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In digital pathology, the final appearance of digitized images is affected by several factors, resulting in stain color and intensity variation. Stain normalization is an innovative solution to overcome stain variability. However, the validation of color normalization tools has been assessed only from a quantitative perspective, through the computation of similarity metrics between the original and normalized images. To the best of our knowledge, no works investigate the impact of normalization on the pathologist's evaluation. The objective of this paper is to propose a multi-tissue (i.e., breast, colon, liver, lung, and prostate) and multi-center qualitative analysis of a stain normalization tool with the involvement of pathologists with different years of experience. Two qualitative studies were carried out for this purpose: (i) a first study focused on the analysis of the perceived image quality and absence of significant image artifacts after the normalization process; (ii) a second study focused on the clinical score of the normalized image with respect to the original one. The results of the first study prove the high quality of the normalized image with a low impact artifact generation, while the second study demonstrates the superiority of the normalized image with respect to the original one in clinical practice. The normalization process can help both to reduce variability due to tissue staining procedures and facilitate the pathologist in the histological examination. The experimental results obtained in this work are encouraging and can justify the use of a stain normalization tool in clinical routine., Competing Interests: M. Salvi, O.A.M. Pennisi and F. Molinari are equity holders in AEQUIP S.r.l, Turin, Italy. Remaining authors declare no competing interests regarding the publication of this article., (© 2022 The Authors.)

Columnar cell lesions of the breast are increasingly recognized at mammography for their tendency to calcify. We studied 392 vacuum-assisted core biopsies performed solely for calcifications to evaluate the frequency of columnar cell lesions, their relationship with radiological risk, appearance of calcifications, and clinical data. Management and follow-up of columnar cell lesions without and with atypia (flat epithelial atypia) was analyzed. Cases with architectural atypia (cribriform spaces and/or micropapillae) were excluded from flat epithelial atypia. Calcifications were within the lumen of acini affected by columnar cell lesions in 137 out of 156 biopsies diagnosed with some columnar cell lesions. These represented 37% of vacuum-assisted core biopsies and 62% of low radiological risk (BI-RADS3) calcifications. High-risk (BI-RADS5) calcifications were never associated with columnar cell lesions. Age and menopausal status were comparable in columnar and in not-columnar cell lesions. Atypia was associated with long-term hormone replacement therapy in both lesions. Surgical biopsy was recommended for all cases with atypia. Flat epithelial atypia, as the only histological findings on vacuum-assisted core biopsies, was never associated with malignancy at surgery. In conclusion, we suggest that surgical excision is not mandatory when flat epithelial atypia is found as the most advanced lesion on vacuum-assisted core biopsy performed for low radiological risk calcifications, and that women should be advised of the possible hormone dependency of this entity.

An estrogen dependency has been suggested for the growth of fibroadenomas: however, thus far, none of the steroid hormone receptors acting on breast tissues has been demonstrated in the stroma of breast fibroepithelial lesions. In this study, the expression of estrogen receptor (ER)-alpha and -beta was investigated by immunohistochemistry in 33 fibroadenomas and in 30 benign, three borderline and seven malignant phyllodes tumors, all with spindle cell growth and in one distant metastasis. In addition, the presence of ER-beta mRNA and its variants was evaluated by RT-PCR in microdissected stroma. The possible correlation between hormone receptor expression and differentiation processes of stromal cells was investigated by smooth muscle actin and calponin immunostaining. ER-beta was the only hormone receptor expressed by stroma of fibroadenomas and phyllodes tumors, both at protein and mRNA level. The highest percentage of ER-beta was observed in fibroadenomas with cellular stroma and in phyllodes tumors. In both lesions, ER-beta-positive stromal cells showed expression of smooth muscle actin and/or calponin, as demonstrated by double immunostaining. In addition, the mean age at diagnosis was significantly lower in patients with ER-beta-positive vs ER-beta-negative fibroadenomas. In contrast, in phyllodes tumors, ER-beta expression was higher in older patients. In conclusion, (i) only ER-beta is detected in the stroma of fibroadenomas and phyllodes tumors; (ii) its expression correlates with the expression of smooth muscle markers and suggests a role of ER-beta in myofibroblastic differentiation of stromal cells. These two results, together with the young age of patients carrying fibroadenomas with highly ER-beta-positive stroma cells, may further indicate a hormone-receptor mechanism involved in regulating the growth of fibroadenomas. Conversely, the older age of patients with ER-beta-rich phyllodes tumors suggests that mechanisms, probably independent from estrogen stimulation, act on the growth of these tumors.