The Prognostic Significance of Cancer-Associated Fibroblasts in Esophageal Squamous Cell Carcinoma

<div><p>Background</p><p>Cancer-associated fibroblasts (CAF) are activated fibroblasts in the cancer stroma and play an important role in cancer progression. Some reports have indicated the correlation between the expression of CAF markers and adverse prognosis in several cancers. However, no reports have studied CAF phenotype and its clinical relevance in esophageal squamous cell carcinoma (ESCC).</p><p>Methods</p><p>We investigated CAF phenotype of ESCC based on histology and immunohistochemical expressions of five CAF markers such as fibroblast activation protein (FAP), smooth muscle actin (SMA), fibroblast-specific protein-1 (FSP1), platelet-derived growth factor receptor (PDGFRα), and PDGFRβ in 116 ESCC tissue samples. Besides, we also examined the correlation of the CAF phenotype with clinical relevance as well as other cancer-microenvironment related factors.</p><p>Results</p><p>Histologically immature CAF phenotype was correlated with poor prognosis (p<0.001) and associated with increased microvessel density, increased tumor associated macrophages, and epithelial to mesenchymal transition. CAF markers were characteristically expressed in stromal fibroblast close to tumor cells and the expression pattern of 5 CAF markers was highly heterogeneous in every individual cases. Of five CAF markers, SMA, FSP1, and PDGFRα were unfavorable prognostic indicators of ESCC. The number of positive CAF markers was greater in ESCC with immature CAFs than in those with mature ones.</p><p>Conclusions</p><p>Our results demonstrate that histologic classification of CAF phenotype is a reliable and significant prognostic predictor in ESCC. CAF markers have the potential to be diagnostic and therapeutic targets in ESCC.</p></div

Overall and disease free survival curves using the Kaplan–Meier method by log-rank test for cancer associated fibroblast (CAF)-activation protein expression: (A, F) SMA; (B, G) FSP1; (C, H) FAP; (D, I) PDGFRα; (E, J) PDGFRβ.

<p>Overall and disease free survival curves using the Kaplan–Meier method by log-rank test for cancer associated fibroblast (CAF)-activation protein expression: (A, F) SMA; (B, G) FSP1; (C, H) FAP; (D, I) PDGFRα; (E, J) PDGFRβ.</p

Immunohistochemical staining of cancer-associated fibroblast related fibroblast markers.

<p>A: Analysis of immunohistochemical staining performed based on both intensity and staining area. B: Demonstration of the immunohistochemical staining of smooth muscle actin (SMA), fibroblast-specific protein-1 (FSP-1), fibroblast activator protein (FAP), platelet-derived growth factor receptor (PDGFR)α, and PDGFRβ.</p

Multivariate Cox proportional hazard model analysis by classification of cancer associated fibroblasts.

a<p>Seven cases of unsatisfactory for minimal number of evaluated lymph nodes, were excluded in the analysis.</p>b<p>FAP expression was not significant in univariate analysis for disease-free survival.</p>c<p>One case with lack of tissue was excluded in the analysis.</p

Histological categorization of stromal fibroblast on hematoxylin and eosin slides.

<p>A. Mature type when fibroblasts show thin, wavy, and small spindle cell morphology; normal fibroblasts; B. Immature type when fibroblasts show large, plump spindle-shaped morphology; C–D. Survival curves using the Kaplan–Meier method by log-rank test for histologic subtype of cancer associated fibroblast.</p

Heatmap of 116 esophageal squamous cell carcinoma cases according to histologic subtype and expression pattern of cancer associated fibroblasts.

<p>Heatmap of 116 esophageal squamous cell carcinoma cases according to histologic subtype and expression pattern of cancer associated fibroblasts.</p

Comparison of clinicopathologic characteristics according to histologic subtypes of cancer associated fibroblast.

a<p>by Fisher’s exact test, otherwise chi square test.</p>b<p>Severn cases of unsatisfactory for minimal number of evaluated lymph nodes, were excluded in the analysis.</p>c<p>One case was excluded in the analysis of microvessel density and macrophages due to lack of tissue.</p

Quantitative real-time PCR results of <i>CDK4</i> amplification by a) well-differentiated (WD) and dedifferentiated (DD) liposarcomas: amplification levels were not different between the two histologic subtypes.

<p>Quantitative real-time PCR results of <i>CDK4</i> amplification in <b>b)</b> WD and <b>c)</b> DD liposarcomas according to tumor recurrence: amplification was higher in WD liposarcomas with recurrence after surgical resection, but not different in DD liposarcomas regardless of tumor recurrence.</p

Predictors of Recurrence-free Survival for Well-differentiated Liposarcoma in Univariate and Multivariate Analyses.

<p>HR, hazard ratio; CI, confidence interval.</p

CDK4 Immunohistochemistry Results According to Tumor Recurrence.

<p>WD, well-differentiated; DD, dedifferentiated; IHC immunohistochemistry.</p