Patient distribution
A total of 13 patients were included in this analysis. In the phase I part of the study, 12 patients were enrolled over a 29 months period (from November of 2017 to March of 2020). All patient data are summarized in Table 1. The median age was 65 years old (range: 48 - 80 years old) and 58% were male (n = 7). Seven patients (58%) were never smokers, whereas the rest had discontinued smoking. Seven patients were currently consuming alcohol (one of them only sporadically and the other 6 daily). Three patients had discontinued the use of alcohol and two reported that they never consumed it. All patients had a primary oral tongue tumor as the oral cavity subsite (n = 11 on the lateral tongue and n = 1 on the midline of the dorsal tongue) with a median size of 2.1 cm (range: 0.5 – 4 cm). Fifty percent (n = 6) of the lesions were exophytic, 25% (n = 3) had a flat morphology, 17% (n = 2) were ulcerative and one patient (8%) presented with a submucosal tumor. Patient 5 had an excisional biopsy of a squamous cell carcinoma of the lateral tongue at an outside hospital prior to referral to MSK. This patient was imaged with PARPi-FL and had a partial glossectomy performed 4 days later. The final pathological report of this patient revealed that there was no residual tumor in the glossectomy specimen. The one patient included in this report from the phase II study was enrolled in October of 2020.
Toxicity assessment
No clinically significant alterations in vital signs were observed in patients after PARPi-FL administration (see Supplementary Table 1). Temperature, blood pressure, respiratory rate and pulse remained similar or underwent changes within the physiological range pre and post swish. Patients who presented with hypertension before PARPi-FL administration continued to show elevated blood pressure after the procedure. The patient’s oral mucosa was checked by the responsible surgeon before PARPi-FL administration, immediately after imaging and 2 - 3 days later. No patients presented any signs of local irritation or reported signs of local toxicity when examined or actively questioned. Clinical blood sample analysis, encompassing complete blood count and differential, routine clinical chemistry and a metabolic panel revealed no pathological alterations related to PARPi-FL topical application.
Analysis of PARPi-FL in Blood
No PARPi-FL was detected in the blood sample taken after the PARPi-FL swish via LCMS analysis in all patients. We determined the detection limit of our LCMS approach at 0.001 ng/mL of PARPi-FL and potential corresponding metabolites. Complete absorption of topically applied PARPi-FL into the bloodstream would correspond to 0.002 ng/μL at the highest dose level (1000 nM, assuming an average blood volume of 5 L), suggesting that measurable amounts of PARPi-FL after topical application are unlikely to enter the blood stream (Supplementary Fig. 2).
Image acquisition optimization
Prior to patient imaging, we conducted tests with PARPi-FL containing agarose phantoms (0 - 5 µM PARPi-FL) to identify suitable settings for patient imaging (Supplementary Fig. 3). We aimed at optimizing the imaging settings towards sensitivity in the relevant concentration range of 100 - 1000 nM. As expected, the recorded signal intensity was lower at longer distance from the object (23 mm vs. 5 mm) and shorter exposure times (12 ms < 30 ms < 50 ms). To achieve a signal to background ratio of 3, which we considered adequate contrast to differentiate tumor from background tissue in vivo, at 23 mm from the object the PARPi-FL concentration needed to be 0.6 µM at 83 ms and 0.8 µM at 50 ms exposure time. Hence, this distance was considered incompatible to achieve sufficient image contrast at relevant concentrations. At 5 mm distance, the values decreased to 0.3 µM at 50 ms and 0.5 µM at 30 ms to achieve a signal to background ratio of 3 (Supplementary Fig. 3A). Since it was difficult to steadily hold and focus the endoscope at a 5 mm distance in patients, we chose a 10 mm distance from object for the clinical study and a 30 ms exposure time, allowing us to conduct lag-free real-time in vivo imaging.
PARP1 expression in patient tissue
Tissue samples from 8 patients were available for PARP1 expression analysis via IHC. Two of these specimens were excluded, since they had no tumor left in the paraffin block after standard-of-care histopathological assessment. PARP1 IHC staining was analyzed and quantified in tumor, mucosal epithelium and deep margin (i.e. healthy muscle tissue) based on the pathological assessment (Fig. 3A). PARP1 expression in tumor,16.35 ± 9.18% PARP1 area (DAB)/total tissue area (tissue), was significantly higher than in the normal mucosal epithelium (8.41 ± 2.91% DAB/tissue) and in the deep muscular margin (3.12 ± 1.79% DAB/tissue) (Fig. 3B; p < 0.0001 using linear regression analysis which considered the repeated measurements from the same patients, Supplementary Tables 2 and 3). On average, the % DAB/tissue was 45% lower in normal squamous epithelium compared to tumor and 81% lower in normal deep muscle compared to tumor.
PARPi-FL imaging protocol
Groups of 3 patients were studied with increasing concentrations of PARPi‑FL (100 nM, 250 nM, 500 nM, and 1000 nM). Patient 5 (250 nM) was analyzed separately, since the final pathology report did not identify any residual viable tumor. Fig. 3C shows an example of tumor enhancement with PARPi-FL at 250 nM. Signal quantification was conducted by placing ROIs on tumor and adjacent non-tumor tissue on still images of the three imaging time points “pre PARPi-FL”, “PARPi-FL pre-wash” and “PARPi-FL post-wash” and analysis of their tumor to normal mucosal ratio (TMR). Fig. 4A shows an example of the signal increase in the tumor area after PARPi-FL application in a patient imaged at 1000 nM.
Intra-patient imaging analysis
We compared TMR values pre PARPi-FL, PARPi-FL pre wash and PARPi-FL post-wash in each patient. A significant increase in TMR from pre PARPi-FL to PARPi-FL pre-wash (9/11 patients), and PARPi-FL post-wash (11/11 patients) was observed. The individual increase in TMR is displayed in Fig. 4B. The values on signal intensity and p-values between imaging time points for each patient can be found in Supplementary Table 4.
Our observations suggest that the clearing step is important in obtaining high contrast images. Without the clearing step, 18% (2/11) of the lesions would have been missed. We believe that this fact is due to the accumulation of unbound PARPi-FL around the tongue’s filiform papillae. The clearing step, associated with the saliva outflow, plays an important role in clearing the unbound compound before signal quantification is performed.
Patient with no residual tumor in the surgical resection specimen
Patient 5 (250 nM), had a previous excisional biopsy of a tongue SCC with compromised margins at an outside institution. The patient presented with a flat residual lesion at the site of the previous resection that was assumed to be a persistent tumor. The patient had a pre‑PARPi-FL lesion to margin ratio (LMR) of 1.20. The contrast increased after PARPi-FL administration (LMR: 2.24) but the contrast decreased after the clearing step (LMR: 1.53) (Fig. 4C).
Patient with contralateral, incidentally identified lesion
Patient 2 had a large lesion on the lateral border of the tongue that was examined with PARPi-FL at the 100 nM dose level. In the process of imaging the contralateral side of the tongue in this patient as the imaging control, we noticed an area of increased PARPi-FL uptake (Fig. 4D). This area did not present any clinical evidence of macroscopic tumor in the pre-surgical setting. This area had TMRs of 1.28 (pre PARPi-FL), 1.33 (PARPi-FL pre-wash) and 1.71 (PARPi-FL post-wash). Post-wash, the TMR was significantly higher (p = 0.03) than pre PARPi-FL. Based on the pre-operative PARPi-FL finding and surgeon’s scrutiny after the patient was under anesthesia, intraoperative biopsy of this lesion was performed. Histopathologic examination confirmed a second primary squamous cell carcinoma at this location measuring 2 mm in the largest diameter.
Inter-patient imaging analysis
We also conducted an analysis between the different PARPi-FL dose groups and observed a consistent increase in TMR in PARPi-FL pre-wash and post-wash images with increasing PARPi-FL dose levels (Fig. 5A). The mean predicted TMRs PARPi-FL post wash were 2.1 (100 nM), 2.1 (250 nM) and 2.8 (500 nM). At the highest dose level of 1000 nM PARPi-FL post wash yielded an average TMR of 3.3, a contrast value that is suitable to clearly distinguish tumor from normal tissue. Considering all time points, when accounting for the repeated measurements per patients (with a random intercept), the TMR was on average 39% higher at 250 nM compared to 100 nM, 62% higher at 500 nM than 100 nM dose, and 92% higher at 1000 nM as compared to 100 nM. These results indicated that 1000 nM was the most suitable concentration for PARPi-FL imaging. For descriptive data on the linear regression analysis, the average values on TMR, and the p-values please see Supplementary Table 5.
Microscopic imaging of PARPi-FL accumulation
To demonstrate that the macroscopically detected increase in fluorescence signal after PARPi-FL application was based on its specific accumulation in cell nuclei, we analyzed a tumor and free-of disease normal tissue sample originating from a patient on phase II. The patient harbored a right buccal mucosal OSCC and was imaged after 1000 nM PARPi-FL and clearing with 30% PEG 300 in water (Fig. 5B). IHC corroborated the difference in PARP1 expression within the tumor and the free-of-disease normal tissue in this patient (Fig 5C). Confocal microscopy images clearly demonstrated that PARPi-FL accumulated exclusively in tumor cell nuclei, while no nuclear signal was observed in the non-tumor tissue (Fig. 5D). Quantification was carried out by verifying the presence of PARPi-FL fluorescence signal inside the nucleus of cells. In the tumor tissue 1061 nuclei were quantified and 45.14% of cells showed PARPi-FL uptake. In the normal tissue 412 nuclei were analyzed) and only 3.89% of cells had PARPi-FL uptake. This difference was statistically significant (p < 0.0001), corroborating the specificity of PARPi-FL for tumor cells. (Fig. 5E).