Objective To examine the significance of susceptible gene detection for hereditary cancer syndrome (HCS) among general population. Methods A total of 2 928 individuals undergoing routine health examinations in Healthcare Center of Zhongshan Hospital, Fudan University, from September 2021 to April 2024 were enrolled retrospectively. Next generation sequencing was employed to identify susceptible genes for HCS. American College of Medical Genetics and Genomics (ACMG) guideline was used to analyze the pathogenicity of variants. Clinical data, imagings, follow-up data were also collected. Results The overall mutation rate of HCS panel was 3.59% (105/2 928), with 0.61% (18/2 928) for MutY DNA glycosylase (MUTYH), 0.27% (8/2 928) for breast cancer susceptibility gene 1/2 (BRCA1/2) and 0.23% (7/2 928) for mismatch repair (MMR) genes. Conclusions Healthy individuals carrying tumor susceptible genes usually lack the relevant clinical phenotypes. Whether comprehensive testing needs to be carried out among healthy people remains to be further explored.

Objective:To analyze the relationship between clinicopathological features and germline mismatch repair (MMR) gene variants in endometrial cancer patients and to evaluate the clinical utility of germline multi-gene panel testing.Methods:This single-center, retrospective case series study included 100 endometrial cancer patients treated in Zhongshan Hospital, Fudan University between July 2022 and February 2024. We collected clinicopathological data and tumor molecular testing results. 61 cancer susceptibility genes were tested using next-generation sequencing, and the associations between the detection rate of germline variants and the clinicopathological characteristics in endometrial cancer patients were explored.Results:Among 100 patients, 28% (28/100) were found to have pathogenic variants in cancer susceptibility genes, of which 20 patients carried germline MMR gene variants and the remaining 8 patients carried variants in other cancer susceptibility genes. Of the 20 patients diagnosed with Lynch syndrome, only 40% (8/20) met the Chinese family history criteria for Lynch syndrome. Among 53 patients with intact MMR protein expression, 1 patient was identified with a germline MMR gene variant. In the 14 Lynch syndrome patients with confirmed microsatellite status, 5/14 of those showed low microsatellite instability or microsatellite stability. Germline multi-gene panel testing in all endometrial cancer patients additionally identified 1 Lynch syndrome patient and 8 patients with non-Lynch hereditary cancers.Conclusion:Current clinical screening criteria may miss some endometrial cancer patients with Lynch syndrome. Compared with traditional screening pattern, germline multi-gene panel testing not only improves the detection rate of Lynch syndrome in endometrial cancer patients but also identifies other hereditary cancer predispositions.

Objective:? To summarize the achievements in improving the consistency of clinical liquid chromatography-tandem mass spectrometry (LC-MS/MS) testing results.Methods:? From 2021 to 2024, Zhongshan Hospital Affiliated to Fudan University recruited laboratories voluntarily participating in the MSHP (Clinical LC-MS/MS Testing Consistency Program). As of Batch 202404, a total of 76 laboratories had enrolled, including 60 medical institutions (all tertiary hospitals) and 16 third-party laboratories. Test items were established, and comparative samples were distributed regularly-each item′s samples covered three concentrations (high, medium, and low). Samples were shipped via cold chain and tested within one week. Our laboratory′s measurements served as the target, with participating labs′ results within ±25% of the target deemed qualified. Passing-Bablok regression and Bland-Altman analysis were used to assess consistency.Results:Taking 3-MT (3-methoxytyramine) as an example, the coefficients of variation (CVs) for the project′s three concentration levels improved from 17.00%, 47.18%, and 4.88% in the first comparative batch to 9.59%, 9.59%, and 6.1% in Batch 202404. Passing-Bablok regression results for the 5 units participating in 3-MT testing showed that Laboratory A had proportional bias but no systematic bias (regression slope [95% CI]: 0.903 [0.862-0.952]; intercept [95% CI]: 0.912 [-1.921-6.073]). The remaining laboratories exhibited no proportional or systematic bias with the target (Laboratory B: slope 1.031 [0.961-1.147], intercept-0.733 [-4.641-8.272]; Laboratory C: slope 0.982 [0.940-1.009], intercept-0.576 [-2.675-1.891]; Laboratory D: slope 0.973 [0.939-1.066], intercept-1.168 [-6.108-1.649]; Laboratory E: slope 0.999 [0.905-1.051], intercept-1.876 [-6.111-3.508]). Bland-Altman analysis indicated that all 5 laboratories′ results generally showed good consistency with the target. Through quality feedback and optimizing sample preparation concentrations, result consistency was enhanced.? Conclusion:? Clinical LC-MS/MS testing consistency programs contribute to improving the comparability of test results.

Objective:To analyze the relationship between clinicopathological features and germline mismatch repair (MMR) gene variants in endometrial cancer patients and to evaluate the clinical utility of germline multi-gene panel testing.Methods:This single-center, retrospective case series study included 100 endometrial cancer patients treated in Zhongshan Hospital, Fudan University between July 2022 and February 2024. We collected clinicopathological data and tumor molecular testing results. 61 cancer susceptibility genes were tested using next-generation sequencing, and the associations between the detection rate of germline variants and the clinicopathological characteristics in endometrial cancer patients were explored.Results:Among 100 patients, 28% (28/100) were found to have pathogenic variants in cancer susceptibility genes, of which 20 patients carried germline MMR gene variants and the remaining 8 patients carried variants in other cancer susceptibility genes. Of the 20 patients diagnosed with Lynch syndrome, only 40% (8/20) met the Chinese family history criteria for Lynch syndrome. Among 53 patients with intact MMR protein expression, 1 patient was identified with a germline MMR gene variant. In the 14 Lynch syndrome patients with confirmed microsatellite status, 5/14 of those showed low microsatellite instability or microsatellite stability. Germline multi-gene panel testing in all endometrial cancer patients additionally identified 1 Lynch syndrome patient and 8 patients with non-Lynch hereditary cancers.Conclusion:Current clinical screening criteria may miss some endometrial cancer patients with Lynch syndrome. Compared with traditional screening pattern, germline multi-gene panel testing not only improves the detection rate of Lynch syndrome in endometrial cancer patients but also identifies other hereditary cancer predispositions.

Objective:? To summarize the achievements in improving the consistency of clinical liquid chromatography-tandem mass spectrometry (LC-MS/MS) testing results.Methods:? From 2021 to 2024, Zhongshan Hospital Affiliated to Fudan University recruited laboratories voluntarily participating in the MSHP (Clinical LC-MS/MS Testing Consistency Program). As of Batch 202404, a total of 76 laboratories had enrolled, including 60 medical institutions (all tertiary hospitals) and 16 third-party laboratories. Test items were established, and comparative samples were distributed regularly-each item′s samples covered three concentrations (high, medium, and low). Samples were shipped via cold chain and tested within one week. Our laboratory′s measurements served as the target, with participating labs′ results within ±25% of the target deemed qualified. Passing-Bablok regression and Bland-Altman analysis were used to assess consistency.Results:Taking 3-MT (3-methoxytyramine) as an example, the coefficients of variation (CVs) for the project′s three concentration levels improved from 17.00%, 47.18%, and 4.88% in the first comparative batch to 9.59%, 9.59%, and 6.1% in Batch 202404. Passing-Bablok regression results for the 5 units participating in 3-MT testing showed that Laboratory A had proportional bias but no systematic bias (regression slope [95% CI]: 0.903 [0.862-0.952]; intercept [95% CI]: 0.912 [-1.921-6.073]). The remaining laboratories exhibited no proportional or systematic bias with the target (Laboratory B: slope 1.031 [0.961-1.147], intercept-0.733 [-4.641-8.272]; Laboratory C: slope 0.982 [0.940-1.009], intercept-0.576 [-2.675-1.891]; Laboratory D: slope 0.973 [0.939-1.066], intercept-1.168 [-6.108-1.649]; Laboratory E: slope 0.999 [0.905-1.051], intercept-1.876 [-6.111-3.508]). Bland-Altman analysis indicated that all 5 laboratories′ results generally showed good consistency with the target. Through quality feedback and optimizing sample preparation concentrations, result consistency was enhanced.? Conclusion:? Clinical LC-MS/MS testing consistency programs contribute to improving the comparability of test results.

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