Clonality analysis of neuroendocrine cells in gastric adenocarcinoma.

AIM: To achieve a better understanding of the origination of neuroendocrine (NE) cells in gastric adenocarcinoma. METHODS: In this study, 120 cases of gastric adenocarcinoma were obtained. First, frozen section-immunohistochemistrical samples were selected from a large quantity of neuroendocrine cells. Second, laser capture microdissection was used to get target cells from gastric adenocarcinoma and whole genome amplification was applied to get a large quantity of DNA for further study. Third, genome-wide microsatellite abnormalities [microsatellite instability (MSI), loss of heterozygosity (LOH)] and p53 mutation were detected by polymerase chain reaction (PCR)-single-strand conformation polymer- phism-silver staining and PCR-sequencing in order to identify the clonality of NE cells. RESULTS: The total incidence rate of MSI was 27.4%, while LOH was 17.9%. Ten cases had a highest concordance for the two types of cells. The other samples had similar microsatellite changes, except for cases 7 and 10. Concordant p53 mutations exhibited in sample 4, 14, 21 and 27, and there were different mutations between two kinds of cells in case 7. In case 17, mutation took place only in adenocarcinoma cells. p53 mutation was closely related with degree of differentiation, tumor-node-metastasis stage, vessel invasion and lymph node metastasis. In brief, NE and adenocarcinoma cells showed the same MSI, LOH or p53 mutation in most cases (27/30). In the other three cases, different MSI, LOH or p53 mutation occurred. CONCLUSION: NE and the gastric adenocarcinoma cells may mainly derive from the same stem cells, but the remaining cases showing different origin needs further investigation.

[Detection and clinicopathologic significance of microsatellite alteration and p53 mutation of neuroendocrine cells in colorectal adenocarcinoma].

OBJECTIVE: To study the possible clonal origin of neuroendocrine cells in colorectal adenocarcinoma. METHODS: Twenty-six microsatellite loci were screened using laser capture microdissection, DNA extraction and whole genome amplification. Microsatellite instability (MSI) and loss of heterozygosity (LOH) in adenocarcinoma cells and neuroendocrine cells amongst 30 cases of colorectal carcinoma with neuroendocrine differentiation were detected using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP)-silver staining. The mutation status of p53 was evaluated by PCR-sequencing. The clonal origin of neuroendocrine cells in colorectal adenocarcinoma was determined. RESULTS: Amongst the 30 cases studied, the prevalence of MSI was 16.9% while that of LOH was 8.5%. The rate showed no statistically significant difference between adenocarcinoma cells and neuroendocrine cells. In 6 cases, the microsatellite alteration was entirely consistent. In 23 cases, the rate of microsatellite alteration consistency was greater than that of inconsistency. In 1 case, the consistency and inconsistency rates were identical. There was statistically significant difference between consistency and inconsistency of microsatellite alteration. The prevalence of p53 mutation was 16.7% which was the same for both adenocarcinoma cells and neuroendocrine cells. CONCLUSIONS: Adenocarcinoma cells and neuroendocrine cells in colorectal adenocarcinoma with neuroendocrine differentiation have similar biologic changes. It is likely that they are of identical origin.

[Regulation and mechanism of Notch signaling pathway in small cell lung cancer].

OBJECTIVE: To investigate the status of Notch signaling pathway in small cell lung cancer (SCLC). METHODS: Expression plasmids of pEFBOS-NIC-MYC and pEFBOS-neo were transfected into NCI-H446 cells. Stably transfected cell lines were selected and their growth rates were examined by MTT method. Expression of downstream genes along the Notch signaling pathway were studied by RT-PCR. Protein expression of euroendocrine markers of CgA and NSE were detected by Western blot analysis and immunocytochemistry. RESULTS: The expression of HES1 was increased in the pEFBOS-NIC-MYC group, but the expression of hASH in the pEFBOS-NIC-MYC group was decreased significantly. The transfected cells with pEFBOS-NIC-MYC plasmid showed a significantly slower growth rate compared with that of two control groups (P < 0.05, Student's t-test). Immunocytochemistry of NSE showed that PUs in the NIC transfected group, sham group and negative control group were 7.21 ± 0.59, 28.25 ± 1.46, 30.57 ± 1.31 respectively, the former one was smaller than the values of the latter two significantly (P < 0.01). Western blot analysis showed the grave scales of CgA in NIC transfected group and sham group to be 0.54 ± 0.03 and 0.99 ± 0.05 respectively (grave scales of the negative control was set as 1.00), the former one significantly smaller than that of the other two groups (P < 0.01). The grave scales of NSE in the NIC transfected group and sham group were 0.43 ± 0.02 and 1.07 ± 0.09 respectively (grave scales of the negative control was set as 1.00) and the former one was significantly smaller than the other two groups (P < 0.01). CONCLUSION: Notch signaling pathway regulates SCLC cells through its inhibitory effect on hASH1 transcription via HES1 along with an expression inhibition of neuroendocrine markers in SCLC.

[Expression and significance of CD147 and E-cadherin in human gastric carcinoma].

OBJECTIVE: To investigate the expression of CD147 and E-cadherin in gastric carcinoma and their correlation with clinicopathological features. METHODS: The expression of CD147 and E-cadherin in gastric cancer tissue chip (TC) was detected by in situ hybridization (ISH) and immunohistochemistry in 220 cases of gastric carcinoma and 31 cases with normal gastric mucosa. RESULTS: The expression rates of CD147 mRNA, E-cadherin mRNA and E-cadherin protein were 50.5%, 17.7% and 15.5%, respectively. The CD147 expression was negatively correlated with E-cadherin expression. There was a significant relationship between CD147 mRNA expression and tumor diameter, TNM stage, invasion depth, vessel invasion, lymph node and distant metastasis. The E-cadherin mRNA expression was closely related with TNM stage, invasion depth and vascular invasion. There was a significant relationship between E-cadherin protein expression and tumor diameter, TNM stage and vascular invasion. The mean survival time in cases with CD147-positive expression was shorter than that in negative cases, while E-cadherin was in an opposite relationship. CONCLUSION: In gastric carcinoma, up-regulation of CD147 and down-regulation of E-cadherin are in a negative correlation. The examination of both these two factors together is useful for predicting the invasion and metastasis of gastric cancer, therefore, can be used as a significant marker to direct clinic therapy and estimation of prognosis.

[Inhibitory effects of Notch1 overexpression on proliferation and neuroendocrine marker expression in a small cell lung cancer cell line].

OBJECTIVE: To investigate the effects of Notch1 signal activation on proliferation and neuroendocrine marker expression in small cell lung cancer cells. METHODS: The active form of Notch1 (NIC) was over-expressed in NCI-H446 cells by constitutive transfection and a stable transfected cell line was established. Proliferation of NCI-H446 cells was analysed by MTT assay on 6 successive days. Expression of neuroendocrine markers (CgA, NSE) was observed by immunohistochemistry and Western blot analysis. Statistical analysis was conducted to compare the results in cells with NIC transfected and those in control groups. RESULTS: MTT assay showed that absorbance (A) of cells overexpressing Notch1 was significantly depressed compared with that of the control cells (P<0.05). Immunohistochemistry of CgA showed that PUs in the NIC transfected group, sham group and negative control group were 8.81 +/- 0.77, 38.10 +/- 1.55, 38.97 +/- 0.80, respectively, the former one was significantly smaller than that of the latter two (P<0.01). Immunohistochemistry of NSE showed that PUs in the NIC transfected group, sham group and negative control group were 7.21 +/- 0.59, 28.25 +/- 1.46, 30.57 +/- 1.31, respectively, the former one was significantly smaller than that in the latter two (P<0.01). Western blot analysis showed that the gray scales of CgA in the NIC transfected group and sham group were 0.54 +/- 0.03 and 0.99 +/- 0.05, respectively, (gray scale of the negative control set as 1.00), the former one was significantly smaller than that of the other two groups (P<0.01). The gray scales of NSE in the NIC transfected group and sham group were 0.43 +/- 0.02 and 1.07 +/- 0.09, respectively (gray scale of the negative control set as 1.00), the former one was significantly smaller than that of the other two groups (P<0.01). CONCLUSION: Notch1 may behave as a tumor suppressor in small cell lung cancer. Notch1 signal activation can inhibit the proliferation and neuroendocrine marker expression in small cell lung cancer cells, suggesting that Notch1 gene could be a new target for small cell lung cancer treatment and probable relief of paraneoplastic syndrome.

[Study on the expression and significance of Galectin-3 and CDC25B mRNA in human gastric carcinoma].

OBJECTIVE: To study the expression of Galectin-3 and CDC25B mRNA in gastric carcinoma and their correlation with clinical-pathological features and the survival time. METHODS: Tissue microarray (TMA) technique and in situ hybridization were used to detect the expression of Galectin-3 and CDC25B mRNA in 220 gastric carcinoma specimens and 31 normal gastric mucosa samples. RESULTS: In situ hybridization results revealed that from the 220 cases, the positive expression rate of Galectin-3 and CDC25B mRNA were 58.6% and 54.1%, respectively. There was significant relationship between the Galectin-3 mRNA expression and tumor diameter, advanced TNM stage, invasion depth, vessel invasion, lymph node and distant metastasis. There was significant relationship between CDC25B mRNA expression and tumor diameter, advanced TNM stage, vessel invasion, lymph node and distant metastasis. In addition, there was apositive relationship of Galectin-3 and CDC25B mRNA expression. Finally, the mean survival time in cases with Galectin-3 and CDC25B mRNA positive expression was significantly shorter than those without Galectin-3 and CDC25B expression. CONCLUSION: The expression of Galectin-3 and CDC25B mRNA appears to act as a promoting factor in the onset and development of gastric cancer. It can be used as a marker of prognosis of gastric carcinoma in clinical practice.

[Microsatellite instability and allele-specific chromosome 3p deletion in breast cancer and precancerous lesions].

OBJECTIVE: To investigate the incidence and clinicopathologic significance of MSI and LOH on 3P in breast carcinoma and its precancerous lesions, intraductal papillary adenoma and ductal carcinoma in situ. METHODS: 41 paired sporadic invasive breast carcinomas, 13 archival precancerous lesion specimens of the breast and 14 couples of benign hyperplasia were collected. Twelve microsatellites on chromosomes 2p, 3p, 5q, 6q, 16q, 17q, eleven markers on chromosome 3p were amplified for MSI and LOH, respectively, by polymerase chain reaction ( PCR ) with designed primers and detecting after polyacrylamide gel electrophoresis. In addition, the expression of protein of hMSH2, hMLHI, FHIT, ER, and PR were detected by immunohistochemistry. RESULTS: MSI was observed, at least two microsatellite markers, in 15 out of 41 (36. 6%) of the carcinomas, almost all belonging to poorly or intermediately differentiated carcinoma. Instability was shown in 9 of the 13 cases of precancerous lesions, but only 2 among them had more than 2 MSI sites. There was no MSI in benign hyperplasia. MSI was targeted predominately at D3S1766, D2S2739 in both carcinomas and precancerous lesions. Of the 11 loci examined, D3S1295, D3S1029 and D3S1038 were identified as the locus with most frequent LOH which were all correlated significantly with some clinicopathological parameters such as histological type, lymph node metastasis in breast cancer, while D3S1295 and D3S1029 were the most frequent markers in precancerous lesions. LOH of D3S1295 had significant correlation with negative expression of FHIT. Positive expression of hMLH1 and hMSH2 protein was detected in breast carcinomas in scattered distribution and their positive rate was 45% and 40% , respectively. In precancerous lesions, hMLH1 and hMSH2 protein showed diffuse expression and their positive rate was 61. 54% and 76. 92% , respectively, significantly lower than that in the control tissues. CONCLUSION: Defective expression of MMR genes is closely associated with the development of breast cancer. Genomic instability might play a role in the early stage during multi-step mammary carcinogenesis. MSI indicates poor histological differentiation in breast carcinoma. D3S1766 and D2S2739 might be the sensitive sites to detect MSI in breast carcinoma and precancerous lesions. The smallest common LOH deletion regions seem likely to be situated between 3p14 and 3p25, indicating the existence of breast tumor related genes in those regions and some of them might affect tumor development.

[Loss of heterozygosity on chromosome 3p in breast cancer and precancerous lesion].

OBJECTIVE: To study the loss of heterozygosity (LOH) on chromosome 3p in breast cancers and precancerous lesion. METHODS: LOH at 11 microsatellite loci was detected in 41 cases of breast cancers and 12 cases of precancerous lesion by polymerase chain reaction and silver stain. The expressions of ER, PR, FHIT and hMLH1 were detected in breast cancer by immunohistochemistry. RESULTS: LOH on 3p was detected in 97% of breast cancers. D3S1295, D3S1029 and D3S1038 located at 3p14, 3p21-p22 and 3p25 were identified as the loci with most frequent LOH (53.1%, 43.6% and 52.5%). LOH of D3S1038 and expression of hMLH1 protein correlated with several clinicopathological features. LOH of D3S1295 had significant negative correlation with the expression of FHIT. In breast precancerous lesions, LOH on 3p was detected in 41.7% lesions. D3S1295 and D3S1029 were also identified as the most frequent LOH locus (27.3% and 16.7%). The smallest common LOH region seems likely lie between 3p14 and 3p25. CONCLUSIONS: The smallest LOH region indicates the existence of breast tumor related genes and some of them affect gene expression.

Correlative studies on bFGF mRNA and MMP-9 mRNA expressions with microvascular density, progression, and prognosis of gastric carcinomas.

AIM: To investigate the mRNA expressions of bFGF and MMP-9 in gastric carcinomas so as to reveal their correlations with tumor microvascular density (MVD), invasion, metastasis, and prognosis. METHODS: In situ hybridization and immunohistochemical techniques were used to detect the expressions of bFGFmRNA and MMP-9mRNA and the proteins of CD34 in 105 specimens of gastric carcinomas. RESULTS: In situ hybridization study showed that positive rates of bFGF mRNA and MMP-9mRNA expressions were 60.95% and 59.19%; the mean MVD was 46.09+/-11.52 and 43.75+/-13.41, respectively in piece/0.72 mm(2) in tumors with bFGFmRNA and MMP-9mRNA positive expressions, which were significantly higher than those with negative expression (29.41+/-12.47; 33.45+/-13.92 piece/0.72 mm(2), respectively). The positive expression rates of bFGFmRNA and MMP-9mRNA were correlated to the tumor invasion depth (r(s) = 0.211, P = 0.031; r(s) = 0.335, P = 0.001), growing pattern (r(s) = 0.324, P = 0.001; r(s) = 0.267, P = 0.006), vessel invasion (r(s) = 0.579, P = 0.001; r(s) = 0.209, P = 0.032), lymph node metastasis (r(s) = 0.405, P = 0.001; r(s) = 0.343, P = 0.001) and distant metastasis (r(s) = 0.474, P = 0.001; r(s) = 0.468, P = 0.001), but not correlated to tumor type (r(s) = 0.134, P = 0.173; r(s) = 0.103, P = 0.145) and differentiations (r(s) = 0.096, P = 0.332; r(s) = 0.102, P = 0.298). The mean MVD was much higher in the tumors with infiltrating growth at stage T(3)-T(4), with vessel invasion, lymph node metastasis and distant metastasis than those with expanding growth type (t = 10.105, P = 0.001) at stage T(1)-T(2) (t = 5.961, P = 0.001), with non-vessel invasion (t = 7.394, P = 0.001), non-lymph node metastasis (t = 3.819, P = 0.01) and non-distant metastasis (t = 10.578, P = 0.001). Positive correlation was observed between MVD and the expressions of bFGFmRNA and MMP-9mRNA (t = 3.207, P = 0.002; t = 7.035, P = 0.001, respectively). The mean survival time and 5-year survival rate were lower in cases with MVD over 39.5 and the positive expressions of bFGFmRNA and MMP-9mRNA than those with MVD less than 39.5 and the negative expressions of bFGFmRNA and MMP-9mRNA. CONCLUSION: bFGF and MMP-9 promote the angiogenesis of the gastric cancers. Detection of the expressions of bFGF and MMP-9 can serve as a useful index to determine the angiogenesis, invasion, metastasis, and prognosis of gastric cancers.

[Relationship between bFGF mRNA and MMP-9 mRNA expression in gastric carcinoma and their clinicopathological features as well as patients survival].

OBJECTIVE: To investigate the mRNA expression of bFGF and MMP-9 in gastric carcinomas and to find their correlation with tumor microvascular density (MVD), invasion, metastasis and patients survival. METHODS: In situ hybridization and immunohistochemistry technique were used to test the expression of bFGF mRNA and MMP-9 mRNA and protein of CD34 in 105 specimens of gastric carcinoma. RESULTS: In situ hybridization revealed that the positive rates of bFGF mRNA and MMP-9 mRNA were 60.95 and 58.1%, respectively; The mean MVD (46.09 +/- 11.52, 43.75 +/- 13.41 piece/0.72 mm(2)) in tumors with bFGF mRNA and MMP-9 mRNA positive expression was significantly higher than that (29.41 +/- 12.47; 33.45 +/- 13.92 piece/0.72 mm(2)) in tumors with their negative expression, respectively; The positive expression rates of bFGF and MMP-9 mRNA were correlated to invasion depth (r(s) = 0.211, P = 0.031; r(s) = 0.335, P = 0.001, respectively), growing pattern (r(s) = 0.324, P = 0.001; r(s) = 0.267, P = 0.006, respectively), vessel invasion (r(s) = 0.579, P = 0.001; r(s) = 0.209, P = 0.032, respectively), lymph node metastasis (r(s) = 0.405, P = 0.001; r(s) = 0.343, P = 0.001, respectively) and distant metastasis (r(s) = 0.474, P = 0.001; r(s) = 0.468, P = 0.001, respectively), but not correlated to tumor type (r(s) = 0.134, P = 0.173; r(s) = 0.103, P = 0.145, respectively) and differentiation (r(s) = 0.096, P = 0.332; r(s) = 0.102, P = 0.298, respectively); And then, the mean MVD in tumors with infiltrating type, stage T(3)-T(4), vessel invasion, lymph node metastasis and distant metastasis was significantly higher than that in tumors with expanding type (t = 10.105, P = 0.001), stage T(1)-T(2) (t = 5.961, P = 0.001), non-vessel invasion (t = 7.394, P = 0.001), non-lymph node metastasis (t = 3.819, P = 0.01) and non-distant metastasis (r = 10.578, P = 0.001); There was a positive relationship between MVD and bFGF mRNA and MMP-9 mRNA (t = 3.207, P = 0.002; t = 7.035, P = 0.001), respectively; the mean survival time in cases with positive bFGF mRNA and MMP-9 mRNA and MVD value >/= 39.5 was significantly shorter than that in cases with their negative expression and MVD value < 39.5. CONCLUSIONS: bFGF and MMP-9 promote angiogenesis in gastric cancer. Test of the expression of bFGF and MMP-9 may act as an useful index to determine angiogenesis, invasion, metastasis and patients survival.

Biological characteristics of breast carcinomas with neuroendocrine cell differentiation.

BACKGROUND: The aim of this study was to investigate DNA content and expression of c-erbB-2, PS2, and prostate-specific antigen (PSA) proteins in breast carcinomas with neuroendocrine (NE) cell differentiation. METHODS: Chromogranin, c-erbB-2, PS2, and PSA in 131 samples of breast cancer were detected immunohistochemically. Classic Feulgen staining image analysis techniques were used to quantify DNA content in 81 of the breast cancer samples. RESULTS: The c-erbB-2 positive rate in breast carcinoma samples containing neuroendocrine cells was 37.5% and the rate of high expression of c-erbB-2 (++ or +++) was 33.3%, both significantly lower than that in breast carcinomas without neuroendocrine cells (62.6% and 68.7%, respectively, P < 0.05). The rates of positive PS2 and PSA expression in breast carcinoma samples containing neuroendocrine cells were 72.2% and 55.0%, respectively, both significantly higher than that in breast carcinoma samples without neuroendocrine cells (45.0% and 16.4%, respectively, P < 0.05). In NE(+) samples, the integral optical density, DNA index, DNA stemline peak, > 5 c aneuploidy cells, and rate of aneuploidy among cells were all lower than that in NE(-) breast carcinomas (P < 0.01). In NE(+) grade I or II breast carcinomas, these indices were also all lower than that in the NE(-) breast carcinoma samples (P < 0.01). CONCLUSION: Breast carcinomas with neuroendocrine differentiation have a lower rate of malignancy. Neuroendocrine differentiation could serve as a prognostic marker in clinical practice.

Neuroendocrine markers in adenocarcinomas: an investigation of 356 cases.

AIM: To investigate the incidence of neuroendocrine (NE) cells and their hormone products in adenocarcinomas and evaluate their significance in clinical pathology and prognosis. METHODS: By using tissue sectioning and immunocyto-chemistry, 356 cases of adenocarcinomas were studied to examine the presence of chromorgranin and polypeptide hormones in adenocarcinoma samples from our hospital. RESULTS: The positive rate of NE cells and hormone products was 41.5 % (54/130) and 59.3 % (32/54), respectively in large intestinal adenocarcinoma cases; 39.6 % (38/96) and 36.8 % (14/38), respectively in gastric cancer cases; 38.1 % (8/21) and 50.0 % (4/8), respectively in prostatic cancer cases; 21.0 % (17/81) and 17.6 % (3/17), respectively in breasr cancer cases; 17.9 % (5/28) and 60.0 % (3/5), respectively in pancreatic cancer cases. Among carcinomas of large intestine, pancreas and breast, the highly differentiated NE cell numbers were higher than the poorly differentiated NE cell numbers; while the gastric carcinoma cases had more poorly differentiated NE cells than highly differentiated NE cells. The higher detection rate of NE cells and their hormone products, the higher 5-year survival rate among the large intestine cancer cases. CONCLUSION: Close correlation was observed between NE cells and their hormone products with the cancer differentiations. For colorectal carcinomas, there is a close correlation of the presence of NE cells and their hormone products with the tumor staging and prognosis.

[Study of neuroendocrine markers in 356 cases of adenocarcinomas]

OBJECTIVE: To ivestigate the incidence of neuroendocrine (NE) cells and hormone products in adenocarcinomas and to explore its clinicopathological significance. METHODS: 356 cases of adenocarcinomas were studied by immunocytochemistry with antibodies for chromorgranin and polypeptide hormones. RESULTS: The prevalence of NE cells and hormone products were detected in 54 of 130(41.5%) and 32 of 54 (59.3%) colorectal carcinomas, 38 of 96(39.6%) and 14 of 38 (36.8%) gastric cancer, 8 of 21(38.1%) and 4 of 8(50.0%) prostatic carcinomas, 17 of 81(21.0%) and 3 of 17(17.6%) breast cancer, 5 of 28(17.9%) and 3 of 5 (60.0%) pancreatic carcinomas, respectively. Among carcinomas of large intestine, pancreas and breast, the incidence of NE cells in well differentiated ones was higher than that in the poorly differentiation. By contrast, NE cells were found more frequently in the letter than in the former in gastric carcinoma. The cases with NE cell (++) or polypeptide positive cells exhibited higher 5-year survival rate than those without NE cells in colorectal carcinomas. CONCLUSION: The presence of neuroendocrine cells and the hormone products may be close correlated with the degree of tumore cell differentiation. For colorectal carcinoms, there is a close correlation of the presence of NE cells and the hormone products with the tumor staging and prognosis.