Simple SummaryMultiple technological solutions are being explored to be used in the intraoperative assessment of resection margins in breast cancer and detection of the residual tumor cells during breast-conserving surgery (BCS) for the purpose of reducing the need for a re-resection. We applied the cross-polarization optical coherence tomography (CP OCT) method for intraoperative ex vivo human breast cancer specimens imaging and performed a qualitative and quantitative assessment of 3D CP OCT data using a depth-resolved approach of measuring the attenuation coefficient estimation in co- and cross-polarization channels.En facecolor-coded attenuation coefficient maps were constructed, and targeted calculations of the characteristic median value of the attenuation coefficients in both channels were performed for different breast tissue regions. As a result, highly accurate differentiation of the tumorous from non-tumorous breast tissue was achieved. This new optical technique with estimation attenuation coefficients of volumetric CP OCT data can be used as an innovative adjunct intraoperative tool to evaluate resection margins during BCS and to perform a targeted histological biopsy.AbstractIntraoperative differentiation of tumorous from non-tumorous tissue can help in the assessment of resection margins in breast cancer and its response to therapy and, potentially, reduce the incidence of tumor recurrence. In this study, the calculation of the attenuation coefficient and its color-coded 2D distribution was performed for different breast cancer subtypes using spectral-domain CP OCT. A total of 68 freshly excised human breast specimens containing tumorous and surrounding non-tumorous tissues after BCS was studied. Immediately after obtaining structural 3D CP OCT images,en facecolor-coded attenuation coefficient maps were built in co-(Att(co)) and cross-(Att(cross)) polarization channels using a depth-resolved approach to calculating the values in each A-scan. We determined spatially localized signal attenuation in both channels and reported ranges of attenuation coefficients to five selected breast tissue regions (adipose tissue, non-tumorous fibrous connective tissue, hyalinized tumor stroma, low-density tumor cells in the fibrotic tumor stroma and high-density clusters of tumor cells). The Att(cross) coefficient exhibited a stronger gain contrast of studied tissues compared to the Att(co) coefficient (i.e., conventional attenuation coefficient) and, therefore, allowed improved differentiation of all breast tissue types. It has been shown that color-coded attenuation coefficient maps may be used to detect inter- and intra-tumor heterogeneity of various breast cancer subtypes as well as to assess the effectiveness of therapy. For the first time, the optimal threshold values of the attenuation coefficients to differentiate tumorous from non-tumorous breast tissues were determined. Diagnostic testing values for Att(cross) coefficient were higher for differentiation of tumor cell areas and tumor stroma from non-tumorous fibrous connective tissue: diagnostic accuracy was 91–99%, sensitivity—96–98%, and specificity—87–99%. Att(co) coefficient is more suitable for the differentiation of tumor cell areas from adipose tissue: diagnostic accuracy was 83%, sensitivity—84%, and specificity—84%. Therefore, the present study provides a new diagnostic approach to the differentiation of breast cancer tissue types based on the assessment of the attenuation coefficient from real-time CP OCT data and has the potential to be used for further rapid and accurate intraoperative assessment of the resection margins during BCS.