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Proton magnetic resonance spectroscopy (1H-MRS) is increasingly used in clinical studies of brain tumor to provide information about tissue metabolic profiles. In this study, we evaluated changes in the levels of metabolites predominant in recurrent glioblastoma (rGBM), to characterize the response of rGBM to anti-angiogenic therapy. We examined thirty-one rGBM patients treated with daily doses of cediranib, acquiring serial chemical shift imaging data at specific time points during the treatment regimen. We defined spectra from three regions of interest (ROIs)—enhancing tumor (ET), peritumoral tissue (PT), and normal tissue on the contralateral side (cNT)—in post-contrast T1-weighted images, and normalized the concentrations of N-acetylaspartate (NAA) and choline (Cho) in each ROI to the concentration of creatine in cNT (norCre). We analyzed the ratios of these normalized metabolites (i.e., NAA/Cho, NAA/norCre, and Cho/norCre) by averaging all patients and categorizing two different survival groups. Relative to pre-treatment values, NAA/Cho in ET was unchanged through day 28. However, after day 28, NAA/Cho significantly increased in relation to a significant increase in NAA/norCre and a decrease in Cho/norCre; interestingly, the observed trend was reversed after day 56, consistent with the clinical course of GBM recurrence. Notably, ROC analysis indicated that NAA/Cho in tumor shows a high prediction to 6-month overall survival. These metabolic changes in these rGBM patients strongly suggest a direct metabolic effect of cediranib, and might also reflect an anti-tumor response to anti-angiogenic treatment during the first two months of treatment. Further study is needed to confirm these findings.
Glioblastoma multiforme (GBM) is a severe and generally fatal brain tumor, with an annual incidence of approximately 9,000 in the United States. Despite aggressive treatment strategies involving surgery, radiation and cytotoxic chemotherapy, the average survival time for a patient with GBM is less than 1 year, and fewer than 5% of patients survive 5 years or more (1). Innovative therapeutic approaches are desperately needed for this patient population. GBM is typically characterized by marked angiogenesis and paradoxically severe hypoxia and necrosis (2-5). Angiogenesis in GBM is mediated by vascular endothelial growth factor (VEGF) (6-8), which leads to dysfunctional and highly permeable microvessels, characterized by abnormalities in pericyte coverage and basement membrane thickness (2, 9-11).
Bevacizumab (Avastin, Genentech/Roche), a humanized monoclonal antibody that targets VEGF-A ligand, was approved by the US Food and Drug Administration in May 2009 for use as a monotherapy for recurrent GBM (rGBM), based on phase II evaluations (12). However, the mechanisms by which anti-angiogenic therapies benefit these patients are not well understood. Jain and colleagues have demonstrated that anti-VEGF therapies “normalize” the tumor vasculature. Their findings in both pre-clinical models and a clinical trial of rectal cancer have indicated that anti-VEGF therapy leads to reductions in microvessel density and mean blood vessel diameter, basement membrane thickness, tumor interstitial pressure and vasculature permeability as well as enhanced pericyte coverage (10, 13, 14).
A phase II clinical trial of cediranib (AZD2171, AstraZeneca Pharmaceuticals, UK), a potent oral pan-VEGF receptor TK inhibitor, demonstrated that vascular normalization was induced at 24 hours and lasted to 28 days in recurrent GBM patients, as determined by structural and functional MRI metrics (15, 16). However, these functional and structural improvements were reversed, and the tumor reverted to an abnormal state with further continuation of cediranib therapy. These findings thus suggest there may be a “normalization window” during which delivery of chemotherapeutics may be optimized.
A preclinical study of GBMs in mice demonstrated that use of cediranib can lead to a decrease in edema, by vascular normalization, as well as prolonged survival even as tumor growth persists (17). The study suggested that the benefits of anti-angiogenic therapies might be partially due to anti-edema effects rather than direct anti-tumor effects. However, other investigators have noted direct anti-tumor effects of VEGF blockade (18), as well as tumoristatic activities—i.e., tumor growth inhibition and tumor cell apoptosis—in a broad range of human tumors (19-21), and a possible inhibitory effect on stem-cell-like glioma cells (22). Thus, although the extent of vascular normalization after one dose of cediranib correlates with both progression-free survival (PFS) and overall survival (OS) in recurrent GBM patients (23), underscoring the clinical importance of vascular normalization, the degree of anti-tumor effect, if any, beyond vascular normalization in this class of therapies remains uncertain in humans.
Early magnetic resonance spectroscopy (MRS) studies showed clear differences between the spectral profiles of tumor and normal brain tissues. For example, choline (Cho) is typically elevated in brain tumors and metastases, potentially because of increased cellular turnover and the accelerated membrane synthesis that occur in rapidly dividing cancer cells (24, 25). Levels of N-acetylaspartate (NAA), regarded as a neuronal marker, decrease in any disease that adversely affects neuronal integrity (26). Hence, relative to normal healthy brain tissue, neoplastic tissue generally exhibits elevated Cho concomitant with decreased NAA (27), a hallmark widely used in clinical practice. Given these metabolic characteristics of tumors, 1H-MRS may be able to improve demarcation of cancerous brain tissue when used in combination with the high-quality anatomical data provided by conventional MRI techniques.
In this study, we compared 1H-MRS data and conventional MRI data in a group of patients undergoing cediranib monotherapy for rGBM. Our results suggest that 1H-MRS confirms the findings from MRI, and provides additional information to improve understanding of cancer responses to anti-angiogenic agents in rGBM patients.
Thirty-one patients (mean age 53.7, range 20-77) with recurrent GBM (rGBM) were recruited for this study. Every patient underwent surgical resection and radiochemotherapy after initial diagnosis and pathologic confirmation of GBM. At the time of enrollment into the study, all patients had tumor recurrence, as determined by MRI and/or neurological deterioration.
All patients received an oral dose of cediranib (45 mg) daily, which was reduced as necessary (15, 16) until there was radiographic or clinical evidence of disease progression. Neurological and physical examinations and MRI studies were performed throughout the course of treatment.
MR studies were performed using a 3T MRI scanner (Tim Trio, Siemens, Malvern, PA) at a series of time points: -5, -1, 1, 26-28, 54-56, and 110-112 days from the start of cediranib treatment.
We performed serial 2D Chemical Shift Imaging (CSI) (28) using Point Resolved Spectroscopy (PRESS) (29) for signal pre-localization and Outer Volume Saturation (OVS) (30) to minimize contamination from subcutaneous fat. Water suppression was achieved with a modified Chemical Selective Saturation (CHESS) (31) method known as water suppression enhanced through T1 effects (WET) (32). Acquisition parameters included weighted k-space sampling TR/TE=1700/135(144) ms, NA=3, nominal resolution 1×1×1.4 cm3. First- and second-order shimming was performed automatically, followed by manual adjustment. Data selected for analysis had a typical Full Width at Half Maximum (FWHM) in the range of 20Hz for the water line.
The MRI protocols used conventional sequences (T1, T2, FLAIR, post-Gd T1, volumetric post-Gd image) and dynamic sequences (DCE, DSC, DTI), as reported in Batchelor et al. (15, 16) (see the details in Appendix A).
For 11 out of 31 patients, MRS quality was inadequate to reliably detect distinct signals from the metabolites in the tumor. In some of these patients, MRS quality was compromised by tumor location near the skull; because in these cases shimming could not be adequately performed, the signal was considerably contaminated by fat (5 of 11 patients). Additionally, in some patients, marked necrosis in the lesion caused indistinguishable peaks in the spectra (8 of 11 patients). Those spectra were objectively excluded from the data before further processing to ensure reliable analysis. The spectra obtained from 20 of the total group of 31 subjects were included in quantitative measurements.
We analyzed the spectroscopic raw data using LC Model 6.1 software (Provencher, Ontario, CA (33)), with a manual script written in Matlab. The spectra were grouped in three regions of interest (ROIs), defined by the corresponding T1-weighted post-contrast images at baseline for: (1) enhancing tumor (ET), (2) non-enhancing surrounding tumor (peritumoral tissue) (PT), and (3) normal tissue on the contralateral side of tumor (cNT). The location and numbers of the voxels in each ROI were serially consistent across all time points, although there were changes in the intensity of enhancement as a consequence of treatment. To accurately assess tumor metabolism, the voxels in the enhancing tumor were selected so as to avoid areas of necrosis, hemorrhage, calcification, cysts, or ventricles. Only fitted spectra with standard deviation (%SD) lower than 25%, per Cramér-Rao lower bounds automatically provided by the LC model, were accepted. There was no subjective spectral apodization. The concentrations of all metabolites were normalized to the normal side creatine concentration (norCre).
We examined the changes in metabolite concentrations during treatment by analyzing the ratio of NAA to Cho and the ratios of NAA and Cho to normal side Cre (norCre). Typical spectra (as shown in Figure 1) demonstrate that the NAA peak is higher than the Cho peak in normal tissue, whereas the ratio is reversed in tumor, i.e. Cho peaks above NAA.
Changes in the MRI parameters were additionally analyzed (see the details in Appendix B), and are displayed in the supplementary data. We assessed the vascular indexes by analyzing changes in the contrast-enhanced T1-weighted tumor volume (CE-T1), vessel size (VS), and Ktrans(in this non-flow-limited state, assumed to mainly represent permeability, P) within regions of enhancement. We quantified the water-related indexes, the functional consequences of vascular normalization, using three different techniques that indicate hydration level. We measured: 1) T2-weighted abnormality fluid-attenuated inversion recovery (FLAIR), 2) trace apparent diffusion coefficient of water (ADC), and 3) extracellular extravascular space fraction (Ve), within regions of enhancement. We also derived the absolute T1 relaxation time constant values from variable flip angle T1 mapping sequences.
We analyzed the MRS/MRI data in relation to overall survival (OS), and based on the six-month survival threshold, categorized all patients as ‘high overall survival’ or ‘low overall survival’ responders. Metabolite ratios on days 1, 28, and 56 were compared with baseline ratios.
We computed Student's t-test p-values against the null hypothesis, which assumes no change in metabolite ratios during treatment. The changes in MRI parameters (i.e., CE-T1, VS, P, FLAIR, ADC, and Ve) were analyzed in a similar way. Statistical significance determined by Student's paired t-test was accepted at a confidence level of 0.95 (* p<0.05). We performed an ROC statistical analysis to determine how predictive the MRS measurements were of 6-month survival. Numerical data were presented as average ± one SD. The number of subjects included in the analysis at each time point is given in Table S1 (Supplemental Data).
No corrections were made for T1 or T2, or for possible variations in water concentration between normal and tumor tissues. Our data analyses are strictly semi-quantitative, as routine clinical studies do not allow for data acquisition to correct for metabolite and water relaxation. In addition, we have assumed tissue water concentration in the tumor is similar to that in normal brain tissue; hence, we calculated only the apparent metabolite concentrations.
Table 1 shows the averaged values of three metabolite ratios (i.e., NAA/norCre, Cho/norCre, and NAA/Cho) with standard deviations, the coefficients of variation, and p values tested by student t-statistics between two pre-treatment visits in three ROIs. Relatively small mean differences were observed between two baselines, with moderate but acceptable coefficients of variation (< 30%).
Figure 2 demonstrates a representative example of serial T1 post-contrast MR images and raw spectra in one representative voxel (denoted by the blue-lined box) of the enhancing tumor region during the time-course of treatment. The spectra display dynamic changes of each metabolite's peak in the range of 0.5–4 ppm.
Figure 3 shows the changes in the NAA/norCre, Cho/norCre, NAA/Cho ratios relative to pretreatment values, as well as lipid and lactate levels normalized by norCre, averaging across all eligible patients.
The primary metabolic index in Figure 3a, NAA/Cho, provides a combined picture of the most commonly used diagnostic criterion of metabolic changes for some types of tumors (27, 34, 35). Many studies have reported lower NAA/Cho ratio in tumors (compared to normal tissue) due to decreased levels of NAA and/or increased levels of Cho (36-39); such findings are frequently interpreted as resulting from the replacement of normal brain tissue by cancerous tissue. Though averaged, NAA/Cho in both enhancing tumor and peritumor regions showed no significant change until 28 days; there was significant increase between days 28 and 56 (p=0.01), then a subsequent decrease. In the contralateral normal tissue, NAA/Cho was relatively constant.
As illustrated in Figure 3b, the ratio of lipids and lactate (including all lipid peaks in the range of 0.5-2 ppm) in enhancing tumor versus Cre on the contalateral normal side (norCre) decreased significantly on day 56. Like the other metabolites, (lipids and lactate)/norCre was relatively stable in the contralateral normal tissue.
Figures 3c and 3d show the individual behavior of the metabolites normalized by Cre in the contralateral tissue (norCre). Figure 3c illustrates a sharp increase in NAA/norCre in the enhancing tumor after a single dose of cediranib. The increase until day 56 (p=0.02), at which time point the value began to decrease until the end of the study (p=0.04). In the peritumoral region, NAA/norCre increased until day 28, and remained relatively constant close to the normal value (i.e., 1.5) until day 112 (p=0.04).
In contrast, Cho/norCre (Figure 3d) in the enhancing tumor showed a different pattern: an increase up to day 28 (p=0.03), a decrease from 28-56 days, then no change until the end of the study (day 112). The decrease in Cho reached statistical significance (p=0.047) between days 28 and 56. A similar trend was found in the peritumoral region. In the contralateral normal tissue, both NAA/norCre and Cho/norCre remained relatively constant.
We also analyzed the changes in the MR parameters relative to baseline for the same subset of 20 patients; for reference, results are shown in Figure S1 (Supplementary Data).
The MRI data from the subset of patients from whom we also acquired analyzable MRS data were similar to the MRI findings of others in the whole 31-patient sample (16). In both the larger data set of 31 patients and the subset of 20 patients from whom we acquired MRS data, the volume of contrast-enhanced tumor (CE-T1) decreased until day 28 and thereafter began to increase. An abrupt and substantial decrease in Ktrans (mean ~ -70%) was noted immediately after the first dose of cedirinib (day 1). The relative tumor vessel size also decreased until day 28, and began to increase after day 28. We observed sustained decrease in vasogenic edema, demonstrated by reduced FLAIR lesion volumes, ADC, and Ve for the duration of the therapy. These findings suggested a high probability of anti-permeability effects of cedranib until day 28.
We analyzed MRS/MRI findings in relation to patients' overall survival times, based on the six-month survival threshold. Early post-treatment time points (i.e., days 1, 28, 56) may be the most important for treatment management because early indications of therapeutic outcome provide better opportunity to optimize therapeutic intervention and improve survival (40).
As shown in Figure S2 (Supplementary Data), the MRS data indicated no significant difference in the ratios of the metabolites, including NAA/norCre, Cho/norCre, NAA in tumor/NAA in the contralateral normal tissue (tumNAA/norNAA), and Cho in tumor/Cho in the contralateral normal tissue (tumCho/norCho); similarly, the group differences in MRI measurements of ‘high overall survival’ and ‘low overall survival’ responders were considered as having no significant effect. (Figure S3 in Supplementary Data). However, NAA/Cho (Figure 4), the most commonly used clinical MRS measure for discriminating normal and abnormal tissues (27), notably showed an increase in the ‘high overall survival’ group (15%, 9%, 40% with p<0.05 on days 1, 28, and 56, respectively), while showing a decrease in the ‘low overall survival’ group (-12%, -10%, -20% on days 1, 28, and 56, respectively). Based on this finding, we performed ROC analysis to determine the probability that NAA/Cho predicts 6-month survival. In Table 2, the values of an area under the ROC curve (AUC) and p-values at early time points, particularly days 28 and 56, demonstrated the high possibilities (74% and 95%) and the significances (0.02 and 0.01).
We compared the relative changes in the ratios of the three metabolites before and after one dose of cediranib to the changes in the absolute values of norCre and the T1 relaxation time constant in Figure S4 (Supplementary Data). The comparison showed subtle changes in norCre and T1 time constant after one dose, confirming that changes in NAA/norCre are independent of norCre and T1 changes.
In this clinical study we sought to identify metabolic changes in rGBM, using MRS to distinguish the changes induced by cediranib treatment. We observed elevated choline, lactate, and lipid peaks, with very low NAA peak, in enhancing lesions as well as a reverse metabolite profile in contralateral normal tissue at baseline. The mean values of NAA/Cho were 2.4 in the enhancing tumor and 5.0 in normal tissue. These results mirror the classic patterns of metabolite peaks seen in high-grade gliomas and the mean values that have been measured in other published reports (27, 39, 41-43). Our results also confirm the ability of MRS to evaluate tumor response to treatment (27, 39, 44-48). Our results both extend earlier studies to track metabolic changes induced by an anti-angiogenic agent in tumor tissue, and report several new findings.
The first new observation is the consistency of the NAA/Cho ratio with increased concentration of both Cho and NAA in tumoral as well as peritumoral regions during the vascular normalization window of 28 days (15). One interpretation of these findings is that tumor cells are not directly killed during the initial normalization window, and the marked changes in tumor enhancement observed in conventional MRI reflect anti-vascular effects of the anti-angiogenic drug. This interpretation is compatible with preclinical results reported (17). The decrease in tumor size and the consequential shift in brain seen until one month after treatment initiation might reasonably prompt one to conclude that NAA/norCre and Cho/norCre increases are attributable to partial volume effects. A statistically significant decrease in hydration level (i.e., vasogenic edema, as seen by ADC) during one month may also account for these changes.
The second observation is a significant increase in NAA/Cho with a significant reduction in Cho and an increase in NAA after 28 days. This finding suggests that cediranib has a direct effect on cellular metabolism in rGBM patients—an effect that is temporally separated from its anti-vascular effects and distinct from preclinical models of cediranib—possibly due to the longer survival times seen in humans compared to animal models of GBM. Further evidence of this direct metabolic effect is supported by a significant decrease in observed lipids and lactate after day 28; it has been previously shown that the spectra from active glioblastoma contain elevated peaks of lipids (27, 42). Snuderal et al. [Cancer Research, submitted] observed reduced cellular density in the central area of the tumor in the autopsies of five cediranib-treated patients included in our study population. This morphological finding is consistent with the metabolic changes we detected. Also of note is that the delayed anti-tumor effect associated with anti-angiogenic treatment has also been reported in other tumor types (49, 50).
Interestingly, ADC in MRI showed a decrease, primarily because of the significant anti-permeability effect of cediranib, overwhelming its cytotoxic effect, as an increase in ADC has been shown to correlate with cell-killing mechanism for cytotoxic therapies. At baseline the value of ADC was already high, and after edema greatly reduced, ADC dropped to a normal level especially within 28 days, indicating a window of vascular normalization. However, between 28 and 56 days, there was no significant decrease in ADC; in fact, we observed a slight increase in the responding group, which was likely related to the cytotoxic mechanism of cediranib. Therefore, considering the different mechanisms of anti-angiogenic and cytotoxic therapies, there would not be a complete discrepancy between ADC and 1H-MRS.
The trend we observed in the MRS data was reversed after day 56, consistent with the general clinical course of tumor recurrence and the eventual death of the patients. These findings might imply that while cediranib does have direct effects on metabolism in some tumor cells, those cells that survive despite blocked angiogenesis are eventually able to continue to grow.
A third observation in our study is the high probability of NAA/Cho to predict 6-month survival of rGBM patients treated by cediranib, as determined by ROC analysis. The changes in NAA/Cho illustrated in Figure 4 showed positive values in ‘high overall survival’ responders compared to negative values in the ‘low overall survival’ group. An ROC analysis of NAA/Cho showed high significances on day 28 (p=0.02, AUC=0.74) and day 56 (p=0.01, AUC=0.95). Together these data might imply that it is the critical time frame between 28 and 56 days that discriminates the tumor response to this anti-angiogenic agent. This finding suggests that NAA/Cho has good correlation with tumor responses for predicting six-month survival on the anti-angiogenic treatment, reflecting a combined picture for the opposite changes of two primary metabolites.
We acknowledge that this study is limited by its small sample size, and more importantly, by our still-incomplete understanding of the correlations between MRS findings and tissue morphology. The conventional interpretations of the MRS findings in glioblastoma were generated primarily in the pre-anti-angiogenic era. Although a direct anti-tumor effect is the interpretation most consistent with our MRS findings in this study, we cannot at this stage rule out other possible interpretations, and additional, larger sample-size studies are warranted.
This prospective study provides preliminary evidence that cediranib elicits direct metabolic effects in rGBM; traditionally such findings have been interpreted as anti-tumor effects resulting from vascular normalization based on the dynamics of the predominant brain metabolites. Our data also suggest that with further technical advances, early changes in metabolites detectable by 1H-MRS might be able to serve as imaging biomarkers, to predict treatment response in patients with recurrent malignant glioblastomas. NAA/Cho changes in enhancing tumor tissues suggest that anti-VEGF therapy not only has an anti-vascular effect, but that such effect modulates tumor and brain tissue both early and late in the disease process. These findings, if confirmed in larger studies, could shed further light on the mechanism of action of this new class of anti-angiogenic agents, and potentially even be used to make treatment management decisions.
We thank Wei-ting Zhang and Meiyun Wang for sharing their MRI data and analysis. We specially appreciate Nichole Eusemann for her excellent help with revising our manuscript. We also acknowledge all financial supports by NIH grants including R21 and K24, and by the Harvard Catalyst grant.
Financial support: NCI grants
- 1R21CA117079-01, Batchelor-PI; ClinicalTrials.gov #NCT00254943
We will disclose any potential conflicts of interest.