SNP (–617C>A) in ARE-Like Loci of the NRF2 Gene: A New Biomarker for Prognosis of Lung Adenocarcinoma in Japanese Non-Smoking Women

<div><p>Purpose</p><p>The transcription factor NRF2 plays a pivotal role in protecting normal cells from external toxic challenges and oxidative stress, whereas it can also endow cancer cells resistance to anticancer drugs. At present little information is available about the genetic polymorphisms of the <i>NRF2</i> gene and their clinical relevance. We aimed to investigate the single nucleotide polymorphisms in the <i>NRF2</i> gene as a prognostic biomarker in lung cancer.</p><p>Experimental Design</p><p>We prepared genomic DNA samples from 387 Japanese patients with primary lung cancer and detected SNP (c.–617C>A; rs6721961) in the ARE-like loci of the human <i>NRF2</i> gene by the rapid genetic testing method we developed in this study. We then analyzed the association between the SNP in the <i>NRF2</i> gene and patients’ overall survival.</p><p>Results</p><p>Patients harboring wild-type (WT) homozygous (c.–617C/C), SNP heterozygous (c.–617C/A), and SNP homozygous (c.–617A/A) alleles numbered 216 (55.8%), 147 (38.0%), and 24 (6.2%), respectively. Multivariate logistic regression models revealed that SNP homozygote (c.–617A/A) was significantly related to gender. Its frequency was four-fold higher in female patients than in males (10.8% female vs 2.7% male) and was associated with female non-smokers with adenocarcinoma. Interestingly, lung cancer patients carrying <i>NRF2</i> SNP homozygous alleles (c.–617A/A) and the 309T (WT) allele in the <i>MDM2</i> gene exhibited remarkable survival over 1,700 days after surgical operation (log-rank p = 0.021).</p><p>Conclusion</p><p>SNP homozygous (c.–617A/A) alleles in the <i>NRF2</i> gene are associated with female non-smokers with adenocarcinoma and regarded as a prognostic biomarker for assessing overall survival of patients with lung adenocarcinoma.</p></div

Clinicopathological profiling of 24 patients harboring homozygous SNP alleles (–617A/A) in the <i>NRF2</i> gene.

<p>Abbreviation: Ad, adenocarcinoma; Mix, adenocarcinoma and squamous cell carcinoma; Ple, pleomorphic carcinoma; Sq, squamous cell carcinoma; F, female; M, male; Wt, wild type.</p>†<p>Patient (case 5) died because of primary pancreatic cancer.</p

Classification of primary lung cancer patients with respect to <i>NRF2</i> genotypes, smoking behavior, adenocarcinoma, and gender.

*<p><i>P</i>-values were calculated by Fisher’s exact test.</p><p>Abbreviation: M, male; F, female.</p

Kaplan-Meier plots showing the overall survival of patients harboring the WT homozygote (–617C/C), WT/SNP heterozygote (–617C/A), or SNP homozygote (–617A/A) in the <i>NRF2</i> gene.

<p>Patients with p-stages I to IV (<b>A</b>) and p-stage I only NSCLC (<b>B</b>). The number of patients at times 0, 500, 1000, or 1500 days after surgical operation is described along with genotypes of the <i>NRF2</i> gene.</p

Frequencies of wild type (–617C) and SNP (–617A) alleles in the <i>NRF2</i> gene among different ethnic groups.

<p>N, the number of subjects.</p>*<p>1000 Genomes. <a href="http://browser.1000genomes.org/Homo_sapiens/Variation/Population?db=corer=2" target="_blank">http://browser.1000genomes.org/Homo_sapiens/Variation/Population?db=corer=2</a>∶178129537–178130537;v = rs6721961;vdb = variation;vf = 4574214.</p

SmartAmp-based detection of SNP (c.–617C>A) in the <i>NRF2</i> gene.

<p>SNP (c.–617C>A) resides in the promoter region of the <i>NRF2</i> gene on chromosome 2q31.2. Panel <b>A</b> presents a schematic illustration of annealing sites of the TP, FP, and OP primers. Panel <b>B</b> shows cDNA encoding a partial sequence of the <i>NRF2</i> gene and primer annealing sites. Panel <b>C</b> depicts the results of SNP detection. a.u. = arbitrary unit.</p

Classification of primary lung cancer patients with respect to genotypes of <i>NRF2</i> and <i>MDM2</i> genes.

<p>N, the number of patients; % in parentheses.</p

Logistic regression analysis for evaluation of the association among homozygous SNP alleles (–617A/A) in the <i>NRF2</i> gene and gender/smoking experience of lung cancer patients.

<p>Abbreviation: CI, confidence interval.</p><p>Gender code: 1 = female; 0 = male.</p><p>Smoking experience code: 1 = smoker; 0 = non-smoker.</p><p>The multivariate logistic regression analysis was performed under two categories, <i>i.e.</i>, the gender (female and male) and the smoking experience (smoker and non-smoker).</p

Clinicopathological characterization of primary lung cancer patients as well as presence of SNP (c.309T>G) in the <i>MDM2</i> gene and allele frequency.

<p>Clinicopathological characterization of primary lung cancer patients as well as presence of SNP (c.309T>G) in the <i>MDM2</i> gene and allele frequency.</p

Analysis of DNA products formed in the Duplex SmartAmp reaction.

<p>A: Time course of typical Duplex SmartAmp reaction by real-time monitoring. B. Agarose gel electrophoresis of DNA products after Duplex SmartAmp reaction. We sampled aliquots of reaction mixtures at different reaction times (t = 0, 13, 16 and 30 minutes) and separated DNA products by agarose gel electrophoresis. The “Maker” represents a 100-bp DNA ladder.</p