A number of groups have documented the utility of miRNA ISH in formalin-fixed paraffin embedded tissue specimens using commercially available, LNA modified and DIG labeled probes (7, 9, 11). However, most studies to date have been descriptive or semiquantitative in small patient cohorts. Here we have developed a method for detecting miRNAs in situ multiplexed with 4’,6-diamidino-2-phenylindole (DAPI) and cytokeratin immunofluorescence to quantify the miRNA signal within tumor epithelia using the AQUA technology (15). By applying cytokeratin and DAPI staining to define cytoplasmic and nuclear compartments and epithelial and stromal compartments, miRNA expression can be measured in a standardized and reproducible manner without the use of feature extraction-based software or using “fold change,” the most common metric for RT-PCR based studies. Considerable effort was spent in optimizing the experimental conditions to maximize signal to noise (as evaluated by ISH of scrambled probe) without sacrificing specificity. We found the additional EDC fixation step, double DIG labeled probes, optimization of the titer of the sheep anti-DIG antibody (Roche Diagnostics), and careful increase of the stringency of post-hybridization wash were all required to provide optimal conditions. To validate our quantitative ISH (qISH) method, we assessed expression of miR-92a, miR-21, miR-34a, and miR-221, all of which have been shown to be deregulated in breast cancer (19-22). In assessment of over 400 breast cancer cases, we found a broad dynamic range in the population with AQUA scores ranging from 0–140 and variable expression patterns specific to each miRNA. Examples are shown in Figure 1. While in most cases miRNA expression was cytoplasmic, miR-221 was often localized to the nucleus, an uncommon but previously described localization for mature miRNAs (23-25). We cannot rule out the possibility of the probe recognizing the immature pre-miRNA in the nucleus (23, 24). It should be noted that the scrambled sequence probe was uniformly negative.
In order to prove specificity, we assessed miR-21 ISH on heart tissue from a miR-21 knockout mouse as shown in Figure 2A (26). No specific signal for miR-21 could be detected in the knockout tissue while normal wild type heart shows moderate levels of expression. We then used a miR-221 specific unlabeled blocking oligo (same sequence as the endogenous mature miR-221) to compete for miR-221 specific DIG labeled probe hybridization, and as shown in Figure 2B, no specific signal was detected in the presence of the blocking oligo. Finally, we transfected MCF-7 and MDA-MB-231, low and high miR-221 expressing cell lines respectively, with anti-miR-221 to determine quantitatively the decrease in signal measured by qISH as compared with the decrease in expression as measured by qRT-PCR. The miR-221 signal was decreased by 55.5% as measured by qISH and 49.9% as measured by qRT-PCR in the MCF-7 cells and was similarly decreased in the MDA-MB-231 cells (Figure 2C-D, and Supplementary Figure 1). To our knowledge this study represents the first time these methods have been used to validate miRNA ISH specificity. As a final validation step, we assessed reproducibility of each assay and found high correlations between near serial sections performed on different days (Figure 2E). The results from these specificity and reproducibility assays gave us the confidence to begin assays on large cohort TMAs.
As a proof of concept for miRNAs as biomarkers, we assessed the prognostic value of each miRNA in breast cancer as evaluated by qISH. The qISH AQUA scores were classified according to quartiles to assess miRNA expression and association with survival. For miR-221, the highest three quartiles were nearly overlapping so they were collapsed and compared with the lowest quartile revealing a significantly shorter disease specific survival for the low expression of miR-221 with a p-value of 0.0210 (Figure 3A). This cutoff between high and low expression was also significant in univariate and multivariate analysis providing discovery level evidence that miR-221 expression is an independent prognostic factor in breast cancer (Figure 3B). High miR-221 expression was also associated with ER status and lymph node negativity (Figure 3 C-D). Proof of prognostic value will require more extensive analysis on larger multi-institutional cohorts along with a prospectively designed hypothesis. The other miRNAs were not significantly prognostic (data not shown).