A phase I study of a PARP1-targeted topical fluorophore for the detection of oral cancer

Abstract

Background: Visual inspection and biopsy is the current standard of care for oral cancer diagnosis, but is subject to misinterpretation and consequently to misdiagnosis. Topically applied PARPi-FL is a molecularly specific, fluorescent contrast-based approach that may fulfill the unmet need for a simple, in vivo, non-invasive, cost-effective, point-of-care method for the early diagnosis of oral cancer. Here, we present results from a phase I safety and feasibility study on fluorescent, topically applied PARPi-FL. Twelve patients with a histologically proven oral squamous cell carcinoma (OSCC) gargled a PARPi-FL solution for 60 s (15 mL, 100 nM, 250 nM, 500 nM, or 1000 nM), followed by gargling a clearing solution for 60 s. Fluorescence measurements of the lesion and surrounding oral mucosa were taken before PARPi-FL application, after PARPi-FL application, and after clearing. Blood pressure, oxygen levels, clinical chemistry, and CBC were obtained before and after tracer administration.

Results: PARPi-FL was well-tolerated by all patients without any safety concerns. When analyzing the fluorescence signal, all malignant lesions showed a significant differential in contrast after administration of PARPi-FL, with the highest increase occurring at the highest dose level (1000 nM), where all patients had a tumor-to-margin fluorescence signal ratio of >3. A clearing step was essential to increase signal specificity, as it clears unbound PARPi-FL trapped in normal anatomical structures. PARPi-FL tumor cell specificity was confirmed by ex vivo tabletop confocal microscopy. We have demonstrated that the fluorescence signal arose from the nuclei of tumor cells, endorsing our macroscopic findings.

Conclusions: A PARPi-FL swish & spit solution is a rapid and non-invasive diagnostic tool that preferentially localizes fluorescent contrast to OSCC. This technique holds promise for the early detection of OSCC based on in vivo optical evaluation and targeted biopsy of suspicious lesions in the oral cavity.

Trial registration: Clinicaltrials.gov -NCT03085147, registered on March 21st, 2017.

Keywords: Fluorescence-guided-detection; Molecular imaging; Oral cancer; PARP1; PARPi-FL; Swish; Topical application.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest. C.B., S.K., S.P. and T.R. are shareholders of Summit Biomedical Imaging, LLC. S.K., S.P. and T.R. are co-inventors on PCT application WO2016164771. T.R. is co-inventor on PCT application WO2012074840. T.R. is a paid consultant for Theragnostics, Inc. All the other authors have no relevant conflict to declare. This arrangement has been reviewed and approved by Memorial Sloan Kettering Cancer Center in accordance with its conflict of interest policies.

© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.

Figures

Figure 1.

Design of phase I dose escalating study NCT03085147 and concept of PARPi-FL imaging for delineation of oral cancer. A, Patients (n = 12) with biopsy proven oral squamous cell carcinoma (OSCC) gargled a solution of PARPi-FL (step 1) at increasing concentrations (100 nM, 250 nM, 500 nM, and 1000 nM; each dose n = 3) for 1 min. Then, patients gargled a clearing solution for 1 min (step 2). Using a Quest Spectrum imaging device with an endoscopic camera and PARPi-FL optimized LED–filter system, the tumor area and surrounding margin of the patient were imaged before PARPi-FL administration, after PARPi-FL administration pre-wash and after PARPi-FL administration post-wash. B, The illustration describes the mechanism-of-action of PARPi-FL based oral cancer detection. After topical application of PARPi-FL in the oral cavity, the imaging agent binds to PARP1 in the nucleus of a cell. PARP1 is overexpressed in OSCC compared to normal mucosal tissue.

Figure 2.

Schematic overview and flow chart of the PARPi-FL clinical trial. Twelve Patients with biopsy proven OSCC were identified and consented to the Phase I dose escalation study.

Figure 3.

PARP1 expression in patients and PARPi-FL mediated contrast. A, Representative PARP1 immunohistochemistry images and H&E image from a biopsy of a patient (slide overview), showing an area of OSCC (orange circle). B, Quantification of PARP1 expression in immunohistochemistry samples (n = 8 patients) was performed and compared between tumor, epithelium, and deep margin. C, Phase I PARPi-FL imaging of the patient shown in panel A.

Figure 4.

Intra-patient analysis after PARPi-FL imaging. A, Tumor area of a patient was imaged using a Quest Spectrum imaging device with an endoscopic camera pre-PARPi-FL, after gargling 1000 nM PARPi-FL solution (PARPi-FL, pre wash), and after gargling 500 nM PARPi-FL solution and a clearing solution of 30% PEG300 in PBS (PARPi-FL, post wash). The 2-D fluorescence intensity histogram illustrates the signal enhancement in the tumor area. B, Tumor-to-margin ratios of fluorescence imaging for each patient with histologically-proven OSCC (n = 11) pre PARPi-FL, after PARPi-FL (pre-wash) and after PARPi-FL (post-wash). Statistical significance was determined using the Wilcoxon matched-pairs signed rank test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are presented as mean ± SEM. C, PARPi-FL post-wash image and quantification of patient 5. Histopathology found no OSCC. D, PARPi-FL imaging and quantification of the tumor-contralateral side of the tongue in patient 2. An intraoperative biopsy of the lesion was performed and confirmed a second primary 2 mm squamous cell carcinoma.

Figure 5.

Inter-patient analysis and subcellular specificity of PARPi-FL. A, Tumor-to-margin ratio of fluorescence imaging for dose groups (100 nM, 250 nM, 500 nM, and 1000 nM). Ratios were calculated from 5 ROIs per FOV and 3 FOVs of each patient. Displayed are individual data points, means and SEM. B, PARPi-FL macroscopic imaging of a patient sample from phase II (PARPi-FL post-wash, 1000 nM), which was selected for microscopic confirmation of PARPi-FL specificity. 1 = area of tumor where the biopsy was taken from, 2 = area where the biopsy of the free-of-disease margin was taken from. C, H&E and PARP1 IHC (overview and zoom-in) of the tumor and margin biopsies from patient in panel B, demonstrating higher PARP1 expression in the tumor area compared to normal adjacent mucosa. D, Microscopic analysis of the tumor and margin biopsies to evaluate the PARPi-FL accumulation following topical 1 min swish & spit application. Fresh tissues underwent nuclear counterstaining with Hoechst 33342 ex vivo prior to microscopy. Orange arrows point to the nuclei of tumor cells, red arrows point to nuclei of cells in a normal (benign) basal layer, and white arrows point to the interstitial layer (collagen autofluorescence). E, Quantification of PARPi-FL fluorescence signal inside the nuclei of cells. Cell nuclei were identified using Hoechst 33342.