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Quantitative analysis of microRNAs in tissue microarrays by in situ hybridization
 
Jason A. Hanna1, Hallie Wimberly1, Salil Kumar1, Frank Slack2, Seema Agarwal1, and David L. Rimm1
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Supplementary Material

As an example of the inverse relationship, there are several papers on miR-221 targeting and down-regulating ERα in cell line model systems (22, 36, 37). In contrast we and others have found a positive association with ERα and miR-221 in patient tissue (38). Yoshimoto et al. found that miR-221, as measured by qRT-PCR, gradually increased in expression as ER-α protein expression increased in 171 breast care patients (38). Similarly we show the same relationship by qISH in 473 patients. These observations suggest that new targets of miRNAs validated by forced-expression model systems may not be generalizable to human tumors and could benefit from more extensive validation in other systems.

Since miRNAs were discovered it has been speculated they could have value as biomarkers and numerous papers have been published where miRNAs, measured by RT-PCR show associations with outcomes (38-41). However, there are still few examples of miRNA biomarkers in widespread clinical usage. One reason for this may be the challenge of high throughput measurement by RT-PCR and the challenge of validation in high quality cohorts such that the level of evidence is sufficient to change practice. In this paper, we describe measurements of miRNAs on over 600 lung and breast cancer patients using this in situ method. We are optimistic that this approach could increase throughput sufficiently to allow the testing of miRNAs necessary for broad clinical adoption.

Although we are optimistic about the potential of this method, there are clearly limitations to the efforts to date. Most significantly, we are unable to report absolute quantification of miRNAs in ng/µg of total RNA or similar. While we are currently working toward this goal, it should be noted that the vast majority of RT-PCR based work also does not achieve absolute standardization. Although we do have a wide range of AQUA scores, the dynamic range of ISH is likely within the range of other hybridization techniques such as microarrays which cover 3–4 logs, whereas qRT-PCR has been reported to span a dynamic range of 6–7 logs (42-45). A second limitation of this method is tumor heterogeneity. While this is often not assessed in efforts using RT-PCR, we are able to compare measurements from multiple histospots from the same tumor when arrays are made at 2-fold or greater redundancy. Compared using linear regression, we find R2 values to be in the 0.4 to 0.6 range, compared with proteins in a comparable assay system where R2 values are seen to be 0.6 to 0.8. It is too early to assess the meaning of the spatial heterogeneity we observe. In the future it may be interesting to correlate this with RT-PCR and tumor geography (i.e., leading edge vs training edge, well vs poorly differentiated regions or perivascular vs hypoxic regions).

In summary, we have developed a method to quantitatively measure miRNA expression within subcellular compartments in tumor epithelia. Compared with other methods for miRNA detection and quantitative analysis, miRNA qISH has the advantages of retaining critical spatial information while increasing throughput when used in combination with tissue microarrays. In developing this method we found that the inverse relationship between miRNAs and their target proteins is difficult to identify in large patient cohorts suggesting the interaction between miRNAs and targets to be more complicated than suggested by in vitro models. Finally, as an example, we found that miR-221 may have value as a prognostic marker. While this is preliminary and exploratory with respect to its prognostic value, it provides evidence for the usage of miRNAs as tissue biomarkers.

Acknowledgments

Thanks to Eric Olson and his group for providing the miR-21 knockout mouse tissue. Thanks to George Vande Woude and his group for providing the Met4 antibody. This work was supported by NIH RO-1 grant number CA 114277 to DLR.

Competing interests

Dr. Rimm is a co-founder, consultant for and stockholder in HistoRx, the exclusive licensee of the Yale-owned AQUA technology.

Correspondence
Address correspondence to David L. Rimm M.D.-Ph.D., Department of Pathology, BML 116, Yale University School of Medicine, 310 Cedar St. PO Box 208023, New Haven, CT, USA. Email: [email protected]


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