miR-21 Expression in Cancer Cells may Not Predict Resistance to Adjuvant Trastuzumab in Primary Breast Cancer

Abstract

Trastuzumab is established as standard care for patients with HER2-positive breast cancer both in the adjuvant and metastatic setting. However, 50% of the patients do not respond to the trastuzumab therapy, and therefore new predictive biomarkers are highly warranted. MicroRNAs (miRs) constitute a new group of biomarkers and their cellular expression can be determined in tumor samples by in situ hybridization (ISH) analysis. miR-21 is highly prevalent and up-regulated in breast cancer and has been linked to drug resistance in clinical and in vitro settings. To determine expression patterns of miR-21 in high-grade breast cancers, we examined miR-21 expression in 22 HER2-positive tumors and 15 HER2-negative high-grade tumors by ISH. The histological examination indicated that patient samples could be divided into three major expression patterns: miR-21 predominantly in tumor stroma, predominantly in cancer cells, or in both stromal and cancer cells. There was no obvious difference between the HER2-positive and HER2-negative tumors in terms of the miR-21 expression patterns and intensities. To explore the possibility that miR-21 expression levels and/or cellular localization could predict resistance to adjuvant trastuzumab in HER2-positive breast cancer patients, we analyzed additional 16 HER2-positive tumors from patients who were treated with trastuzumab in the adjuvant setting. Eight of the 16 patients showed clinical recurrence and were considered resistant. Examination of the miR-21 expression patterns and intensities revealed no association between the miR-21 scores in the cancer cell population (p = 0.69) or the stromal cells population (p = 0.13) and recurrent disease after adjuvant trastuzumab. Thus, our findings show that elevated miR-21 expression does not predict resistance to adjuvant trastuzumab.

Keywords: HER2; biomarker; breast cancer; miR-21; prediction; response; trastuzumab.

Figure 1

MicroRNA-21 expression patterns and scoring in grade 3 breast cancers. Representative miR-21 ISH expression patterns and intensities in HER2-positive (A–F) and HER2-negative tumors (G–I). The miR-21 ISH signal (dark blue staining, examples indicated by arrows in (A)) is seen exclusively in stromal cells (A,D,F, and G), or in both stromal cells and cancer cells (B,C,E,H, and I). Stromal and cancer cell tissue compartments are indicated by St and Ca, respectively. In (D,G, and H), arrows indicate miR-21 positive stromal cells. The variations in miR-21 ISH signal intensity and prevalence of positive cells were scored 0, 1, 2, or 3 by two observers (see Material and Methods section) in both cancer and stromal cells. The miR-21^CC and miR-21^St scores are indicated (miR-21^CC/miR-21^St). All sections were counter stained with nuclear fast red. The examples are from tumors in the reference group and are representative for the individual tumors. Bar: 50 μm.

Figure 2

MicroRNA (miR)-21 scores in the HER2 reference group. The miR-21 scoring was performed as described in Material and Methods section, here presented as average scores of two observers: miR-21^CC (A) and miR-21^St (B).

Figure 3

Double staining for miR-21 and HER2 in HER2-positive breast cancers. Tissue sections were processed first for miR-21 ISH and subsequently for HER2 IHC. The figure shows three cases (A–D), (E–H) and (I–L). miR-21 was detected using TSA-Cy5 substrate (white) and HER2 with Cy3-conjugated anti-rabbit antibody (red). All sections were counterstained with DAPI (blue). miR-21 ISH signal is seen in HER2-positive cancer cells in cases (A–D) and (E–H) [examples are indicated by arrows in (A–D)], whereas HER2-positive cancer cells in case (I–L) are miR-21-negative. The examples are from tumors in the reference group and are representative for the three tumors. Bars: 20 μm.

Figure 4

MicroRNA-21 scores in the HER2 study group. The miR-21 scores for cancer cell-associated miR-21 expression, miR-21^CC (A), miR-21 stromal expression, miR-21^St (B), and the total summarized miR-21 scores (C). The scores are averages of two observers and here presented in scatter grams. Mann–Whitney U test: p = 0.69 for miR-21^CC, p = 0.13 for miR-21^St, and p = 0.20 for total miR-21.