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Multicenter Study
. 2025 Sep 29;27(1):169.
doi: 10.1186/s13058-025-02123-5.

Hsa-miR-155-5p expression in primary breast tissue may have the potential for prediction of breast cancer brain recurrence: results from the multi-institutional exploratory cohort study

Yoichi Koyama  1 , Masako Muguruma  2 , Yoshiya Horimoto  2   3 , Kazutaka Narui  4 , Akimitsu Yamada  5 , Kimito Yamada  6 , Shinya Yamamoto  7 , Shunichiro Orihara  8 , Hiroshi Kaise  2 , Akiko Kogure  9 , Yusuke Yoshioka  9 , Takahiro Ochiya  10 , Takashi Ishikawa  11
Affiliations
Multicenter Study

Hsa-miR-155-5p expression in primary breast tissue may have the potential for prediction of breast cancer brain recurrence: results from the multi-institutional exploratory cohort study

Yoichi Koyama et al. Breast Cancer Res. .

Abstract

Background: Despite the known incidence of brain metastases in breast cancer, no useful biomarker has been clinically established for breast cancer brain metastasis (BCBM). In the present study, we aimed to examine the expression of microRNAs (miRNAs) related to BCBM in formalin-fixed paraffin-embedded (FFPE) tissues to identify their usefulness as predictive biomarkers of breast cancer brain recurrence (BCBR).

Methods: Pairs of primary breast and metastatic site tissue samples were collected from 38 patients who experienced the first recurrence of metastasis to a single distant organ (brain/lungs/liver/bones = 11/12/9/6 patients) between January 2007 and December 2021 at five institutions in Japan. We evaluated the expression of 15 miRNAs in FFPE specimens of untreated breast and metastatic sites from the same patient using quantitative polymerase chain reaction.

Results: Analysis of the selected 15 miRNAs revealed that hsa-miR-155-5p exhibited significant BCBR-specific overexpression in untreated primary breast tissues (p < 0.001). Two other miRNAs, hsa-miR-150-5p and hsa-miR-20b-5p, exhibited moderate (ρ = 0.587) and weak (ρ = 0.350) positive correlations with hsa-miR-155-5p, respectively. The BCBR prediction model demonstrated a high discrimination ability for hsa-miR-155-5p (AUC = 0.960). The localization of hsa-miR-155-5p in primary breast cancer tissue by in situ hybridization confirmed that hsa-miR-155-5p was uniformly stained in the breast cancer cells.

Conclusions: Hsa-miR-155-5p expression in untreated primary breast tissue may be a valuable biomarker for predicting BCBR. A personalized escalation strategy is expected to be helpful in conquering brain metastases.

Keywords: Brain recurrence; Breast cancer; Formalin-fixed paraffin-embedded tissue; Hsa-miR-155-5p; MicroRNA; Polymerase chain reaction.

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Conflict of interest statement

Declarations. Ethics approval and consent to participate: This study was approved by the Ethics Committee of Tokyo Medical University (Approval number; T2022-0044). All procedures involving human participants performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Consent for publication: Due to the retrospective nature of the study, the requirement for informed consent was waived. The opportunity to refuse participation in the study was guaranteed by disclosing information about the research through a public announcement. Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Flow diagram of the study. In this study, we enrolled 38 eligible early breast cancer patients with a single distant organ metastasis as an initial recurrence (11 brain, 12 lung, 9 liver, and 6 bone metastases) from five participating institutes in Japan. Total RNA containing miRNAs was extracted from formalin-fixed, paraffin-embedded tissue specimens, and cDNA was synthesized and subjected to real-time quantitative PCR. We examined the expression levels of 15 miRNAs in untreated primary breast and metastatic tissue specimens from the same patients
Fig. 2
Fig. 2
The box plots of 15 miRNA’s expression compared among initial distant recurrence organs in primary breast cancer specimens (a), and validation of hsa-miR-155-5p expression among molecular subtypes (b). (a) The relative expression of 15 miRNAs (median ± 95% CI) in untreated primary breast specimens is shown. Blue, red, green, and yellow box indicates brain, lungs, liver, and bones only recurrence, respectively. The Kruskal-Wallis test was used to compare the relative expression levels of microRNAs among the four cohorts. Of the 15 miRNAs, only hsa-miR-155-5p showed significantly specific expression to breast cancer brain recurrence (BCBR), i.e., it was over-expressed in patients with brain-only recurrence compared to patients with visceral- or bone-only recurrence. (b) The relative expression of hsa-miR-155-5p in primary breast tissues, according to molecular subtypes, is shown. Blue, red, green, and yellow box indicates brain, lungs, liver, and bones only recurrence, respectively. We used the Kruskal–Wallis test to compare microRNA relative expression levels. We applied the Bonferroni correction for multiple testing (α = 0.05/4 = 0.0125). A similar trend of hsa-miR-155-5p specific expression in breast cancer brain recurrence was obtained across all molecular subtypes; however, it did not reach statistical significance, likely due to the limited sample size
Fig. 3
Fig. 3
The relative expression of 15 miRNAs (median ± 95% CI) in untreated primary breast and brain tissue cancer specimens from the same patients (n = 11) is shown. The pink and ivory boxes indicate primary breast and brain tissues, respectively. The Mann-Whitney U test was used to compare the levels of detected microRNA expression. No miRNAs were over-expressed in brain tissue. Six miRNAs, hsa-miR-10b-5p, hsa-miR-130a-3p, hsa-miR-150-5p, hsa-miR-155-5p, hsa-miR-199a-5p, and hsa-miR-503-5p, showed significantly higher expression in primary breast tissue than in brain tissue
Fig. 4
Fig. 4
Scattered plots of miRNAs that were significantly related to the expression of hsa-miR-155-5p in primary breast cancer specimens, as well as receiver operating characteristic (ROC) curves for predicting breast cancer brain recurrence (BCBR) (Intention to treat; N = 38). (a) The correlation between the expression of hsa-miR-155-5p and that of other miRNAs in primary breast cancer specimens, as determined by Spearman’s rank correlation coefficient, among eligible patients, is shown. Two miRNAs, hsa-miR-150-5p and hsa-miR-20b-5p, showed significant moderate (ρ = 0.587) and weak (ρ = 0.350), respectively, positive correlations with hsa-miR-155-5p. These two miRNAs also showed a significant, moderate positive correlation (ρ = 0.410). A BCBR prediction model (positive for BCBR onset) showed that hsa-miR-155-5p (AUC = 0.960) and hsa-miR-150-5p (AUC = 0.778) significantly predicted BCBR. (b) The BCBR prediction model incorporates established clinical and pathological prognostic factors commonly used in breast cancer, including clinical disease stage and molecular subtypes. Clinical disease stage (AUC = 0.855) and molecular subtypes (AUC = 0.759) significantly predicted BCBR independently. Furthermore, the addition of hsa-miR-155-5p improves the predictive accuracy of BCBR beyond that of these prognostic factors
Fig. 5
Fig. 5
Validation of hsa-miR-155-5p expression in primary breast tissue. a A comparison with primary early breast cancer patients without recurrence. b Localization by in situ hybridization. (a) The relative expression of miR-155 in primary breast tissue compared to early breast cancer patients without recurrence is shown. We examined additional primary breast tissue specimens from patients with brain-only recurrence (n = 13) and from patients without recurrence for at least five years after breast surgery, with similar clinical backgrounds (n = 13). The Mann–Whitney U test was used to compare the levels of detected relative microRNA expression. The trend of miR-155 expression in primary breast tissues from patients with brain-only recurrence supported the initial finding; however, it did not reach statistical significance, likely due to the limited sample size. (b) The localization of hsa-miR-155-5p in primary breast cancer specimens by in situ hybridization (ISH) is shown (up: hematoxylin and eosin (HE) stain; middle: hsa-miR-155-5p probe; down: scramble (negative control) probe). The scale bar indicates 250 μm (left) and 50 μm (right). We selected one of the primary breast tissues from the above patients with higher relative hsa-miR-155-5p expression. ISH confirmed that hsa-miR-155-5p stained uniformly in the nuclei and cell membranes of cancer cells
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