Aberrant methylation of p16(INK4a) is an early event in lung cancer and a potential biomarker for early diagnosis - PubMed (original) (raw)
Aberrant methylation of p16(INK4a) is an early event in lung cancer and a potential biomarker for early diagnosis
S A Belinsky et al. Proc Natl Acad Sci U S A. 1998.
Abstract
The p16(INK4a) (p16) tumor suppressor gene can be inactivated by promoter region hypermethylation in many tumor types including lung cancer, the leading cause of cancer-related deaths in the U.S. We have determined the timing of this event in an animal model of lung carcinogenesis and in human squamous cell carcinomas (SCCs). In the rat, 94% of adenocarcinomas induced by the tobacco specific carcinogen 4-methylnitrosamino-1-(3-pyridyl)-1-butanone were hypermethylated at the p16 gene promoter; most important, this methylation change was frequently detected in precursor lesions to the tumors: adenomas, and hyperplastic lesions. The timing for p16 methylation was recapitulated in human SCCs where the p16 gene was coordinately methylated in 75% of carcinoma in situ lesions adjacent to SCCs harboring this change. Moreover, the frequency of this event increased during disease progression from basal cell hyperplasia (17%) to squamous metaplasia (24%) to carcinoma in situ (50%) lesions. Methylation of p16 was associated with loss of expression in both tumors and precursor lesions indicating that both alleles were functionally inactivated. The potential of using assays for aberrant p16 methylation to identify disease and/or risk was validated by detection of this change in sputum from three of seven patients with cancer and 5 of 26 cancer-free individuals at high risk. These studies show for the first time that an epigenetic alteration, aberrant methylation of the p16 gene, can be an early event in lung cancer and may constitute a new biomarker for early detection and monitoring of prevention trials.
Figures
Figure 1
Detection of p16 methylation in hyperplasias, adenomas, and adenocarcinomas induced by NNK in the rat. Methylation is evident in hyperplasias from lanes 1–5 and 7, in adenomas from lanes 9–11 and 14, and in adenocarcinomas from lanes 15–25. Unmethylated p16 alleles were present in all lesions due to contaminating normal tissue and in normal lung and alveolar type II cells. No methylated alleles are detected in modified DNA from normal lung (lanes 8 and 26) or alveolar type II cells (lane 27).
Figure 2
Rat and human lesions with _p16_methylation. (A) Focal epithelial hyperplasia in the lung of a rat treated with NNK. Note the small size of the lesion and the lack of disruption of the normal alveolar architecture. (B) Higher magnification of the lesion in A. (C) Adenoma in the lung of an NNK-treated rat. The epithelial cells exhibit minimal atypia. Note the large number of neutrophils and macrophages within the luminal spaces and in the stroma (arrow points to one luminal aggregate of inflammatory cells). (D) Minimal basal cell hyperplasia of the bronchial epithelium in a human biopsy specimen. Note the normal appearing ciliated surface. (E) Squamous metaplasia with minimal atypia of the bronchial epithelium in a section obtained at lobectomy. This patient had a SCC distant from this site at the time of lobectomy. (F) Squamous metaplasia with moderate atypia of the bronchial epithelium obtained at lobectomy. This patient also had a SCC distant from this site at the time of lobectomy. (G) CIS of the bronchial epithelium in a human biopsy specimen.
Figure 3
Methylation of p16 in premalignant lesions, CIS, and SCC. (A) Lymphocytes microdissected from in and around the SCC constituted the normal (N) tissue for analysis. Other areas analyzed included normal appearing bronchus (NB) within the cancer field, adjacent CIS, and SCC obtained from three patients. (B) Precursor lesions and SCCs obtained through biopsy or lobectomy from several different cases. Lesions microdissected included basal cell hyperplasia (Hyper), squamous metaplasia (Meta), CIS, and SCC. Cell lines H249 and U172 serve as positive controls for detecting unmethylated (U) and methylated (M) p16 alleles, respectively by MSP. A PCR product of the appropriate molecular weight (151 bp for U, 150 bp for M) indicates the presence of unmethylated and/or methylated p16 alleles in that sample.
Figure 4
Summary of the frequency for _p16_methylation in premalignant lesions, CIS, SCC, and sputum samples. The frequency (expressed as a percentage) for detecting _p16_methylation is depicted for basal cell hyperplasia (Hyper), squamous metaplasia (Meta), CIS, SCC, and in sputum from cancer-free smokers and lung cancers. Above the bar graphs the total number of positive samples per total sample population for a specific lesion or population is indicated.
Figure 5
Immunohistochemistry to detect p16 protein in human lung lesions. (A) Immunohistochemical detection of p16 protein (brown nuclear staining) in an area of bronchiolar squamous metaplasia. (B) Lack of detectable p16 protein in an area of bronchiolar squamous metaplasia with marked atypia. (C) Strong nuclear p16 immunostaining in a SCC. (D) Lack of p16 protein in a SCC.
Figure 6
Methylation of p16 in sputum from smokers. Bisulfite modified DNA was amplified by primers specific to methylated or unmethylated alleles of the p16 gene as described. Unmethylated p16 alleles were detected in all 16 sputum samples and in DNA from normal human bronchial epithelial (NHBE) cells obtained from a never-smoker. The Calu1 cell line (positive control for p16 methylation) is completely methylated. Methylated alleles of p16 were observed in DNA from 4 cancer-free persons (lanes 3, 10, 13, and 14), 1 person diagnosed at the time of sputum induction with a SCLC (lane 2) who developed a NSCLC 2 years later, 1 smoker (lane 15) who was positive for lung cancer at the time of sputum induction, and in the positive control (Calu1). No methylation was observed in the negative control normal human bronchial epithelial cells. Samples negative for_p16_ methylation are shown in lanes 1, 4–9, 11–12, and 16.
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