Prognosis and Progression of ESCC Patients with Perineural Invasion

Abstract

Perineural invasion (PNI) has been recognized as a poor prognostic factor in several malignancies, but the definition and pathogenesis of PNI in esophageal squamous cell carcinoma (ESCC) remains to be defined. PNI was evaluated by H&E staining and S100 immunohistochemistry. The predictive value of PNI in the prognosis of ESCC patients was analyzed. PNI was evaluated in vitro and in vivo. A total of 54 specimens (17.88%) were defined as PNI-a and 99 specimens (32.78%) as PNI-b. S100 staining was superior to H&E staining for PNI detection (50.66% vs 27.15%, P < 0.001, κ = 0.506). Tumor depth (P = 0.001), tumor stage (P = 0.010), and vascular invasion (P < 0.001) were significantly associated with PNI. PIN-a and PNI-b had significant lower disease free survival (DFS) and disease specific survival (DSS) than PNI-0 patients, and the prognosis of PNI-b patients was significantly worse than PNI-a patients for DFS (P = 0.009). PNI was an independent predictor for DFS and DSS in ESCC as evaluated by univariate and multivariate analyses. ESCC cells could metastasize along the nerve in vitro and in vivo, and PNI was a dynamic process. S100 staining significantly improved the accuracy of PNI detection. PNI was associated with local recurrence and poor prognosis of ESCC patients.

Figure 1. Representative histology and ultrastructure of PNI in ESCC.

(a) The nerve fiber was partly embedded by tumor cells and stained by H&E. (b) The nerve fiber was identified in the serial section of A by positive S100 immunohistochemistry (arrow). The arrows indicate the nerve fiber at the same position. (c,d) Cancer cells (arrows) were identified within the perineurium of the nerve. (e) The axon (asterisk) is robustly surrounded by Schwann cells (SC), which constitutes the endoneurium of the nerve (arrow), ×4000. (f) View of the box in (e). The perineurium (red circle) is composed of fibroblasts (F), and tumor cells (green circle) have invaded the perineurium. A gap (arrow) exists between the esophageal cancer cells and the surrounding structures, ×10000.

Figure 2. Prognosis of ESCC patients.

Disease-free survival (a) and disease-specific survival (b) according to different types of PNI.

Figure 3. In vitro co-culture model of cancer cell colonies and nerve.

(a–c) Dynamic process of the co-culture between cancer and DRGs in Matrigel (a: magnification, x40; b and c: magnification, x200). (d) GFP-labeled tumor cells travelled through neurites, which shows the same location as in c (magnification, x200).

Figure 4. Variety patterns of the histologic appearance of PNI in ESCC.

(a) The nerve fiber was tightly surrounded by tumor cells. The perineurium of the nerve exhibited high integrity. (b) Tumor cells invaded the nerve and damaged the perineurium of the nerve. (c) Tumor cells invaded the nerve. (d) The nerve fibers were irregularly damaged by tumor cells, which broke through the perineurium and grew outward.

Figure 5. In vivo model of tumor metastasis along sciatic nerves.

(a) A sciatic nerve injected with EC109. Arrows indicate the tumor implantation sites; arrowheads indicate the proximal nerve to the spine. (b) Isolated image of the sciatic nerve of Figure a. (c) Longitudinal pathological examination of the sciatic nerve by H&E staining. (d–f) Magnified area in the boxes of Figure c.