Kathleen D. Danenberg

ERCC1 and Thymidylate Synthase mRNA Levels Predict Survival for Colorectal Cancer Patients Receiving Combination Oxaliplatin and Fluorouracil Chemotherapy

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.

Quantitation of intratumoral thymidylate synthase expression predicts for disseminated colorectal cancer response and resistance to protracted-infusion fluorouracil and weekly leucovorin.

PURPOSE Response rates to fluorouracil (5-FU)-based therapy remain low. As new, active agents are being tested, information regarding specific intratumoral genetic determinants of chemotherapy sensitivity or resistance can be used to plan therapy rationally. Intratumoral thymidylate synthase (TS) quantitation may be among the most important determinants of sensitivity or resistance to 5-FU. MATERIALS AND METHODS Forty-six disseminated colorectal cancer patients had measurable tumor biopsies for polymerase chain reaction (PCR)-based determination of TS mRNA pretreatment. Protracted infusion of 5-FU 200 mg/m2/d for 21 days with weekly intravenous leucovorin 20 mg/m2 each cycle was given. After two cycles, responses were evaluated. Response data were correlated with independently determined intratumoral ratios of TS/beta-actin mRNA for each patient. RESULTS TS/beta-actin ratios were successfully obtained for 42 patients (91%). TS/beta-actin ratios ranged from 0.3 x 10(-3) to 18.2 x 10(-3) (median, 3.5 x 10[-3]). Twelve patients (26%) responded to treatment (median TS/beta-actin ratio, 1.7 x 10[+3]). Thirty-four patients did not respond (median TS/beta-actin ratio, 5.6 x 10[-3]). No patient with a TS mRNA level greater than 4.1 x 10(-3) responded. The median TS/beta-actin ratio (3.5 x 10[-3]) significantly segregated responders from nonresponders (P = .001). Median survival for patients with TS/beta-actin ratios < or = 3.5 x 10(-3) was 13.6 months; for patients with TS/beta-actin ratios greater than 3.5 x 10(-3), it was 8.2 months (P = .02). CONCLUSION For this cohort, the intratumoral TS/beta-actin ratio had a statistically significant association with response and survival. This relationship for other 5-FU schedules remains unknown. Confirmation of these data in a larger patient population could lead to determination of therapy for disseminated colorectal cancer based on a specific intratumoral molecular parameter.

ERCC1 mRNA levels complement thymidylate synthase mRNA levels in predicting response and survival for gastric cancer patients receiving combination cisplatin and fluorouracil chemotherapy.

PURPOSE We have previously shown that relative thymidylate synthase (TS) mRNA levels in primary gastric adenocarcinomas treated with fluorouracil (5-FU) and cisplatin are inversely associated with response and survival. This is a presumed function of TS as a target for 5-FU activity. We now test the hypotheses that the relative mRNA level of the excision repair cross-complementing (ERCC1) gene is inversely associated with response and survival as an independent function of cisplatin efficacy. PATIENTS AND METHODS Patients had intact, untreated, primary gastric adenocarcinoma cancer and were evaluated for eligibility on a preoperative cisplatin infusion-5-FU protocol. cDNA, derived from primary gastric tumors before chemotherapy, was used to determine ERCC1 mRNA levels, expressed as the ratio of polymerase chain reaction (PCR) product of the ERCC1 gene and the beta-actin gene. RESULTS The median ERCC1 mRNA level from 38 primary gastric cancers (33 assessable for response) was 5.8 x 10(-3) (range, 1.8 x 10(-3) to 19.5 x 10(-3)). Of 17 responding patients, 13 (76%) were less than or equal to 5.8 x 10(-3) and four were greater than 5.8 x 10(-3) (P = .003). The median survival for patients with ERCC1 mRNA levels less than or equal to 5.8 x 10(-3) has not been reached, whereas for those greater than 5.8 x 10(-3) it was 5.4 months (P = .034). The median TS mRNA level, 3.7 x 10(-3) (range, 0.9 to 18.9) also segregated responsive versus resistant tumors (P = .024). With both ERCC1 and TS mRNA levels below their medians, 11 of 13 patients (85%) responded; with both ERCC1 and TS mRNA levels above their medians, two of 10 patients (20%) responded (P = .003). CONCLUSION Considered separately, either ERCC1 or TS mRNA levels in a primary gastric adenocarcinoma has a statistically significant relationship to response. ERCC1 mRNA levels have a statistically significant association with survival; in this cohort TS mRNA levels did not reach statistically significant association with survival as in our previous publication. Whether these molecular parameters are independent of each other as predictors of outcome remains to be determined.

Thymidylate synthase mRNA level in adenocarcinoma of the stomach: a predictor for primary tumor response and overall survival.

PURPOSE We tested the hypothesis that polymerase chain reaction (PCR) quantitation of the enzyme thymidylate synthase (TS) within a primary adenocarcinoma of the stomach, has an inverse relationship to response and survival for patients who receive fluorouracil (5FU)-based chemotherapy. PATIENTS AND METHODS Before systemic chemotherapy, the genetic expression of TS (TSmRNA level) was determined using a PCR method. Gene expression was calculated by determining the ratio between the amount of radiolabeled PCR product with the linear amplification range of the TS gene and the beta-actin gene. Chemotherapy consisted of two cycles of protracted infusion (PI) 5FU 200 mg/m2/d administered for 3 weeks with leucovorin 20 mg/m2/w. Cisplatin 100 mg/m2 was administered on day 1. RESULTS Sixty-five patients with primary gastric cancer had a median TS mRNA level of 4.6 x 10(-3) (range, 0.9 to 20.1 x 10(-3)). Thirty-five percent of patients had measurable responses in their primary tumors. The mean gastric cancer TSmRNA level in responding and resistant patients is statistically significant (P < .001). The median survival time was 43+ months for treated patients with TSmRNA levels less than the median and 6 months for those with TS m-RNA levels greater than the median (P = .003). CONCLUSION The genetic expression of TS (TSmRNA level) influences response to 5FU-based chemotherapy and survival for a cohort of patients with primary gastric cancer. Confirmation of these data could lead to therapeutic decisions based on specific molecular properties within a tumor.

Ribonucleotide reductase messenger RNA expression and survival in gemcitabine/cisplatin-treated advanced non-small cell lung cancer patients

Purpose: No chemotherapy regimen, including the widely used combination of gemcitabine/cisplatin, confers significantly improved survival over any other in metastatic non-small cell lung cancer (NSCLC); however, the selection of patients according to key genetic characteristics can help to tailor chemotherapy. Ribonucleotide reductase subunit M1 (RRM1) is involved in DNA synthesis and repair and in gemcitabine metabolism, and the excision repair cross-complementing group 1 (ERCC1) gene has been related to cisplatin activity. Experimental Design: Patients were part of a large randomized trial carried out from September 1998 to July 2000, comparing gemcitabine/cisplatin versus gemcitabine/cisplatin/vinorelbine versus gemcitabine/vinorelbine followed by vinorelbine/ifosfamide. We analyzed RRM1 and ERCC1 mRNA expression in paraffin-embedded samples obtained from bronchoscopy by real-time quantitative reverse transcription-PCR. Results were correlated with survival using the Kaplan-Meier method. Results: A total of 100 patients were assessed. There was a strong correlation between RRM1 and ERCC1 mRNA expression levels (Spearman r = 0.410; P < 0.001). In the gemcitabine/cisplatin arm, patients with low RRM1 mRNA expression levels had significantly longer median survival than those with high levels [13.7 versus 3.6 months; 95% confidence interval (CI), 9.6–17.8 months; P = 0.009]. Median survival was also significantly longer among patients with low mRNA expression levels of both RRM1 and ERCC1 (not reached), than among those with high levels of both genes (6.8 months; 95% CI, 2.6–11.1 months; P = 0.016). Conclusions: RRM1 mRNA expression is a crucial predictive marker of survival in gemcitabine/cisplatin-treated patients. Genetic testing of RRM1 mRNA expression levels can and should be used to personalize chemotherapy.

An in vivo platform for translational drug development in pancreatic cancer

Effective development of targeted anticancer agents includes the definition of the optimal biological dose and biomarkers of drug activity. Currently available preclinical models are not optimal to this end. We aimed at generating a model for translational drug development using pancreatic cancer as a prototype. Resected pancreatic cancers from 14 patients were xenografted and expanded in successive groups of nude mice to develop cohorts of tumor-bearing mice suitable for drug therapy in simulated early clinical trials. The xenografted tumors maintain their fundamental genotypic features despite serial passages and recapitulate the genetic heterogeneity of pancreatic cancer. The in vivo platform is useful for integrating drug screening with biomarker discovery. Passages of tumors in successive cohorts of mice do not change their susceptibility to anticancer agents and represent a perpetual live bank, facilitating the application of new technologies that will result in the creation of an integrated stable database of tumor-drug response data and biomarkers.

Molecular Determinants of Cetuximab Efficacy

PURPOSE To investigate whether mRNA expression levels of cyclin D1 (CCND1), cyclooxygenase 2 (Cox-2), epidermal growth factor receptor (EGFR), interleukin 8 (IL-8), and vascular endothelial growth factor (VEGF), all members of the EGFR signaling pathway, are associated with clinical outcome in patients with EGFR-expressing metastatic colorectal cancer (CRC) treated with cetuximab. PATIENTS AND METHODS Thirty-nine patients with metastatic CRC, refractory to both irinotecan and oxaliplatin, were enrolled on IMCL-0144 and treated with single-agent cetuximab. The intratumoral mRNA levels of CCND1, Cox-2, EGFR, IL-8, and VEGF were assessed from paraffin-embedded tissue samples using laser-capture microdissection and quantitative real-time polymerase chain reaction. RESULTS There were 21 women and 18 men with a median age of 64 years (range, 35 to 83 years). Higher gene expression levels of VEGF were associated with resistance to cetuximab (P = .038; Kruskal-Wallis test). The combination of low gene expression levels of Cox-2, EGFR, and IL-8 was significantly associated with overall survival (13.5 v 2.3 months; P = .028; log-rank test). Both findings were independent of skin toxicity that was itself significantly correlated to survival. Patients with a lower mRNA amount of EGFR had a longer overall survival compared with patients that had a higher mRNA amount (7.3 v 2.2 months; P = .09; log-rank test). Patients with lower expression of Cox-2 had a significantly higher rate of grade 2 to 3 skin reactions under cetuximab treatment. CONCLUSION This pilot study suggests that gene expression levels of Cox-2, EGFR, IL-8, and VEGF in patients with metastatic CRC may be useful markers of clinical outcome in single-agent cetuximab treatment.

Telomerase reverse transcriptase expression is increased early in the Barrett’s metaplasia, dysplasia, adenocarcinoma sequence

Barrett’s esophagus is a multistage polyclonal disease that is associated with the development of adenocarcinoma of the esophagus and csophagogastric junction. Telomerase activation is associated with cellular immortality and carcinogenesis, and increased expression of the telomerase reverse transcriptase catalytic subunit (hTERT) has been used for the early detection of malignant diseases. To identify’ biomarkers associated with each stage of the Barrett’s process, relative mRNA expression levels of hTERT were measured using a quantitative reverse transcription-polymerase chain reaction method (ABI 7700 Sequence Detector (TaqMan system) in Barrett’s intestinal metaplasia (n —14), Barrett’s dysplasia (n =10), Barrett’s adenocarcinoma (n = 14), and matching normal squamous esophagus tissues (n = 32). hTERT expression was significantly increased at all stages of Barren’s esophagus, including the intestinal metaplasia stage, compared to normal tissues from patients without cancer (intestinal metaplasia vs. normal esophagus, P <0.0001; dysplasia, P = 0.001; adenocarcinoma, P = 0.007; all Alann-Whitney U test). hTERT expression levels were significantly higher in adenocarcinoma tissues than in intestinal metaplasia tissues (P = 0.003), and were higher in dysplasia compared with intestinal metaplasia tissues (P = 0.056). hTERT levels were also significantly higher in histologically normal squamous esophagus tissues from cancer panents than in normal esophagus tissues from patients vrith no cancer (P = 0.013). Very high expression levels ([hTERT × 100: β-actin] >20) were found only in patients with cancer. These findings suggest that telomerase activation is an important early event in the development of Barrett’s esophagus and esophageal adenocarcinoma, that very high telomerase levels may be a clinically useful biomarker for the detection of occult adenocarcinoma, and that a widespread cancer ‘field’ effect is present in the esophagus of patients with Barrett’s cancer.

Transcripts in pretreatment biopsies from a three-arm randomized trial in metastatic non-small-cell lung cancer

Non-small-cell lung cancer patients with locally advanced or metastatic disease at the time of diagnosis show marginal response to chemotherapy in terms of tumor shrinkage, time to progression and median survival. The identification and implementation of predictive genetic markers of response-specific cytotoxic drugs is a priority of current research and future trials. In this study, we have used quantitative PCR to analyse expression of β-tubulin III, stathmin, RRM1, COX-2 and GSTP1 in mRNA isolated from paraffin-embedded tumor biopsies of 75 nonsmall-cell lung cancer patients treated as part of a large randomized trial. In total, 22 patients were treated with gemcitabine/cisplatin, 25 with vinorelbine/cisplatin and 28 with paclitaxel/carboplatin. There were no differences in clinical characteristics and transcript levels in the pretreatment biopsies according to treatment arm. Patients with low β-tubulin III levels had better response in the paclitaxel/carboplatin arm (P=0.05), and those with low RRM1 levels showed a tendency to better response in the gemcitabine/cisplatin arm. Time to progression was influenced by β-tubulin III (P=0.03) and stathmin (P=0.05) levels in the vinorelbine/cisplatin arm, and there was a tendency toward correlation between β-tubulin III levels and time to progression in the paclitaxel/carboplatin arm. RRM1 levels influenced time to progression (P=0.05) and even more so, survival (P=0.0028) in the gemcitabine/cisplatin arm. The predictive value of β-tubulin III, stathmin and RRM1 should be tested in prospective customized chemotherapy trials, the results of which will help tailor chemotherapy to improve patient survival.

Adenomatous polyposis coli gene promoter hypermethylation in non-small cell lung cancer is associated with survival

Methylation of 5′ CpG islands in promoter and upstream coding regions has been identified as a mechanism for transcriptional inactivation of tumor suppressor genes. The purpose of this study was to determine whether hypermethylation of the adenomatous polyposis coli (APC) gene promoter occurs in primary non-small cell lung cancer (NSCLC), and whether hypermethylated APC has any relationship with survival. APC promoter 1A methylation was determined in normal and corresponding tumor tissue from 91 NSCLC patients and in a control group of 10 patients without cancer, using a quantitative fluorogenic real-time PCR (Taqman®) system. APC promoter methylation was detectable in 86 (95%) of 91 tumor samples, but also in 80 (88%) of 91 normal samples of NSCLC patients, and in only two (20%) of 10 normal lung tissues of the control group. The median level of APC promoter methylation was 4.75 in tumor compared to 1.57 in normal lung tissue (P<0.001). Patients with low methylation status showed significantly longer survival than did patients with high methylation status (P=0.041). In a multivariate analysis of prognostic factors, APC methylation was a significant independent prognostic factor (P=0.044), as were pT (P=0.050) and pN (P<0.001) classifications. This investigation shows that APC gene promoter methylation occurs in the majority of primary NSCLCs. High APC promoter methylation is significantly associated with inferior survival, showing promise as a biomarker of biologically aggressive disease in NSCLC.