Nerve fibers in breast cancer tissues indicate aggressive tumor progression

Abstract

Emerging evidence has indicated nerve fibers as a marker in the progression of various types of cancers, such as pancreatic cancer and prostate cancer. However, whether nerve fibers are associated with breast cancer progression remains unclear. In this study, we evaluated the presence of nerve fibers in 352 breast cancer specimens and 83 benign breast tissue specimens including 43 cases of cystic fibrosis and 40 cases of fibroadenoma from 2 independent breast tumor center using immunohistochemical staining for specific peripheral nerve fiber markers.In all, nerve fibers were present in 130 out of 352 breast cancer tissue specimens, while none were detected in normal breast tissue specimens. Among 352 cases, we defined 239 cases from Sun Yat-Sen Memorial Hospital, Guangzhou, China, as the training set, and 113 cases from the First Affiliated Hospital of Shantou University, Guangdong, China, as the validation set. The thickness of tumor-involving nerve fibers is significantly correlated with poor differentiation, lymph node metastasis, high clinical staging, and triple negative subtype in breast cancer. More importantly, Cox multifactor analysis indicates that the thickness of tumor-involving nerve fibers is a previously unappreciated independent prognostic factors associated with shorter disease-free survival of breast cancer patients. Our findings are further validated by online Oncomine database. In conclusion, our results show that nerve fiber involvement in breast cancer is associated with progression of the malignancy and warrant further studies in the future.

FIGURE 1

Nerve fibers present in breast cancer. (A) PGP9.5. (B) NF. (C) Class III-β-tubulin. (D) Isotype-matched antibody, mouse IgG. (E) H&E staining. Represented images of nerve fibers in breast cancer specimens. Nerve fibers were detected in serial sections of breast cancer tissues using IHC staining with 3 different specific neuronal markers. Original magnifications: 100× for the wild view; 400× for the left up corner. Scale bar, 100 μm. H&E = hematoxylin–eosin, IgG = immunoglobulin G, IHC = immunohistochemical, NF = neurofilament, PGP9.5 = protein gene product 9.5.

FIGURE 2

Nerve fibers in breast cancer correlate with high malignancy. Represented images of nerve fibers in different progression of breast tissue. (A) Nerve fibers were absent in cystic fibrosis of the breast and breast fibroadenoma, while they were present in breast carcinoma in situ and invasive ductal breast cancer. The arrow indicates the involvement of nerve fibers in breast tissue specimens. Scale bar, 100 μm. (B) The graph shows that the percentage of nerve fibers involvement in different progression of breast cancer tissues varied from 11.11% to 52.54%. Red bar represents the positive rate of the nerve fibers while blue bar represents the rate of absence of nerve fibers. P values were obtained using χ² test. (^∗∗: grade II vs grade III: P < 0.01 by both 1-sided and 2-sided test; ^∗∗∗: grade I vs grade II/III: P < 0.001 by both 1-sided and 2-sided test; breast cancer in situ vs invasive ductal breast cancer: P = 0.018 by 1-sided test and P = 0.026 by 2-sided test.) (C) The maximum diameter of the nerve fibers in invasive ductal breast cancer samples also varied among cancer in situ and grades I–III cancers. (Mean + Standard error of mean; ^∗∗: P < 0.01 by both 1-sided and 2-sided tests; ^∗∗∗: P < 0.001 by both 1-sided and 2-sided tests.)

FIGURE 3

Diameter of nerve fibers can be a prognostic marker of breast cancer. The diameter of nerve fibers of every breast cancer patients in (A) training set (left) and validation set (right). (B) The cutoff value (d = 0.21 mm) was automatically generated by X-tile plots. Time-dependent ROC curves in the training set (left) and validation set (right). Data are AUC (95% CI) or hazard ratio (95% CI). Upper panel showed data of 1 year follow-up, whereas down panel showed data of 5 years follow-up. (C) Kaplan–Meier survival curve of DFS and OS in training set (left) and validation set (right). AUC = area under the curve, CI = confidence interval, DFS = disease-free survival, OS = overall survival, ROC = receiver operator characteristic.

FIGURE 4

Nerve fibers in breast cancer specimens have different location. (A) Represented images of nerve fibers located in invasive front of breast cancer and the center of breast cancer. Original magnifications: left panel: 40×; right panel: 400×. Scale bar, 100 μm. (B) The proportion of nerve fibers located in invasive front and the center of tissue specimens from different grades of breast cancer. (C) Kaplan–Meier survival curve for patients with nerve fibers located in invasive front and the center of breast cancer.