Objective:The aim of this study was to develop and validate a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantification of melatonin in human serum.Methods:We describe the performance and validation of melatonin by LC-MS/MS. 182 serum samples from the patients diagnosed with Sleep disturbance who visited the Department of Psychiatry at Zhongshan Hospital affiliated to Fudan University from February 2022 to March 2023(56 males,162 females,mean age [45.51±16.31]years), as well as 182 healthy individuals were included(87 males,95 females,mean age [48.55±11.93]years). The two groups were used to assess the application of serum melatonin levels as a diagnostic indicator for sleep disorders (SDs). The liquid chromatography mass spectrometry (LC-MS) system with an chromatography column (2.1×100 mm, 1.8 μm) was used for separation. The column temperature was set at 35 ℃, as well as the mobile phase consisting of a 0.1% formic acid aqueous solution and pure acetonitrile. The flowing rate was set at 0.4 ml/min for gradient elution. The LC-MS/MS method was validated according to guidance documents, including the following parameters: specificity, selectivity, matrix effect, carryover contamination and reproducibility, lower limit of measuring interval, linearity, precision, recovery rate, dilution consistency, and serum sample stability. Then, it was subsequently employed to profile melatonin changes in Sleep disturbance.Results:The lower limit of quantification for melatonin was 1 pg/ml, and the linear range of detection was 1 pg/ml to 500 pg/ml ( r=0.999). The intra-day and intra-batch precision, expressed as the coefficient of variation ( CV), was within the range from 3.07% to 6.86%, which met the requirement of less than 15%. The recovery rate of the spiked samples ranged from 105.91% to 116.30%. The level of serum melatonin in the sleep disturbance group was significantly lower than that in the healthy control group ([2.00(1.00,3.28)] vs [8.35(4.28,14.80)] pg/ml, P<0.001). Conclusions:The LC-MS/MS method we developed for the quantification of melatonin is clinical practicable.

Objective:To explore the risk stratification and prognostic significance of loss of chromosome Y (LOY) in patients with multiple myeloma (MM).Methods:The clinical data of 193 male patients with newly diagnosed MM admitted to Zhongshan Hospital of Fudan University from January 2018 to January 2020 were analyzed retrospectively and divided into a normal karyotype group(178) and a LOY karyotype group (15) according to the results of their primary conventional cytogenetics. Rank sum test, 2×2 chi-square test and independent sample t-test were used to compare laboratory findings, such as liver and kidney function, immunohistochemistry and cytogenetics, treatment efficacy and survival prognosis, between the two groups. The clinical prognostic significance of LOY was summarized through survival analysis and Cox regression. Results:Among the newly diagnosed male MM patients, 8%(15/178) were confirmed with LOY cases. The proportion of patients with Revised International Staging System(R-ISS) stage Ⅲ was significantly higher in the LOY group (8/15) than that in the normal karyotype group (40/178)(χ 2=7.052, P<0.01). A higher proportion of 1q21 amplification also occurred in the LOY group (10/13 vs 77/162)(χ 2=4.159, P<0.05). The proportion of complete response(CR)/stringent complete response(sCR) in the normal karyotype group after the fourth chemotherapy (63/171) was significantly higher than that in the LOY group (1/15)(χ 2=5.564, P<0.05). The proportion of progressive disease (PD) was lower in the normal karyotype group (16/171 vs 4/15) (χ 2=4.306, P<0.05). The 2-year progression-free survival (PFS) of MM patients for the LOY group was significantly shorter compared to that for the normal karyotype group ( Z=?3.201, P<0.01). Univariate survival analysis showed that PFS was significantly shorter in newly diagnosed MM patients with Creatinine(Cr)≥93 μmol/L, β 2-microglobulin (β 2-MG)≥4.0 mg/L, serum free light chain(sFLC)<0.06, bone marrow plasma cells (BMPC)≥30%, R-ISS stage Ⅲ, failure to achieve CR/sCR after the fourth chemotherapy, with LOY, 1q21 amplification, P53 deletion and t(4;14) ( P<0.05). Cox regression analysis showed that Cr≥93 μmol/L( HR=4.460, 95% CI 1.615-12.314, P=0.004), sFLC<0.06( HR=2.873, 95% CI 1.206-6.849, P=0.017), failure to achieve CR/sCR after the fourth chemotherapy( HR=3.522, 95% CI 1.437-8.634, P=0.006)and with LOY( HR=3.485, 95% CI 1.473-8.249, P=0.006)were independent risk factors for PFS in newly diagnosed MM patients. Conclusions:LOY is an independent risk factor for poor prognosis. It is important for the clinical outcome and prognosis of patients with newly diagnosed MM, and may become a novel clinical assessment indicator.

Objective:This work aims to evaluate the clinical values of comprehensive genomic profiling examination based on circulating tumor DNA (ctDNA) in advanced lung cancer patients.Methods:This is a single-center, retrospective study that collected peripheral blood samples from patients with advanced lung cancer and performed gene mutation analysis using the TruSight Oncology 500 ctDNA assay kits. Between February 2022 and March 2023, a total of 82 patients were enrolled in Zhongshan Hospital, Fudan University, and 76 patients were included in the final analysis.According to the AMP/ASCO/CAP guidelines, mutations of targeted genes were divided into four levels (Tier I-IV), and the effectiveness of targeted therapy guided by ctDNA was evaluated. Descriptive statistics were used for basic characteristics, and the analysis of factors related to tumor mutational burden (TMB) was performed using the rank-sum test.Results:The ctDNA detection success rate was 92.7%(76/82).The median turnaround time for ctDNA testing was 10.5 days (9,13 days). At least one actionable mutation (Tier I or Ⅱ) was detected in 82.9%(63/76) of patients, and 28.6% (18/63) of patients received matched therapy, achieving a disease control rate of 18/18 and an objective response rate of 12/18.Conclusion:Comprehensive genomic profiling based on ctDNA can effectively identify actionable alterations in patients with advanced lung cancer and provide valuable information for matched therapy.

Objective:To verify the performance of the next-generation sequencing (NGS) platform and evaluate the application of NGS, droplet digital PCR (ddPCR) and super amplification refractory mutation system (super-ARMS) in the detection of circulating free DNA (cfDNA) mutations in patients with non-small-cell lung cancer (NSCLC).Methods:A total of 75 patients with NSCLC in the respiratory department of Zhongshan Hospital Affiliated to Fudan University were enrolled. The standards, cfDNA from 25 patients with newly diagnosed and untreated NSCLC, and self-made mixed samples mixed with hemoglobin (1 000 mg /dl), bilirubin (500 mg/l), fat emulsion (2%), enterococcus gDNA and Escherichia coli gDNA were used to verify the blank limit, analytical sensitivity, precision, accuracy and specificity of NGS platform. The cfDNA mutations of 75 NSCLC patients were detected by ddPCR and NGS, and the mutation positive rates of the two platforms were compared. The linear relationship between the two platforms was compared by Pearson correlation test. 12 patients were selected by simple random sampling for the detection of plasma super-ARMS platform. The performance of three platforms in the detection of plasma cfDNA mutation in patients with NSCLC was compared.Results:The blank limit of NGS platform was set to 0.00%, the analytical sensitivity was 0.2%, the intra-assay precision and inter-assay precision were 100%. The test results were not affected by endogenous hemoglobin, bilirubin or fat emulsion in plasma or exogenous DNA interference, and the analysis specificity was good. The mutation positive rates of plasma cfDNA in 75 NSCLC patients detected by ddPCR and NGS were 61.33% and 60.00%, respectively. The complete coincidence rate was 89.33%, which suggests there was a positive correlation between the mutation abundance of NGS and ddPCR ( r=0.984, P=0.001). Among the plasma of 12 NSCLC patients, the results of NGS, ddPCR and super-ARMS were completely consistent in 7 cases, including 2 wild-types and 5 mutants. Conclusion:The NGS platform was verified to be useful for cfDNA mutation detection in patients with NSCLC. The ddPCR, NGS and super-ARMS have their own advantages in detecting cfDNA mutations in patients with NSCLC.

BACKGROUND: The detection of driver oncogenes of lung cancer is of great importance. There are various gene detection techniques nowadays which are different from each other. We carried out this study to investigate the specificity and sensitivity of assay panels based on an Amplification Refractory Mutation System-polymerase chain reaction (ARMS-PCR) technique of Amplification Mutation Specific System (AMSS) in detection of lung cancer gene mutation. To estimate the applicable value of assay panels in clinical settings. METHODS: We collected cancer tissue specimens or fluid specimens from 309 patients. Mutation results were presented for those samples previously detected by ARMS-PCR. In comparison, we carried out AMSS-PCR using (epidermal growth factor receptor, EGFR) assay panel and Six-Alliance assay panel as well as Sanger sequencing. Software SPSS 22.0 (SPSS IBM) was used for statistical analysis. RESULTS: The rates of consistency between the results by assay panels and Sanger sequencing or ARMS-PCR were 97.41% and 97.73%, respectively. Besides, EGFR assay panel had higher consistency rates with other detection methods than Six-Alliance assay panel. As for consistency test, the Kappa values of assay panels with Sanger sequencing, assay panels with ARMS-PCR, and ARMS-PCR with Sanger sequencing were 0.946, 0.953, and 0.913, respectively. The receiver operating characteristic curve (ROC) area under curve (AUC) of assay panels was 0.976 referring to Sanger sequencing, and 0.975 as ARMS-PCR was referred to. CONCLUSIONS AMSS-PCR can make an optimal cancer gene mutation detection method for clinical settings.
Adult ; Aged ; Aged, 80 and over ; DNA Mutational Analysis ; methods ; Female ; Humans ; Lung Neoplasms ; genetics ; Male ; Middle Aged ; Polymerase Chain Reaction ; ROC Curve ; Young Adult

Objective To evaluate the short-term prediction of high-sensitivity cardiac troponin T (hs-cTnT) and other cardiovascular risk biomarkers in patients undergoing maintenance hemodialysis (MHD).Methods We conducted a cohort survey in 296 consecutive MHD patients whose clinical data were retrospectively analyzed.Before MHD,hs-cTnT and other relative cardiovascular biomarkers were detected.The end point (all-cause death) and time of occurring were recorded in the next 13 months.The differences between survival and all-cause death were analyzed by t-test,Mann-Whitney test and x2 test.The best two percentile cutoff point was calculated by X-tile and the survival rate was calculated by Kaplan-Meier Logistic regression analysis was applied to analyze the odd ratio between high risk and non-high risk hs-cTnT group.Non-high risk group was divided into intermediate risk and low risk group based on the 99th percentile of hs-cTnT in healthy population,to further evaluate its short-term prediction value for MHD patients.The short-term significance of hs-cTnT was proved to be independently associated with all-cause death by Logistic regression analysis.Results The mean value of serum hs-cTnT in survival group was 0.05 (0.03～0.07) ng/mL,while in the death group it was 0.07 (0.04～0.14) ng/mL,which had statistical significance (P =0.027).The best two percentile cutoff of hs-cTnT in MHD patients was 0.1 ng/mL.The survival rate in high risk group (hs-cTnT＞0.1 ng/mL) is lower than it in non-high risk group (hs-cTnT≤0.1 ng/mL) (76.67％ vs.96.62％,P ＜0.05).The odd ratios for high risk group and non-high risk group was 7.288 (P＜ 0.001).Moreover,further grouping the non-high risk group by hs-cTnT =0.014 ng/mL,intermediate risk group (hs-cTnT＞0.014 ng/mL) group has lower survival rate than low risk group (hs-cTnT≤0.014ng/mL),while there wasn't any death case occurred in the low risk group.Conclusions Hs-cTnT is an independent risk factor to all-cause death.Thus hs-cTnT can be a strong indicator of short-term prediction and prognostic evaluation.

Objective We are going to establish a robust liquid chromatography-tandem mass spectrometric(LC-MS/MS) method for plasma aldosterone assay.Methods 324 healthy individuals were enrolled in Zhongshan Hospital from February to April in 2016 for reference interval survey.The signallinearity,lower limits of quantitation,precision and accuracy of LC-MS/MS have been evaluated.Results from LC-MS/MS and RIA methods were compared.Software SPSS17.0 software was used for statistical analysis.Results The performance characteristics for the method in terms of linearity,lowerlimits of quantitation,precision and accuracy were verified.Linear range of ALD were between 25-2000 pg/ml;the LC-MS/MS assay had a limit of quantitation of 20 pg/ml for ALD;the intra-and inter-assay CV of ALD were ＜10％ and ＜6％,respectively;the recovery of ALD from serum samples ranged between 97.3 and 105.8％ The reference value of ALD in health people ranged between 21-211.6 pg/ml The regression equation by LC-MS/MS (X) and RIA (Y) was:Y =0.271X + 138.900(r=0.43;n=322).Conclusion LC-MS/MS method is robust and reliable for the analysis of aldosterone in plasma and suitable for clinical application.

Objective The aim of our study was to develop a robust LC-MS/MS method for determination of MN and NMN in blood plasma.Methods A liquid chromatography -tandem mass spectrometric ( LC-MS/MS) method was used, with signal linearity, lower limits of quantitation, precision and accuracy being evaluated.The study recruited 126 healthy volunteers, and MN and NMN in blood plasma were determined.At the same time samples from 21 patients ( 17 pheochromocytoma, 4 ectopic pheochromocytoma) , a hypertension group of 108 persons, and a control group of 84 persons were analyzed. A paired T test was used to compare the MN and NMN levels between the different groups.Results The performance characteristics for the method in terms of linearity, lower limits of quantitation, precision and accuracy were verified.Significant differences were found between the concentration levels of MN and NMN in the diseased and healthy groups.Conclusion A robust and reliable LC-MS/MS method for the determination of MN and NMN in blood plasma has been developed and was shown to be suitable for clinical application.

Objective To verify and monitor the performance of accuracy, precision and comparability of 26 clinical biochemical analytes (29 methods) in the six centers involved in multi-centers reference intervals research, and to ensure the reliability of theirmeasurement results.Methods During the period of the systems evaluating, two levels of commercial quality control materials and fresh frozen human serum reference materials were applied to verify the performance of inter-laboratory precision and accuracy of analysis systems. During the period of samples testing, the commercial quality control materials were measured whenever samples were analysed, the fresh frozen serum reference materials were measured once a month.The coefficient of variations (CVs), bias and total errors were calculated to assess the precision, accuracy and comparability.Results Verification of precision and accuracy: ( 1 ) the ranges of CVs of 29 methods in the six laboratory laboratories were 0.4%-6.0%, the CVs of all 29 methods met the criterion . (2) The overall average bias of the analysis systems of 21 analytes (24 methods) ranged from -5.15%( ALT) to 4.46% ( Ur ) .Among 24 methods the overall average bias of TP, Glu-GOD, Ur, Cl, Ca exceeded the acceptable range.The quality assessment during the period of samples testing:(1) The overall average bias ranged from -1.95%(Ca) to 2.92%(Ur), median 1.26%, they all met the requirements of relevant standards.( 2 ) When commercial control materials were tested, the requirements of CVs were fulfilled for most methods in the six laboratories,and the CVs of TP, Alb, Cl, Ca exceeded the acceptable range.The overall average TE of all methods met the quality specification for the C-N controls material.For the C-P control material, only the overall average TE of TP (5.05%) exceeded thearceptable range while the other methods met the requirement in criterion.Conclusions The performance of precision and accuracy of the analysis systems used in the six laboratories passed the verification.During the period of sample testing, the performance of precision and accuracy of the most methods in the 6 laboratories met the requirements of quality specifications, and the overall performance was good.Because of the limitation of current technology the performance of some methods didn't fulfill the requirement of specifications, and need to be improved.

Background and purpose: Uridine diphosphoglucu-ronosyl transferase 1A1 (UGT1A1) is an important enzyme for metabolism of irinotecan. The activity of UGT1A1 enzyme was significantly affected by the gene polymorphism. This study aimed to investigate the correlation of UGT1A1*28 and *6 gene polymorphisms with irinotecan-based chemotherapy in colorectal cancer(CRC). Methods: Analysis of UGT1A1*28 and *6 gene polymorphisms was performed in 160 gastrointestinal cancer patients admitted to Zhongshan Hospital Fudan University from Apr. 2013 to Dec. 2013 by amplifying the gene fragments using PCR, STR and Sanger sequencing. Eighty-two cases with CRC treated with irinotecan were chosen to observe the adverse events during chemotherapy. The incidence of different genotypes was compared. Results:The distribution of the genotypes in 160 gastrointestinal cancer patients was as followed:UGT1A1*28 wild-type genotype TA6/6 (124, 77.5%), heterozygous genotype TA6/7 (33, 20.5%), and homozygous genotype TA7/7 (3, 2.0%);UGT1A1*6 wild-type genotype GG (105, 65.6%), heterozygous genotype GA (48, 30.0%), and homozygous genotype AA (7, 4.4%). In the 82 CRC cases, the incidences of grade 3 and 4 neutropenia in the patients carrying UGT1A1*28 (TA6/7+TA7/7 ) were higher than those in the WT genotype (TA6/6) (58.3%vs 0.0, P<0.001), and increased the total incidence of adverse events (76.0%vs 45.6%, P<0.001). There was no signiifcant relevance between UGT1A1*6 genotype, age, gender chemotherapy and adverse events. Conclusion:In the CRC cases with irinotecan-based chemotherapy, the UGT1A1*28 (TA6/7+TA7/7) genotype signiifcantly increased the risk of grade 3 and 4 neutropenia. Detecting UGT1A1 gene polymorphisms may guide individualized treatment and predict adverse events.