Evaluation of Diagnostic Accuracy Following the Coadministration of Delta-Aminolevulinic Acid and Second Window Indocyanine Green in Rodent and Human Glioblastomas

Abstract

Purpose: Fluorescence-guided-surgery offers intraoperative visualization of neoplastic tissue. Delta-aminolevulinic acid (5-ALA), which targets enzymatic abnormality in neoplastic cells, is the only approved agent for fluorescence-guided neurosurgery. More recently, we described Second Window Indocyanine Green (SWIG) which targets neoplastic tissue through enhanced vascular permeability. We hypothesized that SWIG would demonstrate similar clinical utility in identification of high-grade gliomas compared with 5-ALA.

Procedures: Female C57/BL6 and nude/athymic mice underwent intracranial implantation of 300,000 GL261 and U87 cells, respectively. Tumor-bearing mice were euthanized after administration of 5-ALA (200 mg/kg intraperitoneal) and SWIG (5 mg/kg intravenous). Brain sections were imaged for protoporphyrin-IX and ICG fluorescence. Fluorescence and H&E images were registered using semi-automatic scripts for analysis. Human subjects with HGG were administered SWIG (2.5 mg/kg intravenous) and 5-ALA (20 mg/kg oral). Intraoperatively, tumors were imaged for ICG and protoporphyrin-IX fluorescence.

Results: In non-necrotic tumors, 5-ALA and SWIG demonstrated 90.2 % and 89.2 % tumor accuracy (p value = 0.52) in U87 tumors and 88.1 % and 87.7 % accuracy (p value = 0.83) in GL261 tumors. The most distinct difference between 5-ALA and SWIG distribution was seen in areas of tumor-associated necrosis, which often showed weak/no protoporphyrin-IX fluorescence, but strong SWIG fluorescence. In twenty biopsy specimens from four subjects with HGG, SWIG demonstrated 100 % accuracy, while 5-ALA demonstrated 75-85 % accuracy; there was 90 % concordance between SWIG and 5-ALA fluorescence.

Conclusion: Our results provide the first direct comparison of the diagnostic utility of SWIG vs 5-ALA in both rodent and human HGG. Given the broader clinical utility of SWIG compared with 5-ALA, our data supports the use of SWIG in tumor surgery to improve the extent of safe resections.

Clinical trial: NCT02710240 (US National Library of Medicine Registry; https://www.clinicaltrials.gov/ct2/show/NCT02710240?id=NCT02710240&draw=2&rank=1 ).

Keywords: 5-ALA; Fluorescence-guided surgery; High grade glioma; Near-infrared; Second-window ICG.

Conflict of interest statement

Disclosures: JYKL has had stock options in VisionSense in the past. No other author disclosures or conflicts of interest.

Figures

Figure 1:. Orthotopic Mouse Glioma Model and Ex-Vivo Fluorescence Imaging.

A-D) PpIX and ICG accumulation in non-necrotic tumors: White-light imaging (A) and H&E staining (B) demonstrates a small, non-necrotic tumor (27.3% of coronal section) in the right hemisphere of this mouse with an orthotopic, U87 tumor. 5-ALA and SWIG were administered to this mouse. Under 400nm excitation and 610–690nm emission for protoporphyrin-IX (C), strong PpIX fluorescence is seen throughout the tumor and corpus callosum, resulting in PpIX fluorescence in 29.3% of the cross-sectional area. Under 785nm excitation (D), strong ICG fluorescence is detected throughout the tumor. There is also non-specific fluorescence seen in the ipsilateral and contralateral ventricles, as well as in the right cortex, resulting in ICG fluorescence in 32.7% of the cross-sectional area. On H&E staining, the ventricles and corpus callosum are devoid of neoplastic cells. In the right cortex, few cells with dark nuclei were seen, but their morphology suggested the presence of inflammatory cells rather than neoplastic cells. This was likely in response to the needle tract made for the intracranial implantation. E-H) PpIX and ICG accumulation in necrotic tumors: White-light imaging (E) and H&E staining (F) demonstrates a large, necrotic tumor (44.2% of coronal section) in the right hemisphere of this mouse with an orthotopic, GL261 tumor. 5-ALA and SWIG were administered to this mouse. PpIX fluorescence (G) is seen in the peripheral areas of the tumor but not in the tumor core, where there is a significant amount of hemorrhage and necrosis, resulting in positive fluorescence in only 35.5% of the cross-sectional area. In contrast, ICG fluorescence (H) is observed throughout the tumor, even in the necrotic core, resulting in positive fluorescence in 45.2% of the cross-sectional area.

Figure 2:. Non-Tumor Areas of Fluorescence under Near-Infrared and Blue-Light Excitation

A-D) Brain autofluorescence: White-light imaging (A) and H&E staining (B) demonstrates a large, necrotic tumor (43.7% of coronal section) in the right hemisphere of this mouse with an orthotopic, GL261 tumor. This mouse did not receive any 5-ALA or SWIG, in order to investigate the presence of autofluorescence under blue-light excitation and NIR excitation. Under 400nm excitation and 610–690nm emission (C), diffuse autofluorescence is seen throughout the brain. The overall signal intensity, however, is 8-fold lower than in the mice that received 5-ALA (8-stops on Canon imaging system). Under 785nm excitation (D), no areas of autofluorescence is seen. E-H) PpIX and ICG accumulation in control brain: White-light imaging (E) and H&E staining (F) demonstrates a normal brain with no neoplasm in this control mouse. 5-ALA and SWIG were administered to this mouse. Strong PpIX fluorescence is seen in the hippocampal formation, while the corpus callosum demonstrates moderate PpIX fluorescence (G); the overall fluorescence intensity is 4-fold lower than in tumor-bearing mice that received 5-ALA. Strong ICG fluorescence is observed in the lateral ventricles bilaterally and moderate fluorescence is seen in the third ventricle and the horns of the lateral ventricles (H); H&E staining reveals the choroid plexus in these locations.

Figure 3:. Fluorescence Imaging Accuracy for Detecting Neoplastic Tissue with 5-ALA and SWIG in Murine HGG Model with U87 and GL261 Cell Lines

A) In the 20 coronal slices obtained from U87 tumor-bearing mice, 5-ALA and SWIG had similar accuracies (90.2% vs 89.2%, p-value=0.52) B) In the 17 coronal slices from non-necrotic GL261 tumor-bearing mice, 5-ALA and SWIG had similar accuracies (88.1% vs 87.7%, p-value=0.83) C) In the 10 coronal slices from necrotic GL261 tumor-bearing mice, 5-ALA had significantly lower accuracy compared to SWIG (71.2% vs 86.1%, p-value

Figure 4:. 5-ALA and SWIG Dual-Fluorophore Administration and Intraoperative Visualization.

A 76-year-old female patient undergoing primary resection of a high-grade glioma was administered both 5-ALA and SWIG. A-C) Intraoperatively, white-light and fluorescence imaging was performed over the intact dura. White-light imaging (A) did not demonstrate any areas suspicious for underlying neoplasm. Blue-light excitation (B) did not elicit any red-fluorescence suggestive of neoplasm. NIR excitation (C), however, localized an area of ICG accumulation consistent with the area of neoplasm. D-G) Upon durotomy, the tumor was visualized under white-light (D) as a hypervascular and red mass extending to the cortex. Blue-light excitation (E) elicited strong PpIX fluorescence in the area consistent with neoplasm on white-light imaging. Similarly, NIR excitation (F) detected an area of ICG accumulation in the same location that was visualized over the intact dura in (C). A biopsy specimen from this gross tumor demonstrated high-grade glioma cells on histopathology (G). H-L) In another patient with recurrent HGG, two specimens demonstrated discrepancies between SWIG and 5-ALA fluorescence. One white-matter specimen (white arrow) demonstrated strong red fluorescence (I) but no NIR fluorescence (J). This specimen did not contain any neoplastic cells on histopathology (K). Another specimen (white, notched arrow) demonstrated no PpIX fluorescence (I) but strong NIR fluorescence (J). On histopathology, this specimen contained neoplastic cells mixed in with necrotic tissue (L).