Original Articles Oncol Res Treat DOI: 10.1159/000479068 Received: March 11, 2017 Accepted: June 29, 2017 Published online: October 24, 2017 Evidence for an Association of ERCC1 Expression and Mismatch Repair Status with Overall Survival in Colorectal Cancer Patients Pan Li a Zhi-Tao Xiao b Todd A. Braciak a Qing-Jian Ou b Gong Chen b Fuat S. Oduncu a a Department of Hematology and Oncology, Medizinische Klinik und Poliklinik IV, Ludwig Maximilians University, Munich, Germany; of Colorectal Surgery, State Key Laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Guangzhou, China bDepartment Summary Background: The aim in this study was to determine if an association of excision repair cross-complementing group 1 (ERCC1) gene and mismatch repair (MMR) status with overall survival (OS) could be found from our analysis of a large cohort of Chinese colorectal cancer patients (CRC). Methods: In total, 2,233 tissue samples isolated from individual CRC tumors were assessed by immunohistochemistry for the expression of ERCC1 and 4 MMR genes. Results: The rates of proficient MMR (pMMR) and ERCC1 expression were 89.6 and 90.7%, respectively. We found that patients with positive ERCC1 expression and deficient (d)MMR status had higher overall survival (OS) than those with either positive ERCC1 and pMMR, negative ERCC1 and dMMR, or negative ERCC1 expression and pMMR status (OS 79 vs. 69 vs. 66 vs. 61%, hazard ratio (HR) 0.90, 95% confidence interval (CI) 0.80–1.00; p = 0.043). Despite this finding, we found no statistical difference in OS between ERCC1-positive and -negative CRC patients when ERCC1 expression was considered alone (OS 70 vs. 62%, HR 0.82, 95% CI 0.65– 1.04; p = 0.11). Conclusion: Our results indicate that the combined examination of ERCC1 expression and dMMR status can be used to aid OS assessment in CRC patients. © 2017 S. Karger GmbH, Freiburg Correspondence can also be addressed to: Dr. Gong Chen, Department of Colorectal Surgery, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, East Dongfeng Road 651, 510060 Guangzhou, China, email@example.com © 2017 S. Karger GmbH, Freiburg Fax +49 761 4 52 07 14 Information@Karger.com www.karger.com Accessible online at: www.karger.com/ort Introduction There are at least 4 pathways of DNA repair that operate on specific types of damaged DNA; these comprise the base excision repair (BER), the nucleotide excision repair (NER), the mismatch repair (MMR), and the double-strand break repair (DDSBR) pathway . Mismatch repair defects are produced from germline mutations in any one of the MLH1, MSH2, MSH6, or PMS2 genes. It is estimated that approximately 1 in 3,000 persons within the general population carry a mutation in at least 1 MMR gene and have a deficient (d)MMR status . This is in contrast with observations in CRC patients where a dMMR status has been shown to be present in approximately 15–20% of all CRCs in Western countries [3, 4]. Because of this large discrepancy between healthy individuals and cancer patients, guidelines in the 2009 Evaluation of Genomic Applications in Practice of Prevention (EGAPP) recommended screening of the MMR status for all newly diagnosed CRC patients . MMR corrects mismatched nucleotides and insertion-deletion loops in DNA caused by polymerase errors, chemical modifications, and recombination between heterologous DNA sequences, and therefore could affect both cancer etiology and response to therapy in CRC patients . In support of MMR effects on response to therapy, it was more recently demonstrated that stage II CRC patients with dMMR had a better prognosis than those with proficient (p)MMR status but could actually be harmed by 5-fluorouracil (FU) treatment as treatment-induced DNA damage could go unchecked in these dMMR patients . Another protein that has the potential to impact CRC patient outcomes is ERCC1. ERCC1 is a rate-limiting molecule in the NER pathway, which is responsible for repairing DNA adducts induced by platinum drugs . It has been shown to form a heterodimer with xeroderma pigmentosum group F to execute excision repair of the DNA strand . Because of this important role in Prof. Dr. Dr. Fuat S. Oduncu Department of Hematology and Oncology Medizinische Klinik und Poliklinik IV, Ludwig Maximilians University Ziemssenstr. 1, 80336 Munich, Germany firstname.lastname@example.org Downloaded by: Univ.of Adelaide 220.127.116.11 - 10/26/2017 7:26:40 AM Keywords Colorectal cancer · MMR status · ERCC1 · Biomarker · Prognosis Fig. 1. Immunostainings. Oncol Res Treat 2017;40:1–5 Patients and Methods Patients The ethics committee of the Sun Yat-sen University Cancer Center approved this study, and informed consent for all patients analyzed was obtained at the beginning of the study. A total of 4,500 histologically confirmed consecutive CRC patients were recruited post-surgery from the Sun Yat-sen University Cancer Center between May 2011 and May 2016. All patients were of Chinese origin. The clinical and family histories of each patient were reviewed. Finally, 2,233 cases were selected for analysis after strict exclusion criteria were applied, including age less than 18 years and more than 85 years, severe complications, multiprimary cancer, synchronous and metachronous CRC, family history (first-degree and second-degree relatives with any kind of cancer), familial adenomatous polyposis, and death not from tumor-related causes. The primary tumor site was categorized as right colon if the tumor was located above the splenic flexure, left colon if it was located at or below the splenic flexure, and rectum. The median follow-up for living patients was 4.3 years. Treatment and Follow-Up Stage I (T1–2 N0) (332 cases) and stage II (T3–4 N0) CRC patients (648 cases) without high-risk clinical features (e.g., T4 stage, bowel perforation or clinical bowel obstruction, inadequate lymph node sampling, poorly differentiated histology) were treated with radical surgery or endoscopic removal of the tumor alone. Stage II (T3–4 N0) CRC patients with high-risk clinical features (228 cases) were recommended to follow the XELODA regimen. Stage III (Tx N1–2) patients (606 cases) were designated to receive radical surgery and 12 cycles of adjuvant mFOLFOX/XELOX treatment within a 6-month period. All stage IV (Tx Nx M1) patients received palliative surgery (398 cases) or radical surgery (22 cases). The first-line treatment for stage IV CRC was use of the mFOLFOX/FOLFIRI regimen. 89 patients with rectal cancer within our study also received neoadjuvant chemoradiotherapy. The patients’ clinical responses were evaluated in accordance with the RECIST guidelines. After surgery, tumor recurrence was determined by physical examination, serum carcinoembryonic antigen assay, and abdominal or thoracic imaging every 3–6 months for the following 3 years, then every 6 months for the next 2 years, and finally at annual check-ups. The duration of follow-up was defined as the time between surgery and disease recurrence, death, or last hospital contact (scheduled follow-up or telephone contact). The cutoff date for inclusion in this analysis was May 2016. Li/Xiao/Braciak/Ou/Chen/Oduncu Downloaded by: Univ.of Adelaide 18.104.22.168 - 10/26/2017 7:26:40 AM DNA repair, ERCC1 expression has been studied, and it has been reported that a gene polymorphism of ERCC1 at codon 118 was a predictive factor for tumor response to oxaliplatin/5-FU combination chemotherapy in patients with advanced CRC . In addition, ERCC1 is also now considered a possible predictive biomarker for cisplatin use in non-small cell lung cancer (NSCLC) based on the results obtained from an International Adjuvant Lung Cancer Trial Collaborative Group (IALT) trial . While these results obtained with regard to patient response to therapy and ERCC1 expression suggest the importance of this protein in cancer progression, the definitive prognostic value of ERCC1 expression has not yet been established for patients with CRC in relation to disease outcome. It is likely that defects in the BER and NER pathways can impair DNA repair capacity, resulting in an accumulation of DNA damage, carcinogenesis, and reduced chemotherapeutic sensitivity of cancer cells [12, 13]. Nevertheless, so far, no study has shown a prognostic role for combined ERCC1 and MMR status in CRC patients. Therefore, in this study, we determined whether there was an association of ERCC1 expression and MMR status with overall survival (OS) in CRC patients with stage I–IV disease. We were aided in this analysis by having access to a large cohort of CRC patients who, following the recommendations of the EGAPP , underwent systematic immunohistochemistry (IHC) screening for microsatellite instability. At the same time, testing for ERCC1 expression was also performed, which has become a routine test in combination with IHC analysis with regard to patient prognosis. The results obtained from this study indicate that measurement of ERCC1 and MMR status in CRC patients has prognostic value and can aid patient care. Table 1. Clinicopathological characteristics of patients Results Association of ERCC1 and MMR with OS in CRC Patients, n (%) Sex Male Female Age, years 20–39 40–59 60–85 Location Right colon Left colon Rectum Pathology G1 G2 G3 Mucinous Signet-ring Stage I II III IV Mismatch repair (MMR) status Deficient (d)MMR Proficient (p)MMR ERCC1 Negative Positive MMR+ERCC1 dMMR+ERCC1(–) dMMR+ERCC1(+) pMMR+ERCC1(–) pMMR+ERCC1(+) Alive Yes No Statistical Analyses Patient data are described as frequencies (percentages). Differences in distribution between the variables examined were assessed with either the Χ2 or the Fisher’s exact test. The primary end point was OS defined as the time elapsed from the date of surgery to tumor-induced death. Surviving patients were censored on the last follow-up date. Median follow-up and the 95% confidence interval (CI) were calculated using the reverse Kaplan-Meier method. The survival curve was estimated with the Kaplan-Meier method and compared using the log-rank test. Univariate and multivariable Cox proportional hazards models were used to explore associations of MMR status, age, stage, differentiation grade, and sex. The score and likelihood ratio test p values were used to test the statistical significance of each covariate in the univariate and multivariable Cox models, respectively. All statistical tests were 2-sided, and p values of ≤ 0.05 were considered statistically significant. All statistical analyses were performed using SPSS software (IBM Corp., Armonk, NY, USA). Of the 2,233 patients evaluated, 232 were found to have dMMR with an overall prevalence of 10.4%. 208 patients were found to have negative ERCC1 expression with a prevalence of 9.3%. When patients were assessed with regard to ERCC1 expression in combination with MMR status, 1,839 were found to have positive ERCC1 and pMMR with a prevalence of 82.4%, 162 were found to have negative ERCC1 and pMMR with a prevalence of 7.3%, 184 were found to have positive ERCC1 and dMMR with a prevalence of 8.2%, and finally 47 patients were found to have negative ERCC1 and dMMR with a prevalence of 2.1%. Our study population included 1,316 males and 917 females of which 11.5 and 8.7%, respectively, were categorized as dMMR whereas 9.6 and 8.9%, respectively, were found to be ERCC1-negative. More detailed clinicopathological information for all patients is given in table 1. dMMR versus pMMR CRC were more likely to be stage IIA (16.6%) versus other stages (stage I: 9.1%, stage IIB: 8.0%, stage IIC: 19.0%, stage IIIA: 10.9%, stage IIIB: 8.4%, stage IIIC: 11.9%, stage IVA: 5.3%, stage IVB: 4.3%) (p < 0.001), right colon (22.5%) versus left colon (7.4%) and rectum (6.2%) (p < 0.001), occurring in men (11.6%) versus women (8.7%) (p = 0.031), poorly or undifferentiated (23.6%) versus well or moderately differentiated (9.6%) (p < 0.001), occurring in young age (20–39 years, 13.8%) versus older age (40–59 years, 12.2%; 60–85 years, 8.3%) (p = 0.001), ERCC1- Characteristics 1,314 (58.9) 917 (41.1) 188 (8.4) 1,023 (45.9) 1,021 (45.7) 521 (23.3) 740 (33.1) 972 (43.5) 104 (4.7) 2,002 (89.7) 18 (0.8) 95 (4.3) 14 (0.6) 332 (14.9) 876 (39.2) 606 (27.1) 419 (18.8) 232 (10.4) 2,001 (89.6) 207 (9.3) 2024 (90.7) 47 (2.1) 184 (8.2) 162 (7.2) 1,839 (82.4) 1,542 (69.1) 689 (30.9) Table 2. Multi-analysis of overall survival Variable Age Sex Stage Location Grade ERCC1 MMR Hazard ratio 95% confidence interval lower upper 2.38 1.41 3.19 1.75 2.23 0.92 1.56 2.36 1.39 3.23 1.66 2.07 0.91 1.18 2.42 1.44 3.85 1.86 2.26 0.93 2.06 p value < 0.001 0.032 < 0.001 0.001 0.001 < 0.001 0.002 negative (23.1%) versus ERCC1-positive (9.1%) (p < 0.001). The multi-analysis results are shown in table 2. Overall, we found that most CRC patients with a pMMR status also tended to simultaneously express the ERCC1 protein (p < 0.001). One important finding of this study was that we found dMMR status to be a better predictor for survival benefit than pMMR status (OS 77 vs. 68%, hazard ratio (HR) 0.66, 95% CI 0.50– Oncol Res Treat 2017;40:1–5 Downloaded by: Univ.of Adelaide 22.214.171.124 - 10/26/2017 7:26:40 AM Immunohistochemical Staining for MMR and ERCC1 Proteins Blocks of formalin-fixed, paraffin-embedded adenocarcinoma tissue comprising an area of normal colorectal mucosa adjacent to the tumor were selected in each case. Cases with complete nuclear loss of expression in invasive tumor cells with retained expression in inflammatory cells and/or adjacent normal tissue as positive controls were considered MMR-deficient or ERCC1negative. Staining was performed using the following primary antibodies: mouse anti-human MLH1 (dilution 1: 50, clone OTI1C1, zhongshan jiqiao, Beijing), rabbit anti-human MSH2 (dilution 1: 200, clone ZA0622, zhongshan jiqiao), mouse anti-human MSH6 (dilution 1: 100, clone OTI5D1, zhongshan jiqiao), mouse anti-human PMS2 (dilution 1: 50, clone OTI2G5, zhongshan jiqiao), and mouse anti-human ERCC1 (dilution 1: 200, clone OTI1A3, zhongshan jiqiao). Whole tissue sections were read separately by 2 pathologists blinded to the patients’ clinical characteristics. Discordant cases were reviewed by a supplementary pathologist to reach a consensus. Illustrative immunostainings are shown in figure 1. Fig. 2. Survival plots of patients showing both ERCC1 expression and MMR status. Discussion In this study, we evaluated a large cohort of Chinese CRC patients for any associations of ERCC1 expression and MMR status for possible prognostic application. In adherence to the EGAPP recommendations , we followed the clinical outcomes of 2,233 CRC patients in search for any evidence of an association with DNA repair pathways. From this Chinese patient-based population analysis, we were able to demonstrate that use of the combination of ERCC1 expression and MMR status harbors prognostic value that might aid patient care. MMR is a strand-specific repair pathway which is initiated with the recognition of damaged DNA . When MMR is deficient, unrepaired areas of DNA can accumulate and result in microsatellite instability . It has been found that MMR deficiency is most commonly caused by epigenetic inactivation of the MLH1 gene in sporadic CRCs, with changes in MSH2 and MSH6 accounting for the next smaller percentage of deficiencies in patients . For CRC, it has previously been demonstrated that stage II patients with dMMR have a better prognosis, and in these patients, 5-FU treatment may actually cause additional harm . In our study, we evaluated the OS of 2,233 CRC patients representing all 4 stages of disease (I–IV) and found that some patients with a dMMR status had statistically significantly better OS. However, this association Oncol Res Treat 2017;40:1–5 with better survival required inclusion in the analysis of tumor ERCC1 protein expression. The ERCC1 protein plays many roles in tumorigenesis. ERCC1 has been shown to act as an endonuclease in the NER pathway that incises 5’ to any damaged DNA strand, allowing the removal of the damaged strand, polymerization, and religation . Although ERCC1 has been used as a prognostic marker in NSCLC , only few studies have evaluated the potential role of this protein as a prognostic marker in CRC, and most of the previous data generated about this protein was gathered in the metastatic setting [18, 19] or from the analysis of ERCC1 polymorphisms [20–22]. Previous work suggested that positive tumor expression of ERCC1 can predict inferior survival outcomes for cancer patients . With regard to CRC, the Colorectal Oral Novel Therapy for the Inhibition of Angiogenesis and Retarding of Metastases(CONFIRM)-1 and CONFIRM-2 trials have previously shown that high ERCC1 expression was associated with shorter OS in patients receiving first-line chemotherapy . Yet, another study did not conclude that there was any significant association between ERCC1 expression and the prognosis of CRC . In our current study, we found that most CRC patients with a pMMR status tended to have tumors that simultaneously expressed the ERCC1 protein. Moreover, we found that there was a prognostic effect for patients with dMMR when combined with a positive ERCC1 expression profile. This effect suggests that there may be an interaction between the BER and NER pathways in CRC. While our findings here are novel and possibly useful for use in patient care, there are some limitations with respect to gaining a full understanding within this current study. One possible limitation is that we incorporated all stages (I–IV) of CRC into 1 group to examine the effects of ERCC1 expression and MMR status on OS. The examination of each separate stage of colon or rectal cancer might provide additional prognostic value for ERCC1 and MMR status in tumor pathogenesis. Finally, we only focused on looking for associations with clinical outcome for ERCC1 expression and MMR status. It is likely that other important pathways are involved that could aid the prognosis of CRC patients. Thus, it is important to identify other relevant mechanisms or pathways of action that contribute to the pathogenesis of CRC. Li/Xiao/Braciak/Ou/Chen/Oduncu Downloaded by: Univ.of Adelaide 126.96.36.199 - 10/26/2017 7:26:40 AM 0.87; p = 0.003). Yet, we found no statistical differences in OS between ERCC1-positive and -negative CRC patients (OS 70 vs. 62%, HR 0.82, 95% CI 0.65–1.04; p = 0.11). Most importantly, we were able to show a prognostic effect following the measurement of both ERCC1 and MMR status of patients. Here, we were able to demonstrate that patients with both a positive ERCC1 expression and dMMR status had greater OS than all other patient groups including those with positive ERCC1 and pMMR, patients with negative ERCC1 and dMMR, and finally those with negative ERCC1 expression and pMMR status (OS 79 vs. 69 vs. 66 vs. 61%, HR 0.90, 95% CI 0.80–1.00; p = 0.043). The survival plots of patients showing both ERCC1 expression and MMR status are provided in figure 2. Conclusion Our study represents one of the first to incorporate a large dataset of CRC patients to investigate the effects of ERCC1 and dMMR status on clinical outcomes following the EGAPP recommendations for research in CRC. 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