Magnetic Silica-Coated Fluorescent Microspheres (MagSiGlow) for Simultaneous Detection of Tumor-Associated Proteins.

Multiplexed bead assays for solution-phase biosensing often encounter cross-over reactions during signal amplification steps, leading to unwanted false positive and high background signals. Current solutions involve complex custom-designed and costly equipment, limiting their application in simple laboratory setup. In this study, we introduce a straightforward protocol to adapt a multiplexed single-bead assay to standard fluorescence imaging plates, enabling the simultaneous analysis of thousands of reactions per plate. This approach focuses on the design and synthesis of bright fluorescent and magnetic microspheres (MagSiGlow) with multiple fluorescent wavelengths serving as unique detection markers. The imaging-based, single-bead assay, combined with a scripted algorithm, allows the detection, segmentation, and co-localization on average of 7500 microspheres per field of view across five imaging channels in less than one second. We demonstrate the effectiveness of this method with remarkable sensitivity at low protein detection limits (100 pg/mL). This technique showed over 85 % reduction in signal cross-over to the solution-based method after the concurrent detection of tumor-associated protein biomarkers. This approach holds the promise of substantially enhancing high throughput biosensing for multiple targets, seamlessly integrating with rapid image analysis algorithms.

Metformin and adipose-derived stem cell combination therapy alleviates radiation-induced skin fibrosis in mice.

BACKGROUND: Radiation therapy often leads to late radiation-induced skin fibrosis (RISF), causing movement impairment and discomfort. We conducted a comprehensive study to assess the effectiveness of metformin and adipose-derived stem cells (ASCs), whether autologous or allogeneic, individually or in combination therapy, in mitigating RISF. METHODS: Using a female C57BL/6J mouse model subjected to hind limb irradiation as a representative RISF model, we evaluated metformin, ASCs, or their combination in two contexts: prophylactic (started on day 1 post-irradiation) and therapeutic (initiated on day 14 post-irradiation, coinciding with fibrosis symptoms). We measured limb movement, examined skin histology, and analyzed gene expression to assess treatment efficacy. RESULTS: Prophylactic metformin and ASCs, whether autologous or allogeneic, effectively prevented late fibrosis, with metformin showing promising results. However, combination therapy did not provide additional benefits when used prophylactically. Autologous ASCs, alone or with metformin, proved most effective against late-stage RISF. Prophylactic intervention outperformed late therapy for mitigating radiation skin damage. Co-culture studies revealed that ASCs and metformin downregulated inflammation and fibrotic gene expression in both mouse and human fibroblasts. CONCLUSIONS: Our study suggests metformin's potential as a prophylactic measure to prevent RISF, and the combination of ASCs and metformin holds promise for late-stage RISF treatment. These findings have clinical implications for improving the quality of life for those affected by radiation-induced skin fibrosis.

Human adipose ECM alleviates radiation-induced skin fibrosis via endothelial cell-mediated M2 macrophage polarization.

Radiation therapy can lead to late radiation-induced skin fibrosis (RISF), causing movement restriction, pain, and organ dysfunction. This study evaluated adipose-derived extracellular matrix (Ad-ECM) as a mitigator of RISF. Female C57BL/6J mice that were irradiated developed fibrosis, which was mitigated by a single local Ad-ECM injection, improving limb movement and reducing epithelium thickness and collagen deposition. Ad-ECM treatment resulted in decreased expression of pro-inflammatory and fibrotic genes, and upregulation of anti-inflammatory cytokines, promoting M2 macrophage polarization. Co-culture of irradiated human fibroblasts with Ad-ECM down-modulated fibrotic gene expression and enhanced bone marrow cell migration. Ad-ECM treatment also increased interleukin (IL)-4, IL-5, and IL-15 expression in endothelial cells, stimulating M2 macrophage polarization and alleviating RISF. Prophylactic use of Ad-ECM showed effectiveness in mitigation. This study suggests Ad-ECM's potential in treating chronic-stage fibrosis.

Extracellular vesicle analysis of plasma allows differential diagnosis of atypical pancreatic serous cystadenoma.

Increased use of cross-sectional imaging has resulted in frequent detection of incidental cystic pancreatic lesions. Serous cystadenomas (SCAs) are benign cysts that do not require surgical intervention unless symptomatic. Unfortunately, up to half of SCAs do not have typical imaging findings ("atypical SCAs"), overlap with potentially malignant precursor lesions, and thus pose a diagnostic challenge. We tested whether the analysis of circulating extracellular vesicle (EV) biomarkers using a digital EV screening technology (DEST) could enhance the discrimination of cystic pancreatic lesions and avoid unnecessary surgical intervention in these atypical SCAs. Analysis of 25 different protein biomarkers in plasma EV from 68 patients identified a putative biomarker signature of Das-1, Vimentin, Chromogranin A, and CAIX with high discriminatory power (AUC of 0.99). Analysis of plasma EV for multiplexed markers may thus be helpful in clinical decision-making.

Establishment of a Robust and Reproducible Model of Radiation-Induced Skin and Muscle Fibrosis.

Radiation-induced skin fibrosis (RISF) can result from a plethora of scenarios including cancer therapy, accidental exposure, or acts of terrorism. Radioactive beams can penetrate through the skin and affect the structures in their path including skin, muscles, and internal organs. Skin is the first structure to get exposed to radiation and is susceptible to develop chronic fibrosis, which is challenging to treat. Currently, limited treatment options show moderate efficacy in mitigating radiation-related skin fibrosis. A key factor hindering the development of effective countermeasures is the absence of a convenient and robust model that could allow for translation of the experimental findings to humans. Here, a robust and reproducible murine hind limb skin fibrosis model has been established for prophylactic and therapeutic evaluation of possible agents for functional and molecular recovery. The right hind limb was irradiated using a single dose of 40 (Gray) Gy to induce skin fibrosis. Subjects developed edema and dermatitis in the early stages proceeded by visible skin constriction. Irradiated limbs showed a significantly reduced limb range of motion in the following weeks. In late stages, acute side effects subsided, yet chronic fibrosis persisted. A gait index was performed as an additional functional assay, which demonstrated the development of functional impairment. These non-invasive methods demonstrated reliable measurements for tracing fibrosis progression, which is supported by histological analyses. The radiation dose, application, and post-irradiation analyses employed in this model offer a vigorous and reproducible method for studying radiation-induced skin fibrosis and testing the efficacy of therapeutical agents.

Single extracellular vesicle analysis for early cancer detection.

Extracellular vesicles (EVs) are actively shed into the circulation from both cancer and host cells. EVs are emerging as one of the diagnostic frontrunners for early cancer detection, disease monitoring, and treatment evaluation. The advantages of EVs rely on the fact that vesicles are being shed by dividing tumor cells, with indications that human and viral oncogenes, cellular metabolic rate, and tumor characteristics such as pH and hypoxia contribute to the high shed rates in cancer. In this review, we provide an overview of EVs and the rationale for using them for early cancer detection. We examine emerging technologies for single EV analysis (sEVA) and why these technologies will be necessary for early cancer detection.

Single-EV analysis (sEVA) of mutated proteins allows detection of stage 1 pancreatic cancer.

Tumor cell-derived extracellular vesicles (EVs) are being explored as circulating biomarkers, but it is unclear whether bulk measurements will allow early cancer detection. We hypothesized that a single-EV analysis (sEVA) technique could potentially improve diagnostic accuracy. Using pancreatic cancer (PDAC), we analyzed the composition of putative cancer markers in 11 model lines. In parental PDAC cells positive for KRASmut and/or P53mut proteins, only ~40% of EVs were also positive. In a blinded study involving 16 patients with surgically proven stage 1 PDAC, KRASmut and P53mut protein was detectable at much lower levels, generally in <0.1% of vesicles. These vesicles were detectable by the new sEVA approach in 15 of the 16 patients. Using a modeling approach, we estimate that the current PDAC detection limit is at ~0.1-cm3 tumor volume, below clinical imaging capabilities. These findings establish the potential for sEVA for early cancer detection.

Pragmatic Mentoring Strategies for the Busy Surgeon Scientist.

OBJECTIVE: The value of research mentorship in academic medicine is well-recognized, yet there is little practical advice for how to develop and sustain effective mentoring partnerships. Gaining research skill and mentorship is particularly critical to success in academic surgery, yet surgeon scientists are challenged in their mentorship efforts by time constraints and lack of education on how to mentor. To address this gap, this study explored the strategies that award-winning faculty mentors utilize in collaborating with their medical student mentees in research. DESIGN, SETTING, AND PARTICIPANTS: For this qualitative study, the authors invited physician recipients of an institution-wide mentorship award to participate in individual, semi-structured interviews during July and August 2018. Following interview transcription, the authors independently coded the text and collaboratively identified common mentoring strategies and practices via a process of thematic analysis. RESULTS: Nine physician mentors, representing a mix of genders, medical specialties and types of research (basic science, clinical, translational, and health services), participated in interviews. The authors identified 12 strategies and practices from the interview transcripts that fell into 5 categories: Initiating the partnership; Determining the research focus; Providing project oversight; Developing mentee research competence; and Supporting mentee self-efficacy. CONCLUSION: Award-winning mentors employ a number of shared strategies when mentoring medical trainees in research. These strategies can serve as a guide for academic surgeons who wish to improve their research mentoring skills.

Allogeneic adipose-derived stem cells mitigate acute radiation syndrome by the rescue of damaged bone marrow cells from apoptosis.

Acute radiation syndrome (ARS) is the radiation toxicity that can affect the hematopoietic, gastrointestinal, and nervous systems upon accidental radiation exposure within a short time. Currently, there are no effective and safe approaches to treat mass population exposure to ARS. Our study aimed to evaluate the therapeutic potential of allogeneic adipose-derived stem cells (ASCs) for total body irradiation (TBI)-induced ARS and understand the underlying mitigation mechanism. We employed 9.25 Gy TBI dose to C57BL/6 mice and studied the effect of allogeneic ASCs on mice survival and regeneration of the hematopoietic system. Our results indicate that intraperitoneal-injected ASCs migrated to the bone marrow, rescued hematopoiesis, and improved the survival of irradiated mice. Our transwell coculture results confirmed the migration of ASCs to irradiated bone marrow and rescue hematopoietic activity. Furthermore, contact coculture of ASCs improved the survival and hematopoiesis of irradiated bone marrow in vitro. Irradiation results in DNA damage, upregulation of inflammatory signals, and apoptosis in bone marrow cells, while coculture with ASCs reduces apoptosis via activation of DNA repair and the antioxidation system. Upon exposure to irradiated bone marrow cells, ASCs secrete prosurvival and hematopoietic factors, such as GM-CSF, MIP1α, MIP1ß, LIX, KC, 1P-10, Rantes, IL-17, MCSF, TNFα, Eotaxin, and IP-10, which reduces oxidative stress and rescues damaged bone marrow cells from apoptosis. Our findings suggest that allogeneic ASCs therapy is effective in mitigating TBI-induced ARS in mice and may be beneficial for clinical adaptation to treat TBI-induced toxicities. Further studies will help to advocate the scale-up and adaptation of allogeneic ASCs as the radiation countermeasure.

Reductions in vancomycin and meropenem following the implementation of a febrile neutropenia management algorithm in hospitalized adults: An interrupted time series analysis.

OBJECTIVE: To evaluate broad-spectrum intravenous antibiotic use before and after the implementation of a revised febrile neutropenia management algorithm in a population of adults with hematologic malignancies. DESIGN: Quasi-experimental study. SETTING AND POPULATION: Patients admitted between 2014 and 2018 to the Adult Malignant Hematology service of an acute-care hospital in the United States. METHODS: Aggregate data for adult malignant hematology service were obtained for population-level antibiotic use: days of therapy (DOT), C. difficile infections, bacterial bloodstream infections, intensive care unit (ICU) length of stay, and in-hospital mortality. All rates are reported per 1,000 patient days before the implementation of an febrile neutropenia management algorithm (July 2014-May 2016) and after the intervention (June 2016-December 2018). These data were compared using interrupted time series analysis. RESULTS: In total, 2,014 patients comprised 6,788 encounters and 89,612 patient days during the study period. Broad-spectrum intravenous (IV) antibiotic use decreased by 5.7% with immediate reductions in meropenem and vancomycin use by 22 (P = .02) and 15 (P = .001) DOT per 1,000 patient days, respectively. Bacterial bloodstream infection rates significantly increased following algorithm implementation. No differences were observed in the use of other antibiotics or safety outcomes including C. difficile infection, ICU length of stay, and in-hospital mortality. CONCLUSIONS: Reductions in vancomycin and meropenem were observed following the implementation of a more stringent febrile neutropenia management algorithm, without evidence of adverse outcomes. Successful implementation occurred through a collaborative effort and continues to be a core reinforcement strategy at our institution. Future studies evaluating patient-level data may identify further stewardship opportunities in this population.

Extracellular Vesicle Analysis Allows for Identification of Invasive IPMN.

BACKGROUND AND AIMS: Advances in cross-sectional imaging have resulted in increased detection of intraductal papillary mucinous neoplasms (IPMNs), and their management remains controversial. At present, there is no reliable noninvasive method to distinguish between indolent and high risk IPMNs. We performed extracellular vesicle (EV) analysis to identify markers of malignancy in an attempt to better stratify these lesions. METHODS: Using a novel ultrasensitive digital extracellular vesicle screening technique (DEST), we measured putative biomarkers of malignancy (MUC1, MUC2, MUC4, MUC5AC, MUC6, Das-1, STMN1, TSP1, TSP2, EGFR, EpCAM, GPC1, WNT-2, EphA2, S100A4, PSCA, MUC13, ZEB1, PLEC1, HOOK1, PTPN6, and FBN1) in EV from patient-derived cell lines and then on circulating EV obtained from peripheral blood drawn from patients with IPMNs. We enrolled a total of 133 patients in two separate cohorts: a clinical discovery cohort (n = 86) and a validation cohort (n = 47). RESULTS: From 16 validated EV proteins in plasma samples collected from the discovery cohort, only MUC5AC showed significantly higher levels in high-grade lesions. Of the 11 patients with invasive IPMN (inv/HG), 9 had high MUC5AC expression in plasma EV of the 11 patients with high-grade dysplasia alone, only 1 had high MUC5AC expression (sensitivity of 82%, specificity of 100%). These findings were corroborated in a separate validation cohort. The addition of MUC5AC as a biomarker to imaging and high-riskstigmata allowed detection of all cases requiring surgery, whereas imaging and high-risk stigmata alone would have missed 5 of 14 cases (36%). CONCLUSIONS: MUC5AC in circulating EV can predict the presence of invasive carcinoma within IPMN. This approach has the potential to improve the management and follow-up of patients with IPMN including avoiding unnecessary surgery.

The Impact of Human Lipoaspirate and Adipose Tissue-Derived Stem Cells Contact Culture on Breast Cancer Cells: Implications in Breast Reconstruction.

BACKGROUND: Autologous fat transfer in the form of lipoaspirates for the reconstruction of the breast after breast cancer surgery is a commonly used procedure in plastic surgery. However, concerns regarding the oncologic risk of nutrient-rich fat tissue are widely debated. Previous studies have primarily focused on studying the interaction between adipose-derived stem cells (ASCs) and breast cancer cells. METHODS: In this study, we performed a comprehensive analysis of the paracrine- and contact-based interactions between lipoaspirates, ASCs and breast cancer cell lines. An inverted flask culture method was used to study the contact-based interaction between lipoaspirates and breast cancer cells, while GFP-expressing breast cancer cell lines were generated to study the cell-cell contact interaction with ASCs. Three different human breast cancer cell lines, MCF-7, MDA-MB-231 and BT-474, were studied. We analyzed the impact of these interactions on the proliferation, cell cycle and epithelial-to-mesenchymal (EMT) transition of the breast cancer cells. RESULTS: Our results revealed that both lipoaspirates and ASCs do not increase the proliferation rate of the breast cancer cells either through paracrine- or contact-dependent interactions. We observed that lipoaspirates selectively inhibit the proliferation of MCF-7 cells in contact co-culture, driven by the retinoblastoma (Rb) protein activity mediating cell cycle arrest. Additionally, ASCs inhibited MDA-MB-231 breast cancer cell proliferation in cell-cell contact-dependent interactions. Quantitative real-time PCR revealed no significant increase in the EMT-related genes in breast cancer cells upon co-culture with ASCs. CONCLUSION: In conclusion, this study provides evidence of the non-oncogenic character of lipoaspirates and supports the safety of clinical fat grafting in breast reconstruction after oncological surgical procedures. In vivo studies in appropriate animal models and long-term post-operative clinical data from patients are essential to reach the final safety recommendations.

Bead-Based Extracellular Vesicle Analysis Using Flow Cytometry.

Extracellular vesicles (EVs) represent promising circulating biomarkers for cancers, but their high-throughput analyses in clinical settings prove challenging due to lack of simple, fast, and robust EV assays. Here, a bead-based EV assay detected by flow cytometry is described, which integrates EV capture using microbeads with EV protein analyses by flow cytometry. The assay is fast (<4 h for 48 samples), robust, and compatible with conventional flow cytometry instruments for high-throughput EV analysis. With the method, a panel of pancreatic cancer biomarkers in EVs from plasma samples of pancreatic cancer patients is successfully analyzed. The assay is readily translatable to other biomarkers or cancer types and can be run with standard materials on conventional flow cytometers, making it highly flexible and adaptable to diverse research and clinical needs.

Concordance Between Expert and Nonexpert Ratings of Condensed Video-Based Trainee Operative Performance Assessment.

OBJECTIVE: We examined the impact of video editing and rater expertise in surgical resident evaluation on operative performance ratings of surgical trainees. DESIGN: Randomized independent review of intraoperative video. SETTING: Operative video was captured at a single, tertiary hospital in Boston, MA. PARTICIPANTS: Six common general surgery procedures were video recorded of 6 attending-trainee dyads. Full-length and condensed versions (n = 12 videos) were then reviewed by 13 independent surgeon raters (5 evaluation experts, 8 nonexperts) using a crossed design. Trainee performance was rated using the Operative Performance Rating Scale, System for Improving and Measuring Procedural Learning (SIMPL) Performance scale, the Zwisch scale, and ten Cate scale. These ratings were then standardized before being compared using Bayesian mixed models with raters and videos treated as random effects. RESULTS: Editing had no effect on the Operative Performance Rating Scale Overall Performance (-0.10, p = 0.30), SIMPL Performance (0.13, p = 0.71), Zwisch (-0.12, p = 0.27), and ten Cate scale (-0.13, p = 0.29). Additionally, rater expertise (evaluation expert vs. nonexpert) had no effect on the same scales (-0.16 (p = 0.32), 0.18 (p = 0.74), 0.25 (p = 0.81), and 0.25 (p = 0.17). CONCLUSIONS: There is little difference in operative performance assessment scores when raters use condensed videos or when raters who are not experts in surgical resident evaluation are used. Future validation studies of operative performance assessment scales may be facilitated by using nonexpert surgeon raters viewing videos condensed using a standardized protocol.

Characterization of single microvesicles in plasma from glioblastoma patients.

BACKGROUND: Extracellular vesicles (EV) are shed by tumor cells but little is known about their individual molecular phenotypes and heterogeneity. While exosomes have received considerable attention, much less is known about larger microvesicles. Here we profile single microvesicles (MV) and exosomes from glioblastoma (GB) cells and MV from the plasma of patients. METHODS: EV secreted from mouse glioma GL261 and human primary GBM8 cell lines as well as from the plasma of 8 patients with diagnoses of GB and 2 healthy controls were isolated and processed for single vesicle analysis. EV were immobilized on glass slides and the heterogeneity of vesicle and tumor markers were analyzed at the single vesicle level. RESULTS: We show that (i) MV are abundant, (ii) only a minority of MV expresses putative MV markers, and (iii) MV share tetraspanin biomarkers previously thought to be diagnostic of exosomes. Using MV capture and staining techniques that allow differentiation of host cell and GB-derived MV we further demonstrate that (i) tumoral MV often present as <10% of all MV in GB patient plasma, and (ii) there is extensive heterogeneity in tumor marker expression in these tumor-derived MV. CONCLUSION: These results indicate that single MV analysis is likely necessary to identify rare tumoral MV populations and the single vesicle analytical technique used here can be applied to both MV and exosome fractions without the need for their separation from each other. These studies form the basis for using single EV analyses for cancer diagnostics.

A Patient-derived Xenograft Model of Pancreatic Neuroendocrine Tumors Identifies Sapanisertib as a Possible New Treatment for Everolimus-resistant Tumors.

Patients with pancreatic neuroendocrine tumors (PNET) commonly develop advanced disease and require systemic therapy. However, treatment options remain limited, in part, because experimental models that reliably emulate PNET disease are lacking. We therefore developed a patient-derived xenograft model of PNET (PDX-PNET), which we then used to evaluate two mTOR inhibitor drugs: FDA-approved everolimus and the investigational new drug sapanisertib. PDX-PNETs maintained a PNET morphology and PNET-specific gene expression signature with serial passage. PDX-PNETs also harbored mutations in genes previously associated with PNETs (such as MEN1 and PTEN), displayed activation of the mTOR pathway, and could be detected by Gallium-68 DOTATATE PET-CT. Treatment of PDX-PNETs with either everolimus or sapanisertib strongly inhibited growth. As seen in patients, some PDX-PNETs developed resistance to everolimus. However, sapanisertib, a more potent inhibitor of the mTOR pathway, caused tumor shrinkage in most everolimus-resistant tumors. Our PDX-PNET model is the first available, validated PDX model for PNET, and preclinical data from the use of this model suggest that sapanisertib may be an effective new treatment option for patients with PNET or everolimus-resistant PNET.

Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles.

Binding of programmed death ligand-1 (PD-L1) to programmed cell death protein-1 (PD1) leads to cancer immune evasion via inhibition of T cell function. One of the defining characteristics of glioblastoma, a universally fatal brain cancer, is its profound local and systemic immunosuppression. Glioblastoma has also been shown to generate extracellular vesicles (EVs), which may play an important role in tumor progression. We thus hypothesized that glioblastoma EVs may be important mediators of immunosuppression and that PD-L1 could play a role. We show that glioblastoma EVs block T cell activation and proliferation in response to T cell receptor stimulation. PD-L1 was expressed on the surface of some, but not of all, glioblastoma-derived EVs, with the potential to directly bind to PD1. An anti-PD1 receptor blocking antibody significantly reversed the EV-mediated blockade of T cell activation but only when PD-L1 was present on EVs. When glioblastoma PD-L1 was up-regulated by IFN-γ, EVs also showed some PD-L1-dependent inhibition of T cell activation. PD-L1 expression correlated with the mesenchymal transcriptome profile and was anatomically localized in the perinecrotic and pseudopalisading niche of human glioblastoma specimens. PD-L1 DNA was present in circulating EVs from glioblastoma patients where it correlated with tumor volumes of up to 60 cm3. These results suggest that PD-L1 on EVs may be another mechanism for glioblastoma to suppress antitumor immunity and support the potential of EVs as biomarkers in tumor patients.

In vivo imaging reveals a tumor-associated macrophage-mediated resistance pathway in anti-PD-1 therapy.

Monoclonal antibodies (mAbs) targeting the immune checkpoint anti-programmed cell death protein 1 (aPD-1) have demonstrated impressive benefits for the treatment of some cancers; however, these drugs are not always effective, and we still have a limited understanding of the mechanisms that contribute to their efficacy or lack thereof. We used in vivo imaging to uncover the fate and activity of aPD-1 mAbs in real time and at subcellular resolution in mice. We show that aPD-1 mAbs effectively bind PD-1+ tumor-infiltrating CD8+ T cells at early time points after administration. However, this engagement is transient, and aPD-1 mAbs are captured within minutes from the T cell surface by PD-1- tumor-associated macrophages. We further show that macrophage accrual of aPD-1 mAbs depends both on the drug's Fc domain glycan and on Fcγ receptors (FcγRs) expressed by host myeloid cells and extend these findings to the human setting. Finally, we demonstrate that in vivo blockade of FcγRs before aPD-1 mAb administration substantially prolongs aPD-1 mAb binding to tumor-infiltrating CD8+ T cells and enhances immunotherapy-induced tumor regression in mice. These investigations yield insight into aPD-1 target engagement in vivo and identify specific Fc/FcγR interactions that can be modulated to improve checkpoint blockade therapy.

Multiparametric plasma EV profiling facilitates diagnosis of pancreatic malignancy.

Pancreatic ductal adenocarcinoma (PDAC) is usually detected late in the disease process. Clinical workup through imaging and tissue biopsies is often complex and expensive due to a paucity of reliable biomarkers. We used an advanced multiplexed plasmonic assay to analyze circulating tumor-derived extracellular vesicles (tEVs) in more than 100 clinical populations. Using EV-based protein marker profiling, we identified a signature of five markers (PDACEV signature) for PDAC detection. In our prospective cohort, the accuracy for the PDACEV signature was 84% [95% confidence interval (CI), 69 to 93%] but only 63 to 72% for single-marker screening. One of the best markers, GPC1 alone, had a sensitivity of 82% (CI, 60 to 95%) and a specificity of 52% (CI, 30 to 74%), whereas the PDACEV signature showed a sensitivity of 86% (CI, 65 to 97%) and a specificity of 81% (CI, 58 to 95%). The PDACEV signature of tEVs offered higher sensitivity, specificity, and accuracy than the existing serum marker (CA 19-9) or single-tEV marker analyses. This approach should improve the diagnosis of pancreatic cancer.