Intraoperative near-infrared imaging with receptor-specific versus passive delivery of fluorescent agents in pituitary adenomas

Abstract

Objective: Intraoperative molecular imaging with tumor-targeted fluorescent dyes can enhance resection rates. In contrast to visible-light fluorophores (e.g., 5-aminolevulinic-acid), near-infrared (NIR) fluorophores have increased photon tissue penetration and less contamination from tissue autofluorescence. The second-window ICG (SWIG) technique relies on passive accumulation of indocyanine green (ICG) in neoplastic tissues. OTL38, conversely, targets folate receptor overexpression in nonfunctioning pituitary adenomas. In this study, we compare the properties of these 2 modalities for NIR imaging of pituitary adenomas to better understand the potential for NIR imaging in neurosurgery.

Methods: A total of 39 patients with pituitary adenomas were enrolled between June 2015 and January 2018 in 2, sequential, IRB-approved studies. Sixteen patients received systemic ICG infusions 24 hours prior to surgery, and another 23 patients received OTL38 infusions 2-3 hours prior to surgery. NIR fluorescence signal-to-background ratio (SBR) was recorded during and after resection. Immunohistochemistry was performed on the 23 adenomas resected from patients who received OTL38 to assess expression of folate receptor-alpha (FRα).

Results: All 16 adenomas operated on after ICG administration demonstrated strong NIR fluorescence (mean SBR 4.1 ± 0.69 [SD]). There was no statistically significant difference between the 9 functioning and 7 nonfunctioning adenomas (p = 0.9). After administration of OTL38, the mean SBR was 1.7 ± 0.47 for functioning adenomas, 2.6 ± 0.91 for all nonfunctioning adenomas, and 3.2 ± 0.53 for the subset of FRα-overexpressing adenomas. Tissue identification with white light alone for all adenomas demonstrated 88% sensitivity and 90% specificity. SWIG demonstrated 100% sensitivity but only 29% specificity for both functioning and nonfunctioning adenomas. OTL38 was 75% sensitive and 100% specific for all nonfunctioning adenomas, but when assessment was limited to the 9 FRα-overexpressing adenomas, the sensitivity and specificity of OTL38 were both 100%.

Conclusions: Intraoperative imaging with NIR fluorophores demonstrates highly sensitive detection of pituitary adenomas. OTL38, a folate-receptor-targeted fluorophore, is highly specific for nonfunctioning adenomas but has no utility in functioning adenomas. SWIG, which relies on passive diffusion into neoplastic tissue, is applicable to both functioning and nonfunctioning pituitary adenomas, but it is less specific than targeted fluorophores. Thus, targeted and nontargeted NIR fluorophores play important, yet distinct, roles in intraoperative imaging. Selectively and intelligently using either agent has the potential to greatly improve resection rates and outcomes for patients with intracranial tumors.

Keywords: 5-ALA = 5-aminolevulinic acid; ACTH = adrenocorticotropic hormone; EPR = enhanced permeability and retention; FRα = folate receptor–alpha; GH = growth hormone; ICG = indocyanine green; NIR = near-infrared; NPV = negative predictive value; PPV = positive predictive value; SBR = signal-to-background ratio; SPECT = single-photon emission computed tomography; SWIG = second-window ICG; TSH = thyroid-stimulating hormone; folate receptor; indocyanine-green; near-infrared imaging; pituitary adenoma; pituitary surgery; targeted imaging.

FIG. 1.

Application of SWIG in a patient with a prolactin-staining nonfunctioning pituitary adenoma. A and B: Preoperative coronal (A) and sagittal (B) contrast-enhanced T1-weighted MR images demonstrating a 22-mm adenoma. C and D: Initial view of the tumor after opening the dura as visualized with white light (C) and with NIR imaging (D). NIR imaging delineates the tumor with a high SBR of 4.5. E and F: Margin specimen biopsied during surgery, viewed with white light (E) and with NIR imaging (F). Viewed with white light alone, the specimen did not convincingly appear neoplastic. With NIR imaging, however, the specimen fluoresced with an SBR of 3.2. The final pathological examination demonstrated that the specimen was neoplastic, suggesting that the NIR imaging was more sensitive for neoplasm in this case. Figure is available in color online only.

FIG. 2.

Intraoperative and MR images obtained in the same patient as represented in Fig. 1 after resection of the pituitary adenoma. A and B: Postresection view obtained with white light (A) and NIR (B) imaging prior to closure. The surgeon was satisfied that no residual tumor was present (A). NIR imaging demonstrated no residual areas of fluorescence (B). C and D: Postoperative day 1 coronal (C) and sagittal (D) contrast-enhanced T1-weighted MR images demonstrating gross-total resection. The absence of NIR fluorescence prior to closure correctly predicted absence of residual neoplasm. Figure is available in color online only.

FIG. 3.

Ex-vivo NIR imaging of pituitary adenoma specimens. A: Ex-vivo imaging of a specimen from a pituitary adenoma (middle) shows NIR fluorescence. A meningioma specimen (right) and a glioblastoma specimen (left) are shown for comparison. B and C: Microscopic imaging of the same pituitary adenoma specimen, under white light only (B) and with NIR imaging (C). Original magnification ×100. Figure is available in color online only.

FIG. 4.

Photomicrographs and intraoperative photographs showing correlation of FRα expression with OTL38 imaging signal in pituitary adenomas. A and B: This patient’s adenoma stained for FRα with an H-score of 0, as no cells expressed any FRα (A). The SBR upon tumor exposure was 1.4 (B). C and D: This patient’s adenoma stained for FRα with an H-score of 100, indicating moderate FRα expression in some cells (C). The SBR upon tumor exposure was 1.8 (D). E and F: This patient’s adenoma stained for FRα with an H-score of 270, indicating high FRα expression in nearly all the cells (E). The SBR upon tumor exposure was 3.3 (F). Original magnification ×200. Figure is available in color online only.

FIG. 5.

SWIG in a patient with a null-cell nonfunctioning pituitary adenoma. A and B: Preoperative coronal (A) and sagittal (B) contrast-enhanced T1-weighted MR images demonstrating a 2-mm adenoma. C and D: Initial tumor view after opening the dura, with white light (C) and with NIR imaging (D). The NIR imaging delineates the tumor with a high SBR of 3.5 (D). E and F: Margin specimen biopsied during surgery, viewed with white light (E) and with NIR imaging (F). With white light alone, the specimen did not appear neoplastic. With NIR imaging, the specimen fluoresced with an SBR of 2.5. The finding of final pathological examination was that the specimen was not neoplastic. Thus, the NIR imaging led to a false-positive signal in this case. G and H: Followup coronal (G) and sagittal (H) contrast-enhanced T1-weighted MR images obtained 3 months after surgery, demonstrating gross-total resection. Figure is available in color online only.

FIG. 6.

Passive versus receptor-targeted delivery of NIR agent. A: ICG is hypothesized to be delivered to tumor tissues via the EPR effect. Benign tissue with intact vasculature and endothelium are minimally permeable to the NIR dye, but tumor tissue with disorganized and/or damaged vasculature allows the NIR dye to diffuse into the tissue and be retained. Thus, delayed intraoperative imaging 24 hours after systemic injection of ICG allows visualization of tumor tissue with permeable vasculature. B: OTL38 is a folate analog linked to ICG. Thus, it binds to and is internalized by cells that overexpress folate receptors but does not bind to cells that do not overexpress folate receptors. This allows OTL38 to specifically accumulate within tumor tissue with folate-receptor overexpression, such as nonfunctioning pituitary adenoma cells. Figure is available in color online only.