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© 2001 American Society for Clinical Oncology
ERCC1 and Thymidylate Synthase mRNA Levels Predict Survival for Colorectal Cancer Patients Receiving Combination Oxaliplatin and Fluorouracil ChemotherapyByFrom the University of Southern California/Norris Comprehensive Cancer Center and Response Genetics Inc, Los Angeles, CA. Address reprint requests to Heinz-Josef Lenz, MD, Gastrointestinal-Oncology Program, University of Southern California/Norris Comprehensive Cancer Center, Ste 3456, 1441 Eastlake Ave, Los Angeles, CA 90033; email: lenz{at}hsc.usc.edu
PURPOSE: To test the hypotheses of whether the relative mRNA expression of the thymidylate synthase (TS) gene and the excision cross-complementing (ERCC1) gene are associated with response to and survival of fluorouracil (5-FU)/oxaliplatin chemotherapy in metastatic colorectal cancer. PATIENTS AND METHODS: Patients had progressive stage IV disease after unsuccessful 5-FU and irinotecan chemotherapy. All patients were evaluated for eligibility for a compassionate 5-FU/oxaliplatin protocol. cDNA was derived from paraffin-embedded tumor specimens to determine TS and ERCC1 mRNA expression relative to the internal reference gene beta-actin using fluorescence-based, real-time reverse transcriptase polymerase chain reaction.
RESULTS: The median TS gene expression level from 50 metastasized tumors was 3.4 x 10-3 (minimum expression, 0.18 x 10-3;maximum expression, 11.5 x 10-3), and the median ERCC1 gene expression level was 2.53 x 10-3 (minimum, 0.0; maximum, 14.61 x 10-3). The gene expression cutoff values for chemotherapy nonresponse were 7.5 x 10-3 for TS and 4.9 x 10-3 for ERCC1. The median survival time for patients with TS CONCLUSION: These data suggest that intratumoral ERCC1 mRNA and TS mRNA expression levels are independent predictive markers of survival for 5-FU and oxaliplatin combination chemotherapy in 5-FU–resistant metastatic colorectal cancer. Precise definition of the best TS cut point will require further analysis in a large, prospective study.
FOR MORE THAN 40 years, the mainstay of chemotherapy for treatment of advanced colorectal cancer has been fluorouracil (5-FU). The response rate of 5-FU is about 10% to 20% when it is administrated as a single agent.1-3 The introduction of oxaliplatin into the chemotherapy treatment of metastatic colorectal tumors represents a significant advancement in fighting this disease. Although synergistic effects of 5-FU and oxaliplatin have increased response rates up to 25% even in heavily pretreated relapsing patients, the mechanisms for resistance still remain unknown. Resistance to platinum agents has been attributed to enhanced tolerance to platinum DNA adducts, decreased drug accumulation, and enhanced DNA repair.4 Oxaliplatin, a platinum-based chemotherapeutic agent carrying a 1,2-diamino-cyclohexane ring, has shown antitumor efficacy in vitro and in vivo. This bulkier carrier group is considered to lead to platinum-DNA adducts, which are more cytotoxic than adducts formed from other platinum agents and more effective at blocking DNA replication. Recent data have shown that deficiency in the mismatch repair system and increased ability of the replication complex to synthesize DNA past the site of DNA damage (enhanced replicative bypass) cause resistance to cisplatin but not to oxaliplatin.5 Proteins of the nucleotide excision repair pathway are thought to repair DNA damage caused by platinum agents. The excision repair cross-complementing (ERCC) gene family prevents damage to DNA by nucleotide excision and repair. Modified nucleotides together with adjacent nucleotides are removed from the damaged strand during the first step (excision), which is followed by recovery of an intact strand through DNA polymerase activity (repair synthesis).6,7 The ERCC1 gene encodes a protein of 297 amino acids that is considered to function in a complex with ERCC11, XPF, and ERCC4.8 This complex may be required in both recombinational repair and nucleotide excision repain.9 Thymidylate synthase (TS), the target enzyme of the antimetabolite 5-FU, has been shown to be an independent prognostic marker of 5-FU chemotherapy in gastrointestinal tumors.10-12 It’s role in 5-FU/oxaliplatin combination chemotherapy has not been defined yet. Because of the efficacy of 5-FU/oxaliplatin combination chemotherapy in advanced colorectal cancer, the determination of distinct molecular parameters, which may be at least in part responsible for inherent resistance or sensitivity to each drug, may become a useful tool. It would help to spare already heavily pretreated patients from the side effects of chemotherapy, patients who most likely will not benefit from the treatment. Our group has shown that the relative mRNA level of ERCC1 is inversely associated with survival and response in gastric cancer patients treated with 5-FU and cisplatin.13 On the basis of these findings, we tested the hypothesis of whether TS and ERCC1 mRNA expression levels would predict the clinical outcome of patients with advanced colorectal cancer treated with 5-FU/oxaliplatin.
RNA Extraction and cDNA Synthesis RNA was isolated from paraffin-embedded specimens by Response Genetics Inc (Los Angeles, CA), according to a proprietary procedure (United States patent pending). After RNA isolation, cDNA was prepared from each sample, as described previously.14
Polymerase Chain Reaction Quantification and mRNA Expression
Patient Selection and Chemotherapy Treatment All patients were enrolled onto compassionate oxaliplatin treatment protocol 3C-98-3 at the University of Southern California/Norris Comprehensive Cancer Center from 1998 to 2000 and received the following combination therapy regimen: oxaliplatin 130 mg/m2 every 3 weeks and continuous-infusion 5-FU 200 mg/m2/d. For all patients, prior treatment with 5-FU had failed, and for 60% (30 of 50), an additional second-line treatment with irinotecan had failed. All patients had documented progressive disease before protocol entry. All patients gave written informed consent to participate in the clinical trial and for evaluation of the TS and ERCC1 mRNA expression analysis. Tumor samples were obtained directly before enrollment onto the compassionate 5-FU/oxaliplatin protocol, after patient pretreatment. The tumor samples were taken from metastatic sites of the liver or from a recurrent colorectal tumor mass.
Clinical Evaluation and Response Criteria
Statistical Analysis
Demographics and Patients Available for Response and Survival Evaluation A total of 50 patients, consisting of 14 women (28%) and 36 men (72%) with a median age of 59 years (minimum age, 34 years; maximum age, 83 years), were evaluated in this study. There were 39 whites, six Hispanics, three Asians, and two African-Americans. All 50 patients were assessable to associate TS expression and ERCC1 expression levels with survival. Forty-five patients (90%) were assessable to test the association between the molecular parameters and response by using the above-cited criteria.
TS and ERCC1 Expression Levels
Survival in Relation to TS Expression
Survival in Relation to ERCC1 Expression Using 4.9 x 10-3 as a cutoff, 40 patients (80%) had low ERCC1 expression and 10 (20%) had high ERCC1 expression. Figure 2 displays a Kaplan-Meier plot of the estimated probability of survival versus ERCC1 expression levels and shows a median survival of 10.2 months (95% CI, 7.8 to 15.1 months) for the low-expression group and 1.9 months (95% CI, 1.1 to 4.9 months) for the high-expression group (P < .001; log-rank test). The probability of survival at 6 months was .76 for patients with ERCC1 expression  4.9 x 10-3 compared with .16 for patients with ERCC1 expression greater than 4.9 x 10-3. Patients with ERCC1 levels greater than 4.9 x 10-3 had a 4.8-fold (95% CI, 2.09- to 15.88-fold) increased relative risk of dying compared with patients with ERCC1 levels 4.9 x 10-3 in the univariate analysis (P < .001; Table 2).
Survival in Relation to Combined ERCC1 and TS Expression Low TS and ERCC1 expression levels were detected in 36 (72%) of the patients, and 14 patients (28%) had a high TS and/or ERCC1 expression level. Among the seven patients with high TS expression, three (43%) also showed high ERCC1 expression. Three patients (33%) with high ERCC1 expression also showed high TS expression. Among 43 patients with low TS expression, 36 patients (84%) showed low ERCC1 expression too. Thirty-six patients (90%) with low ERCC1 expression also expressed TS at a low level. Patients with low expression levels for both genes had significantly superior survival. The median survival time was 11.1 months (95% CI, 8.4 to 17.5 months) for the low TS and ERCC1 expressors and 1.9 months (95% CI, 1.1 to 4.9 months) for the high TS and/or ERCC1 expressors (P < .001, log-rank test; Fig 3). Patients with low expression levels for both genes had a probability of survival at 6 months of .85, compared with .10 for the patients with a high expression level for at least one gene, TS or ERCC1. The relative risk of dying for patients with increased expression of at least one gene (TS or ERCC1) was 7.12 (95% CI, 2.60 to 19.52) compared with patients who had low expression levels for both genes in the tumor (P < .001; Table 2). TS and ERCC1 mRNA expression are independent of each other, as revealed by the stratified analysis (Table 3).
Association of Response With TS and ERCC1 Gene Expression Levels The median TS expression level was 3.4 x 10-3 (minimum, 0.18 x 10-3; maximum, 11.50 x 10-3) for the 45 measurable patients and was identical to that of the entire 50-patient cohort. When responses were analyzed by segregating tumors into low and high TS expressors, three (75%) out of four partial responders, 26 (96%) of 27 patients with stable disease, and nine (64%) of 14 patients with progressive disease had low TS expression (P = .02, Fisher’s exact test; Table 4).
The median ERCC1 expression level was 2.7 x 10-3 (minimum, 0.00; maximum, 14.61 x 10-3) for the 45 measurable patients and not significantly different from that of the entire 50-patient cohort. However, the ERCC1 expression level was not statistically significantly associated with response to chemotherapy (P = .29, Fisher’s exact test, data not shown).
It is well known that intrinsic resistance and acquired resistance are critical factors of efficacy of platinum-based chemotherapy. Enhanced tolerance to platinum compounds and a decreased drug uptake may be due in part to an increased platinum resistance.4 Deficiency in the mismatch repair activity (intrinsic resistance) and an enhanced ability of the replication complex to synthesize DNA past the site of DNA damage (enhanced replicative bypass) were found to be responsible for resistance to other platinum agents, eg, cisplatin and carboplatin, but were not found to contribute to oxaliplatin resistance.20-23
Recently, biweekly treatment administration schedules (v triweekly) Patients with low ERCC1 expression (cutoff, 4.9 x 10-3) showed a significantly better survival than patients with high expression. Although the ERCC1 expression was not significantly associated with response, our data support the hypothesis that enhanced DNA repair decreases the benefit of platinum-based treatment. These findings are in agreement with other reports relating ERCC1 mRNA expression level to response and survival in gastric and ovarian cancer treated with platinum compounds. These studies showed a statistically significant lower gene expression level for the excision repair gene (ERCC1) in the responder versus the nonresponder group.13,26 These data indicate that the nucleotide excision repair process is important for DNA adducts formed by platinum agents and that the ERCC1 gene seems to be a critical protein in this pathway. Recent analyses identify the DNA repair pathway as a very complex event involving nucleotide excision repair, mismatch repair, base excision repair, and gene-specific repair.27 A recent case-control study in lung cancer confirmed that xeroderma pigmentosum complementation group G (XPG) and Cockayne’s syndrome complementary group B (CSB), two other members of the ERCC gene family, are important in the DNA repair process related to cancer development.28 The analysis of more genes involved in these DNA repair processes will give us a better understanding of the impact of DNA repair function for sensitivity or resistance of tumors to certain drugs. Different anticancer drugs are combined to increase efficacy of chemotherapy in most solid tumors. Second- or third-line chemotherapy is usually less effective and may be associated with significant toxicities. Identification of molecular predictors of chemotherapy efficacy might become an important tool for designing individualized treatment schedules. Therefore, the mRNA expression of TS, the target enzyme of 5-FU, was also examined in the study.
We found a significant survival benefit for patients who showed intratumoral mRNA TS expression levels Furthermore, in earlier studies, we determined the TS mRNA expression level using fresh-frozen tissue without microdissection. Although the chance of contamination with normal tissue was less than 15%, it still might have altered the measured TS mRNA levels. A study by Miyamoto et al29 supports this theory because it demonstrated decreased TS mRNA levels in normal tissue compared with the tumor tissue of the colorectum. The data suggest that extent of TS upregulation after failure of 5-FU treatment may predict response to 5-FU/oxaliplatin. The molecular basis for this observation is not clear. Furthermore, the literature is inconsistent regarding the effects of oxaliplatin on the pharmacokinetics of 5-FU. Increased 5-FU plasma levels as well as unaffected pharmacokinetic parameters of 5-FU after oxaliplatin infusion have been reported.30 However, since the TS mRNA expression above the cutoff level of 7.5 x 10-3 is associated with chemotherapy resistance, the data suggest the effect of oxaliplatin on the 5-FU pathway may be insufficient to overcome high TS levels. These data support earlier results of our group that identified TS mRNA level as a predictive marker for response and survival in colorectal tumors treated with 5-FU chemotherapy.10,12 Our data demonstrate a significant inverse association for intratumoral mRNA expression of the excision repair gene ERCC1 and intratumoral mRNA expression of the thymidylate synthase gene (TS) with response and survival in 5-FU–refractory patients with metastatic colorectal tumors undergoing 5-FU/oxaliplatin combination chemotherapy. The stratified analysis showed that the intratumoral ERCC1 mRNA and TS mRNA expression levels are independent of each other, which suggests that the expression levels of these genes might be independent predictive markers for survival of 5-FU/oxaliplatin combination chemotherapy in 5-FU–resistant metastatic colorectal cancer. In addition, the mRNA expression level of the TS gene might be a predictive marker for response for this combination chemotherapy. The intratumoral TS or ERCC1 mRNA expression is not the only mechanism that causes sensitivity or resistance to 5-FU and oxaliplatin. Activity differences of other enzymes of the 5-FU pathway might modify the efficacy of 5-FU, as has been shown for dUTPase, dihydropyrimidine dehydrogenase, and thymidine phosphorylase.31,32 Although ERCC1 is a key enzyme of the nucleotide excision repair pathway, other proteins, such as ERCC2 and XPF, may alter the efficacy of the overall pathway to remove DNA adducts caused by platinum.33 Finally, defects in the mismatch repair system (hMutL-alpha, hMutS-alpha) contribute to increased platinum resistance.34 It has also been shown that deletions of chromosome 18q, associated with loss of heterozygosity of the DCC gene and p53 overexpression, were associated with poorer survival in colorectal tumors.35 Modification in the aforementioned genes might help to explain why some patients with low expression showed progression while being treated with 5-FU/oxaliplatin. However, the conclusions are drawn from a limited retrospective study. Prospectively randomized, translational, treatment trials are needed to confirm our results. The goal of these hypothesis-driven clinical trials should be to evaluate whether intratumoral TS and ERCC1 mRNA expression are independent, powerful markers for selecting second- or third-line chemotherapy for patients with metastatic colon cancer resistant to 5-FU and irinotecan. Furthermore, precise definition of the best TS cut point will also require further analysis in a large, prospective study.
Supported by grant nos. R01 CA82655, R01 CA74166, and P30 CA14089 from the National Cancer Institute, Bethesda, MD. J.S. is supported by Dr Mildred Scheel Stiftung, Bonn, Germany. J.B. is supported by the Hubert Burda Foundation for Cancer Research, Munich, Germany.
1. Bleiberg H: Role of chemotherapy for advanced colorectal cancer: New opportunities. Semin Oncol 23: 42-50, 1996 2. De Gramont A, Vignoud J, Tournigand C, et al: Oxaliplatin with high-dose leucovorin and 5-fluorouracil 48-hour continuous infusion in pretreated metastatic colorectal cancer. Eur J Cancer 33: 214-219, 1997 3. Schmoll H-J: Development of treatment for advanced colorectal cancer: Infusional 5-FU and the role of new agents. Eur J Cancer 32A: S18-S22, 1996 (suppl 5) 4. Johnson NP, Hoeschele JD, Rahn RO: Kinetic analysis of an in-vitro binding of radioactive cis- and trans-dichlorodiamminplatinum(II) to DNA. Chem Biol Interact 30: 151-169, 1980[Medline] 5. Raymond E, Faivre S, Woynarowski JM, et al: Oxaliplatin: Mechanism of action and antineoplastic activity. Semin Oncol 25: 4-12, 1998 (suppl 5)[Medline]
6.
Van Houten B: Nucleotide excision repair in Escherichia coli. Microbiol Rev 54: 18-51, 1990 7. Hoeijmakers JHJ: Nucleotide excision repair: II. From yeast to mammals. Trends Genet 9: 211-217, 1993[Medline] 8. Van Duin M, de Wit J, Odjik H, et al: Molecular characterization of the human excision repair gene ERCC-1: cDNA cloning and amino acid homology with the yeast DNA repair gene RAD 10. Cell 44: 913-923, 1986[Medline] 9. Tomkinson AE, Bardwell AJ, Bardwell L, et al: Yeast DNA repair and recombination proteins Rad 1 and Rad 10 constitute a single-strand DNA endonuclease. Nature 362: 860-862, 1993[Medline] 10. Lenz HJ, Hayashi K, Salonga D, et al: p53 point mutations and thymidylate synthase messenger RNA levels in disseminated colorectal cancer: An analysis of response and survival. Clin Cancer Res 4: 1243-1250, 1998[Abstract] 11. Lenz HJ, Leichman CG, Danenberg KD, et al: Thymidylate synthase mRNA level in adenocarcinoma of the stomach: A predictor for primary tumor response and overall survival. J Clin Oncol 14: 176-182, 1996[Abstract] 12. Lenz HJ, Danenberg KD, Leichman CG, et al: p53 and thymidylate synthase gene expression in untreated stage II colon cancer: Association with recurrence, survival, and site. Clin Cancer Res 4: 1227-1234, 1998[Abstract]
13.
Metzger R, Leichman CG, Danenberg KD, et al: ERCC1 mRNA levels complement thymidylate synthase mRNA levels in predicting response and survival for gastric cancer patients receiving combination cisplatin and fluorouracil chemotherapy. J Clin Oncol 16: 309-316, 1998 14. Lord RV, Salonga D, Danenberg KD, et al: Telomerase reverse transcriptase expression is increased early in the Barrett’s metaplasia, dysplasia, carcinoma sequence. J Gastrointest Surg 4: 135-142, 2000[Medline]
15.
Heid CA, Stevens J, Livak KJ, et al: Real time quantitative PCR. Genome Res 6: 986-994, 1996
16.
Gibson UE, Heid CA, Williams PM: A novel method for real time quantitative RT-PCR. Genome Res 6: 995-1001, 1996 17. Miller R, Siegmund D: Maximally selected chi-square statistics. Biometrics 38: 1011-1016, 1982 18. Halpern J: Maximally selected chi-square statistics for small samples. Biometrics 38: 1017-1023, 1982 19. Pike MC: Asymptomatically efficient rank invariant procedures. J R Stat Soc Series A 135: 201-203, 1972 20. Vaisman A, Varchenko M, Chaney SG: Correlation between mismatch repair defects and increased replicative bypass in cisplatin resistant cell lines. Proc Am Assoc Cancer Res 38: A2091, 1997 (abstr) 21. Dunn TA, Schmoll HJ, Grunwald V, et al: Comparative cytotoxicity of oxaliplatin and cisplatin in non-seminomatous germ cancer cell lines. Invest New Drugs 15: 109-114, 1997[Medline]
22.
Fink D, Zheng H, Nebel S, et al: In vitro and in vivo resistance to cisplatin in cells that have lost DNA mismatch repair. Cancer Res 57: 1841-1845, 1997
23.
Fink D, Nebel S, Aebi S, et al: The role of DNA mismatch repair in platinum drug resistance. Cancer Res 56: 4881-4886, 1996 24. Bensmaine MA, de Gramont A, Brienza S, et al: Factors predicting for efficacy of oxaliplatin in combination with 5-fluorouracil (5-FU) ± folinic acid (FA) in a compassionate-use cohort of 370 5-FU resistant advanced colorectal cancer (CRC) patients. Eur J Cancer 36: 2335-2343, 2000
25.
Reardon JT, Vaisman A, Chaney SG, et al: Efficient nucleotide excision repair of cisplatin, oxaliplatin, and bis-aceto-ammine-dichloro-cyclohexylamine-platinum(IV) (JM216) platinum intrastrand DNA diadducts. Cancer Res 59: 3968-3971, 1999
26.
Dabolkar M, Bostick-Bruton F, Weber C, et al: ERCC-1 and ERCC-2 expression in malignant tissues from ovarian cancer patients. J Natl Cancer Inst 84: 1512-1517, 1992 27. Reed E: Platinum-DNA adduct, nucleotide excision repair and platinum based anti-cancer chemotherapy. Cancer Treat Rev 24: 331-344, 1998[Medline]
28.
Cheng L, Spitz MR, Hong WK, et al: Reduced expression of nucleotide excision repair genes in lung cancer: A case-control analysis. Carcinogenesis 21: 1527-1530, 2000 29. Miyamoto S, Ochiai A, Boku N, et al: Discrepancies between the gene expression, protein expression, and enzymatic activity of thymidylate synthase and dihydropyrimidine dehydrogenase in human gastrointestinal cancers and adjacent normal mucosa. Int J Oncol 18: 705-713, 2001[Medline] 30. Culy CR, Clemett D, Wiseman LR: Oxaliplatin: A review of its pharmacological properties and clinical efficacy in metastatic colorectal cancer and its potential in other malignancies. Drugs 60: 895-924, 2000[Medline]
31.
Ladner RD, Lynch FJ, Groshen S, et al: dUTPase nucleotidohydrolase isoform expression in normal and neoplastic tissues: Association with survival and response to 5-fluorouracil in colorectal cancer. Cancer Res 60: 3493-3503, 2000
32.
Salonga D, Danenberg KD, Johnson M, et al: Colorectal tumors responding to 5-fluorouracil have low gene expression levels of dihydropyrimidine dehydrogenase, thymidylate synthase, and thymidine phosphorylase. Clin Cancer Res 6: 1322-1327, 2000 33. Patty DP, Wood RD: Damage recognition in nucleotide excision repair of DNA. Gene 241: 193-204, 2000[Medline]
34.
Drummond JT, Anthoney A, Brown R, et al: Cisplatin and Adriamycin resistance are associated with MutL-alpha and mismatch repair deficiency in an ovarian tumor cell line. J Biol Chem 271: 19645-19648, 1996
35.
Jen J, Kim H, Piantadosi S, et al: Allelic loss of chromosome 18q and prognosis in colorectal cancer. N Engl J Med 331: 213-221, 1995 Submitted March 21, 2001; accepted July 5, 2001.
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Copyright © 2001 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
25% tumor increase). Survival was computed as the number of days from the initiation of chemotherapy with 5-FU/oxaliplatin to death. Patients who were alive at the last follow-up evaluation were censored at that time. 
2 method of Miller and Sigmund
