生长抑素受体亚型SSTR-2和SSTR-3mRNA在原发性肝癌中的表达

目的：了解原发性肝癌组织中生长抑素受体（somatostatin receptor,SSTR）亚型SSTR-2和SSTR-3基因的表达情况。方法：采用逆转录-PCR法检测27例原发性肝癌患者组织和癌旁组织中生长抑素受体亚型SSTR-2和SSTR-3 mRNA的表达。结果：27例原发性肝癌患者的癌组织和癌旁组织SSTR-2 mRNA的表达阳性率分别为81.5%(22/27)和96.3%(26/27);SSTR-3 mRNA在肝癌组织中表达的阳性率为66.7%(18/27),在癌旁组织中的阳性率为51.9%(14/27)。结论：大多数原发性肝癌肿瘤组织中有一种以上生长抑素受体亚型基因的表达。     

Survivin基因在喉癌中的表达及意义

目的研究Survivin基因在喉癌中的表达及其与各临床病理因素的关系.方法应用RT-PCR法检测2株喉癌细胞株,40例喉癌组织标本及相应的癌旁组织中Survivin基因的表达;应用免疫组织化学链霉菌抗生物素蛋白-过氧化酶连接SP法检测Survivin基因在120例喉癌组织中的表达情况.结果 RT-PCR显示喉癌细胞株和67.5%(27例)的喉癌组织表达Survivin mRNA,而癌旁组织内无1例阳性表达.SP法显示在Survivin基因在喉癌中阳性表达率为66.7%,其阳性表达率在有无淋巴结转移、复发、临床分期、5年生存率等方面差异有显著性(P＜0.05).结论 Survivin基因与喉癌的自然的发展过程有关,同时可作为判断肿瘤恶性程度,判断预后的有效指标.Survivin基因可能成为喉癌新的诊断标志及基因治疗的靶点.     

肝细胞癌ASC基因启动子区甲基化及其mRNA表达研究

目的：探讨肝细胞肝癌 （HCC） ASC基因启动子区甲基化与mRNA表达及其临床病理特征的关系。方法： 运用甲基化特异性聚合酶链反应 （MSP） 和实时荧光定量PCR技术, 检测58例肝细胞癌及其相应癌旁组织、 15例肝硬化肝组织、 5例慢性病毒性肝炎肝组织和5例正常肝组织中ASC基因启动子区甲基化状态及其mRNA的表达水平。结果： 58例肝细胞癌组织中有40例（69.0%） 发生ASC基因启动子区甲基化, 其相应癌旁组织中有27例 （46.6%） 发生该基因启动子区甲基化, 而在肝硬化、 肝炎和正常肝组织中均未检测到该基因启动子区甲基化。ASC基因在肝细胞癌组织中甲基化频率高于其相应癌旁组织 （P=0.015）。ASC基因启动子区甲基化与肿瘤直径 （P=0.001）、 生长方式 （P=0.003） 以及国际抗癌联盟第6版TNM （TNM6） 分期 （P=0.001） 有关。以ASC基因在正常肝组织中的表达量为参照, 58例肝细胞癌组织中有33例出现ASC基因mRNA低表达或表达缺失, 其相应癌旁组织中有14例出现低表达或表达缺失, 而在肝硬化及肝炎肝组织中ASC基因mRNA均呈正常表达。与癌旁组织相比, 肝细胞癌组织中ASC基因mRNA表达明显下调 （P<0.05）。在发生ASC基因启动子区甲基化的40例肝细胞癌组织中有26例出现mRNA低表达。结论： ASC基因启动子区甲基化是肝细胞癌中的频发事件, 可能在肝细胞癌的进展中起重要作用。     

KAI1基因在喉鳞癌中的表达及其临床意义

目的探讨KAI1基因在喉鳞状细胞癌中的表达及其临床意义。方法取40例喉鳞癌及9例正常对照喉组织,应用半定量的RT PCR方法,检测KAI1基因mRNA的表达。结果正常喉黏膜KAI1基因mRNA的中、低表达率和阴性率均为33.3%(3/9);无淋巴结转移组喉癌高、中、低表达率和阴性率分别为40.0%(10/25)、28.0%(7/25)、20.0%(5/25)和12.0%(3/25);淋巴结转移组喉癌中、低表达率和阴性率分别为20.0%(3/15)、26.7%(4/15)和53.3%(8/15),无淋巴结转移组喉癌与正常喉组织比较,KAI1mRNA表达升高差异有统计学意义(P<0.05),有淋巴结转移与无淋巴结转移组喉癌比较,KAI1基因mRNA表达下降差异有统计学意义(P<0.05)。病理高分化喉癌KAI1基因mRNA高、中、低表达率分别为50.0%(5/10)、30.0%(3/10)和20.0%(2/10);病理低分化喉癌其高、中、低表达率和阴性率分别为8.3%(1/12)、16.7%(2/12)、16.7%(2/12)和58.3%(7/12);高分化与低分化比较,KAI1基因mRNA表达差异有统计学意义(P<0.05)。结论KAI1基因mRNA表达下降可能与喉鳞状细胞癌的病理低分化和淋巴结转移有关。     

胃癌BRCA1基因甲基化与BRCA1 mRNA表达的关系研究

目的：探讨乳腺癌易感基因1（BRCA1）启动子CpG岛甲基化与BRCA1 mRNA表达的相关性及其意义。方法采用甲基化特异性PCR技术检测37例胃癌组织和对应癌旁组织及6例正常胃组织中BRCA1基因启动子CpG岛甲基化状态;采用逆转录PCR技术检测37例胃癌组织和对应癌旁组织及6例正常胃组织中BRCA1 mRNA 表达水平。结果胃癌组织中 BRCA1基因启动子 CpG 岛总甲基化率为48.6%（18/37）,显著高于癌旁组织[5.4%（2/37）]和正常胃组织[0.0%（0/6）],差异有统计学意义（χ2=17.541、5.021,P均〈0.05）。胃癌组织中BRCA1 mRNA阳性表达率为48.6%（18/37）,显著低于癌旁组织[94.6%（35/37）]和正常胃组织[100.0%（6/6）],差异有统计学意义（χ2=19.215、5.520,P均〈0.05）。BRCA1基因启动子CpG岛甲基化与BRCA1 mRNA表达成负相关（ r=-0.515,P〈0.05）。结论 BRCA1基因启动子CpG岛甲基化可能是导致BRCA1 mRNA失表达的主要原因之一。     

LYVE-1与血管内皮生长因子-C在喉鳞状细胞癌中的表达和意义

目的通过测定喉鳞状细胞癌组织内LYVE-1和血管内皮生长因子-C（VEGF-C）的表达情况,为喉鳞状细胞癌转移和预后的判定以及治疗提供一定的理论依据。方法通过免疫病理学方法检测LYVE-1的表达并计数喉鳞状细胞癌组织中淋巴管密度（LVD）,RT-PCR法检测喉鳞状细胞癌中VEGF-C的表达,使用统计学方法对LVD和VEGF-C表达进行分析。结果喉癌组织内存在LYVE-1（＋）的管腔样结构,LYVE-1在喉癌组织和癌旁正常组织之间表达差异有统计学意义（P〈0．05）,喉癌组织中VEGF-C mRNA的平均水平与正常组织之间差异有统计学意义（P〈0．05）,瘤组织比正常组织高4-5倍,而且喉癌组织内VEGF-C表达与LVD之间存在相关性。结论喉鳞状细胞癌瘤组织中存在淋巴管;VEGF-C mRNA在喉鳞状细胞癌组织中的表达要比正常对照组织高,且VEGF-C mRNA的高表达可能会通过促进瘤内淋巴管的增生来促进喉癌的淋巴结转移。     

喉鳞癌中p73、p63的表达及其临床意义

目的:探讨p73、p63在喉鳞癌(LSCC)组织中的表达及与喉鳞癌发生、发展的关系和生物学意义。方法:应用原位分子杂交方法,检测p73mRNA、p63mRNA在47例喉鳞癌组织、癌旁组织及14例正常喉组织中的表达。结果:p73在正常喉组织中不表达;在喉癌组织、癌旁组织中的阳性表达率分别为57.4%(27/47)、29.8%(14/47),分别与正常喉组织比较,差异有统计学意义(P<0.05);组织分化程度越低、TNM分期越晚、淋巴结转移率越高,则p73 mRNA表达越强(均P<0.05)。p63在喉鳞癌、癌旁组织中的阳性表达率为91.5%(43/47)、93.6%(44/47)、分别与正常喉组织(57.1%)比较,差异有统计学意义(P<0.05);p63与喉癌临床病理特征无关。结论:p73表达有助于判断喉鳞癌的生物学行为和预后。p63对LSCC生物学行为的影响较小。     

喉癌中Omi/HtrA2的表达及作用初探

目的:检测Omi/HtrA2在喉癌组织中的表达,初步探讨其在喉癌发生、发展中的意义。方法:采用RT-PCR与免疫组织化学方法,检测20例喉鳞状细胞癌及12例声带息肉手术切除标本中Omi/HtrA2mRNA与蛋白的表达情况。结果:癌旁正常组织中未检测到Omi/HtrA2蛋白表达,而在喉癌组织与声带息肉组织中均有表达。Omi/HtrA2基因在高分化喉鳞癌组织中的表达水平高于低分化型喉鳞癌组织、声带息肉以及癌旁的正常组织(P<0.01)。结论:Omi/HtrA2在喉鳞状细胞癌中有表达,表明Omi/HtrA2的促凋亡作用参与了喉癌的发生发展过程,为癌症治疗提供了新的理论基础。     

喉癌组织中Klotho基因的表达及其临床意义

目的   探讨喉癌组织中Klotho基因的表达及其与临床病理特征的关系。方法   应用实时荧光定量PCR（QPCR）和Western blotting检测80例喉癌组织及癌旁正常组织中的Klotho mRNA及蛋白表达情况,并探讨两者表达与临床病理特征的关系。采用甲基化特异性PCR（MSP）法检测Klotho基因启动子DNA甲基化状况。结果   80例喉癌组织中的Klotho mRNA 相对表达量为0.2890±0.012,低于癌旁正常组织的0.7908±0.015,差异有统计学意义（P＜0.01）;Klotho蛋白相对表达量为0.2290±0.009,低于癌旁正常组织的0.5368±0.023,差异有统计学意义（P＜0.01）。Klotho mRNA及蛋白表达与肿瘤浸润深度及淋巴结转移有关（P＜0.05）,而与年龄、性别、肿瘤部位、分化程度无关（P＞0.05）。喉癌组织及癌旁正常组织中Klotho基因启动子区DNA甲基化率分别为53.8％ （43／80） 、26.2％ （21／80）,差异有统计学意义（P＜0.05）。结论  Klotho基因在喉癌组织中表达下调,其表达可能受Klotho基因启动子区DNA甲基化水平的调控,且与浸润深度及淋巴结转移密切相关。     

死亡相关蛋白激酶基因启动子区过甲基化与喉癌的关系

目的　探讨死亡相关蛋白激酶 (DAPK)基因启动子区过甲基化与喉癌的关系。方法采用MSP和RT PCR法分析喉部正常黏膜、喉癌及相应癌旁组织中DAPK基因启动子区过甲基化及其mRNA表达状况。结果　 5 8例喉癌组织中 ,有 39(6 7.2 % )例DAPK基因启动子区过甲基化 ,其在各病理分级和T分级之间差异无显著性 (P >0 .0 5 ) ,而在N分级 (N0和N1)之间差异有显著性 (P <0 .0 0 1)。 5 8例癌旁组织中 ,有 6例甲基化 ,且均为喉癌组织中有甲基化的病例。RT PCR结果显示 ,所有甲基化的喉癌组织中均无DAPKmRNA表达 ,而喉部正常组织、非甲基化的喉癌组织和癌旁组织中均有其表达。结论　喉癌中DAPK基因启动子区过甲基化与其mRNA失表达有关 ,可能是喉癌发生、发展的原因之一 ,可作为喉癌诊断和预后分析的检测指标     

Recurrent amplification in the 22q11 region in laryngeal squamous cell carcinoma results in overexpression of the CRKL but not the MAPK1 oncogene

Thirteen laryngeal squamous cell carcinoma cell lines were recently studied by array comparative genomic hybridization (array-CGH) in order to identify recurrent DNA copy number alterations in the tumor genome. A highly amplified region 22q11.2 was found in two of the thirteen cell lines. Two established oncogenes CRKL and MAPK1 are localized in this region, but only CRKL was amplified in both cell lines. Therefore, to check if amplification of either CRKL or MAPK1 genes may be important in the pathogenesis of laryngeal squamous cell carcinoma, the DNA copy number and mRNA expression were measured in a cohort of 17 LSCC cell lines by quantitative real-time PCR (qPCR). For the CRKL gene gains of the copy number were found in 3/17 cell lines, while overexpression was found in 6/17 cell lines. Gains in the copy number for the MAPK1 gene were found in 1/17 cell lines, but overexpression was not detected in any cell line. A highly significant correlation between DNA copy number and expression for CRKL gene, but not for MAPK1 gene was established using the Pearson test. Thereafter, 46 primary samples of laryngeal cancer were tested by qPCR to check for possible gains in copy number of the CRKL gene. Gains were found in 3/46 cases. These results suggest that CRKL, but not MAPK1 is the target oncogene of the rare but recurrent amplification at 22q11.2 in laryngeal squamous cell carcinoma.     

Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis.

PURPOSE: The epidermal growth factor receptor (EGFR) is frequently overexpressed in non-small-cell lung carcinoma (NSCLC), and EGFR inhibitors are promising new therapeutic agents. The molecular mechanisms responsible for EGFR overexpression are poorly understood. Materials and METHODS: Gene copy number and protein status of EGFR were investigated in microarrayed tumors from 183 NSCLC patients, including squamous cell carcinoma (SCC; 89 patients) and non-SCC (94 patients) histologies. Protein expression was assessed by immunohistochemistry on a scale from 0 to 400 (percentage of positive cells x staining intensity). Gene and chromosome 7 copy numbers were identified by fluorescent in situ hybridization (FISH). RESULTS: EGFR protein overexpression was observed in 62% of the NSCLC (25% scored 201 to 300; 37% scored 301 to 400), more frequently in SCC than non-SCC (82% v 44%; P <.001), and in 80% of the bronchioloalveolar carcinomas. The prevalent FISH patterns were balanced disomy (40%) and trisomy (38%) for EGFR gene and chromosome 7 (40%), whereas balanced polysomy was seen in 13% and gene amplification was seen in 9% of the patients. Gene copy number correlated with protein expression (r = 0.4; P <.001). EGFR overexpression or high gene copy numbers had no significant influence on prognosis. CONCLUSION: EGFR overexpression is frequent in NSCLC, is most prominent in SCC, and correlates with increased gene copy number per cell. High gene copy numbers per cell showed a trend toward poor prognosis. It will be important to evaluate EGFR gene and EGFR protein status and signal protein expression to properly interpret future clinical trials using EGFR inhibitors.     

鼻咽癌患者HER-2/neu基因扩增和表达及其临床意义

目的：研究鼻咽癌HER-2／neu基因扩增、表达及其临床意义。方法：采用原位荧光杂交、逆转录多聚链式反应和免疫组化技术检测鼻咽癌组织HER-2／neu基因扩增、表达。结果：HER-2／neu基因在鼻咽癌中无扩增，但有过表达，其原因是由于mRNA高表达所致；这种过表达与鼻咽癌预后之间未显示有相关性。结论：HER-2／neu基因在鼻咽癌无扩增，有过表达，这种过表达未显示预后意义。     

Relationship between epidermal growth factor receptor gene copy number and protein expression in oral cavity squamous cell carcinoma

This study was designed to explore the relationship between epidermal growth factor receptor (EGFR) copy number and EGFR protein expression in oral cavity squamous cell carcinoma (OSCCs) in Taiwan. A total of 160 oral cavity squamous cell carcinomas were examined for EGFR protein overexpression using immunohistochemistry and for copy number using a fluorescence in situ hybridization (FISH) assay. Overexpression and increased gene copy numbers of EGFR were found in 75 (46.88%) and 50 (31.25%) cases, respectively. The concordance rate for EGFR gene amplification and protein overexpression was 100%. EGFR overexpression was associated with a poor prognosis both in terms of disease-free survival (DFS) and overall survival (OS). On the other hand, the association between an increase in EGFR gene copies and DFS or OS was insignificant. This was despite the observed significant associations between gene copy number and tumor stage, depth of tumor invasion, lymph node metastasis, bone invasion and perineural invasion. EGFR protein overexpression is closely related to EGFR copy number. Standard methodological and interpretation criteria need to be established that allows EGFR copy number combined with EGFR protein expression to be determined in a manner that allows individualized EGFR targeted therapy in OSCC patients.     

HER 2/neu expression and gene amplification in colon cancer

HER 2/neu is an important oncogene in breast cancer, but the prevalence and significance of HER 2/neu gene amplification in colon cancer have been poorly documented. We have evaluated HER 2/neu gene amplification and protein overexpression in a series of colon cancers to assess the frequency, concordance and clinical significance of these events. HER 2/neu gene copy number was measured in 154 primary colon tumors, 15 liver metastases and matched normal tissues using a quantitative PCR/ligase detection reaction (LDR) technique developed and validated in our laboratory. HER 2/neu copy number was confirmed by fluorescent in situ hybridization (FISH) in all tumors found to have gene amplification. In an independent and blinded fashion, HER 2/neu expression was assessed in paraffin sections from 139 of the tumor specimens using the HercepTest kit. HER 2/neu gene amplification was observed in 4 (2.4%) of the 169 tumor specimens and in none of the normal tissues. There was no apparent association with stage of disease, tumor grade or patient survival. Among 139 cases evaluated by immunohistochemistry (IHC), HER 2/neu overexpression was seen in 5 cases (3.6%). There was extremely high concordance (kappa = 0.852) between gene amplification and protein overexpression. The low prevalence of HER 2/neu gene amplification and protein overexpression suggests that this oncogene plays an infrequent role in the development and progression of colon cancer. These data indicate that the primary mechanism of dysregulated HER 2/neu expression in colon cancer, as in breast cancer, is gene amplification.     

Amplification of cyclin D1 and MDM-2 in oesophageal carcinoma.

AIMS: This study investigated amplification of the cyclin D1 and MDM-2 genes, and overexpression of the cyclin D1 gene product, in oesophageal carcinoma. METHODS: Paired tumour and normal DNA samples from 26 oesophageal adenocarcinomas and 19 squamous cell carcinomas were analysed by Southern blotting with specific DNA probes for cyclin D1 and MDM-2, and for a control gene (alpha-lactalbumin). The cyclin D1 and MDM-2 gene copy numbers were calculated for each tumour. Expression of the cyclin D1 gene was assessed by immunohistochemical analysis of its protein product. RESULTS: Cyclin D1 gene amplification (by a factor of between two and six) was identified in seven tumours (16%). MDM-2 gene amplification (by a factor of between two and 11) was identified in 10 tumours (22%). Overexpression of cyclin D1 protein was identified in eight tumours and was significantly associated with gene amplification (P=0.04; Fisher's exact test), and with early T stage (P=0.01; Fisher's exact test). CONCLUSIONS: Cyclin D1 and MDM-2 amplification and cyclin D1 overexpression occur, although infrequently, in the development of oesophageal carcinoma. Cyclin D1 overexpression may influence tumour behaviour, causing the disease to present at an earlier T stage. The mechanism for this effect is unclear, and warrants further investigation.     

Evaluation of HER-2/neu gene amplification and protein expression in non-small cell lung carcinomas

HER-2/neu gene amplification and cell surface overexpression are important factors in breast cancer for prognosis and prediction of sensitivity to anti-HER-2/neu monoclonal antibody therapy. In lung cancer, the clinical significance of HER-2/neu expression is currently under evaluation. We investigated 238 non-small lung carcinomas for HER-2/neu protein overexpression by immunohistochemistry using the HercepTest. We found 2+ or 3+ overexpression in 39 patients (16%), including 35% in adenocarcinomas and 20% in large cell carcinomas, but only 1% of squamous cell carcinomas. Marked (3+) overexpression was uncommon (4%). The association between protein expression and gene copy number per cell, as determined by fluorescence in situ hybridisation assay, was investigated in 51 of these NSCLC tumours. Twenty-seven tumours (53%) were negative by both tests. Marked (3+) protein expression and gene amplification were present in only 4% of samples. In 11 tumours (21%), gene gain was accompanied by chromosomal aneusomy and did not result in high protein levels while in 7 (14%) the score 2+ was associated with maximum number of signals per cell <9. The prognostic implication of HER-2/neu protein expression was studied in 187 surgically resected tumours. No statistical difference in survival was observed comparing patients with positive (2+/3+) and negative tumours (0/1+), although 3+ patients showed a tendency to shorter survival. The therapeutic implications of protein expression and gene amplification in lung cancer need to be examined in prospective clinical trials.