Andrea Murray

Autoantibodies in lung cancer: possibilities for early detection and subsequent cure

Background: People with lung cancer usually present at a late stage in the course of their disease when their chances of long-term survival are low. At present there is little to offer for early diagnosis, even in those at high risk of developing the disease. Autoantibodies have been shown to be present in the circulation of people with various forms of solid tumour before cancer-associated antigens can be detected, and these molecules can be measured up to 5 years before symptomatic disease. Objective: To assess the potential of a panel of tumour-associated autoantibody profiles as an aid to other lung cancer screening modalities. Methods: Plasma from normal controls (n = 50), patients with non-small cell lung cancer (n = 82) and patients with small cell lung cancer (n = 22) were investigated for the presence of autoantibodies to p53, c-myc, HER2, NY-ESO-1, CAGE, MUC1 and GBU4-5 by enzyme-linked immunosorbent assay. Results: Raised levels of autoantibodies were seen to at least 1/7 antigens in 76% of all the patients with lung cancer plasma tested, and 89% of node-negative patients, with a specificity of 92%. There was no significant difference between the detection rates in the lung cancer subgroups, although more patients with squamous cell carcinomas (92%) could be identified. Conclusion: Measurement of an autoantibody response to one or more tumour-associated antigens in an optimised panel assay may provide a sensitive and specific blood test to aid the early detection of lung cancer.

Clinical validation of an autoantibody test for lung cancer

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.

EarlyCDT®-Lung test: improved clinical utility through additional autoantibody assays

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 %.

The O-linked glycosylation of secretory/shed MUC1 from an advanced breast cancer patient's serum

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.

EarlyCDT-Lung: An Immunobiomarker Test as an Aid to Early Detection of Lung Cancer

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 = 574) with newly diagnosed lung cancer were identified: group 1 (n = 122), 100% small cell lung cancer (SCLC); group 2 (n = 249), 97% non-small cell lung cancer (NSCLC); group 3 (n = 122), 100% NSCLC; group 4 (n = 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. ©2011 AACR.

Epitope Mapping of Anti-MUC1 Mucin Protein Core Monoclonal Antibodies

A panel of 56 murine monoclonal antibodies against the MUC1 mucin was analysed for reactivity against purified MUC1 mucin and synthetic MUC1 mucin protein core-related peptides. In an ELISA assay, with purified normal urinary mucin as the target antigen, 48/56 (86%) of antibodies showed positive reactivity. A smaller proportion of antibodies, 31/56 (55%), reacted with a bovine serum albumin conjugate containing the synthetic peptide, APDTRPAPG. This peptide represents the hydrophilic region of the tandem repeat sequence of the MUC1 mucin core. These 31 anti-mucin core antibodies were then evaluated for reactivity with a set of overlapping heptamers with sequences based upon that of the 20-amino acid tandem repeat of the MUC1 protein core. For this purpose, the ‘Pepscan’ procedure was employed to detect antibody binding to peptides tethered at their carboxy termini to the polypropylene pins. Synthetic peptides binding to these antibodies were identified for 29 of the samples provided and epitopes or ‘minimum binding units’ were deduced for these. Most antibodies bound to epitopes of 4 amino acid residues with the sequence RPAP occurring most frequently.The results confirm that the short hydrophilic region PDTRPAP in the MUC1 mucin core is immunodominant in the induction of antibodies to the MUC1 mucin since half of the antibodies submitted reacted with this peptide.

Purification of monoclonal antibodies by epitope and mimotope affinity chromatography.

Murine monoclonal antibodies raised against the carcinoma-associated MUC1 mucin have applications in the diagnosis and therapy of human cancer. Many of these antibodies define linear epitopes of three, four or five amino acids within an immunodominant region of the MUC1 protein core. Various synthetic peptides which incorporated this region were prepared and covalently linked to agarose beads for use as affinity matrices. An unrelated peptide was identified as a mimotope for one of the anti-MUC1 antibodies using phage display technologies and this was also evaluated as a potential ligand in an affinity matrix. Epitope affinity chromatographic purification of an anti-MUC1 antibody was performed using hybridoma tissue culture supernatants as sample. Following sample application and column washing, antibody was desorbed from the matrix by gradient elution with increasing concentrations of NaSCN. The procedure has proved efficient for the purification of anti-MUC1 antibodies and the concentration of NaSCN required for antibody desorption gives a measure of the relative binding affinity of the antibody for the peptide epitope matrix so that separation strategies may be optimised.

Use of autoantibodies in breast cancer screening and diagnosis

Breast cancer is the most common cancer among women and accounts for 6% of all cancer deaths. Current screening modalities for breast cancer diagnosis include mammography, digital mammography and magnetic resonance imaging; however, there is still an urgent need to develop an alternative modality of screening for earlier diagnosis. Autoantibodies to tumor-associated autoantigens can be elicited in breast cancer patients. Tumor-associated antigens vary between cancers and can be the result of a number of different events, including mutation, overexpression or altered expression patterns. The inherent amplification of signals provided by the host’s own immune system to low levels of tumor-associated antigens in early disease provides a potential route to the early diagnosis of cancer. In addition, autoantibody responses in breast cancer have been correlated with patient survival and their response to treatment.

Production and characterization of an anti-(MUC1 mucin) recombinant diabody.

Abstract A recombinant diabody fragment based on the anti-MUC1 monoclonal antibody, C595 has been produced in a bacterial expression system. Substitution of a 7-amino-acid linker sequence (Gly6Ser) for the original single-chain (sc)Fv 15-amino-acid linker (Gly4Ser)3, using polymerase-chain-reaction-based strategies, forces variable heavy (VH) and light (VL) domains to pair with complementary domains on neighbouring scFv molecules, forming a scFv dimer (diabody). This recombinant protein shows similar binding characteristics to the parental C595 monoclonal antibody. The ability to bind to MUC1 mucin on carcinoma cell surfaces will allow its potential as a diagnostic and therapeutic reagent of clinical utility to be investigated.

Novel protein targets for organophosphorus pesticides in rat brain

We report preliminary results from a proteomic search for rat brain protein targets adducted by organophosphorous pesticides. Azamethaphos, chlorfenvinphos, diazinon, malathion and chlorpyrifos oxons (in rat brain homogenates) or pirimiphos-methyl (after systemic treatment) were tested at levels producing no more than 30% inhibition of brain acetylcholinesterase. Loss of reactivity with tritiated diisopropylflurophosphate was taken as proof of adduction by the test organophosphate. In addition to acetylcholinesterase other, previously unrecognised, adducted proteins were detected in total brain protein extracts at 30, 32, 41, 71 and 83kDa. Azamethiphos adducted all but the 30 and 32kDa bands, but chlorpyrifos only acetylcholinesterase.