This study evaluated the infiltration of high grade brain tumors using high order diffusion tensor imaging (DTI). Seven patients with glioblastoma underwent MRI with a standard protocol and high order DTI with 20 and 60 diffusion directions. Biopsies of the tumor and adjacent normal-appearing white matter were taken and analyzed histopathologically. Generalized anisotropy (GA) and generalized variance (GV) measured by DTI at the biopsy sites were significantly correlated with the tumor infiltration percentage on histology, suggesting high order DTI may help delineate tumor margins. Further validation with more patients is needed.

1. Histopathologic Evaluation of High
Grade Brain Tumors Infiltration Using
High Order Diffusion Tensor Imaging
T. Stefani, B. Pereira, A. Coste, T. Khalil, E. Chabert, J.L. Kemeny, L. Boyer, B. Jean.
eP-72
University Hospital, Neuroradiology Dept.
58 rue Montalembert,
63003 Clermont-Ferrand, FRANCE
Email: thomas.stefani@aitb.org

2. Summary
• 1 Purpose
• 2 Materials and Methods
• 3 Results
• 4 Discussion
• 5 Conclusion
• 6 Bibliography
The authors of this study have no conflict of interest to disclose.

3. 1 Purpose
• Delineation of high grade brain tumors is critical for treatment
planning, follow up and prognosis. Diffusion Weighted Imaging is
widely used in neuro oncology for this purpose [1-3].
• High grade brain tumors are heterogeneous and infiltrative lesions.
Tumoral cell dissemination is known to be found beyond from the
tumor visible margins therefore analysis of the Brain Adjacent to
Tumor (BAT)remains challenging with conventional MRI sequences [4].
• Diffusion Tensor Imaging (DTI) is sensitive to changes in white matter
fiber bundles microstructure [8].
• The aim of this study was to evaluate the peripheral white matter
infiltration of high grade brain tumors using high order Diffusion
Tensor Imaging.

4. 2 Materials and Methods
• Monocentric prospective study, from April 2012 to April 2013.
• Local IRB approval and informed consent from all patients were obtained.
• All patients from 18 to 85 years of age having a suspected cerebral high grade tumor
(WHO III-IV) on CT scans were screened to be included the protocol.
• Exclusion criteria: MRI contraindication, severe renal failure, cerebral biopsy
contraindication, pregnancy.
TotalTotal
17 patients screened
IncludedIncluded
7 patients
Biopsy RefusalBiopsy Refusal
2 patients
Post-CTSurgicalPost-CTSurgical
Treatment
3 patients
Not WHO III-IVNot WHO III-IV
5 patients
(1 WHO II, 2 metastasis,
1 lymphoma,
1 atypical meningioma)
BiopsyBiopsy
12 patients
DiffusionTensor MRIDiffusionTensor MRI
14 patients
excluded
excluded
excluded

5. 2 Materials and Methods
• Seven patients were prospectively included and underwent 3 T clinical
MRI scans (Signa MR750, GE Medical Systems, Milwaukee, Wis).
– Usual diagnostic MRI protocol included :
• 3D-T1GE (Repetition Time (TR) 8.8 ms, Echo Time (TE) 3.54 ms, 1x1x1.2mm3)
beforeand after contrast media injection
• T2-SE (TR 4923 ms, TE 100.23 ms, 1x1x4mm3)
• Diffusion(TR 8500 ms, TE minimum, 1x1x2mm3, b0-b500-b1000 s/mm2)
• T2 FLAIR (TR 11000 ms, TE 142.97 ms, Inversion Time (TI) 2350 ms, 1x1x4mm3)
• Perfusion Weighted (Echo Planar Imagine (EPI), TR 2000 ms, TE 14.4 ms,
2x2x4mm3)
• Multivoxel Spectroscopy (Point Resolved Spectroscopy (PRESS), TE 144 ms and
TE 35ms, 2x2x2cm3)
– DiffusionTensor Sequence :
• Diffusion Tensor Imaging 20 directions (EPI, TR 8000, TE minimum,
1x1x2mm3, b0-b1000 s/mm2, sequence duration: 8 minutes).
• Diffusion Tensor Imaging 60 directions (EPI, TR 8000, TE minimum,
1x1x2mm3, b0-b3000 s/mm2 sequence duration: 16 minutes).
• The 20 directions sequence was done prior to the 60 directions sequence to decrease the probability of bad
sequences associated with motion artifacts, or premature termination of examination.

6. 2 Materials and Methods
• Patients then underwent surgical CT guided stereotactic tumor biopsy
targeted with Leksell frame (Iplan Stereotaxy 3.0.2,Brainlab), according to
standard procedure, maximum 1 week after MRI procedure (mean delay
= 4 days).
• An extra biopsy (10mm length, 1mm diameter) was performed in normal-appearing
white matter adjacent to the tumor along the biopsy pathway 3 to 8 mm
awayfrom the enhancementarea.
• The coordinates of the center of the biopsy window were noted.
Fig. 1. CT guided stereotactic biopsy planning. Target is located along
the biopsy pathway, 3 to 4 mm away from the enhancement area.

7. 2 Materials and Methods
• Biopsies were fixed with formaldehyde.
• Histopathologicanalysis :
– WHO classification of the tumor.
• Using immunohistochemical markers :
– MAP2 (microtubule associated protein 2), Ki67.
• Semi quantitative estimation of tumor infiltration percentage(IP):
– Number of atypical cells / Total number of cells in the field of view.
– (Field1mm x 2mm, Magnification x10).
Fig. 2. Brain adjacent to tumor biopsy sample, field
1x2mm, magnification 10. Ki67 immunologic marker
showing moderate proliferative rate.

8. 2 Materials and Methods
• Analysis of DT-MRI datasets was done using in-house software using MATLAB scripts (R2012a.
Natick, Massachusetts: The MathWorksInc) courtesy of Angelos Barmpoutis, PhD [5].
– Fourth order tensor resolution :
• 9 x 9 Matrix diagonalisation :
 6 Eigenvalues i
 6 EigenvectorsUi
Fig. 3. Fourth order tensor and its eigenvalues/vectors orientation distribution
fonction (ODF).

9. 2 Materials and Methods
– Computation of the tensor scalars [6], at the biopsy locus (MRI and biopsy CT images
coregistration, 1x1x1mm3 precision, EPI spatial distortions correction using Iplan
Stereotaxy 3.0.2,Brainlab)
• GA (Generalized Anisotropy)
• GV (Variance)
• MD (Mean Diffusion)
with
Fig. 4. Fourth order tensor scalar computation at the biopsy
locus (blue dot).

10. 2 Materials and Methods
• Statistical analysis (Spearman’s rank correlation) was done using STATA
10.0 (Stata Corporation, College Station, TX, USA).
– 30 subjects needed (risk  = 5%, statistical power  = 90%).
• Primary analysis:
– Correlation coefficient was evaluated between the 4th order diffusion Generalized
Anisotropy (GA) distribution and the tumor infiltration percentage (TI).
• Secondary analysis:
– Correlation coefficient was evaluated between the 4th order diffusion tensor metrics
distributions (GV, MD) and the tumor infiltration percentage.

11. 3 Results
• 7 prospective patients(3M, 4F).
• Age ranging from 52 to 86 year old (mean age value = 63).
• Homogeneous population of glioblastoma multiforme (GBM).
• Tumor InfiltrationPercentage(TI) values :
– Mean TI value= 0.16 [0;1].
• Tensor scalars mean values:
– GA (biopsy) = 0.36 [0.18;0.68].
– GV (biopsy)= 0.0028 [0.0009;0.0086].
– MD (biopsy)= 0.00065 [0.00035;0.00075].

12. 3 Results
• We observed a significant correlationbetween :
– GA and tumor infiltration percentage (TI) (r=-0.98 p=0.006).
– GV and tumor infiltration percentage (TI) (r=-0.98 p=0.006).
• MD showed no significant correlation(r=0.11 p=0.76) with TI.
Fig. 5. GA maps.
Left: (patient number: 03) Right frontoparietal
glioblastoma showing low GAsignal in a
peripheral ROI (red arrow) and 0% TI on
histology.
Right: (patient number: 07) Left parietal
glioblastoma showing high GAsignal in a
peripheral ROI (yellow arrow) and an 10% TI
on histology.

13. 4 Discussion
• In this study we introduced 4th order diffusion tensor which offers an
alternative model to conventionnal 2nd order tensor (3 x 3 matrix).
– Jayachandra et al [7], showed that anisotropy could be estimated more accurately for
complex microstructure with 4th order DTI compared to 2nd order DTI.
– Simpler modelisation compared to other techniques (Qball, High Angular Resolution DTI).
• Provenzale et al [8] showed that there is a significant difference in Fractional
Anisotropy (FA) decrease for peritumoral NAWM between gliomas and meningiomas
but not for peritumoral regions (hyperintense areas on T2-weighted MR images),
showing that DTI may be sensitive to subtle microstructure changes.
• Stadlbauer et al [9] showed FA is better than MD for assessment and delineation of
different degrees of pathologic changes (ie, TI) in glioma but their retrospective
population washeterogeneous(WHO Grade II and III gliomas).
• Ellingson et al [10] showed limitations of conventional 2nd order DTI compared to 4th
order DTI when evaluating glioblastomas but needed careful tissue validation as they
didn’tcorrelate their results to a biopsy with cell quantification.

14. 4 Discussion
• In our study we showed that GA and GV were significatively correlated
to Tumor Infiltration Percentage in glioblastoma.
• Butthis study has some limitations:
– Few subjects : only 7 patients (this study is still recruiting).
– Non fixed distance from tumor to biopsy due to surgeon target choice.
– Semi quantitative estimation of percentage infiltration.
– Coregistration accuracy: 1mm accuracy (3mm diameter ROI and correction of EPI
spatial distortions)
– 60 directions GA maps have a lower signal to noise ratio (SNR) compared to 20
directions sequence.

15. 5 Conclusion
•These preliminary results despite the little number of inclusions, show that
GA and GV measurements derived from high order diffusion tensor estimation
are correlated (p=0.006, p=0.006) to tumor infiltration percentage of the local
environment of glioblastoma.
 GA derived from fourth order diffusion tensor could be used for
assessment and delineation of different degrees of pathologic changes (ie,
Tumor Infiltration Percentage) in glioblastoma.
•These results need to be confirmed at the end of the inclusion plan (30
patients completed with DTI and biopsy) in order to validate a clinical non
invasive method to delineate real infiltration margins of GBM without the use
of extra biopsy in the vicinity of high grade gliomas.

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6 Bibliography