Background. KRAS genotyping in tumor samples is a decisive clinical test for the anti-EGFR therapy management. However, the complexity of KRAS mutation landscape across different cancer types and the mosaic effect caused by cancer cellularity and heterogeneity make the choice of KRAS genotyping method a challenging topic in the clinical practice. Methods. We depicted the landscape of somatic KRAS mutation in 7,844 primary tumors and 10,336 metastatic tumors across over 30 types of cancer using the Cancer Genome Atlas (TCGA) and Integrated Mutation Profiling of Actionable Cancer Targets (MSKCC-IM PACT) databases, respectively. A snapback primer assay based on melting curve analysis was developed to detect the most common somatic mutations in KRAS codons 12 and 13. The sensitivity and accuracy of the method was validated by genotyping 100 colorectal cancer (CRC) samples, in comparison with Sanger sequencing and T-A cloning sequencing. Results. Pancreas adenocarcinoma (somatic mutation frequency 90.6%), colorectal adenocarcinoma (42.5%), and lung adenocarcinoma (32.6%) are the top three most KRAS mutant primary cancer types. The metastatic tumors showed a higher prevalence (90.99% versus 66.31%) and diversity of KRAS mutation compared with the primary tumors. Mutations in codons 12 and 13 are the predominant genetic alteration in KRAS (84.15% for TCGA and 86.13% for MSK-IMPACT). Moreover, KRAS mutation is highly correlated with the overall survival of patients with metastatic cancer. The snapback primer assay showed a more favorable performance in enriching and detecting the KRAS codon 12 and 13 mutation (1% mutation load) compared with Sanger sequencing (20% mutation load and 7% false-negative rate). Conclusions. KRAS mutation pattern is highly diverse among different cancer types and is associated with the survival of patients with metastatic cancers. The snapback primer assay is a reliable, sensitive method to detect the major mutant KRAS alleles, which might facilitate the effective cancer treatment decisions.

The ability to simultaneously detect JAK2 V617F and MPL W515K/L mutations would substantially improve the early diagnosis of myeloproliferative neoplasms (MPNs) and decrease the risk of arterial thrombosis. The goal of this study is to achieve a point of care testing platform for simultaneous analysis of major genetic alterations in MPN. Here, we report a microfluidic platform including a glass capillary containing polypropylene matrix that extracts genomic DNA from a drop of whole blood, a microchip for simultaneous multi-gene mutation screening, and a handheld battery-powered heating device. The µmLchip system was successfully used for point-of-care identification of the JAK2 V617F and MPL W515K/L mutations. The µmLchip assays were then validated by mutation analysis with samples from 100 MPN patients who had previously been analyzed via unlabeled probe melting curve analysis or real-time PCR. The results from the µmLchip were in perfect agreement with those from the other methods, except for one discrepant result that was negative in the unlabeled probe melting curve analysis but positive in the µmLchip. After T-A cloning, sequences of cloned PCR products revealed JAK2 V617F mutation in the sample. The portable microfluidic platform may be very attractive in developing point-of-care diagnostics for MPL W515K/L and JAK2 V617F mutations.

Objective To evaluate the sensitivity, repeatability and accuracy of microarray digital PCR system in detecting JAK2 V617F mutation, which was closely related to myeloproliferative neoplasms (MPN).Methods All of the 31 MPN patients with JAK2 V617F mutation, including 18 cases of polycythemia vera(PVs),11 primary thrombocythemias (ETs) and 2 primary myelofibrosis (PMFs), were collected from Huashan Hospital, Fudan University during 2014 -2015, while 10 normal controls and 6 cases with abnormal increased hemoglobin were involved.Human erythroleukemia cell line ( HEL ) and colorectal cancer cell SW480 were used as the mutant and the wild type control, respectively.The sensitivity of microarray digital PCR were verified by detecting the gradient diluted mutation standard harboring 30%, 10%, 1%, 0.1%and 0.01%mutant allele burden, respectively .Repeatability was evaluated by detecting 1%and 10% mutated samples for 5 times, respectively.MGB probe real time PCR was selected as the reference method to verify the accuracy of the digital PCR.Results With digital PCR, the accurate quantitation of JAK2 V617F mutation was achieved down to 0.1%, which is approximate to 0.16 copies per microliter.The results obtained from the two kinds of technique showed a high correlation by linear regression analysis (R2 =0.998 3).The results of repeated samples showed CVs as 17.18% for 1%mutant allele burden and 7.50%for 10%.Among all cases, the 31 patients known mutated were detected as positive and 10 controls as negative by both digital PCR and Real time PCR.In another 6 cases, 2 were found JAK2 V617F mutation of low allele burdens of 0.37% and 0.18% by digital PCR but detected as negative by real time PCR.Conclusions Microarray digital PCR offers a higher sensitivity and better repeatability than real time PCR which could help detect rare JAK2 V617F mutations in MPNs accurately.

Extramammary Paget disease (EMPD) is a rare cutaneous malignant neoplasm. The familial occurrence of EMPD and the high risk of concomitant secondary tumors in EMPD patients have gained much attention. These findings highlight the importance of genetic alterations in the tumorigenesis of this skin cancer. Genetic tests and functional analysis of mismatch repair (MMR) genes were performed in EMPD. The results showed that 8 of 20 cases with germline MMR genes mutations and 5 of them exhibited microsatellite instability (MSI). Immunohistochemical staining showed that the tumor tissues from 20 patients had the normal expression of MLH1 but 5 cases had the reduced expression of MSH2. There is a nearly significant correlation between MSI and germline mutations. In 172 cases, rates of germline and somatic mutations were 34.3% and 13.4%, respectively. The mutations of MLH1 V384D (15.7%), R217C (4.1%), and I219V (5.2%) were common in this cancer. In addition, the yeast 2-hybrid and immunoprecipitation assays exhibited reduced interaction between MLH1 and PMS2 in MLH1 V384D and R217C but not I219V. Moreover, MLH1 V384D and R217C had impaired MMR activity compared with the wild-type and I219V mutation by an in vitro MMR assay. The germline mutations in MMR genes are involved in the pathogenesis of EMPD and partially explain the genetic abnormalities for this disease.

An elevated serum bilirubin has been reported to be associated with a reduced risk of some cancer; however, the prognostic significance of serum bilirubin in colorectal cancer wasn't fully understood. The purpose of this study was to evaluate whether serum bilirubin could predict the prognosis of patients in stages II and III colorectal cancer. A retrospective cohort of 986 patients with colorectal cancer who received surgical resection between January 2005 and December 2010 was included in the study. Levels for serum bilirubin were obtained from medical records. Survival analysis was used to evaluate the predictive value of bilirubin. Serum direct bilirubin (DBIL) was validated as a significant prognostic factor by univariate cox regression test for both overall survival (OS) and disease free survival (DFS) (P < 0.05). X-tile program identified 3.6 as optimal cutoff values for DBIL in terms of OS and DFS. Patients were then divided into DBIL high (DBIL ≥ 3.60 μmol/l) and low group (DBIL < 3.60 μmol/l) according to the optimal cutoff. High DBIL had higher percentage of lymph node metastasis and lymphovascular invasion as compared with low DBIL levels (P < 0.05). Multivariate cox regression analyses confirmed that high DBIL level was an independently prognostic factor for both OS (HR: 1.337, 95% CI: 1.022-1.748, P = 0.034) and DFS (HR: 1.312, 95% CI: 1.049-1.643, P = 0.018). In addition, nomograms on OS and DFS were established according to all significant factors, and c-indexes were 0.715 (95% CI: 0.683-0.748) and 0.704 (95% CI: 0.678-0.730), respectively. Nomograms based on OS and DFS can be recommended as practical models to evaluate prognosis for CRC patients.

Ephrin-A2, a member of the Eph/ephrin family, is associated with tumorigenesis and tumor progression. This study aimed to assess the diagnostic and prognostic value of both serum and tissue levels of Ephrin-A2 in prostate cancer (PCa) management. One hundred and forty-five frozen prostate tissues, 55 paraffin-embedded prostate tissues, 88 serum samples, and seven prostate cell lines (RWPE-1, LNCaP, LNCaP-LN3, PC-3, PC-3M, PC-3M-LN4, and DU145) were examined via quantitative reverse transcription-PCR (qRT-PCR), immunohistochemistry, enzyme-linked immunosorbent assay, and western blotting. Induced Ephrin-A2 messenger RNA (mRNA) or protein expression was detected in 8.6 % (5/58) benign prostatic hyperplasia (BPH), 59.8 % (52/87) PCa, and five prostate cancer cell lines. Ephrin-A2 immunostaining was present in 6.7 % (1/15) patients with BPHs and 62.5 % (25/40) clinically localized PCa. Accordingly, serum Ephrin-A2 was significantly higher in PCa patients compared to those in the BPH patients and controls (P < 0.001). The expression of Ephrin-A2 was higher in tumor patients with an elevated Gleason score or T3-T4 staging. Ephrin-A2 expression was correlated with Ki-67 expression in PCa patients, both at the gene scale and protein level. Our data indicate that Ephrin-A2 is a potential diagnostic and prognostic biomarker and a promising molecular therapeutic target to attenuate prostate cancer progression.

Extramammary Paget's disease (EMPD) is a rare cutaneous neoplasm. The aim of this study was to elaborate the clinical and pathological features of Chinese EMPD male patients. The study comprised 246 patients with EMPD at our institute from January 1993 to December 2012. Scrotum was the most common initial site. The average age of onset was 63.9 years but the mean delay in diagnosis was 3.6 years. EPMD spread exclusively to the inguinal lymph nodes and the right inguinal lymph nodes are more likely to suffered Paget cells infiltration. Accompanying malignancies were found in 20 patients. Pathological examination revealed 63 patients defined as invasive EMPD. Immunohistochemical detection showed various expression levels of EMA, CEA, CK7, HER2/neu, Ki67, P53, CK20 and S100 in tumor tissues, but negative expression of VIM, LCA and HMB45. HER2/neu protein exhibited a significant association with invasive EMPD. A novel histological type of EMPD with CK7-/S100+ was identified. Elevated serum PSA level was observed in only 16% patients. Invasive EMPD often had advanced age of onset. Metastatic EMPD showed significantly shorter in the delay in diagnosis and the greater length of skin lesion in contrast to others. This study demonstrates the clinical and pathological features of Chinese male EMPD patients, and may provide implications for the management of Chinese EMPD patients.

在当前医疗环境下,临床检验项目在疾病诊疗中的重要性日益凸显,而各种负面因素均可造成检验项目的过度使用,这势必浪费宝贵的医疗资源.通常理想化的项目使用模式以科学合理、高品质、高性价比的诊疗服务为目标,以最终提高临床医疗服务质量和效益为目的.首先,检验项目优化使用的关键在于检验医师和临床医生相互合作,及建立合理的检验项目使用模式;其次,检验科需发挥积极的精准检验作用;最后,临床医生和检验医师需通过合作制定检验项目诊断指南和流程,并通过对计算机医嘱录入系统进行特殊设置,实现对检验项目指南应用的科学管理,以避免重复检查或减少不必要的检测项目.

Myeloproliferative neoplasms ( MPN ) are clonal hematopoietic stem cell diseases characterized by proliferation of one or more myeloid cell lineages in the bone marrow and increased mature and immature cells inperipheral blood.In recent years , several molecular markers have been discovered , such as JAK2 (JAK2 V617F and JAK2 exon12),MPL515 and TET2 mutations and so on.The discoveries provide important significance of better understanding of the pathogenesis in MPNs and are helpful for the purposes of both diagnosis and therapy.However , diagnosis remains difficult in the remaining 30%-45% of patients who lack JAK2 or MPL mutations.Recently as was reported , the identification of CALR mutations will partly address a gap above and may be a new biomarker in the molecular diagnosis of MPNs .In this review,the discovery of CALR mutations in MPN will be introduced.

Objective To investigate the infection situation and the gene subtype distribution characteristics of human papilloma-virus(HPV)in Fengxian District of Shanghai to provide the reliable scientific basis for preventing HPV infection and the prevention and treatment of cervical cancer.Methods The PCR combined with reverse dot-blot technique was adopted to analyze the HPV de-tection results among 18 194 women in our hospital from February 2011 to October 2013.Results Among 18 194 cases of sample, 2 986 cases were HPV positive with the total HPV infection rate of 16.41%,in which 21 genotypes were all detected out.Single HPV genotype infection was in 2714 cases,two kinds of HPV genotype infection were in 148 cases(4.96%)and three kinds or more of HPV genotype infection were in 124 cases.The HPV positive rate was 16.88% in the outpatient and 12.76% in the inpa-tients.In the HPV infection,the high-risk type was more than the low-risk type,its detection number was 7.5 times of low-risk type.Most of the high-risk HPV infection were HPV16,52,58;most of the low-risk HPV infection was HPV cp8304,followed by HPV6 and HPV11.Conclusion The HPV prevalence in Fengxian District is generally in line with the Asian population distribution rule,but has its own unique regional characteristics.The infection age peak is 51-65 years old.The high-risk type infection is high-er than the low-risk type infection,which is dominated by the single genotype infection.

OBJECTIVE To investigate the feasibility of a new serological screening model of syphilis which non-treponemal antigen serum test was performed as the first test is positive.METHODS A total of 1584 clinical samples were screened for syphilis with chemiluminescence immunoassay(CLIA) and treponema pallidum gelatin particle agglutination test(TPPA),respectively.The positive samples were retested by toluidine red unheated serum test(TRUST).RESULTS Totally 57 and 56 samples were detected by CLIA and TPPA with positive rate of 3.6% and 3.5%,respectively,there was no significant difference in positive rate between the two methods,and there was 99.9% of the coincidence rate between them(Kappa=0.995,P0.01).Of 57 positive samples,35 were detected by TRUST with detection rate of 61.4%,which was lower than that by CLIA(100.0%) and TPPA(98.2%),there was statistically significant difference(all P0.01).CONCLUSION The new screening model of syphilis with specific antibody of treponema pallidum detected by CLIA as confirmation test and then the positive samples retested by TRUST is feasible,which can increase sensitivity and lower false-negative and is better than currently used traditional screening model of syphilis.

Objective To construct the recombinant plasmid with gene CD24 and to express immunogenic protein in human embryo kidney Hek293 cells.Methods CD24 gene was cloned into the vector pYD5-hFC by PRC method.The required recombinant plasmids pYD5-CD24 transformed into the GH10α component cells.Then the selected recombinant plasmids were transfected with lipofectamine reagent into Hek293 cells.After 72 hours,the protein in the culture supernatant of the Hek293 cells were collected and analyzed by western blot.Results The recombinant plasmid transfected with CD24 gene were constructed.It was detected out that the CD24 protein with hFC sign were expressed in human embryo kidney Hek293 cells.Conclusion The CD24 gene expressed successfully in the transfected Hek293 cells and had good immunogenic activity.

Objective To explore the correlation between apolipoprotein M(apoM) and other blood lipid indicators such as total cholesterol(TC),triglyceride(TG),high-density lipoprotein cholesterol(HDL-C),and low-density lipoprotein cholesterol(LDL-C).Methods Plasma levels of apoM were measured by ELISA in 200 outpatients(101 male and 99 female).The levels of TC,TG,HDL-C and LDL-C were measured by enzymatic method.The correlation between ApoM and other blood lipid indicators was analyzed.Results Male and female data mixed analysis showed that ApoM was significantly and positively correlated with TC(r=0.418,P<0.01),HDL-C(r=0.786,P<0.01) and HDL-C/TC(r=0.671,P<0.01),and negatively related to TG(r=-0.407,P<0.01).Conclusion Level of ApoM is significantly and positively correlated with TC,HDL-C and HDL-C/TC,respectively,while it is negatively correlated with TG.It suggests that ApoM may play a role in the metabolism of lipid.