We report the initial findings from coregistered ALA-induced PpIX fluorescence-enhanced resection of malignant gliomas in 11 patients with newly diagnosed GBM, with the goal of establishing the statistical significance of relationships between 1) quantitative assessments of MR image signals and the spatially coregistered qualitative fluorescence signatures determined intraoperatively, and 2) these same qualitative fluorescence determinations and the subsequent neuropathological evaluations of the biopsy specimens taken from these locations. Previous studies of ALA-induced PpIX fluorescence have reported encouraging results in guiding the neurosurgeon to achieve higher percentages of complete tumor resection,28,29
which has repeatedly been shown to correlate with patient survival.1,2,17,24,29,30
Although pioneering in scope, intent, and positive outcome for high-grade glioma surgery, those studies did not explicitly describe quantitatively (either spatially or in terms of preoperative MR image contrast characteristics) the relationship between the MR imaging signature and the degree of intraoperative PpIX fluorescence observed by the neurosurgeon.
One standard feature used to distinguish pathological tissue pre- and postsurgically is contrast enhancement on MR imaging after Gd injection. In this study we used two measures to quantify contrast enhancement on T1-weighted image volumes: 1) GdE from subtraction image volumes (pre- and postcontrast injection), and 2) nCRs on T1-weighted, postcontrast-injection image volumes. The GdE served as a surrogate measure for Gd proper–induced changes in the MR images, whereas nCR served as a surrogate measure for what the neurosurgeon most often uses in the operating room on T1-weighted, Gd-enhanced images. We found evidence of a significant difference in both GdE and nCR between fluorescing and nonfluorescing tissues. The PpIX fluorescence can provide the neurosurgeon with real-time information for differentiating tumor from normal tissue, independent of any image-guidance system. The degree to which preoperative MR image signatures are predictive of intraoperative PpIX fluorescence is of practical importance in understanding the potential role of fluorescence during surgery.
In this study, the odds ratio for intraoperative fluorescence for tumor tissue was highly favorable (OR 7.32). The PPV for tissue with observable fluorescence to be tumor confirmed the effective accumulation of PpIX in tumor tissue (95%) and abnormal tissue (99%). The sensitivity and specificity measures of intraoperative fluorescence for tumor tissue were also favorable (75 and 71%, respectively). Similar to previous studies, intraoperative macroscopic fluorescence showed sensitivity limitations. Low NPVs (26%) point to the need for improving the detection limit of intraoperative fluorescence imaging. Several groups, including ours, are using intraoperative probes that take advantage of the spectroscopic signature of PpIX (or similar fluorophores) and have signal detection sensitivities that exceed those of the current surgical microscope.3,9,28,29,36,38,40
Selective accumulation of PpIX in neoplastic tissues may involve changes in intracellular metabolism, increased ALA uptake, vascularization, proliferation, differentiation, or blood-brain barrier breakdown.4
Here, histopathological scores used pathological characteristics to categorize specimens into four distinct levels, in a manner similar to WHO grading of a surgical specimen. This analysis provided a semiquantitative assessment, analogous to histopathological grading, of the biological aggressiveness of each specimen, and accounted for other biological characteristics such as degree of differentiation, cellular metabolism, and environmental changes. Tumor burden scores offered another important metric, analogous to tumor infiltration, which was used to relate levels of fluorescence to the presence of tumor cells. These parameters were chosen in our analysis because previous studies have indicated that mitochondrial content and cellular density play a significant role in the selective accumulation of PpIX.4
An analysis of association performed using chi-square statistics demonstrated a significant relationship between the levels of intraoperative fluorescence and histopathological score (χ2
= 58.8, p < 0.001), between fluorescence and tumor burden (χ2
= 42.7, p < 0.001), and between fluorescence and necrotic burden (χ2
= 30.9, p < 0.001). The Spearman rank correlation analysis provided statistically significant correlation coefficients of 0.51 for fluorescence and histopathological score, and 0.49 for fluorescence and tumor burden, but no statistically significant correlation between fluorescence and necrotic burden. This lack of a direct correlation between degree of fluorescence and necrosis could be attributed to poor fluorescence in heavily necrotic tissue as a result of fewer viable PpIX-producing cells.
The neuropathological analysis increases confidence in the accuracy of intraoperative PpIX fluorescence for abnormal tissue. The data support the idea that biological aggressiveness and burden of tumor cells are important, synergistic factors that explain accumulation of PpIX to observable levels. This study uses parameters similar to conventional histopathological characteristics to provide explanatory variables for the selective accumulation of PpIX in resected tumor tissue. These results provide a framework for further understanding of PpIX fluorescence, as observed intraoperatively, relative to conventional notions of tumor grading and infiltration.
The Spearman rank correlation coefficients for histopathological score (0.51) and tumor burden (0.49), however, indicate that although these variables are important in understanding accumulation of PpIX, other biological characteristics, such as levels of cellular growth, endothelial proliferation, blood-brain barrier disruption, and possibly glial-cell phenotype are also likely to be important.4,7,11
This study adds two major contributions to the neurosurgical literature regarding FGR that differ from previous clinical studies in which PpIX fluorescence guidance was used. First, this work provides evidence that a strong correlation exists between features on preoperative MR imaging by using patient-specific image-guidance spatial information and intraoperative fluorescence, to the degree that spatially coregistered features on MR imaging are predictive of corresponding intraoperative fluorescence. Second, this work reports that a strong correlation exists between tumor aggressiveness and corresponding intraoperative fluorescence (using patient-specific image-guidance spatial information for each biopsy specimen), to the degree that spatially coregistered intraoperative PpIX fluorescence is predictive of tumor aggressiveness. These contributions are of practical importance for the neurosurgical community to integrate FGR into conventional image-guidance protocols.
One limitation of our study is registration error of our image-guidance system and intraoperative brain shift and deformation, with subsequent degradation of navigational accuracy over the course of a surgical procedure. In an attempt to reduce errors in analysis that occur as a result of navigational inaccuracies, we constrained data points by using an ROI methodology. Although this methodology reduces this error, it does not completely eliminate it. Second, determination of intraoperative fluorescence in this study is nonquantitative, as it is in all current commercial adaptations of operating microscope systems. Quantitative determination of fluorescence will refine our understanding of the relationship, so that a standard can be applied between patients and across studies. Our group is actively working on this challenge. Furthermore, our group is currently developing a methodology to merge our fluorescence information with our stereovision system34
to create 3D surface maps of the surgical field, with fluorescence map overlays of these reconstructions onto the MR image-guidance cross-sectional images, using both qualitative and quantitative fluorescence information. Third, current fluorescence surgical systems are sometimes not able to detect low levels of fluorescence (for example, NPV 26%), which can lead to a high number of false negatives, as was observed in this study (that is, low NPVs). We have developed and are currently in clinical testing phases of an intraoperative probe that quantifies PpIX levels at submicrogram per milliliter concentrations, with the aim of distinguishing normal from abnormal tumor tissue in a more sensitive manner than is possible with our current fluorescence surgical microscope. Last, current fluorescence systems detect only fluorescence of surface tissue. Overlying nonfluorescent tissue, including blood or necrotic debris, will prevent visualization of deeper, fluorescing tumor. Systems capable of detecting fluorescing tissue at depth are also under development.