Correspondence

ERCC1 and Non–Small-Cell Lung Cancer

N Engl J Med 2007; 356:2538-2541June 14, 2007

Article

To the Editor:

Zheng et al. (Feb. 22 issue)1 conclude that the excision repair cross-complementation group 1 (ERCC1) protein is a determinant of survival after surgical treatment of early-stage non–small-cell lung cancer. The ERCC1 protein associates with the xeroderma pigmentosum group F (XPF) protein to form a nuclease that functions in DNA repair.2 The level of ERCC1 protein was proposed as a useful predictor of response to cisplatin-based chemotherapy and clinical outcome,3 on the basis of immunohistochemical staining of tumors with the use of the monoclonal antibody 8F1. To our knowledge, however, no controlled experiments demonstrating the specificity of this antibody have been reported.

We examined 8F1 and a second commercially available antibody (FL-297) for specificity in detecting ERCC1, using ERCC1-positive normal human fibroblasts and cells from patients with inherited mutations in ERCC1 (Patient 165TOR) and XPF (Patient XP2YO) causing a deficiency of ERCC1–XPF nuclease.2,4 Immunoblotting of cell lysates with FL-297 revealed a single band of appropriate molecular weight in normal fibroblasts and confirmed that the level of ERCC1 protein was reduced in cells from Patients XP2YO and 165TOR (Figure 1AFigure 1Comparison of ERCC1 Antibodies 8F1 and FL-297 for the Detection of ERCC1.). The 8F1 antibody detected ERCC1 and at least one other protein in whole-cell extracts (Figure 1B). Tubulin antibody confirmed equal loading of cell extracts (Figure 1C). The cross-reacting protein is present in normal and ERCC1-deficient cell extracts.

ERCC1-deficient cells from Patient XP2YO and normal human fibroblasts were differentially labeled with cytoplasmic beads and cocultured. Immunostaining with FL-297 discriminated between ERCC1-positive and ERCC1-deficient cells (Figure 1D). In contrast, 8F1 strongly stained the nuclei of all cells. Irradiation of cells with ultraviolet (UV) light through a filter containing 8-μm pores causes subnuclear domains of UV-induced DNA damage that can be identified with an antibody recognizing thymine dimers.5 ERCC1–XPF repair nuclease accumulates at these sites of DNA damage.5 Immunostaining of irradiated fibroblasts with antithymine dimer and FL-297 yielded signals that colocalized (Figure 1E). In contrast, the antigen recognized by 8F1 does not preferentially accumulate at sites of DNA damage (Figure 1F) or give a signal that colocalizes with that of FL-297 (Figure 1G).

These experiments show that ERCC1 is not the principal antigen recognized by the 8F1 antibody on immunostaining of human cells. Furthermore, 8F1 does not discriminate between ERCC1-positive and ERCC1-deficient nuclei. The identity of the antigen recognized by 8F1 and the reason that 8F1 stains some tumors more strongly than others are unknown. These results underscore the point that even monoclonal antibodies raised against recombinant protein are not guaranteed to be specific.

Laura J. Niedernhofer, M.D., Ph.D.
Nikhil Bhagwat, M.B., B.S.
Richard D. Wood, Ph.D.
University of Pittsburgh, Pittsburgh, PA 15213

Support for the 8F1-antibody research was provided by a grant to the University of Pittsburgh Cancer Institute from the Lung Specialized Programs of Research Excellence of the National Institutes of Health. The 8F1 antibody is available for commercial licensing from Cancer Research UK's technology transfer section, Cancer Research Technology. Dr. Wood receives a percentage of any revenues derived from such licensing as part of the Awards to Inventors program. No other potential conflict of interest relevant to this letter was reported.

5 References

1. 1

   Zheng Z, Chen T, Li X, Haura E, Sharma A, Bepler G. DNA synthesis and repair genes RRM1 and ERCC1 in lung cancer. N Engl J Med 2007;356:800-808
   Full Text | Web of Science | Medline

2. 2

   Sijbers AM, de Laat WL, Ariza RR, et al. Xeroderma pigmentosum group F caused by a defect in a structure-specific DNA repair endonuclease. Cell 1996;86:811-822
   CrossRef | Web of Science | Medline

3. 3

   Olaussen KA, Dunant A, Fouret P, et al. DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy. N Engl J Med 2006;355:983-991
   Full Text | Web of Science | Medline

4. 4

   Jaspers NG, Raams A, Silengo MC, et al. First reported patient with human ERCC1 deficiency has cerebro-oculo-facio-skeletal syndrome with a mild defect in nucleotide excision repair and severe developmental failure. Am J Hum Genet 2007;80:457-466
   CrossRef | Web of Science | Medline

5. 5

   Volker M, Mone MJ, Karmakar P, et al. Sequential assembly of the nucleotide excision repair factors in vivo. Mol Cell 2001;8:213-224
   CrossRef | Web of Science | Medline

To the Editor:

Zheng et al. appear to have oversimplified the relationship of regulatory subunit of ribonucleotide reductase (RRM1) and ERCC1 expression levels with prognosis in non–small-cell lung cancer. The authors did not stratify their patients with regard to smoking and other potential covariables and genotyping analysis of RRM1 and ERCC1. To date, 226 and 92 single-nucleotide polymorphisms (SNPs) have been reported in RRM1 and ERCC1, respectively, and many of these SNPs have a functional effect (details are available at www.ncbi.nlm.nih.gov/SNP/). For instance, the SNP in RRM1 (2464G→A) is associated with resistance to gemcitabine therapy,1 whereas the SNP in ERCC1 (118C→T) influences the outcome of cisplatin therapy in patients with non–small-cell lung cancer.2 Smoking is also an important factor that has interplay with the SNP of ERCC1.2

Shu-Feng Zhou, M.D., Ph.D.
Queensland University of Technology, Brisbane 4001, Australia
s4.zhou@qut.edu.au

2 References

1. 1

   Kwon WS, Rha SY, Choi YH, et al. Ribonucleotide reductase M1 (RRM1) 2464G→A polymorphism shows an association with gemcitabine chemosensitivity in cancer cell lines. Pharmacogenet Genomics 2006;16:429-438
   CrossRef | Web of Science | Medline

2. 2

   Isla D, Sarries C, Rosell R, et al. Single nucleotide polymorphisms and outcome in docetaxel-cisplatin-treated advanced non-small-cell lung cancer. Ann Oncol 2004;15:1194-1203
   CrossRef | Web of Science | Medline

To the Editor:

Zheng et al. suggest that using a combination of the gene expression of both ERCC1 and RRM1 can isolate a subgroup of patients with excellent survival. In particular, Figure 5 of the article shows that the subgroup with a high expression of both genes has an overall survival of more than 120 months. It does not appear that the authors analyzed this particular subgroup with regard to whether the patients' tumors were largely in stage pIA. If this is the case, it would be interesting to know what the average size of the tumor was among these 55 patients with high expression of ERCC1 and RRM1. If the tumors were small, it may be that these two markers were just identifying patients who presented with small tumors and thus had a relatively better prognosis.

Lawrence Panasci, M.D.
Victor Cohen, M.D.
Sir Mortimer B. Davis Jewish General Hospital, Montreal, QC H3T 1E2, Canada

Author/Editor Response

Niedernhofer et al. show two distinct proteins with slightly different molecular masses interacting with 8F1, ERCC1 and an unidentified protein. This difference may explain the reported lack of association between ERCC1 messenger RNA (mRNA) and protein expression. Since the identity and pattern of expression of the larger band are obscure, its effect on the prognostic usefulness of ERCC1 levels measured by 8F1 is unknown.

We did not observe a significant difference in RRM1 and ERCC1 levels between tumors measuring less than 3 cm and those measuring more than 3 cm. In our data set, tumor size was not significantly associated with either overall survival (P=0.07 by the log-rank test) or disease-free survival (P=0.09) when categorized into four groups (≤2.0 cm, 2.1 to 3.5 cm, 3.6 to 5.0 cm, and >5.0 cm). We believe that the observed survival advantage for patients with high RRM1 and ERCC1 expression cannot be explained by tumor size.

The Entrez database contains a total of 272 SNPs for RRM1 and 109 for ERCC1. Nine are in the coding region of RRM1; four result in amino acid alterations, and SNP 2464A→G does not (Table 1Table 1 RRM1 Coding-Region Variations in Non–Small-Cell Lung Cancers.). For ERCC1, five coding-region SNPs are described, and two result in amino acid alterations. We sequenced the genomic region of RRM1 and deposited the data in GenBank (AF107045), which provided the reference for some of the reported SNPs.1 In addition, we described SNPs in the RRM1 promoter region that have a substantial effect on in vitro reporter gene transcription; however, we were unable to show their effect on in vivo gene expression.2

We sequenced the coding region of RRM1 in fresh-frozen specimens from 13 white men and women with non–small-cell lung cancer; at least 70% of the cells in the specimens were tumor cells. Sequences of good quality were compared with NM_001033 (Table 1) and all patient sequences were identical to one another.

We had previously reported an A at position 2455. In all specimens, G was the only nucleotide found at this position. For all other SNPs, we found only the nucleotide reported in the reference sequence. We have noted that the sequence chromatograms for the regions containing the SNPs frequently display low nucleotide signal values. The automated base assignment is often ambiguous in particular when double cytosines or guanines precede or follow the referenced SNPs. These technical limitations may account for the reported SNPs and call their existence into question. Given these limitations, it is our opinion that investigations of the correlations between reported SNPs and clinical outcomes are premature.

Gerold Bepler, M.D., Ph.D.
Zhong Zheng, M.D., Ph.D.
Tingan Chen, M.D., Ph.D.
H. Lee Moffitt Cancer Center, Tampa, FL 33612
gerold.bepler@moffitt.org

2 References

1. 1

   Pitterle DM, Kim YC, Jolicoeur EMC, Cao Y, O'Briant KC, Bepler G. Lung cancer and the human gene for ribonucleotide reductase subunit M1 (RRM1). Mamm Genome 1999;10:916-922
   CrossRef | Web of Science | Medline

2. 2

   Bepler G, Zheng Z, Gautam A, et al. Ribonucleotide reductase M1 gene promoter activity, polymorphisms, population frequencies, and clinical relevance. Lung Cancer 2005;47:183-192
   CrossRef | Web of Science | Medline

Citing Articles (16)

Citing Articles

1. 1

   Emre Tepeli, Vildan Caner, Nur Büyükpınarbaşılı, G. Ozan Çetin, Füsun Düzcan, Levent Elmas, Gülseren Bağcı. (2012) Expression of ERCC1 and its clinicopathological correlations in non-small cell lung cancer. Molecular Biology Reports 39:1, 335-341
   CrossRef

2. 2

   I. G. Hwang, J. S. Jang, J. H. Do, J. H. Kang, G. W. Lee, S. Y. Oh, H. C. Kwon, H. J. Jun, H. Y. Lim, S. Lee, K. C. Chi, S. J. Lee. (2011) Different relation between ERCC1 overexpression and treatment outcomes of two platinum agents in advanced biliary tract adenocarcinoma patients. Cancer Chemotherapy and Pharmacology 68:4, 935-944
   CrossRef

3. 3

   Katsuhiro Okuda, Hidefumi Sasaki, Yu Hikosaka, Osamu Kawano, Haruhiro Yukiue, Motoki Yano, Yoshitaka Fujii. (2011) Excision Repair Cross Complementation Group 1 Polymorphisms Predict Overall Survival after Platinum-Based Chemotherapy for Completely Resected Non-Small-Cell Lung Cancer. Journal of Surgical Research 168:2, 206-212
   CrossRef

4. 4

   Desirée Hao, Harold Y. Lau, Misha Eliasziw, Alan Box, Román Diaz, Alexander C. Klimowicz, Brian Shin, Susan P. Lees-Miller, Anthony M. Magliocco. (2011) Comparing ERCC1 protein expression, mRNA levels, and genotype in squamous cell carcinomas of the head and neck treated with concurrent chemoradiation stratified by HPV status. Head & Neckn/a-n/a
   CrossRef

5. 5

   Corinne M. Doll, Michael Prystajecky, Misha Eliasziw, Alexander C. Klimowicz, Stephanie K. Petrillo, Peter S. Craighead, Desiree Hao, Roman Diaz, Susan P. Lees-Miller, Anthony M. Magliocco. (2010) Low ERCC1 mRNA and protein expression are associated with worse survival in cervical cancer patients treated with radiation alone. Radiotherapy and Oncology 97:2, 352-359
   CrossRef

6. 6

   Francesca Toffalorio, Tommaso De Pas, Elisa Giovannetti. (2010) Differences of Gene Expression in Non-small Cell Lung Cancer. Journal of Thoracic Oncology 5:8, 1310-1311
   CrossRef

7. 7

   Xin Wang, Jun Zhao, Lu Yang, Li Mao, Tongtong An, Hua Bai, Shuhang Wang, Xuyi Liu, Guoshuang Feng, Jie Wang. (2010) Positive expression of ERCC1 predicts a poorer platinum-based treatment outcome in Chinese patients with advanced non-small-cell lung cancer. Medical Oncology 27:2, 484-490
   CrossRef

8. 8

   Xin Shelley Wang, Qiuling Shi, Charles Lu, Ethan M. Basch, Valen E. Johnson, Tito R. Mendoza, Gary M. Mobley, Charles S. Cleeland. (2009) Prognostic value of symptom burden for overall survival in patients receiving chemotherapy for advanced nonsmall cell lung cancer. CancerNA-NA
   CrossRef

9. 9

   Jordi Rodon, Maria D Iniesta, Kyriakos Papadopoulos. (2009) Development of PARP inhibitors in oncology. Expert Opinion on Investigational Drugs 18:1, 31-43
   CrossRef

10. 10

    Chiun Hsu, Sung-Hsin Kuo, Fu-Chang Hu, Ann-Lii Cheng, Jin-Yuan Shih, Chong-Jen Yu, Chia-Chi Lin, Tsu-Chen Huang, Pan-Chyr Yang, Chih-Hsin Yang. (2008) Gemcitabine plus conventional-dose epirubicin versus gemcitabine plus cisplatin as first-line chemotherapy for stage IIIB/IV non-small cell lung carcinoma—A randomized phase II trial. Lung Cancer 62:3, 334-343
    CrossRef

11. 11

    Katsuhiro Okuda, Hidefumi Sasaki, Charles Dumontet, Osamu Kawano, Haruhiro Yukiue, Tomoki Yokoyama, Motoki Yano, Yoshitaka Fujii. (2008) Expression of excision repair cross-complementation group 1 and class III β-tubulin predict survival after chemotherapy for completely resected non-small cell lung cancer. Lung Cancer 62:1, 105-112
    CrossRef

12. 12

    Junichi SHIMIZU, Yoshitsugu HORIO, Hirotaka OSADA, Toyoaki HIDA, Yoshinori HASEGAWA, Kaoru SHIMOKATA, Takashi TAKAHASHI, Yoshitaka SEKIDO, Yasushi YATABE. (2008) mRNA expression of RRM1, ERCC1 and ERCC2 is not associated with chemosensitivity to cisplatin, carboplatin and gemcitabine in human lung cancer cell lines. Respirology 13:4, 510-517
    CrossRef

13. 13

    J Wei, Z Zou, X Qian, Y Ding, L Xie, J J Sanchez, Y Zhao, J Feng, Y Ling, Y Liu, L Yu, R Rosell, B Liu. (2008) ERCC1 mRNA levels and survival of advanced gastric cancer patients treated with a modified FOLFOX regimen. British Journal of Cancer 98:8, 1398-1402
    CrossRef

14. 14

    Tetsuya Fujii, Shinichi Toyooka, Kouichi Ichimura, Yoshiro Fujiwara, Katsuyuki Hotta, Junichi Soh, Hiroshi Suehisa, Naruyuki Kobayashi, Motoi Aoe, Tadashi Yoshino, Katsuyuki Kiura, Hiroshi Date. (2008) ERCC1 protein expression predicts the response of cisplatin-based neoadjuvant chemotherapy in non-small-cell lung cancer. Lung Cancer 59:3, 377-384
    CrossRef

15. 15

    Christopher G. Azzoli, Bernard J. Park, William Pao, Maureen Zakowski, Mark G. Kris. (2008) Molecularly Tailored Adjuvant Chemotherapy for Resected Non-small Cell Lung Cancer. Journal of Thoracic Oncology 3:1, 84-93
    CrossRef

16. 16

    Olaussen, Ken A., , Fouret, Pierre, , Kroemer, Guido, . (2007) ERCC1-Specific Immunostaining in Non–Small-Cell Lung Cancer. New England Journal of Medicine 357:15, 1559-1561
    Full Text