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Arterial Spin Labeling and Dynamic Susceptibility Contrast-enhanced MR Imaging for evaluation of arteriovenous shunting and tumor hypoxia in glioblastoma

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults and carries a dismal prognosis. Significant challenges in the care of patients with GBM include marked vascular heterogeneity and arteriovenous (AV) shunting, which results in tumor hypoxia and inadequate delivery of systemic treatments to reach tumor cells. In this study, we investigated the utility of different MR perfusion techniques to detect and quantify arteriovenous (AV) shunting and tumor hypoxia in patients with GBM. Macrovascular shunting was present in 33% of subjects, with the degree of shunting ranging from (37–60%) using arterial spin labeling perfusion. Among the dynamic susceptibility contrast-enhanced perfusion curve features, there were a strong negative correlation between hypoxia score, DSC perfusion curve recovery slope (r = −0.72, P = 0.018) and angle (r = −0.73, P = 0.015). The results of this study support the possibility of using arterial spin labeling and pattern analysis of dynamic susceptibility contrast-enhanced MR Imaging for evaluation of arteriovenous shunting and tumor hypoxia in glioblastoma.


Results
Of the 16 patients who underwent MRI imaging, one patient was excluded due to motion degraded ASL images, one patient declined surgery given the extensive multifocal nature of the tumor, and two patients were excluded because the histopathology demonstrated diffuse midline glioma. Among the 12 included patients, seven were male and five were female (mean age = 63.25 ± 12.1, median age = 62 years).
Macrovascular shunting was present in 4/12 (33%) subjects, with degree of shunting ranging from 37-60% (Figs 1 and 2). The average hypoxia score was higher in patients with macrovascular shunting compared to subjects without shunting (49.25 vs 40.1), but the difference was not statistically significant (P > 0.05). Among the DSC perfusion curve features, there was a strong negative correlation between hypoxia score and DSC perfusion curve recovery slope (r = −0.72, P = 0.018), as well as angle (−0.73, P = 0.015). In addition, the skewness of recovery slope was positively correlated with hypoxia score (r = 0.70, P = 0.023). Among other histopathologic features, Ki-67 was positively correlated with the mean angle (r = 0.71, P = 0.028) and slope (r = 0.82, P = 0.006) of the DSC perfusion curve recovery.
Voxel-based analysis of enhancing tumor demonstrated significant difference between the angle and slope of DSC curve recovery in patients with macrovascular shunting compared to the patients without shunting (P < 0.001), Table 1).
The right panel of Fig. 3 shows the principal components (PC) based on the perfusion signal time-curve of all subjects' enhancing tumor and shunting voxels. The skewness of the third principal component was significantly more negative in patients without shunting (−0.65 vs −0.085, P = 0.006) and was positively correlated with microvascular proliferation in histopathologic analysis (r = 0.67, P = 0.031).
In Fig. 4, the mean of perfusion signals and first three principal components obtained from all voxels multiplied by +/− two standard deviations of the respective principal components are plotted. Principal component 1 was found to relate primarily to the global baseline signal level at each voxel. Principal component 2 has the greatest variability around the baseline and the depth of the curve; therefore, it conveys the depth of the signal decrease in relation to the baseline level, while the third principal component reflects the shape of the perfusion signal and the steepness of the signal recovery to a greater degree.

Discussion
In this study, we showed that ASL can detect macrovascular shunting in a subset of GBM patients, and that tumors with and without shunting have different DSC perfusion curve characteristics. In addition, we also showed that the slope and angle of the DSC perfusion curve are strongly correlated with tumor hypoxia and Ki-67 proliferation index. Although the hypoxia score was higher in patients with macrovascular shunting; however, the difference was not statistically significant, which could be due to lack of adequate statistical power in our study.
Two previous small studies used direct injection of 99mTc-labeled microparticles to quantify AV shunting in gliomas based on the notion that the injected microparticles should be trapped in a normal capillary bed but escape through abnormal arteriovenous channels in the presence of AV shunting, eventually becoming trapped www.nature.com/scientificreports www.nature.com/scientificreports/ and imaged in the lungs. Mariani et al. included two patients with glioma, and both demonstrated AV shunting ranging from 63% to 70% 8 . Another study by the same group included seven patients with high grade glioma, which all demonstrated AV shunting ranging from 47-89% 1 . Yoshikawa et al. studied 26 patients with GBM using cerebral angiography and used early venous filling as an indicator of AV shunting. They found that 53% of GBMs had AV shunting, and that AV shunting is significantly more common in presylvian tumors compared to other locations 2 . The results of our study are more concordant with those of Yoshikawa et al., as we also demonstrated AV shunting in a subset of patients with GBM. This may be due to differences in technique as the nuclear medicine studies described used particles with diameters of 25 to 50 mm, while ASL imaging uses labelled water as a tracer. In addition, our criterion to determine AV shunting was visualization of bright veins, which is an equivalent to early venous filling on cerebral angiography. Improving the spatial resolution and signal to noise ratio of ASL by applying different acquisition techniques such as continuous arterial spin labeling (CASL) and imaging at higher field magnets 15 may be helpful to improve the detection of smaller veins that carry shunted blood. In addition, application of techniques such as velocity selective ASL 16 may have an added value by using velocity cut-offs that can be adjusted based on the degree of tumor vascularity. The strong but reverse correlations between the slope and angle of the DSC perfusion curve with tumor hypoxia and Ki-67 proliferation index in our study is consistent with two prior studies that demonstrated low proliferative activity in hypoxic tumors areas compared to well-oxygenated tumor compartments 17,18 .
The proposed PCA data-driven method uses temporal dynamics of DSC MR imaging to extract information relevant to tumor hypoxia not typically obtained with traditional cerebral blood volume (CBV) maps 14 . Thus, our results suggest that more comprehensive analysis of the DSC perfusion curve can provide additional clinically-useful information about the GBM tumor microenvironment and the PCA extracts information may reflect tumor AV shunting, as evidenced by the close relationship in PCA space with the shunted vessels on ASL (Fig. 3). Our study lends a number of implications for clinical practice. The method described can serve as a tool to compare the efficacy of systemic forms of treatment in patients with glioblastoma with different degrees of shunting. In addition, local forms of treatment may be more suitable for patients with high levels of AV shunting  www.nature.com/scientificreports www.nature.com/scientificreports/ rather than systemic treatments 19 . Finally, to determine the potential effect of AV shunting on drug delivery, quantitative pharmacology studies can be performed to determine the biodistribution of drugs under different degrees of AV shunting. For example, temozolamide, currently the standard of care for all newly diagnosed GBM 20 , can be labelled with experimental PET radiotracers 21 and be used to determine the biodistribution of the drug under different degrees of AV shunting.
The current work has several limitations. The main limitation is the small sample size, noting that this study was prospective and required preoperative consent to perform MR sequences that are not part of the routine brain tumor imaging at our institution, as well as additional histopathology examinations. Another limitation in the widespread use of our approach is the specialized nature of principal component analysis; however, the software package Cancer Imaging Phenomics Toolkit (CaPTk) that is used in this study is freely available for use 22 .

Conclusion
ASL imaging can be used to determine and quantify macrovascular arteriovenous shunting in patients with GBM. The combination of ASL imaging and DSC perfusion curve analysis might provide important information about the tumor microenvironment and tumor hypoxia. This study is limited by small patient numbers and its preliminary nature, but hints to the possibility of a combined approach using DSC and ASL to make noninvasive predictions of arteriovenous shunting and tumor hypoxia. This clinical significance of this approach in the context of systemic therapies need to be investigated in future studies.

Methods
Institutional review board of the University of Pennsylvania approval was obtained for this prospective study and all research was performed in accordance with relevant guidelines and regulations. Informed consent was obtained from the participants. 16 patients with intra-axial brain mass suggestive of high grade glioma were referred to the radiology department of Hospital of University of Pennsylvania from Dec 2016 to May 2018 and underwent brain MRI at 3-Tesla (Magnetom TrioTim; Siemens, Erlangen, Germany) using a 12-channel phased array head coil. Routine sequences were obtained, including pre and post-contrast axial T1-weighted 3D MPRAGE (TR/TE/TI = 1760/3.1/950 ms, 192 × 256 matrix size, 1-mm section thickness) and post contrast axial FLAIR (TR/TE/TI = 9420/141/2500 ms, 3-mm section thickness). SWI scanning parameters were (TR) = 27 ms, (TE) = 20 ms, field of view (FOV) = 220 mm, imaging matrix = 256 × 192, slice thickness = 1.5 mm, and GRAPPA factor = 2. Pseudocontinuous ASL (PCASL) was performed using a labeling time = 1.5 s, PLD = 1.5 s, and labeling plane offset = 8 cm, TR/TE = 9181 ms/11 ms, FOV = 220 × 220 mm 2 , matrix size = 64 × 64, voxel size = 3.4 × 3.4 × 5 mm 3 and 3D spiral read out. For DSC imaging, a bolus of gadobenate dimeglumine (MultiHance; Bracco Diagnostics, Princeton, New Jersey) was injected with a preloading dose of 0.07 mmol/kg, to reduce the effect of contrast agent leakage on CBV measurements 23   www.nature.com/scientificreports www.nature.com/scientificreports/ venous drainage on ASL CBF (cerebral blood flow) maps, confirmed by hyperintensity on SWI images (Figs 1  and 2). Normal veins appear dark on SWI images due to high deoxy-hemoglobin content, while arterialized vein appear bright 11 . ASL images were linearly co-registered with the T1 post-contrast image using an affine www.nature.com/scientificreports www.nature.com/scientificreports/ registration tool FLIRT 24 , available in FSL 25 . Hyperintense draining veins identified on ASL images and confirmed on T1 post-contrast images were segmented in a semi-automated fashion using ITK-SNAP 26 . The enhancing component of the tumor was also segmented in semi-automated fashion. Macrovascular AV shunting was quantified by normalizing the blood flow (BF) in the draining vein to the volume and BF of the tumor and draining vein. The AV shunt fraction was quantified with the following formula: (Vol vein × mean BF vein )/(Vol tumor × mean BF tumor ) + (Vol vein × mean BF vein ).
Analysis of DSC perfusion curve. DSC data was linearly co-registered with the T1 post-contrast image using FLIRT, and was used to extract at each voxel the time to maximum drop of the perfusion signal (T D ), the time to 95% recovery of the perfusion signal (T R ), and the respective signal intensities (I D , I R ). The slope of the recovery curve was calculated by slope = (I R − I D )/(T R − T D ), and recovery angle calculated by Angle = ArcTan(slope). Subsequently, the mean, median, standard deviation, and skewness were calculated for slope of the recovery curve, and recovery angle.
Calculation of Principal Components. Principal component analysis (PCA) is a dimensionality reduction method that has been used 13,14 to distill the DSC-MRI time series down to a few principal components that capture the temporal dynamics of blood perfusion. In order to characterize the perfusion characteristics of enhancing tumor (ET) in shunted and non-shunted patients, we also used the ASL-based ROIs as described before. All voxels of ET of patients with and without macrovascular shunting as well as voxels from the tumor venous drainage that demonstrated shunting were utilized to calculate the first three principal components. We performed PCA using these ROIs and then segmented ET of all subjects 13,14 . Cancer Imaging Phenomics Toolkit (CaPTk) software was used for initial calculations of PC analysis 27 .
Evaluation of Principal Components. The principal components incorporate and convey complex, high-dimensional information from various aspects of the dynamics perfusion time-series, such as baseline signal, depth of signal decrease, slope, and angle of signal recovery. The first principal components were plotted in Fig. 3 (right panel), which displays ET of shunted patients (green) and non-shunted patients (blue), as well as the ROI of tumor venous drainage with shunting (red). The left panel of Fig. 3 shows the mean perfusion curve with two standard deviations bars for the same groups. PCA was subsequently used to capture the information of the perfusion time series in all subjects. Because of the relative consistency in the perfusion pattern of the various regions, a feature vector consisting of first three principal components was sufficient to capture more than 98% of the variance in the perfusion signal for all tissue types and all patients (Fig. 3, Right panel, depicts the first three components).
Histopathological analysis. All surgical specimens were reviewed by a neuropathologist (M.P.N) who was blinded to the shunting data. The tissue was stained with hematoxylin and eosin, as well as with the following antibodies: K-67 (Dako IR62661, clone MIB-1, prediluted), ERG (BIOCARE, PM421AA, prediluted), CAIX (Leica, CAIX-L-CE, 1:100). Unstained slides were pretreated with heat retrieval, epitope retrieval 1, in citrate buffer pH 6.0 (Leica microsystems) for 20 min. Immunohistochemical staining was performed on the Bond 111 Autostainer with the DAB chromogen and a hematoxylin counterstain. The overall tumor Ki-67 proliferation index was semi-quantitatively estimated based on inspection of a stained section from one tumor block. ERG staining for endothelial cells was performed in order to assess vascular density in dense tumor (Fig. 5). Small, medium, and large caliber vessels in the tumor were each scored on a range of 0-6, with 0 referring to rare vessels present, and 6 referring to dense vascularity. Staining for CA IX was performed to evaluate hypoxia. CAIX catalyzes the reversible hydration of carbon dioxide and is considered to be a reliable cellular biomarker of tumor hypoxia 28 . Expression of CAIX was determined by semi-quantitatively assessing the percentage of stained tumor cells and their staining intensity with an H-score ranging from 0-300. The percentages of weak (1+), moderate (2+), and strong (3+) staining were assessed, and the H-score was calculated as: (weak %)*1 + (moderate %)*2 + (strong %)*3.