NSCLC患者血浆p16和MGMT基因甲基化检测及临床意义

目的：检测非小细胞肺癌（non—small cell lung cancer，NSCLC）患者外周血血浆中p16基因、O^6-甲基乌嘌呤-DNA甲基转移酶（O^6-methylguanine—DNA methyhransferase，MGMT）基因启动子的甲基化状态，探讨p16、MGMT基因启动子的异常甲基化在NSCLC筛查及早期诊断中的意义。方法：利用巢式甲基化特异性聚合酶链反应法检测NSCLC患者外周血血浆p16、MGMT基因启动子的甲基化状态。结果：65例NSCLC血浆样品中分别发现19例（29．23％）p16基因启动子异常甲基化和16例（24．62％）MGMT基因启动子异常甲基化，45例正常对照血浆组未检测到p16、MGMT基因启动子的异常甲基化（P〈0．05），血浆中两基因甲基化检出率与NSCLC的分型及临床分期无明显相关性（P〉0．05）。结论：利用巢式甲基化特异性PCR法检测外周血血浆中p16、MGMT基因启动子的甲基化，可为NSCLC的筛查、早期诊断及预后判断提供有价值的信息。     

中国非小细胞肺癌患者血清DAPK基因、p16基因启动子甲基化的分析(英文)

目的分析65名中国南京军区南京总医院的非小细胞肺癌(NSCLC)住院患者血清中DAPK、p16启动子区域甲基化的改变状况及其临床意义。方法运用甲基化特异性PCR技术,检测65例NSCLC患者血清DAPK、p16基因启动子区域甲基化的改变情况,并分析与临床病理资料的关系。结果 NSCLC患者血清DAPK基因甲基化检出率为30．8％(20/65),p16基因甲基化检出率为43．1％(28/65),而正常对照组和良性肺部疾病组血清未检出DAPK基因、p16基因甲基化,DAPK基因、p16基因甲基化检出率与NSCLC病理类型、分期及转移状态无明显关系。结论 DAPK基因、p16基因检出启动子区域异常甲基化是NSCLC早期辅助诊断的分子标志物之一。     

肺癌患者外周血血浆中p16基因异常甲基化的检测

目的 分析肺癌患者外周血血浆中p16基因启动子区异常甲基化发生情况,探讨血浆中p16基因异常改变作为肺癌临床辅助诊断分子生物学标志物的可能性。方法 利用半巢式甲基化特异性PCR技术,检测了137例肺癌患者血浆和112例相对应肿瘤组织DNA p16基因的异常甲基化情况。结果 在血浆和肿瘤组织中的p16基因甲基化检出率分别为75．2％和80．4％;其中鳞癌、腺癌、腺鳞癌和小细胞肺癌患者血浆标本的阳性率分别为77．9％,65．1％,75．1％和91．7％。血浆DNAp16基因甲基化仅在肿瘤组织存在同样甲基化的病例中被检出。血浆和肿瘤组织中,p16基因的甲基化异常改变与肿瘤的分期、分型无明显相关性。结论 分析血浆DNA的p16基因异常甲基化有可能成为辅助肺癌诊断的有效方法之一。     

痰标本p16和MGMT基因甲基化对肺癌的诊断价值

目的：分析肺癌患者痰标本中p16和MGMT基因启动子区甲基化的改变情况,评价该指标作为肺癌辅助诊断分子标志物的价值。方法：运用甲基化特异性PCR技术,检测77例原发性肺癌患者痰标本和部分对应肿瘤组织(53例),以及30例正常对照者痰标本中p16和MGMT基因启动子区域的甲基化改变。结果：49例(63．6%)肺癌患者痰标本中检测到了pi6基因异常甲基化,34例(44．2%)检测到了MGMT基因异常甲基化,77例患者痰标本中2个基因中至少有1个基因出现甲基化为64例(83．1%),对肿瘤的检出灵敏度较高。长期吸烟史是影响肺鳞状细胞癌痰标本p16(P=0．001)基因启动子区甲基化的因素。随TNM分期增高,肺鳞状细胞癌患者痰标本中p16基因甲基化比例增高(P=0．021)；随TNM分期增高,肺腺癌患者痰标本MGMT基因甲基化比例增高(P=0．023)。对照组正常人痰液标本未发现p16和MGMT基因启动子区甲基化。结论：痰标本中p16和MGMT基因甲基化是临床肺癌辅助诊断的有效生物标志物之一。     

胃癌血清肿瘤相关基因超甲基化检测及其意义

目的：探讨检测胃癌患者血清中肿瘤相关基因超甲基化的可行性及其意义。肿瘤患者的血清中游离DNA存在质或量的变化，是可能的肿瘤标志物。DNA甲基化不改变DNA序列和遗传密码，具有可逆性，被认为是基因转录调控的表观遗传机制之一。抑癌基因启动子超甲基化通常导致基因的转录失活，其功能的丧失，参与肿瘤的发生。方法：通过甲基化特异性PCR（MSP）检测52例胃癌组织和配对血清的hMLHl、E—cadherin、GSTPI、P15和P16基因的启动子超甲基化。20例健康体检者血清作为对照。结果：在胃癌组织中检测到hMLH1、E—cadherin、GSTP1、D15和p16基因的启动子超甲基化率分别为28名％、76．9％、23．1％、59．6％和69.2％；在患者血清中检测到hMLH1、E—cadherin、GSTP1、P15和P16基因的启动子超甲基化率分别为13．5％、38．5％、15．4％、25．0％和30．8％。84．7％的患者血清可以检测到异常甲基化．所有血清中检测到异常甲基化的患者，其肿瘤组织也能检测到相应基因的异常甲基化。对照组血清未检测到任何异常甲基化。结论：MSP检测血清异常甲基化是一种具有较高灵敏度和特异性的方法，可以在大部分胃癌患者血清中同时检测到多个肿瘤相关基因的异常甲基化，可以将其作为胃癌诊断的辅助手段。     

非小细胞肺癌病人痰标本p16和MGMT基因的异常甲基化

痰脱落细胞学检查以其方例、无创的特点成为目前诊断肺癌的最基本手段之一,然而它的敏感性比较差。近年来分子肿瘤学的理论和技术进展为我们提供了利用分子病理学标志物发现早期肺癌的可能。研究表明,某些基因的异常甲基化是肺癌中常见的分子水平改变;其包括p16INK4a(p16)和O^6甲基鸟嘌呤－DNA甲基转移酶（O^6-methyl-guanine-DNA methyltransferase,MGMT)。在这项研究中,我们从肺癌病人的痰标本脱落细胞中分离制备DNA,检测了p16和MGMT这两个基因的启动子区的甲基化状态。采用甲基化特异性PCR（methylation-specific PCR,MSP)的方法,我们对71例非小细胞肺癌（non-small cell lung cancer,NSCLC)病的痰标本进行了分析。所有被检NSCLC病人均有明确的组织病理学诊断,其中,肺鳞癌39例,肺腺癌25例,腺鳞癌7例。这71例病人的痰标本仅有31例（43．6％）被细胞学检查诊断为癌。我们的分析资料显示：在71例痰标本中p16基因的高甲基化检出率为83．1％（59／71）,MGMT的高甲基化检出率为59．2％（42／71）。在被测痰细胞DNA中,这两个基因（其中一个或两者）的启动子区异常甲基化的综合检出率高达90．1％（64／71）;而且与相应肿瘤组织中这两个基因的甲基化状态存在很好的符合率。这项研究结果表明,利用MSP技术检查痰脱落细胞DNA中p16和MGMT两基因的异常甲基化是一项敏感、无创、易行的方法。它将有助于非小细胞肺癌早期发现,以及肺癌高危人群的筛查。     

肺癌患者组织样品中p16基因的异常甲基化

目的 分析肺癌患者组织样品中p16基因启动子区域异常甲基化的改变情况,评价该指标作为辅助临床诊断的分子标记物的价值。方法 运用甲基化特异性PCR技术,检测51例原发性肺癌患者的肿瘤组织、外周血血浆和痰标本中p16基因启动子区域的异常甲基化改变。结果 在43例肿瘤组织、36例血浆和39例痰标本中检测到了p16基因异常甲基化。凡在肿瘤组织检出p16基因甲基化阳性的病例,其血浆和(或)痰标本也为阳性;而肿瘤组织p16基因甲基化阴性的病例,其血浆和痰标本也为阴性。将血浆和痰标本的p16甲基化分析与痰细胞学检查相结合,可以发现92．2％的肺癌病例。结论 利用半巢式甲基化特异性PCR进行的血浆和痰标本p16基因甲基化分析,有可能成为辅助肺癌诊断的分子生物学指标。     

非小细胞肺癌病人血浆p16基因异常甲基化的检测

肺癌高死亡率的根本原因在于早期诊断的困难。现代分子肿瘤学的研究进展揭示了在肺支气管上皮癌变过程中发生的多种分子水平异常,使得我们有可能将外周血等容易取得的微创性样品中的分子病理学改变作为肺癌早期诊断的辅助指标。通过以往的研究我们发现,一条等位基因缺失而另一条等位基因异常高甲基化是肺癌组织中抑癌基因p16失活的重要机制之一。在这项研究中,我们利用从非小细胞肺癌（non-small cell lung cancer,NSCLC)病人外周血血浆中提取的DNA分析了P16基因的甲基化状态。采用改进的甲基化特异性PCR（methylation-specific PCR,MSP)的方法,95例NSCLC病人的血浆DNA被检测,其中肺鳞癌59例,肺腺癌36例。作为对照,75例相应的肿瘤组织被同时分析。结果显示,p16基因启动子区高甲基化状态的检出率在血浆中为72．6％（69／95）,而在相应的肺癌组织中为74．7％（56／75）。值得注意的是,血浆DNAp16基因异常甲基化只是从那些肿瘤组织DNA表现p16基因高甲基化的病例中被检出－二者存在极好的符合率。我们还发现,在Ⅰ期病人中,血浆p16基因异常高甲基化在肺鳞癌（82．4％）,比在肺腺癌（33．3％）中要高得多（P＜0．005）。从而提示,血浆p16基因异常甲基化有可能成为一项早期发现非小细胞肺癌（尤其是鳞癌）的、非常重要的分子病理学指标。而血浆DNA的MSP则是检测这一指标的敏感、可靠并且便于操作的技术之一,成为一种可以应用于人群筛查的方法。     

非小细胞肺癌中ASC基因启动子甲基化的意义

目的：探讨非小细胞肺癌（NSCLC）中ASC基因启动子甲基化与患者临床特征之间的关系以及去甲基化试剂对肺癌细胞系中ASC基因甲基化状态的逆转。方法：应用甲基化特异性PCR（MSP）检测48例NSCLC患者肿瘤组织和3株肺癌细胞系中ASC基因的甲基化状态，RT—PCR检测去甲基化试剂诱导肺癌细胞系中甲基化ASC基因的重新表达。结果：NSCLC肿瘤组织和相对应的正常肺组中ASC基因启动子甲基化的发生率分别为49．7％和8-3％，二者之间有显著性差异（P〈0．01）。重度吸烟患者的肿瘤组织中ASC基因启动子甲基化的发生率明显增高（P〈0．05），早期的NSCLC（T1）中ASC基因启动子甲基化也有相对较高的发生率（P〈0．05）。去甲基化试剂5-氮-2’-脱氧胞苷（5-aza-2＇-deoxycytidine）可使甲基化的ASC基因启动子发生逆转而获得重新表达。结论：ASC基因启动子的甲基化可能与吸烟引起的非小细胞肺癌有关．并可能是非小细胞肺癌发展过程中的一个早期事件，甲基化的ASC基因可以成为NSCLC治疗的靶点。     

肺癌血清p16基因启动子畸变甲基化检测

[目的]检测肺癌患者血清DNA中p16基因启动子畸变甲基化,并探讨其在肺癌早期诊断和预后评价中的作用。[方法]收集新鲜的肺癌组织及其对应的血清标本69例,肺良性疾病患者标本25例,健康人血清标本10例。采用PCR技术分别检测肿瘤组织DNA和血清游离DNA中p16基因启动子畸变甲基化的状况。[结果]28.99%(20/69)肺癌组织出现p16高甲基化;出现高甲基化的组织标本相对应的20例血清中有15例(75%)出现p16基因高甲基化。肺良性疾病患者及正常人的血清、DNA中均未见p16基因高甲基化。血清p16基因高甲基化与肺癌TNM分期及分化程度密切相关(P<0.01或P<0.05)。[结论]非小细胞肺癌血清中p16基因甲基化状况的检测,可能有助于非小细胞肺癌的早期诊断或预后评估。     

Alterations of the p16(ink4a) gene in resected nonsmall cell lung tumors and exfoliated cells within sputum

Recently, we reported that p16 protein expression was nondetectable in 49.5% of 107 resected nonsmall cell lung cancers (NSCLCs), suggesting that the p16(INK4a) gene is frequently inactivated in primary NSCLC. To identify the molecular basis for this p16 immunohistochemical negativity further, we performed a genetic and epigenetic study of p16(INK4a) status in a series of 115 NSCLC samples parallel to the clinicopathologic and prognostic analyses. Microdissected tumor DNA samples were screened for homozygous deletion using comparative multiplex-polymerase chain reaction (PCR), for intragenic mutation using direct sequencing and for loss of heterozygosity (LOH) using an intragenic microsatellite marker, D9S942. Of these samples, 67 were further analyzed by SmaI-based PCR methylation assay to evaluate aberrant methylation at the gene. To examine the correlation of aberrant methylation in tumor and sputum samples, sputum samples from 12 matched patients were assessed for this change. We found that methylation of the p16(INK4a) gene was present in 38 of the 67 (56.7%) tumors and was significantly associated with negative p16 protein expression (p = 0.029). A 92% (11/12) concordance of sputum samples with matched resected tumors was found. The survival rates among adenocarcinoma patients with p16(INK4a) methylation were lower, but at a level of borderline significance compared with those patients without methylation (p = 0.071). In addition, 29.4% of the informative cases were found to harbor LOH at D9S942. None of the 115 microdissected tumors exhibited homozygous deletion in the p16(INK4a) gene. Only 1 patient exhibited a complex mutation at the fourth ankyrin repeat consensus sequence and concordantly demonstrated p16 immunohistochemical negativity. Overall, 69% (79/115) of NSCLC tumors had at least 1 type of p16(INK4a) alteration. Our data provide compelling evidence that p16(INK4a) alterations are involved in NSCLC tumorigenesis and that promoter methylation is the predominant mechanism in p16(INK4a) deregulation.     

Promoter hypermethylation patterns of p16, O6-methylguanine-DNA-methyltransferase, and death-associated protein kinase in tumors and saliva of head and neck cancer patients

Aberrant promoter hypermethylation is common in head and neck cancer and may be useful as a marker for cancer cells. We examined whether cells with tumor-specific aberrant DNA-methylation might be found in the saliva of affected patients. We tested 30 patients with primary head and neck tumors using methylation-specific PCR searching for promoter hypermethylation of the tumor suppressor gene p16 (CDKN2A), the DNA repair gene O6-methylguanine-DNA-methyltransferase (MGMT) and the putative metastasis suppressor gene death-associated protein kinase (DAP-K). Aberrant methylation of at least one of these genes was detected in 17 (56%) of 30 head and neck primary tumors; 14 (47%) of 30 at p16, 10 (33%) of 30 at Dap-K and 7 (23%) of 30 at MGMT. In 11 (65%) of 17 methylated primary tumors abnormal methylated DNA was detected in the matched saliva samples. Abnormal promoter methylation in saliva DNA was found in all tumor stages and more frequently in tumors located in the oral cavity. Moreover, none of the saliva from patients with methylation-negative tumors displayed methylation of any marker. Of 30 saliva samples from healthy control subjects (15 smokers and 15 nonsmokers), only one sample from a smoking patient was positive for DNA methylation at two target genes. Detection of aberrant promoter hypermethylation patterns of cancer-related genes in saliva of head and cancer patients is feasible and may be potentially useful for detecting and monitoring disease recurrence. Long-term longitudinal studies are needed to evaluate this approach for early detection of head and neck cancer in at-risk populations.     

Detection of p16 hypermethylation in circulating plasma DNA of non-small cell lung cancer patients

We detected tumor-associated aberrant hypermethylation of the p16 gene in plasma DNA from 105 non-small cell lung cancer (NSCLC) patients (65 squamous cell carcinoma (SCC) and 40 adenocarcinoma (ADC)) and 92 matched tumor DNA samples, using a modified semi-nested methylation-specific PCR (MSP). This technique increased the sensibility of detecting p16 hypermethylation from DNA samples in varying stages. p16 hypermethylation was present in 73.3% (77/105) of the plasma samples, and 79.3% (73/92) of the tumor samples. Among those cases with methylated p16 sequence in tumor samples, 87.7% (64/73) also demonstrated this epigenetic alteration in the corresponding plasma DNA. Only patients whose tumor cells had hypermethylated p16 gene exhibited aberrant methylation in their plasma samples. Regarding different clinical stages of SCC and ADC, the frequencies of p16 hypermethylation in plasma DNA were nearly the same as those in corresponding tumors, except for stage I ADC. Our study indicated that aberrant methylation of p16 may be an excellent biomarker for early diagnosis and follow-up of NSCLC patients, and MSP is a reliable method for these purposes.     

Aberrant promoter methylation of multiple genes in non-small cell lung cancers

Aberrant methylation of CpG islands acquired in tumor cells in promoter regions is one method for loss of gene function. We determined the frequency of aberrant promoter methylation (referred to as methylation) of the genes retinoic acid receptor beta-2 (RARbeta), tissue inhibitor of metalloproteinase 3 (TIMP-3), p16INK4a, O6-methylguanine-DNA-methyltransferase (MGMT), death-associated protein kinase (DAPK), E-cadherin (ECAD), p14ARF, and glutathione S-transferase P1 (GSTP1) in 107 resected primary non-small cell lung cancers (NSCLCs) and in 104 corresponding nonmalignant lung tissues by methylation-specific PCR. Methylation in the tumor samples was detected in 40% for RARbeta, 26% for TIMP-3, 25% for p16INK4a, 21% for MGMT, 19% for DAPK, 18% for ECAD, 8% for p14ARF, and 7% for GSTP1, whereas it was not seen in the vast majority of the corresponding nonmalignant tissues. Moreover, p16INK4a methylation was correlated with loss of p16INK4a expression by immunohistochemistry. A total of 82% of the NSCLCs had methylation of at least one of these genes; 37% of the NSCLCs had one gene methylated, 22% of the NSCLCs had two genes methylated, 13% of the NSCLCs had three genes methylated, 8% of the NSCLCs had four genes methylated, and 2% of the NSCLCs had five genes methylated. Methylation of these genes was correlated with some clinicopathological characteristics of the patients. In comparing the methylation patterns of tumors and nonmalignant lung tissues from the same patients, there were many discordancies where the genes methylated in nonmalignant tissues were not methylated in the corresponding tumors. This suggests that the methylation was occurring as a preneoplastic change. We conclude that these findings confirm in a large sample that methylation is a frequent event in NSCLC, can also occur in smoking-damaged nonmalignant lung tissues, and may be the most common mechanism to inactivate cancer-related genes in NSCLC.     

Aberrant promoter methylation in Chinese patients with non-small cell lung cancer: patterns in primary tumors and potential diagnostic application in bronchoalevolar lavage.

This study was aimed at defining patterns of aberrant gene methylation in non-small cell lung cancer (NSCLC) in Chinese patients and its use in detecting cancer cells in bronchoalveolar lavage (BAL). The methylation-specific PCR (MSP) was used to study methylation of the p16, retinoic acid receptor-beta (RARbeta), death-associated protein (DAP) kinase, and O(6)-methylguanine-DNA-methyltransferase (MGMT) genes in 75 NSCLCs [44 adenocarcinomas and 31 squamous cell carcinomas (SCCs)] and 68 BALs from suspected lung cancers. More females had adenocarcinoma than SCC (11 of 44 versus 2 of 31, P = 0.04). Aberrant methylation in at least one gene was found in 63 of 75 (84%) NSCLCs. p16, RARbeta, DAP kinase, and MGMT methylation was similar in adenocarcinoma and SCC. However, females with NSCLC showed more frequent p16 methylation than males (12 of 13 versus 36 of 62, P = 0.02), because of more frequent p16 methylation in female adenocarcinomas (10 of 11 versus 17 of 33, P = 0.02). This sexual difference was not observed in RARbeta, DAP kinase, and MGMT. At 92%, the frequency of p16 methylation in Chinese female NSCLC is one of the highest known. For BAL, MSP and cytological analysis showed concordant and discordant results in 25 of 68 and 43 of 68 samples. Of 41 MSP+/cytology- cases, 35 were eventually shown to have malignant lung lesions, 4 were at high risk but had no evidence of lung cancer, and 2 were lost to follow-up. There were two MSP-/cytology+ cases. Frequent gene methylations were seen in Chinese NSCLC patients. More frequent p16 methylation was seen in female patients. MSP is a useful molecular adjunct for cancer cell detection in BAL samples.     

Methylation assay for the diagnosis of lung cancer on bronchial aspirates: a cohort study.

PURPOSE: Recent studies have detected aberrant promoter methylation of adenomatous polyposis coli promoter 1 A (APC), cyclin-dependent kinase inhibitor-2A (p16(INK4a)), retinoic acid receptor beta2, and RAS association domain family protein 1 (RASSF1A) in bronchial aspirates and suggested their use as biomarkers for lung cancer diagnostics. The purpose of this study was to validate these candidate marker genes in a retrospective cohort study. EXPERIMENTAL DESIGN: Bronchial aspirates collected from a cohort comprising 247 patients with suspected lung cancer were investigated retrospectively regarding aberrant promoter methylation using a quantitative methylation-specific real-time PCR (QMSP). RESULTS: Eighty-nine patients were diagnosed with primary lung cancer, 102 had benign lung disease, and 56 showed miscellaneous other conditions. A panel consisting of APC, p16(INK4a), and RASSF1A emerged as useful combination. This panel detected aberrant methylation in bronchial aspirates of 22 of 35 (63%) and 21 of 44 (44%) centrally and peripherally located primary lung cancers, respectively. Bronchial aspirates also showed aberrant methylation in 5 of 7 (71%) patients with a recurrent lung cancer and in 8 of 30 (27%) cases without tumor recurrence. In contrast, only 1 of 102 patients with benign lung disease displayed a (false) positive test result. Rarely, aberrant methylation was found in patients with other malignancies (3 of 16). The QMSP assay correctly confirmed lung cancer in 8 of 12 (67%) cases with an ambiguous cytology. Moreover, it disclosed 9 of 26 (35%) of peripheral tumors lacking simultaneous cytologic or histologic diagnosis of malignancy. CONCLUSIONS: Our findings suggest that the QMSP assay could be applied as a reflex test in cases of suspected lung cancer that defy a definite diagnosis by conventional methods. Thus, the assay could be a useful diagnostic adjunct especially regarding peripheral tumors.     

Aberrant promoter methylation in bronchial epithelium and sputum from current and former smokers

Recent studies from our laboratory suggest that gene-specific methylation changes in sputum could be good intermediate markers for the early detection of lung cancer and defining the efficacy of chemopreventive interventions. The purpose of our study was to determine the prevalence for aberrant promoter methylation of the p16, O(6)-methylguanine-DNA methyltransferase (MGMT), death-associated protein (DAP) kinase, and Ras effector homologue (RASSFIA) genes in nonmalignant bronchial epithelial cells from current and former smokers in a hospital-based, case control study of lung cancer. The relationship between loss of heterozygosity, at 9p and p16 methylation in bronchial epithelium and the prevalence for methylation of these four genes in sputum from cancer-free, current and former smokers were also determined. Aberrant promoter methylation of p16 was seen in at least one bronchial epithelial site from 44% of cases and controls. Methylation of the DAP kinase gene was seen in only 1 site from 5 cases and 4 controls, whereas methylation of the RASSFIA was not detected in the bronchial epithelium. Promoter methylation for p16 and DAP kinase was seen as frequently in bronchial epithelium from current smokers as from former smokers. No promoter methylation of these genes was detected in bronchial epithelium from never-smokers. Methylation of the p16 gene was detected in sputum from 23 of 66 controls. DAP kinase gene promoter methylation was also seen in sputum from 16 controls, and 8 of these subjects were positive for p16 methylation. Methylation of the MGMT gene was seen in sputum from 9 controls, whereas RASSFIA promoter methylation was only seen in 2 controls. The correlation between p16 status in the bronchial epithelium obtained from lung lobes that did not contain the primary tumor and the tumor itself was examined. Seventeen of 18 tumors (94%) showed an absolute concordance, being either methylated in the tumor and at least 1 bronchial epithelial site, or unmethylated in both tumor and bronchial epithelium. These results indicate that aberrant promoter hypermethylation of the p16 gene, and to a lesser extent, DAP kinase, occurs frequently in the bronchial epithelium of lung cancer cases and cancer-free controls and persists after smoking cessation. The strong association seen between p16 methylation in the bronchial epithelium and corresponding primary tumor substantiates that inactivation of this gene, although not transforming by itself, is likely permissive for the acquisition of additional genetic and epigenetic changes leading to lung cancer.     

Hypermethylation of p16(INK4a) and p15(INK4b) genes in non-small cell lung cancer.

Hypermethylation of CpG island is a common mechanism by which tumor suppressor genes are inactivated. The tumor suppressor genes p16(INK4a) and p15(INK4b) are important components of the cell cycles. We have studied the feasibility of detecting tumor-associated aberrant p16(INK4a) and p15(INK4b) methylation in non-small cell lung cancer (NSCLC) using methylation-specific PCR. We found a high frequency of hypermethylation of the p16(INK4a) gene in 17 of 45 cases of NSCLC. In this study, there was no difference between the clinicopathological features or overall survival of patients with and without p16(INK4a) methylation. On the other hand, p15(INK4b) promoter hypermethylation is rare (5/45) in lung cancer and occurs in association with p16(INK4a) methylation. The overall survival of patients with p15(INK4b) methylation was markedly shortened in this series. We also analyzed cells in bronchial washings, and p16(INK4a) methylation was detected in 4 of 17 cases of NSCLC. Moreover, 1 of 10 plasma samples from patients with NSCLC was positive for p16(INK4a) methylation. Our results suggest a possible prognostic role of p15(INK4b) methylation in NSCLC, and that the detection of aberrant p16(INK4a) methylation in both bronchial washings and plasma may be useful for cancer diagnosis.