MAGE‑A genes as predictors of the outcome of laryngeal squamous cell carcinoma
- Authors:
- Published online on: July 29, 2020 https://doi.org/10.3892/ol.2020.11920
- Article Number: 59
Abstract
Introduction
Cancer is a major public health problem in China. Laryngeal squamous cell carcinoma (LSCC) is the most common malignant disease of the head and neck area, and is responsible for 99% of primary laryngeal carcinoma worldwide (1). Due to the increasing incidence rate (7 cases/100,000) (2), LSCC has gradually caused extensive concern among researchers and medical professionals for intensified research (3,4) and clinical trials (5–8). Although surgery combined with chemoradiotherapy has significantly improved the survival rate of patients (5-year survival rate is 63%) (2), recurrence and metastasis are still common and the prognosis remains poor, particularly in patients at the advanced stage. Therefore, the discovery of novel specific markers for early diagnosis and prognosis is urgently needed to improve patient survival.
Melanoma-associated antigen (MAGE), which was first discovered by Van der Bruggen (9) and termed MAGE-I, is a group of well-differentiated members of cancer/testicular antigens (CTA) (9). Thus far, ~60 members of MAGE have been discovered and investigated (10). The MAGE gene family encodes tumor antigens recognized by autologous cytotoxic T lymphocytes (11,12). Based on the difference in gene expression and genetic structure, the MAGE family is categorized into two subfamilies, MAGE-I (MAGE-A, MAGE-B and MAGE-C) and MAGE-II (MAGE-D) (13). The most widely studied gene is MAGE-A, which is strictly tumor-specific, and includes 12 family members, termed MAGE-A1-12 (13). MAGE-A is expressed in several types of tumors, such as breast and gastric cancer as well as glioma (12,14).
The expression of MAGE-A genes in the peripheral blood of patients with LSCC remains unclear. Due to the high similarity in the sequences of MAGE-A1, -A2, -A3, -A4 and-A6, it is difficult to design a unique primer to detect the different genes. In order to investigate the expression of the MAGE-A family genes in the peripheral blood of patients with LSCC and its association with prognosis, multiple MAGE-A genes in the peripheral blood of 104 patients with LSCC and 30 healthy volunteers were detected by multiple nested reverse transcription (RT)-PCR and restriction endonuclease treatment. The aim of the present study was to explore whether the expression of MAGE-As in the peripheral blood circulating tumor cells (CTCs) may be used as a biomarker for guiding clinical treatment and monitoring prognosis in patients with LSCC.
Materials and methods
Patients and clinical parameters
A total of 104 patients with LSCC were recruited from the Department of Otolaryngology, The Fourth Hospital of Hebei Medical University between June 2011 and June 2012. In addition, 30 healthy volunteers with no history of carcinoma were enrolled in the present study during the same period. None of the patients underwent chemotherapy or radiotherapy prior to surgery. Written informed consent was provided by all participants before enrollment. The study protocol was approved by the Medical Ethics Committee of The Fourth Hospital of Hebei Medical University (approval no. 2011KY112).
The clinicopathological data of the patients were retrospectively collected, including age, smoking history, tumor size, clinical stage (8th edition of the American Joint Committee on Cancer) (15), clinical classification, pathological degree and lymph node metastasis.
Blood and tissue sample collection
Fresh blood samples (5 ml) were collected from the patients before surgery, as well as from the volunteers. All blood samples were immediately stored at 4°C, and RNA extraction was performed on the day of sample collection. Blood samples were processed within 1 to 4 h after collection. Blood samples of healthy volunteers were used as the negative control for the RT-PCR assay.
Normal testicular tissue samples were collected from two patients undergoing castration at the Department of Urinary Surgery, The Fourth Hospital of Hebei Medical University. Written informed consent was provided by the two patients. The samples were stored at −80°C until subsequent experiments.
RNA extraction and cDNA synthesis
Red blood cell lysis buffer was used to collect the peripheral blood cells. Total RNA was extracted from the peripheral blood cells and tissues using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Ultraviolet spectrophotometry and agarose gel electrophoresis were used to determine the quality of the RNA. The isolated RNA was stored at −80°C. RNA (2 µg) was used to synthesize the first-strand cDNA using the RevertAid First Strand cDNA Synthesis kit (Fermentas; Thermo Fisher Scientific, Inc.) at 42°C for 1 h.
RT-PCR
cDNA was used for PCR amplification by using Go Taq Green Master Mix (Promega Corporation) with primers for the MAGE-A9 and MAGE-A11 genes. RNA integrity was confirmed by performing PCR amplification with the primer for the GAPDH gene. Agarose gel electrophoresis (2%) was used to identify the RT-PCR products, and the bands were observed under ultraviolet light. Each sample were measured three times. GAPDH was used as the internal reference gene. The primer sequences, product lengths, PCR cycle conditions, initial denaturation and final extension steps are presented in Table I.
Multiplex semi-nested PCR
GoTaq® Green Master mix (Promega Corporation) was used to amplify the cDNA. In the first cycle of PCR, the reaction mixture comprised 5 µl cDNA product of the reverse transcription, 0.5 µl MAGE-F1 (10 µM), 1 µl MAGE-R1 (10 µM), 0.2 µl MAGE-F2 (10 µM), 0.2 µl MAGE-R2 (10 µM), 25 µl 2X PCR Master mix and deionized water added up to 50 µl. In the second cycle of PCR, the reaction mixture comprised 5 µl external PCR product, 0.5 µl MAGE-F3 (10 µM), 1 µl MAGE-R3 (10 µM), 0.2 µl MAGE-F4 (10 µM), 0.2 µl MAGE-R4 (10 µM), 50 µl 2X PCR Master mix and deionized water added up to 100 µl. The primers are presented in Table II. GAPDH was used as the internal reference gene. The thermocycling conditions were as follows: (1)95°C for 5 min; (2)32 cycles of 95°C for 45 sec, 65°C for 45 sec and 72°C for 90 sec and 31 cycles of (2); (3)72°C for 6 min. PCR product (6 µl) was used for electrophoresis on a 1.5% agarose gel. The amplification results were observed in the gel imaging system (Syngene Inc.).
Restriction endonuclease treatment
The products of multiplex semi-nested PCR were purified using the QIAquick PCR Product Purification kit (Qiagen China Co., Ltd.) according to the manufacturer's instructions. The purified products were digested by restriction endonucleases BclI, SphI, EcoRI, Eco47III and AflIII, and gene fragments of MAGE-A1, -A2, -A3, -A4 and -A6 were obtained, respectively (Table III). The restriction fragment (6 µl) was analyzed by electrophoresis using a 1.5% agarose gel. The bands were observed in the gel imaging system.
 | Table III.Restriction endonuclease mixtures, multi-MAGE-A products and restriction fragments for each tested MAGE-A gene. |
Statistical analysis
SPSS v20.0 software (IBM Corp.) was used to analyze the data. The display strip is defined as high expression group and vice versa. χ2 or Fisher's exact test were used to evaluate the potential association between the expression of all MAGE-A genes or single MAGE-A genes and patient clinicopathological characteristics. The Kaplan-Meier method was used to estimate the overall survival time of patients with LSCC. The Cox regression model was used for univariate and multivariate analysis of overall survival and prognostic factors. P<0.05 was considered to indicate a statistically significant difference.
Results
Expression of MAGE-A genes in the peripheral blood of patients with LSCC and healthy donors
Multiplex semi-nested RT-PCR and RT-PCR were used to detect the expression of MAGE-A genes, including MAGE-A1, -A2, -A3, -A4, -A6, -A9 and -A11, in two normal testicular tissue samples (Fig. 1A). As the MAGE-A gene belongs to the CTA gene family, it is only expressed in normal testis and other germ cells, but not in other normal tissues (9). Therefore, testicular tissue was used in the present study as a positive control. Subsequently, the expression of MAGE-A in the blood samples of 104 patients with LSCC and 30 healthy donors was examined. The representative samples of the control and MAGE-A products in the peripheral blood of healthy donors (negative control) and patients with LSCC, as well as normal testicular tissue (positive control) are presented in Fig. 1A. The MAGE-A product was observed in the normal testicular tissues and in the blood samples of a number of patients with LSCC, but not in the blood samples from the healthy donors. The representative LSCC blood samples with positive MAGE-A gene expression after internal PCR (second PCR cycle) are presented in Fig. 1B.
As presented in Fig. 2, 31 of 104 patients with LSCC (29.8%) exhibited MAGE-A gene expression in the peripheral blood. The expression of MAGE-A9 and -A11 mRNA was detected by RT-PCR, whereas the expression pattern of other individual MAGE-A genes was identified by restriction endonuclease treatment. MAGE-A1 expression was positive in 19 of 104 (18.3%), MAGE-A2 expression was positive in 21 of 104 (20.2%), MAGE-A3 expression was positive in 21 of 104 (20.2%), MAGE-A4 expression was positive in 16 of 104 (15.4%), MAGE-A6 expression was positive in 12 of 104 (11.5%), MAGE-A9 expression was positive in 27 of 104 (26.0%) and MAGE-A11 expression was positive in 29 of 104 (27.9%) patients with LSCC. The frequency of individual MAGE-A gene expression was in the following order: A11 > A9 > A2 = A3 > A1 > A4 > A6. A total of 18 patients were positive for only one MAGE-A gene, 12 patients were positive for two genes, seven patients were positive for three genes, eight patients were positive for four genes, eight patients were positive for five genes and two patients were positive for six genes. The genomic information of MAGE-A1-12 for all 104 patients with LSCC is presented in Table SI.
Fig. 3 demonstrates the expression of the MAGE-A gene products from two patients following restriction endonuclease treatment. A multiple MAGE-As product was observed in the peripheral blood of patients no. 17 and 45. Subsequently, the MAGE-A product (second PCR cycle) was digested with BclI, SphI, EcoRI, Eco47III and AflIII, and the digested products were separated by agarose gel electrophoresis. The individual MAGE-A genes were identified by observing the fragment pattern. For patient 17 (Fig. 3A), the fragments of 787 bp was observed after BclI digestion, 720 bp was observed after SphI digestion, 747 bp was observed after EcoRI digestion, and 438 bp was observed after AflIII digestion, indicating the presence of MAGE-A1, -A2, -A3 and -A6 in the PCR product. The expression pattern of MAGE-As in patient 45 is presented in Fig. 3B; the expression of MAGE-A1 and -A6 was not observed, but the 720 bp band was observed after SphI digestion, and the fragment of 747 bp was observed after EcoRI digestion, indicating the presence of MAGE-A2 and -A3 in the PCR product.
Association between MAGE-A gene expression in the peripheral blood and the clinicopathological characteristics of patients with LSCC
The association between MAGE-A gene expression in the peripheral blood and the clinicopathological characteristics of patients with LSCC was evaluated (Table IV). The expression of MAGE-A genes (MAGE-As, -A1, -A2, -A3, -A4, -A6, -A9 and -A11) were not associated with age, smoking history, tumor size and location, but was positively associated with lymph node metastasis (P=0.001, P=0.022, P<0.001, P=0.001, P=0.016, P<0.001, P=0.001 and P=0.016, respectively). High expression levels of MAGE-As in LSCC were associated with the histological degree (P=0.007). Among individual MAGE-As, positive MAGE-A1, -A3, -A4 and -A6 expression was more frequent in patients with histological grade G3 compared with those with histological grades G1/G2 (P=0.005, P=0.013, P=0.001 and P=0.001, respectively). In addition, more frequent positive expression of MAGE-A3, -A6, -A9 and -A11 was observed in patients with high clinical stage compared with that in patients with low clinical stage (P=0.044, P=0.048, P=0.01 and P=0.045, respectively). No associations were observed between each individual MAGE-A genes expression and other clinicopathological factors.
| Table IV.Clinicopathological characteristics and MAGE-A gene expression in the peripheral blood from 104 patients with laryngeal squamous cell carcinoma. |
Association between MAGE-A gene expression in the peripheral blood and the overall survival of patients with LSCC
All 104 patients with LSCC were followed up for 18–65 months. Kaplan-Meier analysis was performed to determine the association between MAGE-A gene (MAGE-A1, -A2, -A3, -A4, -A6, -A9 and -A11) expression levels and the overall survival of patients with LSCC. Overall survival of patients with high MAGE-A gene expression in the peripheral blood was significantly lower compared with those with low expression (P=0.020, P=0.061, P=0.010, P=0.036, P=0.039, P<0.001, P<0.001 and P<0.001, respectively; Fig. 4). To further evaluate the prognostic significance of MAGE-A expression, univariate analysis of clinicopathological factors and overall survival was performed. High expression of MAGE-As (P=0.024), -A2 (P=0.014), -A3 (P=0.041), -A4 (P=0.046), -A6 (P<0.001), -A9 (P<0.001) and -A11 (P<0.001), as well as lymph node metastasis (P<0.01), low clinical stage (P=0.013) and high histological grade (P=0.028) were demonstrated to be predictors of poor overall survival (Table V). clinical stage, which includes tumor size and lymph node metastasis, and MAGE-As, which includes MAGE-A1, -A2, -A3, -A4 and -A6, were not considered as independent prognostic factors. Lymph node metastasis, histological grade and the expression of MAGE-A2, -A3, -A4, -A6, -A9 and -A11 were further analyzed by multivariate Cox regression analysis. As demonstrated in Table V, high expression of MAGE-A11 (P=0.032) and lymph node metastasis (P<0.01) were determined to be independent prognostic factors for poor prognosis of patients with LSCC.
| Table V.Univariate and multivariate analyses of prognostic factors for overall survival of patients with laryngeal squamous cell carcinoma. |
Discussion
Clinical imaging is often the first to identify the tumor and is important for the diagnosis of recurrence and metastasis of the auxiliary technology (2). Failure to identify the primary or metastatic tumors by imaging means that the best time for diagnosis and treatment is missed, and late metastasis of the tumor has no effective treatment and the prognosis is poor (2). The process by which tumor cells invade the body remains unclear and may involve the active invasion of cells via epithelial-to-mesenchymal transition (16) and passively shed individual cells or clusters of tumor cells from impaired tumor blood vessels (17,18). The presence of CTCs in the peripheral blood is considered to be the key factor of tumor metastasis (19). A number of studies have evaluated the clinical application value of CTCs for metastatic breast (20,21), prostate (22), colon (23) and lung (24,25) cancer. Using the FDA-approved Cell Search system (Veridex), the peripheral blood CTC count test and analysis of the prognosis of patients suggest that CTCs can be used as an independent prognostic indicator (26,27). The technology detects the biomarkers on the cell surface to identify the CTCs (26,27). The downregulation or absence of epithelial markers should have an effect on detecting CTCs (19). Thus, the discovery of new tumor markers may help identify CTCs in the peripheral blood of patients with malignant tumors.
RT-PCR, which has a wide range of applications, is considered the most sensitive method for detecting CTCs in the peripheral blood (28). However, the following issues may exist: i) Large amounts of water in the peripheral blood may dilute the normal mRNA and cause false negative results (29); ii) contamination of the target gene in the peripheral blood, leading to DNA amplification, may result in false positive results (30), and iii) low levels of abnormal transcription and amplification of target genes in the peripheral blood tumor cells may also cause false-positive results (30). Improving the detection technology, designing more suitable primers and selecting accurate tumor markers may help avoid the aforementioned issues. Previous studies have used RT-nested PCR to detect the mRNA expression of CTCs in the peripheral blood of non-cancerous and colorectal cancer cells, and demonstrated high sensitivity and specificity (31,32). It has been demonstrated that the expression of MAGE-A genes can be used as a biomarker of CTCs in colorectal, breast and gastric cancer (33). The results of the previous study demonstrated that the MAGE-A antigen is a tumor-associated antigen of LSCC (34). Therefore, MAGE-A mRNA may be used to detect CTCs in the peripheral blood of patients with LSCC as a specific tumor marker, which may guide the diagnosis and prognosis of LSCC. In the present study, fresh blood was used for further analysis of MAGE-A gene expression; fresh blood is easy to collect and observe, allowing for timely guidance on clinical diagnosis and treatment.
Due to the high homology, it is difficult to design specific primers for a single MAGE-A gene. Therefore, a pair of primer sequences containing two forward primers and two reverse primers were designed in the present study for the amplification of a mixture of MAGE-A1, -A2, -A3, -A4 and -A6 genes by multi-RT-nested PCR. The expression of MAGE-A9 and -A11 mRNA was detected by RT-PCR. MAGE-A mRNA was expressed in different degrees in peripheral blood of patients with LSCC, while MAGE-A expression was not detected in the healthy donors. These are consistent with previous studies (35,36). A total of 18 patients were positive for only one MAGE-A gene, 12 patients were positive for two genes, seven patients were positive for three genes, eight patients were positive for four genes, eight patients were positive for five genes, and two patients were positive for six genes. Since the MAGE-A gene only exists in tumor cells (9), if MAGE-A gene expression is detected in peripheral blood, they must be expressed in tumor cells. In view of the definition of CTC, the results of the present study indicated that MAGE-A is expressed in CTCs. The results of the present study suggested that MAGE-A mRNA was only expressed in tumor cells and may be detected only in the presence of tumor cells in the peripheral blood of patients with LSCC. Therefore, the expression of MAGE-A genes may be used as a specific marker for detecting CTCs in the peripheral blood of patients with LSCC.
The association between MAGE-A gene expression in the peripheral blood and the clinicopathological features of patients with LSCC was statistically analyzed in the present study. The expression of MAGE-A1, -A3, -A4 and -A6 were more frequently detected in patients with histological grade G3 tumors compared with patients with tumor grades G1/G2. A previous study has demonstrated that histological grade and tumor prognosis are significantly associated in breast cancer (37). Therefore, the expression of MAGE-A1, -A3, -A4 and -A6 genes may be an important indicator of the prognosis of LSCC. In the present study, the expression of MAGE-A3, -A6, -A9 and -A11 was observed in patients with a high clinical stage more frequently compared with that in patients with a low clinical stage. Patients with late clinical staging usually have a poor prognosis (37). In addition, the expression of MAGE-As in the peripheral blood of patients with LSCC was positively associated with the lymph node metastasis status in the present study. For each individual MAGE-A gene, including MAGE-A1, -A2, -A3, -A4, -A6, -A9 and -A11, the expression frequency in patients with lymph node metastasis was significantly higher compared with that in patients without lymph node metastasis. Previous studies have demonstrated that CTCs are commonly present in advanced metastatic malignancies, with more CTCs in the peripheral blood of distantly metastatic breast cancer compared with early-stage breast cancer (38), and similar observations in ovarian cancer (39). In the present univariate analysis, individual MAGE-A expression, metastatic state of the lymph nodes, clinical stage and histological grade qualified to enter the regression model; however, as clinical stage includes the metastatic state of the lymph nodes and distant metastasis, only the metastatic state of the lymph nodes was entered into the regression model. The results demonstrated that multiple MAGE-As expression, the metastatic state of the lymph nodes and distant metastasis were risk factors for the 5-year survival of patients with LSCC.
To date, serological hallmark and high-resolution imaging technology still cannot identify micrometastasis and reflect the efficacy of treatments. CTCs detected in the peripheral blood suggest the possibility of early occult micrometastasis (26,27). CTCs cannot only be used to study the biological characteristics of malignant tumors, but they overcome the disadvantage of traditional tissue biopsy and allow real-time dynamic monitoring of the changes in the tumor (40). Previous studies have demonstrated that the changes in the number of CTCs can reflect the efficacy of treatments and provide the basis for individual treatment. Qiao et al (41), reported the quantitative variation of CTCs in a patient with ESCC before and after surgery and during a 5-year follow-up period; the results demonstrated that the number of CTCs before and the initial period after surgery remained high. By contrast, following combined treatment, the number of CTCs deceased, and after 117 weeks, the number gradually stabilized at a low level (41). Thus, a change in the number of peripheral blood CTCs may be used to monitor disease status and treatment efficacy. Another previous study reported that clinically acquired drug resistance did not become resistant at the cellular level during the course of treatment, but clinically acquired drug resistance was the selective reaction of heterogeneous cancer cells to target cells (42). Through the dynamic supervision of certain molecular indicators of CTCs, the endpoint for therapy can be determined (43). CTCs in the peripheral blood of patients with LSCC can be examined to dynamically monitor the changes and development of the tumor in order to achieve an improved understanding of personalized therapy. CTCs in the peripheral blood of patients with LSCC may be monitored by detecting the MAGE-A genes to guide clinical treatment and the judgement of prognosis.
In conclusion, the results of the present study identified the expression patterns of multiple MAGE-A genes in the peripheral blood of patients with LSCC. MAGE-A gene expression in the peripheral blood may therefore be used as a molecular marker for guiding the treatment and monitoring the prognosis of patients with LSCC.
Supplementary Material
Supporting Data
Acknowledgements
Not applicable.
Funding
This study was supported by the Financial Supporting Program of Hebei Province (grant no. 2014]1257), the Financial Department of Hebei Province (grant no. 2016]361006) and the Hebei Health Department (grant no. 20150786).
Availability of data and materials
The datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.
Authors' contributions
BS and MS contributed to the experimental design and fundraising. YZ and YX performed some of the expriments. RZ and LG assisted in the data analysis. SL analyzed the data and drafted the initial manuscript.
Ethics approval and consent to participate
This study was approved by the Medical Ethics Committee of The Fourth Hospital of Hebei Medical University (Shijiazhuang, China; approval no. 2011KY112). Written informed consent was provided by all participants before enrollment.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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