Bevacizumab-IRDye800CW for tumor detection in fluorescence-guided meningioma surgery (LUMINA trial): a single-center phase I study.

OBJECTIVE: Meningiomas are one of the most frequently occurring brain tumors and can be curatively treated with gross-total resection. A subtotal resection increases the chances of recurrence. The intraoperative identification of invisible tumor remnants by using a fluorescent tracer targeting an upregulated biomarker could help to optimize meningioma resection. This is called molecular fluorescence-guided surgery (MFGS). Vascular endothelial growth factor α (VEGFα) has been identified as a suitable meningioma biomarker and can be targeted with bevacizumab-IRDye800CW. METHODS: The aim of this prospective phase I trial was to determine the safety and feasibility of bevacizumab-IRDye800CW for MFGS for intracranial meningiomas by administering 4.5, 10, or 25 mg of the tracer 2-4 days prior to surgery. Fluorescence was verified during the operation with the standard neurosurgical microscope, and tissue specimens were postoperatively analyzed with fluorescence imaging systems (Pearl and Odyssey CLx) and spectroscopy to determine the optimal dose. Uptake was compared in several tissue types and correlated with VEGFα expression. RESULTS: No adverse events related to the use of bevacizumab-IRDye800CW occurred. After two interim analyses, 10 mg was the optimal dose based on ex vivo tumor-to-background ratio. Although the standard intraoperative imaging revealed no fluorescence, postoperative analyses with tailored imaging systems showed high fluorescence uptake in tumor compared with unaffected dura mater and brain. Additionally, tumor invasion of the dura mater (dural tail) and invasion of bone could be distinguished using fluorescence imaging. Fluorescence intensity showed a good correlation with VEGFα expression. CONCLUSIONS: Bevacizumab-IRDye800CW can be safely used in patients with meningioma; 10 mg bevacizumab-IRDye800CW provided an adequate tumor-to-background ratio. Adjustments of the currently available neurosurgical microscopes are needed to achieve visualization of targeted IRDye800CW intraoperatively. A phase II/III trial is needed to methodically investigate the benefit of MFGS with bevacizumab-IRDye800CW for meningioma surgery in a larger cohort of patients.

Feasibility of bevacizumab-IRDye800CW as a tracer for fluorescence-guided meningioma surgery.

OBJECTIVE: Meningiomas are frequently occurring, often benign intracranial tumors. Molecular fluorescence can be used to intraoperatively identify residual meningioma tissue and optimize safe resection; however, currently no clinically approved agent is available for this specific tumor type. In meningiomas, vascular endothelial growth factor α (VEGFα) is upregulated, and this biomarker could be targeted with bevacizumab-IRDye800CW, a fluorescent agent that is already clinically applied for the resection of other tumors and neoplasms. Here, the authors investigated the feasibility of using bevacizumab-IRDye800CW to target VEGFα in a CH-157MN xenografted mouse model. METHODS: Five mice with CH-157MN xenografts with volumes of 500 mm3 were administered intravenous bevacizumab-IRDye800CW. Mice were imaged in vivo at 24 hours, 48 hours, and 72 hours after injection with the FMT2500 fluorescence imaging system. Biodistribution was determined ex vivo using the Pearl fluorescent imager at 72 hours after injection. To mimic a clinical scenario, 2 animals underwent postmortem xenograft resection using both white-light and fluorescence guidance. Lastly, fresh and frozen human meningioma specimens were incubated ex vivo with bevacizumab-IRDye800CW, stained with anti-VEGFα, and microscopically examined. RESULTS: In vivo, tumors fluoresced at all time points after tracer administration and background fluorescence decreased with time. Ex vivo analyses of tracer biodistribution showed the highest fluorescence in resected tumor tissue. Brain, skull, and muscle tissue showed very low fluorescence. Microscopically, fluorescence was observed in the cytoplasm and was correlated with VEGFα expression patterns. During postmortem surgery, both the tumor bulk and a small tumor remnant were detected. Bevacizumab-IRDye800CW bound specifically to all tested human meningioma samples, as indicated by a high fluorescent signal in the tumor bulk compared with the surrounding healthy dura mater. CONCLUSIONS: Bevacizumab-IRDye800CW showed meningioma specificity, as illustrated by high VEGFα-mediated uptake in the meningioma xenograft mouse model. Small tumor lesions were detected using fluorescence guidance. Thus, the next step will be to assess the feasibility of using already available clinical grade bevacizumab-IRDye800CW to optimize meningioma resection in a human trial.

Necrosis binding of Ac-Lys0(IRDye800CW)-Tyr3-octreotate: a consequence from cyanine-labeling of small molecules.

BACKGROUND: There is a growing body of nuclear contrast agents that are repurposed for fluorescence-guided surgery. New contrast agents are obtained by substituting the radioactive tag with, or adding a fluorescent cyanine to the molecular structure of antibodies or peptides. This enables intra-operative fluorescent detection of cancerous tissue, leading to more complete tumor resection. However, these fluorescent cyanines can have a remarkable influence on pharmacokinetics and tumor uptake, especially when labeled to smaller targeting vectors such as peptides. Here we demonstrate the effect of cyanine-mediated dead cell-binding of Ac-Lys0(IRDye800CW)-Tyr3-octreotate (800CW-TATE) and how this can be used as an advantage for fluorescence-guided surgery. RESULTS: Binding of 800CW-TATE could be blocked with DOTA0-Tyr3-octreotate (DOTA-TATE) on cultured SSTR2-positive U2OS cells and was absent in SSTR2 negative U2OS cells. However, strong binding was observed to dead cells, which could not be blocked with DOTA-TATE and was also present in dead SSTR2 negative cells. No SSTR2-mediated binding was observed in frozen tumor sections, possibly due to disruption of the cells in the process of sectioning the tissue before exposure to the contrast agent. DOTA-TATE blocking resulted in an incomplete reduction of 61.5 ± 5.8% fluorescence uptake by NCI-H69-tumors in mice. Near-infrared imaging and dead cell staining on paraffin sections from resected tumors revealed that fluorescence uptake persisted in necrotic regions upon blocking with DOTA-TATE. CONCLUSION: This study shows that labeling peptides with cyanines can result in dead cell binding. This does not hamper the ultimate purpose of fluorescence-guided surgery, as necrotic tissue appears in most solid tumors. Hence, the necrosis binding can increase the overall tumor uptake. Moreover, necrotic tissue should be removed as much as possible: it cannot be salvaged, causes inflammation, and is tumorigenic. However, when performing binding experiments to cells with disrupted membrane integrity, which is routinely done with nuclear probes, this dead cell-binding can resemble non-specific binding. This study will benefit the development of fluorescent contrast agents.

Evaluation of Ac-Lys0(IRDye800CW)Tyr3-octreotate as a novel tracer for SSTR2-targeted molecular fluorescence guided surgery in meningioma.

PURPOSE: Meningioma recurrence rates can be reduced by optimizing surgical resection with the use of intraoperative molecular fluorescence guided surgery (MFGS). We evaluated the potential of the fluorescent tracer 800CW-TATE for MFGS using in vitro and in vivo models. It targets somatostatin receptor subtype 2 (SSTR2), which is overexpressed in all meningiomas. METHODS: Binding affinity of 800CW-TATE was evaluated using [177Lu] Lu-DOTA-Tyr3-octreotate displacement assays. Tumor uptake was determined by injecting 800CW-TATE in (SSTR2-positive) NCI-H69 or (SSTR2-negative) CH-157MN xenograft bearing mice and FMT2500 imaging. SSTR2-specific binding was measured by comparing tumor uptake in NCI-H69 and CH-157MN xenografts, blocking experiments and non-targeted IRDye800CW-carboxylate binding. Tracer distribution was analyzed ex vivo, and the tumor-to-background ratio (TBR) was calculated. SSTR2 expression was determined by immunohistochemistry (IHC). Lastly, 800CW-TATE was incubated on frozen and fresh meningioma specimens and analyzed by microscopy. RESULTS: 800CW-TATE binding affinity assays showed an IC50 value of 72 nM. NCI-H69 xenografted mice showed a TBR of 21.1. 800CW-TATE detection was reduced after co-administration of non-fluorescent DOTA-Tyr3-octreotate or administration of IRDye800CW. CH-157MN had no tumor specific tracer staining due to absence of SSTR2 expression, thereby serving as a negative control. The tracer bound specifically to SSTR2-positive meningioma tissues representing all WHO grades. CONCLUSION: 800CW-TATE demonstrated sufficient binding affinity, specific SSTR2-mediated tumor uptake, a favorable biodistribution, and high TBR. These features make this tracer very promising for use in MFGS and could potentially aid in safer and a more complete meningioma resection, especially in high-grade meningiomas or those at complex anatomical localizations.

ER stress and UPR activation in glioblastoma: identification of a noncanonical PERK mechanism regulating GBM stem cells through SOX2 modulation.

Patients with aggressive brain tumors, named glioblastoma multiforme (GBM), have a poor prognoses. Here we explored if the ER stress/unfolded protein response (UPR) is involved in the pathophysiology of GBM and may provide novel therapeutic targets. Immunohistochemical analyses of a tissue microarray containing primary GBM specimens showed strong variability in expression of the UPR markers GRP78/BiP, XBP1, and ATF4. Interestingly, high ATF4 expression was associated with poor overall survival suggesting involvement of PERK signaling in GBM progression. In vitro experiments using patient-derived neurospheres, enriched for GBM stem cells (GSCs), showed high sensitivity for the ER stressor thapsigargin (Tg) mainly via PERK signaling. In contrast, neurospheres-derived differentiated GBM cells were less sensitive likely due to lower UPR activity as indicated by comparative transcriptional profiling. Tg and Tunicamycin strongly reduced neurosphere forming ability of GSCs that was linked with potent PERK-dependent downregulation of SOX2 protein. Interestingly, SOX2 downregulation occurred directly via PERK, not requiring downstream activation of the PERK-UPR pathway. Moreover, PERK inactivation resulted in aberrant serum-induced differentiation of GBM neurospheres accompanied by persistent SOX2 expression, delayed upregulation of GFAP and reduced cell adherence. In conclusion, we provide evidence that PERK signaling contributes to the prognoses of primary GBM patients and identified PERK as a novel regulator of SOX2 expression and GSC differentiation. The role of PERK appeared to be pleiotropic involving UPR-dependent, as well as novel identified noncanonical mechanisms regulating SOX2. ER stress and PERK modulation appear to provide promising therapeutic targets for therapy in GBM.

The application of fluorescence techniques in meningioma surgery-a review.

Surgical resections of meningiomas, the most common intracranial tumor in adults, can only be curative if radical resection is achieved. Potentially, the extent of resection could be improved, especially in complex and/or high-grade meningiomas by fluorescence-guided surgery using 5-aminolevulinic acid (5-ALA), indocyanine green (ICG), or fluorescein. This review aims to summarize and evaluate these fluorescence-guided meningioma surgery techniques. PubMed and Embase were searched for relevant articles. Additionally, we checked reference lists for further studies. Forty-eight articles were included in the final analysis. 5-ALA fluoresced with varying sensitivity and selectivity in meningiomas and in invaded bone and dura mater. Although ICG was mainly applied for video angiography, one report shows tumor fluorescence 18-28 h post-ICG injection. Lastly, the use of fluorescein could aid in the identification of tumor remnants; however, detection of dural tail is highly questionable. Fluorescence-guided meningioma surgery should be a reliable, highly specific, and sensitive technique. Despite numerous studies reporting the use of fluorescent dyes, currently, there is no evidence that these tools improve the radical resection rate and long-term recurrence-free outcome in meningioma surgery without neurological deficits. Evidence regarding the effectiveness and increased safety of resection after the application of these fluorophores is currently lacking. Future research should focus on the development of a meningioma-targeted, highly sensitive, and specific fluorophore.