Nuclear S100A7 is associated with poor prognosis in head and neck cancer

Abstract

Background: Tissue proteomic analysis of head and neck squamous cell carcinoma (HNSCC) and normal oral mucosa using iTRAQ (isobaric tag for relative and absolute quantitation) labeling and liquid chromatography-mass spectrometry, led to the identification of a panel of biomarkers including S100A7. In the multi-step process of head and neck tumorigenesis, the presence of dysplastic areas in the epithelium is proposed to be associated with a likely progression to cancer; however there are no established biomarkers to predict their potential of malignant transformation. This study aimed to determine the clinical significance of S100A7 overexpression in HNSCC.

Methodology: Immunohistochemical analysis of S100A7 expression in HNSCC (100 cases), oral lesions (166 cases) and 100 histologically normal tissues was carried out and correlated with clinicopathological parameters and disease prognosis over 7 years for HNSCC patients. Overexpression of S100A7 protein was significant in oral lesions (squamous cell hyperplasia/dysplasia) and sustained in HNSCC in comparison with oral normal mucosa (p(trend)<0.001). Significant increase in nuclear S100A7 was observed in HNSCC as compared to dysplastic lesions (p = 0.005) and associated with well differentiated squamous cell carcinoma (p = 0.031). Notably, nuclear accumulation of S100A7 also emerged as an independent predictor of reduced disease free survival (p = 0.006, Hazard ratio (HR = 7.6), 95% CI = 1.3-5.1) in multivariate analysis underscoring its relevance as a poor prognosticator of HNSCC patients.

Conclusions: Our study demonstrated nuclear accumulation of S100A7 may serve as predictor of poor prognosis in HNSCC patients. Further, increased nuclear accumulation of S100A7 in HNSCC as compared to dysplastic lesions warrants a large-scale longitudinal study of patients with dysplasia to evaluate its potential as a determinant of increased risk of transformation of oral premalignant lesions.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. (A) Box-Plot analysis: Box plots showing distribution of total scores based on immunohistochemistry of S100A7 protein in paraffin-embedded sections of oral normal tissues, squamous cell hyperplasia, dysplasia and HNSCC.

The vertical axis shows the total immunostaining score, obtained as described in the Methods section. (i) Nuclear S100A7 expression in squamous cell hyperplasia (IHC scoring range 0–7), dysplasia (range 0–7) and HNSCC (range 0–7) (b) cytoplasmic S100A7 in normal (range 0–7), squamous cell hyperplasia (range 0–7), dysplasia (range 0–7) and HNSCC (range 0–7). (B)Immunohistochemical analysis of S100A7 in head-and-neck tissues. Paraffin-embedded sections of histologically normal mucosa, squamous cell hyperplasia or with dysplasia, and HNSCC were stained using anti-S100A7 monoclonal antibody as described in the Methods section. (i) Normal oral mucosa showing no S100A7 immunostaining; (ii) squamous cell hyperplasia showing nuclear and cytoplasmic S100A7 immunostaining; (iii) dysplasia depicting nuclear and cytoplasmic S100A7 immunostaining in epithelial cells; (iv) HNSCC illustrating both intense cytoplasmic and nuclear staining in tumor cells; (v) HNSCC section with a dysplasia showing S100A7 immunostaining in epithelial cells (original magnification ×100); (vi) HNSCC used as a negative control, showing no S100A7 immunostaining in tumor cells; and (vii) ER-negative breast cancer tissue showing S100A7 immunostaining. Arrows show nuclear and cytoplasmic localization (i-v, vii original magnification ×200).

Figure 2. Receiver operating characteristic curves of S100A7 (nuclear/cytoplasmic) in (a) normal vs. squamous cell hyperplasia; (b) normal vs. dysplasia; (c) normal vs. HNSCC.

Bold line shows ROC analysis for nuclear S100A7. Dashed line shows ROC analysis for cytoplasmic S100A7. Y-axis of the plot shows true-positive fraction and X-axis shows false positive fraction.

Figure 3. Evaluation of S100A7 overexpression (nuclear/cytoplasmic) as a prognostic marker in HNSCC.

Kaplan–Meier estimation of cumulative proportion of disease-free survival: (a) Median time for disease-free survival (DFS; no recurrence/metastasis) in HNSCC patients showing nuclear immunostaining of S100A7 was 13 months, whereas in patients showing no/faint S100A7 immunostaining in nucleus median DFS was 70 months (p = 0.016); (b) In patients showing increased cytoplasmic S100A7 expression the median DFS was 14 months compared with HNSCC that showed mild or moderate cytoplasmic immunostaining (median DFS  = 70 months, p = 0.055). Time-dependent Positive and Negative Predictive Values (PPV(t), NPV(t)) of nuclear S100A7 expression. (c) PPV(t) for time to cancer relapse for 49 HNSCC patients with S100A7+ (solid line) and for all 77 HNSCC patients with survival data (dashed line); d) NPV(t) for time to cancer relapse for 28 HNSCC patients with S100A7− (solid line).

Figure 4. Verification of S100A7 expression in tissues.

(a) RT-PCR analysis of S100A7 in oral normal mucosa, squamous cell hyperplasia, dysplasia and HNSCC tissues. For RT-PCR analysis and Western blot analysis, we used normal (n = 5), hyperplasia (n = 5), dysplasia (n = 5) and HNSCC (n = 5) tissues. Panel shows increased levels of S100A7 transcripts in oral lesions -squamous cell hyperplasia (H), dysplasia (D) and HNSCC (T) compared with the oral normal mucosa (N) that showed basal levels of S100A7 transcripts. β-actin used as a control to normalize the quantity of RNA used for each RT-PCR reaction is shown in the lower panel. (b) Western blot analysis of S100A7 in oral normal mucosa (N), squamous cell hyperplasia (H), dysplasia (D) and HNSCC tissues. Equal amount of protein lysates from these tissues were electrophoresed on 12% SDS-PAGE and transferred to PVDF membrane. The membrane was incubated with respective primary and secondary antibodies as described in the Methods section and the signal detected by enhanced chemiluminescence method. Panel shows increased expression of S100A7 protein in oral lesions - squamous cell hyperplasia (H), dysplasia (D) and HNSCC (T) compared with oral normal mucosa (N). GAPDH was used as loading control.