癌基因组学研究进展

癌是一种基因组疾病,其主要的特征是基因组不稳定性。癌基因组不稳定性表现为单核苷酸突变、微卫星不稳定性、基因结构和拷贝数改变、染色体杂合性和纯合性丢失以及表基因组效应等。癌基因组不稳定性来源于胚系突变和体细胞突变。搞清基因组不稳定性与癌发生发展的关系对于癌的诊断、治疗、预防和药物研制具有重要意义。本文介绍癌基因组学的几个主要研究领域,包括癌基因组不稳定性、癌易感基因的筛查与鉴定以及基因表达谱与临床表型的关系等。

癌基因组学研究的新趋势

2007年国际人类基因组大会（HGM2007）于2007年5月21—24日在加拿大蒙特利尔召开，来自全球数十个国家的科学家会聚一堂，探讨共同感兴趣的基因组科学的相关议题。正如大家所期待的那样，癌基因组学作为大会的主要讨论专题之一，受到了广泛关注。笔者综合从大会现场得到的信息，将癌基因组学研究的新趋势归纳为下列几个方面：

癌基因组学研究新进展

第六届人类基因组太平洋会议与第七届亚太人类遗传学大会联合会议于2006年3月6-10日在我国台湾省台北市召开，来自亚太地区15个国家和地区的数百名科学家会聚一堂，讨论相关领域共同感兴趣的学术问题，交流各自的研究成果，总结现阶段的最新进展，提出今后发展的走向，商讨合作事宜。我国约20位有关领域的科学家参加了此次会议。现以个人之管见，简要介绍此次大会的基本情况，重点介绍与癌基因组学与癌遗传学相关的内容，以供参考。

蛋白激酶基因在不同癌基因组中的变异频率及表达变化

目的探讨蛋白激酶基因突变与癌症发生的关系。方法本研究通过搜索癌基因组数据库,获得人类全部514个蛋白激酶基因在不同癌基因组中的变异频率及表达变化。结果基因表达搜索结果显示,绝大多数基因的表达上调。基因变异搜索结果显示,512个成员发生变异,变异频率最高为90%。其中,基因扩增频率要高于突变频率和删除频率,且删除频率最低。12个成员的突变频率>30%,功能研究相关的文献资料显示,其中的8个成员可能与癌症产生密切相关。结论笔者推测高频突变的蛋白激酶基因可能具有重要功能,其很可能是癌基因或者抑癌基因。本研究结果为进一步深入研究相关蛋白激酶基因的功能,尤其是功能未知的成员,及其与癌症发生的关系提供重要参考。

Genetics of hepatocellular carcinoma

The completely assembled human genome has made it possible for modern medicine to step into an era rich in genetic information and high-throughput genomic analysis. These novel and readily available genetic resources and analytical tools may be the key to unravel the molecular basis of hepatocellular carcinoma (HCC). Moreover, since an efficient treatment for this disease is lacking, further understanding of the genetic background of HCC will be crucial in order to develop new therapies aimed at selected targets. We report on the current status and recent developments in HCC genetics. Special emphasis is given to the genetics and regulation of major signalling pathways involved in HCC such as p53, Wnt- signalling, TGFβ, Ras, and Rb pathways. Furthermore, we describe the influence of chromosomal aberrations as well as of DNA methylation. Finally, we report on the rapidly developing field of genomic expression profiling in HCC, mainly by microarray analysis.

Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis

AIM:To investigate the expression of co-stimulatorymolecule B7-H3 in gastric carcinoma and adenomatissue as well as normal gastric tissue and to explore therelationship between B7-H3 expression and pathologicalfeatures and prognosis of gastric carcinoma.METHODS:B7-H3 expression was detected in 102samples of human gastric carcinoma and 10 samples ofgastric adenoma and 10 samples of normal gastric tissueby immunohistochemical assay.Correlation betweenthe expression of B7-H3 and the patients'age,sex,gastric carcinoma locus,tumor size,tissue type,tumorinfiltration depth,differentiation degree,lymph nodemetastasis,and survival time was analyzed.RESULTS:B7-H3 was expressed in all gastric adenomasamples and in 58.8% samples of gastric carcinoma.B7-H3 expression in gastric carcinoma samples wasnot related with the patients'age,sex,lymph nodemetastasis,and tumor size(P>0.05),but with thesurvival time,infiltration depth of tumor and tissue type.CONCLUSION:Detection of B7-H3 expression in gastriccarcinoma tissue is beneficial to the judgment of theprognosis of gastric carcinoma patients and the choice oftreatment.

Lymph node micrometastasis and its correlation with MMP-2 expression in gastric carcinoma

AIM： To examine matrix metalloproteinase-2 （MMP-2） expression in gastric cancer tissues and to evaluate its relationship with lymph node micrometastasis. MATERIALS： The authors studied 850 lymph nodes resected from 30 patients with gastric carcinoma who underwent gastrectomy with lymphadenetomy using reverse transcription polymerase chain reaction （RT-PCR） assay in addition to H-E staining. MMP-2 expression of the tumor tissues was detected by immunohistochemical technique （EliVision^TM plus）. RESULTS： MMP-2 expression was positive in 21 （70%） cases and negative in g （30%） cases. No significant correlations were found between MMP-2 expression and other variables such as age, gender, tumor location, tumor diameter, Lauren classification and lymphatic invasion. In contrast, MMP-2 expression correlated significantly with depth of tumor infiltration （P = 0.022）, lymph node metastasis （P = 0.030） and tumor differentiation （P = 0.043）. Lymph node micrometastases were detected in 77 （12.5%） lymph nodes of 14 （46.7%） gastric carcinoma patients. MMP-2 expression was positive in 12 （85.7%） of the 14 patients with lymph node micrometastasis, and in g （56.3%） of the 16 patients without lymph node micrometastasis （P = 0.118）. CONCLUSIONS： Our results demonstrate that MMP-2 expression has significant correlation with tumor invasion, tumor differentiation and lymph node metastases. MMP-2 expression may be an important biological characteristics and significant prognostic parameter of gastric carcinoma. We also conclude that MMP-2 may participate in the development of lymph node micrometastasis of gastric carcinoma. Further investigations are needed to draw a conclusion.

Reduced expression of E-cadherin/catenin complex in hepatocellular carcinomas

AIM： TO examine the immunoreactivity of E-cadherin and four subtypes of catenin family in human hepatocellular carcinomas （HCCs） and to investigate the correlation between expression of E-cadherin/ catenin complex and clinicopathologic parameters of HCC patients. METHODS： An immunohistochemical study for E-cadherin and catenins was performed on 97 formalin-fixed, paraffin-embedded specimens of HCC. RESULTS： Reduced expression of E-cadherin, ^-, 13-, y-catenin and p120 was observed in 69%, 76%, 63%, 71% and 73%, respectively. Both expressions of E-cadherin and catenin components were significantly correlated with tumor grade （P = 0.000）. It showed significant difference between expression of catenin members and tumor stage （P = 0.003, P = 0.017, P = 0.007 and P = 0.000, respectively）. The reduced expression of E-cadherin in HCCs was significantly correlated with intrahepatic metastasis （IM） and capsular invasion （P = 0.008, P = 0.03, respectively）. A close correlation was also observed between the expression of catenins and the tumor size （P = 0.002, P = 0.034, P = 0.016 and P = 0.000, respectively）. In addition, the expression of each catenin was found correlated with IM （P = 0.012, P = 0.049, P =0.026 and P = 0.014, respectively）. No statistically significant difference was observed between the expression level of E-cadherin/catenin complex and lymph node permission, vascular invasion and satellite nodules. Interestingly, only expression of p120 showed correlation with AFP value （P = 0.035）. The expression of E-cadherin was consistent with α-, β-, γ-catenin and p120 expression （P = 0.000）. Finally, the abnormal expression of E-cadherin/catenin complex was significantly associated with patients＇ survival （P = 0.0253, P = 0.0052, P = 0.003, P = 0.0105 and P = 0.0016, respectively）. Nevertheless, no component of E-cadherin/catenin complex was the independent prognostic factor of HCC patients. CONCLUSION： Down-regulated expressions of E-cadherin, catenins and p120 occur frequently in HCCs and contribute to the progression and development of tumor. It may be more exact and valuable to detect the co-expression of E-cadherin/catenin complex than to explore one of them in predicting tumor invasion, metastasis and patient＇s survival.

Clinicopathologic significance of BAG1 and TIMP3 expression in colon carcinoma

AIM: To explore the expression of BAG1 and tissue inhibitor of metalloproteinase 3 (TIMP3) in colon carcinoma and their correlation and clinicopathologic significance. METHODS: SABC immunohistochemistry was used to detect the expression of BAG1 and TIMP3 in 80 colon carcinoma tissues and 20 normal colonic mucosa. RESULTS: Positive rate of BAG1 in colon carcinoma tissue (80%) was notably higher compared to normal colonic mucosa (10%) (P < 0.05). However, no significant difference was observed in positive rate of TIMP3 in colon carcinoma tissue (43.75%) as compared with normal colonic mucosa (60%) (P > 0.05). Expression of BAG1 and TIMP3 was strongly associated with colon carcinoma differentiation, Duke's staging, lymph node metastasis and survival rate (P < 0.05), but not associated with gender and age. Moreover, BAG1 expression was not correlated with TIMP3. CONCLUSION: Our results suggest that over-expression of BAG1 or attenuated expression of TIMP3 may play an important role in genesis and development of colon carcinoma. The protein expression levels of BAG1 and TIMP3 are related to the malignant degree, infiltration and metastasis of colon carcinoma. BAG1 and TIMP3 might be new biological parameters in predicting invasion and metastasis of colon carcinoma.

Changes of histology and expression of MMP-2 and nm23-H1 in primary and metastatic gastric cancer

AIM: To investigate the changes of histology and expression of MMP-2 and nm23-H1 in primary and metastatic gastric cancer. METHODS: One hundred and seventy-seven gastric cancer patients with lymph node and/or distal metastasis between 1997 and 2001 were reviewed. Differences in histology of the primary and metastatic gastric cancer were assessed. MMP-2 and nm23-H1 immunoreactivity was compared in 44 patients with tumor infiltration to the serosa layer. RESULTS: Poorly and moderately differentiated metastatic gastric cancer was found in 88.7% (157/177) and primary gastric cancer in 75.7% (134/177) of the patients. The histological type of metastatic gastric cancer that was not completely in accordance with the preponderant histology of primary gastric cancer was observed in 25 patients (14.1%). MMP-2 immunoreactivity in metastatic gastric cancer was significantly stronger than that in primary gastric cancer, while nm23-H1 immunoreactivity showed no difference in primary and metastatic gastric cancer. CONCLUSION: Metastatic gastric cancer presents more aggressive histological morphology and higher MMP-2 immunoreactivity than primary gastric cancer. This heterogeneity may elicit a possible mechanism of gastric cancer metastasis.

Expression and hypermethylation of p27^(kip1) in hepatocarcinogenesis

AIM： To study the expressions of p27^kip1 protein and p27mRNA, the hypermethylation of p27^kip1 and the relation between them in various stages of hepatocarcinogenesis. METHODS： p27 protein and p27mRNA were detected by immunohistochemical staining and in situ hybridization respectively in 68 cases of normal liver, liver cirrhosis, pericancerous cirrhosis and hepatocellular carcinoma （HCC）. The hypermethylation of p27^kip1 was detected by methylation-specific PCR （MSP） in 44 cases of normal liver, liver cirrhosis, and HCC. RESULTS： The positive rate of p27 protein was 66.7% （4/6） in normal liver, 60.0% （6/10） in liver cirrhosis, 50.0% （12/24） in pericancerous cirrhosis and 21.4% （6/28） in HCC. There were no statistical differences in normal liver, liver cirrhosis and pericancerous cirrhosis, but the positive rate of p27 protein significantly decreased in HCC compared to that in the other groups （P = 0.006, %2 = 7.664）. The positive rate of p27^kip1 mRNA was 83.3% （5/6） in normal liver, 70.0% （7/10） in liver cirrhosis, 75.0% （18/24） in pericancerous cirrhosis and 25.0% （7/28） in HCC. There were no statistical differences in normal liver, liver cirrhosis and pericancerous cirrhosis, but the positive rate of p27^kip1 mRNA also significantly decreased in HCC compared to that in the other groups （P = 0.000, %2 = 16.600）. In addition, there was a significant correlation between the expression of p27 protein and p27mRNA in the integrated group of normal liver and liver cirrhosis. However, no significant correlation was found between pericancerous cirrhosis and HCC. Using MSP, we found that 1 HCC in 44 cases （including 6 cases of normal liver, 10 cases of liver cirrhosis and 28 cases of HCC） was methylated, whose p27 protein and p27mRNA were negative. CONCLUSION： The reduction or loss of p27 protein and p27mRNA are potentially involved in hepatocarcinogenesis. The hypermethylation of p27 might lead to the loss of p27mRNA transcription.

Clinicopathological significance of LRP16 protein in 336 gastric carcinoma patients

AIM： To investigate the expression of leukemia related protein 16 （LRP16）, and the possible relationship between LRP16 expression and clinicopathological indices in 336 gastric carcinoma patients. METHODS： Immunohistochemistry was used to detect LRP16 expression in 336 cases of paraffin-embedded gastric carcinoma tissues and 60 cases of distal normal mucosa. The relationships between LRP16 expression and patients＇ age, tumor size, histological grade, clinical stage, metastatic status and prognosis were analysed. RESULTS： The expression of LRP16 was 58.6% （197/336） in gastric carcinoma and 31.7% （19/60） in distal normal gastric mucosa. The expression of LRP16 in carcinoma was significantly higher than that in normal mucosa tissues （x^2 = 14.929, P = 0.001）. LRP16 protein expression was found in 44.1% （63/143） carcinomas at stage Ⅰ and Ⅱ, and 69.4% （134/193） carcinomas at stage Ⅲ and Ⅳ （Z2 = 21.804, P = 0.001）, and in 56.9% （182/320） of cancers without metastasis but 93.8% （15/16） of those with metastasis （2 = 8.543, P = 0.003）. The expression of LRP16 was correlated with tumor size, infiltrative depth, clinical stage, lymphatic invasion and distant metastasis （all P 〈 0.05）. Follow-up data showed that there was a significant difference in median survival time between cancer patients with expression of LRP16 （27.0 mo） and those without （48.0 mo, Log rank =31.644, P = 0.001）. CONCLUSION： The expression of LRP16 may be associated with invasion, metastasis and prognosis of gastric cancer.

Expression of pituitary homeobox 1 gene in human gastric carcinogenesis and its clinicopathological significance

AIM： To investigate the effect of pituitary homeobox 1 （PITX1） expression in cases of human gastric cancer on cancer differentiation and progression, and carcinogenesis. METHODS： Using polyclonal PITX1 antibodies, we studied the expression of PITX1 in normal gastric mucosa, atypical hyperplasia, intestinal metaplasia, and cancer tissue samples from 83 gastric cancer patients by immunohistochemistry. Moreover, semi-reverse transcription polymerase chain reaction （semi-RT-PCR） was performed to detect the mRNA level of PITX1 in three gastric cancer cell lines and a normal gastric epithelial cell line. Subsequently, somatic mutations of the PITX1 gene in 71 gastric cancer patients were analyzed by a combination of denaturing high performance liquid chromatography （DHPLC） and DNA sequencing. RESULTS： Immunohistochemistry showed that PITXl was strongly or moderately expressed in the parietal cells of normal gastric mucosa （100%）, while 55 （66.3%） out of 83 samples of gastric cancers showed decreased PITXl expression. Moreover, PITXl expression was reduced in 20 out of 28 cases （71.5%） of intestinal metaplasia, but in only 1 out of 9 cases （11%） of atypical hyperplasia. More importantly, PITXl expression was significantly associated with the differentiation, position and invasion depth of gastric cancers （r = -0.316, P 〈 0.01; r = 0.213, P 〈 0.05; r = -0.259, P 〈 0.05, respectively）. Similarly, levels of PITXl mRNA were significantly decreased in 2 gastric cancer cell lines, BGC-823 and SGC-7901, compared with the normal gastric epithelial cell line GES-1 （0.306 ± 0.060 vs 0.722 ± 0.102, P 〈 0.05; 0.356 ± 0.081 vs 0.722 ± 0.102, P 〈 0.05, respectively）. Nevertheless, no somatic mutation of PITX1 gene was found in 71 samples of gastric cancer by DHPLC analysis followed by sequencing. CONCLUSION： Down-regulation of PITX1 may be a frequent molecular event in gastric carcinogenesis. Aberrant levels of PITXl expression may be closely correlated with the progression and differentiation of gastric cancer,

Multi-Omics and Its Clinical Application in Hepatocellular Carcinoma:Current Progress and Future Opportunities

Hepatocellular carcinoma(HCC)is the sixth most common malignancy and the fourth leading cause of cancer related death worldwide.China covers over half of cases,leading HCC to be a vital threaten to public health.Despite advances in diagnosis and treatments,high recurrence rate remains a major obstacle in HCC management.Multi-omics currently facilitates surveillance,precise diagnosis,and personalized treatment decision making in clinical setting.Non-invasive radiomics utilizes preoperative radiological imaging to reflect subtle pixel-level pattern changes that correlate to specific clinical outcomes.Radiomics has been widely used in histopathological diagnosis prediction,treatment response evaluation,and prognosis prediction.High-throughput sequencing and gene expression profiling enabled genomics and proteomics to identify distinct transcriptomic subclasses and recurrent genetic alterations in HCC,which would reveal the complex multistep process of the pathophysiology.The accumulation of big medical data and the development of artificial intelligence techniques are providing new insights for our better understanding of the mechanism of HCC via multi-omics,and show potential to convert surgical/intervention treatment into an antitumorigenic one,which would greatly advance precision medicine in HCC management.

Expressions of chemokine receptor CXCR4 and its ligand CXCL12 in salivary adenoid cystic carcinoma

Objective: To examine expressions of chemokine receptor CXCR4 and its ligand CXCL12 in primary focus and lymphogenous metastasis of salivary adenoid cystic carcinoma (ACC) with lung metastasis. Methods: Using immunohistochemical hypersensitivity catalyzed signal amplification (CSA), expressions of chemokine receptor CXCR4 and ligand CXCL12 were detected in tissue specimens from 20 cases of primary cancer focus and lymphogenous metastasis of salivary adenoid cystic carcinoma, of which 7 cases were associated with lung metastasis and 3 with lympogenous metastasis. Twenty cases of tongue carcinoma (including 10 cases with lymphogenous metastasis) and 15 cases of mucoepidermoid carcinoma (including 5 cases with lymphogenous metastasis) were used as the malignant control group;and salivary mixed tumor (n=10), tongue leukoceratosis (n=10) and cervical lymph node reactive hyperplasia (n=10) were used as the benign control group. Results: Expression of CXCR4 in the tissues and lymph metastases of oral and maxillofacial salivary ACC, mucoepidermoid carcinoma and tongue carcinoma was significantly higher than that of the benign control group (P<0.05); expression of CXCR4 in the primary focus of ACC was significantly higher than that of the malignant control group; and expression of CXCR4 in the ACC with lung metastasis was 87.1% (6/7), significantly higher than that without lung metastasis(P<0.01). There was evident positive expression of CXCL12 in endotheliocytes of microvessels within cancer and paracancer tissues and significantly high expression of CXCL12 in lymphogenous metastasis(P<0.05). Conclusion: Chemokine receptor CXCR4 and its ligand CXCL12 may be associated with local invasion and lymphogenous metastasis of oral and maxillofacial cancer, especially with lung metastasis of salivary ACC.

HER2 gene status and the relationship with p21 protein expression in gastric cancer

Objective： We aimed to analysis the HER2 gene status and its relationship with p21 protein expression in gastric carcinoma. Methods： Fluorescence in situ hybridisation （FISH） and immunohistochemistry （IHC） techniques were used to detect HER2 gene status and p53 protein in 59 cases of gastric cancer. Results： FISH detection of HER2 gene amplification rate was 16.9% （10/59）, HER2 gene amplification in 49 cases without copy number gain and gene amplification were a total of 49.2% （29/59）. HER2 protein expression was 42.4% （25/59）, HER2 gene amplification rates in patients with ＋＋＋, ＋＋ HER2 protein expression were 3/3 and 5/8, while in patients with ＋ HER2 protein expression, it was 2/14, there was significant difference （P 0.05）. p21 protein expression rate was 49.2% （29/59）, HER2 gene amplification rates and p21 protein expression had significant difference in tumor invasion depth, lymph node metastasis （P 0.05）; had no statistical significance in histological type, age, gender differences （P 0.05）. Conclusion： HER2 gene amplification rate and gene copy number had positively correlation with p21 protein expression, HER2 gene status and expression of p21 protein combined detection can provide a reference value in gastric cancer metastasis, patient’s condition development and prognosis, it also can guide clinical development of individual treatment.

The relationship between Bag-1 and gastric cancer clinicopathologic characteristic

Objective： The aim of our study was to investigate Bcl-2 antanogene-1 （Bag-l） expressions in gastric cancer and evaluate their clinical significance. Methods： Bag-1 expressions were studied by the immunohistochemical streptavidin- perosidase （S-P） method in 62 samples of gastric cancer tissues, 11 normal gastric tissues, and their clinical significance was analyzed. Results： The positive rate of Bag-1 expression in the gastric carcinoma tissues was 41.94% （26/62）, while it was 9.09% （1/11） in the normal gastric tissues （P = 0.046）. Though Bag-1 was positively related to the tumour pathological grade, lymph node metastasis and prognosis （P 〈 0.05）, it was not related to the sex, age, location, tumour diameter, depth of invasion （P 〉 0.05）. Conclusion： There are over expression of Bag-1 gene protein in gastric cancer, it is significance in prognosis of gastric cancer.

On the origin of cardiac mucosa: A histological and immunohistoc-hemical study of cytokeratin expression patterns in the developing esophagogastric junction region and stomach

AIM： To examine the fetal and neonatal esophagogastric junction region （EGJ） histologically for the presence of an equivalent to adult cardiac mucosa （CM）; to study the expression patterns of all cytokeratins （CK） relevant to the EGJ during gestation; to compare the CK profile of the gestational and the adult EGJ; and to determine the degree of development in the adult EGJ histology and CK profile during gestation. METHODS： Forty-eight fetal autopsy specimens of the EGJ were step-sectioned and stained with hematoxylin and eosin （H＆E） to select sections showing the mucosal lining. Immunohistochemistry for CK5, 7, 8, 13, 18, 19, and 20 was performed. Antibody staining was then graded for location, intensity, and degree. RESULTS： The distal esophagus was lined by simple columnar epithelium from 12-wk gestational age （GA）. The proximal part of this segment consisted of mucusproducing epithelium, devoid of parietal cells. CK5 and 13 were present exclusively in multilayered epithelia and CK8, 18, and 19 predominantly in simple columnar epithelium. There were no differences in the frequencies of the coordinate CK7＋/20＋ and the CK7-/20- immunophenotypes between different locations. The prevalence of the CK7＋/ 20- immunophenotype decreased, and that of the CK7-/ 20＋ immunophenotype increased significantly from the distal esophagus to the distal stomach. CONCLUSION： Fetal columnar-lined lower esophagus （fetal CLE） may be the equivalent and precursor of the short segments of columnar epithelium found in the distal esophagus of some normal adult subjects. Esophageal simple columnar epithelium without parietal cells （ESN） may be the precursor of adult CM. The similarities between the fetal and adult EGJ and stomach CK expression pattems support the conclusion that adult CN has an identifiable precursor in the fetus. This would then indicate that at least a part of the adult CM has a congenital origin.