Jan Brabender

PURPOSE: Overexpression of the excision repair cross-complementing 1 (ERCC1) gene, which is crucial in the repair of cisplatin (CDDP)-DNA adducts, is reported to negatively influence the effectiveness of CDDP-based therapy for gastric and ovarian cancers. Recent evidence indicates that Gemcitabine (Gem) may modulate ERCC1 nucleotide excision repair activity, and down-regulation of DNA repair activity by ERCC1 antisense RNA reportedly inhibits synergism of CDDP/Gem. We investigated whether ERCC1 mRNA expression levels were associated with clinical outcomes after treatment with a combination Gem/CDDP regimen for patients with advanced stage non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Response and survival were correlated with the level of ERCC1 expression in 56 patients with advanced (stage IIIb or IV) NSCLC treated as part of a multicenter randomized trial with Gem 1250 mg/m(2) days 1 and 8 plus CDDP 100 mg/m(2) on day 1 every 3 weeks. mRNA was isolated from paraffin-embedded pretreatment primary tumor specimens, and relative expression levels of ERCC1/beta-actin were measured using a quantitative reverse transcription-PCR (Taqman) system. RESULTS: ERCC1 expression was detectable in all tumors. There were no significant differences in ERCC1 levels by gender, age, performance status, weight loss, or tumor stage. The overall response rate was 44.7%. There were no significant associations between ERCC1 expression and response. Median overall survival was significantly longer in patients with low ERCC1 expression tumors (61.6 weeks; 95% confidence interval, 42.4-80.7 weeks) compared to patients with high expression tumors (20.4 weeks, 95% confidence interval, 6.9-33.9 weeks). ERCC1 expression, Eastern Cooperative Oncology Group performance status, and presence of weight loss were significant prognostic factors for survival in a Cox proportional hazards multivariable analysis. CONCLUSIONS: These data suggest that ERCC1 expression is a predictive factor for survival after CDDP/Gem therapy in advanced NSCLC. Although there was a trend toward decreased response with high ERCC1 mRNA levels, this difference failed to reach statistical significance. This result may reflect the impact of Gem and the requirement for ERCC1 expression for CDDP/Gem synergism or may be attributable to the relatively small patient sample size in this study. Prospective studies of ERCC1 as a predictive marker for activity of CDDP-based regimens in NSCLC are warranted.

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 <or= 7.5 x 10(-3) (43 of 50 patients) was 10.2 months, compared with 1.5 months for patients with TS greater than 7.5 x 10(-3) (P < .001). Patients with ERCC1 expression <or= 4.9 x 10(-3) (40 of 50 patients) had a median survival time of 10.2 months, compared with 1.9 months for patients with ERCC1 expression greater than 4.9 x 10(-3) (P < .001). A TS of 7.5 x 10(-3) segregated significantly into response, stable disease, and progression (P = .02), whereas the association between ERCC1 and response did not reach statistical significance (P = .29). 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.

The prognostic role of epidermal growth factor receptor (EGFR) and HER2-neu remains controversial in patients with non-small cell lung cancer (NSCLC). We studied the association between the mRNA expression of EGFR, HER2-neu, and survival in primary tumor and matching nonmalignant tissues from 83 patients with NSCLC. Analysis was performed using a quantitative real-time PCR system (Taqman). EGFR and HER2-neu mRNA expression was detectable in all (100%) specimens analyzed. Twenty-nine (34.9%) patients had high HER2-neu expression, and 28 (33.7%) patients had high EGFR expression. A high HER2-neu and EGFR coexpression was detectable in 14 (16.9%) patients. High HER2-neu expression was associated with inferior survival (P = 0.004), whereas high EGFR expression showed a trend toward inferior survival (P = 0.176). The impact of HER2-neu and EGFR coexpression on patients' survival was additive (P = 0.003). Multivariate analysis determined high HER2-neu expression (P = 0.041), and high EGFR/HER2-neu coexpression (P = 0.030) as significant and independent unfavorable prognostic factors. These findings indicate that HER2-neu and EGFR play a crucial role in the biological behavior of NSCLCs. Testing of molecular marker coexpression (EGFR and HER2-neu) improves the estimation of prognosis and appears to define low- and high-risk groups for treatment failure in curatively resected NSCLC.

The serum of cancer patients often harbors increased free DNA levels, which can potentially be used for cancer detection. Because genetic and epigenetic alterations of the adenomatous polyposis coli (APC) gene are common events in gastrointestinal tumor development, we sought to investigate the frequency and level of aberrant APC promoter methylation in primary tumors and paired preoperative serum or plasma samples of lung cancer patients by semiquantitative methylation-specific fluorogenic real-time PCR. We detected methylation of APC in 95 of 99 (96%) primary lung cancer tissues. Forty-two of 89 (47%) available serum and/or plasma samples from these cases carried detectable amounts of methylated APC promoter DNA. In contrast, no methylated APC promoter DNA was detected in serum samples from 50 healthy controls. A highly elevated APC methylation level in lung cancer tissue was the only independent factor predicting inferior survival in this cohort (P = 0.015). APC methylation analysis appears to be promising as a prognostic factor in primary lung cancer and as a noninvasive tumor marker in plasma and/or serum DNA.

PURPOSE: Thymidylate synthase (TS) is a target enzyme of 5-fluorouracil. Recently, the TS gene has been shown to contain a polymorphic tandem repeat sequence. The aim of this study was to determine whether differences in the number of tandem repeats could affect gene expression or mRNA translation. EXPERIMENTAL DESIGN: We quantified TS mRNA isolated from 130 colorectal cancer tissues by real-time reverse transcription-PCR and TS protein in 92 available samples by the fluoro-dUMP binding assay. These values were compared with TS genotypes of the samples determined by a PCR assay. RESULTS: There was no relation between TS genotype and mRNA expression level. On the other hand, cancer tissues with the 3R/3R genotype had a significantly higher TS protein expression level than did those with the 2R/3R genotype. These results suggest that the efficiency of TS mRNA translation is responsible for the genotype-dependent difference in TS protein expression. Further analysis using TS 5'-untranslated region-luciferase reporter constructs showed that the RNA with the three-repeat sequence was translated three to four times more efficiently than that with two-repeat sequence. CONCLUSIONS: From the results of both in vitro and in vivo study, we conclude that TS mRNA with a three-repeat sequence has greater translation efficiency than that with the two-repeat sequence. The results provide the rationale for comprehensive usage of TS genotyping with quantitation of TS mRNA or TS protein to predict the patient's response to 5-fluorouracil-based chemotherapy.