[Analyzed the molecular interaction network of tumor suppressor gene 14-3-3 sigma in lung cancer cell based on stable isotope labeling by amino acids in cell culture technology].

OBJECTIVE: To analysis the molecular interaction network of 14-3-3 sigma in non small cell lung cancer (NSCLC) cells. METHODS: Established stable over-expressed 14-3-3 sigma protein PG cells, MTT assay was used to assess the growth rate of PG cells. Though stable isotope labeling by amino acids in cell culture (SILAC) and Mass spectrometry (MS) technology, to identify difference expressed proteins caused by over expressed 14-3-3 sigma. The protein expressed >2 or <0.5 times was termed as the differential protein. By searching Human protein reference database (HPRD) and Kyoto encyclopedia of genes and genomes (KEGG), established the molecular interaction network of tumor suppressor gene 14-3-3 sigma. RESULTS: The growth rate of over-expressed 14-3-3 sigma PG cell was obviously slower down compared to vector PG cells. A database including 147 differential protein was established. And a molecular interaction network of 14-3-3 sigma containing 26 protein was constructed.In this network, the expression of CSNK2A1 (casein kinase II subunit alpha), involved in numerous cellular processes, such as cell cycle progression, apoptosis and transcription, was the most significantly increased. A DNA repair protein, MEN1 (Menin) which functions as a transcriptional regulator was the most significantly decreased. CONCLUSION: After stable transfected with 14-3-3 sigma gene, growth rate of PG cells was inhibited, the proteins associated with cell cycle, DNA damage repair mechanisms were significantly changed, and constructed the molecular interaction network.

Radiomics Signature to Predict Prognosis in Early-Stage Lung Adenocarcinoma (≤3 cm) Patients with No Lymph Node Metastasis.

OBJECTIVES: To investigate the predictive ability of radiomics signature to predict the prognosis of early-stage primary lung adenocarcinoma (≤3 cm) with no lymph node metastasis (pathological stage I). MATERIALS AND METHODS: This study included consecutive patients with lung adenocarcinoma (≤3 cm) with no lymph node metastasis (pathological stage I) and divided them into two groups: good prognosis group and poor prognosis group. The association between the radiomics signature and prognosis was explored. An integrative radiomics model was constructed to demonstrate the value of the radiomics signature for individualized prognostic prediction. RESULTS: Six radiomics features were significantly different between the two prognosis groups and were used to construct a radiomics model. On the training and test sets, the area under the receiver operating characteristic curve value of the radiomics model in discriminating between the two groups were 0.946 and 0.888, respectively, and those of the pathological model were 0.761 and 0.798, respectively. A radiomics nomogram combining sex, tumor size and rad-score was built. CONCLUSION: The radiomics signature has potential utility in estimating the prognosis of patients with pathological stage I lung adenocarcinoma (≤3 cm), potentially enabling a step forward in precision medicine.

[Analysis of first-line chemoresistance and prediction of chemo-response in non-small cell lung cancer by comparative genomic hybridization].

OBJECTIVE: To explore the association between chromosomal disequilibrium and chemoresistance/chemosensitivity in non-small cell lung cancer (NSCLC) using comparative genomic hybridization (CGH). METHODS: Genomic DNA samples were prepared from the tumor tissues in paraffin-embedded sections derived from 88 patients with advanced NSCLC (18 with chemosensitivity and 16 with chemoresistance). The DNAs were first amplified by a degenerate oligonucleotide prime-polymerase chain reaction protocol and then labeled with fluorescence as probes for CGH analyses. The correlations of the resulting chromosomal imbalances with the chemo-sensitivity and other pathological features of the patients were analyzed. RESULTS: A total of 640 abnormal chromosome regions including 96.12% gains and 3.88% losses were detected in 88 specimens. The results indicated that the most frequently gained chromosome regions were 19p13.1-13.3 (39/88, 44.12%), followed by 9q12-q22 (26/88, 29.41%), 22q12-q13 (26/88, 29.41%), and Xq (29/88, 32.35%). The total number of abnormal regions related with chemo-sensitivity was 188( 182 gains and 6 losses), while the number of the abnormal regions linked to the chemoresistance was 452 (431 gains and 21 losses) (P=0.005). Gains of 14p12-p13 and 19p were significantly correlated with the chemosensitivity of the NSCLC (P=0.006). Gains of 1q12-q22, 10q25-q26, 5p15.1-p15.3, 19q13.2-13.4, 20p11.2-p12, 21q22, and Xp 21-p22.1 were also significantly correlated with the chemoresistance (P]0.005, 0.029, 0.039, 0.029, 0.039, 0.016, and 0.006, respectively). No correlation between the chromosome abnormalities and other clinical features was observed. CONCLUSIONS: The specific gains and losses of chromosome region is correlated with platinum-based first-line chemotherapy in NSCLC patients,as confirmed by CGH detection. This finding is useful for further identifying the chemosensitivity-related functional genes, predicting clinical effectiveness, and achieve individualized treatment in the future.

[DNA microarrays-based microRNA expression profiles derived from formalin-fixed paraffin-embedded tissue blocks of squammous cell carcinoma of larynx].

OBJECTIVE: To establish DNA microarrays-based microRNA (miRNA) expression profiles of squamous cell carcinoma of larynx, using archived formalin-fixed paraffin-embedded tissue blocks, and to screen out and identify the differentially expressed miRNAs associated with the biological characteristics of this malignant disease. METHODS: Total RNA was prepared from the formalin-fixed paraffin-embedded tissue blocks. After quality identification and fluorescent labeling, the RNA samples were hybridized with the Agilent human miRNA microarrays which contains 723 probes for human miRNAs. The data was processed with the softwares GeneSpring GX and R-Project. RESULTS: From the formalin-fixed paraffin-embedded tumor blocks collected, 24 RNA samples were obtained with the quality accorded to the requirement of miRNA microarray analysis, and both the hybridization and consequent data processing were accomplished. A total of 319 miRNAs were identified and among them 96 were detected in all the 24 formalin-fixed paraffin-embedded blocks of laryngeal carcinoma; and 5 differentially expressed miRNAs (false discovery rate < 0.05) were found to be associated significantly with the lymphatic metastasis of laryngeal squamous cell carcinoma (P < 0.05), including miR-23a(*), miR-28-5p, miR-15a, miR-16 and miR-425. CONCLUSIONS: Histopathological archives of well-annotated formalin-fixed paraffin-embedded tissue specimens are the valuable resources for miRNA study including to collect RNA samples for miRNA microarray analysis. A panel of differentially expressed miRNAs (miR-23a(*), miR-28-5p, miR-15a, miR-16 and miR-425) derived from the miRNA expression profile may serve as the potential molecular biomarkers for the prediction of metastasis development in laryngeal squamous cell carcinoma.

[Application of protein markers in combination with ThinPrep bronchial brush cytology in classification of lung cancer subtypes].

OBJECTIVE: To evaluate the value of application of cellular protein markers stained by immunocytochemistry in combination with ThinPrep bronchial brush cytology in classification of lung cancer subtypes. METHODS: Remaining bronchial brush cytology samples from 206 lung cancer patients with positive cytological diagnosis and 45 fine needle aspiration samples of resected lung carcinomas were collected. The expressions of CK10/13, CK7, CK18, CD56 and SYN in those samples were detected by immunocytochemistry (ICC) using corresponding antibodies. RESULTS: The sensitivity and specificity of CK10/13 were 94.7% and 72.0%, respectively, in diagnosis of squamous cell carcinoma. The sensitivity and specificity of CK7 were 98.6% and 61.5%, and those of CK18 were 98.6% and 37.5%, respectively, in diagnosis of adenocarcinoma. The sensitivity and specificity of CD56 were 86.3% and 82.9%, and those of SYN were 81.6% and 93.5%, respectively, in diagnosis of small cell lung cancer. No significant difference was found in the expressions of CK10/13, CK7 and CK18 protein markers among differently differentiated lung squamous cell carcinomas and adenocarcinomas (P > 0.05). The classification rate of cytology in combination with ICC in differential diagnosis for 44 cases of unclassified lung cancer reached 90.0% for squamous cell carcinoma, 96.3% for adenocarcinoma, and 100.0% for small cell lung carcinoma. CONCLUSION: Application of cellular protein markers in combination with ThinPrep bronchial brush cytology is helpful to improve the differential diagnosis of lung cancer subtypes, and may become a supplementary diagnostic method in subclassification of lung cancer.

Amplification and overexpression of Aurora-A in esophageal squamous cell carcinoma.

Aurora-A/BTAK/STK15 gene which encodes a centrosome-associated kinase is located on chromosome 20q13.2, a highly amplified region in various human tumors. Recent studies have demonstrated the overexpression and amplification of Aurora-A in many malignant human cancers. The purpose of this study was to investigate the amplification and expression of Aurora-A in esophageal squamous cell carcinoma. Amplification of Aurora-A was determined by fluorescence in situ hybridization in 7 esophageal cancer cell lines and real-time PCR in 29 esophageal cancer samples. We detected Aurora-A expression in 7 esophageal cancer cell lines and 38 esophageal cancers samples by semi-quantitative reverse transcription-PCR and Western blot hybridization. The amplification of Aurora-A was detected in 27 of 29 (93.1%) esophageal cancer samples and 6 of 7 (85.7%) cancer cell lines. Aurora-A was overexpressed in 27 of 38 (71.1%) esophageal cancer samples and all 7 esophageal cancer cell lines. We conclude that Aurora-A is amplified and overexpressed in esophageal squamous cancer.

[Over-expression of osteopontin in non-small cell lung cancers: its clinical significance].

OBJECTIVE: Data obtained from a differentially expressed cDNA library constructed previously in this laboratory demonstrated that the extracellular matrix molecule osteopontin (OPN) is one of most considerably over-expressed genes in non-small cell lung cancers (NSCLCs). The purpose of the present study was to explore the expression status of OPN in a large scale NSCLC tissue samples, and estimate its significance in progression of the malignant disease. METHODS: RT-PCR was performed with the tumor and adjacent normal tissues from 35 patients with NSCLC, at transcriptional levels of OPN. To determine the expression of OPN protein in the tumor tissues, immunohistochemical (IHC) staining was subsequently carried out on paraffin-embedded sections in tissue microarrays containing 662 samples derived from NSCLC cases. The correlation between the expression level of OPN and clinical characteristics was analyzed statistically. RESULTS: Comparing with the paired normal lung tissue, high level RNA of OPN was detected in 80.0% (28/35) of the NSCLC tumor tissues by RT-PCR, which confirmed the information obtained previously by our differentially expressed cDNA library. The results of IHC analysis showed that positively stained OPN protein was observed in 59.6% (331/555) of the tumor tissues, which was remarkably higher than that (25.2%, 27/107) detected in the normal control tissues (P < 0.001). Among the NSCLCs investigated, over-expressed OPN was more frequently found in squamous cell carcinomas (SCCs) than in adenocarcinomas. A further analysis on SCCs demonstrated that the rate of over-expressed OPN was significantly different between the primary tumors with and without lymphatic metastases (68.6% vs. 49.7%, P = 0.001), but similar in the primary tumors and their corresponding metastases in lymph nodes (68.6% vs. 75.5%, P = 0.171). CONCLUSION: Expression of OPN protein is distinctly increased in NSCLCs, particularly in SCCs. OPN over-expression is considerably correlated with lymph node metastasis, increasing the risk of tumor metastasis (OR = 2.212). The resulting data suggest that OPN facilitates the progression of NSCLCs.

[Expression of serum breast drug-resistance protein in predicting chemosensitivity of NSCLC].

OBJECTIVE: To investigate expression of serum breast cancer resistance protein (BCRP) in non-small cell lung cancer patient (NSCLC) and healthy adult, and its correlation with chemosensitivity as one passible value of BCRP in clinical application. METHODS: Venous blood specimens of 44 advanced NSCLC patients and 30 healthy adults were collected. Antibody of BCRP was used to detect its expression in the experiment. Part of venous specimens were randomly selected for Western-blot, and all specimens were examined by ELISA at last. Chemotherapy response of these patients was observed in order to analyze the correlation between BCRP expression level and chemosensitivity. RESULTS: Western blot result showed that BCRP expression can be detected both in NSCLC patient and normal adult. The expression level in NSCLC patients detected by ELISA was significantly higher than that in the healthy adults (P = 0.00); which was also significantly higher in chemo-resistant patients than that in the chemosensitive (P = 0.02) and the healthy adults (P = 0.00); however, BCRP expression in chemo-sensitive patients was not significantly different from that in the healthy adults (P = 0.08). CONCLUSION: Breast cancer resistance protein (BCRP) is found to be expressed at high level in the serum of NSCLC patient, the intensity of BCRP expression may be correlated with chemotherapy resistance in NSCLC, and the high level expressing of BCRP may indicate resistance to the platinum-based chemotherapy regimen. Detection of serum BCRP may someday become a useful bio-marker in predicting chemosensitivity of NSCLC.

[Correlation between the expression of PCDGF in serum and the chemotherapeutic sensitivity in NSCLC].

OBJECTIVE: To investigate the expression of PC cell-derived growth factor (PCDGF) in the serum of non-small cell lung cancer (NSCLC) patients and healthy adults, and it's correlation with chemotherapeutic sensitivity. METHODS: The venous blood samples of 44 advanced NSCLC patients and 30 healthy adults were collected, and PCDGF mono-antibody was used for detection in the experiment. A part of specimens were randomly selected for Western-blot, and all specimens were eventually examined by ELISA. Chemotherapeutic response of these patients was recorded in order to analyze the correlation between PCDGF expression level and chemotherapeutic sensitivity. RESULTS: Western blot results indicated that there was PCDGF expression both in NSCLC patients and healthy adults, and the expressing intensity of PCDGF in NSCLC patients was higher than that in healthy adults. The result of ELISA showed PCDGF expression in the patients whoever was chemoresistant or chemosensitive was significantly higher than that in healthy adults (P < 0.01), However, in chemoresistant patients, it was significantly higher than that in chemosensitive with a borderline statistical difference (P < 0.05). CONCLUSION: PC cell-derived growth factor is found to be not only expressed in healthy adult but also in NSCLC patient at a high level in the serum, which may indicate metastasis and active proliferation in NSCLC. The intensity of PCDGF expression may be correlated with chemotherapy response and the high level expressing of PCDGF may indicate resistant to platinum-based chemotherapeutic regimen.

[TPX2 expression and its significance in squamous cell carcinoma of lung].

OBJECTIVE: To study the expression of targeting protein for Xklp2 (TPX2) and its significance in squamous cell carcinoma (SCC) of the lung. METHOD: Two SCC cell lines and 4 immortalized bronchial epithelial cell lines (as a precancerous model) were examined by Western blot for TPX2 expression. Reverse transcription-polymerase chain reaction analysis for TPX2 was also performed using tumor tissues from 21 patients with SCC of the lung. The expression of TPX2 was studied by immunohistochemistry (using tissue microarray) on paraffin-embedded sections of pulmonary SCC and corresponding precancerous lesions from a group of 319 patients. RESULTS: TPX2 was variably expressed in all the cell lines studied. Compared with matched controls using normal lung tissue, high level of TPX2 mRNA was detected in 16 of the 21 SCC tumor tissue samples analyzed. Immunohistochemical study showed that TPX2 was mainly present in tumor tissues but not in normal controls. The expression of TPX2 correlated with tumor grade, stage and nodal status. As for precancerous lesions, the level of TPX2 was also increased, in accordance with the degree of dysplasia. CONCLUSIONS: Expression of TPX2 may play a role in carcinogenesis of bronchial epithelium and tumor progression of pulmonary SCC. It may also represent a potential biomarker for surveillance of SCC of lung.

Highly sensitive determination of the methylated p16 gene in cancer patients by microchip electrophoresis.

The p16 tumor suppressor gene is inactivated by promoter region hypermethylation in many types of tumor. Recent studies showed that aberrant methylation of the p16 gene is an early event in many tumors, especially in lung cancer, and may constitute a new biomarker for early detection and monitoring of prevention trials. We detected tumor-associated aberrant hypermethylation of the p16 gene in plasma and tissue DNA from 153 specimens using a modified semi-nested methylation-specific PCR (MSP) combining plastic microchip electrophoresis or slab gel electrophoresis, respectively. Specimens were from 79 lung cancer patients, 15 abdominal tumor patients, 30 positive controls and 30 negative controls. The results showed that the positive rate obtained by microchip electrophoresis was more than 26.6% higher and the same specificity was kept when compared with slab gel electrophoresis. The microchip electrophoresis can rapidly and accurately analyze the PCR products of methylated DNA and obviously improve the positive rate of diagnosis of cancer patients when compared with gel electrophoresis. This method with the high assay sensitivity might be used for detection of methylation of p16 gene and even to facilitate early diagnosis of cancer patients.

[Multivariate analysis of prognosis in gastrointestinal stromal tumor].

OBJECTIVE: To identify prognostic factors in patients with gastrointestinal stromal tumors (GIST). METHODS: Hematoxylin and eosin (H&E) stained histopathological slides of tumors from patients with mesenchymal neoplasms growing in the gastrointestinal tract and abdomen were reviewed. Two histologically representative areas were identified and chosen for tissue microarray. Immunohistochemical staining was performed to demonstrate c-kit protein (CD117), CD34, smooth muscle actin, desmin and S-100 protein. The relations of various clinicopathologic features to outcome were analyzed. RESULTS: The overall disease-specific survival of 194 patients was 93.5% at 1 year, 72.1% at 3 years and 63.2% at 5 years. Univariate analysis indicated that the tumor size, mitotic count, primary location, necrosis, high cellularity, mucosal invasion, mixed cell type, hemorrhage, direct tumor invasion of surrounding tissue, male sex, incompleteness of resection, cytologic atypia were significant predictors of survival. Multivariate analysis showed that tumor size, mitotic count, necrosis, direct tumor invasion of surrounding tissue and male sex were poor prognostic signs. CONCLUSION: Tumor size and mitotic count are important prognostic factors. However, to evaluate the prognosis of these tumors, a surgical pathologist should incorporate multiple parameters into their histologic evaluation in attempt to reach an appropriate opinion on the aggressiveness of GIST.

[Abnormalities of microsatellite in transitional cell carcinoma of urinary bladder related with aromatic amine exposure].

OBJECTIVE: To study the microsatellite abnormalities of the aromatic amine exposure-associated transitional cell carcinoma (TCC) and sporadic TCC of urinary bladder, and to evaluate the potential of microsatellite analysis on detection of this diseases. METHODS: Based on our previous investigations, 5 microsatellite markers (D17S695, D9S162, D3S1295, DBH and D3S1234) that had high frequencies of loss of heterozygosity (LOH) in sporadic TCC, were selected for analysis with the bladder lesions derived from 16 patients with aromatic amine exposure history. The microsatellite analysis with urine sediments from the post-operated patients was also carried out. RESULTS: There was at least one informative marker out of the 5 microsatellite foci showed polymorphism in the DNA derived from 16 patients examined. Within 87.50% (14/16) patients, LOH was detected in the bladder lesions at least with one microsatellite marker. The LOH frequency of D3S1295 was higher in occupational TCC patients than that in sporadic TCC patients. The diagnostic accordance rate of patients showed LOH in at least one microsatellite marker with patients diagnosed by pathology was 81.25% (13/16). In the urine sediments from 8 TCC post-operated patients, LOH was found at least with one microsatellite marker. CONCLUSION: There could be a different LOH pattern in aromatic amine exposure-associated TCC, and genes near D3S1295 might play a role in the occupational exposure-associated TCC.

Detailed deletion mapping of loss of heterozygosity on 9p13-23 in laryngeal squamous cell carcinoma by microsatellite analysis.

BACKGROUND: This study was designed to investigate the hot spots of microsatellite loss of heterozygosity (LOH) on 9p13-23 in laryngeal squamous cell carcinoma and to find out the correlation between the incidence of microsatellite LOH and the clinicopathological parameters. METHODS: Tumor tissues were obtained from paraffin embedded sections with microdissection. Genomic DNA was extracted from tumor tissues and peripheral blood lymphocytes with the phenol-chloroform. Polymerase chain reaction (PCR) amplification and denaturing gel electrophoresis were carried out in a set of 42 squamous cell carcinoma (SCC) of larynx and corresponding peripheral blood lymphocytes using 13 highly polymorphic microsatellite markers on 9p13-23. The correlation was analyzed between microsatellite LOH at the high frequency on 9p13-23 and clinicopathological parameters in the patients with squamous cell carcinoma of larynx. RESULTS: Of the 42 laryngeal cancers, 41 (97.6%) showed LOH in at least one of the microsatellite markers tested on 9p13-23. The most frequently deleted marker was D9S162 in 17 of the 19 (89.5%) informative samples. The marker D9S171, which is located on 9p21, had LOH detected in 12 of the 15 informative cases (80.0%). LOH at the D9S1748 marker (closest to the p16 gene locus) was detected in 18 of the 36 informative cases (50.0%). Allelic deletion mapping revealed two minimal regions of LOH encompassing markers D9S161-D9S171 on 9p21 and IFNA-D9S162 on 9p22-23. Multiple LOH (> or = 4) on 9p21-23 was found more frequently in the patients under 60 years, with supraglottic SCC or cervical lymph node metastasis than those over 60 years, with glottic SCC or without cervical lymph node metastasis (P < 0.01 or 0.01, 0.05, respectively). On the contrary, there was no correlation between T stages or pathologic classification and the frequency of LOH on 9p21-23 in 42 SCC of Larynx. CONCLUSIONS: These findings imply the presence of at least two putative tumor suppressor genes on 9p13-23 in laryngeal SCC. Multiple genetic alterations are probably implicated in supraglottic SCC with cervical lymph node metastasis in younger patients.

Quantification of plasma DNA as a screening tool for lung cancer.

BACKGROUND: Recent studies suggest that circulating DNA may be a potential tumor marker for lung cancer, but most of these studies are conducted between healthy controls and lung cancer patients, with few or no benign lung disease patients included. The objective of this study was to evaluate the performance of plasma DNA quantification in discriminating lung cancer from the healthy and benign lung disease. METHODS: Plasma DNA was extracted with a QIAamp DNA Blood Midi kit and quantified by a PicoGreen dsDNA quantitation kit in 44 healthy individuals, 36 benign lung disease patients and 67 lung cancer patients. Discrimination power was evaluated by the receiver operating characteristic curve. RESULTS: Plasma DNA values were significantly increased in lung cancer patients, especially in those with metastases, and in benign lung disease patients compared with that in the healthy individuals (P < 0.001, respectively). The values in lung cancer patients were significantly increased compared with that in the benign lung disease patients (P < 0.001). The area under the curve was 0.96 [95% confidence interval (CI) 0.92 - 0.99] for the healthy versus lung cancer, 0.73 (95% CI 0.64 - 0.83) for lung cancer versus benign lung disease, and 0.86 (95% CI 0.80 - 0.91) for lung cancer versus the healthy and benign lung disease. CONCLUSIONS: Plasma DNA quantification has a strong power to discriminate lung cancer from the healthy and from the healthy and benign lung disease, less power to discriminate lung cancer from benign lung disease. Plasma DNA quantification may be useful as a screening tool for lung cancer.

[Hypermethylation of p16 gene in clinical specimens of patients with lung cancer].

OBJECTIVE: To evaluate aberrant methylation of the p16 promoter as a useful biomarker of lung cancer. METHODS: A modified methylation-specific semi-nested PCR was performed to detect p16 hypermethylation in the matched samples of tumor tissue, blood plasma and sputum derived from 51 cases of lung cancer patients. RESULTS: Hypermethylation of p16 promoter was demonstrated in 84.3% of the tumor tissues, 70.6% of the blood plasma and 76.5% of the sputum specimens, respectively. Only the patients whose tumor tissues had p16 hypermethylation exhibited aberrant methylation in their plasma and/or sputum specimens. Combining with cytological examination, 92.2% of the patients with lung cancer could be detected by p16 hypermethylation assay in both sputum and plasma samples. CONCLUSION: The results indicate that p16 hypermethylation in plasma and sputum identified by semi-nested PCR is a biomarker of lung cancer which can be useful as an auxillary diagnostic parameter.

[Screening of hypermethylated DNA fragments in tumor tissue derived from patients with lung cancer].

The investigations on the role of DNA methylation in carcinogenesis have been mainly focused on promoter hypermethylation of tumor suppressor genes. As a number of genes associated with cancer development may be influenced by DNA methylation, identification of these genes is of great importance for understanding the epigenetic alteration in carcinogenesis. In this study, hypermethylated regions of genomic DNA from Chinese lung cancer patients were identified by a modified methylation-sensitive arbitrarily primed PCR (MS-AP-PCR). Eight hypermethylated DNA fragments (HMDF) were separated from a PCR product region between 300 and 500bp in size. After cloning, sequencing and searching with Blast and NewCpGseek programs,the result showed that all of them were typical CpG island sequences, four fragments had 99% approximately 100% homology to regions on human chromosome 2, 7, 9 and 10, respectively,but only one revealed to be known gene. Neural Network Promoter Prediction, TSSG and TSSW programs were run to analyze possible functions of the rest 7 fragments, of which 4 were identified as candidate promoter regions, indicating that they might belong to new genes. The hypermethylated DNA fragments identified in this study might be specific epigenetic alterations in the Chinese lung cancer.

[Detection of hypermethylation of p16 gene in plasma DNA from lung cancer patients].

OBJECTIVE: To detect hyper methylation of p16 gene in plasma DNA from patients with lung cancer, and to assess its potential as a malignant marker. METHODS: Using a modified semi-nested methylation-specific PCR (MSP), the status of methylation of the p16 was investigated in plasma DNA from 137 lung cancer patients and 112 matched tumor tissues. RESULTS: Hypermethylation of the p16 was present in 75.2% (103/137) of the plasma samples and 80.4% (90/112) of the tumor tissues. Hypermethylation of the p16 in the plasma was detected in 77.9% squamous-cell carcinoma, 65.1% adenocarcionma, 75.1% adeno-squamous-cell carcinoma, and 91.7% small-cell lung cancer. Only in those patients whose tumor tissues had hypermethylation of p16 gene, similar changes could be detected in their plasma samples. Hypermethylation of the p16 in plasma and the corresponding tumor tissues was not significantly correlated with the clinical stage and pathological type of the tumor. CONCLUSION: The result indicates that hypermethylation of the p16 may be a useful marker in the auxiliary diagnosis of lung cancer.

[The detection of Rb2/p130 and p53 gene mutations by denaturing high-performance liquid chromatography analysis of sputum of lung cancer patients and its clinical implications].

OBJECTIVES: To probe Rb2/p130 and p53 gene mutations at their hot-spots by denaturing high-performance liquid chromatography (DHPLC) analysis in sputum samples from lung cancer patients and to evaluate the feasibility of these gene markers in the clinical diagnosis of lung cancer. METHODS: Genomic DNAs were extracted from 47 sputum samples (35 of lung cancer, 12 of benign lung disease) and their parallel peripheral blood lymphoid cells. The genomic DNAs were subjected to PCR amplification of Rb2/p130 gene at exon 19 - 22 and p53 gene at exon 5 - 9. The mutations of Rb2/p130 and p53 were detected by DHPLC by analysis of the PCR products. RESULTS: Of the 47 sputum samples, the Rb2/p130 gene mutation detection rates were 22.86% (8/35) in the lung cancer group and 0 (0/12) in the control group (P = 0.049). The sensitivity and specificity were 22.86% and 100% respectively by using Rb2/p130 gene mutation detection as a diagnostic marker for lung cancer. p53 gene mutation detection rates were 28.57% (10/35) in the lung cancer group and 0 (0/12) in the control group (P = 0.046). The sensitivity and specificity were 28.57% and 100% respectively by using p53 gene mutation detection as a diagnostic marker for lung cancer. The sensitivity and specificity reached 51.43% and 100% respectively when Rb2/p130 and p53 gene mutations were combined as diagnostic markers for lung cancer. CONCLUSIONS: Because of the low sensitivity, Rb2/p130 or p53 gene mutation detection alone is not feasible as a gene marker in the clinical diagnosis of lung cancer. The increased sensitivity, by combining the two gene markers, suggests that it may be feasible to use a panel of molecular markers such as p53, Rb2/p130, and others as diagnostic markers for lung cancer.

[Expression of Survivin in transitional cell carcinoma of urinary bladder and its clinical significance].

OBJECTIVE: Survivin is a member of the inhibitors of apoptosis protein (IAP) family. Recent researches had shown that survivin plays an important role in oncogenesis. This study was designed to investigate the expression of survivin in transitional cell carcinoma (TCC) of urinary bladder and its clinical significance. METHODS: Immunohistochemical assay was used to detect the expression of survivin in 75 cases of tumor tissue and 7 cases of normal bladder tissue. RESULTS: The expression rates of survivin were 77.3% (58/75) in TCC of urinary bladder, whereas no expression of survivin was detected in the 7 cases of normal bladder tissue. CONCLUSIONS: Expression of survivin protein was observed in the tumor tissue derived from the patients with bladder TCC, indicating that this protein may play an important rule in carcinogenesis of human urinary bladder. The expression of survivin was statistically significant associated with tumor grade. Our results suggested that the expression of survivin may be considered as a prognostic factor for bladder TCC.