Pre-treatment Biomarker for Clinical Response to Neuronavigation Repetitive Transcranial Magnetic Stimulation (rTMS) in the Acute Phase Treatment of Refractory Major Depressive Episode- Role of Intrinsic Functional Connectivity
Phase: NA
Conditions: Depression
Sponsor: Chinese University of Hong Kong

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Project title of main study: Pre-treatment biomarker for clinical response to 
neuronavigated repetitive transcranial magnetic stimulation (rTMS) in the acute 
phase treatment of refractory major depressive episode- role of intrinsic functional 
connectivity 
Project title of sub-study: Clinical utility of Functional near infrared spectroscopy 
(FNIRS) to define biomarkers for predicting and monitoring treatment response to 
neuro-navigated rTMS in Major Depressive Disorder 
Research Plan and Methodology 
i. 
Sampling     
Participants will be referred by psychiatrists from specialist outpatient clinics based in the 
regional hospitals of New Territories East Cluster under the Hospital Authority of Hong 
Kong. Written informed consents will be obtained from all participants according to the 
Declaration of Helsinki.  
Inclusion criteria: 1) Ages 18-64 years with DSM-IV major depressive disorder 
(MDD), currently in a moderate to severe depressive episode, scoring > 20 on 
Montgomery-Asberg Depression Rating Scale (MADRS)1, >18 on Hamilton 
Depression Rating Scale (HDRS) 17-item2; 2) failed at least one full course of 
antidepressant medication given at adequate dosage for at least six weeks in the current 
episode, 3) right-handed 
Exclusion criteria: 1)History of significant head trauma; 2)active abuse of alcohol or 
illegal substances; 3)current psychotic symptoms and suicide ideation; 4)recent suicide 
attempt, 5) any other DSM-IV Axis I and Axis II psychiatric diagnosis other than Major 
Depressive Disorder; 6)significant neurologic history such as dementia, stroke, seizure, 
Parkinson’s disease, multiple sclerosis; 7) contraindications to fMRI (e.g. pace makers, 
metal implants, pregnancy etc). 
ii. 
 Pre-treatment assessment 
 
A) 
Baseline clinical assessment 
 
At baseline (before treatment), subjects’ age, level of education, handedness will be 
noted by research assistant.  A research psychiatrist will administer MADRS1, HDRS2, 
DSM-IV SCID-I/II 3-4 to ascertain current/ lifetime Axis I and II psychiatric diagnosis and 
score on Global Assessment of Functioning, age at onset of MDD and number of major 
depressive episodes, number of failed antidepressants, current life stressor(s) and the 
perceived severity on a visual analog scale of 0-10, and clinical global impression scale 
(CGI).5  
Subjects are asked to complete self-administered questionnaires including 1) Beck 
Depression Inventory II 6 that has been validated in local Chinese dialect 7; 2) a 60-item 
NEO Five Factor Inventory that measures five personality traits on neuroticism, 
extraversion, openness to experience, agreeableness and conscientiousness;8 3) 
ruminative response subscale (Chinese version) (RRS) of the response style 

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questionnaire, a 22-item questionnaire comprising of two distinct factors on brooding and 
reflection.9 Published data suggest rumination may be correlated positively with duration 
of depressive episode and predict onset of depressive episode10.   
             
B1) 
Structural Magnetic Resonance Imaging (MRI) of brain for neuro-navigated 
rTMS and resting state functional MRI (rs-fMRI) to map intrinsic functional 
connectivity (baseline,  2 weeks and 8 weeks post-treatment) 
A whole brain anatomical data set is acquired with T1-weighted sequence (repetition 
time(TR)/echo time(TE): 7.6/3.5ms, field of view 230mm, 250 contiguous slices, 0.6mm 
thickness, reconstruction matrix 224 x 224) for voxel-based morphometry (VBM) analysis 
and functional to anatomical image co-registration. 
A resting-state functional MRI will be taken for either six (phase I) or twelve minutes 
(phase II). Subjects will be instructed with written cues projected (for 5 seconds) on the 
screen to stay relaxed and look at the fixation cross hair. Stimuli are projected on a TV 
monitor equipped by MRI-compatible Esys (Invivo) system. Subjects view the screen 
through mirror glasses. The functional scans are acquired on a 3.0-T whole-body scanner 
(Achieva TX, Philips Healthcare, Best, the Netherlands) using PRESTO (principles of echo 
shifting with a train of observation) sequence (TR28ms, TE 12ms, flip angle 7°. Slab 
thickness 125mm, 230mm field of view, data matrix 80 x 51 x 25) with a nominal in-plane 
resolution of 2.8 x 2.8mm and a temporal resolution of 2.7 sec/scan. Slices will be tilted 
about 30º clockwise along the AC-PC plane to obtain better signal in the orbitofrontal 
cortex.  
All the functional and anatomical MRI data are transferred to an offline workstation for 
image analysis. MRI data will be preprocessed using the default pipeline of the CONN 
toolbox v18.b11, which is an open-source MATLAB (2019B, Mathworks, Natick, MA, 
USA) and SPM-based (Statistical Parametric Mapping, www.fil.ion.ucl.ac.uk/spm/) cross-
platform software1. The preprocessing steps included realignment and unwarping, 
temporal slice time correction, functional outlier detection (ART-based identification of 
outlier scans), segmentation, normalization and smoothing (8 mm Gaussian kernel). The 
best way to denoise resting-state fMRI data is currently still a controversial topic12. 
Therefore, component based noise correction with and without global signal regression11,13. 
will be applied. For denoising with component based noise correction a principal 
component analysis was used to identify the time series data from the noise regions, 
including white matter and cerebrospinal fluid. Significant principal components are then 
introduced as covariates in the general linear model as an estimate of the physiological 
noise signal space. Regressors: white matter (10 dimensions), cerebrospinal fluid (5 
dimensions), realignment parameters (6 dimensions), scrubbing (61 dimensions). Further, 
a default temporal band-pass filter (.008-.09 Hz) and detrending. For global signal 
regression the same procedure was performed, but an additional regressor of the global 
signal (1 dimension) was added. The Subject-level correlation coefficients of the regions 
of interest and seed maps were converted to normally distributed Fisher-transformed Z 
scores to conform to the assumptions of generalised linear models. These connectivity 
values and statistical seed maps were subsequently used for the second-level analyses to 
examine group differences (responders versus nonresponders) and perform correlational 

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analyses to examine whether either pretreatment connectivity or change in functional 
connectivity is associated with TMS treatment response.  
B2) 
Offline resting state FNIRS (baseline and serial measures) 
In a sub-sample of subjects who agree for extra brain mapping sessions, offline resting 
state FNIRS measures will be taken in pre-treatment phase (within 7-10 days before first 
rTMS session), week 2 and week 4 in the course of rTMS treatment, 2 weeks and 8 weeks 
post-treatment.  
The FNIRS measurements will be taken in a specialized laboratory situated in the Center 
for Cognition and Brain Studies, Department of Psychology of CUHK (Sino Building of 
Chung Chi College), which is in close proximity to University MTR station and inside the 
same amenity as the CU Clinical Psychology Department.  
All subjects will be instructed to keep still with eyes closed, relax their mind and remain 
motionless for two blocks of 15-minute resting state session (totaling 30 minutes). The 
target mapping areas are bilateral fronto-parietal areas for their roles in default mode 
network (DMN). Optical signals will be recorded on a two-wavelength (690 and 830 nm, 
110MHz) frequency-domain oximeter (Imagent, ISS Inc., Champaign, IL, USA). 
Offline mode is opted in this study as clinical applicability is our main concern. 
Simultaneous FNIRS-rTMS is theoretically possible but the set-up will substantially 
increase session time, compromising the tolerability of rTMS session and likely 
compliance. Aside, previous studies have shown the FNIRS signal changes are sustainable 
and detectable with offline measures after TMS. 
 The FNIRS are measured with source fibers emitting light at the two different wavelengths 
that are co-localized as a pair, giving rise to 24 source locations. Photons that scatter 
through the head are collected by 16 detector fiber bundles connected to photomultiplier 
tubes (PMTs). These sources and detectors are held on the participants’ scalp with a rigid 
custom-made head-mount system (montage).  
Sources and detectors are arranged in groups of two parallel rows (1.7 cm to 2.5 cm apart 
from each other) to create overlapping light channels so as to increase the spatial resolution 
and signal-to-noise ratio.  
Source-detector placement in each of the four mapping areas (left and right frontal, left and 
right parietal) is opted. After the montage is positioned, coordinates of 230 points on the 
scalp, including the locations of source-detector pairs and three fiducial points (nasion, left 
and right pre-auricular points), are digitized using ASA 4.5 software (ANT, Netherlands) 
in conjunction with an infrared camera and 3-dimensional digitization system (Visor, ANT 
BV, 1mm spatial resolution). These points are used for calculation of the exact source-
detector distance for data analysis and, more importantly, for co-registration of functional 
optical data with each participant’s structural MRI.  
The individually co-registered data are then Talairach-transformed to permit comparison 
across subjects. The ISS Imagent control box is connected to a computer that controls and 
records the intensity of emitted and recorded signals. The sampling rate used in this study 

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is 19.5Hz, giving rise to a 51.3ms sampling interval.  During this interval, light sources are 
time multiplexed to allow different sources to be distinguished from each other. Light 
intensity and phase-delay data are computed and stored in ASCII format in the computer 
during the recording. Similar to fMRI analyses, predicted time series are generated by 
convolving the hemodynamic response function (SPM 8) with the impulse function for 
each event, i.e., from the onset to the offset of one sequence trial.  
The pulse-corrected bandpass-filtered oxy-haemoglobin concentration change from each 
channel is then correlated with the predicted time series for repeated and random conditions 
separately for each recording block and each participant. The Beta weight for each 
condition is estimated, Fisher-transformed and averaged across each recording block for 
each participant in each session. The averaged data are analyzed using Opt-3 for generation 
of statistical maps. In Opt-3D, the optical signal, in this case hemoglobin concentration 
change, for a given voxel is defined as the mean value of the channels that overlap at that 
particular voxel. Channels with source-detector distances shorter than 20mm and longer 
than 60mm are excluded from statistical map analysis as the light path would be too short 
to have reached the cortical surface or too long to have enough light reaching the detector, 
respectively. Channels will be removed if the standard deviation of the phase delay data is 
greater than 230 picseconds (i.e. the channel is excessively noisy). 
C) Neuro-navigated rTMS protocol 
rTMS treatment suite and personnel: the treatment facilities are based in Chen Wai Wai 
Vivien Foundation Therapeutic Physical Mental Exercise Centre, which is a CU clinical 
and research centre sponsored by private fund where the physicians delivering the 
treatment (PI and co-PI) are serving on site. The treatment centre is equipped with 
resuscitation facilities.  Full-time research staff and physiotherapist are available to handle 
medical and behavioral emergencies. 
Stimulator set-up: Neuro-navigated high-frequency rTMS over a specific area on left 
DLPFC (between BA9 and BA46, junction of middle and anterior two-thirds of the middle 
frontal gyrus) will be provided using Magstim Super Rapid2 (Phase I) or Neurosoft Neuro-
MS/D Advanced Therapeutic Transcranial Magnetic Stimulator (Phase II) that both 
generate short duration biphasic pulses. A figure-of-eight coil will be held in place with a 
custom-made stand tangential to the scalp with the handle pointing back and away from 
the midline at 45 degrees. The resting motor threshold will be determined Motor Evoked 
Potential or the standard visual method (whichever revealed the lowest motor threshold). 
Stimulation parameters Phase I: 10 hertz, 120% motor threshold, thirty trains of 5 
seconds with 25 seconds between trains, 1500 pulses per day delivered 5 days per week, 
totaling 30000 pulses over 4 weeks. 
Stimulation parameters Phase II: triplet 50 hertz, repeated at 5 hertz, 120% motor 
threshold, twenty trains of 2 seconds with 8 seconds between trains, 600 pulses per day 
delivered 5 days per week, totaling 12000 pulses over 4 weeks. 
Stimulation localization: The DLPFC coordinates of stimulation target site is  determined 
individually for each participant using a structural T1-weighted MRI scan. This area is 

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defined around the junction of Brodmann area 9 and 46. To coregister the scan with the 
patient’s head, advanced stereotactic techniques incorporated into Brainsight TMS (Rogue 
Research Inc., Canada) for phase I of the study and the Neural Navigator (Brain Science 
Tools BV, Netherlands) for phase II of the study were used. 
Clinical follow-up and outcome assessments 
All participants are evaluated by a research psychiatrist who is blinded to the functional 
connectivity maps of the participants and other potential clinical predictors of response 
measured at baseline. Follow-up assessments are scheduled at the end of week 2, week 4, 
week 6, week 8, and week 12.  
Primary outcome measures are the percentage change in symptom scores on MADRS and 
CGI at week 4, week 6, week 8, and week 12 compared to baseline. Secondary outcome 
measures include scores on GAF, BDI and RRS. 
Clinical response is defined as CGI= 2 and 50% reduction of MADRS score from baseline, 
respectively at the end of six weeks and twelve weeks. 
Clinical remission criteria is MADRS <7 and CGI of 1, respectively at the end of six weeks 
and twelve weeks. 
iv. 
Management of decompensated mental states, serious adverse somatic 
reactions to rTMS and drop-outs 
 
The research psychiatrists (PI and co-I) will be responsible for the detection of high-risk 
mental states (emergence of psychotic symptoms, catatonic state, suicide ideation and para-
suicide state) at the regular follow-up assessments.  Physical adverse effects will be 
checked at each follow-up and evaluated on the severity. In times of emergent risky mental 
state and adverse somatic reactions, the research psychiatrists will work collaboratively 
with the participant and if necessary, the treating psychiatrists on the necessary psychiatric 
and medical management plans. All adverse effects will be reported to the “serious adverse 
effect monitoring sub-committee” under the local ethics committee. All drop-out 
participants will be assessed by phone followed by clinic-based interviews to work out the 
care plan as indicated clinically. At the end of the study, the subjects will receive the 
generic psychiatric service as they are originally entitled to. The research team will ensure 
a seamless aftercare will be arranged for all participants. 
 
v. 
Sample size estimation 
 
Sample size estimation is based on the parameter estimate that addresses the main objective. 
In our study it is the expected Pearson correlation coefficient between functional 
connectivity of the ROIs and quantitative measure of clinical efficacy (percentage drop in 
MADRS score). In Fox et al’s study (2012)14, the correlation coefficient is -0.355 (p<0.05 
one-tailed). According to Friedman’s partial power table, the required sample size is at 
least 58 at alpha=0.8 and Pearson r= 0.35. Taking into account the drop-out rate of 20% 
reported in the active rTMS group of a prior randomized controlled trial comparing efficacy 

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of neuro-navigated rTMS and conventional targeting protocol, the required sample size 
that is sufficiently powered to examine the main objective is adjusted to 128. 
 
vi. 
Data analysis  
 
Intention-to-treat analysis will be adopted with the last observation carried forward for 
participants with at least one post-baseline observation. To describe the trajectory of 
clinical response over the course of follow-up, survival analysis will be used to track the 
proportions of responders and remitters respectively at 2, 4, 6, 8, 10, 12 weeks.  
 
To test the main objectives, we plan to perform: 
1) Group comparison of functional connectivity between ROIs between clinically 
defined responders and non-responders at 4 weeks, 6 weeks and 12 weeks, 
respectively. We hypothesize pre-treatment anti-correlation between the left 
DLPFC target and subgenual cingulate is stronger in responders than non-
responders at the respective time points.  
2) Group comparison of functional connectivity between ROIs in clinically defined 
remitters and non-remitters at 4 weeks, 6 weeks and 12 weeks, respectively. We 
hypothesize pre-treatment anti-correlation between the left DLPFC target and 
subgenual cingulate is stronger in remitters than non-remitters at the respective time 
points. 
3) Correlations analyses of the functional connectivity between ROIs and percentage 
drop in MADRS scores at 4 weeks, 6 weeks and 12 weeks compared to baseline, 
respectively. We hypothesize larger percentage drop in MADRS from baseline 
correlates with greater anti-correlation between the left DLPFC target and 
subgenual cingulate, at the respective time points. 
4) multiple linear regression of functional connectivity between ROIs on percentage 
drop in MADRS score from baseline at 4 weeks, 6 weeks and 12 weeks respectively, 
in the presence of other baseline demographic and clinical characteristics. We 
hypothesize higher percentage drop in MADRS from baseline correlates with 
greater anti-correlation between the left DLPFC target and subgenual cingulate, at 
the respective time points after adjusting for baseline demographic and clinical 
characteristics. 
5) Correlate FNIRS signals in left DLPFC detected in week 1 and 2 with depressive 
symptom scores (MADRS) in week 6, 8, and 12 in order to look for early 
biomarkers for treatment response. 
6) Group comparison of FNIRS signals in left DLPFC detected 7-10 days before 
rTMS treatment in clinically defined responders and non-responders at 6 weeks and 
12 weeks, respectively. 
 
Declaration: 
The research protocol fully complies with the Declaration of Helsinki and guidelines of 
ICH-GCP 
References: 

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