Feasibility Study of Motility Contrast Tomography for Predicting Therapeutic Response
Conditions: Bladder Cancer
Sponsor: Animated Dynamics, Inc.

 
 
 
 
 
 
 
 
 
 
 
 
 
 
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NOTIFICATION TO SPONSOR/CRO OF BOARD ACTION                                 
 
 
 
 
 
 
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SPONSOR:   Animated Dynamics, Inc. 
PROTOCOL NUM:   002 
AMD. PRO. NUM:    
TITLE:  
Feasibility Study of Motility Contrast Tomography for Predicting Therapeutic Response Among Bladder Cancer Patients Receiving 
Neoadjuvant Chemotherapy 
 
APPROVAL INCLUDES:  
Revised Protocol (03-01-2017)  
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Page 2 of 2
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DISTRIBUTION OF COPIES: 
Contact, Company 
Travis Morgan, MBA, Animated Dynamics, Inc.  

 
 
 
Protocol Title: 
Feasibility Study of Motility Contrast Tomography for Predicting 
Therapeutic Response Among Bladder Cancer Patients Receiving 
Neoadjuvant Chemotherapy 
 
Date: 
March 1, 2017 
 
Principal Investigator: 
Multiple 
 
Co-investigators: 
Ran An, Ph.D. 
Animated Dynamics, Inc. (“ADI”) 
 
David D. Nolte, Ph.D. 
Department of Physics 
Purdue University 
 
John J. Turek, Ph.D. 
Department of Basic Medical Sciences 
Purdue University 
 
Resources: 
Funded by Animated Dynamics, Inc. 
 
 
 

Page 2 of 17 
 
PROJECT SUMMARY 
In the US alone, millions of cancer patients every year receive chemotherapy with only a 20-60% probability of 
pathological response, while some experience adverse side effects that lower quality of life without necessarily 
prolonging it.  Reliable identification of ineffective therapies can eliminate needless human suffering while 
increasing overall probability of positive response to treatment.  Chemotherapy resistance profiling entails testing 
whether a patient exhibits strong resistance to a therapy prior to its final selection by the oncologist.  However, 
there are no effective methods for quickly assessing patient chemotherapy resistance.  Patient Derived Xenograft 
(PDX) models have replaced older Chemotherapy Sensitivity and Resistance Assays (CSRAs) to some degree, but 
both technologies suffer from long testing times, high cost, and/or low accuracy.   
Motility Contrast Tomography (MCT) has recently emerged as a technology that measures the biodynamic 
response of intact tumor biopsies to applied therapeutics by using Doppler detection of infrared light scattered 
from intracellular motions inside living tissue.  Several small scale animal, xenograft, and human studies have 
shown this phenotypic profiling technique to be highly accurate in prediction of response and resistance to 
chemotherapy.  This project will be the first human trial of biodynamic phenotyping to predict chemotherapy 
response among bladder cancer patients.  Specifically, the study cohort will include patients selected for 
neoadjuvant chemotherapy treatment, because this setting offers the opportunity for near-term outcome 
measurement at the time of post-chemo surgery.  Pre-therapy fresh tumor specimens will be imaged using MCT, 
and the resulting bio-dynamic signatures will be compared to confirmed pathological response at the time of 
surgery.  Observation of a high predictive value will provide the basis for expanded clinical trials and prompt 
commercialization of a biodynamic chemotherapy selection assay for bladder and other cancer patients.  
 
 
 
 
Study Schema:
Ex vivo MCT Study
Ex vivo MCT imaging assay 
on excess tumor specimen
Compare MCT pattern vs 
confirmed response per 
RECIST v1.1 criteria
Cystectomy
Pathologically Confirmed 
Response/ Non-response
Dropped from Study Cohort
Standard of Care
Transurethral Resection of Bladder 
Tumor (TURBT) performed per 
standard of care.  Excess tissue 
not required for pathology sent 
for MCT imaging.
Cystectomy
Neoadjuvant chemo

Page 3 of 17 
 
RATIONALE & BACKGROUND 
STUDY RATIONALE: The demonstrated ability of MCT to accurately assess tumor xenografts may establish it as a 
reliable technique for patient tumor stratification based on predicted response to therapy, which could enable a 
treatment selection based on the personal needs of an individual patient.  This study is designed to assess MCT as 
an assay for predicting chemosensitivity to treatment with chemotherapy agents routinely used in the neoadjuvant 
and adjuvant treatment of bladder cancer.  If positive, the results of this study will provide the basis for expanded 
clinical trials and use of MCT in therapy selection. 
BACKGROUND: Live cell imaging has become the standard for high-content analysis and drug discovery 
applications. The most common assays on live cells include viability, proliferation and cytotoxicity assays as cellular 
physiology and function are measured responding to applied perturbations of xenobiotics. Cellular and tissue 
viability assays are typically measured using exogenous vital dyes as biomarkers of membrane integrity or cellular 
metabolic activity. However, dyes are invasive, potentially toxic, and often require fixing of the tissue or 
permeabilization of the membranes [1, 2]. Furthermore, the common format of high content analysis and flow 
cytometry requires isolated cells, or cells distributed on flat hard surfaces. Isolated cells lack many of the 
biologically-relevant intercellular connections and communications that are hallmarks of healthy tissue [3, 4], and 
flattened cells on plates have pathological shapes and anisotropic cellular adhesions [5]. 
Discovery of technology that can predict response to cancer therapy is an urgent priority.  While many 
technologies exist to evaluate early response to drugs ex vivo, the need to perform viability, cytotoxicity and 
proliferation assays in three-dimensional tissue or culture has become increasingly urgent [6, 7], as drug response 
in 2D is often not the same as drug response in biologically-relevant three dimensional culture. This is in part 
because genomic profiles are not preserved in monolayer cultures [8-10]. There have been several studies that 
have tracked the expression of genes associated with cell survival, proliferation, differentiation and resistance to 
therapy that are expressed differently in 2D cultures relative to three-dimensional culture. For example, cell lines 
of epithelial ovarian cancer [11, 12], hepatocellular carcinoma [13-15] or colon cancer [16] display expression 
profiles more like those from tumor tissues when measured in three-dimensional culture, but not when grown in 
2D. In addition, the three-dimensional environment of 3D culture presents different pharmacokinetics than 2D 
monolayer culture and produce differences in cancer drug sensitivities [17-20].  Finally, most current technologies 
rely on destructive end-point assessment, preventing meaningful longitudinal observation of therapy response 
over time. 
One of the main challenges to migrating drug-response assays to the third and fourth dimensions has been to find 
a means to extract vital information from deep (up to a millimeter) inside living tissue. Tissue is translucent and 
light can propagate diffusively many centimeters. Furthermore, the dynamic motions of living cells cause dynamic 
light scattering that produces phase fluctuations on the scattered light fields that can be measured as dynamic 
speckle in diffusely reflected light from tissue. This is the basis of diffusing wave spectroscopy (DWS) [21, 22] and 
diffusion correlation spectroscopy (DCS) [23-26], but these techniques lack depth resolution. A powerful tool in the 
characterization of light propagation in tissues is the use of interferometry [27]. Interferometric detection is the 
underlying process in optical coherence tomography (OCT) [28-31], which is a point-scanning technique that 
suppresses speckle [32-34], although speckle decorrelation in OCT data can provide similar information as 
provided by DCS. This has been used to measure intracellular rheology [35] and to find dynamic signatures of 
apoptosis [36]. Transport also can be detected at cellular resolution using phase contrast microscopy [38], but this 
approach cannot be used in thick tissues. 

Page 4 of 17 
 
Dr. Nolte and colleagues have developed volumetrically-resolved tissue dynamic imaging that uses the advantages 
of depth selectivity from low-coherence interferometry, combined with high speckle contrast in broad-illumination 
digital holography. The technique is called Motility Contrast Tomography (MCT) and uses low-coherence digital 
holography to penetrate up to 1 millimeter into living tissue to measure speckle dynamics from light scattering 
from dynamic motion in living cells [37]. It was previously applied as a cytotoxicity assay to study the efficacy of 
anti-mitotic drugs [40].  In essence, the technology works by profiling the ‘movement’ of cellular organelles.  
Specific changes in organelle motion are detectable very early in cells undergoing response to chemotherapy 
treatment, and may be usable as an early predictor of chemotherapy response. 
MCT is based on optical coherence imaging (OCI) [38]. OCI is a full-field short-coherence holography [39] that 
collects backscattered speckle. With the help of coherence gating, OCI can optically section tissue up to 1 mm 
deep. MCT specifically uses intracellular motion as the endogenous contrast to characterize submicron subcellular 
motion inside three-dimensional living tissue [42]. 
Figure 1 shows the holographic recording principle of MCT. After calibration, the short coherence light is first 
divided into an object beam and a reference beam. The object beam hits the living tissue sample, and 
backscattered speckles from the tissue are collected by the lens L1. The living tissue sample locates at the focal 
plane of the lens L1, so L1 also performs an optical Fourier transform of the backscattered light. The charge 
coupled device (CCD) locates at the other focal plane of L1, so it captures the Fourier transformed scattering light 
from the tissue. The reference beam is controlled by a delay stage (not shown in the figure) to adjust the path 
length of the reference beam to perform a zero-path match between the object and reference beams. The beam 
splitter combines both beams and because they are zero-path matched, they interfere at the CCD plane. The 
reference beam is tilted by 20° in an off-axis configuration, and the spacing of the interference fringes (Λ) is 3 
pixels (24 μm). The speckle size (aspec) is adjusted to be 3 fringes wide (70 μm). Additional details about the 
experimental setup can be found in reference [41]. 
Fig.1. The principle of MCT on 
multicellular tumor spheroids. The 
biological sample is located at the 
image plane of lens L1. The back 
scattered light from the sample is 
Fourier transformed by L1 and 
interfered with reference beam on 
the CCD chip. The speckle hologram 
is recorded on the Fourier plane with 
a 20 crossing angel with the 
reference beam.  Examples of a) Raw 
digital hologram; b) reconstructed 
image; c) MCI image.  O.A.: optical 
axis; I.P.: image plane; L1: lens; BS: 
beam splitter; CCD: charge coupled 
device. 
EX VIVO CANCER CHEMOSENSITIVITY ANALYSIS  MCT was previously applied to study the efficacy of anti-mitotic 
drugs using multicellular tumor spheroids [40]. When applying MCT to tumor xenografts, it is also capable of 
showing a significantly different response between two cell lines under cisplatin.  After applying the drug, the 
normalized standard deviation (NSD) value of the platinum-sensitive cell line (A2780) drops from 0.7 to 0.1 in 8 

Page 5 of 17 
 
hours. In contrast, the NSD value of the platinum-resistant cell line (A2780-CP70) remains nearly constant (0.81 to 
0.80) 9 hours after applying drug. The NSD value of normal mouse tissue attached to the tumor xenograft 
decreases only a little (0.6 to 0.52) compared with A2780. Fig. 2 shows the cisplatin drug response curves. The NSD 
value of each point is averaged over the entire target. The time between measurements is 24 minutes for A2780-
CP70 and normal mouse tissue and is 12 minutes for A2780. The 20 μM cisplatin was applied at time t = 0, and the 
measurements lasted 9 hours for A2780-CP70 and normal mouse tissue, and lasted 8 hours for A2780. At time=0, 
the aggressive cell line A2780-CP70 has the highest NSD and the normal mouse mesenterium tissue has lowest 
NSD (0.6). The NSD of the platinum-sensitive cell line A2780 lies in the middle: 0.7. After applying cisplatin, the NSD 
curve of A2780 drops immediately. The NSD value of the A2780-CP70 almost doesn't change. 
Fig. 2 Motility metric (NSD) of ovarian cancer tumor 
xenografts responding to 20 μM cisplatin. The     x-
axis is time (minute), the y-axis is NSD value. The 
sensitive tumor is A2780, while the insensitive tumor 
A2780-CP70. Both tumor tissues begin with higher 
motility than normal mouse tissue. The cisplatin was 
added at time t=0. The NSD of A2780 dropped very 
fast and after 8 hours it dropped to 0.1. The NSD of 
the insensitive tumor A2780-CP70 didn't change 
during 9 hour peroid. The NSD of normal mouse 
tissue dropped a little compared with the A2780. 
 
In a further study in a veterinary clinical setting, MCT 
has been used to predict patient outcome for canine 
non-Hodgkin’s lymphoma.  Canine non-Hodgkin’s lymphomas are initially characterized by tumoral infiltration of 
peripheral lymph nodes. Canine non-Hodgkin’s lymphomas are diverse in their clinical aggressiveness and response 
to chemotherapy.  The only current biomarker for chemoresponsiveness is tumor cell immunophenotype (i.e. T-
cell vs. B-cell origin), but chemoresponsiveness varies tremendously within immunophenotype, which reduces the 
clinical utility of this biomarker.  In our study, we used MCT to measure the heterogeneous response of canine 
lymphoma biopsies to the standard-of-care doxorubicin.  The biodynamic signatures of doxorubicin responsivity ex 
vivo were correlated with canine patient outcome.  These studies have demonstrated, for the first time, the utility 
of label-free intracellular biodynamic markers to predict therapeutic efficacy for cancer treatment in dogs. 
SPECIFIC AIMS 
The primary study objective is to examine the feasibility of using MCT as a chemosensitivity assay among bladder 
cancer patients receiving neoadjuvant chemotherapy by comparing MCT patterns consistent with chemotherapy 
response or resistance ex-vivo to confirmed response or resistance to chemotherapy as measured by Response 
Evaluation Criteria in Solid Tumors (RECIST) v1.1 criteria.   
PRIMARY ENDPOINT: Objective pathological response measured at the time of surgery. 
RESEARCH DESIGN AND METHODS 
STUDY DESIGN: Retrospective correlation study. 

Page 6 of 17 
 
PARTICIPANT IDENTIFICATION: Patients of all races and ethnic groups are eligible for this study.  Patients will be 
recruited from participating sites after interpretation of initial cystoscopy and/or radiological screening, but prior 
to performance of a second staging biopsy and/or Transurethral Resection of Bladder Tumor (TURBT) procedure 
per standard of care.  Pre-screening of potential participants may be performed by the principal investigator or 
delegates under the principal investigator’s direct supervision.  Once all eligibility criteria are confirmed, potential 
participants will be offered the opportunity to participate.  Potential participants will be presented with the 
purpose of the study and the potential risks and benefits of participation.  All potential participants who are 
interested in participation will undergo the informed consent process privately at the time of their appointment or 
during a return appointment after they have an opportunity to review the consent.  Participants will be considered 
registered to the study upon receipt of a signed informed consent statement and determination by the principal 
investigator post-cystoscopy procedure that sufficient tumor tissue will likely be available to allow for excess 
specimen to be used in the study.   
Registration information will be maintained by the Principal Investigator.  All participants will be assigned a unique 
study ID derived from the bar-coded specimen collection kit used to collect all specimens in the study.  All subjects 
will adhere to inclusion and exclusion criteria as listed below.   
INCLUSION CRITERIA: To be eligible for the study, patients must meet the following criteria: 
1. 
Ability to understand and willingness to sign an informed consent and authorization for release of 
tissue not required for pathologic diagnosis to be used for research purposes 
2. 
≥ 18 years old at time of consent 
3. 
Suspected muscle-invasive cancer and/or high probability of receiving neoadjuvant chemotherapy 
and a cystectomy 
EXCLUSION CRITERIA: To be eligible for the study, patients must not have any of the following:  
1. 
Women who are pregnant or breastfeeding 
2. 
Known tumor genetics, comorbitities or other factors, which in the treating physician’s professional 
judgement, make the patient an unlikely candidate to receive neoadjuvant chemotherapy or a 
cystectomy. 
METHODS:  See Appendix B for a provider workflow checklist.  Patients will be identified as noted above in 
“Participant Identification.”  Patient confidentiality will be maintained during all chart reviews and data collection.   
PRE-TREATMENT BIOPSY PLAN  
The standard of care for newly diagnosed or recurrent transitional cell carcinoma in the bladder is pathologic 
confirmation by TUR-BT (TransUrethral Resection-Bladder Tumor).  This is done for both tissue confirmation and 
staging.  Typically, a tumor that is likely to be muscle invasive is usually larger and more sessile than small and 
papillary.  The most important part of the specimen procured is the base to confirm muscle invasive disease. 
Often, urologist procure enough tissue to make the diagnosis without resecting the entire tumor.  Hence, often 
times there is plenty of tissue remaining or that can be procured for testing the study hypothesis.  We suggest the 
surgeon procure at least 80mg of fresh tissue for this study (approximately 3-4 loops) then continue the TUR for 
pathologic confirmation as is standard of care. 

Page 7 of 17 
 
BIOPSY SAMPLE COLLECTION PARAMETERS 
Tumor biopsy samples will be collected using standardized specimen collection kits, each containing a unique bar 
code that will serve as the patient’s primary study ID.  Upon completion of a TURBT procedure on a consenting 
patient where sufficient excess tissue is available for MCT imaging (at least 80mg of excess tumor), the excess 
specimen will be placed in a bar coded 15mL vial containing transport medium, chilled using an ice pack provided 
with each collection kit, and conveyed to FedEx for next day delivery to Animated Dynamics’ laboratory in 
Indianapolis.  See Appendix B. 
BIOPSY SAMPLE PROCESSING AND ANALYSIS 
Upon receipt of tissue, researchers at Animated Dynamics will dissect prepared samples into small pieces up to 1 
mm3 in volume.  Sample pieces will be immobilized in multi-well specimen plates using agarose.  Prepared plates 
will be imaged using MCT.  After a 2-4 hour baseline period, selected anti-cancer drugs will be applied.  The 
dynamic spectra are then acquired for at least 9 hours to evaluate dynamic response to the applied therapeutics.  
Samples are then removed from the MCT system, fixed, and stored at 4° C.   
CHEMOTHERAPY TREATMENT PLAN 
Administration and treatment schedule: Registered participants will be treated per routine care guidelines and 
subject to treating physician discretion.  Patients may be treated with an additional biologic agent if done within 
the confines of a separate clinical trial. 
General concomitant medication and support care guidelines: Routine care guidelines should be followed at 
investigator discretion. 
Dose delays/modifications:  Dose adjustments for toxicity will be based on recommendations per the package 
insert and at the treating investigator’s discretion.  
Duration of follow up: Participants will be followed per routine care guidelines for progression.  
Criteria for removal: Participants may withdraw from the study at any time at their own request, or they may be 
withdrawn at any time at the discretion of the investigator for safety, behavioral, or administrative reasons.  
Reasons for participant removal include: noncompliance with study procedures or follow-up procedures; 
participant withdrawal of consent and election to discontinue participation in the trial; any other reason which in 
the opinion of the investigator, would justify removing the participant from the study.  
MEASURMENT OF EFFECT - CHEMOTHERAPY 
Timing of response assessment:  In addition to a baseline scan (obtained within 35 days prior to registration), 
patients’ response to chemotherapy will be evaluated immediately upon surgical tumor resection per routine care 
guidelines and according to institutional practice. 
Methods of response assessment:  Response and progression will be evaluated in this study using the new 
international criteria proposed by the revised RECIST guideline (version 1.1).  Changes in the largest diameter 
(unidimensional measurement) of the tumor lesions are used in the RECIST criteria.   
Evaluable for objective response:  Only those patients who have measurable disease present at baseline, have 
received at least one cycle of neoadjuvant chemotherapy, and have had their disease re-evaluated upon surgical 
tumor resection will be considered evaluable for response.  These patients will have their response classified 

Page 8 of 17 
 
according to the definitions stated below.  (Note: Patients who exhibit objective disease progression prior to the 
end of cycle 1 will also be considered evaluable.) 
Evaluable Non-Target Disease Response: Patients who have lesions present at baseline that are evaluable but do 
not meet the definitions of measurable disease, have received at least one cycle of therapy, and have had their 
disease re-evaluated will be considered evaluable for non-target disease.  The response assessment is based on the 
presence, absence, or unequivocal progression of the lesions.  
Measurable disease: Measurable lesions are defined as bladder lesions that can be accurately measured in at least 
one dimension (longest diameter to be recorded) as ≥2 cm by cystoscopy or CT scan, MRI, or calipers by clinical 
exam.  All tumor measurements must be recorded in millimeters (or decimal fractions of centimeters).  
Non-measurable disease:  All other lesions (or sites of disease), including small lesions (longest diameter <2 cm), 
are considered non-measurable disease for purposes of this study.   
Target lesions: Any measurable bladder lesions up to a maximum of 2 lesions per bladder and 4 lesions in total, 
should be identified as target lesions and recorded and measured at baseline.  Target lesions should be selected on 
the basis of their size (lesions with the longest diameter), be representative of all lesions, but in addition should be 
those that lend themselves to reproducible repeated measurements.  It may be the case that, on occasion, the 
largest lesion does not lend itself to reproducible measurement in which circumstance the next largest lesion 
which can be measured reproducibly should be selected.  A sum of the diameters (longest for non-nodal lesions, 
short axis for nodal lesions) for all target lesions will be calculated and reported as the baseline sum diameters.  
The baseline sum diameters will be used as reference to further characterize any objective tumor regression in the 
measurable dimension of the disease. 
Non-target lesions:  All other lesions (or sites of disease) including any measurable lesions over and above the 
target lesions should be identified as non-target lesions and should also be recorded at baseline.  Measurements of 
these lesions are not required, but the presence, absence, or in rare cases unequivocal progression of each should 
be noted throughout follow-up.  
Methods for Evaluation of Measurable Disease:  All measurements should be taken and recorded in metric 
notation using a ruler or calipers.  All baseline evaluations should be performed as closely as possible to the 
beginning of treatment and never more than 4 weeks before the beginning of the treatment. 
RESPONSE CRITERIA  
Complete Response (CR):  Disappearance of all target lesions.  Any pathological lymph nodes (whether target or 
non-target) must have reduction in short axis to <10 mm. 
Partial Response (PR):  At least a 30% decrease in the sum of the diameters of target lesions, taking as reference 
the baseline sum diameters. 
Progressive Disease (PD):  At least a 20% increase in the sum of the diameters of target lesions, taking as reference 
the smallest sum on study (this includes the baseline sum if that is the smallest on study).  In addition to the 
relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm.  (Note: the 
appearance of one or more new lesions is also considered progression.) 
Stable Disease (SD):  Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as 
reference the smallest sum diameters while on study. 

Page 9 of 17 
 
MEASUREMENT OF EFFECT – MOTILITY CONTRAST TOMOGRAPHY 
The foundational data structure that is obtained by MCT is a time-frequency spectrogram.  This spectrogram 
captures how the Doppler frequencies of intracellular motion respond to the applied drug.  This data structure is 
two-dimensional and can exhibit a variety of patterns that have been associated with mechanistic response of the 
tissue to the drug.  To further improve the interpretation of the MCT results, numerous metrics have been 
developed that quantify the cellular response.  These include: Backscatter brightness, NSD, change in NSD, dipole 
sine response, dipole cosine response, and quadrapole response.  A further set of metrics, that are linearly 
dependent on the previous metrics, have more direct cellular mechanistic basis.  These include: mitotic index, 
apoptotic index, necrotic index, metabolic activity, and membrane ruffling.  These metrics are non-orthogonal, and 
a patient will often show strongly correlated responses among these metrics.  Multiparameter logistic regression is 
performed on these metrics to provide a single-valued predictor of therapy response.   
DRUG FORMULATION AND PROCUREMENT 
NOTE: All therapy in this trial is standard of care.  All drugs to be tested in this study are commercially available 
formulations procured, stored, and administered in accordance with routine care standards and institutional 
policy.  The primary therapies that will be screened using MCT in this study will include the following list, but may 
be supplemented from time to time at the investigators discretion.  Therapies are listed in order of descending 
priority, with lower priority therapies tested subject to availability of sufficient tissue quantity.  When tissue 
quantity permits, both combination and monotherapies will be imaged using MCT. 
• DDMVAC (dose-dense methotrexate, vinblastine, doxorubicin, and cisplatin) 
• Gemcitabine and cisplatin 
• CMV (cisplatin, methotrexate, and vinblastine) 
STUDY COSTS: All non-routine care procedures including the preparation, handling, and transport of MCT 
specimens, MCT assay procedures, and reporting of outcome data will be paid for by the study. 
SAFETY CONSIDERATIONS: NOTE: All procedures and therapy in this trial are standard of care.  Since the study does 
not entail any intervention in patients’ care, only adverse events related to the loss of patient confidentiality will 
be reported to the IRB at the time of continuing review.  
STATISTICAL CONSIDERATIONS 
STUDY DESIGN: This is a non-randomized, multi-arm, multi-center, feasibility study, designed to examine the 
feasibility of using MCT as a chemosensitivity assay among bladder cancer patients by comparing ex-vivo MCT 
patterns consistent with chemotherapy response and resistance to confirmed pathological response or resistance 
as measured by Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 criteria.   
DEFINITION OF PRIMARY OUTCOME ENDPOINT: Feasibility is defined as a high statistical correlation between MCT-
predicted response, and surgically confirmed response to neoadjuvant chemotherapy. 
ANALYSIS PLAN FOR PRIMARY OBJECTIVE: The study will be considered successful, if a majority of specimens yield 
interpretable MCT results, and if MCT signatures are identified which demonstrate a high statistical correlation to 
confirmed patient response. 

Page 10 of 17 
 
EXPLORATORY ANALYSIS: Due to the inadequate samples size, no formal analysis is planned.  Descriptive analysis 
of the correlation between MCT parameters and clinical outcome measures will be performed.   
DETERMINATION OF SAMPLE SIZE: A target sample size of 500 patients was selected for initial enrollment using 
the investigators’ previous research experience to approximate the appropriate number needed to demonstrate 
the feasibility of this technique.  It is anticipated that only 20-30% of enrolled patients will yield specimens 
evaluable for response.  It is estimated that it will take approximately 6-12 months to complete accrual.  
EVALUATION FOR RESPONSE: Participants who provide a bladder tumor specimen, have received at least one cycle 
of chemotherapy, and have had their disease re-evaluated upon surgical resection will be considered evaluable for 
response. 
REPLACEMENTS: Participants who withdraw from the study prior to having the TURBT procedure or fail to 
complete at least one evaluation for response will be replaced.  No reported data will be included for participants 
who are replaced.   
DATA AND RECORD KEEPING 
DATA COLLECTION: Each subject will be assigned a unique study ID number once it has been determined they 
meet inclusion criteria.  Subject outcome data will be collected using a secure web-based data collection form 
which will identify subjects only by their unique study ID and procedure date.  Only the study investigators will 
have access to the list matching each subject ID to the patient’s medical records, and only the PI and his immediate 
staff will enter follow-up data into the data collection form.  The following data points will be collected for this 
study:  
• 
pre-treatment clinical/ pathological description of disease 
• 
TURBT date, location, and clinical description 
• 
objective response, as confirmed upon surgical tumor resection, 
• 
Neoadjuvant chemotherapy agent(s) and regimen used 
• 
Pre and post-treatment CT scans (if available) 
RECORD KEEPING/CONFIDENTIALITY:  This information will be stored on password protected electronic systems 
managed by the study team.  All hardcopies of study related documents will be kept in locked and secure files 
accessible only to the study team.  The potential risk to subjects is the loss of confidentiality.  To mitigate this risk, 
the data collection form contains no patient identifiers.  As data are entered into the data collection form, the PI 
will create a master list that links the patient to her study identification number.  This master list will be stored in a 
locked room in a locked cabinet with limited public access.  The electronic files will be stored on a secure computer 
with password protection.  To minimize the risk of lost confidentiality, only study investigators will have access to 
the list that links patient identifiers to the assigned study number.  Once the patient is assigned a study 
identification number, that number will be used without additional patient identifiers whenever possible.  All 
collected information will be stored electronically on an encrypted and password protected laptop.   
RISK ASSESSMENT:  This study involves only procedures and commercial therapeutic agents used in accordance 
with routine care practice and institutional guidelines.  No increase in patient health risk is anticipated as a result 
of this study.  There is a slight risk of loss of confidentiality of participant information.  Every effort will be made to 

Page 11 of 17 
 
keep participant information confidential.  The PI is responsible for conducting continuous review of data and 
participant safety.  At any time during the conduct of the trial, if it is the opinion of the investigators that the risk 
(or benefits) to the participants warrant early closure of the study, the study will be closed.  
CHANGES TO THE PROTOCOL: Study procedures will not be changed without the mutual agreement of the 
Principal Investigator and co-investigators. If it is necessary for the study protocol to be amended, the amendment 
or a new version of the study protocol (amended protocol) will be generated by the Principal Investigator and must 
be approved by an IRB.  Local requirements must be followed.  If a protocol amendment requires a change to the 
Written Informed Consent Form, then the IRB must be notified.  Approval of the revised Written Informed Consent 
Form by the IRB is required before the revised form is used.  The Principal Investigator is responsible for the 
distribution of these documents to his IRB, and to the staff at his center.  The distribution of these documents to 
the regulatory authority will be handled according to local practice. 
ETHICS: 
The final study protocol, including the final version of the Written Informed Consent Form, must be approved by 
an IRB.  The Principal Investigator is responsible for informing the IRB of any amendment to the protocol in 
accordance with local requirements.  In addition, the IRB must approve any advertising used to recruit participants 
for the study.  The protocol must be periodically re-approved by the IRB annually as local regulations may require.  
Progress reports and notifications of serious unexpected adverse events will be provided to the IRB according to 
local regulations and guidelines.  The study will be performed in accordance with ethical principles originating from 
the Declaration of Helsinki, which are consistent with ICH/Good Clinical Practice, and applicable regulatory 
requirements. 
WRITTEN INFORMED CONSENT: The investigator will ensure the participant is given full and adequate oral and 
written information about the nature, purpose, possible risks and benefits of the study.  Participants must also be 
notified they are free to discontinue participation in the study at any time.  The participant should be given the 
opportunity to ask questions and allowed time to consider the information provided.  The participant’s signed and 
dated informed consent must be obtained before utilizing any of the patient’s health information or specimens for 
the study.  The investigator must store the original, signed Written Informed Consent Form.  A copy of the signed 
Written Informed Consent Form must be given to the participant.  A copy of the Written Informed Consent Form is 
included in Appendix A. 
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25. D. A. Boas, L. E. Campbell, and A. G. Yodh, "Scattering and Imaging with Diffusing Temporal Field 
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26. T. Durduran, R. Choe, W. B. Baker, and A. G. Yodh, "Diffuse optics for tissue monitoring and tomography," 
Reports on Progress in Physics, vol. 73, Jul 2010. 
27. D. D. Nolte, Optical Interferometry for Biology and Medicine vol. 1: Springer, 2012. 
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Page 15 of 17 
 
Appendix A – Written Informed Consent Form 
 
 

Page 16 of 17 
 
Appendix B – Provider Workflow Checklist 
1) Patient Screening 
a) Based on known tumor genetics, comorbidities, or other factors, is the patient highly unlikely to receive 
neoadjuvant chemotherapy? If “YES” then stop. 
b) Obtain patient informed consent document 
2) Specimen Screening 
a) Is there at least 80mg of excess tumor specimen (approximately 3-4 loops) after preserving that which is 
necessary for routine pathology?  If “YES” then proceed. 
3) Specimen Preparation 
a) Open an Onco4D specimen collection kit (provided by Animated Dynamics) and apply the kit’s unique bar 
code identifier to patient paperwork using provided bar code labels.  Place remaining bar code labels in 
patient file for use with additional paperwork in the future.  This bar code will serve as the patient’s 
unique “Code” for purposes of identification throughout the study period, to reduce the risk of 
compromising protected health information. 
b) Place one copy of the patient informed consent signature in the collection kit (retaining one copy for the 
patient, and one for the practice). 
c) Collect two buccal swab samples using the matching bar coded swabs provided, replace in the zip top 
sleeve, and return to the collection kit. 
d) Dissect approximately 80mg of excess lesion: 
i) 
Approximately 3-4 “loops” of tissue should yield sufficient specimen size 
e) Place fresh specimen(s) into bar coded vial in specimen collection kit ensuring that specimen is submerged 
in transport media, and seal vial. 
f) 
Place vial in zip top bag provided and seal bag. 
g) Place sealed bag in cooling kit along with buccal swabs. 
h) Place frozen ice pack in kit, close, and seal kit. 
i) 
Deliver Onco4D kit to Fedex courier as soon as possible (same day) for delivery to Animated Dynamics.). 
j) 
Record the association of the patient and the assigned Code for safekeeping. 
k) Forward pathology reports related to the TURBT procedure, as well as recommended chemotherapy 
regimen (if any) as soon as available.  
4) Outcome Data 
a) As soon as practical after cystectomy, provide: 

Page 17 of 17 
 
i) 
neoadjuvant chemotherapy regimen given 
ii) pre and post-chemotherapy CT studies 
iii) pathology report from cystectomy specimen, including confirmed pathological response per RECIST 
v1.1 (if available)