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Clinical and postmortem diffusion MRI for deep brain stimulator electrode localization in Essential Tremor patients

by

Jingxian Zhang

Trinity College Department of Neuroscience

Duke University

Thesis committee

Nandan Lad, MD, PhD; Supervisor

G. Allan Johnson, PhD

Evan Calabrese, PhD

Thesis submitted in fulfillment of the requirements for the degree of

Graduation with Distinction in Bachelor of Science in the Department of Neuroscience in Trinity College

of Duke University

2015

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Dedication

This thesis is dedicated to my dearest siblings Alicia and Michael Zhang.

It is also dedicated to Karishma Popli, carry on Karishma!

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Contents Abstract .................................................................................................................................... 5

List of Tables ............................................................................................................................ 6

List of Figures .......................................................................................................................... 7

Acknowledgements................................................................................................................. 8

1. Introduction ......................................................................................................................... 9

2. Methods...............................................................................................................................12

2.1 DBS patient data .........................................................................................................12

2.1.1 Pre-operative anatomic MRI.............................................................................13

2.1.2 Pre-operative diffusion tensor MRI .................................................................13

2.1.3 Post-operative CT..............................................................................................14

2.1.4 Post-operative electrode testing and outcomes...............................................14

2.2 Fiber tractography......................................................................................................14

2.2.1 Deterministic fiber tractography in clinical diffusion MRI ............................15

2.2.2 Analysis of DBS contact proximity with clinical DRT tractography .............15

2.2.3 Probabilistic fiber tractography in postmortem diffusion MRI .....................16

2.2.4 Analysis of DBS contact proximity with postmortem DRT tractography ....17

2.2.5 Analysis of DBS contact proximity with postmortem rendering of Vim......18

3. Results .................................................................................................................................18

3.1 DBS Lead and Contact segmentation and modeling ...............................................19

3.2 Diffusion Tensor Imaging and Fiber tractography ..................................................20

3.3 Deterministic tractography........................................................................................21

3.4 Probabilistic tractography..........................................................................................21

3.5 Registration of postmortem MRI to clinical datasets...............................................22

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3.6 Statistical analysis of DBS contact position and clinical efficacy ............................23

4. Discussion ...........................................................................................................................24

4.1 In vivo patient dataset analysis ..................................................................................24

4.2 Ex vivo patient dataset analysis .................................................................................26

References ...............................................................................................................................28

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Abstract:

Deep brain stimulation (DBS) is now the preferred surgical treatment for a variety of movement disorders. We propose standardized protocols for modeling implanted

DBS electrodes to better visualize and understand the correlation between contact

positioning, underlying tractography and efficacy of treatment outcomes. Imaging

datasets (stereotactic CT, MRI-FLAIR and DTI) of patients treated for essential tremor with bilateral ventral intermediate (Vim) nucleus DBS were analyzed, and a standardized protocol was developed to accurately model the placement of DBS leads

and individual contacts. This was paired with consistent fiber tractography of the

relevant dentatorubrothalamic tract in each patient dataset: deterministic fiber tracking

was performed on clinical MRI in Brainlab neuronavigation software, while probabilistic

fiber tracking was performed on a postmortem diffusion MRI template of the brainstem

and thalamus in Avizo 3D imaging software. A reliable and reproducible method to

segment DBS lead and contact positions in relation to the DRT validates the feasibility

of including DTI fiber tractography-based analyses when studying targeting, lead

location and programming for DBS. This work could provide further insight into circuit

modulation of underlying white matter pathways that appear to be the true targets of

neuromodulation by DBS.

Keywords: Deep brain stimulation; diffusion tensor imaging; fiber tractography;

Essential Tremor

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List of Tables1

Table 1: Stereotaxic coordinates of DBS electrode contacts for each patient......................40

Table 2: Patient Demographics..............................................................................................43

Table 3: Summary of post-operative electrode testing outcomes .......................................44

Table 4: Results of Spearman rank correlation.....................................................................47

1 Table formatting credit to Dr. Evan Calabrese; Duke University Medical Center Dept. of Radiology CIVM

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List of Figures

Figure 1: DRT mapped through three ROIs in the clinical MRI image set..33

Figure 2: Contact creation and fiber tractography...............................................................34

Figure 3: Fiber tractography through contact ROI in 10 patients .......................................35

Figure 4: Probabilistic tractography of postmortem diffusion MRI DRT36

Figure 5: Registration between in vivo and postmortem MRI.............................................37

Figure 6: Probabilistic DRT shown with red nuclei, Vim, and implanted leads ...............38

Figure 7: DBS electrode position relative to postmortem template DRT in 12 patients....39

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Acknowledgements:

This work was supported by the National Institutes of Health and the National

Institute of Biomedical Imaging and Bioengineering (grant number P41 EB015897). I would like to sincerely thank and credit my team members and major contributors from their various departments in Duke University Medical Center: Dr. Evan Calabrese and

Dr. G. Allan Johnson in Radiology, Mr. Peter Masso from Brainlab, Dr. Patrick Hickey in

Neurology, Dr. Christine Hulette in Pathology, Ms. Beth Parente in Surgery, Dr. Dennis

Turner in Surgery, Dr. Allen Song in Brain Imaging, and Dr. Guillermo Sapiro in Duke

Biomedical Engineering. I would also like to thank Percy Rochelle and Robert

Satterwhite for their help in procuring brain specimens, Mark Martin for help with clinical

image data, Porsche Atwater and Anne Jarvis for their guidance and support, and Dr.

Shouyin Zhang and Ms. Xiaoling Lu.

Most importantly, I would like to thank my principal investigator Dr. Nandan Lad

in Surgery, who has been an unwavering source of inspiration and support, for his

mentorship, time, and encouragement these past two years.

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1. Introduction

Essential tremor (ET) is one of the most common neurological disorders occurring in approximately 4.0% of individuals aged 40 years and older (Dogu et al., 2003). ET is characterized by action and postural tremor of the arms, and may involve the head and voice as well. Most patients experience progression in tremor severity

over time and other neurological signs such as rest tremor and ataxia may also arise.

Many patients with ET report only mild symptoms, however, about three quarters of

patients have significant disability and decreased quality of life. More than 90% of

patients who seek medical care report disability and 10% of patients who present to a

movement disorder clinic report severe motor disabilities, including tremor that

interferes with eating, drinking, writing, or communication (Louis et al., 2001) . First-line medical treatments include propanolol and primidone, though medications are typically

effective in only 50% of patients and rarely reduce the tremor to asymptomatic levels

(Deuschl et al., 2011; Elble and Deuschl, 2009). For the subset of medically refractory cases surgery is an effective option.

The ventralis intermedius (Vim) nucleus of the thalamus is the typical intervention target structure in the ventral thalamus for patients with ET (Deuschl et al., 1998; Hassler et al., 1979; Schaltenbraaand et al., 1977). Two primary surgical procedures have been performed in patients with ET: thalamotomy and high frequency deep brain

stimulation (DBS) (Flora et al., 2010; Lehericy et al., 2001; Schuurman et al., 2008). Both procedures target the Vim, however equal success has been reported with

posterior subthalamic area (PSA) modulation, prompting further investigation regarding the optimal neuromodulation target for tremor suppression.

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The Vim receives its major afferent projections from the deep cerebellar nuclei, which then project to the motor cortex. Microelectrode recording of the Vim in patients with ET identifies cells discharging in bursts that are time locked to the patients tremor,

indicating that tremor is associated with an abnormal discharge in the cerebellothalamic

pathway (Benabid et al., 1996). An interruption in this pathway from lesion or stimulation provides some theoretical basis for the empirical observation of tremor

improvement, but a more precise understanding is still lacking.

High frequency deep brain stimulation (DBS) has largely replaced the ablative procedures used in the past to treat such movement disorders (Flora et al., 2010). DBS has been shown to be a reproducible, adjustable, and reversible neuromodulation technique (Barkhoudarian et al., 2010; Kumar et al., 2003). Accurate targeting and selective stimulation are essential in optimizing symptom alleviation and minimizing

potential side effects. The size and position of the neural targets is variable, and

indirect targeting is based on atlas-defined coordinates rather than patient-specific

anatomy, although new acquisition and processing approaches are addressing this

target localization challenge (Duchin et al., 2012; Kim et al., 2014; Lenglet et al., 2012). Historically, various imaging modalities and targeting methods have been used to

achieve successful clinical outcomes, including MR imaging, CT scanning,

ventriculography, and microelectrode recording (De Salles et al., 2004; Lee et al., 2005; Mori et al., 1999; Sedrak et al., 2008). Recent advances in imaging modalities have refined the visualization of surgical targets and landmarks. Multi-modal and advanced

neuroimaging techniques such as diffusion tensor imaging (DTI) show great promise for providing increased sensitivity and specificity of the underlying structurefunction

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relationships (Basser et al., 2000; Coenen et al., 2011; Hyam et al., 2012; Mori et al., 1999; Poupon et al., 2000). These techniques are also being explored for the more immediate clinical needs of DBS lead targeting, intraoperative testing, postoperative

programming, patient-specific maps, algorithms and treatment strategies (Lenglet et al., 2012; Kim et al., 2014).

Patients undergoing DBS offer a unique opportunity to study the functional

anatomy of stimulation targets in humans. Here, protocols are presented for modeling

implanted DBS electrode leads and evaluating electrode contact positions relative to

key fiber tracts mapped by diffusion tensor imaging and fiber tractography (FT). This has utility and implications not only for understanding circuit modulation of current grey

matter DBS targets (Vim, subthalamic nucleus, internal globus pallidus) in movement disorder surgery, but is also critical for future white matter targets (e.g. fornix in memory disorders, cingulate in mood disorders) (Gutman et al., 2009; Laxton et al., 2010; Lozano et al., 2012; Mayberg et al., 2005). The analysis has been implemented on individual clinical patient datasets as well as on a postmortem diffusion MRI template of

the brainstem and thalamus transformed into patient image space. The purpose of

developing these protocols was to standardize the imaging of these patients and better

visualize and understand the correlation between DBS electrode contact positioning and

efficacy of treatment outcomes in individual patients. The high resolution diffusion MRI

postmortem template with 3D nuclei and DRT tract reconstruction can be registered to

individual in vivo clinical images of DBS patients to correlate electrode proximity to the

DRT and test for surgical outcomes. Likewise, the direct analysis of electrode

positioning with DRT tractography in clinical images using Brainlab software can shed

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light on how DBS lead localization in individual patient anatomy affects treatment

efficacy and potential side effects, while expanding the analysis pre-operatively will

pave the way for even better surgical results and more precise understanding of

structure-function relationships.

2. Methods

2.1. DBS patient data

All experiments on patient image datasets were approved by the Duke University

institutional review board (IRB). Twelve patients with medically refractory ET received DBS targeted to the ventralis intermedius nucleus of the thalamus (Vim). Atlas based targeting of the Vim nuclei was performed according to standard neuroanatomical

targets relative to the AC-PC plane (Schaltenbrand et al., 1977). The Vim target was identified on FLAIR imaging using target coordinates 13 to 15 mm lateral to the anterior

commissure-posterior commissure (AC-PC) line, 0 mm below the AC-PC plane, and 30% of the total AC-PC distance posterior to the midpoint of AC-PC.

All patients underwent pre-operative structural and diffusion tensor MRI, as well

as post-operative x-ray computed tomography (CT) scans to localize electrode placement. Patients undergoing bilateral VIM DBS were implanted with two quadripolar

electrodes (model 3389, Medtronic, Minneapolis, Minnesota) for a total of eight contacts per patient. Contacts measure 1.5 mm in diameter with a 0.5 mm tip before the most

distal contact and 0.5 mm spacing between contacts. Final stereotaxic coordinates for

each electrode contact are provided in Table 1. Patient demographics for both studies

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included 4 males and 8 females with average age 65 15 years (Table 2); two of the patients from the postmortem diffusion MRI study were not used in the clinical study due

to CT slicing above 1mm in thickness.

Patients also underwent detailed preoperative, intraoperative and postoperative

neurological testing for motor and tremor control, sensory and cranial nerve testing,

muscle tone and spasticity, overall flexibility and reflexes, as well as cognitive

assessment. The physical examinations were performed by two senior clinicians

experienced with clinical care of patients with ET.

2.1.1 Pre-operative anatomic MRI2

Pre-operative MR imaging was performed on a 3 Tesla GE Discovery MR750

scanner (Waukesha, WI). T1-weighted structural images were obtained with a 3D fast

spoiled-gradient-recalled (FSPGR) pulse sequence (TR = 6.5 ms, TE = 2.5 ms, = 12,

BW = 140 kHz), at 1 mm isotropic resolution. T2 fluid attenuated inversion recovery (FLAIR) images were acquired with an inversion-prepared spin echo pulse sequence (TR = 10,000 ms, TE = 148 ms, TI = 2,250 ms, BW = 781 Hz/pixel) at 1 x 1 mm in-plane resolution with 1 mm slice thickness and 1 mm spacing between slices.

2.1.2 Pre-operative diffusion tensor MRI

Diffusion tensor data were acquired with an echo-planar imaging sequence (TR = 8,000 ms, TE = 84.9 ms, BW = 1562 Hz/pixel) using a 30-direction gradient encoding scheme at b = 1000 s/mm2 with 2 non-diffusion-weighted images. The acquisition

2 Sections 2.1.1 to 2.1.3 credit to Dr. Nandan Lad, Ms. Beth Parente, Dr. Allen Song; Duke University

Medical Center Dept. of Surgery; Duke-UNC Brain Imaging and Analysis Center

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matrix was 128 x 128 over a 240 x 240 mm field of view (FOV) with a slice thickness of 2 mm. Data were zero-filled in k-space to a matrix size of 256 x 256 prior to

reconstruction for a final nominal voxel size of 0.94 x 0.94 x 2 mm.

2.1.3 Post-operative CT

CT images were acquired on a Siemens SOMATOM Definition Flash scanner

with a spiral scan using a 512 x 512 Matrix over a 250 x 250 mm FOV for an in-plane

resolution of 0.484 mm. Approximately 300 contiguous, non-overlapping, 0.625 mm

thick slices were acquired covering the entire neurocranium, Additional scan parameters

include MA setting = 250 and kVp = 120. The standard reconstruction algorithm was

used.

2.1.4 Post-operative electrode testing and outcomes3

Post-operative electrode testing was performed on DBS patients after initial

surgical recovery. Each contact was tested independently at voltages ranging from 0.5

to 3 volts dependent on patient tolerance, with frequency between 135-185 Hz and

pulse width between 60-90 microseconds. For each contact, treatment efficacy was

recorded on a three level subjective scale that included no effect, mild/moderate control, and good/excellent control. Patients experiencing persistent undesired side

effects were recorded on as having significant side effects, while patients that did not

experience any side effects, experienced transient side effects, or only presented side

effects at very high testing voltages that were not used in the final voltage setting were

recorded as no significant side effects. Typical side effects included paresthesias,

3 Section credit to Dr. Patrick Hickey; Duke University Medical Center Dept. of Neurology

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worsening of tremor, and/or dysarthria. In some cases an effective contact was

identified early in testing and the remaining contacts were not tested to avoid

unnecessary patient discomfort. The corresponding data points were recorded as n/a.

Outcome testing results are summarized in Table 3.

2.2 Fiber tractography

2.2.1 Deterministic fiber tractography in clinical diffusion MRI

Deterministic fiber tracking was performed using iPlan stereotaxy software

(Brainlab, Feldkirchen, Germany). Future research will consider further validation with other techniques (Aganj et al, 2010; Aganj et al., 2011; Lenglet et al., 2012). All imaging datasets were individually loaded into Brainlab iPlan. After ensuring accurate and

overlapping fusion of all dataset image pairs in the Image Fusion function, regions of

interest (ROI) were mapped under Fiber Tracking using the FLAIR MRI image set for clear structural contrast. Fractional anisotropy threshold and minimum length were

standardized for all patients at 0.2 and 75 mm, while maximal angle change of fibers

was set at the default value of 70 degrees.

In the case of ET patients, the putative mechanism of action of DBS of the Vim

thalamus is the modulation of the underlying white matter fiber tract termed the

dentatorubrothalamic (DRT) tract (Groppa et al., 2014). The DRT tract is the primary fiber bundle forming the superior cerebellar peduncle, which is one of the largest

efferent connections of the cerebellum and consists of axon fibers arising from cells

located in the dentate, emboliform, and globose nuclei. These fibers then project to the thalamus and terminate in the ventral lateral and ventral posterolateral thalamic nuclei,

which go on to project to the primary motor cortex (Habas and Cabanis, 2007; Kwon et

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al., 2011). As such, this system is functionally relevant for somatomotor coordination and tremor control. Three cubic ROIs were marked for the DRT tract (Figure 1): 1) in axial section, ROI around the red nucleus at its widest diameter, 2) in axial section, ROI around resulting fibers which reached the precentral gyrus containing the primary motor

cortex (M1), 3) in axial section, end region ROI around the ipsilateral dentate nucleus to include resulting fibers from the first ROI which traveled along the superior cerebellar

peduncle (Yousry et al., 1997). In addition, mapping of the most distal DBS active contact (contact 0) and its relation to the DRT tract was performed by establishing two cubic ROIs: 1) in axial cut, the cubic ROI generated for the most distal DBS active contact (contact 0) and 2) in axial cut, ROI around the precentral gyrus to include resulting fibers from the first ROI which traveled along the DRT tract.

2.2.2 Analysis of DBS contact proximity with clinical DRT tractography

The relevant active contact as determined by post-operative testing outcomes

was established as the sole ROI for fiber tracking. Tractography was performed on 10

ET patients using the FLAIR MRI image set for clear structural contrast at fractional

anisotropy threshold of 0.2 and minimum length 75 mm, while maximal angle change of

fibers was set at the default value of 70 degrees.

2.2.3 Probabilistic fiber tractography in clinical diffusion MRI4

Tractography regions of interest (ROIs) were manually segmented from both anatomic and tensor-derived image data, using a histology-based human brainstem

4Section credit to Dr. Evan Calabrese; Duke University Medical Center Dept. of Radiology CIVM

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atlas for reference (Paxinos and Huang, 1995). ROIs for tracking the DRT included the superior cerebellar peduncles, the red nuclei, and Vim nuclei of the thalamus.

Tractography fiber data were reconstructed using FSLs BedpostX, a direct,

multi-fiber orientation estimation algorithm that provides estimates of fiber distribution

error for probabilistic tractography (Behrens et al., 2007). These data were used for probabilistic fiber tractography using FSLs ProbtrackX. Tractography of the bilateral

DRTs was generated using the superior cerebellar peduncle as a seed region, and the

contralateral red nucleus and Vim nucleus of the thalamus as waypoints. Tracking

parameters included 5000 seeds per voxel, a step size of 100 m, and a curvature

threshold of 45 per voxel. Resulting tractography data were thresholded at > 2000

tracks per voxel, or roughly 1%, consistent with previous work (Jbabdi et al., 2013).

2.2.4 Analysis of DBS contact proximity with postmortem DRT tractography

In order to validate the anatomic accuracy of postmortem tractography results,

we assessed the proximity of each DBS lead to dentatorubrothalamic tract tractography

from postmortem brainstem datasets after spatial transformation into the anatomic

space of each patient dataset. Individual DBS leads were clearly differentiated and

identified on the post-operative CT scan, and the proximity of each spherical lead to the

postmortem dentatorubrothalamic tract tractography were classified in a four rank

system. The eight leads per patient were analyzed as separate data points because

each lead was tested independently for treatment efficacy. DBS leads directly inside

the tract were ranked as inside, leads contacting the surface of the rendered tract

were ranked as touching, leads in close proximity to the tract but not contacting it were

ranked as close, and leads not in close vicinity of the tract were ranked as not

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touching. The shape and size of the dentatorubrothalamic tract was highly dependent

on the threshold used for tractography. We determined the area inside the DRT tract to

be at a threshold of greater than 2000 fibers per voxel, which is consistent with optimal

threshold recommendations for probabilitstic tractography. The rank system for DBS

lead proximity was evaluated independently by two observers and then compiled into a

single numerically ranked datasheet and evaluated with a Spearman rank correlation.

2.2.5 Analysis of DBS contact proximity with postmortem rendering of Vim

We also assessed the proximity of each DBS lead to a rendering of the Vim from

the postmortem brainstem datasets to gauge the correlation between patient treatment

outcomes and stimulation of the Vim of the thalamus. Vim was segmented bilaterally

for the postmortem brainstem template referencing anatomy from the Mai-Paxinos

Human Nervous System atlas. The high contrast resolution of the postmortem

brainstem datasets allowed for accurate definition of the Vim apart from the surrounding

thalamic nuclei. Vim segmentation was transformed into each individual patients

anatomic space using the spatial transformations detailed earlier. A similar four rank

system as the analysis of DRT tract proximity was used respective to the Vim, after

which correlation was evaluated with a Spearman statistic test.

3. Results

Postoperative lead visualization and overlapping of contacts with their underlying

DTI target has been done in a limited fashion to date. We utilized the currently

commercially available DBS lead 3389 (Medtronic, Inc., Minneapolis, MN) to examine the underlying fiber connectivity and putative circuits being modulated by stimulation.

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The function of these circuits has been extensively studied in preclinical models,

however visualization in humans has been limited due to inability to reliably visualize the

lead and its contacts and their relation to associated fiber tractography in stereotactic

space.

Furthermore, the ability to visualize the underlying circuit being modulated and

regional neuroanatomy is critical for optimal clinical results (Coenen et al., 2011; Henderson, 2012; Hyam et al., 2012). Specifically, it was found that the DRT tract was reproducibly visualized passing through the active DBS contacts of 10 ET patients

undergoing Vim stimulation. These findings are discussed in detail as follows.

3.1. DBS Lead and Contact segmentation and modeling

Leads and contacts were segmented and modeled using the Object Creation function after alignment in the View and Adjustment function in Brainlab software. The post-operative CT image set was used to align the length of the lead parallel to the

vertical axis of the screen, with the bottom of the lead (where the contacts are located) centered right on the horizontal axis.

The lead can then be made into an object using auto segmentation in Object Creation; a histogram is provided in this function so that the lead can be outlined by its

difference in radiopacity. This provides a standardized way to accurately depict the

leads actual shape and slight curvature in its final location instead of relying on a linear

model as has been done previously (Coenen et al., 2011).

Contacts are 1.5 mm in diameter, and created as new objects standardized using the brush function; this function allows users to adjust size of the brush diameter to highlight and create an object. To correctly model the spacing of the contacts on the

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lead in relation to each other, a void object was also made using brush size 0.5 mm; this object is used in the sagittal view to fill out the 0.5 mm dead space at the tip of the lead as well as adjusted to fill out space between contacts. The electrode pattern used for patients had between contact spacing fixed at 0.5 mm for typical Vim DBS leads

(Model 3389). The standardized brush sizes used for contact and spacing coupled with the exact outline of the lead by density allows for correct positioning of the dead space

at the lead tip and subsequent contacts/contact spacing in the simulation. To model

each contact as consistently as possible to their actual shape, the brush function set at

1.5 mm was used in the sagittal, coronal, and axial planes to create an approximate

sphere of volume 0.002 cm,verified in the Plan Content section.

3.2. Diffusion Tensor Imaging and Fiber tractography

To visualize relevant fiber tracts that cross through the individual DBS lead

contacts, the Fiber Tracking function was utilized to make a new region of interest from

the previously created contact using the Existing 3D Object selection. It is then possible to track fibers that run through this contact to brain structures of interest (Figure 2A).

Figure 2B shows a sample ET patient treated with bilateral Vim DBS using

modeled electrodes created as regions of interest in the Fiber Tracking function. To

account for the specificity and small size of this ROI, minimum fiber length was adjusted to 30 mm, while fractional anisotropy threshold and maximal angle change of fibers

were held constant at 0.2 and 70 degrees, respectively.

3.3 Deterministic tractography

Deterministic tractography through the active DBS contact set as the sole

region of interest yielded consistent fiber tracking patterns across the patient datasets

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(Figure 3). It can be observed that these fiber tracts directly contacting the DBS electrodes closely follow the DRT tract modeled previously, and this was consistently

seen throughout the 10 ET patients. As the DBS electrode itself is set as region of

interest, there is no need for a correlation analysis of the results: the fiber tracts shown

are in overlapping voxels as the active contact that is used for treatment. Overlay of

fiber tracts contacting the DBS electrodes with the target DRT tract can provide analysis

on correlation of electrode positioning with treatment outcomes for each patient.

3.4 Probabilistic tractography5

Probabilistic tractography with multiple fiber orientations was used to reconstruct

DRT probability maps in the postmortem brainstem dataset. The DRT courses from the

dentate nucleus of the cerebellum through the superior cerebellar peduncle, crosses the

midline in the mid pons, passes through and around the contralateral red nucleus, and

finally relays in the Vim nucleus of the thalamus before continuing to cortical motor

areas (e.g. Figure 4A). Importantly, routine clinical diffusion tractography data are poorly suited for accurately representing crossing fibers, such as those present in the

midline crossing of the DRT between the dentate nucleus and the red nucleus.

Probabilistic tractography of the postmortem template correctly represented the midline

crossing of the DRT as well as its connections to the contralateral red nuclei and Vim

nuclei (Figure 4AC). Probabilistic tractography also revealed minor branches of the DRT coursing along the medial aspect of the red nuclei (Figure 4B). These medial pathways have been observed in histology studies of the human brainstem (Massion,

5 Sections 3.4 to 3.6 referenced from manuscript submitted to Human Brain Mapping co-authored

under Dr. Evan Calabrese

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1967), but have not previously been demonstrated with diffusion tractography. Visualization of probabilistic DRT tractography with surface renderings of the Vim nuclei

and red nuclei clearly demonstrates the close relationship between these structures in

the human brainstem, and highlights the potential difficulty in accurately targeting DBS

electrodes in this complex brain region (Figure 4C).

3.5 Registration of postmortem MRI to clinical datasets

Postmortem brainstem data were non-linearly registered to 12 patient datasets.

We observed considerable variation in patient brain anatomy, particularly with regard to

ventricle size. Nonetheless, registration of postmortem brainstem data to patient

datasets yielded good visual alignment (Figure 5). Major borders, such as the anterior surface of the pons, the dorsal surface of the thalamus, and the posterior surface of the

tectum showed strong agreement between patient datasets and registered postmortem

data (Figure 5AB). Smaller features, such as the optic chiasm, were also very closely aligned after registration (Figure 5BC).

Additional assets from the postmortem dataset, including probabilistic

tractography and 3D segmentations of the Vim and red nuclei, were also transformed

into patient image space. These data, combined with post-operative CT data for

electrode localization, allowed 3D visualization of the complex spatial relationships

between DBS contacts and the relevant nuclei and white matter tracts (Figure 6AD). In most clinical datasets, DBS contacts were clearly visible in post-operative CT data as

four discrete bulges at the distal end of the electrode (e.g. Figure 6B). In cases where contacts were not clearly visible in CT data, they could be inferred based on the specific

geometry of the electrodes used (Coenen et al., 2011b). Using these data, we

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measured the position of each electrode contact with respect to the DRT and Vim from

the postmortem template. Across all patient datasets, 22/24 electrodes had a least one

contact directly touching the Vim nucleus segmented from the postmortem template

(e.g. Figure 6AB). In contrast, only 18/24 electrodes had at least one contact directly touching the thresholded DRT model (Figure 7).

3.6 Statistical analysis of DBS contact position and clinical efficacy

In order to assess the accuracy of our postmortem template, we tested for

statistically significant correlation between electrode proximity to the DRT and Vim, and

clinical outcomes including treatment efficacy and the presence of side effects. The

non-parametric Spearman rank correlation was used because clinical outcomes were

assessed as ordinal variables. The calculated p-values and R-values (i.e. Spearman correlation coefficients) for each comparison are presented in Table 4. We observed no significant correlation between contact proximity to the DRT or Vim and the presence of

side effects. Despite the fact that the Vim nucleus of the thalamus was the explicit target

for DBS electrodes, we did not detect a statistically significant correlation between

treatment outcome and contact proximity to the Vim from the postmortem template. We

did, however, detect a highly significant, yet weak, positive correlation between

treatment efficacy and contact proximity to the DRT from the postmortem template (p = 0.005, R = 0.336). This correlation remained significant after Bonferroni correction for multiple comparisons (pcorrected = 0.02). This correlation suggests that our postmortem DRT model has at least some degree of anatomic relevance for DBS electrode targeting

in ET patients.

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4. Discussion

Our results describe new protocols for both in vivo and ex vivo MRI to

consistently model DBS electrode position in a standardized fashion relative to

visualizations of relevant pathways mapped by diffusion tensor imaging and fiber

tractography. Reliable fiber tracts and modulation of the associated sensorimotor

networks in patients with ET was seen, a finding that is consistent with prior studies

(Benabid et al., 1996; Coenen et al., 2011). The most striking findings in our analysis are the intimate positioning of DBS leads with surrounding fiber tracts previously shown

to respond to neuromodulation of the sensorimotor system by modulating distinct white

matter circuits in ET. In particular, 1) we demonstrate using individual patient MRI that fiber tracts approximating the DRT tract in ET patients are stimulated by their active

DBS contact in the thalamus, and 2) we analyze the correlation between patient DBS contact positioning to probabilistic DRT tractography and 3D modeling of the Vim in a

high resolution postmortem brainstem and thalamus template.

4.1 In vivo patient dataset analysis

Seeding of the DRT pathway in patients undergoing Vim DBS for tremor showed

connections from the primary motor cortex (M1), ipsilateral red nucleus and cerebellum, which is consistent with the published literature (Coenen et al., 2011). The Vim is generally accepted to be the cerebellar receiving area of the thalamus before the fibers

are projected to the primary motor cortex (Hasslet et al., 1979; Carpenter, 1991). Retrograde tracer studies of herpes simplex virus-1 from M1 injections in macaques stains both the cerebellum and globus pallidus (Hyam et al., 2012).

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In the in vivo portion of this study, we used physical localization of contacts from

individual cases to generate consistent fiber tractography of tracts directly touching the

contact. The tracts were then visually assessed to approximate the shape and position

of the DRT tract. Benefits of this approach include bypassing the statistic correlation

analysis of tract proximity to contact positioning, as the tracts shown are mapped

through the active contact as ROI. However, this approach can be greatly refined with 1) overlaying the DRT tract generated through its three relevant ROIs (red nuclei, dentate nucleus, precentral gyrus) on top of the active contact generated tractography to determine if they indeed have significant overlap, 2) quantizing this overlap numerically with fiber counts or another thresholded method, and 3) analyzing this degree of overlap for each patient with their patient outcomes. Generation of connectomic maps of

cortical connectivity by selecting the entire thalamus or entire cortex relative to the DBS

contact of interest is an alternative to the approach we have used (Henderson, 2012). Further study is required to test these methods in other regions of surgical interest such

as the sensory and anterior thalamic nuclei for pain and epilepsy surgery, respectively

(Bittar et al., 2005; Lega et al., 2010). By combining patient dataset models with intraoperative and postoperative

clinical results, detailed analyses can be made for each patient to pinpoint correlations

in electrode placement and treatment outcome that can serve as a guide for

programming as well as creation of patient-specific models (Hagmann et al., 2010; Lenglet et al., 2012). Further studies examining electrical fields of stimulation and surrounding pathways will be critical to dissect therapeutic stimulation from potential

stimulation side effects. The ability to visualize the fiber target of interest in relation to

26

the surgical implant allows not only for more accurate sub-millimiter lead placement, but

also postoperative programming, current steering and novel stimulation algorithms. DTI

fiber tractography will likely play an increasingly important role for mapping proposed

white matter DBS targets for large neurological disorders including Alzheimers Disease

(Fornix stimulation) and Depression (Cingulate Cg25 stimulation).

4.2 Ex vivo patient dataset analysis6

In the ex vivo portion of this study, we present a high spatial and angular

resolution diffusion MRI template of the postmortem human brainstem and thalamus

with 3D reconstructions of the nuclei and white matter tracts involved in ET circuitry. We

demonstrate accurate registration of these data to in vivo, clinical images from patients

receiving DBS therapy, and correlate electrode proximity to tractography of the DRT

with improvement of ET symptoms. This serves as a proof of concept for using high-

resolution postmortem diffusion MRI reference atlases for DBS targeting. Our results

show that; 1) postmortem diffusion MRI can be used to create a high-quality, high-resolution template of the human brainstem and thalamus; 2) these data can be accurately aligned to patient datasets using automated image registration; and 3) that electrode position within the registered template has a significant correlation with

treatment efficacy.

Postmortem diffusion tractography can provide increased anatomic accuracy

through improved image quality, increased spatial and angular (diffusion) resolution,

6 Section referenced from manuscript submitted to Human Brain Mapping co-authored under Dr.

Evan Calabrese

27

and reduced image artifacts. High angular resolution diffusion datasets also allow

advanced tractography methods including probabilistic tractography with multiple fiber

orientations, which may be more sensitive than standard deterministic tractography

(Behrens et al., 2007). We were able to achieve good registration between our postmortem template and 12 patient datasets, particularly with regard to ventricular

and/or exterior borders of the brainstem and thalamus. We were able to show a

statistically significant correlation between treatment efficacy and contact proximity to

the DRT of our postmortem template. Although this correlation was highly significant, it

was relatively weak, suggesting that other factors play a role in treatment efficacy. One

interesting result of our study was the lack of a significant correlation between treatment

efficacy and contact proximity to the Vim, which was the intended electrode target.

There is increasing evidence that the anti-tremor effects of Vim DBS are related to

modulation of the DRT rather than the Vim itself. DRT fibers pass through a portion of

the Vim, and it is possible that stimulation of this area is principally responsible for

tremor control.

High-resolution MRI-based reference atlases, like the postmortem data

presented here, could improve on conventional histology-based atlases by incorporating

accurate volumetric imaging and 3D connectivity mapping from diffusion tractography.

Usage of such postmortem atlases in conjunction with individual deterministic tractography of in vivo MRI datasets can serve as valuable tools to evaluating and

improving patient treatment efficacy.

28

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Figure 1. A) The dentatorubrothalamic tract (DRT) was mapped through three cubic regions of interest (ROIs) in the FLAIR MRI image set; B) first ROI around the red nucleus in axial view; C) the second ROI around the precentral gyrus; D) the third ROI around the ipsilateral dentate nucleus.

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Figure 2. Each contact object was standardized using the Axial Custom View in Object Creation with brush size 1.5 mm to consistently model contacts as spheres with volume 0.002 cm. Contact objects were set as ROIs in the Fiber Tracking section; this enables tracking of fibers running through each contact to target brain structures. A) Fibers through contacts in sampled ET patients closely follow the relevant DRT pathway. B) Close up of fibers directly passing through contacts 0 and 1 are shown.

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Figure 3. Relevant active contact as determined by post-operative testing outcomes was established as the sole region of interest for fiber tracking in each patient FLAIR MRI for 10 ET patients; visualization of fiber tractography with the DBS electrodes (red).

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Figure 4. Probabilistic tractography of the DRT from postmortem diffusion MRI of the brainstem and thalamus. A) A schematic of the DRT is shown for reference. B) Probabilistic tractography shows the course of the DRT, including minor branches that pass medially to the red nucleus (arrowheads). C) Visualization of the spatial relationships between the DRT, the red nucleus (red), and Vim nucleus of the thalamus (blue).

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Figure 5. Representative data showing registration between in vivo and postmortem MRI datasets. A) A parasagittal in vivo image with a surface rendering of the registered postmortem dataset superimposed. BD) Sagittal, axial and coronal slices, respectively, of an in vivo dataset with the corresponding slices from the registered postmortem anatomic image superimposed. Arrowheads indicate major borders that demonstrate the accuracy of registration.

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Figure 6. Visuospatial integration of image data including in vivo MRI, CT, and registered postmortem data. In each panel, a single slice of the pre-operative FSPGR image is shown with the corresponding slice of the registered postmortem dataset superimposed. Probabilistic tractography of the DRT (orange) is shown along with surface renderings of the red nuclei (red), the Vim nuclei of the thalamus (blue), and the implanted DBS leads derived from post-operative CT data (green). AB) Posterior and anterior views, respectively, of a coronal slice through the red nucleus. C) Mid-sagittal slice. D) Oblique view of an axial slice through the red nucleus.

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Figure 7. Visualization of DBS electrode position relative to the DRT from the registered postmortem template for all 12 patient datasets examined in this study. For each patient, we show a single oblique slice, roughly corresponding to the diagram in the top left, along with probabilistic tractography of the DRT derived from the registered postmortem template (orange), and a surface rendering of the implanted DBS electrodes derived from post-operative CT data (green).

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Table 1: Stereotaxic coordinates of each electrode contact for each patient included in the study. Stereotaxic coordinates are provided in millimeters from the mid-commissural point (MCP). Distance from the third ventricle (3V) in millimeters is also included. n/a indicates missing data.

MCP Coordinates (mm) Electrode Contact

Lateral (X) AP (Y) Vertical (Z) Distance

From 3V

Patient 01 Contact 0 12.88 -5.63 -1.98 12.3 Patient 01 Contact 1 13.57 -4.68 0.00 12.0 Patient 01 Contact 2 14.94 -3.30 1.59 13.0 Patient 01 Contact 3 15.07 -2.80 3.97 13.0 Patient 01 Contact 8 11.56 -6.91 0.49 9.5 Patient 01 Contact 9 12.79 -5.93 2.51 10.0 Patient 01 Contact 10 13.04 -4.18 4.63 11.6 Patient 01 Contact 11 13.79 -3.81 6.51 12.6 Patient 02 Contact 0 13.66 -5.79 -0.80 11.1 Patient 02 Contact 1 14.46 -4.99 1.80 11.0 Patient 02 Contact 2 14.66 -5.06 2.61 11.8 Patient 02 Contact 3 -15.54 -3.97 4.30 13.4 Patient 02 Contact 8 15.77 -3.89 1.77 11.6 Patient 02 Contact 9 16.33 -3.08 3.67 14.2 Patient 02 Contact 10 17.13 -2.08 6.07 15.5 Patient 02 Contact 11 17.30 -1.08 7.97 15.7 Patient 03 Contact 0 13.85 -8.09 -3.43 12.1 Patient 03 Contact 1 14.48 -6.76 -1.33 11.5 Patient 03 Contact 2 14.48 -5.94 -0.06 10.2 Patient 03 Contact 3 15.19 -5.15 1.12 11.8 Patient 03 Contact 8 12.68 -8.42 -0.89 8.1 Patient 03 Contact 9 13.13 -7.79 0.56 9.0 Patient 03 Contact 10 13.67 -6.66 1.93 10.6 Patient 03 Contact 11 14.49 -5.66 2.84 10.5 Patient 04 Contact 0 13.37 -8.47 3.14 9.3 Patient 04 Contact 1 13.66 -7.19 5.00 9.9 Patient 04 Contact 2 13.52 -6.33 7.00 11.4 Patient 04 Contact 3 14.66 -5.05 8.85 12.7 Patient 04 Contact 8 15.75 -3.44 2.69 10.7 Patient 04 Contact 9 16.60 -3.21 4.83 12.5 Patient 04 Contact 10 16.60 -2.07 5.97 13.0 Patient 04 Contact 11 18.32 -1.78 7.97 13.8 Patient 05 Contact 0 13.39 -7.39 -1.59 11.1 Patient 05 Contact 1 13.89 -6.13 -0.04 11.0 Patient 05 Contact 2 14.16 -5.02 2.63 10.6 Patient 05 Contact 3 15.05 -3.57 4.63 13.7 Patient 05 Contact 8 12.20 -5.56 -1.30 11.0 Patient 05 Contact 9 13.22 -4.62 0.25 10.6 Patient 05 Contact 10 13.22 -3.17 2.37 12.7 Patient 05 Contact 11 15.43 -1.91 4.37 13.1 Patient 06 Contact 0 10.40 -5.54 -0.27 9.0

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Patient 06 Contact 1 11.35 -5.22 1.18 8.3 Patient 06 Contact 2 11.79 -4.35 3.07 10.6 Patient 06 Contact 3 12.32 -3.17 5.83 11.3 Patient 06 Contact 8 14.19 -4.68 -1.69 12.2 Patient 06 Contact 9 14.09 -4.14 -0.44 12.2 Patient 06 Contact 10 15.12 -2.57 2.32 13.5 Patient 06 Contact 11 16.44 -1.53 4.99 14.1 Patient 07 Contact 0 13.73 -10.95 -2.66 11.7 Patient 07 Contact 1 13.51 -10.73 1.21 10.8 Patient 07 Contact 2 13.68 -9.81 0.79 9.7 Patient 07 Contact 3 14.01 -8.14 3.23 10.0 Patient 07 Contact 8 11.09 -8.79 -1.73 9.4 Patient 07 Contact 9 12.20 -7.75 0.82 8.6 Patient 07 Contact 10 12.12 -6.53 2.16 8.0 Patient 07 Contact 11 12.78 -6.09 4.16 8.9 Patient 08 Contact 0 n/a n/a n/a n/a Patient 08 Contact 1 n/a n/a n/a n/a Patient 08 Contact 2 n/a n/a n/a n/a Patient 08 Contact 3 n/a n/a n/a n/a Patient 08 Contact 8 n/a n/a n/a n/a Patient 08 Contact 9 n/a n/a n/a n/a Patient 08 Contact 10 n/a n/a n/a n/a Patient 08 Contact 11 n/a n/a n/a n/a Patient 09 Contact 0 13.32 -8.37 -3.41 11.3 Patient 09 Contact 1 13.83 -6.86 0.70 10.2 Patient 09 Contact 2 14.96 -5.29 2.22 11.4 Patient 09 Contact 3 15.56 -4.16 4.93 12.4 Patient 09 Contact 8 12.59 -8.23 -2.58 10.7 Patient 09 Contact 9 13.16 -7.11 -0.77 10.0 Patient 09 Contact 10 13.72 -5.98 1.15 10.5 Patient 09 Contact 11 14.40 -4.62 3.63 12.2 Patient 10 Contact 0 13.64 -5.82 -3.34 12.0 Patient 10 Contact 1 14.42 -4.91 -0.44 10.6 Patient 10 Contact 2 15.15 -3.60 -1.74 12.2 Patient 10 Contact 3 16.32 -1.94 4.21 13.6 Patient 10 Contact 8 11.96 -6.01 -2.03 10.3 Patient 10 Contact 9 11.81 -5.49 -0.87 9.3 Patient 10 Contact 10 12.74 -4.11 2.03 10.7 Patient 10 Contact 11 14.10 -2.59 2.76 13.3 Patient 11 Contact 0 16.35 -7.32 -5.18 15.1 Patient 11 Contact 1 16.41 -7.09 -4.00 14.9 Patient 11 Contact 2 16.55 -6.38 -2.05 14.5 Patient 11 Contact 3 16.82 -5.42 0.73 14.7 Patient 11 Contact 8 9.49 -8.81 -3.68 8.4 Patient 11 Contact 9 9.86 -7.97 -1.64 8.0 Patient 11 Contact 10 10.23 -7.14 0.29 8.6 Patient 11 Contact 11 10.73 -6.46 2.11 9.5 Patient 12 Contact 0 14.34 -7.58 -1.38 12.4 Patient 12 Contact 1 14.51 -6.91 -0.03 11.0

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Patient 12 Contact 2 14.85 -6.06 2.18 11.7 Patient 12 Contact 3 15.18 -5.04 4.38 12.9 Patient 12 Contact 8 12.17 -9.66 -2.06 10.7 Patient 12 Contact 9 12.66 -8.94 -0.40 9.6 Patient 12 Contact 10 13.38 -7.58 1.97 10.3 Patient 12 Contact 11 14.40 -6.56 3.32 12.0

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Table 2: Demographic information for the 12 patients included in this study.

Patient Gender Age Patient 01 F 32 Patient 02 F 75 Patient 03 M 74 Patient 04 F 39 Patient 05 F 61 Patient 06 F 68 Patient 07 F 71 Patient 08 F 84 Patient 09 M 72 Patient 10 F 65 Patient 11 M 72 Patient 12 M 69

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Table 3: Treatment efficacy, side effects, and proximity to the DRT and Vim from the postmortem model for each DBS contact (eight per patient). + indicates mild/moderate tremor reduction and the presence of side effects. ++ indicates good/excellent tremor reduction. - indicates no tremor reduction and the absence of side effects. n/a denotes missing data points.

Electrode Contact Efficacy Side Effects DRT Proximity Vim

Proximity Patient 01 Contact 0 ++ + 0 mm 0 mm Patient 01 Contact 1 ++ + 0 mm 0 mm Patient 01 Contact 2 + + 0 mm >1 mm Patient 01 Contact 3 + + 0 mm 0 mm Patient 01 Contact 8 ++ + 1 mm Patient 01 Contact 11 + + >1 mm 0 mm Patient 02 Contact 0 ++ + 0 mm 0 mm Patient 02 Contact 1 ++ + 0 mm 0 mm Patient 02 Contact 2 ++ + >1 mm >1 mm Patient 02 Contact 3 + + 1 mm 0 mm Patient 03 Contact 2 - + >1 mm >1 mm Patient 03 Contact 3 - + >1 mm 1 mm Patient 04 Contact 3 + + 1 mm Patient 04 Contact 8 ++ - 1 mm 0 mm Patient 05 Contact 8 ++ - 1 mm >1 mm Patient 06 Contact 0 + - >1 mm 0 mm

45

Patient 06 Contact 1 ++ - 0 mm 0 mm Patient 06 Contact 2 n/a n/a 0 mm >1 mm Patient 06 Contact 3 n/a n/a >1 mm >1 mm Patient 06 Contact 8 + - 0 mm 1 mm 1 mm 0 mm Patient 07 Contact 1 ++ - >1 mm 0 mm Patient 07 Contact 2 n/a n/a >1 mm >1 mm Patient 07 Contact 3 n/a n/a >1 mm 0 mm Patient 07 Contact 8 ++ - >1 mm 0 mm Patient 07 Contact 9 + - >1 mm 0 mm Patient 07 Contact 10 n/a n/a >1 mm 0 mm Patient 07 Contact 11 >1 mm 0 mm Patient 08 Contact 0 ++ - 0 mm >1 mm Patient 08 Contact 1 ++ - 0 mm 0 mm Patient 08 Contact 2 n/a n/a 1 mm Patient 08 Contact 9 - - >1 mm 0 mm Patient 08 Contact 10 n/a n/a >1 mm 0 mm Patient 08 Contact 11 n/a n/a >1 mm 0 mm Patient 09 Contact 0 ++ - 1 mm Patient 09 Contact 1 + - 0 mm 1 mm 0 mm Patient 11 Contact 8 ++ + >1 mm 0 mm Patient 11 Contact 9 ++ + >1 mm 0 mm Patient 11 Contact 10 n/a n/a >1 mm 0 mm Patient 11 Contact 11 n/a n/a 1 mm 0 mm

46

Patient 12 Contact 2 n/a n/a >1 mm 0 mm Patient 12 Contact 3 n/a n/a >1 mm 0 mm Patient 12 Contact 8 - - >1 mm >1 mm Patient 12 Contact 9 ++ - 0 mm >1 mm Patient 12 Contact 10 n/a n/a 1 mm 0 mm

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Table 4: Results of Spearman rank correlation. * denotes statistical significance.

Spearman Correlation Correlation Coefficient p-value Contact Proximity to Vim vs Side Effects 0.015 0.902 Contact Proximity to DRT vs Side Effects 0.029 0.815 Contact Proximity to Vim vs Efficacy 0.043 0.726 Contact Proximity to DRT vs Efficacy 0.336 0.005*