鼻咽癌组织中p16基因的缺失、高甲基化和蛋白的表达及其临床意义

目的探讨鼻咽癌组织中p16基因的缺失、高甲基化和蛋白的表达及其临床意义。方法应用免疫组织化学EnVision法检测90例鼻咽非角化性癌(NKC)组织p16蛋白的表达缺失,聚合酶链反应和甲基化酶切方法检测23例NKC组织p16基因缺失和高甲基化。结果90例NKC中p16蛋白表达阴性率为46.7%(42/90),而对照组的阴性率为0(0/30),P<0.05。p16蛋白表达与NKC患者的5年生存率有明显的相关性,生存期5年内者,其缺失率为60.0%(36/60);生存期5年以上者,其缺失率为20.0%(6/30),P<0.05。有、无远处器官转移的病例p16蛋白阴性率分别为81.8%(9/11)和41.8%(33/79),P<0.05。而有、无颅底破坏和(或)颅神经侵犯病例p16蛋白阴性率分别为41.7%(10/24)和48.5%(32/66),P>0.05。23例NKC中未检测到p16基因外显子1的缺失,但有10例p16基因外显子2的缺失,缺失率为43.4%(10/23);同时检测到2例外显子1的异常甲基化,高甲基化率为8.7%(2/23);总突变率为52.1%(12/23)。结论在NKC的发生发展过程中,p16基因缺失起着重要的作用。p16蛋白表达与NKC患者的5年生存率和远处转移有一定的相关性,而与NKC的局部组织侵犯无明显相关性。     

家族性腺瘤性息肉病患者APC基因启动子区异常甲基化及大片段结构异常

目的 研究3个家族性腺瘤性息肉病(familial adenomatous polyposis,FAP)家系的腺瘤样息肉病(adenomatus polyposis coli)基因(APC)启动子1A区异常甲基化及DNA大片段结构异常.方法 对3个FAP家系成员的肿瘤组织标本和正常组织标本DNA进行化学修饰,应用甲基化特异PCR(methylation-speeif-ic PCR,MsP)和DNA序列分析方法筛查APC基因启动子1A区甲基化情况.采用多重连接依赖性探针扩增(multiplex ligation-dependent probe amplification,MIPA)分析系统检测5例FAP患者肿瘤组织标本和正常标本的APC基因的15个外显子及启动子区DNA大片段结构异常.结果 在1个家系中发现2例患者存在APC基因启动子1A区异常甲基化.同一个家系中另1例患者存在APC基因全基因杂合性缺失.结论 APC基因启动子1A区异常甲基化可影响APC功能,可能是结直肠癌进展过程中的早期事件;大片段缺失可能是导致典型FAP的一个因素.     

原发胃癌中p16和p15基因表达及甲基化异常

为检测胃癌组织中抑癌基因p16,p15及其启动子区甲基化状态和P16、P15蛋白表达情况。选择p16、p15基因及启动子区域，用PCR－SSCP、MSP（甲基化特异的PCR）和测序法对100例胃癌患者的癌组织、癌旁正常组织和5例正常组织进行检测，同时用免疫组化法检测了癌组织和正常对照组织的P16和P15的表达。结果发现癌组织p16和p15基因启动子区甲基化率显著高于癌旁正常组织和正常对照；胃癌组织中，71％的病例P16表达阴性，54％的病例具有p16基因启动子区的高甲基化，无突变和纯合缺失检出；11％的病例P15表达阴性，9％的病例具有p15基因启动子区的高甲基化，p15异常与低分化胃癌有关，p15基因内含子1和外显子1内各发现1例DNA序列改变；癌组织中p16和p15基因启动子区甲基化与其蛋白表达密切相关。结果显示p16基因启动子区域高甲基化是胃癌中p16基因失活的关键因素之一，并在胃癌的发生发展中发挥重要作用；p15基因启动子区域高甲基化在胃癌中起一定作用。     

Detection of loss of heterozygosity of p53 gene in paraffin-embedded breast cancers by non-isotopic PCR–SSCP

A rapid non-isotopic PCR-SSCP (polymerase chain reaction-single-stranded conformation polymorphism) method was developed in this study to detect polymorphism and loss of heterozygosity (LOH) of p53 in formalin-fixed and paraffin-embedded samples of normal breast tissue and of breast cancer. p53 expression was also examined by immunohistochemistry. In 35 paired samples, heterozygosity in exon 4 of p53 was detected in 17 cases (49 per cent) and LOH of the p53 gene in breast cancer tissues was observed in 7 out of 15 informative cases (47 per cent). The correlation of LOH of p53 with positive p53 immunostaining did not reach statistical significance, but all immunostaining-positive tumours among informative cases had LOH of p53. The results support the hypothesis that in most cases the allelic deletion of p53 may uncover the ‘recessive mutation’ in the remaining allele. However, LOH of p53 was more frequent than positive immunostaining and was significantly associated with poor differentiation of breast cancer (P<0·05). The results suggest that the allelic deletion of p53 may also contribute to the development and progression of breast cancer by reducing the amount of normal p53 protein. These results show that non-isotopic PCR-SSCP is a simple, fast, and effective method for detecting polymorphism and LOH of the p53 gene, which is especially useful for retrospective studies.     

胃癌中p16INK4a基因启动子高甲基化及其蛋白表达

目的探讨胃癌中p16^INK4a基因失活的主要分子机制及其与胃癌发生、发展的关系。方法采用甲基化特异性PCR技术检测62例胃癌和癌旁组织及10例慢性胃炎黏膜p16^INK4a基因启动子区CpG岛甲基化状态,用免疫组化EnVision两步法检测p16^INK4a蛋白的表达。结果62例胃癌和癌旁组织p16^INK4a基因启动子高甲基化率分别为51．6％（32／62）和19.4％（12／62）,10例正常对照胃黏膜未发现高甲基化,胃癌组织p16^INK4a基因启动子高甲基化率高于癌旁组织和正常对照（P〈0．05）。58.1％（36／62）的胃癌组织p16^INK4a蛋白表达阴性,其中72.2％（26／36）的病例具有p16^INK4a基因启动子的高甲基化,p16^INK4a基因启动子的高甲基化与其蛋白失表达密切相关（P〈0.05）。结论胃癌中p16^INK4a基因启动子的高甲基化是p16^INK4a基因失活的主要机制,并可能是胃癌发生中的早期分子事件。     

Inactivation of the p16 gene in human pituitary nonfunctioning tumors by hypermethylation is more common in null cell adenomas

Recent studies have shown that methylation of the CpG island within the p16/CDKN2A/MTS1 (p16) gene is associated with loss of expression of p16 protein in pituitary tumors. We analyzed a series of 21 pituitary adenomas and three normal pituitaries along with a human pituitary cell line (HP75) for methylation of exon 1 by methylation-specific PCR, immunohistochemistry, and Western blotting. PCR analysis showed that 5/7 (71%) of null cell adenomas, but only 2/7 (29%) gonadotroph tumors were hypermethylated. In addition, 1 of 2 ACTH tumors but no GH (n=4) or PRL (n=1) adenoma examined were hypermethylated. Immunostaining and Western blot analysis of protein expression supported the methylation-specific PCR analyses. These results show that p16 gene silencing by hypermethylation is more common in null cell adenomas compared to other nonfunctioning adenomas such as gonadotroph tumors and that the role of p16 in the pathogenesis of pituitary adenomas is restricted to specific tumor subtypes. Supported in part by NIH CA90249 and by a grant from the Jarislowsky Foundation.     

低剂量螺旋CT扫描联合血清p16基因甲基化检测在肺癌早期诊断中的应用

目的研究在无症状的肺癌高危人群中利用低剂量CT（LDCT）联合血清p16基因甲基化检测进行肺癌早期诊断的可行性。方法肺癌高危人群入组标准：男性，年龄55～75岁；吸烟指数≥400支／年，目前仍在吸烟或戒烟不超过10年。共893例受检者被随机分为两组。一组为447例（LDCT—p16组），平均年龄66岁，进行LDCT联合血清p16基因甲基化检测；另一组为446例（CXR组），平均年龄67岁，接受后前位胸片检查。两组检查阳性病例将接受进一步组织病理学检查。并分别统计两组阳性结节检出率及肺癌检出率，并行X2检验。结果LDCT—p16组与CXR组分别有96．8％和92．8％的受检者完成了检查。LDCT—p16组中1113％病人可疑肺癌，明显高于CXR组的6．5％（P〈0．05）。其中LDCT—p16组中有7例，CXR组中有2例确诊为肺癌。LDCT—p16组肺癌检出率高于CXR组，但无统计学意义（P〉0．05）。结论低剂量CT联合血清p16基因甲基化检测是一种敏感、安全、可行的筛查早期肺癌的方法，能够取代胸片筛查早期肺癌。     

CDKN 2/p16基因甲基化失活与肺癌关系的研究

目的 研究ＣＤＫＮ２／ｐ１６基因５’端调控序列区ＣｐＧ岛甲基化的状态与肺癌发展的关系。方法 采用甲基化敏感性核酸酶切基因组ＤＮＡ的方法,对８９例肺癌的ＣＤＳＮ２／ｐ１６基因进行了Ｓｏｕｔｈｅｒｎ杂交分析。结果 ８９例肺癌发现该基因甲基化２１例,甲基化频率为２３．６％（２１／８９）,其中１７例发生于４２例Ｐ１６蛋白 阴性表达的患者。结论 ＣＤＫＮ２ｐ／１６基因５’端ＣｐＧ岛甲基化可能是该基因失活的重     

Characteristics of loss of heterozygosity in large cell neuroendocrine carcinomas of the lung and small cell lung carcinomas

Large cell neuroendocrine carcinoma (LCNEC) of the lung is a new entity. Besides morphological characteristics, its molecular biological features have been investigated by many researchers and compared to those of other neuroendocrine carcinomas, small cell lung carcinoma (SCLC) and carcinoid tumor (CT). However, there are few reports that show the significantly different genetic characteristics between them. The purpose of the present paper was to study the frequency of loss of heterozygosity (LOH) at chromosome 3p (3p14.2) in 38 neuroendocrine carcinomas of the lung (13 LCNEC, 11 SCLC and 14 CT) and 10 large cell carcinomas (LCC). The frequencies of LOH at 3p14.2 were 69.2% in LCNEC, 81.8% in SCLC, 50.0% in LCC and 7.14% in CT. Those at 22q13.3 were 30.8% in LCNEC, 72.7% in SCLC, 45.5% in LCC and 7.14% in CT. In particular, the frequency of SCLC with LOH at both 3p14.2 and 22q13.3 (63.6%) was significantly higher than that of LCNEC (15.4%). LCNEC and SCLC had different characteristics of LOH patterns at 3p14.2 and 22q13.3. The combined analysis of the LOH at 3p14.2 and 22q13.3 is thought to be useful for differential diagnosis between LCNEC and SCLC.     

p16 expression in colorectal adenocarcinoma: marker of aggressiveness and morphological types

Aim: The aim of the present study was to investigate the clinicopathological roles of p16 expression in a large cohort of patients with colorectal adenocarcinoma with tight methodology and close follow-up. Methods: p16 protein expression was investigated in 194 patients (102 men, 92 women) with colorectal adenocarcinomas by immunohistochemistry. The findings were correlated with their clinicopathological features. Results: p16 protein was detected in 80% (155 of 194) of patients with colorectal carcinoma. The p16 protein was more often detected in male patients with colorectal cancers (86% versus 73%, p = 0.03). p16 protein expression was more often seen in carcinomas in the rectum, sigmoid and descending colon compared with more proximal colon (90% versus 61%, p = 0.001). The p16 protein was more often detected in well or moderately differentiated colorectal adenocarcinoma than poorly differentiated colorectal adenocarcinoma (84% versus 63%, p = 0.009). The level of expression of p16 protein is related to the lymph nodal status (p = 0.004) and the TNM staging of the colorectal carcinoma (p = 0.008). Conclusion: p16 protein expression was common in colorectal adenocarcinomas. The expression correlated with gender of the patient, distal location, differentiation and staging of the tumour. The findings suggest that p16 plays an important role in cancer pathogenesis and has implications for improving the clinical management.     

髓母细胞瘤8号染色体短臂的杂合性丢失

目的研究髓母细胞瘤8号染色体的遗传学异常,寻找与该肿瘤发病机制有关的杂合性丢失位点。方法通过微卫星分析（microsatellite analysis）方法,应用19个位于8号染色体短臂（8p）上的多态性标记物,检测髓母细胞瘤的杂合性丢失（loss of heterozygosity,LOH）。结果在所检测的23例髓母细胞瘤中,21例为原发肿瘤,2例为复发肿瘤。染色体8p总的LOH比率为51%（124个LOH/243个可分析位点）。我们在8p22-23.2之间发现了一个高比率的共同丢失区,其长度为18.14 cM。结论染色体8p22-23.1上很可能存在重要的抑癌基因,该基因的丢失可能与髓母细胞瘤发病有关。     

No evidence of microsatellite instability but frequent loss of heterozygosity in primary resected lung cancer

We examined microsatellite instability and loss of heterozygosity (LOH) in primary lung tumors from 93 cancer patients, using 16 microsatellite markers. The cases studied included 87 non-small-cell lung cancers (NSCLC) and six small-cell lung cancers (SCLC). All the patients except two were current or former smokers. The microsatellite markers were all dinucleotide repeat sequences from chromosomal locations 1p, 3p, 5q, 8p, 9p, 10p, 11p, 13q, and 17q. None of the tumors showed microsatellite instability (0/93). In NSCLC, 28% (24/87) of the cases showed LOH in at least one locus, whereas, in SCLC, 67% (4/6) had allelic losses. The frequency of LOH differed between the various cell types of NSCLC. The highest frequency was seen in large cell carcinoma (3/6, 50%) followed by squamous cell carcinoma (16/43, 37%) and adenocarcinoma (5/35, 14%). The most common site of LOH was 3p, where markers D3S1284, D3S659, D3S1289, D3S966, D3S647, and D3S1038 were studied. LOH, studied with 9p markers (D9S126, D9S171, D9S162), was less common. The present results, together with earlier reports, suggest that smoking-related primary lung cancers seldom show microsatellite instability but are characterized by frequent LOH. Environ. Mol. Mutagen. 30:217–223, 1997. © 1997 Wiley-Liss, Inc.     

非小细胞肺癌中p16基因的突变研究

目的　探讨ｐ１６ 基因在非小细胞肺癌发生发展过程中所起作用。方法　应用 Ｐ Ｃ Ｒ 与双链 Ｄ Ｎ Ａ 直接测序技术对４０ 例非小细胞肺癌中ｐ１６ 基因外显子２ 的纯合缺失与序列改变进行了研究。结果４０ 例非小细胞肺癌中有２ 例存在ｐ１６ 基因外显子２ 的纯合缺失；１４ 例肿瘤 Ｄ Ｎ Ａ 样品中检出ｐ１６ 基因外显子２ 的１９ 个点突变和１ 个移码突变。其中８ 个突变位于１２１ 位密码子中３８０ 位碱基处。结论　ｐ１６ 基因点突变在非小细胞肺癌中发生频率较高，是参与非小细胞肺癌发生发展的主要突变形式。非小细胞肺癌中ｐ１６ 基因的突变热点为１２１ 位密码子中３８０ 位碱基的转换与颠换。     

Loss of heterozygosity during the development and progression of differentiated adenocarcinoma of the stomach

In a recent allelotypic analysis of differentiated adenocarcinoma of the stomach, loss of heterozygosity (LOH) was found frequently on chromosomes 2q, 4p, 5q, 6p, 7q, 11q, 14q, 17p, 18q, and 21q. To clarify the sequence of these chromosomal losses during gastric carcinogenesis, microsatellite analysis of the chromosome arms described above was performed in 25 early and 29 advanced differentiated adenocarcinomas of the stomach. LOH on these chromosome arms fell within a range of 20–50 per cent. On 4p, 7q, 14q, 17p, and 21q, LOH was detected at a similar frequency in both early and advanced carcinomas, while LOH on 2q, 5q, 6p, 11q, and 18q was observed more than twice as frequently in advanced than in early lesions. Mean fractional allelic losses (FALs) were 0·221 in early and 0·413 in advanced carcinomas, representing a significant difference (P<0·05). These results suggest that LOH on 4p, 7q, 14q, 17p, and 21q is a relatively early event, while LOH on 2q, 5q, 6p, 11q, and 18q typically accumulates during the progression of gastric carcinogenesis. © 1998 John Wiley & Sons, Ltd.     

The influence of anthracosis and p16 ink4a gene aberrant methylation on small-sized pulmonary adenocarcinoma

Aims

Anthracosis is the deposition of black dusty material in the pulmonary parenchyma. Previous reports showed anthracosis and p16^ink4a gene aberrant methylation are closely related to the promotion and progression of small-sized pulmonary adenocarcinoma. In this study, we investigated the influence of anthracosis and p16^ink4a gene aberrant methylation on clinical samples from patients with small-sized adenocarcinoma.

Methods and results

DNA was bisulfite modified and methylation-specific PCR was performed to detect p16^ink4a gene aberrant methylation; black dusty material was extracted from lung tissues. Anthracotic index (AI) was defined as the absolute absorbance by densitometry. The histopathological diagnosis was concluded according to Noguchi's classification for small-sized pulmonary adenocarcinoma. The mean AI and the frequency of p16^ink4a gene aberrant methylation of heavy smokers were significantly higher than that of nonsmokers (Ｐ < 0.01 andＰ < 0.05, respectively). The frequency of p16^ink4a gene aberrant methylation of early stage small-sized adenocarcinoma was lower than that of advanced and poorly differentiated, while p16^ink4a protein expression level of early stage small-sized adenocarcinoma was significantly higher than that of poorly differentiated small-sized adenocarcinoma (P < 0.05).

Conclusions

AI and p16^ink4a gene aberrant methylation may provide a potential universal biomarker for small-sized adenocarcinoma.     

High promoter hypermethylation frequency of p14/ARF in supratentorial PNET but not in medulloblastoma

AIMS: Medulloblastoma (MB) is the most common primitive neuroectodermal tumour (PNET) of the central nervous system. Although supratentorial PNET (sPNET) and MB are histologically similar, their clinical behaviour differs, sPNET being more aggressive than MB. The aim of this study was to determine whether sPNET and MB are genetically different entities. METHODS AND RESULTS: We investigated 32 PNET primary tumour samples (23 MB and nine sPNET) and four PNET cell lines, for the presence of CDKN2A homozygous deletions at exon 1-alpha of p16/INK4 and exon 1-beta of p14/ARF, and promoter hypermethylation of both genes. No homozygous deletion of either p16/INK4 or p14/ARF was demonstrated in any of the PNET primary tumour samples. Methylation of p16/INK4 was found in one of six sPNET and in one of 23 MB, while p14/ARF methylation was observed in three of six sPNET and in three of 21 MB. No methylation of p16/INK4 or p14/ARF was found in any of the PNET cell lines analysed. The three MB cell lines did not show p16/INK4 expression, and only the MB Daoy cell line (homozygously deleted at CDKN2A) presented loss of p14/ARF expression. CONCLUSIONS: Our results in this limited series of central PNET show that p14/ARF is frequently involved in PNET carcinogenesis, with a higher frequency, but not statistically significant, for sPNET than for MB.     

肺癌组织p16和Rb基因mRNA表达的双重原位杂交观察

目的：研究Ｐ１６和Ｒｂ基因在ｍＲＮＡ表达及其与肺癌的关系。方法：制备ｐ１６和Ｒｂ基因ｃＤＮＡ探针,采用双重原位杂交的方法,地８９例肺癌和正常肺组织进行了Ｐ１６ 一Ｒｂ基因ｍＲＮＡ表达的定位研究。结果：小细胞肺癌ｐ１６ｍＲＮＡ阳性表达率高达８２．４％,而非小细胞肺癌阳性率为４５．８％,两者差异有显著性。非小细胞肺癌ＲｂｍＲＮＡ阳性表达率为８１．９％,而小细胞肺癌一率仅５．９％,两者差异有高度显著性,     

p16和Rb基因蛋白在肺癌中的表达

目的：研究ｐ１６和Ｒｂ基因蛋白的表达与肺癌临床病理学特征的关系。方法：采用免疫组织化学方法对８９例肺癌进行了ｐ１６和Ｒｂ蛋白的定位观察。结果：肺癌；ｐ１６基因蛋白的总丢失率为４７．２％，且与肺癌的组织学类型，淋巴结转移，临床病理分期有关。Ｒｂ基因蛋白的总丢失率为３１．５％，与组织学类型有关。     

外源性野生型p53基因在两个肺腺癌细胞系中表达及对p16和p21基因的调控

目的 探讨p53基因在肺癌细胞周期中的作用机制，以及裸鼠体内基因治疗的作用。方法 用以腺病毒为载体的野生型p53基因pAdCMV-p53(Ad-p53)感染高转移肺腺癌95D细胞系和高浸润肺腺癌L-18系，对感染前后各细胞系的细胞生长曲线、p53、p16和p21基因的表达以及调亡进行分析。此外，用Ad-p53对两细胞系进行了裸鼠体内感染实验。结果 体外实验中，导入p53基因细胞系的生长均得到抑制，并且最终都出现凋亡，感染后的细胞中p53和p21基因的mRNA表达量明显增高，p16基因的mRNA表达量则无明显变化。p53基因治疗后的裸鼠，其中95D细胞系的肿瘤全部消失；L-18细胞系的腹腔注射组肿瘤全部消失，而皮下注射组无明显变化。结论 p53基因是一个有效的肿瘤抑制基因，其诱导细胞凋亡的途径与p16基因不同。腺病毒介导的野生型p53基因可以有效地控制肺癌细胞的发展和转移。     

原发性胃癌中19p部分微卫星多态位点杂合性缺失分析

目的 筛选胃癌19p部分微卫星多态位点的杂合性缺失（loss of heterozygosite,LOH)频率,以初步确定19p上与胃癌相关基因连锁最密切的微卫星多态位点。方法 采用聚合酶链反应-单链长度多态（polymerase chain reaction-single strand length polymorophism,PCR-SSLP)-银染法选取19p上9对微卫星多态标记（D19S424,D19S216,D19S406,D19S413。D19S221,D19S226,D19S411,D19S883,D19S886）,对43例原发性胃癌的杂合性缺失情况进行了分析。结果 43例中22例至少在1个位点发生LOH,总缺失率为48.88%,这9个位点的LOH频率分别为29.63%,11.53%,33.33%,8.57%,13.15%,8.00%,6.45%,6.89%,10.71%,在D19S886也同时出现微卫星不稳定性（microsatellite instability,MSI)17.85%。结论 提示19p上的LOH缺失频发区域可能涉及与人类原发性胃癌发生发展相关基因的存在。