Detection of EGFR and K-ras mutations in non-small cell lung cancer using cytological specimens / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To validate the feasibility for detecting EGFR and k-ras mutations using cytological specimens.</p><p><b>METHODS</b>Cytological specimens including fine-needle aspiration (FNA), pleural effusion (PLE) and fiberoptic bronchoscopic (FOB) brushing were collected from patients with non-small cell lung cancer (NSCLC ) from January 2011 to July 2011 at the Department of Cytology, Cancer Hospital, Chinese Academy of Medical Sciences (CHCAMS). Polymerase chain reaction (PCR) was carried out to amplify EGFR exons 18-21 and k-ras codons 12-13, and then the PCR products sequencing and analysis were performed.</p><p><b>RESULTS</b>Fifty cytological specimens were collected including 19 cases of FOB, 9 cases of FNA, 22 cases of PLE. Of them DNA was successfully extracted in 43 cases, and specific PCR amplification products sequencing were performed in 42 cases. EGFR mutations were detected in 14 of 42 specimens (33.3%), the frequencies of EGFR mutations in exons 19, 20 and 21 were 16.7% (7/42), 4.8% (2/42) and 11.9% (5/42), respectively, and no mutation was found in exon 18. Higher frequencies of EGFR mutations were detected in exons 19 and 21 (85.7%). Mutations were identified in 38.7% (12/31) cases of adenocarcinoma. K-ras mutations were found in 2 of 42 specimens (4.8%). EGFR and K-ras mutations were not found in the same case.</p><p><b>CONCLUSIONS</b>Cytological specimens are feasible for detecting EGFR and K-ras mutation. This is especially beneficial in patients in whom histological materials can not be obtained.</p>

Overexpression of p-Stat3 and Mcl-1, and their correlation with differentiation and apoptotic resistance in esophageal squamous cell carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To detect the expression of phosphorylated-signal transducer and activator of transcription 3 (p-Stat3) and myeloid leukemia-1 (Mcl-1) as well as their correlation, and to investigate the functional role of Stat3 and Mcl-1 in the pathogenesis of esophageal squamous cell carcinoma (ESCC).</p><p><b>METHODS</b>Stat3 activity in ESCC cells was inhibited with JAK/Stat3 inhibitors (AG490 or JSI-124). Specific siRNA was used to inhibit the Stat3 expression. Cell apoptosis was detected by flow cytometry. Expression of Mcl-1 protein was determined by Western blotting. Expression of phospho-Stat3 (Tyr705) and myeloid leukemia-1 (Mcl-1) proteins in ESCC tissues was detected by tissue microarray and immunohistochemistry. The relationship between p-Stat3 or Mcl-1 aberrant expression and clinicopatholohical features of ESCC was analyzed. The correlation of their expression was also analyzed.</p><p><b>RESULTS</b>Suppression of the Stat3 signaling activation in ESCC cells led to marked apoptosis, and dramatic reduction of Mcl-1 protein. The positive rate of phospho-Stat3 (Tyr705) expression was 45.0% in 50/111 of the ESCC tissue samples. The lower the degree of tumor differentiation, the higher the positive rate of phospho-Stat3 (Tyr705), showing a significant difference (P = 0.018). The positive rate of Mcl-1 protein expression was 72.1% (80/111), and the lower the degree of tumor differentiation was, the higher there was the positive rate of Mcl-1, with a significant difference (P = 0.026). There was a positive correlation between the expressions of p-Stat3 and Mcl-1 proteins (P = 0.012).</p><p><b>CONCLUSIONS</b>In a subset of ESCC tissues, p-Stat3 (Tyr705) and Mcl-1 are overexpressed and positively correlated with each other, and both are correlated with tumor differentiation. Persistent activation of Stat3 contributes to apoptotic resistance in ESCC cells, and may be at least partly mediated through upregulation of Mcl-1.</p>

Gene construction and screening of humanized single chain antibody library against VSTM1-v2 cytokine / 药学学报

To rapidly select potent anti-VSTM1-v2 scFv (single-chain antibody fragment) by construction and screening of a humanized scFv library in which a murine VH-CDR3 library was grafted onto a human scFv framework. A murine VH-CDR3 library was amplified from anti-VSTM1-v2 murine cDNA and grafted on human scFv (VH3-VK1) framework. Anti-VSTM1-v2 scFv templates were selected and enriched through ribosome display, TA-cloned into expression vector, and transformed into BL21 (DE3) for soluble expression of target scFv. A total of 1000 clones were randomly picked. Positive ones were first identified using colony PCR, indirect ELISA, Western blotting and then verified with sequencing and dose response ELISA. At last an anti-VSTM1-v2 humanized scFv with good binding affinity (EC50 = 21.35 nmol x L(-1)) was selected from the humanized library of 10(12) members generated in this study. This scFv antibody might have potential applications. This study provides a new approach for rapid screening of humanized antibodies.

Current applications of high-throughput DNA sequencing technology in antibody drug research / 药学学报

Since the publication of a high-throughput DNA sequencing technology based on PCR reaction was carried out in oil emulsions in 2005, high-throughput DNA sequencing platforms have been evolved to a robust technology in sequencing genomes and diverse DNA libraries. Antibody libraries with vast numbers of members currently serve as a foundation of discovering novel antibody drugs, and high-throughput DNA sequencing technology makes it possible to rapidly identify functional antibody variants with desired properties. Herein we present a review of current applications of high-throughput DNA sequencing technology in the analysis of antibody library diversity, sequencing of CDR3 regions, identification of potent antibodies based on sequence frequency, discovery of functional genes, and combination with various display technologies, so as to provide an alternative approach of discovery and development of antibody drugs.

Ribosome display screening of a novel human anti-IgE scFv fragment / 药学学报

Total mRNA was extracted from lymphocytes separated from the peripheral blood of allergic patients, and then variable region of heavy chain (VH) and variable region of light chain (VL) cDNA library were constructed by RT-PCR. Human scFv templates for rabbit reticulocyte lysate ribosome display were assembled by primers and linker peptide (Gly4Ser)3. mRNA bound in antibody-ribosome-mRNA complexes was recovered using in-situ single primer RT-PCR, and three rounds of anti-IgE scFv DNA were enriched. The target DNA fragments were double enzyme digested and ligated into plasmid pET22b (+), followed by transformation in E. coli Rosseta (DE3). Positive clones were screened using clone PCR, Dot blotting and antigen ELISA. The correct lengths of VH (400 bp) and VL (710 bp) PCR products were obtained. The expected 1,000 bp ribosome display templates were also observed in agarose gel electrophoresis. After three rounds of ribosome display target sequences were effectively enriched, leading to a library of 10(13) members. Antibodies with the highest ELISA value for IgE were generated in the strain pET-IgE-6. A human anti-IgE scFv library was successfully constructed as described herein. Ribosome display using single primer in-situ RT-PCR as the recovery procedure effectively enriched target sequences. Anti-IgE scFv with high affinity and specificity were identified. The prepared human anti-IgE scFv fragment might be self-developed to a lead drug for treating asthma. Our study provides an alternative method for rapid discovery of human antibodies of therapeutic importance.

Expression pattern of E-cadherin and p120-catenin in infiltrating lobular carcinoma and ductal carcinoma of the breast and its significance / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To determine how patients with infiltrating lobular carcinoma (ILC) differ from patients with the more common infiltrating ductal carcinoma (IDC), and observe the different expression patterns of E-cadherin and p120-catenin proteins in both ILCs and IDCs.</p><p><b>METHODS</b>The patients with ILC admitted to our hospital from Jan 1999 to Dec 2006 and patients with IDC from Jan 2000 to Dec 2000 were included in this study. All their pathological slides were reviewed, and their clinical data and treatment variables were analyzed retrospectively. Then the expression patterns of E-cadherin and p120-catenin proteins in both ILCs and IDCs were detected by immunohistochemistry on tissue microarray.</p><p><b>RESULTS</b>The 5-year overall survival was 81.7% for ILCs and 79.1% for IDCs (P = 0.055). The 5-year disease-free survival was 61.8% for ILCs and 83.7% for IDCs (P < 0.001). Cytoplasmic localization of p120-catenin and loss of E-cadherin expression were more common in ILCs than in IDCs. The complete losses of E-cadherin in ILCs and IDCs were 55.6% (20/36) and 20.4% (45/221, P < 0.001), respectively. The p120-catenin showed a diffuse cytoplasmic localization in 66.7% (24/36) of ILCs and 16.3% (36/221) of IDCs (P < 0.001). Interestingly, the cytoplasmic localization of p120-catenin was clearly associated with the absence of E-cadherin expression in ILCs (P = 0.002), cytoplasmic localization of p120-catenin and absence of E-cadherin expression were observed 55.6% (20/36) in ILCs compared with 4.1% (9/221) in IDCs (P < 0.001).</p><p><b>CONCLUSION</b>ILC has several specific biological and prognostic characteristics which are different in IDC. Different expression patterns of E-cadherin and p120-catenin proteins can be helpful to recognize ILC from IDC.</p>

Application of interphase fluorescence in situ hybridization in the diagnosis of lymphoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of interphase FISH in the routine clinicopathological practice and its values in the differential diagnosis of lymphomas.</p><p><b>METHODS</b>A total of 74 fresh tissue samples clinically suspicious of lymphoma were investigated by FISH using three probes including IgH/bcl-2, IgH/CCND1 and API2/MALT1, corresponding the translocation t(14;18), t(11;14) and t(11;18) respectively. The results of FISH were analyzed and compared with the histopathologic diagnosis.</p><p><b>RESULTS</b>Histological evaluation eventually confirmed that there were 62 cases of lymphoma and 12 cases of reactive lymphoid processes. The translocations were detected in 7 cases in 62 cases of lymphoma: 3 demonstrated t(14;18) including 2 cases of follicular lymphomas and 1 nodular sclerosing Hodgkin lymphoma. Four cases had t(11;14) including mantle cell lymphoma (2 cases), follicular lymphoma (1 case) and small cell lymphoma (1 case). A lymphoid hyperplasia case showed detectable t(14;18). All 25 cases of DLBCL showed no evidence of t(14;18). Amplification or loss of regional genes was seen more often in malignant than in the benign cases.</p><p><b>CONCLUSION</b>Interphase FISH offers useful ancillary technology that plays an important role in differential diagnosis and classification of lymphoma.</p>

Correlation between aneuploidy of chromosome 17, over-expression of TP53 and TOPIIalpha, and the clinicopathological features and diagnosis of gastric adenocarcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>The purpose of this study was to investigate the markers which can be used in auxiliary diagnosis of gastric adenocarcinoma (GAC), and their correlation with their clinicopathological features.</p><p><b>METHODS</b>122 surgical specimens including 99 gastric adenocarcinoma (GAC), 18 adjacent mucosa and 5 distal normal mucosa were collected, and analyzed by in situ hybridization (FISH). The centromere probe cen17, specific for chromosome 17, which was reported to be frequently amplified in GAC, was selected for the FISH analysis. The clinicopathological features of the 99 GAC cases were reviewed, and the level of TP53 and TOPIIalpha gene expression, located in chromosome 17, was detected using tissue micro-array (TMA), compared with that of corresponding adjacent normal mucosa. Data were analyzed with SPSS 11.5 for Windows.</p><p><b>RESULTS</b>The statistical results of FISH and TMA showed that 58.6% of cen17 in tumor tissues were aneuploid, and 45.5% of TP53 and 84.7% of TOPIIalpha were over-expressed in GAC samples, significantly higher than those in non-tumor gastric mucosa (0, 12.1% and 14.1%, respectively) (P = 0.000). 58 GAC tissues were aneuploid of cen17, including 26 cases TP53-positive and 49 cases TOPIIalpha-positive. The expression of TP53 in non-tumor gastric mucosa with dysplasia was significantly higher than that in the mucosa without dysplasia (P = 0.009). Aneuploidy of cen17 was more frequent in grade 1 or 2 than in grade 3 GAC (P < 0.05). Higher frequency of aneuploidy of cen17 was also observed in the gastric cardia than in pylorus (P < 0.05), while no correlation was found between aneuploidy of cen17 and age, sex of patients, lymph node metastasis, and clinical stage of tumors. Over-expression of TP53 protein was associated with the size of tumors (P < 0.05). In addition, a negative correlation was observed between over-expression of TOPIIalpha and lymph node metastasis (LNM) as well as TNM classification (P < 0.05).</p><p><b>CONCLUSION</b>Detection of aneuploidy of cen17 as well as over-expression of TP53 and TOPIIalpha may be helpful in the diagnosis and prognostic prediction of gastric adenocarcinoma.</p>

Up-regulated manganese superoxide dismutase expression increases apoptosis resistance in human esophageal squamous cell carcinomas / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Esophageal cancer is one of the most common malignancies in the world. In order to identify the proteins associated with esophageal squamous cell carcinomas (ESCC), we analyzed the protein profiles of ESCC cases with tumor and matched adjacent normal tissues.</p><p><b>METHODS</b>Two-dimensional electrophoresis (2-DE) was carried out to analyze the protein profiles. Dysregulated protein spots were identified by Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) and verified by liquid chromatography/electrospray ionization ion trap-mass spectrometry/mass spectrometry (LC-ESI-IT MS). RT-PCR and immunohistochemistry on tissue microarray were performed to confirm the gene dysregulation in esophageal cancerous tissues. RNA interference (RNAi) was used to knock down the gene expression in ESCC cell lines. Apoptosis assay with annexin V-FITC/PI staining was conducted and cells were analyzed by flow cytometry.</p><p><b>RESULTS</b>2-DE showed that two protein spots with approximate molecular weights and different pI were elevated in 12 out of 18 ESCCs as compared to the corresponding normal tissues. Both the two spots were identified as MnSOD by MALDI-TOF and were verified by LC-ESI-IT MS. MnSOD overexpression was detected in 14 tumors out of 24 cases by RT-PCR and 52 tumors out of 116 cases by immunohistochemistry comparing to normal epithelia. siRNA-mediated silencing of MnSOD in KYSE450 and KYSE150 cell lines revealed that MnSOD protected ESCC cells from apoptosis induced by ultraviolet (UV) and doxorubicin (DOX).</p><p><b>CONCLUSIONS</b>These findings suggest that there existed two isoforms of MnSOD protein in normal and tumor esophageal tissues. MnSOD was overexpressed in ESCC and its up-regulation in esophageal cancer cells was associated with apoptosis resistance.</p>

Influence of interphase nuclei preparation techniques on fluorescence in situ hybridization results in solid tumors / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a technology platform for the preparation of interphase nuclei for the fluorescence in situ hybridization (FISH) detection of solid tumor tissues.</p><p><b>METHODS</b>The centromere probe of chromosome 3 was labeled by the random primer technique, and then hybridized to interphase nuclei prepared by six different methods in order to study the influence on FISH detection.</p><p><b>RESULTS</b>Each method of slide preparation had its own characteristic, and could be used according to different needs. As regards to FISH, collagenase method got the best results. Whereas for frozen samples or small tissues, to prepare printing slides was more applicable.</p><p><b>CONCLUSION</b>The comparison of different slide preparation methods lays a technology foundation for the FISH application in cancer researches and clinical diagnosis of solid tumors.</p>

Highly expressed protein in cancer (Hec 1) and chromosome instability / 中国医学科学院学报

Highly expressed in cancer (Hec 1), locating at centromere during cell mitosis, plays an important role in the pathway of spindle checkpoint. Hec 1-Nuf 2 complex is the structural basis for the recruitment of Mad 1/Mad 2 complex of spindle checkpoint. Hec 1 can interact with the subunit of 26S proteasome and inhibit the degradation of cyclins. It was initially identified as a protein interacting with Rb by yeast two-hybridization assay. Rb interacts with Hec 1 to regulate the binding ability of Smc 1 with DNA and participates in the regulation of M phase. Hec 1 mainly expresses at G2/M phase and functions through the phosphorylation by kinase Nek 2. Hec 1 is over expressed in some cancer cell lines and amplified in tumor tissues. The dysfunction of Hec 1 gene may cause severe impediment of chromosome separation and finally lead to chromosome instability, which is closely associated with the occurrence and development of tumors. Therefore, Hec 1 may become a new target of tumor gene therapy.

Altered expression of the HSD17B4 gene in esophageal squamous cell carcinoma and loss of heterozygosity analysis / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the alteration of the gene HSD17B4 in esophageal squamous cell carcinoma and its potential significance.</p><p><b>METHODS</b>The mRNA expression and loss of heterozygosity (LOH) of HSD17B4 in 40 primary esophageal tumors were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and microsatellite analysis with the intragenic marker D5S1384 of the gene.</p><p><b>RESULTS</b>The frequencies of allelic loss of D5S1384 and the rate of down-regulation of gene HSD17B4 were 46.2% and 62.5%, respectively.</p><p><b>CONCLUSION</b>HSD17B4 may be a candidate tumor suppressor gene associated with esophageal squamous cell carcinoma.</p>

Strategy for the identification of amplified genes in tumors / 中华医学遗传学杂志

Amplification of genomic DNA is often observed in tumors. The identification of genes in amplified regions may be helpful to the discovery of oncogenes associated with a specific tumor. Array-based comparative genomic hybridization and restriction landmark genomic scanning are applicable to the definition of such regions. The copy number alterations of target genes present in these regions and the levels of their mRNA and protein can be characterized by quantitative PCR and tissue microarray staining with immunohistochemistry. Transfection, RNA interference, cDNA microarray or reverse transcription-multiplex ligation-dependent probe amplification will facilitate demonstrating the role of such amplified genes in the pathogenesis of a specific tumor.

Clinical analysis of coronary artery bypass graftin g in 13 patients / 第三军医大学学报

Objective　Coronary artery bypass grafting (CABG)i n 13 patients was analyzed. Methods　9 patients were performed C ABG with cardiopulmonary bypass, 4 patients undergone off-pump coronary artery bypass(OPCAB). Among the 4 patients, 3 undergone transmyocardial laser revascula rization concomitantly. 2 patients with single-vessel disease, 3 with double-v essel disease, 7 with triple-vessel disease and 1 with left main coronary arter y disease. The average bypass per patient was 2.3. Results　All patients survived, 11 patients were angina free, 2 were angina relief. C onclusion　CABG is a safe operation, OPCAB may reduce blood transfusion and complication, patients recover more quickly after OPCAB compared with those with CABG.

Study of the inhibitory effect of suramin on the angiogenesis of lung cancer / 第三军医大学学报

Objective　To investigate the role of suramin in inhibiting the angiogenesis of lung cancer. Methods　The protei n expression of VEGF and its receptor fms-like tyrosine kinase(Flt), kinase ins ert domain-containing receptor(KDR) in lung cancer cell A549 and ECV-304 was studied with immunohistochemical technique. The anti-angiogenesis effect of su ramin was evaluated with immunohistochemical technique and cell culture. Results　Suramin showed no effect on the production of lung cancer cel ls and their secretion of VEGF. No possitive expression of Flt, KDR in A549 was found in the suramin interfered group and non-interfered group, but VEGF w as e xpressed in both groups. The Flt and KDR expressions in ECV-304 cells were decr eased significantly after the treatment of suramin at 0.25 ng/ml and 2.5 ng/ml r espectively. There was no effect on proliferetion of A549 and ECV-304 after sur amin in terference. Conclusion　The mechanism of inhibiting-angiogenes is of suramin is pro bably due to reducing the VEGF receptor expression in vascular endothelial cells and inhibiting the binding of VEGF and its receptors.

Clinical analysis of coronary artery bypass graftin g in 13 patients / 第三军医大学学报

Objective　Coronary artery bypass grafting (CABG)i n 13 patients was analyzed. Methods　9 patients were performed C ABG with cardiopulmonary bypass, 4 patients undergone off-pump coronary artery bypass(OPCAB). Among the 4 patients, 3 undergone transmyocardial laser revascula rization concomitantly. 2 patients with single-vessel disease, 3 with double-v essel disease, 7 with triple-vessel disease and 1 with left main coronary arter y disease. The average bypass per patient was 2.3. Results　All patients survived, 11 patients were angina free, 2 were angina relief. C onclusion　CABG is a safe operation, OPCAB may reduce blood transfusion and complication, patients recover more quickly after OPCAB compared with those with CABG.

Study of the inhibitory effect of suramin on the angiogenesis of lung cancer / 第三军医大学学报

Objective　To investigate the role of suramin in inhibiting the angiogenesis of lung cancer. Methods　The protei n expression of VEGF and its receptor fms-like tyrosine kinase(Flt), kinase ins ert domain-containing receptor(KDR) in lung cancer cell A549 and ECV-304 was studied with immunohistochemical technique. The anti-angiogenesis effect of su ramin was evaluated with immunohistochemical technique and cell culture. Results　Suramin showed no effect on the production of lung cancer cel ls and their secretion of VEGF. No possitive expression of Flt, KDR in A549 was found in the suramin interfered group and non-interfered group, but VEGF w as e xpressed in both groups. The Flt and KDR expressions in ECV-304 cells were decr eased significantly after the treatment of suramin at 0.25 ng/ml and 2.5 ng/ml r espectively. There was no effect on proliferetion of A549 and ECV-304 after sur amin in terference. Conclusion　The mechanism of inhibiting-angiogenes is of suramin is pro bably due to reducing the VEGF receptor expression in vascular endothelial cells and inhibiting the binding of VEGF and its receptors.