Establishment of a 1, 4, 7, 10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS-ester) based lectin microarray for efficiently detecting serum glycans in gastric cancers.

Development of cancers is involved in changes of a variety of glycans. Lectin microarray is one of the most powerful methodologies for investigation of glycan alterations in biological samples with its advantages of high through-put, selectivity and specificity of the technique. However, utilization of lectin microarrays available commercially keeps of great challenges. In this study, we took use of the molecular self-assembled monolayer technique to modify a gold surface with the reagent 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS-ester) in combination with 16-amino-1-hexadecanethiol hydrochloride. Cross-linking effect of DOTA-NHS-ester is brought about via activating three -OH ends to three terminals of succinylimidines, making selective binding of the terminal amino groups in proteins possible. We immobilized ten commercial lectins on the platform and measured changes of serum lectin-matched glycans in patients with gastric cancer. The results demonstrated that this biochip modification platform conferred impressive chemical surface stabilization, sensitivity and geometric images. We observed that all the serum glycans tested in the patients were significantly higher than those in the controls (P < 0.05). The biochip would provide a versatile platform for investigation of potential glycan biomarkers in making tumor diagnosis decision and analyzing escape of tumors from immunity.

Integrative multi-omics analysis depicts the methylome and hydroxymethylome in recurrent bladder cancers and identifies biomarkers for predicting PD-L1 expression.

BACKGROUND: Urinary bladder cancer (UBC) is a common malignancy of the urinary tract; however, the mechanism underlying its high recurrence and responses to immunotherapy remains unclear, making clinical outcome predictions difficult. Epigenetic alterations, especially DNA methylation, play important roles in bladder cancer development and are increasingly being investigated as biomarkers for diagnostic or prognostic predictions. However, little is known about hydroxymethylation since previous studies based on bisulfite-sequencing approaches could not differentiate between 5mC and 5hmC signals, resulting in entangled methylation results. METHODS: Tissue samples of bladder cancer patients who underwent laparoscopic radical cystectomy (LRC), partial cystectomy (PC), or transurethral resection of bladder tumor (TURBT) were collected. We utilized a multi-omics approach to analyze both primary and recurrent bladder cancer samples. By integrating various techniques including RNA sequencing, oxidative reduced-representation bisulfite sequencing (oxRRBS), reduced-representation bisulfite sequencing (RRBS), and whole exome sequencing, a comprehensive analysis of the genome, transcriptome, methylome, and hydroxymethylome landscape of these cancers was possible. RESULTS: By whole exome sequencing, we identified driver mutations involved in the development of UBC, including those in FGFR3, KDMTA, and KDMT2C. However, few of these driver mutations were associated with the down-regulation of programmed death-ligand 1 (PD-L1) or recurrence in UBC. By integrating RRBS and oxRRBS data, we identified fatty acid oxidation-related genes significantly enriched in 5hmC-associated transcription alterations in recurrent bladder cancers. We also observed a series of 5mC hypo differentially methylated regions (DMRs) in the gene body of NFATC1, which is highly involved in T-cell immune responses in bladder cancer samples with high expression of PD-L1. Since 5mC and 5hmC alternations are globally anti-correlated, RRBS-seq-based markers that combine the 5mC and 5hmC signals, attenuate cancer-related signals, and therefore, are not optimal as clinical biomarkers. CONCLUSIONS: By multi-omics profiling of UBC samples, we showed that epigenetic alternations are more involved compared to genetic mutations in the PD-L1 regulation and recurrence of UBC. As proof of principle, we demonstrated that the combined measurement of 5mC and 5hmC levels by the bisulfite-based method compromises the prediction accuracy of epigenetic biomarkers.

PSA density in the diagnosis of prostate cancer in the Chinese population: results from the Chinese Prostate Cancer Consortium.

We performed this study to investigate the diagnostic performance of prostate-specific antigen density (PSAD) in a multicenter cohort of the Chinese Prostate Cancer Consortium. Outpatients with prostate-specific antigen (PSA) levels ≥4.0 ng ml-1 regardless of digital rectal examination (DRE) results or PSA levels <4.0 ng ml-1 and abnormal DRE results were included from 18 large referral hospitals in China. The diagnostic performance of PSAD and the sensitivity and specificity for the diagnosis of prostate cancer (PCa) and high-grade prostate cancer (HGPCa) at different cutoff values were evaluated. A total of 5220 patients were included in the study, and 2014 (38.6%) of them were diagnosed with PCa. In patients with PSA levels ranging from 4.0 to 10.0 ng ml-1, PSAD was associated with PCa and HGPCa in both univariate (odds ratio [OR] = 45.15, P < 0.0001 and OR = 25.38, P < 0.0001, respectively) and multivariate analyses (OR = 52.55, P < 0.0001 and OR = 26.05, P < 0.0001, respectively). The areas under the receiver operating characteristic curves (AUCs) of PSAD in predicting PCa and HGPCa were 0.627 and 0.630, respectively. With the PSAD cutoff of 0.10 ng ml-2, we obtained a sensitivity of 88.7% for PCa, and nearly all (89.9%) HGPCa cases could be detected and biopsies could be avoided in 20.2% of the patients (359/1776 cases). Among these patients who avoided biopsies, only 30 cases had HGPCa. We recommend 0.10 ng ml-2 as the proper cutoff value of PSAD, which will obtain a sensitivity of nearly 90% for both PCa and HGPCa. The results of this study should be validated in prospective, population-based multicenter studies.

Expression of Narcissus pseudonarcissus lectin and mannose receptor positive macrophages predict progression and prognosis of patients with gastric cancer.

BACKGROUND: Mannose receptor (MR) is an immune adhesion molecule and is mainly expressed in macrophages and nonmature dendritic cells. The ligand mannose, one of the natural ligands of MR, is a monosaccharide, which is localized in the envelope or cytoplasm of macrophages. The aim of this study was to investigate expression of MR and its ligand mannose in tumor tissues of primary advanced gastric cancer and to evaluate the predictive and prognostic value of the positive cells in gastric cancer patients. METHODS: Histochemical staining for Narcissus pseudonarcissus lectin (NPL) and immunohistochemical envision two-step assay for MR were used to detect expression of NPL and MR in primary advanced gastric adenocarcinoma tissues. Adjacent non-cancerous gastric tissues of the patients were used as controls. Relationship of NPL and MR expression in the tumor tissues with clinicopathological features and survival time of the gastric cancer patients were analyzed. RESULTS: Numbers of NPL+ and MR+ macrophages in stromal tissues of gastric cancer were significantly higher than those in the adjacent non-cancerous gastric tissues (P=0.006; P<0.001). NPL expression in the primary tumor tissues was significantly more dominant than that in the adjacent non-cancerous gastric tissues (P=0.003). Expression of both the molecules in macrophages in tumor tissues was negatively correlated (r=-0.363, P=0.009). TNM stage of the patients was closely correlated to number of MR+ macrophages and NPL expression in the stromal tissues of gastric cancer (P=0.009 and P=0.020). Kaplan-Meier survival model data showed that the patients with low counting of NPL+ macrophages and high counting of MR+ macrophages significantly led to worse disease progression and poorer prognosis (P=0.008). Cox regression analysis further demonstrated that high expression of MR+ macrophages was an independent predictor of poor prognosis in patients with gastric cancer (P=0.033). CONCLUSIONS: Occurrence of mannose and MR in tumor tissues of gastric cancer might be prognostic factors for estimating risk of gastric cancer patients.

Development of a dendrimer PAMAM­based gold biochip for rapid and sensitive detection of endogenous IFN­Î³ and anti­IFN­Î³ IgG in patients with hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a rare but severe disease characterized by immune hyperactivation and cytokine storm. Given the high mortality rate of HLH, there is a need for more effective diagnostic tools and treatments. The present study developed a dendrimer­based protein biochip for rapid, sensitive and simultaneous detection of serum interferon (IFN)­Î³ and endogenous anti­IFN­Î³ antibody (Ab) in patients with HLH. A gold biochip was modified with 1, 4­phenylene diisothiocyanate (PDITC), polyamidoamine (PAMAM) or PDITC­activated PAMAM. The optimal immobilization concentration for Ab capture and the reaction concentration for detecting Ab on the PDITC­activated PAMAM­modified biochip were 6.25 and 3.12 µg/ml, respectively; the limit of detection of IFN­Î³ protein was 50 pg/ml. The efficiency of the protein­probed biochip in detecting IFN­Î³ and anti­IFN­Î³ Ab in serum samples from 77 patients with HLH was evaluated; the positive rates for IFN­Î³ and anti­IFN­Î³ IgG Ab were 63.6% (49/77) and 61.0% (47/77), respectively. The present results demonstrated that the PDITC­activated PAMAM­modified biochip might be a sensitive tool for the specific detection of IFN­Î³ and anti­IFN­Î³ Ab in serum, and might have clinical applicability for the diagnosis of HLH.

Establishment of a protein biochip to detect serum IgG antibodies against IL-2 and soluble CD25 in hemophagocytic lymphohistiocytosis.

BACKGROUND: Interleukin-2 (IL-2) and soluble CD25 (sCD25) are among the most important cytokines and diagnostic biomarkers in hemophagocytic lymphohistiocytosis (HLH). Detecting serum level of IL-2 and sCD25 is valuable for making clinical diagnosis and treatment decision in HLH. METHODS: Since tests showing serum IgG antibody against IL-2 or sCD25 have never been carried out, a new protein biochip, which was modified with cysteine and activated sophorolipid (Cys-SL), was developed. RESULTS: Limits of detection on the biochip were 78 pg/ml for IL-2 and 39 pg/ml for sCD25, respectively. The data showed that on-chip seroimmunological responses to IL-2 and sCD25 proteins were 20.8% and 83.1% and the seroprevalence of IL-2 and sCD25 IgG antibodies were 45.5% and 57.2%, respectively. Data collection for the seroprevalence of serum antigen-antibody complex of sCD25 was 68.8%. The new biochip model shared similar sensitivity and specificity to chemiluminescent immunoassay (CLIA) in its measuring capacity of serum sCD25. CONCLUSIONS: We addressed and confirmed the involvement of serum IgG antibodies against IL-2 and sCD25 as well as Ag-Ab complex of sCD25 in HLH patients. Therefore, this biochip platform would offer a new technological substitution for clinical serological diagnosis of HLH.