Low expression of PTK6/Brk predicts poor prognosis in patients with laryngeal squamous cell carcinoma

Abstract

Background: Protein tyrosine kinase 6 (PTK6), also known as breast tumor kinase (Brk), was a nonreceptor tyrosine kinase containing SH3, SH2, and tyrosine kinase catalytic domains. The deregulated expression of PTK6 was observed in various human cancers. However, little was known about PTK6 expression and its clinicopathological significance in human laryngeal squamous cell carcinoma (LSCC).

Materials: PTK6 expression was evaluated in 7 pairs of surgically resectable laryngeal tissues by Western blotting and in 13 pairs of surgically resectable laryngeal tissues by reverse transcription-PCR (RT-PCR). Using immunohistochemistry, we performed a retrospective study of the PTK6 expression levels on 134 archival LSCC paraffin-embedded samples. Prognostic outcomes correlated with PTK6 were examined using Kaplan-Meier analysis and Cox proportional hazards model.

Results: The PTK6 expression level was lower in LSCC tissues than in the adjacent noncancerous epithelial laryngeal tissues by Western blots and RT-PCR. By immunohistochemical analysis, we observed high expression of PTK6 in 25 of 76 (32.9%) adjacent noncancerous epithelial laryngeal tissues and in 39 of 134 (29.1%) of LSCC, respectively. Multivariate analysis demonstrated that pN status and the expression level of PTK6 (P < 0.05) were independent and significant prognostic factors. In the primary LSCC category, median DFS (disease free survival) of high, medium and low PTK6 expression patients were 88.5 months ,74.5 months and 49.0 months (log-rank test, P = 0.002); median OS (overall survival) of high, medium and low PTK6 expression patients were 88.5 months ,76.3 months and 65.7 months (log-rank test, P = 0.002). Reduced cytoplasmic PTK6 expression in LSCC was significantly associated with late pN status (P =0.005, r = 0.27), advanced pTNM stages (III and IV) (P =0.027, r = 0.147), and poor differentiated LSCC (P <0.0001, r = 0.486). In adjacent paracancerous laryngeal epithelial samples, median DFS of high, medium and low PTK6 expression patients were 92.6 months ,75.6 months and 48.5 months (log-rank test, P = 0.020); median OS of high, medium and low PTK6 expression patients were 92.9 months ,78.9 months and 74.6 months (log-rank test, P = 0.042).

Conclusion: The present findings indicated that cytoplasmic PTK6 expression is a potential prognostic factor for survival in LSCC patients. High expression of PTK6 was associated with favorable OS and DFS in LSCC patients.

Figure 1

Expression levels of PTK6 in laryngeal squamous cell carcinoma tissues. A. Expression levels and quantitative analysis of PTK6 protein in 7 paired laryngeal squamous cell carcinoma tissues by Western blotting. N: paracarcinoma (normal) laryngeal epithelial tissues. T: laryngeal squamous cell carcinoma tissues. The numbers under each PTK6 lane indicated relative expression of each sample by normalizing to GADPH which serves as an internal control. B: The relative quantity of PTK6 protein was determined by densitometric analysis of the Western blots. C: mRNA level of PTK6 in 13 paired laryngeal squamous cell carcinoma tissues by real-time PCR. N: paracarcinoma (normal) laryngeal epithelial tissues. T: laryngeal squamous cell carcinoma tissues.

Figure 2

PTK6 expression in the LSCC tissues by immunohistochemistry analysis. A. PTK6 protein expression was higher in the normal laryngeal epithelial tissues than in LSCC tissues (original magnification: A, x 100); B and C were higher magnification of part of A, B. PTK6 immunohistochemical staining in paracarcinoma laryngeal epithelial tissues (original magnification: x 400) C. PTK6 immunohistochemical staining in the LSCC epithelial tissues (original magnification:, x 400) ; D and E. PTK6 immunohistochemical staining in the well differentiated LSCC epithelial tissues (original magnification: D, x 100; E, x 400); F and G. PTK6 immunohistochemical staining in the mediate differentiated LSCC epithelial tissues (original magnification: F, x100; G, x 400); H and I. PTK6 immunohistochemical staining in the poor differentiated LSCC epithelial tissues (original magnification: H, x 100; I, x 400).

Figure 3

The Kaplan-Meier survival analysis in different PTK6 expression levels of laryngeal squamous cell carcinoma (LSCC) patients. A and B: The survival curve of high, medium and low PTK6 expression LSCC patients in the primary LSCC category (A: DFS, P = 0.002; B: OS P = 0.002); C and D: The survival curve of high, medium and low PTK6 expression LSCC patients in adjacent paracancerous laryngeal epithelial sample (C: DFS, P = 0.02; D: OS, P = 0.042).