Mutations of K-ras oncogene in primary colorectal tumors,related liver metastases and portal vein blood of patients with colorectal cancer

AIM: To evaluate the concordance of K-ras oncogene mutations in primary colorectal tumors, liver metastases and portal vein blood of the patients with colorectal cancer, and to find out the relationship between mutated K-ras oncogene and liver metastases in colorectal cancer.METHODS: Fifty-nine patients with colorectal cancer were screened for the mutations of K-ras oncogene in the tissue samples of primary tumors, portal vein blood and liver metastases (only 15 cases of the 59 patients) by real-time fluorescence quantitative PCR and DNA sequencing. The results were also analyzed with the clinical data of the patients.RESULTS: Point mutations of K-ras were found in the primary tumors in 20 (33.9%) of the 59 patients with colorectal cancer, and 18 (30.5%) of the 59 patients in their portal vein blood. K-ras mutations in 8 (53.3%) of 15 liver metastases were also detected. No significant difference among the rates of K-ras mutation in primary tumor tissues, portal vein blood and related liver metastases was observed (P>0.05). Eighteen cases with mutated K-ras gene in portal vein blood showed the mutations in primary tumor tissues. The patients without mutated K-ras gene in primary tumor tissue also showed negative mutation of K-ras in the portal vein blood. The mutated K-ras gene in both liver metastase and portal vein blood were detected in 8 of the 15 cases with liver metastases, and no mutated K-ras gene was detected in the others with liver metastases. The main types of K-ras mutations found in primary tumors, liver metastases (5 simultaneous, 2 metachronous) and portal vein blood were GGT to GAT and GGT to GTT at codon 12. A K-ras mutation at codon 13 (GGC to GAC) was found in one case with metachronous liver metastases. The rate of concordance of K-ras status between primary tumors and portal vein blood was 96.6%. Detection of K-ras mutations in liver metastases was accordant with that in portal vein blood, but the type of K-ras mutation was partially discordant.CONCLUSION: The K-ras mutations in primary tumors, liver metastases and portal vein blood of patients with colorectal cancer are concordant, and mutated K-ras detected in both cancer tissue and portal vein blood may indicate liver micrometastases from colorectal cancer.     

Relationship between expression of COX-2 and clinicopathological features in esophageal carcinoma

AIM:To examine COX-2 expression in esophageal carcinoma, and to study relationships between COX-2 expression and clinicopathological features and prognosis of esophageal carcinoma patients. METHODS:89 paraffin - embedded tissue samples from patients with esophageal carcinoma were collected, its clinicopathological features such as tumour differentiation, depth of invasion, length and site of the tumor, regional lymph node metastases, distant metastasis were recorded. Survival time of 81 cases were also recorded. By SP immunohistochemistry method, the expression of COX-2 in tumor samples was examined. RESULTS:COX-2 expression in esophageal carcinoma was markedly higher than that in nomal esophagus, the expression was higher in less differentiated and deeper invaded cases (P<0.05), but it had no correlations with other clinicopathological features such as age,sex, length and site of the tumor, regional lymph node metastases, and distant metastasis (P>0.05). Cases of esophageal carcinoma with lower COX-2 expression had longer survival time than those with higher COX-2 expression (P<0.01). CONCLUSIONS:COX-2 expression is higher in esophageal carcinoma than normal esophagus. COX-2 expression of esophageal carcinoma is higher in less differentiated and deeper invaded cases, but it has no correlation with age, sex, length and site of the tumor, regional lymph node metastases, and distant metastasis. Patients with lower COX-2 expression have longer survival time than those with higher COX-2 expression.     

Methylation of TIMP-3 gene and their relationships with their clinical-pathological characteristics of colorectal cancers

AIM: To investigate whether methylation of the TIMP-3 gene is associated with clinical-pathological characteristics, recurrence and metastasis of the colorectal cancer. METHODS: Nest methylation specific PCR (nMSP) and RT-PCR techniques were used to detect methylation of TIMP-3 gene and its mRNA expression in the colorectal cancer specimen and adjacent non-cancerous tissues. RESULTS: The expression of TIMP-3 mRNA in tumor tissues was distinctly reduced (P<0.01). The expression of TIMP-3 mRNA in those without lymph node metastasis was higher than those with lymph node metastasis (P<0.01). The patients with Duke's C, D and lymph node metastasis were more to contain methylated TIMP-3 compared to those with Duke's A, B and no lymph node metastasis (P<0.05). Statistical differences in pathological characteristics such as tumor site, Duke's stage, histological differentiation and type between TIMP-3 methylation positive group and negative group were observed (P<0.05). CONCLUSION: Methylation of the TIMP-3 gene promoter usually occurs in the proximal site, infiltrating type, poor cellular differentiation, lymph node metastasis and advanced stage of colorectal cancers patients.     

Expression of Tpl-2 in colorectal carcinogenesis

AIM: To analyze the relationship between Tpl-2 (tumor progression locus 2)expression and clinicopathological parameters of colorectal carcinoma by investigating the expression of Tpl-2 in adjacent normal mucosa, colorectal adenomas and colorectal carcinoma. METHODS: Tpl-2 expression in normal mucosa, adenoma and carcinoma was examined and compared in a set of tissue microarrays by immunohistochemistry. The potential relationship between Tpl-2 expression and clinicopathological features was analyzed. RESULTS: The expression of Tpl-2 in carcinoma was significantly increased compared to the adenoma and normal mucosa (P<0.01). No significant difference was detected between the adenoma and normal mucosa (P>0.05). Meanwhile, the correlation between Tpl-2 expression and lymph node metastasis (N stage) and TNM stage (P<0.05) was observed. However, the correlation between the Tpl-2 expression and clinicopathological features of colorectal cancer including sex, age, body mass index (BMI), tumor size, histological differentiation, invasive depth (T stage),distant metastasis(M stage) and K-ras mutation (P>0.05) was not found. CONCLUSION: Tpl-2 has a relevance to the development of colorectal cancer as a promotive factor in the colorectal carcinogenesis.     

Expression and significance of T-cell immunoglobulin and ITIM domain in colorectal cancer

AIM: To study the expression and clinical significance of T-cell immunoglobulin and ITIM domain (TIGIT) in colorectal cancer. METHODS: The patients with colorectal cancer (n=80) from January 2016 to June 2018 were selected. The expression of TIGIT and CD155 in the colorectal cancer tissues and adjacent normal tissues were detected by immunohistochemical staining method. The expression of TIGIT and CD155 was also determined by Western blot and ELISA. RESULTS: The positive expression rates of TIGIT and CD155 were 78.8% (63/80) and 83.8% (67/80) in the colorectal cancer tissues, significantly higher than that in the paracancerous tissues of 8.8% (7/80) and 18.8% (15/80), respectively (P<0.05). There was a positive correlation between TIGIT and CD155 expression (r=0.867, P<0.01). The expression levels of TIGIT and CD155 were increased as the stage evolved. The positive rates of TIGIT and CD155 in the colorectal cancer tissues were correlated with the degree of differentiation, pathological stage and lymph node metastasis (P<0.05). CONCLUSION: TIGIT and CD155 are correlated with the occurrence and development of colorectal cancer, and can be used as one of the prognostic indicators of colorectal cancer.     

Mutation of the transforming growth factor-β typeⅡ receptor gene in sporadic colorectal cancers with microsatellite instability

AIM: To determine the relationship between the mutation of the RII gene and RER status in the tumorigenesis of sporadic colorectal cancer. METHODS: We screened RER status and mutation of the RII gene from 50 sporadic colorectal cancers (19 in the proximal colon, 31 in the distal colorectum). RESULTS: RER was found in 13 cases (8 in the proximal colon, 5 in the distal colorectum), and 5 of them showed mutations of the RII gene. All 5 cancers carrying a TGF-β RII gene mutation showed RER+, but there wasn't any mutation of RII gene in RER(-) cases. Four of 5 RII mutation were located at the cecum. CONCLUSION: These data indicate that the TGF- βRII gene is a major target of microsatellite instability and mutation of the RII gene play an important role in carcinogenesis of sporadic colorectal cancer with microsatellite instability , especially at the cecum.     

Expression and clinical significance of chemokine receptor CXCR6 in primary colorectal cancer

AIM: To investigate the expression of chemokine receptor CXCR6 in primary colorectal cancer and determine the association between CXCR6 expression and synchronous liver metastasis/prognosis. METHODS: The colorectal cancer tissues from 143 patients were collected from August 2004 to December 2008 in the First Affiliated Hospital, Sun Yat-sen University. Twenty-night cases of the adjacent normal colorectal tissues were enrolled as controls. The expression of CXCR6 was detected by immunohistochemistry and the mean intergrated absorbance ( mIA ) was calculated by Image-Pro Plus 6.0 software. The relationship between CXCR6 expression and synchronous liver metastasis/prognosis was analyzed. RESULTS: The CXCR6 staining was mainly positive in colorectal cancer tissues but not in adjacent normal colorectal tissues. The mIA of CXCR6 in colorectal cancer was 1.54±0.04 (range: 0.41~2.84), and was 1.63±0.05 and 1.41±0.08 (P<0.05) in the cases with (n=83) or without (n=60) synchronous liver metastasis, respectively. According to the mean mIA of CXCR6 (1.54), the cases was divided into high CXCR6 group (mIA≥1.54) and low CXCR6 group ( mIA <1.54). The overall survival rate in high CXCR6 group was significantly lower than that in low CXCR6 group (P<0.05). In multivariate Cox regression models, age (P<0.05), lymph node metastasis (P<0.05) and synchronous liver metastasis (P<0.01) but not CXCR6 were identified as independent risk factors for poor outcome. In subgroup analysis, high CXCR6 expression was associated with poorer survival in the patients with stage I~III colorectal cancer (P<0.01) but not those with synchronous liver metastasis (P>0.05).CONCLUSION: CXCR6 in primary colorectal cancer tissues is associated with liver metastasis. It may become a potential target for the treatment of colorectal cancer liver metastasis.     

Significance of CK19 expression in detecting lymph node micrometastases in patients with laryngeal carcinoma

AIM: To evaluate the significance of cytokeratin19 (CK19) expression in diagnosing micrometastases in lymph nodes in patients with laryngeal carcinoma. METHODS: Forty cases of laryngeal carcinoma together with 163 lymph nodes were studied by the staining with hematoxylin and eosin(HE)and immunostaining with antibody against cytokeratin 19 (CK19) on histological sections of the primary tumor and regional lymph nodes. RESULTS: In all cases, CK19 was positively expressed in the primary tumor. Among 163 lymph nodes, metastases were confirmed by HE in 23 lymph nodes, and in 42 lymph nodes staining with immunohistochemistry, micrometastases were found in 19 lymph nodes. Micrometastases in the lymphonodes were significantly related to the T staging. CONCLUSION: Immunohistochemical staining is a valuable method for the detection of node micrometastases in patients with laryngeal carcinoma.     

Relationship between activity of matrix metalloproteinases-2 and invasion,metastasis of breast cancer

AIM: To investigate relationship between activity of matrix metalloproteinases-2 (MMP-2, 72 kD) and invasion, metastasis of breast cancer. METHODS: Useing zymography and computer software assisted analysis, the activitive levels of MMP-2 (72 kD) in tissues from breast cancer were measeured. RESULTS: Mean activitive levels of MMP-2 72 kD (13.93±3.60) in breast cancer were lower than those in benign disease (21.43±8.31), P<0.05. There was no difference (P>0.05) in MMP-2 62 kD+72 kD of benign and malignant disease, but MMP-2 62 kD (13.83±4.53) and MMP-2 62 kD/62 kD+72 kD(0.48) respectively were significantly higher in malignant disease (P<0.01). It was also found that MMP-2 62 kD/62 kD+72 kD were apparently higher in invasive carcinomas (0.48) and lymph node metastases (0.61), P<0.01, respectively. CONCLUSION: These results demonstrated that a clear relationship between MMP-2 activity and the invasion and metastasis of breast carcinoma.     

Expression and clinical significance of Bmi-1 in colorectal cancer

AIM:This study was to investigate the expression and significance of Bmi-1 in colorectal carcinoma (CRC), and to explore the effect of Bmi-1 on Ki67 expression in human CRC.METHODS:The samples from sixty CRC, thirty adenomas and twenty normal colorectal mucosal tissues were used in this study.The expression of Bmi-1 protein was detected by immunohistochemistry.The clinicopathological features and survival rate of patients were also analyzed.RESULTS:The overexpression of Bmi-1 was respectively 25.0％, 6.7％and 0% in CRC and adenomas as well as normal colorectal mucosal tissues.The results showed that the expression of Bim-1 was significantly higher in CRC, compared with that in adenomas and normal colorectal mucosal tissues (P<0.05).The overexpression of Bmi-1 protein in CRC was obviously associated with distant metastasis and TNM stage (P<0.05), but not with gender, age, tumor size, tumor site, histological type, differentiation degree and lymph node metastasis (P>0.05).Kaplan-Meier survival analysis showed that the overexpression of Bmi-1 reduced significantly survival of CRC patients (P<0.05).No statistical relation between expression of Bmi-1 and Ki67 in CRC was observed.CONCLUSION:The overexpression of Bmi-1 protein is significantly correlated with tumorigenesis, metastasis and prognosis of CRC.Bmi-1 might be regarded as a parameter in evaluating prognosis of CRC.     

Expression of midkine and its clinical significance in transitional cell carcinoma of human urinary bladder

AIM: To investigate the expression of midkine in bladder transitional cell carcinoma and to analyze its relationship with the features of clinical pathology and prognosis.METHODS: The expressions of midkine protein in 50 cases of bladder transitional cell carcinoma samples were detected by SP immunohistochemical method using polyclonal antibodies against human midkine.Survival time of 40 cases was recorded.RESULTS: The protein expression of midkine was found in cytoplasm of tumor cells.The overall positive rate of midkine in 50 cases of bladder carcinoma was 90% (45/50).The positive degree of midkine showed a trend of increasing in grade and stage.There was statistically significant difference among them (P<0.05),but not with sex,age,treatment or tumor number and size (P>0.05).Patients with high expression of MK predicted a poor clinical outcome.CONCLUSION: Midkine is overexpressed in bladder transitional cell carcinoma than that in normal bladder.MK expression in bladder cancer is higher in less differentiated and deeper invaded cases,but it has no correlation with age,sex,treatment,tumor number and size.Patients with higher MK expression have shorter survival time than those with lower MK expression.     

Analyzing clinical characteristic of miR-196b in human colorectal cancer using TCGA and effect of miR-196b on 5-FU resistance of CRC cell line

AIM: To investigate the clinical characteristics of microRNA (miR)-196b in colorectal cancer (CRC) and to study its biological function in 5-fluorouracil (5-FU) resistance. METHODS: miRNA sequence dataset and the corresponding clinical data of CRC patients were downloaded from The Cancer Genome Atlas (TCGA). Expression level and clinical characteristics of miR-196b in CRC patients were analyzed using SPSS 17.0. CRC cell line overexpres-sing miR-196b was established using transient transfection method. MTS test was used to evaluate the effect of miR-196b overexpression on 5-FU resistance. RESULTS: miR-196b expression was associated with lymph node metastasis and TNM stage (P<0.05), but not related with age and sex. Lymph node metastasis and distant metastasis were independent prognostic factors for rectal patients (P<0.05). The expression level of miR-196b was not associated with survival condition of rectal patients. The viability of the cells overexpressing miR-196b treated with different concentrations of 5-FU was significantly higher than that in control group (P<0.05). CONCLUSION: miR-196b may be a potential biomarker of TNM stage and lymph node metastasis in CRC. miR-196b increases the 5-FU resistance of CRC cells.     

Study on the expression of pituitary tumor-transforming (PTTG) and fragile histidine triad (FHIT) in gastric cancer and pericancerous tissues

AIM:To Study on the expressive levels of PTTG and FHIT and detect their clinicopathological significances in gastric cancer and pericancerous tissues.METHODS:Immunohistochemical method was used on routinely paraffin-embedded sections of 49 cases with gastric cancer and 20 subjects with pericancerous tissues. RESULTS:The positive rate and score of PTTG in gastric cancer were significantly higher than those in pericancerous tissues (P<0.05,P<0.01),but those of FHIT were opposite (P<0.05,P<0.01),the positive cases of PTTG and negative subjects of FHIT showed severely-atypical hyperplasis. The positive rate and score of PTTG were significantly lower in the cases of infiltrating depth T₁-T₂ and without-metastasis of distant organs than those in the subjects of infiltrating depth T₃-T₄ and with-metastasis of distant organs(P<0.05,P<0.01). The positive rate and score of FHIT were significantly higher in the cases of infiltrating depth T₁-T₂,without-metastasis of lymphnodes,with-metastasis of the first site lymphnodes,and without metatstasis of distant organs than those in the subjects of infiltrating depth T₃-T₄,with-metastasis of lymphnodes,with-metastasis of the third site lymphnodes,and with-metastasis of distant organs(P<0.05,P<0.01). The significantly negative correlation was found between the score of PTTG and FHIT in gastric cancer tissues(r=-0.36,P<0.01). CONCLUSION:The expression of PTTG and FHIT might be important biological markers for reflecting the carcinogenesis,progression,invasive potential,metastastic status and prognosis of gastric cancer. The assays of PTTG and FHIT in benign lesions might have clinical values for the prevention and early-stage finding of gastric cancer.     

Quantitative perfusion parameters of dynamic contrast-enhanced magne-tic resonance imaging in patients with rectal cancer: estimation of the microvascular perfusion and permeability

AIM: To investigate the perfusion parameters using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in rectal cancer patients so as to explore its potential value in estimating the microvascular condition including perfusion and permeability. METHODS: The data of 38 rectal cancer patients examined with DCE-MRI was retrospectively analyzed. The perfusion parameters of carcinoma and normal rectal wall in each case were calculated, including volume transfer constant (K^trans), rate constant of back flux (K_ep), extravascular extracellular space fractional volume (V_e) and initial area under curve (iAUC). The mean values of tumor and normal rectal wall, mucinous and nonmucinous carcinoma, poorly and moderately-to-well differentiated carcinoma, case with or without lymph node metastasis were compared. RESULTS: All the parameters of rectal cancer were higher than normal rectal wall (P<0.01). No significant difference was found between poorly and moderately-to-well differentiated carcinoma in terms of K^trans, K_ep and V_e, neither was the case with or without lymph node metastasis. The cases with lymph node metastasis had lower iAUC than those without (P<0.05). CONCLUSION: Quantitative perfusion DCE-MRI answered the microvascular perfusion and permeability change of rectal cancer compared with normal rectal wall, besides it could be used to distinguish between mucinous and nonmucinous carcinoma, which demonstrated its value in the evaluation of rectal cancer. However, it should not be recommended to predict the degrees of tumor cell differentiation and lymph node metastasis just according to the perfusion parameters.     

DNA ploidy analysis and the expression of TIMP-2 and E-cadherin in gastric carcinoma

AIM:To investigate DNA ploidy and the expression of TIMP-2 and E-cadherin in gastric carcinoma in order to understand its molecular basis and probable mechanism of invasion and metastasis. METHODS:Immunohistochemical methods were used to detect the expression for TIMP-2 and E-cadherin in 99 cases of gastric carcinoma, 16 cases of adjacent noncancerous mucosa, 16 cases of distant metastases and 25 cases of metastatic lymph nodes. Flow cytometry DNA ploidy and S-phase fraction (SPF) analysis was performed on 47 cases of gastric cancer, 6 cases of adjacent noncancerous mucosa and 4 cases of distant metastasis cancer with the use of formalin-fixed paraffin embedded specimens. RESULTS:The expression of TIMP-2 was significantly correlated with Borrmann’s classification, LN metastasis and the depth of invasion. The expression of E-cadherin was significantly correlated with tumor cell differentiation, Lauren’s classification, Borrmann’s classification, LN metastasis and the depth of invasion. There was a positive relationship between DNA aneuploid rate and differentiation and LN metastasis. There was a positive relationship between SPF that is higher than 15% and tumor size, differentiation and LN metastasis. And there was a significantly difference between carcinoma and noncarcinoma when the expression of E-cadherin, DNA aneuploid rate and SPF were analyzed. There was no correlation between TIMP-2 and E-cadherin. There was a positive relationship between DNA ploidy or SPF and the expression of E-cadherin. CONCLUSION:As the development of tumor progression and heterogeneity, the abnormal expression of TIMP-2 or E-cadherin or the rate of DNA aneupoid or higher SPF gradually correspondingly increases, suggesting that they play a crucial role in gastric carcinoma progression. Furthermore, each factor influences one another and further accelerates the process of tumor progression.     

Expression and prognostic significance of SLP-2 in gastric cancer

AIM: To investigate the expression of the red cell membrane integration protein SLP-2 (stomatin-like protein 2) in gastric cancer tissues and to analyze its correlation with clinicopathological manifestations and prognosis. METHODS: One hundred and ninety gastric cancer tissue samples with detailed clinical information were collected from the Department of Pathology, Sun Yat-sen University Cancer Center. The protein expression of SLP-2 in ganstric cancer was detected by the method of immunohistochemistry. The relationships between SLP-2 expression and the clinicopathological manifestations were evaluated. RESULTS: The positive rate of SLP-2 in gastric cancer tissue was 63.2% (120/190). SLP-2 expression was relevant to infiltration depth, TNM stage and lymph node metastasis (P<0.05). However, no statistical difference was observed in the SLP-2 expression associated with sex, age, differentiation, tumor size and distant metastases. Kaplan-Meier survival curves revealed that increased expression of SLP-2 was associated with poor prognosis in gastric adenocarcinoma patients (P<0.01). Based on the univariate analysis, 7 factors were found to have statistical significance of associations with overall survival, including SLP-2 expression, lymph node metastasis, histological grade, tumor size, invasive depth, distant metastases and the 7th edition of the UICC TNM classification. Only the tumor size and the 7th edition of the UICC TNM classification were independent prognostic factors for overall survival in the multivariate analysis. CONCLUSION: SLP-2 is highly expressed in gastric adenocarcinoma tissues and may play an important role in tumor progression and metastasis. Although SLP-2 is not an independent prognostic factor, it may influence the prognosis of gastric cancer. Increased expression of SLP-2 can be used for predicting unfavorable prognosis in gastric adenocarcinoma patients.     

Identification and establishment of sorting method for isolating CD11b+ myeloid cells in human hepatic metastases from colorectal cancer

AIM: To establish a method for obtaining specific cells in solid tumor tissue by sorting of CD11b⁺ myeloid cells in hepatic metastases from colorectal cancer.METHODS: Tumor tissues were prepared into single cell suspension by mechanical method combined with enzyme digestion, and then the CD11b⁺ myeloid cells were isolated by flow cytometry. The sorted cells were identified by immunocytochemistry. The viability and morphologiy of the sorted cells were evaluated by Giemsa and Typan blue staining. The cell purity was evaluated by flow cytometry.RESULTS: Sufficient numbers of CD11b⁺ cells with high purity were isolated by sorting with flow cytometry from the single cell suspension prepared by mechanical and enzyme digestion. The purity of the cells was confirmed by statistical analysis (P<0.05). The positive rates of the cells before and after sorting were significantly different (P<0.01). The positive cells were verified by immunocytochemical method. Meanwhile, the sorted cells had complete morphology and good activity.CONCLUSION: The CD11b⁺ myeloid cells in solid tumor tissue can be isolated by flow cytometry from the machine-enzyme digestion suspension with high purity, good activity and complete morphology.