In this study we prospectively collected tissue specimens from CE and NE regions in patients with untreated GBM utilizing MRI-guided neurosurgical techniques to evaluate their physiologic MRI and histopathologic characteristics and to assess the correlation of in vivo MR parameters with histologic features of GBM. We observed that a number of histopathologic features and quantitative MRI parameters differ between CE and NE regions. These variables also demonstrate significant correlations suggesting that DSC perfusion and DW MRI are capable of detecting heterogeneous histopathologic features within anatomically distinct portions of treatment-naive GBM.
The results of our study suggest that the tissue sampling site criteria of either rCBV > 3, ADC < 1200, or CNI > 2 within CE and NE regions are highly effective in identifying tumor-containing regions in patients with treatment-naive GBM. This is of particular interest for defining tumor burden in NE regions, where being able to distinguish reactive edema from biologically active infiltrative tumor is clinically important. Previous studies in patients with newly diagnosed GBM have shown that increased volumes of ADC < 1200 or CNI > 2 within the T2 hyperintense lesion were associated with poor overall survival prior to initial surgical resection.41
Additionally, Saraswathy et al.42
have demonstrated that increased volumes of these 3 physiologic MR parameters within the T2 hyperintense lesion at the postsurgery and pretreatment examinations are associated with decreased survival. Future studies will follow up on these findings by considering these measures as candidates for defining physiologic as opposed to anatomic tumor burden in serial studies of response to therapy.
From the comparison of histologic parameters within CE versus NE regions, it was clear that the hallmark histopathologic features of GBM, including necrosis and complex microvascular hyperplasia, were present in a significant number of CE regions but either absent or rare in NE regions. Despite this, the majority of NE regions (81%) did contain tumor cells, as indicated by a positive tumor score. Additionally, a median Ki-67 score of 4.55% was observed among the NE regions. Interestingly, the hypoxia score was zero for all except one of the NE tumor specimens. Given that well-oxygenated tissues are sensitive to radiation damage, this finding raises the possibility that tumor in NE regions of GBM may be more effectively treated by radiation or DNA damaging chemotherapies than is tumor in CE regions. This has significant implications for planning boost volumes for radiation therapy. Of further interest in designing therapy protocols is the presence of simple microvascular hyperplasia in 65% of the samples from NE regions, which indicates that although there is increased vasculature in these areas, the vessel permeability is limited.
The relationship between increased intratumoral perfusion and malignant growth has been associated with hypoxic cellular conditions within GBM.43–49
The morphologic feature of CE on T1-weighted MRI, which reflects the disruption of the blood–brain barrier, has previously been thought of as a marker of elevated complex microvasculature, but several studies have demonstrated a discrepancy between regions of CE and microvascular density. In this study, a strong positive correlation was observed for rCBV and rPH in CE regions with the histopathologic features of proliferation, cell density, and microvascular hyperplasia. This suggests that DSC MR imaging is capable of capturing a clinically useful, noninvasive assessment of the CE lesion for newly diagnosed GBM that can be utilized on an individual basis to provide quantifiable biomarkers.7,12,18–21,32,50–52
The pattern of microvascular proliferation can vary widely in GBM.53–60
Areas of infiltrative tumor often contain delicate vasculature that resembles normal cerebral vasculature. Regions with more malignant tumor features contain both simple and complex microvascular hyperplasia. Simple microvascular hyperplasia has been characterized by hyperplastic capillaries with increased endothelial cellularity and luminal patency.32,53–60
Complex microvascular hyperplasia is identified by large collections of capillaries with partially to completely thrombosed slit-like lumen, resulting in minimal perfusion to the surrounding tissue.32,53–60
The correlation of rCBV and rPH with microvascular morphology suggests that DSC perfusion imaging is sensitive to all 3 types of vascular morphology and can be used to guide the acquisition of tumor samples, irrespective of the patterns of CE.
The results of our study challenge the widely held concept that T2 FSE and FLAIR hyperintense non-enhancing regions have limited malignant potential. By using physiologic MRI parameters to target tissue specimens from NE regions, we were able to identify areas with histopathologic features corresponding to biologically active tumor. Interestingly, rADC and rFSE were found to be associated with tumor score in the NE region, but this was not the case in CE regions. This was thought to be due to partial volume averaging of tumor and necrosis within voxels that correspond to the CE region, which makes the image intensities higher than would have otherwise been observed. Previous investigations have also yielded mixed results. Sadeghi et al.,18
in their investigation of untreated GBM, reported an inverse correlation of rADC with microvascular density but no inverse correlation between rADC and tumor cell density in their examination of 33 tumor tissue specimens. On the other hand, several other groups have reported finding an inverse correlation between rADC and cell density.29–32
These conflicting findings are likely due to the differences in sampling strategies for obtaining tissue samples, as well as the complex interactions of water diffusivity within the heterogeneous cellular environment of GBM.
Our study was specifically designed to look at regions within the enhancing and non-enhancing lesions with physiologic imaging parameters suggestive of tumor. Given that it was not always possible to exactly target the areas requested, as well as the differences in scale of the image resolution versus the amount of tissue evaluated using histology, the fact that 90% of the enhancing samples and 81% of the non-enhancing samples had a positive tumor score is encouraging. To determine whether the imaging data can accurately define lesion boundaries would require a sampling strategy that also targeted regions of non-enhancing tumor with physiologic parameters unlikely to correspond to tumor—however, this may not be practical from the patient's perspective given the risk of taking tissue from regions of normal brain.
One limitation of this study is that the potential exists for misregistration between biopsy sites and MRI uploaded to the neuronavigational device theatrically when significant amounts of brain shift occur. Such a mismatch could lead to inaccuracy in DSC and DWI measurements—however, several steps were utilized to negate any possible brain shift that may have occurred following dural opening. We sought to minimize significant amounts of brain shift by (a) performing accurate intraoperative neuronavigational system registration to the patient's facial anatomy, (b) avoiding substantial loss of cerebrospinal fluid, (c) watching for intraoperative brain swelling, (d) testing registration accuracy against visible cortical landmarks immediately prior to biopsy sampling, and (e) using standardized ROIs of sufficient size to compensate for any minimal shift in brain location. We believe that any minimal amount of brain shift that may have occurred prior to biopsy sampling did not result in significant sampling error or adversely affect the results of this study. Further prospective studies may consider implementing the use of closed brain biopsies to negate this potential limitation.
Our investigation suggests that the heterogeneous distribution of histopathologic features within GBM can be reliably identified by physiologic MRI. The associations of imaging and histologic parameters observed indicate that these physiologic imaging variables are likely to be of interest for defining residual postsurgical tumor burden and may need to be considered in expanding the definition of the Revised Assessment in Neuro-Oncology criteria for assessing response to therapy.61