Intraoperative spectroscopic evaluation of sentinel lymph nodes in breast cancer surgery

Background: Sentinel lymph node (SLN) biopsy is a standard procedure for patients with breast cancer. Positive SLNs on histological examination can lead to a second surgery for axillary lymph node clearance (ALNC). Here we report a non-destructive technique based on autofluorescence (AF) imaging and Raman spectroscopy for intra-operative assessment of SLNs excised in breast cancer surgery.Methods: A microscope integrating AF imaging and Raman spectroscopy modules was built to allow scanning of lymph node biopsy samples. AF imaging was utilised to determine optimal sampling locations for Raman spectroscopy measurements, such that scanning was completed within 20-30 minutes. After optimisation of the AF image analysis and training of classification models based on data from 85 samples, the AF-Raman technique was tested on an independent set of 81lymph nodes. Sensitivity and specificity were calculated using post-operative histology as a standard of reference.Results: The area under the receiver operating characteristic (ROC) curve for the AF-Raman analysis for bisected lymph nodes was 0.93. For a regime that maximised specificity (reduced risk of false positive detections), a 97% specificity and a 80% sensitivity was achieved. The main confounders for metastasis were areas rich in histiocytes, for which only few Raman spectra had been included in the training dataset.Conclusions: This preliminary study indicates that with further development and extension of the training dataset (inclusion of Raman spectra of histiocytes), the AF-Raman is a promising technique for intra-operative assessment of SLNs. Intra-operative detection of metastatic SLNs could greatly reduce additional surgery for axillary clearance

Combined total internal reflection AF spectral-imaging and Raman spectroscopy for fast assessment of surgical margins during breast cancer surgery

The standard treatment for breast cancer is surgical removal mainly through breast conserving surgey (BCS). We developed a new technique based on auto-fluorescence (AF) spectral imaging and Raman spectroscopy for fast intraoperative assessment of excision margins in BCS. A new wide-field AF imaging unit based on total internal reflection (TIR) was combined with a Raman spectroscopy microscope equipped with a 785 nm laser. The wavelength of the AF excitation was optimized to 365 nm in order to maximize the discrimination of adipose tissue. This approach allows for the non-adipose regions of tissue, which are at higher-risk of containing tumor, to be targeted more efficiently by the Raman spectroscopy measurements. The integrated TIR-AF-Raman was tested on small tissue samples as well as fresh wide local excisions, delivering the analysis of the entire cruciate surface of BCS specimens (5.1 × 7.6 cm 2) in less than 45 minutes and also providing information regarding the location of the tumour in the specimen. Full automation of the instrument and selection of a faster translation stage would allow for the measurement of BCS specimens within intraoperative time scale (20 minutes). This study demonstrates that the TIR-AF Raman microscope represents a feasible step towards the development of a technique for intraoperative assessment of large WLE within intraoperative timescales

Intraoperative spectroscopic evaluation of sentinel lymph nodes in breast cancer surgery

Background and objectives: Sentinel lymph node (SLN) biopsy is a standard procedure for patients with breast cancer and normal axilla on imaging. Positive SLNs on histological examination can lead to a subsequent surgery for axillary lymph node clearance (ALNC). Here we report a non-destructive technique based on autofluorescence (AF) imaging and Raman spectroscopy for intra-operative assessment of SLNs excised in breast cancer surgery.Methods: A microscope integrating AF imaging and Raman spectroscopy modules was built to allow scanning of lymph node biopsy samples. During AF-Raman measurements, AF imaging determined optimal sampling locations for Raman spectroscopy measurements. After optimisation of the AF image analysis and training of classification models based on data from 85 samples, the AF-Raman technique was tested on an independent set of 81 lymph nodes comprised of 58 fixed and 23 fresh specimens. The sensitivity and specificity of AF-Raman were calculated using post-operative histology as a standard of reference.Results: The independent set contained 66 negative lymph nodes and 15 positive lymph nodes according to the reference standard, collected from 78 patients recruited randomly. For this set of specimens, the area under the receiver operating characteristic (ROC) curve for the AF-Raman technique was 0.93 [0.83-0.98]. AF-Raman was then operated in a regime that maximised detection specificity, producing a 94% detection accuracy: 80% sensitivity and 97% specificity. The main confounders for SLN metastasis were areas rich in histiocytes clusters, for which only few Raman spectra had been included in the training dataset.Discussion: This preliminary study indicates that with further development and extension of the training dataset by inclusion of additional Raman spectra of histiocytes clusters and capsule, the AF-Raman may become a promising technique for intra-operative assessment of SLNs. Intra-operative detection of positive biopsies could avoid second surgery for axillary clearance

Real-time ex-vivo assessment of basal cell carcinoma surgical margins in Mohs surgery by autofluorescence-Raman spectroscopy: a pilot study

BackgroundAutofluorescence (AF)-Raman spectroscopy is a technology that can detect tumour tissue in surgically excised skin specimens. The technique does not require tissue fixation, staining, labelling or sectioning, and provides quantitative diagnosis maps within 30 minutes.ObjectivesTo explore the clinical application of AF-Raman microscopy to detect residual basal cell carcinoma (BCC) positive margins in ex-vivo skin specimens excised during real-time Mohs surgery. To investigate the ability to analyse skin specimens from different parts of the head-and-neck areas and detect nodular, infiltrative and superficial BCC.MethodsFifty Mohs tissue layers (50 patients) were investigated: 27 split samples (two halves) and 23 full-face samples. The AF-Raman results were compared to frozen section histology, carried out intra-operatively by the Mohs surgeon and post-operatively by dermato-pathologists. The latter was used as the standard of reference.ResultsThe AF-Raman analysis was completed within the target time of 30 minutes and was able to detect all sub-types of BCC. For the split specimens, the AF-Raman analysis covered 97% of the specimen surface area and detected 8 out of 9 BCC positive layers (similar to Mohs surgeons). For the full-face specimens, poor contact between tissue and cassette coverslip led to lower coverage of the specimen surface area (92%), decreasing the detection rate (4 out of 6 positives for BCC).ConclusionsThese preliminary results for the split specimens demonstrate the feasibility of AF-Raman microscopy for rapid assessment of Mohs layers for BCC presence. However, for full-face specimens, further work is required to improve the contact between the tissue and the coverslip to increase sensitivity

Diagnostic accuracy of autofluorescence-Raman microspectroscopy for surgical margin assessment during Mohs micrographic surgery of basal cell carcinoma

BackgroundAutofluorescence (AF)–Raman microspectroscopy is a technology that can detect residual basal cell carcinoma (BCC) on the resection margin of fresh, surgically excised tissue specimens. The technology does not require tissue fixation, staining, labelling or sectioning, and provides quantitative diagnosis maps of the surgical margins in 30 min.ObjectivesTo determine the accuracy of the AF–Raman instrument in detecting incomplete BCC excisions during Mohs micrographic surgery (MMS), using histology as the reference standard.MethodsSkin layers from 130 patients undergoing MMS at the Nottingham University Hospitals NHS Trust (September 2022–July 2023) were investigated with the AF–Raman instrument. The layers were measured when fresh, immediately after excision. The AF–Raman results and the intraoperative assessment by Mohs surgeons were compared with a postoperative consensus-derived reference produced by three dermatopathologists. The sensitivity, specificity, and positive and negative predictive values were calculated. The study was registered with ClinicalTrials.gov (NCT03482622).ResultsAF–Raman analysis was successfully completed for 125 of 130 layers and, on average, covered 91% of the specimen surface area, with the lowest surface area covered being 87% for the eyelid and the highest being 94% for forehead specimens. The AF–Raman instrument identified positive margins in 24 of 36 BCC-positive cases [67% sensitivity, 95% confidence interval (CI) 49–82] and negative margins in 65 of 89 BCC-negative cases (73% specificity, 95% CI 63–82). Only one of 12 false-negative cases was caused by misclassification by the AF–Raman algorithm. The other 11 false-negatives cases were a result of no valid Raman signal being recorded at the location of the residual BCC due to either occlusion by blood or poor contact between tissue and the cassette window. The intraoperative diagnosis by Mohs surgeons identified positive margins in 31 of 36 BCC-positive cases (86% sensitivity, 95% CI 70–95) and negative margins in 79 of 89 BCC-negative cases (89% specificity, 95% CI 81–95).ConclusionsThe AF–Raman instrument has the potential to provide intraoperative microscopic assessment of surgical margins in BCC surgery. Further improvements are required for tissue processing, to ensure complete coverage of the surgical specimens

Diagnostic accuracy of autofluorescence-Raman microspectroscopy for surgical margin assessment during Mohs micrographic surgery of basal cell carcinoma

Abstract: Background Autofluorescence (AF)-Raman microspectroscopy is a technology that can detect residual basal cell carcinoma (BCC) on the resection margin of fresh, surgically excised tissue specimens. The technology does not require tissue fixation, staining, labelling or sectioning, and provides quantitative diagnosis maps of the surgical margins in 30 min.Objectives To determine the accuracy of the AF-Raman instrument in detecting incomplete BCC excisions during Mohs micrographic surgery (MMS), using histology as the reference standard.Methods Skin layers from 130 patients undergoing MMS at the Nottingham University Hospitals NHS Trust (September 2022-July 2023) were investigated with the AF-Raman instrument. The layers were measured when fresh, immediately after excision. The AF-Raman results and the intraoperative assessment by Mohs surgeons were compared with a postoperative consensus-derived reference produced by three dermatopathologists. The sensitivity, specificity, and positive and negative predictive values were calculated. The study was registered with ClinicalTrials.gov (NCT03482622).Results AF-Raman analysis was successfully completed for 125 of 130 layers and, on average, covered 91% of the specimen surface area, with the lowest surface area covered being 87% for the eyelid and the highest being 94% for forehead specimens. The AF-Raman instrument identified positive margins in 24 of 36 BCC-positive cases [67% sensitivity, 95% confidence interval (CI) 49-82] and negative margins in 65 of 89 BCC-negative cases (73% specificity, 95% CI 63-82). Only one of 12 false-negative cases was caused by misclassification by the AF-Raman algorithm. The other 11 false-negatives cases were a result of no valid Raman signal being recorded at the location of the residual BCC due to either occlusion by blood or poor contact between tissue and the cassette window. The intraoperative diagnosis by Mohs surgeons identified positive margins in 31 of 36 BCC-positive cases (86% sensitivity, 95% CI 70-95) and negative margins in 79 of 89 BCC-negative cases (89% specificity, 95% CI 81-95).Conclusions The AF-Raman instrument has the potential to provide intraoperative microscopic assessment of surgical margins in BCC surgery. Further improvements are required for tissue processing, to ensure complete coverage of the surgical specimens. Autofluorescence (AF)-Raman microspectroscopy has been shown to identify residual basal cell carcinoma on both frozen and fresh skin specimens immediately after excision by Mohs micrographic surgery. This first diagnostic test of accuracy of AF-Raman microspectroscopy on full-face Mohs tissue layers from 130 patients shows that, with improvements in tissue processing, the AF-Raman instrument is a viable technique for intraoperative assessment of surgical margins