Esophageal cancer is the sixth and seventh most common cause of cancer-related mortality and malignancy worldwide, respectively (1). Esophageal adenocarcinoma and esophageal squamous cell carcinoma (ESCC) are the major histopathological types of esophageal cancer, and ESCC is common in Eastern and Central Asia (2). Given the difficulty of early screening for ESCC, most patients are diagnosed at a locally advanced stage.

Among the treatment modalities for esophageal cancer, concurrent chemoradiotherapy (CCRT), as a definitive or neoadjuvant therapy, is beneficial for improving the disease prognosis (3–5). Definitive CCRT and neoadjuvant CCRT followed by surgery are recommended for treating unresectable or locally advanced esophageal cancer, including ESCC. For locally advanced ESCC, the effectiveness of neoadjuvant CCRT on improving survival have been demonstrated in NEOCRTEC 5010 trial (focusing on ESCC) and CROSS trial (with 23% ESCC patients) (6, 7). The definitive CCRT for locally advanced ESCC with response to initial chemoradiation has similar clinical outcome in comparison to initial chemoradiation followed by surgery (8). Taken together, both definitive and neoadjuvant CCRT are optional treatment modalities for locally advanced ESCC. However, long-term survival rate of patients with locally advanced ESCC remains less than 30% (9). Therefore, the development of informative predictors of the prognosis of patients with ESCC is clinically important.

In recent years, advances in technologies for high-throughput genomic surveys, including next-generation sequencing (NGS) of DNA, have enabled comprehensive characterization of somatic mutations in clinical specimens. Through whole-exome or whole-genome sequencing, differential mutations in matched DNA between normal and ESCC tissues have been identified. Among the reported mutations, those in TP53, CDKN2A, FAT1, NOTCH1, PIK3CA, KMT2D, and NFE2L2 were validated as candidate biomarkers for ESCC development. However, there are no biomarkers for predicting the clinical outcomes of ESCC treatment. Therefore, in this study, we evaluated the predictive power of a 35-gene mutation profile, clinicopathological characteristics of patients with ESCC, and radiation parameters in assessing the clinical outcomes in patients with ESCC treated with CCRT.

Using a combination of a 35-gene mutation profile, clinical nodal staging, and RT dosimetry, mutations in FSIP2 and SYNE1 were identified as potential predictors of outcomes of definitive CCRT in patients with ESCC.

Conventional clinicopathological factors and RT dosimetry parameters have been found to be correlated with prognosis in terms of the tumor spreading extent and RT toxicity to the lung. A previous report indicated that in patients with esophageal cancer treated with CCRT and concurrent CCRT, the radiation pneumonitis rate was significantly increased when lung V30 ≥13% (10), and dosimetric variables, including lung V30 >8%, were associated with worse OS in univariate analysis (11), respectively. Therefore, we included lung V30 ≥10% in the analysis model and found that it was a predominant prognostic factor compared with the T and N stages, for PFS and OS. Radiation-induced lung injury including radiation pneumonitis and pulmonary fibrosis are major, sometimes fatal, dose-limiting toxicities of thoracic RT, which may affect the prognosis of patients (12). Regarding the intrinsic characteristics of tumors, gene mutations or mutation profiles examined using the NGS panel revealed a distinct correlation between the clinical outcomes of definitive CCRT. FSIP2, located at 2q32.1, encodes a fibrous sheath-interacting protein. The fibrous sheath is a cytoskeletal structure in the sperm flagellum (13). Recurrent amplification of FSIP2 has been reported in 22% of seminomas (14) and 15.3% of testicular germ cell tumors (15). A higher FSIP2 mutation rate was reported in metastatic breast cancer compared to that in early stage breast cancer (16). Additionally, FSIP2 shows high expression in patients with clear cell renal cell carcinoma and is associated with poor survival outcomes and prognosis (17). Collectively, these results indicate that FSIP2 plays a role in metastasis, tumor invasion, and chemotherapeutic resistance in cancer. A mutation may cause the loss of FSIP2 expression and therefore act as a favorable PFS marker for ESCC. SYNE1 and forkhead box protein E1 promoter methylation have been identified as candidate biomarkers in colorectal cancer plasma DNA (18). A high promoter hypermethylation rate of up to 80% was detected in the biopsy samples of patients with colitis-associated colorectal cancer (19). In addition, cumulative evidences suggest that changes in SYNE1 expression levels, somatic mutations, promoter methylation level, and single-nucleotide polymorphisms are related to the occurrence and development of lung cancer (20), oral cancer (21), hepatocellular carcinoma (22), and gastric cancer (23). Furthermore, SYNE1 was found to be frequently mutated in an Indian ESCC cohort (24). In the present study, we reported SYNE1 mutations associated with worse prognosis in patients with ESCC, which is consistent with a previous report of patients with clear cell renal cell carcinoma showing that SYNE1 mutations correlate with a higher tumor mutation burden and poorer outcomes (25). Additionally, SYNE1 mutations in patients with clear cell renal cell carcinoma are involved in immune response signal and alterations based on the profiles of infiltrating immune cells (25). As radiation is known to trigger the immunologic response, SYNE1 mutation may involve the radioresistant signal. Our results showed that mutations in FSIP2 and SYNE1 have opposite effects on the survival of patients with ESCC treated with definitive CCRT. Further investigations are needed to explore the role of FSIP2 and SYNE1 mutations in the development of biomarkers or treatment targets.

The mutation rates of several genes differed between our ESCC cohort and TCGA cohort (26, 27). As two TCGA cohorts comprised patients from southern and northern China, the different gene mutation rates may not be due to ethnic difference. However, the frequency of locally advanced stage (stage III and IV) in our cohort (86.4%) was significantly higher than in TCGA cohort (50.2%), which may have led to the different mutation rates. It is unclear whether the different mutation rates were related to the tobacco smoking status, consumption of alcoholic beverages, and exposure to fine particulate matters (such as PM_2.5) or indoor air pollutants (such as polycyclic aromatic hydrocarbons) (2, 28). A previous study of a Chinese cohort reported no significant differences in the rate or composition of mutations between smokers and non-smokers and suggested that smoking contributes to the ESCC risk via mechanisms distinct from those in other smoking-related cancers (29). Different allele frequency thresholds of mutations in targeted genes, disease etiology, or disease stage in different studies also cause differences in the mutation rate. These observations indicate that the gene mutation profiles among different sources have significant variations; hence, NGS data should be interpreted with caution.

A limitation of this study was the lack of germline mutation data for comparison with somatic mutations to identify the actual somatic mutations. In addition, the number of samples, particularly those from females, used for NGS was relatively small; therefore, the reported mutation frequencies may not be fully representative of a larger population. Inclusion of adequate numbers of male and female patients with ESCC is required in further studies. Mutant genes may generate chemoresistant or radioresistant tumor cells and alter the chemosensitivity or radiosensitivity of patients with ESCC. Further validation of the biological functions and clinical roles of FSIP2 and SYNE1 in both ESCC experimental models and patients is warranted.

In conclusion, a combination of a 35-gene mutation profile and RT dosimetry identified mutations in FSIP2 and SYNE1 as well as lung V30 and clinical nodal staging as potential predictors for developing a prediction model for clinical outcomes of patients with ESCC treated with definitive CCRT.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/sra/PRJNA742478.

The studies involving human participants were reviewed and approved by Ethics Committee of Tianjin Medical University Institute and Hospital (Documentation number #bc2018057). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

Y-JC conceived and supervised all works. Y-CH and Y-JC designed, analyzed and drafted the article. PT, CT, QP, YW and ZY collected the patient samples and clinical data. C-WC participated interpretation the data. All authors contributed to the article and approved the submitted version.

This research was funded in part by research grants from the MacKay Memorial Hospital, Taipei, Taiwan (MMH-E-109-13 and MMH-E-110-13), Ministry of Science and Technology, Taiwan (MOST 108-2320-B-039-023-MY3), and China Medical University, Taiwan (CMU109-MF-22).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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