Search

Context.— The process for identifying patients with monoclonal gammopathies is complex. Initial detection of a monoclonal immunoglobulin protein (M protein) in the serum or urine often requires compilation of analytical data from several areas of the laboratory. The detection of M proteins depends on adequacy of the sample provided, available clinical information, and the laboratory tests used. Objective.— To develop an evidence-based guideline for the initial laboratory detection of M proteins. Design.— To develop evidence-based recommendations, the College of American Pathologists convened a panel of experts in the diagnosis and treatment of monoclonal gammopathies and the laboratory procedures used for the initial detection of M proteins. The panel conducted a systematic literature review to address key questions. Using the Grading of Recommendations Assessment, Development, and Evaluation approach, recommendations were created based on the available evidence, strength of that evidence, and key judgements as defined in the Grading of Recommendations Assessment, Development, and Evaluation Evidence to Decision framework. Results.— Nine guideline statements were established to optimize sample selection and testing for the initial detection and quantitative measurement of M proteins used to diagnose monoclonal gammopathies. Conclusions.— This guideline was constructed to harmonize and strengthen the initial detection of an M protein in patients displaying symptoms or laboratory features of a monoclonal gammopathy. It endorses more comprehensive initial testing when there is suspicion of amyloid light chain amyloidosis or neuropathies, such as POEMS (polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes) syndrome, associated with an M protein.

PURPOSE This guideline provides recommendations for available tissue-based prostate cancer biomarkers geared toward patient selection for active surveillance, identification of clinically significant disease, choice of postprostatectomy adjuvant versus salvage radiotherapy, and to address emerging questions such as the relative value of tissue biomarkers compared with magnetic resonance imaging. METHODS An ASCO multidisciplinary Expert Panel, with representatives from the European Association of Urology, American Urological Association, and the College of American Pathologists, conducted a systematic literature review of localized prostate cancer biomarker studies between January 2013 and January 2019. Numerous tissue-based molecular biomarkers were evaluated for their prognostic capabilities and potential for improving management decisions. Here, the Panel makes recommendations regarding the clinical use and indications of these biomarkers. RESULTS Of 555 studies identified, 77 were selected for inclusion plus 32 additional references selected by the Expert Panel. Few biomarkers had rigorous testing involving multiple cohorts and only 5 of these tests are commercially available currently: Onco type Dx Prostate, Prolaris, Decipher, Decipher PORTOS, and ProMark. With various degrees of value and validation, multiple biomarkers have been shown to refine risk stratification and can be considered for select men to improve management decisions. There is a paucity of prospective studies assessing short- and long-term outcomes of patients when these markers are integrated into clinical decision making. RECOMMENDATIONS Tissue-based molecular biomarkers (evaluating the sample with the highest volume of the highest Gleason pattern) may improve risk stratification when added to standard clinical parameters, but the Expert Panel endorses their use only in situations in which the assay results, when considered as a whole with routine clinical factors, are likely to affect a clinical decision. These assays are not recommended for routine use as they have not been prospectively tested or shown to improve long-term outcomes—for example, quality of life, need for treatment, or survival. Additional information is available at www.asco.org/genitourinary-cancer-guidelines .

IgA nephropathy (IgAN) is an autoimmune disease and the most common primary glomerulonephritis. The four-hit hypothesis describes mechanism of the disease, from synthesis of galactose deficient IgA (GD-IgA), to recognition of GD-IgA by anti-glycan antibodies and deposition of the formed immune complex in the mesangium. Complement and coagulation cascade activation ensues, resulting in mesangial activation and cytokine release, podocyte injury, mesangial sclerosis and tubulointerstitial damage. Currently, there is no disease cure, and 30-40% of patients progress to end stage renal disease.Using complementary bioinformatic approaches, we demonstrate different levels of deviation of the transcriptome of the glomerulus in IgAN from normal, with the aim of identifying therapeutic targets. Approaches used herein include, deconvolution of the transcriptome to estimate immune constitution, co-regulation-based functional analysis of differentially expressed genes, modular co-expression analysis, network analysis of metabolic pathways and differential gene correlation analysis.We describe the immune composition in IgAN and the relatively low fold changes of the abundance of different immune cells and strength of immune signatures compared with control. Additionally, we identify enrichment of the intestinal network for IgA synthesis, repression of expression and dysregulation of networks of amino acid metabolism and PPAR signaling pathways in IgAN glomeruli. We also find loss of correlation between expression of matrix synthesizing and matrix degrading genes in IgAN.We conclude by discussing how therapies based on some nodes in these altered pathways described have been shown to be efficacious in IgAN and/or other inflammatory diseases and the potential of others in effective treatment.

ObjectivesLaboratory testing and the measurement of appropriate biomarkers play a critical role in managing patients with coronavirus disease 2019 (COVID-19), allowing for disease diagnosis, monitoring progression, prognostication, prediction of treatment response, and risk stratification. We sought to characterize these effects on a more detailed, mechanistic level.MethodsWe reviewed the literature and identified a multitude of reports that describe the unique effects of this virus and its devastating consequences to multiple organ systems in COVID-19 patients.ResultsThere are specific alterations in biomarkers related to coagulation, depopulation of T-cell subtypes, the cytokine storm and inflammation, and kidney and cardiac dysfunction.ConclusionsLaboratory measurement of specific parameters and the use of appropriate prognostic, predictive, and monitoring biomarkers afford clinicians the ability to make informed medical decisions and guide therapy for patients afflicted with this dreaded disease.

Current serological immunoassays have inherent limitations for certain infectious diseases such as Lyme disease, a bacterial infection caused by Borrelia burgdorferi in North America. Here we report a novel method of manufacturing high-density multiplexed protein microarrays with the capacity to detect low levels of antibodies accurately from small blood volumes in a fully automated system. A panel of multiple serological markers for Lyme disease are measured using a protein microarray system, Lyme Immunochip, in a single step but interpreted adhering to the standard two-tiered testing algorithm (enzyme immunoassay followed by Western blot). Furthermore, an enhanced IgM assay was supplemented to improve the test’s detection sensitivity for early Lyme disease. With a training cohort (n = 40) and a blinded validation cohort (n = 90) acquired from CDC, the Lyme Immunochip identified a higher proportion of Lyme disease patients than the two-tiered testing (82.4% vs 70.6% in the training set, 66.7% vs 60.0% in the validation set, respectively). Additionally, the Immunochip improved sensitivity to 100% while having a lower specificity of 95.2% using a set of investigational antigens which are being further evaluated with a large cohort of blinded samples from the CDC and Columbia University. This universal microarray platform provides an unprecedented opportunity to resolve a broad range of issues with diagnostic tests, including multiplexing, workflow simplicity, and reduced turnaround time and cost.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma, and it constitutes biologically heterogeneous entities. Standard first-line therapies cure ~60% of patients, the rest being either refractory or experiencing relapse. Currently, there are no robust predictive biomarkers of therapeutic response. Heterogeneity of DLBCL is partly explained by the cell of origin (COO), ie, germinal centre B cell or activated B cell, with the latter exhibiting worse prognosis. While gene expression profiling (GEP) is the gold standard for determining COO, surrogate immunohistochemical algorithms are used clinically, but show significant discordance with GEP. Recently, additional genetic subgroups with different prognoses have been reported. However, the tools/expertise required for analysis prohibit widespread deployment. Liquid biopsy-based assays show promise in providing clinically actionable information, are noninvasive and facilitate serial sampling to assess mechanisms of therapy resistance. Circulating, cell-free DNA analysis has shown enhanced sensitivity for detecting molecular alterations, but this modality cannot determine alterations of the tumor proteome or on signalling pathways. Exosomes are endosomally derived vesicles, are found in high abundance in body fluids and are readily isolated using a variety of methods. Tumour-derived exosomes can yield data regarding genetic, transcriptional, and proteomic changes useful for diagnosis, prognosis, and therapy of DLBCL. At present, standardized techniques for isolating exosomes are lacking and discriminating between exosomes from neoplastic and normal B cells is challenging. Refinements in isolation procedures are required to realize their full potential as precision medicine tools to provide comprehensive information on disease subtypes, identify prognostic factors, allow real-time monitoring of therapy response and delineate novel drug targets.

Exosomes are small (30-200nm) membrane-bound vesicles released by all cells. They have been found in many clinical sample types, particularly those that are obtained in minimally-invasive fashion, including serum/plasma, urine, cerebrospinal and other fluids. They serve as an ideal source of biomarkers as their contents reflect the cell of origin and disease status of patients. Exosomes can serve as a "liquid biopsy," a non-invasive means to assess disease/health status in real-time. They can provide insights into disease mechanisms as they carry and transfer important signaling molecules, which may induce changes in the recipient cells. This is particularly relevant for metastatic cancers, as exosomes can prime the pre-metastatic niche. Many different approaches can be used to characterize the effects of the transfer of exosome content into the recipient cells, including global, untargeted approaches and protein-specific, targeted approaches. We describe herein our studies on the use of antibody arrays to probe for protein expression changes in hepatocytes that result from treating these cells with exosomes derived from uveal melanoma cells.

Uveal melanoma (UM) is a rare melanoma subtype with an estimated annual incidence of approximately 2000 in the United States. Despite excellent rates of local disease control with surgery or radiotherapy, nearly 50% of patients will develop metastatic disease, mostly to the liver, with a life expectancy of 12 months. The receptor tyrosine kinase MET is overexpressed in 60-86% of cases and, together with its ligand HGF, it plays a significant role in UM migration and metastasis. In recent years, it has been demonstrated that tumor cells communicate with the microenvironment at distant organs by secreting exosomes, extracellular small vesicles released into body fluids, to support cancer development, immune evasion and metastatic progression. Exosomes are able to stably transfer their unique cargos of proteins and RNA from the primary tumor to distant sites, such as the pre-metastatic “niche”. The functional role of UM exosomes in the tumor microenvironment cross-talk is largely unknown. To get deeper insight into UM exosome biology, we have isolated exosomes from UM cells before and after treatment with the MET inhibitor crizotinib and examined their content by proteomic analysis. Approximately 600 proteins were identified from each of these samples, with 444 proteins in common and 145 and 167 proteins uniquely detected in exosomes from untreated and treated cells, respectively. The expression of several exosome-associated proteins, including MET, were suppressed by crizotinib, suggesting that the exosomal cargo can be pharmacologically manipulated. Exposure of normal hepatocytes to UM-derived exosomes results in activation of MET in a dose dependent fashion, with induction of both the MAPK and AKT/PI3K pathways. Exosomes derived from serum of patients with advanced uveal melanoma also express MET and induced similar effects in hepatocytes. Furthermore, UM-derived exosomes activate the expression of pro-angiogenic and pro-inflammatory factors in normal hepatocytes, while exosomes isolated from crizotinib-treated UM cells reduced these effects. UM-derived exosomes also stimulated immune cells to release cytokines and chemokines, including IL-6, IL-8 CCL2, CCL3, CCL5 and CXCL1. Functionally, we demonstrated that conditioned media from exosome-treated hepatocyte highly stimulated UM cell migration, which could be repressed by the combination of MET and cytokine receptor inhibitors. These findings provide evidence that UM derived exosomes play a role in reprogramming the recipient cells to facilitate the establishment of metastasis, and the inhibition of exosome-mediated signaling may provide a potential therapeutic strategy in the early stages of metastasis. Citation Format: Grazia Ambrosini, Alex J. Rai, Blake Ebert, Gary K. Schwartz. Uveal melanoma exosomes express MET and re-program the recipient cells [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 6082.

Purpose.—Clinical use of analytical tests to assess genomic variants in circulating tumor DNA (ctDNA) is increasing. This joint review from the American Society of Clinical Oncology and the College of American Pathologists summarizes current information about clinical ctDNA assays and provides a framework for future research.Methods.—An Expert Panel conducted a literature review on the use of ctDNA assays for solid tumors, including preanalytical variables, analytical validity, interpretation and reporting, and clinical validity and utility.Results.—The literature search identified 1338 references. Of those, 390, plus 31 references supplied by the Expert Panel, were selected for full-text review. There were 77 articles selected for inclusion.Conclusions.—The evidence indicates that testing for ctDNA is optimally performed on plasma collected in cell stabilization or EDTA tubes, with EDTA tubes processed within 6 hours of collection. Some ctDNA assays have demonstrated clinical validity and utility with certain types of advanced cancer; however, there is insufficient evidence of clinical validity and utility for the majority of ctDNA assays in advanced cancer. Evidence shows discordance between the results of ctDNA assays and genotyping tumor specimens, and supports tumor tissue genotyping to confirm undetected results from ctDNA tests. There is no evidence of clinical utility and little evidence of clinical validity of ctDNA assays in early-stage cancer, treatment monitoring, or residual disease detection. There is no evidence of clinical validity or clinical utility to suggest that ctDNA assays are useful for cancer screening, outside of a clinical trial. Given the rapid pace of research, reevaluation of the literature will shortly be required, along with the development of tools and guidance for clinical practice.

Exosomes are RNA and protein-containing nanovesicles secreted by all cell types and found in abundance in body fluids, including blood, urine and cerebrospinal fluid. These vesicles seem to be a perfect source of biomarkers, as their cargo largely reflects the content of parental cells, and exosomes originating from all organs can be obtained from circulation through minimally invasive or non-invasive means. Here we describe an optimized procedure for exosome isolation and analysis using clinical samples, starting from quick and robust extraction of exosomes with Total exosome isolation reagent, then isolation of RNA followed by qRT-PCR. Effectiveness of this workflow is exemplified by analysis of the miRNA content of exosomes derived from serum samples – obtained from the patients with metastatic prostate cancer, treated prostate cancer patients who have undergone prostatectomy, and control patients without prostate cancer. Three promising exosomal microRNA biomarkers were identified, discriminating these groups: hsa-miR375, hsa-miR21, hsa-miR574.

Prostate cancer is a complex disease, with multiple subtypes and clinical presentations. Much progress has been made in recent years to understand the underlying genetic basis that drives prostate cancer. Such mechanistic information is useful for development of novel therapeutic targets, to identify biomarkers for early detection or to distinguish between aggressive and indolent disease, and to predict treatment outcome. Multiple tests have become available in recent years to address these clinical needs for prostate cancer. We describe several of these assays, summarizing test details, performance characteristics, and acknowledging their limitations. There is a pressing unmet need for novel biomarkers that can demonstrate improvement in these areas. We introduce one such candidate biomarker, RAD9, describe its functions in the DNA damage response, and detail why it can potentially fill this void. RAD9 has multiple roles in prostate carcinogenesis, making it potentially useful as a clinical tool for men with prostate cancer. RAD9 was originally identified as a radioresistance gene, and subsequent investigations revealed several key functions in the response of cells to DNA damage, including involvement in cell cycle checkpoint control, at least five DNA repair pathways, and apoptosis. Further studies indicated aberrant overexpression in approximately 45% of prostate tumors, with a strong correlation between RAD9 abundance and cancer stage. A causal relationship between RAD9 and prostate cancer was first demonstrated using a mouse model, where tumorigenicity of human prostate cancer cells after subcutaneous injection into nude mice was diminished when RNA interference was used to reduce the normally high levels of the protein. In addition to activity needed for the initial development of tumors, cell culture studies indicated roles for RAD9 in promoting prostate cancer progression by controlling cell migration and invasion through regulation of ITGB1 protein levels, and anoikis resistance by modulating AKT activation. Furthermore, RAD9 enhances the resistance of human prostate cancer cells to radiation in part by regulating ITGB1 protein abundance. RAD9 binds androgen receptor and inhibits androgen-induced androgen receptor's activity as a transcription factor. Moreover, RAD9 also acts as a gene-specific transcription factor, through binding p53 consensus sequences at target gene promoters, and this likely contributes to its oncogenic activity. Given these diverse and extensive activities, RAD9 plays important roles in the initiation and progression of prostate cancer and can potentially serve as a valuable biomarker useful in the management of patients with this disease.

TPS3154 Background: Assessing treatment response in castrate resistant prostate cancer (CRPC), remains a challenge due to the limited sensitivity and specificity of existing imaging modalities. Understanding prostate cancer biology with tumor biopsies does not address the issue of tumor heterogeneity or cellular degradation during the decalcification process of bone biopsies. Next generation positron emission tomography (PET) imaging and circulating biomarkers might provide additional insights on treatment responses and inform clinical decision-making earlier in therapy. 18F-DCFPyL (PyL) is a second-generation fluorinated PSMA PET tracer that has superior sensitivity and specificity to detect prostate cancer compared to standard imaging. Its role in assessing tumor response to therapy has not been evaluated. Circulating tumor DNA (ctDNA) in blood can provide tumor genomic information, while exosomes in serum and urine may provide data on the proteomic landscape of tumors. Methods: We are conducting a prospective study of 15 men with metastatic CRPC who are scheduled to start a new systemic therapy for their disease. Upon enrollment, subjects will have baseline assessments with standard cross-sectional imaging, 99mTc bone scan, and blood work. Standard scans will be performed every 8-12 weeks until progression of disease. PyL PET/CT scans and liquid biopsies (ctDNA and exosomes) will occur at baseline, 6 weeks after starting their new therapy, and at disease progression. Lesions seen on PET/CT images will be identified by a certified reader. The maximum standardized uptake value (SUVmax) will be measured and recorded in up to the five hottest lesions and normalized to a background SUVmean measured in the liver, spleen, kidney, mediastinum, and parotid glands. Changes in the normalized SUV from baseline to the 6-week PyL PET scan will be correlated to PSA response by the Pearson’s correlation coefficient. The Kaplan-Meier method will be used to evaluate progression free survival and overall survival dichotomized by the median value of SUV change. Blood for ctDNA and exosomes will be stored for future analysis. The study is open with two patients enrolled at the time of submission. One patient has completed his initial PyL PET/CT scan. Clinical trial information: NCT03585114.

Purpose Mesothelin is currently considered the best available serum biomarker of malignant pleural mesothelioma. To examine the diagnostic accuracy and use of serum mesothelin in early diagnosis, we performed an individual patient data (IPD) meta-analysis. Methods The literature search identified 16 diagnostic studies of serum mesothelin, measured with the Mesomark enzyme-linked immunosorbent assay. IPD of 4,491 individuals were collected, including several control groups and 1,026 patients with malignant pleural mesothelioma. Mesothelin levels were standardized for between-study differences and age, after which the diagnostic accuracy and the factors affecting it were examined with receiver operating characteristic (ROC) regression analysis. Results At a common diagnostic threshold of 2.00 nmol/L, the sensitivities and specificities of mesothelin in the different studies ranged widely from 19% to 68% and 88% to 100%, respectively. This heterogeneity can be explained by differences in study population, because type of control group, mesothelioma stage, and histologic subtype significantly affected the diagnostic accuracy. The use of mesothelin in early diagnosis was evaluated by differentiating 217 patients with stage I or II epithelioid and biphasic mesothelioma from 1,612 symptomatic or high-risk controls. The resulting area under the ROC curve was 0.77 (95% CI, 0.73 to 0.81). At 95% specificity, mesothelin displayed a sensitivity of 32% (95% CI, 26% to 40%). Conclusion In patients suspected of having mesothelioma, a positive blood test for mesothelin at a high-specificity threshold is a strong incentive to urge further diagnostic steps. However, the poor sensitivity of mesothelin clearly limits its added value to early diagnosis and emphasizes the need for further biomarker research.

ACADEMIC EMERGENCY MEDICINE 2012; 19: 56–62 © 2012 by the Society for Academic Emergency Medicine Abstract Objectives: The objective was to describe the association between two novel biomarkers, calprotectin and leucine-rich alpha glycoprotein-1 (LRG), and appendicitis in children. Methods: This was a prospective, cross-sectional study of children 3 to 18 years old presenting to a pediatric emergency department (ED) with possible appendicitis. Blood and urine samples were assayed for calprotectin and LRG via enzyme-linked immunosorbent assay (ELISA). Final diagnosis was determined by histopathology or telephone follow-up. Biomarker levels were compared for subjects with and without appendicitis. Recursive partitioning was used to identify thresholds that predicted appendicitis. Results: Of 176 subjects, mean (±SD) age was 11.6 (±4.0) years and 52% were male. Fifty-eight patients (34%) were diagnosed with appendicitis. Median plasma calprotectin, serum LRG, and urine LRG levels were higher in appendicitis versus nonappendicitis (p

Tumor markers are molecules derived from patient body fluids or tissues that are measured and can provide information useful for the management of cancer patients. Applications include detection of cancer, monitoring of disease progression, and evaluation of the effectiveness of therapeutic regimens, among others. Henry Bence-Jones discovered the first tumor marker in 1846—a protein seen in acidified urine of patients with multiple myeloma (1). Since that time, many more tumor markers have been described for a variety of other cancers. The Food and Drug Administration (FDA) has approved several of these markers (Table 1), and many more are being investigated and evaluated for clinical use. The goal of this chapter is to review how tumor marker assays are evaluated, describe the applications of tumor markers, provide details on the detection of tumor markers using immunoassays, and then list examples of clinically useful tumor markers that are used in the clinical laboratory today. Although the last few years have witnessed a plethora of developments in molecular testing of tumor markers, these will not be discussed here, as we will restrict our discussion to immunoassay-based detection methods.

This chapter reviews the design, performance, and application of tumor marker assays; interferences; specific tumor markers; and the impact of new technologies on the discovery of novel tumor markers. In most populations, there is an overlap between the noncancer and cancer groups, since individuals without cancer may exhibit elevated levels of a marker and some individuals with cancer may not show elevations in a particular marker. The possible applications for tumor markers include screening, diagnosis, monitoring of therapy, detection of disease recurrence, and determination of prognosis. The prevalence of most cancers in the population is low, but with the inappropriate sensitivity and specificity of some tumor marker assays, false-positive results may be generated. Surgical removal of the tumor should result in a dramatic fall of the tumor marker, consistent with its half-life. Antibodies can be employed for specific targeting of analytes in many complex biological matrices, including blood (whole or a portion thereof), urine, and other fluids. The specificity of the antigen-antibody interaction, coupled with the exquisite sensitivity of enhanced signal detection methods available today, makes immunoassays the method of choice in laboratory medicine.

Early detection remains the most promising approach to improve long-term survival of patients with ovarian cancer. In a five-center case-control study, serum proteomic expressions were analyzed on 153 patients with invasive epithelial ovarian cancer, 42 with other ovarian cancers, 166 with benign pelvic masses, and 142 healthy women. Data from patients with early stage ovarian cancer and healthy women at two centers were analyzed independently and the results cross-validated to discover potential biomarkers. The results were validated using the samples from two of the remaining centers. After protein identification, biomarkers for which an immunoassay was available were tested on samples from the fifth center, which included 41 healthy women, 41 patients with ovarian cancer, and 20 each with breast, colon, and prostate cancers. Three biomarkers were identified as follows: (a) apolipoprotein A1 (down-regulated in cancer); (b) a truncated form of transthyretin (down-regulated); and (c) a cleavage fragment of inter-α-trypsin inhibitor heavy chain H4 (up-regulated). In independent validation to detect early stage invasive epithelial ovarian cancer from healthy controls, the sensitivity of a multivariate model combining the three biomarkers and CA125 [74% (95% CI, 52–90%)] was higher than that of CA125 alone [65% (95% CI, 43–84%)] at a matched specificity of 97% (95% CI, 89–100%). When compared at a fixed sensitivity of 83% (95% CI, 61–95%), the specificity of the model [94% (95% CI, 85–98%)] was significantly better than that of CA125 alone [52% (95% CI, 39–65%)]. These biomarkers demonstrated the potential to improve the detection of early stage ovarian cancer.

Urine is a noninvasive sample that is ideal for screening, because it is easy to collect, cost-effective, and can provide a wealth of information on a patient's health status. We provide a brief discussion on the anatomy and physiology of the kidney, a concise overview on B and T cells as key mediators of the immune system, and then delve into the various B-cell neoplasms. This discussion details Waldenstrom's macroglobulinemia, plasmacytoma, heavy chain disease, amyloidosis, and multiple myeloma. Of primary clinical importance from a technical perspective, two commonly applied techniques for the separation and characterization of urine proteins include urine protein electrophoresis and urine protein immunofixation. Procedural details for both techniques are provided herein.

Background: There is a need to identify reliable markers for malignant mesothelioma. Soluble mesothelin related peptides (SMRP) and osteopontin (OPN) have gained interest in recent years for this purpose. Methods: SMRP (Fujirebio Diagnostics Inc.) and OPN (R&D Inc.) ELISA methods were evaluated for multiple parameters. Concentrations were measured in blood from patients with mesothelioma, normal healthy volunteers, and patients with other (non-mesothelioma) cancers. In silico analysis was performed using the GeoProfiles database. At the protein level, SMRP and OPN were measured in cell culture supernatants, and values were compared in patients pre- and post-extrapleural pneumonectomy. Results: The SMRP assay demonstrates intra-assay CVs of 2.3% and 3% (at 4.6 nM and 13.7 nM, respectively), and inter-assay CVs of 3.5% and 3.7% at the same concentrations. The limit of detection (LOD) is 0.182 nM. The OPN assay demonstrates intra-assay CVs of 5.8%, 4.1%, and 5.2% (at 1.9, 5.1, and 11.1 ng/mL, respectively), and inter-assay CVs of 8.5%, 8.4%, and 12.1% at the same concentrations. The LOD is 0.032 ng/mL. Both SMRP and OPN in mesothelioma patients were significantly higher than in patients with other (non-mesothelioma) cancer and in healthy volunteers. The two markers do not appear to correlate with each other and exhibit different tissue expression patterns. Protein concentrations of these markers are highest in different sets of cell lines. Finally, SMRP but not OPN concentrations were decreased in five of seven consecutive patients after extrapleural pneumonectomy, compared to their respective pre-operative values. Conclusions: These assays provide reliable and reproducible quantitation of SMRP and OPN proteins. Both are increased in mesothelioma patients compared to non-mesothelioma controls. However, the two analytes do not correlate with each other and show distinct expression profiles and protein expression. Concentrations of SMRP but not OPN are decreased in post-surgical samples. Our results further characterize these markers, establish assay performance characteristics, and lay the groundwork for further studies to measure these markers in blood. Clin Chem Lab Med 2010;48:271–8.

The screening of prostate cancer is usually based on serum prostate-specific antigen (PSA) level. However, PSA is a marker with low specificity and poor sensitivity for early diagnosis and risk stratification of prostate cancer. We performed a systematic review of publications that evaluated the accuracy and reliability of novel exosomal …

Biomarkers have become invaluable tools in the clinical space for cancer detection, diagnosis, patient prognosis and treatment selection. An ideal biomarker should be easily assayed with minimally invasive medical procedures but possess high sensitivity and specificity. Although many candidate biomarkers for various diseases have been proposed in the literature, very few have made their way to clinical use, and for certain types of cancer there are no reliable biomarker options. The use of prostate specific antigen (PSA) has led to a dramatic increase in the diagnosis of prostate cancer - over 230,000 new cases in 2014. However, most of these cases are of low risk disease that is unlikely to cause significant morbidity and mortality, leading to the accepted notion that prostate cancer is overdiagnosed. On the other hand, while it is a sensitive test, less than ¼ of patients undergoing a prostate biopsy based on PSA testing are actually found to have the disease. The identification of biomarkers that are more senstitive and specific predictors for the presence of prostate cancer, as well as for the aggressiveness of the disease, could decrease the complications and morbidity related to prostate biopsies and definitive treatment. The circulating miRNA in blood could potentially fill this void, providing biomarkers suitable for real-time detection and application to the management of prostate cancer patients. The main advantages of miRNAs versus longer molecules (mRNA, lncRNA) include: (i) smaller number of sequences The objective of this project was to develop a set of reagents and tools for efficient recovery and analysis of circulating miRNAs. Our team has built a complete workflow to provide: (1) high throughput isolation of total serum RNA using a new MagMaxTM mirVanaTM reagent kit for the KingFisherTM Flex and Duo instruments, and (2) downstream analysis of microRNA profiles by qRT-PCR using next generation TaqMan® microRNA assays. The effectiveness of this workflow is exemplified by analysis of a panel of miRNAs in samples derived from serum of patients with metastatic prostate cancer versus control groups. Following this fast and easy workflow suitable for serum and other body fluids, disease-specific RNA signatures can be identified and used as biomarkers. TaqMan® is a registered Trademark of Roche Molecular Systems, Inc. used under permission and license. Citation Format: Emily M. Zeringer, Alex J. Rai, Joel DeCastro, Luming Qu, Marie Gonzalez, Laura Chapman, Alexander V. Vlassov, Susan M. Magdaleno, Chunmei Liu, Fangqi Hu, Shoulian Dong, Linda Wong. A complete workflow for high throughput isolation of serum microRNAs and downstream analysis by qRT-PCR: application to cancer biomarker discovery. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3387. doi:10.1158/1538-7445.AM2015-3387

Cancer proteomics is an exciting field that is witnessing many new developments in recent years. It is hoped that these advances will result in decreased cancer death rates, which have not declined dramatically in the last several decades. Some of the problems with current tumor markers include the lack of sensitivity and specificity, factors that prevent their use in population-based screening of disease. Thus, there is an urgent need to identify novel biomarkers that can faithfully detect the disease state. As we are now in the post-genome era, many opportunities have been created. Genomic sequence data are available for human, as well as several other species. We are now poised to mine these data and to determine the functions of the encoded proteins constituting the human genome. Proteomics affords this opportunity by providing enhanced procedures and tools for discovery and also a framework for understanding these components in terms of pathogenesis. New technologies and improvements in existing methodologies will allow for the rapid growth in the identification and characterization of peptides and proteins that are unique to various clinical states. This technology can be successfully applied to clinical specimens for the identification of new tumor markers.

Context.—Current tumor markers for ovarian cancer still lack adequate sensitivity and specificity to be applicable in large populations. High-throughput proteomic profiling and bioinformatics tools allow for the rapid screening of a large number of potential biomarkers in serum, plasma, or other body fluids. Objective.—To determine whether protein profiles of plasma can be used to identify potential biomarkers that improve the detection of ovarian cancer. Design.—We analyzed plasma samples that had been collected between 1998 and 2001 from patients with sporadic ovarian serous neoplasms before tumor resection at various International Federation of Gynecology and Obstetrics stages (stage I [n = 11], stage II [n = 3], and stage III [n = 29]) and from women without known neoplastic disease (n = 38) using proteomic profiling and bioinformatics. We compared results between the patients with and without cancer and evaluated their discriminatory performance against that of the cancer antigen 125 (CA125) tumor marker. Results.—We selected 7 biomarkers based on their collective contribution to the separation of the 2 patient groups. Among them, we further purified and subsequently identified 3 biomarkers. Individually, the biomarkers did not perform better than CA125. However, a combination of 4 of the biomarkers significantly improved performance (P ≤ .001). The new biomarkers were complementary to CA125. At a fixed specificity of 94%, an index combining 2 of the biomarkers and CA125 achieves a sensitivity of 94% (95% confidence interval, 85%–100.0%) in contrast to a sensitivity of 81% (95% confidence interval, 68%–95%) for CA125 alone. Conclusions.—The combined use of bioinformatics tools and proteomic profiling provides an effective approach to screen for potential tumor markers. Comparison of plasma profiles from patients with and without known ovarian cancer uncovered a panel of potential biomarkers for detection of ovarian cancer with discriminatory power complementary to that of CA125. Additional studies are required to further validate these biomarkers.

Exosomes are small vesicles (30-150 nm) found in abundance in human body fluids which function as carriers of different species of RNA and protein between diverse locations in the body. The spectrum of current scientific interest in exosomes is wide and ranges from studying their functions and pathways to utilizing them in diagnostics and therapeutics development. As such, there is a growing need for quick and easy methods for both isolation of exosomes and analysis of their cargo. We present herein a workflow for exosome isolation and analysis which entails: (i) fast and efficient isolation of exosomes from serum, plasma, and urine of both healthy donors and patients with prostate cancer, using Total Exosome Isolation reagents; (ii) characterization of their size distribution and count with Nanosight LM10 instrument; (iii) extraction of exosome “cargo” with Total Exosome RNA and Protein Isolation kit; (iv) characterization of exosomal RNA content using the Ion Torrent PGM sequencing and qRT-PCR. The protocol described herein lays the groundwork for the development of a standardized operating procedure (SOP) for isolation of exosomes and downstream analysis of their constituents, using clinical samples. We demonstrate that cancer-specific RNA signatures residing within the exosomes can be delineated from different patient cohorts. This is the first step towards developing a method whereby performance characteristics can be measured and used to optimize a validated assay useful for routine testing of clinical samples. Citation Format: Robert A. Setterquist, Alex J. Rai, Emily Zeringer, Mu Li, Tim Barta, Jeoffrey Schageman, Susan Magdaleno, Alexander V. Vlassov. Development of a standard operating procedure for exosome isolation and analysis using clinical samples: Application to cancer biomarker discovery. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1870. doi:10.1158/1538-7445.AM2014-1870