Investigating Trk Protein Expression between Oropharyngeal and Non-oropharyngeal Squamous Cell Carcinoma: Clinical Implications and Possible Roles of Human Papillomavirus Infection

Abstract

Purpose: The relationship between head and neck squamous cell carcinoma (HNSCC) and subtypes of tropomyosin-related kinase (Trk) has not been studied in-depth. In this study, we evaluated the expression patterns of TrkA, TrkB, and panTrk and their clinicopathological significance as well as association with p16 expression and human papilloma virus (HPV) status.

Materials and methods: Total of 396 radically resected oropharyngeal (n=121) and non-oropharyngeal (n=275) HNSCCs were included. Immunohistochemistry for TrkA, TrkB, and panTrk was performed. In addition, p16 immunohistochemistry was performed to assess the HPV status. Using HPV-negative HNSCC cell lines, FaDu and CAL27, HPV type 16 E6/E7 gene was transfected, and then changes of TrkA and TrkB expression were analyzed.

Results: In the clinical samples of HNSCC, high expression of TrkA and panTrk were more associated with oropharyngeal and p16 positive squamous cell carcinoma (SCC). In patients with completely resected (R0-resected) oropharyngeal SCC, high TrkA expression was related to superior overall survival and recurrence-free survival (RFS). In patients with R0-resected oral cavity SCC, high panTrk was related to poor RFS. In HPV type E6/E7 gene-transfected FaDu and CAL27 cell lines, increase of TrkA expression was observed.

Conclusion: It seems that expression pattern of panTrk and TrkA differed according to anatomical sites of HNSCC and was closely related to p16 expression and patient prognosis. Trk expression should be considered in the context of anatomical site, p16 expression or HPV status and Trk subtypes.

Keywords: Head and neck squamous cell carcinoma; Human papillomavirus; Trk proteins; TrkA; TrkB; panTrk.

Fig. 1.

Expression pattern of Trk protein and p16 using immunohistochemistry (×200). Representative expression as assessed by Trk immunohistochemistry: Trk showed diffuse cytoplasmic staining in tumor cells. The four cases shown had different intensities of Trk expression in all tumor cells. (A) Negative, H-score 0. (B) Intensity 1, H-score 100. (C) Intensity 2, H-score 200. (D) Intensity 3, H-score 300. Strong and diffuse nuclear and cytoplasmic staining of p16 immunohistochemical staining (E) and negative staining was observed (F).

Fig. 2.

Expression of Trk protein in head and neck squamous cell carcinomas according to anatomical location and p16 status. (A) H-scores of TrkA and panTrk were significantly higher at oropharyngeal sites than non-oropharyngeal areas (p < 0.001). However, there was no significant difference in H-scores of TrkB according to anatomical site. (B) H-scores of TrkA and panTrk were higher in p16 positive cases than p16 negative cases. No significant difference was observed in the H-score of TrkB between p16 positive cases and p16 negative cases.

Fig. 3.

Alteration of TrkA and TrkB expression and proliferation ability by HPV-16 oncogene. (A) Increased level of TrkA mRNA 48 hours after transfection in FaDu cell line. (B) Increased level of TrkA mRNA 48 hours after transfection in CAL-27 cell-line. (C) Slight decreased level of TrkB mRNA in FaDu cell line. (D) Decreased level of TrkB in CAL27 cell line. Increased level of TrkC mRNA 48 hours after transfection in FaDu cell line (E) and CAL27 cell line (F). CTR, transfection with empty vector; HPV, transfection with HPV-16 oncogene.

Fig. 4.

Kaplan-Meier analysis of R0-resected head and neck squamous cell carcinomas (SCCs) in aspect of anatomical sites. (A) In the analysis of 300 R0 resected cases, TrkA high expression group tended to have a longer overall survival (OS) than the Trk low expression group (p=0.060). (B) No significant difference was observed in recurrence-free survival (RFS) according to TrkA expression status (p=0.348). (C) In the analysis of 82 R0 resected oropharyngeal SCCs, TrkA high group had a significantly longer OS than the TrkA low expression group (p=0.044). (D) In addition, the TrkA high group showed longer RFS than the TrkA low expression group (p=0.032). (E) In 180 R0 resected oral cavity SCCs, high panTrk expression tended to be related to poor OS (p=0.065). (F) Furthermore, the high panTrk group showed a significantly inferior RFS than the low panTrk group (p=0.001).