Autumn Gaither Davis

Molecular Analysis of Plasma DNA for the Early Detection of Lung Cancer by Quantitative Methylation Specific PCR

Purpose: Aberrant promoter hypermethylation of tumor suppressor genes is a promising marker for lung cancer detection. We investigated the likelihood of detecting aberrant DNA methylation of tumor suppressor genes in plasma samples of patients with abnormalities of the lung detected upon computed tomography (CT) scan. Experimental Design: In a small evaluation cohort, four gene promoters (DCC, Kif1a, NISCH, and Rarb) were found to be methylated with increased frequency in samples from cancer patients specifically. We then examined DNA from 93 plasma samples from patients with abnormal findings in the lung detected upon CT scan for aberrant methylation of these four gene promoters by quantitative fluorogenic real-time PCR. The patients were divided into two groups, ground glass opacity (n = 23) and cancerous tumors (n = 70). Plasma DNA from age-matched nodule-free individuals were used as controls (n = 80). Results: In plasma, 73% of patients with cancerous tumors showed methylation of at least one gene with a specificity of 71% (P = 0.0001). Only 22% patients with ground glass opacity exhibited methylation of at least one gene. When smoking history was taken into account, 72% of cancer patients with no smoking history or those who smoked <20 pack-years showed methylation of at least one gene with 100% specificity (P = 0.05) when compared with matched controls. Among heavy smokers with 20+ pack-years of smoking history, 30% of the control group and 73% of the patients with cancerous tumors showed methylation (P = 0.0001). Conclusions: These biomarkers can distinguish between cancerous and noncancerous abnormal CT findings. Clin Cancer Res; 16(13); 3463–72. ©2010 AACR.

Association of polymorphisms in the cyclin D1 and XPD genes and susceptibility to cancers of the upper aero-digestive tract.

DNA repair enzyme genetic polymorphisms have been postulated to increase the risk of certain cancers in the presence of tobacco carcinogen exposures. The XPD protein is an important component of the TFIIH transcription factor complex. XPD genetic polymorphisms resulting in amino acids substitutions may lead to alterations in TFIIH helicase activity, resulting in repair and transcription defects. Cyclin D1 is a key regulatory protein for the transition of cells from the G1‐S cell cycle phase. The CCND1 G870A polymorphism has been reported to enhance alternate splicing of a stable mRNA variant, which may result in the bypass of the G1/S cell cycle checkpoint. In this study, XPD G23591A (Asp312Asn) and A35931C (Lys751Gln) polymorphisms and the CCND1 G870A splice variant frequencies were determined in 273 upper aero‐digestive tract cancer cases and 269 controls. The XPD Asp312Asn variant frequency was significantly different among cases and controls and conferred an odds ratio (OR) of 1.3 (95% CI 1.0–1.8). However, individuals with the CCND1 G870A and XPD Lys751Gln variants had higher age adjusted ORs of 3.2 (95% CI 2.2–4.6) and 2.2 (95% CI 1.5–3.2), respectively. Furthermore, a significant gene–gene interaction was observed among cases with at least two variant alleles for both CCND1 and XPD genes [OR 7.09 (95% CI 4.03–12.5)]. Smokers with a combination of at least one variant allele of both CCND1 and XPD genes also had an elevated risk as compared to nonsmokers. This is the first study to suggest an associative interaction between XPD and CCND1 genetic polymorphisms, tobacco exposure, and cancer risk.

Gastrin-releasing peptide receptor expression in non-cancerous bronchial epithelia is associated with lung cancer: a case-control study

BackgroundNormal bronchial tissue expression of GRPR, which encodes the gastrin-releasing peptide receptor, has been previously reported by us to be associated with lung cancer risk in 78 subjects, especially in females. We sought to define the contribution of GRPR expression in bronchial epithelia to lung cancer risk in a larger case-control study where adjustments could be made for tobacco exposure and sex.MethodsWe evaluated GRPR mRNA levels in histologically normal bronchial epithelial cells from 224 lung cancer patients and 107 surgical cancer-free controls. Associations with lung cancer were tested using logistic regression models.ResultsBronchial GRPR expression was significantly associated with lung cancer (OR = 4.76; 95% CI = 2.32-9.77) in a multivariable logistic regression (MLR) model adjusted for age, sex, smoking status and pulmonary function. MLR analysis stratified by smoking status indicated that ORs were higher in never and former smokers (OR = 7.74; 95% CI = 2.96-20.25) compared to active smokers (OR = 1.69; 95% CI = 0.46-6.33). GRPR expression did not differ by subject sex, and lung cancer risk associated with GRPR expression was not modified by sex.ConclusionsGRPR expression in non-cancerous bronchial epithelium was significantly associated with the presence of lung cancer in never and former smokers. The association in never and former smokers was found in males and females. Association with lung cancer did not differ by sex in any smoking group.

15q12 Variants, Sputum Gene Promoter Hypermethylation, and Lung Cancer Risk: A GWAS in Smokers

BACKGROUND Lung cancer is the leading cause of cancer-related mortality worldwide. Detection of promoter hypermethylation of tumor suppressor genes in exfoliated cells from the lung provides an assessment of field cancerization that in turn predicts lung cancer. The identification of genetic determinants for this validated cancer biomarker should provide novel insights into mechanisms underlying epigenetic reprogramming during lung carcinogenesis. METHODS A genome-wide association study using generalized estimating equations and logistic regression models was conducted in two geographically independent smoker cohorts to identify loci affecting the propensity for cancer-related gene methylation that was assessed by a 12-gene panel interrogated in sputum. All statistical tests were two-sided. RESULTS Two single nucleotide polymorphisms (SNPs) at 15q12 (rs73371737 and rs7179575) that drove gene methylation were discovered and replicated with rs73371737 reaching genome-wide significance (P = 3.3×10(-8)). A haplotype carrying risk alleles from the two 15q12 SNPs conferred 57% increased risk for gene methylation (P = 2.5×10(-9)). Rs73371737 reduced GABRB3 expression in lung cells and increased risk for smoking-induced chronic mucous hypersecretion. Furthermore, subjects with variant homozygote of rs73371737 had a two-fold increase in risk for lung cancer (P = .0043). Pathway analysis identified DNA double-strand break repair by homologous recombination (DSBR-HR) as a major pathway affecting susceptibility for gene methylation that was validated by measuring chromatid breaks in lymphocytes challenged by bleomycin. CONCLUSIONS A functional 15q12 variant was identified as a risk factor for gene methylation and lung cancer. The associations could be mediated by GABAergic signaling that drives the smoking-induced mucous cell metaplasia. Our findings also substantiate DSBR-HR as a critical pathway driving epigenetic gene silencing.

Ability to culture resectable non-small cell lung carcinomas is correlated with recurrence

Present clinicopathologic staging of non-small cell lung cancer is limited in its ability to provide more than a general prognostic estimate in patients with lung cancer who have resectable disease. This study was performed to identify whether the ability to adapt tumor tissue from resectable (stage I to IIIa) non-small cell lung cancer was associated with a poorer prognosis and an increased risk for early tumor recurrence. We attempted to culture a tumor specimen obtained from 90 patients with resectable non-small cell lung cancer. We used a culture medium conditioned by exposure to the lung cancer cell line A549-1, a known producer of autocrine lung cancer growth factors, and provided tumor colony scaffolding using a feeder layer of inactivated fibroblasts, and found these measures improved tumor culture yields. Twenty-two cell lines were obtained, a success rate of 24.4%. Tumor recurrences were more common (79%) among the culture-positive patients than among the culture-negative patients (37.5%; p < 0.002). For all patients, survival at 19 months in the culture-positive patients was 50.0%, compared with 83.6% in the culture-negative patients (p < 0.005). The median survival for the culture-positive patients was 15 months, versus 21.7 months for the culture-negative patients (p < 0.004). The establishment of a culture was a predictor of shortened survival for patients with stage I disease. In patients with stage I disease, survival at 19 months was 54.5% for the culture-positive patients, versus 89% for the culture-negative patients (p < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Elevated gastrin‐releasing peptide receptor mRNA expression in buccal mucosa: Association with head and neck squamous cell carcinoma

Expression of gastrin‐releasing peptide receptor (GRPR) is elevated in mucosa adjacent to head and neck squamous cell carcinoma (HNSCC) compared with mucosa from cancer‐free controls, suggesting elevated GRPR expression may indicate presence of HNSCC.

Abstract 4661: Decreased epidermal growth factor receptor plasma levels as an indicator for head and neck squamous cell carcinoma case status and disease progression

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The epidermal growth factor receptor (EGFR), c-Met and G-protein-coupled receptor signaling pathways contribute to head and neck squamous cell carcinoma (HNSCC) growth. To assess these pathways in HNSCC patients, we measured plasma levels of EGFR, the EGFR ligands transforming growth factor alpha (TGF-α) and amphiregulin (AR), the c-Met ligand hepatocyte growth factor (HGF) and COX-2, a component of prostaglandin production, in HNSCC patients (n=22) and cancer-free controls (n=32) enrolled through an Early Detection Research Network- (EDRN-) sponsored effort. Analytes were measured using a multiplex enzyme-linked immunosorbent assay. Differences in analyte levels between cases and controls and the prognostic potential of each analyte were evaluated. EGFR plasma levels were significantly decreased (P=0.04, rank sum test (RST)) and HGF plasma levels significantly increased (P<0.01, RST) in HNSCC cases compared to cancer-free controls. TGF-α levels tended to be higher in HNSCC cases (P=0.07, RST) while AR and COX-2 levels did not differ between cases and controls (P=0.32 and 0.32, respectively RST). Area under the Receiver Operating Characteristic (ROC) curve for HGF levels in subjects with low EGFR plasma levels was 0.92. EGFR plasma levels were significantly reduced in HNSCC cases who were former or active smokers compared to never smokers (P = 0.009, Kruskal-Wallis test (KWT)), but no difference in EGFR plasma levels by smoking status was observed among cancer-free controls (P = 0.97, KWT). HGF plasma levels tended to be lower in HNSCC cases who were never smokers (P=0.06 KWT) but did not differ by smoking status among cancer-free controls (P=0.16, KWT). Low EGFR plasma levels were associated with significantly reduced progression-free survival (PFS) in EDRN HNSCC cases (P = 0.03, log rank test); no other analyte tested was associated with PFS. We noted a negative correlation between EGFR plasma levels in EDRN HNSCC cases and previously reported EGFR tumor levels assessed by immunohistochemical staining that was not significant (Spearmans rho=-0.41, P=0.10, n=17). Plasma EGFR and HGF levels may provide indications of HNSCC cancer status, and EGFR plasma levels may be a valuable prognostic indicator for patients with HNSCC. Decreased plasma EGFR levels in HNSCC patients were likely not due to general effects related to smoking and instead may reflect tissue characteristics specific to tobacco-related cancers. We found EGFR plasma levels in HNSCC patients were not positively correlated with EGFR tumor levels. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4661.