Positive Esophageal Squamous Cell Carcinoma Research Articles

Updated results of first-line serplulimab versus placebo plus chemotherapy in PD-L1–positive esophageal squamous cell carcinoma: A randomized, double-blind, multicenter phase 3 study (ASTRUM-007).

e16016 Background: Immune checkpoint inhibitors plus chemotherapy (chemo) improve antitumor efficacy as first-line treatment in patients with esophageal squamous cell carcinoma (ESCC). However, the optimal target population for this combination therapy remains uncertain. At ESMO Asia Congress 2022, we presented results from the interim analysis of the phase 3 study of serplulimab (anti-PD-1 antibody) plus chemo as first-line treatment for PD-L1–positive (CPS ≥1) ESCC patients. Here we report the results from an updated analysis, which is also the final analysis of overall survival (OS). Methods: In this randomized, double-blind, multicenter phase 3 study, patients with histologically confirmed locally advanced or distantly metastatic, PD-L1–positive ESCC who had no prior systemic antitumor therapy were randomized 2:1 to receive serplulimab 3 mg/kg or placebo plus chemo (5-FU + cisplatin) intravenously Q2W. Randomization was stratified by PD-L1 expression level (CPS ≥10 vs CPS < 10), age (≥65 vs < 65 years), and disease status (locally advanced vs distantly metastatic). The primary endpoints were IRRC-assessed progression-free survival (PFS) per RECIST v1.1 and OS. Secondary endpoints included other efficacy measures, safety, quality of life, and biomarkers. Results: Between June 19, 2019 and December 17, 2021, 976 patients were screened and 551 were randomized (serplulimab-chemo, n = 368; placebo-chemo, n = 183). At this updated analysis, median follow-up duration was 24.2 months. Median OS was significantly longer in the serplulimab-chemo group than the placebo-chemo group (14.6 vs 11.2 months; hazard ratio [HR] 0.70, 95% CI 0.57–0.86; P = 0.0006). IRRC-assessed median PFS per RECIST v1.1 was prolonged with the addition of serplulimab (6.5 vs 5.3 months; HR 0.58, 95% CI 0.47–0.72). Efficacy improvements were also observed in confirmed objective response rate (58.7% vs 42.1%) and duration of response (median, 7.1 vs 4.6 months) as assessed by IRRC per RECIST v1.1. Grade ≥3 treatment-related adverse events were reported in 203 (53.1%) and 82 (48.8%) patients in the respective groups. Incidence of grade ≥3 immune-related adverse events was higher in the serplulimab-chemo group compared to the placebo-chemo group (9.2% vs 3.0%). Fifteen deaths (3.1% in the serplulimab-chemo group and 1.8% in the placebo-chemo group) that might be related to study treatment were reported. Conclusions: With another 9.3 months of follow-up, the encouraging efficacy and manageable safety of serplulimab plus chemo as first-line treatment in patients with PD-L1–positive advanced ESCC were maintained, further supporting the development of serplulimab plus 5-FU and cisplatin for previously untreated ESCC patients. Clinical trial information: NCT03958890 .

Association of supraclavicular node metastasis with survival in node positive esophageal squamous cell carcinoma patients treated using definitive chemoradiation

Association of supraclavicular node metastasis with survival in node positive esophageal squamous cell carcinoma patients treated using definitive chemoradiation

Chloroquine Inhibits Stemness of Esophageal Squamous Cell Carcinoma Cells Through Targeting CXCR4-STAT3 Pathway.

Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent cancers worldwide. Recent studies have shown that cancer stem cells (CSCs) are present in ESCC, are thought to lead to aggressive tumor behavior and the prognosis. The CXC chemokine receptor 4 (CXCR4), is regarded as a putative CSCs marker in various malignancies. Here, we demonstrate that CXCR4 played a key role in ESCC progression and CXCR4 positive ESCC cells possessed stem-like properties. Furthermore, the anti-malarial agent chloroquine (CQ) targeted CXCR4-positive ESCC cells via STAT3 pathway. Therefore, CQ with anti-CSCs effects may be an effective adjunct to current ESCC chemotherapy regimens.

Abstract 5309: Inhibition of cancer stem-like cells in esophageal squamous cell carcinoma with SOCS-1 gene therapy

Abstract INTRODUCTION: Cancer stem-like cells are thought to be one of the causes of cancer initiation and progression. They are associated with the resistance to radiotherapy and chemotherapy for various types of tumor including esophageal squamous cell carcinoma (ESCC). Therefore, cancer stem-like cells are promising therapeutic targets of cancer. Suppressor of cytokine signaling-1 (SOCS-1) was identified as a negative regulator of various cytokine signaling. We have previously demonstrated that overexpression of SOCS-1 showed a potent anti-tumor effect on ESCC through inhibiting of the JAK/STAT signaling pathway. This study aimed to evaluate the anti-tumor effect of SOCS-1 gene therapy using adenoviral vector (AdSOCS-1) against cancer stem-like cells in ESCC. METHODS: ESCC cell lines were separated into aldehyde dehydrogenase (ALDH) positive and ALDH negative cells by ALDEFLOUR, and then sphere formation assays were performed. The inhibitory effects of AdSOCS-1 and AdLacZ on the sphere formation were evaluated using TE8, TE14 ESCC cell lines. The inhibition of sphere formation was also evaluated using ESCC cells obtained from ESCC PDX mice (ESCC8). Next, the expression of transcription factors associated with cancer stem-like cells in ESCC was evaluated by RT-PCR. The inhibitory effects on tumor formation by AdSOCS-1 were evaluated by tumorigenicity assay using TE8 xenograft mice. RESULTS: In the sphere formation assay, ALDH positive TE8 cells and TE14 cells represented strong sphere formation capacity. Compared with AdLacZ infected ALDH positive cells, sphere formation of AdSOCS-1 infected ALDH positive cells was significantly inhibited. The inhibition of sphere formation was also confirmed in ESCC cells obtained from ESCC8 PDX mouse tumor tissue. Interestingly, the expression level of Sox2, a critical stem cell factor regulated by STAT3, was also decreased by AdSOCS-1. Furthermore, AdSOCS-1 decreased the tumorigenic activity of the ALDH positive ESCC tumor cells. CONCLUSIONS: A potent anti-tumor effect of SOCS-1 gene therapy on ESCC may be partially explained by inhibiting the expression of a STAT3 regulated transcription factor critical for cancer stem-like cells. Citation Format: Ayumi Yamamoto, Eri Munekage, Satoshi Serada, Shigehiro Tsujii, Kosuke Hiramatsu, Minoru Fujimoto, Kazuhiro Hanazaki, Tetsuji Naka. Inhibition of cancer stem-like cells in esophageal squamous cell carcinoma with SOCS-1 gene therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5309.

Abstract 5421: Roles of sex hormone pathways in the gender disparity of esophageal squamous-cell carcinoma

Abstract Objectives: Esophageal squamous-cell carcinoma (ESCC) incidence has been increasing rapidly, with an imbalanced ratio of occurrence in men more often than in women. The expression of sex hormone receptors is associated with the prognosis of ESCC. However, the exact roles of sex hormones and hormone receptors in ESCC remain unclear. We systematically evaluated the effects and the underlying mechanism of sex hormones and their receptors on esophageal carcinogenesis and malignant proliferation to clarify the extent to which sex hormones pathways contribute to the gender disparity of ESCC. Methods: To determine the roles of hormones in vivo, we used a 4NQO-induced esophageal carcinogenesis model, a xenograft model and the castrated BALB/c Nude mouse model. To observe the function of hormones on the proliferation of ESCC cells in vitro, we performed colony formation, Cell Counting Kit-8, and EdU assays. To elucidate the precise molecular mechanism by which hormone receptors exert their functions, we performed ChIP-seq and RNA-seq to comprehensively identify the ESCC-specific hormone responsive genes and regulatory networks. Results: There is a large male predominance in the 4NQO-induced esophageal carcinogenesis C57BL/6J mouse model. AR-positive esophageal cancer cells grow more rapidly in the male BALB/c Nude mice than the female mice. ERα-positive esophageal cancer cells possess faster proliferative capability in the female BALB/c Nude mice compared to the male mice, whereas ERβ-positive cells exhibit the opposite gender disparity in phenotypes. Moreover, the above proliferative effect is attenuated in castrated or ovariectomized mice. Furthermore, exogenous supplementation of estrogen pellet in ovariectomized female mice enhances ERα-positive cell growth but suppresses ERβ-positive cell growth. In vitro experimental data show that dihydrotestosterone treatment promotes the growth of AR positive ESCC cells, while 17-β estradiol (E2) exerts opposite functions depending on the estrogen receptor subtypes. Integrative analysis of ChIP-seq and RNA-seq data reveals that AR/ER is transactivated upon hormone treatment and binds to the promoter or enhancer of hormone responsive genes to induce differential gene expression. These genes are responsible for the malignant development of ESCC and may provide a new explanation of the gender disparity of ESCC. Conclusions: Androgen promotes the proliferation of ESCC cells via AR while estrogen exerts dual functions dependent on the specific estrogen receptor subtypes, which may provide new insight into the etiology, prevention, and hormone-based treatment of ESCC. Citation Format: Furong Huang, Hongyan Chen, Qianben Wang, Zhihua Liu. Roles of sex hormone pathways in the gender disparity of esophageal squamous-cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5421.

Glypican-1 targeted antibody-based therapy induces preclinical antitumor activity against esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) has a poor prognosis despite the development of multimodal therapy. Expression of glypican-1 (GPC1) has been reported to be elevated in a subset of patients with ESCC and associated with chemoresistance. This study aimed to determine the association of GPC1 with ESCC growth and potential usefulness of the GPC1 targeted therapy by monoclonal antibody (mAb) in ESCC. Expression of GPC1 was higher in ESCC tumor tissues than in adjacent non-tumoral tissues and normal tissues. Knockdown of GPC1 decreased growth of ESCC cells and induced apoptosis via inhibition of EGFR, AKT and p44/42-MAPK signaling pathways in vitro. Anti-GPC1 mAb strongly inhibited tumor growth via antibody-dependent cellular cytotoxicity dependent and independent manner in GPC1-positive ESCC xenograft models. Anti-GPC1 mAb also inhibited tumor growth of GPC1 positive ESCC patients derived tumor xenograft models. Furthermore, anti-GPC1 mAb showed a significant tumor growth inhibition with decreased angiogenesis compared with IgG treated controls in ESCC xenografted mice. Treatment with anti-GPC1 mAb was not toxic in mice. Anti-GPC1 mAb may have a potent anti-tumor effect and represent a novel treatment option for patients with GPC1-positive ESCC.

MT3-MMP down-regulation promotes tumorigenesis and correlates to poor prognosis in esophageal squamous cell carcinoma.

The membrane‐type matrix metalloproteinases (MT‐MMPs) play an important role in degrading the extracellular matrix (ECM) and facilitating protease‐dependent tumor progression and invasion. Here, we report that unlike MT1‐MMP, MT3‐MMP was down‐regulated in esophageal squamous cell carcinoma (ESCC) as detected by real‐time PCR (qPCR), Western blot analysis, and immunohistochemistry (IHC). Down‐regulation of MT3‐MMP was observed at protein level in 66.3% of ESCC specimens (by IHC, n = 86) for routine pathologic diagnosis, as well as at mRNA level in 63.3% of surgically resected ESCC tumors paired with surrounding nontumor tissues (by qPCR, n = 30). Notably, MT3‐MMP down‐regulation significantly correlated with lymph node metastasis and poor overall survival of patients with ESCC (median 5‐year survival = 50.69 vs. 30.77 months for patients with MT3‐MMP‐negative and ‐positive ESCC, respectively). Mechanistically, MT3‐MMP negatively regulated proliferation, colony formation, and migration of ESCC cells, in association with cell cycle arrest at G1, due to up‐regulation of p21Cip1 and p27Kip1. Together, as a tumor suppressor in ESCC, MT3‐MMP down‐regulation represents an unfavorable factor for prognosis of patients with ESCC.

Contribution of nestin positive esophageal squamous cancer cells on malignant proliferation, apoptosis, and poor prognosis.

BackgroundThe stem cell-associated intermediate filament nestin has recently been linked with neoplastic transformation, but the specific mechanism by which nestin positive tumor cells leads to malignant invasion and metastasis behaviors of esophageal squamous cell carcinoma (ESCC) remains unclear.MethodsTo obtain insight into the biological role of nestin in ESCC, we explored the association of the nestin phenotype with malignant proliferation and apoptosis in esophageal squamous cancer cells. Nestin expression was determined in ESCC specimens and cell lines, and correlated with clinicopathological properties, including clinical prognosis and proliferative markers. The association of the nestin phenotype with apoptotic indicators was also analyzed.ResultsNestin was expressed in ESCC specimens and cell lines. ESCC patients with nestin-positive tumors had significantly shorter median survival and progression-free survival times than those with nestin-negative tumors. Positive staining for the proliferation markers Ki67 and PCNA (proliferating cell nuclear antigen) was detected in 56.9% and 60.2% of ESCC specimens, respectively, and was strongly correlated with the nestin phenotype. Notably, expression of cyclin dependent kinase-5 (CDK5) and P35 was detected in 53.8% and 48.4% of ESCC specimens, respectively, and was strongly associated with the nestin phenotype.ConclusionOur data demonstrated nestin expression in ESCC specimens and cell lines, and revealed a strong association of the nestin phenotype with poor prognosis in ESCC patients. Furthermore, we showed that nestin positive ESCC cells played an important role in the malignant proliferation and apoptosis.

Mitogen activated protein kinase kinase kinase 3 (MAP3K3/MEKK3) overexpression is an early event in esophageal tumorigenesis and is a predictor of poor disease prognosis

BackgroundMitogen-activated protein kinase kinase kinase3 (MAP3K3/MEKK3) was identified to be differentially expressed in esophageal squamous cell carcinoma (ESCC) using cDNA microarrays by our laboratory. Here in we determined the clinical significance of MEKK3 in ESCC.MethodsImmunohistochemical analysis of MEKK3 expression was carried out in archived tissue sections from 93 ESCCs, 47 histologically normal and 61 dysplastic esophageal tissues and correlated with clinicopathological parameters and disease prognosis over up to 7.5 years for ESCC patients.ResultsMEKK3 expression was significantly increased in esophageal dysplasia and ESCC in comparison with normal mucosa (ptrend < 0.001). Kaplan Meier survival analysis showed significantly reduced median disease free survival median DFS = 10 months in patients with MEKK3 positive ESCCs compared to patients with no immunopositivity (median DFS = 19 months, p = 0.04). ESCC patients with MEKK3 positive and lymph node positive tumors had median DFS = 9 months, as compared to median DFS = 21 months in patients who did not show the alterations (p = 0.01). In multivariate Cox regression analysis, combination of MEKK3 overexpression and node positivity [p = 0.015, hazard ratio (HR) = 2.082, 95% CI = 1.154 - 3.756] emerged as important predictor of reduced disease free survival and poor prognosticator for ESCC patients.ConclusionsAlterations in MEKK3 expression occur in early stages of development of ESCC and are sustained during disease progression; MEKK3 in combination with lymph node positivity has the potential to serve as adverse prognosticator in ESCC.

Sa1787 Regulation of Chemotherapy Resistance Through Epithelial-Mesenchymal Transition by Zinc Finger E-Box-Binding Proteins

Introduction: Zinc finger E-box-binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in transforming growth factor (TGF)-β-mediated epithelial-to-mesenchymal transition (EMT) and cancer stem cell functions. We have demonstrated recently that ZEBs are upregulated in esophageal squamous cell carcinoma (ESCC) cells with mesenchymal characteristics and increased malignant potential such as anchorage independent growth, invasiveness and tumorigenicity upon xenograft transplantation (Cancer Res. 2011;71:683647). However, the functional roles of ZEBs in chemotherapeutic resistance remain to be elucidated. Methods: Primary tumor tissues from patients who underwent surgery with or without neoadjuvant chemotherapy were analyzed. ESCC cell lines and In Vitro transformed human esophageal cells EPC2T were treated with either 5-fluorouracil or cisplatin, and subjected to WST-1 colorimetric cell proliferation assays, phase contrast imaging, flow cytometry, quantitative RT-PCR andWestern blotting analyses. Short hairpin RNA was stably transduced by lentivirus to knockdown ZEB1 and ZEB2 in a regulatable manner (Tet-On system). Results: 70% of primary tumor tissues from patients who received neoadjuvant chemotherapy (n=10) contained ZEB1 positive ESCC cells, reminiscent of EMT. By contrast, ZEB1 positive ESCC cells were found in 33% of patients without neoadjuvant chemotherapy (n=51). EMT was present in a subset of cultured ESCC cells. The percentage of mesenchymal cells correlated with downregulation of E-cadherin and upregulation of N-cadherin, Vimentin and ZEBs. Moreover, flow cytometry revealed that the mesenchymal ESCC cells express a high level of CD44, a cancer stem cell marker, implicated in invasion, metastasis and chemoresistance. Interestingly, chemotherapeutic drug sensitivity was reduced as a function of the frequency of mesenchymal subset of the cells when four different cell lines were compared. In two cell lines (TE1 and TE5) with moderate levels of mesenchymal cells, the cells surviving 5-fluorouracil treatment were predominantly mesenchymal. In HCE7 cells displaying fully mesenchymal characteristics, knockdown of either ZEB1 or ZEB2 increased 5-fluorouracil sensitivity. Moreover, TGF-β induced EMT in EPC2T cells, resulted in a significantly increased cell viability rate upon cisplatin treatment and that was antagonized by knockdown of either ZEB1 or ZEB2. Conclusions: These data demonstrate that ZEB1 and ZEB2 confer chemotherapy resistance through EMT-mediated enrichment of a unique subset of ESCC cells defined by a CD44 upregulation. Neoadjuvant therapy may induce ZEB1 positive cells, thus having broader implications upon EMT and cancer therapy.

Expression of p21/WAF‐1, status of apoptosis and p53 mutation in esophageal squamous cell carcinoma with HPV infection

Human papilloma virus (HPV) is regarded as a causative carcinogenic agent in anogenital squamous cell carcinoma (SCC), but there is controversy about its etiologic role in esophageal SCC (ESCC). In this study, we attempted to clarify whether HPV infection plays a crucial role in the development of ESCC by analysis of multiple factors. These included: detection of HPV DNA; evaluation of immunohistochemical assays for HPV-related cell cycle regulators and apoptosis by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling method; and genetic analysis of the p53 gene. Twenty of the 48 ESCC examined (42%) were found to be positive for the HPV genome by polymerase chain reaction. They comprised 16 cases with the HPV16 subtype, three with the HPV18 subtype, and one with both HPV16 and 18. Immunohistochemical analysis revealed that the expression of p21/WAF-1 was significantly decreased in HPV-positive cases (chi2 = 9.2614; P = 0.0023). Furthermore, the 10 apoptosis-negative (< or =10%) cases of HPV-positive SCC were almost exclusively p21/WAF-1-negative (chi2 = 12.1406; P = 0.0005), indicating the significance of the relationship between HPV infection and the phenotype that is expected from HPV-induced inhibition of p53. Although 14 cases possessed missense and deletion mutations of the p53 gene (of which four mutations were found in HPV-positive ESCC), no accumulation of the mutation was defined in the phenotype, suggesting that distinct mutation processes might be involved in HPV-negative and -positive ESCC. The data provide significant support for the hypothesis that HPV infection may play a crucial role in the oncogenesis of some ESCC.