p53基因点突变与胃粘膜细胞癌变的关系

目的研究ｐ５３基因在胃粘膜细胞癌变过程中的变异方式和类型。方法选取５６例胃镜活检胃粘膜病变组织标本（胃癌２３例,异型增生１６例,肠上皮化生１７例）,应用单链构象多态性（ＳＳ－ＣＰ）和ＤＮＡ序列分析等技术,对ｐ５３基因点突变的方式和类型进行检测和分析。结果ｐ５３基因在胃癌、异型增生和肠上皮化生组织中点突变频率较高,分别为６０．１％、３１．３％和１１．８％,点突变分布在第５～８外显子,无突变热点。错义突变（７３．９％）和移码突变占全部突变的８６．９％。结论ｐ５３基因点突变是胃粘膜细胞癌变过程中多因素作用的结果,错义和移码突变可能是其失活的主要原因,ｐ５３基因点突变频率在肠型胃癌发展进程中呈渐进趋势,进一步明确了ｐ５３基因变异是胃粘膜细胞癌变过程中的一个重要因素。     

p53基因突变及染色体倍性与大肠癌转移之间关系初探

目的研究大肠癌转移与ｐ５３基因突变及染色体倍性之间的关系。方法采用特异合成引物对ｐ５３基因的有关外显子进行ＰＣＲ扩增，结合单链构象多态性（ＳＳＣＰ）和银染技术，检测１５例大肠癌手术标本ｐ５３基因的突变；使用流式细胞仪（ＦＡＣＳ）分析其染色体的倍性。结果８例有转移灶大肠癌病人中７例表现有ｐ５３基因的突变，而７例未转移大肠癌病人只显示３例有ｐ５３基因的突变。异倍体的９例病人中７例有ｐ５３基因突变，二倍体的６例病人中有３例有ｐ５３基因突变。结论大肠癌转移与ｐ５３基因突变细胞染色体异倍性三者相互存在着密切联系     

p53基因突变与人非小细胞肺癌临床病理生理分析

目的 探讨ｐ５３基因点突变与人非小细胞肺癌临床生理特征的联系。方法 应用聚合酶反应－单链构象多态性分析方法（ＰＣＲ－ＳＳＣＰ０检测原发性非小细胞肺癌癌组织的ｐ５３基因第５～８外显子点突变。结果 ４０例肺癌组织中１９例（４７．５％）有点突变发生,８例良性肿瘤组织均无ｐ５３基因点突变发生,点突变发生与病理分期和淋巴结转移有明显关系（Ｐ〈０．０２５）,结论ｐ基因估变在非小细胞肺癌的发生和进展中可能起重要作用。     

非小细胞肺癌p53基因突变检测及临床病理分析

目的　探讨ｐ５３基因点突变在非小细胞肺癌中的发生情况及临床病理联系。方法　运用聚合酶链反应（ＰＣＲ）－单链构象多态性分析方法（ＳＳＣＰ）检测原发性非小细胞肺癌的ｐ５３ 基因第５－８外显子点突变。结果　８例良性肿瘤均无点突变发生,４０ 例肺癌中１９例（４７．５％ ）有点突变发生,点突变发生与临床分期和淋巴结累及程度明显相关（Ｐ＜ ０．０２）。结论　ｐ５３基因点突变在非小细胞肺癌的发生和进展中起着相当重要的作用。     

肝细胞癌肿瘤抑制基因p53过度表达及点突变的研究

应用一种敏感的ＡＲＦ免疫组经和ＰＣＲ、银染ＰＣＲ－ＳＳＣＰ方法检测了本地区３８例肝细胞癌组织中肿瘤抑制基因ｐ５３的过度表达及点突变，结果发现１６例有ｐ５３蛋白过度表达（４１．２％），７例有ｐ５３基因２４９位密码子点突变，２例２４９位密码子外第７外显子点突变。９例ｐ５３基因有突变的肝癌中８例ｐ５３蛋白阳性，两者符合率为８８．９％，ｐ５３基因蛋白过度表达和点突点与ＨＣＣ分化和转移有关。本组ＨＣＣ ｐ５     

溶瘤腺病毒ＳＧ６００-ｐ５３对裸鼠胃癌移植瘤的抑制作用

目的：利用我们前期构建的溶瘤腺病毒ＳＧ６００技术平台,研究其携带野生型ｐ５３基因对裸鼠胃癌移植瘤模型抗肿瘤活性。方法：克隆ｐ５３基因全长ｃＤＮＡ,插入到溶瘤腺病毒ＳＧ６００基因组中,同时建立裸鼠胃癌移植瘤模型,体内观察ＳＧ６００-ｐ５３对胃癌移植瘤模型的抗肿瘤疗效。结果：成功构建溶瘤腺病毒ＳＧ６００-ｐ５３,裸鼠胃癌移植瘤的ｐ５３基因获增强表达。在胃癌移植瘤裸鼠模型中,经４周治疗,重组ＳＧ６００-ｐ５３抑瘤率达 ７５.５９％,优于Ａｄ-ｐ５３（４５.００％）和ＳＧ６００（５６.９５％）。结论：ＳＧ６００-ｐ５３作为一种有效的抗肿瘤新型制剂,对胃癌移植瘤有很好的特异性抗肿瘤活性,可能具有良好的临床应用前景。     

肺癌患者癌组织和痰液细胞中p53和K-ras基因突变的研究

目的 检测肺癌组织和肺癌患者痰液脱落细胞中ｐ５３、Ｋ－ｒａｓ基因突变情况,比较联合检测ｐ５３、Ｋ－ｒａｓ和单一检测ｐ５３或Ｋ－ｒａｓ基因在肺癌诊断中的价值。方法 应用ＰＣＲ－ＳＳＣＰ－银染法检测了５９例肺癌组织、癌旁肺组织、１４全肺部良性病变肺组织及患者痰液脱落细胞中ｐ５３基因第５～８外显子、Ｋ－ｒａｓ基因第１外显子突变。结果 肺癌组织中ｐ５３基因突变率为３７．３％（２２／５９）,痰液脱落细胞为３     

非小细胞肺癌患者ＸＲＣＣ１基因多态性对ｐ５３基因突变发生的影响

目的：观察非小细胞肺癌（ＮＳＣＬＣ）患者ＸＲＣＣ１基因３９９位点多态性以及吸烟对ｐ５３基因突变发生的影响。方法：运用不对称ＰＣＲ技术扩增ｐ５３基因的第５～８外显子,用聚合酶链反应 单链构象多态性分析法（ＰＣＲ-ＳＳＣＰ）检测ｐ５３基因突变,并对有差异的结果测序验证;运用ＰＣＲ技术扩增ＸＲＣＣ１基因的第１０外显子,从正反两个方向对扩增片段进行ＤＮＡ测序分析其多态性;同时了解吸烟与它们的关系。结果：３８例可手术的ＮＳＣＬＣ患者中发现１６例有ｐ５３基因的突变,发生突变的１６例中６例至少携带１个ＸＲＣＣ１-３９９Ｇｌｎ等位基因,１０例为野生基因型ＸＲＣＣ１-３９９Ａｒｇ／Ａｒｇ,用二项分类Ｌｏｇｉｓｔｉｃ回归模型分析ＸＲＣＣ１-３９９Ｇｌｎ多态性与ｐ５３基因突变两者的关系,优势比（ＯＲ）为１.５３６（９５％ＣＩ：０.３７６～６.２８０）;该模型中吸烟与ｐ５３基因突变的ＯＲ为１.５２４（９５％ＣＩ：０.２５０～９.２９５）。结论：ＮＳＣＬＣ患者中,ＸＲＣＣ１基因３９９位点多态性和吸烟情况对ｐ５３基因突变发生无明显影响。     

外源p53对人肺癌细胞生长的抑制

目的 观察外源性野外型ｐ５３对有ｐ５３基因突变的人肺癌细胞系生长的影响。方法 用ＰＣＲ－ＳＳＣＰ及ＤＮＡ测序，选择ｐ５３突变的人肺巨细胞癌系８０１－Ｄ。构建野生型ｐ５３表达质粒ＰＺｉＰ－ｐ５３。用基因枪介导外源基因。经Ｇ４１８筛选得到转染细胞系８０１－Ｄ－ｐ５３。用ＰＣＲ检测外源基因，观察转染细胞恶性生长的变化。结果 转染细胞系８０１－Ｄ－ｐ５３体外长期传代有外源性ｐ５３基因存在，转染细胞生长明显     

p53基因缺失与非小细胞肺癌的相关研究

为探讨ｐ５３基因缺失在非小细胞肺癌（ＮＳＣＬＣ）发病机制中的作用，应用Ｓｏｕｔｈｅｒｎ印迹杂交技术检测１７例ＮＳＣＬＣｐ５３基因缺失情况，发现７例（４１．２％）存在ｐ５３基因缺失，发生淋巴结扩散的ＮＳＣＬＣ，其ｐ５３基因缺失率较高。结果提示ｐ５３基因的缺失与ＮＳＣＬＣ的发生、发展相关。     

Analysis of p53 mutations in cells taken from paraffin-embedded tissue sections of ductal carcinoma in situ and atypical ductal hyperplasia of the breast

Mutations in the p53 tumor suppressor gene are frequent in breast tumors but the implication of p53 mutations in breast cancer development remains poorly understood. In this study, we applied laser capture microdissection (LCM) microscope to histologically review and sample cells from paraffin-embedded breast tissue sections obtained from six cases of ductal carcinoma in situ (DCIS) and ten cases of atypical ductal hyperplasia (ADH). p53 mutations were detected, using single stranded conformational polymorphism (SSCP) and sequencing, in cell samples of three cases with DCIS and five cases with ADH. p53 mutations are therefore present in DCIS and ADH of the breast, considered as pre-malignant precursors to breast cancer, and some of them may represent early events in breast cancer development.     

Predictive value of Ki-67, p53 protein, and DNA content in the diagnosis of gastric carcinoma.

BACKGROUND: The ability to make a precise preoperative diagnosis is a valuable and effective method in improving the prognosis of patients with gastric carcinoma. The authors examined retrospectively whether preoperative histopathologic analysis with p53 protein, Ki-67 labeling index, and DNA ploidy along with preoperative radiographic and endoscopic findings led to a precise preoperative diagnosis of patients with gastric carcinoma. METHODS: Histopathologic analysis of p53 protein, Ki-67 labeling index, and DNA content was performed on formalin fixed, paraffin embedded tissue. Tissue sections from endoscopic and surgically resected specimens were stained immunohistochemically for p53 protein and Ki-67 labeling index, and the cell nuclear DNA content of the surgically resected primary lesion was measured using a microspectrophotometer. These analyses were performed on 16 patients with early gastric carcinoma (EGC) who were diagnosed with advanced gastric carcinoma (AGC) based on the preoperative imaging findings and on 15 patients with AGC who were diagnosed preoperatively with EGC. RESULTS: Overexpression of p53 in the AGC group was significantly more frequent compared with that in the EGC group (P = 0.0386). With regard to the correlation between lymph node metastases and p53 overexpression, there was no apparent relation in either the AGC group (P = 0.648) or the EGC group (P = 0.726). The AGC group had significantly higher Ki-67 labeling indices compared with the EGC group (P = 0.0195). There was complete concordance between endoscopic and surgically resected specimens with regard to the p53 and Ki-67 labeling index findings. DNA ploidy in the primary tumor did not differ between the AGC and EGC groups. The survival rates for the EGC group were significantly superior to those for the AGC group (P = 0.0312). CONCLUSIONS: The findings of the current study suggest that in routine clinical practice, the combination of preoperative imaging findings in addition to Ki-67 labeling indexes, and p53 protein analyses may be useful for the accurate diagnosis of EGC; however, DNA ploidy did not appear to reflect the growth potential of gastric carcinoma.     

应用PCR-SSCP银染技术分析胃癌p53基因的点突变

目的 分析胃癌中p53基因第5－8外显子点突变的发生及其意义。方法 应用聚合酶链反应－单链构象多态性分析（PCR－SSCP）和银染技术，对40例胃良性疾病和30例胃癌标本中p53基因第5－8外显子的点突变情况进行检测。结果 40例胃良性疾病均无点突变发生，30例胃癌中有12例发生p53基因的点突变，突变率为40．00％（12／30）。结论 p53基因突变与胃癌的发生密切相关；PCR－SSCP银染技术不仅灵敏安全，而且简便、快速、重复性好，可用于临床的基因诊断。     

Relationship between E-cadherin gene mutation and p53 gene mutation,p53 accumulation,Bcl-2 expression and Ki-67 staining in diffuse-type gastric carcinoma

E-cadherin mutations are found in 50% of diffuse-type gastric carcinoma, but not in intestinal gastric carcinoma. Because cell-cell adhesion mediated by E-cadherin plays an important role in epithelial cell survival, E-cadherin mutations could alter the apoptotic behavior of tumor cells. p53 and Bcl-2 family members are also important regulators of cellular apoptosis. This is the first study that investigates the relationship between E-cadherin gene mutation and p53 gene mutation, p53 accumulation, Bcl-2 expression, and Ki-67 expression in diffuse-type gastric carcinoma (24 cases, E-cadherin mutation status: wild-type in 8 patients and mutant in 16 patients). The mutation status of exons 5-8 of p53 was analyzed by denaturing high pressure liquid chromatography (DHPLC) in formalin-fixed, paraffin-embedded tumor sections, followed by direct sequencing of cases with aberrant chromatographic patterns. p53 mutations were found in 1 of 8 tumors without E-cadherin mutation (12.5%) and in 1 of 16 tumors with E-cadherin mutation (6.3%), a difference that was not statistically significant (p = 1.00). p53 accumulation was found in 8 of 24 tumors (33.3%) by immunohistochemical staining. p53 accumulation was significantly more frequent in tumors without E-cadherin mutations (5 of 8 tumors, 62.5%) than in gastric carcinoma tissues with E-cadherin mutations (3 of 16 tumors, 18.8%, p = 0.03). Bcl-2 staining was not observed in gastric carcinoma cells without E-cadherin mutations, but was detectable in 5 of 16 tumors with E-cadherin mutations (31.3%), a difference that was not statistically significant (p = 0.13). No relationship was observed between Ki-67 staining and the E-cadherin mutation status (p = 1.00). These data suggest that the presence of E-cadherin mutations can significantly alter the accumulation of the apoptosis-regulating p53 protein, whereas no correlation with the p53 mutation status or with Ki-67 staining was observed.     

The detection of p53 gene mutation using a microdissection technique in primary intracranial germ cell tumors

Using a microdissection technique, the contribution of the p53 mutation to tumorigenesis and prognosis in each histological subtype of the intracranial germ cell tumors (GCTs) was evaluated. Nineteen patients had primary intracranial GCTs, including 4 germinomas (GEs), 4 teratomas (TEs), 1 mixed tumor of GE and TE, and 10 mixed GCTs containing non-germinomatous malignant germ cell tumors (NG-MGCTs). After microdissection of specific subtypes, genomic DNA was screened for mutations in exons 5-8 of the p53 gene, using the dideoxyfingerprinting (ddF) followed by direct DNA sequencing. The direct sequencing revealed a total of six mutations in PCR products derived from the five cases (26%) which showed mobility shifts in ddF. Among the six mutations detected, four were missense mutations and two were silent. Missense mutations of the p53 gene tended to occur more frequently in the NG-MGCT component than in the GE or TE components (3/15 vs. 1/12 vs. 0/13). The incidence of missense mutations was not different between the survivors (3/13) and the deceased (1/6). This study suggests the possible role of the p53 gene in the tumori-genesis of NG-MGCT. However, p53 gene mutation did not correlate with the prognosis of NG-MGCT.     

Genetic mutual relationship between PTEN and p53 in gastric cancer

Both PTEN (encoding phosphate and tensin homologue) and p53 are known as cancer suppressor genes, and they are assumed that their gene mutations and loss of heterozygosity (LOH) occur frequently in various types of carcinoma. In the present study, we investigated both the p53 mutation and LOH of PTEN in 113 gastric cancer patients. We observed the LOH of PTEN in 11.1% of the patients with normal p53s and 46.2% of the patients with p53 gene mutations. The result that LOH of PTEN was frequently observed in the cases with p53 gene mutations and other data in this study suggested that both PTEN and p53 have complimentary roles in gastric carcinoma development.     

Carcinogenesis of gastric endocrine cell carcinoma: analysis of histopathology and <Emphasis Type="Italic">p53</Emphasis> gene alteration

Background Endocrine cell carcinoma of the stomach is characterized by endocrine differentiation and aggressive biological behavior, and is frequently accompanied by an adenocarcinoma component. Because the carcinogenic pathway and genetic alterations remain unclear, we investigated the histogenesis of this tumor by histopathological and p53 gene analysis.Methods The materials were 68 gastric endocrine cell carcinomas and 30 carcinoid tumors, which were resected from 93 Japanese patients for histopathological and immunohistochemical investigation. We also analyzed the concordance of p53 mutational status between the associated adenocarcinoma and endocrine cell carcinoma components, using microdissection and direct sequencing techniques.Results An adenocarcinoma component was associated with 70.6% (48/68) of endocrine cell carcinomas, of which 42 (87.5%) were of well- to moderately differentiated type, while 36 of these 42 (85.7%) demonstrated histological continuity with the endocrine cell carcinoma components. Overexpression of p53 protein was observed in 58.8% (20/34) of cases. Common p53 mutational status between the two components was revealed in 73.3% (11/15) of cases analyzed. In contrast, carcinoid tumors did not exhibit p53 protein overexpression (0/15) or gene mutation (0/5).Conclusions These data suggest that gastric endocrine cell carcinomas predominantly arise from endocrine precursor cell clones occurring in preceding adenocarcinoma components (particularly the differentiated type), transforming into endocrine cell carcinoma during rapid clonal expansion under the influence of p53 gene alteration.     

High-throughput mutation profiling identifies novel molecular dysregulation in high-grade intraepithelial neoplasia and early gastric cancers

Background

There is still no widely accepted molecular marker available to distinguish between gastric high-grade intraepithelial neoplasia (HG-IEN) and invasive early gastric cancer (EGC).

Methods

HG-IEN and EGC lesions coexisting in the same patient were manually microdissected from a series of 15 gastrectomies for EGC; 40 ng DNA was used for multiplex PCR amplification using the Ion AmpliSeq Cancer Panel, which explores the mutational status of hotspot regions in 50 cancer-associated genes.

Results

Of the 15 EGCs, 12 presented at least one somatic mutation among the 50 investigated genes, and 6 of these showed multiple driver gene somatic mutations. TP53 mutations were observed in 9 cases; APC mutations were identified in 3 cases; and ATM and STK11 were mutated in 2 cases. Seven HG-IEN lesions shared an identical mutational profile with the EGC from the same patient; 13 mutations observed in APC, ATM, FGFR3, PIK3CA, RB1, STK11, and TP53 genes were shared by both HG-IEN and ECG lesions. CDKN2A, IDH2, MET, and RET mutations were observed only in EGC. TP53 deregulation was further investigated in an independent series of 75 biopsies corresponding to all the phenotypic lesions occurring in the EGC carcinogenetic cascade. p53 nuclear immunoreaction progressively increased along with the dedifferentiation of the lesions (P < 0.001). Overall, 18 of 20 p53-positive lesions showed a TP53 mutated gene.

Discussion

Our results support the molecular similarity between HG-IEN and EGC and suggest a relevant role for TP53 in the progression to the invasive phenotype and the use of immunohistochemistry as a surrogate to detect TP53 gene mutations.     

p53 mutation profiling of multiple esophageal carcinoma using laser capture microdissection to demonstrate field carcinogenesis

Inactivation of the p53 tumor suppressor gene is one of the most frequent genetic alterations observed in human esophageal carcinomas. In patients with esophageal carcinoma, one of the significant pathological features of the tumor is the presence of multiple lesions within the esophagus. However, the molecular mechanisms involved in the occurrence of multiple lesions have remained elusive. To characterize p53 alterations in multiple esophageal carcinomas and to study their roles in carcinogenesis, we performed p53 immunohistochemical and p53 mutation analyses using laser capture microdissection on surgically resected human esophageal carcinomas from 11 patients: 9 patients with multiple esophageal carcinomas, 1 with an intramural metastasis lesion within the esophagus and 1 with an intraepithelial carcinoma lesion contiguous to the main lesion. In each of the patients with multiple esophageal carcinomas, we examined samples from 1 main lesion and 1 representative concomitant lesion. Molecular analyses of samples from fresh-frozen normal tissues and tumor tissues of the main lesion (whole tumor) were also performed by the same method. p53 protein accumulation was observed in 16 (72.7%) of 22 lesions from the 11 cases. No p53 mutation was found in normal esophageal tissues. In the 9 cases of multiple esophageal carcinomas, point mutations were detected in the whole tumor in 1 (11.1%) case, in the microdissected tumor samples of main lesions in 3 (33.3%) cases and in the microdissected tumor samples of concomitant lesions in 3 (33.3%) cases. For the microdissected tumor samples, there was a 54.5% (12/22) concordance rate between the results of immunostaining and molecular analysis. In the 8 cases of whole tumors in which a p53 mutation was not observed, 2 cases revealed p53 mutation in the microdissected tumor samples of the main lesion. All 6 cases of multiple esophageal carcinomas that showed a p53 mutation in the microdissected tumor sample had a discordant p53 mutational status between the main and concomitant lesions. In contrast, both the intramural metastasis lesion and the intraepithelial carcinoma contiguous to the main lesion showed p53 mutational patterns identical to those of the main lesions. In conclusion, the analysis of microdissected DNA by laser capture microdissection is useful for characterizing the heterogeneity of the p53 gene mutation in multiple carcinoma lesions that cannot be accurately analyzed in whole esophageal tumor DNA. The finding of different p53 gene mutations among multiple esophageal carcinoma lesions suggest further evidence of multicentric or field carcinogenesis of the human esophagus.     

Occurrence of p53 gene abnormalities in gastric carcinoma tumors and cell lines.

We explored the state of the p53 gene in gastric cancer. Using one or more methods, we examined 15 specimens from primary carcinomas (14 tumors, one cell line), five cell lines derived from metastases, and seven paired samples of nonmalignant gastric mucosa. Sequence analyses of complementary DNA containing the entire p53 gene open reading frame demonstrated abnormalities in one of five samples from primary tumors and in all five samples from metastases. The single cell line derived from a primary carcinoma had no abnormality of the gene. The six abnormalities included four point mutations, one base-pair deletion resulting in a frame shift, and a 24 base-pair deletion caused by an intronic point mutation (as determined by sequence analysis of genomic DNA). Four of the six mutations mapped to regions highly conserved among species or involved in simian virus 40 T-antigen binding. Restriction fragment length polymorphism studies confirmed that chromosome 17p allelic deletions occur only in a minority of primary tumors, but that they may occur more frequently in metastases. Northern blotting and ribonuclease protection assays detected only a fraction of the p53 gene abnormalities detected by sequencing. Our findings indicate that mutations of the p53 gene are relatively rare in primary gastric tumors but appear to be relatively frequent in cell lines derived from metastatic lesions. Our results may help in understanding the molecular events associated with progression and metastasis in gastric carcinoma.