Significance:Current standard practice for margin assessment in solid tumor resection often leads to suboptimal results due to the inability to assess margins completely in a time-efficient manner. On the other hand, for small skin cancers, peripheral and deep en face margin assessment (PDEMA) offers 100% assessment of margins while sparing the utmost amount of normal surrounding tissues. Nonetheless, PDEMA is limited in its use owing to its lengthy tissue processing and imaging time as well as its requirement for high-quality frozen sections and real-time histologic analysis. Aim:We aim to explore fluorescence molecular imaging (FMI) as a tool for resolving obstacles and integrating PDEMA into the surgeon-to-pathologist workflow for large solid tumors. Approach:A review of recent pre-clinical and clinical studies using FMI to assess surgical margins was conducted to highlight promising fluorescence imaging technologies utilized in the surgical suite and laboratory. Results:FMI techniques that provide macroscopic resolution are efficient in time and have a notable ability to identify true negative tissue yet have limited capability in identifying true positive tissues. Moreover, meso- and microscopic FMI methods require additional time to attain a higher resolution but deliver an enhanced sensitivity in detecting true positive tissues. In both cases, experts are still required to learn to interpret the FMI signals, which prohibits a seamless clinical integration. Conclusions:Our proposed margin assessment platform (MAP) incorporates both macroscopic and, meso- or microscopic imaging with post-processing and machine learning for interpretation, to enable the application of PDEMA into solid tumor surgery. MAP leverages the advantages of each technique and thoroughly tackles the limitations of time and expertise to optimize the efficiency and accuracy of margin assessment and ultimately improve clinical outcomes.

Background Although polioviruses (PVs) replicate in lymphoid tissue of both the pharynx and ileum, research on polio vaccine-induced mucosal immunity has predominantly focused on intestinal neutralizing and binding antibody levels measured in stool.Methods To investigate the extent to which routine immunization with intramuscularly injected inactivated polio vaccine (IPV) may induce nasal and pharyngeal mucosal immunity, we measured PV type-specific neutralization and immunoglobulin (Ig) G, IgA, and IgM levels in nasal secretions, adenoid cell supernatants, and sera collected from 12 children, aged 2-5 years, undergoing planned adenoidectomies. All participants were routinely immunized with IPV and had no known contact with live PVs.Results PV-specific mucosal neutralization was detected in nasal and adenoid samples, mostly from children who had previously received 4 IPV doses. Across the 3 PV serotypes, both nasal (Spearman rho >= 0.87, P <= .0003 for all) and adenoid (Spearman rho >= 0.57, P <= .05 for all) neutralization titers correlated with serum neutralization titers. In this small study sample, there was insufficient evidence to determine which Ig isotype(s) was correlated with neutralization.Conclusions Our findings provide policy-relevant evidence that routine immunization with IPV may induce nasal and pharyngeal mucosal immunity. The observed correlations of nasal and pharyngeal mucosal neutralization with serum neutralization contrast with previous observations of distinct intestinal and serum responses to PV vaccines. Further research is warranted to determine which antibody isotype(s) correlate with polio vaccine-induced nasal and pharyngeal mucosal neutralizing activity and to understand the differences from intestinal mucosal immunity. This study provides policy-relevant evidence that children routinely immunized with inactivated polio vaccine in the United States and who had no known contact with a live poliovirus had detectable nasal and pharyngeal mucosal immunity to poliovirus.

Paired-Agent Imaging (PAI) is a quantitative fluorescence imaging technique that estimates the drug target concentration. It involves the co-administration of a targeted and an untargeted imaging probe to correct for nonspecific uptake and to quantify the available receptor concentration, known as the binding potential. PAI has been demonstrated in a pre-clinical setting using a 1:1 molar concentration of the targeted, ABY-029, and untargeted, IRDYE 680LT, imaging agent. However, the effects of different molar concentrations of imaging agents on the binding potential have not been studies thus far. In this study, we examined this relationship in tissue-mimicking liquid phantoms with varying molar concentration ratios. The phantom fluorescence was measured using the Pearl Imaging system and then the binding potential was quantified using MATLAB. We determined that the binding potential remains stable across concentration but increases for dye ratios where the targeted dye is higher.

We report a first-in-humans clinical study of EPR oximetry using the OxyChip to establish its feasibility and utility for clinically useful tumor oxygen measurements in cancer patients. Repeated measurements from a cohort of 11 cancer patients in 33 sessions over a long period of time revealed variable levels of clinically significant hypoxia as well as variable responses to a hypoxia-mitigation intervention. Overall, in light of this variability, this study further underscores the need to provide individualized repeatable assessment of tumor oxygenation in the context of planned hyperoxygenation interventions to optimize clinical outcomes.

Supplementary Table 1 from Identification of Hypoxia-Regulated Proteins in Head and Neck Cancer by Proteomic and Tissue Array Profiling

Paired Agent Imaging (PAI) is a fluorescence imaging technique where a targeted probe is co-administered with an untargeted probe. PAI has been successfully demonstrated in a pre-clinical setting and its clinical translation is in progress. The tissue distribution and excretion of the two fluorescent dyes, ABY-029 and IRDY680LT, must display similar kinetics in order for the PAI model to hold. To study the excretion of the dyes, plasma studies need to be conducted to examine the presence of fluorescence in vivo over a select period of time. The current method of measuring plasma fluorescence involves centrifuging blood to isolate plasma and them measuring on a fluorometer which can be time consuming and inefficient. In this study, we examine multiple methods for visualizing and quantifying plasma fluorescence using blood and plasma phantoms at multiple concentrations. The phantom fluorescence was measured using the Pearl Imaging system and the Fluoromax-3. We have determined that imaging blood directly in a fluorescence imaging system provides the same information as plasma alone.

BackgroundMohs micrographic surgery is a procedure used for non-melanoma skin cancers that has 97-99% cure rates largely owing to 100% margin analysis enabled by en face sectioning with real-time, iterative histologic assessment. However, the technique is limited to small and aggressive tumors in high-risk areas because the histopathological preparation and assessment is very time intensive. To address this, paired-agent imaging (PAI) can be used to rapidly screen excised specimens and identify tumor positive margins for guided and more efficient microscopic evaluation.MethodsA mouse xenograft model of human squamous cell carcinoma (n = 8 mice, 13 tumors) underwent PAI. Targeted (ABY-029, anti-epidermal growth factor receptor (EGFR) affibody molecule) and untargeted (IRDye 680LT carboxylate) imaging agents were simultaneously injected 3-4 h prior to surgical tumor resection. Fluorescence imaging was performed on main, unprocessed excised specimens and en face margins (tissue sections tangential to the deep margin surface). Binding potential (BP) – a quantity proportional to receptor concentration – and targeted fluorescence signal were measured for each, and respective mean and maximum values were analyzed to compare diagnostic ability and contrast. The BP and targeted fluorescence of the main specimen and margin samples were also correlated with EGFR immunohistochemistry (IHC).ResultsPAI consistently outperformed targeted fluorescence alone in terms of diagnostic ability and contrast-to-variance ratio (CVR). Mean and maximum measures of BP resulted in 100% accuracy, while mean and maximum targeted fluorescence signal offered 97% and 98% accuracy, respectively. Moreover, maximum BP had the greatest average CVR for both main specimen and margin samples (average 1.7 ± 0.4 times improvement over other measures). Fresh tissue margin imaging improved similarity with EGFR IHC volume estimates compared to main specimen imaging in line profile analysis; and margin BP specifically had the strongest concordance (average 3.6 ± 2.2 times improvement over other measures).ConclusionsPAI was able to reliably distinguish tumor from normal tissue in fresh en face margin samples using the single metric of maximum BP. This demonstrated the potential for PAI to act as a highly sensitive screening tool to eliminate the extra time wasted on real-time pathological assessment of low-risk margins.

Intraoperative margin analysis is crucial for the successful removal of cutaneous squamous cell carcinomas (cSCC). Artificial intelligence technologies (AI) have previously demonstrated potential for facilitating rapid and complete tumour removal using intraoperative margin assessment for basal cell carcinoma. However, the varied morphologies of cSCC present challenges for AI margin assessment. The aim of this study was to develop and evaluate the accuracy of an AI algorithm for real-time histologic margin analysis of cSCC. To do this, a retrospective cohort study was conducted using frozen cSCC section slides. These slides were scanned and annotated, delineating benign tissue structures, inflammation and tumour to develop an AI algorithm for real-time margin analysis. A convolutional neural network workflow was used to extract histomorphological features predictive of cSCC. This algorithm demonstrated proof of concept for identifying cSCC with high accuracy, highlighting the potential for integration of AI into the surgical workflow. Incorporation of AI algorithms may improve efficiency and completeness of real-time margin assessment for cSCC removal, particularly in cases of moderately and poorly differentiated tumours/neoplasms. Further algorithmic improvement incorporating surrounding tissue context is necessary to remain sensitive to the unique epidermal landscape of well-differentiated tumours, and to map tumours to their original anatomical position/orientation.

Fluorescence paired-agent imaging combined with en face margin analysis can detect positive margins in low tumor-bearing (<1% tumor volume) whole tissue samples, as demonstrated in xenograft murine models.

In nonmetastatic head and neck cancer treatment, surgical margin status is the most important prognosticator of recurrence and patient survival. Fresh frozen sectioning (FFS) of tissue margins is the standard of care for intraoperative margin assessment. However, FFS is time intensive, and its accuracy is not consistent among institutes. Mapping the epidermal growth factor receptor (EGFR) using paired-agent imaging (PAI) has the potential to provide more consistent intraoperative margin assessment in a fraction of the time as FFS. PAI was carried out through IV injection of an anti-epidermal growth factor receptor (EGFR) affibody molecule (ABY-029, eIND 122,681) and an untargeted IRDye680LT carboxylate. Imaging was performed on 4 µm frozen sections from three oral squamous cell carcinoma xenograft mouse models (n = 24, 8 samples per cell line). The diagnostic ability and tumor contrast were compared between binding potential, targeted, and untargeted images. Confidence maps were constructed based on group histogram-derived tumor probability curves. Tumor differentiability and contrast by confidence maps were evaluated. PAI outperformed ABY-029 and IRDye 680LT alone, demonstrating the highest individual receiver operating characteristic (ROC) curve area under the curve (PAI AUC: 0.91, 0.90, and 0.79) and contrast-to-noise ratio (PAI CNR: 1, 1.1, and 0.6) for FaDu, Det 562, and A253. PAI confidence maps (PAI CM) maintain high tumor diagnostic ability (PAI CMAUC: 0.91, 0.90, and 0.79) while significantly enhancing tumor contrast (PAI CMCNR: 1.5, 1.3, and 0.8) in FaDu, Det 562, and A253. Additionally, the PAI confidence map allows avascular A253 to be differentiated from a healthy tissue with significantly higher contrast than PAI. Notably, PAI does not require additional staining and therefore significantly reduces the tumor delineation time in a 5 × 5 mm slice from 35 min to under a minute. This study demonstrated that PAI improved tumor detection in frozen sections with high diagnostic accuracy and rapid analysis times. The novel PAI confidence map improved the contrast in vascular tumors and differentiability in avascular tumors. With a larger database, the PAI confidence map promises to standardize fluorescence imaging in intraoperative pathology-assisted surgery (IPAS).

Thorough tumor resection is crucial for successful treatment of squamous cell carcinomas (SCCs) because positive surgical margins are associated with poor patient prognosis. Current methods of margin analysis, however, are limited by inefficient pathological read-times that increase exponentially with tissue size. Here, a fluorescence paired-agent imaging (PAI) approach is presented to identify regions of tumor burden in whole, thick tissue margins to act as a rapid screening tool and help focus pathological evaluation. The approach was applied to mouse models of head and neck SCC, and positive tumor burden was detected and localized in deep tissue margins up to 1.3 mm thick. Serial sections with hematoxylin and eosin and EGFR-immunostaining demonstrated good correlation with binding potential (BP: proportional to targeted biomolecule concentration) maps generated from PAI fluorescence slices and confirmed the presence of positive margins suggested by high intensity regions in the whole tissue BP maps. Findings support the use of PAI as a rapid screening method for detecting regions of tumor burden in large, en face tumor margin sections.

Lipoblastoma/lipoblastomatosis presents some unique diagnostic and therapeutic challenges when encountered in the oral cavity. In these rare cases, diagnostic confirmation with molecular testing and a conservative surgical resection can contribute to successful management.

The surgeon's role in the management of lymphoma is typically limited to performing biopsies for diagnosis. Most patients with lymphoma are treated with chemotherapy and/or radiation, but in rare cases, lymphoma can be primarily treated with surgery. We present a case of nodular lymphocyte-predominant Hodgkin lymphoma in a 4-year-old child with cervical adenopathy and discuss initial treatment with surgery alone. Surgery as primary treatment avoids the serious long-term sequelae of chemotherapy and radiation, and reserves those options for possible future recurrences; however, this approach should be reserved for patients with limited and low-risk disease. This case report reviews the pros and cons of treating early-stage nodular lymphocyte-predominant Hodgkin lymphoma in a pediatric patient with surgery alone.

Objective The overall objective of this clinical study was to validate an implantable oxygen sensor, called the ‘OxyChip’, as a clinically feasible technology that would allow individualized tumor-oxygen assessments in cancer patients prior to and during hypoxia-modification interventions such as hyperoxygen breathing. Methods Patients with any solid tumor at ≤3-cm depth from the skin-surface scheduled to undergo surgical resection (with or without neoadjuvant therapy) were considered eligible for the study. The OxyChip was implanted in the tumor and subsequently removed during standard-of-care surgery. Partial pressure of oxygen (pO 2 ) at the implant location was assessed using electron paramagnetic resonance (EPR) oximetry. Results Twenty-three cancer patients underwent OxyChip implantation in their tumors. Six patients received neoadjuvant therapy while the OxyChip was implanted. Median implant duration was 30 days (range 4–128 days). Forty-five successful oxygen measurements were made in 15 patients. Baseline pO 2 values were variable with overall median 15.7 mmHg (range 0.6–73.1 mmHg); 33% of the values were below 10 mmHg. After hyperoxygenation, the overall median pO 2 was 31.8 mmHg (range 1.5–144.6 mmHg). In 83% of the measurements, there was a statistically significant (p ≤ 0.05) response to hyperoxygenation. Conclusions Measurement of baseline pO 2 and response to hyperoxygenation using EPR oximetry with the OxyChip is clinically feasible in a variety of tumor types. Tumor oxygen at baseline differed significantly among patients. Although most tumors responded to a hyperoxygenation intervention, some were non-responders. These data demonstrated the need for individualized assessment of tumor oxygenation in the context of planned hyperoxygenation interventions to optimize clinical outcomes.

The goal of fluorescence-guided surgery (FGS) in oncology is to improve the surgical therapeutic index by enhancing contrast between cancerous and healthy tissues. However, optimal discrimination between these tissues is complicated by the nonspecific uptake and retention of molecular targeted agents and the variance of fluorescence signal. Paired-agent imaging (PAI) employs co-administration of an untargeted imaging agent with a molecular targeted agent, providing a normalization factor to minimize nonspecific and varied signals. The resulting measured binding potential is quantitative and equivalent to in vivo immunohistochemistry of the target protein. This study demonstrates that PAI improves the accuracy of tumor-to-healthy tissue discrimination compared to single-agent imaging for in vivo FGS. PAI using a fluorescent anti-epidermal growth factor receptor (EGFR) affibody molecule (ABY-029, eIND 122,681) with untargeted IRDye 700DX carboxylate was compared to ABY-029 alone in an oral squamous cell carcinoma xenograft mouse model at 3 h after dye administration (n = 30). PAI significantly enhanced tumor discrimination, as compared to ABY-029 alone in low EGFR-expressing tumors and highly heterogeneous populations including multiple cell lines with varying expression (diagnostic accuracy: 0.908 vs. 0.854 and 0.908 vs. 0.822; and ROC curve AUC: 0.963 vs. 0.909 and 0.957 vs. 0.909, respectively) indicating a potential for universal FGS image thresholds to determine surgical margins. In addition, PAI achieved significantly higher diagnostic ability than ABY-029 alone 0.25–5-h post injection and exhibited a stronger correlation to EGFR expression heterogeneity. The quantitative receptor delineation of PAI promises to improve the surgical therapeutic index of cancer resection in a clinically relevant timeline.

Non-specific uptake and retention of molecular targeted agents and heterogeneous tissue optical properties diminish the ability to differentiate between tumor and normal tissues using molecular targeted fluorescent agents. Paired-agent imaging (PAI) can increase the diagnostic ability to detect tumor tissue by mitigating these non-specific effects and providing true molecular contrast by co-administration of an untargeted control imaging agent with a targeted agent. This study evaluates the suitability of available clinically translatable untargeted agents for the translation of PAI in fluorescence-guided surgery using an affibody-based targeted imaging agent (ABY-029). Three untargeted agents that fluoresce near 700 nm and exhibit good clinical safety profiles (methylene blue, IRDye 700DX, and IRDye 680LT) were tested in combination with the clinically tested IRDye 800CW–labeled anti-epidermal growth factor receptor (EGFR) affibody molecule, ABY-029 (eIND 122,681). Properties of the untargeted agent important for human use and integrity of PAI were tested: (1) plasma protein binding; (2) fluorescence signal linearity in in vitro whole blood dilution; (3) in vivo pharmacokinetic matching to targeted agent in negative control tissue; and (4) in vivo diagnostic accuracy of PAI vs single agent imaging (SAI) of ABY-029 alone in orthotopic oral head and neck squamous cell carcinomas. IRDye 680LT outperformed IRDye 700DX and methylene blue with the highest signal linearity (R2 = 0.9998 ± 0.0002, 0.9995 ± 0.0004, 0.91 ± 0.02, respectively), the highest fluorescence yield in whole blood at 1 μM (104.42 ± 0.05, 103.68 ± 0.09, 101.9 ± 0.2, respectively), and the most closely matched ABY-029 pharmacokinetics in EGFR-negative tissues (binding potential error percentage = 0.31

During resection of head and neck cancers, surgeons collect multiple small tissue samples from the excision wound bed for “frozen section” analysis by a clinical pathologist to determine clear margins. However, this process is disruptive to work flow, time-consuming, and expensive. We have validated Paired-Agent Imaging (PAI) in in vivo xenograft models using epidermal growth factor receptor and demonstrated that PAI exhibits higher diagnostic accuracy targeted agent alone – tested in both fresh tissue (~3-mm) and frozen sections (10-m). Here PAI will be extended to screening of tissue biopsies to quantify tumor burden in comparison to standard frozen section pathology.

Paired agent imaging demonstrates comparable accuracy with immunohistochemistry, but more time-efficient, and labor-saving. It may open up a new chapter for biopsy screening and intra-operative margin detection.