Increased serum levels of complement C3a anaphylatoxin indicate the presence of colorectal tumors

Abstract

Background & aims: Late diagnosis of colorectal carcinoma results in a significant reduction of average survival times. Yet despite screening programs, about 70% of tumors are detected at advanced stages (International Union Against Cancer stages III/IV). We explored whether detection of malignant disease would be possible through identification of tumor-specific protein biomarkers in serum samples.

Methods: A discovery set of sera from patients with colorectal malignancy (n = 58) and healthy control individuals (n = 32) were screened for potential differences using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Candidate proteins were identified and their expression levels were validated in independent sample sets using a specific immunoassay (enzyme-linked immunosorbent assay).

Results: By using class comparison and custom-developed algorithms we identified several m/z values that were expressed differentially between the malignant samples and the healthy controls of the discovery set. Characterization of the most prominent m/z values revealed a member of the complement system, the stable form of C3a anaphylatoxin (ie, C3a-desArg). Based on a specific enzyme-linked immunosorbent assay, serum levels of complement C3a-desArg predicted the presence of colorectal malignancy in a blinded validation set (n = 59) with a sensitivity of 96.8% and a specificity of 96.2%. Increased serum levels were also detected in 86.1% of independently collected sera from patients with colorectal adenomas (n = 36), whereas only 5.6% were classified as normal.

Conclusions: Complement C3a-desArg is present at significantly higher levels in serum from patients with colorectal adenomas (P < .0001) and carcinomas (P < .0001) than in healthy individuals. This suggests that quantification of C3a-desArg levels could ameliorate existing screening tests for colorectal cancer.

Figure 1

A: Flow-chart of experimental and analytical procedures for identification of colorectal cancer specific serum markers. The first step focused on SELDI-TOF MS-based profiling of a discovery set (red). The reproducibility of the data set was explored with an independent validation set (red). These steps were followed by protein characterization of features in prominent m/z values (green). An ELISA test for complement C3a-desArg was used to validate all SELDI-TOF MS-based results (blue) and to predict samples from a validation set and a set of sera from patients with colorectal adenomas (blue) based on serum level thresholds derived from the discovery set, or a combination of the discovery and training set (gray). B: Examples of SELDI-TOF spectra (IMAC3 ProetinChip Array) from a healthy individual (normal) and a patient with colorectal carcinoma (malignancy). The dotted lines indicate peaks at prominent m/z values at 8941.1 and 9148.7. These peaks show higher expression in the cancer sample.

Figure 2

A: Scatter plot of SELDI-TOF MS-based m/z intensities at 8941.1 and age of patients. A Pearson’s correlation coefficient of expression levels and age of r = 0.204 indicated that there is no correlation. B: Summary of the SELDI-TOF MS-based values for the peak intensity at 8941.1 of the discovery and the validation set. Note that the peak intensities do not correlate with the UICC stage in either of the samples. SELDI-TOF MS values for 8941.1 are lower in healthy individuals (N).

Figure 3

A: Immunoassay with an antibody against complement C3a-desArg reveals the identity of this protein at the prominent SELDI-TOF MS-derived m/z values of 8941.1 and 9148.7. The analysis confirms increased expression of complement C3a-desArg (8933.28) in serum from cancer patients compared to serum from healthy individuals (Normal). The peak at 9141.19 is the expected matrix-induced adduct. B: Scatter plot of SELDI-TOF MS-based measured intensities for the m/z intensities at 8941.1 and ELISA data for C3a-desArg. The regression analysis revealed good correlation between the SELDI-TOF MS-derived data and quantification of protein concentration with the immunoassay (r=0.71). The green squares indicate values from normal individuals, while the red squares those from patients with colorectal cancer. C: Summary of all ELISA values for complement C3a-desArg for the healthy individuals (N), sera from patients with colorectal adenomas (P), and with colorectal carcinomas according to UICC stage.