Early-stage Esophageal Squamous Cell Carcinoma Research Articles

235. A NOVEL TUMOR STAGING SYSTEM INCORPORATING CN STATUS FOR STRATIFYING EARLY STAGE ESOPHAGEAL SQUAMOUS CELL CARCINOMA PATIENTS AFTER TRIMODAL THERAPY

Abstract Background This study aims to assess the prognostic significance of cN status in early-stage esophageal squamous cell carcinoma (ESCC) patients following neoadjuvant chemoradiotherapy (nCRT) and develop a novel staging model for personalized survival prediction. Methods Patients with ESCC who underwent nCRT and esophagectomy were included. The Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) were employed to evaluate the validity of various oncological staging systems. A reduction in AIC and BIC values indicates improved predictive capability and accuracy. Results We established a novel staging model by incorporating ypTNM stage and cN status, effectively stratifying ypI ESCC patients. Cox regression analysis showed that only cN status was an independent prognostic factor for both overall survival (p = 0.030) and disease-free survival (p = 0.019) in patients with stage ypI. The newly developed ypTNM-cN staging exhibited a superior alignment with overall survival trends compared to the AJCC 8th ypTNM staging, with a significantly lower AIC of 3143.014 versus 3149.950. This superiority was further supported by a BIC of 3146.605 compared to 3153.541. In terms of disease-free survival outcomes, the emerging ypTNM-cN staging, with AIC and BIC values of 3196.057 and 3199.648, respectively, outperformed the AJCC 8th ypTNM staging, which recorded values of 3203.853 and 3207.444. Conclusion We have devised a novel staging system based on ypTNM stage and cN status to accurately categorize patients with ypI stage. Our ypTNM-cN staging system offers valuable insights into the classification of ypI ESCC and demonstrates reliable classification efficacy for all ESCC patients following nCRT and surgery.

799. OPTIMUM NUMBERS OF DISSECTED LYMPH NODES FOR ACCURATE NODAL STAGING IN CT1-2N0 ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Abstract Background Patients with esophageal squamous cell carcinoma (ESCC) with lymph node metastasis may be misclassified as pN0 due to an inadequate lymph node dissection. The purpose of this study was to confirm that patients with cT1-2N0 ESCC are indeed pN0 and to propose an adequate number of lymph nodes for correct nodal staging. Methods A total of 1,855 patients who underwent upfront surgery for cT1-2N0 ESCC at two tertiary academic hospital in Korea between 1995 and 2019 were included in the study. The probability of false-negative lymphadenectomy and NSS (nodal staging score) was calculated to assess the probability of pN0 status. Results The mean age was 63.36 ± 8.01 years, with 1,726 (93.05%) male patients. The mean number of dissected lymph nodes was 34.69 ± 13.98. The clinical T stages were as follows: cTis & cT1a 354 (19.08%), cT1b 1,053 (56.77%), and cT2 448 (24.15%). Pathologic nodal upstaging occurred in 443 (23.91%); pN1 352 (19.0%), pN2 78 (4.21%), and pN3 13 (0.7%). By calculating the probability of false-negative lymphadenectomy, removing fewer than 33 lymph nodes was associated with a 20% risk of missing a positive lymph node, whereas removing 53 lymph nodes was associated with a 10% risk of missing a positive lymph node. The NSS, which reflects the probability that a patient with ypN0 status is actually free of occult nodal disease after surgery, increased as the number of dissected lymph nodes increased. Conclusions Extensive lymph node dissection is mandatory for accurate staging and reducing the risk of missing positive lymph nodes in early-stage ESCC. The survival benefits of extensive lymph node dissection need to be evaluated in further analysis.

ESCCPred: a machine learning model for diagnostic prediction of early esophageal squamous cell carcinoma using autoantibody profiles.

Esophageal squamous cell carcinoma (ESCC) is a deadly cancer with no clinically ideal biomarkers for early diagnosis. The objective of this study was to develop and validate a user-friendly diagnostic tool for early ESCC detection. The study encompassed three phases: discovery, verification, and validation, comprising a total of 1309 individuals. Serum autoantibodies were profiled using the HuProtTM human proteome microarray, and autoantibody levels were measured using the enzyme-linked immunosorbent assay (ELISA). Twelve machine learning algorithms were employed to construct diagnostic models, and evaluated using the area under the receiver operating characteristic curve (AUC). The model application was facilitated through R Shiny, providing a graphical interface. Thirteen autoantibodies targeting TAAs (CAST, FAM131A, GABPA, HDAC1, HDGFL1, HSF1, ISM2, PTMS, RNF219, SMARCE1, SNAP25, SRPK2, and ZPR1) were identified in the discovery phase. Subsequent verification and validation phases identified five TAAbs (anti-CAST, anti-HDAC1, anti-HSF1, anti-PTMS, and anti-ZPR1) that exhibited significant differences between ESCC and control subjects (P < 0.05). The support vector machine (SVM) model demonstrated robust performance, with AUCs of 0.86 (95% CI: 0.82-0.89) in the training set and 0.83 (95% CI: 0.78-0.88) in the test set. For early-stage ESCC, the SVM model achieved AUCs of 0.83 (95% CI: 0.79-0.88) in the training set and 0.83 (95% CI: 0.77-0.90) in the test set. Notably, promising results were observed for high-grade intraepithelial neoplasia, with an AUC of 0.87 (95% CI: 0.77-0.98). The web-based implementation of the early ESCC diagnostic tool is publicly accessible at https://litdong.shinyapps.io/ESCCPred/ . This study provides a promising and easy-to-use diagnostic prediction model for early ESCC detection. It holds promise for improving early detection strategies and has potential implications for public health.

Development and validation of predictive models for esophageal squamous cell carcinoma and its precancerous lesions using terminal motif analysis in circulating cell-free DNA

Objectives: To develop and validate predictive models for esophageal squamous cell carcinoma (ESCC) using circulating cell-free DNA (cfDNA) terminal motif analysis. The goal was to improve the non-invasive detection of early-stage ESCC and its precancerous lesions. Methods: Between August 2021 and November 2022, we prospectively collected plasma samples from 448 individuals at the Department of Endoscopy, Cancer Hospital, Chinese Academy of Medical Sciences for cfDNA extraction, library construction, and sequencing. We analyzed 201 cases of ESCC, 46 high-grade intraepithelial neoplasia (HGIN), 46 low-grade intraepithelial neoplasia (LGIN), 176 benign esophageal lesions, and 29 healthy controls. Participants, including ESCC patients and control subjects, were randomly assigned to a training set (n=284) and a validation set (n=122). The training cohort underwent z-score normalization of cfDNA terminal motif matrices and a selection of distinctive features differentiated ESCC cases from controls. The random forest classifier, Motif-1 (M1), was then developed through principal component analysis, ten-fold cross-validation, and recursive feature elimination. M1's efficacy was then validated in the validation and precancerous lesion sets. Subsequently, individuals with precancerous lesions were included in the dataset and participants were randomly allocated to newly formed training (n=243), validation (n=105), and test (n=150) cohorts. Using the same procedure as M1, we trained the Motif-2 (M2) random forest model with the training cohort. The M2 model's accuracy was then confirmed in the validation cohort to establish the optimal threshold and further tested by performing validation in the test cohort. Results: We developed two cfDNA terminal motif-based predictive models for ESCC and associated precancerous conditions. The first model, M1, achieved a sensitivity of 90.0%, a specificity of 77.4%, and an area under the curve (AUC) of 0.884 in the validation cohort. For LGIN, HGIN, and T1aN0 stage ESCC, M1's sensitivities were 76.1%, 80.4%, and 91.2% respectively. Notably, the sensitivity for jointly predicting HGIN and T1aN0 ESCC reached 85.0%. Both the predictive accuracy and sensitivity increased in line with the cancer's progression (P＜0.001). The second model, M2, exhibited a sensitivity of 87.5%, a specificity of 77.4%, and an AUC of 0.857 in the test cohort. M2's sensitivities for detecting precancerous lesions and ESCC were 80.0% and 89.7%, respectively, and it showed a combined sensitivity of 89.4% for HGIN and T1aN0 stage ESCC. Conclusions: Two predictive models based on cfDNA terminal motif analysis for ESCC and its precancerous lesions are developed. They both show high sensitivity and specificity in identifying ESCC and its precancerous stages, indicating its potential for early ESCC detection.

Prognostic factors for patients with pathologic T1-T2N+ esophageal squamous cell carcinoma: A retrospective study with external validation

BackgroundLymph node metastasis is significantly associated with a worse prognosis in patients with localized early-stage esophageal squamous cell carcinoma. This study aimed to explore the prognostic factors and develop a nomogram for predicting survival in patients with pathologic T1-2N+ esophageal squamous cell carcinoma. MethodsBetween 2014 and 2022, patients with pT1-2N+ esophageal squamous cell carcinoma who underwent esophagectomy with lymphadenectomy at 2 institutes were reviewed and assigned to training and external validation cohorts. Independent prognostic factors were identified via univariate and multivariate Cox regression analyses. The nomogram model was developed and evaluated by the area under the receiver operating characteristic curve and calibration curve. ResultsIn total, 268 patients with a median age of 65 years (range, 40–82) were included and assigned to training (n = 190) and external validation (n = 78) cohorts. The Cox proportional hazards model demonstrated that body mass index (P = .031), surgical approach (P < .001), T stage (P = .015), and Clavien-Dindo classification (P < .001) were independent prognostic factors in the training cohort. The nomogram showed good discrimination, with an area under the receiver operating characteristic curve for 1-year, 3-year, and 5-year of 0.810, 0.789, and 0.809 in the training cohort and 0.782, 0.679, and 0.698 in the validation cohort. The calibration curve showed that the predicted survival probability was in good agreement with the actual survival probability. ConclusionLower body mass index, left surgical approach, T2 stage, and Clavien–Dindo classification grade III to V were related to worse prognosis in patients with pT1-T2N+ esophageal squamous cell carcinoma. The developed nomogram may predict individual survival accurately.

Lysophosphatidic acid promotes ESCC progression by increasing the level of CCL2 secreted by esophageal epithelial cells.

Lysophosphatidic acid (LPA) is a small bioactive lipid which acts as a potent regulator in various tumor progressions through six G-protein-coupled receptors (LPA1-LPA6). Our previous study demonstrated that the LPA-producing enzyme, autotaxin (ATX), was upregulated in esophageal squamous cell carcinoma (ESCC) and ATX high expression levels indicated a poor prognosis. Esophageal squamous cell carcinoma is a type of malignant tumor which originates from epithelial cells. Its progression can be affected by the interaction between cancer cells and normal cells. However, the impact of LPA on the interaction between esophageal epithelial cells and cancer cells in the development of ESCC remains uncertain. MTS and Edu assays were performed to determine ESCC cell proliferation in culture medium (CM) derived from LPA-stimulated esophageal epithelial cells (Het-1a). A wound healing assay, transwell migration and an invasion assay were performed to assess the metastatic ability of ESCC cells. Cytokine array analysis was conducted to detect the differentially secreted cytokines in CM. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were utilized to uncover the pathways and cytokines that are influenced by LPA in ESCC. Immunohistochemical staining was employed to measure the expression of ATX and CCL2 in early-stage ESCC. Quantitative real-time PCR, western blot, enzyme-linked immunosorbent assay and an antibody neutralization assay were employed to measure the mechanism of LPA-mediated communication between epithelial cells and cancer cells. Functional experiments showed that exposing ESCC cancer cells to CM from LPA-treated Het-1a results in promoting proliferation, migration, invasion and epithelial-mesenchymal transition processes. Using cytokine array analysis, we discovered that LPA triggers the release of multiple cytokines from epithelial cells. After screening of the TCGA and GEO databases, CCL2 was identified and found to be correlated with ATX expression in ESCC. Furthermore, CCL2 levels in both mRNA expression and secretion were observed to be upregulated in epithelial cells upon stimulation with LPA. Blocking CCL2 effectively reduced the pro-migration influence of CM derived from LPA-treated Het-1a. Mechanism studies have demonstrated that LPA activated the NF-κB signaling pathway through LPA1/3, ultimately causing an increase in CCL2 expression and secretion in Het-1a. Our findings, taken together, demonstrate that CM from LPA-treated esophageal epithelial cells plays a significant role in promoting the progression of ESCC, with CCL2 acting as the primary regulator.

Multimodal analysis of cfDNA methylomes for early detecting esophageal squamous cell carcinoma and precancerous lesions

Detecting early-stage esophageal squamous cell carcinoma (ESCC) and precancerous lesions is critical for improving survival. Here, we conduct whole-genome bisulfite sequencing (WGBS) on 460 cfDNA samples from patients with non-metastatic ESCC or precancerous lesions and matched healthy controls. We develop an expanded multimodal analysis (EMMA) framework to simultaneously identify cfDNA methylation, copy number variants (CNVs), and fragmentation markers in cfDNA WGBS data. cfDNA methylation markers are the earliest and most sensitive, detectable in 70% of ESCCs and 50% of precancerous lesions, and associated with molecular subtypes and tumor microenvironments. CNVs and fragmentation features show high specificity but are linked to late-stage disease. EMMA significantly improves detection rates, increasing AUCs from 0.90 to 0.99, and detects 87% of ESCCs and 62% of precancerous lesions with >95% specificity in validation cohorts. Our findings demonstrate the potential of multimodal analysis of cfDNA methylome for early detection and monitoring of molecular characteristics in ESCC.

Abstract 1075: Buccal mucosal remodeling predicts esophageal cancer risk

Abstract Lifestyle habits, including alcohol consumption, tobacco smoking, and germline variants of ALDH2 and ADH1B, increase the risk of developing esophageal squamous cell carcinoma (ESCC). However, current prediction methods that rely on self-reported lifestyle information lack accuracy. We previously demonstrated that driver-mutated clones remodel the esophageal epithelium with age and that this remodeling is accelerated in heavy drinkers and smokers. Nonetheless, predicting cancer from clonal expansion in normal tissues remains challenging, due to limited sample availability beyond the blood. The squamous epithelium continuously covers the esophagus, throat, and oral cavity. Therefore, we evaluated buccal mucosal remodeling by mutant clones and its potential as a predictive biomarker of ESCC. A total of 387 buccal mucosa samples were collected from 110 subjects, including 62 with ESCC and 48 without, using swabs of three different sizes, followed by error-corrected sequencing targeting 26 driver genes that showed positive selection in samples from ESCC and physiologically normal esophageal tissues, as well as ALDH2 and ADH1B. Somatic mutations were detected in all samples (median, 27 (1-134)). In total, 13,235 mutations, including 12,085 nonsynonymous and 1,150 synonymous mutations, with VAFs ranging from 0.0016-0.12 (median, 0.0025), were detected. Ten of the 26 genes, TP53, CHEK2, NOTCH1, CDKN2A, NOTCH2, FAT1, AJUBA, PPM1D, ARID1A, and ZNF750 showed positive selection (dN/dS &amp;gt;1, q&amp;lt;0.1). Correlation analysis demonstrated that driver mutations in TP53, FAT1, and PPM1D were positively correlated with not only germline risk variants of ALDH2 but also alcohol consumption. The impact of alcohol consumption on the number and sum of VAFs of mutations in each of the three genes was analyzed and stratified by the presence or absence of the germline risk variant of ALDH2, and positive correlations were observed only among subjects with risk variants. Compared to subjects without ESCC, patients with early-stage or advanced ESCC displayed significantly higher numbers and sums of VAFs of driver mutations (p&amp;lt;0.05). These results indicated that buccal mucosal remodeling driven by positively selected mutations is associated with lifestyle and germline risk factors for ESCC, thus motivating the establishment of a predictive model for ESCC using genetic swab data. The prediction model for ESCC using sequencing data outperformed a model using self-reported lifestyle and alcohol flushing reaction and a model using lifestyle and germline risk variants (AUC of 0.94, 0.72, and 0.88, respectively). In conclusion, this is the first study to use clonal expansion in normal tissues to predict the presence of solid organ cancer, and our study successfully established a prediction model for ESCC based on buccal mucosal remodeling and germline variants. Citation Format: Akira Yokoyama, Tomonori Hirano, Koichi Watanabe, Masashi Tamaoki, Kenshiro Hirohashi, Yoshihiro Ishida, Yoshikage Inoue, Yasuhide Takeuch, Yo Kishimoto, Soo Ki Kim, Chikatoshi Katada, Yasuhito Nanya, Hiroshi Seno, Seishi Ogawa, Manabu Muto, Nobuyuki Kakiuchi. Buccal mucosal remodeling predicts esophageal cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1075.

NMR and MS reveal characteristic metabolome atlas and optimize esophageal squamous cell carcinoma early detection

Metabolic changes precede malignant histology. However, it remains unclear whether detectable characteristic metabolome exists in esophageal squamous cell carcinoma (ESCC) tissues and biofluids for early diagnosis. Here, we conduct NMR- and MS-based metabolomics on 1,153 matched ESCC tissues, normal mucosae, pre- and one-week post-operative sera and urines from 560 participants across three hospitals, with machine learning and WGCNA. Aberrations in ‘alanine, aspartate and glutamate metabolism’ proved to be prevalent throughout the ESCC evolution, consistently identified by NMR and MS, and reflected in 16 serum and 10 urine metabolic signatures in both discovery and validation sets. NMR-based simplified panels of any five serum or urine metabolites outperform clinical serological tumor markers (AUC = 0.984 and 0.930, respectively), and are effective in distinguishing early-stage ESCC in test set (serum accuracy = 0.994, urine accuracy = 0.879). Collectively, NMR-based biofluid screening can reveal characteristic metabolic events of ESCC and be feasible for early detection (ChiCTR2300073613).

Expression of SOX4 Significantly Predicts the Risk of Lymph Node Metastasis for Patients With Early-Stage Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma stands as a notably aggressive malignancy within the digestive system. In cases of early esophageal cancer without lymph node metastasis, endoscopic surgical resection offers a viable alternative, often resulting in improved patient quality of life. However, the paucity of methods to preoperatively ascertain lymph node involvement complicates surgical planning. SOX4 gene was previously found to be highly associated with invasive metastasis in our work through single-cell RNA sequencing on 5 paired tumor/peritumor tissues. This research included the collection of 124 tissue samples from 106 patients (106 tumor and 18 lymph node specimens). Samples were methodically arranged into a tissue microarray and treated with immunohistochemical staining. Statistical analysis was conducted to assess the relationship between them. In the univariate analysis, 3 factors were identified as statistically significant in relation to lymph node metastasis: T category (P = .014), vascular invasion (P < .001), and SOX4 intensity (P = .001). Additionally, when evaluating SOX4 intensity alongside other clinical indicators, SOX4 was shown to independently influence lymph node metastasis. Further, the multivariate analysis revealed that vascular invasion (P < .001) and SOX4 intensity (P = .003) were significantly associated with lymph node metastasis, exhibiting hazard ratios of 10.174 and 7.142, respectively. The results of our study indicate that both SOX4 expression and vascular invasion serve as predictors of lymph node metastasis in patients diagnosed with category T1 esophageal squamous cell carcinoma, underscoring the potential utility of SOX4 in prognostic evaluations.

Validation of serum cystatin SN detection for diagnosis and poor prognosis of esophageal squamous cell carcinoma.

The identification of effective tumor markers is of paramount importance for the early diagnosis, treatment, and prognosis of esophageal squamous cell carcinoma (ESCC). The present study endeavors to identify efficacious serological markers that can differentiate patients with early-stage ESCC from those with benign esophageal lesions and healthy controls (HC). Cystatin-SN (CST1), an active cysteine protease inhibitor belonging to the Cystatin (CST) superfamily, is implicated in the pathogenesis of inflammation and tumorigenesis. The objective of this investigation is to assess the diagnostic, therapeutic, and prognostic potential of serum CST1 in ESCC. In our prior RNA sequencing and screening endeavors, we have identified ten genes that are up-regulated in relation to esophageal cancer. Subsequently, we have verified the gene CST1 from the transcriptome data of the The Cancer Genome Atlas Program (TCGA) and Gene Expression Profiling Interactive Analysis (GEPIA) database. Following this, we conducted an enzyme-linked immunosorbent assay (ELISA) to ascertain the expression levels of CST1 in serum samples from clinical cohorts. The study revealed a significant elevation in serum CST1 levels among patients with early-stage esophageal squamous cell carcinoma (ESCC) (7.41 ± 4.32 ng/ml) compared to those with esophageal benign lesions (4.67 ± 2.43 ng/ml) (p < 0.0001) and healthy controls (4.87 ± 2.77 ng/ml) (p < 0.0001). The diagnostic sensitivity of CST1 for ESCC was 75.68% (specificity 70.83%, AUC 0.775). Combination of CST1 and SCC-Ag exhibited the AUC up to 0.819. Additionally, serum CST1 levels exhibited a significant decrease at 1-2 weeks post-surgery (4.49 ± 3.31 ng/ml) compared to pre-surgery levels (7.68 ± 3.71 ng/ml) (p<0.0001). Survival analysis demonstrated a strong association between high (844/415-1543 d) or low (1490/645-1710 d) serum CST1 levels at diagnosis and overall survival time (p < 0.001). Furthermore, multivariate regression analysis confirmed CST1 (p=0.024, HR=2.023, 95%CI 1.099-3.725) as an independent prognostic factor. Serum CST1 has the potential to function as a diagnostic indicator for distinguishing early-stage esophageal squamous cell carcinoma (ESCC) from individuals with benign esophageal lesions and healthy individuals. Additionally, it could serve as a prognostic predictor and therapeutic efficacy indicator for patients with ESCC.

Postoperative Radiotherapy in Curatively Resected Esophageal Squamous Cell Carcinoma With Occult Recurrent Laryngeal Nerve Lymph Node Metastasis.

Surgery is the mainstream treatment for early-stage esophageal squamous cell carcinoma (ESCC) and occult recurrent laryngeal nerve lymph node metastasis (RLNM) is not uncommon among those with R0 resection. The clinical value of postoperative radiotherapy (PORT) in patients with RLNM only is still controversial. Consecutive patients with early-stage ESCC treated with R0 resection and pathologically confirmed RLNM only from June 2012 to July 2022 were retrospectively reviewed. PORT, covering the supraclavicular and superior mediastinum area (small T-field) at a dose of 50.4Gy for 28 fractions, was performed in some patients. Propensity score matching (PSM) was performed to balance the baseline characteristics between patients with or without PORT. Pattern of failure, disease-free survival (DFS), and overall survival (OS) were compared. Among the 189 patients identified, 69 (35.5%) received PORT and the other 120 (63.5%) did not. After PSM, 154 patients were included in the matched cohort, including 62 in the PORT group and 92 in the non-PORT group. With a median follow-up of 48 (95% CI: 40.3-55.7) months, 69 patients developed their initial disease recurrence in the whole population and PORT significantly decreased the frequency of local recurrence (61.2% vs 21.4%) among those with recurrent disease. Additionally, in the PSM matched cohort, PORT significantly prolonged patients' DFS (HR 0.393, P = 0.002) and OS (HR 0.462, P = 0.020). Moreover, PORT remained as the independent factor associated with improved DFS (HR 0.360, P = 0.001) and OS (HR 0.451, P = 0.021) after multivariate Cox analyses. In addition, tumor location and pathological TNM stage were found to be independent prognostic factors associated with survival outcomes. PORT is associated with improved DFS and OS in ESCC patients with R0 resection and RLNM only, which warrants future validation.

Outcomes following hypofractionated radiation therapy alone for surgically unfit early esophageal squamous cell carcinoma patients; a retrospective single center analysis

Background and purposeTo report the feasibility of hypofractionated radiation therapy (RT) alone for early stage esophageal squamous cell carcinoma (ESCC) patients. Materials and methodsThe oncologic outcomes of 60 cT1-2 N0 ESCC patients who received hypofractionated RT (54 ∼ 60 Gy by 3.0 Gy per fraction) from 2004 to 2018 were retrospectively evaluated. ResultsThe 5-year rates of local control (LC), progression-free survival, cancer-specific survival, and overall survival were 81.1 %, 44.2 %, 73.7 %, and 54.5 %, respectively. In Cox regression analysis, tumor length < 3 cm was correlated with favorable LC (HR 0.167, p = 0.090), and the 5-year LC rates were 95.7 % and 72.0 % in < 3 cm and ≥ 3 cm subgroups, respectively (p = 0.053). Grade ≥ 2 esophagitis was observed in 44 patients (73.3 %) and grade ≥ 2 esophageal strictures developed in five (8.3 %), respectively. The patients with ≥ 3 cm tumor more frequently suffered from grade ≥ 2 esophagitis (13/24 vs. 31/36, p = 0.006) and grade ≥ 2 esophageal stricture (0/24 vs. 5/36, p = 0.056), respectively. The patients with cT2 tumor suffered from grade ≥ 2 esophagitis more frequently than those with T1 tumor (29/44 vs. 15/16, p = 0.03). ConclusionsHypofractionated RT alone, with the merit of short treatment course, could be used as feasible option in treating the early stage ESCC patients who are unfit for surgical resection or chemoradiation. Especially, tumor length < 3 cm seems a good indication of this treatment scheme based on favorable LC rate with low incidence of esophageal toxicities.

Secreted proteins encoded by super enhancer-driven genes could be promising biomarkers for early detection of esophageal squamous cell carcinoma

BackgroundEarly detection of cancer remains an unmet need in clinical practice, and high diagnostic sensitivity and specificity biomarkers are urgently required. Here, we attempted to identify secreted proteins encoded by super-enhancer (SE)-driven genes as diagnostic biomarkers for esophageal squamous cell carcinoma (ESCC). MethodsWe conducted an integrative analysis of multiple data sets including ChIP-seq data, secretome data, CCLE data and GEO data to screen secreted proteins encoded by SE-driven genes. Using ELISA, we further identified up-regulated secreted proteins through a small size of clinical samples and verified in a multi-centre validation stage (345 in test cohort and 231 in validation cohort). Receiver operating characteristic curves were used to calculate diagnostic accuracy. Artificial intelligence (AI) method named gradient boosting machine (GBM) were applied for model construction to enhance diagnostic accuracy. ResultsSerum EFNA1 and MMP13 were identified, and showed significantly higher levels in ESCC patients compared to normal controls. An integrated Five-Biomarker Panel (iFBPanel) established by combining EFNA1, MMP13, carcino-embryonic antigen, Cyfra21-1 and squmaous cell carcinoma antigen had AUCs of 0.881 and 0.880 for ESCC in test and validation cohorts, respectively. Importantly, the iFBPanel also exhibited good performance in detecting early-stage ESCC patients (0.872 and 0.864). Furthermore, the iFBPanel was further empowered by AI technology which showed excellent diagnostic performance in early-stage ESCC (0.927 and 0.907). ConclusionsOur study suggested that serum EFNA1 and MMP13 could potentially assist ESCC detection, and provided an easy-to-use detection model that might help the diagnosis of early-stage ESCC.

Usefulness of the aldehyde breath test for predicting metachronous recurrence in patients with esophageal squamous cell carcinoma and hypopharyngeal squamous cell carcinoma.

The rate of metachronous recurrence after endoscopic submucosal dissection for early-stage esophageal squamous cell carcinoma and hypopharynx squamous cell carcinoma is as high (10-15%). The acetaldehyde breath test may detect acetaldehyde dehydrogenase 2 gene polymorphisms. Therefore, we evaluated its usefulness in assessing metachronous recurrence in patients with esophageal squamous cell carcinoma and hypopharynx squamous cell carcinoma. A total of 76 patients underwent endoscopic submucosal dissection for esophageal squamous cell carcinoma and hypopharynx squamous cell carcinoma and were followed up for at least 3years (non-recurrence group: 52 patients; recurrence group: 24 patients). The risk factors for carcinogenesis were compared between the recurrence and non-recurrence groups, and the acetaldehyde-to-ethanol ratio was assessed. The cutoff acetaldehyde-to-ethanol ratio that correlated with recurrence was established, and the cumulative recurrence rate was evaluated. The recurrence group had a higher acetaldehyde-to-ethanol ratio, daily alcohol consumption, and Lugol-voiding lesion grade than the non-recurrence group in the univariate analysis. The cutoff acetaldehyde-to-ethanol ratio for recurrence was 28.1 based on the receiver operating characteristic curve. The multivariate analysis revealed an acetaldehyde-to-ethanol ratio of > 28.1 and a Lugol-voiding lesion grade associated with carcinogenesis. Patients with an acetaldehyde-to-ethanol ratio of ≥ 28.1 had a significantly high recurrence rate using the Kaplan-Meier method. The acetaldehyde-to-ethanol ratio detected using the acetaldehyde breath test could be a novel biomarker of metachronous recurrence after endoscopic submucosal dissection in patients with esophageal squamous cell carcinoma and hypopharynx squamous cell carcinoma. UMIN000040615.

Salivary Extracellular MicroRNAs for Early Detection and Prognostication of Esophageal Cancer: A Clinical Study

Early detection of esophageal squamous cell carcinoma (ESCC) will facilitate curative treatment. We aimed to establish a microRNA (miRNA) signature derived from salivary extracellular vesicles and particles (EVPs) for early ESCC detection and prognostication. Salivary EVP miRNA expression was profiled in a pilot cohort (n = 54) using microarray. Area under the receiver operator characteristic curve (AUROC) and least absolute shrinkage and selector operation regression analyses were used to prioritize miRNAs that discriminated patients with ESCC from controls. Using quantitative reverse transcription polymerase chain reaction, the candidates were measured in a discovery cohort (n= 72) and cell lines. The prediction models for the biomarkers were derived from a training cohort (n= 342) and validated in an internal cohort (n= 207) and an external cohort (n= 226). The microarray analysis identified 7 miRNAs for distinguishing patients with ESCC from control subjects. Because 1 was not always detectable in the discovery cohort and cell lines, the other 6 miRNAs formed a panel. A signature of this panel accurately identified patients with all-stage ESCC in the training cohort (AUROC= 0.968) and was successfully validated in 2 independent cohorts. Importantly, this signature could distinguish patients with early-stage (stage Ⅰ/Ⅱ) ESCC from control subjects in the training cohort (AUROC= 0.969, sensitivity= 92.00%, specificity= 89.17%) and internal (sensitivity= 90.32%, specificity= 91.04%) and external (sensitivity= 91.07%, specificity= 88.06%) validation cohorts. Moreover, a prognostic signature based on the panel was established and efficiently predicted the high-risk cases with poor progression-free survival and overall survival. The salivary EVP-based 6-miRNA signature can serve as noninvasive biomarkers for early detection and risk stratification of ESCC. Chinese Clinical Trial Registry, ChiCTR2000031507.

AI-assisted identification of intrapapillary capillary loops in magnification endoscopy for diagnosing early-stage esophageal squamous cell carcinoma: a preliminary study.

Esophageal squamous cell carcinoma (ESCC) is one of the most common histological types of esophageal cancers. It can seriously affect public health, particularly in Eastern Asia. Early diagnosis and effective therapy of ESCC can significantly help improve patient prognoses. The visualization of intrapapillary capillary loops (IPCLs) under magnification endoscopy (ME) can greatly support the identification of ESCC occurrences by endoscopists. This paper proposes an artificial-intelligence-assisted endoscopic diagnosis approach using deep learning for localizing and identifying IPCLs to diagnose early-stage ESCC. An improved Faster region-based convolutional network (R-CNN) with a polarized self-attention (PSA)-HRNetV2p backbone was employed to automatically detect IPCLs in ME images. In our study, 2887 ME with blue laser imaging (ME-BLI) images of 246 patients and 493 ME with narrow-band imaging (ME-NBI) images of 81 patients were collected from multiple hospitals and used to train and test our detection model. The ME-NBI images were used as the external testing set to verify the generalizability of the model. The experimental evaluation revealed that the proposed method achieved a recall of 79.25%, precision of 75.54%, F1-score of 0.764 and mean average precision (mAP) of 74.95%. Our method outperformed other existing approaches in our evaluation. It can effectively improve the accuracy of ESCC detection and provide a useful adjunct to the assessment of early-stage ESCC for endoscopists.

Eosinophils Exert Antitumorigenic Effects in the Development of Esophageal Squamous Cell Carcinoma

Eosinophils are present in several solid tumors and have context-dependent function. Our aim is to define the contribution of eosinophils in esophageal squamous cell carcinoma (ESCC), as their role in ESCC is unknown. Eosinophils were enumerated in tissues from 2 ESCC cohorts. Mice were treated with 4-NQO for 8 weeks to induce precancer or 16 weeks to induce carcinoma. The eosinophil number was modified by a monoclonal antibody to interleukin-5 (IL5mAb), recombinant IL-5 (rIL-5), or genetically with eosinophil-deficient (ΔdblGATA) mice or mice deficient in eosinophil chemoattractant eotaxin-1 (Ccl11-/-). Esophageal tissue and eosinophil-specific RNA sequencing was performed to understand eosinophil function. Three-dimensional coculturing of eosinophils with precancer or cancer cells was done to ascertain direct effects of eosinophils. Activated eosinophils are present in higher numbers in early-stage vs late-stage ESCC. Mice treated with 4-NQO exhibit more esophageal eosinophils in precancer vs cancer. Correspondingly, epithelial cell Ccl11 expression is higher in mice with precancer. Eosinophil depletion using 3 mouse models (Ccl11-/- mice, ΔdblGATA mice, IL5mAb treatment) all display exacerbated 4-NQO tumorigenesis. Conversely, treatment with rIL-5 increases esophageal eosinophilia and protects against precancer and carcinoma. Tissue and eosinophil RNA sequencing revealed eosinophils drive oxidative stress in precancer. Invitro coculturing of eosinophils with precancer or cancer cells resulted in increased apoptosis in the presence of a degranulating agent, which is reversed with NAC, a reactive oxygen species scavenger. ΔdblGATA mice exhibited increased CD4 T cell infiltration, IL-17, and enrichment of IL-17 protumorigenic pathways. Eosinophils likely protect against ESCC through reactive oxygen species release during degranulation and suppression of IL-17.

A matched cohort comparison of endoscopic resection, chemoradiation and esophagectomy in the treatment of early-stage esophageal squamous cell carcinoma

BackgroundThe efficacy of endoscopic resection in early-stage esophageal squamous cell carcinoma has not been defined. MethodsWe queried the National Cancer Database to identify patients presenting with cT1N0M0 esophageal squamous cell cancer between 2004 and 2017. Transitive match methods were used to 1:1:1 propensity match patients undergoing endoscopic resection to patients undergoing esophagectomy and those undergoing definitive chemoradiotherapy. Kaplan Meier method was used to compare 5-year overall survival profiles for matched cohorts. Results301 patients (19%) underwent endoscopic resection; 497 (32%) esophagectomy; 767 (49%) chemoradiation. On comparison of matched cohorts, patients undergoing chemoradiation demonstrated lower rates of survival than those undergoing esophagectomy (32% vs. 59%, p < 0.0001) while those undergoing endoscopic resection demonstrated rates comparable to patients undergoing esophagectomy (53% vs. 59%, p = 0.77). ConclusionsFor cT1N0M0 esophageal squamous cell cancer, endoscopic resection is associated with rates of survival similar to those following esophagectomy and better than those following definitive chemoradiation.

ClusterNet: a clustering distributed prior embedded detection network for early-stage esophageal squamous cell carcinoma diagnosis.

Early and accurate diagnosis of esophageal squamous cell carcinoma (ESCC) is important for reducing mortality. Analyzing intrapapillary capillary loops' (IPCLs) patterns on magnification endoscopy with narrow band imaging (ME-NBI) has been demonstrated effective in the diagnosis of early-stage ESCC. However, even experienced endoscopists may face difficulty in finding and classifying countless IPCLs onME-NBI. We propose a novel clustering prior embedded detection network: ClusterNet. ClusterNet is capable of analyzing the distribution of IPCLs on ME-NBI automatically and enables endoscopists to overview multiple types of visualization. With ClusterNet assisting, endoscopists may observe ME-NBI images more efficiently, thus they may also predict the pathology and make medical decisions more easily. We propose the first large-scale ME-NBI dataset with fine-grained annotations by consensus of expert endoscopists. The dataset is splitted into a training set and an independent testing set based on patients. With two strategies for embedding, ClusterNet can automatically take the clustering effect into consideration. Prior to this work, none of the existing approaches take the clustering effect, which is rather important in classifying the IPCLs, into account. ClusterNet achieves an average precision of 81.2% and an average recall of 90.0% for the detection of IPCLs patterns on each patient of the independent testing set. We also compare ClusterNet with other state-of-the-art detection approaches. The performance of ClusterNet with embedding strategies is consistently superior to that of other approaches in terms of average precision, recall andF2-Score. Experiments demonstrate that our proposed method is able to detect almost all the IPCLs patterns on ME-NBI and classify them according to the Japanese Endoscopic Society (JES) classificationaccurately.