Nerve Fibers in the Tumor Microenvironment as a Novel Biomarker for Oncological Outcome in Patients Undergoing Surgery for Perihilar Cholangiocarcinoma

Abstract

Introduction: Perihilar cholangiocarcinoma (pCCA) is a biliary tract cancer with a dismal prognosis, with surgery being the only chance of cure. A characteristic aggressive biological feature of pCCA is perineural growth which is defined by the invasion of cancer cells to nerves and nerve fibers. Recently, nerve fiber density (NFD) was linked to oncological outcomes in various malignancies; however, its prognostic role in pCCA remains to be elucidated.

Materials and methods: Data of 101 pCCA patients who underwent curative-intent surgery between 2010 and 2019 were included in this study. Extensive group comparisons between patients with high and low NFD were carried out, and the association of cancer-specific survival (CSS) and recurrence-free survival with NFD and other clinicopathological characteristics was assessed using univariate and multivariable cox regression models.

Results: Patients with high NFD showed a median CSS of 90 months (95% CI: 48-132, 3-year CSS = 77%, 5-year CSS = 72%) compared to 33 months (95% CI: 19-47, 3-year CSS = 46%, 5-year CSS = 32%) in patients with low NFD (p = 0.006 log rank). Further, N1 category (HR = 2.84, p = 0.001) and high NFD (HR = 0.41, p = 0.024) were identified as independent predictors of CSS in multivariable analysis. Patients with high NFD and negative lymph nodes showed a median CSS of 90 months (3-year CSS = 88%, 5-year CSS = 80%), while patients with either positive lymph nodes or low NFD displayed a median CSS of 51 months (3-year CSS = 59%, 5-year CSS = 45%) and patients with both positive lymph nodes and low NFD a median CSS of 24 months (3-year CSS = 26%, 5-year CSS = 16%, p = 0.001 log rank).

Conclusion: NFD has been identified as an important novel prognostic biomarker in pCCA patients. NFD alone and in combination with nodal status in particular allows to stratify pCCA patients based on their risk for inferior oncological outcomes after curative-intent surgery.

Keywords: Biomarker; Cholangiocarcinoma; Nerve fiber density; Oncological outcome.

Fig. 1

Workflow and study cohort. a Overview of all steps from tissue to biomarker. FFPE, formalin-fixed paraffin-embedded. b Study cohort. CSS, cancer-specific survival; NFD, nerve fiber density; pCCA, perihilar cholangiocarcinoma.

Fig. 2

Oncological survival in pCCA. a CSS and OS in pCCA. The median CSS was 49 months (95% CI: 29–69) and the median OS 33 months (95% CI: 19–47), respectively. b RFS in pCCA. The median RFS was 37 months (95% CI: 18–56). c PGP staining of pCCA with high NFD. Zoomed in image of the tumor with a lot of small nerves in the stroma between the tumor glands (blue arrows). On the zoomed in image of the routine HE staining, these small nerve fibers are not visible. These results occur in patients corresponding to the blue line in the Kaplan-Meier curve in d and f (high NFD). d CSS in pCCA stratified by NFD. The median CSS was 90 months (95% CI: 48–132) in patients with high NFD compared to 33 months (95% CI: 19–47) in patients with low NFD (p = 0.006 log rank). e PGP staining of pCCA with low NFD. Zoomed in image of the tumor without any small nerves in the stroma between the tumor glands. The zoomed in image of PGP shows a positive bigger nerve trunk with perineural invasion. These big nerve fibers are also easily recognized on the zoomed in routine HE staining. f RFS in pCCA stratified by NFD. The median RFS was 83 months (95% CI: 34–132) in patients with high NFD compared to 24 months (95% CI: 13–35) in patients with low NFD (p = 0.004 log rank). g CSS in pCCA stratified by NFD and pN category. The median CSS was 90 months (95% CI: 57–123) in patients with high NFD and negative lymph nodes, 51 months (95% CI: 38–64) in patients with either positive lymph nodes or low NFD but not both, and 24 months (95% CI: 14–32) in patients with both positive lymph nodes and low NFD (p = 0.001 log rank). h RFS in pCCA stratified by NFD and pN category. The median RFS was 83 months (95% CI: 42–124) in patients with high NFD and negative lymph nodes, 45 months (95% CI: 8–82) in patients with either positive lymph nodes or low NFD but not both, and 10 months (95% CI: 0–21) in patients with both positive lymph nodes and low NFD (p = 0.001 log rank). CI, confidence interval; CSS, cancer-specific survival; RFS, recurrence-free survival; OS, overall survival; pCCA, perihilar cholangiocarcinoma; PGP, protein gene product 9.5; NFD, nerve fiber density.

Fig. 3

Origin of the nerve fibers. Schematic overview of tissue with cancers cells invading the nerve. a Routine HE staining showing perineural invasion of cancer cells invading a large nerve trunk (red arrow). b PGP immunohistochemistry staining being expressed in this cancer-invaded nerve (yellow arrow). NPY immunohistochemistry (sympathetic origin) is expressed (c), and VIP immunohistochemistry (parasympathetic) is not expressed in this large nerve trunk (d). Schematic overview of tissue with cancer cells not invading the nerve. e Routine HE staining showing the yellow arrow to the localization of the small nerve fibers that are not visible on the HE staining. f Those small nerve fibers stain positive in the PGP immunohistochemistry. g Negative staining of these small nerve fibers in the VPN immunohistochemistry. h Yellow arrow points to the positive staining in the VIP immunohistochemistry of these small nerve fibers. NPY, neuropeptide Y; VIP, vasoactive intestinal peptide.