In the recent years promising results have been shown by the use of Compression optical coherence elastography (C-OCE) as a new optical biopsy approach to morphological assessment/diagnostics of human breast cancer using differences in elastic properties of morphological components of cancerous tissues. In this study, for the first time, a relationship was established between microstructural organization and biomechanical properties of breast-cancer tissue with pathomorphological changes caused by chemotherapy. To characterize texture of collagen fibers in the microenvironment of breast cancer, high-resolution visualization by Second-harmonic generation (SHG) microscopy was used. A side-by-side C-OCE and SHG imaging of patients' breast cancer tissues before and after chemotherapy was carried out. Regions of the cancer stroma (collagen fibers outside aggregates of cancer cells) were assessed separately from regions of cancer cell clusters penetrated by collagen fibers. For cancer stroma areas after chemotherapy, a statistically significant decrease in stiffness values was found. Simultaneously, parameters of collagen texture in SHG images (mean intensity, "coherency" and "energy") indicated increase in the collagen content, orientational orderliness, and collagen-texture heterogeneity. In contrast, cancer-cell areas post chemotherapy showed a statistically significant increase in stiffness. Analysis of SHG images of these regions indicated decrease in the inter-cellular collagen content and heterogeneity of its texture, whereas its orientational orderliness somewhat increased. The established negative correlation between stiffness and SHG parameters of collagen in cancer stroma indicates the contribution of the increase in orientational orderliness and total collagen content to the reduction in stiffness of breast cancer stroma after chemotherapy. For cancer-cell regions, significantly lower correlation between stiffness and SHG parameters (especially for coherency) was found, indicating stronger role of chemotherapy-induced changes in cancer cells themselves. These results give a deeper insight in the role of collagen texture organization in biomechanics of breast cancer tissues and contribute to a more detailed substantiation of the morphological characterization of breast cancer by C-OCE imaging.

We report the first application of compression optical coherence elastography (C-OCE) to evaluate both linear and nonlinear elastic properties of lymph nodes (LNs) for assessing their status, which is an important factor for detecting metastasis and staging breast cancer. The reported study involved 27 excised sentinel and axillary LNs from 24 patients with breast cancer, including normal LNs ( n = 6), reactive LNs with follicular hyperplasia ( n = 7) and sinus histiocytosis ( n = 8), and metastatic LNs ( n = 6). C-OCE data were compared with co-registered gold-standard histopathology and demonstrated a significantly higher contrast in differentiation of LNs as compared to structural optical coherence tomography imaging. Normal LNs are characterized by low stiffness values in the areas of LN cortex (<200 kPa). C-OCE revealed that abnormal reactive LNs with follicular hyperplasia exhibit moderately increased stiffness (200–300 kPa) in the cortex areas and more pronounced stiffness increase (400–600 kPa) in the areas of sinus histiocytosis. LNs with total metastasis exhibit fairly uniformly distributed highest stiffness values (>600 kPa). Since in terms of the tangent Young’s modulus there remained an appreciable overlap among these types of LNs, we also evaluated their elastic nonlinearity parameters. Complementary usage of both linear and nonlinear elastic parameters enabled very clear differentiation of all four main states of LNs. Thus, C-OCE imaging demonstrates high potential for future intraoperative usage to rapidly determine the LN status during breast-conserving surgery and to assess the extent of the cancer cells propagation in regional lymphatic collectors for preserving benign LNs. This is important for reducing the associated risks and complications (such as lymphedema) from excessive surgical removal of lymphoid structures.

Currently, optical biopsy technologies are being developed for rapid and label-free visualization of biological tissue with micrometer-level resolution. They can play an important role in breast-conserving surgery guidance, detection of residual cancer cells, and targeted histological analysis. For solving these problems, compression optical coherence elastography (C-OCE) demonstrated impressive results based on differences in the elasticity of different tissue constituents. However, sometimes straightforward C-OCE-based differentiation is insufficient because of the similar stiffness of certain tissue components. We present a new automated approach to the rapid morphological assessment of human breast cancer based on the combined usage of C-OCE and speckle-contrast (SC) analysis. Using the SC analysis of structural OCT images, the threshold value of the SC coefficient was established to enable the separation of areas of adipose cells from necrotic cancer cells, even if they are highly similar in elastic properties. Consequently, the boundaries of the tumor bed can be reliably identified. The joint analysis of structural and elastographic images enables automated morphological segmentation based on the characteristic ranges of stiffness (Young's modulus) and SC coefficient established for four morphological structures of breast-cancer samples from patients post neoadjuvant chemotherapy (residual cancer cells, cancer stroma, necrotic cancer cells, and mammary adipose cells). This enabled precise automated detection of residual cancer-cell zones within the tumor bed for grading cancer response to chemotherapy. The results of C-OCE/SC morphometry highly correlated with the histology-based results (r =0.96-0.98). The combined C-OCE/SC approach has the potential to be used intraoperatively for achieving clean resection margins in breast cancer surgery and for performing targeted histological analysis of samples, including the evaluation of the efficacy of cancer chemotherapy.

Intraoperative 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 face color-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.

Soft biological tissues, breast cancer tissues in particular, often manifest pronounced nonlinear elasticity, i.e., strong dependence of their Young’s modulus on the applied stress. We showed that compression optical coherence elastography (C-OCE) is a promising tool enabling the evaluation of nonlinear properties in addition to the conventionally discussed Young’s modulus in order to improve diagnostic accuracy of elastographic examination of tumorous tissues. The aim of this study was to reveal and quantify variations in stiffness for various breast tissue components depending on the applied pressure. We discussed nonlinear elastic properties of different breast cancer samples excised from 50 patients during breast-conserving surgery. Significant differences were found among various subtypes of tumorous and nontumorous breast tissues in terms of the initial Young’s modulus (estimated for stress < 1 kPa) and the nonlinearity parameter determining the rate of stiffness increase with increasing stress. However, Young’s modulus alone or the nonlinearity parameter alone may be insufficient to differentiate some malignant breast tissue subtypes from benign. For instance, benign fibrous stroma and fibrous stroma with isolated individual cancer cells or small agglomerates of cancer cells do not yet exhibit significant difference in the Young’s modulus. Nevertheless, they can be clearly singled out by their nonlinearity parameter, which is the main novelty of the proposed OCE-based discrimination of various breast tissue subtypes. This ability of OCE is very important for finding a clean resection boundary. Overall, morphological segmentation of OCE images accounting for both linear and nonlinear elastic parameters strongly enhances the correspondence with the histological slices and radically improves the diagnostic possibilities of C-OCE for a reliable clinical outcome.

In the study the multimodal optical coherence tomography (MM OCT) including microstructural cross-polarization OCT (CP OCT) imaging with the application of attenuation coefficients combined with compression OCT-elastography (OCE) with quantitative morphological segmentation based on specific stiffness ranges for delineation of breast cancer margins was applied. The research was carried out on different morphological and molecular subtypes of human breast cancer. The findings of this study suggest that OCE and CP OCT of breast cancer images may, in the future, enable real-time feedback to the surgeon about accurate resection margin location in patients with breast cancer.

The aims of this study are (i) to compare ultrasound strain elastography (US-SE) and compression optical coherence elastography (C-OCE) in characterization of elastically linear phantoms, (ii) to evaluate factors that can cause discrepancy between the results of the two elastographic techniques in application to real tissues, and (iii) to compare the results of US-SE and C-OCE in the differentiation of benign and malignant breast lesions. On 22 patients, we first used standard US-SE for in vivo assessment of breast cancer before and then after the lesion excision C-OCE was applied for intraoperative visualization of margins of the tumors and assessment of their type/grade using fresh lumpectomy specimens. For verification, the tumor grades and subtypes were determined histologically. We show that in comparison to US-SE, quantitative C-OCE has novel capabilities due to its ability to locally control stress applied to the tissue and obtain local stress-strain curves. For US-SE, we demonstrate examples of malignant tumors that were erroneously classified as benign and vice versa. For C-OCE, all lesions are correctly classified in agreement with the histology. The revealed discrepancies between the strain ratio given by US-SE and ratio of tangent Young's moduli obtained for the same samples by C-OCE are explained. Overall, C-OCE enables significantly improved specificity in breast lesion differentiation and ability to precisely visualize margins of malignant tumors compared. Such results confirm high potential of C-OCE as a high-speed and accurate method for intraoperative assessment of breast tumors and detection of their margins.

The aim of this study is to compare the results of tissue stiffness estimates given by compression (strain) ultrasound elastography (S-USE) and compression optical coherence elastography (C-OCE) beyond the difference in resolution and penetration depth. Namely, the focus of this work is on the contribution of elastic nonlinearity and mechanical inhomogeneity of the tissues to the stiffness estimates and resultant diagnostic performance of these techniques. We demonstrate that in comparison with S-USE despite basically the same compression principle, the applied realization of quantitative C-OCE have novel capabilities due to its ability to obtain spatially-resolved local pressure control and the local stress-strain curve mapping within the tissue.

The aim of the study We compare the effectiveness of multimodal optical coherence tomography (MM OCT) in the traditional structural OCT mode and the OCT elastography (OCE) mode in addressing two clinically important tasks: (1) detecting groups of tumor cells at surgical margins during breast-сonserving surgery (BСS) in breast cancer (BC) and (2) identifying breast tumor margins. The obtained results were correlated with corresponding histological sections. Materials and Methods The study was performed on 100 surgical margin samples (top, bottom, medial, and lateral — four samples from each patient in total) obtained from 25 patients with BC who underwent BCS (lumpectomy), and on 25 postoperative tumor samples (to determine tumor margins). With MM OCT method, we visually and numerically assessed the scattering (level and depth of OCT signal penetration) and elastic (stiffness values, or Young’s modulus (kPa)) properties of the tumor and non-tumor breast tissue and the obtained values were compared with the results of postoperative histological examination. Results In 4 surgical margin samples (out of 100), with the OCE method we identified groups of histologically confirmed tumor cells (“positive” resection margins) at the distance of about 5 mm from the visible tumor margin. The identified zones were larger than 0.5 mm with stiffness of more than 400 kPa in all these cases. However, the structural OCT could not identify these groups of tumors and they were not distinguishable from the surrounding fibrous tissue. In the areas of tumor into non-tumor tissue transition, structural OCT images detected tumor margins only if they were adjacent to adipose tissue and did not detect them if there were adjacent to non-tumor fibrous tissue. OCE images with high stiffness values (more than 400 kPa) and high contrast showed a clear tumor margin with both adipose and fibrous tissue. Conclusion The study demonstarets the potential of MM OCT, particularly its OCE mode, as a real-time method for intraoperative tumor margin and surgical margin assessment in BCS. OCE images compared to structural OCT images visualize higher contrast between different types of breast tissue (adipose tissue, fibrous stroma, hyalinized stroma, tumor cell clusters), as well as more accurate identification of the tumor border and detection of small groups of tumor cells at surgical margins. An algorithm for intraoperative MM OCT examination of the state of the resection margin is proposed in accordance with standard clinical guidelines for achieving clean surgical margins in breast cancer patients.

The purpose of this study is to compare the results of ultrasound strain elastography (US-SE) with compression optical coherence elastography (C-OCE) measurements in breast cancer, using histologic analysis as the reference standard, and to assess factors that can affect the results of the two elastography techniques. We demonstrate that in comparison to US-SE, C-OCE have novel capabilities due to its ability to locally control pressure on the breast tissue and obtain local stress-strain curves. The obtained results confirm high potential of using C-OCE in intraoperative search for resection breast cancer margins.

We demonstrate possibilities of multimodal OCT for quick distinguishing of uninformative necrotic zones from other morphological structures in freshly-excised breast cancer samples to improve information value of subsequent histological examination.

In this study multiphoton tomography, based on second harmonic generation (SHG), and two-photon-excited fluorescence (TPEF) was used to visualize both the extracellular matrix and tumor cells in different morphological and molecular subtypes of human breast cancer. It was shown, that quantified assessment of the SHG based imaging data has great potential to reveal differences of collagen quantity, organization and uniformity in both low- and highly- aggressive invasive breast cancers. The values of quantity and uniformity of the collagen fibers distribution were significantly higher in low-aggressive breast cancer compared to the highly-aggressive subtypes, while the value representing collagen organization was lower in the former type. Additionally, it was shown, that TPEF detection of elastin fibers and amyloid protein may be used as a biomarker of detection the low-aggressive breast cancer subtype. Thus, TPEF/SHG imaging offers the possibility of becoming a useful tool for the rapid diagnosis of various subtypes of breast cancer during biopsy as well as for the intraoperative determinination of tumor-positive resection margins.

The status of sentinel lymph nodes (SLNs) has a substantial prognostic value because these nodes are the first place where cancer cells accumulate along their spreading route. Routine SLN biopsy ("gold standard") involves peritumoral injections of radiopharmaceuticals, such as technetium-99m, which has obvious disadvantages. This review examines the methods used as "gold standard" analogs to diagnose SLNs. Nonradioactive preoperative and intraoperative methods of SLN detection are analyzed. Promising photonic tools for SLNs detection are reviewed, including NIR-I/NIR-II fluorescence imaging, photoswitching dyes for SLN detection, in vivo photoacoustic detection, imaging and biopsy of SLNs. Also are discussed methods of SLN detection by magnetic resonance imaging, ultrasonic imaging systems including as combined with photoacoustic imaging, and methods based on the magnetometer-aided detection of superparamagnetic nanoparticles. The advantages and disadvantages of nonradioactive SLN-detection methods are shown. The review concludes with prospects for the use of conservative diagnostic methods in combination with photonic tools.

The aim of this study was to evaluate the application of attenuation coefficients calculated from the cross-polarization optical coherence tomography (CP OCT) data for differentiation of breast cancer from non-tumorous breast tissues. For this purpose, surgically obtained breast specimens from 45 patients were examined using CP OCT in order to construct color-coded en-face OCT maps based on calculation of three optical coefficients (the commonly used rate of attenuation in the co- polarization channel; and, additionally, the attenuation in the cross- polarization channel; and the interchannel attenuation difference). It was shown that the use of these optical coefficients significantly increased the amount of obtained information from the OCT data in comparison with unprocessed images, enabling objective quantification for differentiating non-tumorous and tumorous tissue (adipose tissue, normal stroma, tumor stroma and agglomerates of tumor cells). The attenuation in the cross- polarization channel and the interchannel attenuation difference provided greater contrast for the visualization of the different breast cancer structures compared to the attenuation coefficient in the co- polarization channel. The findings of this study suggest that assessment of CP OCT images of breast cancer based on optical coefficients calculation may in perspective enable real-time feedback about accurate resection margin in patient with breast cancer to the surgeon.

Breast conserving surgery (BCS) has become increasingly accepted as the surgical management of breast cancer in clinical practice. The main goal of BCS is the complete removal of cancer with clear surgical margins. This study demonstrates the results of the diagnostic accuracy of compression optical coherence elastography (C-OCE) method in ex vivo determining of “positive” and “negative” surgery margins in BCS. The studies were carried out by multimodal spectral optical coherence tomograph (IAP RAS, Russia) in elastographic mode. C-OCE can in real-time determine absolute stiffness values (Young's modulus, kPa) of the tissue with a resolution of 40-50 μm at a depth up to 2 mm. 25 female patients who underwent BCS for invasive ductal or lobular carcinomas were enrolled to the study. Four fresh tissue samples per patient were harvested <5 mm from the tumor (upper, lower, lateral, medial). Sensitivity, specificity and diagnostic accuracy were calculated by comparing the predicted margin status from the OCE imaging with the true margin status by histology. Based on the sensitivity and specificity values, the Receiver operating characteristic (ROC) curves and the area under ROC curve were calculated. Histological examination revealed 4 of 100 specimens with tumor cells (“positive” surgical margins) from in situ and invasive carcinoma. In 96 of 100 specimens adipose, adipose with streaks of connective tissue or fibrosis were found by histology (“negative” surgical margins). C-OCE method detected “positive” and “negative” surgical margins at the same specimens. The good agreement between C-OCE and histology was established. Young's modulus in the frame 500х500 μm on C-OCE images was chosen to characterize breast tissue elasticity properties. For detecting “positive” surgery margins, threshold equal to >159kPa was proposed. This threshold provides sensitivity = specificity = 94.4% (94.2% diagnostic accuracy). The area under the ROC curve was equal to 0.98. Adjusted threshold for determining tumor areas on C-OCE images is characterized by high diagnostic efficiency and can be used to identify in real-time residual tumor cells at the surgical margins during breast conserving surgery.

Breast cancer is a genetically heterogeneous disease characterized by various biomolecular and morphological features that affect the diagnosis, prognosis, and treatment response. In this study we combined cross-polarization optical coherence tomography (CP OCT) and multiphoton tomography (MPT), based on second harmonic generation (SHG), and two-photon-excited fluorescence (TPEF) to visualize tumor stroma and tumor cells in specimens of a human breast tissue. The data obtained by both techniques were compared with histopathology. The CP OCT and MPT images were quantitatively assessed to distinguish a breast normal tissue from a cancer as well as between a low and a high grade of cancer. Quantitative assessment of the CP OCT image included the calculation of signal attenuation coefficients separately for co- and cross- polarization channels and the formation the color-coded en face distribution maps of these coefficients. The attenuation coefficient in cross- polarization channel showed better difference between breast cancer of low and high grades and distinguish them from normal tissue. The SHG images were processed using texture analysis techniques to quantify the density of collagen fibers in normal tissue and tumor. Thus, both imaging techniques have great potential to distinguish nontumorous and tumorous human breast tissue of varying degrees of malignancy and could provide identifying breast cancer margins for in surgery.

The aim of this study was to investigate the application of optical coefficients obtained from cross-polarization optical coherence tomography (CP OCT) data to differentiate breast cancer tissues from normal breast tissues. For this, surgically obtained breast specimens from 35 patients were investigated using CP OCT in order to construct pseudocoloren faceOCT maps based on a calculation of three optical coefficients: the commonly used, rate of attenuation in the co-channel (Att(co-)) and, additionally, the attenuation in the cross-channel (Att(cross-)); and the interchannel attenuation difference (uD). It was shown that the use of these optical coefficients significantly increased the information available from the OCT data in comparison with unprocessed images, and that this enabled objective quantification for differentiating non-tumorous and tumorous tissue (adipose tissue, normal stroma, tumor stroma and agglomerates of tumor cells). The Att(cross-)and the uD provided greater contrast for the visualization of the different breast cancer structures compared to the Att(co-)coefficient. While the Att(co-)of tumor cells was significantly higher (p < 0.05) than that of adipose tissue, the Att(co-)difference between normal or tumor stroma and tumor cells was not observed. On the other hand, normal or tumor stroma was associated with significantly higher (p < 0.05) Att(cross-)and significantly lower (p < 0.05) uD as compared to tumor cells. Furthermore, Att(cross-)of tumor stroma was significantly higher (p < 0.05) than that of normal (non-tumor) stroma. The findings of this study suggest that CP OCT and an assesment of the optical coefficients of such breast cancer images may, in the future, enable real-time feedback to the surgeon about accurate resection margin locations in patients with breast cancer.

The possibility to assess molecular-biological and morphological features of particular breast cancer types can improve the precision of resection margin detection and enable accurate determining of the tumor aggressiveness, which is important for treatment selection. To enable reliable differentiation of breast-cancer subtypes and evaluation of resection margin, without performing conventional histological procedures, here we apply cross-polarization optical coherence tomography (CP-OCT) and compare it with a novel variant of compressional optical coherence elastography (C-OCE) in terms of the diagnostic accuracy (Ac) with histological verification. The study used 70 excised breast cancer specimens with different morphological structure and molecular status (Luminal A, Luminal B, Her2/Neo+, non-luminal and triple-negative cancer). Our first aim was to formulate convenient criteria of visual assessment of CP-OCT and C-OCE images intended (i) to differentiate tumorous and non-tumorous tissues and (ii) to enable more precise differentiation among different malignant states. We identified such criteria based on the presence of heterogeneities and characteristics of signal attenuation in CP-OCT images, as well as the presence of inclusions/mosaic structures combined with visually feasible assessment of several stiffness grades in C-OCE images. Secondly, we performed a blinded reader study of the Ac of C-OCE versus CP-OCT, for delineation of tumorous versus non-tumorous tissues followed by identification of breast cancer subtypes. For tumor detection, C-OCE showed higher specificity than CP-OCT (97.5% versus 93.3%) and higher Ac (96.0 versus 92.4%). For the first time, the Ac of C-OCE and CP-OCT were evaluated for differentiation between non-invasive and invasive breast cancer (90.4% and 82.5%, respectively). Furthermore, for invasive cancers, the difference between invasive but low-aggressive and highly-aggressive subtypes can be detected. For differentiation between non-tumorous tissue and low-aggressive breast-cancer subtypes, Ac was 95.7% for C-OCE and 88.1% for CP-OCT. For differentiation between non-tumorous tissue and highly-aggressive breast cancers, Ac was found to be 98.3% for C-OCE and 97.2% for CP-OCT. In all cases C-OCE showed better diagnostic parameters independently of the tumor type. These findings confirm the high potential of OCT-based examinations for rapid and accurate diagnostics during breast conservation surgery.