WFDC2 is a potential prognostic and immunotherapy biomarker in lung adenocarcinoma.

OBJECTIVE: The prognosis of lung adenocarcinoma (LUAD), which is the most common type of lung cancer, remains poor. Little is known about the function and mechanism of whey acidic protein four-disulfide core domain 2 (WFDC2) in LUAD. METHODS: In this study, we used online databases to compare WFDC2 expression between LUAD and normal tissues, to analyze the relationship between WFDC2 and overall survival, and to investigate the potential roles of WFDC2. RESULTS: We found that WFDC2 protein and mRNA expression levels were significantly higher in LUAD tissue than in normal tissue, and high WFDC2 mRNA expression was associated with better overall survival. WFDC2 mRNA expression was correlated with the mutation status of TP53. The biological function of WFDC2 was associated with the cell cycle, and low WFDC2 mRNA expression was associated with an elevated tumor mutational burden and neoantigen levels. A negative relationship was observed between WFDC2 and immune gene expression, and high WFDC2 mRNA expression was found in patients with LUAD and low programed cell death 1 mRNA expression. CONCLUSIONS: We propose that WFDC2 is associated with clinical benefits of immune checkpoint inhibitors in LUAD.

Large-scale proteomics reveals precise biomarkers for detection of ovarian cancer in symptomatic women.

Ovarian cancer is the 8th most common cancer among women and has a 5-year survival of only 30-50%. While the survival is close to 90% for stage I tumours it is only 20% for stage IV. Current biomarkers are not sensitive nor specific enough, and novel biomarkers are urgently needed. We used the Explore PEA technology for large-scale analysis of 2943 plasma proteins to search for new biomarkers using two independent clinical cohorts. The discovery analysis using the first cohort identified 296 proteins that had significantly different levels in malign tumours as compared to benign and for 269 (91%) of these, the association was replicated in the second cohort. Multivariate modelling, including all proteins independent of their association in the univariate analysis, identified a model for separating benign conditions from malign tumours (stage I-IV) consisting of three proteins; WFDC2, KRT19 and RBFOX3. This model achieved an AUC of 0.92 in the replication cohort and a sensitivity and specificity of 0.93 and 0.77 at a cut-off developed in the discovery cohort. There was no statistical difference of the performance in the replication cohort compared to the discovery cohort. WFDC2 and KRT19 have previously been associated with ovarian cancer but RBFOX3 has not previously been identified as a potential biomarker. Our results demonstrate the ability of using high-throughput precision proteomics for identification of novel plasma protein biomarker for ovarian cancer detection.

Knockdown of HE4 suppresses tumor growth and invasiveness in lung adenocarcinoma through regulation of EGFR signaling.

It has been shown that the high expression of human epididymis protein 4 (HE4) in most lung cancers is related to the poor prognosis of patients, but the mechanism of pathological transformation of HE4 in lung cancer is still unclear. The current study is expected to clarify the function and mechanism of HE4 in the occurrence and metastasis of lung adenocarcinoma (LUAD). Immunoblotting evaluated HE4 expression in lung cancer cell lines and biopsies, and through analysis of The Cancer Genome Atlas (TCGA) dataset. Frequent HE4 overexpression was demonstrated in LUAD, but not in lung squamous cell carcinoma (LUSC), indicating that HE4 can serve as a biomarker to distinguish between LUAD and LUSC. HE4 knockdown significantly inhibited cell growth, colony formation, wound healing, and invasion, and blocked the G1-phase of the cell cycle in LUAD cell lines through inactivation of the EGFR signaling downstream including PI3K/AKT/mTOR and RAF/MAPK pathways. The first-line EGFR inhibitor gefitinib and HE4 shRNA had no synergistic inhibitory effect on the growth of lung adenocarcinoma cells, while the third-line EGFR inhibitor osimertinib showed additive anti-proliferative effects. Moreover, we provided evidence that HE4 regulated EGFR expression by transcription regulation and protein interaction in LUAD. Our findings suggest that HE4 positively modulates the EGFR signaling pathway to promote growth and invasiveness in LUAD and highlight that targeting HE4 could be a novel strategy for LUAD treatment.

Diagnostic Utility of Selected Matrix Metalloproteinases (MMP-2, MMP-3, MMP-11, MMP-26), HE4, CA125 and ROMA Algorithm in Diagnosis of Ovarian Cancer.

Ovarian cancer (OC) has an unfavorable prognosis. Due to the lack of effective screening tests, new diagnostic methods are being sought to detect OC earlier. The aim of this study was to evaluate the concentration and diagnostic utility of selected matrix metalloproteinases (MMPs) as OC markers in comparison with HE4, CA125 and the ROMA algorithm. The study group consisted of 120 patients with OC; the comparison group consisted of 70 patients with benign lesions and 50 healthy women. MMPs were determined via the ELISA method, HE4 and CA125 by CMIA. Patients with OC had elevated levels of MMP-3 and MMP-11, similar to HE4, CA125 and ROMA values. The highest SE, SP, NPV and PPV values were found for MMP-26, CA125 and ROMA in OC patients. Performing combined analyses of ROMA with selected MMPs increased the values of diagnostic parameters. The topmost diagnostic power of the test was obtained for MMP-26, CA125, HE4 and ROMA and performing combined analyses of MMPs and ROMA enhanced the diagnostic power of the test. The obtained results indicate that the tested MMPs do not show potential as stand-alone OC biomarkers, but can be considered as additional tests to raise the diagnostic utility of the ROMA algorithm.

Elucidating the role of liver enzymes as markers and regulators in ovarian cancer: a synergistic approach using Mendelian randomization, single-cell analysis, and clinical evidence.

OBJECTIVE: To investigate the association between liver enzymes and ovarian cancer (OC), and to validate their potential as biomarkers and their mechanisms in OC. Methods Genome-wide association studies for OC and levels of enzymes such as Alkaline phosphatase (ALP), Aspartate aminotransferase (AST), Alanine aminotransferase, and gamma-glutamyltransferase were analyzed. Univariate and multivariate Mendelian randomization (MR), complemented by the Steiger test, identified enzymes with a potential causal relationship to OC. Single-cell transcriptomics from the GSE130000 dataset pinpointed pivotal cellular clusters, enabling further examination of enzyme-encoding gene expression. Transcription factors (TFs) governing these genes were predicted to construct TF-mRNA networks. Additionally, liver enzyme levels were retrospectively analyzed in healthy individuals and OC patients, alongside the evaluation of correlations with cancer antigen 125 (CA125) and Human Epididymis Protein 4 (HE4). RESULTS: A total of 283 single nucleotide polymorphisms (SNPs) and 209 SNPs related to ALP and AST, respectively. Using the inverse-variance weighted method, univariate MR (UVMR) analysis revealed that ALP (P = 0.050, OR = 0.938) and AST (P = 0.017, OR = 0.906) were inversely associated with OC risk, suggesting their roles as protective factors. Multivariate MR (MVMR) confirmed the causal effect of ALP (P = 0.005, OR = 0.938) on OC without reverse causality. Key cellular clusters including T cells, ovarian cells, endothelial cells, macrophages, cancer-associated fibroblasts (CAFs), and epithelial cells were identified, with epithelial cells showing high expression of genes encoding AST and ALP. Notably, TFs such as TCE4 were implicated in the regulation of GOT2 and ALPL genes. OC patient samples exhibited decreased ALP levels in both blood and tumor tissues, with a negative correlation between ALP and CA125 levels observed. CONCLUSION: This study has established a causal link between AST and ALP with OC, identifying them as protective factors. The increased expression of the genes encoding these enzymes in epithelial cells provides a theoretical basis for developing novel disease markers and targeted therapies for OC.

Human epididymis protein 4, a novel potential biomarker for diagnostic and prognosis monitoring of lung cancer.

OBJECTIVE: This study aimed to explore the application value of human epididymis protein 4 (HE4) in diagnosing and monitoring the prognosis of lung cancer. METHODS: First, TCGA (The Cancer Genome Atlas) databases were used to analyze whey-acidic-protein 4-disulfide bond core domain 2 (WFDC2) gene expression levels in lung cancer tissues. Then, a total of 160 individuals were enrolled, categorized into three groups: the lung cancer group (n = 80), the benign lesions group (n = 40), and the healthy controls group (n = 40). Serum HE4 levels and other biomarkers were quantified using an electro-chemiluminescent immunoassay. Additionally, the expression of HE4 in tissues was analyzed through immunohistochemistry (IHC). In vitro cultures of human airway epithelial (human bronchial epithelial [HBE]) cells and various lung cancer cell lines (SPC/PC9/A594/H520) were utilized to detect HE4 levels via western blot (WB). RESULTS: Analysis of the TCGA and UALCAN (The University of Alabama at Birmingham Cancer Data Analysis Portal) databases showed that WFDC2 gene expression levels were upregulated in lung cancer tissues (p < 0.01). Compared with the control group and the benign group, HE4 was significantly higher in the serum of patients with lung cancer (p < 0.001). Receiver operating characteristic (ROC) analysis confirmed that HE4 had better diagnostic efficacy than classical markers in the differential diagnosis of lung cancer and benign lesions and had the highest diagnostic value in lung adenocarcinoma (area under the ROC curve [AUC] = 0.826). HE4 increased in early lung cancer and positively correlated with poor prognosis (p < 0.001). Moreover, the results of WB and IHC revealed that the expression of HE4 was increased in lung cancer cells (SPC/A549/H520) and lung cancer tissues but decreased in PC9 cells with a lack of exon EGFR19 (p < 0.05). CONCLUSION: Serum HE4 emerges as a promising novel biomarker for the diagnosis and prognosis assessment of lung cancer.

Human epididymis protein 4: Analysis of national health and nutrition examination survey data.

BACKGROUND: Human epididymis protein 4 (HE4) is a tumor marker overexpressed in ovarian cancer and is commonly utilized to aid with diagnosis of an adnexal mass. HE4 levels vary based on pregnancy, age, menopausal status, and tobacco use. OBJECTIVE(S): The objective of this study was to evaluate population-based data to examine factors that affect HE4 among adult women in the United States and stratify levels of HE4 by demographic and gynecologic factors. STUDY DESIGN: A retrospective analysis was conducted using data from 2,480 women aged 20 + who participated in the National Health and Nutrition Examination Survey (2001-2002). From these cross-sectional data, serum HE4 and cotinine, a marker of tobacco exposure, were combined with demographic and interview data. Estimated glomerular filtration rates (eGFR) were based on serum creatinine, age, sex, and race. Other variables of interest included menopausal status, pregnancy, and various gynecologic factors. Summary HE4 data are provided as geometric means with associated 95 % confidence intervals. RESULTS: HE4 levels were independently associated with age, renal function, and nicotine use, all p < 0.001. Pre-menopausal women with a history of endometriosis were found to have elevated HE4 levels compared to those without, p < 0.01; however, we found no such difference among post-menopausal women. Adjusting for age, no differences in HE4 were found based on race/ethnicity, p = 0.29. HE4 levels showed statistically significant associations with income level; however, these were small and clinically irrelevant. CONCLUSION: This study provides evaluation of HE4 levels among a data set representative of 98.5 million non-institutionalized women in the United States and gives insight into extraneous factors that may influence these levels.

Human epididymal protein 4 and its combined detection show good diagnostic value in lung cancer: A retrospective study.

OBJECTIVES: This study aimed to assess the diagnostic value of human epididymal protein 4 (HE4), a potential novel biomarker for lung cancer, and its combined detection with five other conventional biomarkers in lung cancer diagnosis and subtyping. METHODS: In this retrospective study, 115 lung cancer patients, 50 patients with benign pulmonary disease, and 50 healthy controls were included. Serum HE4, progastrin-releasing peptide (ProGRP), squamous cell carcinoma (SCC) antigen, cytokeratin-19 fragment (CYFRA21-1), neuron-specific enolase (NSE), and carcinoembryonic antigen (CEA) were analyzed using the electrochemiluminescence immunoassay and chemiluminescence immunoassay. The receiver operating characteristic curve was performed to analyze the diagnostic efficacy of individual biomarkers in identifying both lung cancer and its histologic subtypes. RESULTS: All six biomarkers showed significantly elevated levels in the lung cancer group compared to both benign pulmonary disease and control groups (P < 0.05). Among the biomarkers evaluated, HE4 exhibited the highest diagnostic performance for lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma with area under the curve (AUC) values of 0.921, 0.891, and 0.937, respectively. ProGRP was the optimal biomarker for small cell lung cancer with an AUC of 0.973. The combination of all six biomarkers yielded the largest AUCs in the diagnosis of lung cancer subtypes (0.937 for lung adenocarcinoma, 0.998 for lung squamous cell carcinoma, and 0.985 for small cell lung cancer). Furthermore, specific combinations, such as HE4 + CEA, HE4 + SCC, and ProGRP + HE4 + NSE, showed strong diagnostic performance in lung cancer. CONCLUSIONS: HE4 and its combined detection held substantial clinical significance in the diagnosis of lung cancer and its histologic subtyping, especially for lung adenocarcinoma and lung squamous cell carcinoma.

Relationship between human epididymal protein 4 and depth of tumor invasion, postoperative recurrence, and metastasis of epithelial epithelial ovarian cancer.

This study aimed to analyze the relationship between human epididymal protein 4 (HE4) and infiltration depth, postoperative recurrence, and metastasis of epithelial ovarian cancer (OVCA). Immunohistochemistry was used to detect the expression level of HE4 in cancer tissues and adjacent tissues of 90 patients with epithelial OVCA admitted to our hospital from May 2017 to January 2018. Cox regression was used to analyze the factors affecting the prognosis of epithelial OVCA. The relationship between HE4 and the prognosis of epithelial OVCA was analyzed by the receiver operating characteristic curve and Kaplan-Meier survival curve. The positive expression rate of HE4 in epithelial OVCA was 85.56%, which was higher than 34.44% in adjacent tissues (p < 0.01). The International Federation of Gynecology and Obstetrics stage, infiltration depth, lymph node metastasis, postoperative recurrence and metastasis, and HE4 positivity were independent risk factors for the prognosis, and platinum-based chemotherapy sensitivity was an independent protective factor for the prognosis of patients with epithelial OVCA (p < 0.05). The area under the curve of HE4 in diagnosing epithelial OVCA and predicting recurrence was 0.863 and 0.700, the sensitivity was 91.60% and 85.60%, and the specificity was 90.20% and 65.60%. The median progression-free survival and overall survival were 26.1 and 30.2 months in HE4-positive epithelial OVCA patients, while these were 31.4 and 35.6 months in HE4-negative epithelial OVCA patients (p < 0.05). In conclusion, HE4 was highly expressed in epithelial OVCA tissues. Its expression level was related to the depth of tumor invasion, postoperative recurrence and metastasis, and other clinicopathological characteristics of patients with epithelial OVCA.

Evaluating the effectiveness of pre-operative diagnosis of ovarian cancer using minimally invasive liquid biopsies by combining serum human epididymis protein 4 and cell-free DNA in patients with an ovarian mass.

OBJECTIVE: To assess the feasibility of scalable, objective, and minimally invasive liquid biopsy-derived biomarkers such as cell-free DNA copy number profiles, human epididymis protein 4 (HE4), and cancer antigen 125 (CA125) for pre-operative risk assessment of early-stage ovarian cancer in a clinically representative and diagnostically challenging population and to compare the performance of these biomarkers with the Risk of Malignancy Index (RMI). METHODS: In this case-control study, we included 100 patients with an ovarian mass clinically suspected to be early-stage ovarian cancer. Of these 100 patients, 50 were confirmed to have a malignant mass (cases) and 50 had a benign mass (controls). Using WisecondorX, an algorithm used extensively in non-invasive prenatal testing, we calculated the benign-calibrated copy number profile abnormality score. This score represents how different a sample is from benign controls based on copy number profiles. We combined this score with HE4 serum concentration to separate cases and controls. RESULTS: Combining the benign-calibrated copy number profile abnormality score with HE4, we obtained a model with a significantly higher sensitivity (42% vs 0%; p<0.002) at 99% specificity as compared with the RMI that is currently employed in clinical practice. Investigating performance in subgroups, we observed especially large differences in the advanced stage and non-high-grade serous ovarian cancer groups. CONCLUSION: This study demonstrates that cell-free DNA can be successfully employed to perform pre-operative risk of malignancy assessment for ovarian masses; however, results warrant validation in a more extensive clinical study.

Human Epididymis Protein 4 (HE4) and Cancer Antigen 125 (CA125) for Prediction of Optimal Primary Surgery in Non-Mucinous Epithelial Ovarian Cancer.

OBJECTIVE: To determine the relationship between pre-operative HE4 and CA125 levels in non-mucinous epithelial ovarian cancer cases (EOC) and outcomes of primary surgery for prediction of optimal surgery. METHODS: A retrospective study was performed on non-mucinous EOC who underwent primary surgery at King Chulalongkorn Memorial Hospital from 2016 to 2020. Demographic and clinical characters were collected. Histopathology and pre-operative tumor markers namely HE4 and CA125 were also recruited. Primary surgical outcomes were classified as optimal (OS) and suboptimal surgery (SS). RESULTS: One hundred and seventy patients were enrolled in the study. There were 130 and 40 cases in OS and SS, respectively. Average age and body mass index (BMI) of EOC were 54.2 years old and 23.1 Kg/m2, respectively. Both groups had comparable demographic characteristics. Two-thirds (103/170) and one-third (63/170) had early stage and clear cell histopathology, respectively. The median level of HE4 were 118.60 and 603.45 pmol/L in OS and SS, respectively. OS and SS had average CA125 at 146.95 and 814.70 U/L, respectively. The best cut-off point of HE4 and CA125 less than 170.95 pmol/L and 316.4 U/mL gave predicting OS with area under curve (AUC) at 0.78 and 0.75, respectively. HE4 and CA125 cut-off point had sensitivity, specificity, positive predict value (PPV) and negative predictive value (NPV) at percentage of 60.8/60.8, 87.5/82.5, 94.1/91.9 and 40.7/39.3, respectively. CONCLUSION: HE4 and CA125 of non-mucinous EOC among OS had significantly less than SS and could be the predicting of optimal surgery.

Silencing HE4 alleviates the renal fibrosis in lupus nephritis mice by regulating the C3/MMPs/prss axis.

To explore the regulatory effect of human epididymis protein 4 (HE4) on renal fibrosis in mice with lupus nephritis (LN) and the underlying mechanism. Ten-week old MRL/LPR mice were injected with HE4 shRNA adenovirus vector through the renal pelvis for 5 days. Renal tissues were extracted for HE and Masson staining to evaluate pathological changes and fibrosis in lupus nephritis mice. The level of urine protein was measured using a biochemical analyzer, while the expression level of HE4 and p-NF-κB p65 in renal tissues was visualized using an immunofluorescence assay. The level of ß2-microglobulin (ß2-MG), neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule 1 (Kim-1) was determined by the immunohistochemical assay. Western blotting was used to determine the levels of C3, HE4, matrix metalloproteinase-2 (MMP2), MMP9, p-p65, prss23, and prss35 in renal tissues. Compared to wild-type C57BL/6 mice, MRL/LPR mice showed a marked increase in the number of glomeruli, hyperplasic basement membrane, severe infiltration of inflammatory cells in renal tubules and glomeruli, obvious necrosis in glomeruli, elevated fibrosis levels, and increased levels of urine protein, ß2-MG, NGAL, Kim-1, C3, HE4, MMP2, MMP9, and p-p65; and decreased levels of prss23 and prss35 were observed in MRL/LPR mice. After the administration of the HE4 shRNA adenovirus vector, the repaired structure of renal tubules and glomeruli improved infiltration of inflammatory cells, reduced collagen fiber and urine protein, suppressed levels of C3, HE4, MMP2, MMP9, and p-P65, and facilitated the expression of prss23 and prss35 which were observed. Silencing HE4 improved renal fibrosis and inhibited inflammation in mice with lupus nephritis, which may play a role in inhibiting C3/MMPs and promoting prss-related protein expression.

[Delivery of epididymis-specific mRNAs from mouse epididymosomes into N2a and TM4 cells].

OBJECTIVE: To investigate whether mouse epididymis-specific mRNAs Adam7 and Crisp1 can be delivered into N2a and TM4 cells, and to provide an experimental basis for exploring the function of epididymal mRNAs. METHODS: Using RT-PCR, we detected the presence of epididymis-specific genes (Adam7, Crisp1, Defb22, Wfdc2, and Wfdc9) in the testis, epididymis, epididymosome and sperm of adult male BALB/c mice as well as in the human testis, seminal vesicles and sperm. We isolated epididymosomes of BALB/c mice by low-speed centrifugation, filtration and ultracentrifugation, fluorescently labeled them by PKH26, co-incubated them for 1 hour with the N2a and TM4 cells after 24 hours of starvation culture, and observed whether they were fused with the N2a and TM4 cells and ingested using the epididymosomes without PKH26 labeling, PKH26 dye without epididymosomes, and non- epididymosome or -PKH26 dye as controls. Then we detected the epididymis-specific genes in the N2a and TM4 cells after 1-hour co-incubation by RT-PCR. RESULTS: Adam7 and Crisp1 were present in the mouse epididymis, epididymosomes and sperm, and in the human seminal vesicles and sperm as well, but not in the testes of either the mice or men. PKH26 and Hoechst33258 fluorescence double-labeling showed that the mouse epididymosomes were fused with the N2a and TM4 cells and ingested; RT-PCR revealed the mRNAs of Adam7 and Crisp1 in the N2a and TM4 cells after 1-hour co-incubation; and Western blot exhibited the CRISP1 protein in the N2a and TM4 cells incubated with epididymosomes. CONCLUSION: Epididymosomes can deliver epididymis-specific mRNAs Adam7 and Crisp1 into N2a and TM4 cells, where Crisp1 may be translated into proteins, though their function and significance need to be further studied.

Analysis of potential biomarkers for diabetic kidney disease based on single-cell RNA-sequencing integrated with a single-cell sequencing assay for transposase-accessible chromatin.

Diabetic kidney disease (DKD) is a renal microvascular disease caused by hyperglycemia that involves metabolic remodeling, oxidative stress, inflammation, and other factors. The mechanism is complex and not fully unraveled. We performed an integrated single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq) and single-cell RNA-sequencing (scRNA-seq) analyses of kidneys from db/db and db/m mice to identify differential open chromatin regions and gene expression, particularly in genes related to proximal tubular reabsorption and secretion. We identified 9,776 differentially expressed genes (DEGs) and 884 cell type-specific transcription factors (TFs) across 15 cell types. Glucose and lipid transporters, and TFs related to the circadian rhythm in the proximal tubules had significantly higher expression in db/db mice than in db/m mice (P<0.01). Crosstalk between podocytes and tubular cells in the proximal tubules was enhanced, and renal inflammation, oxidative stress, and fibrosis pathways were activated in db/db mice. Western blotting and immunohistochemical staining results showed that Wfdc2 expression in the urine and kidneys of DKD patients was higher than that in non-diabetic kidney disease (NDKD) controls. The revealed landscape of chromatin accessibility and transcriptional profiles in db/db mice provide insights into the pathological mechanism of DKD.

The First Transcriptomic Atlas of the Adult Lacrimal Gland Reveals Epithelial Complexity and Identifies Novel Progenitor Cells in Mice.

The lacrimal gland (LG) secretes aqueous tears. Previous studies have provided insights into the cell lineage relationships during tissue morphogenesis. However, little is known about the cell types composing the adult LG and their progenitors. Using scRNAseq, we established the first comprehensive cell atlas of the adult mouse LG to investigate the cell hierarchy, its secretory repertoire, and the sex differences. Our analysis uncovered the complexity of the stromal landscape. Epithelium subclustering revealed myoepithelial cells, acinar subsets, and two novel acinar subpopulations: Tfrchi and Car6hi cells. The ductal compartment contained Wfdc2+ multilayered ducts and an Ltf+ cluster formed by luminal and intercalated duct cells. Kit+ progenitors were identified as: Krt14+ basal ductal cells, Aldh1a1+ cells of Ltf+ ducts, and Sox10+ cells of the Car6hi acinar and Ltf+ epithelial clusters. Lineage tracing experiments revealed that the Sox10+ adult populations contribute to the myoepithelial, acinar, and ductal lineages. Using scRNAseq data, we found that the postnatally developing LG epithelium harbored key features of putative adult progenitors. Finally, we showed that acinar cells produce most of the sex-biased lipocalins and secretoglobins detected in mouse tears. Our study provides a wealth of new data on LG maintenance and identifies the cellular origin of sex-biased tear components.

New insights into the diagnostic characteristics and clinical application of serum biomarkers for lung cancer, and human epididymis protein 4 as a new biomarker?

The value of serum tumor biomarkers used for lung cancer diagnosis is still controversial in clinical practice. This study aimed to further dissect and evaluate the clinical value of serum progastrin-releasing peptide (ProGRP), neuron-specific enolase (NSE), squamous cell carcinoma antigen (SCC-Ag), carcinoembryonic antigen (CEA), cytokeratin-19 fragment (CYFRA21-1) together with a potential new biomarker, the human epididymis protein 4 (HE4) for lung cancer diagnosis, in a large cohort of a Chinese population. Ostensibly healthy individuals, as well as those with benign non-cancerous diseases, benign tumors, lung cancers, and other types of malignancies, were enrolled in the study. Serum ProGRP, NSE, SCC-Ag, CEA, CYFRA21-1, and HE4 were analyzed using the chemiluminescence immunoassay. Data were analyzed utilizing the SPSS and GraphPad Prism software. Detailed dissection of the diagnostic characteristics of serum 6 biomarkers on lung cancer was performed. All 6 biomarkers showed capabilities in characterizing lung cancer from other diseases. ProGRP and NSE were highly specific to small cell lung cancer (SCLC); SCC-Ag was a fair biomarker for NSCLC, specifically SCC histotype; CEA showed specificity to SCLC, followed by NSCLC; CYFRA21-1 was a good biomarker for both SCLC and NSCLC; HE4 showed high specificity to SCLC. For NSCLC characterization, CYFRA21-1+HE4+CEA was the best combinatory pattern in the terms of diagnostic performance (AUC=0.8110). The best combinatory analysis for SCLC was ProGRP+NSE+HE4 (AUC=0.9282). Patients with advanced stage, larger tumor, males, and age 50 or older had higher serum biomarkers levels than those with early stage, smaller tumor, females, and age under 50. Six biomarkers had capabilities in characterizing lung cancer with high or fair diagnostic performance. HE4 is a potential biomarker for both SCLC and NSCLC diagnosis, which merits further investigation.

Human epididymis protein 4 is associated with severity and poor prognosis of connective tissue disease-associated interstitial lung disease with usual interstitial pneumonia pattern.

BACKGROUND: Overexpression of human epididymis protein 4 (HE4) was previously described in idiopathic pulmonary fibrosis (IPF), but whether serum HE4 can be considered as a potential biomarker in connective tissue disease-associated interstitial lung disease (CTD-ILD) with usual interstitial pneumonia (UIP) pattern was still unknown. METHOD: A total of 55 CTD-ILD patients with UIP pattern (UIP-CTD) and 52 healthy controls were enrolled in this study. The serum levels of HE4 and Krebs von den Lungen-6 (KL-6) were evaluated in both cohorts. In addition, immunohistochemistry analysis for HE4 was performed on the lung sections of 6 patients with rheumatoid arthritis-associated UIP (UIP-RA) and 6 patients with early-stage lung cancer as normal control. RESULTS: The levels of serum HE4 and KL-6 were higher in patients with UIP-CTD than in healthy controls (292.3 pmol/L versus 79.5 pmol/L for HE4, p < 0.001; 1091.0 IU/mL versus 171.5 IU/mL for KL-6, p < 0.001). Significant correlations between serum HE4 levels and percentpredicted forced vital capacity (FVC%) (r = -0.425, p = 0.004), percent predicted diffusing capacity of the lung for carbon monoxide (DLCO%) (r = -0.447, p = 0.003), and Gender-Age-Physiology (GAP) index (r = 0.494, p < 0.001) were observed in UIP-CTD patients. In immunohistochemistry analysis, elevated expression of HE4 in bronchiolar epithelium and mesenchyme was observed in patients with UIP-RA compared with controls. The serum levels of HE4 (≥277.5 pmol/L) and GAP index were related to an increased risk of mortality (HR = 3.884, p = 0.034; HR = 1.480, p = 0.028, respectively). CONCLUSION: The expression of HE4 in serum and lung specimens was significantly elevated in UIP-CTD patients. Moreover, serum HE4 may be utilized as a biomarker to evaluate the severity of disease and predict the prognosis of UIP-CTD patients.

Detection of plasma exosomal miRNA-205 as a biomarker for early diagnosis and an adjuvant indicator of ovarian cancer staging.

BACKGROUND: Ovarian cancer (OC) is one of the serious threats to the health of women worldwide, and accurate biomarkers are urgently demanded for early diagnosis of OC. We have previously confirmed that miR-205 promotes the invasion and metastasis of OC cells by inhibiting the expression of the tumor suppressor gene TCF21. In this study, we used liquid biopsy technology to detect the expression levels of the four genes, miR-205, CA125, HE4 and TCF21, in the exosomes of plasma of OC patients. Combined with analysis of clinicopathological parameters of OC patients, we aimed to provide efficient and non-invasive laboratory biomarkers for early diagnosis of OC. METHODS: 36 OC patients who were diagnosed in local hospitals from September 2020 to July 2021 were selected as OC group, 31 cases of surgically diagnosed with ovarian benign lesions were selected as benign group, and 32 healthy people who underwent physical examination during the same period were selected as a control group. We employed transmission electron microscope (TEM), Western blotting (WB), and nanoparticle tracking analysis (NTA) to identify biomarkers in the exosomes extracted from plasma of the three groups. The RNA levels of miR-205, CA125, HE4 and TCF21 genes in plasma exosomes were detected by real-time quantitative PCR (qRT-PCR) method. We used clinical pathological parameters and the Receiver Operating Characteristic (ROC) curves to evaluate the diagnostic efficacy for the genes detected in plasma exosomes. RESULTS: We found that the expression level of miR-205 in plasma exosomes of the OC group was significantly higher than that of the benign and control groups (P <  0.05), and the level of miR-205 was elevated during the III-IV periods of OC and lymph node metastasis. CONCLUSION: The level of miR-205 in plasma exosomes is a valuable tumor biomarker to improve OC diagnosis.

Recurrence monitoring for ovarian cancer using a cell phone-integrated paper device to measure the ovarian cancer biomarker HE4/CRE ratio in urine.

Ovarian cancer has a poor cure rate and rates of relapse are high. Current recurrence detection is limited by non-specific methods such as blood testing and ultrasound. Based on reports that human epididymis four (HE4) / creatinine (CRE) ratios found in urine are elevated in ovarian cancers, we have developed a paper-based device that combines lateral flow technology and cell phone analysis to quantitatively measure HE4/CRE. Surrogate samples were used to test the performance over clinically expected HE4/CRE ratios. For HE4/CRE ratios of 2 to 47, the percent error was found to be 16.0% on average whether measured by a flatbed scanner or cell phone. There was not a significant difference between the results from the cell phone or scanner. Based on published studies, error in this method was less than the difference required to detect recurrence. This promising new tool, with further development, could be used at home or in low-resource settings to provide timely detection of ovarian cancer recurrence.

Elevated Serum Human Epididymis Protein 4 Is Associated With Disease Activity and Systemic Involvements in Primary Sjögren's Syndrome.

Background: We aimed to investigate the clinical utility of human epididymis protein 4, a tumor biomarker being widely utilized in clinical practice in the diagnosis of ovarian cancer, in primary Sjögren's Syndrome (pSS). Methods: A total of 109 pSS patients and 113 healthy controls (HCs) were included in the study. HE4 were determined by Roche Cobas E601 electrochemical luminescence analyzer. Clinical and laboratory findings were reviewed, and the relationships between HE4 and clinical parameters were determined by Spearman's correlation test. The European league against rheumatism Sjögren's syndrome disease activity index (ESSDAI) was utilized to evaluate disease activity. Findings: The levels of HE4 were significantly elevated in patients with pSS compared to HCs (103.65 pmol/L vs. 46.52 pmol/L, p<0.001). The levels of HE4 were positively correlated with ESSDAI scores (r=0.462, p<0.001). Significant positive correlations between the levels of HE4 with pulmonary involvements (r=0.442, p<0.001) and renal involvements (r=0.320, p=0.001) were observed. Receiver operating curve (ROC) analysis revealed an optimal cut-off value of 104.90 pmol/L and 128.05 pmol/L for distinguishing patients with pulmonary and renal involvements, with the areas under the ROC curve (AUCs) of 0.778 (95%CI 0.685-0.870, p<0.001) and 0.768 (95%CI 0.646-0.891, p=0.001), respectively. Among patients with pulmonary involvement, the levels of HE4 were positively correlated with the semiquantitative HRCT grade (r=0.417, p=0.016), and negatively correlated with the percentage of forced vital capacity (FVC) (r= -0.460, p=0.047) and diffusing capacity of the lung for carbon monoxide (DLco) (r= -0.623, p=0.004). For patients with renal involvement, HE4 was positively correlated with creatinine (r=0.588, p=0.021) and negatively correlated with estimated glomerular filtration rate (r= -0.599, p=0.030). Conclusions: Our findings demonstrated a novel role of HE4 in clinical stratification of pSS, suggesting that introducing HE4 to the current pSS test panel may provide additional diagnostic value, particularly in evaluating disease activity and pulmonary/renal involvements.