Towards targeted colorectal cancer biopsy based on tissue morphology assessment by compression optical coherence elastography

Identifying the precise topography of cancer for targeted biopsy in colonoscopic examination is a challenge in current diagnostic practice. For the first time we demonstrate the use of compression optical coherence elastography (C-OCE) technology as a new functional OCT modality for differentiating between cancerous and non-cancerous tissues in colon and detecting their morphological features on the basis of measurement of tissue elastic properties. The method uses pre-determined stiffness values (Young’s modulus) to distinguish between different morphological structures of normal (mucosa and submucosa), benign tumor (adenoma) and malignant tumor tissue (including cancer cells, gland-like structures, cribriform gland-like structures, stromal fibers, extracellular mucin). After analyzing in excess of fifty tissue samples, a threshold stiffness value of 520 kPa was suggested above which areas of colorectal cancer were detected invariably. A high Pearson correlation (r =0.98; p <0.05), and a negligible bias (0.22) by good agreement of the segmentation results of C-OCE and histological (reference standard) images was demonstrated, indicating the efficiency of C-OCE to identify the precise localization of colorectal cancer and the possibility to perform targeted biopsy. Furthermore, we demonstrated the ability of C-OCE to differentiate morphological subtypes of colorectal cancer – low-grade and high-grade colorectal adenocarcinomas, mucinous adenocarcinoma, and cribriform patterns. The obtained ex vivo results highlight prospects of C-OCE for high-level colon malignancy detection. The future endoscopic use of C-OCE will allow targeted biopsy sampling and simultaneous rapid analysis of the heterogeneous morphology of colon tumors

Post-Operational Photodynamic Therapy of the Tumor Bed: Comparative Analysis for Cold Knife and Laser Scalpel Resection

In this paper, we report on a study regarding the efficiency of the post-operational phototherapy of the tumor bed after resection with both a cold knife and a laser scalpel in laboratory mice with CT-26 tumors. Post-operational processing included photodynamic therapy (PDT) with a topically applied chlorin-based photosensitizer (PS), performed at wavelengths of 405 or 660 nm, with a total dose of 150 J/cm2. The selected design of the tumor model yielded zero recurrence in the laser scalpel group and 92% recurrence in the cold knife group without post-processing, confirming the efficiency of the laser scalpel in oncology against the cold knife. The application of PDT after the cold knife resection decreased the recurrence rate to 70% and 42% for the 405 nm and 660 nm procedures, respectively. On the other hand, the application of PDT after the laser scalpel resection induced recurrence rates of 18% and 30%, respectively, for the considered PDT performance wavelengths. The control of the penetration of PS into the tumor bed by fluorescence confocal microscopy indicated the deeper penetration of PS in the case of the cold knife, which presumably provided deeper PDT action, while the low-dose light exposure of deeper tissues without PS, presumably, stimulated tumor recurrence, which was also confirmed by the differences in the recurrence rate in the 405 and 660 nm groups. Irradiation-only light exposures, in all cases, demonstrated higher recurrence rates compared to the corresponding PDT cases. Thus, the PDT processing of the tumor bed after resection could only be recommended for the cold knife treatment and not for the laser scalpel resection, where it could induce tumor recurrence

Intraoperative Assessment of Breast Cancer Tissues after Breast-Conserving Surgery Based on Mapping the Attenuation Coefficients in 3D Cross-Polarization Optical Coherence Tomography

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

Histological validation of in vivo assessment of cancer tissue inhomogeneity and automated morphological segmentation enabled by Optical Coherence Elastography

Abstract We present a non-invasive (albeit contact) method based on Optical Coherence Elastography (OCE) enabling the in vivo segmentation of morphological tissue constituents, in particular, monitoring of morphological alterations during both tumor development and its response to therapies. The method uses compressional OCE to reconstruct tissue stiffness map as the first step. Then the OCE-image is divided into regions, for which the Young’s modulus (stiffness) falls in specific ranges corresponding to the morphological constituents to be discriminated. These stiffness ranges (characteristic "stiffness spectra") are initially determined by careful comparison of the "gold-standard" histological data and the OCE-based stiffness map for the corresponding tissue regions. After such pre-calibration, the results of morphological segmentation of OCE-images demonstrate a striking similarity with the histological results in terms of percentage of the segmented zones. To validate the sensitivity of the OCE-method and demonstrate its high correlation with conventional histological segmentation we present results obtained in vivo on a murine model of breast cancer in comparative experimental study of the efficacy of two antitumor chemotherapeutic drugs with different mechanisms of action. The new technique allowed in vivo monitoring and quantitative segmentation of (1) viable, (2) dystrophic, (3) necrotic tumor cells and (4) edema zones very similar to morphological segmentation of histological images. Numerous applications in other experimental/clinical areas requiring rapid, nearly real-time, quantitative assessment of tissue structure can be foreseen

Simultaneous Probing of Metabolism and Oxygenation of Tumors In Vivo Using FLIM of NAD(P)H and PLIM of a New Polymeric Ir(III) Oxygen Sensor

Tumor cells are well adapted to grow in conditions of variable oxygen supply and hypoxia by switching between different metabolic pathways. However, the regulatory effect of oxygen on metabolism and its contribution to the metabolic heterogeneity of tumors have not been fully explored. In this study, we develop a methodology for the simultaneous analysis of cellular metabolic status, using the fluorescence lifetime imaging microscopy (FLIM) of metabolic cofactor NAD(P)H, and oxygen level, using the phosphorescence lifetime imaging (PLIM) of a new polymeric Ir(III)-based sensor (PIr3) in tumors in vivo. The sensor, derived from a polynorbornene and cyclometalated iridium(III) complex, exhibits the oxygen-dependent quenching of phosphorescence with a 40% longer lifetime in degassed compared to aerated solutions. In vitro, hypoxia resulted in a correlative increase in PIr3 phosphorescence lifetime and free (glycolytic) NAD(P)H fraction in cells. In vivo, mouse tumors demonstrated a high degree of cellular-level heterogeneity of both metabolic and oxygen states, and a lower dependence of metabolism on oxygen than cells in vitro. The small tumors were hypoxic, while the advanced tumors contained areas of normoxia and hypoxia, which was consistent with the pimonidazole assay and angiographic imaging. Dual FLIM/PLIM metabolic/oxygen imaging will be valuable in preclinical investigations into the effects of hypoxia on metabolic aspects of tumor progression and treatment response

Nonlinear Elasticity Assessment with Optical Coherence Elastography for High-Selectivity Differentiation of Breast Cancer Tissues

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

Diagnostic Accuracy of Cross-Polarization OCT and OCT-Elastography for Differentiation of Breast Cancer Subtypes: Comparative Study

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

Tomorrow's Europe : the views of those concerned

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