Cancer cell metabolism leads to a uniquely acidic microenvironment in solid tumors, but exploiting the labile extracellular pH differences between cancer and normal tissues for clinical use has been challenging. Here we describe the clinical translation of ONM-100, a nanoparticle-based fluorescent imaging agent. This is comprised of an ultra-pH sensitive amphiphilic polymer, conjugated with indocyanine green, which rapidly and irreversibly dissociates to fluoresce in the acidic extracellular tumor microenvironment due to the mechanism of nanoscale macromolecular cooperativity. Primary outcomes were safety, pharmacokinetics and imaging feasilibity of ONM-100. Secondary outcomes were to determine a range of safe doses of ONM-100 for intra-operative imaging using commonly used fluorescence camera systems. In this study (Netherlands National Trial Register #7085), we report that ONM-100 was well tolerated, and four solid tumor types could be visualized both in- and ex vivo in thirty subjects. ONM-100 enables detection of tumor-positive resection margins in 9/9 subjects and four additional otherwise missed occult lesions. Consequently, this pH-activatable optical imaging agent may be clinically beneficial in differentiating previously unexploitable narrow physiologic differences.

Inadequate surgical margins occur frequently in oral squamous cell carcinoma surgery. Fluorescence molecular imaging (FMI) has been explored for intraoperative margin assessment, but data are limited to phase-I studies. In this single-arm phase-II study (NCT03134846), our primary endpoints were to determine the sensitivity, specificity and positive predictive value of cetuximab-800CW for tumor-positive margins detection. Secondary endpoints were safety, close margin detection rate and intrinsic cetuximab-800CW fluorescence. In 65 patients with 66 tumors, cetuximab-800CW was well-tolerated. Fluorescent spots identified in the surgical margin with signal-to-background ratios (SBR) of ≥2 identify tumor-positive margins with 100% sensitivity, 85.9% specificity, 58.3% positive predictive value, and 100% negative predictive value. An SBR of ≥1.5 identifies close margins with 70.3% sensitivity, 76.1% specificity, 60.5% positive predictive value, and 83.1% negative predictive value. Performing frozen section analysis aimed at the fluorescent spots with an SBR of ≥1.5 enables safe, intraoperative adjustment of surgical margins.

Fluorescence molecular imaging (FMI) is becoming a powerful tool to improve surgical precision and diagnostics. Although significant advances have been made in imaging technology and contrast agents, debate continues regarding suitable standards for FMI. Standardizing all FMI aspects is crucial for adoption in clinical procedures and routine practice. Currently, the lack of clear-cut guidelines on FMI study reporting complicates comparison between studies and impedes standardization. This work presents community-driven REFLECT guidelines for FMI study reporting for fluorescence-guided surgery and interventions, covering preclinical, translational, and clinical studies. It aims to improve quality of reporting, enable comparison between studies, and enhance interpretation of results. The resulting structured checklist encompasses all essential details for reporting on the contrast agent, imaging device, imaging protocol, and image processing and analysis methods. Adoption of this framework is encouraged to enhance reproducibility and establish alignment with standardization efforts in the field - thus fostering advancements in FMI.

SignificanceShortwave-infrared (SWIR) imaging is reported to yield better contrast in fluorescence-guided surgery than near-infrared (NIR) imaging, due to a reduction in scattering. This benefit of SWIR was shown in animal studies, however not yet in clinical studies with patient samples.AimWe investigate the potential benefit of SWIR to NIR imaging in clinical samples containing cetuximab-IRDye800CW in fluorescence-guided surgery.ApproachThe potential of the epidermal growth factor-targeted NIR dye cetuximab-IRDye800CW in the shortwave range was examined by recording the absorption and emission spectrum. An ex vivo comparison of NIR and SWIR images using clinical tumor samples of patients with penile squamous cell carcinoma (PSCC) and head and neck squamous cell carcinoma (HNSCC) containing cetuximab-IRDye800CW was performed. The comparison was based on the tumor-to-background ratio and an adapted contrast-to-noise ratio (aCNR) using the standard of care pathology tissue assessment as the golden standard.ResultsBased on the emission spectrum, cetuximab-IRDye800CW can be detected in the SWIR range. In clinical PSCC samples, overall SWIR imaging was found to perform similarly to NIR imaging (NIR imaging is better than SWIR in the 2/7 criteria examined, and SWIR is better than NIR in the 3/7 criteria). However, when inspecting HNSCC data, NIR is better than SWIR in nearly all (5/7) examined criteria. This difference seems to originate from background autofluorescence overwhelming the off-peak SWIR fluorescence signal in HNSCC tissue.ConclusionSWIR imaging using the targeted tracer cetuximab-IRDye800CW currently does not provide additional benefit over NIR imaging in ex vivo clinical samples. Background fluorescence in the SWIR region, resulting in a higher background signal, limits SWIR imaging in HNSCC samples. However, SWIR shows potential in increasing the contrast of tumor borders in PSCC samples, as shown by a higher aCNR over a line.

Over 370,000 new cases of oral squamous cell carcinomas (OSCC) were reported in 2020.¹ The primary treatment for these tumors is surgical resection. ² Yet, owing to the complex anatomy of the head and neck, complete resection of primary tumors is challenging and inadequate margins are observed in up to 40% of cases.^{3 – 5} These include both positive margins (tumor within 1 mm of the edge of the resected specimen) and close margins (tumor between 1 and 5 mm of the edge of the resected specimen).^6,7 In cases where inadequate margins are found (any $<5\mathrm{mm}$ thick), patients often require intensive adjuvant therapy⁸ because a secondary surgery is typically not possible due to altered anatomy or the proximity of vital structures. In either scenario, patient outcomes are generally worse than if clear margins were obtained on the initial surgery.⁹