XPG mRNA Expression Levels Modulate Prognosis in Resected Non-small-cell Lung Cancer in Conjunction With BRCA1 and ERCC1 Expression

Roberta Bartolucci; Jia Wei; Jose Javier Sanchez; Laia Perez-Roca; Imane Chaib; Francesco Puma; Raffaele Farabi; Pedro Mendez; Fausto Roila; Tatsuro Okamoto; Miquel Taron; Rafael Rosell

Disclosures

Clin Lung Cancer. 2009;10(1):47-52. 

In This Article

Patients and Methods

Patients

Tumor samples from 54 patients with stage IB-IIB NSCLC who had undergone surgical resection at the Azienda Ospedaliera Santa Maria (Terni, Italy) between February 1997 and December 2003 were examined for gene expression after obtaining approval from the Institutional Review Board and patients' signed informed consent. Patient characteristics are shown in Table 1.

Laboratory Methods

ERCC1, RRM1, XPG, and BRCA1 gene expression was assessed in formalin-fixed, paraffin-embedded surgical specimens from 54 patients with lung cancer. By using laser capture microdissection (PALM Microlaser, Oberlensheim, Germany), a minimum of 80% of tumor tissue was ensured. After standard tissue sample deparaffinization using xylene and alcohols, samples were lysed in a Tris-chloride, ethylenediaminetetraacetic acid, sodium dodecyl sulfate, and proteinase K-containing buffer. RNA was then extracted with phenol/chloroform/isoamyl alcohol followed by precipitation with isopropanol in the presence of glycogen and sodium acetate. RNA was treated with DNAse I (Ambion Inc, Austin, TX) to avoid DNA contamination. cDNA was synthesized using Moloney murine leukemia virus retrotranscriptase enzyme followed by quantitative PCR (ABI PRISM 7900HT, Applied Biosystems, Austin, TX), using β-actin as the endogenous reference gene. Primers and probes are listed in Table 2.

Statistical Analysis

The Mann-Whitney U test was used to assess significant association between gene expression levels and dichotomous variables. The Spearman's rho method was used to correlate expression levels of ERCC1, RRM1, XPG, and BRCA1. Median survival and disease-free survival (DFS) were calculated using Kaplan-Meier estimates, and differences between curves were tested using the log-rank test. A univariate and multivariate stepwise procedure using Cox regression analysis was used to assess the association between each potential prognostic factor and median survival and DFS. All statistical analyses were carried out at a 5% level of significance and with a power of 80%, using the Statistical Package for the Social Sciences, version 13 (SPSS Inc, Chicago, IL).

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