Histology and Lung Nodule Fluorescence in Intraoperative Molecular Imaging With Pafolacianine

Abstract

Background: Intraoperative molecular imaging (IMI) uses a cancer-targeted fluorescent agent injected into patients to localize tumor nodules. Pafolacianine is a folate receptor (FR)-targeted near-infrared fluorescent probe. Almost 10% of patients have false negative fluorescence findings intraoperatively. We hypothesized that tumor histology explains why lung cancer may not fluoresce.

Methods: Adenocarcinoma (AC) (A549, LKR) and squamous cell carcinoma (SCC) (H127, H1264) cell lines were stained with pafolacianine. Near-infrared fluorescent microscopy was used to quantify mean fluorescence intensity. Tissue microarray slides of patients with AC and SCC were evaluated by immunohistochemistry for FR alpha (FRα) and beta (FRβ) expression. Finally, we retrospectively analyzed IMI data from clinical trials of patients with AC and SCC receiving pafolacianine.

Results: AC (intensity 30.31) cell lines have a higher fluorescence intensity than SCC cell lines (intensity 5.4) (P < .001). On slide analysis, 93.8% of ACs expressed FRα compared with 44.4% of SCCs (P = .002). Finally, there were 326 patients enrolled in clinical trials: 211 had lesions localized in vivo, and 134 of these patients had pure AC or SCC. All 9 patients with SCC have a positive smoking history and a mean pack-year of 60.2 (SD 3,6), whereas 76% of patients with AC have a history of smoking and a mean pack-year of 29.3 (P = .02). The odds ratio for fluorescence of (AC/SCC) was 2.05 (P = .004) and 2.01 (P = .02) on univariate and multivariate logistic regression, respectively.

Conclusions: During IMI with pafolacianine, a nonfluorescent nodule is more likely to be SCC than AC. AC has a high probability of fluorescing because of higher expression of FRα or FRβ, or both.

Figure 1

(A) Comparison of adenocarcinoma (AC) cell lines (A549 and LKR) with squamous cell carcinoma (SCC) cell lines (H1264 and H2170) when stained by pafolacianine (OTL-38). The first 2 rows are the negative control, and the fourth row is competitive inhibition. (B) Comparison of mean fluorescence intensity (MFI) among different cell lines. All images are at 40× magnification. The asterisks (∗,∗∗,∗∗∗,∗∗∗∗) denote significance at P < .001. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 2

Immunohistochemistry (IHC) of adenocarcinoma (AC) and squamous cell carcinoma (SCC) cell lines for folate receptor alpha (FRα) and folate receptor beta (FRβ). The first column represents hematoxylin and eosin (H&E) staining, the second column antibodies against FRα, and the third column antibodies against FRβ. The first 2 rows represent AC cell lines, and the third and fourth row are SCC. All images are at 40× magnification. Nuclei are purple; FR stain is brown. (ACC, adenocarcinoma; SCC, squamous cell carcinoma.)

Figure 3

(A) Comparison of tissue microassay (TMA) slides of adenocarcinoma (AC) and squamous cell carcinoma (SCC) using immunohistochemistry (IHC). (First row) Hematoxylin and eosin (H&E) sections of AC and SCC. (Second row) (left) AC and (right) SCC TMA section stained with antibody against folate receptor alpha (FRα). (Third row) (left) AC and (right) SCC TMA section stained with antibody against folate receptor beta (FRβ). (B) Distribution of FRα and FRβ expression in both AC and SCC.