Field Of Liquid Biopsy Research Articles

Circulating tumor DNA-based predictive biomarkers in breast cancer clinical trials: a narrative review.

Breast carcinoma is the most frequent and the second leading cause of cancer mortality in women worldwide. Current treatment decisions are based on tumor profiling of the initial tissue biopsy. Cancer though evolves both spatially and temporarily in a significant percentage of patients during treatment. However, sequential biopsies from the primary tumor or its metastatic sites are not either convenient or feasible in the majority of cases. In the era of precision medicine, analysis of circulating blood-based biomarkers in the field of liquid biopsies provides an insight into the dynamic molecular profiling of the primary tumor and its metastases, in a relatively non-invasive way. The latter permits not only patient stratification but also longitudinal evaluation of treatment response, when incorporated into clinical trials. This review summarizes the results from recent and ongoing circulating tumor DNA (ctDNA)-based biomarker-driven clinical trials, with respect to ctDNA analysis’ predictive role, both in adjuvant, neo-adjuvant, and metastatic setting. Furthermore, current challenges in ctDNA analysis applications are critically discussed, including pre-analytical and analytical issues, and future perspectives in this field, through the conduct of well-designed, multicenter, randomized, large-scale, biomarker-stratified trials, with robust statistical methods. Despite in its infancy, ctDNA analysis holds great promise as a minimally invasive tool regarding tailored, personalized treatment guidance for breast cancer patients.

The main sources of circulating cell-free DNA: Apoptosis, necrosis and active secretion.

Cell-free DNA (cfDNA) as an emerging biomarker with huge potential for clinical application, especially in the field of liquid biopsy. The field is now in a critical transitional period in which cfDNA-based analysis is developing rapidly. No doubt learning more about the biological knowledge of cfDNA is beneficial to catalyze this transformation process. Therefore, in this review we have summarized the characteristics of cfDNA, including its structure and origin of tissues, in order to provide researchers with a more holistic insight of cfDNA. Subsequently, we focused on the pathways that cfDNA releases from cells, such as apoptosis, necrosis, and active secretion. Additionally, the clearance of cfDNA derived from both cellular death and active secretion in the physiological environment is also discussed. Finally, we have mentioned the link between cfDNA active secretion and tumor microenvironment.

Liquid Biopsy perspectives theranostics and personalized oncology

The current limitations of cancer diagnosis and molecular profiling based on invasive tissue biopsies or clinical imaging have led to the development of the Liquid Biopsy field (LB),

Methylation Taking Center Stage: Some in the liquid biopsy field are turning from mutation to methylation to improve their assays, with an eye on early cancer detection

Methylation Taking Center Stage: Some in the liquid biopsy field are turning from mutation to methylation to improve their assays, with an eye on early cancer detection

DNA in extracellular vesicles: biological and clinical aspects.

The study of extracellular vesicles (EVs), especially in the liquid biopsy field, has rapidly evolved in recent years. However, most EV studies have focused on RNA or protein content and DNA in EVs (EV-DNA) has largely been unnoticed. In this review, we compile current evidence regarding EV-DNA and provide an extensive discussion on EV-DNA biology. We look into EV-DNA biogenesis and mechanisms of DNA loading into EVs, as well as describe the particularly significant function of DNA-carrying EVs in the maintenance of cellular homeostasis, intracellular communication, and immune response modulation. We also examine the current role of EV-DNA in the clinical setting, specifically in cancer, infections, pregnancy, and prenatal diagnosis.

Abstract 6452: Exosomal Let 7 and its downstream protein targets may serve as predictive biomarkers in peritoneal carcinomatosis

Abstract The prognosis for patients with colorectal cancer with peritoneal carcinomatosis (PC) is dismal. Current biomarkers and imaging studies lack sensitivity and specificity to identify PC at early stages. Predictive and diagnostic serum biomarkers that help diagnose PC at early stages is necessary to improve patient outcomes. In the rapidly evolving field of liquid biopsy, exosomes show significant promise. The objective of our study is to identify exosomal biomarkers that are differentially expressed in patients with PC compared to visceral metastases (VM). Proteomic and miRNA analysis were performed using mass spectrometry and next generation sequencing on plasma exosomes from ten patients with colorectal cancer VM and nine patients with PC. Multiple members of the Let-7 family had differential levels of expression between these two patient groups. Specifically, Let-7 isoforms (Let-7a, Let-7d, Let-7e, Let-7f, Let-7g, Let-7i) were upregulated in plasma exosomes of patients with PC compared to those with VM. In addition, 33 exosomal proteins that are downstream targets of Let-7 were also found to be differentially expressed. Correlative investigation was conducted using cell lines representative of PC, VM and control to investigate the possible origins for this exosomal profile. RT-PCR was performed on cell lysates taken from normal colon (CCD-33-CO) and colorectal cancer (SK-CO-1, SNU-C1, and T84) cell lines to further identify Let-7 isoforms in these samples. Let-7 isoforms (Let 7a, Let-7b, Let-7d, Let-7e, Let-7f, Let-7i) were identified at increased levels in the colorectal cancer PC cell line in comparison to cells lines of non-PC colorectal cancer and normal colon. In-vivo and in-vitro studies demonstrate plasma exosomal Let-7 and its downstream protein targets are differentially expressed in colorectal PC compared to VM. Let-7 has been previously implicated in cancer progression and chemoresistance. While this differential expression suggests possible mechanistic differences between colorectal PC and VM, it may also offer a potential liquid biopsy for predicting peritoneal metastasis at a much earlier and treatable time. Citation Format: Mei Li M. Kwong, Paul A. Vallejos, Vola-Masoandro Andrianarijaona, Audrey Choi, Matthew J. Selleck, Tiantian Liu, Janviere Kabagwira, Charles Wang, William Langridge, Nathan R. Wall, Maheswari Senthil. Exosomal Let 7 and its downstream protein targets may serve as predictive biomarkers in peritoneal carcinomatosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6452.

Abstract 773: Tumor-derived Extracellular Vesicles (EVs) expressing TGFb as potential biomarkers for anti-TGFb antibody activity and drug activity in liquid biopsy

Abstract The field of liquid biopsies is of enormous interest in cancer using a noninvasive process. There are currently three major approaches: Circulating Cell Tumors (CTCs), circulating cell free DNA (cfDNA) and Extracellular Vesicles (EVs) such as exosomes, microvesicles and apoptotic bodies. The EVs are cell-derived small membrane vesicles secreted by cells that convey biological messages, either by surface-to-surface interaction or by shuttling bioactive molecules to a recipient cell's cytoplasm. The EVs released by cells harbor the cargos of their original cells and could be a good tool to follow drug activity. We are developing anti-TGFβ antibody SAR439459 for the treatment of cancer (NCT03192345), and in this vitro study we explore the effect of anti-TGFβ antibody in EVs carrying TGFβ. The cargo of EVs were analyzed by an Elisa test for both latent and active free TGFβ and by miRNA expression. Cell lines were selected based on their expression of TGFβ (A549, DU45, PC3, MCF7 and Caco2), treated for 72h and compared with respective untreated controls. The EVs were isolated from the supernatant and purified. Nanoparticle Tracking Analysis was used to quantify and determine size distribution of EVs. The expression of active free and latent TGFβ was done using Elisa tests. TGFβ antibody-coated magnetic beads were used to capture EVs, and miRNA expression sequencing was done using the Illumina NextSeq. Anti-TGFβ treatment reduced notably the expression of latent TGFβ in the supernatant of cultured tumor cells; the active free TGFβ was very low expressed or not detected in the supernatants. EVs express both latent and active free TGFβ; the anti-TGFβ antibody treatment significantly reduced the active free TGFβ and altered miRNA expression in EVs. Finally, using TGFβ Antibody-coated magnetic beads, we demonstrate that intact EVs express TGFβ at their surface. In conclusion, these results suggest that EVs express TGFβ at their surface and carry the active free TGFβ. Finally, the expression of active free TGFβ in EVs is decreased with anti-TGFβ antibody, suggesting that it might be a biomarker for monitoring anti-TGFβ antibody activity in patients by liquid biopsies. Citation Format: Manoel Nunes, Camille Bernard, Gaelle Muzard, Wilson Dos-Santos-Bele, Jack Pollard, Alexei Protopopov, Colette Dib. Tumor-derived Extracellular Vesicles (EVs) expressing TGFb as potential biomarkers for anti-TGFb antibody activity and drug activity in liquid biopsy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 773.

Analysis of cancer-related mutations in extracellular vesicles RNA by Droplet Digital™ PCR.

Extracellular vesicles (EVs) are taking their place as potential biomarkers in the field of liquid biopsy. In this study, EVs were isolated from plasma samples of 31 patients with colorectal cancer and melanoma via differential centrifugation and Droplet Digital™ PCR (Bio-Rad, CA, USA) was used to profile BRAF V600E/K, KRAS G12A/C/D/V and KRASG13D mutations from EV-derived cDNA. The concordance rates with corresponding tissue were 54%and 44% in the colorectal cancer and melanoma cohort, respectively. Two patients displayed mutations in EVs not previously detected in tissue as evidence for emerging molecular resistance to anti-EGFR and BRAF/MEK inhibitor therapy prior to radiological evidence of tumor progression. We concluded that EV-derived nucleic acids may provide clinically relevant diagnostic information and mirror evolution of the disease.

Abstract B14: Artificial intelligence assists automation and high performance of circulating tumor cells enumeration and circulating tumor microemboli characterization in fluorescence microscopy images

Abstract Analysis of circulating tumor cells (CTCs) provides exclusive benefits compared with other liquid biopsy techniques due to their advanced clinical applications including single-cell multiomics (DNA, RNA, proteins, and metabolites) analysis and the ability to generate CTCs-derived xenograft models. Current CTC analysis primarily relies on labor-intensive and human-biased manual CTC enumeration or semiautomated work, hindering clinical adaptation of CTC analysis into practice. Beyond CTC enumeration, recent studies focused on detection and characterized features of circulating tumor microemboli (CTM) to improve cancer monitoring and diagnosis, but the added complexity of CTM makes CTC analysis even more challenging. Development of an automated, unbiased, and specialized computation method for CTC analysis remains a clinical unmet need. We developed a deep learning-based framework by applying computer vision to efficiently automate CTC enumeration and characterization with high throughput and accuracy. Fluorescence microscopy CTCs images (CK+/CD45-/DAPI+) have been prepared from blood samples of non-small cell lung cancer patients by the “CMx” CTC capture platform. This study applied an “active learning” model that only requires a small upfront training dataset while being able to accumulate more training data to make improvements with minimal annotation efforts. We have collected 20 images annotated by subject matter experts to train the initial model. Using an iterative process where feedback from the subject matter experts was used to influence the model, we achieved continuous improvements using adaptive annotations. After the model had been well trained and validated by 4 extra images, we used 18 new test images to benchmark the model performance. Preliminary results demonstrated that our AI model outperformed current semiautomated methods having higher sensitivity and reduced time for CTC enumeration in a lung cancer CTC sample analysis. In 18 test samples, the model predicted 34% more total CTCs than the current method (1,775 versus 1,328 counts). By applying the model to the 24 training samples, the model also recovered 45% more total CTCs absent from the original human annotation (2,507 versus 1,732 counts). In some samples, enumeration assisted by the model saved 90% of time over the current method (<20 min vs ~4 hrs.) for CTC enumeration, which would result in an enormous advantage if the datasets being analyzed included a large number of samples. Furthermore, our AI model was able to characterize features of CTM, including CTC clusters and CTC-associated immune cells, in addition to enumerating the samples. The AI model developed in this study is a promising method for CTC analysis that significantly improved throughput, accuracy, and reproducibility, leading to an approach of CTC analysis better suited for clinical adoption. Development of AI-driven CTC/CTM analysis provides additional resources and advancements to the field of liquid biopsy for the diagnosis and treatment of disease. Citation Format: Wei-Lei Yang, Chi-Bin Li, Jia-Yang Chen, Samuel Chen, Ten-Jen Liu, Ying-Chih Chang. Artificial intelligence assists automation and high performance of circulating tumor cells enumeration and circulating tumor microemboli characterization in fluorescence microscopy images [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B14.

Plasma-Derived Extracellular Vesicle Phosphoproteomics through Chemical Affinity Purification.

The invasive nature and the pain caused to patients inhibit the routine use of tissue biopsy-based procedures for cancer diagnosis and surveillance. The analysis of extracellular vesicles (EVs) from biofluids has recently gained significant traction in the liquid biopsy field. EVs offer an essential "snapshot" of their precursor cells in real time and contain an information-rich collection of nucleic acids, proteins, lipids, and so on. The analysis of protein phosphorylation, as a direct marker of cellular signaling and disease progression could be an important stepping stone to successful liquid biopsy applications. Here we introduce a rapid EV isolation method based on chemical affinity called EVtrap (extracellular vesicle total recovery and purification) for the EV phosphoproteomics analysis of human plasma. By incorporating EVtrap with high-performance mass spectrometry (MS), we were able to identify over 16 000 unique peptides representing 2238 unique EV proteins from just 5 μL of plasma sample, including most known EV markers, with substantially higher recovery levels compared with ultracentrifugation. Most importantly, more than 5500 unique phosphopeptides representing almost 1600 phosphoproteins in EVs were identified using only 1 mL of plasma. Finally, we carried out a quantitative EV phosphoproteomics analysis of plasma samples from patients diagnosed with chronic kidney disease or kidney cancer, identifying dozens of phosphoproteins capable of distinguishing disease states from healthy controls. The study demonstrates the potential feasibility of our robust analytical pipeline for cancer signaling monitoring by tracking plasma EV phosphorylation.

The Blood Circulating Rare Cell Population. What is it and What is it Good For?

Blood contains a diverse cell population of low concentration hematopoietic as well as non-hematopoietic cells. The majority of such rare cells may be bone marrow-derived progenitor and stem cells. This paucity of circulating rare cells, in particular in the peripheral circulation, has led many to believe that bone marrow as well as other organ-related cell egress into the circulation is a response to pathological conditions. Little is known about this, though an increasing body of literature can be found suggesting commonness of certain rare cell types in the peripheral blood under physiological conditions. Thus, the isolation and detection of circulating rare cells appears to be merely a technological problem. Knowledge about rare cell types that may circulate the blood stream will help to advance the field of cell-based liquid biopsy by supporting inter-platform comparability, making use of biological correct cutoffs and “mining” new biomarkers and combinations thereof in clinical diagnosis and therapy. Therefore, this review intends to lay ground for a comprehensive analysis of the peripheral blood rare cell population given the necessity to target a broader range of cell types for improved biomarker performance in cell-based liquid biopsy.

Refining Cancer Management Using Integrated Liquid Biopsy.

Liquid biopsy has emerged in the last ten years as an appealing noninvasive strategy to support early cancer diagnosis and follow-up interventions. However, conventional liquid biopsy strategies involving specified biomarkers have encountered unexpected inconsistencies stemming from the use of different analytical methodologies. Recent reports have repeatedly demonstrated that integrated detection of multiple liquid biopsy biomarkers can significantly improve diagnostic performance by eliminating the influence of intratumoral heterogeneity. Herein, we review the progress in the field of liquid biopsy and propose a novel integrated liquid biopsy framework consisting of three categories: elementary, intermediate, and advanced integration. We also summarize the merits of the integration strategy and propose a roadmap toward refining cancer diagnosis, metastasis surveillance, and prognostication.

The Translational Status of Cancer Liquid Biopsies

Precision oncology aims to tailor clinical decisions specifically to patients with the objective of improving treatment outcomes. This can be achieved by leveraging omics information for accurate molecular characterization of tumors. Tumor tissue biopsies are currently the main source of information for molecular profiling. However, biopsies are invasive and limited in resolving spatiotemporal heterogeneity in tumor tissues. Alternative non-invasive liquid biopsies can exploit patient’s body fluids to access multiple layers of tumor-specific biological information (genomes, epigenomes, transcriptomes, proteomes, metabolomes, circulating tumor cells, and exosomes). Analysis and integration of these large and diverse datasets using statistical and machine learning approaches can yield important insights into tumor biology and lead to discovery of new diagnostic, predictive, and prognostic biomarkers. Translation of these new diagnostic tools into standard clinical practice could transform oncology, as demonstrated by a number of liquid biopsy assays already entering clinical use. In this review, we highlight successes and challenges facing the rapidly evolving field of cancer biomarker research.Lay SummaryPrecision oncology aims to tailor clinical decisions specifically to patients with the objective of improving treatment outcomes. The discovery of biomarkers for precision oncology has been accelerated by high-throughput experimental and computational methods, which can inform fine-grained characterization of tumors for clinical decision-making. Moreover, advances in the liquid biopsy field allow non-invasive sampling of patient’s body fluids with the aim of analyzing circulating biomarkers, obviating the need for invasive tumor tissue biopsies. In this review, we highlight successes and challenges facing the rapidly evolving field of liquid biopsy cancer biomarker research.

Detection of incipient tumours by screening of circulating plasma DNA: hype or hope?

ABSTRACTBackground: The last half-decade has been marked by a rapid expansion of research efforts in the field of so-called liquid biopsies, thereby investigating the potential of blood-derived cell-free tumour DNA (ctDNA) markers for application in clinical oncological management. The analysis of cfDNA appears to be particularly attractive for therapy monitoring purposes, while in terms of early cancer diagnosis and screening the potentials are just starting to be explored. Challenges, both of biological and technical nature, need to be addressed. One such challenge is to overcome the low levels of ctDNA in the circulation, intrinsic to many early-stage cancers. Methods: Here, we give an overview of the features of ctDNA and the approaches that are currently being applied with the ultimate aim to detect tumours in a presymptomatic stage. Conclusion: Although many studies report encouraging results, further technical development and larger studies are warranted before application of ctDNA analysis may find its place in clinic.

The Different Facets of Liquid Biopsy: A Kaleidoscopic View.

The current limitations of cancer diagnosis and molecular profiling based on invasive tissue biopsies or clinical imaging have led to the development of the liquid biopsy field. Liquid biopsy includes the isolation of circulating tumor cells (CTCs), circulating free or tumor DNA (cfDNA or ctDNA), extracellular vesicles (EVs), and tumor-educated platelets (TEPs) from body fluid samples and their molecular characterization to identify biomarkers for early cancer diagnosis, prognosis, therapeutic prediction, and follow-up. These innovative biosources show similar features as the primary tumor from where they originated or interacted. This review describes the different technologies and methods used for processing these biosources as well as their main clinical applications with their advantages and limitations.

Magnetic nanotechnology for circulating tumor biomarkers screening: Rational design, microfluidics integration and applications

Magnetic nanotechnology represents a major and promising frontier with great potential to significantly advance the field of liquid biopsies. The last decade has witnessed considerable progress in the research and development of magnetic nanosystems for circulating tumor biomarkers screening. With the emergence of microfluidics, both rational design of magnetic nanomaterials from microfluidic reactors and efficient magnetic screening of circulating tumor biomarkers from microfluidic chips become available. This review focuses on recent advances of magnetic nanoparticles for the screening of circulating tumor biomarkers including circulating tumor cells, exosomes, and nucleic acids. We summarize the established conventional magnetic nanosystems for circulating tumor biomarkers screening, highlight microfluidic reactors-enabled magnetic nanoparticles synthesis, and discuss the emerging roles of microfluidic chips in magnetic screening of circulating tumor biomarkers. In addition, the current challenges and opportunities are provided for guiding future studies.

The future of cell-free DNA testing to guide therapeutic decisions in B-cell lymphomas.

Tissue biopsy is the current gold standard technique for diagnosis and molecular profiling of lymphomas, but it carries several disadvantages in terms of procedural risks (infectious and haemorrhagic complications, anaesthesiologic risks) and analytic aspects (heterogeneity of tumors, low representation of tumor cells in the tissue). Noninvasive genotyping of B-cell lymphomas through circulating tumor DNA (ctDNA) is emerging as a practical tool to monitor the genetics and course of the disease from diagnosis to eventual relapse.This review will explore recent advances in the field of liquid biopsy in lymphomas, highlighting their clinical implications. ctDNA has been recently proposed an alternative source of tumor DNA for genotyping purposes, especially for those samples having low tumor representation or when longitudinal genetic monitoring is limited by the inaccessibility of relapsed tumor tissues. Also, ctDNA has been recently proposed radiation-free tool for the early identification of chemorefractory lymphoma patients. The detection of ctDNA circulating in the bloodstream of lymphoma patients can inform about the genetics of the disease at diagnosis identifying druggable alterations, detect the onset of mutation of resistance during treatment, anticipate about relapse earlier than standard methods [e.g. PET associated with computed tomography (PET/CT)] during follow-up.

CtDNA as a noninvasive monitoring tool in metastatic melanoma.

9548 Background: The field of liquid biopsy provides a promising alternative to standard tissue biopsies. Previous work has shown that plasma circulating cell-free DNA (ctDNA) can reflect the heterogeneous spectrum of mutations in cancer including metastatic melanoma. Our project aimed to establish and statistically validate plasma-based assays for tumour load and therapy monitoring in melanoma. Methods: On a large cohort of stage III and stage IV melanoma patients (N = 96) who received signalling targeted or immune checkpoint inhibitors we showed that the most common oncogenic drivers of this disease such as the BRAFV600E, NRASQ61 and the TERTC250T and TERTC228T promoter mutations (termed TERTprom) can be analysed in ctDNA with highly sensitive droplet digital PCR technology (detection of mutant ctDNA down to 0.01% analytical sensitivity). Results: Our research has demonstrated that ctDNA (irrespective of the genotype) significantly correlates with tumour stage (P < 0.05). Using receiver operating characteristics (ROC) analyses thresholds were established for risk stratification and response prediction. Elevated ctDNA at baseline was a significant predictor of disease progression compared to elevated LDH or S100 in multivariable cox proportional hazards model (Hazard ratio [HR] 7.43, P = 0.05). During therapy, patients with low ctDNA load (below the ROC threshold) had significantly better radiological outcomes and prolonged progression free survival (PFS) compared to patients with high ctDNA load (P < 0.0001). Our findings were confirmed on an independent cohort of metastatic melanoma patients (N = 35) treated with immune checkpoint inhibitors, where also during therapy low ctDNA load correlated with prolonged PFS (P = 0.003). An added benefit of ctDNA was demonstrated in about 80% of the patients, where ctDNA analyses preceded the radiological diagnosis of response or relapse. Progression was detected in plasma ctDNA in average 3.5 months earlier as compared to routine imaging techniques. Finally, we demonstrated that the occurrence of NRASQ61 mutation in BRAFV600-inhibitor treated patients at therapy baseline was associated with treatment failure. The sub-clonal NRASQ61 mutation at therapy baseline was an independent predictor of shorter PFS (HR 2.69, P = 0.02) as compared to BRAFV600E patients without the NRASQ61 mutation at therapy baseline. Conclusions: In sum, our results support the value of ctDNA as a sensitive biomarker for real-time therapy monitoring and early detection of disease progression.

Role of Liquid Biopsy in Clinical Decision-Making for Breast Cancer

Liquid biopsies are easily obtainable, non-invasive, longitudinal snapshots that can be used to measure micrometastatic disease burden, monitor disease progression, and provide genomic assessments of primary tumor/metastatic lesions. To date, most published studies have focused on circulating tumor cells (CTCs) and cell-free circulating tumor DNA (ctDNA); however, the liquid biopsy field is expanding exponentially and new blood components are currently under investigation. CTCs and ctDNA remain the most extensively studied liquid biopsy components to date. Several additional blood-based components are the basis of active, ongoing investigations. Some on the horizon include serum/plasma exosomes, platelet-mRNA, miRNA characterization, and global proteomic studies. In the era of individualized medicine, liquid biopsy has potential to improve upon current breast cancer management by offering dynamic monitoring possibilities as well as novel targets for therapy.

Tumor-Derived Extracellular Vesicles as a Novel Source of Protein Biomarkers for Cancer Diagnosis and Monitoring.

"Liquid biopsies" have received attention as a complementary tool for traditional tissue biopsies that may enhance the spectrum of analysis for tumor-derived factors. One such factor gaining prominence in the liquid biopsy field is extracellular vesicles (EVs), membrane-bound nanovesicles which are secreted by cells into biofluids such as blood, urine, and saliva. EVs are released in both physiological and pathological conditions and can transport a variety of molecules, including proteins, metabolites, RNA, microRNAs, and DNA, to distant sites throughout the body. As such, they are emerging as a promising source of tumor biomarkers for the noninvasive diagnosis, prognosis, and monitoring of cancer patients. In particular, the wealth of tumor-related information that can be gleaned from the EV proteomic cargo has become apparent through mass spectrometric analysis, which has provided new benchmarks for clinically focused biomarker research. In this review, the current achievements in the use of MS for identifying potential EV-derived protein biomarkers of cancer are explored, and the techniques and challenges involved in this pursuit are summarized.