The CA 19-9 assay detects a carbohydrate antigen on multiple protein carriers, some of which may be preferential carriers of the antigen in cancer. We tested the hypothesis that the measurement of ...the CA 19-9 antigen on individual proteins could improve performance over the standard CA 19-9 assay. We used antibody arrays to measure the levels of the CA 19-9 antigen on multiple proteins in serum or plasma samples from patients with pancreatic adenocarcinoma or pancreatitis. Sample sets from three different institutions were examined, comprising 531 individual samples. The measurement of the CA 19-9 antigen on any individual protein did not improve upon the performance of the standard CA 19-9 assay (82% sensitivity at 75% specificity for early-stage cancer), owing to diversity among patients in their CA 19-9 protein carriers. However, a subset of cancer patients with no elevation in the standard CA 19-9 assay showed elevations of the CA 19-9 antigen specifically on the proteins MUC5AC or MUC16 in all sample sets. By combining measurements of the standard CA 19-9 assay with detection of CA 19-9 on MUC5AC and MUC16, the sensitivity of cancer detection was improved relative to CA 19-9 alone in each sample set, achieving 67-80% sensitivity at 98% specificity. This finding demonstrates the value of measuring glycans on specific proteins for improving biomarker performance. Diagnostic tests with improved sensitivity for detecting pancreatic cancer could have important applications for improving the treatment and management of patients suffering from this disease.
The validation of candidate biomarkers often is hampered by the lack of a reliable means of assessing and comparing performance. We present here a reference set of serum and plasma samples to ...facilitate the validation of biomarkers for resectable pancreatic cancer. The reference set includes a large cohort of stage I-II pancreatic cancer patients, recruited from 5 different institutions, and relevant control groups. We characterized the performance of the current best serological biomarker for pancreatic cancer, CA 19-9, using plasma samples from the reference set to provide a benchmark for future biomarker studies and to further our knowledge of CA 19-9 in early-stage pancreatic cancer and the control groups. CA 19-9 distinguished pancreatic cancers from the healthy and chronic pancreatitis groups with an average sensitivity and specificity of 70-74%, similar to previous studies using all stages of pancreatic cancer. Chronic pancreatitis patients did not show CA 19-9 elevations, but patients with benign biliary obstruction had elevations nearly as high as the cancer patients. We gained additional information about the biomarker by comparing two distinct assays. The two CA 9-9 assays agreed well in overall performance but diverged in measurements of individual samples, potentially due to subtle differences in antibody specificity as revealed by glycan array analysis. Thus, the reference set promises be a valuable resource for biomarker validation and comparison, and the CA 19-9 data presented here will be useful for benchmarking and for exploring relationships to CA 19-9.
Cancer serum protein profiling by mass spectrometry has uncovered mass profiles that are potentially diagnostic for several common types of cancer. However, direct mass spectrometric profiling has a ...limited dynamic range and difficulties in providing the identification of the distinctive proteins. We hypothesized that distinctive profiles may result from the differential expression of relatively abundant serum proteins associated with the host response.
Eighty-four antibodies, targeting a wide range of serum proteins, were spotted onto nitrocellulose-coated microscope slides. The abundances of the corresponding proteins were measured in 80 serum samples, from 24 newly diagnosed subjects with lung cancer, 24 healthy controls, and 32 subjects with chronic obstructive pulmonary disease (COPD). Two-color rolling-circle amplification was used to measure protein abundance.
Seven of the 84 antibodies gave a significant difference (p < 0.01) for the lung cancer patients as compared to healthy controls, as well as compared to COPD patients. Proteins that exhibited higher abundances in the lung cancer samples relative to the control samples included C-reactive protein (CRP; a 13.3 fold increase), serum amyloid A (SAA; a 2.0 fold increase), mucin 1 and alpha-1-antitrypsin (1.4 fold increases). The increased expression levels of CRP and SAA were validated by Western blot analysis. Leave-one-out cross-validation was used to construct Diagonal Linear Discriminant Analysis (DLDA) classifiers. At a cutoff where all 56 of the non-tumor samples were correctly classified, 15/24 lung tumor patient sera were correctly classified.
Our results suggest that a distinctive serum protein profile involving abundant proteins may be observed in lung cancer patients relative to healthy subjects or patients with chronic disease and may have utility as part of strategies for detecting lung cancer.
Changes to the glycan structures of proteins secreted by cancer cells are known to be functionally important and to have potential diagnostic value. However, an exploration of the population ...variation and prevalence of glycan alterations on specific proteins has been lacking because of limitations in conventional glycobiology methods. Here we report the use of a previously developed antibody-lectin sandwich array method to characterize both the protein and glycan levels of specific mucins and carcinoembryonic antigen-related proteins captured from the sera of pancreatic cancer patients (n = 23) and control subjects (n = 23). The MUC16 protein was frequently elevated in the cancer patients (65% of the patients) but showed no glycan alterations, whereas the MUC1 and MUC5AC proteins were less frequently elevated (30 and 35%, respectively) and showed highly prevalent (up to 65%) and distinct glycan alterations. The most frequent glycan elevations involved the Thomsen-Friedenreich antigen, fucose, and Lewis antigens. An unexpected increase in the exposure of α-linked mannose also was observed on MUC1 and MUC5ac, indicating possible N-glycan modifications. Because glycan alterations occurred independently from the protein levels, improved identification of the cancer samples was achieved using glycan measurements on specific proteins relative to using the core protein measurements. The most significant elevation was the cancer antigen 19-9 on MUC1, occurring in 19 of 23 (87%) of the cancer patients and one of 23 (4%) of the control subjects. This work gives insight into the prevalence and protein carriers of glycan alterations in pancreatic cancer and points to the potential of using glycan measurements on specific proteins for highly effective biomarkers.
The CA19-9 biomarker is elevated in a substantial group of patients with pancreatic ductal adenocarcinoma (PDAC), but not enough to be reliable for the detection or diagnosis of the disease. We ...hypothesized that a glycan called sTRA (sialylated tumor-related antigen) is a biomarker for PDAC that improves upon CA19-9.
We examined sTRA and CA19-9 expression and secretion in panels of cell lines, patient-derived xenografts, and primary tumors. We developed candidate biomarkers from sTRA and CA19-9 in a training set of 147 plasma samples and used the panels to make case-control calls, based on predetermined thresholds, in a 50-sample validation set and a blinded, 147-sample test set.
The sTRA glycan was produced and secreted by pancreatic tumors and models that did not produce and secrete CA19-9. Two biomarker panels improved upon CA19-9 in the training set, one optimized for specificity, which included CA19-9 and 2 versions of the sTRA assay, and another optimized for sensitivity, which included 2 sTRA assays. Both panels achieved statistical improvement (
< 0.001) over CA19-9 in the validation set, and the specificity-optimized panel achieved statistical improvement (
< 0.001) in the blinded set: 95% specificity and 54% sensitivity (75% accuracy), compared with 97%/30% (65% accuracy). Unblinding produced further improvements and revealed independent, complementary contributions from each marker.
sTRA is a validated serological biomarker of PDAC that yields improved performance over CA19-9. The new panels may enable surveillance for PDAC among people with elevated risk, or improved differential diagnosis among patients with suspected pancreatic cancer.
Molecular markers to detect subtypes of cancer cells could facilitate more effective treatment. We recently identified a carbohydrate antigen, named sTRA, that is as accurate a serological biomarker ...of pancreatic cancer as the cancer antigen CA19-9. We hypothesized that the cancer cells producing sTRA are a different subpopulation than those producing CA19-9. The sTRA glycan was significantly elevated in tumor tissue relative to adjacent pancreatic tissue in 3 separate tissue microarrays covering 38 patients. The morphologies of the cancer cells varied in association with glycan expression. Cells with dual staining of both markers tended to be in well-to-moderately differentiated glands with nuclear polarization, but exclusive sTRA staining was present in small clusters of cells with poor differentiation and large vacuoles, or in small and ill-defined glands. Patients with higher dual-staining of CA19-9 and sTRA had statistically longer time-to-progression after surgery. Patients with short time-to-progression (<2 years) had either low levels of the dual-stained cells or high levels of single-stained cells, and such patterns differentiated short from long time-to-progression with 90% (27/30) sensitivity and 80% (12/15) specificity. The sTRA and CA19-9 glycans define separate subpopulations of cancer cells and could together have value for classifying subtypes of pancreatic adenocarcinoma.
Cystic lesions of the pancreas are increasingly being recognized due to the widespread use of high resolution abdominal imaging. Since certain cyst types are precursors to invasive cancer, this ...situation presents an opportunity to intervene prior to malignant progression. Effective implementation of that strategy has been hampered by difficulties in clearly distinguishing cystic lesions with no malignant potential from those with malignant potential. Here we explored whether glycosylation variants on specific proteins in cyst fluid samples could serve as biomarkers to aid in this diagnosis.
We used a novel antibody-lectin sandwich microarray method to measure the protein expression and glycosylation of mucin (MUC)1, MUC5AC, MUC16, carcinoembryonic antigen, and other proteins implicated in pancreatic neoplasia in cyst fluid samples. Fifty-three cyst fluid samples were obtained from patients with mucinous cystic neoplasms (n=17), intraductal papillary mucinous neoplasms (n=15), serous cystadenomas (n=12), or pseudocysts (n=9), with confirmation of histologic diagnosis at surgical resection.
The detection of a glycan variant on MUC5AC using the lectin wheat-germ agglutinin discriminated mucin-producing cystic tumors (mucinous cystic neoplasms+intraductal papillary mucinous neoplasms) from benign cystic lesions (serous cystadenomas+pseudocysts) with a 78% sensitivity at 80% specificity, and when used in combination with cyst fluid CA 19-9 gave a sensitivity of 87% at 86% specificity. These biomarkers performed better than cyst fluid carcinoembryonic antigen (37%/80% sensitivity/specificity).
These results demonstrate the value of glycan variants for biomarker discovery and suggest that these biomarkers could greatly enhance the accuracy of differentiating pancreatic cystic tumors. Validation studies will be required to determine the clinical value of these markers.
Abstract
Glycan arrays continue to be the primary resource for determining the glycan-binding specificity of proteins. The volume and diversity of glycan-array data are increasing, but no common ...method and resource exist to analyze, integrate, and use the available data. To meet this need, we developed a resource of analyzed glycan-array data called CarboGrove. Using the ability to process and interpret data from any type of glycan array, we populated the database with the results from 35 types of glycan arrays, 13 glycan families, 5 experimental methods, and 19 laboratories or companies. In meta-analyses of glycan-binding proteins, we observed glycan-binding specificities that were not uncovered from single sources. In addition, we confirmed the ability to efficiently optimize selections of glycan-binding proteins to be used in experiments for discriminating between closely related motifs. Through descriptive reports and a programmatically accessible Application Programming Interface, CarboGrove yields unprecedented access to the wealth of glycan-array data being produced and powerful capabilities for both experimentalists and bioinformaticians.
Glycan arrays have enabled detailed studies of the specificities of glycan-binding proteins. A challenge in the interpretation of glycan array data is to determine the specific features of glycan ...structures that are critical for binding. To address this challenge, we have developed a systematic method to interpret glycan array data using a motif-based analysis. Each glycan on a glycan array is classified according to its component sub-structures, or motifs. We analyze the binding of a given lectin to each glycan in terms of the motifs in order to identify the motifs that are selectively present in the glycans that are bound by the lectin. We compared two different methods to calculate the identification, termed intensity segregation and motif segregation, for the analysis of three well-characterized lectins with highly divergent behaviors. Both methods accurately identified the primary specificities as well as the weaker, secondary specificities of all three lectins. The complex binding behavior of wheat germ agglutinin was reduced to its simplified, independent specificities. We compiled the motif specificities of a wide variety of plant lectins, human lectins, and glycan-binding antibodies to uncover the relationships among the glycan-binding proteins and to provide a means to search for lectins with particular binding specificities. This approach should be valuable for rapidly analyzing and using glycan array data, for better describing and understanding glycan-binding specificities, and as a means to systematize and compare data from glycan arrays.
Profiling Bladder Cancer Using Targeted Antibody Arrays Sanchez-Carbayo, Marta; Socci, Nicholas D.; Lozano, Juan Jose ...
The American journal of pathology,
2006, 20060101, 2006-Jan, 2006-01-00, Letnik:
168, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Bladder cancer is a common malignancy requiring a high degree of surveillance because of the frequent recurrences and the poor clinical outcome of invasive disease. To date, serum biomarkers for ...bladder cancer lack optimal sensitivity and specificity to assist in diagnosis and disease categorization. Here, we designed antibody arrays for bladder cancer by selecting antibodies against targets differentially expressed in bladder tumors. Serum protein profiles measured by an antibody array containing 254 antibodies discriminated bladder cancer patients from controls (
n
= 95) with a correct classification rate of 93.7%. A second independent antibody array containing 144 antibodies revealed that protein profiles provide predictive information by stratifying patients with bladder tumors (
n
= 37) based on their overall survival (
P
= 0.0479). In addition, serum proteins, such as c-met, that were top ranked at identifying bladder cancer patients were associated with pathological stage, tumor grade, and survival when validated by immunohistochemistry of tissue microarrays containing bladder tumors (
n
= 173). This study provides experimental evidence for the use of several integrated technologies strengthening the process of biomarker discovery. Serum protein profiles obtained by antibody arrays represent comprehen-sive means for bladder cancer diagnosis and clinical outcome stratification, which could potentially assist in selection of cancer patients who would benefit from early, individualized therapeutic intervention.
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