SPRAYDYE: a phase I/II randomised pilot study to assess the safety and feasibility of topically applied AKRO-6qcICG, a cathepsin-activatable fluorescent imaging agent, for real-time intraoperative resection margin assessment in breast-conserving surgery.

INTRODUCTION: The incidence of tumour-positive surgical resection margins (TPRMs) after breast-conserving surgery (BCS) remains high, ranging from 10 to 40%. A TPRM, defined as breast cancer cells at the edge of the resected specimen at pathological evaluation, implies residual tumour and necessitates re-resection or boost radiation. To prevent these additional treatments, intraoperative near-infrared (NIR) fluorescence imaging with the topically applied, fluorescently quenched, cathepsin-activatable imaging agent AKRO-6qcICG might be used to detect residual cancer in the surgical cavity and guide additional resection during BCS. Cathepsins are proteolytic enzymes that are upregulated by breast cancer (associated) cells and therefore are suitable targets for tumour imaging. Ex vivo validation studies have shown that topically applied AKRO-6qcICG allows for clear breast cancer visualization and the detection of TPRMs. The proposed phase I/II study in healthy volunteers and breast cancer patients will assess the local and systemic safety of a single, topical dose of AKRO-6qcICG and its feasibility for intraoperative margin assessment during BCS. METHODS AND ANALYSIS: A total of six healthy volunteers (Part A) and 16 breast cancer patients (Part B) will be enrolled. In Part A, AKRO-6qcICG will be topically applied randomly on drawn blisters in two doses as will the vehicle compound, and one blister will be untreated functioning as a negative control. Physician and subject will remain blinded. In Part B, a single dose of AKRO-6qcICG will be topically applied in the surgical cavity. The primary objective is, with the occurrence of treatment-emergent (serious) adverse events as primary outcome measure. Secondary outcome measures include local and systemic tolerability parameters such as wound healing, numeric rating scales of pain and pruritus, vital signs, electrocardiogram parameters, clinical laboratory tests and pharmacokinetic parameters. Among the exploratory outcome measures are the diagnostic accuracy of the imaging agent to detect residual tumour in the surgical cavity and the tumour-to-background ratio of the fluorescent signal. ETHICS AND DISSEMINATION: This protocol has been approved by the Medical Ethical Committee Leiden-Den Haag- Delft (METC- LDD). The protocol is registered at EU Clinical Trials Register number 2025-523166-24-00. The results of this study will be reported through peer- reviewed publications and conference presentations. CLINICAL TRIAL REGISTRATION: https://ctis.eu/trial/2025-523166-24-00?from=search.