The determination of prognosis in patients with esophageal squamous cell carcinoma (ESCC) is primarily based on staging according to the TNM-classification, whereas conventional grading is of minor clinical importance because of its deficiencies in prognostic patient stratification. Recently, a novel, highly prognostic grading scheme based on budding activity and cell nest size has been proposed for squamous cell carcinoma (SCC) of both pulmonary as well as oral origin. In order to investigate the utility and transferability of this approach to ESCC, we evaluated budding activity and cell nest size, as well as other histomorphologic characteristics, in a cohort of 135 primarily resected tumors and correlated the results with clinicopathologic and outcome parameters. High budding activity and small cell nest size showed a strong association with reduced overall, disease-specific, and disease-free survival (P<0.001, respectively) in ESCC. The combination of both markers in a 3-step grading system showed excellent prognostic separation of well-differentiated (G1), moderately differentiated (G2), and poorly differentiated (G3) carcinomas (P<0.001). The hazard ratio for disease-free survival in multivariate analysis under inclusion of stage was 2.97 for G2 and 5.42 for G3 ESCC (P<0.001). World Health Organization-based grading had no prognostic impact. Taken together, our data prove the value of tumor budding and cell nest size as excellent outcome predictors in ESCC and validate the utility of a previously established grading scheme proposed for oral and pulmonary SCC in this tumor entity. Ultimately, these combined efforts may result in a universal grading system for SCC regardless of the site of origin.

Esophageal squamous cell carcinoma (ESCC) is the most common esophageal cancer associated with poor prognosis and additional therapeutic strategies must be implemented to optimize ESCC treatment. Meanwhile, the important biologic role and potential prognostic and therapeutic implications of a tumors immunologic microenvironment (IM) have been recognized in various cancers.In order to investigate the contexture and the prognostic relevance of the IM in ESCC, we immunohistochemically evaluated the extent of overall/intraepithelial TILs (CD3+/CD8+) and of PD-1 / PD-L1 expression in a cohort of 125 therapy-naive ESCCs, additionally assessing PD-L1 copy number status via fluorescence in-situ hybridization.High intraepithelial CD3+ TILs (CD3ihigh) and high PD-L1 expression on tumor cells (PD-L1high) were each significantly associated with improved overall- (OS) (CD3+: p = 0.019; PD-L1: p = 0.028), disease specific- (DSS) (CD3+: p = 0.05; PD-L1: p = 0.006) and disease free survival (DFS) (CD3+: p = 0.009; PD-L1: p < 0.001). CD3ihigh- and PD-L1high cases were significantly associated with one another (p < 0.001). Subgrouping of ESCC revealed decreased OS (p = 0.031), DSS (p = 0.012) and DFS (p < 0.001) for CD3ilow/PD-L1low cancers.Our data not only associate CD3ihigh- and PD-L1high ESCC with a beneficial outcome, but also demonstrate PD-L1high- and CD3ihigh status to be closely intertwined. Furthermore, our study demarcates a prognostically unfavorable, “non-immunoreactive” CD3ilow / PD-L1low ESCC-subgroup, potentially forming the basis for an immune-based stratification of ESCC.

Barrett's columnar epithelium with dysplasia is the most important risk factor for adenocarcinoma of the distal esophagus. The molecular mechanisms responsible for progression of columnar metaplasia to dysplasia and invasive carcinoma are mostly unknown. We investigated expression of the tumor suppressor gene p53, E-cadherin expression and cell proliferation in the metaplasia-dysplasia-carcinoma sequence of esophageal adenocarcinoma. In 24 patients with R0-resected adenocarcinomas of the distal esophagus we evaluated the expression of E-cadherin (antibody HECD-1), mutated p53 (antibody DO1) and cell proliferation (antibody MiB1) by immunohistochemistry in sections of adenocarcinoma, columnar metaplasia, with and without dysplasia, and in squamous epithelium of the esophagus. No p53 immunoreactivity was seen in sections of normal squamous epithelium or columnar metaplasia. Fifty per cent of invasive adenocarcinomas stained positive for mutated p53. The p53 expression correlated with the T-category (P = 0.048) and the N-category (P = 0.024). There was a significant decrease in the expression of E-cadherin from columnar metaplasia to dysplasia and to esophageal adenocarcinoma (P < 0.0001). Expression of E-cadherin in columnar metaplasia without dysplasia was similar to that seen in normal squamous epithelium of the esophagus. The Ki-67 proliferation fraction increased significantly from normal squamous epithelium to columnar metaplasia to dysplasia and to invasive carcinoma (P < 0.001), with a marked expansion of the proliferative component. There was no correlation between cell proliferation, E-cadherin expression and the tumor stage. In contrast to the alterations in the p53 expression, a decreased E-cadherin expression and the expansion of the proliferative component represent an early phenomenon in the malignant degeneration of Barrett's esophagus. This might aid in the early detection of esophageal adenocarcinoma.

Initial treatment planning in esophageal squamous cell carcinoma mainly relies on clinical staging. Recently, a highly prognostic grading system based on the cellular dissociation parameters Tumor Budding and Cell Nest Size has been proposed for resected esophageal squamous cell carcinoma. To probe for the transferability and relevance of this established novel grading system in the pretreatment setting, we evaluated Tumor Budding/Cell Nest Size in pretherapeutic biopsies of either primarily resected (cohort 1, n=80) or neoadjuvantly treated (cohort 2, n=75) esophageal squamous cell carcinoma. Grading data were correlated with clinicopathologic and survival parameters. High Tumor Budding Activity and small Cell Nest Size in pretherapeutic biopsies were strongly associated with shortened overall survival, disease-free survival, and disease-specific survival in both cohorts. A modified histopathologic grading system incorporating both factors termed “Cellular Dissociation Grade” showed excellent prognostic demarcation between well (G1), moderately (G2), and poorly differentiated (G3) carcinomas in both scenarios (overall survival: cohort 1: P<0.001; cohort 2: P=0.009) and was predictive for a high pathologic tumor stage and the presence of nodal metastases in primarily resected patients. Multivariate analyses revealed the Cellular Dissociation Grade to be a predictor of poor outcome in the pretherapeutic setting independent of clinical stage (overall survival, disease-free survival, and disease-specific survival: P<0.001). Hazard ratio for disease-free survival was 3.19 for G2 and 5.66 for G3 carcinomas compared with G1 neoplasms. Our data not only prove the transferability of histopathologic grading based on Tumor Budding/Cell Nest Size to biopsy specimens in esophageal squamous cell carcinoma, but also demonstrate that the Cellular Dissociation Grade is a strong outcome predictor in this entity even in the pretreatment scenario. Therefore, we believe that this novel type of grading has the ability to serve as a powerful histology-based pretherapeutic biomarker, that might supplement clinical staging for choosing the most suitable therapy decision.