.2] after adjustment for variables linked to the smoking status, including age and the presence of bronchioloalveolar (BAC) features. The smoking status was also predictive of both RPA (OR 6.7, 95% CI 1.5-33.3) and XPF levels (OR 12.5, 95% CI 2.9-50) after adjusting for age, sex and BAC features. In patients with resected lung AC, ERCC1, RPA and XPF expression levels are higher in NS compared with ES.

Protein expression levels of excision repair cross-complementation group 1 and xeroderma pigmentosum D correlate with response to platinum-based chemotherapy in the patients with advanced epithelial ovarian cancer

This study was undertaken to examine whether there is an association between excision repair cross-complementation group 1 (ERCC1) and xeroderma pigmentosum D (XPD) protein expression levels and response to platinum-based chemotherapy in epithelial ovarian cancer (EOC). The study cohort consisted of 91 consecutive patients suffering from stage III or IV disease of primary EOC from 1999 to 2004 at the Women's Hospital, School of Medicine, Zhejiang University. There were 36 sensitive cases of serous ovarian cancer, 27 resistant cases of serous ovarian cancer, 15 cases of clear cell cancer, and 13 cases with serous ovarian cancer receiving neoadjuvant chemotherapy. The ovarian tissue microsections were stained by standard immunohistochemical techniques to show ERCC1 and XPD protein expression levels. In resistance group of serous ovarian cancer, ERCC1 and XPD protein expression levels were significantly higher than those of sensitivity group, and after receiving neoadjuvant chemotherapy, they showed 23% and 32% higher than before. Meanwhile, their levels of clear cell cancer group were significantly higher than serous ovarian cancer group's. Upregulation of ERCC1 and XPD protein expression was associated with resistance process to platinum-based chemotherapy in advanced EOC. This study provided evidence that differences of nucleotide excision repair-related genes expression may have an effect on the observed differences in clinical behavior of EOC.

ERCC1 protein expression predicts the response of cisplatin-based neoadjuvant chemotherapy in non-small-cell lung cancer

The excision repair cross-complementation group 1 (ERCC1) and BRCA1 have been identified as predictors of clinical outcomes among patients with non-small-cell lung cancer (NSCLC) treated with cisplatin-based chemotherapy. In this study, we immunohistochemically examined the ERCC1 and BRCA1 protein expression levels in 35 patients with metastatic mediastinal lymph nodes obtained prior to treatment as retrospective study. These patients had been enrolled in our studies on neoadjuvant chemotherapy with cisplatin and irinotecan (15 patients) or chemoradiotherapy with cisplatin and docetaxel plus concurrent thoracic radiation (20 patients). The relations between the ERCC1 or BRCA1 protein expression and the clinical outcomes of the patients were then examined. The rates of radiological response and pathological effectiveness were significantly higher among patients with ERCC1-negative tumors, compared with those with positive tumors in the neoadjuvant chemotherapy group (radiological response rates; 100% vs. 42.8%, P=0.013; pathological effectiveness; 100% vs. 47.1%, P=0.038), but no associations were observed in the neoadjuvant chemoradiotherapy group. Regarding survival, no significant differences in overall survival or disease-free survival were observed between patients with ERCC1-negative and positive tumors in both the neoadjuvant chemotherapy and chemoradiotherapy groups. In summary, we showed that a ERCC1-negative protein status was significantly related to tumor responsiveness to neoadjuvant chemotherapy with cisplatin and irinotecan, but such a status was not a clear prognostic predictor to cisplatin-based neoadjuvant therapy in NSCLC patients. Further study is needed to clarify the value of molecular predictors for customizing therapy for patients with NSCLC.