PCa Has Higher Expression of
PCa Has Higher Expression of miR-182 but Low miR-182 Associated with Biochemical Recurrence
The 4-Phenylbutyric acid of miR-182 was further examined in a TMA of prostatectomy samples (Outcome TMA), which was created by the National Cancer Instituteesponsored Coop-erative Prostate Cancer Tissue Resource.25,26 This TMA used a case-control design in which patients with biochemical recurrence were matched to control patients who did not have disease recurrence by age, Gleason grade
sum score, pTNM stage, year of prostatectomy, and ethnicity.25,26 Usable ISH data were obtained for 133 of the
patients on the TMA. Numerous tissue cores had been depleted, limiting the ability for paired case-control anal-ysis. Clinical information for the patients analyzed are provided in Table 2. Using inForm software version 2.4.1 (PerkinElmer) and an H&E reference slide, a pathologist (A.M.A.) segmented the miR-182 ISH images into benign and cancer regions and objectively quantified the mean pixel intensity of miR-182 within these regions. Similar to other reports,14e18 miR-182 expression was higher in PCa epithelium than benign epithelium by both an unpaired analysis (Figure 2A), and a paired analysis of 56 patients with benign and cancer epithelium available (Figure 2B).
Table 2 Clinical Information for TMA Patients Analyzed from the Outcome and Murphy TMAs
All patients measured
Cancer/benign ratio patients
Murphy TMA for
Recurrence No recurrence
Recurrence No recurrence
Race/ethnicity, % (SNP analysis)
Other NA NA
Unknown NA NA
Gleason score, %
Unknown NA NA
AA, African American; EA, European American; NA, not applicable; SNP, single-nucleotide polymorphism; TMA, tissue microarray.
The American Journal of Pathology - ajp.amjpathol.org 915
Baumann et al
Figure 2 miR-182 levels are higher in prostate cancer epithelium, but low cancer/benign ratio of miR-182 expression is associated with biochemical recurrence. A and B: miR-182 in situ hybridization (ISH) quantification in the prostate by an unpaired analysis (two-tailed t-test; A) and a paired analysis (two-tailed paired t-test; B). Lines in B connect benign and cancer data points of the same patient, with red lines indicating an increase and blue lines a decrease. C: In the Murphy validation tis-sue microarray, miR-182 levels increase signifi-cantly in cancer epithelium by ISH (two-tailed paired t-test). Lines connect benign and cancer data points of the same patient, with red lines indicating an increase and blue lines a decrease.
D: miR-182 in a representative core that contains prostatic intraepithelial neoplasia (PIN). Left column shows miR-182 ISH. Right column shows hematoxylin and eosin (H&E) of adjacent section. Boxed areas in top right panel are shown in higher magnification in three rows below; the second row shows benign epithelium; the third row shows PIN; the fourth row shows cancer. E: In the Outcome TMA, cancer/benign ratios of miR-182 expression stratified by biochemical recur-rence (U-test). Data are expressed as means 5th to 95th percentiles. n Z 133 (A); n Z 56 (B); n Z 55 (C); n Z 34 (D, recurred); n Z 22 (D, did not recur). *P < 0.05, ****P < 0.0001. Original magnification: 5 (D, first row) 20 (D, second, third, and fourth rows).
Validation using a second prostatectomy TMA (Murphy TMA) also found significantly higher miR-182 in cancer by
a paired analysis of 55 patients (Figure 2C and Table 2). miR-182 levels consistently appeared to be increased in PIN, equal to that of PCa (Figure 2D), but there were not sufficient regions for statistical analysis. Although the TMAs were not powered to study PIN, it suggests that up-regulation of miR-182 is an early event in PCa.
As a measure of data consistency, the interclass correla-tion coefficient of the mean miR-182 pixel intensity be-tween multiple cores from the same patient on the Outcome TMA was high in both benign epithelium (0.67; 95% CI, 0.51e0.80) and cancer epithelium (0.77; 95% CI,
0.68e0.85). Although intrapatient variance was low, it is unclear whether the higher interpatient variance we observed in miR-182 signal is attributable to true biological variation or differing RNA preservation among the speci-mens. Importantly, age of the samples did not correlate with ISH signal intensity (Supplemental Figure S2A), but sample fixation and preservation may still differ. Without a robust normalization method available for ISH, the absolute
intensities of miR-182 ISH did not correlate with clinical parameters (Supplemental Figure S2B and data not shown). Therefore, internal normalization was applied through the cancer/benign ratio of miR-182 expression per patient, to reduce potential confounding due to interpatient differences in RNA quality.