Patients afflicted with pancreatic ductal adenocarcinoma (PDAC) face a dismal prognosis, but headway could be made if physicians could identify the disease earlier. A compelling strategy to broaden ...the use of surveillance for PDAC is to incorporate molecular biomarkers in combination with clinical analysis and imaging tools. This article summarizes the components involved in accomplishing biomarker validation and an analysis of the requirements of molecular biomarkers for disease surveillance. We highlight the significance of consortia for this research and highlight resources and infrastructure of the Early Detection Research Network (EDRN). The EDRN brings together the multifaceted expertise and resources needed for biomarker validation, such as study design, clinical care, biospecimen collection and handling, molecular technologies, and biostatistical analysis, and studies coming out of the EDRN have yielded biomarkers that are moving forward in validation. We close the article with an overview of the current investigational biomarkers, an analysis of their performance relative to the established benchmarks, and an outlook on the current needs in the field. The outlook for improving the early detection of PDAC looks promising, and the pace of further research should be quickened through the resources and expertise of the EDRN and other consortia.
The current best serum marker for pancreatic cancer, CA 19‐9, detects a carbohydrate antigen on multiple protein carriers. Better knowledge of the protein carriers of the CA 19‐9 antigen in various ...disease states may lead to improved diagnostic tests. To identify proteins that carry the CA 19‐9 antigen, we immunoprecipitated the CA 19‐9 antigen from pooled sera and identified the associated proteins using MS. Among the high‐confidence identifications, we confirmed the presence of the CA 19‐9 antigen on Apolipoprotein B‐100 by antibody arrays and Western blot and on kininogen, ARVCF, and Apolipoprotein E by antibody arrays. We characterized the frequency and levels of the CA 19‐9 antigen on the four proteins across various patient groups (pancreatic cancer, pancreatitis, and healthy controls) using antibody arrays. Nearly, 10–25% of the subjects showed elevations of the antigen on each protein, but the elevations were not associated with disease state or total CA 19‐9 levels. These results contribute to our knowledge of the carrier proteins of an important functional glycan and the rate at which the glycan is displayed. This work also demonstrates a strategy for using the complementary methods of MS and antibody microarrays to identify protein carriers of glycans and assess the diagnostic value of measuring glycans on individual proteins.
The antibody-lectin sandwich arrays (ALSA) is a powerful new tool for glycoproteomics research. ALSA enables precise measurements of the glycosylation states of multiple proteins captured directly ...from biological samples. The platform can be used in a high-throughput mode with low sample consumption, making it well suited to biomarker research exploring glycan alterations on specific proteins. This article provides detailed descriptions of the use of ALSA, with a particular focus on biomarker research. The preparation and selection of antibodies and lectins, the preparation and use of the arrays and samples, and special considerations for using the platform for biomarker research are covered.
To better understand the molecular mechanisms that underlie the tumorigenesis and progression of clear cell renal cell carcinoma (ccRCC), we studied the gene expression profiles of 29 ccRCC tumors ...obtained from patients with diverse clinical outcomes by using 21,632 cDNA microarrays. We identified gene expression alterations that were both common to most of the ccRCC studied and unique to clinical subsets. There was a significant distinction in gene expression profile between patients with a relatively non-aggressive form of the disease 100% survival after 5 years with the majority (15/17 or 88%) having no clinical evidence of metastasis versus patients with a relatively aggressive form of the disease (average survival time 25.4 months with a 0% 5-year survival rate). Approximately 40 genes most accurately make this distinction, some of which have previously been implicated in tumorigenesis and metastasis. To test the robustness and potential clinical usefulness of this molecular distinction, we simulated its use as a prognostic tool in the clinical setting. In 96% of the ccRCC cases tested, the prediction was compatible with the clinical outcome, exceeding the accuracy of prediction by staging. These results suggest that two molecularly distinct forms of ccRCC exist and that the integration of expression profile data with clinical parameters could serve to enhance the diagnosis and prognosis of ccRCC. Moreover, the identified genes provide insight into the molecular mechanisms of aggressive ccRCC and suggest intervention strategies.
Altered glycosylation on the surfaces or secreted proteins of tumor cells is common in pancreatic cancer and is thought to promote cancer progression, but the factors leading to the changes in ...carbohydrate structures are incompletely understood. We hypothesized that pro-inflammatory conditions can lead to alterations in cancer-associated glycans on mucins produced by pancreatic-cancer cells. With the use of a novel antibody-glycan microarray method, we measured the effects of pro-inflammatory stimuli (oxidative stress and treatment with the cytokines IFNγ, IL-1α, and TNFα) on the expression and glycosylation of the mucins MUC1, MUC5AC, and MUC16 in multiple pancreatic cancer cell lines. Mucin glycosylation was significantly affected in specific cell lines, particularly in structures involving terminal galactose or N-acetylgalactosamine. In addition, the responses of the cell lines grouped according to the expression of cell-surface markers that are associated with tumorigenicity, as cell lines bearing minimal surface markers, showed evidence of increased O-glycan extension and decreased presentation of terminal β1,4-linked galactose, opposite to cell lines bearing multiple markers. These results suggest mechanisms whereby inflammation might influence tumor behavior in a cell-type specific manner through modulating the presentation of cancer-associated glycans.
The CA 19‐9 antigen is currently the best individual marker for the detection of pancreatic cancer. In order to optimize the CA 19‐9 assay and to develop approaches to further improve cancer ...detection, it is important to understand the specificity differences between CA 19‐9 antibodies and the consequential affect on biomarker performance. Antibody arrays enabled multiplexed comparisons between five different CA 19‐9 antibodies used in the analysis of plasma samples from pancreatic cancer patients and controls. Major differences were observed between antibodies in their detection of particular patient samples. Glycan array analysis revealed that certain antibodies were highly specific for the canonical CA 19‐9 epitope, sialyl‐Lewis A, while others bound sialyl‐Lewis A in addition to a related structure called sialyl‐Lewis C and modification with Nue5Gc. In a much larger patient cohort, we confirmed the binding of sialyl‐Lewis C glycan by one of the antibodies and showed that the broader specificity led to the detection of an increased number of cancer patients without increasing detection of pancreatitis patient samples. This work demonstrates that variation between antibody specificity for cancer‐associated glycans can have significant implications for biomarker performance and highlights the value of characterizing and detecting the range of glycan structures that are elevated in cancer.
The sialyl-Lewis A (sLeA) glycan forms the basis of the CA19–9 assay and is the current best biomarker for pancreatic cancer, but because it is not elevated in ∼25% of pancreatic cancers, it is not ...useful for early diagnosis. We hypothesized that sLeA-low tumors secrete glycans that are related to sLeA but not detectable by CA19–9 antibodies. We used a method called motif profiling to predict that a structural isomer of sLeA called sialyl-Lewis X (sLeX) is elevated in the plasma of some sLeA-low cancers. We corroborated this prediction in a set of 48 plasma samples and in a blinded set of 200 samples. An antibody sandwich assay formed by the capture and detection of sLeX was elevated in 13 of 69 cancers that were not elevated in sLeA, and a novel hybrid assay of sLeA capture and sLeX detected 24 of 69 sLeA-low cancers. A two-marker panel based on combined sLeA and sLeX detection differentiated 109 pancreatic cancers from 91 benign pancreatic diseases with 79% accuracy (74% sensitivity and 78% specificity), significantly better than sLeA alone, which yielded 68% accuracy (65% sensitivity and 71% specificity). Furthermore, sLeX staining was evident in tumors that do not elevate plasma sLeA, including those with poorly differentiated ductal adenocarcinoma. Thus, glycan-based biomarkers could characterize distinct subgroups of patients. In addition, the combined use of sLeA and sLeX, or related glycans, could lead to a biomarker panel that is useful in the clinical diagnosis of pancreatic cancer.
Précis: This paper shows that a structural isomer of the current best biomarker for pancreatic cancer, CA19–9, is elevated in the plasma of patients who are low in CA19–9, potentially enabling more comprehensive detection and classification of pancreatic cancers.
DNA separations have been performed using microfabricated capillary electrophoresis chips and detected using a single-molecule fluorescence burst counting technique. We enhanced the percentage of ...electromigrating DNA molecules that were detected by focusing the sample through the 1-μm-diameter focused laser beam. The sample was focused by introducing a taper in the separation channel and by a sheath flow delivered from cross channels. The sample stream width, single-molecule velocities, and single-molecule count rates varied linearly with the current density ratio as expected. The optimal laser power for each focusing condition was investigated using dilute solutions of pBluescript DNA. Although fluorescence burst heights and background varied with laser power, the signal-to-noise ratio was only weakly dependent on this parameter using our single-molecule counting technique. Focused single-molecule counting was used to detect separations of a 100−1000-bp DNA sizing ladder. The increase in molecular detection efficiency was quantified by applying a focusing current midway through the ladder separation and comparing observed count rates to the known molecular concentration in the bands. The molecular detection efficiency varied linearly with the applied current density ratio, and greater than 3-fold enhancements in detection efficiency were achieved.
Four different immunoassay and antibody microarray methods performed at four different sites were used to measure the levels of a broad range of proteins (N = 323 assays; 39, 88, 168, and 28 assays ...at the respective sites; 237 unique analytes) in the human serum and plasma reference specimens distributed by the Plasma Proteome Project (PPP) of the HUPO. The methods provided a means to (1) assess the level of systematic variation in protein abundances associated with blood preparation methods (serum, citrate‐anticoagulated‐plasma, EDTA‐anticoagulated‐plasma, or heparin‐anticoagulated‐plasma) and (2) evaluate the dependence on concentration of MS‐based protein identifications from data sets using the HUPO specimens. Some proteins, particularly cytokines, had highly variable concentrations between the different sample preparations, suggesting specific effects of certain anticoagulants on the stability or availability of these proteins. The linkage of antibody‐based measurements from 66 different analytes with the combined MS/MS data from 18 different laboratories showed that protein detection and the quality of MS data increased with analyte concentration. The conclusions from these initial analyses are that the optimal blood preparation method is variable between analytes and that the discovery of blood proteins by MS can be extended to concentrations below the ng/mL range under certain circumstances. Continued developments in antibody‐based methods will further advance the scientific goals of the PPP.
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