Necroptosis in Esophageal Squamous Cell Carcinoma: An Independent Prognostic Factor and Its Correlation with Tumor-Infiltrating Lymphocytes.

Takuro Yamauchi,Naoki Nakaya,Hironobu Sasano,Yohei Ozawa,Takashi Kamei,Yusuke Gokon,Hiroshi Okamoto,Fumiyoshi Fujishima,Toshiaki Fukutomi,Tomohiro Nakamura,Yusuke Taniyama,Junichi Tsunokake,Masatoshi Hashimoto,Chiaki Sato,Ryujiro Akaishi,Shunsuke Ueki

doi:10.3390/cancers13174473

Abstract

Simple SummaryNecroptosis is a regulated form of necrotic cell death that plays pivotal roles in cancer biology, including tumorigenesis, metastasis, and cancer immunity. However, the significance of necroptosis in esophageal squamous cell carcinoma has remained largely unknown. In addition, its correlation with the tissue microenvironment has not yet been explored. In this study, we first investigated the diagnostic and prognostic significance of mixed lineage kinase domain-like protein (MLKL) and phosphorylated MLKL (pMLKL), both of which are currently considered the most reliable markers for detecting necroptosis. We also investigated the correlations between the status of MLKL/pMLKL and tumor-infiltrating lymphocytes) in esophageal squamous cell carcinoma patients.Necroptosis is a pivotal process in cancer biology; however, the clinical significance of necroptosis in esophageal squamous cell carcinoma (ESCC) has remained unknown. Therefore, in this study, we aimed to verify the potential involvement of necroptosis in the clinical outcome, chemotherapeutic resistance, and tumor microenvironment of ESCC. Mixed lineage kinase domain-like protein (MLKL) and phosphorylated MLKL (pMLKL) were immunohistochemically examined in 88 surgically resected specimens following neoadjuvant chemotherapy (NAC) and 53 pre-therapeutic biopsy specimens, respectively. Tumor-infiltrating lymphocytes (TILs) were also evaluated by immunolocalizing CD3, CD8, and forkhead box protein 3 (FOXP3) in the residual tumors after NAC. High pMLKL status in the post-NAC resected specimens was significantly correlated with worse prognosis in ESCC patients. Multivariate analysis demonstrated that a high pMLKL status was an independent prognostic factor. In pre-NAC biopsy specimens, a high pMLKL status was significantly associated with a lower therapeutic efficacy. CD8+ TILs were significantly lower in the high-pMLKL group. FOXP3+ TILs were significantly higher in both high-MLKL and high-pMLKL groups. We first demonstrated pMLKL status as an independent prognostic factor in ESCC patients. Our study revealed the possible involvement of necroptosis in the immunosuppressive microenvironment, resulting in the attenuated therapeutic efficacy of NAC and eventual adverse clinical outcomes in ESCC.

Highlights

Esophageal cancer is the eighth most common cancer and the sixth leading cause of cancer-related death worldwide [1]
We examined the correlation of these immunoreactivities with clinicopathological factors of the patients, including age and sex, pT, pN, and pStage; tumor differentiation; resected margin; Response Evaluation Criteria in Solid Tumors (RECIST) grade; and histopathological tumor regression grade (Table 1)
In the surgically resected specimens following neoadjuvant chemotherapy (NAC), a high status of phosphorylated MLKL (pMLKL) (detected in 21.6% (19/88)) was significantly correlated with age (p = 0.029) and pT status (p = 0.009)

Summary

Introduction

Esophageal cancer is the eighth most common cancer and the sixth leading cause of cancer-related death worldwide [1]. Esophageal squamous cell carcinoma (ESCC) is the most common esophageal cancer in Japan, and the standard treatment for patients with locally advanced ESCC is radical surgical resection following neoadjuvant chemotherapy (NAC) [2]. It is true that the therapeutic efficacy of NAC exhibits large individual differences, and non–responders experience adverse effects before surgery without any clinical benefit [3,4]. It is important to accurately predict the therapeutic response before the start of chemotherapy in order to establish the optimal treatment strategy for individual ESCC patients. Necroptosis, a molecularly regulated cell death, demonstrates all the morphological features of necrosis, including cell swelling, collapse of plasma membrane, and release of intracellular contents, all of which could subsequently induce secondary inflammatory responses [5,6,7]. Several studies on cell lines, animal models, and human tissue have indicated that necroptosis is a pivotal process in cancer biology, including tumorigenesis, metastasis, and cancer immunity [8,9]

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