Excision Repair Cross-complementation Group 1 mRNA Research Articles

Osteopontin enhances cisplatin resistance of human A549 lung cancer cells via stimulating the PI3K signaling pathway and upregulating ERCC1 expression.

BackgroundTo investigate the regulatory role of osteopontin (OPN) in cisplatin (DDP) resistance in non-small cell lung cancer (NSCLC).MethodsOPN over-expression and interference vectors were constructed. Human NSCLC A549 cells were divided into normal control, shRNA-NC, shRNA-OPN, OPN, negative control (NC), control+DDP, shRNA-NC+DDP, shRNA-OPN+DDP, OPN+DDP, and NC+DDP groups. Cell proliferation was measured by MTT assay. Cell apoptosis was detected by flow cytometry. PI3K (phosphoinositide 3-kinase), p-ERK1/2 (phospho-extracellular signal-regulated kinases) and ERCC1 (excision repair cross-complementation group 1) mRNA and protein expression was determined by real-time PCR and Western blot.ResultsOPN overexpression and interference vectors were successfully constructed. When compared with control group, OPN group had significantly stimulated cell viability, reduced cell apoptosis, and increased expression of PI3K, p-ERK1/2 and ERCC1 (all P<0.05), whereas all other groups had lower cell viability, higher cell apoptosis, and inhibited protein expression. Moreover, OPN+DDP group had significantly higher proliferation, lower cell apoptosis, and enhanced PI3K, p-ERK1/2 and ERCC1 mRNA and protein expression compared with shRNA-OPN+DDP (all P<0.05).ConclusionsOPN can promote tumor cell growth and reduce cell apoptosis by activating PI3K pathway, which may induce the resistance of NSCLC to cisplatin. Moreover, the stimulation of ERCC1 expression may also increase the cisplatin resistance of NSCLCs.

Construction of warning system for lung cancer by whole genome sequencing screening for sensitive methylation sites of non-small-cell lung carcinoma

Objective To explore the construction of early warning system for non-small cell lung cancer (NSCLC) by whole genome sequencing. Methods The experiment was divided into two groups: normal tissue and NSCLC tissue. The expression of excision repair cross-complementation group 1 (ERCC1), tubb3 and RRM1 mRNA in NSCLC cells was detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR). The distribution of methylation in NSCLC cells was detected by whole genome sequencing. CpG methylation analysis was performed using sequencing data. Histone modification data were used to analyze the relationship between histone modification and DNA methylation. The expression of histone methylated eggs and methylated related enzymes in normal and NSCLC tissues were detected by protein immunoassay. Results Compared with the control cells, ERCC1 mRNA [(0.78±0.14) vs. (0.12±0.04), χ2=6.370, P<0.05] and RRM1 mRNA [(0.52±0.07) vs. (0.05±0.01), χ2=5.360, P<0.05] expression in NSCLC cells was down-regulated compared with control cells. Methylation level in NSCLC group increased at transcription start site and decreased in intergene region (χ2=3.140, P<0.05). Nine CpG methylation sensitive sites were found in the NSCLC group (RUNX3, MIR196A1, HOXA11, OTP, GATA4, PTPRU, SLC15A3, ZIC1 and TFAP2B). NSCLC group compared with control group, the lower histone acetylation [(H3K9ac [(43.57±8.84) vs. (10.64±4.35), χ2=8.730, P<0.05] and H3K27ac [(40.52±8.64) vs. (9.67±3.58), χ2=5.470, P<0.05) ] and histone methylation [(H2az [(42.56±9.74) vs. (12.47±6.05), χ2=7.420, P<0.05]. H3K4me1 [(37.47±6.42) vs. (15.46±7.34), χ2=5.380, P<0.05], H3K4me2 [(50.37±10.24) vs. (9.47±6.54), χ2=9.270, P<0.05]. H3K4me3 [(52.37±6.49) vs. (10.58±5.88), χ2=1.690, P<0.05] and H3K79me2 [(34.55±6.42) vs. (11.23±6.94), χ2=3.450, P<0.05]. Compared to the control group, NSCLC group DNMT1 express cut [(1.88±0.24) vs. (0.12±0.01), χ2=5.430, P<0.05], DNMT3a expression cut [(1.75±0.36) vs. (0.49±0.11), χ2=7.890, P<0.05], expression of DNMT3b cut [(0.88±0.14) vs. (0.13±0.05), χ2=1.360, P<0.05], expression of H3K4me3 cut [(2.53±0.35) vs. (0.35±0.08), χ2=5.440, P<0.05), H3K9me2 expression cut (0.55±0.07) vs. (0.05±0.01), χ2=3.270, P<0.05]. Conclusion Histone modification is closely related to DNA methylation, and the expression of histone methylation and methylation-related enzymes is also affected. Methylation sensitive sites can be used as biomarkers for early detection of NSCLC. Key words: Whole genome sequencing; Non-small-cell lung carcinoma; Methylation; Histone modification; Lung cancer cells

Could excision repair cross-complementing group-1 mRNA expression from peripheral blood lymphocytes predict locoregional failure with cisplatin chemoradiation for locally advanced laryngeal cancer?

The association of excision repair cross-complementing 1 mRNA (ERCC-1 mRNA) expression with the outcome has been reported with immunohistochemistry (IHC) using tumor tissue in head and neck cancer. We evaluated ERCC-1 mRNA expression by reverse transcription polymerase chain reaction (RT-PCR) from peripheral blood lymphocytes (PBLs) as bio-predictor of locoregional failure (LRF) to chemoradiation (CRT) for locally advanced laryngeal squamous cell cancer (LALSCC). A total of 107 male patients with LALSCC were enrolled in this prospective study. ERCC-1 mRNA expression by PBLs was determined by RT-PCR. Definitive CRT was delivered with 35mg/m2 weekly cisplatin. Response Evaluation Criteria in Solid Tumor 1.1 (RECIST 1.1) were used in evaluating treatment response. The primary objective was to assess LRF. The influence of patient characteristics, treatment response, weekly cisplatin cycles, ERCC mRNA expression was determined for LRF, progression-free survival (PFS) and overall survival (OS). A total of 98 patients completed definitive CRT. The median value of 2-ΔΔCT ERCC-1 mRNA expression was 3.9; based on which it was categorized as low and high. Correlation of ERCC-1 expression with treatment response was insignificant (P- .38). With a median follow-up of 33 months; 2-year LRF, PFS, and OS was 63.3%, 34.7% and 79.4%. The 2-year LRF, PFS and OS for low versus high expression were 53.1%versus 73.5% (P-value = 0.036), 44.9%versus 24.4% (P-value = 0.047) and 81.6%versus 77.2% (P-value = 0.33), respectively. In multivariate analysis, ERCC-1 expression, T-stage, N-stage and tumor subsite are predictive factors for LRF; T-stage and nodal recurrence for OS; stage and treatment response for PFS. LALSCC patient with ERCC-1 mRNA low expression was associated with lower LRF rate, and improved PFS.

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.

Expression of tumor heterogeneity-related genes in esophageal squamous cell carcinoma and its predictive value for chemotherapy sensitivity

Objective To investigate the expression of excision repair cross-complementation group 1 (ERCC1) and ribonucleotide reductase subunit M1 (RRM1) in patients with esophageal squamous cell carcinoma, and the relationship between the expression of ERCC1 and RRM1 genes and chemotherapy sensitivity. Methods A total of 77 patients with esophageal squamous cell carcinoma admitted in hospital from Jan. 2008 to Dec. 2012 were recruited. ERCC1 and RRM1 mRNA levels in the cancerous tissue were detected by real-time fluorescent quantitative RT-PCR. The relationship between short-term effects of chemotherapy and ERCC1 and RRM1 mRNA levels was analyzed. Results Levels of mRNA in stages Ⅰa, Ⅰb, Ⅱa, and Ⅱb were (0.578±0.081), (0.560±0.084), (0.521±0.080), (0.464±0.091) for ERCC1 and( 0.511±0.089), (0.539±0.086), (0.584±0.092), (0.637±0.101 )for RRM1, respectively. As the clinical stage advanced, the ERCC1 mRNA level declined and the RRM1 mRNA level increased (t=2.679 and 2.952, P=0.034 and 0.025, respectively). Levels of mRNA in patients with complete remission, partial remission, stable disease and progressive disease were (0.487±0.097, 0.511±0.095, 0.552±0.086, 0.568±0.088)for ERCC1 and(0.504±0.091, 0.544±0.095, 0.595±0.093, 0.616±0.097)for RRM1, respectively. The clinical effect of chemotherapy was negatively correlated with mRNA expression of ERCC1 and RRM1 (r=0.567, P=0.032). Conclusions Levels of ERCC1 and RRM1 mRNA expression are correlated with the staging of esophageal squamous cell carcinoma and chemotherapy sensitivity, and can be used as a predictive parameter for chemotherapy sensitivity in patients with esophageal squamous cell carcinoma. Key words: DNA-Repair; Esophageal neoplasms; Antineo plastic combined chemotherapy Protocols; Genes, tumor suppressor

Comparison of lung cancer A549 cell inhibition treated by zoledronic acid different schedule combined with cisplatin

Objective To compare lung cancer A549 cell inhibition treated by zoledronic acid different schedule combined with cisplatin.Methods According to different schedule of drug treatment,lung cancer A549 cells were divided into 4 groups (drug concentrations of zoledronic acid and cisplatin were 1.560 0,0.312 5 mg/L):(1) A group (control group):A549 cells were cultured,no drug treatment; (2) B group (simultaneously treated group):zoledronic acid and cisplatin were added simultaneously; (3) C group (cisplatin + zoledronic acid group):cells were incubated with cisplatin for 24 h followed by with zoledronic acid for 24 h; (4) D group (zoledronic acid + cisplatin group):cells were incubated with zoledronic acid for 24 h followed by with cisplatin.Methyl thiazol tetrazolium (MTT) assay and annexin V/ propidium iodide (PI) double staining were used to examine each groups A549 cells proliferation and apoptosis respectively.Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was applied to detected mRNA expression of mediator of DNA damage checkpoint protein 1 (MDC1),excision repair cross complementation group 1 (ERCC1) and receptor-associated protein 80 (RAP80) in A549 cells.Results Inhibition rates of A,B,C,D groups A549 cells were 0％,(39.16±4.94)％,(46.94 ±3.45) ％,(38.43 ± 4.84) ％,respectively.Rates of B,C,D groups were significantly higher than that of A group (all P ＜ 0.05),Rates of B,D group were significantly lower than that of C group (all P ＜ 0.05) ; Apoptosis rates of each group were (0.53 ±0.15)％,(32.3 ±0.50)％,(36.53 ±0.31)％,(29.33 ± 2.48) ％,respectively.Rates of B,C,D groups were significantly higher than that of A group (all P ＜ 0.05).Rates ofB,D group were significantly lower than that of C group (all P＜0.05); MDC1 and RAP80 gene expressions of B,C,D groups (0.374 ± 0.022 and 1.127 ± 0.018,0.320 ± 0.023 and 1.036 ±0.027,0.416 ±0.016 and 1.181 ±0.041,respectively) were significantly lower than that of A group (0.677 ±0.028 and 1.548 ±0.038) (all P ＜0.05),Expressions of B,D group were significantly higher than that of C group (all P ＜ 0.05) ; Expressions of ERCC1 mRNA had no significantly difference (all P ＞ 0.05).Conclusion Zoledronic acid combined with cisplatin can inhibit lung cancer A549 cells proliferation and induce cells apoptosis synergistically with schedule-dependent.The most remarkable effect was found in cells treated with cisplatin followed by with zoledronic acid.The downregulated expression of MDC 1 and RAP80 may be involved in the mechanism. Key words: Cisplatin ; Zoledronic acid ; A549 cell; Mediator of DNA damage checkpoint protein 1 ; Excision repair cross complementation group 1 ; Receptor-associated protein 80

Cell cycle association and hypoxia regulation of excision repair cross complementation group 1 protein (ERCC1) in tumor cells of head and neck cancer.

Excision repair cross complementation group 1 (ERCC1) is a key component of homologous recombination-based repair of interstrand DNA cross-links (ICLs). As a consequence, ERCC1 mediates resistance to mitomycin C (MMC) and platinum chemotherapeutic agents and may predict treatment failure. Clinical response to MMC or cisplatin (CDDP)-based radiochemotherapy (RCT) was assessed in 106 head and neck squamous cell carcinoma (HNSCC) patients and correlated with cell nuclear immunoreactivity of the mouse monoclonal (clone: 8 F1) ERCC1 antibody in tumor tissue samples. BEAS-2B epithelial and Detroit 562 pharyngeal squamous carcinoma cells were treated with CDDP, MMC, and 5-fluorouracil (5-FU) at 50 % growth inhibitory (IC-50) concentrations. ERCC1 protein synthesis was compared with cell cycle distribution using combined immunocytochemistry and flow cytometry. ERCC1 messenger RNA (mRNA) and protein expression was investigated in normoxic and hypoxic conditions in Detroit 562 cells. Clinically, the nonresponder revealed significantly lower HNSCC tissue ERCC1 immunoreactivity than the responder (p = 0.0064) or control normal mucosa, which led to further mechanistic investigations. In vitro, control cells and cells treated with cytotoxic agents showed increasing ERCC1 levels from the G1 through S and G2 phases of the cell cycle. In CDDP-treated cells, ERCC1 mRNA and protein expression increased. Under hypoxic conditions, ERCC1 gene expression significantly decreased. Although ERCC1+ cells show increased chemoresistance, they might be particularly radiosensitive, representing G2 cell cycle phase and less hypoxic. ERCC1 expression might be indirectly related with some conditions important for RCT treatment, but it is not a clear predictor for its failure in HNSCC patients.Electronic supplementary materialThe online version of this article (doi:10.1007/s13277-014-2001-2) contains supplementary material, which is available to authorized users.

ERCC1 mRNA expression is associated with the clinical outcome of non-small cell lung cancer treated with platinum-based chemotherapy.

We conducted a prospective study to analyze the expression of the excision repair cross-complementing group 1 (ERCC1) and ribonucleotide reductase subunit M1 (RRM1) genes in 297 Chinese patients with advanced non-small cell lung cancer (NSCLC). The goal of this study was to evaluate these genes as potential biomarkers for prediction of tumor response and clinical outcome. Patients with unresectable, locally advanced or metastatic NSCLC were enrolled between September 2007 and September 2009, and they were followed up until September 2012. A fluorescence-based real-time detection method was used to quantify relative levels of ERCC1 and RRM1 cDNA. Relative amounts of ERCC1 and RRM1 cDNA were calculated by comparing to actin. By the end of follow-up, 132 patients had died and 165 patients experienced progression. The median overall survival time was 18.7 months (range, 1-60 months). The median levels of ERCC1 and RRM1 were 2.46 x 10(-2) and 0.97 x 10(-2), respectively. Patients with low ERCC1 expression had a significantly higher rate of complete response to chemotherapy, with an OR (95%CI) of 1.56 (1.03-2.47). Moreover, individuals with low levels of ERCC1 had longer overall survival than patients with high expression, with an adjusted hazard ratio (95%CI) of 0.57 (0.35-0.93). In summary, low ERCC1 mRNA expression was associated with better response to chemotherapy and correlated with longer survival in advanced NSCLC patients treated with platinum-based chemotherapy.

Effect of TUBB3, TS and ERCC1 mRNA expression on chemoresponse and clinical outcome of advanced gastric cancer by multiplex branched-DNA liquid chip technology

To analyze the impact of β-tubulin-III (TUBB3), thymidylate synthase (TS) and excision repair cross complementation group 1 (ERCC1) mRNA expression on chemoresponse and clinical outcome of patients with advanced gastric cancer treated with TXT/CDDP/FU (DCF) regimen chemotherapy. The study population consisted of 48 patients with advanced gastric cancer. All patients were treated with DCF regimen palliative chemotherapy. The mRNA expressions of TUBB3, TS and ERCC1 of primary tumors were examined by multiplex branched-DNA liquid chip technology. The patients with low TUBB3 mRNA expression had higher response rate to chemotherapy than patients with high TUBB3 expression (P=0.011). There were no significant differences between response rate and TS or ERCC1 expression pattern. Median overall survival (OS) and median time to progression (TTP) were significantly longer in patients with low TUBB3 mRNA expression (P=0.002, P<0.001). TS or ERCC1 expression was not correlated with TTP and OS. In the combined analysis including TUBB3, TS and ERCC1, the patients with 0 or 1 high expression gene had better response rate, TTP and OS than the remaining patients (all P<0.001). Multivariate analysis revealed that ECOG (Eastern Cooperative Oncology Group)≥2 (HR=2.42, P=0.009) and TUBB3 (HR=2.34, P=0.036) mRNA expression significantly impacted on OS. High TUBB3 mRNA expression is correlated with resistance to DCF regimen chemotherapy. TUBB3 might be a predictive and prognostic factor in patients with advanced gastric cancer treated with TXT-based chemotherapy. The combined evaluation of TUBB3, TS and ERCC1 expression can promote the individual treatment in advanced gastric cancer.

Excision repair cross-complementation group 1 codon 118 polymorphism, micro ribonucleic acid and protein expression, clinical outcome of the advanced gastric cancer response to first-line FOLFOX-4 in Qinghai-Tibetan plateau population

The excision repair cross-complementation group 1 (ERCC1) codon 118 C/T polymorphism has been associated with clinical outcome in cancer patients treated with platinum chemotherapy. Ethnic differences in the frequency of this polymorphism have been observed in Caucasian and African populations. The aim of this study was to evaluate the frequency and survival benefit of the ERCC1 codon 118 C/T polymorphism in a high-altitude population with advanced gastric cancer. Polymerase chain reaction-restriction fragment length polymorphism was used to determine the frequency of ERCC1 118 codon C/T polymorphism in 206 advanced gastric cancer patients residing in the high-altitude Qinghai-Tibetan plateau. The influence of the ERCC1 codon 118 C/T polymorphism on its micro ribonucleic acid (mRNA) and protein expression, clinicopathological features; response to the platinum-based combination chemotherapy, and the outcome was evaluated. The Kaplan-Meier method was used for survival analysis. The correlation of ERCC1 codon 118 polymorphism with ERCC1 mRNA and protein expression, clinicopathological characteristics, and first-line oxaliplatin, 5-fluorouracil, and leucovorin (FOLFOX-4) response was determined by χ(2)-test. ERCC1 codon 118 C/T polymorphism was not associated with ERCC1 mRNA and protein expression, FOLFOX-4 response, and progression-free survival (PFS) or overall survival (OS). High ERCC1 mRNA and protein expression levels were associated with significantly lower FOLFOX-4 responses, PFS, and OS. ERCC1 codon 118 C/T polymorphism is not an important prognostic marker for advanced gastric cancer. Determination of ERCC1 mRNA and protein levels may be beneficial in predicting the response and outcome of FOLFOX-4 therapy in gastric cancer.

ERCC1 mRNA levels can predict the response to cisplatin-based concurrent chemoradiotherapy of locally advanced cervical squamous cell carcinoma

BackgroundThe purpose of this study was to investigate whether the excision repair cross-complementation group 1 (ERCC1) mRNA expression could predict treatment response of patients with locally advanced cervical squamous cell carcinoma (LACSCC) who underwent cisplatin-based concurrent chemoradiotherapy (CCCRT).MethodsA total of sixty LACSCC patients, treated with radical CCCRT from a single institution were evaluated. ERCC1 mRNA expression was determined by quantitative real-time RT-PCR in pre-treatment tumor tissues. The association of ERCC1 status with clinicopathological characteristics (age, histological grade, tumor size, parametrial invasion, lymph node metastasis and FIGO stage) and treatment response were analyzed.ResultsNo significant association between ERCC1 mRNA expression and clinicopathological characteristics were observed. Patients with low ERCC1 mRNA level had a significantly higher rate of complete response (86.21%) than patients with high level of ERCC1 expression (19.36%; p < 0.001). In the logistic regression analysis, low ERCC1 mRNA level retained an independent role in predicting complete response to CCCRT (P < 0.001). An ERCC1 expression level of 0.0901 was determined as an optimal cutoff value to identify complete response patients to CCCRT treatment. The sensitivity for detection of a complete response was 81.48% with a specificity of 96.97% (area under the curve, 0.893; 95% confidence interval, 0.804–0.983).ConclusionsThis is the first analysis of the association between ERCC1 mRNA levels and treatment response in patients with LACSCC. Low ERCC1 mRNA level appears to be a highly specific predictor of response to CCCRT in LACSCC.

KRAS mutational status affects oxaliplatin-based chemotherapy independently from basal mRNA ERCC-1 expression in metastatic colorectal cancer patients

Background:In this study, we evaluated the possibility that KRAS mutational status might be predictive of oxaliplatin (OXA) efficacy. We also explored the role of excision repair cross complementing group-1 (ERCC-1).Methods:Ninety anti-epidermal growth factor receptor-naive advanced colorectal cancer patients were retrospectively analysed. In all patients KRAS mutational status was assessed. In 60 patients mRNA ERCC-1 expression was also investigated. Response rate (RR) and progression-free survival (PFS) after FOLFOX-6±bevacizumab were evaluated according to KRAS status and mRNA ERCC-1 expression.Results:Among 90 patients 47% wild-type (wt) and 53% mutated (mt) KRAS tumours were found. Response rate was 26% in the wt KRAS group, whereas it was 56% in the mt KRAS group; the difference is statistically significant in the total sample (P=0.008) and when only patients receiving FOLFOX-6±bevacizumab as first-line are considered (P=0.01). Progression-free survival was longer in mt than in wt KRAS patients over all patients (10 vs 8 months, respectively, P=0.001) and in those treated as first-line (10 vs 8 months, respectively, P=0.0069). Mt KRAS patients experienced a longer survival (24 vs 18 months; P=0.01). ERCC-1 mRNA expression was not found to correlate with FOLFOX activity in our analysis.Conclusion:Our results suggest that activating mutation of KRAS oncogene may predict response to OXA. Basal expression of ERCC-1 mRNA does not explain the high efficacy of FOLFOX-6 in mt KRAS patients.

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 determination of relationship between “excision repair cross-complementing group 1” (ERCC1) gene T19007C and C8092A single nucleotide polymorphisms and clinicopathological parameters in non-small cell lung cancer

DNA repair plays a key role in prevention of carcinogenesis and one of the most important DNA repair mechanisms is nucleotide excision repair (NER) pathway. This pathway includes a number of genes such as excision repair cross-complementing group 1 (ERCC1) gene which are responsible for the 5' incision of damaged DNA. A reduced DNA repair capacity associated with ERCC1 mRNA level has been observed in lung carcinogenesis. Two single nucleotide polymorphisms (SNPs) in ERCC1 gene, T19007C (rs11615) and C8092A (rs3212986), reportedly predict to affect the mRNA of ERCC1 in non-small cell lung cancer (NSCLC). To examine the role of two common SNPs in ERCC1 gene further, we conducted this study where 80 cases histopatologically diagnosed as NSCLC were genotyped. Genomic DNA was extracted from formalin-fixed, paraffin embedded tissues and two SNPs were analyzed using real-time PCR. The distributions of TT, TC, and CC genotypes of the T19007C SNP were 40, 44 and 16%, respectively. Significantly increased frequency of the patients carrying at least one 19007C allele was observed in early stage compared to advanced stage (P=0.002). And also, the frequency of TC and CC genotypes significantly increased in younger patients compared to older patients (P=0.035). Regarding C8092A SNP, the distribution of CC, CA, and AA genotypes was 38, 51 and 11%, respectively. There was no significant difference in the genotype distribution between C8092A SNP and clinicopathological parameters. This study indicated that harboring at least one 19007C allele may have protective effect in NSCLC.

Abstract 2475: Excision repair cross complementing group 1 (ERCC1) predicts the efficacy of chemoradiotherapy for invasive bladder cancer

Abstract Purpose: Chemoradiation therapy (CRT) is now widely recognized as bladder-preserving therapy for muscle-invasive bladder cancer (MIBC). However, some patients who fail CRT may miss the chance to be cured by cystectomy. Therefore, it is important to select patients with MIBC who are expected to have a good response to CRT. Several reports indicate that the excision repair cross-complementing group 1 (ERCC1) gene is associated with resistance to cisplatin and radiation therapy. In this study, we examined the correlation between ERCC1 and CRT in vitro and in vivo in bladder cancer. Experimental Design: Bladder cancer cell lines T24, 5637, Cl8-2 (multi-drug-resistant subline of T24), and CDDP10-3 (cisplatin-resistant subline of T24) were used for in vitro assays to measure ERCC1 expression level and growth inhibition with cisplatin or irradiation (IR). To clarify the association between ERCC1 and cisplatin resistance in bladder cancer cells, we knocked down ERCC1 in Cl8-2 and CDDDP10-3 with siRNA (C18-2ΔERCC1 and CDDP10-3ΔERCC1). To further prove the cause of the radiation sensitivity of ERCC1 knockdown cells, we measured the phosphorylated histone variant H2A.X, a marker of DNA damage. In the clinical study, we then examined by immunohistochemistry whether ERCC1 nuclear staining correlates with the efficacy of CRT using cisplatin in 22 patients with MIBC. Results: Cl8-2 cells expressed ERCC1 mRNA 5.96-fold higher than did T24. Cl8-2 and CDDP10-3 were more resistant to cisplatin or IR than was T24. Our ERCC1 knockdown experiments showed that there was statistical difference in the resistance to IR exposure not cisplatin compared with C18-2CTL and CDDP10-3CTL. C18-2ΔERCC1 and CDDP10-3ΔERCC1 recovered more slowly in terms of the number of phospho-H2A.X foci than did C18-2cont and CDDP10-3cont, suggesting continued accumulation or persistence of DSBs. In immunohistochemistry with ERCC1, six of eight positive cases did not have complete response to CRT, whereas 12 of 14 negative cases had complete response. Sensitivity and specificity were 75% and 85.7%, respectively (p = 0.008). Conclusion: Our results suggest that in some bladder cancer cells, ERCC1 expression correlates with IR resistance but not with cisplatin resistance. Moreover, the lack of ERCC1 expression correlated well with the efficacy of CRT, and especially with that of IR, in our clinical study. Although further study is needed, ERCC1 expression level may predict the efficacy of CRT for MIBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2475. doi:10.1158/1538-7445.AM2011-2475

Thymidylate Synthase and Excision Repair Cross-Complementing Group-1 as Predictors of Responsiveness in Mesothelioma Patients Treated with Pemetrexed/Carboplatin

The pemetrexed/platinum agent combination represents the standard of care in first-line treatment for malignant pleural mesothelioma (MPM). However, there are no established indicators of responsiveness that can be used to optimize the treatment. This retrospective study aimed to assess the role of excision repair cross-complementing group-1 (ERCC1) and thymidylate synthase (TS) in tumors, and correlate expression levels and polymorphisms of these key determinants of drug activity with the outcome of MPM patients treated with carboplatin/pemetrexed in first-line setting. Analysis of TS and ERCC1 polymorphisms, mRNA and protein expression was done by PCR and immunohistochemistry [with the H-score (histologic score)] in tumor specimens from 126 MPM patients, including 99 carboplatin-/pemetrexed-treated patients. A significant correlation between low TS protein expression and disease control (DC) to carboplatin/pemetrexed therapy (P = 0.027), longer progression-free survival (PFS; P = 0.017), and longer overall survival (OS; P = 0.022) was found when patients were categorized according to median H-score. However, patients with the higher tertile of TS mRNA expression correlated with higher risk of developing progressive disease (P = 0.022), shorter PFS (P < 0.001), and shorter OS (P < 0.001). At multivariate analysis, the higher tertile of TS mRNA level and TS H-score confirmed their independent prognostic role for DC, PFS, and OS. No significant associations were found among ERCC1 protein expression, TS and ERCC1 polymorphisms, and clinical outcome. In our series of carboplatin-/pemetrexed-treated MPM patients, low TS protein and mRNA levels were significantly associated to DC, improved PFS, and OS. Prospective trials for the validation of the prognostic/predictive role of TS in MPM patients treated with pemetrexed-based regimens are warranted.

Excision Repair Cross-Complementing Group 1 May Predict the Efficacy of Chemoradiation Therapy for Muscle-Invasive Bladder Cancer

Chemoradiation therapy (CRT) is now widely recognized as bladder-preserving therapy for muscle-invasive bladder cancer (MIBC). However, some patients who fail CRT may miss the chance to be cured by cystectomy. Therefore, it is important to select patients with MIBC who are expected to have a good response to CRT. Several reports indicate that the excision repair cross-complementing group 1 (ERCC1) gene is associated with resistance to cisplatin and radiation therapy. In this study, we examined the correlation between ERCC1 and CRT in vitro and in vivo in bladder cancer. Bladder cancer cell lines T24, 5637, Cl8-2 (multidrug-resistant subline of T24), and CDDP10-3 (cisplatin-resistant subline of T24) were used for in vitro assays to measure ERCC1 expression level and growth inhibition with cisplatin or ionizing radiation (IR). We then examined by immunohistochemistry that whether ERCC1 nuclear staining correlates with the efficacy of CRT using cisplatin in 22 patients with MIBC. Cl8-2 cells expressed ERCC1 mRNA 5.96-fold higher than did T24. Cl8-2 and CDDP10-3 were more resistant to cisplatin or IR than was T24. Resistance to IR, but not to cisplatin, was removed by suppressing ERCC1 using siRNA in both Cl8-2 and CDDP10-3 cells. In immunohistochemistry with ERCC1, 6 of 8 positive cases did not have complete response to CRT, whereas 12 of 14 negative cases had complete response. Sensitivity and specificity were 75% and 85.7%, respectively (P = 0.008). Although further study is needed, ERCC1 expression level may predict the efficacy of CRT for MIBC.

Expression of xeroderma pigmentosum complementation group C protein predicts cisplatin resistance in lung adenocarcinoma patients

DNA repair has been suggested to be a major cause of spontaneous drug resistance in patients with lung adenocarcinomas (LADC). Among the DNA repair-related proteins, excision repair cross-complementation group 1 (ERCC1) has been shown to be essential for repairing cisplatin-induced interstrand cross-linkage. However, the role of other DNA repair-related proteins in drug resistance has not been clearly elucidated. In this study, we used suppression subtractive hybridization and microarray analysis to identify the DNA repair-related genes associated with cisplatin resistance. We focused on the association of XPC protein expression, which plays a pivotal role in the earliest response to global genomic repair, with the survival of LADC patients. Using suppression subtractive hybridization and a microarray analysis to identify drug resistance-associated DNA repair-related genes, we found that the mRNA levels of ERCC1, MSH-3, MSH-6 and XPC were significantly increased in LADC patients. Since the results of ERCC1 mRNA expression corresponded well with those in previous reports, in this study we focused on the clinical correlation between XPC expression and patient survival. The level of XPC protein was determined by immunohistochemical and immunoblotting analyses. We detected the XPC protein in 46 (43%) of 107 pathological LADC samples. XPC protein expression correlated with tumor stage, cigarette smoking and poor survival. In the in vitro experiments with LADC cell lines, increased XPC expression was associated with elevated drug resistance, and silencing of XPC expression reduced cisplatin resistance. Our results suggest that XPC expression predicts drug resistance in LADC.

ERCC1 protein, mRNA expression and T19007C polymorphism as prognostic markers in head and neck squamous cell carcinoma patients treated with surgery and adjuvant cisplatin-based chemoradiation

Adjuvant cisplatin-based chemoradiation improves survival in HNSCC patients presenting with risk features. ERCC1 (excision repair cross-complementation group 1) is associated with resistance to chemo- and radiation therapy and may have a prognostic value in HNSCC patients. Here we studied ERCC1 expression and the polymorphism T19007C as prognostic markers in these patients. This is a retrospective and translational analysis, where ERCC1 protein expression was evaluated by immunohistochemistry, using an H-score, and mRNA expression was determined by RT-PCR. T19007C genotypes were detected by PCR-RFLP carried out using DNA template extracted from normal lymph nodes. A high H-score was seen in 32 patients (54%), who presented better 5-year overall survival (5-y OS: 50% vs. 18%, HR 0.43, p=0.026). Fifteen out of 45 patients (33%), with high mRNA expression, presented better 5-year overall survival (OS) (86% vs. 30%, HR 0.26, p=0.052). No OS difference was detected among T19007C genotypes. High H-score and mRNA expression remained significant as favorable prognostic factors in a multivariate analysis. Collectively, our results suggest that high ERCC1 expression seems to be associated with better OS rates in HNSCC patients submitted to adjuvant cisplatin-based chemoradiation.

Low ERCC1 mRNA and protein expression are associated with worse survival in cervical cancer patients treated with radiation alone

Purpose To evaluate the association of excision repair cross-complementation group 1 (ERCC1) expression, using both mRNA and protein expression analysis, with clinical outcome in cervical cancer patients treated with radical radiation therapy (RT). Experimental design Patients ( n = 186) with locally advanced cervical cancer, treated with radical RT alone from a single institution were evaluated. Pre-treatment FFPE biopsy specimens were retrieved from 112 patients. ERCC1 mRNA level was determined by real-time PCR, and ERCC1 protein expression (FL297, 8F1) was measured using quantitative immunohistochemistry (AQUA®). The association of ERCC1 status with local response, 10-year disease-free (DFS) and overall survival (OS) was analyzed. Results ERCC1 protein expression levels using both FL297 and 8F1 antibodies were determined for 112 patients; mRNA analysis was additionally performed in 32 patients. Clinical and outcome factors were comparable between the training and validation sets. Low ERCC1 mRNA expression status was associated with worse OS (17.9% vs 50.1%, p = 0.046). ERCC1 protein expression using the FL297 antibody, but not the 8F1 antibody, was significantly associated with both OS ( p = 0.002) and DFS ( p = 0.010). After adjusting for pre-treatment hemoglobin in a multivariate analysis, ERCC1 FL297 expression status remained statistically significant for OS [HR 1.9 (1.1–3.3), p = 0.031]. Conclusions Pre-treatment tumoral ERCC1 mRNA and protein expression, using the FL297 antibody, are predictive factors for survival in cervical cancer patients treated with RT, with ERCC1 FL297 expression independently associated with survival. These results identify a subset of patients who may derive the greatest benefit from the addition of cisplatin chemotherapy.