Molecular characterization and functional roles of circulating cell-free extrachromosomal circular DNA.

Circular DNA segments isolated from chromosomes are known as extrachromosomal circular DNA (eccDNA). Its distinct structure and characteristics, along with the variations observed in different disease states, makes it a promising biomarker. Recent studies have revealed the presence of eccDNAs in body fluids, indicating their involvement in various biological functions. This finding opens up avenues for utilizing eccDNAs as convenient and real-time biomarkers for disease diagnosis, treatment monitoring, and prognosis assessment through noninvasive analysis of body fluids. In this comprehensive review, we focused on elucidating the size profiles, potential mechanisms of formation and clearance, detection methods, and potential clinical applications of eccDNAs. We aimed to provide a valuable reference resource for future research in this field.

Genomic characterization and immunotherapy for microsatellite instability-high in cholangiocarcinoma.

BACKGROUND: Microsatellite instability-high (MSI-H) is a unique genomic status in many cancers. However, its role in the genomic features and immunotherapy in cholangiocarcinoma (CCA) is unclear. This study aimed to systematically investigate the genomic characterization and immunotherapy efficacy of MSI-H patients with CCA. METHODS: We enrolled 887 patients with CCA in this study. Tumor samples were collected for next-generation sequencing. Differences in genomic alterations between the MSI-H and microsatellite stability (MSS) groups were analyzed. We also investigated the survival of PD-1 inhibitor-based immunotherapy between two groups of 139 patients with advanced CCA. RESULTS: Differential genetic alterations between the MSI-H and MSS groups included mutations in ARID1A, ACVR2A, TGFBR2, KMT2D, RNF43, and PBRM1 which were enriched in MSI-H groups. Patients with an MSI-H status have a significantly higher tumor mutation burden (TMB) (median 41.7 vs. 3.1 muts/Mb, P < 0.001) and more positive programmed death ligand 1 (PD-L1) expression (37.5% vs. 11.9%, P < 0.001) than those with an MSS status. Among patients receiving PD-1 inhibitor-based therapy, those with MSI-H had a longer median overall survival (OS, hazard ratio (HR) = 0.17, P = 0.001) and progression-free survival (PFS, HR = 0.14, P < 0.001) than patients with MSS. Integrating MSI-H and PD-L1 expression status (combined positive score ≥ 5) could distinguish the efficacy of immunotherapy. CONCLUSIONS: MSI-H status was associated with a higher TMB value and more positive PD-L1 expression in CCA tumors. Moreover, in patients with advanced CCA who received PD-1 inhibitor-based immunotherapy, MSI-H and positive PD-L1 expression were associated with improved both OS and PFS. TRIAL REGISTRATION: This study was registered on ClinicalTrials.gov on 07/01/2017 (NCT03892577).

Radiation Therapy With Combination Therapy of Immune Checkpoint Inhibitors and Antiangiogenic Therapy for Hepatocellular Carcinoma.

PURPOSE: Immune checkpoint inhibitors (ICIs) combined with antiangiogenic therapy have limited efficacy in treating advanced hepatocellular carcinoma (HCC). The synergistic effect of systemic therapy and radiation therapy (RT) might resolve this problem. We aimed to investigate the effect of RT on the treatment outcomes of ICIs and antiangiogenic combination therapy in patients with advanced-stage HCC. METHODS AND MATERIALS: This retrospective observational study analyzed the medical records of 194 patients with Barcelona Clinic Liver Cancer stage C HCC who were admitted to our center from August 2018 to June 2022 and received ICIs combined with antiangiogenic therapy as the first-line treatment. Patients who were administered RT for tumor thrombus or symptomatic metastases within 8 weeks of the commencement of combination therapy were allocated to the RT group, whereas those who did not receive RT were assigned to the non-radiation therapy (NRT) group. Propensity score matching was used to mitigate selection bias. The primary endpoints were progression-free survival (PFS) and overall survival (OS). The secondary endpoints included objective response rate, disease control rate (DCR), local PFS, out-of-field PFS, and treatment-related adverse events. RESULTS: A total of 76 patients diagnosed with advanced-stage HCC and treated with ICIs and antiangiogenic therapy were included in the study, with 33 patients in the RT group and 43 patients in the non-RT group. After propensity score matching, 29 matched patient pairs were generated. The median follow-up was 15.5 months, and the RT sites were mainly located on the tumor thrombus (55.2%) and extrahepatic metastatic lesions (48.3%). The median PFS was 8.3 months (95% CI, 5.4-11.3) in the RT group and 4.2 months (95% CI, 3.4-5.0) in the NRT group (P < .001). The median OS was not reached in the RT group and was 9.7 months (95% CI, 4.1-15.3) in the NRT group (P = .002). The objective response rate was 75.9% (95% CI, 56.5-89.7) in the RT group and 24.1% (95% CI, 10.3-43.5) in the NRT group (P < .001). The DCR was 100% in the RT group and 75.9% (95% CI, 56.5-89.7) in the NRT group (P = .005). The median local PFS and out-of-field PFS were 13.2 months (95% CI, 6.3-20.1) and 10.8 months (95% CI, 7.0-14.7), respectively. RT was an independent prognostic factor for PFS (hazard ratio = 0.33; 95% CI, 0.17-0.64; P < .001) and OS (hazard ratio = 0.28; 95% CI, 0.11-0.68; P = .005), respectively. The rates of any grade treatment-related adverse events were similar between the 2 groups. CONCLUSIONS: In comparison to the combination of ICIs and antiangiogenic therapy, the inclusion of RT has been observed to improve the DCR and survival outcomes in patients with advanced-stage HCC. The safety profile of this triple therapy was satisfactory.

The efficacy and safety of bevacizumab as a salvage therapy for patients with advanced hepatocellular carcinoma targeting immune tolerance.

As is well understood that malignant tumour progression requires additional blood vessels to provide the nutrients necessary for growth. Many patients with advanced hepatocellular carcinoma (aHCC) experience disease progression after treatment with lenvatinib (Lenva) and immune checkpoint inhibitors (ICIs). Therefore, we designed a double-arm retrospective study to evaluate the antitumour activity of additional bevacizumab (Beva, an anti-vascular endothelial growth factor-targeting drug) as a means to reduce the blood vessels needed for tumour growth. Compared with the control group, the group that received Beva had prolonged progression-free survival (PFS) and a trend toward a benefit for overall survival duration. This study aimed to evaluate the anticancer effect of Beva in patients with aHCC who experienced tumour progression after treatment with Lenva+ICIs. From April 2021 to March 2023, we retrospectively included 20 patients as the experimental group and 21 patients as the control group. The patients in the experimental group experienced disease progression after receiving targeted therapy and ICIs, after which we added Beva to the treatment. The patients in the control group only received targeted therapy and ICIs. The efficacy endpoints were overall survival (OS), progression-free survival (PFS), objective response rate (ORR), and disease control rate (DCR), which were evaluated according to RECIST v1.1. Adverse events were assessed using NCI-CTCAE v5.0. Ultimately, 20 patients with aHCC in the experimental group of received Beva after disease progression, compared with 21 patients in the control group. The median OS was 12.6 mo (95% CI: 6.8-18.7) vs. 9.3 mo (95% CI: 4.3-14.4), and the median PFS was 6.9 mo (95% CI: 6.4-7.4) vs. 4.1 mo (95% CI: 2.4-5.8). The ORR for all patients was 5%, and the DCR for all patients was 70.0%. The median follow-up time for all patients was 7.5 mo (95% CI: 5.0-10.0). All patients had adverse events, but no fatal adverse events were observed. In conclusion, Bevacizumab is a drug resistant treatment option for patients with advanced hepatocellular carcinoma after Lenva+PD-1/PD-L1 treatment.

Development and validation of a selenium metabolism regulators associated prognostic model for hepatocellular carcinoma.

BACKGROUND: Selenium metabolism has been implicated in human health. This study aimed to identify a selenium metabolism regulator-based prognostic signature for hepatocellular carcinoma (HCC) and validate the role of INMT in HCC. METHODS: Transcriptome sequencing data and clinical information related to selenium metabolism regulators in TCGA liver cancer dataset were analysed. Next, a selenium metabolism model was constructed by multiple machine learning algorithms, including univariate, least absolute shrinkage and selection operator, and multivariate Cox regression analyses. Then, the potential of this model for predicting the immune landscape of different risk groups was evaluated. Finally, INMT expression was examined in different datasets. After knockdown of INMT, cell proliferation and colony formation assays were conducted. RESULTS: A selenium metabolism model containing INMT and SEPSECS was established and shown to be an independent predictor of prognosis. The survival time of low-risk patients was significantly longer than that of high-risk patients. These two groups had different immune environments. In different datasets, including TCGA, GEO, and our PUMCH dataset, INMT was significantly downregulated in HCC tissues. Moreover, knockdown of INMT significantly promoted HCC cell proliferation. CONCLUSIONS: The current study established a risk signature of selenium metabolism regulators for predicting the prognosis of HCC patients. INMT was identified as a biomarker for poor prognosis of HCC.

Clinical outcomes of lenvatinib plus transarterial chemoembolization with or without programmed death receptor-1 inhibitors in unresectable hepatocellular carcinoma.

BACKGROUND: Programmed death receptor-1 (PD-1) inhibitors have been approved as second-line treatment regimen in hepatocellular carcinoma (HCC), but it is still worth studying whether patients can benefit from PD-1 inhibitors as first-line drugs combined with targeted drugs and locoregional therapy. AIM: To estimate the clinical outcome of transarterial chemoembolization (TACE) and lenvatinib plus PD-1 inhibitors for patients with unresectable HCC (uHCC). METHODS: We carried out retrospective research of 65 patients with uHCC who were treated at Peking Union Medical College Hospital from September 2017 to February 2022. 45 patients received the PD-1 inhibitors, lenvatinib, TACE (PD-1-Lenv-T) therapy, and 20 received the lenvatinib, TACE (Lenv-T) therapy. In terms of the dose of lenvatinib, 8 mg was given orally for patients weighing less than 60 kg and 12 mg for those weighing more than 60 kg. Of the patients in the PD-1 inhibitor combination group, 15 received Toripalimab, 14 received Toripalimab, 14 received Camrelizumab, 4 received Pembrolizumab, 9 received Sintilimab, and 2 received Nivolumab, 1 with Tislelizumab. According to the investigators' assessment, TACE was performed every 4-6 wk when the patient had good hepatic function (Child-Pugh class A or B) until disease progression occurred. We evaluated the efficacy by the modified Response Evaluation Criteria in Solid Tumors (mRECIST criteria). We accessd the safety by the National Cancer Institute Common Terminology Criteria for Adverse Events, v 5.0. The key adverse events (AEs) after the initiation of combination therapy were observed. RESULTS: Patients with uHCC who received PD-1-Lenv-T therapy (n = 45) had a clearly longer overall survival than those who underwent Lenv-T therapy (n = 20, 26.8 vs 14.0 mo; P = 0.027). The median progression-free survival time between the two treatment regimens was also measured {11.7 mo [95% confidence interval (CI): 7.7-15.7] in the PD-1-Lenv-T group vs 8.5 mo (95%CI: 3.0-13.9) in the Lenv-T group (P = 0.028)}. The objective response rates of the PD-1-Lenv-T group and Lenv-T group were 44.4% and 20% (P = 0.059) according to the mRECIST criteria, meanwhile the disease control rates were 93.3% and 64.0% (P = 0.003), respectively. The type and frequency of AEs showed little distinction between patients received the two treatment regimens. CONCLUSION: Our results suggest that the early combination of PD-1 inhibitors has manageable toxicity and hopeful efficacy in patients with uHCC.

Real-world efficacy and prognostic factors of lenvatinib plus PD-1 inhibitors in 378 unresectable hepatocellular carcinoma patients.

INTRODUCTION: Combining lenvatinib with a programmed cell death protein-1 (PD-1) inhibitor has been explored for the treatment of un-resectable hepatocellular carcinoma (uHCC). This study aimed to investigate the real-world efficacy of and prognostic factors for survival associated with lenvatinib plus PD-1 inhibitor treatment in a large cohort of Asian uHCC patients even the global LEAP-002 study failed to achieve the primary endpoints. METHODS: Patients with uHCC treated with lenvatinib and PD-1 inhibitors were included. The primary endpoints were overall survival (OS) and progression-free survival (PFS), and the secondary endpoints were the objective response rate (ORR) and adverse events (AEs). Prognostic factors for survival were also analyzed. RESULTS: A total of 378 uHCC patients from two medical centers in China were assessed retrospectively. The median patient age was 55 years, and 86.5% of patients were male. Hepatitis B virus (HBV) infection (89.9%) was the dominant etiology of uHCC. The median OS was 17.8 (95% confidence interval (CI) 14.0-21.6) months. The median PFS was 6.9 (95% CI 6.0-7.9) months. The best ORR and disease control rate (DCR) were 19.6% and 73.5%, respectively. In multivariate analysis, Child‒Pugh grade, Barcelona Clinic Liver Cancer stage, Eastern Cooperative Oncology Group performance status score, involved organs, tumor burden score, and combination with local therapy were independent prognostic factors for OS. A total of 100% and 57.9% of patients experienced all-grade and grade 3/4 treatment-emergent AEs, respectively. CONCLUSION: This real-world study of lenvatinib plus PD-1 inhibitor treatment demonstrated long survival and considerable ORRs and DCRs in uHCC patients in China. The tolerability of combination therapy was acceptable but must be monitored closely.

Efficacy of immune checkpoint inhibitors plus molecular targeted agents after the progression of lenvatinib for advanced hepatocellular carcinoma.

Background: Lenvatinib is a standard first-line systemic therapy in advanced hepatocellular carcinoma (aHCC) and is widely used in all lines. However, the efficacy and safety of immune checkpoint inhibitors (ICIs) plus molecular targeted agents (MTAs) after the progression of lenvatinib treatment are unclear. Objective: The aim of this study was to evaluate the anticancer effects of ICI plus MTA in patients with aHCC who progressed after lenvatinib. Methods: We retrospectively included aHCC patients treated with ICI plus MTA after the progression of lenvatinib from two medical centers. Participants who continued lenvatinib treatment were classified into the "ICI+Lenva" group, while the "ICI+Others" group included patients receiving other MTAs. The efficacy endpoints were progression-free survival (PFS), post-progression survival (PPS), overall survival (OS), and tumor response following RECIST v1.1. Safety was evaluated according to Common Terminology Criteria for Adverse Events v5.0. Results: In this study, 85 eligible aHCC patients were enrolled, including 58 in the ICI+Lenva group and 27 in the ICI+Others group. At a median follow-up time of 22.8 months, the median PPS and PFS were 14.0 (95% CI: 9.0-18.2) and 4.5 months (95% CI: 3.5-8.3), respectively. The objective response and disease control rates were 10.6% and 52.9%, respectively. No significant differences were observed in any of the efficacy endpoints between the two groups. Prolonged PPS was associated with Child-Pugh grade A, AFP < 400 IU/ml, and concomitant locoregional treatment. All patients experienced adverse events (AEs), but no fatal AEs were observed. Conclusion: ICI plus MTA in aHCC patients after the progression of lenvatinib presented high antitumor activity and safety. Patients could continue lenvatinib treatment and receive ICIs as well as locoregional treatment to achieve better OS.

High incidence combination of multiple primary malignant tumors of the digestive system.

BACKGROUND: Clinical reports of multiple primary malignant tumors (MPMTs) in the digestive system are increasing. In China, although the survival rate of patients with MPMTs is increasing, the quality of life is very low. Many patients have reached the advanced stage when the second primary tumor is found, resulting in no early intervention and treatment. This is due to the misunderstanding of MPMTs by clinicians, who treat such tumors as metastases. Therefore, before a patient has a second primary tumor, doctors should understand some common combinations of digestive system MPMTs to provide clinical guidance to the patient. AIM: To explore the high incidence combination of digestive system MPMTs under heterochronism and synchronization. METHODS: A total of 1902 patients with MPMTs at Peking Union Medical College Hospital were analyzed retrospectively. They were divided into metachronous MPMT and synchronous MPMT groups, and then the high incidence combinations of the first primary cancer and the second primary cancer in metachronous cancer and synchronous cancer were sorted. Sex and age differences between metachronous and synchronous tumors were tested by the chi square test and t test, respectively. A P value < 0.05 was considered as statistically significant, and SPSS version 26.0 (SPSS Inc., Chicago, Illinois, United States) was used for statistical analysis. RESULTS: Among the 1902 patients with MPMTs confirmed by pathology, 1811 (95.2%) cases were secondary primary cancers, 89 (4.7%) cases were tertiary primary cancers, and 2 (0.1%) cases were quaternary primary cancers. Most (88.2%) of the secondary primary cancers were identified as metachronous multiple primary cancers six months after diagnosis of the first primary cancer. The top ten most common MPMTs in the first primary cancer group ranged from high to low as follows: Breast cancer, thyroid cancer, nonuterine cancer, lung cancer, colon cancer, kidney cancer, uterine cancer, bladder cancer, rectal cancer, and gastric cancer. The highest incidence rate of the first primary cancer in male metachronous cancer was lung cancer (11.6%), the highest incidence rate of the second primary cancer was still lung cancer (24.9%), the highest incidence rate of the first primary cancer in female metachronous cancer was breast cancer (32.7%), and the highest incidence rate of the second primary cancer was lung cancer (20.8%). Among them, breast cancer, nonuterine cancer and uterine cancer were female-specific malignant tumor types, and thyroid cancer also accounted for 79.6% of female patients. The top five metachronous cancer combinations, independent of female-specific malignant tumor types and thyroid cancer, were colon cancer and lung cancer (26 cases), kidney cancer and lung cancer (25 cases), rectal cancer and lung cancer (20 cases), gastric cancer and lung cancer (17 cases), and bladder cancer and lung cancer (17 cases). The most common synchronous cancer combination was colon cancer and rectal cancer (15 cases). CONCLUSION: Screening for lung cancer should be performed six months after the detection of colon cancer while rectal cancer screening should be performed within six months.

Differentially expressed liver exosome-related genes as candidate prognostic biomarkers for hepatocellular carcinoma.

Background: Exosomes are involved in cell-to-cell communication, neovascularization, cancer metastasis, and drug resistance, which all play an important role in the occurrence and progression of hepatocellular carcinoma (HCC). Because there are few mechanistic studies about the function of exosomes in HCC, the goals of this study were to identify exosome-related genes in HCC, to establish a reliable prognostic model for HCC, and to explore underlying mechanisms. Methods: The exoRBase and The Cancer Genome Atlas (TCGA) databases were used to analyze differentially expressed genes (DEGs). Cox regression and least absolute shrinkage and selection operator analyses were used to identify DEGs closely related to the overall survival of patients with HCC. An exosome-related prognostic model was then constructed in TCGA and validated in the International Cancer Genome Consortium database. A nomogram was developed to predict survival. CIBERSORT was used to estimate the abundance of different types of immune cells. Immunotherapy-related DEGs were used to predict the effect of immunotherapy. Results: Forty-eight exosome-related DEGs were obtained; of them, five [exportin 1 (XPO1), lysosomal thiol reductase (IFI30), F-box protein 16 (FBXO16), calmodulin 1 (CALM1), MORC family CW-type zinc finger 3 (MORC3)] were selected to construct a predictive model. Patients with HCC were then divided into low- and high-risk groups using the best cut-off value, as determined by the X-tile software. Prognosis was significantly poorer in the high-risk than in the low-risk group (P=0.009; hazard ratio =2.65). Features related to exosomes were found to positively regulate immune response. Further analysis showed a higher risk score was associated with higher expression of immune checkpoint molecules, including programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), T cell Ig and ITIM domain (TIGIT), and indoleamine-2,3-dioxygenase 1 (IDO1). Conclusions: This study has identified a novel signature based on exosome-related genes that has potential as a prognostic biomarker for HCC. Our research provides an immunological perspective for the development of precision treatment for HCC.

Development and validation of a genomic instability-related lncRNA prognostic model for hepatocellular carcinoma.

Genomic instability is a characteristic of tumors, and recent studies have shown that it is related to a poor prognosis of multiple cancers. Long non-coding RNAs (lncRNAs) have become a research hotspot in recent years, and many unknown biological functions are being explored. For example, some lncRNAs play a critical role in the initiation and progression of multiple cancer types by modulating genomic instability. However, the role of genomic instability-related lncRNAs in liver cancer remains unclear. Therefore, we screened genomic instability-related lncRNAs by combining somatic mutation data and RNA-Seq data in The Cancer Genome Atlas (TCGA) database. We established a genomic instability-related lncRNA model (GLncM) involving ZFPM2-AS1 and MIR210HG to predict the hepatocellular carcinoma (HCC) prognosis and further explore the clinical significance of these lncRNAs, and the robustness of the model was validated in the verification set. Thereafter, we calculated the immune score for each patient and explored the relationship between genome instability and the immune microenvironment. The analysis indicated that this model was better than the immune microenvironment in predicting the prognosis of HCC patients, suggesting that the GLncM may be an effective indicator of HCC prognosis and providing a new direction and strategy for estimating the prognosis of HCC patients.

Gut microbiome is associated with the clinical response to anti-PD-1 based immunotherapy in hepatobiliary cancers.

BACKGROUND: The gut microbiome is associated with the response to immunotherapy for different cancers. However, the impact of the gut microbiome on hepatobiliary cancers receiving immunotherapy remains unknown. This study aims to investigate the relationship between the gut microbiome and the clinical response to anti-programmed cell death protein 1 (PD-1) immunotherapy in patients with advanced hepatobiliary cancers. METHODS: Patients with unresectable hepatocellular carcinoma or advanced biliary tract cancers who have progressed from first-line chemotherapy (gemcitabine plus cisplatin) were enrolled. Fresh stool samples were collected before and during anti-PD-1 treatment and analyzed with metagenomic sequencing. Significantly differentially enriched taxa and prognosis associated taxa were identified. The Kyoto Encyclopedia of Genes and Genomes database and MetaCyc database were further applied to annotate the differentially enriched taxa to explore the potential mechanism of the gut microbiome influencing cancer immunotherapy. RESULTS: In total, 65 patients with advanced hepatobiliary cancers receiving anti-PD-1 treatment were included in this study. Seventy-four taxa were significantly enriched in the clinical benefit response (CBR) group and 40 taxa were significantly enriched in the non-clinical benefit (NCB) group. Among these taxa, patients with higher abundance of Lachnospiraceae bacterium-GAM79 and Alistipes sp Marseille-P5997, which were significantly enriched in the CBR group, achieved longer progression-free survival (PFS) and overall survival (OS) than patients with lower abundance. Higher abundance of Ruminococcus calidus and Erysipelotichaceae bacterium-GAM147 enriched in the CBR group was also observed in patients with better PFS. In contrast, worse PFS and OS were found in patients with higher abundance of Veillonellaceae, which was significantly enriched in the NCB group. Functional annotation indicated that the taxa enriched in the CBR group were associated with energy metabolism while the taxa enriched in the NCB group were associated with amino acid metabolism, which may modulate the clinical response to immunotherapy in hepatobiliary cancers. In addition, immunotherapy-related adverse events were affected by the gut microbiome diversity and relative abundance. CONCLUSIONS: We demonstrate that the gut microbiome is associated with the clinical response to anti-PD-1 immunotherapy in patients with hepatobiliary cancers. Taxonomic signatures enriched in responders are effective biomarkers to predict the clinical response and survival benefit of immunotherapy, which might provide a new therapeutic target to modulate the response to cancer immunotherapy.

p53 transcriptionally regulates SQLE to repress cholesterol synthesis and tumor growth.

Cholesterol is essential for membrane biogenesis, cell proliferation, and differentiation. The role of cholesterol in cancer development and the regulation of cholesterol synthesis are still under active investigation. Here we show that under normal-sterol conditions, p53 directly represses the expression of SQLE, a rate-limiting and the first oxygenation enzyme in cholesterol synthesis, in a SREBP2-independent manner. Through transcriptional downregulation of SQLE, p53 represses cholesterol production in vivo and in vitro, leading to tumor growth suppression. Inhibition of SQLE using small interfering RNA (siRNA) or terbinafine (a SQLE inhibitor) reverses the increased cell proliferation caused by p53 deficiency. Conversely, SQLE overexpression or cholesterol addition promotes cell proliferation, particularly in p53 wild-type cells. More importantly, pharmacological inhibition or shRNA-mediated silencing of SQLE restricts nonalcoholic fatty liver disease (NAFLD)-induced liver tumorigenesis in p53 knockout mice. Therefore, our findings reveal a role for p53 in regulating SQLE and cholesterol biosynthesis, and further demonstrate that downregulation of SQLE is critical for p53-mediated tumor suppression.

Label-Free Quantitative Proteomics Identifies Novel Plasma Biomarkers for Distinguishing Pulmonary Tuberculosis and Latent Infection.

The lack of effective differential diagnostic methods for active tuberculosis (TB) and latent infection (LTBI) is still an obstacle for TB control. Furthermore, the molecular mechanism behind the progression from LTBI to active TB has been not elucidated. Therefore, we performed label-free quantitative proteomics to identify plasma biomarkers for discriminating pulmonary TB (PTB) from LTBI. A total of 31 overlapping proteins with significant difference in expression level were identified in PTB patients (n = 15), compared with LTBI individuals (n = 15) and healthy controls (HCs, n = 15). Eight differentially expressed proteins were verified using western blot analysis, which was 100% consistent with the proteomics results. Statistically significant differences of six proteins were further validated in the PTB group compared with the LTBI and HC groups in the training set (n = 240), using ELISA. Classification and regression tree (CART) analysis was employed to determine the ideal protein combination for discriminating PTB from LTBI and HC. A diagnostic model consisting of alpha-1-antichymotrypsin (ACT), alpha-1-acid glycoprotein 1 (AGP1), and E-cadherin (CDH1) was established and presented a sensitivity of 81.2% (69/85) and a specificity of 95.2% (80/84) in discriminating PTB from LTBI, and a sensitivity of 81.2% (69/85) and a specificity of 90.1% (64/81) in discriminating PTB from HCs. Additional validation was performed by evaluating the diagnostic model in blind testing set (n = 113), which yielded a sensitivity of 75.0% (21/28) and specificity of 96.1% (25/26) in PTB vs. LTBI, 75.0% (21/28) and 92.3% (24/26) in PTB vs. HCs, and 75.0% (21/28) and 81.8% (27/33) in PTB vs. lung cancer (LC), respectively. This study obtained the plasma proteomic profiles of different M.TB infection statuses, which contribute to a better understanding of the pathogenesis involved in the transition from latent infection to TB activation and provide new potential diagnostic biomarkers for distinguishing PTB and LTBI.

Evidence for a direct cross-talk between malic enzyme and the pentose phosphate pathway via structural interactions.

Recent studies have revealed that the oxidative pentose phosphate pathway (PPP), malic enzyme (ME), and folate metabolism are the three major routes for generating cellular NADPH, a key cofactor involved in redox control and reductive biosynthesis. Many tumor cells exhibit altered NADPH metabolism to fuel their rapid proliferation. However, little is known about how NADPH metabolism is coordinated in tumor cells. Here we report that ME1 increases the PPP flux by forming physiological complexes with 6-phosphogluconate dehydrogenase (6PGD). We found that ME1 and 6PGD form a hetero-oligomer that increases the capability of 6PGD to bind its substrate 6-phosphogluconate. Through activating 6PGD, ME1 enhances NADPH generation, PPP flux, and tumor cell growth. Interestingly, although ME1 could bind either the dimer-defect mutant 6PGD (K294R) or the NADP+-binding defect 6PGD mutants, only 6PGD (K294R) activity was induced by ME1. Thus, ME1/6PGD hetero-complexes may mimic the active oligomer form of 6PGD. Together, these findings uncover a direct cross-talk mechanism between ME1 and PPP, may reveal an alternative model for signaling transduction via protein conformational simulation, and pave the way for better understanding how metabolic pathways are coordinated in cancer.

Diagnostic performance of interferon-γ release assay for lymph node tuberculosis.

The aim of the study was to evaluate the performance of interferon-γ (IFN-γ) release assay (IGRA) (T-SPOT.TB) for patients with suspected lymph node tuberculosis (TB). Of the 405 patients with suspected lymph node TB, enrolled from Beijing Chest Hospital between July 2011 and April 2015, 83 (20.5%) were microbiologically/histopathologically confirmed lymph node TB, and 282 (69.6%) did not have active TB. The remaining 21 inconclusive TB and 19 clinical TB were excluded from the final analysis (9.9%). T-SPOT.TB using peripheral blood mononuclear cells was performed to examine the IFN-γ response to the Mycobacterium tuberculosis-specific antigens early secretory antigenic target 6 and culture filtrate protein 10. The overall sensitivity and specificity for T-SPOT.TB were 90.4% and 70.5%, respectively. Spot-forming cells in the lymph node TB group (184 [48-596/10(6) peripheral blood mononuclear cells {PBMCs}]) were significantly higher than that in the nonactive TB group (0 [0-41]/10(6) PBMCs) (P<0.001). These results suggest that the IGRA assay could be a useful aid in the diagnosis of lymph node TB.

Risk factors for false-negative T-SPOT.TB assay results in patients with pulmonary and extra-pulmonary TB.

OBJECTIVES: To investigate the risk factors for false-negative T-SPOT.TB results in patients with pulmonary TB (PTB) and extra-pulmonary TB (EPTB). METHODS: Patients with suspected TB who underwent valid T-SPOT.TB tests were prospectively enrolled at Beijing Chest Hospital between November 2012 and November 2013. Basic characters and clinical laboratory findings were compared between true-positive and false-negative T-SPOT.TB groups. RESULTS: Of 1928 suspected TB patients, 774 (530 PTB and 244 EPTB) microbiologically/histopathogenically-confirmed patients (636 culture-confirmed) were analyzed. Forty-six PTB patients (8.7%) and 32 EPTB patients (13.1%) had negative T-SPOT.TB results. Multivariate analysis showed that increased age [odds radio (OR) 2.26, 95% confidence interval (CI) 1.11-4.58], over-weight (BMI ≥ 25 kg/m(2), OR 2.43, 95% CI 1.05-5.63), and a longer period of illness before hospitalization (>6 months, OR 2.46, 95% CI 1.24-4.92) were independent risk factors for false-negative T-SPOT.TB results in PTB patients. In EPTB patients, increased age (OR 2.42, 95% CI 1.09-5.35) also showed an independent association with false-negative T-SPOT.TB results. CONCLUSION: Careful interpretation of negative T-SPOT.TB results is necessary in older patients with suspected PTB or EPTB, and in PTB patients who are over-weight or have had longer periods of illness before hospitalization.

[Evaluation of T-SPOT.TB assay in the diagnosis of pulmonary tuberculosis within different age groups].

OBJECTIVE: To evaluate the value of T-SPOT.TB assay in the diagnosis of pulmonary tuberculosis within different age groups. METHODS: We analyzed 1 518 suspected pulmonary tuberculosis (PTB) patients who were admitted to the Beijing Chest Hospital from November 2012 to February 2014 and had valid T-SPOT.TB tests before anti-tuberculosis therapy. The 599 microbiologically and/or histopathologically-confirmed PTB patients (16-89 years old, 388 males and 211 females) and 235 non-TB patients (14-85 years old, 144 males and 91 females) were enrolled for the analysis of diagnostic performance of T-SPOT.TB, while patients with uncertain diagnosis or diagnosis based on clinical impression (n=684) were excluded from the analysis. The sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio of the T-SPOT.TB were analyzed according to the final diagnosis. Furthermore, the diagnostic performance of T-SPOT.TB assay in the younger patients (14-59 years old) and elderly patients (60-89 years old) were also analyzed respectively. Categorical variables were compared by Pearson's Chi-square test, while continuous variables were compared by the Mann-Whitney U-test. RESULTS: The sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio of the T-SPOT.TB in diagnosis of PTB were 90.1% (540/599), 65.5% (154/235), 86.9% (540/621), 72.3% (154/213), 2.61, and 0.15, respectively. The sensitivity and specificity of T-SPOT.TB assay were 92.6% (375/405) and 75.6% (99/131), respectively in the younger patients, and 85.0% (165/194), 52.9% (55/104) respectively in the elderly patients. The sensitivity and specificity of T-SPOT.TB assay in the younger patients were significantly higher than those in the elderly patients (P<0.01), and the spot forming cells in the younger PTB patients were significantly higher than in the elderly PTB patients [300 (126, 666)/10(6) PBMCs vs. 258 (79, 621)/10(6) PBMCs, P=0.037]. CONCLUSION: T-SPOT.TB is a promising test in the diagnosis of younger patients (14-59 years old) with suspected PTB, but the diagnostic performance in elderly patients (60-89 years old) is relatively reduced.