ERCC1 and RRM1 in the International Adjuvant Lung Trial by Automated Quantitative in Situ Analysis
2011; Elsevier BV; Volume: 178; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2010.11.029
ISSN1525-2191
AutoresGerold Bepler, Ken A. Olaussen, A.L. Vataire, Jean‐Charles Soria, Zhong Zheng, Ariane Dunant, Jean‐Pierre Pignon, Michael J. Schell, Pierre Fouret, Robert Pirker, Martin Filipits, Élisabeth Brambilla,
Tópico(s)Lung Cancer Treatments and Mutations
ResumoThe excision repair cross completing group 1 gene product (ERCC1) and the regulatory subunit of ribonucleotide reductase (RRM1) have been reported as being prognostic of outcome and predictive of therapeutic efficacy in patients with non–small cell lung cancer. Routinely processed surgical specimens from 784 patients from the International Adjuvant Lung Trial were arrayed as tissue microarrays. In situ protein levels were scored with an automated, quantitative analysis system, dichotomized into high and low marker categories, and analyzed for associations with patients' characteristics, survival, and benefit from adjuvant chemotherapy. Scores for both markers were significantly associated with contributing center (P < 0.001) and skewed, with the bulk of scores being low. High scores were more frequent in women for ERCC1 and RRM1 and in older patients and those with adenocarcinoma for RRM1. Low ERCC1 scores indicated significant benefit from adjuvant chemotherapy [hazard ratio (HR) = 0.73 for chemotherapy versus control, P = 0.02]. Although all other survival associations were not statistically significant, low RRM1 scores trended to indicate benefit from adjuvant chemotherapy (HR = 0.84, P = 0.25), and ERCC1 scores were marginally prognostic of survival (HR = 0.77 for high versus low scores, P = 0.10). We conclude that contributing center and specimen quality substantially affect the levels of both markers. Future trials should incorporate the collection and processing of tumor specimens prospectively on standardized protocols to better reveal the impact of biomarkers on clinically relevant outcomes. The excision repair cross completing group 1 gene product (ERCC1) and the regulatory subunit of ribonucleotide reductase (RRM1) have been reported as being prognostic of outcome and predictive of therapeutic efficacy in patients with non–small cell lung cancer. Routinely processed surgical specimens from 784 patients from the International Adjuvant Lung Trial were arrayed as tissue microarrays. In situ protein levels were scored with an automated, quantitative analysis system, dichotomized into high and low marker categories, and analyzed for associations with patients' characteristics, survival, and benefit from adjuvant chemotherapy. Scores for both markers were significantly associated with contributing center (P < 0.001) and skewed, with the bulk of scores being low. High scores were more frequent in women for ERCC1 and RRM1 and in older patients and those with adenocarcinoma for RRM1. Low ERCC1 scores indicated significant benefit from adjuvant chemotherapy [hazard ratio (HR) = 0.73 for chemotherapy versus control, P = 0.02]. Although all other survival associations were not statistically significant, low RRM1 scores trended to indicate benefit from adjuvant chemotherapy (HR = 0.84, P = 0.25), and ERCC1 scores were marginally prognostic of survival (HR = 0.77 for high versus low scores, P = 0.10). We conclude that contributing center and specimen quality substantially affect the levels of both markers. Future trials should incorporate the collection and processing of tumor specimens prospectively on standardized protocols to better reveal the impact of biomarkers on clinically relevant outcomes. Tumor progression and resistance to therapeutic interventions are major obstacles to improving the outcome for patients with non–small cell lung cancer (NSCLC). Although notable progress has been made in the treatment of NSCLC, it is still associated with a poor prognosis for most patients. Modern techniques have facilitated the identification of genes and their products that may play a role in disease progression and patient response to therapy. Among these are the excision repair cross completing group 1 gene product (ERCC1) and the regulatory subunit of ribonucleotide reductase (RRM1). ERCC1 is a component of the 5′ endonuclease of the nucleotide excision repair complex, and it is crucial for the repair of DNA damage caused by interstrand and intrastrand cross-links that prevent both replication and transcription.1Reed E. ERCC1 and clinical resistance to platinum-based therapy.Clin Cancer Res. 2005; 11: 6100-6102Crossref PubMed Scopus (120) Google Scholar Previous work in patient-derived specimens has shown that elevated levels of ERCC1 are associated with improved outcomes in patients treated with surgical resection alone.2Simon G.R. Sharma S. Cantor A. Smith P. Bepler G. ERCC1 expression is a predictor of survival in resected patients with non-small cell lung cancer.Chest. 2005; 127: 978-983Crossref PubMed Scopus (259) Google Scholar, 3Olaussen K.A. Dunant A. Fouret P. Brambilla E. Andre F. Haddad V. Taranchon E. Filipits M. Pirker R. Popper H.H. Stahel R. Sabatier L. Pignon J- P. Tursz T. Le Chavalier T. Soria J.-C. DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy.N Engl J Med. 2006; 355: 983-991Crossref PubMed Scopus (1569) Google Scholar It is presumed that this improvement in survival is a result of increased DNA damage repair capacity, and evidence of a reduced lung cancer risk in individuals with increased levels for nucleotide excision repair genes exists.4Wei Q. Cheng L. Hong W.K. Spitz M.R. Reduced DNA repair capacity in lung cancer patients.Cancer Res. 1996; 56: 4103-4107PubMed Google Scholar, 5Cheng L. Spitz M.R. Hong W.K. Wei Q. Reduced expression levels of nucleotide excision repair genes in lung cancer: a case-control analysis.Carcinogenesis. 2000; 8: 1527-1530Crossref Google Scholar However, in patients who require cytotoxic chemotherapy, in particular with agents that induce DNA adducts and cross-links, high levels of ERCC1 are associated with reduced efficacy presumably through the increased efficiency of repair of platinum-induced DNA damage.1Reed E. ERCC1 and clinical resistance to platinum-based therapy.Clin Cancer Res. 2005; 11: 6100-6102Crossref PubMed Scopus (120) Google Scholar, 3Olaussen K.A. Dunant A. Fouret P. Brambilla E. Andre F. Haddad V. Taranchon E. Filipits M. Pirker R. Popper H.H. Stahel R. Sabatier L. Pignon J- P. Tursz T. Le Chavalier T. Soria J.-C. DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy.N Engl J Med. 2006; 355: 983-991Crossref PubMed Scopus (1569) Google Scholar, 6Bepler G. Kusmartseva I. Sharma S. Gautam A. Cantor A. Sharma A. Simon G. RRM1-modulated in vitro and in vivo efficacy of gemcitabine and platinum in non-small cell lung cancer.J Clin Oncol. 2006; 24: 4731-4737Crossref PubMed Scopus (309) Google Scholar RRM1 functions as the regulatory subunit of ribonucleotide reductase and controls substrate specificity and the on/off function of ribonucleotide reductase, whereas the catalytic subunit (RRM2) converts nucleoside diphosphates to the corresponding deoxynucleotides.7Stubbe J. Ribonucleotide reductases in the twenty-first century.Proc Natl Acad Sci U S A. 1998; 95: 2723-2724Crossref PubMed Scopus (87) Google Scholar Results from multiple independent laboratories and clinical studies have shown that RRM1 is the dominant determinant of efficacy for the chemotherapeutic agent gemcitabine, a nucleoside analog.6Bepler G. Kusmartseva I. Sharma S. Gautam A. Cantor A. Sharma A. Simon G. RRM1-modulated in vitro and in vivo efficacy of gemcitabine and platinum in non-small cell lung cancer.J Clin Oncol. 2006; 24: 4731-4737Crossref PubMed Scopus (309) Google Scholar, 8Davidson J.D. Ma L. Flagella M. Geeganage S. Gelbert L.M. Slapak C.A. An increase in the expression of ribonucleotide reductase large subunit 1 is associated with gemcitabine resistance in non-small cell lung cancer cell lines.Cancer Res. 2004; 64: 3761-3766Crossref PubMed Scopus (268) Google Scholar, 9Rosell R. Danenberg K.D. Alberola V. Bepler G. Sanchez J.J. Camps C. Provencio M. Isla D. Taron M. Diz P. Artal A. Ribonucleotide reductase messenger RNA expression and survival in gemcitabine/cisplatin-treated advanced non-small cell lung cancer patients.Clin Cancer Res. 2004; 10: 1318-1325Crossref PubMed Scopus (347) Google Scholar, 10Bergman A. Eijk P. van Haperen V. Smid K. Veerman G. Hubeek I. van den Ijssel P. Ylstra B. Peters G. In vivo induction of resistance to gemcitabine results in increased expression of ribonucleotide reductase subunit M1 as a major determinant.Cancer Res. 2005; 65: 9510-9516Crossref PubMed Scopus (166) Google Scholar, 11Ceppi P. Volante M. Novello S. Rapa I. Danenberg K.D. Danenberg P.V. Cambieri A. Selvaggi G. Saviozzi S. Calogero R. Papotti M. Scagliotti G.V. ERCC1 and RRM1 gene expressions but not EGFR are predictive of shorter survival in advanced non-small-cell lung cancer treated with cisplatin and gemcitabine.Ann Oncol. 2006; 17: 1818-1825Crossref PubMed Scopus (297) Google Scholar, 12Nakahira S. Nakamori S. Tsujie M. Takahashi Y. Okami J. Yoshioka S. Yamasaki M. Marubashi S. Takemasa I. Miyamoto A. Takeda Y. Nagano H. Dono K. Umeshita K. Sakon M. Monden M. Involvement of ribonucleotide reductase M1 subunit overexpression in gemcitabine resistance of human pancreatic cancer.Int J Cancer. 2007; 120: 1355-1363Crossref PubMed Scopus (170) Google Scholar, 13Kim S.O. Jeong J.Y. Kim M.R. Cho H.J. Ju J.Y. Kwon Y.S. Oh I.J. Kim K.S. Kim Y.I. Lim S.C. Kim Y.C. Efficacy of gemcitabine in patients with non-small cell lung cancer according to promoter polymorphisms in the ribonucleotide reductase M1 gene.Clin Cancer Res. 2008; 14: 3083-3088Crossref PubMed Scopus (48) Google Scholar In addition, RRM1 suppresses carcinogen-induced lung tumorigenesis in experimental animal models, which appears to be a result of increased DNA damage repair capacity in the presence of elevated RRM1.14Gautam A. Bepler G. Suppression of lung tumor formation by the regulatory subunit of ribonucleotide reductase.Cancer Res. 2006; 66: 6497-6502Crossref PubMed Scopus (68) Google Scholar It also suppresses tumor progression as shown by decreased cellular migration and invasion in vitro and suppression of metastasis formation in mouse models, resulting in increased animal survival.15Fan H. Huang A. Villegas C. Wright J.A. The R1 component of mammalian ribonucleotide reductase has malignancy-suppressing activity as demonstrated by gene transfer experiments.Proc Natl Acad Sci U S A. 1997; 94: 13181-13186Crossref PubMed Scopus (99) Google Scholar, 16Gautam A. Li Z.R. Bepler G. RRM1-induced metastasis suppression through PTEN-regulated pathways.Oncogene. 2003; 22: 2135-2142Crossref PubMed Scopus (137) Google Scholar In patients with NSCLC and pancreatic cancer who had a complete surgical resection and no adjuvant therapy, survival was longer for those with high levels of expression compared with those with low levels of expression.17Bepler G. Sharma S. Cantor A. Gautam A. Haura E. Simon G. Sharma A. Sommers E. Robinson L. RRM1 and PTEN as prognostic parameters for overall and disease-free survival in patients with non-small-cell lung cancer.J Clin Oncol. 2004; 22: 1878-1885Crossref PubMed Scopus (175) Google Scholar, 18Zheng Z. Chen T. Li X. Haura E. Sharma A. Bepler G. The DNA synthesis and repair genes RRM1 and ERCC1 in lung cancer.N Engl J Med. 2007; 356: 800-808Crossref PubMed Scopus (448) Google Scholar, 19Akita H. Zheng Z. Takeda Y. Chiwan K. Kittaka N. Kobayashi S. Marubashi S. Takemasa I. Nagano H. Dono K. Nkamori S. Monden M. Mori M. Doki Y. Bepler G. Significance of RRM1 and ERCC1 expression in resectable pancreatic adenocarcinoma.Oncogene. 2009; 28: 2903-2909Crossref PubMed Scopus (103) Google Scholar Although little controversy exists regarding the association between ERCC1 and RRM1 with platinum and gemcitabine efficacy, there are recent reports of ERCC1 and RRM1 either not being significantly associated or potentially being inversely associated with the survival of NSCLC patients after a complete surgical resection.20Rosell R. Skrzypski M. Jassem E. Taron M. Bartolucci R. Sanchez J.J. Mendez P. Chaib I. Perez-Roca L. Szymanowska A. Rzyman W. Puma F. Kobierska-Gulida G. Farabi R. Jassem J. BRCA1: a novel prognostic factor in resected non-small-cell lung cancer.PLoS One. 2007; 2: e1129Crossref PubMed Scopus (196) Google Scholar, 21Saviozzi S. Ceppi P. Novello S. Ghio P. Lo Iacono M. Borasia P. Cambieri A. Volante M. Papotti M. Calogero R.A. Scagliotti G.V. Non-small cell lung cancer exhibits transcript overexpression of genes associated with homologous recombination and DNA replication pathways.Cancer Res. 2009; 69: 3390-3396Crossref PubMed Scopus (53) Google Scholar Knowing the prognostic impact of both genes on the survival of patients not undergoing chemotherapy with a platinum agent and/or gemcitabine is important for the design and interpretation of clinical trials that incorporate both genes into a treatment decision algorithm, in particular if this treatment is given in a curative attempt in completely resected patients. Two clinical studies in patients with advanced NSCLC have already used this approach and reported a favorable disease response when treatment is selected based on gene expression.22Simon G. Sharma A. Li X. Hazelton T. Walsh F. Williams C. Chiappori A. Haura E. Tanvetyanon T. Antonia S. Cantor A. Bepler G. Feasibility and efficacy of molecular analysis-directed individualized therapy in advanced non-small-cell lung cancer.J Clin Oncol. 2007; 25: 2741-2746Crossref PubMed Scopus (177) Google Scholar, 23Cobo M. Isla D. Massuti B. Montes A. Sanchez J.M. Provencio M. Vinolas N. Paz-Ares L. Lopez-Vivanco G. Munoz M.A. Felip E. Alberola V. Camps C. Domine M. Sanchez J.J. Sanchez-Ronco M. Danenberg K. Taron M. Gandara D. Rosell R. Customizing cisplatin based on quantitative excision repair cross-complementing 1 mRNA expression: a phase III trial in non-small-cell lung cancer.J Clin Oncol. 2007; 25: 2747-2754Crossref PubMed Scopus (436) Google Scholar Finally, most clinico-correlative investigations involving both genes have reported a highly significant coexpression between ERCC1 and RRM1.6Bepler G. Kusmartseva I. Sharma S. Gautam A. Cantor A. Sharma A. Simon G. RRM1-modulated in vitro and in vivo efficacy of gemcitabine and platinum in non-small cell lung cancer.J Clin Oncol. 2006; 24: 4731-4737Crossref PubMed Scopus (309) Google Scholar, 9Rosell R. Danenberg K.D. Alberola V. Bepler G. Sanchez J.J. Camps C. Provencio M. Isla D. Taron M. Diz P. Artal A. Ribonucleotide reductase messenger RNA expression and survival in gemcitabine/cisplatin-treated advanced non-small cell lung cancer patients.Clin Cancer Res. 2004; 10: 1318-1325Crossref PubMed Scopus (347) Google Scholar, 11Ceppi P. Volante M. Novello S. Rapa I. Danenberg K.D. Danenberg P.V. Cambieri A. Selvaggi G. Saviozzi S. Calogero R. Papotti M. Scagliotti G.V. ERCC1 and RRM1 gene expressions but not EGFR are predictive of shorter survival in advanced non-small-cell lung cancer treated with cisplatin and gemcitabine.Ann Oncol. 2006; 17: 1818-1825Crossref PubMed Scopus (297) Google Scholar, 18Zheng Z. Chen T. Li X. Haura E. Sharma A. Bepler G. The DNA synthesis and repair genes RRM1 and ERCC1 in lung cancer.N Engl J Med. 2007; 356: 800-808Crossref PubMed Scopus (448) Google Scholar, 20Rosell R. Skrzypski M. Jassem E. Taron M. Bartolucci R. Sanchez J.J. Mendez P. Chaib I. Perez-Roca L. Szymanowska A. Rzyman W. Puma F. Kobierska-Gulida G. Farabi R. Jassem J. BRCA1: a novel prognostic factor in resected non-small-cell lung cancer.PLoS One. 2007; 2: e1129Crossref PubMed Scopus (196) Google Scholar, 24Reynolds C. Obasaju C. Schell M.J. Li X. Zheng Z. Boulware D. Caton J.R. DeMarco L.C. O'Rourke M.A. Shaw Wright G. Boehm K.A. Asmar L. Bromund J. Peng G. Monberg M.J. Bepler G. Randomized phase III trial of gemcitabine-based chemotherapy with in situ RRM1 and ERCC1 protein levels for response prediction in non-small-cell lung cancer.J Clin Oncol. 2009; 27: 5808-5815Crossref PubMed Scopus (166) Google Scholar We investigated tumor specimens from the International Adjuvant Lung Trial (IALT25The International Adjuvant Lung Cancer Trial Collaborative GroupCisplatin-based adjuvant chemotherapy in patients with completely resected non-small-cell lung cancer.N Engl J Med. 2004; 350: 351-360Crossref PubMed Scopus (2096) Google Scholar) for ERCC1 and RRM1 in situ protein expression using accurate quantitative analysis (AQUA), a recently developed automated immunofluorescence-based technology. One goal was to compare results obtained for ERCC1 by AQUA on tissue microarrays (TMAs) with those previously reported and obtained by standard manual immunohistochemical analysis (IHC) on full-section specimens. Another goal was to corroborate the role of RRM1 alone and combined with ERCC1 on survival of patients treated with surgery alone and those who had received adjuvant chemotherapy. The IALT-biospecimen collection is the largest existing repository of surgically resected NSCLC specimens with prospectively collected clinical outcomes data on a phase III randomized clinical trial.3Olaussen K.A. Dunant A. Fouret P. Brambilla E. Andre F. Haddad V. Taranchon E. Filipits M. Pirker R. Popper H.H. Stahel R. Sabatier L. Pignon J- P. Tursz T. Le Chavalier T. Soria J.-C. DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy.N Engl J Med. 2006; 355: 983-991Crossref PubMed Scopus (1569) Google Scholar, 25The International Adjuvant Lung Cancer Trial Collaborative GroupCisplatin-based adjuvant chemotherapy in patients with completely resected non-small-cell lung cancer.N Engl J Med. 2004; 350: 351-360Crossref PubMed Scopus (2096) Google Scholar Approval was obtained from the local institutional review boards, according to the legal regulations in each participating country. The collection consists of paraffin blocks from 784 patients with stage I to III (stage I, 270; stage II, 180; stage III, 334) cancer who had been randomly assigned to observation (N = 382) or adjuvant chemotherapy with cis-platin plus etoposide (N = 218), cis-platin plus vinorelbine (N = 122), cis-platin plus vindesine (N = 25), or cis-platin plus vinblastine (N = 37). The specimens were from 28 centers in 14 different countries in Europe and South America that contributed 1042 patients to the IALT. Initially, blocks from a total of 867 patients were collected. Blocks from 43 patients could not be used because they contained insufficient material. A central pathologic review was performed on full sections stained with H&E, and the quality of each specimen was rated as poor, average, or good. Specimens with poor quality (N = 40) were removed from the collection. In addition, information on pleural, vascular, and lymphatic invasion and lymphoid infiltration was collected. The results on the level of expression of ERCC1 by standard IHC on full-section specimens using an H-scoring scale from 0 to 3 [staining intensity (0, 1, 2, 3) × proportion score (0, 0.1, 0.5, 1.0)] have been previously reported.3Olaussen K.A. Dunant A. Fouret P. Brambilla E. Andre F. Haddad V. Taranchon E. Filipits M. Pirker R. Popper H.H. Stahel R. Sabatier L. Pignon J- P. Tursz T. Le Chavalier T. Soria J.-C. DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy.N Engl J Med. 2006; 355: 983-991Crossref PubMed Scopus (1569) Google Scholar Specimen cores from patients with average or good rating (N = 784) were arrayed as triplicate spots of 0.6-mm diameter in a total of 13 TMA blocks. A pathology review of the selected cores was performed (E.B.) to verify the presence of tumor tissue and tissue quality (classified as present or absent and average or good). From two patients two separate blocks were arrayed for a total of six spots (782 patients with three tumor spots and two patients with six tumor spots). All specimens from each patient were on the same slide (including the two patients who had two blocks), and patients from the 28 centers were clustered on slides (ie, they were not randomly distributed in the 13 TMA blocks). The following variables were prospectively collected on all patients: disease stage by TNM classification (AJCC sixth edition, 2002), tumor histologic findings, sex, age, performance status, and type of surgery. In addition, data on whether patients received adjuvant radiation and the random assignment to observation or adjuvant chemotherapy, including the type of chemotherapy, were known. The outcomes data used for this analysis were those originally reported with a median follow-up time of 56 months.3Olaussen K.A. Dunant A. Fouret P. Brambilla E. Andre F. Haddad V. Taranchon E. Filipits M. Pirker R. Popper H.H. Stahel R. Sabatier L. Pignon J- P. Tursz T. Le Chavalier T. Soria J.-C. DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy.N Engl J Med. 2006; 355: 983-991Crossref PubMed Scopus (1569) Google Scholar, 25The International Adjuvant Lung Cancer Trial Collaborative GroupCisplatin-based adjuvant chemotherapy in patients with completely resected non-small-cell lung cancer.N Engl J Med. 2004; 350: 351-360Crossref PubMed Scopus (2096) Google Scholar Immunofluorescence combined with AQUA was used to assess in situ expression of the target molecules.26Camp R.L. Chung G.G. Rimm D.L. Automated subcellular localization and quantification of protein expression in tissue microarrays.Nat Med. 2002; 8: 1323-1327Crossref PubMed Scopus (657) Google Scholar Microtome sections of 4-μm thickness were microwaved in 10 mmol/L Tris-EDTA, pH 9.0 (for ERCC1 staining), or 10 mmol/L Tris-HCl, pH 12.0 (for RRM1 staining), for 10 minutes, cooled to room temperature, and rinsed with PBS. Endogenous peroxidase was inactivated with 3% hydrogen peroxide for 20 minutes. Slides were then incubated overnight at 4°C in appropriately diluted primary antiserum (R1AS-6, 1:800, pH 12.0, for RRM1) or antibody (clone 8F1, 1:300, pH 9.0, for ERCC1; cat D8903, lot 9475; Sigma-Aldrich, St. Louis, MO) as described.18Zheng Z. Chen T. Li X. Haura E. Sharma A. Bepler G. The DNA synthesis and repair genes RRM1 and ERCC1 in lung cancer.N Engl J Med. 2007; 356: 800-808Crossref PubMed Scopus (448) Google Scholar Cells of epithelial origin, consisting mostly of malignant cells, were identified by cytokeratin staining. The primary antibody was then visualized with different fluorochrome-labeled secondary antibodies (Envision labeled polymer horseradish peroxidase antirabbit or antimouse for RRM1 or ERCC1 detection; Alexa 555 goat antimouse or goat antirabbit for cytokeratin detection). Fluorescence of the target signals was amplified with Cy5-tyramide. 4′,6-diamidine-2′-phenylindole, which strongly binds to DNA minor groove, was added to the coverslip mounting solution and used for identification of nuclei. The final TMA slides were scanned with SpotGrabber, and image data were analyzed with AQUA (PM-2000, HistoRx, New Haven, CT). Software version 1.2 was used with an exposure time of 30 milliseconds. The maximal range of the AQUA scores with this software version is 0 to 255. The analysis was performed at two different levels. One was solely of the AQUA scores taking the characteristics of the TMA into account, and the other was to compare the individual patients' average scores with clinical outcomes parameters. The primary intent was to test for the prognostic value of RRM1, ERCC1, and both combined on patient survival in the group who did not receive chemotherapy and for interaction between these markers and benefit from adjuvant chemotherapy using the raw ERCC1 and RRM1 values with dichotomization of patients into high and low marker level categories using the median values as threshold. Provisions for preplanned optimal cut point analyses and normalization of marker values had not been made a priori. AQUA scores were analyzed for skewness, kurtosis, and other summary statistics, and they were compared by slides, centers, and specimen quality. The relationships between marker categories and histologic findings, tumor size, lymph node involvement, and tumor stage were analyzed using logistic models stratified by center. The agreement between IHC H-scores3Olaussen K.A. Dunant A. Fouret P. Brambilla E. Andre F. Haddad V. Taranchon E. Filipits M. Pirker R. Popper H.H. Stahel R. Sabatier L. Pignon J- P. Tursz T. Le Chavalier T. Soria J.-C. DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy.N Engl J Med. 2006; 355: 983-991Crossref PubMed Scopus (1569) Google Scholar and AQUA scores was studied by Spearman's correlation coefficient on the continuous values and by κ coefficient using the high and low expression categories. A Cox model that included the clinical prognostic factors isolated in the main analysis of the IALT together with the treatment was used for the prognostic analysis, and it was adjusted for the factors used in the randomization process (including contributing center and specimen quality). All other factors statistically related to the marker in the multivariate logistic model were added to the Cox model. The predictive value of the marker was studied by testing the interaction between the marker category (high or low) and the attributed treatment (chemotherapy or observation) in the same Cox model. Survival curves by marker levels and treatment groups were generated using the Kaplan-Meier method. All reported P values were two-sided. All analyses were performed using SAS software, version 8.2 (SAS Institute Inc., Cary, NC). The expression of both proteins was predominantly nuclear as previously described3Olaussen K.A. Dunant A. Fouret P. Brambilla E. Andre F. Haddad V. Taranchon E. Filipits M. Pirker R. Popper H.H. Stahel R. Sabatier L. Pignon J- P. Tursz T. Le Chavalier T. Soria J.-C. DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy.N Engl J Med. 2006; 355: 983-991Crossref PubMed Scopus (1569) Google Scholar, 18Zheng Z. Chen T. Li X. Haura E. Sharma A. Bepler G. The DNA synthesis and repair genes RRM1 and ERCC1 in lung cancer.N Engl J Med. 2007; 356: 800-808Crossref PubMed Scopus (448) Google Scholar and displayed a fine granular pattern for ERCC1 and a coarse granular pattern for RRM1. ERCC1 values were obtained on 763 of 784 patients. Unavailability of values was a result of absence of tissue in the assigned location. Replicate core values (2× in 77, 3× in 672 and 6× in 1) were averaged. A histopathologic review of H&E-stained tissue cores confirmed the presence of NSCLC in 747 cases, which were used for further analyses and specimen quality (classified as average or good). Expression levels ranged from 2.2 to 149.1, with a median of 9.0, a mean of 13.2, and an SD of 12.8. They were highly skewed to the right (skewness, 4.3; kurtosis, 29.1) (ie, high scores were much farther away from the median than low scores). RRM1 values were obtained on 752 patients, and values on patients with replicates were averaged (2× in 112, 3× in 610 and 6× in 1). Presence of NSCLC in tissue cores was confirmed in 738 cases. Levels ranged from 0.2 to 91.8, with a median of 9.7, a mean of 15.1, and an SD of 15.0. The RRM1 levels were skewed toward the right (skewness, 2.3; kurtosis, 6.0). ERCC1 values in patients with three replicates were highly correlated (Spearman's r = 0.78 to 0.84; P < 0.0001), and the coefficients of variation were below 1.4%. Likewise, replicate RRM1 values were highly correlated (r = 0.76 to 0.80; P < 0.0001), and the coefficients of variation were below 1.6%. ERCC1 and RRM1 expression levels were modestly correlated (N = 730, r = 0.21; P < 0.001) (Figure 1). Specimen origin by center and specimen quality contributed significantly to the differences seen for both ERCC1 and RRM1 values (Table 1), a result that had previously been observed for ERCC1 using full-section specimens and standard immunohistochemical analysis.3Olaussen K.A. Dunant A. Fouret P. Brambilla E. Andre F. Haddad V. Taranchon E. Filipits M. Pirker R. Popper H.H. Stahel R. Sabatier L. Pignon J- P. Tursz T. Le Chavalier T. Soria J.-C. DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy.N Engl J Med. 2006; 355: 983-991Crossref PubMed Scopus (1569) Google Scholar Center accounted for 11% of the overall variation in ERCC1 AQUA levels (P < 0.0001), 10% in ERCC1 IHC levels (P < 0.0001), and 33% in the variation of RRM1 levels (P < 0.001).Table 1ERCC1 and RRM1 Expression Categories by Patient Demographics and Disease Characteristics Stratified by Contributing CenterP value (adjusted by center)CharacteristicsERCC1RRM1Sex Male0.040.04 Female(F > M)(F > M)Age, y 64(older > young)WHO-PS 0 10.790.06 2(0 > 1 > 2)Stage I II0.530.75 IIITumor by TNM 1 20.930.82 3 and 4Nodes by TNM 0 10.650.82 2Histopathologic findings Adenocarcinoma Squamous cell carcinoma0.700.02 Other(adenocarcinoma > squamous cell carcinoma)Pleural invasion Absent0.680.46 PresentVascular invasion Absent0.060.13 Present(absent > present)Lymphatic invasion Absent0.550.40 PresentLymphoid infiltration Not intense0.660.38 IntenseType of surgery Lobe/segmentectomy0.720.18 PneumonectomyQuality of specimen Good0.0070.78 Average (poor specimens excluded)(average > good)WHO-PS, World Health Organization Performance Scale; F, female; M, male. Open table in a new tab WHO-PS, World Health Organization Performance Scale; F, female; M, male. According to the prespecified analysis plan, ERCC1 expression was dichotomized into high versus low using 10 or greater versus less than 10 (high in 331 patients, low in 416). The threshold of 10, rather than the sample median of 9, was used because this resulted in a proportional cohort split similar to the one previously used using IHC-based H-scores (H-score of 1.5 to 3.0 in 335 and 0.0 to 1.0 in 426 patients). For RRM1 dichotomization, the sample median was used as threshold (10 or greater versus less than 10; high in 363 patients, low in 375). We observed that high ERCC1 and RRM1 levels
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