Overexpression of prothymosin alpha predicts poor disease outcome in head and neck cancer

Abstract

Background: In our recent study, tissue proteomic analysis of oral pre-malignant lesions (OPLs) and normal oral mucosa led to the identification of a panel of biomarkers, including prothymosin alpha (PTMA), to distinguish OPLs from histologically normal oral tissues. This study aimed to determine the clinical significance of PTMA overexpression in oral squamous cell hyperplasia, dysplasia and head and neck squamous cell carcinoma (HNSCC).

Methodology: Immunohistochemistry of PTMA protein was performed in HNSCCs (n = 100), squamous cell hyperplasia (n = 116), dysplasia (n = 50) and histologically normal oral tissues (n = 100). Statistical analysis was carried out to determine the association of PTMA overexpression with clinicopathological parameters and disease prognosis over 7 years for HNSCC patients.

Results: Our immunohistochemical analysis demonstrated significant overexpression of nuclear PTMA in squamous cell hyperplasia (63.8%), dysplasia (50%) and HNSCC (61%) in comparison with oral normal mucosa (p(trend)<0.001). Chi-square analysis showed significant association of nuclear PTMA with advanced tumor stages (III+IV). Kaplan Meier survival analysis indicated reduced disease free survival (DFS) in HNSCC patients (p<0.001; median survival 11 months). Notably, Cox-multivariate analysis revealed nuclear PTMA as an independent predictor of poor prognosis of HNSCC patients (p<0.001, Hazard's ratio, HR = 5.2, 95% CI = 2.3-11.8) in comparison with the histological grade, T-stage, nodal status and tumor stage.

Conclusions: Nuclear PTMA may serve as prognostic marker in HNSCC to determine the subset of patients that are likely to show recurrence of the disease.

Figure 1

(a) Box-Plot analysis: Box plots showing distribution of total scores based on immunohistochemistry of PTMA protein in paraffin-embedded sections of oral normal tissues, squamous cell hyperplasia, dysplasia and HNSCC. The vertical axis shows total immunostaining score, obtained as described in the Methods section. Figure shows total score distribution of nuclear PTMA expression in squamous cell hyperplasia (score range 0–7), dysplasia (score range 0–7) and HNSCC (score range 0–7). (b) Immunohistochemical analysis of PTMA in head and neck tissues: Paraffin-embedded sections of histologically oral normal mucosa, squamous cell hyperplasia, dysplasia and HNSCC were stained using anti-PTMA polyclonal antibody as described in the Methods section. (i) normal oral mucosa showing no PTMA immunostaining; (ii) squamous cell hyperplasia showing no immunostaining for PTMA; (iii) squamous cell hyperplasia showing nuclear PTMA immunostaining in epithelial cells; (iv) dysplasia showing no nuclear PTMA immunostaining; (v) dysplasia depicting nuclear PTMA immunostaining in epithelial cells; (vi) HNSCC section showing no nuclear PTMA staining; (vii) HNSCC section illustrating nuclear PTMA staining in tumor cells; (viii) bladder cancer tissue section showing nuclear PTMA immunostaining; (ix) HNSCC section used as a negative control, showing no PTMA immunostaining in tumor cells; (i–ix original magnification ×200).

Figure 2. Survival analysis in HNSCC patients:

(a) Kaplan–Meier estimation of cumulative proportion of disease-free survival, median time for disease-free survival (DFS: no recurrence/metastasis) in HNSCC patients showing nuclear immunostaining of PTMA was 11 months as compared to the patients showing no nuclear PTMA immunostaining (p<0.001); (b) Positive Predictive Values: Positive Predictive Values [PPV(t)] for time to cancer relapse for 51 HNSCC patients with PTMA(+) (solid line) and for all 77 HNSCC patients with survival data (dashed line); (c) Negative Predictive Values: Negative Predictive Values [NPV(t)] for time to cancer relapse for 26 patients with PTMA (−) (solid line), and for all 77 patients (dashed line).

Figure 3. Verification of PTMA overexpression:

(a) RT-PCR analysis of PTMA in normal mucosa, squamous cell hyperplasia, dysplasia and HNSCC tissues. Panel shows increased levels of PTMA transcripts in squamous cell hyperplasia (H1, H2), dysplasia (D1, D2) and HNSCC (T1, T2, T3) compared with the normal oral mucosa (N1, N2) that showed basal levels of PTMA transcripts. β-actin was used as a control to normalize the quantity of RNA used for each RT-PCR reaction is shown in the lower panel. (b) Immunoblot analysis: Immunoblot analysis of PTMA in normal oral mucosa (N1, N2), squamous cell hyperplasia (H1, H2), dysplasia (D1, D2) and HNSCC (T1, T2). Equal amount of protein lysates were electrophoresed on 12% SDS-PAGE and transferred to PVDF membrane. The membrane was incubated with respective primary antibodies and secondary antibodies as described in the Methods section and the signal detected by enhanced chemiluminescence method. Panel shows increased expression of PTMA in squamous cell hyperplasia (H1, H2), dysplasia (D1, D2) and HNSCC (T1, T2) compared with normal oral mucosa (N1, N2). GAPDH was used as loading control (lower panel).