ABSTRACT
Objective To investigate the clinical significance of plasma methylated Septin 9 (SEPT9)gene test for colorectal cancer(CRC).Methods Clinical data of this retrospective study were obtained from Huashan Hospital of Fudan University(2016-2017).The subjects were divided into three groups,84 patients in CRC group,50 patients with adenoma in precancerous group,and 20 cases as healthy controls.A fluorescent PCR assay was used to analyze SEPT 9 methylation in DNA extracted from plasma. Chi square test was used for statistical analysis.Results The positive incidence of SEPT9 gene methylation in plasma was 63.1%(53/84)in CRC group,significantly higher than 10%(5/50)in precancerous group (χ2=35.993, P<0.001), and undetectable in healthy group.The sensitivity of the methylated SEPT9 gene test was 63.1%(53/84), and the sensitivity of a joint detection combined with carcinoembryonic antigen(CEA)was 75%(63/84).The receiver operating characteristic curve(ROC)showed that methylated SEPT9 gene test had 0.828 in the area under the curve(AUC),higher than 0.795 in the AUC of CEA test.In CRC patients,51.4%(19/37)in the stage Ⅰ-Ⅱand 72.3%(34/47)in the stage Ⅲ-Ⅳ were positive for methylated SEPT9 gene test(χ2=3.917, P<0.05).There were no significant differences in gender,age and primary tumor site.Conclusion The SEPT9 gene methylation in plasma is helpful for early screening for CRC,and is associated with CRC progression.
ABSTRACT
Objective To establish a rapid, accurate and low-cost screening method for the detection of calreticulin (CALR) mutations in myeloproliferative neoplasms (MPN).Methods Seventy cases diagnosed with MPN were collected from 2012 to 2016. PCR combined with high resolution melting (HRM) analysis were used to screen the CALR mutations, and Sanger sequencing and T-A sequencing were applied to verify the HRM positive samples. CALR wild type DNA, type 1 and type 2 mutant DNA samples were selected and analyzed 4 times/day for 5 days to detected the CVs of Tm (melting temperature) respectively. JAK2 mutations were also analyzed in MPN patients to compare the association between JAK2 and CALR mutations.Results PCR-HRM analysis showed 7 cases (26.9%) and 5 cases (20.8%) patients with CALR mutations were screened out from 26 essential thrombocythaemia (ET) cases and 24 primary myelofibrosis (PMF) cases, but no CALR mutations were found in cases with polycythaemia vera (PV). All mutations were confirmed by direct sequencing or cloning sequencing. The CVs for HRM analysis of CALR wild type DNA, type 1 and type 2 mutant DNA samples were 1.91%,1.59% and 1.43%, respectively.There were 47 cases with JAK2 V617F and 1 case with exon12 mutation. No coexistence of JAK2 mutation and CALR mutations were found in a single sample.Conclusion PCR-HRM can be used for rapid screening of CALR mutation. Subsequent sequencing can be applied for rapid diagnosis of MPN patients in clinical practice.