MGMT autoantibodies as a potential prediction of recurrence and treatment response biomarker for glioma patients

Abstract

Background: Cancer-specific autoantibodies found in serum of cancer patients have been characterized as potential predictors of the high risk of recurrence and treatment response. The objective of this study is to investigate the clinical utility of serum O-6-methylguanine-DNA methyltransferase (MGMT) autoantibodies as novel biomarkers for prediction of recurrence and treatment response for glioma through MGMT peptides microarray.

Methods: A total of 201 serum samples of glioma patients with various WHO grade and 311 serum samples of healthy donors were examined for the detection of MGMT autoantibodies by peptides microarray. The clinical value of MGMT autoantibodies was studied through univariable and multivariable analyses.

Results: Autoantibodies to MGMT peptides were detected in sera from glioma patients and five highly responsive autoantibodies to peptides were identified in the glioma group. The positive rate of MGMT autoantibody to 20 peptides in glioma groups is compared with healthy individuals, the positive rate of MGMT-02 (45%), MGMT-04 (27%), MGMT-07 (21%), MGMT-10 (13%), and MGMT-18 (24%) were significantly elevated in patients with glioma. MGMT autoantibody and its protein expression exhibited a significant correlation. The levels of MGMT autoantibodies decreased on the 30th day after operation, reaching preoperative levels, similar to those when tumor recurrence developed. Univariable and multivariable analyses revealed that the only preoperative autoantibodies to MGMT-02 peptide were independently correlated with recurrence-free survival. Preoperative seropositive patients were more likely than seronegative patients to have shorter recurrence times and to be resistant to chemoradiotherapy or chemotherapy with temozolomide.

Conclusion: Monitoring the levels of preoperative serum autoantibodies to MGMT-02 peptide was useful for predicting patients at high risk of recurrence and treatment response.

Keywords: MGMT; autoantibodies; prediction of recurrence; treatment response.

Figure 1

Flow chart of serological screening of MGMT peptides. Preparation of MGMT peptide microarray were divided in four steps (1‐4): (1) MGMT proteins were selected; (2) proteins were cut into peptides that were 20 mers with 10aa overlap peptides (blue and red); (3) MGMT peptides were chemically synthesized; (4) MGMT peptides were designed for the microarray. All spots were organized as a 9 × 9 array with peptides probes (blank circle), positive controls (red circle), and negative controls (blue circle). (4) Printed on iPDMS membrane to form microarrays. MGMT peptide microarray experiment (6‐7): (6) Incubation with serum samples; (7) HRP‐labeled secondary antibody. MGMT peptide microarray analysis by chemiluminescence (8‐10): (8) Antibody binding to target peptide was acquired and processed to chemiluminescence immunoassay picture by CCD camera; (9) Chemiluminescence immunoassay picture shown is the result of one of the glioma patients; (10) Further analysis by computer

Figure 2

Cluster analysis of the normalized IgG responses. Normalization operation of the SNR value of each peptide was carried out in order to adjust the data for systematic errors that arose from experimental and not biological variation. Normalized IgG responses of glioma and healthy individuals to 20 MGMT peptides were clustered, respectively. Only one recognition pattern emerged peptides having significantly higher response in glioma individuals. Heatmap responses from 378 serum samples (67 glioma patients and 311 healthy control samples) to the identified 5 peptides. The responses were obtained and clustered by SNR. We could easily identify the differences between the two groups in the serological screening of MGMT peptides from the heatmap

Figure 3

Autoantibodies to MGMT peptides response to glioma in different time points (preoperative, postoperative and recurrence). The coverage ratio of autoantibodies to MGMT peptide response to the sera collected at different times

Figure 4

The changing of SNR values in the autoantibodies to 5 peptides to the sera collected before and 30 days after operation in 10 glioma patients. Autoantibodies to MGMT‐02 (A, n = 6), MGMT‐04 (B, n = 4), MGMT‐07 (C, n = 1), MGMT‐10 (D, n = 2), MGMT‐18 (E, n = 1)

Figure 5

The changing of SNR values in the autoantibodies status to MGMT‐02 peptide before and after operation, and recurrence in 10 glioma patients. Seropositive patients (A, n = 5), seronegetive patients (B, n = 5). The dotted line indicates the cut‐off value. Abbreviations: TTR, Time to Recurrence

Figure 6

Recurrence‐free survival curves of patients according to autoantibodies to MGMT‐02 peptide levels after combined radio‐chemotherapy with temozolomide. Negative group (SNR value <5.5), positive group (SNR value ≥5.5). In the univariate analysis (A) and multivariate analysis (B), MGMT‐02 peptide was significantly correlated to the recurrence‐free survival of patients. P < 0.05 was considered statistically significant