ERCC1 Levels Research Articles

MSH3 expression does not influence the sensitivity of colon cancer HCT116 cell line to oxaliplatin and poly(ADP-ribose) polymerase (PARP) inhibitor as monotherapy or in combination

Defective expression of the mismatch repair protein MSH3 is frequently detected in colon cancer, and down-regulation of its expression was found to decrease sensitivity to platinum compounds or poly(ADP-ribose) polymerase inhibitors (PARPi) monotherapy. We have investigated whether MSH3 transfection in MSH3-deficient colon cancer cells confers resistance to oxaliplatin or PARPi and whether their combination restores chemosensitivity. MSH3-deficient/MLH1-proficient colon cancer HCT116(MLH1) cells were transfected with the MSH3 cDNA cloned into the pcDNA3.1(-) vector. MSH3/MLH1-deficient HCT116, carrying MLH1 and MSH3 mutations on chromosome 3 and 5, respectively, and HCT116 in which wild-type MLH1 (HCT116+3), MSH3 (HCT116+5) or both genes (HCT116+3+5) were introduced by chromosome transfer were also tested. Sensitivity to oxaliplatin and to PARPi was evaluated by analysis of clonogenic survival, cell proliferation, apoptosis and cell cycle. MSH3 transfection in HCT116 cells did not confer resistance to oxaliplatin or PARPi monotherapy. MSH3-proficient HCT116+5 or HCT116+3+5 cells, which were more resistant to oxaliplatin and PARPi in comparison with their MSH3-deficient counterparts, expressed higher levels of the nucleotide excision repair ERCC1 and XPF proteins, involved in the resistance to platinum compounds, and lower PARP-1 levels. In all cases, PARPi increased sensitivity to oxaliplatin. Restoring of MSH3 expression by cDNA transfection, rather than by chromosome transfer, did not affect colon cancer sensitivity to oxaliplatin or PARPi monotherapy; PARP-1 levels seemed to be more crucial for the outcome of PARPi monotherapy.

Oncogenic driver mutations in patients with non-small-cell lung cancer at various clinical stages

BackgroundOncogenic driver mutations are responsible for the initiation and maintenance of non-small-cell lung cancer (NSCLC). Elucidation of driver mutation occurrence in NSCLC has important clinical implications. Patients and methodsNSCLC at various clinical stages were studied for their oncogenic mutations and their association with patients' disease-free survival (DFS). ResultsOf 488 patients with NSCLC, 28 had EML4-ALK fusions. Female, young age (<60 years old), and nonsmoker patients had significant greater mutation frequencies than male, old age (≥60 years old), and smoker patients, respectively (P<0.05). Of 392 patients with NSCLC, 13 had PIK3CA mutations and 3 had MEK1 mutations. EML4-ALK, PIK3CA, and MEK1 mutations were mutually exclusive. EML4-ALK fusion was found to be of coexistence with EGFR and KRAS mutations in two cases. In stage IA NSCLC, EML4-ALK-positive patients had longer DFS than EML4-ALK-negative patients (P = 0.04). However, in stage IIIA NSCLC, EML4-ALK-positive patients had poorer DFS than EML4-ALK-negative patients (P < 0.01). Moreover, multivariate analysis indicated that in stage IIIA NSCLC EML4-ALK fusion was the only significant indicator for poor DFS (P < 0.001). Furthermore, tumors with EML4-ALK fusions had significantly higher levels of ERCC1, a molecule with a key role in platinum drug efficacy, than tumors without EML4-ALK fusions. ConclusionEML4-ALK, PIK3CA, and MEK1 mutations occurred in NSCLC with various distinct clinicopathological characteristics. EML4-ALK fusions could serve as a significant prognostic indicator for locally advanced NSCLC.

A pilot study: sequential gemcitabine/cisplatin and icotinib as induction therapy for stage IIB to IIIA non-small-cell lung adenocarcinoma

BackgroundA phase II clinical trial previously evaluated the sequential administration of erlotinib after chemotherapy for advanced non-small-cell lung cancer (NSCLC). This current pilot study assessed the feasibility of sequential induction therapy in patients with stage IIB to IIIA NSCLC adenocarcinoma.MethodsPatients received gemcitabine 1,250 mg/m2 on days 1 and 8 and cisplatin 75 mg/m2 on day 1, followed by oral icotinib (125 mg, three times a day) on days 15 to 28. A repeatcomputed tomography(CT) scan evaluated the response to the induction treatment after two 4-week cycles and eligible patients underwent surgical resection. The primary objective was to assess the objective response rate (ORR), while EGFR and KRAS mutations and mRNA and protein expression levels of ERCC1 and RRM1 were analyzed in tumor tissues and blood samples.ResultsEleven patients, most with stage IIIA disease, completed preoperative treatment. Five patients achieved partial response according to the Response Evaluation Criteria in Solid Tumors (RECIST) criteria (ORR=45%) and six patients underwent resection. Common toxicities included neutropenia, alanine transaminase (ALT) elevation, fatigue, dry skin, rash, nausea, alopecia and anorexia. No serious complications were recorded perioperatively. Three patients had exon 19 deletions and those with EGFR mutations were more likely to achieve a clinical response (P= 0.083). Furthermore, most cases who achieved a clinical response had low levels of ERCC1 expression and high levels of RRM1.ConclusionsTwo cycles of sequentially administered gemcitabine/cisplatin with icotinib as an induction treatment is a feasible and efficacious approach for stage IIB to IIIA NSCLC adenocarcinoma, which provides evidence for the further investigation of these chemotherapeutic and molecularly targeted therapies.

Abstract 336: Genomic annotation of non-small cell lung cancer patient-derived xenograft models for personalized cancer therapy.

Abstract Background: Recent studies have characterized non-small cell lung cancer (NSCLC) as one of the most genomically deranged of all cancers, necessitating that both new drug development and patient therapy account for intra- and inter-patient tumor heterogeneity. Clinically annotated NSCLC patient-derived xenograft (PDX) models represent a novel approach to integrate this genomic complexity into a clinically relevant pre-clinical platform. We here describe molecular characterization to profile all currently “druggable” oncogenes for NSCLC in paired PDXs and original patient NSCLC tumor (PT). Method: Genomic DNA from archival formalin-fixed, paraffin-embedded (FFPE) PT and fresh first human-to-mouse (P0) NSCLC PDX tumors were isolated and subjected to oncogene mutational profiling using Sequenom's OncoCarta Panel v1. This panel detects 238 mutations in 19 genes commonly altered in cancer. RT-PCR-based molecular analyses of EGFR and KRAS mutations, EML4-ALK fusion transcripts, and RNA expression levels of ERCC1, RRM1 and TS genes were performed by Response Genetics, Inc. Genomic DNA from 3 serially passaged NSCLC PDX tumors (2 KRAS and 1 EGFR mutation models) up to 5 passages were also analyzed. Results: In the first 7 of 9 patient-PDX NSCLC models tested, oncogene mutational fidelity was preserved between PDX and PT with a good correlation of molecular biomarker expression (p&amp;lt;0.01). Two paired models had discrepancies in genotyping: from harboring 2 or 3 oncomutations at a frequency of 5-17% in PT to no mutation detected in P0 tumors), likely due to intra-patient tumor heterogeneity from clonal evolution. Of 3 models that have serial passaged tumors, the frequencies of oncomutation in each model were similar among the same passage (P0) or serial passage (P0 to P5) tumors. In several models tested for in vivo drug efficacy based on the molecular biomarker expression, results matched treatment outcome of the original patients., Conclusion: Our results validate the overall genomic fidelity of PDX tumors compared to original PT. Molecular characterization of individual tumor results in a clinically and genomically annotated PDX model with potential utility for selecting and validating clinically relevant drug target(s) for personalized cancer therapy. Acknowledgement: Supported by UC Davis Comprehensive Cancer Center Developmental Award (NIH/NCI P30CA093373), UL1 RR024146 from the National Center for Research Resources, the Jackson Laboratory, Response Genetics Inc., and the Addario Foundation. Citation Format: Sonal J. Desai, Neal Goodwin, Regina Gandour-Edwards, Royce F. Calhoun, David T. Cooke, Laurel A. Beckett, Martin K.H. Maus, Stephanie H. Astrow, Philip C. Mack, Ralph deVere White, David R. Gandara, Tianhong Li. Genomic annotation of non-small cell lung cancer patient-derived xenograft models for personalized cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 336. doi:10.1158/1538-7445.AM2013-336

Abstract 2489: Proteomic profiling identifies PI3K and DNA repair pathways as potential markers of response to PARP inhibitor BMN673 in SCLC.

Abstract Background: Small cell lung cancer (SCLC) is an aggressive malignancy that accounts for 13% of lung cancers in the US and has a 5-year survival rate &amp;lt;10%. Novel therapeutic approaches are critically needed to improve clinical outcomes. Using proteomic profiling, we previously identified high levels of PARP1 expression in SCLC cell lines and tumors. We also demonstrated in vitro sensitivity of SCLC to two PARP inhibitors, olaparib and rucaparib. Here we explore markers of response and pathways modulated following PARP1 inhibition as a basis for developing potential predictive markers and rational drug combinations. Methods: Sensitivity of SCLC lines to BMN673 and cisplatin was determined by CellTiter-Glo cell viability assay. Total and phospho-protein expression of 200 markers was measured at baseline and following drug treatment in SCLC cell lines by reverse phase protein array (RPPA). Correlations between baseline protein expression and drug sensitivity were determined by Spearman rank correlation. Modulation of protein expression post treatment was assessed by ANOVA. Results: The novel PARP inhibitor BMN673 had potent in vitro activity in a panel of 12 SCLC cell lines with IC50’s ranging from 1.7-15nM. A higher expression level of several DNA repair proteins was associated with greater BMN673 sensitivity. For example, protein expression levels of ERCC1, DNA PKcs, ATM, FANCD2, and pChk2 were inversely correlated with IC50 values (Rho values -0.93 to -0.81; p≤0.02). In contrast, high baseline expression of phosphorylated Akt (T308) (R=0.91, p=0.005) and pAkt (S473) (R=0.81, p=0.02) were associated with decreased sensitivity to BMN673. We also observed increased phosphorylation of mTOR, Akt, and S6 (p&amp;lt;0.02) at 24h following PARP inhibitor treatment, suggesting that PI3K/Akt/mTOR pathway activation may be associated with both inherent and acquired resistance. Consistent with clinical studies suggesting greater PARP inhibitor activity in platinum-sensitive tumors, markers of BMN673 response overlapped with markers of cisplatin sensitivity, with higher DNA repair protein levels and lower pAkt levels associated with greater cisplatin activity in vitro in the same SCLC cell line panel. Conclusions: Here we demonstrate significant, single-agent in vitro activity of the PARP inhibitor BMN673 in SCLC and potential predictive markers of response. Interestingly, for the first time we show an inverse correlation between PARP inhibitor sensitivity and PI3K/Akt/mTOR pathway activation. This observation parallels recent data suggesting that PI3K inhibition may sensitize breast cancer to PARP inhibition and suggests a potential coordinated regulation between DNA repair and PI3K pathways. The activity of BMN673 and these candidate response biomarkers will be further investigated in a Phase I cohort expansion of BMN673 in patients with SCLC. Citation Format: Robert J. Cardnell, Ying Feng, Lixia Diao, You Hong Fan, Jing Wang, Yuqiao Shin, John D. Minna, John V. Heymach, Lauren A. Byers. Proteomic profiling identifies PI3K and DNA repair pathways as potential markers of response to PARP inhibitor BMN673 in SCLC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2489. doi:10.1158/1538-7445.AM2013-2489

Predictive models for customizing chemotherapy in advanced non-small cell lung cancer (NSCLC).

The backbone of first-line treatment for Epidermal Growth Factor (EGFR) wild-type (wt) advanced Non-small cell lung cancer (NSCLC) patients is the use of a platinum-based chemotherapy combination. The treatment is characterized by great inter-individual variability in outcome. Molecular predictive markers are extremely needed in order to identify patients most likely to benefit from platinum-based treatment and resistant ones, thus optimizing chemotherapy approach in NSCLC. Several components of DNA repair response (DRR) have been investigated as potential predictive markers. Among them, high levels of expression of ERCC1, both at protein and mRNA levels, have been associated with resistance to cisplatin in NSCLC. In addition, low levels of expression of RRM1, a target for gemcitabine, have been associated with improved OS in advanced NSCLC patients treated with cisplatin and gemcitabine. Preclinical data and retrospective analyses showed that BRCA1 is able to induce resistance to cisplatin and sensitivity to antimicrotubule agents. In addition, the mRNA levels of expression of RAP80, encoding for a protein cooperating with BRCA1 in homologous recombination (HR), have demonstrated to further sub-classify low BRCA1 NSCLC tumors, improving the predictive model. On the basis of biological knowledge on DNA repair pathway and recent controversial results from clinical validation of potential molecular markers, integrated analysis of multiple DNA repair components could improve predictive information and pave the way to a new approach to customized chemotherapy clinical trials.

A three-gene signature as potential predictive biomarker for irinotecan sensitivity in gastric cancer

ObjectivePersonalized chemotherapy based on molecular biomarkers can maximize anticancer efficiency. We aim to investigate predictive biomarkers capable of predicting response to irinotecan-based treatment in gastric cancer.MethodsWe examined gene expression of APTX, BRCA1, ERCC1, ISG15, Topo1 and methylation of SULF2 in formalin-fixed paraffin-embedded gastric cancer tissues from 175 patients and evaluated the association between gene expression levels or methylation status and in vitro sensitivity to irinotecan. We used multiple linear regression analysis to develop a gene-expression model to predict irinotecan sensitivity in gastric cancer and validated this model in vitro and vivo.ResultsGene expression levels of APTX, BRCA1 and ERCC1 were significantly lower in irinotecan-sensitive gastric cancer samples than those irinotecan-resistant samples (P < 0.001 for all genes), while ISG15 (P = 0.047) and Topo1 (P = 0.002) were significantly higher. Based on those genes, a three-gene signature were established, which was calculated as follows: Index =0.488 - 0.020× expression level of APTX + 0.015× expression level of Topo1 - 0.011 × expression level of BRCA1. The three-gene signature was significantly associated with irinotecan sensitivity (rho = 0.71, P < 0.001). The sensitivity and specificity for the prediction of irinotecan sensitivity based on the three-gene signature reached 73% and 86%, respectively. In another independent testing set, the irinotecan inhibition rates in gastric samples with sensitive-signature were much higher than those with resistant-signature (65% vs. 22%, P < 0.001). Irinotecan therapy with 20 mg/kg per week to immunodeficient mice carrying xenografts with sensitive-signature dramatically arrested the growth of tumors (P < 0.001), but had no effect on mice carrying xenografts with resistant-signature.ConclusionsThe three-gene signature established herein is a potential predictive biomarker for irinotecan sensitivity in gastric cancer.

Molecular profiles of gastric cancer: The UCSD experience.

29 Background: Gastric cancer is the 2nd leading cause of cancer mortality globally. A small number of studies reported that low TS and ERCC1 mRNA levels are associated with improved survival, and may be important as prognostic factors. Methods: Intratumoral gene expression levels were assessed using laser-captured micro-dissection and quantitative Real-Time PCR on formalin fixed paraffin embedded tumor samples from 22 gastric adenocarcinomas. A retrospective chart review was performed to measure clinical parameters. Primary objectives were to determine the range of expression of TS, ERCC1, and HER2, and to investigate if these biomarkers are predictive of survival. Results: TS, ERCC1, and HER2 median expression levels using RT-PCR were 3.56, 1.54, and 0.085, respectively. Median OS was 62 months. Subjects with low TS trended towards improved survival (92 months vs. 34 months, p: 0.17), as did subjects with low ERCC1 (92 months vs. 55 months, p: 0.44). There was no association between HER2 expression and OS. All subjects had HER2 levels below 0.55. With regard to treatment, 90.9% of patients received platinum-based chemotherapy and 4.5% received HER2-guided therapy. Conclusions: Our results are consistent with prior reports that associate low TS and low ERCC1 with improved OS, and may imply prognostic significance. Although these trends did not reach statistical significance, they are consistent with prior studies. Further molecular studies are needed to better assess the utility of these markers as prognostic indicators. There was no clear trend between HER2 levels and OS, most likely because all subjects had low HER2 levels. However, as accurate determination of HER2 expression is becoming increasingly important in the management of gastric cancer patients, further research into the degree of concordance between RT-PCR assessment and FISH assessment of HER2 gene amplication is warranted. Overall, more molecular studies are needed to better stratify this disease into subtypes, and identify new drug targets for chemo-resistant subtypes.

Correlation of messenger RNA expression patterns of ERCC1, TS, EGFR, and VEGFR2 with KRAS and BRAF mutational status in advanced colorectal cancer: Implications for targeted therapies.

383 Background: Gene expression levels of ERCC1, TS, EGFR and VEGFR2 may have predictive value for the personalized use of standard chemotherapeutics as well as agents targeting the EGFR and VEGF pathways and the efficacy of EGFR directed monoclonal antibodies like panitumumab and cetuximab has been confirmed to be dependent on wt KRAS and wt BRAF in patients with advanced colorectal cancer. We investigated the correlations between KRAS/BRAF mutational status and the mRNA expression levels of these genes. Methods: Formalin-fixed paraffin-embedded tumor specimens from 600 patients with advanced colorectal adenocarcinoma were microdissected and DNA and RNA was extracted. Specifically designed primers and probes were used to detect 7 different base substitutions in codon 12 and 13 of KRAS, V600E mutations in BRAF and the expression levels of ERCC1, TS, EGFR and VEGFR2 by RT-PCR. Results: Mt KRAS tumors had significantly lower TS and EGFR gene expression levels compared with wt KRAS (p&lt;0,001), whereas mt BRAF tumors showed significantly increased TS and EGFR mRNA levels compared to wt BRAF (p&lt;0,001). Mt BRAF tumors showed significantly higher mRNA levels than mt KRAS tumors (p&lt;0,001). ERCC1 and VEGFR2 mRNA levels were significantly down-regulated in mt KRAS specimen (p&lt;0,001), but showed no significant correlation with BRAF mutational status. Conclusions: KRAS and BRAF mutations are associated with opposite mRNA expression levels for TS and EGFR. Recently, resistance to BRAF inhibition in mt BRAF colorectal tumors has been shown in preclinical models to be associated with up-regulation of EGFR. Our data suggests that BRAF mutants are associated with high EGFR levels at the time of diagnosis, and not necessarily part of an acquired mechanism of resistance. Significantly lower mRNA expression levels of VEGFR2 in mt KRAS tumors may explain lower response to angiogenesis inhibition seen in the TML study.

Molecular profiles of appendiceal adenocarcinoma: The UCSD experience.

397 Background: Appendix cancer is rare, which precludes its study in randomized trials. As such, no evidence-based guidelines currently exist regarding the optimum systemic therapy for this disease entity. Typically, these patients are treated with regimens for colorectal carcinoma. Previous studies have shown high intra-tumoral mRNA levels of EGFR were significantly associated with response to irinotecan based chemotherapy, and that mucinous colorectal cancer overexpresses markers of resistance to 5-FU and oxaliplatin. We examined pharmacogenomics markers for appendiceal cancer and colon cancer to understand underlying similarities and differences between these tumor types. Methods: Intratumoral gene expression levels were assessed from paraffin-embedded tissue samples, using laser capture microdissection and quantitative real-time PCR from 69 colorectal and 34 appendiceal adenocarcinomas. A retrospective chart review was performed to correlate gene expression with overall survival. KRAS and BRAF mutational analyses and gene expression levels of ERCC1, TS, and EGFR were correlated with overall survival. Results: Appendiceal tumors had significantly higher expression of EGFR, (2.66 vs 1.4, p&lt;.0001). No BRAF V600E mutations were found in the appendiceal tumors, incidence was 8.97% of the colon patients. UPDATE: In appendix cancer, KRAS mutations were noted in 65.5% of patients, there were no BRAF mutations. Median ERCC1, TS, EGFR, and VEGFR2A expression was 1.4, 1.21, 1.57, 2.19 respectively. Patients with metastatic appendiceal cancer had a significantly longer median OS than metastatic colon patients, (113 mo vs 43.9 mo, p = .0154). For appendiceal cancer patients, there was no significant correlation between any of the biomarkers and OS, although the sample size was underpowered for such an analysis. Conclusions: Metastatic appendiceal cancer patients have significantly better outcomes than metastatic colon cancer patients. Molecular analyses reveal significant differences between these tumor types. Further molecular study of appendiceal cancer is needed, as this study and others suggest fundamental differences in biology from colon cancer.

Biomarker distribution and characteristics for the first 100 patients in MAVERICC, a randomized phase II study of mFOLFOX6-bevacizumab (BV) versus FOLFIRI-BV in previously untreated metastatic colorectal cancer (mCRC).

483^ Background: Retrospective analyses have suggested that ERCC1 is a chemo-resistance marker to platinum compounds; a median tumoral ERCC1 level of 1.7 × 10–3ERCC1/β-actin mRNA has been reported in metastatic colon cancer (Grimminger et al, Pharmacogenomics J, 2011). MAVERICC is the first prospective study of tumoral ERCC1and plasma VEGF-A as potential biomarkers for oxaliplatin- and BV-containing regimens, respectively. We present data from a planned interim biomarker distribution assessment of the first 100 patients (pts) in MAVERICC. Methods: In this randomized, open-label, phase II study, pts (N=360) with histologically or cytologically confirmed CRC and ≥1 measurable metastatic lesion are stratified at screening by tumoral ERCC1 mRNA expression (low vs high, cutoff of ≤ vs &gt;1.7). Stratified pts are randomized 1:1 to mFOLFOX6-BV or FOLFIRI-BV administered in 2-week cycles. The primary objectives are: 1) to assess ERCC1 and VEGF-A as biomarkers of progression-free survival (PFS) for oxaliplatin- and BV-containing 1st-line regimens, and 2) within ERCC1 high pts, to test whether FOLFIRI-BV is associated with a prolonged 1st-line PFS vs mFOLFOX6-BV. Results: With a data cutoff of June 19 2012, 100 pts have been enrolled. Of these, 75 pts were stratified to the ERCC1 high group and 25 pts to the ERCC1 low group. The median ERCC1 mRNA expression was 3.00 (range, 1.73–13.16) and 1.32 (range, 0.70–1.70) in the ERCC1 high and low stratification groups, respectively. Among all pts, the median ERCC1 mRNA expression was 2.37. Pt demographics by group are shown (Table). Plasma VEGF-A evaluation is ongoing. Conclusions: An analysis of the first 100 pts in MAVERICC showed that the median tumoral ERCC1 expression level was higher than the predefined cutoff, providing an adequate population of ERCC1 high pts to assess PFS by treatment regimen. Clinical trial information: NCT01374425. [Table: see text]

Role of Conventional Chemosensitivity Test and Tissue Biomarker Expression in Predicting Response to Treatment of Peritoneal Carcinomatosis From Colon Cancer

Background5-Fluorouracil- or oxaliplatin-based regimens are the treatments of choice in patients with PC from colon cancer. There are currently no useful preclinical evaluations to guide the decision-making process for tailored therapy. The aim of the present study was to compare the advantages and limits of a conventional in vitro chemosensitivity test with those of a panel of biomolecular markers in predicting clinical response to different drugs used to treat colon cancer-derived PC. Patients and MethodsFresh surgical biopsy specimens were obtained from 28 patients with peritoneal carcinomatosis from colon cancer. TS, TP, DPD, MDR1, MRP-1, MGMT, BRCA1, ERCC1, GSTP1, and XPD gene expression levels were determined by real-time reverse transcription polymerase chain reaction. An in vitro chemosensitivity test was used to define a sensitivity or resistance profile to the drugs used to treat each patient. ResultsExpression levels of the genes analyzed were generally poorly related to each other. TS and ERCC1 expression was inversely related to response to 5-FU-and/or oxaliplatin-containing regimens. Significant predictivity in terms of sensitivity but poor predictivity of resistance (56.2%) (P = .037) were observed for ERCC1 expression (90%), and high predictivity of resistance (100%) but very low predictivity of sensitivity (40%) (P = .014) were registered for TS. The best overall and significant predictivity was observed for chemosensitivity test results (62.5% sensitivity and 89% resistance; P = .005). ConclusionsSensitivity and resistance to drugs used in vivo was better defined by the chemosensitivity test than by biomarker expression.

Association between clinical pathology and multiple genes mRNA expression in Chinese patients with NSCLC

The aim of this study was to evaluate whether there was an association between pathology type and ERCC1, BRCA1, RRM1, TUBB3, STMN1, TOP2A and epidermal growth factor receptor (EGFR) messenger ribonucleic acid (mRNA) expression level in Chinese patients with non-small cell lung carcinoma (NSCLC). mRNA expression level of these genes was analyzed in 181 cancer tissues by using xTAG-step liquid-chip array. The mRNA expression level of the seven genes was evaluated in association with the clinical pathology type. The average mRNA expression level of the seven genes were ERCC1 (1.02 ± 0.03), BRCA1 (0.15 ± 0.04), RRM1 (0.19 ± 0.05), TUBB3 (0.31 ± 0.06), STMN1 (2.78 ± 0.42), TOP2A (3.04 ± 0.42) and EGFR (0.58 ± 0.09), respectively in Chinese patients with NSCLC. The mRNA expression level of ERCC1, STMN1 and TOP2A genes were statistical different with different pathology type (p(a) < 0.05); STMN1 and TOP2A genes mRNA expression were much higher in squamous cell lung carcinoma than that in non-squamous cell lung carcinoma (p(a) < 0.05). And ERCC1 gene expression was much lower in squamous cell carcinoma than that in non-squamous cell carcinoma (p(a) < 0.05). mRNA expression level of STMN1, TOP2A and ERCC1 were correlated with the clinical pathology type.

SLUG silencing increases radiosensitivity of melanoma cells in vitro

Melanoma radioresistance has been attributed to the presence of tumor cells with highly efficient DNA damage repair mechanisms. We examined the expression of genes involved in DNA damage repair and DNA damage sensing, and assessed their modulation by SLUG silencing, which is potentially capable of increasing radiosensitivity. Two melanoma cell lines (M14 and M79) were used to evaluate in vitro radiation-induced cytotoxicity before and after SLUG silencing. mRNA expression levels of BRCA1, ERCC1, DNA-PK, PARP, MGMT, ATM and TGM2 were determined by real-time RT-PCR, and protein expression levels of SLUG, caspase 3, p21, PUMA and pMAPK by Western blotting. The cytotoxic effect of radiation was high in M14 and low in M79 cells. SLUG silencing increased the interference of radiation on cell cycle distribution and cell killing by 60 % and 80 % in M79 cells after a 2.4 Gy and 5 Gy radiation dose, respectively. It also led to a significant inhibition of expression of genes involved in DNA damage repair and DNA damage sensing in all cell lines maintained after radiation. An almost total inhibition was observed for TGM2, which is expressed at a high basal level in the most radioresistant cell line (M79). Protein expression of PUMA was induced by radiation and was enhanced after SLUG silencing. Our results reveal a pivotal role of SLUG in regulating a cellular network involved in the response to DNA damage, and highlight the importance of TGM2 in radiosensitivity modulation. SLUG silencing appears to increase radiation sensitivity of the melanoma cells tested.

Specific Biomarkers Are Associated with Docetaxeland Gemcitabine-Resistant NSCLC Cell Lines

Five-year survival rate for lung cancer is limited to 10% to 15%. Therefore, the identification of novel therapeutic prognostic factors is an urgent requirement. The aim of this study is thus to highlight specific biomarkers in chemoresistant non-small cell lung cancer cell lines. Therefore, we checked—in the control condition as well as after short-term pharmacological treatment with either docetaxel or gemcitabine—the expression of genes such as tumor suppressor genes (CDKN2A, DAPK, FHIT, GSTP1, MGMT, RARβ2, RASSF1A, and TIMP3), genes associated with drug resistance (BRCA1, COX2, ERCC1, IGFBP3, RRM1, and TUBB3), and stemness-related genes (CD133, OCT4, and SLUG) in two cellular models of squamous carcinoma (CAEP) and adenocarcinoma (RAL) of the lung originally established. Their promoter methylation profile was also evaluated. Drug-related genes were upregulated. Cisplatin resistance matched with high levels of BRCA1 and ERCC1 in both cell lines; docetaxel sensitivity of CAEP cells was associated to levels of TUBB3 lower than RAL cells. Although CAEP cells were more sensitive to gemcitabine, both cell lines showed high levels of RRM1. Stemness-related genes were downregulated in the control condition but became upregulated in docetaxel-resistant cells, indicating the selection of a population with stemness features. We did not find an unequivocal correspondence between gene expression and respective DNA promoter methylation status, suggesting the involvement of additional mechanisms of gene expression regulation. These results highlight specific biomarkers consistent with the different responses of the two cell lines to standard pharmacological treatments and indicate specific molecular traits for their chemoresistance.

Assessment of ERCC1 and XPF Protein Expression Using Quantitative Immunohistochemistry in Nasopharyngeal Carcinoma Patients Undergoing Curative Intent Treatment

We sought to evaluate the prognostic/predictive value of ERCC1 and XPF in patients with nonmetastatic nasopharyngeal carcinoma (NPC) treated with curative intent. ERCC1 and XPF protein expression was evaluated by immunofluorescence combined with automated quantitative analysis (AQUA) using the FL297 and 3F2 antibodies, respectively. ERCC1 and XPF protein expression levels were correlated with clinical outcomes. Patient characteristics were as follows: mean age 52 years (range, 18-85 years), 67% male, 72% Karnofsky performance status (KPS) ≥ 90%, World Health Organization (WHO) type 1/2/3 = 12%/28%/60%, stage III/IV 65%. With a median follow-up time of 50 months (range, 2.9 to 120 months), the 5-year overall survival (OS) was 70.8%. Median standardized nuclear AQUA scores were used as cutpoints for ERCC1 (n=138) and XPF (n=130) protein expression. Agreement between dichotomized ERCC1 and XPF scores was high at 79.4% (kappa = 0.587, P<.001). Neither biomarker predicted locoregional recurrence, DFS, or OS after adjustment for age and KPS, irrespective of stratification by stage, WHO type, or treatment. Neither ERCC1 nor XPF, analyzed by quantitative immunohistochemistry using the FL297 and 3F2 antibodies, was prognostic or predictive in this cohort of NPC patients.

RASSF1A and ERCC1 Expression Levels Might Be Predictive of Prognosis in Advanced, Recurrent, and Metastatic Squamous Cell Carcinoma of the Head and Neck Treated with Docetaxel and Cisplatin

Background: The purpose of this study was to test the hypothesis that the immunohistochemical expression of ERCC1 and RASSF1A would predict both response to and survival after docetaxel and cisplatin combination chemotherapy in inoperable or recurrent head and neck squamous cell carcinoma. Patients and Methods: A total of 54 patients were treated with frontline systemic chemotherapy composed of docetaxel (60 mg/m²) and cisplatin (65 mg/m²), every 3 weeks for up to 6 cycles. The expression levels of ERCC1 and RASSF1A were evaluated in the available 36 prechemotherapy samples. Results: The overall objective response rate was 35% (complete remission 12% and partial remission 23%). The median progression-free survival and overall survival (OS) times were 5.0 months (95% confidence interval (CI), 3.7–6.4 months) and 24.2 months (95% CI, 3.5–45.0 months), respectively. The status of low ERCC1 and high RASSF1A expression was an independent favorable prognostic factor for OS in multivariate analysis (p = 0.043; hazard ratio, 7.224; 95% CI, 1.060–49.217). Toxicities were comparable with those of previously reported trials. Conclusions: Less intensive doses of cisplatin and docetaxel are active but not effective in reducing toxicity. Also, both ERCC1 and RASSF1A might be useful prognostic markers in this regimen.

Association of EGFR mutations with low BRCA1 gene expression in non-small cell lung cancer

Clinical studies suggest that the mRNA expression level of excision repair cross complementing group 1 gene (ERCC1) is associated with epidermal growth factor receptor (EGFR) mutation and breast cancer susceptibility 1 gene (BRCA1) mRNA expression in non-small cell lung cancer (NSCLC). In this study, the correlation between EGFR mutation status and ERCC1 and BRCA1 gene expression in Chinese NSCLC patients was examined. Real-time polymerase chain reaction (PCR) and direct sequencing were used to detect mRNA expression levels and EGFR mutation status, respectively in microdissected formalin-fixed paraffin-embedded non-small cell lung cancer tissues. EGFR mutations were detected in 27/103 patients (26.2%) and were found to be gender-related (P=0.001). The BRCA1 mRNA expression level was associated with histology, while there was no association with ERCC1. For the EGFR mutant-type, a high BRCA1 gene expression was detected in 2 cases (20.0%) and a low expression in 8 cases (80.0%), while for EGFR wild-type, a high BRCA1 gene expression was detected in 20 cases (43.5%) and a low expression in 26 cases (56.5%). There was no difference in the one-year survival period, according to results obtained for either the ERCC1 or BRCA1 mRNA expression levels. EGFR mutations in NSCLC samples are more likely to express low ERCC1 and BRCA1 mRNA levels. In these latter samples, a statistically significant difference was observed. However, to examine their correlation and clinical outcomes, additional studies are required.

The Predictive and Prognostic Significance of c-erb-B2, EGFR, PTEN, mTOR, PI3K, p27, and ERCC1 Expression in Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma (HCC) is the fifth most common fatal cancer and an important healthcare problem worldwide. There are many studies describing the prognostic and predictive effects of epidermal growth factor receptor 2 (c-erb-B2) and epidermal growth factor receptor 1 (EGFR), transmembrane tyrosine kinases that influence cell growth and proliferation in many tumors.ObjectivesThe current study aimed to investigate the expression levels of c-erb-B2, EGFR, PTEN, mTOR, PI3K, p27, and ERCC1 in hepatocellular carcinoma (HCC) and their correlation with other clinicopathologic features.Patients and MethodsFifty HCC cases were stained immunohistochemically with these markers. Correlations between the markers and clinicopathologic characteristics and survival rates were analyzed.ResultsNo membranous c-erb-B2 staining was seen, whereas cytoplasmic positivity was present in 92% of HCC samples, membranous EGFR was observed in 40%, PI3K was found in all samples, and mTOR was seen in 30%, whereas reduced or absent PTEN expression was observed in 56% of samples and loss of p27 was seen in 92% of the cases. c-erb-B2 and mTOR overexpression, as well as reduced expression of p27, all correlated with multiple tumors (P = 0.041, P < 0.001, and P < 0.001, respectively). P27 loss, and mTOR and EGFR positivity were significantly correlated with AFP (P = 0.047, P = 0.004, and P = 0.008, respectively). Angiolymphatic invasion was more commonly seen in EGFR- and ERCC1-positive cases (P = 0.003 and P = 0.005). EGFR was also correlated with histological grade (P = 0.039). No significant correlations were found among PTEN , PI3K, and the clinicopathological parameters. Disease-free or overall survival rates showed significant differences among therapy modalities, AFP levels, angiolymphatic or lymph node invasions, and ERCC1 and p27 expression levels (P < 0.05).Conclusionsc-erb-B2, EGFR, mTOR, ERCC1 overexpression levels, and loss of p27 may play roles in hepatocarcinogenesis and may be significant predictors of aggressive tumor behavior. These markers were found to be correlated with certain clinicopathologic features, therapy modalities, and survival rates in the current study. These findings may help in planning new, targeted treatment strategies .

207P - Kras Mutational Status and Oxaliplatin Sensitivity: The Other Side of the Moon?

ABSTRACT Background Oxaliplatin is a milestone of colorectal cancer therapy, but it is still lacking of a validated predictive biomarker of response. In a recent retrospective study we found a greater efficacy of oxaliplatin in KRAS mutated patients with metastatic colorectal cancer. Aim of the present study is to investigate “in vitro” the molecular basis of this finding and the possible role of ERCC1, the main mechanism of oxaliplatin resistance. Methods We selected four colorectal cancer cell lines, two KRAS wild type (wt)(HCT-8, HT-29) and two KRAS mutated (mt)(SW620, SW480). The sensitivity of these cell lines to oxaliplatin was evaluated by MTT-test. ERCC1 levels before and after exposure to oxaliplatin were determined by RT-PCR. KRAS was silenced in a KRAS mt cell line (SW620) in order to evaluate the effect on oxaliplatin sensitivity and on ERCC1 levels. ERCC1 was also silenced in all cell lines to confirm his role in the KRAS-mediated oxaliplatin resistance pathway. Results KRAS mt cell lines were more sensitive to oxaliplatin (OR 2,68; IC 95% 1.511-4.757 p Conclusions KRAS mt cell lines were more sensitive to oxaliplatin probably due to their inability to upregulate ERCC1 after exposure to this drug. Therefore, KRAS mutational status could represent a predictor of response to Oxaliplatin in colorectal cancer patients, as a surrogate for ERCC1 modulation. Disclosure All authors have declared no conflicts of interest.