Arterial spin-labeled magnetic resonance perfusion imaging in prediction of pediatric brain tumors grading: inter-observer agreement

Pediatric brain tumors are a major concern with many variable management options. Arterial spin-labeled magnetic resonance perfusion imaging is a relatively new and noninvasive technique that can help in predicting tumor grades and provide us with physiological data about the tumors which significantly aids in all stages of tumor care, including diagnosis, therapy, and follow-up. To determine agreement between independent observers in the assessment and prediction of brain tumor grading in pediatrics by arterial spin-labeled (ASL) magnetic resonance perfusion imaging. Thirty-two patients (21 boys and 11 girls; mean age of (10.28 ± 4.31) years) with brain tumors were evaluated by ASL MRI perfusion. Image analysis was performed by two reviewers for quantifying absolute and relative tumoral blood flow (aTBF and rTBF) as well as qualitative assessment of the tumors in ASL color map images. The inter-observer agreement for the mean aTBF and the mean rTBF values of the studied lesions was almost perfect (inter-class correlation coefficient (ICC) = 0.978, 0.997). There was substantial agreement between both observers for the qualitative assessment of the studied lesions in color ASL images (Kappa = 0.779, with % of agreement = 87.0%). The mean aTBF for grade I tumors was (24.64 ± 3.45 ml/100 g/min), for grade II tumors it was (33.81 ± 3.59 ml/100 g/min), while for high-grade tumors (grade III and IV tumors) it was (75.60 ± 20.0 ml/100 g/min) with (p < 0.001). The mean rTBF of grade I, II, and III/IV tumors was (1.01 ± 0.17); (1.07 ± 0.31); and (3.12 ± 0.24) with (p < 0.001). Arterial spin-labeled perfusion MRI can help in the challenge of prediction of brain tumor grading in pediatrics with accurate quantitative and semi-quantitative measurements of perfusion parameters of the tumors as well as qualitative and visual assessment of the tumors.

The clinical presentation of pediatric brain tumors is also widely variable and affected by the patient's age, tumor location, effect on adjacent structures, and pattern of growth.They may be presented by focal symptoms, symptoms of increased intracranial pressure, or hydrocephalus.Infants may also be presented with failure to thrive [6][7][8].
In this context, imaging has a vital role in preoperative diagnosis and planning of those cases.Computed tomography has a limited diagnostic value and is not preferred in pediatrics because of radiation exposure, so it should be used only in emergencies or if MRI is not available [9,10].For the diagnosis of pediatric brain tumors, MRI is the gold standard method.Although conventional MRI sequences are useful in the detection of brain tumors in children, they frequently lack the sensitivity and specificity for determining the histopathological type of the tumor as there is a wide range of histopathological features included in pediatric brain tumors.Special MRI sequences such as functional MRI, perfusion-weighted images, and diffusion-weighted images help in determining the tumor type and grade [11,12].
Arterial spin labeling is a method for measuring tissue perfusion using magnetic resonance imaging which is rapid, noninvasive, and applicable in routine brain MRI imaging [13][14][15].It allows the quantification of tissue perfusion by the use of flowing arterial blood as a freely diffusible tracer.It uses radiofrequency pulses to alter the longitudinal magnetization of the blood before it reaches the region of interest to obtain a label image, and then it is repeated without magnetization of the blood to obtain the control image, and finally subtracting both images provides us with the ASL perfusion-weighted images [16][17][18].
The aim of our work was to highlight the role of the clinical application of ASL perfusion MRI in the grading of pediatric brain tumors as they have variable pathological features, which cause diagnostic challenges.Our study was based on agreement between independent observers and correlation with histopathological findings in order to determine the role of ASL perfusion study in the prediction of brain tumor grading in children.Therefore, better planning and management for those patients would be available.

Patients
The study was approved by the institutional review board of our institution, and informed verbal consent was obtained from the patient's parents.A prospective study was conducted on 35 consecutive children who were referred from the neurosurgery department and outpatient clinic in the duration between December 2020 and December 2022.The inclusion criteria were: (1) patients with a pathologically proven or provisionally diagnosed to have a brain tumor, (2) patients with a known history of previous excision of a brain tumor and proven or provisionally diagnosed to have a recurrent brain tumor, and (3) patients 18 years old or younger of both genders were included.We excluded 3 patients from the study (2 cases showed marked motion artifacts and one case proved to be an abscess).The final patients enrolled in our study were 32 children (11 girls and 21 boys).Their ages range from 1 to 17 years.All patients were subjected to a clinical neurological examination and underwent MRI.

MRI technique
The technique was performed using a 1.5 Tesla MRI scanner (Ingenia, Philips).All patients were examined in the supine position using a head and neck coil, with the head maintained in a neutral position.Non-cooperative patients (7 patients) were subjected to sedation by oral chloral hydrate syrup at a dose of 50-75 mg per kilogram (mg/kg), with a maximum dose of 2000 mg.Those patients were fasting for 3-6 h before the examination because of the sedation's potential aspiration risk.
The planes obtained were axial, sagittal, and coronal.The sequences obtained were: T1-weighted images (T1WIs), T2-weighted images (T2WIs), fluid-attenuated inversion recovery (FLAIR), post-intravenous (IV) contrast T1WI study (gadoterate meglumine was administered at a dose of 0.5 ml per kilogram (ml/kg) body weight with a maximum dose of 10 ml at a flow rate of about 2 ml/s using a 20-to 22-gauge venous cannula) as an intravenous bolus injection at a flow rate of approximately 2 ml/s except in two cases (a 16-year-old pregnant female and a 4-year-old boy with poor renal function)), diffusion-weighted images (DWI), and ASL perfusion MRI imaging.
Diffusion-weighted images were done in the axial plane before contrast medium administration by using a single-shot echo-planar imaging sequence.The following parameters were used: TR = 5000 ms, TE = 80 ms, inter-slice gap = 1 mm, slice thickness = 6 mm, and FOV = 240 × 240 mm.Lesions of interest were scanned using the b values: zero, 500, and 1000 s/mm 2 .

ASL data acquisition
Arterial spin-labeled perfusion imaging was carried out with a pseudo-continuous labeling technique.Following the labeling pulse, numerous time points were obtained.Fast spin echo single-shot echo planner imaging parallel sequences, maximum water fat shift, and a short TE to keep a good signal to noise ratio.The following parameters were used: a 1.5 mm inter-slice gap, 6 slices with an axial slice thickness of 6 mm, Voxel size was 3.6 × 3.5 × 3 mm, field of view (FOV) was 240 × 240 mm, data matrix was 68 × 68, TR/TE was 250/20, and the entire scan duration was four minutes.

Image analysis
Conventional MR images were reviewed for the detection of space occupying lesions (SOLs) and the primary characterization of detected lesions.The SOLs were reviewed for their site, size, consistency, signal intensity, contrast enhancement, and associated mass effect and edema.Automatically generated apparent diffusion coefficient (ADC) maps were generated and interpreted with correlation with DWIs to classify lesions into free or restricted diffusion.

Post-processing
The vendor's provided workstation (extended MR Workspace 2.6.3.5, Philips Medical Systems Nederland B.V.) was used to transfer the DICOM pictures.Subtraction of alternating tag and control image pairs, motion correction, and development of an ASL grayscale and colored map were the steps used in the postprocessing of arterial spin-tagging data.Regions of interest (ROIs) were manually placed within the high signal area on the ASL grayscale map.The size of ROIs in both regions was similar to each other (ranging from 0.25 to 2.0 cm 2 ).Compared to other conventional MR images, areas of arteries, calcification, hemorrhage, cysts, and necrosis were avoided.

Quantitative absolute TBF analysis
To quantify absolute TBF values, the following equation must be solved [19]: where CBF stands for cerebral blood flow, M (TI 2 ) for the mean signal intensity difference between label and control images, M 0,blood for the equilibrium magnetization of blood, is the tagging efficiency, TI 1 for the duration of the tagging bolus, TI 2 for each slice's inversion time, T 1 , blood for the longitudinal relaxation of blood, and qp for (1) a correction factor that takes into account the difference between the T 1 of blood and the T 1 of brain tissue.The T 1 of the tissue is one of many variables that are directly measured before being added to the equation.Standard values from the published literature are represented by other variables in the equation.Although it would be ideal to gather each patient's variables de novo, this is not always feasible or practical, and the quantification may contain mistakes as a result of these assumptions.The T 1 levels of the blood and tissue are the most important values.Interpretations of the quantified CBF readings will be more accurate if you are aware of the influences and factors in the CBF quantification equation.

Semi-quantitative relative TBF analysis
A region of interest (ROI) was placed over the tumor region as in quantitative analysis, and another ROI was placed over the contralateral normal-appearing gray matter.Then the value of tumor ROI was divided by the value of ROI in the contralateral normal gray matter to estimate relative tumoral blood flow (rTBF) values.

Qualitative analysis of ASL color-coded maps
Color-coded ASL maps were visually analyzed by both observers, and the tumor region's perfusion was classified into 3 main categories: hyper-perfusion, hypo-perfusion, and mixed perfusion according to the color scale.

Statistical analysis and data interpretation
The images were reviewed by two radiologists (RA 16 years of experience, ME 9 years of experience in diagnostic radiology) who independently reviewed the images.Each reader was blinded to the results of the other reader.The data were then fed to the computer and analyzed using IBM SPSS Corp., Released in 2013.IBM SPSS Statistics for Windows, version 22.0 Armonk, NY: IBM Corp. Qualitative data were described using numbers and percentages.Quantitative data were described using the mean and standard deviation for normally distributed data.The significance of the obtained results was evaluated at the (≤ 0.05) level.Additionally, Chi-square and Fischer exact tests were used to compare qualitative data between groups as appropriate.
Receiver operating characteristic (ROC) curve analysis was used for evaluation of validity and cutoff point in comparison with ensured diagnostic test.Sensitivity and Specificity were detected from the curve, and positive predictive value (PPV), negative predictive value (NPV), and accuracy were calculated through cross-tabulation.
For the determination of the agreement, we investigated; • Bland altman analysis and inter-class correlation for continuous variables • Kappa agreement was calculated by cross-tabulation for categorical variables with Kappa Kappa < 0: No agreement Kappa between 0.00 and 0.20: Slight agreement Kappa between 0.21 and 0.40: Fair agreement Kappa between 0.41 and 0.60: Moderate agreement Kappa between 0.61 and 0.80: Substantial agreement Kappa between 0.81 and 1.00: Almost perfect agreement.

Absolute and relative tumoral blood flow
The mean absolute tumoral blood flow (aTBF) of the studied lesions was 28.9 (18.45-103.52)ml/100 g/min as measured by the first observer and 28.45 (3.51-102.88)ml/100 g/min by the second observer.The mean relative tumoral blood flow (rTBF) of the studied lesions was 1.01 (0.81-3.56) by the first observer and 1.11 (0.85-3.42) by the second observer.There was excellent inter-observer agreement as regards both the mean aTBF (Fig. 6) and rTBF (Fig. 7), with the inter-class correlation coefficient (ICC) (95% CI) being 0.978 for the aTBF and 0.997 for the rTBF (Table 2).

Qualitative assessment
Regarding the qualitative assessment of the studied lesion in both gray and color ASL maps, the lesions were classified by both observers into hypo-perfused (n = 20, 18), hyper-perfused (n = 7, 7), and mixed perfusion (n = 5, 7).There was substantial agreement between both observers (Kappa = 0.779), with a percentage of agreement of 87.0% (Table 3).

Tumor grading
According to the 2021 WHO Classification of Tumors of the Central Nervous System, we classified the studied lesions into low-grade (WHO grades 1 and 2) and high-grade (WHO grades 3 and 4) tumors.Both absolute and relative TBF were significantly higher in high-grade tumors than in low-grade tumors.The mean aTBF for grade I, II, and III/IV tumors was (24.64 ± 3.45 ml/100g/ min), (33.81 ± 3.59 ml/100 g/min), and (75.60 ± 20.0 ml/100 g/min).The mean rTBF for grade I, II, and III/IV tumors was (1.01 ± 0.17), (1.07 ± 0.31), and (3.12 ± 0.24).There was a good correlation between calculated absolute and relative TBF and the WHO grades of the tumors with (P < 0.001) (Table 4).

Discussion
Preoperative radiological assessment and accurate diagnosis and grading of brain tumors are very important in guiding therapeutic decisions.The strategy of treatment varies widely according to the type, location, and grade of the tumor.Although histopathologic evaluation is believed to be the gold standard tool in the diagnosis and grading of brain tumors, it has many limitations, such as its dependence on invasive biopsies or surgical removal of a tumor, sampling errors, and inaccurate grading caused by the heterogeneity of the tumors due to internal necrotic components [20,21].Our study aimed to emphasize the role of ASL perfusion MRI in the prediction of the grading of pediatric brain tumors which may help in the management and follow-up of those patients.Many studies have discussed the role of perfusion studies in adult brain tumors.However, not as many studies have been conducted on pediatric brain tumors which have unique features different from adult tumors [22,23].Most of these studies have largely concentrated on dynamic susceptibility contrast (DSC) perfusion imaging, the most widely used technique for assessing brain tumor perfusion [24].This study included 32 patients in the pediatric age group (less than 18 years old).The patients were grouped according to the pathological findings into low-grade tumors (including WHO grades I and II, including 23 patients: 18 patients with grade I and 5 patients with grade II) and high-grade tumors (including WHO grades III and IV, including 9 patients: 1 patient with grade III and 8 patients with grade IV).All lesions in the current study had histopathologically proven diagnoses.
In our study, quantification of absolute tumor blood flow (aTBF) was done by placing ROIs manually by both observers within the high signal area on the ASL grayscale map, avoiding the regions of vessels, calcification, hemorrhage, cystic, and necrotic areas.The values obtained from both observers were analyzed and compared, and there was an excellent inter-observer agreement (ICC = 0.978).
A previous study had found that aCBF values varied between both observers as the CBF max must be normalized to normal-appearing gray matter, the measurement of which is subject to fluctuation between readers.It is difficult and subjective for a reader to identify the maximum CBF location of a tumor "by eye." However, they Fig. 6 Bland-altman analysis for agreement between first and second observers as regard aTBF stated that for readers 1 and 2, personally measured values yielded low-/high-grade prediction accuracy of 92% and 96%, respectively.Therefore, it does not seem that the low precision and accuracy of manually measured data significantly reduce their diagnostic relevance, and their use is justified in the absence of automated tumor sampling techniques [25].There was also excellent agreement between both observers in the assessment of relative tumor blood flow by obtaining the ratio between the ROI placed by both observers in the tumor region and the other ROI placed in the contralateral normal gray matter.
Another study instead preferred placing the other ROI in the contralateral healthy brain tissue, which contains white matter, gray matter, and vascular structures, to calculate the average mean signal intensity of the sizematched contralateral normal brain tissue (normal ROI).This normalization was applied to remove signals from normal vascular and parenchymal structures [26].
On the other side, normal appearing white matter has a long transit time and a higher water content in the case of  brain tumors, so that some researches claim that utilizing white matter as a reference ROI is still debatable.This, in turn, could lead to an underestimation of white matter CBF value by ASL [27].
A study was done by Testud et al. [28] on the role of both dynamic susceptibility contrast (DSC) and ASL MRI perfusion, which concluded that low-and high-grade pediatric brain tumors can be distinguished with DSC and ASL perfusion techniques; however, the accuracy is location-and field-dependent.For supra-tentorial malignancies, DSC outperforms ASL with excellent results, while in posterior fossa lesions, ASL seems to function more effectively.
Arterial spin-labeled perfusion study enabled us to predict tumor grades in studied cases by quantitative assessment of absolute and relative tumoral flow.Compared to low-grade tumors, values were higher in high-grade tumors.In ROC analysis, the AUC of absolute and relative TBF to differentiate low-from high-grade tumors was (0.979 and 0.969, respectively).The calculated cutoff points to discriminate between high-and low-grade tumors were (54.29 ml/100 g/min) for aTBF and (2.84) for rTBF which revealed accuracy of (93.8%), sensitivity of (87.5%), specificity of (95.8%),PPV of (87.5%), and NPV of (95.8%).
In keeping with our results, other studies were done using ASL techniques for differentiating high-(III, IV) from low-grade (I, II) tumors revealing higher CBF in high-than low-grade brain tumors with a high significant difference [29,30].
A similar study was conducted by Morana et al. [31] on 37 children with pathologically proven astrocytomas of different tumor grades, in which ROC analysis demonstrated the AUC of rTBF to differentiate low-from high-grade astrocytomas was 0.96, which showed a significant correlation with DSC calculated values.
Another study done by Dangouloff-Ros et al. [32] on 129 children with brain tumors demonstrated that the cutoff points of aTBF and rTBF were (50 mL/min/100 g) and (0.95), respectively, to distinguish low-from high-grade tumors.
We found, in keeping with previous studies, that ASLweighted perfusion maps and CBF maps are really useful for investigating, evaluating, and quantifying brain tumors in pediatrics.They have many advantages, such as the limitation of the irradiation dose to some vulnerable tissues and organs in children.Additionally, there is no contrast medium that permits intraoperative imaging of pediatric brain tumors as well as repetition of the study for follow-up [33,34].

Limitations
There were some limitations in our study, which we can conclude in a few points.First, the current study enrolled a small number of patients in a single center, which has limited the statistical results.It is advised to do future research with a larger number of patients who have a variety of pathological findings and to conduct multicenter studies.Second, our study was conducted using a 1.5 Tesla MR machine, so we faced a problem of low SNR and motion artifacts in some cases.Further studies using 3 Tesla MR machines may help improve the results.Third, as regards the grading of brain tumors we depended on measuring TBF.Further studies with the application of diffusion tensor imaging and proton MR spectroscopy, as well as molecular and genetic tumor classification, will improve the prediction of pediatric brain tumors grades.

Conclusions
It was found that high-grade tumors have higher absolute and relative TBF than low-grade tumors.Therefore, MR imaging with ASL can be used to classify pediatric brain tumors relying on TBF.The relationship between ASL and WHO tumor grade adds further insight into the understanding of pediatric brain tumors.
Arterial spin labeling is a noninvasive, promising, and recent perfusion imaging technique that offers some merits.Firstly, it does not require the use of an exogenous contrast agent, which is beneficial in the pediatric population.Secondly, it is also a fast and repeatable technique, which is an advantage in the follow-up of those patients throughout their treatment journey.Finally, it may produce results that would be a helpful alternative technique for assessment of brain tumors perfusion, predicting its grade and guiding histopathological sampling in those patients.Hence, we recommend that it be included in the routine MRI protocol.

Fig. 1 Fig. 2 AFig. 5 A
Fig. 1 A 6-year-old boy patient with a WHO grade I adamantinomatous craniopharyngioma.Images show a well-defined partially cystic, partially solid supra-sellar mass complicated by supratentorial hydrocephalic changes.The cystic parts display high signal intensity on axial T1WI (A) and mixed high signal intensities on axial T2WI (B) denoting a high proteinaceous content.The solid parts show strong enhancement on post contrast sagittal T1WI (C).Free diffusion of the lesion is seen in the form of low SI on DWI (b1000 image) and high SI on axial ADC grayscale map (D, E).Hypo-perfusion of the lesion (white arrows) is demonstrated in the axial ASL grayscale and color CBF maps (F, G)

Fig. 7
Fig. 7 Bland-altman analysis for agreement between first and second observers as regard rTBF

Table 1
Site of the studied lesions

Table 2
Agreement between first and second observers as regard absolute and relative TBF (ICC: inter-class correlation coefficient)

Table 3
Inter-observer agreement as regard qualitative assessment of the studied lesions

Table 4
Mean absolute and relative TBF according to WHO grade of the studied lesions * statistically signifcant Similar superscripted letters in same row denote significant difference between groups ROC curve of aTBF and rTBF in differentiating low from high grade

Table 5
ROC curve of TBF, RTBF in differentiating low-from high-grade tumors (AUC: area under curve, PPV: Positive predictive value, NPV: Negative predictive value)