Jerry Haney

Promoter Hypermethylation of Multiple Genes in Sputum Precedes Lung Cancer Incidence in a High-Risk Cohort

A sensitive screening approach for lung cancer could markedly reduce the high mortality rate for this disease. Previous studies have shown that methylation of gene promoters is present in exfoliated cells within sputum prior to lung cancer diagnosis. The purpose of the current study is to conduct a nested case-control study of incident lung cancer cases from an extremely high-risk cohort for evaluating promoter methylation of 14 genes in sputum. Controls (n = 92) were cohort members matched to cases (n = 98) by gender, age, and month of enrollment. The comparison of proximal sputum collected within 18 months to >18 months prior to diagnosis showed that the prevalence for methylation of gene promoters increased as the time to lung cancer diagnosis decreased. Six of 14 genes were associated with a >50% increased lung cancer risk. The concomitant methylation of three or more of these six genes was associated with a 6.5-fold increased risk and a sensitivity and specificity of 64%. This is the first study to prospectively examine a large panel of genes for their ability to predict lung cancer and shows the promise of gene promoter hypermethylation in sputum as a molecular marker for identifying people at high risk for cancer incidence.

EGFR regulation by microRNA in lung cancer: correlation with clinical response and survival to gefitinib and EGFR expression in cell lines

BACKGROUND Allelic loss in chromosome 3p is one of the most frequent and earliest genetic events in lung carcinogenesis. We investigated if the loss of microRNA-128b, a microRNA located on chromosome 3p and a putative regulator of epidermal growth factor receptor (EGFR), correlated with response to targeted EGFR inhibition. Loss of microRNA-128b would be equivalent to losing a tumor suppressor gene because it would allow increased expression of EGFR. PATIENTS AND METHODS We initially showed that microRNA-128b is a regulator of EGFR in non-small-cell lung cancer (NSCLC) cell lines. We tested microRNA-128b expression levels by quantitative RT-PCR, genomic copy number by quantitative PCR, and mutations in the mature microRNA-128b by sequencing. We determined whether microRNA-128b loss of heterozygosity (LOH) in 58 NSCLC patient samples correlated with response to gefitinib and evaluated EGFR expression and mutation status. RESULTS We determined that microRNA-128b directly regulates EGFR. MicroRNA-128b LOH was frequent in tumor samples and correlated significantly with clinical response and survival following gefitinib. EGFR expression and mutation status did not correlate with survival outcome. CONCLUSION Identifying microRNA regulators of oncogenes could have far-reaching implications for lung cancer patients including improving patient selection for targeted agents, development of novel therapeutics, or development as early biomarkers of disease.

KRAS Mutation: Comparison of Testing Methods and Tissue Sampling Techniques in Colon Cancer

Treatment of colon carcinoma with the anti-epidermal growth factor receptor antibody Cetuximab is reported to be ineffective in KRAS-mutant tumors. Mutation testing techniques have therefore become an urgent concern. We have compared three methods for detecting KRAS mutations in 59 cases of colon carcinoma: 1) high resolution melting, 2) the amplification refractory mutation system using a bifunctional self-probing primer (ARMS/Scorpion, ARMS/S), and 3) direct sequencing. We also evaluated the effects of the methods of sectioning and coring of paraffin blocks to obtain tumor DNA on assay sensitivity and specificity. The most sensitive and specific combination of block sampling and mutational analysis was ARMS/S performed on DNA derived from 1-mm paraffin cores. This combination of tissue sampling and testing method detected KRAS mutations in 46% of colon tumors. Four samples were positive by ARMS/S, but initially negative by direct sequencing. Cloned DNA samples were retested by direct sequencing, and in all four cases KRAS mutations were identified in the DNA. In six cases, high resolution melting abnormalities could not be confirmed as specific mutations either by ARMS/S or direct sequencing. We conclude that coring of the paraffin blocks and testing by ARMS/S is a sensitive, specific, and efficient method for KRAS testing.

Abstract 1492: Histological and molecular characteristics of 29 cases of carcinoma in situ from a prospective bronchoscopic cohort

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: The identification of carcinoma in situ (CIS) in a bronchial biopsy may indicate 1.) an adjacent invasive Lung Cancer (LC) 2.) a premalignant lesion that will progress to invasive LC or 3.) a lesion that can regress to a lower histologic grade or normal mucosa. The study of markers that identify lesions at high risk for invasive LC is of critical importance for early detection and treatment. Methods: Over a sixteen year period more than one thousand smokers underwent autofluorsecence bronchoscopy and biopsy. Biopsies were serially sectioned and graded for level of dysplasia according to standard WHO histological criteria. The distribution of the CIS lesions was assessed with the aid of a web-based bronchial mapping tool. Immunohistochemical stains for for Ki-67 (Ki-67 growth fraction) and p53 and FISH tests for seven DNA markers including centromere 6, EGFR, 5p15.2, MYC, NKX21, TP63 and KRAS were performed on subsets of biopsies. Finally, areas of mucosa in a subset of biopsies with features of CIS were marked and microdissected for mutational analysis of TP53 (exons 5-8). Histologic slides and immunohistochemical stains for all biopsies from the date of CIS diagnosis and follow up studies were reviewed, imaged and entered on a web-based bronchial map to visually track histological and molecular changes in the airways. Results: CIS was identified 29 subjects from the more than 1000 who were bronchoscopically examined. Fifty-eight bronchoscopies were conducted on these individuals and 435 biopsies examined. Maximum follow-up time averaged 13 mos. with a maximum of 94 mos. Among the twenty nine 65% had an associated invasive LC at the time of biopsy or during a short period of follow up whereas CIS lesions in 35% did not progress to invasive carcinoma during the follow-up period. All CIS had high Ki-67 growth fraction; p53 staining was variable. FISH analysis showed higher degrees of aneusomy in CIS lesions than lower histological grades and multiple chromosomal gains in CIS lesions shared among subjects and within a given lesion. In one patient, a CIS lesion with a TP53 mutation (G245V, GGC-GTC) spread intramucosally from the site of origin in the right lung to the contralateral lung over a period of 2 years. In another patient, nearly diffuse CIS with extremely high Ki-67 growth fraction was absent on rebiopsy of several bronchial sites three years later. Conclusions: These data indicate that a clonal subset of cells with histologic features of CIS may spread across bronchial mucosal surfaces and be detected by fluorescence bronchoscopy and available histological and molecular studies. CIS lesions with TP53 mutation and multiple genomic gains by FISH analysis are associated with an invasive LC at the time of diagnosis or during follow up. Molecular testing of CIS may assist in determining which lesions will progress. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 1492.