Human blood autoantibodies in the detection of colorectal cancer

Colorectal cancer (CRC) is the second most common malignancy in the western world. Early detection and diagnosis of all cancer types is vital to improved prognosis by enabling early treatment when tumours should be both resectable and curable. Sera from 3 different cohorts; 42 sera (21 CRC and 21 matched controls) from New York, USA, 200 sera from Pittsburgh, USA (100 CRC and 100 controls) and 20 sera from Dundee, UK (10 CRC and 10 controls) were tested against a panel of multiple tumour-associated antigens (TAAs) using an optimised multiplex microarray system. TAA specific IgG responses were interpo- lated against the internal IgG standard curve for each sample. Individual TAA specific responses were examined in each cohort to determine cutoffs for a robust initial scoring method to establish sensitivity and specificity. Sensitivity and specificity of combinations of TAAs provided good discrimination between cancer-positive and normal serum. The overall sensitivity and specificity of the sample sets tested against a panel of 32 TAAs were 61.1% and 80.9% respectively for 6 antigens; p53, AFP, K RAS, Annexin, RAF1 and NY-CO16. Furthermore, the observed sensitivity in Pittsburgh sample set in different clinical stages of CRC;stageI(n=19),stageII(n=40),stageIII(n=34)andstageIV(n=6)wassimilar (73.6%, 75.0%, 73.5% and 83.3%, respectively), with similar levels of sensitivity for right and left sided CRC. We identified an antigen panel of sufficient sensitivity and specificity for early detection of CRC, based upon serum profiling of autoantibody response using a robust multiplex antigen microarray technology. This opens the possibility of a blood test for screening and detection of early colorectal cancer. However this panel will require further validation studies before they can be proposed for clinical practice

The detection of ADAM8 protein on cells of the human immune system and the demonstration of its expression on peripheral blood B cells, dendritic cells and monocyte subsets

A disintegrin and metalloprotease (ADAM) proteins have wide ranging functions, including proteolytic cleavage of cell surface molecules, cell fusion, cell adhesion and intracellular signalling. Recent evidence suggests the involvement of ADAM8 in allergic responses. For instance, ADAM8 is amongst a number of genes up-regulated in experimentally induced asthma in animals. In order to further define the involvement of ADAM8 in allergic responses, we sought in the first instance to examine its distribution on human peripheral blood B cells, resting and activated T cells, monocyte subsets and monocyte derived dendritic cells. Here we demonstrate for the first time ADAM8 protein expression on B cells and dendritic cells, and its higher expression on CD142+CD16- monocytes compared to CD14+CD16+ cells. Immature dendritic cells expressed low levels of ADAM8 when treated with a combination of GM-CSF and IL-4, but stimulation with LPS resulted in a higher level of expression, which was TLR-4 independent. Up-regulation of ADAM8 expression on dendritic cells was also observed after stimulation with TNF-α, but not after stimulation with anti-CD40. The demonstration of ADAM8 expression on these cells provides an opportunity for addressing the potential role of inhaled protease allergens, such as Der p 1, in modulating ADAM8 functions, particularly with regards to innate immune responses by dendritic cells and IgE synthesis by B cells. © 2006 Elsevier GmbH. All rights reserved

Development and validation of a high throughput system for discovery of antigens for autoantibody detection.

An assay employing a panel of tumor-associated antigens has been validated and is available commercially (EarlyCDT®-Lung) to aid the early detection of lung cancer by measurement of serum autoantibodies. The high throughput (HTP) strategy described herein was pursued to identify new antigens to add to the EarlyCDT-Lung panel and to assist in the development of new panels for other cancers. Two ligation-independent cloning vectors were designed and synthesized, producing fusion proteins suitable for the autoantibody ELISA. We developed an abridged HTP version of the validated autoantibody ELISA, determining that results reflected the performance of the EarlyCDT assay, by comparing results on both formats. Once validated this HTP ELISA was utilized to screen multiple fusion proteins prepared on small-scale, by a HTP expression screen. We determined whether the assay performance for these HTP protein batches was an accurate reflection of the performance of R&D or commercial batches. A HTP discovery platform for the identification and optimal production of tumor-associated antigens which detects autoantibodies has been developed and validated. The most favorable conditions for the exposure of immunogenic epitopes were assessed to produce discriminatory proteins for use in a commercial ELISA. This process is rapid and cost-effective compared to standard cloning and screening technologies and enables rapid advancement in the field of autoantibody assay discovery. This approach will significantly reduce timescale and costs for developing similar panels of autoantibody assays for the detection of other cancer types with the ultimate aim of improved overall survival due to early diagnosis and treatment

Serum anti-toxin IgG.

<p>Serum anti-toxin A (A) and anti-toxin B (A) IgG levels in healthy controls (n=19), patients with cystic fibrosis (CF; with no previous history of <i>C. difficile</i> infection; n=16), patients with inflammatory bowel disease (IBD) and <i>C. difficile</i> infection (n=10) and patients with <i>C. difficile</i>-associated diarrhoea (CDAD; n=53). If more than one serum sample was studied per subject, the mean antibody concentration was used for the calculation. Anti-toxin A and B levels in patients with cystic fibrosis were significantly higher than those observed in healthy controls and patients with <i>C. difficile</i>-associated diarrhoea.</p

Toxin A- and B-specific ASCs in patients with cystic fibrosis with and without previous <i>C. difficile</i>.

<p>Peripheral blood mononuclear cells were isolated from a total of 6 and 24 samples collected at varying time intervals from patients with cystic fibrosis with (n=2) and without (n=13) previous history of <i>C. difficile</i> infection, respectively. The frequencies of toxin A- and B-specific antibody secreting cells were significantly higher in those patients with previous <i>C. difficile</i> infection than those without. (<b>A</b>) toxin A-specific ASCs (p=0.05); (<b>B</b>) toxin B-specific ASCs (p=0.026).</p

Toxin A and B-specific ASC frequencies in patients with <i>C. difficile</i>-associated diarrhoea.

<p>Peripheral blood mononuclear cells from 39 blood samples, collected at varying time intervals after onset of <i>C. difficile</i>-associated diarrhoea in 16 patients. A significantly higher proportion of toxin B-specific antibody secretory cells (than toxin A-specific antibody secretory cells) was detected [0.82 (0 - 4.93) % vs 0.33 (0 - 2.12) %; p<0.0001].</p