Alexander, DC

Bailey, C

Bourne, RM

Freeman, A

Hawkes, DJ

Heavey, S

Johnston, EW

Mertzanidou, T

Panagiotaki, E

Patel, D

Punwani, S

Pye, H

Shaw, G

Siow, B

Sridhar, A

Whitaker, H

UCL Discovery

Validation of VERDICT MRI using fresh and ᝐxed prostate specimens with alignedhistological slicesColleen Bailey , Roger M Bourne , Bernard Siow , Edward W Johnston , Hayley Pye , Susan Heavey , ThomyMertzanidou , Hayley Whitaker , Alexander Freeman , Dominic Patel , Greg Shaw , Ashwin Sridhar , Shonit Punwani ,David J Hawkes , Daniel C Alexander , and Eleftheria PanagiotakiCentre for Medical Image Computing, University College London, London, United Kingdom, Discipline of Medical Radiation Sciences, University of Syney,Sydney, Australia, Centre for Advianced Biomedical Imaging, University College London, London, United Kingdom, Imaging, Francis Crick Insitute,London, United Kingdom, Centre for Medical Imaging, University College London, London, United Kingdom, Division of Surgery and InterventionalScience, University College London, London, United Kingdom, Department of Urology, University College London Hospitals, London, United Kingdom,Department of Research Pathology, University College London, London, United KingdomSynopsisThis study provides the ᝐrst step in validating the VERDICT diᝨusion model of tissue microstructure by examining the eᝨects of ᝐxation on tissuemicrostructure and comparing VERDICT parameter maps to histological features. Fresh and ᝐxed parameter maps showed similar spatial trends:ᝐxation decreased the extracellular volume fraction parameter and decreased the cell radius parameter slightly, consistent with water eᝨux.Intracellular volume fraction was lower in regions with lower cellularity, such as the peripheral zone, and directions of diᝨusion anisotropycorresponded with collagen and smooth muscle orientation patterns in the stroma.PurposeBiophysical models like VERDICT  successfully distinguish benign from malignant prostate tissue in vivo, but the accuracy with which modelparameters reᝨect the underlying tissue characteristics is unknown. Traditional histology, which is often used as the gold standard for validation,suᝨers from ᝐxation eᝨects. This study proposes a method to mitigate this problem by comparing VERDICT parameters in fresh and ᝐxedprostatectomy specimens before comparison with corresponding histological slices.MethodsSpecimensTwo prostatectomy specimens were scanned fresh (within 8 hours of surgery) and following formalin ᝐxation (~24 hours later). Fixed specimenswere rehydrated for ~10 hours in saline.Scan protocolMRI scans were carried out at 9.4 T (Varian Inc). All diᝨusion images used a PGSE sequence with 1.25 x 1.25 x 2.5 mm  resolution. Diᝨusion scanparameters are listed in Table 1 (3 orthogonal directions + 1 unweighted image each). Additionally, high-angular resolution DWIs (b=938 s/mm )were acquired (20 directions fresh, 30 directions ᝐxed). The fresh protocol contained fewer points (VERDICT parameters agreed within 10%, exceptD , which has greater variability for the fresh protocol).Data analysisModels combining VERDICT compartments with diᝨerent shapes were ᝐtted voxelwise .Compartments: Ball (unrestricted isotropic diᝨusion), Tensor (unrestricted anisotropicdiᝨusion described by three directions), Zeppelin (cylindrically symmetric tensor) andSphere (isotropically restricted diᝨusion).Parameters ᝐtted include the intracellular volume fraction (f ), intracellular diᝨusioncoeᝨcient (D ), perpendicular diᝨusion coeᝨcients (D , D ), sphere radius (R), and thedirections for anisotropic components. The normalization constant and T2 were also ᝐtted.The primary extracellular diᝨusion coeᝨcient (D ) was ᝐxed to 1.5 μm /ms.Model selection used the Akaike Information Criterion, , where L isthe maximum likelihood obtained from the ᝐt and k is the number of parameters.Landmarks in a 3D-printed mold were used to select the same slice for fresh and ᝐxedscans, then aligning by manual rotation and translation.HistologyGuides in the 3D mold were used to cut 3 µm-sections 5 mm apart, aligned with the imagingplane. Slices were stained with H&E, then digitised (Hamamatsu NanoZoomer) using a 20xobjective.Results and Discussion1 2 3,4 5 6,7 6,71 6 8 8 6,7 6,7 51 1 11 23 45 6781,232I3II ⊥1 ⊥2E2  Ã MO	  'Results and DiscussionFigure 1 shows ᝐts for a voxel with restriction. The Tensor-Ball model did not capture the trends at high b-values (explanation in Figure caption).The data also indicated anisotropy (Fig 1 right), previously observed ex vivo .Model selection (via lowest AIC) output is shown in Figure 2. Fresh specimens displayed some voxels where models without restriction (Ball-Ball andTensor-Ball) were selected. The smaller number of points in the fresh protocol, particularly at high b-values, may reduce the sensitivity to restrictioneᝨects. In ᝐxed specimens, the Zeppelin-Sphere and Tensor-Sphere models best explained the data in most voxels, consistent with other studies .Figure 3 shows the eᝨects of ᝐxation on Tensor-Sphere parameters. Spatial trends were similar between fresh and ᝐxed specimens. R was larger infresh specimens and f  was smaller, an additional reason that models without restriction may be selected more frequently for fresh tissue: lesswater experienced the sphere boundary during measurement, which limited the precision of R. The changes in R and f  may result from an eᝨux ofwater and cell shrinkage during ᝐxation.The extracellular perpendicular diᝨusion coeᝨcients increased slightly from fresh to ᝐxed (a decrease in anisotropy). The apparent decrease in Dfrom 0.63 to 0.22 µm /ms may be due to the lower precision of this parameter in the fresh protocol, but the value from ᝐxed voxels agreed withprevious high-resolution studies .Figure 4 aligns the f  and R ᝐxed parameter maps with histology. The zoomed portion for Specimen 1 highlights trends that can be seen across thesample: regions with lower cellularity had lower f  and R hit the upper limit allowed by the ᝐt in more voxels compared to the transition zone.The colour FA map indicates the primary diᝨusion direction and demonstrated stronger anisotropy in stromal regions. The orientation of thecollagen and smooth muscle aligned with the primary diᝨusion direction, a correspondence previously observed using a simple diᝨusion tensormodel .ConclusionsMicrostructural features from histology corresponded to features of the VERDICT parameter maps, including fewer cells in regions with low f , suchas the peripheral zone, and orientation patterns in the stromal regions that corresponded to the orientation of the extracellular anisotropy. Smallchanges in f  and R, which can be explained by water eᝨux, were observed between fresh and ᝐxed tissue maps, but relative trends within aspecimen were unaᝨected. The next step for validation involves clinical VERDICT data and these results emphasise that comparison needs toaccount for the eᝨects of tissue ᝐxation on microstructure.AcknowledgementsThis work was supported by funding from EPSRC platform grant EP/M020533/1, the CRUK-EPSRC Cancer Imaging Centre at KCL/UCL and by EPSRCfellowship EP/N021967/1.References1. Panagiotaki, E. et al. Noninvasive quantiᝐcation of solid tumor microstructure using VERDICT MRI. Cancer Res. 74, 1902–1912 (2014).2. Panagiotaki, E. et al. Microstructural Characterization of Normal and Malignant Human Prostate Tissue With Vascular , Extracellular , andRestricted Diᝨusion for Cytometry in Tumours Magnetic Resonance Imaging. Invest. Radiol. 50, 218–227 (2015).3. Panagiotaki, E. et al. Compartment models of the diᝨusion MR signal in brain white matter: a taxonomy and comparison. Neuroimage 59, 2241–54 (2012).4. Liang, S. et al. Information-based ranking of 10 compartment models of diᝨusion-weighted signal attenuation in ᝐxed prostate tissue. NMRBiomed. 29, 660-671 (2016).5. Bourne, R. M. et al. Eᝨect of formalin ᝐxation on biexponential modeling of diᝨusion decay in prostate tissue. Magn. Reson. Med. 70, 1160–6(2013).6. Bourne, R. M., Bongers, A., Chatterjee, A., Sved, P. & Watson, G. Diᝨusion anisotropy in fresh and ᝐxed prostate tissue ex vivo. Magn. Reson. Med.634, 626–634 (2015).44III24II6IIFiguresTable 1 Scan parameters for the fresh and ᝐxed protocols. The value in each gradient separation (Δ) + duration (δ)/gradient strength (G) boxcorresponds to the b-value (s/mm ) for that scan. Empty boxes indicate that gradient strength was not included in the protocol. Values inparentheses show the number of averages for a particular scan (no parentheses=1 average). 2Figure 1 Data (points) with ᝐts (lines) using (a) the Tensor-Ball and (b) Tensor-Sphere models. Left graphs plot signal attenuation with b-value fordiᝨerent Δ. Vertical lines in (a) result from an incorrect primary diᝨusion direction that attempts to compensate for mis-ᝐt of high-b data for Tensor-Ball. Right graphs plot signal variation with gradient direction for the high angular resolution scan. ψ is the angle between the diᝨusion gradient andprimary diᝨusion direction of the model (this may diᝨer for diᝨerent ᝐts, which is why the points have diᝨerent positions along the x-axes in thetop and bottom right plots). Figure 2 Results of the model selection using the AIC. Zeppelin-Sphere and Tensor-Sphere best explained the data in most voxels of the ᝐxedspecimens. Some voxels in the fresh specimens are best explained by models without restriction, as discussed in the text. Figure 3 Parametric maps and histograms for all voxels in the prostate for one specimen, fresh (left) and ᝐxed (right). The fresh specimen containsvoxels where R hits the maximum of 20 µm allowed by the ᝐt, particularly in the low-cellularity peripheral zone, indicating restriction could not bereliably modelled; these regions correspond to the regions where Ball-Ball and Tensor-Ball are selected in Fig 2. Trends were similar in the secondspecimen. Figure 4 (a) Intracellular volume fraction, f , and (b) cell radius, R, maps from the ᝐxed MRI alongside (c) histology for the corresponding slice. (d) Thecolour FA map for the Tensor portion of the Tensor-Sphere model indicates the primary diᝨusion direction, with colour and brightnesscorresponding to the directions and strength of the anisotropy as indicated in the legend at the top right. The boxes on Specimen 1 indicate thezoomed region displayed in the middle row, demonstrating variations in cellularity, collagen alignment and lumen space on histology. I

Validation of VERDICT MRI using fresh and fixed prostate specimens with aligned histological slices

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Validation of VERDICT MRI using fresh and fixed prostate specimens with aligned histological slices

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