A new biomarker panel for differential diagnosis of cholangiocarcinoma: Results from an exploratory analysis.

INTRODUCTION: Diagnosis of cholangiocarcinoma (CCA) can be challenging due to unclear imaging criteria and difficulty obtaining adequate tissue biopsy. Although serum cancer antigen 19-9 and carcinoembryonic antigen have been proposed as potential diagnostic aids, their use remains limited by insufficient sensitivity and specificity. This exploratory analysis aimed to identify individual- and combinations of serum biomarkers to distinguish CCA from hepatocellular carcinoma (HCC) and chronic liver disease (CLD) controls using samples from a published study. METHODS: This prospective, multicenter, case-control study included patients aged ≥18 years at high-risk of HCC. Serum and ethylene diamine tetraacetic acid-plasma samples were collected prior to any treatment and confirmed diagnosis of HCC or CCA. Fourteen biomarkers (measured by electrochemiluminescence immunoassays or enzyme-linked immunosorbent assays) were subjected to univariate analysis and 13 included in a multivariate analysis (per selected combinations and exhaustive search). RESULTS: Overall, 55 CCA, 306 HCC, and 733 CLD control samples were analyzed. For distinguishing CCA from HCC, alpha-fetoprotein and matrix metalloproteinase-2 (MMP-2) showed the best individual performance (area under the curve (AUC) 86.6% and 84.4%, respectively); tissue inhibitor of metalloproteinase-1 (TIMP-1) was most able to distinguish CCA from CLD (AUC 94.5%) and from HCC + CLD (AUC 88.6%). The combination of MMP-2 and TIMP-1 was the best-performing two-marker panel, with AUC >90% for all comparisons. CONCLUSION: MMP-2 and TIMP-1 are promising biomarkers that could support differential diagnosis of CCA. Incorporating these assays into the diagnostic algorithm could provide additional diagnostic information in a non-invasive, rapid manner, and could supplement existing diagnostic methods.

VWD domain stabilization by autocatalytic Asp-Pro cleavage.

Domains known as von Willebrand factor type D (VWD) are found in extracellular and cell-surface proteins including von Willebrand factor, mucins, and various signaling molecules and receptors. Many VWD domains have a glycine-aspartate-proline-histidine (GDPH) amino-acid sequence motif, which is hydrolytically cleaved post-translationally between the aspartate (Asp) and proline (Pro). The Fc IgG binding protein (FCGBP), found in intestinal mucus secretions and other extracellular environments, contains 13 VWD domains, 11 of which have a GDPH cleavage site. In this study, we investigated the structural and biophysical consequences of Asp-Pro peptide cleavage in a representative FCGBP VWD domain. We found that endogenous Asp-Pro cleavage increases the resistance of the domain to exogenous proteolytic degradation. Tertiary structural interactions made by the newly generated chain termini, as revealed by a crystal structure of an FCGBP segment containing the VWD domain, may explain this observation. Notably, the Gly-Asp peptide bond, upstream of the cleavage site, assumed the cis configuration in the structure. In addition to these local features of the cleavage site, a global organizational difference was seen when comparing the FCGBP segment structure with the numerous other structures containing the same set of domains. Together, these data illuminate the outcome of GDPH cleavage and demonstrate the plasticity of proteins with VWD domains, which may contribute to their evolution for function in a dynamic extracellular environment.

Thyroglobulin Cutoff Values for Detecting Excellent Response to Therapy in Patients With Differentiated Thyroid Cancer.

Context: Serum thyroglobulin (Tg) is a biochemical marker for detecting persistent or recurrent differentiated thyroid carcinoma (DTC) post-thyroidectomy. Tg can indicate DTC before structural disease (SD) is visible with imaging procedures. Objective: This work aimed to evaluate the clinical performance of the Elecsys® Tg II assay at a Tg cutoff of 0.2 ng/mL for ruling out SD in adults with DTC after total/near-total thyroidectomy, with or without radioiodine ablation (RAI). Methods: Patients were enrolled into 2 cohorts: longitudinal (Tg assessed every 6 months over 2 years under thyroid-stimulating hormone [TSH] suppression therapy following thyroidectomy with or without RAI) and cross-sectional with confirmed SD (Tg assessed once >12 weeks after thyroidectomy). Analyses were performed for both cohorts combined and in the longitudinal cohort. Results: The study included 530 clinically evaluable samples, the majority (n = 424 samples) from patients who had not received RAI treatment. Following correction for SD prevalence (4.97% in the longitudinal cohort), an Elecsys Tg II cutoff of 0.2 ng/mL ruled out SD with a negative predictive value of 99.9% (95% CI, 99.5%-100%). The assay had excellent sensitivity (98.5%-100%) and acceptable specificity (53.4%-53.5%) for detecting SD (Tg ≥ 0.2 ng/mL) for both cohorts combined and in the longitudinal cohort, with similar findings in RAI-treated and non-RAI-treated subgroups. Conclusion: In this cohort of DTC patients post-thyroidectomy, a Tg cutoff of 0.2 ng/mL was highly effective for ruling out the presence of SD under TSH-suppressed conditions, including in patients who had not received RAI treatment.

Deciphering the involvement of the Hippo pathway co-regulators, YAP/TAZ in invadopodia formation and matrix degradation.

Invadopodia are adhesive, actin-rich protrusions formed by metastatic cancer cells that degrade the extracellular matrix and facilitate invasion. They support the metastatic cascade by a spatially and temporally coordinated process whereby invading cells bind to the matrix, degrade it by specific metalloproteinases, and mechanically penetrate diverse tissue barriers by forming actin-rich extensions. However, despite the apparent involvement of invadopodia in the metastatic process, the molecular mechanisms that regulate invadopodia formation and function are still largely unclear. In this study, we have explored the involvement of the key Hippo pathway co-regulators, namely YAP, and TAZ, in invadopodia formation and matrix degradation. Toward that goal, we tested the effect of depletion of YAP, TAZ, or both on invadopodia formation and activity in multiple human cancer cell lines. We report that the knockdown of YAP and TAZ or their inhibition by verteporfin induces a significant elevation in matrix degradation and invadopodia formation in several cancer cell lines. Conversely, overexpression of these proteins strongly suppresses invadopodia formation and matrix degradation. Proteomic and transcriptomic profiling of MDA-MB-231 cells, following co-knockdown of YAP and TAZ, revealed a significant change in the levels of key invadopodia-associated proteins, including the crucial proteins Tks5 and MT1-MMP (MMP14). Collectively, our findings show that YAP and TAZ act as negative regulators of invadopodia formation in diverse cancer lines, most likely by reducing the levels of essential invadopodia components. Dissecting the molecular mechanisms of invadopodia formation in cancer invasion may eventually reveal novel targets for therapeutic applications against invasive cancer.

Post-translational modifications reshape the antigenic landscape of the MHC I immunopeptidome in tumors.

Post-translational modification (PTM) of antigens provides an additional source of specificities targeted by immune responses to tumors or pathogens, but identifying antigen PTMs and assessing their role in shaping the immunopeptidome is challenging. Here we describe the Protein Modification Integrated Search Engine (PROMISE), an antigen discovery pipeline that enables the analysis of 29 different PTM combinations from multiple clinical cohorts and cell lines. We expanded the antigen landscape, uncovering human leukocyte antigen class I binding motifs defined by specific PTMs with haplotype-specific binding preferences and revealing disease-specific modified targets, including thousands of new cancer-specific antigens that can be shared between patients and across cancer types. Furthermore, we uncovered a subset of modified peptides that are specific to cancer tissue and driven by post-translational changes that occurred in the tumor proteome. Our findings highlight principles of PTM-driven antigenicity, which may have broad implications for T cell-mediated therapies in cancer and beyond.

Multimarker Panels for Detection of Early Stage Hepatocellular Carcinoma: A Prospective, Multicenter, Case-Control Study.

Hepatocellular carcinoma (HCC), the sixth most common cancer worldwide, has an incidence rate equal to mortality. Over 80% of HCC cases occur within a high-risk population, mainly patients with both cirrhosis and chronic hepatitis B or C. With a 5-year survival rate ranging from <16% for advanced HCC to >90% for early stage HCC, there is a high medical need for the early detection of HCC. In this study, we systematically evaluated biomarkers mentioned in international guidelines and peer-reviewed literature for HCC surveillance and diagnosis with the aim of identifying combinations that display high sensitivity and specificity for early stage HCC. Fifty biomarkers were measured in the first sample panel, panel A (n = 110), and subjected to univariate analysis. Of these, 35 biomarkers (38 assays) from panel A and an additional 13 biomarkers from the literature were prioritized for subsequent multivariate evaluation with lasso regression and exhaustive search of two- to four-biomarker combinations (panel B). Panel B included 1,081 samples from patients with HCC (n = 308) or with chronic liver diseases (n = 740). Among all patients, 61.0% had hepatitis B, 32.9% had hepatitis C, and 60.5% had cirrhosis; 40.6% of patients with HCC had early stage cancer. Protein induced by vitamin K absence-II (PIVKA-II; also known as des-gamma-carboxy prothrombin [DCP]) and alpha-fetoprotein (AFP) demonstrated the best clinical performance, both individually and in combination, and the addition of a third biomarker (Lens culinaris agglutinin-reactive fraction of AFP [AFP-L3], cartilage oligomeric matrix protein [COMP], insulin-like growth factor-binding protein 3 [IGFBP3], or matrix metalloproteinase 3 [MMP3]) further increased sensitivity for the detection of both early stage and all-stage HCC. The addition of age and sex to the three-biomarker panel resulted in an improved diagnostic performance. Conclusion: The combination of AFP and PIVKA-II, with either IGFBP3, COMP or MMP3, plus age and sex, demonstrated the best performance for the detection of early- and all-stage HCC. These novel panels performed similar to that of the GALAD score (sex [gender], age, plus serum levels of AFP, AFP-L3 and DCP [PIVKA-II]), a promising screening tool developed for HCC detection.

The coding capacity of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic1. To understand the pathogenicity and antigenic potential of SARS-CoV-2 and to develop therapeutic tools, it is essential to profile the full repertoire of its expressed proteins. The current map of SARS-CoV-2 coding capacity is based on computational predictions and relies on homology with other coronaviruses. As the protein complement varies among coronaviruses, especially in regard to the variety of accessory proteins, it is crucial to characterize the specific range of SARS-CoV-2 proteins in an unbiased and open-ended manner. Here, using a suite of ribosome-profiling techniques2-4, we present a high-resolution map of coding regions in the SARS-CoV-2 genome, which enables us to accurately quantify the expression of canonical viral open reading frames (ORFs) and to identify 23 unannotated viral ORFs. These ORFs include upstream ORFs that are likely to have a regulatory role, several in-frame internal ORFs within existing ORFs, resulting in N-terminally truncated products, as well as internal out-of-frame ORFs, which generate novel polypeptides. We further show that viral mRNAs are not translated more efficiently than host mRNAs; instead, virus translation dominates host translation because of the high levels of viral transcripts. Our work provides a resource that will form the basis of future functional studies.

Assembly Mechanism of Mucin and von Willebrand Factor Polymers.

The respiratory and intestinal tracts are exposed to physical and biological hazards accompanying the intake of air and food. Likewise, the vasculature is threatened by inflammation and trauma. Mucin glycoproteins and the related von Willebrand factor guard the vulnerable cell layers in these diverse systems. Colon mucins additionally house and feed the gut microbiome. Here, we present an integrated structural analysis of the intestinal mucin MUC2. Our findings reveal the shared mechanism by which complex macromolecules responsible for blood clotting, mucociliary clearance, and the intestinal mucosal barrier form protective polymers and hydrogels. Specifically, cryo-electron microscopy and crystal structures show how disulfide-rich bridges and pH-tunable interfaces control successive assembly steps in the endoplasmic reticulum and Golgi apparatus. Remarkably, a densely O-glycosylated mucin domain performs an organizational role in MUC2. The mucin assembly mechanism and its adaptation for hemostasis provide the foundation for rational manipulation of barrier function and coagulation.

Characterization of the First Conotoxin from Conus ateralbus, a Vermivorous Cone Snail from the Cabo Verde Archipelago.

Conus ateralbus is a cone snail endemic to the west side of the island of Sal, in the Cabo Verde Archipelago off West Africa. We describe the isolation and characterization of the first bioactive peptide from the venom of this species. This 30AA venom peptide is named conotoxin AtVIA (δ-conotoxin-like). An excitatory activity was manifested by the peptide on a majority of mouse lumbar dorsal root ganglion neurons. An analog of AtVIA with conservative changes on three amino acid residues at the C-terminal region was synthesized and this analog produced an identical effect on the mouse neurons. AtVIA has homology with δ-conotoxins from other worm-hunters, which include conserved sequence elements that are shared with δ-conotoxins from fish-hunting Conus. In contrast, there is no comparable sequence similarity with δ-conotoxins from the venoms of molluscivorous Conus species. A rationale for the potential presence of δ-conotoxins, that are potent in vertebrate systems in two different lineages of worm-hunting cone snails, is discussed.

Hip Arthroscopy: extra-articular Procedures.

Hip preservation is one of the fastest growing fields in orthopaedics and indications of intra-articular procedures are well established. In the last decade, extra-articular procedures have gained momentum and arthroscopic solutions to peri-articular hip pathologies have been offered. It should be noted that many of these pathologies are well-treated conservatively and only those who fail conservative management should be treated operatively. These indications can be divided into 5 categories: greater trochanteric pain syndrome; internal hip snapping; anterior inferior iliac spine/sub-spine impingement; sciatic nerve entrapment; and proximal hamstring injuries. This article reviews the anatomy, patient history and physical examination, imaging, non-operative treatment, endoscopic operative treatment and outcomes of each category. While indications for hip arthroscopy, specifically extra-articular procedures, are rising steadily, there is not enough data to support its superiority over open procedures. Current literature consists of case studies, case reports, and expert opinions and lacks large, randomised control studies.

Revealing the cellular degradome by mass spectrometry analysis of proteasome-cleaved peptides.

Cellular function is critically regulated through degradation of substrates by the proteasome. To enable direct analysis of naturally cleaved proteasomal peptides under physiological conditions, we developed mass spectrometry analysis of proteolytic peptides (MAPP), a method for proteasomal footprinting that allows for capture, isolation and analysis of proteasome-cleaved peptides. Application of MAPP to cancer cell lines as well as primary immune cells revealed dynamic modulation of the cellular degradome in response to various stimuli, such as proinflammatory signals. Further, we performed analysis of minute amounts of clinical samples by studying cells from the peripheral blood of patients with systemic lupus erythematosus (SLE). We found increased degradation of histones in patient immune cells, thereby suggesting a role of aberrant proteasomal degradation in the pathophysiology of SLE. Thus, MAPP offers a broadly applicable method to facilitate the study of the cellular-degradation landscape in various cellular conditions and diseases involving changes in proteasomal degradation, including protein aggregation diseases, autoimmunity and cancer.

Oligomerization and Auto-methylation of the Human Lysine Methyltransferase SETD6.

Signaling via lysine methylation by protein lysine methyltransferases (PKMTs), has been linked to diverse biological and disease processes. The mono-methyltransferase SETD6 (SET-domain-containing protein 6) is a member of the PKMT family and was previously shown to regulate essential cellular processes such as the NF-κB, WNT and the oxidative stress pathways. However, on the biochemical level, little is known about the enzymatic mode of action of SETD6. Here we provide evidence that SETD6 forms high-molecular-weight structures. Specifically, we demonstrate that SETD6 monomeric, dimeric and trimeric forms are stabilized by the methyl donor, S-adenosyl-l-methionine. We then show that SETD6 has auto-methylation activity at K39 and K179, which serves as the major auto-methylation sites with a moderate auto-methylation activity toward K372. A point mutation at K179 but not at K39 and K372, located at the SET domain of SETD6, impaired SETD6 ability to form a trimer, strongly implying a link between the auto-methylation and the oligomerization state. Finally, by radioactive in vitro methylation experiments and biochemical kinetics analysis, we show that the auto-methylation at K39 and K179 increases the catalytic rate of SETD6. Collectively, our data support a model by which SETD6 auto-methylation and self-interaction positively regulate its enzymatic activity in vitro and may suggest that other PKMTs are regulated in the same manner.

Observation versus treatment among men with favorable risk prostate cancer in a community-based integrated health care system: a retrospective cohort study.

BACKGROUND: The objective of this study was to describe overall survival and the management of men with favorable risk prostate cancer (PCa) within a large community-based health care system in the United States. METHODS: A retrospective cohort study was conducted using linked electronic health records from men aged ≥40 years with favorable risk PCa (T1 or 2, PSA ≤15, Gleason ≤7 [3 + 4]) diagnosed between January 2005 and October 2013. Cohorts were defined as receiving any treatment (IMT) or no treatment (OBS) within 6 months after index PCa diagnosis. Cohorts' characteristics were compared between OBS and IMT; monitoring patterns were reported for OBS within the first 18 and 24 months. Cox Proportional Hazards models were used for multivariate analysis of overall survival. RESULTS: A total of 1425 men met the inclusion criteria (OBS 362; IMT 1063). The proportion of men managed with OBS increased from 20% (2005) to 35% (2013). The OBS group was older (65.6 vs 62.8 years, p < 0.01), had higher Charlson comorbidity index scores (CCI ≥2, 21.5% vs 12.2%, p < 0.01), and had a higher proportion of low-risk PCa (65.2% vs 55.0%, p < 0.01). For the OBS cohort, 181 of the men (50%) eventually received treatment. Among those remaining on OBS for ≥24 months (N = 166), 88.6% had ≥1 follow-up PSA test and 26.5% received ≥1 follow-up biopsy within the 24 months. The unadjusted mortality rate was higher for OBS compared with IMT (2.7 vs 1.3/100 person-years [py]; p < 0.001). After multivariate adjustment, there was no significant difference in all-cause mortality between OBS and IMT groups (HR 0.73, p = 0.138). CONCLUSIONS: Use of OBS management increased over the 10-year study period. Men in the OBS cohort had a higher proportion of low-risk PCa. No differences were observed in overall survival between the two groups after adjustment of covariates. These data provide insights into how favorable risk PCa was managed in a community setting.

Quiescin sulfhydryl oxidase 1 (QSOX1) glycosite mutation perturbs secretion but not Golgi localization.

Quiescin sulfhydryl oxidase 1 (QSOX1) catalyzes the formation of disulfide bonds in protein substrates. Unlike other enzymes with related activities, which are commonly found in the endoplasmic reticulum, QSOX1 is localized to the Golgi apparatus or secreted. QSOX1 is upregulated in quiescent fibroblast cells and secreted into the extracellular environment, where it contributes to extracellular matrix assembly. QSOX1 is also upregulated in adenocarcinomas, though the extent to which it is secreted in this context is currently unknown. To achieve a better understanding of factors that dictate QSOX1 localization and function, we aimed to determine how post-translational modifications affect QSOX1 trafficking and activity. We found a highly conserved N-linked glycosylation site to be required for QSOX1 secretion from fibroblasts and other cell types. Notably, QSOX1 lacking a glycan at this site arrives at the Golgi, suggesting that it passes endoplasmic reticulum quality control but is not further transported to the cell surface for secretion. The QSOX1 transmembrane segment is dispensable for Golgi localization and secretion, as fully luminal and transmembrane variants displayed the same trafficking behavior. This study provides a key example of the effect of glycosylation on Golgi exit and contributes to an understanding of late secretory sorting and quality control.

Degradation of Bcl-2 by XIAP and ARTS Promotes Apoptosis.

We describe a mechanism by which the anti-apoptotic B cell lymphoma 2 (Bcl-2) protein is downregulated to induce apoptosis. ARTS (Sept4_i2) is a tumor suppressor protein that promotes cell death through specifically antagonizing XIAP (X-linked inhibitor of apoptosis). ARTS and Bcl-2 reside at the outer mitochondrial membrane in living cells. Upon apoptotic induction, ARTS brings XIAP and Bcl-2 into a ternary complex, allowing XIAP to promote ubiquitylation and degradation of Bcl-2. ARTS binding to Bcl-2 involves the BH3 domain of Bcl-2. Lysine 17 in Bcl-2 serves as the main acceptor for ubiquitylation, and a Bcl-2 K17A mutant has increased stability and is more potent in protection against apoptosis. Bcl-2 ubiquitylation is reduced in both XIAP- and Sept4/ARTS-deficient MEFs, demonstrating that XIAP serves as an E3 ligase for Bcl-2 and that ARTS is essential for this process. Collectively, these results suggest a distinct model for the regulation of Bcl-2 by ARTS-mediated degradation.

Healthcare costs among men with favorable risk prostate cancer managed with observation strategies versus immediate treatment in an integrated healthcare system.

OBJECTIVE: This study explored short-term healthcare costs of men managed with observation strategies (OBS) vs immediate treatment (IMT) for favorable risk prostate cancer (PCa) from the Geisinger Health System, a single integrated health system in Pennsylvania, as evidence from the community setting is limited. METHODS: A retrospective cohort study was conducted using electronic health records from men aged ≥40 years diagnosed with favorable risk PCa (T1 or 2, PSA ≤15 ng/mL, Gleason ≤7 [3 + 4]) between January 2005 and October 2013. Prostate-specific healthcare costs were compared between the OBS and IMT cohorts in men with ≥3 years of follow-up and available linked claims data. Sub-group analyses focused on those men with low-risk PCa (T1-2a, PSA ≤10 ng/mL, Gleason ≤6). Sensitivity analysis stratified the study sample in three cohorts: OBS, switched from OBS to definitive treatment (OBS switch), and IMT. RESULTS: A total of 352 patients were included (OBS = 70 and IMT = 282). Compared with IMT, OBS resulted in significantly lower cumulative PCa-related healthcare costs for the first 3 years ($15,785 vs $23,177; p-value <.001). The main cost drivers were outpatient procedures. The OBS cohort had the lowest incremental PCa-related healthcare costs in the first 3 years (OBS: $5,011 vs OBS switch: $26,040, net cost savings = $21,029, p < .001; OBS: $5,011 vs IMT: $24,064, net cost savings = $19,053, p < .001). CONCLUSIONS: In favorable risk PCa, half of the patients who initially chose OBS eventually underwent treatment after their PCa diagnosis. As expected, OBS was associated with reduced disease management costs compared with IMT.

Phosphoproteomics of Fibroblast Growth Factor 1 (FGF1) Signaling in Chondrocytes: Identifying the Signature of Inhibitory Response.

Fibroblast growth factor (FGF) signaling is vital for many biological processes, beginning with development. The importance of FGF signaling for skeleton formation was first discovered by the analysis of genetic FGFR mutations which cause several bone morphogenetic disorders, including achondroplasia, the most common form of human dwarfism. The formation of the long bones is mediated through proliferation and differentiation of highly specialized cells - chondrocytes.Chondrocytes respond to FGF with growth inhibition, a unique response which differs from the proliferative response of the majority of cell types; however, its molecular determinants are still unclear. Quantitative phosphoproteomic analysis was utilized to catalogue the proteins whose phosphorylation status is changed upon FGF1 treatment. The generated dataset consists of 756 proteins. We could localize the divergence between proliferative (canonical) and inhibitory (chondrocyte specific) FGF transduction pathways immediately upstream of AKT kinase. Gene Ontology (GO) analysis of the FGF1 regulated peptides revealed that many of the identified phosphorylated proteins are assigned to negative regulation clusters, in accordance with the observed inhibitory growth response. This is the first time a comprehensive subset of proteins involved in FGF inhibitory response is defined. We were able to identify a number of targets and specifically discover glycogen synthase kinase3ß (GSK3ß) as a novel key mediator of FGF inhibitory response in chondrocytes.

Diagnostic Performance of Risk of Ovarian Malignancy Algorithm Against CA125 and HE4 in Connection With Ovarian Cancer: A Meta-analysis.

OBJECTIVES: The aim of this study was to determine whether the Risk of Ovarian Malignancy Algorithm (ROMA) is more accurate than the human epididymis 4 (HE4) or carbohydrate antigen 125 (CA125) biomarkers with respect to the differential diagnosis of women with a pelvic mass. The secondary objective is to assess the performance of ROMA in early-stage ovarian cancer (OC) and late-stage OC, as well as premenopausal and postmenopausal patient populations. METHODS/MATERIALS: The PubMed and Google Scholar databases were searched for relevant clinical studies. Eligibility criteria included comparison of ROMA with both HE4 and CA125 levels in OC (unspecified, epithelial, and borderline ovarian tumors), use of only validated ROMA assays, presentation of area under the curve and sensitivity/specificity data, and results from early-stage OC, late-stage OC and premenopausal and postmenopausal women. Area under the curve (AUC), sensitivity/specificity, and the diagnostic odds ratio (DOR) results were summarized. RESULTS: Five studies were selected comprising 1975 patients (premenopausal, n = 1033; postmenopausal, n = 925; benign, n = 1387; early stage, n = 192; and late stage, n = 313). On the basis of the AUC (95% confidence interval) data for all patients, ROMA (0.921 [0.855-0.960]) had a numerically greater diagnostic performance than CA125 (0.883 [0.771-0.950]) and HE4 (0.899 [0.835-0.943]). This was also observed in each of the subgroup populations, in particular, the postmenopausal patients and patients with early OC. The sensitivity and specificity (95% confidence interval) results showed ROMA (sensitivity, 0.873 [0.752-0.940]; specificity, 0.855 [0.719-0.932]) to be numerically superior to CA125 (sensitivity, 0.796 [0.663-0.885]; specificity, 0.825 [0.662-0.919]) and HE4 (sensitivity, 0.817 [0.683-0.902]; specificity, 0.851 [0.716-0.928]) in all patients and for the early- and late-stage OC subgroups. Finally, the ROMA log DOR results were better than HE4 and CA125 log DOR results especially for the early-stage patient group. CONCLUSIONS: The results presented support the use of ROMA to improve clinical decision making, most notably in patients with early OC.

De Novo Sequencing and Resurrection of a Human Astrovirus-Neutralizing Antibody.

Monoclonal antibody (mAb) therapeutics targeting cancer, autoimmune diseases, inflammatory diseases, and infectious diseases are growing exponentially. Although numerous panels of mAbs targeting infectious disease agents have been developed, their progression into clinically useful mAbs is often hindered by the lack of sequence information and/or loss of hybridoma cells that produce them. Here we combine the power of crystallography and mass spectrometry to determine the amino acid sequence and glycosylation modification of the Fab fragment of a potent human astrovirus-neutralizing mAb. We used this information to engineer a recombinant antibody single-chain variable fragment that has the same specificity as the parent monoclonal antibody to bind to the astrovirus capsid protein. This antibody can now potentially be developed as a therapeutic and diagnostic agent.