Contributed equally
Melanoma-associated antigen A (MAGEA) represent a class of tumor antigens that are expressed in a variety of malignant tumors, however, their expression in healthy normal tissues is restricted to germ cells of testis, fetal ovary and placenta. The restricted expression and immunogenicity of these antigens make them ideal targets for immunotherapy in human cancer. In the present study the presence of naturally occurring antibodies against two MAGEA subfamily proteins, MAGEA4 and MAGEA10, was analyzed in patients with melanoma at different stages of disease. Results indicated that the anti-MAGEA4/MAGEA10 immune response in melanoma patients was heterogeneous, with only ~8% of patients having a strong response. Comparing the number of strongly responding patients between different stages of disease revealed that the highest number of strong responses was detected among stage II melanoma patients. These findings support the model that the immune system is involved in the control of melanoma in the early stages of disease.
Melanoma-associated antigen A (MAGEA) subfamily proteins are members of cancer/testis antigens (CTAs), whose normal expression is limited to germ cells, but ectopic expression can be observed in tumor cells of different origins (
MAGEA expression is observed mainly in cancers that have acquired malignant phenotypes, invasiveness or metastasis, and the expression of MAGEA family proteins has been linked to poor prognosis in cancer patients. MAGEA family proteins have oncogenic functions, including support of growth, survival and metastasis, and are thought to contribute actively to malignancy (
MAGEA proteins are known to be highly expressed in a wide range on cancers including bladder, lung, skin and breast malignancies (
Melanoma is the most serious type of skin cancer and its incidence has risen over the years. The etiology of melanoma is multi-factorial, resulting from gene-environment interactions, with the main environmental factor for melanoma development being exposure to sunlight and UV radiation (
The aim of this study was to evaluate the presence of naturally occurring antibodies against two MAGEA proteins in the blood samples of melanoma patients with different stages of disease. MAGEA proteins have oncogenic functions contributing to malignancy, and they are known to be immunogenic proteins. The MAGEA4 and MAGEA10 proteins were expressed in bacteria, purified and used in the enzyme-linked immunosorbent assay (ELISA) for detection of antibodies. We were curious to know i) whether the melanoma patients have antibodies against these proteins, and ii) whether these antibodies can be treated as a potential prognostic marker.
Human sera were obtained from 185 patients with melanoma attending the North Estonian Medical Centre (Tallinn, Estonia) within two years (2013–2014). The melanoma stage was assigned based on tumor thickness, ulceration and the involvement of lymph nodes or organs. The characteristics of patients are shown in
MAGEA4 and MAGEA10 coding sequences from pQMCF-MAGEA4 and pQMCF-MAGEA10 vectors (
Recombinant MAGEA4 or MAGEA10 protein (2 µg/ml) in phosphate-buffered saline (PBS) containing 0.1% of Tween-20 was adsorbed onto 96-well MaxiSorp NUNC-immunoplates (Sigma-Aldrich; Merck KGaA) and incubated overnight at 4ºC. Plates were washed with PBS/0.1% Tween-20 and blocked with 2% BSA in PBS/0.1% Tween-20. Serial dilutions of human serum in 100 µl of 0.4% BSA/PBS/0.1% Tween-20 were added to each well and incubated for one hour at room temperature on the shaker (Titertek-Berthold; Berthold Detection Systems GmbH, Pforzheim, Germany). Horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Zymax/Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used as a secondary antibody for 45 min. After washing four times, the reaction was developed with the TMB Peroxidase E1A substrate kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) for 10 min. and stopped with H2SO4. The absorbance at 450 nm was measured spectrophotometrically using the ELISA plate reader Sunrise™ (Tecan, Männedorf, Switzerland). For quality control, we included three reference sera which were analyzed on every ELISA plate. The CVs of their ODs did not exceed 20%.
The data were analyzed in R (version 3.3.0). Parameter estimates and corresponding CI (credible intervals) were calculated using the BayesFirstAid package (
The patients with positive antibody response were defined as follows: pooled MAGEA4 and MAGEA10 response values obtained from the blood bank donors were log-transformed to ensure normality, after which the mean and the standard deviation was calculated from the control subjects only. Then the melanoma patients, whose log-response value > mean + 2* SD, were redefined as having a strong response. To classify subjects based on their MAGEA protein levels a logistic regression model, including both MAGEA proteins, sex, and age as additive predictors, was trained on the subset of data containing stage 0, I, and II patients. The pROC package was used to calculate the receiver operating characteristic (ROC) curve (
We measured the anti-MAGEA4 and anti-MAGEA10 antibody levels by ELISA from 185 stage 0 (
We first compared the OD values of the controls with the melanoma patients separately for anti-MAGEA4 and anti-MAGEA10 response (
To follow the antibody response from limited to advanced disease, we divided the patients into subgroups, depending on the status of their disease. In
As our samples were not balanced for age and sex (
Next, we focused on patients with strong anti-MAGEA4 and/or anti-MAGEA10 immune responses. Here we included patients whose OD values were higher than the mean OD of the healthy blood bank donors plus 2 SD-s (
MAGEA proteins are highly similar to each other with half of the amino acids identical between MAGEA4 and MAGEA10 proteins. We have analyzed the sera separately for MAGEA4 and MAGEA10 response, and the statistics was performed and cut-off values calculated independently of each other. Interestingly, there were 12 anti-MAGEA10 responses and 5 anti-MAGEA4 responses, out of the total of 17 strong responses (estimated relative frequency of MAGEA4 is 0.31; 95% CI: 0.13, 0.52; 5% probability of relative frequency >0.5) (
Comparing the number of strongly responding patients between different stages of disease revealed that the highest number of strong responses was detected among stage II melanoma patients (
To explore the potential diagnostic value of anti-MAGEA antibodies, we classified all stage 0 vs. pooled stage I and II patients using an additive logistic regression model that includes both MAGEA proteins, age, and sex. We summarized the model performance in a ROC curve where we plotted the sensitivity (true positive rate) values against 1-specificity (false positive rate) values for each possible cut-point (
MAGEA proteins are cancer-testis antigens (CTAs), which elicit both cellular and humoral responses. In this study, we have analyzed the presence of naturally occurring antibodies against two MAGEA family proteins, MAGEA4 and MAGEA10, in melanoma patients with different stages of disease. Our data showed that sera of 15 patients out of 185 (8%) had a strong antibody response against the MAGEA4 and/or MAGEA10 protein. The highest antibody response was detected in stage II melanoma patients.
CTAs are named after their typical pattern of expression in a variety of malignant tumors. Their expression in normal tissues is restricted to germ cells of the testis. Male germ cells are devoid of HLA-class I molecules and cannot present antigens to T cells. Therefore, MAGEA antigens can be considered neo-antigens when expressed in cancer cells (
Our study revealed that 8% of patients had strong antibody responses against the MAGEA4 or/and MAGEA10 protein. When we grouped patients according to the level of the disease, then stage II patients had more antibodies than others, reaching to 16% in case of MAGEA10. Scultz-Thater
In our study, some patients had strong antibody response against both MAGE-A proteins, the others exhibited antibodies against either MAGEA4 or MAGEA10 protein. One of the limitations of this study is that we do not have biopsies of patients and we were not able to perform neither qPCR nor immunochemical analysis to confirm that the antibodies are specific to MAGEA4 or MAGEA10. However, tumor cells very often express more than one MAGEA protein. Simultaneous expression of five or more MAGEA proteins occurs in more than half of oral squamous cell carcinomas (
The existence of strongly responding patients suggests that their immune system has been activated and has started to generate antibodies against the primary tumor. So, our data support the hypothesis that the immune system is involved in the control of melanoma, at least in the early stages. Several studies have shown spontaneous regression of primary melanomas, but regression of metastatic tumors is very rare. A good antibody response at early stages can stop the growth of primary tumor and further spreading to the lymph nodes and other organs. Among the 15 patients of our study with the positive status for MAGEA antibodies, only one has died and one has disease progression during the 2-year post-study follow-up period (data not shown). So, the disease of the majority of patients with strong antibody response is under control. However, this cohort is too small to make long-term conclusions about the prognosis. Longitudinal time-course studies on larger cohorts are needed to establish the prognostic significance of the presence of MAGEA antibodies in patients. We plan to follow the patients and their antibody response for at least five more years and perform then the survival analysis.
The sensitivity and specificity calculations suggest that the anti-MAGEA antibody response can be treated as a potential diagnostic biomarker. One of the limitations for use in clinics is that MAGE-A proteins are expressed only in a portion of cancer cells; different works have shown that the amount of expressing cells is between 25 and 50% (
In addition, from the clinical aspect, the longitudinal detection of MAGEA antibody levels could be utilized for profiling of disease status or of effectiveness of novel immunotherapies, as there exists a great need for biomarkers which could assist in discrimination of patients suitable for immunotherapy or for monitoring the therapy effectiveness of these expensive drugs. For example, it has been shown that during immunotherapy with ipilimumab the MAGEA protein levels declined and elevation correlated with either treatment response or failure, respectively (
In summary, our study supports the role of the host immune response in the progression of melanoma. To the best of our knowledge, the present study is the first report on following the antibody response against MAGEA-s and comparing it with the disease progression. A healthy immune system enables to create antibodies against cancer antigens that are expressed specifically by tumor cells. The link between MAGEA antigens and cancer is widely known and accepted; several works have shown a good cellular and humoral response against MAGEAs (
Not applicable.
This study was supported by institutional research funding (IUT20-27) of the Estonian Ministry of Education and Research and by the European Regional Development Fund through the Center of Excellence in Molecular Cell Engineering, and by Estonian Health Program TerVe project IMGEMEL.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
KÕ, KK and RK designed the experiments; KÕ, KK and LV conducted the experiments; MT and AP collected the clinical data and were responsible for ethics approvals and consent of patients to participate in the study; ÜM performed the statistical analysis; MU was responsible for overall the design and funding of the project and KÕ, ÜM, AP and RK prepared the figures and wrote the manuscript. All authors read and have approved the final manuscript.
Approval no. 2781 (from June 21, 2012) for the use of blood samples of melanoma patients, and no. 254 (from December 13, 2012) for controls were obtained from the Tallinn Medical Research Ethics Committee of Estonian National Institute for Health Development. All the patients, whose blood samples have been used, had signed the consent to participate in the study.
The patients have provided written informed consent that the results of the study are published.
The authors declare that they have no competing interests.
Antibody response against MAGEA4 and MAGEA10 proteins in melanoma patients and controls. A comparison of the magnitude of the anti-MAGEA4 and anti-MAGEA10 immune responses of blood bank controls vs. melanoma patients (stages 0–IV combined) by Tukey box plots showing median and interquartile ranges. Dots correspond to individual blood bank controls and patients. The Y-axis denotes optical density values obtained from the ELISA assay. The number of sera is shown in the parentheses. MAGEA, melanoma-associated antigen A.
A subgroup analysis of the magnitude of the anti-MAGEA4 and anti-MAGEA10 immune responses of melanoma patients (stages 0–IV). Tukey box plots with median and interquartile ranges are shown, as well as dots corresponding to individual blood bank controls and patients. The Y-axis denotes optical density values at 495 nm. The number of sera is shown in the parenthesis. MAGEA, melanoma-associated antigen A.
Comparison of OD values of MAGEA4 and MAGEA10 among the strongly responding patients. Lines correspond to mean values of ELISA assay for MAGEA4 (blue) and MAGEA10 (orange), respectively. Error bars show the SD of at least three different experiments performed on separate ELISA plates. MAGEA, melanoma-associated antigen A.
The fraction of strongly responding patients in relation to the melanoma stage. Patients with OD values higher than the mean OD of the healthy blood bank donors plus 2 SD-s are included. The number of sera is shown in the parenthesis. MAGEA, melanoma-associated antigen A.
ROC curve for anti-MAGE antibody detection. Antibody levels among 185 melanoma patients and 43 blood bank controls were determined by ELISA. The AUC value was 0.74. ROC, receiver operating characteristic; MAGE, melanoma-associated antigen; AUC, area under the curve.
Characteristics of the melanoma patients.
Number | Stage 0 24 | Stage I 67 | Stage II 43 | Stage III 30 | Stage IV 21 | Total 185 |
---|---|---|---|---|---|---|
Sex | ||||||
Male (%) | 4 (16.7%) | 17 (25.4%) | 14 (32.6%) | 12 (40%) | 9 (42.9%) | 56 (30.3%) |
Female (%) | 20 (83.3%) | 50 (74.6%) | 29 (67.4%) | 18 (60%) | 12 (57.1%) | 129 (69.7%) |
Disease duration | ||||||
<5 years | 21 (87.5%) | 47 (70.1%) | 29 (67.4%) | 20 (66.7%) | 18 (85.7%) | 135 (73%) |
≥5 years | 3 (12.5%) | 20 (29.9%) | 14 (32.6%) | 10 (33.3%) | 3 (14.3%) | 50 (27%) |
Mean (range) | 2.0 (0–13) | 4.5 (0–26) | 3.7 (0–18) | 4.5 (0–19) | 2.8 (0–25) | 3.8 (0–26) |
Median | 1 | 2 | 3 | 3 | 1 | 2 |
Age | ||||||
Mean (range) | 51.9 (18–87) | 61.3 (28–87) | 64.3 (33–90) | 63.4 (43–82) | 73.1 (35–92) | 62.5 (18–92) |
Median | 51 | 65 | 66 | 65 | 78 | 65 |
Patients with strong antibody response.
Patient | Gender | Age (years) | Disease duration (years) | Stage | Protein | OD value |
---|---|---|---|---|---|---|
M35 | F | 61 | 2 | IIB | MAGEA4 | 2.05 |
M38 |
F | 73 | 18 | II | MAGEA4 | 2.27 |
M111 |
M | 67 | 1 | IIIC | MAGEA4 | 1.49 |
M123 | F | 65 | 0 | IB | MAGEA4 | 1.48 |
M162 | M | 64 | 3 | IA | MAGEA4 | 1.75 |
M3 | F | 57 | 1 | IB | MAGEA10 | 1.49 |
M38 |
F | 73 | 18 | II | MAGEA10 | 1.67 |
M47 | F | 71 | 4 | IIB | MAGEA10 | 2.09 |
M63 | F | 80 | 3 | IIIA | MAGEA10 | 1.48 |
M70 | F | 64 | 9 | IIB | MAGEA10 | 1.43 |
M76 | F | 61 | 6 | IIB | MAGEA10 | 1.77 |
M99 | F | 66 | 5 | IIB | MAGEA10 | 1.88 |
M111 |
M | 67 | 1 | IIIC | MAGEA10 | 1.55 |
M115 | M | 72 | 1 | IIB | MAGEA10 | 1.95 |
M119 | F | 76 | 11 | IB | MAGEA10 | 1.45 |
M137 | F | 72 | 0 | IIB | MAGEA10 | 1.58 |
M144 | F | 52 | 1 | IIIA | MAGEA10 | 1.77 |
These patients have a strong antibody response against MAGEA4 and MAGEA10 protein. MAGEA, melanoma-associated antigen A.