Abstract
The characterization of the humoral response in cancer patients is becoming a practical alternative to improve early detection. We prepared phage microarrays containing colorectal cancer cDNA libraries to identify phage-expressed peptides recognized by tumor-specific autoantibodies from patient sera. From a total of 1536 printed phages, 128 gave statistically significant values to discriminate cancer patients from control samples. From this, 43 peptide sequences were unique following DNA sequencing. Six phages containing homologous sequences to STK4/MST1, SULF1, NHSL1, SREBF2, GRN, and GTF2I were selected to build up a predictor panel. A previous study with high-density protein microarrays had identified STK4/MST1 as a candidate biomarker. An independent collection of 153 serum samples (50 colorectal cancer sera and 103 reference samples, including healthy donors and sera from other related pathologies) was used as a validation set to study prediction capability. A combination of four phages and two recombinant proteins, corresponding to MST1 and SULF1, achieved an area under the curve of 0.86 to correctly discriminate cancer from healthy sera. Inclusion of sera from other different neoplasias did not change significantly this value. For early stages (A+B), the corrected area under the curve was 0.786. Moreover, we have demonstrated that MST1 and SULF1 proteins, homologous to phage-peptide sequences, can replace the original phages in the predictor panel, improving their diagnostic accuracy.
Highlights
From the ‡Functional Proteomics Laboratory, Centro de Investigaciones Biologicas (CIB-CSIC), 28040 Madrid, Spain; §Centro Nacional de Investigaciones Oncologicas, 28029 Madrid, Spain; ¶IDIBELLInstitut Catalad’Oncologia, 08907 Barcelona, Spain; ʈFacultad de Medicina, Universidad de Barcelona, 08907 Barcelona, Spain; **Hospital de Cabuenes, 33394 Gijon, Spain; ‡‡Fundacion Jimenez Díaz, 28040, Madrid, Spain
We decided to test Colorectal cancer (CRC) cDNA libraries displayed in T7 phages in microarray format for autoantibody screening in colorectal cancer patients’ sera
Screening of colorectal cancer sera with phage display libraries grown in Petri dishes was reported by Ran et al [17], that screening was based on visual interpretation of antibody binding to nitrocellulose lifts of phage plaques using pooled sera, making objective quantification quite difficult
Summary
C-terminal end of the capsid 10B protein of the phage. phage libraries are selected through biopanning procedures involving normal and patient’s serum [8]. Combination of phage display with microarray technologies considerably improved the objective evaluation and throughput of the assays, allowing the testing of thousands of phages with only a few microliters of serum [6, 8, 15] This strategy, presents some limitations, such as the sequence of peptides that are displayed on the surface of the phage capsid [16], the presence of mimotopes [6] and the batch to batch reproducibility in microarray production, which is a common problem to other protein microarray formats. We decided to test CRC cDNA libraries displayed in T7 phages in microarray format for autoantibody screening in colorectal cancer patients’ sera. The combination of both proteomic strategies should increase the number of candidate biomarkers and the diagnostic accuracy. The final TAA candidates showed a significant accuracy for CRC diagnosis
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