Aberrant glycosylation of α3 integrins as diagnostic markers in epithelial ovarian cancer.

BACKGROUND: Glycans are strongly involved in stability and function of integrins (ITG) and tetraspanin protein CD63 and their respective interaction partners as they are dysregulated in the tumorigenic processes. Glycosylation changes is a universal phenomenon of cancer cells. In this study, glycosylation changes in epithelial ovarian cancer (EOC) are explored using tetraspanin and integrin molecules. METHODS: ITG and CD63 were immobilized from 10 EOC and 5 benign ovarian cyst fluid on microtiter wells and traced with 3 glycan binding proteins (STn, WGA, UEA) conjugated on europium nanoparticles. Total protein measurements (ITG & CD63 immunoassays) were also performed. The most promising glycovariant candidates identified were then clinically evaluated on the whole cohort of 77 ovarian cyst fluids. Additional testing was performed in ascites fluid samples of liver cirrhosis (n = 2) and EOC (n = 4). RESULTS: Sialylated Tn antibody based glycovariants of ITGα3 (ITGα3STn) and CD63 (CD63STn) performed better than corresponding protein epitope-based immunoassays, ITGα3IA and CD63IA respectively. Combined ITGα3 based assays (ITGα3IA + ITGα3STn) detected 49 out of 55 malignant & borderline cases without detecting any of the 22 benign and healthy cysts. CONCLUSION: Our findings indicate the potential diagnostic application of ITGα3STn along with total ITGα3IA, which could help reduce the unnecessary surgeries. The results encourage studying further the potential use of these novel assays to detect EOC at earlier clinical stages.

Time-resolved fluorescence based direct two-site apoA-I immunoassays and their clinical application in patients with suspected obstructive coronary artery disease.

Objective: High-density lipoprotein (HDL) is a heterogeneous group of subpopulations differing in protein/lipid composition and in their anti-atherogenic function. There is a lack of assays that can target the functionality of HDL particles related to atherosclerosis. The objective of this study was to construct two-site apolipoprotein A-I (apoA-I) assays and to evaluate their clinical performance in patients with suspected obstructive coronary artery disease (CAD). Approach and results: Direct two-site apoA-I assays (named 109-121 and 110-525) were developed to identify the presence of apoA-I in the HDL of patients with CAD using apoA-I antibodies as a single-chain variable fragment fused with alkaline phosphatase. ApoA-I109-121 and apoA-I110-525 were measured in 197 patients undergoing coronary computed tomography angiography (CTA) and myocardial positron emission tomography perfusion imaging due to suspected obstructive CAD. Among patients not using lipid-lowering medication (LLM, n = 125), the level of apoA-I110-525 was higher in the presence than in the absence of coronary atherosclerosis [21.88 (15.89-27.44) mg/dl vs. 17.66 (13.38-24.48) mg/dl, P = 0.01)], whereas there was no difference in apoA-I109-121, HDL cholesterol, and apoA-I determined using a polyclonal apoA-I antibody. The levels of apoA-I109-121 and apoA-I110-525 were similar in the presence or absence of obstructive CAD. Among patients not using LLM, apoA-I110-525 adjusted for age and sex identified individuals with coronary atherosclerosis with a similar accuracy to traditional risk factors [area under the curve [AUC] (95% CI): 0.75(0.66-0.84) 0.71 (0.62-0.81)]. However, a combination of apoA-I110-525 with risk factors did not improve the accuracy [AUC (95% CI): 0.73 (0.64-0.82)]. Conclusion: Direct two-site apoA-I assays recognizing heterogeneity in reactivity with apoA-I could provide a potential approach to identify individuals at a risk of coronary atherosclerosis. However, their clinical value remains to be studied in larger cohorts.

Diagnostic potential of nanoparticle aided assays for MUC16 and MUC1 glycovariants in ovarian cancer.

Our study reports the discovery and evaluation of nanoparticle aided sensitive assays for glycovariants of MUC16 and MUC1 in a unique collection of paired ovarian cyst fluids and serum samples obtained at or prior to surgery for ovarian carcinoma suspicion. Selected glycovariants and the immunoassays for CA125, CA15-3 and HE4 were compared and validated in 347 cyst fluid and serum samples. Whereas CA125 and CA15-3 performed poorly in cyst fluid to separate carcinoma and controls, four glycovariants including MUC16MGL , MUC16STn , MUC1STn and MUC1Tn provided highly improved separations. In serum, the two STn glycovariants outperformed conventional CA125, CA15-3 and HE4 assays in all subcategories analyzed with main benefits obtained at high specificities and at postmenopausal and early-stage disease. Serum MUC16STn performed best at high specificity (90%-99%), but sensitivity was also improved by the other glycovariants and CA15-3. The highly improved specificity, excellent analytical sensitivity and robustness of the nanoparticle assisted glycovariant assays carry great promise for improved identification and early detection of ovarian carcinoma in routine differential diagnostics.

Primary breast cancer biomarkers based on glycosylation and extracellular vesicles detected from human serum.

BACKGROUND: Breast cancer is a very common cancer that can be severe if not discovered early. The current tools to detect breast cancer need improvement. Cancer has a universal tendency to affect glycosylation. The glycosylation of circulating extracellular vesicle-associated glycoproteins, and mucins may offer targets for detection methods and have been only explored in a limited capacity. AIM: Our aim was to develop an approach to detect the aberrant glycosylation of mucins and extracellular vesicle-associated glycoproteins from human sera using fluorescent nanoparticles, and preliminarily evaluate this approach for the differential diagnosis of breast cancer. METHODS AND RESULTS: The assay involved immobilizing glycosylated antigens using monoclonal antibodies and then probing their glycosylation by using lectins and glycan-specific antibodies coated on Eu+3 -doped nanoparticles. Detection of mucin 1 and mucin 16 glycosylation with wheat germ agglutinin, and detection of the extracellular vesicle-associated CD63 were found to have better diagnostic ability for localized breast cancer than the conventional assays for mucin 1 and mucin 16 based tumor markers when the receiver operating characteristics were compared. CONCLUSIONS: These results indicate that successful differential diagnosis of primary breast cancer may be aided by detecting cancer-associated glycosylation of mucin 1 and mucin 16, and total concentration of CD63, in human serum.

Detection of Prostate Cancer Using Biparametric Prostate MRI, Radiomics, and Kallikreins: A Retrospective Multicenter Study of Men With a Clinical Suspicion of Prostate Cancer.

BACKGROUND: Accurate detection of clinically significant prostate cancer (csPCa), Gleason Grade Group ≥ 2, remains a challenge. Prostate MRI radiomics and blood kallikreins have been proposed as tools to improve the performance of biparametric MRI (bpMRI). PURPOSE: To develop and validate radiomics and kallikrein models for the detection of csPCa. STUDY TYPE: Retrospective. POPULATION: A total of 543 men with a clinical suspicion of csPCa, 411 (76%, 411/543) had kallikreins available and 360 (88%, 360/411) did not take 5-alpha-reductase inhibitors. Two data splits into training, validation (split 1: single center, n = 72; split 2: random 50% of pooled datasets from all four centers), and testing (split 1: 4 centers, n = 288; split 2: remaining 50%) were evaluated. FIELD STRENGTH/SEQUENCE: A 3 T/1.5 T, TSE T2-weighted imaging, 3x SE DWI. ASSESSMENT: In total, 20,363 radiomic features calculated from manually delineated whole gland (WG) and bpMRI suspicion lesion masks were evaluated in addition to clinical parameters, prostate-specific antigen, four kallikreins, MRI-based qualitative (PI-RADSv2.1/IMPROD bpMRI Likert) scores. STATISTICAL TESTS: For the detection of csPCa, area under receiver operating curve (AUC) was calculated using the DeLong's method. A multivariate analysis was conducted to determine the predictive power of combining variables. The values of P-value < 0.05 were considered significant. RESULTS: The highest prediction performance was achieved by IMPROD bpMRI Likert and PI-RADSv2.1 score with AUC = 0.85 and 0.85 in split 1, 0.85 and 0.83 in split 2, respectively. bpMRI WG and/or kallikreins demonstrated AUCs ranging from 0.62 to 0.73 in split 1 and from 0.68 to 0.76 in split 2. AUC of bpMRI lesion-derived radiomics model was not statistically different to IMPROD bpMRI Likert score (split 1: AUC = 0.83, P-value = 0.306; split 2: AUC = 0.83, P-value = 0.488). DATA CONCLUSION: The use of radiomics and kallikreins failed to outperform PI-RADSv2.1/IMPROD bpMRI Likert and their combination did not lead to further performance gains. LEVEL OF EVIDENCE: 1 TECHNICAL EFFICACY: Stage 2.

Nanoparticle-Aided Detection of Colorectal Cancer-Associated Glycoconjugates of Extracellular Vesicles in Human Serum.

Extracellular vesicles (EVs) are found in all biological fluids, providing potential for the identification of disease biomarkers such as colorectal cancer (CRC). EVs are heavily glycosylated with specific glycoconjugates such as tetraspanins, integrins, and mucins, reflecting the characteristics of the original cell offering valuable targets for detection of CRC. We report here on europium-nanoparticle (EuNP)-based assay to detect and characterize different surface glycoconjugates of EVs without extensive purification steps from five different CRC and the HEK 293 cell lines. The promising EVs candidates from cell culture were clinically evaluated on small panel of serum samples including early-stage (n = 11) and late-stage (n = 11) CRC patients, benign condition (n = 11), and healthy control (n = 10). The majority of CRC cell lines expressed tetraspanin sub-population and glycovariants of integrins and conventional tumor markers. The subpopulation of CD151 having CD63 expression (CD151CD63) was significantly (p = 0.001) elevated in early-stage CRC (8 out of 11) without detecting any benign and late-stage samples, while conventional CEA detected mostly late-stage CRC (p = 0.045) and with only four early-stage cases. The other glycovariant assays such as CEACon-A, CA125WGA, CA 19.9Ma696, and CA 19.9Con-A further provided some complementation to the CD151CD63 assay. These results indicate the potential application of CD151CD63 assay for early detection of CRC patients in human serum.

Sensitive and quantitative detection of cardiac troponin I with upconverting nanoparticle lateral flow test with minimized interference.

Measurement of cardiac troponin I (cTnI) should be feasible for point-of-care testing (POCT) to diagnose acute myocardial infarction (AMI). Lateral flow immunoassays (LFIAs) have been long implemented in POCT and clinical settings. However, sensitivity, matrix effect and quantitation in lateral flow immunoassays (LFIAs) have been major limiting factors. The performance of LFIAs can be improved with upconverting nanoparticle (UCNP) reporters. Here we report a new methodological approach to quantify cTnI using UCNP-LFIA technology with minimized plasma interference. The performance of the developed UCNP-LFIA was evaluated using clinical plasma samples (n = 262). The developed UCNP-LFIA was compared to two reference assays, the Siemens Advia Centaur assay and an in-house well-based cTnI assay. By introducing an anti-IgM scrub line and dried EDTA in the LFIA strip, the detection of cTnI in plasma samples was fully recovered. The UCNP-LFIA was able to quantify cTnI concentrations in patient samples within the range of 30-10,000 ng/L. The LoB and LoD of the UCNP-LFIA were 8.4 ng/L and 30 ng/L. The method comparisons showed good correlation (Spearman's correlation 0.956 and 0.949, p < 0.0001). The developed UCNP-LFIA had LoD suitable for ruling in AMI in patients with elevated cTnI levels and was able to quantify cTnI concentrations in patient samples. The technology has potential to provide simple and rapid assay for POCT in ED setting.

Detection of bladder cancer with aberrantly fucosylated ITGA3.

We describe a simple, non-invasive assay to identify fucosylated-glycoisoform of integrin alpha-3 (ITGA3) directly from unprocessed urine. ITGA3 was detected directly from the urine of bladder cancer (BlCa) (n = 13) and benign prostatic hyperplasia (BPH) (n = 9) patients with the use of lectins coated on europium-doped-nanoparticles (Eu3+-NPs). Lectin Ulex europaeus agglutinin-I (UEA) showed enhanced binding with BlCa-derived ITGA3. The evaluation with individual samples showed that a glycovariant ITGA3-UEA assay could significantly discriminate BlCa from BPH patients (p = 0.007). The detection of aberrantly fucosylated-isoform of ITGA3 from urine can be used to distinguish BlCa from age-matched benign controls in a simple sandwich assay.

CA125: A superior prognostic biomarker for colorectal cancer compared to CEA, CA19-9 or CA242.

OBJECTIVES: The tumor stage represents the single most important prognostic factor for colorectal cancer (CRC), although more accurate prognostics remain much needed. Previously, we identified CA125 as an independent significant prognostic factor, which we have further validated along with CEA, CA19-9, and CA242 in a large cohort of CRC patients. METHODS: Using enzyme-linked immunosorbent assays, we analyzed preoperative serum samples in 322 CRC patients operated on between 1998 and 2003. RESULTS: Using the Spearman's rho model, we calculated the correlation between our previous findings on MUC16 and CA125, for which the correlation coefficient was 0.808 (p < 0.001). The Cox regression analysis of the linear and logarithmic values of CEA, CA125, CA242, and CA19-9 identified only CA125 (hazard ratio [HR] 1.03; 95% confidence interval [95% CI] 1.02-1.04; p < 0.001) as significant when using the linear values. Survival among CRC patients with a high CA125 level was poor compared with CRC patients with a low CA125 level (HR 2.48; 95% CI 1.68-3.65; p < 0.001). In subgroup analyses, patients with high CA125 levels and aged ≤67 or >67, with stage I-II or III-IV, and both colon and rectal cancer exhibited poor prognoses. In the multivariate analysis, we used clinical pathological variables in the model, where age, gender, and stage served as the background characteristics. We dichotomized CA125 using the Youden maximal cutoff point, and the median values for CEA, CA19-9, and CA242. CA125 emerged as the only marker remaining significant and independent together with stage, location, and age (HR 1.91; 95% CI 1.24-2.95; p 0.003). CONCLUSIONS: CA125 represents a significant and independent prognostic factor in CRC patients, superior to CEA. Furthermore, CA242 served as a better prognostic marker than both CEA and CA19-9. We recommend including both CA125 and CA242 in prognostic clinical trials among CRC patients.

A prognostic model for colorectal cancer based on CEA and a 48-multiplex serum biomarker panel.

Mortality in colorectal cancer (CRC) remains high, resulting in 860,000 deaths annually. Carcinoembryonic antigen is widely used in clinics for CRC patient follow-up, despite carrying a limited prognostic value. Thus, an obvious need exists for multivariate prognostic models. We analyzed 48 biomarkers using a multiplex immunoassay panel in preoperative serum samples from 328 CRC patients who underwent surgery at Helsinki University Hospital between 1998 and 2003. We performed a multivariate prognostic forward-stepping background model based on basic clinicopathological data, and a multivariate machine-learned prognostic model based on clinicopathological data and biomarker variables, calculating the disease-free survival using the value of importance score. From the 48 analyzed biomarkers, only IL-8 emerged as a significant prognostic factor for CRC patients in univariate analysis (HR 4.88; 95% CI 2.00-11.92; p = 0.024) after correcting for multiple comparisons. We also developed a multivariate model based on all 48 biomarkers using a random survival forest analysis. Variable selection based on a minimal depth and the value of importance yielded two tentative candidate CRC prognostic markers: IL-2Ra and IL-8. A multivariate prognostic model using machine-learning technologies improves the prognostic assessment of survival among surgically treated CRC patients.

Double-Antigen Lateral Flow Immunoassay for the Detection of Anti-HIV-1 and -2 Antibodies Using Upconverting Nanoparticle Reporters.

Rapid diagnostic tests (RDTs) are often used for the detection of anti-human immunodeficiency virus (HIV) antibodies in remote locations in low- and middle-income countries (LMIC) with low or limited access to central laboratories. The typical format of an RDT is a lateral flow assay (LFA) with visual interpretation prone to subjectivity. This risk of misinterpretation can be overcome with luminescent upconverting nanoparticle reporters (UCNPs) measured with a miniaturized easy-to-use reader instrument. An LFA with UCNPs for anti-HIV-1/2 antibodies was developed and the assay performance was evaluated extensively with challenging patient sample panels. Sensitivity (n = 145) of the UCNP-LFA was 96.6% (95% CI: 92.1-98.8%) and specificity (n = 309) was 98.7% (95% CI: 96.7-99.7%). Another set of samples (n = 200) was used for a comparison between the UCNP-LFA and a conventional visual RDT. In this comparison, the sensitivities for HIV-1 were 96.4% (95% CI: 89.8-99.3%) and 97.6% (95% CI: 91.6-99.7%), for the UCNP-LFA and conventional RDT, respectively. The specificity was 100% (95% CI: 96.4-100%) for both assays. The developed UCNP-LFA demonstrates the applicability of UCNPs for the detection of anti-HIV antibodies. The signal measurement is done by a reader instrument, which may facilitate automated result interpretation, archiving and transfer of data from de-centralized locations.

Three two-site apoA-I immunoassays using phage expressed detector antibodies - Preliminary clinical evaluation with cardiac patients.

High density lipoproteins (HDL) are a heterogenous group of subpopulations differing in protein/lipid composition and in their anti-atherogenic function. There is a lack of specific and robust assays which can target the functionality of HDL with respect to atherosclerosis. With recently generated CAD HDL targeted, single chain recombinant antibodies (scFvs) we set out to design and optimize apo A-I tests to compare it with conventional HDL-C and apo A-I analyses for diagnosis and risk assessment of coronary artery disease (CAD) and its outcome. Three highly sensitive two-site apo A-I assays: 022-454, 109-121 and 110-525 were optimized. A preliminary clinical evaluation of these assays, after proper sample dilution procedure, was performed using samples derived from 195 chest pain patients (myocardial infarction (MI), n = 86 and non-MI, n = 109), collected at the time of admission and at discharge from hospital (hospital stay ≤ 24 h). The clinical performance of the assays was compared with apo A-I measured with polyclonal anti-apo A-I antibody using conventional ELISA. Apo A-I data was in addition compared with HDL-C concentration of the samples. The concentration of apo A-I was significantly lower in MI patients than in non-MI individuals with assay 022-454 (admission and discharge samples, P < 0.0001 and = 0.004); assay 109-121 (admission and discharge samples, P = 0.04 and 0.0009), and, ELISA based apo A-I test (admission and discharge samples, P = 0.008 and < 0.0001). HDL-C (admission and discharge samples, P = 0.002 and P = 0.01) was also significantly lower in MI patients. In Kaplan- Meier analysis, two-site assay 109-121 assay predicted mortality from admission samples at 1.5 yrs (whole cohort, P = 0.01 and in MI patients, P = 0.05) and at 6 months (whole cohort, P = 0.04). Assay 110-525 predicted mortality at 1.5 yrs from admission samples of non-MI patients (P = 0.01) and at 6 months from whole discharge sample cohort (P = 0.04). Polyclonal anti-apo A-I based conventional assay predicted mortality at 1.5 yrs from admission samples of whole cohort (P = 0.03). Two-site apo A-I assay 022-454 and HDL-C provided no capability of predicting mortality in the whole cohort or any sub-group. In conclusion, two of the tested recombinant apo A-I antibody combinations (sc 109-121 and sc 110-525) display promising outcome to improve diagnosis and prediction of future cardiac events in cardiac patients over polyclonal apo A-I ELISA and HDL-C assays. The noted differences, while interesting, are preliminary and need however to be verified in extensive cohorts of pathological cardiac conditions and healthy controls.

Upconverting nanoparticle reporter-based highly sensitive rapid lateral flow immunoassay for hepatitis B virus surface antigen.

Detection of hepatitis B Virus surface antigen (HBsAg) is an established method for diagnosing both acute and chronic hepatitis B virus (HBV) infection. In addition to enzyme immunoassays (EIAs), rapid diagnostic tests (RDTs) are available for the detection of HBsAg in resource-poor settings. However, the available RDTs have inadequate sensitivity and therefore are not suitable for diagnosis of patients with low levels of HBsAg and for blood screening. To provide a high-sensitivity RDT, we developed a lateral flow immunoassay (LFIA) for HBsAg utilizing upconverting nanoparticle (UCNP) reporter. The UCNP-LFIA can use whole blood, serum, or plasma and the results can be read in 30 min using a reader device. When compared with a commercial conventional visually read LFIA, the developed UCNP-LFIA had a Limit of Detection (LoD) of 0.1 IU HBsAg/ml in spiked serum, whereas the LoD of the conventional LFIA was 3.2 IU HBsAg/ml. The developed UCNP-LFIA fulfills the WHO criterion for blood screening (LoD ≤ 0.13 IU HBsAg/ml) in terms of LoD. The UCNP-LFIA and conventional LFIA were evaluated with well-characterized sample panels. The UCNP-LFIA detected 20/24 HBsAg-positive samples within the HBsAg Performance Panel and 8/10 samples within the Mixed Titer Performance Panel, whereas the conventional LFIA detected 8/24 and 4/10 samples in these panels, respectively. The performance of the assays was further evaluated with HBsAg-positive (n = 108) and HBsAg-negative (n = 315) patient samples. In comparison with a central laboratory test, UCNP-LFIA showed 95.4% (95% CI: 89.5-98.5%) sensitivity whereas sensitivity of the conventional LFIA was 87.7% (95%CI: 79.9-93.3%).

Prospective validation of microseminoprotein-ß added to the 4Kscore in predicting high-grade prostate cancer in an international multicentre cohort.

OBJECTIVES: To prospectively evaluate the performance of a pre-specified statistical model based on four kallikrein markers in blood (total prostate-specific antigen [PSA], free PSA, intact PSA, and human kallikrein-related peptidase 2), commercially available as the 4Kscore, in predicting Gleason Grade Group (GG) ≥2 prostate cancer at biopsy in an international multicentre study at three academic medical centres, and whether microseminoprotein-ß (MSP) adds predictive value. PATIENTS AND METHODS: A total of 984 men were prospectively enrolled at three academic centres. The primary outcome was GG ≥2 on prostate biopsy. Three pre-specified statistical models were used: a base model including PSA, age, digital rectal examination and prior negative biopsy; a model that added free PSA to the base model; and the 4Kscore. RESULTS: A total of 947 men were included in the final analysis and 273 (29%) had GG ≥2 on prostate biopsy. The base model area under the receiver operating characteristic curve of 0.775 increased to 0.802 with the addition of free PSA, and to 0.824 for the 4Kscore. Adding MSP to the 4Kscore model yielded an increase (0.014-0.019) in discrimination. In decision-curve analysis of clinical utility, the 4Kscore showed a benefit starting at a 7.5% threshold. CONCLUSION: A prospective multicentre evaluation of a pre-specified model based on four kallikrein markers (4Kscore) with the addition of MSP improves the predictive discrimination for GG ≥2 prostate cancer on biopsy and could be used to inform biopsy decision-making.

Ultrasensitive and Robust Point-of-Care Immunoassay for the Detection of Plasmodium falciparum Malaria.

Plasmodium falciparum malaria is widespread in the tropical and subtropical regions of the world. There is ongoing effort to eliminate malaria from endemic regions, and sensitive point-of-care (POC) diagnostic tests are required to support this effort. However, current POC tests are not sufficiently sensitive to detect P. falciparum in asymptomatic individuals. After extensive optimization, we have developed a highly sensitive and robust POC test for the detection of P. falciparum infection. The test is based on upconverting nanophosphor-based lateral flow (UCNP-LF) immunoassay. The developed UCNP-LF test was validated using whole blood reference panels containing samples at different parasite densities covering eight strains of P. falciparum from different geographical areas. The limit of detection was compared to a WHO-prequalified rapid diagnostic test (RDT). The UCNP-LF achieved a detection limit of 0.2-2 parasites/µL, depending on the strain, which is 50- to 250-fold improvement in analytical sensitivity over the conventional RDTs. The developed UCNP-LF is highly stable even at 40 °C for at least 5 months. The extensively optimized UCNP-LF assay is as simple as the conventional malaria RDTs and requires 5 µL of whole blood as sample. Results can be read after 20 min from sample addition, with a simple photoluminescence reader. In the absence of a reader device at the testing site, the strips after running the test can be transported and read at a central location with access to a reader. We have found that the test and control line signals are stable for at least 10 months after running the test. The UCNP-LF has potential for diagnostic testing of both symptomatic and asymptomatic individuals.

HE4 in the evaluation of tumor load and prognostic stratification of high grade serous ovarian carcinoma.

OBJECTIVE: Human epididymis protein 4 (HE4) is a validated, complementary biomarker to cancer antigen 125 (CA125) for high grade serous ovarian carcinoma (HGSC). Currently, there are insufficient data on the utility of longitudinal HE4 measurement during HGSC treatment and follow up. We set to provide a comprehensive analysis on the kinetics and prognostic performance of HE4 with serial measurements during HGSC treatment and follow up. METHODS: This prospective study included 143 patients with advanced HGSC (ClinicalTrials.gov identifier: NCT01276574). Serum CA125 and HE4 were measured at baseline, before each cycle of chemotherapy and during follow up until first progression. Baseline biomarker values were compared to the tumor load assessed during surgery and to residual disease. Biomarker nadir values and concentrations at progression were correlated to survival. RESULTS: The baseline HE4 concentration distinguished patients with a high tumor load from patients with a low tumor load assessed during surgery (p<.0001). The baseline CA125 level was not associated with tumor load to a similar extent (p=.067). At progression, the HE4 level was an independent predictor of worse survival in the multivariate analysis (p=.002). All patients that were alive 3 years post-progression had a serum HE4 concentration below 199.20 pmol/l at the 1st recurrence. CONCLUSION: HE4 is a feasible biomarker in the treatment monitoring and prognostic stratification of patients with HGSC. Specifically, the serum level of HE4 at first relapse was associated with the survival of patients and it may be a useful complementary tool in the selection of second line treatments. This is to the best of our knowledge the first time this finding has been reported.

Glycovariant-based lateral flow immunoassay to detect ovarian cancer-associated serum CA125.

Cancer antigen 125 (CA125) is a widely used biomarker in monitoring of epithelial ovarian cancer (EOC). Due to insufficient cancer specificity of CA125, its diagnostic use is severely compromised. Abnormal glycosylation of CA125 is a unique feature of ovarian cancer cells and could improve differential diagnosis of the disease. Here we describe the development of a quantitative lateral flow immunoassay (LFIA) of aberrantly glycosylated CA125 which is widely superior to the conventional CA125 immunoassay (CA125IA). With a 30 min read-out time, the LFIA showed 72% sensitivity, at 98% specificity using diagnostically challenging samples with marginally elevated CA125 (35-200 U/mL), in comparison to 16% sensitivity with the CA125IA. We envision the clinical use of the developed LFIA to be based on the substantially enhanced disease specificity against the many benign conditions confounding the diagnostic evaluation and against other cancers.

Exploratory Analysis of CA125-MGL and -STn Glycoforms in the Differential Diagnostics of Pelvic Masses.

BACKGROUND: The cancer antigen 125 (CA125) immunoassay (IA) does not distinguish epithelial ovarian cancer (EOC) from benign disease with the sensitivity needed in clinical practice. In recent studies, glycoforms of CA125 have shown potential as biomarkers in EOC. Here, we assessed the diagnostic abilities of two recently developed CA125 glycoform assays for patients with a pelvic mass. Detailed analysis was further conducted for postmenopausal patients with marginally elevated conventionally measured CA125 levels, as this subgroup presents a diagnostic challenge in the clinical setting. METHODS: Our study population contained 549 patients diagnosed with EOC, benign ovarian tumors, and endometriosis. Of these, 288 patients were postmenopausal, and 98 of them presented with marginally elevated serum levels of conventionally measured CA125 at diagnosis. Preoperative serum levels of conventionally measured CA125 and its glycoforms (CA125-MGL and CA125-STn) were determined. RESULTS: The CA125-STn assay identified EOC significantly better than the conventional CA125-IA in postmenopausal patients (85% vs. 74% sensitivity at a fixed specificity of 90%, P = 0.0009). Further, both glycoform assays had superior AUCs compared to the conventional CA125-IA in postmenopausal patients with marginally elevated CA125. Importantly, the glycoform assays reduced the false positive rate of the conventional CA125-IA. CONCLUSIONS: The results indicate that the CA125 glycoform assays markedly improve the performance of the conventional CA125-IA in the differential diagnosis of pelvic masses. This result is especially valuable when CA125 is marginally elevated.

Evaluation of a New Skeletal Troponin I Assay in Patients with Idiopathic Inflammatory Myopathies.

BACKGROUND: The current biomarkers for diagnosis and monitoring of injured and diseased skeletal muscles, such as creatine kinase (CK), have limited tissue specificity and incapability to differentiate between pathological and physiological changes. Thus, new biomarkers with improved diagnostic accuracy are needed. Our aim was to develop and validate a novel assay for skeletal troponin I (skTnI), and to assess its clinical performance in patients with idiopathic inflammatory myopathies (IIM). METHODS: A two-step fluoroimmunoassay was used to analyze samples from healthy reference individuals (n = 140), patients with trauma (n = 151), and patients with IIM (n = 61). RESULTS: The limit of detection was 1.2 ng/mL, and the upper reference limit (90th percentile) was 5.2 ng/mL. The median skTnI concentrations were

Prostate cancer risk SNP rs10993994 is a trans-eQTL for SNHG11 mediated through MSMB.

How genome-wide association studies-identified single-nucleotide polymorphisms (SNPs) affect remote genes remains unknown. Expression quantitative trait locus (eQTL) association meta-analysis on 496 prostate tumor and 602 normal prostate samples with 117 SNPs revealed novel cis-eQTLs and trans-eQTLs. Mediation testing and colocalization analysis demonstrate that MSMB is a cis-acting mediator for SNHG11 (P < 0.01). Removing rs10993994 in LNCaP cell lines by CRISPR/Cas9 editing shows that the C-allele corresponds with an over 100-fold increase in MSMB expression and 5-fold increase in SNHG11 compared with the T-allele. Colocalization analysis confirmed that the same set of SNPs associated with MSMB expression is associated with SNHG11 expression (posterior probability of shared variants is 66.6% in tumor and 91.4% in benign). These analyses further demonstrate variants driving MSMB expression differ in tumor and normal, suggesting regulatory network rewiring during tumorigenesis.