Evaluation of Pafolacianine (Cytalux®) for Fluorescence-Guided Surgery in Head and Neck Squamous Cell Carcinoma: A Negative Study with Important Clinical Implications

TL;DR

A study found that pafolacianine, a fluorescence imaging agent, is not effective for guiding head and neck cancer surgery due to poor tumor targeting.

Contribution

This is the first clinical evaluation of pafolacianine in head and neck squamous cell carcinoma, revealing its limited utility due to insufficient receptor expression.

Findings

Pafolacianine showed minimal binding to HNSCC cell lines and poor tumor localization in mice.

FR-α expression was not significantly higher in tumor tissue compared to normal tissue.

Panitumumab-IRDye800CW demonstrated much stronger tumor targeting than pafolacianine.

Abstract

Pafolacianine (Cytalux®) represents the first FDA-approved tumor-specific fluorescence imaging agent, demonstrating efficacy in ovarian cancer through folate receptor-α (FR-α) targeting. Given the need for improved intraoperative margin assessment in head and neck squamous cell carcinoma (HNSCC), where positive surgical margins occur in 10–30% of cases, we investigated the potential utility of pafolacianine for fluorescence-guided surgery in HNSCC models.

To evaluate the feasibility of visualizing HNSCC using pafolacianine in vitro, in vivo, and clinical tissue analysis, with comparison to fluorescence-guided surgery agents that have been successful in patients.

HNSCC cell lines (FaDu, UMSCC47) were treated with escalating concentrations of pafolacianine (0–500 nM) and assessed for binding at 1 and 24 h. Nude mice bearing HNSCC xenografts (FaDu, UMSCC47) received intraperitoneal injection of pafolacianine (10 nmol) with fluorescence imaging at multiple timepoints. Immunohistochemistry analysis of patient samples (n = 8 tumor, n = 8 normal) evaluated FR-α and FR-β expression. Panitumumab-IRDye800CW served as a positive control for comparison.

In vitro analysis demonstrated minimal pafolacianine binding across all HNSCC cell lines, with fluorescence intensities similar to or lower than the FR-α-negative A549 control cell line. In vivo imaging revealed poor tumor localization with mean fluorescence intensity (MFI) of 7.39 (FaDu) and 6.97 (UMSCC47), substantially lower than non-target tissues including skin. Immunohistochemistry analysis showed no statistically significant difference in FR-α expression between tumor and normal tissue (p > 0.05). For comparison, panitumumab-IRDye800CW demonstrated robust tumor targeting with MFI of 32.14 (FaDu) and 14.98 (UMSCC47).

This study demonstrates that pafolacianine exhibits limited utility for fluorescence-guided surgery in HNSCC due to insufficient FR-α expression and poor tumor-to-background contrast. These negative findings provide crucial evidence against the clinical translation of pafolacianine for HNSCC applications and highlight the importance of target expression validation in precision medicine approaches.

Negative studies such as this are essential for evidence-based clinical decision-making, preventing unnecessary resource allocation and potential patient exposure to ineffective interventions. These findings inform the broader fluorescence-guided surgery field and support continued investigation of alternative targeting strategies for HNSCC.

The online version contains supplementary material available at 10.1007/s11307-025-02068-3.

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