Proteogenomics of different urothelial bladder cancer stages reveals distinct molecular features for papillary cancer and carcinoma in situ.

The progression of urothelial bladder cancer (UC) is a complicated multi-step process. We perform a comprehensive multi-omics analysis of 448 samples from 190 UC patients, covering the whole spectrum of disease stages and grades. Proteogenomic integration analysis indicates the mutations of HRAS regulated mTOR signaling to form urothelial papilloma rather than papillary urothelial cancer (PUC). DNA damage is a key signaling pathway in the progression of carcinoma in situ (CIS) and related to APOBEC signature. Glucolipid metabolism increase and lower immune cell infiltration are associated with PUC compared to CIS. Proteomic analysis distinguishes the origins of invasive tumors (PUC-derived and CIS-derived), related to distinct clinical prognosis and molecular features. Additionally, loss of RBPMS, associated with CIS-derived tumors, is validated to increase the activity of AP-1 and promote metastasis. This study reveals the characteristics of two distinct branches (PUC and CIS) of UC progression and may eventually benefit clinical practice.

Case Report: A novel IRF2BP2 mutation in an IEI patient with recurrent infections and autoimmune disorders.

Introduction: Inborn errors of immunity (IEI) are a heterogeneous group of disorders characterized by increased risk of infections, autoimmunity, autoinflammatory diseases, malignancy and allergy. Next-generation sequencing has revolutionized the identification of genetic background of these patients and assists in diagnosis and treatment. In this study, we identified a probable unique monogenic cause of IEI, and evaluated the immunological methods and pathogenic detections. Methods: A family with a member with a clinical diagnosis of IEI was screened by whole genomic sequencing (WGS). Demographic data, clinical manifestations, medical history, physical examination, laboratory findings and imaging features of the patient were extracted from medical records. Comprehensive immune monitoring methods include a complete blood count with differential, serum levels of cytokines and autoantibodies, T-cell and B-cell subsets analysis and measurement of serum immunoglobulins. In addition, metagenomic sequencing (mNGS) of blood, cerebrospinal fluid and biopsy from small intestine were used to detect potential pathogens. Results: The patient manifested with recurrent infections and autoimmune disorders, who was eventually diagnosed with IEI. Repetitive mNGS tests of blood, cerebrospinal fluid and biopsy from small intestine didn't detect pathogenic microorganism. Immunological tests showed a slightly decreased level of IgG than normal, elevated levels of tumor necrosis factor and interleukin-6. Lymphocyte flow cytometry showed elevated total B cells and natural killer cells, decreased total T cells and B-cell plasmablasts. WGS of the patient identified a novel heterozygous mutation in IRF2BP2 (c.439_450dup p. Thr147_Pro150dup), which was also confirmed in his father. The mutation was classified as variant of uncertain significance (VUS) according to the American College of Medical Genetics and Genomics guidelines. Conclusion: We identified a novel IRF2BP2 mutation in a family with a member diagnosed with IEI. Immune monitoring and WGS as auxiliary tests are helpful in identifying genetic defects and assisting diagnosis in patients with clinically highly suspected immune abnormalities and deficiencies in inflammation regulation. In addition, mNGS techniques allow a more comprehensive assessment of the pathogenic characteristics of these patients. This report further validates the association of IRF2BP2 deficiency and IEI, and expands IEI phenotypes.

Frequent EGFR exon 20 insertion in the so-called peripheral-type squamous cell neoplasm of uncertain malignant potential: a variant of bronchiolar adenoma or under-recognised entity?

INTRODUCTION: Herein we describe a series of rare peripheral pulmonary neoplasms temporarily termed "peripheral type squamous cell neoplasm of uncertain malignant potential (PSCN-UMP)" and investigate their relationship to bronchiolar adenoma (BA) and squamous cell carcinoma (SCC). MATERIALS AND METHODS: The histologic and immunohistochemical features of 10 PSCN-UMPs and six BAs were compared. Whole exome sequencing (WES) and bioinformatics analysis were performed to further compare the genetic features of PSCN-UMPs, BAs, and NSCLCs. RESULTS: All PSCN-UMPs were peripherally located and histologically characterised by the lepidic, nested, and papillary proliferation of relatively bland squamous cells, accompanied by entrapped hyperplastic reactive pneumocytes. The basal squamous cells coexpressed TTF1 and squamous markers. Both cellular components exhibited bland morphology and a low proliferative activity. The six BAs met the morphologic and immunophenotypic features of proximal-type BA. Genetically, driver mutations, including frequent EGFR exon 20 insertions, were found in PSCN-UMPs, while the KRAS mutation, BRAF mutation, and ERC1::RET fusion were detected in BAs. PSCN-UMPs also shared some alterations with BAs in mutational signatures, while copy number variants (CNV) were enriched in MET and NKX2-1 in PSCN-UMP and MCL1, MECOM, SGK1, and PRKAR1A in BA. CONCLUSION: PSCN-UMPs exhibited the proliferation of bland squamous cells accompanied by entrapped pneumocytes and frequent EGFR exon 20 insertions, which showed distinct features from BAs and SCCs. Recognition of this specific entity will help to expand the morphologic and molecular spectrum of peripheral lung squamous neoplasms.

scSTAR reveals hidden heterogeneity with a real-virtual cell pair structure across conditions in single-cell RNA sequencing data.

Cell-state transition can reveal additional information from single-cell ribonucleic acid (RNA)-sequencing data in time-resolved biological phenomena. However, most of the current methods are based on the time derivative of the gene expression state, which restricts them to the short-term evolution of cell states. Here, we present single-cell State Transition Across-samples of RNA-seq data (scSTAR), which overcomes this limitation by constructing a paired-cell projection between biological conditions with an arbitrary time span by maximizing the covariance between two feature spaces using partial least square and minimum squared error methods. In mouse ageing data, the response to stress in CD4+ memory T cell subtypes was found to be associated with ageing. A novel Treg subtype characterized by mTORC activation was identified to be associated with antitumour immune suppression, which was confirmed by immunofluorescence microscopy and survival analysis in 11 cancers from The Cancer Genome Atlas Program. On melanoma data, scSTAR improved immunotherapy-response prediction accuracy from 0.8 to 0.96.

Integrated proteogenomic characterization of urothelial carcinoma of the bladder.

BACKGROUND: Urothelial carcinoma (UC) is the most common pathological type of bladder cancer, a malignant tumor. However, an integrated multi-omics analysis of the Chinese UC patient cohort is lacking. METHODS: We performed an integrated multi-omics analysis, including whole-exome sequencing, RNA-seq, proteomic, and phosphoproteomic analysis of 116 Chinese UC patients, comprising 45 non-muscle-invasive bladder cancer patients (NMIBCs) and 71 muscle-invasive bladder cancer patients (MIBCs). RESULT: Proteogenomic integration analysis indicated that SND1 and CDK5 amplifications on chromosome 7q were associated with the activation of STAT3, which was relevant to tumor proliferation. Chromosome 5p gain in NMIBC patients was a high-risk factor, through modulating actin cytoskeleton implicating in tumor cells invasion. Phosphoproteomic analysis of tumors and morphologically normal human urothelium produced UC-associated activated kinases, including CDK1 and PRKDC. Proteomic analysis identified three groups, U-I, U-II, and U-III, reflecting distinct clinical prognosis and molecular signatures. Immune subtypes of UC tumors revealed a complex immune landscape and suggested the amplification of TRAF2 related to the increased expression of PD-L1. Additionally, increased GARS, related to subtype U-II, was validated to promote pentose phosphate pathway by inhibiting activities of PGK1 and PKM2. CONCLUSIONS: This study provides a valuable resource for researchers and clinicians to further identify molecular pathogenesis and therapeutic opportunities in urothelial carcinoma of the bladder.

Samonella typhi infection-related appendicitis: A case report.

BACKGROUND: Acute appendicitis is one of the most common acute abdominal emergencies around the world, which is always associated with infection. Infection with Salmonella typhi, an enteric pathogen, is a rare cause of acute appendicitis. We here report a patient with acute appendicitis associated with Samonella typhi infection, accompanied with spleen and kidney infarction, providing a rare example for a common surgical emergency. CASE SUMMARY: A 25-year-old Pakistani man presented to the hospital with a 3-d history of fevers, vomiting, and abdominal pain. Computed tomography (CT) revealed a thickened intestinal wall of the ileocecal junction with multiple enlarged lymph nodes nearby. He was diagnosed with acute appendicitis and received laparoscopic appendectomy, which showed mild inflammation of the appendix. After the surgery, the patient presented again with a high fever (> 39 °C) and diarrhea. A CT angiography scan indicated spleen and kidney infarction. According to the blood culture, the diagnosis was finally clear to be Samonella typhi infection. The pyrexia and enteric symptoms were relieved after the application of intravenous levofloxacin. CONCLUSION: This case, characterized by the combination of Salmonella typhi infection, acute appendicitis, and renal and splenic infraction, serves as a rare example for a common surgical emergency.

Perivenous Stellate Cells Are the Main Source of Myofibroblasts and Cancer-Associated Fibroblasts Formed After Chronic Liver Injuries.

BACKGROUND AND AIMS: Studies of the identity and pathophysiology of fibrogenic HSCs have been hampered by a lack of genetic tools that permit specific and inducible fate-mapping of these cells in vivo. Here, by single-cell RNA sequencing of nonparenchymal cells from mouse liver, we identified transcription factor 21 (Tcf21) as a unique marker that restricted its expression to quiescent HSCs. APPROACH AND RESULTS: Tracing Tcf21+ cells by Tcf21-CreER (Cre-Estrogen Receptor fusion protein under the control of Tcf21 gene promoter) targeted ~10% of all HSCs, most of which were located at periportal and pericentral zones. These HSCs were quiescent under steady state but became activated on injuries, generating 62%-67% of all myofibroblasts in fibrotic livers and ~85% of all cancer-associated fibroblasts (CAFs) in liver tumors. Conditional deletion of Transforming Growth Factor Beta Receptor 2 (Tgfbr2) by Tcf21-CreER blocked HSC activation, compromised liver fibrosis, and inhibited liver tumor progression. CONCLUSIONS: In conclusion, Tcf21-CreER-targeted perivenous stellate cells are the main source of myofibroblasts and CAFs in chronically injured livers. TGF-ß signaling links HSC activation to liver fibrosis and tumorigenesis.

Analysis of the clinicopathological characteristics, genetic phenotypes, and prognostic of pure mucinous adenocarcinoma.

BACKGROUND: Primary pure mucinous adenocarcinoma of the lung (PMA) is a rare subtype. However, correlations between clinicopathological features and genetic phenotypes with survival have not been described comprehensively. METHODS: Pure mucinous adenocarcinoma patient information collected from the Surveillance, Epidemiology, and End Results (SEER) database, the Department of Thoracic Surgery, Zhongshan Hospital, Fudan University (FDZSH), and the Cancer Genome Atlas (TCGA) were extracted, evaluated, and compared with other lung adenocarcinomas (LUAD) patient data. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed to explore the functional importance of underlying molecular changes. Overall survival (OS) was evaluated with the Kaplan-Meier method. Univariate and multivariate analysis through Cox proportional hazard regression identified risk factors that predicted OS, and the results were used to construct a nomogram to predict OS for PMA patients. RESULTS: Overall, 3622 patients, 41 patients, and 15 patients with PMA were identified from the SEER, FDZSH, and TCGA databases, respectively. There were 345 differentially expressed genes, 30 differentially mutated genes and 72 differentially methylated genes were identified between PMA and other LUAD samples. In the SEER database, PMA had a better prognosis compared to other LUAD. Compared with patients with other LUAD, patients with PMA exhibited unique clinicopathological features, including fewer grade III/IV tumors, less pleural invasion, more early-stage cancer, and more lower lobe carcinomas. Multivariate analyses showed that age, race, T stage, N stage, surgery, and chemotherapy were independent risk factors. The nomogram had a calibration index of 0.724. CONCLUSIONS: Our research identified unique clinicopathological characteristics and genetic phenotypes for PMA and other LUAD. The nomogram accurately predicted OS.

Histology and oncogenic driver alterations of lung adenocarcinoma in Chinese.

Little is known about association of mucin abundancy with oncogenic-driver alterations, immunohistochemical and clinicopathologic features in lung adenocarcinomas among Chinese. We here retrospectively examined the clinicopathologic and molecular characteristics of pulmonary mucin-producing adenocarcinoma (PMPA) and previously-reported non-mucinous lung adenocarcinomas collected at our institution. Among the 897 non-mucinous adenocarcinomas, 61 PMPA with ≤90% mucin and 39 PMPA with >90% mucin, ALK rearrangements were found in 47 (5.2%) non-mucinous adenocarcinomas, 9 (14.8%) PMPA with ≤90% mucin and 12 (30.8%) PMPA with >90% mucin, respectively, with an ordinal association (coefficient, 95% CI=0.11, 0.06 to 0.17). Similarly, KRAS mutations was found in 53 (5.9%) non-mucinous adenocarcinomas, 7 (11.5%) PMPA with ≤90% mucin and 14 (35.9%) PMPA with >90% mucin (coefficient, 95% CI=0.11, 0.05 to 0.16). However, mucinous abundancy was inversely, ordinally linked to the EGFR mutations (coefficient, 95% CI=-0.28, -0.33 to -0.22). Mucin abundancy seemed not associated with the alterations of HER2, BRAF, ROS1, MET and RET. We divided PMPA with >90% mucin into three histologic types, namely columnar mucinous cell with basal nuclei (type I, n=11), cuboidal cell with goblet cell feature (type II, n=16) and mucinous cribriform pattern (type III, n=12). These histologic subtypes were associated with alterations of ALK, KRAS and MET, and the immunohistochemical reactivity of MUC1, MUC2, MUC5ac, MUC6, TTF-1 and CK20, including high positive rate of MUC6 (90.9%) and CK20 (36.4%) in type I, MUC2 (50%) in type II and MUC1 (100%) in type III. In summary, mucin abundancy is associated with immunohistochemical and oncogenic-driver profiles of lung adenocarcinomas among Chinese.

Prediction of renal allograft chronic rejection using a model based on contrast-enhanced ultrasonography.

OBJECTIVE: To evaluate the application of contrast-enhanced ultrasonography (CEUS) for the diagnosis of renal allograft chronic rejection (CR). METHODS: A total of 104 patients who were suspected to have AR or CR were enrolled in this study (derivation group, n = 66; validation group, n = 38). Before biopsy, all patients received an ultrasound examination. RESULTS: In the CR group, rising time (RT) and time to peak (TTP) of medulla (RTm and TTPm, respectively) were significantly longer compared to those in the AR group. The kidney volume was significantly decreased in the CR group but was increased in the AR group. In the derivation group, age, change in kidney volume, and TTPm were identified as independent predictors by multivariate analysis. Based on the multivariate analysis results and area under receiver operating characteristic (ROC) curves (AUROCs) of individual markers, we constructed a new index as follows: P = -5.424 + 0.074 × age -9.818 × kidney volume change + 0.115 × TTPm; New Index = eP /(1 + eP ). The new index discriminates CR from AR and had better AUROCs than any other parameters. CONCLUSION: In conclusion, the new index provides a new diagnosis model for CR.

Sulforaphane attenuation of experimental diabetic nephropathy involves GSK-3 beta/Fyn/Nrf2 signaling pathway.

Sulforaphane (SFN), the bioactive component of cruciferous vegetables, is a potent indirect antioxidant. Oxidative stress and activation of glycogen synthase kinase 3beta (GSK3ß) are two major contributors to the pathogenesis of diabetic nephropathy (DN). Here, we investigated whether and how SFN affected GSK3ß in experimental models of DN in vivo and in vitro. SFN treatment obviously prevented the increase in urine albumin excretion, matrix expansion, transforming growth factor-ß1 expression, fibronectin and type IV collagen deposition in the diabetic kidney. Simultaneously, the level of 8-oxo-deoxyguanosine, an indicator of oxidative damage, was markedly lowered in SFN-treated diabetic rats, together with a significant reduction in activity of the GSK-3ß/Fyn axis and an evident activation of Nrf2 signaling. Similarly, antifibrotic effects of SFN, parallel to enhanced inhibitory Ser9-phosphorylation of GSK3ß and Fyn/Nrf2 nuclear export/import, were observed in the cultured rat mesangial cells (RMC) exposed to high glucose. The salutary effects of SFN on high-glucose-stimulated RMC were abolished by overexpression of GSK3ß while being rescued by lithium chloride, a well-known GSK3ß inhibitor. Taken together, our findings suggested that SFN ameliorated experimental diabetic nephropathy, at least in part, via GSK3ß/Fyn/Nrf2 signaling pathway.

Nrf2 ameliorates diabetic nephropathy progression by transcriptional repression of TGFß1 through interactions with c-Jun and SP1.

Diabetic nephropathy (DN) is one of the major complications in diabetes patients. Reactive oxygen species (ROS) play key roles in DN progression. As a primary transcription factor, Nrf2 controls the antioxidant response to maintain cellular redox homeostasis. Herein we systemically examined the role of Nrf2 in DN progression and its regulatory mechanism in a mouse model bearing type II diabetes and in cultured human renal mesangial cells (HRMCs). We found that Nrf2 could ameliorate DN progression by transcriptional repression of TGFß1 in vivo and in vitro. Moreover, Nrf2 bound to the specific region in TGFß1 promoter by interactions with transcription factors c-Jun and SP1. Significant abolishment of Nrf2-mediated TGFß1 transcriptional repression could be accomplished by knockdown of either c-Jun or SP1, and site-directed mutagenesis of c-Jun and SP1 binding sites in the TGFß1 promoter specific region. Moreover, after interacting with c-Jun and SP1, Nrf2 inhibited c-Jun and SP1 activations, and thus reversed c-Jun- and SP1-promoted TGFß1 transcription. In all, Nrf2 could slow down DN progression by repression of TGFß1 in a c-Jun and SP1-dependent way. Our findings may provide novel clues for DN preventions and interventions in clinic.

Nrf2 pathway regulates multidrug-resistance-associated protein 1 in small cell lung cancer.

Although multidrug-resistance-associated protein-1 (MRP1) is a major contributor to multi-drug resistance (MDR), the regulatory mechanism of Mrp1 still remains unclear. Nrf2 is a transcription factor that regulates cellular defense response through antioxidant response elements (AREs) in normal tissues. Recently, Nrf2 has emerged as an important contributor to chemo-resistance in tumor tissues. In the present study, the role of Nrf2-ARE pathway on regulation of Mrp1 was investigated. Compared with H69 lung cancer cells, H69AR cells with MDR showed significantly higher Nrf2-ARE pathway activity and expression of Mrp1 as well. When Nrf2 was knocked down in H69AR cells, MRP1's expression decreased accordingly. Moreover, those H69AR cells with reduced Nrf2 level restored sensitivity to chemo-drugs. To explore how Nrf2-ARE pathway regulates Mrp1, the promoter of Mrp1 gene was searched, and two putative AREs--ARE1 and ARE2--were found. Using reporter gene and ChIP assay, both ARE1 and ARE2 showed response to and interaction with Nrf2. In 40 cases of cancer tissues, the expression of Nrf2 and MRP1 was measured by immunohistochemistry (IHC). As the quantitive data of IHC indicated, both Nrf2 and MRP1 showed significantly higher expression in tumor tissue than adjacent non-tumor tissue. And more important, the correlation analysis of the two genes proved that their expression was correlative. Taken together, theses data suggested that Nrf2-ARE pathway is required for the regulatory expression of Mrp1 and implicated Nrf2 as a new therapeutic target for MDR.

3,5-Diiodo-l-thyronine ameliorates diabetic nephropathy in streptozotocin-induced diabetic rats.

3, 5-Diiodothyronine (T2), a natural metabolite of triiodothyronine (T3) from deiodination pathway, can mimic biologic effects of T3 without inducing thyrotoxic effects. Recent studies revealed T3 acted as a protective factor against diabetic nephropathy (DN). Nevertheless, little is known about the effect of T2 on DN. This study was designed to investigate whether and how T2 affects experimental models of DN in vivo and in vitro. Administration of T2 was found to prevent significant decrease in SIRT1 protein expression and activity as well as increases in blood glucose, urine albumin excretion, matrix expansion, transforming growth factor-ß1 expression, fibronectin and type IV collagen deposition in the diabetic kidney. Concordantly, similar effects of T2 were exhibited in the cultured rat mesangial cells (RMC) exposed to high glucose and that could be abolished by a known SIRT1 inhibitor, sirtinol. Moreover, enhanced NF-κB acetylation and JNK phosphorylation present in both diabetic rats and high glucose-treated RMC were distinctly dampened by T2. Collectively, these results suggested that T2 was a protective agent against renal damage in diabetic nephropathy, whose action involved regulation of SIRT1.

Increased expression of neutrophil gelatinase-associated lipocalin receptor by interleukin-1ß in human mesangial cells via MAPK/ERK activation.

Neutrophil gelatinase-associated lipocalin (NGAL), one of the most promising next-generation biomarkers in clinical nephrology, has received extensive attention. However, the basic role of its receptor (NGALR) remains unclear. Here, we have assessed the expression pattern of NGALR in injured glomeruli and explored the possible mechanism of the NGALR involvement in inflammation in human mesangial cells (HMC). The expression pattern of NGALR was detected by immunohistochemistry in biopsy samples of 93 glomerulonephritis patients and healthy controls, and the regulation of NGALR by the proinflammatory cytokines, TGF-ß1, TNF-α and IL-1ß in HMC was analyzed by real-time PCR and Western blotting. NGALR was found to be expressed in glomeruli. Its expression was significantly higher in acute proliferative glomerulonephritis and lupus nephritis than that in other types of glomerulonephritis or healthy kidney tissues. In in vitro experiments, both mRNA and protein levels of NGALR were dramatically induced by treatment of IL-1ß, whereas TGF-ß1 or TNF-α did not have the same effect. Furthermore, it was shown that the IL-1ß-induced NGALR expression is mediated via the MAPK/ERK signaling pathway by using pharmacological inhibitors. Interestingly, the basal mRNA levels of NGAL detected in HMC, could be induced by IL-1ß. However, NGAL protein could not be detected, even with IL-1ß treatment. The ability of HMC to express NGAL protein was ascertained by exogenous administration of NGAL. In conclusion, the data show that NGALR is differentially expressed in human glomerular disease and is significantly up-regulated by Il-1ß in HMC via MAPK/ERK activation. Furthermore, exogenous NGAL can be uptaken into HMC.