Autoantibodies against one or more of these tumour-associated antigens appears to indicate the presence of early-stage breast cancers. Autoantibody assays against a panel of antigens could be used as an aid to mammography in the detection and diagnosis of early primary breast cancer, especially in younger women at increased risk of breast cancer where mammography is known to have reduced sensitivity and specificity.
Background: Autoantibodies may be present in a variety of underlying cancers several years before tumours can be detected and testing for their presence may allow earlier diagnosis. We report the clinical validation of an autoantibody panel in newly diagnosed patients with lung cancer (LC).Patients and methods: Three cohorts of patients with newly diagnosed LC were identified: group 1 (n = 145), group 2 (n = 241) and group 3 (n = 269). Patients were individually matched by gender, age and smoking history to a control individual with no history of malignant disease. Serum samples were obtained after diagnosis but before any anticancer treatment. Autoantibody levels were measured against a panel of six tumour-related antigens (p53, NY-ESO-1, CAGE, GBU4-5, Annexin 1 and SOX2). Assay sensitivity was tested in relation to demographic variables and cancer type/stage.Results: The autoantibody panel demonstrated a sensitivity/specificity of 36%/91%, 39%/89% and 37%/90% in groups 1, 2 and 3, respectively, with good reproducibility. There was no significant difference between different LC stages, indicating that the antigens included covered the different types of LC well.Conclusion: This assay confirms the value of an autoantibody panel as a diagnostic tool and offers a potential system for monitoring patients at high risk of LC.
Tumor-associated autoantibodies (AAbs) have been described in patients with lung cancer, and the EarlyCDT®-Lung test that measures such AAbs is available as an aid for the early detection of lung cancer in high-risk populations. Improvements in specificity would improve its cost-effectiveness, as well as reduce anxiety associated with false positive tests. Samples from 235 patients with newly diagnosed lung cancer and matched controls were measured for the presence of AAbs to a panel of six (p53, NY-ESO-1, CAGE, GBU4-5, Annexin I, and SOX2) or seven (p53, NY-ESO-1, CAGE, GBU4-5, SOX2, HuD, and MAGE A4) antigens. Data were assessed in relation to cancer type and stage. The sensitivity and specificity of these two panels were also compared in two prospective consecutive series of 776 and 836 individuals at an increased risk of developing lung cancer. The six-AAb panel gave a sensitivity of 39 % with a specificity of 89 %, while the seven-AAb panel gave a sensitivity of 41 % with a specificity of 91 % which, once adjusted for occult cancers in the population, resulted in a specificity of 93 %. Analysis of these AAb assays in the at-risk population confirmed that the seven-AAb panel resulted in a significant increase in the specificity of the test from 82 to 90 %, with no significant change in sensitivity. The change from a six- to a seven-AAb assay can improve the specificity of the test and would result in a PPV of 1 in 8 and an overall accuracy of 92 %.
Sixteen research groups participated in the ISOBM TD-4 Workshop in which the reactivity and specificity of 56 monoclonal antibodies against the MUC1 mucin was investigated using a diverse panel of target antigens and MUC1 mucin- related synthetic peptides and glycopeptides. The majority of antibodies (34/56) defined epitopes located within the 20-amino acid tandem repeat sequence of the MUC1 mucin protein core. Of the remaining 22 antibodies, there was evidence for the involvement of carbohydrate residues in the epitopes for 16 antibodies. There was no obvious relationship between the type of immunogen and the specificity of each antibody. Synthetic peptides and glycopeptides were analyzed for their reactivity with each antibody either by assay of direct binding (e.g. by ELISA or BiaCore) or by determining the capacity of synthetic ligands to inhibit antibody binding interactions. There was good concordance between the research groups in identifying antibodies reactive with peptide epitopes within the MUC1 protein core. Epitope mapping tests were performed using the Pepscan analysis for antibody reactivity against overlapping synthetic peptides, and results were largely consistent between research groups. The dominant feature of epitopes within the MUC1 protein core was the presence, in full or part, of the hydrophilic sequence of PDTRPAP. Carbohydrate epitopes were less easily characterized and the most useful reagents in this respect were defined oligosaccharides, rather than purified mucin preparations enriched in particular carbohydrate moieties. It was evident that carbohydrate residues were involved in many epitopes, by regulating epitope accessibility or masking determinants, or by stabilizing preferred conformations of peptide epitopes within the MUC1 protein core. Overall, the studies highlight concordance between groups rather than exposing inconsistencies which gives added confidence to the results of analyses of the specificity of anti-mucin monoclonal antibodies.
MUC1 is a high molecular weight glycoprotein that is overexpressed in breast cancer. Aberrant O-linked glycosylation of MUC1 in cancer has been implicated in disease progression. We investigated the O-linked glycosylation of MUC1 purified from the serum of an advanced breast cancer patient. O-Glycans were released by hydrazinolysis and analyzed by liquid chromatography-electrospray ionization-mass spectrometry and by high performance liquid chromatography coupled with sequential exoglycosidase digestions. Core 1 type glycans (83%) dominated the profile which also confirmed high levels of sialylation: 80% of the glycans were mono-, di- or trisialylated. Core 2 type structures contributed approximately 17% of the assigned glycans and the oncofoetal Thomsen-Friedenreich (TF) antigen (Galbeta1-3GalNAc) accounted for 14% of the total glycans. Interestingly, two core 1 type glycans were identified that had sialic acid alpha2-8 linked to sialylated core 1 type structures (9% of the total glycan pool). This is the first O-glycan analysis of MUC1 from the serum of a breast cancer patient; the results suggest that amongst the cell lines commonly used to express recombinant MUC1 the T47D cell line processes glycans that are most similar to patient-derived material.
Recent publications have reported the technical and clinical validation of EarlyCDT-Lung, an autoantibody test which detected elevated autoantibodies in 40% of lung cancers at diagnosis. This manuscript reports the results of EarlyCDT-Lung run on four new (postvalidation) data sets. The following four cohorts of patients (n ¼ 574) with newly diagnosed lung cancer were identified: group 1 (n ¼ 122), 100% small cell lung cancer (SCLC); group 2 (n ¼ 249), 97% non-small cell lung cancer (NSCLC); group 3 (n ¼ 122), 100% NSCLC; group 4 (n ¼ 81), 62% NSCLC. Serum samples were obtained after diagnosis, prior to any anticancer treatment. Autoantibody levels were measured against a panel of six tumor-related antigens (p53, NY-ESO-1, CAGE, GBU4-5, Annexin 1, and SOX2) in the EarlyCDT-Lung panel and previously established cutoffs applied. In groups 2, 3, and 4, patients were individually matched by gender, age, and smoking history to a control individual with no history of malignant disease. Assay sensitivity was tested in relation to cancer type and stage, and in the matched normals to demographic variables. The autoantibody panel showed sensitivity/specificity of 57%/n.d (not done) for SCLC in group 1, 34%/87% for NSCLC in group 2, 31% and 84% for NSCLC in group 3, and 35%/89% for NSCLC and 43%/89% for SCLC in group 4. There was no significant difference in positivity of EarlyCDT-Lung and different lung cancer stages. These studies confirm the value of an autoantibody assay, EarlyCDT-Lung, as an aid to detecting lung cancer in patients at high risk of the disease. Cancer Prev Res; 4(7); 1126-34. Ó2011 AACR.
Background: Publications on autoantibodies to tumour-associated antigens (TAAs) have failed to show either calibration or reproducibility data. The validation of a panel of six TAAs to which autoantibodies have been described is reported here.Materials and methods: Three separate groups of patients with newly diagnosed lung cancer were identified, along with control individuals, and their samples used to validate an enzyme-linked immunosorbant assay. Precision, linearity, assay reproducibility and antigen batch reproducibility were all assessed.Results: For between-replicate error, samples with higher signals gave coefficients of variation (CVs) in the range 7%–15%. CVs for between-plate variation were only 1%–2% higher. For between-run error, CVs were in the range 15%–28%. In linearity studies, the slope was close to 1.0 and correlation coefficient values were generally >0.8. The sensitivity and specificity of individual batches of antigen varied slightly between groups of patients; however, the sensitivity and specificity of the panel of antigens as a whole remained constant. The validity of the calibration system was demonstrated.Conclusions: A calibrated six-panel assay of TAAs has been validated for identifying nearly 40% of primary lung cancers via a peripheral blood test. Levels of reproducibility, precision and linearity would be acceptable for an assay used in a regulated clinical setting.
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