Background: Glioma is a highly aggressive brain cancer with a poor prognosis. Necroptosis is a form of programmed cell death occurring during tumor development and in immune microenvironments. The prognostic value of necroptosis in glioma is unclear. This study aimed to develop a prognostic glioma model based on necroptosis.Methods: A necroptosis-related risk model was constructed by Cox regression analysis based on The Cancer Genome Atlas (TCGA) training set, validated in two Chinese Glioma Genome Atlas (CGGA) validation sets. We explored the differences in immune infiltration and immune checkpoint genes between low and high risk groups and constructed a nomogram. Moreover, we compiled a third validation cohort including 43 glioma patients. The expression of necroptosis-related genes was verified in matched tissues using immunochemical staining in the third cohort, and we analyzed their relationship to clinicopathological features.Results: Three necroptosis-related differentially expressed genes (EZH2, LEF1, and CASP1) were selected to construct the prognostic model. Glioma patients with a high risk score in the TCGA and CGGA cohorts had significantly shorter overall survival. The necroptosis-related risk model and nomogram exhibited good predictive performance in the TCGA training set and the CGGA validation sets. Furthermore, patients in the high risk group had higher immune infiltration status and higher expression of immune checkpoint genes, which was positively correlated with poorer outcomes. In the third validation cohort, the expression levels of the three proteins encoded by EZH2, LEF1, and CASP1 in glioma tissues were significantly higher than those from paracancerous tissues. They were also closely associated with disease severity and prognosis.Conclusions: Our necroptosis-related risk model can be used to predict the prognosis of glioma patients and improve prognostic accuracy, which may provide potential therapeutic targets and a theoretical basis for treatment.

For patients who treated with tacrolimus after kidney transplant, therapeutic drug monitoring is essential to improve their prognosis. However, previous detection methods have limitations, such as the overestimation and unacceptable bias in the immunoassays. Precision medicine has been challenged. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is recognized as the gold standard due to its accuracy and specificity, but lack of throughput and complex process limits its clinical application. Therefore, an accurate, simple and high throughput method for tacrolimus monitoring is needed for clinical practice. A modified LC-MS/MS method was introduced and validated. Whole blood samples were prepared by a one-step protein precipitation method. Chromatographic separation was achieved using a Phenomenex Kinetex 2.6 μm XB-C18 2.1 × 50 mm column with a total run time of 3.5 min to avoid matrix effect. An electrospray ionization source (ESI) was used in positive ion multiple reaction monitoring (MRM) mode for mass spectrometric detection. In order to protect the mass spectrometer, only part of the sample after LC separation was allowed to enter the mass spectrum, through a two HPLC systems coupled one mass spectrometry design. In this way, the instrument throughput is also improved and realizing the detection of 2 samples within 3.5 min and carried out a shorter analyzing time for each sample of 1.75 min. Additionally, we calculated tacrolimus-intrapatient variant (Tac-IPV) based on this modified method and assessed the prognostic value of Tac-IPV in Chinese kidney transplant patients. The LC-MS/MS was modified by streamlining the procedure and increasing the throughput. The method proved to be accurate and reproducible with all performance parameters suitably meeting the clinical requirements over a calibration ranged from 0.37 to 42.90 ng/mL. Parameters such as linearity, limit of quantification (LoQ) and dilution integrity were validated with a clinical reportable range from 0.37 to 343.20 ng/mL, which was particularly useful for high drug concentrations patients (rare but very serious). Both cross-contamination and matrix effects were negligible. Clinical data of 83 patients showed that Tac-IPV was associated with poor kidney transplant outcome in Chinese (Hazard Ratio (HR) = 3.96, 4.75; 95% Cl: 1.10–14.21, 1.23–18.36; P < 0.05). This modified LC-MS/MS method possessed high throughput and simple sample preparation, allowing it to meet daily clinical needs. At the same time, Tac-IPV based on this modified LC-MS/MS had excellent prognostic value in kidney transplantation. These advantages have great significance for the individualized treatment of Chinese kidney transplant patients and broad application of Tac-IPV.