We retrospectively analyzed 84 cases of TETs (56 cases of thymoma and 28 cases of TC) who were diagnosed by pathological examination from January 2010 to December 2020 and were treated in the Second Affiliated Hospital of Jiaxing University. Clinical data were recorded and retrieved from the electronic database of the hospital. The present study was approved by the Ethics Committee of the Second Affiliated Hospital of Jiaxing University (Ethical Code number: LWSC041), and written informed consent was obtained from all patients.

Among these 84 patients, blood samples were obtained from only 33 cases, which were used to detect MTHFR gene polymorphism in this study. Samples were collected from 21 men and 11 women, ranging from 32 to 74 years old between January 2015 and January 2018. A total of 72 healthy individuals (38 men and 34 women) ranging from 51 to 75 years old were recruited as controls. Genomic DNA was extracted from peripheral venous blood (2 ml) using a commercial kit according to the manufacturer’s instructions (BaiO Technology Co, Ltd., Shanghai, China). The DNA purity (A260/A280 ratio) was determined by using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The chips that can detect MTHFR gene C667T polymorphism were purchased from Shanghai BaiO Company and hybridized by using a BaiO biochip hybridization instrument (BaiO Technology Co., Ltd., China). Data were recorded by a BaiO biochip reader and were analyzed by using a BaiO analysis software (BaiO Technology Co., Ltd., China).

The MTHFR transcriptional expression data (123 patients with TETs) from the TCGA database were obtained from the cbioportal platform (http://www.cbioportal.org). Clinical data showed that among these 123 patients with TETs, 64 (52.0%) cases were men and 59 (48.0%) cases were women, with a median age of 60.5 years. Thymectomy was performed in all patients, 41 of whom underwent postoperative radiotherapy and 6 of whom underwent postoperative chemotherapy. The correlation between the mRNA levels of MTHFR and its methylation was analyzed in this platform.

Surgical resection was performed in 76 patients in this retrospective study. The complete resection rate was 77.6%. Among them, 5 patients with incomplete surgical resection underwent multiple therapies. Among a total of 84 patients, 10 patients with thymoma and 22 patients with TC were treated by platinum-based chemotherapy with an average of 4 cycles. A total of 3 patients with thymoma and 6 patients with TC received postoperative adjuvant radiotherapy. The dose of radiotherapy was approximately 54 Gy (1.8 ~ 2.0 Gy/f).

The follow-up time was from the time of pathological diagnosis to the death or last follow-up time of the patients, recorded every month. The contents of follow-up included the progression of the disease, stability of the disease, death of the patient, and so on. Overall survival (OS) is defined as the time of the first diagnosis to death. The cut-off time of the study was 2020-12-31.

All statistical analyses were performed by using Statistical Package for the Social Sciences (SPSS), version 25.0 (IBM Inc., Chicago, IL, USA). Log-rank analysis was used for the comparisons between the different factors affecting prognosis. Multivariate analysis for OS was performed using a Cox proportional hazards model. All significant variables in the univariate analysis including sex, age, smoking status, maximum tumor diameter, chemotherapy, radiotherapy, WHO histological classification, Masaoka–Koga stage, 8th UICC/AJCC TNM staging, and clinical symptoms were used in the multivariate analysis. The Kaplan–Meier survival rate was calculated and drawn by the SPSS software. The chi-square test was used to compare the MTHFR C667T genotype and allele distribution between patients with TETs and healthy controls. Logistic regression analysis was used to analyze the correlation between different genotypes MTHFR C667T and TET susceptibility. Spearman correlation analysis was performed to analyze the correlations of the mRNA levels of MTHFR expression and its methylation by using the TCGA database. P<0.05 was considered to be statistically significant.

In total, 84 patients diagnosed with thymomas (n=56) and TCs (n=28) who met predefined criteria were recruited in this study. General characteristics were listed in Table 1 . The study consisted of 51 (60.7%) men and 33 (39.3%) women, with a median age of 58 years (range 22–88 years). In addition, 31 cases were asymptomatic at the first visit, 21 cases complained of chest pain or chest distress, 11 cases had respiratory symptoms (including cough and pulmonary infection), and 12 cases had MG. A total of 9 cases were accompanied by other clinical symptoms, such as lumbago and trauma. At the end of follow-up time on December 31, 2020, 19 patients with TETs were deceased, including 4 patients with thymomas and 15 patients with TCs.

The median follow-up period was 36 months. The 5-year survival rate of patients with TETs was 71.9% ( Figure 1 ). Univariate analysis showed that sex (P = 0.005), age (P = 0.019), the maximum tumor diameter (P < 0.001), surgery (P < 0.001), chemotherapy (P < 0.001), radiotherapy (P = 0.001), WHO histological classification (P < 0.001), Masaoka–Koga stage (P < 0.001), and 8th UICC/AJCC TNM staging TNM stage (P < 0.001) were associated with TET prognosis ( Table 2 ). Other variables, including clinical symptoms and the smoking status, were not associated to prognosis. Among these factors, male sex, older age (>58 years old), the maximum tumor diameter (>5 cm), no surgery, no chemotherapy, no radiotherapy, a higher grade of WHO histological classification and advanced tumor stage (both the Masaoka–Koga stage and the 8th UICC/AJCC TNM staging) were correlated with poor prognosis. The survival curves of different variables are shown in Figure 2 : sex (A), age (B), the maximum tumor diameter (C), surgery (D), chemotherapy I, radiotherapy (F), WHO histological classification (G), Masaoka–Koga stage (H), and 8th UICC/AJCC TNM staging (I). The Masaoka–Koga stage and 8th UICC/AJCC TNM staging were strongly correlated with each other in this study (r=0.925, P<0.001). These univariate analysis data were used to establish a subsequent multivariate Cox proportional hazards regression model on the basis of the Masaoka–Koga stage and UICC/AJCC TNM staging. The results showed that the maximum tumor diameter (P = 0.040, hazard ratio [HR]: 3.623, 95%CI: 1.058~12.403 on the basis of Masaoka–Kaga stage; P = 0.045, HR:3.629, 95%CI: 1.027~12.822 on the basis of UICC/AJCC TNM staging), Masaoka–Koga stage (P = 0.008, HR: 5.513, 95%CI: 1.564~19.434), and 8th UICC/AJCC TNM staging (P = 0.007, HR: 8.476, 95%CI:1.815~39.596) were identified as independent prognostic factors of patients with TETs ( Tables 3 , 4 ).The 5-year survival rates of TET patients with Masaoka–Koga stage I, II, III, and IV were 100%, 95.8%, 78.6%, and 20.5%, respectively ( Figure 2H ). The 5-year survival rates for UICC/AJCC TNM stages I, II, III, and IV were 100%, 100%, 73.3%, and 20.5%, respectively ( Figure 2I ).

MTHFR polymorphism in healthy individuals and patients with TETs was further investigated in this study. The results of the chi-square test showed that the genotype (χ ² = 7.987, P = 0.018) and allele distribution (χ ² = 5.750, P = 0.016) statistically significantly differed between patients and healthy controls ( Table 5 ). In logistic regression analysis, heterozygous and homozygous genotypes (CT+TT) showed higher TET susceptibility (odds ratio [OR] = 4.721, P = 0.008, Table 6 ). The analysis also revealed that, compared to the T allele, the presence of the MTHFR C allele at this position was adversely associated with the incidence of TETs (T vs. C: OR = 2.067, 95% CI: 1.136–3.758, P = 0.017, Table 6 ). Kaplan–Meier univariate survival analysis was further conducted, and data showed that there was no correlation between different genotypes and the prognosis of TETs (CC vs. CT + TT, χ ² = 0.003, P = 0.959, Figure 3 ).

Using the gene expression profiles of TETs from the TCGA database, a negative correlation was observed between MTHFR mRNA levels and their methylation by using Spearman correlation analysis (r= -0.24, P = 0.010, Figure 4 ).

In the present study, the median age of the patients at the time of TET diagnosis was 58 years. Using univariate analysis, we observed that the patients with TETs who were older than 58 years or were men had a poor prognosis. As shown in Figure 1 , the OS of the TET patients gradually decreased with an increase in age. These data were supported by the results of a previous study suggesting that young patients with thymoma have a better performance status and less comorbidities and have a better prognosis than older patients (23). Liu et al. reported similar results in patients with thymoma but not in patients with TC (24). In our study, we analyzed thymoma and TC as a single group, and found that age tended to be a prognostic factor. Sex was not identified as a predictive factor in several studies (25, 26); however, in our study, we observed a worse prognosis in male patients with TETs than in their female counterparts. We consider this to be due to the majority of TC patients in our cohort being men with a reduced survival time.

A tumor diameter greater than 5 cm was observed to be an independent prognostic value of TETs in our study, resulting in a poor prognosis. This conclusion was supported by another study, which showed that the tumor diameter is an independent prognostic factor and may even be an indication for surgical treatment (27). Based on the study by Okumura et al., a tumor diameter ≥5 cm was suggested to be an independent risk factor, which was consistent with our findings (28).

Most patients in our cohort were asymptomatic, which may be related to the improved auxiliary inspection technologies and health awareness in our population. The association between autoimmune diseases (ADs) and thymoma has been well established, particularly MG, which has been accompanied in 20%–25% of thymoma patients based on a previous report (29). In our cohort, approximately 22.9% of thymomas were complicated by MG, but only 7% of TC patients had MG. A further analysis of our data showed that MG did not affect the prognosis of TETs, which was inconsistent with previous studies showing that MG is not an independent or direct prognostic factor of TETs, but it may be helpful in an earlier diagnosis of TETs, leading to a better prognosis (30, 31). However, controversial data showed that patients with MG symptoms had a worse prognosis than patients without MG symptoms (32). The relative small numbers of each cohort due to the low incidence of TETs may contribute to the variance. In addition, the data may present a confounding bias as they were generated from a single center lacking external verification, and therefore, multicenter studies may be needed to draw a comprehensive conclusion.

In our study, the cut-off time was 2020-12-31, and therefore, the 2015 WHO histological classification system was used in this study. Our results demonstrated that this classification was associated with the OS of TET patients, but was not an independent prognostic factor by Cox multivariate survival analysis, which was consistent with previous studies (33, 34). An increased WHO histological grade showed more malignant behavior and was associated with a poor prognosis (33). We also reviewed our data using the new 2021 WHO histological classification and observed no change in our results because the new classification system did not affect our data on the pathological diagnosis of TETs (35).

The Masaoka–Koga stage was established by Masoka in a retrospective analysis of 93 patients with TETs and was further modified by Koga in 1994 (36, 37). Many studies have shown that the Masaoka–Koga stage is a prognostic factor for predicting the survival time of TETs (38–40). In addition to the Masaoka–Koga stage, the 8th UICC/AJCC TNM staging for TET patients is also used in clinical practice. In our study, both the Masaoka–Koga and the 8th UICC/AJCC TNM staging served as independent prognostic factors for TETs as supported by published literatures (41, 42).

Thymectomy is the first choice of treatment advocated for all patients with TETs and has been shown to be an independent prognostic factor. According to Okereke et al. (43), early-stage thymoma and even advanced TETs with pleural metastasis can benefit from surgical resection (44). In our study, although there was only a correlation in the univariate analysis, the data suggested that most patients who have undergone surgery have a good prognosis. Chemotherapy is not recommended for patients who have undergone complete thymoma resection at an early stage (45). Advanced TETs are prone to invading adjacent tissues and organs, resulting in difficulties associated with incomplete resection. Postoperative adjuvant chemotherapy is recommended in patients with advanced TET to reduce the regional recurrence and distant metastasis (46). Therefore, these patients are likely to benefit from cisplatin-based chemotherapy (47). In our study, the patients with TETs, and apparently those with advanced disease, appeared to benefit from chemotherapy, especially in advanced patients. In addition to chemotherapy, radiotherapy has been proposed to improve the survival of patients with advanced TETs (48). Better outcomes have been observed in patients with TETs receiving postoperative radiotherapy (PORT) than in those who only underwent surgery (48). In our study, patients with TC who were treated with PORT showed remarkable local control with mild toxicities. For patients with advanced TETs, particularly TC patients, it may be valuable to explore novel treatment strategies, such as immunotherapy.

Evidence from previous studies implicates the gene encoding the folate-metabolizing enzyme MTHFR in cancer, such as gastric cancer and colorectal cancer (CRC) (49, 50). Our study demonstrates the increased risk of TETs in individuals with heterozygous or homozygous T genotypes (CT+TT) at the C667 polymorphism. Patients with TETs are likely to have CT and TT genotypes, but no correlation was observed between the genetic alteration and prognosis of TETs. According to published data from a meta-analysis of 5,423 CRC patients with CT+TT at the MTHFR C667T polymorphism, no prognostic association was observed with either the CC or CT+TT genotypes at the MTHFR C667T polymorphism (50). Another study concluded that the MTHFR C677T polymorphism is unlikely to be a prognostic factor for esophagogastric cancer (51). These data were consistent with our findings. However, interestingly, data indicated that the MTHFR C667T polymorphism affected the prognosis of patients with CRC who were treated with chemotherapeutic drugs including pemetrexed and 5-fluorouracil (52, 53), suggesting that the impaired the folic acid metabolic pathway may affect the sensitivity to chemotherapy drugs and consequent prognostic events.

MTHFR C677T polymorphisms decrease the enzyme activity of the MTHFR enzyme as compared to the wild type and are associated with DNA hypomethylation (54, 55). This can lead to an increased expression of oncogenes, increased DNA chain breaks, and impaired DNA repair (56). MTHFR methylation levels were negatively correlated with the mRNA level of MTHFR using the data derived from the TCGA database. Aberrant DNA methylation may be the etiology and molecular pathogenesis of malignancies including TETs. Chen et al. reported that the degree of hypomethylation was associated with increased disease severity in TETs (1).