Glypican 3 expression in hepatoblastoma and its diagnostic implication / 中华病理学杂志

<p><b>OBJECTIVE</b>To explore the expression and diagnostic significance of glypican-3 (GPC3) in hepatoblastoma.</p><p><b>METHODS</b>Five tissue microarray paraffin blocks were constructed to include 54 cases of hepatoblastoma. The tumor tissue samples were obtained from 3 surgical biopsies, 33 needle biopsies, 5 stage I resection tumors, and 13 stage II resection tumors after transcatheter arterial chemoembolization. Ten samples of non-neoplastic hepatic tissue adjacent to tumor were used as control. Immunohistochemical staining of GPC3 (clone 1G12) was performed. Among the 54 cases of hepatoblastoma, 22 cases were fetal subtype, 24 cases were mixed fetal and embryonal subtype and 8 cases were mixed epithelial and mesenchymal type.</p><p><b>RESULTS</b>GPC3 was positive in fetal epithelial cells (54/54, 100%), but negative or weakly positive in embryonic epithelial cells in all cases of hepatoblastoma. Undifferentiated small cells and all mesenchymal components were negative for the expression. Non-neoplastic hepatocytes adjacent to tumor were negative for GPC3 expression (0/10) .</p><p><b>CONCLUSIONS</b>Fetal epithelial components of hepatoblastoma express GPC3 protein detectable by immunohistochemistry. Normal hepatocytes after birth, small cell undifferentiated and embryonic epithelial components of hepatoblastoma do not or weakly express GPC3 protein. Therefore, GPC3 immunohistochemistry offers a valuable aid to the diagnosis of hepatoblastoma in infants and children.</p>

Amplification of HER2 gene in gastric carcinoma detected by dual in-situ hybridization / 中华病理学杂志

<p><b>OBJECTIVE</b>To explore the status of HER2 gene amplification and its product HER2 protein expression in gastric carcinoma, so as to aid in patient selection for anti-HER2 targeted chemotherapy.</p><p><b>METHODS</b>Eighty-five cases of gastric carcinoma biopsy tissues were collected. The status of HER2 gene amplification was detected by dual in situ hybridization (dual-ISH). And HER2 protein was detected by immunohistochemistry.</p><p><b>RESULTS</b>HER2 gene amplification was detected in 10/85 (11.8%) cases of gastric carcinoma, and no amplification was detected in 75/85 (88.2%) cases. In the 10 cases with HER2 amplification, HER2 immunoreaction scorings of 3+, 2+ and 0/1+ were present in 7, 2 and 1 cases, respectively. In the 75 cases without HER2 amplification, HER2 immunoreaction scorings of 3+, 2+ and 0/1+ were present in 0, 18 (24.0%) and 57 (76.0%) cases, respectively. Histologically, most gastric carcinoma with amplification of HER2 gene was moderately differentiated tubular adenocarcinoma.</p><p><b>CONCLUSIONS</b>HER2 gene dual-ISH technique is a reliable and objective method for detecting HER2 gene amplification in gastric carcinoma biopsy. Clinically, only few gastric carcinomas show HER2 gene amplification and are suitable candidates for anti-HER2 targeted chemotherapy.</p>

Significance of glypican-3 immunohistochemistry in diagnosis of hepatocellular carcinoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To explore the diagnostic significance of glypican-3 (GPC3) immunohistochemistry in hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>Fourteen tissue microarray paraffin blocks were constructed, which comprised 731 samples from hepatic tumors and paratumor tissues, including 357 cases of HCC, 26 cholangiocarcinoma, 171 HCC adjacent hepatic tissue including cirrhosis, 93 hemangioma adjacent hepatic tissues, and 84 carcinomas metastatic to liver. GPC3 (Clone 1G12) protein was detected immunohistochemically in all of cases with positive controls.</p><p><b>RESULTS</b>GPC3 protein was positive in 72.0% HCC (257/357), but negative in the rest 374 of non-HCC cases, including cholangiocarcinoma, HCC adjacent hepatic tissue including cirrhosis, hemangioma adjacent hepatic tissues and metastatic carcinomas. GPC3 positive percentage was significantly correlated with histological grading of HCC (P < 0.01), highest in grade 3 (77.1%, 64/83) followed by grade 2 (73.3%, 187/255), grade 1 (6/12) and grade 4 (0).</p><p><b>CONCLUSIONS</b>GPC3 is a valuable diagnostic marker for hepatocellular carcinoma with sensitivity of 72.0%, and a differential diagnostic marker from tumor adjacent hepatic tissue and carcinomas metastatic to liver with specificity of 100%.</p>

Role of AIB1 gene in development of esophageal squamous cell carcinoma and its clinicopathologic significance / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the role of AIB1 gene in the development of esophageal squamous cell carcinoma (ESCC) and its clinicopathologic significance.</p><p><b>METHODS</b>Two tissue microarrays, including 203 cases of ESCC, were prepared. Fluorescence in-situ hybridization (FISH) and immunohistochemistry were performed to detect the amplification of AIB1 gene and expression of its encoded protein in ESCC. The results were correlated with various clinicopathologic parameters.</p><p><b>RESULTS</b>In the 203 cases of ESCC studied, FISH was successful in 115 cases. Amongst those, amplification/gain of AIB1 gene was observed in 15 cases, including high-level amplification in 5 cases (4.3%) and low-level gain in 10 cases (8.7%). As for immunohistochemical study, AIB1 protein was overexpressed in 94 cases of ESCC. There was a significant association of AIB1 overexpression and tumor staging. AIB1 was overexpressed in 66 of the 123 cases in advanced T stages (T3 to 4), compared with 25 of the 80 cases in early T stages (P = 0.008). Those cases with high-level amplification of AIB1 also showed overexpression of its encoded protein. On the other hand, 8 of the 10 cases with low-level gain of AIB1 showed protein overexpression. The remaining 41 of the 100 cases which did not have AIB1 gene amplification/gain demonstrated overexpression of AIB1 protein.</p><p><b>CONCLUSION</b>Overexpression of AIB1 protein caused by gene amplification/gain or other molecular mechanisms may play an important role in the development and/or progression of a subset of ESCC.</p>

Construction of an eukaryotic expression vector for PRL-2 and its effect on human hepatocellular carcinoma cell invasiveness and migration in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct an eukaryotic expression vector for PRL-2 and evaluate its effect on the invasiveness and migration of a human hepatocellular carcinoma cell line.</p><p><b>METHODS</b>RT-PCR was performed to amplify the complete PRL-2 open reading frame using the total mRNA of hepatocellular carcinoma HepG2 cells as the template. PRL-2 gene was inserted into the pGEM T easy vector and sequenced, and the correct PRL-2 sequence was subcloned into the mammalian expression vector pcDNA3.1+. The constructed PRL-2 vector was transfected into CL1 cells via lipofectamine, and the stable expression of PRL-2 mRNA was detected by RT-PCR, the expressed protein identified by immunohistochemistry and Western blotting, and the effect of PRL-2 on the adhesion ability of CL-1 cell evaluated with MTT assay 20 and 120 min after transfection. The effect of PRL-2 on the invasive migration of CL-2 cells was evaluated according to the number of cells penetrating the Matrigel layer of polycarbonate membrane of Boyden chamber.</p><p><b>RESULTS</b>RT-PCR yielded a fragment of 504 bp and the inserted PRL-2 sequence was verified by sequence analysis. The subclones were identified by restriction endonuclease digestion, and a G418-resistant clone, PRL-2-CL1, was obtained after 8 weeks of selection. RT-PCR showed stable expression of PRL-2 mRNA, and Western blotting confirmed overexpression of PRL-2 protein in the transfected cells. PRL-2 increased the adhesion rate of CL-1 cells to fibronectin at 20 min and 120 min after transfection (P<0.05), and also the number of CL-1 cells penetrating the polycarbonate membrane from 10.0+/-3.7 to 44.8+/-2.6 (P<0.05).</p><p><b>CONCLUSION</b>An eukaryotic expression vector of PRL-2 has been successfully constructed, which allows stable and efficient expression in CL-1 cell line. PRL-2 can promote cell adhesion and invasion activity of human hepatocellular carcinoma cells.</p>

Study of expression of CD138 and heparinase in hepatocellular carcinoma by tissue microarray / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the expression of CD138 and heparinase in hepatocellular carcinoma (HCC) and its relationship with tumor development, progression, metastasis and recurrence.</p><p><b>METHODS</b>Tissue microarray and immunohistochemical study (EnVision method) for CD138 and heparinase was performed on tissue microarray which consisted of 197 cases of HCC, including adjacent non-neoplastic liver tissues, and 66 cases of HCC metastases.</p><p><b>RESULTS</b>The rates of CD138 expression in HCC and adjacent non-neoplastic liver tissues were 48.7% (96/197) and 65.0% (128/197, P < 0.05) respectively. In early-stage and late-stage tumors, the expression rates were 61.7% (29/47) and 44.7% (67/150, P < 0.05) respectively. The rate in patients with metastasis was 33.3% (22/66), as compared with 53.6% (45/84, P < 0.05) in patients without metastasis. In patients with tumor recurrence occurring within or after 1 post-operative year, the expression rates were 23.3% (7/30) and 61.1% (11/18, P < 0.05) respectively. On the other hand, the rates of expression of heparinase in HCC and adjacent non-neoplastic liver tissues were 35.5% (70/197) and 12.7% (25/197, P < 0.05) respectively. In early-stage and late-stage tumors, the expression rates were 29.8% (14/47) and 37.3% (56/150, P > 0.05) respectively. The rate in patients with metastasis was 48.5% (32/66), as compared with 28.6% (24/84, P < 0.05) in patients without metastasis. In patients with tumor recurrence occurring within or after 1 post-operative year, the expression rates were 50.0% (15/30) and 44.4% (8/18, P > 0.05) respectively. In the 66 cases of metastatic HCC studied, the expression rate of CD138 was lower in the heparinase-positive subgroup (P < 0.05).</p><p><b>CONCLUSIONS</b>Loss of CD138 expression is related to HCC development, progression, metastasis and recurrence. Overexpression of heparinase, when coupled with loss of CD138 expression, may take part in tumor metastasis of HCC.</p>

Study on genetic polymorphisms of CYP2F1 gene in Guangdong population of China / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the genetic polymorphism of CYP2F1 gene, a member of CYP450 gene family in the healthy population and the patients with nasopharyngeal carcinoma (NPC) of Guangdong province, and furthermore analyze the relationship between CYP2F1 genetic polymorphism and the risk of developing NPC.</p><p><b>METHODS</b>By direct gene sequencing, all of 10 exons of CYP2F1 gene were detected in 40 peripheral blood specimens of patients with primary NPC. For the genetic polymorphism with high allelic frequency, mismatch PCR-RFLP technique was developed to identify the different frequency between 368 NPC cases and 344 cancer-free controls.</p><p><b>RESULTS</b>There were totally 35 SNPs identified in all of 10 exons and exon-intron junctions of CYP2F1 gene from 40 NPC patients, which included 10 missense mutations and 1 frame shift mutation. The most important mutation was C insertion located in 15-16 bp, which caused the frame shift. The allelic frequency of C insertion was 25%. However, there was no significant difference found between 368 NPC cases and 344 controls in allelic frequency of 15-16 bp C insertion mutation (P>0.05).</p><p><b>CONCLUSION</b>A lot of genetic polymorphism of CYP2F1 gene is found in Guangdong population of China. However, no single genetic polymorphism associated with the individual susceptibility to NPC can be identified. The cooperated operations with multiple genetic polymorphisms of one or more genes may be critical factors contributing to the development and progression of NPC.</p>

Expression of NY-ESO-1 and LAGE-1 cancer-testis antigens in hepatocellular carcinoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the role of NY-ESO-1 and LAGE-1 cancer-testis antigens as targets for immunotherapy and the relationship between corresponding gene expression and biologic behavior of hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>The expression of NY-ESO-1 and LAGE-1 was studied in frozen tumor tissues from 30 cases of HCC by reverse transcriptase-polymerase chain reaction and immunohistochemistry. NY-ESO-1 expression and its distribution were further studied by immunohistochemistry in a tissue array contained 191 cases of HCC.</p><p><b>RESULTS</b>NY-ESO-1 and LAGE-1 mRNAs were expressed in 33.3% (10/30) and 16.7% (5/30) of HCC respectively. Either NY-ESO-1 or LAGE-1 was expressed in 36.7% (11/30) cases. NY-ESO-1 was expressed mainly in the cytoplasm of tumor cells. It was positive in 13.8% (24/174) cases of HCC. There was an increased expression of NY-ESO-1 from 6.8%, 3/44 in small HCC, 16.2%, 21/130 in advanced HCC and 23.1%, 12/52 in metastatic HCC. The expression in the non-metastatic group was 9.8% (12/122). The differences between the metastatic group and non-metastatic group (< 0.05) and between normal liver tissue and HCC (< 0.01) were statistically significant. There was no relationship between NY-ESO-1 expression and tumor size. NY-ESO-1 and LAGE-1 were not detected in adjacent normal liver tissue.</p><p><b>CONCLUSIONS</b>NY-ESO-1 and LAGE-1 are expressed in a high percentage of HCC, especially in cases with metastasis. It is thus possible that NY-ESO-1/LAGE-1 can serve as targets for antigen-specific immunotherapy in HCC and NY-ESO-1 peptide vaccination may be of use for patients with advanced HCC.</p>

Therapy of Duchenne muscular dystrophy with umbilical cord blood stem cell transplantation / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze a Duchenne muscular dystrophy(DMD) patient's muscular regeneration, dystrophin expression and locomotive variation before and after he underwent umbilical cord blood stem cell transplantation in order to assess the therapeutic effect.</p><p><b>METHODS</b>A 12-year-old DMD boy who could not walk for 3 years was confirmed by gene analysis and dystrophin protein immune test on his muscle. He had no other chronic disease. By HLA matching, a piece of umbilical cord blood stem cell with 6 HLA sites matching to the boy was found in Guangdong Umbilical Cord Blood Bank. The number of the nucleated cells of the umbilical cord blood stem cell was 24.08x 10(8). After pretreatment for the DMD boy with busulfan, cyclophosphamide and rabbit anti-human thymocyte globulin, the allergenic cord blood stem cells were transplanted into him by intravenous injection. Cyclosporin A, methylprednisolone, MMF, prostaglandin E1 and ganciclovir were given after the transplantation. At the same time, Gran, the granulocytic cell stimulating factor, and gamma globulin were administered. The biochemistry profile including serum creatine kinase (CK), the reconstruction of blood making, the deletion exon of DMD gene, the regenerating muscles, the dystrophin protein expression, and the locomotive function of the DMD boy were tested regularly.</p><p><b>RESULTS</b>(1) The white blood cells (WBC) of peripheral blood decreased gradually to zero after pretreatment. In a period of 15 days after transplantation, the neutrophil increased to 0.5x 10(9)/L; at 25 days, WBC increased to normal level. Blood platelet was more than 20x 10(9)/L at 22 days. The hemoglobin rose to 85-100 g/L. At 140 days, sternal puncture revealed the rapid growth of neutrophil, blood platelet and hemoglobin. (2)At 140 days, the blood type of the DMD boy transformed from type O to type AB (the donor's blood type being AB). There was no grafe versus host reaction. (3) At 18, 30, 43, 55, 74 and 233 days after transplantation, the PCR-short tandem repeat test of the boy's peripheral blood DNA showed that his genotype was completely the same as the donor's. The results of PCR-short tandem repeat tests of the bone marrow cells DNA by sternal puncture at 140, 183 and 235 days were the same as those of the blood DNA. (4) At 60 days, DMD gene analysis by PCR showed that the defected DMD gene (exon 19 deletion) had been corrected by the umbilical cord stem cells transplantation. (5) At 75 days, the biopsy of calf muscle showed there were myoblast cells and muscular tubes growing. The dystrophin expressions were weak, but a few of them were strong. DNA analysis showed that the donor's gene DNA accounted for 1%-13%. At 126 days, obviously increased dystrophin positive muscular fibers of the boy were found. The donor's fibers rose to 2.5%-25%. (6) The serum CK of the boy declined from 5735 U/L to 274 U/L. (7) At 100 days, physical examination revealed improvement in his arms and legs.</p><p><b>CONCLUSION</b>The therapy of Duchenne muscular dystrophy with allogeneic umbilical cord blood hematopoietic stem cell transplantation may reset up the blood-making function, decrease the serum CK level, restore the dystrophin in muscles, and improve the locomotive function of the DMD boy. These data suggest that the allogeneic umbilical cord blood hematopoietic stem cell transplantation may benefit the DMD boys.</p>

A study of gene amplification and expression of cyclin D1 in hepatocellular carcinoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate gene amplification of CCND1 and expression of cyclin D1 in hepatocellular carcinoma (HCC) and to explore the possible relationship between CCND1 gene status and carcinogenesis of HCC.</p><p><b>METHODS</b>Differential PCR, RT-PCR and immunohistochemistry were used to detect gene amplification, mRNA and protein expression of cyclin D1 in 20 HCC cases respectively. The relationship between the gene amplification rate and the expression level of cyclin D1 and the histological grades of HCC was analyzed.</p><p><b>RESULTS</b>CCND1 gene amplification was detected in 30% of the cases HCC. An overexpression of cyclin D1 mRNA and protein could be demonstrated in 45% and 70% cases respectively. The expression of cyclin D1 mRNA correlated with its gene amplification status (P < 0.05) and was responsible for the protein expression level (P < 0.05). There was a close relationship between the expression level of cyclin D1 protein and HCC histological grades (P < 0.05).</p><p><b>CONCLUSIONS</b>CCND1 gene amplification is a common phenomenon in HCC and may be directly responsible for the cyclin D1 mRNA and protein overexpression. Cyclin D1 protein expression level is directly related to HCC histological grades. Therefore, CCND1 amplification and cyclin D1 overexpression may play an important role in development and differentiation of HCC.</p>

Antisense hTERT inhibits gene expression and functional activity of telomerase in leukemia cell lines / 中华病理学杂志

<p><b>OBJECTIVE</b>To explore the inhibitory effects of gene expression and functional activity of telomerase in leukemia cell lines by in vitro antisense hTERT treatment.</p><p><b>METHODS</b>An antisense hTERT eukaryotic expression vector was constructed by using gene recombination technique, targeting the 5' end mRNA sequence of the telomerase catalytic subunit. The vector expression in leukemia cell lines (HL60 and K562) was achieved by transfection using the SuperFect transfection reagent (Qiagen). After transfection, ectopic expression of the telomerase catalytic subunit was analyzed by quantitative fluorescence real-time RT-PCR, and cellular apoptosis and cell cycle parameters were evaluated by flow cytometry respectively.</p><p><b>RESULTS</b>An antisense pcDNA-hTERT eukaryotic expression vector was successfully constructed. Leukemia cell lines transfected with antisense hTERT constructed displayed a significant inhibition of gene expression of telomerase and its activity in vitro, as compared with the result of the control groups (without transfection and vector control).</p><p><b>CONCLUSION</b>In-vitro antisense hTERT expression may down-regulate the gene expression and biological activity of telomerase in leukemia cells, suggesting a possibility of gene therapy against human malignancy through the telomerase-targeted molecular mechanism.</p>

Relationship between TIMP-3 expression and promoter methylation of TIMP-3 gene in hepatocellular carcinoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate further the possible mechanism of carcinogenesis and portal invasion of hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>Samples of the primary tumors, cancer cells emboli in the portal veins and normal liver tissues adjacent to the tumor were collected from 20 cases of primary HCC. Expression of TIMP-3 (tissue inhibitor of metalloproteinases-3) protein was detected using Western blot. Expression of TIMP-3 mRNA was detected by RT-PCR. Methylation of TIMP-3 gene promoter was detected using methylation-specific PCR (MSP).</p><p><b>RESULTS</b>Expression of TIMP-3 protein and mRNA were obtained in all of the normal liver tissues adjacent to tumor. However, loss of TIMP-3 protein expression was found in 5 and 36 cases respectively in the primary tumors and tumor cell emboli in portal veins. Expression of TIMP-3 protein and mRNA in primary tumors and tumor emboli were significantly lower than that in the normal liver tissues. Promoter methylation of TIMP-3 gene could be detected in primary tumors (7 cases) and cancerous emboli (9 cases) in HCC, while no methylation found in normal liver tissues. In all the HCC cases with promoter gene methylation including primary tumors and cancerous emboli in portal veins, 13 cases showed complete loss and 6 cases showed low expression of TIMP-3 protein and mRNA. Promoter methylation of TIMP-3 was noticed not related with the histological grading of HCC.</p><p><b>CONCLUSIONS</b>There is a close relationship between loss or low expressions of TIMP-3 and carcinogenesis and portal invasion of HCC. The loss and low expression of TIMP-3 gene and protein were caused by methylation of the gene promoter.</p>