MicroRNAs (miR) are a class of short non-coding RNAs that regulate gene expression and are commonly dysregulated in cancers, including those of the breast. MiR-92 is thought to function as an oncogene, promoting cell proliferation and reducing apoptosis. We have previously shown that expression of ERβ1 is negatively regulated by miR-92 in unselected non-microdissected breast cancers, providing a mechanism for down-regulation of this putative tumour suppressor gene. Here, we aimed to confirm the function of ERβ1 in breast cancer cells and examined expression of miR-92, specifically in the breast epithelium, during breast cancer progression. We also investigated the role of miR-92 in fibroblasts and examined whether differential expression in these cells might modify the behaviour of breast cancer epithelial cells. Stable ERβ1 overexpression was achieved using a MoMLV-based vector (pFBneo) or silenced by siRNA and effects were examined using various functional assays. We used laser microdissection (LMD) to isolate matched normal, co-incident DCIS and invasive tissue from 10 breast cancer patients. RNA was extracted, cDNA synthesised and preamplified using MegaPlex PreAmp Primers. MiR-92 expression was analysed by real-time PCR. Expression of ERβ1 protein was concurrently analysed for each patient by immunohistochemistry. LMD was also used to isolate matched normal and cancer-associated fibroblasts from 21 breast cancer patients and miR-92 expression was examined as above. Loss and gain of miR-92 in fibroblasts was achieved using anti-miR-92 miRNA and miRNASelect pEP-hsa-mir-92a-1 expression vector, respectively. The invasive potential of these fibroblasts was examined using a 2D modified Boyden chamber assay. ERβ1 overexpression decreased growth and induced cell death in T47D and BT-20 breast cancer cells. Surprisingly, we found that whilst absolute miR-92 expression levels varied across patients the expression pattern of miR-92 was consistent in all cases; miR-92 levels decreased during breast cancer progression with highest levels in normal breast epithelium, decreasing in DCIS (p<0.01) and lowest in invasive breast tissue (p<0.01). Expression of ERβ1 and miR-92 were not inversely correlated as expected. Further examination of the staining patterns of ERβ1 showed nuclear expression in normal breast epithelium with increased cytoplasmic immunoreactivity during progression to DCIS and invasive breast cancer. ERβ1 immunoreactivity was also seen in stromal fibroblasts in tissues, however miR-92 expression levels remained unchanged between matched microdissected adjacent normal and cancer-associated fibroblasts. Despite this, we demonstrated that fibroblasts can contribute to breast tumour progression as manipulation of miR-92 levels influenced the invasive potential of breast cancer epithelial cells. In summary, whilst confirming a potential tumour suppressor role for ERβ1 we have shown that expression is not inversely correlated with its negative regulator miR-92 in breast epithelium, suggesting other methods of regulation. Expression of miR-92 was lost, rather than gained, in breast epithelial cells during breast cancer progression correlating with a shift in ERβ1 staining from nuclei to the cytoplasm. While miR-92 expression levels remained unchanged in stromal fibroblasts, our data support a functional role in fibroblasts and show that differential miR-92 expression in these cells can influence the invasive capacity of breast cancer epithelial cells. Finally, our study highlights the importance of isolating specific cell types prior to expression analyses since changes in the epithelium may be masked by expression from the stroma and vice versa. Citation Format: Laura Smith, Euan Baxter, Andrew Hanby, Thomas Hughes, Rebecca Millican-Slater, Eldo Verghese, Valerie Speirs. Loss of miR-92 expression in breast epithelial cells is associated with cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B127.

Background Breast cancer patients with estrogen receptor (ER)+/HER2- (and usually node-negative) tumors can avail themselves of Oncotype DX Breast Recurrence Score (ODXRS) testing to predict their risk of distant recurrence within 9 years and, consequently, putative chemotherapy benefit. However, ODXRS testing requires sufficient tumour availability and specimen shipping, which imposes time and financial burdens to testing which have to be met by healthcare systems. The advent of digital pathology offers a potential avenue for exploring computer-aided diagnostic solutions which may overcome these hurdles by extracting the requisite information from hematoxylin and eosin (H&E)-stained tissue whole slide images (WSIs) alone. In turn, this technology could significantly reduce diagnostic turnaround times and cost, and improve accessibility and test reproducibility, thereby enabling healthcare systems to run more efficiently and offer patients more timely results. Ideally, such a platform should incorporate a measure of the underlying tumor biology to provide a fully explainable, white box solution, and may offer further insights into the identification of early recurrence events. Aims The aim of this study was to establish whether our computer-aided solution’s (Q-Plasia OncoReader Breast, QPORB) digital biomarker representing G1/S cell cycle deformations extracted from H&E WSIs was prognostic for disease-free survival (DFS) and could predict disease recurrence, particularly in the setting of low risk ODXRS breast cancers. Methods Primary breast cancer resection/excision specimens (n=70 cases) sent for ODXRS testing from St James’s University Hospital, UK (2016-2019) were collected. Anonymised diagnostic glass slides (n=198 slides) of H&E-stained tumors were scanned at x20 magnification on an Aperio AT2 scanner. In parallel, relevant clinical and histological data were collected from pathology reports and electronic patient records, including both ODXRS and recurrence events during follow-up. The QPORB recurrence scale (QPORB-RS), which combines statistical physics and tumor biology to identify image-based malignant cell cycle deformation, extracts prognostic information from WSIs. The contribution of potential confounders (age, stage, grade, lesion size, Nottingham prognostic index and Charlson score) were accounted for. Results The QPORB-RS was prognostic for DFS for patients with predominantly node-negative (including node micro-metastases) HR+/HER2- tumors over a median follow-up period of 5 years (P=0.02; dichotomized Kaplan Meyer with median cut-off). The QPORB-RS concurred with ODXRS’s high vs. low recurrence risk in 73% (19/26) and 61% (27/44) of cases, respectively, with an overall agreement of 66% (46/70). Moreover, the QPORB-RS identified all 5 patients who had recurrences (with ODXRS of 6, 9, 10, 21 and 26, and ages of 55, 66, 42, 35 and 50 years, respectively) as being high risk in the subset of those given a low (including historically intermediate) ODXRS and who did not receive chemotherapy. Conclusion The QPORB-RS is a good prognostic test of risk of disease recurrence in breast cancer patients with predominantly node-negative (including node micro-metastases) HR+/HER2- tumors within a median 5-year follow-up period. Our efforts are now focussed on extending this cohort and establishing the prognostic value of the QPORB-RS across all breast carcinomas, regardless of molecular subtype, stage/node positivity and menopausal status. Citation Format: Satabhisa Mukhopadhyay, Tathagata Dasgupta, Elizabeth Walsh, Rebecca Millican-Slater, Andrew hanby, Joanne Stephenson, Craig A. Bunnell, Nicolas M. Orsi. Prediction of disease recurrence in low risk Oncotype Dx breast cancers from digital H&E-stained whole slide images of pre-treatment resections alone [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-48.

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Breast cancer is the commonest cancer to affect women in developed countries and is increasing in frequency in the Western world. Approximately 50,000 women and 400 men are diagnosed with breast cancer in the United Kingdom each year. Eighty per cent of these individuals will survive for at least 5 years after diagnosis. In 2012, 11,762 women died of breast cancer in the United Kingdom. Age-standardized rates of new invasive breast cancer diagnosis have increased from 75 to 126 per 100,000 population in the United Kingdom between 1977 and 2010.

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