Association between low estrogen receptor positive breast cancer and staining performance

Abstract

Estrogen receptor (ER) expression in breast carcinomas, determined by immunohistochemistry, indicates statistically significant benefit to endocrine therapy in patients with tumors expressing ER in ≥1% of tumor cells. Rare cases with low ER expression (1-10%) lead to the dilemma of treating these tumors as ER positive or negative. We hypothesize that low ER positive result from poor staining performance and that we may detect this artefact by assessing the average dynamic range of normal ducts adjacent to low ER positive tumors. Using quantitative tools, we compare the dynamic range of normal background ER expression in patients with low (1-10%) ER tumors to dynamic range of ER expression in normal epithelium from control patient populations, to determine if low ER cases are accompanied by decreased dynamic range. Low ER cases were infrequent (1% of invasive breast carcinomas). Twenty-one cases with low ER staining and two control cohorts, including a tissue microarray (TMA) of 10 benign breast sections and a group of 34 control breast carcinomas (reported as ER negative or >10% ER positive) with normal background epithelium, were digitally scanned. QuPath was utilized to quantify ER staining for each cell as the mean optical density of nuclear DAB staining. The dynamic range of ER expression in normal epithelium surrounding low ER tumors was significantly lower (range 2-240, median 16.5) than that of the benign epithelium in the control tumors (range 3-475, median 30.8; p < 0.001) and benign TMA sections (range 38-212, median 114; p < 0.001) suggesting inconsistent stainer performance.

Keywords: Breast cancer; Predictive markers; Tumour biomarkers.

Fig. 1

Consort diagram of clinical case selection.

Fig. 2. Mean Nuclear OD in Low ER and control groups.

The mean nuclear OD values were log transformed. Cases of Low ER (1–10% staining) are compared to normal epithelium in the matched clinical full section control cases and in core tissue on YTMA-55. The lower and upper bars represent the minimum and maximum, respectively; the lower and upper edges of the box represent the 25th and 75th percentiles, respectively; the line in the middle of the box represents the median; the gray dots represent log transformed mean nuclear OD values.

Fig. 3. Cell detection and ER quantification on normal ducts using the open-source platform QuPath.

a Non-neoplastic breast duct epithelium manually demarcated on a whole-slide image, outlined in yellow. b Initial detection of luminal nuclei circled in red. c Following manual elimination of objects deemed to represent non-luminal nuclei or incorrect partitioning of luminal nuclei. d Dynamic range of ER expression plotted with each bar representing the mean DAB for individual nuclei (*100) and the red bar representing the lowest limit of detection. OD = optical density.