A New Look into Cancer—A Review on the Contribution of Vibrational Spectroscopy on Early Diagnosis and Surgery Guidance

In 2020, approximately 10 million people died of cancer, rendering this disease the second leading cause of death worldwide. Detecting cancer in its early stages is paramount for patients’ prognosis and survival. Hence, the scientific and medical communities are engaged in improving both therapeutic strategies and diagnostic methodologies, beyond prevention. Optical vibrational spectroscopy has been shown to be an ideal diagnostic method for early cancer diagnosis and surgical margins assessment, as a complement to histopathological analysis. Being highly sensitive, non-invasive and capable of real-time molecular imaging, Raman and Fourier transform infrared (FTIR) spectroscopies give information on the biochemical profile of the tissue under analysis, detecting the metabolic differences between healthy and cancerous portions of the same sample. This constitutes tremendous progress in the field, since the cancer-prompted morphological alterations often occur after the biochemical imbalances in the oncogenic process. Therefore, the early cancer-associated metabolic changes are unnoticed by the histopathologist. Additionally, Raman and FTIR spectroscopies significantly reduce the subjectivity linked to cancer diagnosis. This review focuses on breast and head and neck cancers, their clinical needs and the progress made to date using vibrational spectroscopy as a diagnostic technique prior to surgical intervention and intraoperative margin assessment.

Concept for using magnetic particle imaging for intraoperative margin analysis in breast-conserving surgery

Breast-conserving surgery (BCS) is a commonly utilized treatment for early stage breast cancers but has relatively high reexcision rates due to post-surgical identification of positive margins. A fast, specific, sensitive, easy-to-use tool for assessing margins intraoperatively could reduce the need for additional surgeries, and while many techniques have been explored, the clinical need is still unmet. We assess the potential of Magnetic Particle Imaging (MPI) for intraoperative margin assessment in BCS, using a passively or actively tumor-targeted iron oxide agent and two hardware devices: a hand-held Magnetic Particle detector for identifying residual tumor in the breast, and a small-bore MPI scanner for quickly imaging the tumor distribution in the excised specimen. Here, we present both hardware systems and demonstrate proof-of-concept detection and imaging of clinically relevant phantoms.

Specimen oriented intraoperative margin assessment in oral cavity and oropharyngeal squamous cell carcinoma

Objective Evaluate the oncologic outcomes and cost analysis of transitioning to a specimen oriented intraoperative margin assessment protocol from a tumour bed sampling protocol in oral cavity (OCSCC) and oropharyngeal squamous cell carcinoma (OPSCC). Study design Retrospective case series and subsequent prospective cohort study Setting Tertiary care academic teaching hospital Subjects and methods Retrospective case series of all institutional T1-T2 OCSCC or OPSCC treated with primary surgery between January 1st 2009 – December 31st 2014. Kaplan-Meier survival estimates with log rank tests were used to compare patients based on final margin status. Cost analysis was performed for escalation of therapy due to positive final margins. Following introduction of a specimen derived margin protocol, successive prospective cohort study of T1-T4 OCSCC or OPSCC treated with primary surgery from January 1st 2017 – December 31st 2018. Analysis and comparison of both protocols included review of intraoperative margins, final pathology and treatment cost. Results Analysis of our intra-operative tumour bed frozen section protocol revealed 15 of 116 (12.9%) patients had positive final pathology margins, resulting in post-operative escalation of therapy for 14/15 patients in the form of re-resection (7/14), radiation therapy (6/14) and chemoradiotherapy (1/14). One other patient with positive final margins received escalated therapy for additional negative prognostic factors. Recurrence free survival at 3 years was 88.4 and 50.7% for negative and positive final margins respectively (p = 0.048). Implementation of a specimen oriented frozen section protocol resulted in 1 of 111 patients (0.9%) having positive final pathology margins, a statistically significant decrease (p < 0.001). Utilizing our specimen oriented protocol, there was an absolute risk reduction for having a final positive margin of 12.0% and relative risk reduction of 93.0%. Estimated cost avoidance applying the specimen oriented protocol to our previous cohort was $412,052.812017 CAD. Conclusion Implementation of a specimen oriented intraoperative margin protocol provides a statistically significant decrease in final positive margins. This change in protocol leads to decreased patient morbidity by avoiding therapy escalation attributable only to positive margins, and avoids the economic costs of these treatments. Graphical abstract

H&E-like histology of unstained fresh and formalin fixed breast tissue with Photo Acoustic Remote Sensing (PARS) microscopy.

e12590 Background: Approximately 30% of breast cancer patients undergoing breast-conserving surgery (BCS) require re-excision(s) to obtain clear margins, causing delays in adjuvant treatment, poor aesthetic results, and increased infection rates, costs, and patient anxiety. Frozen section pathology assessment (FSA) reduces re-excision rates from 27% to 6%, but FSA extends operative times and has a false negative rate of 17%. Photo-Acoustic Remote Sensing (PARS) is a new laser-based light microscope that permits non-contact cellular resolution imaging of unstained tissues. Methods: We used an ultraviolet reflection-mode PARS microscope to study i) formalin-fixed, paraffin embedded (FFPE) breast tissue blocks, ii) unstained thin sections of FFPE invasive ductal carcinoma and ductal carcinoma in situ, iii) formalin fixed bulk surgical specimens of benign and malignant human breast tissue, and iv) benign and malignant fresh rodent mammary tissue without fixation, processing, or staining. Fresh tissues were imaged after a range of warm ischemic times and across a range of cold ischemic times in neutral phosphate buffered saline at 4°C. For each condition, an ultraviolet laser was used to excite DNA and other cellular components in these samples, and signals were captured using a continuous-wave detection laser. The PARS signal was false-colored to closely replicate traditional hematoxylin and eosin staining. Results: In all fixed tissues, PARS virtual histology images were of diagnostic quality, permitted margin assessment, and analogous to transmission light microscopy of standard hematoxylin and eosin-stained FFPE slides, achieved without tissue sectioning or tissue staining. Furthermore, PARS microscopy also provided cellular level virtual histology images in fresh breast tissue with warm and cold ischemic times ranging from twenty minutes to two hours; these images have no available clinical comparators as non-contact cellular level imaging of unprocessed fresh tissue has not previously been reported. In aggregate, we demonstrate the feasibility of PARS to provide diagnostic and margin assessment images across a range of tissues including formalin fixed and freshly resected, unstained, unprocessed breast tissue. Conclusions: PARS is a new microscope technology addressing the practical needs of intraoperative margin assessment during BCS: i) no requirement for tissue staining, ii) rapid acquisition of hematoxylin and eosin-like images without the requirement for tissue freezing, embedding, or sectioning, iii) diagnostic quality cellular resolution, and iv) assessment of resected bulk tissue margins. In principle, this technology may also permit label-free non-contact intraoperative margin assessment of the surgical cavity. These data support the clinical development and evaluation of PARS microscopic intraoperative assessment of BCS margins.

Accuracy of frozen section in intraoperative margin assessment for breast-conserving surgery: A systematic review and meta-analysis

Background and objectives It is well established that tumor-free margin is an important factor for reducing local recurrence and reoperation rates. This systematic review with meta-analysis of frozen section intraoperative margin assessment aims to evaluate the accuracy, and reoperation and survival rates, and to establish its importance in breast-conserving surgery. Methods A thorough review was conducted in all online publication-databases for the related literature up to March 2020. MeSH terms used: “Breast Cancer”, “Segmental Mastectomy” and “Frozen Section”. We included the studies that evaluated accuracy of frozen section, reoperation and survival rates. To ensure quality of the included articles, the QUADAS-2 tool (adapted) was employed. The assessment of publication bias by graphical and statistical methods was performed using the funnel plot and the Egger’s test. The review protocol was registered in PROSPERO (CRD42019125682). Results Nineteen studies were deemed suitable, with a total of 6,769 cases. The reoperation rate on average was 5.9%. Sensitivity was 0.81, with a Confidence Interval of 0.79–0.83, p = 0.0000, I2 = 95.1%, and specificity was 0.97, with a Confidence Interval of 0.97–0.98, p = 0.0000, I-2 = 90.8%, for 17 studies and 5,615 cases. Accuracy was 0.98. Twelve studies described local recurrence and the highest cumulative recurrence rate in 3 years was 7.5%. The quality of the included studies based on the QUADAS-2 tool showed a low risk of bias. There is no publication bias (p = 0.32) and the funnel plot showed symmetry. Conclusion Frozen section is a reliable procedure with high accuracy, sensitivity and specificity in intraoperative margin assessment of breast-conserving surgery. Therefore, this modality of margin assessment could be useful in reducing reoperation rates.

Intraoperative [18F]FDG flexible autoradiography for tumour margin assessment in breast-conserving surgery: a first-in-human multicentre feasibility study

Introduction In women undergoing breast-conserving surgery (BCS), 20–25% require a re-operation as a result of incomplete tumour resection. An intra-operative technique to assess tumour margins accurately would be a major advantage. A novel method for intraoperative margin assessment was developed by applying a thin flexible scintillating film to specimens—flexible autoradiography (FAR) imaging. A single-arm, multi-centre study was conducted to evaluate the feasibility of intraoperative [18F]FDG FAR for the assessment of tumour margins in BCS. Methods Eighty-eight patients with invasive breast cancer undergoing BCS received ≤ 300 MBq of [18F]FDG 60–180 min pre-operatively. Following surgical excision, intraoperative FAR imaging was performed using the LightPath® Imaging System. The first 16 patients were familiarisation patients; the remaining 72 patients were entered into the main study. FAR images were analysed post-operatively by three independent readers. Areas of increased signal intensity were marked, mean normalised radiances and tumour-to-tissue background (TBR) determined, agreement between histopathological margin status and FAR assessed and radiation dose to operating theatre staff measured. Subgroup analyses were performed for various covariates, with thresholds set based on ROC curves. Results Data analysis was performed on 66 patients. Intraoperative margin assessment using FAR was completed on 385 margins with 46.2% sensitivity, 81.7% specificity, 8.1% PPV, 97.7% NPV and an overall accuracy of 80.5%, detecting both invasive carcinoma and DCIS. A subgroup analysis based on [18F]FDG activity present at time of imaging revealed an increased sensitivity (71.4%), PPV (9.3%) and NPV (98.4%) in the high-activity cohort with mean tumour radiance and TBR of 126.7 ± 45.7 photons/s/cm2/sr/MBq and 2.1 ± 0.5, respectively. Staff radiation exposure was low (38.2 ± 38.1 µSv). Conclusion [18F]FDG FAR is a feasible and safe technique for intraoperative tumour margin assessment. Further improvements in diagnostic performance require optimising the method for scintillator positioning and/or the use of targeted radiopharmaceuticals. Trial registration: Identifier: NCT02666079. Date of registration: 28 January 2016. URL: https://clinicaltrials.gov/ct2/show/NCT02666079. ISRCTN registry: Reference: ISRCTN17778965. Date of registration: 11 February 2016. URL: http://www.isrctn.com/ISRCTN17778965.