Scanning fiber endoscope improves detection of 5-ALA induced protoporphyrin IX fluorescence at the boundary of infiltrative glioma (original) (raw)
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Neuro-Oncology, 2011
Extent of resection is a major goal and prognostic factor in the treatment of gliomas. In this study we evaluate whether quantitative ex vivo tissue measurements of d-aminolevulinic acid-induced protoporphyrin IX (PpIX) identify regions of increasing malignancy in low-and high-grade gliomas beyond the capabilities of current fluorescence imaging in patients undergoing fluorescence-guided resection (FGR). Surgical specimens were collected from 133 biopsies in 23 patients and processed for ex vivo neuropathological analysis: PpIX fluorimetry to measure PpIX concentrations (C PpIX ) and Ki-67 immunohistochemistry to assess tissue proliferation. Samples displaying visible levels of fluorescence showed significantly higher levels of C PpIX and tissue proliferation. C PpIX was strongly correlated with histopathological score (nonparametric) and tissue proliferation (parametric), such that increasing levels of C PpIX were identified with regions of increasing malignancy. Furthermore, a large percentage of tumor-positive biopsy sites (∼40%) that were not visibly fluorescent under the operating microscope had levels of C PpIX greater than 0.1 mg/mL, which indicates that significant PpIX accumulation exists below the detection threshold of current fluorescence imaging. Although PpIX fluorescence is recognized as a visual biomarker for neurosurgical resection guidance, these data show that it is quantitatively related at the microscopic level to increasing malignancy in both low-and high-grade gliomas. This work suggests a need for improved PpIX fluorescence detection technologies to achieve better sensitivity and quantification of PpIX in tissue during surgery. G liomas account for over 70% of all primary brain tumors. 1 -3 Currently brain tumor research seeks to find diagnostic and prognostic biomarkers for gliomas that would inform both surgical and/or medical treatment. Extent of resection is increasingly accepted as critical to optimal surgical treatment and patient prognosis. 4,5 Image guidance facilitates neurosurgical resection but is subject to intraoperative brain shift, which degrades the accuracy of relating navigational information with the surgical field presented to the surgeon. 6 -12 The use of d-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence-guided resection (FGR) of brain tumors has gained increased clinical interest. 13 FGR provides the operating surgeon with an intuitive, real-time surgical guidance tool for delineating tumor tissue that mitigates some of the challenges of image-guided neuro-navigation caused by intraoperative brain shift and deformation. ALA-induced PpIX FGR studies support its utility as a surgical tool for intraoperative delineation of a tumor. For example, the largest study to date, a multicenter, randomized phase III clinical study of ALA-induced PpIX for FGR of malignant gliomas, showed a significant improvement in gross total resection of the contrast-enhancing tumor (65% vs 36%), as well as a higher 6-month progression-free survival (41.0% vs 21.1%), in the FGR group compared with the conventional white-light treatment group. 14 -16 In a recent study of malignant gliomas, we found that visible levels of ALA-induced PpIX fluorescence correlate with tumor burden and World Health Organization (WHO) histopathological score of resected specimens. 17 To date, intraoperative detection of PpIX fluorescence for tumor delineation has largely been subjective (i.e., a modified surgical microscope is used to visualize "pink" fluorescence). 15 -21 This approach is limited in its sensitivity for identifying low (but significant) levels of accumulated PpIX in a tumor, potentially leaving some amount of resectable tumor unidentified. 22 Indeed, we recently reported preliminary in vivo results in human brain tumors 23 that indicate PpIX fluorescence may be a tumor-targeting biomarker with a diagnostic performance that exceeds subjective visible assessments when measured quantitatively with a new fiber optic approach. 24 Thus, even though ALA-induced PpIX fluorescence has been shown to be a successful biomarker for surgical resection of malignant glioma, the subjective assessments used to date do not appear to be sufficiently sensitive or quantitative to exhaust its full potential. As a result, establishing the underlying relationships between ALA-induced PpIX concentration in brain tumor tissues and their histologically determined malignancy profile is critical to further development and optimization of FGR.
The use of 5-aminolevulinic acid induced protoporphyrin IX fluorescence in cases of suspected malignant glioma helps surgeon to perform a more radical but safe surgery with preservation of neurological functions and improvement in progression-free survival periods. The Department of Neurosurgery, Hospital Sungai Buloh adopted this new technique with neuronavigation. We did a retrospective analysis of confirmed malignant glioma cases done at our centre over a period of 5 years, from 2008 till 2012. All patients with malignant glioma were divided into 2 groups; with and without 5-ALA fluorescence assisted surgeries. The maximum thickness of post-operative residual tumour was measured. From a total of 23 cases, 10 cases (5 grade III and 5 grade IV) were 5-ALA fluorescence assisted surgery and remaining 13 cases (1 grade III and 12 grade IV) were performed without the fluorescence. The mean maximum thickness of residual tumour in the 5-ALA fluorescence group was 26.1mm and the non-5-ALA fluorescence group was 31.2mm, but statistically the difference was not significant (p=0.431). In the 5-ALA fluorescence group, four patients (40 percent) with post-operative maximum thickness residual tumour ≤2cm, while remaining 6 patients (60 percent) with residual tumour >2cm. In the non-5-ALA fluorescence group, only 2 patients (15.4 percent) with post-operative maximum thickness of residual tumour ≤2cm and the remaining 11 patients (84.6 percent) with residual tumour >2cm (p=0.341). The use of 5-ALA fluorescence in malignant glioma surgery does not significantly extend the degree of cytoreduction compared to conventional method without the use of 5-ALA fluorescence.
Cancers
5-aminolevulinic acid (5-ALA)-induced PpIX fluorescence is used by neurosurgeons to identify the tumor cells of high-grade gliomas during operation. However, the issue of whether 5-ALA-induced PpIX fluorescence consistently stains all the tumor cells is still debated. Here, we assessed the cytoplasmatic signal of 5-ALA by fluorescence microscopy in a series of human gliomas. As tumor markers, we used antibodies against collapsin response-mediated protein 5 (CRMP5), alpha thalassemia/mental retardation syndrome X-linked (ATRX), and anti-isocitrate dehydrogenase 1 (IDH1). In grade III–IV gliomas, the signal induced by 5-ALA was detected in 32.7–75.5 percent of CRMP5-expressing tumor cells. In low-grade gliomas (WHO grade II), the CRMP5-expressing tumor cells did not fluoresce following 5-ALA. Immunofluorescence with antibodies that stain various components of the blood–brain barrier (BBB) suggested that 5-ALA does not cross the un-breached BBB, in spite of its small dimension. To conc...
2011
Object-Accurate discrimination between tumor and normal tissue is crucial for optimal tumor resection. Qualitative fluorescence of protoporphyrin IX (PpIX), synthesized endogenously following δ-aminolevulinic acid (ALA) administration, has been used for this purpose in highgrade glioma (HGG). The authors show that diagnostically significant but visually imperceptible concentrations of PpIX can be quantitatively measured in vivo and used to discriminate normal from neoplastic brain tissue across a range of tumor histologies. Methods-The authors studied 14 patients with diagnoses of low-grade glioma (LGG), HGG, meningioma, and metastasis under an institutional review board-approved protocol for fluorescence-guided resection. The primary aim of the study was to compare the diagnostic capabilities of a highly sensitive, spectrally resolved quantitative fluorescence approach to conventional fluorescence imaging for detection of neoplastic tissue in vivo. Results-A significant difference in the quantitative measurements of PpIX concentration occurred in all tumor groups compared with normal brain tissue. Receiver operating characteristic (ROC) curve analysis of PpIX concentration as a diagnostic variable for detection of neoplastic tissue yielded a classification efficiency of 87% (AUC = 0.95, specificity = 92%, sensitivity = 84%) compared with 66% (AUC = 0.73, specificity = 100%, sensitivity = 47%) for conventional fluorescence imaging (p < 0.0001). More than 81%
Clinical cancer research : an official journal of the American Association for Cancer Research, 2016
While extent of tumor resection is an important predictor of outcome in glioma, margin delineation remains challenging due to lack of inherent contrast between tumor and normal parenchyma. Fluorescence-guided surgery is promising for its ability to enhance contrast through exogenous fluorophores, however, the specificity and sensitivity of the underlying contrast mechanism and tumor delivery and uptake vary widely across approved and emerging agents. Rats with orthotopic F98 wild-type and F98 EGFR-positive gliomas received in vivo administration of IRDye680RD, 5-aminioleuvulinic acid, and ABY-029-markers of perfusion, protoporphyrin metabolism, and EGFR expression, respectively. Ex vivo imaging demonstrates the contrast mechanism-dependent spatial heterogeneity and enables within-animal comparisons of tumor-to-background ratio (TBR). Generally, ABY-029 outperformed PpIX in F98EGFR orthotopic tumor margins and core (50% and 60% higher TBR, respectively). PpIX outperformed ABY-029 in ...
Progress in Biomedical Optics and Imaging - Proceedings of SPIE, 2009
The highly malignant brain tumor, glioblastoma multiforme (GBM), is difficult to fully delineate during surgical resection due to its infiltrative ingrowth and morphological similarities to surrounding functioning brain tissue. Selectiveness of GBM to 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX (PpIX) is reported by other researchers to visualize tumor margins under blue light microscopy. To allow objective detection of GBM, a compact and portable fiber optic based fluorescence spectroscopy system is developed. This system is able to deliver excitation laser light (405 nm) in both the continuous and pulsed mode. PpIX fluorescence peaks are detected at 635 and 704 nm, using a fiber-coupled spectrometer. It is necessary to optimize the detection efficiency of the system as the PpIX quickly photobleaches during the laser illumination. A light dose of 2.5 mJ (fluence rate = 9 mJ/mm 2) is experimentally approved to excite an acceptable level of fluourescence signal arising from glioblastoma. In pulsed illumination mode, an excitation dose of 2.5 mJ, with a dark interval of 0.5 s (duty cycle 50%) shows a significantly shorter photobleaching time in comparison to the continuous illumination mode with the same laser power (p < 0.05). To avoid photobleaching (the remaining signal is more than 90% of its initial value) when measuring with 2.5 mJ delivered energy, the time for continuous and pulsed illumination should be restricted to 2.5 and 1.1 s, respectively.
Protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (5-ALA) is increasingly used as a fluorescent marker for fluorescence-guided resection of malignant gliomas. Understanding how the properties of the excitation light source and PpIX fluorescence interact with the surgical microscope is critical for effective use of the fluorescence-guided tumor resection technique. In this study, we performed a detailed assessment of the intensity of the emitted blue light and white light and the light beam profile of clinical grade operating microscopes used for PpIX visualization. These measurements revealed both recognized fluorescence photobleaching limitations and unrecognized limitations that may alter quantitative observations of PpIX fluorescence obtained with the operating microscope with potential impact on research and clinical uses. We also evaluated the optical properties of a photostable fluorescent standard with an excitation-emission profile similar to PpIX. In addition, we measured the time-dependent dynamics of 5-ALA-induced PpIX fluorescence in an animal glioma model. Finally, we developed a ratiometric method for quantification of the PpIX fluorescence that uses the photostable fluorescent standard to normalize PpIX fluorescence intensity. This method increases accuracy and allows reproducible and direct comparability of the measurements from multiple samples. Operating microscopes are commonplace within the neurosurgical operating theater and are a mainstay of surgical procedures for brain tumor removal 1. As the most important visualization tool in daily use for neurosurgery, operating microscopes are gaining advanced functionality by means of innovative illumination modes. To ensure surgical success, the neurosurgeon must fully understand the illumination properties and functionality of the microscope, especially within the context of fluorescence-guided tumor resection. The principle of fluorescence-guided tumor resection relies on the use of targeting agents with fluorescent properties that can be administered to patients before or during surgery. These agents are intended to accumulate within and around the tumor tissue or within the cells of the tumor, depending on the selectivity and actions of the fluorophore. The desired diagnostic result is to augment visual differentiation and detection of the tumor tissue margins during surgery based on fluorescence. The most notable recent example of a fluorescent agent developed for tumor detection in neurosurgery is 5-aminolevulinic acid (5-ALA), which is used to indicate the presence of tumors and the border regions of malignant gliomas 2. 5-ALA was recently designated as the first US Food and Drug Administration-approved agent for fluorescence-guided resection of high-grade gliomas 3. This prodrug results in fluorescent PpIX accumulation in tumors through a distortion in the metabolic conversion of 5-ALA to heme 4. Understanding both the nuances of its fluorescent properties and the effects that occur with changes in excitation intensity and duration of light
Biomedical Optics 2014, 2014
5-ALA-induced protoporphyrin IX (PpIX) fluorescence enables to guiding in intra-operative surgical glioma resection. However at present, it has yet to be shown that this method is able to identify infiltrative component of glioma. In extracted tumor tissues we measured a two-peaked emission in low grade gliomas and in the infiltrative component of glioblastomas due to multiple photochemical states of PpIX. The second emission peak appearing at 620 nm (shifted by 14 nm from the main peak at 634 nm) limits the sensibility of current methods to measured PpIX concentration. We propose new measured parameters, by taking into consideration the two-peaked emission, to overcome these limitations in sensitivity. These parameters clearly distinguish the solid component of glioblastomas from low grade gliomas and infiltrative component of glioblastomas.