Univariate analysis of factors associated with disease free survival in OSCC.

<p>Footnote: * significant factors associated with survival.</p

miR-21 is mainly expressed in the tumor stroma in OSCC.

<p>(A) An overview image of miR-21 expression where the areas marked by rectangles are magnified in, (B) normal oral mucosa, (C) mild epithelial dysplasia and (D) oral squamous cell carcinoma. Mir-21 appears as a blue stain. In normal oral mucosa, (B) no miR-21 expression is observed, whereas in dysplasia, (C) a weak expression is seen in the subepithelial connective tissue, and a widespread and intense expression is found in the tumor stromal compartment (D). Panels (E), (F) and (G) illustrate combined FISH and IF for miR-21 (E), α-SMA (F) and a composite image (G) showing a high degree of overlap between the two markers.</p

The miR-21 in situ hybridization is specific.

<p>Comparison between the miR-21 probe (A) and the scrambled probe (B) in the same tumor. The scrambled probe (B) gave no expression. Comparison between miR-21 probe (C) and U6 probe (D) in the same tumor. The U6 probe gives a positive nuclear expression in virtually every cell.</p

High miR-21 expression is associated with poor survival.

<p>Top: Waterfall plot of miR-21 expression in 86 OSCCs. miR-21 expression was assessed by in situ hybridization and converted to the area occupied by miR-21 (blue ) times the mean intensity of the blue signal. The cut-off between the upper, middle and lower tertile are depicted by blue, yellow and green bars, respectively. Bottom: Cox regression survival curve for disease free survival, based on miR-21 expression tertiles. Low miR-21 expression is associated with a better survival (p = 0.032).</p

Digital image analysis of in situ hybridization.

<p>The miR-21 expression in the stroma was estimated using digital image analysis. Image (A), (B), (C) and (D) overview the image-analysis process, where (A) is the original cytokeratin stained image, which is used to delineate epithelial tumor cells, seen as green color in (B). Original image of miR-21 ISH on serial section from same tumor, where the ISH signal is quantitated specifically in the stroma area (blue signal). The red color in (B) represents stroma area negative for miR-21. See methods for a detailed description of the image analysis process.</p

miR-21 expression is found in the tumor, endothelium and acinar cells in a subset of OSCCs.

<p>(A) An overview image showing the distribution of the miR-21 expression, which is highest in the central portion of the tumor (B) and is lower at the invasive tumor front (C). We found a strong miR-21 expression in the tumor cells from one specimen (D), which was also present in the budding tumor cells (arrow). miR-21 expression was also observed in the endothelial cells (E, arrow) and in the degenerated acinar cells (F, arrow).</p

Hazard ratios from univariate analysis for disease free survival in OSCC.

<p>On the right hazard ratios including 95% confidence intervals are displayed. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095193#pone-0095193-t001" target="_blank">Table 1</a> for details.</p

Multivariate model of factors associated with disease free survival.

<p>Legend: The remaining factors in multivariate Cox regression after backwards elimination. Only miR-21 TBS and perineural invasion remained as independent factors influencing survival.</p