Prospective validation of tumor folate receptor expression density with the association of pafolacianine fluorescence during intraoperative molecular imaging-guided lung cancer resections

Abstract

Purpose: Pafolacianine, a folate receptor alpha-targeted NIR tracer, has demonstrated clear efficacy in intraoperative molecular imaging-guided (IMI) lung cancer surgery. However, the selection of patients who would benefit from IMI remains challenging given the variability of fluorescence with patient-associated and histopathologic factors. Our goal in this study was to prospectively evaluate whether preoperative FRα/FRβ staining can predict pafolacianine-based fluorescence during real-time lung cancer resections.

Methods: This was a prospective study conducted between 2018 and 2022 that reviewed core biopsy and intraoperative data from patients with suspected lung cancer. A total of 196 patients were deemed eligible, of whom core biopsies were taken from 38 patients and assessed for FRα and FRβ expression by immunohistochemistry (IHC). All patients underwent infusion of pafolacianine 24 h prior to surgery. Intraoperative fluorescence images were captured with the VisionSense bandpass filter-enabled camera. All histopathologic assessments were performed by a board-certified thoracic pathologist.

Results: Of the 38 patients, 5 (13.1%) were found to have benign lesions (necrotizing granulomatous inflammation, lymphoid aggregates) and 1 had metastatic non-lung nodule. Thirty (81.5%) had malignant lesions, with the vast majority (23, 77.4%) being lung adenocarcinoma (7 (22.5%) SCC). None of the benign tumors (0/5, 0%) exhibited in vivo fluorescence (mean TBR of 1.72), while 95% of the malignant tumors fluoresced (mean TBR of 3.11 ± 0.31) compared to squamous cell carcinoma (1.89 ± 0.29) of the lung and sarcomatous lung metastasis (2.32 ± 0.09) (p < 0.01). The TBR was significantly higher in the malignant tumors (p = 0.009). The median FRα and FRβ staining intensities were both 1.5 for benign tumors, while the FRα and FRβ staining intensities were 3 and 2 for malignant tumors, respectively. Increased FRα expression was significantly associated with the presence of fluorescence (p = 0.01), CONCLUSION: This prospective study sought to determine whether preoperative FRα and FRβ expression on core biopsy IHC correlates with intraoperative fluorescence during pafolacianine-guided surgery. These results, although of small sample size, including limited non-adenocarcinoma cohort, suggest that performing FRα IHC on preoperative core biopsies of adenocarcinomas as compared to squamous cell carcinomas could provide low-cost, clinically useful information for optimal patient selection which should be further explored in advanced clinical trials.

Keywords: Fluorescence-guided surgery; Folate receptor; Intraoperative molecular imaging; Lung cancer; Pafolacianine.

Fig. 1

Malignant lesions demonstrate pafolacianine labeling. A Patient with a diagnosis of adenocarcinoma on preoperative biopsy demonstrating pafolacianine-guided in situ localization during IMI-guided resection. B Fluorescence microscopy analysis demonstrates NIR localization of pafolacianine to areas of adenocarcinoma on (inset) with C areas of the tumor demonstrating uptake on pafolacianine at 63 × magnification. D Adenocarcinoma lesions demonstrate the highest tumor-to-background ratio (TBR) compared to squamous cell carcinoma of the lung and lung metastases, but malignant lesions overall have higher fluorescence intensity than benign lesions. E The preoperative FDG PET standard uptake value did not correlate with malignant lesion fluorescence

Fig. 2

FRα presence is associated with pafolacianine NIR labeling intraop. A Analysis of all lesions demonstrates that any FRα presence is associated with statistically higher fluorescence versus minimal to no fluorescence when there is no FRα expression by the tumor (p < 0.05). B There was a correlation of a higher tumor-to-background ratio with increased expression of FRα, but this correlation did not reach statistical significance. Furthermore, there was no difference in fluorescence intensity between 2 + and 3 + FRα scoring. C There is a strong correlation with FRα presence and increased fluorescence when analyzing patients with adenocarcinoma diagnosis on preoperative sampling. D Representative images of a patient with adenocarcinoma diagnosed on core biopsy with a 3 + FRα score by a pathologist and observation of pafolacianine NIR labeling

Fig. 3

Squamous cell cancer lesion with negative FRα staining and lack of fluorescence during pafolacianine-guided resection

Fig. 4

FRβ weakly correlated with NIR fluorescence. A Regardless of the fluorescence intensity that was observed, there was no correlation with FRβ presence (B), with an R² of only 0.02. B In the subgroup analysis, only strong FRβ expression (3 +) was noted to be associated with TBR > 2. C Representative images of malignant and benign lesions showing FRβ not correlating with detection or lack of detection during IMI

Fig. 5

Representative core biopsy samples from various patients with different pathologies. Patient 3 (bottom) and sample 5 did not stain for FRα, whereas the previous 4 biopsies had a strong presence of FRα on IHC