Immune checkpoint blockade therapy, particularly those targeting programmed death 1/programmed cell death ligand 1 (PD-1/PD-L1), has been extensively employed to treat various human cancers, significantly improving clinical outcomes. Increasing evidence reveals that the therapeutic efficacy of PD-1/PD-L1 inhibitors depends on the abundance of PD-L1 on cancer cells and tumor-associated stromal cells. Here, we demonstrated that F-box protein 9 (FBXO9) is a novel regulator of PD-L1. We found that increased expression of FBXO9 suppresses tumor growth and promotes cytotoxic T cell activation in vivo. Mechanistically, FBXO9 directly binds to PD-L1 protein and enhances its degradation via ubiquitination, thereby impeding PD-L1 maturation and tumor immune evasion. Meanwhile, the expression of FBXO9 is decreased in pancreatic cancer tissues in comparison to normal tissues. Furthermore, FBXO9 expression correlates inversely with PD-L1 levels, with lower FBXO9 expression being associated with worse clinical outcome. These findings identify FBXO9 as a tumor suppressor via its facilitation of PD-L1 degradation, underscoring the potential of targeting FBXO9 in immunotherapeutic approaches for treating cancers, particularly in combination with anti-PD-L1 therapy.

BACKGROUND:The aim of this study is to assess the comparability of three commercial procalcitonin (PCT) immunoassays, including Kryptor BRAHMS PCT sensitive, Beckman Coulter DXI 800 Access PCT and Roche BRAHMS PCT, and to evaluate the commutability of four types of External Quality Assessment/Proficiency Tests (EQA/PT) materials among the three assays. METHODS:The limits of quantitation or functional sensitivity (LoQ/FS) for the three PCT assays were validated according to the CLSI EP17 guidelines. Precision was verified at 0.5 μg/L and 2 μg/L according to CLSI EP15 document. A method comparison study was conducted using 506 clinical serum samples from individual patients. Seventeen EQA/PT materials, including frozen pooled human serum (FFS), lyophilized materials (Lyo), bovine serum albumin solution (BSA) with recombinant PCT, and human serum (HS) with recombinant PCT, were tested together with 50 clinical individual serum samples. The commutability of EQA/PT materials was evaluated using the difference in bias approach. RESULTS:The LoQ/FS and imprecision of all three assays met the claimed standards. Pairwise comparisons demonstrated high correlations ranging from 0.994 to 0.998 (all p < 0.0001), with relative biases between -13.9% and 23.3%. Clinical consistency at thresholds of 0.1 μg/L, 0.25 μg/L, 0.5 μg/L, 2.0 μg/L, and 10 μg/L exceeded 95% across all methods. The commutability of the four EQA/PT material types was generally poor. The proportions of commutable results were 6/18 for Lyo, 1/15 for BSA, 5/15 for HS, and 7/12 for FFS, with FFS showing the highest commutability among the materials tested. CONCLUSIONS:The assays meet clinical requirements for PCT testing. Among the four EQA/PT materials evaluated, the HS, Lyo, and BSA materials demonstrated limited commutability. Although FFS does not exhibit full commutability, it is considered the most suitable EQA/PT material among those tested. Some residual non-commutability still exists and warrants further investigation.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the preferred method for vitamin K quantification, yet conventional workflows are labor-intensive and scale poorly. We developed and validated a simple, high-throughput automated magnetic bead-assisted LC-MS/MS method for simultaneous quantification of vitamin K1 (VK1), menaquinone-4 (MK-4), and menaquinone-7 (MK-7) in human serum according to Clinical and Laboratory Standards Institute (CLSI) C62 (2nd ed.) guidelines. This method used MSi050/PLS magnetic beads (5 mg/mL) for extraction via methanol loading and acetonitrile elution, requiring only 100 μL of serum to complete sample pretreatment within 17 min. All three calibration curves were linear from 0.20 to 10.00 ng/mL (r > 0.99), with a lower limit of quantification of 0.10 ng/mL. Total coefficients of variation for VK1, MK-4, and MK-7 ranged from 1.98% to 3.54%, 2.53% to 3.73%, and 2.77% to 4.59%, respectively. The recoveries of VK1, MK-4, and MK-7 were 94.8% to 112%, 98.0% to 113%, and 90.3% to 106%, respectively. Both matrix effects and carryover met CLSI acceptance criteria. Because vitamin K2 is photosensitive, all samples were processed under light-protected conditions. Unextracted samples were stable for 14 days at -20 °C, and extracted samples were stable for 48 h at 4 °C. Additionally, our method was applied to 245 healthy volunteers to characterize serum VK distribution profiles. By integrating automated magnetic bead extraction with rapid LC-MS/MS detection, this robust strategy markedly improves analytical throughput and reproducibility while maintaining high accuracy, offering a practical solution for large-scale clinical vitamin K assessment.

Prostate cancer (PCa) is the most prevalent malignancy among men worldwide. Advanced prostate cancer is characterized by aggressive progression, limited therapeutic response, and poor prognosis. Elucidating its oncogenic mechanisms may provide new opportunities for targeted intervention. Increasing evidence suggests that modulating cytoprotective autophagy represents a promising strategy for improving cancer treatment efficacy and overcoming drug resistance. Here, we identified the G protein subunit GNG4 as a crucial regulator of prostate cancer development. GNG4 expression was markedly elevated in advanced prostate cancer phenotypes and positively correlated with tumor survival, apoptosis, and migration. Further analysis demonstrated that GNG4 depletion suppressed autophagy and enhanced cellular sensitivity to enzalutamide. Mechanistically, GNG4 interacts with GNB1 to stabilize the downstream effector protein GNAI3 through the ubiquitination-proteasome pathway. These three distinct G protein subunits form a functional complex that regulates intracellular autophagy and subsequently influences the malignant behavior of prostate cancer. Furthermore, inhibition of autophagy or GNG4 knockdown significantly increased the antitumor efficacy of enzalutamide both in vitro and in vivo. Our findings identified GNG4 as a pivotal modulator of prostate cancer progression and proposed it as a promising therapeutic target to enhance the clinical response to enzalutamide.

Kidney cancer is one of the most common malignancies of the urinary system, with early surgical resection and molecular targeted therapy being the primary treatment options for clear cell renal cell carcinoma (ccRCC). Nitric oxide synthase interacting protein (NOSIP) has been implicated in several types of malignancies; however, its role in ccRCC remains elusive. In this study, we employed a variety of techniques, including transfection, co-immunoprecipitation (co-IP), real-time polymerase chain reaction (RT-PCR), Western blotting, ubiquitin assay, and animal experiments, to explore the role of NOSIP in renal cancer cells. Our results demonstrated that NOSIP interacts with SPTAN1 and promotes the progression of ccRCC by facilitating the ubiquitination and degradation of SPTAN1, thus downregulating its expression. Elevated SPTAN1 levels were found to inhibit the proliferation and metastasis of ccRCC, while the downregulation of SPTAN1 reversed the inhibition of cell survival caused by NOSIP knockdown. Moreover, xenograft studies in nude mice confirmed that NOSIP promotes tumor growth in vivo. This work identifies NOSIP as a key player in the proliferation and apoptosis of ccRCC and suggests that it contributes to malignancy of ccRCC by modulating SPTAN1 expression in an ubiquitination-dependent manner. Our findings provide a theoretical basis and experimental foundation for early diagnosis and molecular targeted therapy of ccRCC.

Background: Renal cell carcinoma (RCC) is a malignant tumor of the urinary system with a high incidence. Due to limited treatment options and poor prognosis, novel therapeutic strategies are urgently needed. Recent studies revealed that Nosip participates in cell growth regulation by activating endothelial nitric oxide synthase (eNOS) and mediating the ubiquitination of erythropoietin receptor, suggesting its potential role as an oncogene in RCC progression. Methods: Nosip expression was analyzed using reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Functional assays including Cell Counting Kit-8 (CCK-8), wound healing, Transwell, and apoptosis assays were performed to assess cell proliferation, migration, invasion, and apoptosis. Bioinformatics analysis was conducted using The Cancer Genome Atlas Kidney Clear Cell Carcinoma (TCGA-KIRC) dataset. Results: Nosip was significantly upregulated in RCC tissues and cells. Knockdown of Nosip inhibited RCC cell proliferation, migration, and invasion, while overexpression promoted these malignant phenotypes. Tea polyphenols (TPs) treatment suppressed RCC cell viability, induced apoptosis, and inhibited clonogenic capacity in a concentration-dependent manner. Mechanistically, TPs downregulated Nosip expression at both messenger RNA (mRNA) and protein levels. Rescue experiments demonstrated that Nosip overexpression attenuated, while Nosip knockdown enhanced, the anti-tumor effects of TPs. Conclusions: This study identifies Nosip as a proto-oncogene in RCC and demonstrates that TPs suppress RCC progression by directly targeting and downregulating Nosip. These findings establish Nosip as a potential therapeutic target and provide a mechanistic basis for developing TPs as a Nosip-targeted therapy for RCC.

Primary aldosteronism (PA) is a common cause of secondary hypertension, and accurate screening is essential for early detection and intervention. The aldosterone-to-renin ratio (ARR) is the primary screening tool, but most diagnostic laboratories currently rely on immunoassays, which may have limitations in accuracy and reliability. To address this, we investigated the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure plasma aldosterone concentration (PAC) and plasma renin activity (PRA) and to establish an optimal ARR cutoff value for PA screening. In this study, 123 patients from Zhejiang Provincial People's Hospital in China were recruited, and receiver operating characteristic (ROC) analyses were performed to compare the performance of LC-MS/MS-based ARR with chemiluminescent immunoassay (CLIA)-based ARR. Our results showed that LC-MS/MS outperformed CLIA, achieving an area under the curve (AUC) of 0.899 compared with 0.780. The optimal ARR cutoff value was determined to be 20.8, yielding an AUC of 0.927 with 90.00% sensitivity and 83.50% specificity in a different group of patients (n = 201). Notably, this cutoff remained valid in patients taking calcium channel blockers (CCBs), as PAC and PRA levels did not differ significantly between those treated with CCBs and untreated patients. These findings suggest that LC-MS/MS offers superior diagnostic accuracy for PA screening compared with immunoassays and provide a validated ARR cutoff value of 20.8 that can be applied to the screening of PA in the Chinese Han population. Importantly, the results indicate that discontinuation of CCBs may not be necessary during PA screening, which has practical implications for patient management. Taken together, this study highlights the potential of LC-MS/MS to improve the reliability of PA detection, promote earlier diagnosis, and inform clinical decision-making across different populations.

OBJECTIVES:Uric acid (UA), the end-product of purine metabolism, is linked to various medical conditions such as gout and cardiovascular diseases. The increasing incidence of hyperuricaemia and gout in China highlights the need to establish accurate reference intervals for UA. This study uses data from a real-world population to examine the distribution of UA levels across various demographic groups, with a specific focus on seasonal variations in UA concentrations. Through the application of an indirect statistical method, the research aims to establish more precise UA reference intervals for clinical use. METHODS:We analysed 749,739 serum UA data from adults (≥18 years) over 2 years, 2021-2022, studying seasonal, gender, and age effects. The reference intervals were established via the indirect method, and the differences from the manufacturer's ranges were assessed using the reference change value (RCV). RESULTS:UA levels showed seasonal variations with significant gender disparities. In winter/spring, males 18-44: 262-539 µmol/L, females: 175-385 µmol/L; males 45+: 243-525 µmol/L, females: 185-406 µmol/L. In summer/autumn, males 18-44: 273-552 µmol/L, females: 185-395 µmol/L; males 45+: 250-537 µmol/L, females: 192-410 µmol/L. A significant difference was observed in the reference range for males compared to the manufacturer's range, but no difference for females. CONCLUSIONS:Extensive data analysis has uncovered seasonal fluctuations and notable gender- and age-related effects on serum UA concentrations. Using the indirect method to establish more refined reference intervals for serum UA levels confers practical relevance in clinical practice.

Background:Hepatocellular carcinoma (HCC) ranks among the leading causes of cancer-related deaths globally, with current treatments offering limited efficacy. FBXO5 has emerged as a potential oncogene in multiple cancers, yet its role in HCC remains incompletely understood. Notably, tea polyphenols (TPs) are natural compounds with known antitumor properties, but their molecular targets in HCC remain unclear. This study aims to investigate the role of FBXO5 in HCC and explore whether TPs exert their antitumor effects by targeting FBXO5. Methods:FBXO5 expression in HCC was analyzed using The Cancer Genome Atlas (TCGA) data. Functional analysis of FBXO5 in HCC cell lines was performed using small interfering RNA (siRNA) knockdown and overexpression techniques. Cell phenotypes were assessed via Cell Counting Kit-8 (CCK-8), colony formation, scratch healing, and Transwell assays. Cells treated with varying concentrations of TPs were evaluated for effects on viability, proliferation, migration, and invasion, with FBXO5 expression changes analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot. TPs' effects were further validated in FBXO5-overexpressing cells. Results:FBXO5 was significantly upregulated in HCC tissues and correlated with poor patient prognosis. Silencing FBXO5 inhibited HCC cells proliferation, migration, and invasion, whereas its overexpression promoted these malignant behaviors. TPs treatment dose-dependently suppressed HCC cells viability, proliferation, and motility. TPs downregulated FBXO5 expression at both messenger RNA (mRNA) and protein levels. Overexpression of FBXO5 attenuated the antitumor effects of TPs, indicating that TPs partially inhibit HCC progression by suppressing FBXO5. Conclusions:FBXO5 functions as an oncogene in HCC, and TPs may serve as potential therapeutic agents for inhibiting HCC progression by targeting FBXO5.

PURPOSE:Although trimodal therapy is currently the standard organ-sparing approach for muscle-invasive bladder cancer (MIBC), its clinical benefit is limited, and noninvasive biomarkers to guide dynamic decision-making are lacking. Here, we present a proof-of-concept study evaluating disitamab vedotin (RC48, a HER2-targeted antibody-drug conjugate) combined with toripalimab (JS001, anti-PD-1) and radiotherapy for bladder preservation in localized HER2-positive MIBC. PATIENTS AND METHODS:In the first-stage of an open-label phase II clinical trial (ClinicalTrials.gov identifier: NCT05979740), six patients were enrolled and received disitamab vedotin, toripalimab, and radiotherapy. Adverse events were documented according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE v5.0). Tumor response was evaluated every 12 weeks by radiographic imaging, cystoscopy with biopsies, and urine cytology. In parallel, we performed longitudinal liquid biopsy analyses of circulating tumor DNA (ctDNA) and urinary tumor DNA (utDNA) using PredicineCARE assay. RESULTS:The combination was overall tolerable, with no grade 4 treatment-related adverse events or deaths. Five patients (83.3 %) achieved a complete response and remained recurrence-free. Notably, utDNA testing showed high accuracy in monitoring therapeutic effectiveness and enabled early detection of tumor relapse, whereas ctDNA was largely undetectable across blood samples. CONCLUSIONS:These findings establish the feasibility, efficacy, and potential biomarker utility of a novel bladder-preserving regimen, setting the stage for a paradigm shift in MIBC management.

Thioredoxin1 (TRX1) and telomerase are both attractive oncology targets that are tightly implicated in tumor initiation and development. Here, we reported that the 6-dithio-2-deoxyguanosine analog thiotert exhibits an effective cytotoxic effect on myelodysplastic syndromes (MDS) cell SKM-1 and lymphoma cell U-937. Further studies confirmed that thiotert effectively disrupts cellular redox homeostasis, as evidenced by elevated intracellular reactive oxygen species (ROS) levels, increased MnSOD, accelerated DNA impairment, and activated apoptosis signal. Mechanistically, our present study revealed that thiotert treatment effectively inhibited the function of the TRX1/TRXR1 system and telomerase reverse transcriptase (TERT), rendering oxidative damage and impairment of telomeres. Meanwhile, pharmacological administration of glutathione (GSH), N-acetylcysteine (NAC), and mitoquinone (MitoQ), or genetic overexpression of TRX1 or TERT in MDS and cells could dampen the toxicity caused by thiotert. Remarkably, the in vivo mouse model of MDS demonstrated that thiotert administration exhibited greater efficacy in tumor reduction compared to the conventional chemotherapy drug cytarabine. Collectively, these results provide experimental insights into the mechanism of thiotert-induced MDS and lymphoma cell death and unveil that thiotert may be an effective and promising new drug for future MDS and lymphoma treatment.

Latent autoimmune diabetes in adults (LADA) is a slowly progressing form of diabetes that develops in adulthood, characterized by autoimmune destruction of pancreatic β-cells and subsequent insulin deficiency, akin to type 1 diabetes (T1D). Due to its shared genetic, immunological, and metabolic features with both T1D and type 2 diabetes (T2D), LADA is frequently misdiagnosed and inappropriately treated as T2D. To address this, we developed the A.NG algorithm, which identifies serum glycopatterns by calculating the ratio of upregulated to downregulated N-glycans, thereby facilitating the detection of subtle glycan alterations specific to each diabetes subtype. Our method, which utilizes matrix-assisted laser desorption ionization (MALDI) for N-glycan profiling, revealed distinct glycan patterns across T1D, T2D, and LADA, with observed correlations achieving an AUC of 0.918 in this cohort. While these findings demonstrate the technical feasibility of detecting subtype-associated glycosylation changes, their clinical utility for subtype differentiation requires validation in larger studies with refined quantification approaches. Furthermore, complementary ELISA and intact glycopeptide analyses showed that enzymes like FUT8 and FUCA1 contribute to altered glycan expression patterns on specific glycoproteins, which could serve as potential biomarkers for LADA. In conclusion, the A.NG algorithm represents a promising novel approach for distinguishing between LADA and T1D or T2D, with the potential to significantly improve the diagnosis and management of these diabetes subtypes.

Protein lipoylation, a mitochondria-specific post-translational modification (PTM) evolutionarily conserved from bacteria to mammals, plays critical role in metabolic processes. In humans, four identified lipoylated proteins serve as essential components of key enzymes involved in glycolysis, the tricarboxylic acid (TCA) cycle, and amino acid metabolism. The dynamic addition or removal of lipoylation modifications critically regulates the functional activity of these enzymes, with dysregulation strongly associated with cancers. Notably, cancer-associated metabolic reprogramming frequently coincides with functional impairment of lipoylated proteins, which subsequently modulates tumor growth through metabolic adaptation. In this review, we systematically summarized the biosynthesis of lipoic acid (LA), introduced the basic structure of lipoylated protein and presented the regulation of lipoylation. Since metabolic reprogramming is an important feature of tumorigenesis, we discussed the relationship between protein lipoylation and tumor metabolic reprogramming. Cuproptosis is a novel form of cell death characterized by copper-mediated lipoylation, which disrupts mitochondrial metabolism and induces cell death through the aggregation of lipoylated proteins in the TCA cycle. We highlighted the therapeutic potential of targeting lipoylation to disrupt cancer cell energy metabolism, particularly through cuproptosis. These insights reveal the intricate interplay between lipoylation and cancer progression and open new avenues for developing targeted therapies. Furthermore, we proposed innovative combinatorial strategies leveraging the crosstalk between cuproptosis and ferroptosis to overcome tumor drug resistance. These insights establish lipoylation as a promising therapeutic axis for developing precision cancer therapies targeting metabolic vulnerabilities.

112 Background: Cystectomy (RC) with or without (neo)adjuvant cisplatin-based chemotherapy [(N)AC] is standard in fit patients (pts) with MIBC. For those who decline or are ineligible for RC, trimodality bladder preservation therapy (TMT) is recommended. However, about 40% of MIBC pts are cisplatin-ineligible or refuse chemotherapy. A new bladder-preserving therapy is urgently needed. DV is an antibody-drug conjugate directed to HER2, which is highly expressed in urothelial cancer(UC). In a combined analysis of two phase II clinical trials, DV demonstrated a promising efficacy with a manageable safety profile in patients with HER2 overexpression (immunohistochemistry 3+ or 2+) locally advanced or metastatic UC who had progressed on at least one line of systemic chemotherapy (Xinan Sheng JCO 2023). Here, we introduce a prospective study on the safety and preliminary efficacy of Disitamab VEdotin Combined with torIpalimab and radiotherapy as blaDder-preservING therapy in HER2 overexpression muscle-invasive bladder cancer(DECIDING study-stage I). Methods: This was a two stage designed trial which planned to include 6 pts in stage I as safety introduction cohort and 45 pts in stage II as efficacy assessment expansion cohort. Major including criteria includes staging of cT2-T4aN0M0 MIBC, declining or ineligible for RC, HER2 overexpression, and ECOG PS 0/1. Pts receive DV 2.0 mg/kg and toripalimab 3.0 mg/kg in day1, every 14 days for 12 cycles. Radiotherapy performed simultaneously during drug treatment, with a total dose over 56 Gy/23 fx. CT/MRI, cystoscopy with biopsy and cytology were performed to evaluate response. Up to 6 pts were enrolled into stage I cohort. The primary endpoint were dose-limiting toxicity (DLT) in stage I. Secondary endpt were complete response (CR) rate at 3 months, (defined as negative urine cytology and no visible tumors on imaging and negative biopsies in the ITT population), CR rate at 12m and 24m, bladder-intact disease-free survival, cancer specific survival, and overall survival. Results: From 7/2023 to 8/2023, 6 pts of stage I cohort were enrolled. Median age 69 (60-79), 100% MIBC and declined RC, 3/6 and 3/6 of pts confirmed with HER2 IHC 2+ and 3+, respectively. All 6 pts completed first therapy. As of 28 days after first therapy, no DLTs in stage I. 1/6 of pts had grade 3 (Gr 3) TRAE (Hypertriglyceridemia), 2/6 of pts had Gr 2 TRAE (Hyperglycemia and Radiocystitis), and all 6 pts had Gr 1 TRAE(3/6 AST increased; 2/6 ALT increased, 2/6 rash, 1/6 GGT increased, 1/6 diarrhea, 1/6 pruritus, 1/6 nausea). Conclusions: DV combined with toripalimab instead of chemotherapy in TMT was well-tolerated in this early analysis. TRAE was consistent with prior trials and no DLTs in 28 days recived first drug therapy. Clinical trial information: NCT05979740 .

Lung cancer remains a global health crisis, responsible for significant morbidity and mortality. Late-stage diagnosis often limits treatment options and patient survival. Therefore, identifying reliable and sensitive biomarkers for early detection is crucial. Glycosylation, the addition of glycans to protein/RNA/lipid, is a vital cellular process. Normal glycosylation regulates healthy cell function, while alterations, particularly in fucosylation and sialylation, contribute to lung cancer development and progression. These aberrant glycosylation patterns are associated with processes such as immune modulation, cell migration, proliferation, and cell-cell recognition. Fucosylation, a specific type of glycosylation, is frequently altered in lung cancer, with high levels detected in tumors. Understanding the mechanisms behind this altered fucosylation holds immense potential. It can pave the way for the development of novel therapeutic and diagnostic tools for lung cancer. By analyzing specific fucosylation patterns in bodily fluids, it could lead to early-stage diagnosis. This review delves into the mechanisms of fucosylation in lung cancer initiation and metastasis, proposing promising strategies to target the mechanisms, aiming to inhibit tumor growth and disease progression.

Mass spectrometry analysis has been applied in many important diagnostic fields of laboratory medicine. However, there is little literature to guide quality management systems for LC-MS/MS methods. In this study, LC-MS/MS 25-hydroxyvitamin D (25(OH)D) was used as an example to establish internal quality control strategies to ensure the accuracy of clinical vitamin D results. A total of 141 batches of samples were analyzed. Sample internal standard peak area variability, ion pair ratio, and physical examination population data were monitored as quality control strategies for 25(OH)D results. The analytical performance was evaluated by calculated Sigma metrics. Applying our quality control strategies, several abnormal data were monitored in the routine analysis. The daily peak area CV of 25(OH)D fluctuated within a certain range. By selecting P99 CV as the control target, two abnormal batches were found. The ratio of 25(OH)VD3 ion pairs was relatively stable. Among them, batch20230120 had a high CV value, which may be due to the bias caused by the limited number. According to the physical examination data, batch20220913 and batch20220919 exceeded the alarm limit. Sigma level of 25(OH)VD3 in the laboratory was 6.52, which achieved “excellent” performance. In conclusion, we established comprehensive quality control strategies for the determination of 25(OH)D by LC-MS/MS, which has high analytical performance and can provide more accurate reports for the clinic.

p53 expression and mutational landscape in NEBC.

NMF analysis of NEBC mutational signatures.