Lung cancer is one of the most malignant tumors that damage human health and life.Metastasis and recurrence of lung cancer are the leading causes of death.Developing techniques that can detect lung cancer,particularly at an early stage,as well as predict recurrence and metastasis,is an important strategy to improve the outcome.Circulating tumor cell (CTC),regarded as liquid biopsy,open a new field for early diagnosis of lung cancer,as well as for prediction of metastasis,evaluation of prognosis and monitoring of recurrence.

Objective To investigate the clinical value of whole blood red cell distribution width ( RDW) in discriminating lung cancer metastasis.Methods A retrospective analysis was conducted on the patients who were initially diagnosed as primary lung cancer.A total of 525 patients were included for analysis between January 2012 and July 2013,stratified by different stages and metastasis scenarios.RDW data was investigated.Kruskal-Wallis H tests were performed to know the difference of RDW without and within groups.Spearman correlation test was done subsequently to further analyze the correlationship among RDW and clinical parameters.Results RDW was14.5 ( 13.0-15.4 )%in patients with metastasis , which was significantly higher than those without metastasis [12.7 (12.3-13.0)%].Further analysis indicated a similar ascending trend in cases that already had distant or multiple organ invasion.For example,RDW was 14.6 (12.9-15.4) %in patients of stage ⅢtoⅣ,while was 12.6 (12.2-13.1) %in patients of stageⅠtoⅡ.RDW was correlated to lung cancer metastasis and stage advancement.Areas Under Curve ( AUCs) of ROC for lung cancer metastasis and distant metastasis were 0.823 ( 95% CI:0.787-0.859 ) and 0.710 (95%CI:0.655-0.765) respectively,indicating a promising accuracy.The Cut-off value for discriminating lung cancer with/without metastasis was 14.25% with sensitivity being 56.8% and specificity being 98.3%.Conclusion RDW may be a novel biomarker for auxiliary diagnosis of lung cancer metastasis and could be useful to understand state of illness.

Objective To evaluate the application value of circulating tumor cell ( CTC ) in the differential diagnosis of solitary pulmonary nodule ( SPN ) . Methods Peripheral blood samples were collected from 134 patients with solitary pulmonary nodule in Shanghai Chest Hospital from September 2013 to January 2015, including 80 patients with malignant nodule and 54 with benign nodule.CTC levels of the above subjects were detected by ligand-targeted polymerase chain reaction ( LT-PCR ) assay, and serum carcinoembryonic antigen ( CEA ) and cytokeratin 19 fragment ( CYFRA21-1 ) were detected by flow fluorescence assay.Results By Mann-Whitney U Test, the CTC levels of malignant SPN patients [11.06 (8.77-14.41)units/3 ml] were significantly higher than those of benign SPN patients [6.65(4.49 -7.84)units/3 ml] (Z=-6.217,P<0.001).The sensitivity and specificity of differential diagnosis of SPN for CTC were 80%(64/80) and 85%(46/54) respectively.According to the diameter of SPN, the patients were divided into three groups to evaluate the diagnostic value of CTC in SPN with different size .For SPN with diameter less than 8 mm, the sensitivity and specificity of CTC were 6/9 and 4/5 respectively .For SPN with diameter between 8 mm and 20 mm, the sensitivity and specificity of CTC were 83%(35/42) and 85%(29/34).For SPN with diameter greater than 20 mm, the sensitivity and specificity of CTC were 79%(23/29) and 13/15.Conclusion Comparing with the traditional tumor markers, CTC could provide more clinical value in the differential diagnosis of solitary pulmonary nodule .

Objective To explore the clinical diagnostic value of serum human chorionic gonadotropin beta subunit (β-HCG) and alpha fetoprotein ( AFP) in mediastinal germ cell tumors .Methods A retrospective analysis was conducted on the patients who were definitely diagnosed as mediastinal tumors or mediastinal neoplastic lesions .A total of 133 patients were included for analysis between January 2008 and May 2014, divided into two groups.42 cases of mediastinal germ cell tumor patients were marked as case group while 91 cases of other mediastinal tumor or mediastinal neoplastic lesion patients were marked as control group ( including 31 cases of thymoma , 10 cases of mediastinal neurogenic tumor , 2 cases of intrathoracic goiter , 25 cases of mediastinal cyst , 2 cases of mediastinal lipoma , 11 cases of mediastinal lymphoma and 10 cases of thymic carcinoma ) .AFP was detected by chemiluminescence detection , and -HCG was detected by electrochemical luminescence .K-S test was performed to investigate normality of data , non-normally distributed data were described as Median ( interquartile range ) .Mann-Whitney U test was done for measurement of data between two groups .Logistic regression analysis was performed as multivariate analysis.Receiver operating characteristic curve ( ROC) was used to determine the cut-off values.Results The levels of serum AFP and β-HCG in case group were 13.26 (2.39-48.09) ng/ml and 1.99 (0.10-15.7) IU/L, respectively, significantly higher than those in control group [AFP:2.47 (1.78-3.16) ng/ml,β-HCG:0.10 (0.10-0.55) IU/L].The difference of levels of AFP and β-HCG between the case group and the control group were statistically significant ( P=0.000 ) .There were no significant difference when it comes to β-HCG between the case group and intrathoracic goiter patients in control group .Apart from it, the difference of levels of AFP and β-HCG between the case group and every single control group were statistically significant .Cut-off values of AFP and β-HCG for distinguishing mediastinal germ cell tumors from mediastinal tumors were 5.07 ng/ml and 2.32 IU/L.In this scenario, for AFP and β-HCG, sensitivity were 57.1%and 50%, specificity were 97.8%and 96.7%, accuracy were 54.9%and 46.7%, area under the curve ( AUC ) were 0.773 and 0.755, positive likelihood ratios were 26.00 and 15.17respectively.Parallel experiments contributed to increase the sensitivity to 71.4%. Predictive probability (P) =1/[1+exp ( -0.319AFP-0.253HCG+2.850)] was obtained by logistic regression model.When cut-off value of predictive probability ( P ) was 0.30, specificity, AUC, and positive likelihood ratio were increased to 98.9%, 0.835 and 65.00respectively, negative likelihood ratio was decreased to 0.29, positive predictive value and negative predictive value were increased also (96.8%and 88.2%respectively).Conclusion Serum β-HCG, AFP and predictive probability ( P ) is valuable in the diagnosis of mediastinal germ cell tumor .

Observation revealed the abnormalities (increased CD 4 and Th, decreased Tc) in patients of Graves′ disease, and these abnormalities were not significantly adjusted after 6 weeks of treatment with methimazole (MMI), or with MMI and methotrexate (MTX), though the soluble interleukin-2 receptor and thyroid autoantibodies slightly decreased in both groups. However, the addition of MTX did not show any better effects than MMI alone in the treatment.

Objective To estimate the diagnostic value of circulating tumor cell detection for non-small cell lung cancer.Methods A Non-intervention clinical study was conducted in this research.From October 2014 to April 2015, totally 162 NSCLC who presented at Thoracic Surgery Department, 119 benign pulmonary disease and 52 healthy individuals were collected from Shanghai Chest Hospital.Folate receptor ( FR) based polymerase chain reaction ( PCR) method was used to detect the circulating tumor cell ( CTC) level, CEA and CYFRA21-1 was detected by the flowcytometry fluorescence luminance method, SCC was detected with Chemiluminescent microparticle immunoassay.The differences among groups were analyzed by the Kruskal-Wallis test( multi group comparison) and the Mann-Whitney U test( two group comparison) , and the chi-square test was used in the positive rate comparison;the Receiver Operating Characteristics ( ROC) curve was established.Results The median level of CTC in NSCLC patients was 11.90 Units/3 ml, which was significantly higher than those of benign pulmonary disease ( 6.72 CTC Units/3 ml ) and healthy individuals (5.82 CTC Units/3 ml,χ2 =125.990, P<0.01).Areas Under Curve ( AUCs) of ROC curve for NSCLC was 0.853 2(95% CI: 0.809 5,0.896 9).The cut-off value for discriminating NSCLC with benign pulmonary disease/healthy people was 8.74 CTC Units/3 ml with sensitivity being 77.16% and specificity being 90.06%.The positive rate of CTC in Stage I NSCLC patients was 68.7%, which was much higher than that of the combination of tumor markers(χ2 =32.98,P<0.01).Conclusion With relatively high sensitivity and specificity, the detection of circulating tumor cell may has a clinical value of application and extension.

Objective To evaluate the performance of self‐established angiotensin converting enzyme (ACE) detection system . Methods The performance evaluation of ACE reagent kit produced by the Jiuqiang Company including precision ,linearity ,correla‐tion with the reference reagent and reportable range were conducted by using the automatic biochemical analyzer according to the re‐quirements of the EP5‐A2 and EP9‐A documentation in the Clinical and Laboratory Standards Institute (CLSI) .Results Intra‐as‐say CV of the system were 6 .87% ,2 .39% and inter‐assay CV were 6 .09% ,1 .81% ,respectively .During the day CV were 8 .00 %and 2 .8% respectively ,which were less than those provided by the manufacturer (<10% );the lenearity result was R2 =0 .99 .The correlation coefficient (r) of the system comparing with the reference reagent was 0 .990 56 ,moreover the average bias was 8 .55% , showing good correlation ;the repotable range was 9 .0U/L‐600U/L .Conclusion Self‐established ACE detection system can meet the requirements for clinical application .

Objective A new method for detecting K-ras mutations based liquid chip was used to evaluate K-ras mutations associated with non-small cell lung cancer (NSCLC) patients,to direct the personalized treatment and prognosis evaluation.Methods Take the diagnosis technology research methods,the sensitivity and repeatability of the liquid chip K-ras gene mutation detection method were assessed.A total of 100 NSCLC patients from Nov 2011 to Feb 2012 in Shanghai Chest hospital were included in this study,the fresh tumor tissues were collected for DNA extraction.The 2nd exon 12 and 13 codons,containing 8 K-ras mutations occuring in high frequency were amplified by polymerase chain reaction (PCR),followed by ligation of the PCR products to a series of special probes using ligase detection reaction (LDR),then the PCR-LDR products were analyzed by liquid chip platform.Direct sequencing was applied to compare with the detection results.Results The sensitivity of liquid chip technology detection was 10％-20％,higher than the traditional sequencing method by 1％.Average CV value was 4％-15％ and showed good repeatability.5 K-ras mutations in 100 patients (5％) were detected using multiplex PCR-LDR combined fluid chip methods,including 3 Glyl2Val and 2 Gly12Asp mutations in exon 2.The 5 K-ras mutations were verified accurately by direct sequencing.Conclusions The novel detection method of K-ras mutations based PCRLDR and fluid chip shows high throughput,high sensitivity,good repeatability and the results are reliable and accurate.This method can be used to accurately identified K-ras mutations for NSCLC patients prior to their targeted therapy with TKIs.

Objective To explore a new high-throughput method with internal standards for analyzing the methylation profiles of lung cancer related genes. Methods The promoter sequences of 7 lung cancer related genes were cloned into plasmids and the target segments were amplified by their special primers respectively. The products were treated with M. Sss Ⅰ methylase and bisulfite. The multiplex ligation PCR method was established by designing probes containing CpGpCpG(for methylatedsequence) at the 3' ends and choosing the optimal ligation enzyme, annealing and ligation temperatures. The standard calibrators and clinic samples were tested by fluid chip platform. The results were validated by methylationspecific PCR. Results We successfully set up the standard calibrators for methylation and unmethylaiton of 7 lung cancer related genes and established a multiplex ligation PCR combined with fluid chip method, which was used to detect methylation status of 7 genes simultaneously. The fluorescence value of p16INK4A, APC,DAPK, RARIβ, RASSF1 A, MGMT and GSTP1 methylation standard calibrators were 863,909,703,701,901,1 060 and 885, much higher than that of unmethylation standard calibrators. The results were consistent with the results of methylation-specific PCP. ConclusionThe new high-throughput method can be used to evaluate the methylation status of 7 lung cancer related genes simultaneously and might be useful for clinical practice.

Objective:To predict and identify HLA-A * 0201 restricted CD8+ CTL epitopes in Mycobacterium tuberculosis (Mtb) antigen Ag85C, so as to provide evidence for epitope-based study for tuberculosis (TB) vaccine. Methods: The online database SYFPEITHI was applied to predict the potential HLA-A * 0201 restricted epitopes from Ag85C, an antigen of Mycobacterium tuberculosis. T2 cell line was used to assay the affinity between the predicted peptides and HLA-A * 0201 molecules. The specific CTL lines were induced from peripheral blood mononuclear cells (PBMCs) of HLA-A * 0201 positive TB patients and PPD+ healthy donors by peptides with high binding affinity to HLA-A * 0201 molecules. IFN-?production, in vitro proliferation and cytotoxicity of peptide-induced CTL were determined to screen HLA-A * 0201 restricted CD8+ CTL epitopes from those candidates. Results: Fourteen potential epitopes were identified from the SYFPEITHI database. After binding affinity assay, 3 of the 14 peptides (170-178 aa, 317-325 aa, and 144-153 aa) were found to have high binding affinity to HLA-A* 0201 molecules. However, only one peptide (144-153 aa) stimulated its specific CTL to release IFN-y, proliferate in vitro and produce specific cytotoxicity. Conclusion: We have successfully identified a HLA-A * 0201 restricted CD8+ CTL epitope of Mtb Ag85C-FLTREMPAWL( 144-153 aa) , which might be a candidate epitope for TB vaccine designing. Our findings provides a basis for developing novel and effective anti-TB vaccine.