肺癌支气管活检组织及痰液中p53基因突变的检测及临床应用

采用聚合酶链反应－单链构象多态性分析（ＰＣＲ－ＳＳＣＰ）方法，从分子水平探讨了ｐ５３基因突变与肺癌发生之间的关系。对４３例肺癌支气管活检组织及１０例痰液标本进行了ｐ５３基因第５～８外显子突变的检测，发现４３例中有１７例发生了ｐ５３突变，突变率为３９．５％（１７／４３），其中鳞癌为３０．８％（４／１３），腺癌为３５．３％（６／１７），小细胞癌为５８．３％（７／１２），大细胞癌为０％（０／１）。本文对同一患者的痰液标本检测结果与组织标本中ｐ５３基因突变检出率一致，均为３０．０％（３／１０）。采用χ２检验表明，ｐ５３突变在性别构成、临床分期、组织类型及淋巴结转移等方面两组均无显著性差异（Ｐ＞０．０５）。但吸烟组ｐ５３突变率（５３．８％）明显高于非吸烟组（１７．６％）；低中分化组ｐ５３突变率（５１．７％）明显高于高分化组（１４．３％），提示ｐ５３基因突变与吸烟及细胞的分化程度存在相关性（Ｐ＜０．０５）。     

小细胞肺癌组织中p53基因改变的研究

为探讨小细胞肺癌（ＳＣＬＣ）发生的分子机制，应用免疫组化和聚合酶链反应—单链构象多态性分析的方法，对１４例ＳＣＬＣ组织进行ｐ５３基因突变研究。结果显示，有９例出现ｐ５３蛋白阳性，阳性率为６４．３％（９／１４）。ｐ５３基因第５，６，７，８外显子突变率分别为２１．４％（３／１４），１４．３％（２／１４），１４．３％（２／１４），７．１％（１／１４），总突变率为５７．１％（８／１４）。结果表明，ＳＣＬＣ组织中存在较高的ｐ５３基因突变率，故ｐ５３基因可能是人类肺癌发生的关键基因。     

大肠癌的p53基因点突变

作者以改良的ＰＣＲ－ＳＳＣＰ的ＰＣＲ直接测序方法，分析了２２例大肠癌原发灶和１例转移淋巴结组织中ｐ５３基因第七外显子的基因结构变化。发现２７％（６／２２）大肠癌原发灶和检测的１例转移淋巴结中存在ｐ５３基因第七外显子点突变，突变位点分别为２４５、２５１、２５９和２６０密码子。５０％点突变是２４５密码子中碱基Ｇ∶Ｃ向Ａ∶Ｔ转换，其它突变形式是碱基插入和碱基缺失。本研究ｐ５３点突变阳性标本均为结肠癌，其中低分化腺癌突变率高于高、中分化腺癌（Ｐ＝０．０１７８），ＤｕｋｅｓＣ１期突变率高于ＤｕｋｅｓＡ２、Ｂ期（Ｐ＝０．０３６１）。此结果提示，临床检测大肠癌原发灶和转移淋巴结中是否存在ｐ５３基因第七外显子突变，可有助于识别高度恶性的大肠癌和判断大肠癌病员的预后。     

大肠息肉与大肠癌p53及c—erbB—2蛋白表达

应用免疫组织化学ＳＰ法对１６例良性大肠息肉，９６例大肠癌组织中抑癌基因ｐ５３蛋白及原癌基因ｃｅｒｂＢ２蛋白表达进行研究。结果表明，幼年性息肉，增生性息肉均无ｐ５３蛋白表达，而ｃｅｒｂＢ２蛋白可以有一定程度的表达。ｐ５３、ｃｅｒｂＢ２在腺瘤中的表达阳性率分别为２０．０％（２／１０）及３０．０％（３／１０），其中ｐ５３阳性细胞呈散在或灶性分布，但阳性细胞数不超过５％。ｐ５３及ｃｅｒｂＢ２在腺癌中的表达阳性率分别为４３．８％（４２／９６）及５０．６％（４０／７９），其中同时表达两种蛋白者占２９．１％，且ｐ５３蛋白表达与肿瘤进展有关，ｐ５３阳性者多为Ｂ期或Ｃ、Ｄ期癌，更易于发生淋巴结转移。     

大肠癌的RFLP分析及其p53异质性变化的研究

应用检测肿瘤ＤＮＡ多态性的探针，对１５例大肠癌进行了ＬＯＨ分析。结果，探针ｐ１０－５（１７ｐ１３．１）在检出的８个信息个体中有５例发生了ＬＯＨ（占６２．５％）。而ｐＹＮＺ２２．１（１７ｐ１３．３），ｐ５３，ｐＭＳ１－３７（３ｐ１４．２）和ＤＲ７８（１ｐ２１－ｑｔｅｒ）探针的同源区并未检出ＬＯＨ。对１０例大肠癌还进行了ｐ５３ｍＲＮＡ的Ｎｏｒｔｈｅｒｎ印迹分析，发现全部被检测样品均有一条２．８ｋｂ的ｐ５３ｍＲＮＡ杂交带。１０例中６例ｐ５３ｍＲＮＡ表达水平高于配对的正常组织，３例则降低。本研究提示，ｐ５３基因突变存在明显的异质性，同时还说明１７ｐ的ＬＯＨ可能是大肠癌发生过程中的一个至关重要的分子事件。     

大肠癌患者及其家族成员外周血淋巴细胞p53基因突变研究

本文应用聚合酶链反应－单链构象多态（ＰＤＲ－ＳＳＣＰ）银染技术对２０例大肠癌患者和２０例大肠癌家族成员的外周血淋巴细胞ｐ５３基因第５，６，７和８外显子进行检测，发现有６例大肠癌患者和５例家族成员存在基因突变，突变率分别为３０％和２５％，这一结果表明ｐ５３基因突变在大肠癌中的普遍存在，并在大肠癌的发生过程中起着重要作用；而家族成员中发现ｐ５３基因突变将可为癌症的癌前监控和预防提供重要佐证。     

乳腺癌p53基因突变规律及其预后意义

目的 探讨ｐ５３基因突变在乳腺癌发生发展中的演变规律及其与预后的关系。方法 应用ＰＣＲ－单链构象多态性（ＰＣＲ－ＳＳＣＰ）分析方法检测５５例原发性乳腺癌患者ｐ５３基因５～８外显子的突变情况。结果 ｐ５３基因突变发生的频率为３６．４％（２０／５５）,Ｉ～Ⅱ期和Ⅲ～Ⅳ期乳腺癌中ｐ５３基因突变阳性率分别为４７．２％（１７／３６）和１５．８％（３／１９）,而且突变多发生在５～６外显子。突变阳性患者其无病生     

非小细胞肺癌中p53基因突变的初步研究

为了解非小细胞肺癌中ｐ５３基因突变的情况，应用改良聚合酶链反应－单链构象多态（ＰＣＲ－ＳＳＣＰ）银染技术检测１７例非小细胞肺癌中ｐ５３基因第５～８外显子的突变情况，有８例存在ｐ５３基因突变，突变率为４７．００％（８／１７），其中鳞癌为５５．５６％（５／９），腺癌为３７．５０％（３／８）。本研究结果表明：非小细胞肺癌中ｐ５３基因突变较为常见；改良后的ＰＣＲ－ＳＳＣＰ银染技术对非小细胞肺癌的基因诊断具有潜在性应用价值     

卵巢上皮性癌p53基因改变,蛋白过度表达及临床意义

目的：检测卵巢上皮性癌ｐ５３基因改变、蛋白过度表达，探讨其与临床病理和预后的关系。方法：应用ＡＢＣ免疫组化法检测５６例卵巢上皮性肿瘤ｐ５３蛋白表达，对其中４０例应用聚合酶链反应单链构象多态性（ＰＣＲＳＳＣＰ）溴乙锭染色法检测ｐ５３基因５～８外显子突变及杂合性缺失（ＬＯＨ）。结果：（１）ｐ５３蛋白表达阳性率：良性肿瘤０／１０；交界性肿瘤１／４；卵巢上皮性癌２２／４２（５２．３８％），浆液性与粘液性癌表达相似，且３例原发灶与转移灶表达一致。（２）ｐ５３基因突变率：良性肿瘤０／１０；交界性肿瘤１／３；卵巢上皮性癌１３／２７（４８．１５％），其中８例杂合型基因的病例中４例基因突变伴等位基因杂合性缺失。本研究１３例ｐ５３基因突变者中１２例ｐ５３蛋白过度表达。ｐ５３蛋白过度表达与卵巢上皮性癌组织学类型、临床分期及预后无关（Ｐ＞０．０５）。而与卵巢上皮性癌病理有关（Ｐ＜０００５）。结论：ｐ５３基因改变及蛋白过度表达在卵巢癌中较常发生，且与卵巢上皮性癌病理分级有关，这些改变可能是卵巢上皮性癌的早发事件，亦在卵巢上皮性癌的发生发展过程中起重要作用。     

聚合酶链式反应-单链构象多态性分析法检测大肠腺瘤及大肠癌APC基因突变

目的对１５例散发性大肠腺瘤组织和４５例大肠癌组织及其各自的正常肠粘膜组织，进行家族性腺瘤息肉病基因（ａｄｅｎｏｍａｔｏｕｓｐｏｌｙｐｏｓｉｓｃｏｌｉ，ＡＰＣ）第１５外显子ＭＣＲ区域的基因突变检测。方法应用聚合酶链式反应单链构象多态性分析（ＰＣＲＳＳＣＰ）技术进行检测。结果在散发性大肠腺瘤组织中检出突变率为２０．０％（３／１５），在大肠癌组织中的突变率为２２．２％（１０／４５）。实验结果表明，ＡＰＣ基因在散发性大肠腺瘤与大肠癌中均有较高突变频率，两者差异无显著性；ＡＰＣ基因突变在大肠癌中与肿瘤的大小、位置、组织分化程度和有无淋巴结转移无关；ＡＰＣ基因第１５外显子的ＭＣＲ区域是该基因突变的集中区域，且以１３３９～１４３６密码子区域突变率最高，１２６０～１３５９密码子区域突变率最低。结论证实了ＡＰＣ基因突变发生于腺瘤形成阶段，属于大肠癌发生的早期事件。     

大肠癌 p53 抑癌基因突变与临床病理特点的关系

目的 :探讨p53基因突变与大肠癌临床病理特点的关系。方法 :应用PCR SSCP技术检测 135例大肠癌患者p53基因第 5、6、7、8外显子的突变。结果 :153例大肠癌患者p53基因的突变率为54 0 7% ,p53基因突变与大肠癌的病理分级、临床分期、淋巴结转移及年龄均有关。结论 :p53基因与大肠癌的发生、发展及转归均有明显的关系。检测p53基因突变既可作为大肠癌的诊断方法 ,也可作为大肠癌预后的判定指标。     

KRAS, BRAF and PIK3CA mutations in human colorectal cancer: relationship with metastatic colorectal cancer

Many abnormal gene expressions and dysregulated signaling pathways have been found in human colorectal cancer. Activating mutations of the KRAS, BRAF or PIK3CA oncogenes are frequently found in colorectal cancer. The aim of the study was to investigate the molecular occurrence of KRAS, BRAF and PIK3CA mutations in the colorectal tumorigenesis and to study the association of these events with clinicopathological parameters. In our study, DNA was extracted from 200 cases of human colorectal cancer tissue samples. KRAS, BRAF and PIK3CA mutation analysis was performed by PCR and pyrosequencing. Using statistical methods, we analyzed the relationships between the gene mutations and clinicopathological parameters. KRAS point mutations were detected in 63/200 patients (31.5%), with codon 12 mutations in 52/200 patients (26%), codon 13 mutations in 10/200 patients (5%) and codon 12.13 bi-mutations in 1/200 patients (0.5%). The V600E mutations of BRAF were detected in 14/200 patients (7%). PIK3CA point mutations (exon 9, exon 20) were detected in 25/200 (12.5%) patients, exon 9 mutatons in 12/200 patients (6%) and exon 20 mutations in 13/200 (6.5%). Our study suggested that both KRAS and BRAF mutations are exclusive, but KRAS and PIK3CA mutations are coexistent. The mutational status of BRAF did not correlate with Dukes' staging, histological type, age and gender. However, strong associations were found between KRAS, PIK3CA mutations and Dukes' staging (staging D, 12/25, 48%). Notably, our data indicated that colorectal cancers with KRAS and PIK3CA bi-mutations are more likely to develop into liver metastasis.     

p53 and Ki-ras as prognostic factors for Dukes' stage B colorectal cancer

Mutations of the TP53 and Ki-ras genes have been reported to be of prognostic importance in colorectal carcinomas. An increased intracellular concentration of the p53 protein, although not identical to, is sometimes seen in tumours with TP53 mutation and has been correlated with poor prognosis in some tumour types. Previous colorectal cancer studies, addressing the prognostic importance of Ki-ras mutation and TP53 aberrations, yielded contradictory results. The aim of this study was to determine in a clinically and therapeutically homogeneous group of 122 sporadic Dukes' B colorectal carcinomas with a median follow-up of 67 months (3-144 months) whether or not p53 protein expression, TP53 mutation and K-ras mutation correlated with prognosis. p53 staining was performed by immunohistochemistry, using the monoclonal antibody DO7 on paraffin-embedded tissue. Mutations in exons 5-8 of the TP53 gene and in codons 12 and 13 of the K-ras gene were assayed in paraffin-embedded tissue by the single-strand conformation polymorphism (SSCP) assay. Nuclear p53 staining was found in 57 (47%) tumours. Aberrant migration patterns indicating mutation of the TP53 gene were found in 39 (32%) tumours. Forty-six carcinomas (38%) showed a mutation of the Ki-ras codons 12 or 13. In a univariate analysis, patients with wild-type TP53 status showed a trend towards better survival, compared with those with mutated TP53 (log-rank test, P = 0.051). Likewise, tumours immunohistochemically positive for p53 showed a worse prognosis than p53-negative tumours (P = 0.010). The presence or absence of mutations in Ki-ras did not correlate with prognosis (P = 0.703). In multivariate analysis, only p53 immunoreactivity emerged as an independent marker for prognosis hazard ratio (HR) = 2.16, 95% confidence interval (CI) 1.12-4.11, P = 0.02). Assessment of p53 protein expression is more discriminative than TP53 mutation to predict the outcome of Dukes' stage B tumours and could be a useful tool to identify patients who might benefit from adjuvant therapy.     

p53 mutation found to be a significant prognostic indicator in distal colorectal cancer

There has been few reports describing that the prognostic significance of p53 alteration in colorectal tumors depended on the site of origin. Therefore, in this study, we examined whether there is a valid association between the p53 alterations and the prognosis of colorectal cancer patients, especially distal colorectal cancer cases. Tumor samples were collected from 110 patients resected for colorectal cancer between 1989 and 1997. The entire coding region of the p53 gene was analyzed by automated direct sequencing. In addition, the DO-7 monoclonal antibody was used in the immunohistochemical (IHC) assessments. By the Cox univariate analyses in all tumors, Dukes' stage, lymph node metastasis, liver metastasis, p53 mutation and p53 protein overexpression were significant predictors of survival. The cases without p53 mutation showed significantly improved prognosis compared to the cases with p53 mutation (p = 0.0085). In distal tumors, Dukes' stage, liver metastasis, p53 mutation and p53 protein overexpression were significant predictors of survival. Multivariate analysis of all tumors, p53 mutation and liver metastasis were independent indicators of poor survival (p = 0.0223 for p53 mutation, p = 0.0254 for liver metastasis). Also in distal tumors, p53 mutation was an independent indicator (p = 0.0011). Furthermore, the relative risk associated with p53 mutation in distal tumors was much higher than that in all tumors (8.260 vs. 1.796). However, p53 protein overexpression was not an independent indicator of survival in all tumors as well as distal tumors (p = 0.1918 in all tumors, p = 0.0607 in distal tumors). In proximal tumors, p53 mutation was not an independent indicator (p = 0.6673). We think that p53 mutation is a very useful prognostic indicator when distal colorectal cancers are considered.     

THE SIGNIFICANCE OF P53 GENE MUTATIONS AND EXPRESSIONS IN HUMAN COLORECTAL TUMORS

ＴＨＥＳＩＧＮＩＦＩＣＡＮＣＥＯＦＰ５３ＧＥＮＥＭＵＴＡＴＩＯＮＳＡＮＤＥＸＰＲＥＳＳＩＯＮＳＩＮＨＵＭＡＮＣＯＬＯＲＥＣＴＡＬＴＵＭＯＲＳＱｉａｎＨｕａ钱桦；ＹｕＢａｏｍｉｎｇ郁宝铭；ＺｈｏｕＸｉｇｅｎｇ周锡庚；ＷａｎｇＲｕｉｎｉａｎ王瑞年；Ｈｕａ...     

Dysfunction of p53 pathway in human colorectal cancer: analysis of p53 gene mutation and the expression of the p53-associated factors p14ARF, p33ING1, p21WAF1 and MDM2

Mutations of p53 tumor suppressor gene increase with tumor progression in colorectal cancers. In this study, we examined the expressions of p33ING1, p14ARF, MDM2 and p21WAF1 mRNA in 25 advanced colorectal cancers by quantitative RT-PCR method, and compared the expression levels of p33ING1, p14ARF, p21WAF1 and MDM2 in relation to p53 status in the tumors. Fifteen of 25 colorectal cancers (60%) showed abnormal accumulation of p53 protein in the nucleus, and the remaining 10 colorectal cancers (40%) were negative for p53 immunostaining. We found a G --> T transition (nonsense mutation) at the first nucleotide of codon 298 (exon 8) in one p53-negative case, and a frame shift mutation on exon 7 in another p53-negative case. In remaining eight p53-negative cases, there was no mutation in the entire open reading frame of p53 cDNA. Interestingly, in eight cases with p53 wild-type gene, 6 cases (75%) showed a marked down-regulation of p14ARF mRNA, and three cases (37.5%) over-expressed MDM2 mRNA. Only one case with wild-type p53 gene showed normal level expression of p53 regulatory-factors (p33ING1, p14ARF, and MDM2). Thus, p53 tumor suppressor pathway was disrupted in 24 of 25 colorectal cancers (96%).     

Fighting colorectal cancer: molecular epidemiology differences among Ashkenazi and Sephardic Jews and Palestinians.

BACKGROUND: To evaluate and compare differences in the molecular genetics among high-risk (Ashkenazi Jews), intermediate-risk (Sephardic Jews) and low-risk (Palestinians) groups for colorectal cancer who live in the same geographical region. PATIENTS AND METHODS: The 1995-1996 records from the Tel Aviv Medical Center and Muqased hospital (East Jerusalem) randomly identified patients with colorectal cancer. There were 25 patients from each ethnic group. Epidemiological data were obtained from interviews with the patients and from their hospital charts. The levels of cyclin D1, beta-catenine, p27, p53, Ki-67 and Her-2/neu proteins were determined by immunohistochemistry. The main outcome measures were the association between gene expression and colorectal incidence in the different ethnic groups. RESULTS: Ashkenazi Jews have the highest rate of colorectal cancer, and are diagnosed at an early stage compared with Palestinians (72% and 33% of the cases are in Dukes' A and B, respectively), and, hence, this may explain the better 5-year survival rate among this group. Sephardic Jews are diagnosed at a more advanced stage, the tumors are poorly differentiated and they lack p27. Palestinians have significantly higher cyclin D1 levels. There was a statistically significant inverse correlation between the expression of beta-catenine and cyclin D1, as well as p53 and p27 (P <0.05). CONCLUSIONS: Increased expression of cyclin D1, p53, Ki-67, beta-catenine and Her-2/neu, and decreased expression of p27 may be important events in the three ethnic groups with colorectal cancer. The lower mortality rate among Ashkenazi Jews may be partially explained by their better molecular biology profile.     

大肠肿瘤组织中人乳头状瘤病毒感染与P53基因表达的研究

目的探讨HPV16和HPV18感染及P53基因异常表达与大肠肿瘤发生间的关系。方法采用地高辛标记的HPV16和HPV18DNA深外分别在40例大肠癌和30例大肠腺瘤组织石蜡切片上进行原位杂交探测HPVDNA。同时,采用SABC法检测大肠癌和大肠腺瘤的P53表达水平。结果受检大肠腺瘤组织HPVDNA阳性8例（27％）,其中HPV16DNA5例,HPV18DNA3例,主要见于管状绒毛状腺癌和绒毛状腺瘤;大肠腺癌组织中HPVDNA阳性19例（4％）,其中HPV16DNA14例,HPV18DNA5例。HPVDNA主要见于肿瘤细胞核中,少部分见于胞浆中。大肠癌组织中HPV16．18DNA检出阳性率明显高于大肠腺癌,且腺痛中HPV16DNA阳性率明显高于其他类型腺瘤。大肠癌P53蛋白阳性率为48％;腺癌的阳性率则为16．2％。结论HPV16．18型感染并整合至宿主细胞DNA中可能导致P53基因突变与大肠腺癌发生有密切关系。