The Biomarker Potential of Caveolin-1 in Penile Cancer

Abstract

Various types of human cancers were characterized by an altered expression of epithelial or stromal caveolin-1 (CAV1). However, the clinical significance of CAV1 expression in penile cancer remains largely unknown. Here the expression patterns of CAV1 were analyzed in a retrospective cohort (n=43) of penile squamous cell carcinomas (SCC). Upon penile cancer progression, significantly increased CAV1-levels were determined within the malignant epithelium, whereas within the tumor stroma, namely the fibroblastic tumor compartment harboring activated and/or cancer associated fibroblasts, CAV1 levels significantly decline. Concerning the clinicopathological significance of CAV1 expression in penile cancer as well as respective epithelial-stromal CAV1 distributions, high expression within the tumor cells as well as low expression of CAV1 within the stromal compartment were correlated with decreased overall survival of penile cancer patients. Herein, CAV1 expressions and distributions at advanced penile cancer stages were independent of the immunohistochemically proven tumor protein p53 status. In contrast, less differentiated p16-positive tumor epithelia (indicative for human papilloma virus infection) were characterized by significantly decreased CAV1 levels. Conclusively, we provide further and new evidence that the characteristic shift in stromal-epithelial CAV1 being functionally relevant to tumor progression even occurs in penile SCC.

Keywords: biomarker; caveolin-1; microenvironment; p16; p53; penile cancer; tumor stroma.

Figure 1

Immunohistological analysis of CAV1 levels and epithelial-stromal CAV1 distributions in correlation with p16 and p53 levels in human penile tumor tissues. Paraffin-sections of human penile carcinomas were stained for p16, p53 and CAV1 in combination with DAB (brown). Representative images of p16-positive [p16(+)] and p16-negative [p16(-)] tumor specimen are shown. The 53 status (overexpression, wild-type and loss) is indicated. Asteriks (*) mark stromal compartments and bold arrows point to epithelial structures. Sections were counterstained using hematoxylin. # indicates different patients. Scale bar: 200 µm, scale bar of higher magnification images (CAV1): 75 µm.

Figure 2

Correlation of epithelial-stromal CAV1 expressions in penile cancer as related to p16 and p53 expressions. (A) Overall survival curves for patients with p16 expressing (n= 17; dashed line) or p16-negative (n=26; black line) tumors. HR, hazard ratio and log-rank P values are indicated. (B) p16 expressing tumors were classified according to their p53 immunoreactivity (p53 status: overexpression, wild-type, loss). (C) p16 expression in tumors were related to their grade of differentiation: low grade (G1; n= 8), intermediate grade (G2; n=23) and high grade (G3; n=9). (D) Tumor grades (displayed by numbers) were further analyzed according to the respective patient age (at the time of surgery) and the overall survival time (in days) post-surgery. The blue color depicts patient’s death. p16-positivity is indicated by rectangles. Pearson correlation coefficient (Pearson r), 95% confidence interval (CI) and two-tailed P as analyzed for tumor grade versus days post-surgery were indicated (R squared = 0.213). Pearson r = -0.2205 (95%CI: -0.4885 to 0.0855; R squared = 0.049; P = 0.155) for the patients age versus days post-surgery (GX; n=3: The tumor grade could not be identified.) (E) p16 expressing tumors were classified according to their CAV1 immunoreactivity score for low (0-0.5), moderate (>0.5-1.5), and high (>1.5-2) CAV1 expression levels within the tumor epithelium. **P < 0.01 by unpaired t-test (two-tailed). (F) Overall survival curves for patients with low (n=17), moderate (n=15) and high (n=11) CAV1 expressions in tumor cells. HR, hazard ratio and log-rank P (low versus high CAV1 levels) are indicated. HR=0.36 (0.12-1.11), log-rank P (Mantel-Cox) =0.047 (moderate vs. high CAV1). (G) Overall survival curves for patients with p16-negative tumors and low (n=8), moderate (n= 6) and high (n=12) CAV1 content within tumor cells. HR and log-rank P (low versus high CAV1 levels) are indicated. HR=0.22 (0.058-0.80), log-rank P=0.014 (moderate versus high). (H) The differential CAV1 scores within the epithelial compartment were correlated to the respective CAV1 immunoreactivity scores of the tumor stroma (n=17 for low, n=15 for moderate, and n=11 for high epithelial CAV1). p16-positivity is indicated by the blue color. *P<0.05, **P<0.01 by one-way ANOVA followed by post-hoc Tukey’s multiple comparisons test.

Figure 3

Overall survival as related to and p53 expressions and according to the expression of stromal CAV1 in penile cancer. (A) Differential p53 expression levels were classified according to their CAV1 immunoreactivity score for low (0-0.5), moderate (>0.5-1.5), and high (>1.5-2) CAV1 expression levels of respective tumor cells (p53 overexpression n=10, p53 wild-type n= 26, p53 low n= 7). (B) Overall survival curves for patients according to the p53 expression in tumors. HR and log-rank P (wild-type: blue line versus loss: black line) are indicated. HR=2.28 (0.80-6.49) log-rank P=0.053 (overexpression: red line versus loss). (C) Overall survival curves for patients with low (n=12), moderate (n=13) and high (n=18) CAV1 content within the tumor stroma. HR and log-rank P (low versus high CAV1 levels) are indicated. HR=2.33 (0.92-5.91), log-rank P=0.056 (moderate versus high). (D) Overall survival curves for patients with p16-negative tumors and low (n=8), moderate (n= 6) and high (n=12) CAV1 content within the tumor stroma. HR and log-rank P (low versus high CAV1 levels) are indicated. HR=6.22 (1.21-32.02) log-rank P=0.0025 (moderate versus high).

Figure 4

Epithelial-stromal CAV1 distributions in correlation with malignant epithelial differentiation patterns. Paraffin-sections of human penile carcinomas were stained for CAV1 and analyzed according to the tumor grade. (A) Representative images of well differentiated (low-grade, G1), moderately differentiated (G2) and poorly differentiated (high grade, G3) tumors are shown. P16 status is indicated. Scale bar: 200 µm, higher magnifications: 50 µm. (B) Representative images of different areas with variable (decreasing tumor cell) differentiation patterns within the same (p16-negative) tumor specimen are shown. Sections were counterstained using hematoxylin. Asterisks (*) mark stromal compartments and bold arrows point to epithelial structures. # indicates different patients.

Figure 5

Prognostic impacts of epithelial-stromal CAV1 expressions in penile cancer in relation to the tumor grade. (A) Overall survival curves for penile cancer patients according to the tumor grade (low-grade/G1 n=8; moderate/G2 n=23; high grade/G3 n=9). HR and log-rank P (G1 versus G3) are indicated. HR=0.46 (0.17-1.24), log-rank P=0.20 (G1 versus G2); HR=0.35 (0.12-1.08), log-rank P = 0.010 (G2 versus G3). (B) According to the tumor grade, the CAV1 immunoreactivity score for low (0-0.5), moderate (>0.5-1.5), and high (>1.5-2) CAV1 expression levels of respective tumor cells was estimated. (C) Further on, the CAV1 immunoreactivities of the tumor stroma were determined in correlation with the tumor grades. P16-positivities are indicated by the blue color. According to the tumor grade, CAV1 scores only of the p16-negative tumor epithelium (D) and of the tumor stroma (E) were analyzed. * P<0.05 by one-way ANOVA followed by post-hoc Dunnett’s multiple comparisons test. (F) Overall survival curves for penile cancer patients according to the tumor stage (pT1 n=21; pT2 n=16; pT3 n=6). HR and log-rank P (pT1 versus pT3) are indicated. HR=0.23 (0.053-1.01), log-rank P=0.20 (pT2 versus pT3). CAV1 immunoreactivities of the tumor epithelium (G) and of the stroma(H) were determined in correlation with the tumor stages. *P<0.05, by one-way ANOVA followed by post-hoc Tukey’s multiple comparisons test.