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 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.

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.

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.

[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.

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.

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.

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.

Kinetics of HE4 and CA125 as prognosis biomarkers during neoadjuvant chemotherapy in advanced epithelial ovarian cancer.

BACKGROUND: Ovarian cancer (OC) is considered the most lethal gynecological cancer, of which more than 65% cases are diagnosed in advanced stages, requiring platinum-based neoadjuvant chemotherapy (NACT). METHODS: A prospective-longitudinal study was conducted among women with advanced epithelial ovarian cancer (AEOC), III and IV stages, and treated with NACT, at the National Cancer Institute - Mexico, from July 2017 to July 2018. Serum samples were obtained for quantification of CA125 and HE4 using ELISA at the first and in each of the three NACT cycles. The therapeutic response was evaluated through standard tomography. We determined whether CA125 and HE4, alone or in combination, were associated with TR to NACT during follow up. RESULTS: 53 patients aged 38 to 79 years were included, 92.4% presented papillary serous subtype OC. Higher serum HE4 levels were observed in patients with non-tomographic response (6.89 vs 5.19 pmol/mL; p = 0.031), specially during the second (p = 0.039) and third cycle of NACT (p = 0.031). Multivariate-adjusted models showed an association between HE4 levels and TR, from the second treatment cycle (p = 0.042) to the third cycle (p = 0.033). Changes from baseline HE4 levels during the first cycle was negative associated with TR. No associations were found between CA125 and TR. CONCLUSIONS: Serum HE4 levels were independently associated with TR among patients with AOEC treated with NACT, also a reduction between baseline HE4 and first chemotherapy levels was also independently associated with the TR. These findings might be relevant for predicting a lack of response to treatment.

The diagnostic accuracy of human epididymis protein 4 (HE4) for discriminating between benign and malignant pelvic masses: a systematic review and meta-analysis.

INTRODUCTION: Many women with benign pelvic masses, suspected of ovarian cancer, are unnecessarily referred for treatment at specialized centers. There is an unmet clinical need to improve diagnostic assessment in these patients. Our objective was to obtain summary estimates of the accuracy of human epididymis protein (HE4) for diagnosing ovarian cancer and to compare the performance of HE4 with that of cancer antigen 125 (CA125). MATERIAL AND METHODS: We searched PubMed, Ovid and Scopus using search terms for "pelvic masses" and "HE4", to identify studies that evaluated HE4 for diagnosing malignant ovarian masses, in adult women presenting with a pelvic mass, suspected of ovarian cancer, and with diagnosis confirmed by histopathology. Screening, data extraction and Risk of Bias assessment with the QUADAS-2 tool were done independently by two authors. We performed a meta-analysis of the accuracy of HE4 and CA125 using a random-effects bivariate logit-normal model. A study protocol was registered at PROSPERO (CRD42020158073). RESULTS: In the 17 eligible studies, which included 3404 patients, ovarian cancer prevalence ranged from 15% to 71%. Overall, the studies were heterogeneous. All studies seemed to have recruited patients in specialized settings. A meta-analysis of seven HE4 studies resulted in a mean sensitivity of 79.4% (95% confidence interval [CI] 74.1%-83.8%) and a mean specificity of 84.1% (95% CI 79.6%-87.8%), for cut-off values of 67-72 pmol/L. Based on eight studies, the mean sensitivity of CA125 was 81.4% (95% CI 74.6%-86.2%) and the mean specificity was 56.8% (95% CI 47.9%-65.4%), at a cut-off of 35 U/ml. Given a 40% ovarian cancer prevalence, the positive predictive value (PPV) for HE4 would be 76.9% (71.9%-81.2%) vs 55.6% (50.2%-60.9%) for CA125. The negative predictive value (NPV) would be 85.9 (82.8%-88.6%) and 81.9% (76.2%-86.4%), respectively. At a 15% prevalence, the NPV would be 95.8% (95% CI 94.4%-96.7%) for HE4 and 94.4% (95% CI 92.3%-96.0%) for CA125. The PPV would be 46.9% (40.4%-53.4%) and 24.9% (21.1%-29.2%), respectively. CONCLUSIONS: HE4 had higher specificity and similar sensitivity compared with CA125. At high prevalence, PPV was also higher for HE4, but at low prevalence, it had a similar NPV to CA125. The field would benefit from studies conducted in general settings.

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.

HE4 Predicts Progressive Fibrosis and Cardiovascular Events in Patients With Dilated Cardiomyopathy.

Background Cardiac fibrosis plays a crucial role in the pathogenesis of dilated cardiomyopathy (DCM). HE4 (human epididymis protein 4) is a secretory protein expressed in activated fibroblasts that exacerbates tissue fibrosis. In the present study, we investigated the clinical utility of HE4 measurement in patients with DCM and its pathophysiological role in preclinical experiments in vivo and in vitro. Methods and Results We measured serum HE4 levels of 87 patients with DCM. Endomyocardial biopsy expressed severe fibrosis only in the high HE4 group (P<0.0001). Echocardiography showed that left ventricular end-diastolic diameter tends to decrease over time (58±7.3 to 51±6.6 mm; P<0.0001) in the low HE4 group (<59.65 pmol/L [median value]). HE4 was significantly associated with risk reduction of mortality and cardiovascular hospitalization in multivariate Cox model. In vivo, HE4 was highly expressed in kidney and lung tissue of mouse, and scarcely expressed in heart. In genetically induced DCM mouse model, HE4 expression increased in kidney but not in heart and lung. In vitro, supernatant from HE4-transfected human embryonic kidney 293T cells enhanced transdifferentiation of rat neonatal fibroblasts and increased expression of fibrosis-related genes, and this was accompanied by the activation of extracellular signal-regulated kinase signaling in cardiac fibroblasts. Treatment with an inhibitor of upstream signal of extracellular signal-regulated kinase or a neutralizing HE4 antibody canceled the profibrotic properties of HE4. Conclusions HE4 functions as a secretory factor, activating cardiac fibroblasts, thereby inducing cardiac interstitial fibrosis. HE4 could be a promising biomarker for assessing ongoing fibrosis and a novel therapeutic target in DCM. Registration URL: https://upload.umin.ac.jp/cgi-open-bin/ctr; Unique identifier: UMIN000043062.

WFDC2 Promotes Spasmolytic Polypeptide-Expressing Metaplasia Through the Up-Regulation of IL33 in Response to Injury.

BACKGROUND & AIMS: WAP 4-disulfide core domain protein 2 (WFDC2), also known as human epididymis protein 4, is a small secretory protein that is highly expressed in fibrosis and human cancers, particularly in the ovaries, lungs, and stomach. However, the role of WFDC2 in carcinogenesis is not fully understood. The present study aimed to investigate the role of WFDC2 in gastric carcinogenesis with the use of preneoplastic metaplasia models. METHODS: Three spasmolytic polypeptide-expressing metaplasia (SPEM) models were established in both wild-type and Wfdc2-knockout mice with DMP-777, L635, and high-dose tamoxifen, respectively. To reveal the functional role of WFDC2, we performed transcriptomic analysis with DMP-777-treated gastric corpus specimens. RESULTS: Wfdc2-knockout mice exhibited remarkable resistance against oxyntic atrophy, SPEM emergence, and accumulation of M2-type macrophages in all 3 SPEM models. Transcriptomic analysis revealed that Wfdc2-knockout prevented the up-regulation of interleukin-33 (IL33) expression in the injured mucosal region of SPEM models. Notably, supplementation of recombinant WFDC2 induced IL33 production and M2 macrophage polarization, and ultimately promoted SPEM development. Moreover, long-term treatment with recombinant WFDC2 was able to induce SPEM development. CONCLUSIONS: WFDC2 expressed in response to gastric injury promotes SPEM through the up-regulation of IL33 expression. These findings provide novel insights into the role of WFDC2 in gastric carcinogenesis.

MRPL15 is a novel prognostic biomarker and therapeutic target for epithelial ovarian cancer.

PURPOSE: To analyze the role of six human epididymis protein 4 (HE4)-related mitochondrial ribosomal proteins (MRPs) in ovarian cancer and selected MRPL15, which is most closely related to the tumorigenesis and prognosis of ovarian cancer, for further analyses. METHODS: Using STRING database and MCODE plugin in Cytoscape, six MRPs were identified among genes that are upregulated in response to HE4 overexpression in epithelial ovarian cancer cells. The Cancer Genome Atlas (TCGA) ovarian cancer, GTEX, Oncomine, and TISIDB were used to analyze the expression of the six MRPs. The prognostic impact and genetic variation of these six MRPs in ovarian cancer were evaluated using Kaplan-Meier Plotter and cBioPortal, respectively. MRPL15 was selected for immunohistochemistry and GEO verification. TCGA ovarian cancer data, gene set enrichment analysis, and Enrichr were used to explore the mechanism of MRPL15 in ovarian cancer. Finally, the relationship between MRPL15 expression and immune subtype, tumor-infiltrating lymphocytes, and immune regulatory factors was analyzed using TCGA ovarian cancer data and TISIDB. RESULTS: Six MRPs (MRPL10, MRPL15, MRPL36, MRPL39, MRPS16, and MRPS31) related to HE4 in ovarian cancer were selected. MRPL15 was highly expressed and amplified in ovarian cancer and was related to the poor prognosis of patients. Mechanism analysis indicated that MRPL15 plays a role in ovarian cancer through pathways such as the cell cycle, DNA repair, and mTOR 1 signaling. High expression of MRPL15 in ovarian cancer may be associated with its amplification and hypomethylation. Additionally, MRPL15 showed the lowest expression in C3 ovarian cancer and was correlated with proliferation of CD8+ T cells and dendritic cells as well as TGFßR1 and IDO1 expression. CONCLUSION: MRPL15 may be a prognostic indicator and therapeutic target for ovarian cancer. Because of its close correlation with HE4, this study provides insights into the mechanism of HE4 in ovarian cancer.

Human epididymis protein 4 promotes P­glycoprotein­mediated chemoresistance in ovarian cancer cells through interactions with Annexin II.

The aim of the present study was to investigate the effects of human epididymis protein 4 (HE4) on drug resistance and its underlying mechanisms. The associations among proteins were detected by immunoprecipitation and immunofluorescence assays. Then, stably transfected cell lines CAOV3­HE4­L and CAOV3­A2­L expressing HE4 short hairpin (sh)RNAs and ANXA2 shRNAs, respectively, were constructed. MTT assay, immunocytochemistry, western blotting, reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and flow cytometry were employed to examine drug sensitivity, as well as the expression and activity of P­glycoprotein (P­gp). HE4 and P­gp in epithelial ovarian cancer tissues were assessed via immunohistochemistry. MicroRNAs that targeted the P­gp gene, ABCB1, were predicted using bioinformatics methods, and their expression was evaluated by RT­qPCR. The common signaling pathways shared by HE4, ANXA2 and P­gp were selected by Gene Set Enrichment Analysis (GSEA). The interaction of HE4, ANXA2 and P­gp were confirmed. P­gp expression was positively associated with HE4 and ANXA2 expression, respectively. Moreover, it was observed that there was no significant rescue of P­gp expression in CAOV3­A2­L cells following the administration of active HE4 protein. In addition, the expression of HE4 and P­gp in ovarian cancer tissues of drug­resistant patients were higher compared with that of the drug­sensitive group (P<0.05). Furthermore, the results revealed that hsa­miR­129­5p was significantly increased accompanied by decreased HE4 or ANXA2 expression and P­gp expression in CAOV3­HE4­L and CAOV3­A2­L cells. GSEA analyses disclosed that HE4, ANXA2 and P­gp genes were commonly enriched in the signaling pathway involved in regulating the actin cytoskeleton. These results indicated that HE4 promotes P­gp­mediated drug resistance in ovarian cancer cells through the interactions with ANXA2, and the underlying mechanism may be associated with decreased expression of hsa­miR­129­5p and dysregulation of the actin cytoskeleton signaling pathway.

HE4 Overexpression by Ovarian Cancer Promotes a Suppressive Tumor Immune Microenvironment and Enhanced Tumor and Macrophage PD-L1 Expression.

Ovarian cancer is a highly fatal malignancy characterized by early chemotherapy responsiveness but the eventual development of resistance. Immune targeting therapies are changing treatment paradigms for numerous cancer types but have had minimal success in ovarian cancer. Through retrospective patient sample analysis, we have determined that high human epididymis protein 4 (HE4) production correlates with multiple markers of immune suppression in ovarian cancer, including lower CD8+ T cell infiltration, higher PD-L1 expression, and an increase in the peripheral monocyte to lymphocyte ratio. To further understand the impact that HE4 has on the immune microenvironment in ovarian cancer, we injected rats with syngeneic HE4 high- and low-expressing cancer cells and analyzed the differences in their tumor and ascites immune milieu. We found that high tumoral HE4 expression promotes an ascites cytokine profile that is rich in myeloid-recruiting and differentiation factors, with an influx of M2 macrophages and increased arginase 1 production. Additionally, CTL activation is significantly reduced in the ascites fluid, and there is a trend toward lower CTL infiltration of the tumor, whereas NK cell recruitment to the ascites and tumor is also reduced. PD-L1 expression by tumor cells and macrophages is increased by HE4 through a novel posttranscriptional mechanism. Our data have identified HE4 as a mediator of tumor-immune suppression in ovarian cancer, highlighting this molecule as a potential therapeutic target for the treatment of this devastating disease.