Clinical trial NCT02181075
Conditions: Liver Tumour
Keywords: ThermoDox High Intensity Focused Ultrasound Lyso thermosensitive Liposomal Non invasive drug delivery Hepatic metastatic disease Liver tumour(s) Targeted Release of Chemotherapy
Sponsor: University of Oxford
Start date: 2014-07-01; Completion date: 2017-04-01
Enrollment: 10 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This proof of concept study proposes targeted delivery of a broad-spectrum cytotoxic agent (doxorubicin), via a specially formulated LTSL (ThermoDox®) activated by mild hyperthermia, by using focused ultrasound (FUS), to achieve enhanced intra-tumoural doxorubicin concentrations for the same systemic dose.

Adult patients with incurable confirmed hepatic primary or secondary tumours received a single cycle of LTLD, followed by ultrasound-mediated hyperthermia to a single target liver tumour. The primary endpoint relates to evidencing enhanced delivery of doxorubicin from LTLD at the target tumour site, by comparing intratumoural concentrations of the drug before and after focused ultrasound (FUS) exposure.

Detailed description:
To date, purely pharmacological approaches have failed to address what is essentially a threefold challenge: (i) to deliver therapeutically significant concentrations of active agents to the tumour vasculature while minimizing off target effects; (ii) to release the therapeutic agent 'on-demand' at the target site; and, (iii) to improve the distribution and spread of the therapeutic agent against the intra-tumoural pressure gradient in order to achieve a therapeutically relevant concentration throughout the tumour.

Recent pre-clinical studies performed at Oxford using ThermoDox® released using FUS has shown that increased uptake at the target site is achievable. Hence there is great promise in using this combination therapy to achieve increased tumour uptake and local dose for the equivalent dose of doxorubicin used in systemic chemotherapy for human subjects, which has a well established and safe toxicity profile. The first extracorporeal FUS device in Europe was used for a study performed at Oxford between 2002 and 2004.

This single centre trial was sponsored by the University of Oxford. The recruiting study site was Oxford University Hospitals NHS Trust, where there is extensive clinical FUS experience.

The study is split into two parts. Part I identified optimal FUS exposure parameters for a range of patient BMIs and tumour locations within the liver using real time thermometry data from an implanted thermistor. After at least 5 and no more than 14 participants have had the intervention using real-time thermometry, data was reviewed by the Trial Management Group (TMG) to confirm readiness to proceed without real-time thermometry. Part II, which did not require thermistor implantation, is designed to reflect how the therapy would be implemented in clinical practice.

Participants received treatment for 1 day and are followed up for 30 days. All evaluable participants from both Part I and Part II were included in the endpoint analysis. Doxorubicin concentrations were directly determined from tissue biopsies of the target tumour, using a Good Laboratory Practice-validated high performance liquid chromatography (HPLC) assay, based on previously published methods.

If this study demonstrates successful targeted drug delivery in human subjects using LTSLs released by mild-hyperthermia, this could potentially transform the future of chemotherapy in clinical practice; targeted therapy using LTSLs containing other chemotherapeutic agents triggered non-invasively by mild hyperthermia could be applied to any solid organ cancer.

Arms: arm 1: All participants in Part I received:

Pre-LTLD Biopsy of Target Liver Tumour ThermoDox® (LTLD) Post-LTLD Biopsy of Target Liver Tumour Focused Ultrasound of Target Liver Tumour Thermometry of Target Tumour Post-LTLD+FUS (Post-FUS) Biopsy of Target Liver Tumour

Part I of the study was designed to identify optimal focused ultrasound (FUS) exposure parameters for a range of tumour locations within the liver, using real-time thermometry data from an implanted thermometry device (a thermistor or thermocouple). Plasma and biopsy samples of the target liver tumour were taken pre-LTLD, post-LTLD and post-LTLD+FUS. arm 2: All participants in Part II received:

ThermoDox® (LTLD) Focused Ultrasound of Target Liver Tumour Post-LTLD Biopsy of Target Liver Tumour

Following a minimum of 5 Part I cases, and subject to Trial Management Group approval, Part II of the study was opened to run in parallel to Part I. Part II did not require implantation of a thermometry device, and instead used predictions from Part I data to set the FUS parameters. Targeted drug delivery in Part II thus proceeded completely non-invasively, and this part of the study was designed to more closely reflect how the therapy might be implemented in routine clinical practice. Plasma samples were taken pre-LTLD, post-LTLD and post-LTLD+FUS. Biopsy samples of the target liver tumour were taken only post-LTLD+FUS.
Interventions: intervention 1: ThermoDox® (LTLD) intervention 2: Focused Ultrasound of Target Liver Tumour intervention 3: Pre-LTLD Biopsy of Target Liver Tumour intervention 4: Post-LTLD Biopsy of Target Liver Tumour intervention 5: Post-LTLD+FUS (Post-FUS) Biopsy of Target Liver Tumour intervention 6: Thermometry of Target Tumour
Intervention descriptions: intervention 1: ThermoDox® (LTLD) infusion at a dose of 50mg/m2 whilst under general anaesthetic during intervention (Day 1) intervention 2: Whilst the ThermoDox® was circulating in the blood stream, the JC200 Therapeutic Ultrasound device was used to induce mild hyperthermia in a single (region of) a target liver tumour. intervention 3: None intervention 4: None intervention 5: None intervention 6: A clinically approved thermistor or thermocouple was placed in the target liver tumour for real-time thermometry.

Protocol documents:
Prot_000.pdf: 
 
 
TARDOX_V3.0_06Apr2016                                                   
                                                                                Protocol Template V3.0_18Feb2013 
Page 1 of 59 
 
 
 
Full title: A Proof of Concept Study to Investigate the Feasibility of Targeted 
Release of Doxorubicin from Lyso-thermosensitive Liposomal (LTSL) Doxorubicin 
(ThermoDox®) Using Focused Ultrasound in Patients with Primary or Secondary 
Liver Tumours 
Short title: Targeted chemotherapy using focused ultrasound for liver tumours 
 
 
Protocol Version & date: 
Version 3.0 06Apr2016 
Sponsor Protocol Number: 
OCTO_040  
Ethics Number: 
14/NE/0124 
EudraCT Number: 
2014-000514-61 
Clintrials.gov. No. 
NCT02181075 
 
 
 
 
 
 
 
 
     
 
 
 
Sponsored by the University of Oxford 
 
Funders: 
NIHR Oxford Biomedical Research Centre 
Joint Research Office, Block 60, Churchill Hospital, Old Road, Headington, Oxford OX3 7LE 
 
Celsion Corporation 
Lawrenceville, NJ, USA 
 
 
Confidentiality Statement 
This document contains confidential information that must not be disclosed to anyone other than the Sponsor, the 
trials office, the Investigator Team, host NHS Trust(s), regulatory authorities, and members of the Research Ethics 
Committee.   


 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 2 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
 
General contact information 
 
Trial Office (OCTO) 
TARDOX Trial Office 
Oncology Clinical Trials Office (OCTO) 
Department of Oncology, The University of Oxford 
Oxford Cancer and Haematology Centre 
Churchill Hospital 
Oxford 
Tel: +44 (0)1865 227190 
Fax: +44 (0)1865 227039 
Email: OCTO-TARDOX@oncology.ox.ac.uk 
Website: http://www.octo-oxford.org.uk  
Clinical Trial Coordinator: Miss Lucy Boyle 
 
 
Chief Investigator 
Professor Mark Middleton 
Consultant Medical Oncologist 
Department of Oncology, University of Oxford 
Oxford Cancer and Haematology Centre Churchill Hospital Oxford OX3 7LE 
Tel: +44 (0)1865-235315 
Fax: +44 (0)1865-235981 
Email: mark.middleton@oncology.ox.ac.uk 
 
 
Lead Senior Scientific Investigator 
Professor Fergus Gleeson 
Consultant Radiologist 
Department of Radiology Churchill Hospital  Oxford OX3 7LE 
Tel.: +44 (0) 28 95048492 
Email: fergus.gleeson@oncology.ox.ac.uk 
 
 
Lead Senior Scientific Investigator 
Professor Constantin Coussios 
Professor of Biomedical Engineering 
Head of the Biomedical Ultrasonics, Biotherapy and Biopharmaceuticals 
Laboratory  Institute of Biomedical Engineering 
Old Road Campus Research Building University of Oxford OX3 7DQ 
Tel: +44 (0) 1865-617726 
Fax: +44 (0) 1865-617728 
Email: constantin.coussios@eng.ox.ac.uk 
 
 
Sponsor 
Ms Heather House 
 
University of Oxford 
Joint Research Office, Block 60, Churchill Hospital 
Oxford OX3 7LE 
Tel: +44 (0) 1865-572245 
E-mail: heather.house@admin.ox.ac.uk  
 
 
Lead NHS Trust 
 
 
 
Ms Heather House 
 
R&D Department, Oxford University Hospitals NHS Trust 
Joint Research Office, Block 60, Churchill Hospital Oxford OX3 7LE 
Tel: +44 (0) 1865-572245 
E-mail: heather.house@admin.ox.ac.uk 
 
 
Investigational Drug Supplier 
Celsion Corporation, Lawrenceville, NJ, USA 
Celsion Key Contacts 
Dr Nicholas Borys, Senior Vice President & CMO  
Michael DeSalvo, Manufacturing & Quality 
Lauren Musso, Project Manager, Clinical Operations 
 
 


 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 3 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
 
Patient  
Registration 
 
To register a patient on the trial: 
Please fill in the TARDOX registration form and send in to the trial office. 
Mon-Fri 09:00-17:00 UK Time, Fax & Email 24 hours* 
Fax: 01865 227039  
Email: octo-TARDOX@oncology.ox.ac.uk  (preferred) 
*Faxes & emails received outside office hours will be dealt with the next 
working day 
 
 
Clinical Queries and Emergency 
Contact Details 
During office hours: Clinical Queries should be directed to the research 
office.  The call will be passed on to the Chief Investigator or to the Clinical 
Fellow assigned to TARDOX. 
 
Out of office hours: call the Oxford University Hospital Trust Churchill and 
John Radcliffe switchboard on Tel: +44 (0) 1865 741 841 and ask to call the 
TARDOX Study Oncologist on call. 
 
 
Trial Statistician 
Susan Dutton  
Senior Medical Statistician and OCTRU Lead Statistician 
Oxford Clinical Trials Research Unit 
Centre for Statistics in Medicine 
Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences 
University of Oxford 
Botnar Research Centre 
Windmill Road 
Oxford OX3 7LD 
Susan.Dutton@csm.ox.ac.uk 
 
Dr Lang’O Odondi 
 
Medical Statistician 
Oncology Clinical Trials Office (OCTO) 
 
Department of Oncology & Centre for Statistics in Medicine 
 
Botnar Research Centre 
 
Windmill Road 
 
Oxford OX3 7LD 
Lango.Odondi@oncology.ox.ac.uk 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 4 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
TABLE OF CONTENTS 
PROTOCOL SYNOPSIS ................................................................................................................. 8 
SUMMARY OF SCHEDULE EVENTS ............................................................................................. 13 
KEY DEFINITIONS ...................................................................................................................... 15 
ABBREVIATIONS ....................................................................................................................... 15 
1 
INTRODUCTION ........................................................................................................ 16 
1.1 
BACKGROUND ON LIVER METASTASES ......................................................................................... 17 
1.1.1 Epidemiology .................................................................................................................. 17 
1.1.2 Treatment Modalities .................................................................................................... 17 
1.2 
BACKGROUND ON PRIMARY LIVER CANCER ................................................................................... 18 
1.2.1 Epidemiology .................................................................................................................. 18 
1.2.2 Treatment Modalities .................................................................................................... 18 
1.3 
INVESTIGATIONAL MEDICINAL PRODUCTS USED IN THE STUDY .......................................................... 18 
1.3.1 Lyso-Thermosensitive Liposomal Doxorubicin (LTLD, ThermoDox®) ............................. 18 
1.3.2 Current ThermoDox® Trials ............................................................................................ 19 
1.4 
OTHER RESEARCH INTERVENTIONS .............................................................................................. 19 
1.4.1 Mild Hyperthermia Using a HIFU Therapeutic Device ................................................... 19 
1.5 
RATIONALE FOR THE STUDY ....................................................................................................... 20 
1.5.1 Pre-Clinical Studies ......................................................................................................... 20 
1.5.2 Clinical Studies ................................................................................................................ 20 
2 
STUDY DESIGN .......................................................................................................... 20 
2.1 
STUDY FLOW CHARTS ............................................................................................................... 21 
 ....................................................................................................... Error! Bookmark not defined. 
2.2 
DURATION OF PATIENT PARTICIPATION ........................................................................................ 24 
2.3 
POST-TRIAL CARE AND FOLLOW-UP ............................................................................................. 24 
3 
OBJECTIVES AND ENDPOINTS .................................................................................... 24 
4 
PATIENT SELECTION .................................................................................................. 26 
4.1 
ELIGIBILITY CRITERIA FOR ENTRY INTO THE MAIN STUDY ................................................................... 26 
4.1.1 Inclusion criteria: ............................................................................................................ 26 
4.1.2 Exclusion criteria: ........................................................................................................... 26 
4.2 
PROTOCOL DEVIATIONS AND WAIVERS TO ENTRY CRITERIA ............................................................... 27 
4.3 
RE-SCREENING IF PATIENT DOES NOT MEET INCLUSION/EXCLUSION CRITERIA FIRST TIME ROUND .............. 27 
4.4 
PATIENT REGISTRATION PROCEDURE ............................................................................................ 27 
5 
TRIAL ASSESSMENTS AND PROCEDURES .................................................................... 28 
5.1 
SCREENING (D-30 TO D-2) ........................................................................................................ 28 
5.1.1 Optional Two Stage Screening ....................................................................................... 28 
5.1.2 Informed Consent ........................................................................................................... 28 
5.1.3 Screening Assessments................................................................................................... 29 
5.2 
EVALUATIONS DURING THE INTERVENTION (D-1 TO D2) .................................................................. 30 
5.3 
FOLLOW-UP AND END OF STUDY EVALUATIONS (WITHIN 60 DAYS)..................................................... 31 
EVALUATIONS ON EARLY WITHDRAWAL .................................................................................................... 32 
5.4 
TRIAL PROCEDURES ................................................................................................................. 32 
5.4.1 Preparation for Intervention .......................................................................................... 32 


 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 5 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
5.4.2 Intervention .................................................................................................................... 32 
5.4.3 Focused Ultrasound Procedure ...................................................................................... 35 
5.4.4 Passive Acoustic Monitoring .......................................................................................... 35 
5.4.5 Participant Monitoring ................................................................................................... 35 
6 
EARLY PATIENT WITHDRAWAL .................................................................................. 35 
6.1 
PATIENT EVALUABILITY AND REPLACEMENT ................................................................................... 36 
7 
SAMPLES FOR LABORATORY ANALYSIS ...................................................................... 36 
7.1 
SAMPLES TO BE ANALYSED IN LOCAL TRUST’S LABORATORIES ............................................................ 36 
7.1.1 Diagnostic Laboratories ................................................................................................. 36 
7.1.2 Pathology ....................................................................................................................... 36 
7.2 
SAMPLES TO BE SENT TO AND ANALYSED IN A CENTRAL LABORATORY ................................................. 36 
7.3 
PHARMACOKINETIC ASSAYS ....................................................................................................... 36 
7.4 
PHARMACODYNAMIC ASSAYS ..................................................................................................... 37 
7.5 
SUMMARY OF SAMPLES/ASSAYS TO BE TAKEN DURING THE STUDY ..................................................... 37 
7.6 
SAMPLES FOR BIOBANKING ....................................................................................................... 37 
7.7 
LABELLING AND CONFIDENTIALITY OF SAMPLES SENT ....................................................................... 37 
7.8 
CLINICAL REPORTING OF EXPLORATORY RESEARCH ASSAY RESULTS ..................................................... 37 
7.9 
TRIAL SAMPLE RETENTION AT END OF STUDY ................................................................................. 37 
7.10 
WITHDRAWAL OF CONSENT FOR SAMPLE COLLECTION AND/OR RETENTION ......................................... 37 
8 
INVESTIGATIONAL MEDICINAL PRODUCTS (IMPS) ..................................................... 37 
8.1 
NAME OF IMPS ...................................................................................................................... 37 
8.2 
TREATMENT DOSE ................................................................................................................... 37 
8.3 
DURATION OF TREATMENT ........................................................................................................ 38 
8.4 
MANAGEMENT OF DRUG ADMINISTRATION ................................................................................... 38 
8.5 
SPECIAL PRECAUTIONS .............................................................................................................. 38 
8.6 
ACTUAL VERSUS IDEAL BODY WEIGHT ........................................................................................... 38 
8.7 
DOSE MODIFICATION ................................................................................................................ 38 
8.8 
CALCULATING AND RECALCULATING BSA/DOSES ........................................................................... 38 
8.9 
DOSE CAPPING ........................................................................................................................ 39 
8.10 
DOSE-BANDING ...................................................................................................................... 39 
8.11 
COMPLIANCE .......................................................................................................................... 39 
8.12 
MANAGEMENT OF OVERDOSE .................................................................................................... 39 
9 
OTHER TREATMENTS (NON-IMPS) ............................................................................. 39 
9.1 
FOCUSED ULTRASOUND ............................................................................................................ 39 
9.2 
BACKGROUND SYSTEMIC THERAPY............................................................................................... 39 
9.3 
SUPPORT MEDICATION ............................................................................................................. 39 
9.4 
CONCOMITANT MEDICATION AND NON-DRUG THERAPIES ................................................................ 40 
9.5 
PROHIBITED THERAPIES ............................................................................................................. 40 
9.6 
(POTENTIAL) DRUG INTERACTIONS ............................................................................................. 40 
10 
RADIOTHERAPY ........................................................................................................ 40 
11 
DRUG MANAGEMENT ............................................................................................... 40 
11.1 
DRUG SUPPLIES ....................................................................................................................... 40 
11.2 
DRUG ORDERING ..................................................................................................................... 41 


 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 6 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
11.3 
IMP RECEIPT ......................................................................................................................... 41 
11.4 
HANDLING AND STORAGE .......................................................................................................... 41 
11.5 
LABELLING ............................................................................................................................. 41 
11.6 
DOSING DISPENSING ................................................................................................................ 42 
11.7 
DRUG ACCOUNTABILITY ............................................................................................................ 42 
11.8 
DRUG RETURNS FROM PATIENTS ................................................................................................. 42 
11.9 
DRUG DESTRUCTION ................................................................................................................ 42 
11.10 OCCUPATIONAL SAFETY ............................................................................................................ 42 
12 
EVALUATION OF RESPONSE ...................................................................................... 42 
12.1 
MEASUREMENT OF DISEASE FOR SOLID TUMOUR............................................................................ 43 
12.2 
TUMOUR ASSESSMENT ............................................................................................................. 43 
12.2.1 
Baseline evaluations................................................................................................... 43 
12.2.2 
Evaluations during treatment and at off-study ......................................................... 43 
13 
ASSESSMENT OF SAFETY ........................................................................................... 43 
13.1 
ADVERSE EVENT DEFINITIONS .................................................................................................... 43 
13.2 
CLINICAL LABORATORY ABNORMALITIES AND OTHER ABNORMAL ASSESSMENTS AS AES AND SAES ........... 44 
13.3 
DETERMINING ADVERSE EVENT CAUSALITY .................................................................................... 44 
13.4 
EXPECTED ADVERSE EVENTS ....................................................................................................... 45 
13.5 
SUSPECTED UNEXPECTED SERIOUS ADVERSE DRUG REACTIONS (SUSARS) ......................................... 47 
13.6 
EXPEDITED REPORTING OF SAES ................................................................................................. 47 
13.7 
FOLLOW-UP OF SERIOUS ADVERSE EVENTS ................................................................................... 47 
13.8 
ADVERSE EVENTS THAT SHOULD BE NOTIFIED TO TRIAL OFFICE........................................................... 48 
13.9 
REPORTING ADVERSE EVENTS ON THE CRF................................................................................... 48 
13.10 EVENTS EXEMPT FROM BEING REPORTED AS AE/ SAES ................................................................... 48 
13.10.1 Progression of underlying disease.............................................................................. 48 
13.10.2 Death on study ........................................................................................................... 49 
13.10.3 Elective admissions and supportive care ................................................................... 49 
13.11 DEVELOPMENT SAFETY UPDATE REPORTS .................................................................................... 49 
13.12 INFORMING INVESTIGATORS OF NEW SAFETY INFORMATION ............................................................. 49 
14 
PREGNANCY ............................................................................................................. 49 
15 
DEFINING THE END OF TRIAL ..................................................................................... 49 
16 
STATISTICAL CONSIDERATIONS ................................................................................. 50 
16.1 
SAMPLE SIZE AND POWER .......................................................................................................... 50 
17 
STATISTICAL ANALYSIS PLAN ..................................................................................... 50 
17.1 
INCLUSION IN ANALYSIS ............................................................................................................. 50 
17.2 
SUBGROUP ANALYSIS ................................................................................................................ 50 
17.3 
INTERIM ANALYSES .................................................................................................................. 50 
17.4 
PROCEDURES FOR REPORTING ANY DEVIATION(S) FROM THE ORIGINAL STATISTICAL PLAN ....................... 50 
17.5 
FINAL ANALYSIS ....................................................................................................................... 50 
18 
TRIAL COMMITTEES .................................................................................................. 51 
18.1 
TRIAL MANAGEMENT GROUP (TMG) ......................................................................................... 51 
18.2 
DATA AND SAFETY MONITORING ................................................................................................ 51 
18.3 
TRIAL STEERING COMMITTEE ..................................................................................................... 51 


 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 7 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
DATA MANAGEMENT ............................................................................................................... 51 
18.4 
DATABASE CONSIDERATIONS ...................................................................................................... 51 
18.5 
CASE REPORTS FORMS (CRFS) ................................................................................................... 51 
18.6 
ACCOUNTING FOR MISSING, UNUSED, OR SPURIOUS DATA. .............................................................. 52 
19 
CLINICAL STUDY REPORT ........................................................................................... 52 
20 
STUDY SITE MANAGEMENT ....................................................................................... 52 
20.1 
STUDY SITE RESPONSIBILITIES ..................................................................................................... 52 
20.2 
STUDY SITE SET UP AND ACTIVATION ............................................................................................ 52 
20.3 
ARRANGEMENTS FOR SITES OUTSIDE THE UK ................................................................................ 52 
20.4 
STUDY DOCUMENTATION .......................................................................................................... 52 
21 
REGULATORY AND ETHICAL CONSIDERATIONS .......................................................... 52 
21.1 
ETHICAL CONDUCT OF THE TRIAL AND ETHICS APPROVAL .................................................................. 52 
21.2 
REGULATORY AUTHORITY APPROVAL ........................................................................................... 53 
21.3 
NHS RESEARCH GOVERNANCE................................................................................................... 53 
21.4 
PROTOCOL AMENDMENTS ......................................................................................................... 53 
21.5 
URGENT SAFETY MEASURES ....................................................................................................... 53 
21.6 
TEMPORARY HALT ................................................................................................................... 54 
21.7 
SERIOUS BREACHES.................................................................................................................. 54 
21.8 
REPORTS: PROGRESS, SAFETY AND END OF STUDY REPORTS .......................................................... 54 
22 
EXPENSES AND BENEFITS .......................................................................................... 54 
23 
QUALITY ASSURANCE ................................................................................................ 54 
23.1 
RISK ASSESSMENT .................................................................................................................... 54 
23.2 
ON-SITE MONITORING ............................................................................................................. 54 
23.3 
CENTRAL MONITORING ............................................................................................................ 54 
23.4 
AUDIT AND REGULATORY INSPECTION ......................................................................................... 55 
24 
RECORDS RETENTION & ARCHIVING .......................................................................... 55 
25 
PATIENT CONFIDENTIALITY ....................................................................................... 55 
26 
STUDY FUNDING ....................................................................................................... 55 
27 
SPONSORSHIP AND INDEMNITY ................................................................................ 55 
27.1 
SPONSORSHIP ......................................................................................................................... 55 
27.2 
INSURANCE ............................................................................................................................ 56 
27.3 
CONTRACTS/AGREEMENTS ........................................................................................................ 56 
28 
PUBLICATION POLICY ................................................................................................ 56 
29 
REFERENCES ............................................................................................................. 56 
APPENDIX 1: WHO PERFORMANCE SCALE ................................................................................. 59 
APPENDIX 2: AMERICIAN SOCIETY OF ANAESTHESIOLOGISTS (ASA) CLASSIFICATION SYSTEM ... 59 
APPENDIX 3: CHILD-PUGH SCORE ............................................................................................. 59 
 
 


 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 8 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
PROTOCOL SYNOPSIS 
Full Title of study: 
A proof of concept study to investigate the feasibility of targeted release of doxorubicin 
from lyso-thermosensitive liposomal (LTSL) doxorubicin (ThermoDox®) using ultrasound-
guided focused ultrasound in patients with primary or secondary liver tumours 
Short Title: 
A Phase 1 Clinical Trial of targeted chemotherapy using focused ultrasound for liver 
tumours 
Trial Acronym: 
TARDOX 
Study Design: 
Phase 1 prospective non-randomised safety cohort study with all patients recruited from 
a single UK site (Oxford). The study has an open label design with all participants 
receiving ThermoDox® and FUS. The study will run in two parts. Part I is required to 
identify optimal FUS exposure parameters using real time thermometry data from an 
implanted thermometry device, such as a thermistor. Part II does not require 
implantation of thermometry device and is designed to more accurately reflect how the 
therapy would be implemented in clinical practice. All evaluable participants from both 
Part I and Part II will be included in the endpoint analysis. 
 
Part I 
For each participant, a single liver tumour will receive the intervention and follow-up 
imaging. The ability to achieve targeted release of doxorubicin from ThermoDox® at the 
tumour site will be determined using three tumour biopsies with corresponding 
peripheral blood samples taken during intervention and/or using a radiological method 
(MR-Spectroscopy). 
 
Part II 
For each participant, a single liver tumour will receive the intervention and follow-up 
imaging.. The optimal FUS exposure parameters determined in Part I will be selected for 
Part II interventions, accounting for differences in propagation path length identified in 
the Ultrasound Session. The ability to achieve targeted release of doxorubicin from 
ThermoDox® at the tumour site will be determined using the same methods as for Part I, 
with the exception of requiring only two tumour biopsies. 
 
In the 30 days following intervention, both Part I and II participants will be followed up 
with a maximum of two CTs, two PET-CTs and three MRI/MR-Spectroscopy scans. There 
is however built in flexibility for the scans to be performed at different timepoints up to 
60 days post intervention should the patient provide this optional consent and not have 
started another treatment. If available, other non-FUS control tumour(s) in the liver 
exposed to ThermoDox® alone will be selected as controls against which radiological 
response in the FUS-targeted tumour (target tumour) will be compared. 
 
Detailed criteria for evaluation of efficacy and safety are described in subsequent 
sections. 
Patient Numbers: 
A maximum of 28 evaluable participants will be recruited to the study overall. 
Target Population: 
Adult patients with incurable (unresectable, non-ablatable-for-cure) confirmed hepatic 
primary (hepatocellular carcinoma or cholangiocarcinoma) or secondary (metastatic) 
tumour. All patients will have progressed or maintained stability on conventional 
chemotherapy. To be suitable, patients must have liver tumour(s) in the liver of size 1cm 
or greater, which are amenable to ultrasound-guided biopsy. 
Objectives: 
PRIMARY OBJECTIVE: 
To determine whether targeted release of doxorubicin from ThermoDox® (‘drug’) using 
mild hyperthermia generated non-invasively by focused ultrasound (FUS) is feasible in 
cancer patients. 
 
SECONDARY OBJECTIVES: 
• 
Part I only: To determine optimal FUS exposure parameters for a range of 
participant Body Mass Indices (BMIs) and tumour locations within the liver 


 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 9 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
• 
To assess the safety of FUS-induced mild hyperthermia for drug delivery 
• 
To assess the local and systemic cytotoxic effects of ThermoDox® in this setting  
 
TERTIARY (EXPLORATORY) OBJECTIVES: 
Regarding those participants for which doxorubicin has been released by optimal FUS 
exposure parameters: 
• 
To establish the validity of other minimally-invasive and non-invasive methods 
of evaluating intratumoural uptake of doxorubicin as an alternative to that used 
in primary endpoint: 
a) Fluorescence microscopy of biopsy 
b) MR-Spectroscopy 
c) Plasma pharmacokinetics  
• 
To determine whether the dose of doxorubicin released has a therapeutically 
significant effect on the target tumour. 
Rationale: 
The development of distant spread (metastases) is a leading cause of death in patients 
with advanced solid tumours. The liver is one of the most common sites for metastatic 
disease. Studies have shown that the treatment of distant metastases improves survival. 
Life expectancy for patients with metastatic colorectal cancers without treatment ranges 
from 5 to 9 months with a median survival after diagnosis of 6 months [1-3]. Surgical 
resection remains the only treatment shown to have long-term curative potential [4]. 
However, only a minority (10-20%) of patients are surgical candidates [5]. Systemic 
chemotherapy is the current treatment of choice for patients with inoperable liver 
metastasis. However, long-term survival is rare. The majority of primary liver cancers 
(hepatocellular carcinoma (HCC) and cholangiocarcioma) are unresectable and survial 
remains dismal [6-9]. Post-hoc analysis of the Phase III HEAT study is indicative that 
ThermoDox® treatment improves survival in patients with HCC, where Radiofrequency 
Ablation (RFA) was optimal. 
 
This proof of concept study proposes targeted delivery of broad-spectrum cytotoxic 
agent (doxorubicin), via a specially formulated LTSL (ThermoDox®) activated by mild 
hyperthermia, as both a feasible and effective solution to the drug-delivery problem for 
the same systemic dose. Recent animal work performed at Oxford using ThermoDox® 
released using FUS has shown that increased uptake at the target site is achievable. It is 
proposed that this method of targeted drug delivery is clinically applicable, and may 
achieve increased tumour uptake and local dose for the equivalent dose of doxorubicin 
used in systemic chemotherapy, which has a well-established and safe toxicity profile. 
 
Clinical studies have demonstrated: 
1. The safety profile of ThermoDox® has been shown to be consistent with that of 
doxorubicin (HEAT study) 
2. ThermoDox® treatment shows promise for improved progression-free survival 
and overall survival in the subset HCC of patients having optimal RFA (HEAT 
study) 
3. ThermoDox® has demonstrated a dose-response effect against metastatic liver 
tumours [10] 
 
If this study demonstrates successful targeted drug delivery in human subjects using 
ThermoDox® combined with mild-hyperthermia, this could potentially transform the 
future of chemotherapy in clinical practice; targeted therapy using LTSLs containing 
other chemotherapeutic agents triggered non-invasively by mild hyperthermia could be 
applied to any solid tumour. 
Primary Endpoint: 
 
A demonstrable two-fold increase in, or value exceeding 10μg/g of, the concentration 
of intra-tumoural doxorubicin at the treated tumour site following FUS-induced mild 
hyperthermia, in at least 50% of evaluable participants. 
Secondary Endpoints: 
1. Part I only: Achievement of the desired range of mild hyperthermia in the target 
tissue as monitored by the implanted thermometry device during FUS exposure 


 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 10 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
2. Persistence of cell viability stain or percentage of cell or tissue necrosis under 30% 
following FUS exposure, as assessed by cytological or histological methods  
3. Significant bone marrow suppression, deranged liver function and liver toxicity. 
4. Adverse events up to 30 days post-intervention: 
a. Deemed related to ThermoDox® 
b. Deemed related to FUS 
Tertiary 
(Exploratory) 
Endpoints: 
Regarding doxorubicin released by optimal FUS exposure parameters: 
1. Combined analysis of relevant end points to establish most effective alternative 
analytical method(s) for quantifying drug release: 
a. Released biopsy samples exhibiting averaged regions of nuclear or 
background fluorescence levels at least twice that of a suitable control, 
as assessed by fluorescence microscopy 
b. Liver MRI/MR-Spectroscopy indicating targeted delivery of doxorubicin 
to the tumour by demonstrating a statistically significant increase in 
the tumour:background ratio of signal intensity following intervention 
compared to baseline  
c. 
A significant decrease of in total doxorubicin plasma levels over time-
matched clearance levels, following FUS-induced release 
2. Radiological evidence of tumour response at timepoints up to 60 days post-
intervention in the FUS-targeted tumour and control(s), as assessed by 
Principles of CHOI and RECIST response evaluation using MRI and CT [11, 12] 
and SUVmax using PET-CT [13] 
Inclusion Criteria 
 
 
Patients will only be eligible for inclusion in this study if all of the following criteria apply: 
1. Pathologically confirmed advanced solid tumour with liver metastasis suitable 
for intervention (as assessed by ultrasound or other radiological methods). In 
addition confirmed primary liver tumours (hepatocellular carcinoma or 
cholangiocarcinoma) can be included. 
2. Will have progressed or remained stable on conventional chemotherapy. 
3. Male or Female, Age ≥ 18 years. 
4. Have life expectancy of ≥ 3 months. 
5. Left Ventricular Ejection Fraction (LVEF) ≥ 50% on echocardiogram.  
6. Have not received radiotherapy to the target area within the preceding 12 
months. 
7. A World Health Organisation (WHO) performance status of ≤ 1 (Appendix 1). 
8. Able and willing to give written informed consent, indicating that they are 
aware of the investigational nature of this study and potential risks, and able to 
comply with the protocol for the duration of the study, including scheduled 
follow-up visits and examinations.  
Exclusion criteria 
Patients will be ineligible for recruitment to the study if any of the following conditions 
hold: 
1. Have surgery or other procedure requiring general anaesthesia planned to be 
undertaken during the period of the study. 
2. Have serious illnesses including, but not limited to, congestive heart failure 
(NYHA class III or IV functional classification); life threatening cardiac 
arrhythmia; or myocardial infarction or cerebral vascular accident within the 
last 6 months. 
3. Have on going significant infection (chest, urine, blood, intra-abdominal). 
4. Have uncontrolled diabetes. 
5. Have received a life-time dose of doxorubicin > 450mg/m2 or a life-time dose of 
epirubicin > 900mg/m2 or any dose of both. 
6. Pregnant or breast-feeding. In women of childbearing potential, a negative 


 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 11 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
pregnancy test (serum) is required within 30 days prior to study intervention. 
7. Female participants of child bearing potential and male participants whose 
partner is of child bearing  potential who are not willing to practice an 
acceptable form of contraception (i.e. oral contraceptive, diaphragm, cervical 
cap, condom, surgical sterility) during the study and for 6 months thereafter. 
Women whose partner has or men who have undergone a vasectomy must use 
a second form of birth control. 
8. Have any known allergic reactions to any of the drugs or liposomal components 
or intravenous imaging agents to be used in this study. 
9. Have portal or hepatic vein tumour invasion/thrombosis. 
10. Inadequate haematological and biochemical function (section 4.1.2)  
11. Have contraindications to receiving doxorubicin including prior sensitivity (rash, 
dyspnoea, wheezing, urticarial or other symptoms) attributed to anthracyclines 
or other liposomal drugs. 
12. Use of chemotherapy or of an investigational drug within 30 days or 5 half-lives, 
whichever is longer, preceding the intervention. 
13. Have medically significant active infection. 
14. Have Child-Pugh Class C liver disease, or Class A-B with encephalopathy and/or 
refractory ascites (Appendix 3). 
15. Documented HIV positive. 
16. Documented diagnosis of haemochromatosis. 
17. Documented history of contrast-induced nephropathy. 
18. Have any of the following contraindications for liver biopsy: 
a. Suspected liver haemangioma or other vascular tumour 
b. Tense ascites 
c. 
Known cystic liver disease* 
d. Extra-hepatic biliary obstruction* 
(* Relative contraindications only and may be non-exclusive at discretion of the 
study team) 
19. Other medical or psychiatric conditions or laboratory abnormalities that the 
investigator considers would make the patient a poor trial candidate. 
Trial 
dose 
and 
administration: 
The investigative medicinal product is ThermoDox® LTSL doxorubicin. It is supplied in 
20cc vials each vial containing 30mg doxorubicin in 15ml of solution. A carton containing 
4 vials is normally sufficient for one treatment of one subject. 
One single dose of ThermoDox® with a doxorubicin concentration of 50mg/m2 will be 
given during the FUS exposure session as an intravenous infusion for both Part I & II. 
Duration on study: 
30 days from intervention (Day 1) with a maximum of 60 days of radiological follow-up 
Study 
Procedures 
and 
frequency: 
Part I 
Part I is concerned with optimisation of the FUS acoustic parameters. The optimal FUS 
exposure parameters to achieve the desired range of mild hyperthermia in the target 
tumour (and consequent ThermoDox® release) will be determined. A single target liver 
tumour will have already been identified. The participant requires insertion of a co-axial 
needle during intervention for monitoring and biopsy; consequently Part I is minimally 
invasive. 
 
Day -1: Attend for oral premedication and for baseline radiological assessment 
(Liver MRI/MR-Spectroscopy, Liver pCT and whole body FDG PET-CT scans) 
 
Day 1: Target tumour exposed to a combination of FUS and ThermoDox® under 
General Anaesthetic. Three liver tumour biopsies and temperature readings are 
taken via a single co-axial needle. Three peripheral blood samples are taken for 


 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 12 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
plasma pharmacokinetics. 
 
Day 2: Peripheral blood samples (FBC, U&E, LFT) are taken. Repeat Liver 
MRI/MR-Spectroscopy is performed within 36 hours of intervention. Participant 
is discharged after a minimum of 24 hours of observation post-intervention. 
 
Follow-up: Clinical reviews and repeat set of blood samples (FBC, U&E, LFT) at 
Days 15 and 30. A maximum of two further sets of scans (Liver MRI/MR-
Spectroscopy, Liver pCT and whole body FDG PET-CT) are performed within 60 
days post-intervention (one at 4 week follow-up, the other being flexible).  
 
Part II 
Part II is concerned with monitoring efficacy of the intervention (ThermoDox® release by 
mild hyperthermia), using the optimised FUS acoustic parameters identified in Part I. In 
particular, the release of doxorubicin and its therapeutic effect will be investigated. A 
single liver tumour will have been identified for intervention.. The participant requires 
insertion of a co-axial needle during intervention for a biopsy of the tumour area 
receiving FUS exposure for HPLC and microscopy analysis, and consequently Part II is 
minimally invasive. 
 
Day -1:  As for Part I. 
 
Day 1: Target tumour exposed to a combination of FUS and ThermoDox® under 
General Anaesthetic. Two biopsies of the tumour receiving intervention are 
taken via a single co-axial needle. Peripheral blood samples are taken for plasma 
pharmacokinetics. 
 
Day 2: As for Part I. 
 
Follow-up: As for Part I. 
 
Patient care post-trial: 
Patients will be managed as per standard of care. 
Criteria for 
evaluation 
Safety: 
Safety and toxicity will be reported using NCI CTCAE (currently version 4.03).  
Efficacy: 
Efficacy will be assessed by evidencing targeted ThermoDox® release, as per the primary 
endpoint. In addition radiological response will be assessed as a tertiary endpoint. 
Pharmacokinetic assays: 
Analysis of plasma samples to capture plasma pharmacokinetics of doxorubicin. 
Histopathology: 
Histological analysis of biopsy samples obtained from the targeted liver tumour may be 
performed by microscopy where tissue mass allows  
No. of Study Site(s) 
Single Centre UK (OXFORD)  
End of study 
Last Patient Last Visit 
Publication policy 
The intention is to publish this research in a specialist peer reviewed scientific journal on 
completion of the study. The results may also be presented at scientific meetings and/or 
used for a thesis.  The Investigators will be involved in reviewing drafts of the 
manuscripts, abstracts, press releases and any other publications arising from the trial 
and retain final editorial control. 
 


 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
        V3.0_18Feb2013 
Page 13 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver metastases 
SUMMARY OF SCHEDULE EVENTS 
Visit Description 
Ultrasound Screening1 
Full Screening & Main 
Study 
Intervention 
2wk Follow-up 4wk Follow-up* 
Flexible 
Radiological 
Follow-up 
Consent 
Screening 
Consent 
Screening 
Visit No. 
1a 
1b 
2a2 
2b3 
3 
4 
5 
Flexible 
Visit Type 
Outpatient 
Outpatient 
Outpatient 
Outpatient 
Outpatient then Inpatient from Day -1 or Day 14  
Outpatient 
Outpatient 
Outpatient 
Admission Duration (days) 
0 
0 
0 
0 
3 
0 
0 
0 
Day 
 
From -40 to -2 
 
From -30 to -2 
(-7 to -1) 
(Pre-
intervention) 
-1 
(Pre-
intervention) 
1 
(Intervention 
Day) 
2 
(Recovery Day) 
15 
30 
12-60** 
Visit window (days) 
 
After 1a 
 
After 2a 
After 2b 
±3 
+7 not <30 
Not >60 
 
 
 
 
 
 
 
 
 
 
Informed Consent 
X 
 
X 
 
 
 
 
 
 
 
 
Inclusion / Exclusion 
Criteria 
 
 
 
X 
 
 
 
 
 
 
 
Demographics, Medical & 
Surgical History 
 
 
 
X 
 
 
 
 
 
 
 
Concomitant medication 
 
 
 
X 
 
 
 
 
 
X 
 
Physical Exam & Vital 
Signs 
 
 
 
X 
 
 
 
X 
 
 
 
Height 
 
 
 
X 
 
 
 
 
 
 
 
Weight 
 
 
 
X 
 
X 
 
 
 
 
 
Urinalysis ± Pregnancy 
Test 
 
 
 
X 
 
 
 
 
 
 
 
ECG 
 
 
 
X 
 
 
 
 
 
 
 
Echocardiogram5 
 
 
 
X 
 
 
 
 
 
 
 
                                                     
 
1 The ultrasound screening may optionally be performed in advance of screening for the main study, or during the study screening. 
2 Study consent is usually taken during the screening visit but occasionally in advance (e.g. in clinic), but must occur before study procedures are performed outside of standard care. 
3 Best efforts will be made to ensure all screening procedures & required tests occur during a single screening visit but, but according to resources and patient preference, one or more 
additional screening visit(s) may occasionally be required. 
4 It is not compulsory for participants to be admitted on day -1. I.e. the patient can attend to complete pre-intervention assessments and return on day 1 to be admitted for the 
intervention.  
5 Echocardiogram will only be repeated if clinically indicated. 


 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
        V3.0_18Feb2013 
Page 14 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver metastases 
WHO PS 
 
 
X 
 
 
 
 
 
 
 
Haematology tests6 
 
 
 
X 
 
 
 
X 
X 
X 
 
Biochemistry tests 7 
 
 
 
X 
 
 
 
X 
X 
X 
 
Group & Save 8 
 
 
 
X 
 
 
 
 
 
 
 
Coagulation studies9 
 
 
 
X 
 
 
 
 
 
 
 
Pre-operative Anaesthetic 
Assessment (ASA 
Classification) 
 
 
 
X 
 
 
 
 
 
 
 
Ultrasound Session10 
 
X 
 
X 
 
 
 
 
 
 
 
Liver MRI/MR 
Spectroscopy11 
 
 
 
 
X 
 
 
X12 
 
X 
X 
Liver pCT 13 
 
 
 
 
X 
 
 
 
 
X 
X 
Whole Body FDG PET-CT 
 
 
 
 
X 
 
 
 
 
X 
X 
Pre-medication 
 
 
 
 
 
X 
 
 
 
 
 
FUS + ThermoDox® 
 
 
 
 
 
 
X 
 
 
 
 
ThermoDox® Plasma 
Samples 
 
 
 
 
 
 
X14 
 
 
 
 
Tumour Biopsy Samples 
 
 
 
 
 
 
X15 
 
 
 
 
Adverse Event Review 
 
 
 
 
 
 
 
X 
X 
X 
X 
 
* The 4 week follow-up visit will mark the end of the study and the participant will be free to consider other studies from this point, however scans may be performed after this time point in the 
flexible scan follow-up visit if that particant is not already enrolled on another study at that time. 
** The flexible radiology follow-up visit will be chosen based on patient preference, available imaging slots and emerging radiological data. Patient visits will be minimised where possible. The flexible 
scan visit will be performed at least a week either side the 4 week follow-up visit, and may coincide with the 2 week follow-up visit. 
In Part II, the number of follow-up MRI/MR-Spectroscopy, Liver pCT and whole body FDG PET-CT scans may be reduced,  if the data from Part I informs that a more efficient (in terms of reducing 
patients & site requirements)  schedule is possible.
                                                     
 
6 Full Blood Count (FBC); including Haemoglobin (Hb), white blood cells (WBC) with differential count (neutrophils and lymphocytes) and platelets.  
7 Biochemical profile to include U&Es, (Na+, K+, Ur, Cr), phosphate, calcium, LFTs (total protein, ALP, Albumin, ALT or AST, Bili, LDH) (validity period for bloods is 3 days). 
8 Group & Save, as standard for surgical candidates, date of valid test to be recorded on CRF, not blood group. Can alternatively be performed on d-1. 
9 Coagulation studies are performed at baseline only (PT & APTT). 
10 During the Ultrasound Session feasibility of intervention is confirmed and a target tumour will be selected by ultrasound. Only one screening ultrasound is required. 
11 MRI includes the abdomen. Measurements made using the modified RECIST 1.1 criteria.  
12 Liver MRI includes the abdomen. The first Liver MRI/MR Spectroscopy following intervention can alternatively be performed on the same day as intervention, if intervention is in the 
morning. It should occur within 36 hours of the completion of intervention, and ideally within 24 hours where possible.  
13 Liver pCT includes the abdomen. Measurements made using CHOI and the modified RECIST 1.1 criteria. 
14 Three blood samples for pharmacokinetics in both Phases I and II (Pre-Treatment, Unreleased drug, Released drug). 
15 Three biopsies in Part I (Pre-Treatment, Unreleased drug, Released drug), two biopsies in Part II (Released drug). 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 15 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
KEY DEFINITIONS 
“Intervention” refers to attempted targeted release of ThermoDox® by mild hyperthermia using a focused ultrasound. 
 
The “FUS-targeted tumour” or “target tumour” refers to the liver tumour treated with FUS following ThermoDox® 
delivery. This is in contrast to the RECIST definition, which defines target tumours as those measurable lesions which 
are followed through the patient’s treatment course. 
 
A “non-FUS control tumour” refers to a liver tumour receiving ThermoDox® alone which has been selected for 
radiological follow-up and comparision with the target tumour. 
 
The “desired range of mild hyperthermia” is used in the context of ThermoDox® release in this study and is defined as 
achieved bulk tissue temperature in the range of 41-47oC 
 
“Optimal FUS exposure parameters” are defined as those combinations of settings on the HIFU device (power, duty 
cycle and transducer motion plan) which result in an adequate temperature rise to successfully release doxorubicin 
from the LTSLs in the target tumour, without causing direct thermal tissue damage. 
 
An “evaluable participant” is defined as a participant who has received FUS following delivery of ThermoDox® in Part I 
or II, and for whom it has been possible to evaluate the outcome of the intervention based on biopsy samples, or 
radiologically. 
 
An “evaluable sample” is defined as a sample for which sufficient material was available for analysis and the analytical 
technique has provided quantifiable data. 
ABBREVIATIONS 
AE 
 
Adverse Event 
ADL 
 
Activities of Daily Living 
ADR 
 
Adverse Drug Reaction 
ALT 
 
Alanine Transaminase 
ANC 
 
Absolute Neutrophil Count 
ASA 
 
American Society of Anaesthesiologists  
AST 
 
Aspartate Transaminase 
BD 
 
Bi Daily 
BMI 
 
Body Mass Index 
CCA 
 
Cholangiocarcinoma 
CI 
 
Chief Investigator 
CR 
 
Complete Response 
CRC 
 
Colorectal cancer 
CRF 
 
Case Report Form 
CT 
 
Computerized Tomography 
CTA 
 
Clinical Trials Authorisation 
CTA 
 
Clinical Trial Agreement 
CTCAE  
Common Toxicity Criteria Adverse Events 
CTIMP  
Clinical Trial of an Investigational Medicinal Product 
DLT 
 
Dose Limiting Toxicity 
DNA 
 
Deoxyribonucleic Acid 
DSUR 
 
Development Safety Update Report 
ECG 
 
Electrocardiogram 
eCRF 
 
Electronic Case Report Form 
EMA 
 
European Medicines Agency 
EP 
 
Early Progression 
FBC 
 
Full Blood Count 
FDA 
 
Federal Drug Administration 
FDG-PET                Fluorodeoxyglucose Positron Emission Tomography 
FUS 
 
Focused Ultrasound 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 16 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
GCP 
 
Good Clinical Practice 
GI 
 
Gastro-Intestinal 
GMP 
 
Good Manufacturing Practices 
GP 
 
General Practitioner 
Hgb 
 
Haemoglobin 
HCC 
 
Hepatocellular Carcinoma 
HIFU 
 
High Intensity Focused Ultrasound 
HIV 
 
Human Immunodeficiency Virus 
IB 
 
Investigators Brochure 
ICH 
 
International Conference of Harmonisation 
IMP 
 
Investigational Medicinal Product 
LDH 
 
Lactate Dehydrogenase 
LVEF 
 
Left Ventricular Ejection Fraction  
LTLD 
 
Lyso-Thermosensitive Liposomal Doxorubicin 
LTSL 
 
Lyso-Thermosensitive Liposome 
mCRC 
 
Metastatic Colorectal Cancer 
MedDRA 
Medical Dictionary for Regulatory Activities 
MHRA  
Medicines and Healthcare products Regulatory Agency 
MRI 
 
Magnetic Resonance Imaging 
MTD 
 
Maximum Tolerated Dose 
NCI 
 
National Cancer Institute  
NE 
 
Not Evaluable 
NHS 
 
National Health Service 
NIMP 
 
Non-Investigational Medicinal Product 
OCTO  
 
Oncology Clinical Trials Office 
OCTRU  
Oxford Clinical Trials Research Unit 
OD 
 
Once Daily 
OS 
 
Overall survival 
pCT 
 
Perfusion CT 
PD 
 
Pharmacodynamic or Progressive Disease 
PFS 
 
Progression Free Survival 
PI 
 
Principal Investigator 
PICs 
 
Participant Identification Centres (PICs) 
PIS 
 
Patient Information Sheet 
PK 
 
Pharmacokinetic 
PO 
 
Per Os (oral) 
PR 
 
Partial Response 
QT 
 
QT interval 
QTc 
 
Corrected QT interval 
REC 
 
Research Ethics Committee 
RECIST  
Response Evaluation Criteria In Solid Tumours 
RFA 
 
Radio-frequency Ablation 
RR 
 
Response Rate 
SAE 
 
Serious Adverse Event 
SD 
 
Stable Disease 
SOP 
 
Standard Operating Procedure 
SUSAR  
Suspected Unexpected Serious Adverse Reaction 
SUVmax  
Standardised Uptake Value Maximum [13] 
TSC 
 
Trial Steering Committee 
TSL 
 
Thermosensitive Liposome 
WBC 
 
White Blood Count 
 
1 
INTRODUCTION 
This proof of concept study proposes targeted delivery of a broad-spectrum cytotoxic agent (doxorubicin), via a 
specially formulated LTSL (ThermoDox®) activated by mild hyperthermia, to achieve enhanced intratumoural 
doxorubicin concentrations for the same systemic dose. 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 17 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
1.1 
Background on Liver Metastases 
1.1.1 
Epidemiology 
The development of distant spread (metastases) is a leading cause of death in patients with malignant solid tumours. 
Lung, breast and colorectal cancer are, in that order, the three most common cancers worldwide. These three cancer 
sites account for 40% of the world’s total cases diagnosed, amounting to 4 million a year. They are also the leading 
cause of cancer death, resulting in almost 2.5 million deaths each year worldwide (International Agency for Research 
on Cancer: Fact Sheet, 2012). 
 
The liver is one of the most common sites for metastatic disease. Liver metastases are diagnosed in 10–25% of 
patients at the time of resection of their primary colorectal tumour and, eventually, up to 70% of patients develop 
liver metastases [15, 16]. In patients with breast cancer, 15% of patients have liver metastasis at the time of diagnosis, 
and as many as 50% of patients with stage IV disease will develop metastases [17-21]. The most common sites for 
metastatic spread of breast cancer are the lung, bone and liver (50-71%) [22]. Approximately a third of patients with 
non-small cell lung cancer have liver metastases at the time of diagnosis [23]. 
 
In a significant proportion of patients, the liver is the only site of distant disease. In 30% of the patients with colorectal 
and breast cancer, the metastases are confined to the liver [15, 16, 24]. Studies have shown that the treatment of 
distant metastases improves survival. Life expectancy for patients with colorectal metastases without treatment is 
dismal and ranges from 5 to 9 months with a median survival after diagnosis of 6 months [1-3]. 
 
The presence of liver metastasis is an important prognostic factor in patients with lung cancer [25] with a reduction in 
the five-year survival rate from 36% to only 1% [25, 26]. Although recent advances in breast cancer treatment have 
significantly improved survival rates in recent years, metastatic breast cancer as a whole has a median survival time of 
two years which is largely unchanged [22]. Life expectancy of breast cancer patients with liver metastases improves 
from 3-6 months without treatment to 15-32 months with treatment [18, 20, 21, 27]. 
1.1.2 
Treatment Modalities 
Surgical resection remains the gold standard in the treatment of colorectal liver metastasis, as it is the only treatment 
shown to have long-term curative potential [4]. A large series of over one thousand patients followed up after liver 
resection for colorectal metastases demonstrated an operative mortality rate of 2.8% and a five-year survival of 37% 
for resections that involved a lobectomy or more [28]. However, only a minority (10-20%) of patients are surgical 
candidates due to either the cancer being too locally advanced, extra-hepatic, or being unfit for surgery due to co-
morbidities [5]. Furthermore, the majority of patients undergoing liver resection develop new, non-resectable hepatic 
or extrahepatic metastases after hepatic resection [29, 30]. With the advent of neoadjuvant chemotherapy it is 
possible to convert some previously inoperable patients to the resectable group. A large trial of neoadjuvant systemic 
chemotherapy for colorectal liver metastasis looked at 330 patients with primarily unresectable liver disease from a 
cohort of 434 patients referred over a six year period. Of this group, 16% responded and went on to have liver 
resections with a comparable survival benefit to that of a primary liver resection (40% at 5 years). However, of the 
majority group with primarily unresectable tumours the vast majority (84%) remained inoperable [31]. 
 
Systemic chemotherapy remains the treatment of choice for patients with inoperable liver metastasis. Despite 
advances in systemic chemotherapy it remains a palliative treatment, as long-term survival is rare. In patients with 
liver metastasis from colorectal cancer, systemic chemotherapy with 5-fluorouracil (5-FU) has a response rate of only 
20% with a median response duration of 6 months [32]. In the same patient group, combined chemotherapy 
regimens, such as 5-FU and leucovorin, have improved the median overall survival to approximately 12 months [33, 
34] but with a trade-off of increased systemic toxicity.  
 
Minimally invasive or non-invasive techniques in the treatment of patients with radiologically detectable and discrete 
metastatic liver disease are in demand, driven by surgical inoperability of the majority of liver metastases. Such non-
invasive techniques are also in increasing demand among an ageing population. Ablative technologies are now 
becoming established and are showing encouraging outcomes for the treatment of metastatic liver disease. Such 
technologies are primarily being employed for palliation of patients with inoperable liver tumours who are 
unresponsive to chemotherapy. Modalities of ablative therapy include radiofrequency and microwave ablation, high 
intensity focused ultrasound (HIFU), laser and cryoablation. Overall, such techniques have the advantage over hepatic 
resection of shorter length of hospital stay and lower complication rates but the disadvantage of less proven survival 
benefits, although much clinical research activity is ongoing in this regard. Most of the current literature for ablative 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 18 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
therapies relates to treatment of hepatocellular carcinoma (HCC) rather than metastatic liver disease and to date 
there are no trials which compare outcomes between the various ablative technologies [35].  
 
Trans-arterial techniques offer an alternative to ablative techniques. However no survival benefit has been shown in 
the treatment of colorectal metastases with embolization alone [36, 37] and several Phase III trials have failed to 
demonstrate a survival benefit in hepatic arterial chemotherapy over systemic chemotherapy [38]. Hepatic artery 
chemoembolization delivers a dual ischaemic and cytotoxic insult with theoretical benefits beyond either of the 
individual components of treatment [38]. However, multiple chemoembolization procedures are often required and 
reports of treatment response vary greatly among available studies. A previous study included 30 patients who 
received between one and three chemoembolization procedures using a combination of cytotoxic agents. Radiological 
response was seen in 63% of the cases but all responses were transient. Median survival was extended to 29 months 
after initial diagnosis of liver metastasis. Mild-to-moderate toxicity was seen and ‘post-embolization syndrome’ (fever, 
right upper quadrant pain, nausea and vomiting) was common [38]. 
 
Although targeted treatment of liver metastases with ablative or trans-arterial techniques allows localized, minimally 
invasive therapy without significant toxicity, the majority of patients with metastatic liver disease from malignant solid 
tumours are unsuitable for these therapies; thus palliative chemotherapy has been the mainstay of treatment. 
Nevertheless, the availability of these ablative modalities has stimulated interest in developing an additive or 
synergistic combination therapy for patients with metastatic disease using chemotherapy and ablation. The treatment 
of colorectal liver metastases with Radio-Frequency Ablation (RFA) provides palliation by extending life expectancy to 
a median survival of 36 months [39], representing a significant improvement over chemotherapy alone. However, 
such treatments must be radiologically amenable to accurate targeting of the tumour or tumours and thus only a 
limited number of metastatic tumours in the liver can be successfully treated by RFA. 
1.2 
Background on Primary Liver Cancer 
1.2.1 
Epidemiology 
Primary liver cancers are the third highest cause of cancer-related death globally [40]. Although incidence is lower in 
the UK, it is increasing, with around 4300 new cases per year at present. In England, primary liver cancers account for 
around 1% of all cancer cases and have a 20% survival rate at one year (Cancer Research UK Liver Cancer: Key Facts, 
2014). HCC, also known as malignant hepatoma, is the most commonly occurring primary liver cancer, accounting for 
45% of cases in England (National Cancer Intelligence Network: Trends in incidence of primary liver cancer subtypes), 
and has risk factors including hepatitis B and C and cirrhosis. Cholangiocarcinoma (CCA), cancer of the biliary tract, 
accounts for 40% of primary liver cancers in England and is the most common cause of death from primary liver 
cancer in the UK, where it has an annual mortality rate of 1500 [41]. 
1.2.2 
Treatment Modalities 
Available treatment options for primary liver cancers largely mirror those previously discussed for metastatic liver 
tumours, and are dependent on staging. In HCC, again resection is the gold standard for localised disease, but is not an 
option in cirrhosis, often the underlying cause for HCC. Consequently there is the additional option for total 
hepatectomy and liver transplant in cirrhosis, should a suitable donor be found. Early diagnosis and treatment of HCC 
can achieve survival rates of 60-70%. However, due to a lack of alternative treatment options, prognosis in advanced 
disease or progressive disease following local therapy is dismal [6-8]. Sorafenib has been shown to increase median 
survival in those with advanced HCC by almost three months over placebo in a Phase III trial of over 600 patients [42]. 
In the case of CCA, again surgery offers the only chance of cure, but the majority of cases are inoperable due to 
presence of extra-hepatic metastasis at diagnosis. A study looking at survival rates in CCA diagnosed in European 
adults between 1990-1999 demonstrated age-standardised five-year relative survival rate of around 12% [9]. A recent 
UK study has demonstrated a six-month progression free survival of 57% using combination chemotherapy [43]. 
 
1.3 
Investigational Medicinal Products used in the study  
1.3.1 
Lyso-Thermosensitive Liposomal Doxorubicin (LTLD, ThermoDox®) 
ThermoDox® (or Lyso-Thermosensitive Liposomal Doxorubicin – LTLD) is being developed within an Investigational 
New Drug programme in the USA by Celsion Corporation (Celsion). Celsion will provide ThermoDox® as the 
Investigational Medicinal Product (IMP) for this study. ThermoDox® is a liposomal drug delivery system that releases a 
broad-spectrum cytotoxic drug (doxorubicin) when exposed to mild hyperthermia as the trigger. Drug release occurs 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 19 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
in the range of 39-45°C and maximal activation of the LTSLs occurs around 42°C. In this study, using FUS, we aim to 
achieve the desired range of mild hyperthermia for ThermoDox® release. 
1.3.2 
Current ThermoDox® Trials 
The ongoing clinical program is currently investigating ThermoDox in two indications: hepatocellur carcinoma and 
recurrent chest wall breast cancer.  
 
The lead clinical study is A Phase III, Randomized, Double Blind, Dummy-Controlled Study of ThermoDox® (Lyso-
Thermosensitive Liposomal Doxorubicin-LTLD) in Hepatocellular Carcinoma (HCC) using standardized Radiofrequency 
Ablation (sRFA) treatment time ≥ 45 minutes for intermediate size solitary lesions ≥ 3 cm to ≤ 7 cm. (Celsion Protocol 
104-13-302, The OPTIMA Study). This trial is designed to evaluate an optimized RFA dwell time ≥ 45 minutes based on 
results from the prior clinical program in HCC. The OPTIMA study was initiated in June 2014 and is designed to enroll 
about 550 patients.  The primary endpoint is overall survival with progression free survival as a secondary endpoint. 
 
The HEAT Study, a pivotal Phase III trial enrolled 701 newly diagnosed HCC subjects with unresectable hepatocellular 
carcinoma (HCC) lesions, with at least one ≥ 3.0 cm and none > 7.0 cm. The study demonstrated that ThermoDox can 
be safely administered in this patient population but failed to meet the primary endpoint of Progression Free Survival 
and Overall Survival.  A Post hoc analysis of the data from the HEAT Study was undertaken in order to better 
understand the results and to identify any subgroups that may have benefited from the treatment.  The analysis 
suggests that ThermoDox efficacy for the treatment of single lesions is greatly improved when RFA dwell time is ≥ 45 
minutes which has formed the basis of the hypothesis driving the current prospective pivotal study OPTIMA.  
 
A Phase II open label study in subjects with unresectable loco-regional recurrent breast cancer of the chest wall (RCW) 
administered ThermoDox in conjuction with mild local hyperthermia using microwave or ultrasound heating devices is 
on-going. The study has completed enrollment of 17 subjects to evaluate bioequivalence, safety and efficacy of 
ThermoDox manufactured at two manufacturing sites.  Additionally, a Phase II open label study in subjects with RCW 
administering ThermoDox in conjuction with mild local hyperthermia and radiation therapy will enroll patients 
throughout Europe and Israel.  
 
The most frequent adverse events in patients treated with ThermoDox® at a 50 mg/m2 dose are myelosuppression 
(60%), haematological adverse event (related or unrelated) (45%) and alopecia (40%). Other toxicities reported in 
greater than 5% of patients include pyrexia, fatigue, nausea and decreased appetite. To date, no significant 
hypersensitivity or anaphylactoid reactions have been reported in clinical studies (refer to the ThermoDox® 
Investigator’s Brochure [44], referred to as the ‘ThermoDox® IB’ from hereon). 
 
Of particular relevance to this study, the safety profile of ThermoDox® in the HEAT study has been shown to be 
consistent with that of doxorubicin, with neutropenia being the most important adverse event. ThermoDox® was well 
tolerated with no unexpected serious adverse events.  
 
 
1.4 
Other research interventions  
1.4.1 
Mild Hyperthermia Using a HIFU Therapeutic Device 
HIFU therapeutic devices employ focused ultrasound, usually through the medium of water, to provide a thermal 
effect, typically ablation, at a precise anatomical location (section 5.4.3). Although less commonly performed for 
hepatic metastatic disease than either RFA or microwave ablation, ablative HIFU has the advantage of being 
completely non-invasive. The University of Oxford and the Oxford University Hospitals NHS Trust have extensive 
clinical HIFU experience. The first extracorporeal HIFU device in Europe was used for a study performed at the 
Churchill Hospital between 2002 and 2004 [45]. The study used an ultrasound-guided HIFU system (Model-JC Tumour 
Therapy System, HAIFU Technology Company Ltd, Chongqing, China) to treat 30 patients with tumour deposits in the 
kidney or liver, and demonstrated both safety and efficacy. This was followed by the first clinical trial of HIFU ablation 
of tumours in the Western world carried out in Oxford [46]. This non-randomised Phase II clinical trial compared 
radiological response against histological findings and again demonstrated safety and feasibility for non-invasive 
treatment of solid tumour deposits. These studies have led to CE-marking of the JC and JC-200 treatment devices for 
non-invasive treatment of solid tumours such as liver cancer, kidney tumours, breast cancer, bone tumours, 
pancreatic tumours and uterine fibroids.  


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 20 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
 
Experiments have been performed in our laboratory using gel based tissue-mimicking model phantoms, 
demonstrating that the temperature rises required for release of the drug from the LTSLs can be achieved using a 
known clinical ultrasound therapy system at a fraction of the power used for thermal ablation. The resultant bulk 
heating effect on the model is well below the established thresholds for thermal cell death. It follows that drug 
release from LTSLs under mild hyperthermia is theoretically achievable without any significant thermal damage to the 
tissue.  
 
In this study, using focused ultrasound (FUS) to induce mild hyperthermia in participants under controlled limits with 
real-time temperature monitoring, we aim to remain well below thresholds for ablation or cell death of the targeted 
tumour cells by thermal damage.  The aim of using FUS in this study is to investigate the potential for triggered drug-
release by mild hyperthermia at the targeted tumour site and not its direct ablative effect as a cancer treatment itself. 
1.5 
Rationale for the study 
To date, purely pharmacological approaches have failed to address what is essentially a three-fold challenge: (i) to 
deliver therapeutically significant concentrations of active agents to the tumour vasculature while minimizing off-
target effects; (ii) to release the therapeutic agent ‘on-demand’ at the target site; and, (iii) to improve the distribution 
and spread of the therapeutic agent against the intra-tumoural pressure gradient in order to achieve a therapeutically 
relevant concentration throughout the tumour. 
 
1.5.1 
Pre-Clinical Studies 
Recent animal work performed at Oxford using ThermoDox® released using FUS has shown that increased uptake at 
the target site is achievable. It is proposed that this method of targeted drug delivery is clinically applicable, and may 
achieve increased tumour uptake and local dose for the equivalent dose of doxorubicin used in systemic 
chemotherapy, which has a well-established and safe toxicity profile. 
1.5.2 
Clinical Studies 
Clinical studies have demonstrated: 
1. The safety profile of ThermoDox® has been shown to be consistent with that of doxorubicin (HEAT study) 
2. ThermoDox® treatment shows promise for improved progression-free survival and overall survival in the 
subset of HCC patients having optimal RFA (HEAT study) 
3. ThermoDox® has demonstrated a dose-response effect against metastatic liver tumours [10] 
 
If this study demonstrates successful targeted drug delivery in human subjects using ThermoDox® combined with 
mild-hyperthermia, this could potentially transform the future of chemotherapy in clinical practice; targeted therapy 
using LTSLs containing other chemotherapeutic agents triggered non-invasively by mild hyperthermia could be applied 
to any solid tumour. 
2 
STUDY DESIGN 
Phase 1 prospective non-randomised open label single site study to determine whether targeted release of 
doxorubicin from ThermoDox® (‘drug’) using mild hyperthermia generated non-invasively by FUS is possible in cancer 
patients. All participants will receive ThermoDox® and FUS.  
 
The study will run in two parts. Part I will identify optimal FUS exposure parameters for a range of patient BMIs and 
tumour locations within the liver using real time thermometry data from an implanted thermometry device, such as a 
thermistor or thermocouple. After at least 5 participants have had the intervention using real-time thermometry, data 
will be reviewed by the Trial Management Group (TMG) to confirm readiness to proceed without real-time 
thermometry (Part II). Part II, which does not require implantation of a thermometry device, is designed to reflect how 
the therapy would be implemented in clinical practice. All evaluable participants from both Part I and Part II will be 
included in the endpoint analysis.  
 
 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
        V3.0_18Feb2013 
Page 21 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
2.1 
Study Flow Charts 
 
Figure 1: Recruitment, Screening and Intervention Preparation flow chart. Note that the ultrasound screening session can optionally be consented for in advance of the main study consent. 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
        V3.0_18Feb2013 
Page 22 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
 
 
Figure 2: Part I Intervention Plan, Recovery & Follow-up flow chart 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
        V3.0_18Feb2013 
Page 23 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
 
 
 
Figure 3: Part II Intervention Plan, Recovery & Follow-up flow chart 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 24 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
2.2 
Duration of patient participation 
Participants will be in the study for less than three months following entry on study and the participant will be 
followed up clinically for a maximum of 37 days and radiologically for a maximum of 60 days after intervention (Day 
1). 
 
2.3 
Post-trial care and follow-up 
Following the completion of the study, patients will receive standard care until disease progression or death. 
3 
OBJECTIVES AND ENDPOINTS 
The objectives and endpoints for the study are detailed in the following tables. Only evaluable samples will be 
included in endpoint analysis. All endpoints will be evaluated at the end of Phase II. 
 
The following qualifiers (*,†, ‡ and §) apply to Table 1, Table 2 and Table 3: 
 
* In Part II the Unreleased biopsy is not performed and therefore the average value for all evaluable tumours receiving 
intervention in Part I is used as a comparison for the two-fold increase from Unreleased to Released biopsy. 
 
† Duration refers to the completion of sample processing but not necessarily the sample analysis. The sample may be 
stored in a fixed or frozen state and analysed at some time period beyond the specified duration, for example to 
facilitate batch processing. 
 
‡ Analytical method will only be performed where tissue mass allows. It is expected at most one technique can be 
performed in addition to the primary endpoint for a given sample. Where biopsy mass falls short, the primary 
endpoint will be prioritised. 
 
§ Must be shown in at least 50% of evaluable participants.  
 
Primary Objective 
Primary Endpoint 
How and when data is 
captured 
To determine whether targeted 
release of doxorubicin from 
ThermoDox® (‘drug’) using mild 
hyperthermia generated non-
invasively by focused ultrasound 
(FUS) is feasible in cancer 
patients 
 
A demonstrable two-fold increase in*, or 
value exceeding 10μg/g of, the concentration 
of intra-tumoural doxorubicin at the treated 
tumour 
site 
following 
FUS-induced 
mild 
hyperthermia  §  
 
Quantification of intra-tumoural drug release 
will be achieved by direct analysis of the intra-
tumoural biopsy samples using standardised 
curve(s) 
Analytical chemistry performed 
on a recorded mass of biopsy 
sample processed within 12 
hours of intervention† 
Table 1: Primary Objective and Endpoints for the TARDOX Study 
 
 
 
Secondary Objectives 
Secondary Endpoints 
How and when data is captured 
Part I only: To determine 
optimal 
FUS 
exposure 
parameters for a range of 
participant Body Mass Indices 
(BMIs) and tumour locations 
within the liver 
Achievement of the desired range of mild 
hyperthermia 
in 
the 
target 
tissue 
as 
monitored by the implanted thermometry 
device during FUS exposure 
Real-time 
thermometry 
monitoring during intervention 
To assess the safety of FUS-
induced mild hyperthermia for 
drug delivery 
 
1. Persistence of cell viability stain or 
percentage of cell or tissue necrosis 
under 30% following FUS exposure, as 
assessed by cytological or histological 
1. Cytological or histological 
analysis of biopsy samples 
processed within 96hr of 
intervention† 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 25 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
methods16 ‡,§ 
 
2. Adverse events deemed related to FUS 
up to 30 days post-intervention 
 
2. Adverse event recording for 
30 days post-intervention 
To assess the local and systemic 
cytotoxic 
effects 
of 
ThermoDox® in this setting 
1. Significant bone marrow suppression, 
deranged liver function and liver toxicity 
 
2. Adverse events deemed related to 
ThermoDox® up to 30 days post-
intervention 
1. Grade 3 and 4 laboratory 
results from blood tests at 
Day 1 and Day 15 post 
intervention 
 
2. Adverse event recording for 
30 days post-intervention 
Table 2: Secondary Objectives and Endpoints for Parts I & II of the TARDOX Study 
 
 
Regarding participants for which doxorubicin has been released by optimal FUS exposure parameters: 
Tertiary 
(Exploratory) 
Objectives 
Tertiary (Exploratory) Endpoints 
How and when data is captured 
1. To establish the validity of 
other minimally-invasive and 
non-invasive 
methods 
of 
evaluating 
intratumoural 
uptake of doxorubicin as an 
alternative to that used in 
primary endpoint  
Combined analysis of relevant end points to 
establish most effective alternative analytical 
method(s) for quantifying drug release 
 
See below 
a. Fluorescence microscopy of 
biopsy  
Released biopsy samples exhibiting regions 
of nuclear or background fluorescence 
levels at least twice that of a suitable 
control, as assessed by fluorescence 
microscopy ‡,§ 
Microscopic analysis of biopsy 
samples processed within 96hr of 
intervention† 
b. MR-Spectroscopy 
 
Liver 
MRI/MR-Spectroscopy 
indicating17 
targeted delivery of doxorubicin to the 
tumour by demonstrating a statistically 
significant increase in the tumour:background 
ratio of signal intensity following intervention 
compared to baseline 
MRI/MR-Spectroscopy 
signal 
within 36 hours of intervention 
c. 
Plasma pharmacokinetics 
A statistically significant decrease in total 
doxorubicin plasma levels over time-matched 
clearance levels, following HIFU-induced 
release 
Analytical chemistry of plasma 
samples processed within 12hr of 
intervention† 
2. To determine whether the 
dose of doxorubicin released 
has a therapeutically significant 
effect  on the target tumour 
Radiological evidence of tumour response at 
timepoints over 30 up to 60 days in the FUS-
targeted tumour and control(s), as assessed 
by principles of CHOI and RECIST response 
evaluation using MRI and CT [11, 12] and 
SUVmax using PET-CT [13] § 
Scans to be performed within 60 
days of intervention. 
Table 3: Tertiary (Exploratory) Objectives and Endpoints for Parts I & II of the TARDOX Study 
 
                                                     
 
16 In some necrotic tumours, base line necrosis may be high (e.g. >30%) even prior to treatment. Consequently an increase in 
necrosis by no more than 25% over that of a suitable control can be used as an alternative endpoint. 
17 The spectroscopy signal analysed may not be doxorubicin itself; metabolites or breakdown products of ThermoDox® may be 
utilized. 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 26 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
4 
PATIENT SELECTION 
Written informed consent must be obtained before any study specific procedures are performed. 
4.1 
Eligibility criteria for entry into the main study 
The Investigator will determine patient eligibility based on the following inclusion/exclusion criteria.  
4.1.1 
Inclusion criteria:  
Patients will only be eligible for inclusion in this study if all of the following criteria apply: 
1. Pathologically confirmed advanced solid tumour with liver metastasis suitable for intervention (as assessed 
by ultrasound or other radiological methods). In addition confirmed primary liver tumours (hepatocellular 
carcinoma or cholangiocarcinoma) can be included. 
2. Will have progressed or remained stable on conventional chemotherapy. 
3. Male or Female, Age ≥ 18 years. 
4. Have life expectancy of ≥ 3 months. 
5. Left Ventricular Ejection Fraction (LVEF) ≥ 50% on echocardiogram.  
6. Have not received radiotherapy to the target area within the preceding 12 months. 
7. A World Health Organisation (WHO) performance status of ≤ 1 (Appendix 1). 
8. Able and willing to give written informed consent, indicating that they are aware of the investigational nature 
of this study and potential risks, and able to comply with the protocol for the duration of the study, including 
scheduled follow-up visits and examinations.  
4.1.2 
Exclusion criteria: 
Patients will be ineligible for recruitment to the study if any of the following conditions hold: 
1. Have surgery or other procedure requiring general anaesthesia planned to be undertaken during the period of 
the study. 
2. Have serious illnesses including, but not limited to, congestive heart failure (NYHA class III or IV functional 
classification); life threatening cardiac arrhythmia; or myocardial infarction or cerebral vascular accident within 
the last 6 months. 
3. Have on going significant infection (chest, urine, blood, intra-abdominal). 
4. Have uncontrolled diabetes. 
5. Have received a life-time dose of doxorubicin > 450 mg/m2 or a life-time dose of epirubicin > 900 mg/m2 or any 
dose of both. 
6. Pregnant or breast-feeding. In women of childbearing potential, a negative pregnancy test (serum) is required 
within 30 days prior to study intervention. 
7. Female participants of child bearing potential and male participants whose partner is of child bearing  potential 
who are not willing to practice an acceptable form of contraception (i.e. oral contraceptive, diaphragm, cervical 
cap, condom, surgical sterility) during the study and for 6 months thereafter. Women whose partner has or 
men who have undergone a vasectomy must use a second form of birth control. 
8. Have any known allergic reactions to any of the drugs or liposomal components or intravenous imaging agents 
to be used in this study. 
9. Have portal or hepatic vein tumour invasion/thrombosis. 
10. Inadequate Haematological and Biochemical indices:  
 
Lab Test 
Exclude if: 
INR 
> 1.5 times the institution’s upper normal limit (UNL)*  
Absolute neutrophil count  
<1,500/mm3 (or < 1.5 (10^9/L))   
Platelet count 
< 60,000 /mm3  (or <60 (10^9/L)) 
Hgb 
< 9.0 g/dL** 
Serum creatinine, or 
Calculated creatinine clearance (CrCl) 
≥ 2.5 mg/dL (or ≥ 221 µmol/L)  
≤ 25.0 mL/min. 
Serum bilirubin 
> 3.0 mg/dL (or >51 µmol/L ) 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 27 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
Serum albumin 
< 2.8 g/dL  (or <28 g/L) 
* Except in patients who are therapeutically anticoagulated for medical conditions unrelated to cancer such 
as atrial fibrillation. Subjects may be re-screened after the condition is treated or anticoagulant is withheld 
** Unless the haemoglobin value has been stable, the patient is cardiovascularly stable, asymptomatic, and 
judged to withstand the FUS procedure 
 
11. Have contraindications to receiving doxorubicin including prior sensitivity (rash, dyspnoea, wheezing, urticarial 
or other symptoms) attributed to anthracyclines or other liposomal drugs. 
12. Use of chemotherapy or of an investigational drug within 30 days or 5 half-lives, whichever is longer, preceding 
the intervention. 
13. Have medically significant active infection. 
14. Have Child-Pugh Class C liver disease, or Class A-B with encephalopathy and/or refractory ascites (Appendix 3). 
15. Documented HIV positive. 
16. Documented diagnosis of haemochromatosis. 
17. Documented history of contrast-induced nephropathy. 
18. Have any of the following contraindications for liver biopsy: 
a. Suspected liver haemangioma or other vascular tumour 
b. Tense ascites 
c. 
Known cystic liver disease* 
d. Extra-hepatic biliary obstruction* 
       (* Relative contraindications only and may be non-exclusive at discretion of the study team) 
19. Other medical or psychiatric conditions or laboratory abnormalities that the investigator considers would make 
the patient a poor trial candidate. 
4.2 
Protocol deviations and waivers to entry criteria 
Protocol adherence is a fundamental part of the conduct of a clinical study.  Changes to the approved protocol need 
prior approval unless for urgent safety reasons. 
 
Investigators must contact the TARDOX Trial Office to obtain guidance and/or clarification as necessary if unsure 
whether the patient satisfies all the entry criteria and to clarify matters of clinical discretion.  The trial office will 
contact the chief investigator or clinical coordinators as necessary. Requests for protocol waivers to enter a patient 
that is clearly ineligible will not be granted. 
 
Investigators should not deviate from the protocol for the management of enrolled subjects deliberately unless 
essential to protect the rights or safety of the individual. Examples might include the addition or deletion of tests, 
dosing, duration of treatment etc. It may be necessary to withdraw the patient from further study.  All deviations 
should be fully documented/ justified and reported to the trial office without delay. Deviations should be discussed 
with the trial office and/or the Chief Investigator prior to being actioned where possible. 
4.3 
Re-screening if patient does not meet inclusion/exclusion criteria first time round 
If a patient does not meet the inclusion/exclusion criteria first time round, he/she can be re-screened. Patients who 
fail at re-screening are ineligible for the TARDOX study and may not be re-screened again. 
4.4 
Patient registration procedure 
Participants will be recruited from patients referred to oncology services for the management of metastatic cancer 
from solid tumour primaries. A screening log must be kept of all patients considered for the study including any that 
are subsequently excluded; the reason for exclusion must be recorded on this form. A copy of the screening log should 
be sent to the trial office on request, but without patient identifiers. The original must be retained on site. Before 
entering a patient onto the study the Principal Investigator or designee will confirm eligibility. If in any doubt the Chief 
Investigator must be consulted before entering the patient.  
 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 28 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
A completed patient registration / eligibility checklist along must be sent to the TARDOX Trial Office. Each patient 
must be registered with the TARDOX Trial Office using this Registration Form and receive a unique trial number by the 
TARDOX Trial Team. 
 
Patients should be registered by the site by faxing the registration form to the TARDOX Trial Office using fax number 
01865 227039.  Alternatively the scanned Registration Form can be emailed to: octo-TARDOX@oncology.ox.ac.uk. The 
Fax/Email service is available 24 hours a day (Faxes/emails received outside office hours, 09:00 – 17:00 Monday to 
Friday, will be dealt with the next working day). Registration Forms will be provided in the Investigator Site File. 
Following registration, the site will be notified by fax or email that a patient has been successfully entered into the 
trial and of the patient’s unique trial number. 
The original Registration Form should be passed to the TARDOX Trial Office once complete. 
 
5 
TRIAL ASSESSMENTS AND PROCEDURES 
Please refer to the Summary Schedule of Events given at the front of this protocol. Details of all protocol evaluations 
and investigations must be recorded in the patient’s medical record for extraction onto the CRF. 
 
5.1 
Screening (d-30 to d-2) 
The following must be performed within days -30 to -2, where day -1 is the day of admission for intervention. The 
exception is the Ultrasound Session (section 5.1.3.1) which can take place as early as day -40 if separate consent is 
obtained. Tests performed as part of routine standard of care need only be repeated if outside the time window.   
 
Consenting patients will be enrolled into the study once all eligibility has been confirmed. In the patient’s interests, 
best efforts will be made to ensure that consent and screening procedures are performed in the least number of visits 
possible (sections 5.1.2 and 5.1.3).  
 
5.1.1 
Optional Two Stage Screening 
Informed consent by patients to screen for the trial may be sought in one process using the main Consent Form (CF) 
and the main PIS*. Or, optionally, informed consent can be separated into two stages; ultrasound screening followed 
by, if suitable, informed consent for the full study and remaining screening assessments.  
 
The two stage process allows the ultrasound screening session to be performed and the feasibility of intervention 
decided in advance of commencing other screening assessments. This can avoid the patient having to complete 
informed consent for the full study and potentially undergoing other screening tests before ultrasound screening.  A 
separate Ultrasound Screening CF and Patient Information Sheet (PIS) are available for this purpose. Potential 
participants who consent for ultrasound screening who are successfully screened will then be provided with the main 
PIS and, after informed consent, be offered to complete consent for the other screening assessments. If eligible the 
patient can then be enrolled on the study.   
 
*The main PIS and main CF encompasses the ultrasound session screening assessment, therefore it is not necessary to 
complete the separate ultrasound screening CF and PIS if the one stage process is planned and the patient has 
provided informed consent on the main CF. 
 
5.1.2 
Informed Consent 
Potential participants will be given a current, approved version of the PIS. They will also receive clear verbal 
information about the study detailing no less than: the nature of the study; the implications and constraints of the 
protocol; the known side effects and any risks involved in taking part.  It will be explained that they will be free to 
withdraw from the study at any time, for any reason, without prejudice to future care, and with no obligation to give a 
reason for withdrawal. They will have at least 24 hours to consider the information provided and the opportunity to 
question the Investigator, their GP or other independent parties before deciding whether to participate. The patient 
must personally sign and date the latest approved version of the relevant CF before any study procedures are 
performed, outside of standard care.  


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 29 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
 
The Investigator (or delegate) who obtains consent must be suitably qualified and experienced. All delegates working 
on behalf of the investigator must be authorised by the PI.  The Investigator is responsible for ensuring that the trial 
consent procedures comply with current applicable GCP Regulatory and ethical requirements. Informed consent 
discussions and outcomes must be well documented in the medical record. The Investigator (or delegate) must be 
satisfied that the patient has made an informed decision before taking consent.  The patient and the Investigator (or 
delegate) must personally sign and date the current approved version of the informed consent form in each other’s 
presence. A copy of the information and signed consent form will be given to the participant. The original signed form 
will be retained in the medical record and a copy is to be filed in the Investigator Site File.  
 
Contraceptive/ Pregnancy counselling 
Doxorubicin, the active chemotherapy agent of the study drug (ThermoDox®) can cause foetal harm when 
administered to a pregnant woman. Animal studies have demonstrated teratogenic and embryotoxic effects at low 
doses. There are no adequate and well-controlled studies in pregnant women. 
 
All participants must be advised on the need to use reliable methods of contraception during the study and for six 
months after intervention. The advice should include:  
(1) The acceptable methods, including: male or female sterilization, implants, injectables, combined oral 
contraceptives, some intrauterine devices (IUDs), and abstinence.  
(2) The recommendation that a barrier method should be used in addition to another form of contraception. 
(3) Males should continue to take these precautions for a minimum six months after the intervention.  
(4) Females should continue to take these precautions a minimum of six months after the intervention. 
(5) That any pregnancy (also applies to females partners of male trial subjects) occurring within six months of the 
trial intervention will be followed up and the outcome of all pregnancies (spontaneous miscarriage, elective 
termination, normal birth or congenital abnormality) will be reported and followed up even if participant is 
discontinued from the study.  
 
Breast-feeding counselling 
There is the potential for serious adverse effects in breast-feeding infants from doxorubicin administered to nursing 
mothers and consequentially, breast-feeding women are excluded from this study (see exclusion criteria). 
5.1.3 
Screening Assessments 
Inclusion / Exclusion Criteria 
Sections 4.1.1 & 4.1.2. 
 
Medical History 
Medical History including prior diagnosis, prior oncological disease, prior treatment (including chemotherapy & 
surgery) and concomitant diseases will be recorded. 
 
Demographics 
The date of birth and gender will be recorded.  
 
The WHO performance status (Appendix 1). 
 
Concomitant Medication  
These will be recorded in CRFs as per section 9.4. 
 
Physical Examination  
An abdominal exam and cardiorespiratory exam will be performed. 
 
Vital Signs 
Temperature will be recorded. Resting pulse and blood pressure (BP) measurements will be taken after the participant 
has been in a sitting position for at least five minutes.  
 
Height & Weight  
Body Surface Area (BSA) and Body Mass Index (BMI) will be calculated 
 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 30 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
Haematology & Biochemistry Tests 
• 
Full Blood Count (FBC); including Haemoglobin (Hb), white blood cells (WBC) with differential count 
(neutrophils and lymphocytes), and platelets 
• 
Biochemical profile; including sodium, potassium, calcium, phosphate, urea, creatinine 
• 
Liver profile; including total protein, albumin, bilirubin, ALP, AST and/or ALT and LDH 
• 
Group & Save (as standard for surgical candidates, date of valid test to be recorded on CRF, not blood group) 
• 
Clotting Studies; including PT and APPT 
 
Urinary Tests +/- Pregnancy Test 
Tests to be performed will be: 
• 
Urinalysis by dipstick.  
• 
In women of childbearing age, a serum or urine Human Chorionic Gonadotropin (HCG) pregnancy test 
 
ECG Test 
A 12-lead ECG will be taken for each participant. The eligibility of participants with abnormal AND clinically significant 
results must be confirmed. 
 
Echocardiogram 
An echocardiogram must be performed at baseline to assess LVEF. The test will only be repeated if clinically indicated. 
 
Pre-operative Anaesthetic Assessment (ASA Classification) 
A pre-operative assessment will be performed by a suitably trained doctor or nurse, usually in consultant anaesthetist-
lead clinic. During this time the previous investigations (as per standard departmental practice) will be reviewed and a 
decision made if the participant can proceed with a General Anaesthetic.  The date and outcome of the assessment 
(ASA classification) will be recorded. 
 
5.1.3.1 
Ultrasound Screening Assessment  
A focused diagnostic ultrasound scan will be performed in order to choose suitable tumour area(s), which are easily 
identified on ultrasound and amenable for biopsy. For participants in both Parts I and II, a single liver tumour 
amenable to intervention will be targeted. The tumour selected for intervention will be treated with a combination of 
ThermoDox® and FUS.  Refer to section 5.4.3 for more information on the FUS intervention. 
 
As assessed at the Ultrasound session, in order to proceed with the study, the participant must: 
1. Have one or more incurable (unresectable, non-ablatable-for cure) liver tumours of size 1cm or greater, 
which is amenable to ultrasound-guided biopsy 
2. Have the total volume to be treated no larger than either removal of 3 hepatic segments or removal of more 
than 30% of total liver volume (as per maximum surgical limit) 
3. Be an appropriate candidate for FUS with consideration given to the following factors: 
a. Overall health of liver 
b. Absence of significant cirrhosis 
c. 
Absence of significant ascites 
d. Ultrasound path free of previous radiotherapy or scar tissue 
e. Ultrasound path free of overlying skin ulceration 
 
In some circumstances, if a participant is found to be ineligible for Part I of the study due to anatomical tumour 
location, they may still be eligible for Part II. This is because real-time temperature monitoring is not required in Part II 
and thus the anatomical position of a given tumour area may be more amenable to delivery of the intervention. 
 
5.2 
Evaluations during the intervention (d-1 to d2) 
Evaluations on day -1 (pre-Intervention day) 
• 
Dose of oral dexamethasone given as pre-medication (section 5.5.2 & 9.3) 
• 
Baseline scans (Usually on d-1 but may be performed from day -7  to day 1 provided before intervention): 
o 
Liver MRI/MR -Spectroscopy   
o 
Liver pCT 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 31 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
o 
Whole body FDG PET-CT  
 
Evaluations on day 1 (intervention day) 
• 
Confirm d-1 pre-medications were given. 
• 
Day 1 pre-medications given 
• 
Confirmatory portable ultrasound to confirm tumour location before patient anaesthetised  
• 
General anaesthetic start and finish time 
• 
Dose of ThermoDox® administered, start and finish time 
• 
Start and finish time of final FUS exposure and FUS parameters 
• 
Thermometry data (Part I only) 
• 
Bloods samples for pharmacokinetics (Pre-Treatment, Unreleased and Released plasma samples) 
• 
Biopsy samples for assay quantification ± microscopy: 
o 
Part I: Pre-Treatment, Unreleased, Released 
o 
Part II: Released 
• 
Adverse event monitoring 
 
Evaluations on day 2 (recovery day) 
• 
Physical exam 
• 
Vital signs: Temperature, resting pulse and blood pressure (BP) after participant has been in a sitting position 
for at least five minutes 
• 
Full Blood Count (FBC); including Haemoglobin (Hb), white blood cells (WBC) with differential count 
(neutrophils and lymphocytes), and platelets 
• 
Biochemical profile; including sodium, potassium, calcium, phosphate, urea, creatinine 
• 
Liver profile; including total protein, albumin, bilirubin, ALP, AST and/or ALT and LDH 
• 
Post-treatment Liver MRI/MR-Spectroscopy (within 36 hours, and ideally within 24 hours, of intervention18) 
• 
Adverse event monitoring 
5.3 
Follow-up and end of study evaluations (within 60 days) 
 
Follow-up 1: Day 15 (±3 day tolerance) 
Parts I & II: 
• 
Full Blood Count (FBC); including Haemoglobin (Hb), white blood cells (WBC) with differential count 
(neutrophils and lymphocytes), and platelets 
• 
Biochemical profile; including sodium, potassium, calcium, phosphate, urea, creatinine 
• 
Liver profile; including total protein, albumin, bilirubin, ALP, AST and/or ALT and LDH 
• 
Adverse event monitoring 
 
Follow-up 2: Day 30 (+7 not <30) 
Parts I & II: 
• 
Adverse event monitoring 
• 
Concomitant medication recording 
• 
Full Blood Count (FBC); including Haemoglobin (Hb), white blood cells (WBC) with differential count 
(neutrophils and lymphocytes), and platelets 
• 
Biochemical profile; including sodium, potassium, calcium, phosphate, urea, creatinine 
• 
Liver profile; including total protein, albumin, bilirubin, ALP, AST and/or ALT and LDH 
 
Follow-up Scans: From Day 12 - Day 60 post-intervention as defined in the summary of schedule events (one set of 
scans at 4 weeks follow-up, the other being flexibly timed in accordance with emerging radiological data from 
previous study participants) 
Parts I & II: 
• 
Maximum of two follow-up Liver MRI/MR-Spectroscopy scans 
• 
Maximum of two follow-up Liver pCT scans 
• 
Maximum of two follow-up whole body FDG PET-CT scans 
                                                     
 
18 The time of intervention is defined as the time at which the final FUS exposure is complete 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 32 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
If a participant is withdrawn from the study following the intervention, adverse event recording should continue for 
30 days post-intervention. No other follow-up evaluations will be required. 
5.4 
Trial Procedures 
5.4.1 
Preparation for Intervention 
Part I and Part II 
 
Day -1 (pre-Intervention): 
• 
The participant attends the day before intervention and is given the first dose of oral premedication 
within 24 hours of the procedure. If required, the patient may be admitted for the pre-medication and 
stay in hospital until completion of evaluations on Day 2 (see section 5.2 & 5.3 for evaluations). 
 
Day -1 (pre-Intervention) or Day 1 (Intervention): 
• 
The liver tumour identified for intervention in the Ultrasound Session is correlated with both MRI and CT 
images and may be re-examined by ultrasound in advance of anaethetising for the intervention. 
 
5.4.2 
Intervention 
During the intervention the following people are usually present: the Clinical Investigator and/or Co-investigators, 
anaesthetist, anaesthetic Operating Department Practitioner  (ODP), specialist oncology nurse, trained HIFU specialist, 
Radiologist for biopsy, Radiology nurse and, in Part I, research engineer for real-time data acquisition. 
 
The participant is intubated and anaesthetised. Antibiotic prophylaxis is given on induction. The participant is 
positioned over the HIFU machine optimally to allow access via the HIFU transducer across the water-bath and access 
to insert the coaxial needle for taking biopsy or monitoring temperature.  At the time of coaxial needle insertion the 
skin over the puncture area is shaved if necessary, prepared appropriately and a sterile drape is applied. 
 
A detailed Ultrasound Session will have taken place previously (section 5.1.3.1). Further ultrasound planning occurs 
immediately prior to intervention. This includes three-dimensional tumour evaluation using the diagnostic ultrasound 
component of the device that will be used to administer FUS exposure. The non-mobile HIFU therapeutic device is 
located in a purpose-built unit on the Churchill Hospital site and consequently all intervention will take place in this 
unit. 
 
The HIFU therapeutic device is used to generate hyperthermia in the contoured region of the target tumour using 
optimised levels of FUS. More information on the FUS procedure can be found in section 5.4.3. 
 
The participants’ liver tumours are treated with a combination of FUS and/or ThermoDox® as determined in the 
Ultrasound Session (section 5.1.3.1). Intervention is performed under General Anaesthetic and the participant is kept 
in overnight for monitoring following the procedure. 
 
Part I 
For each participant in Part I, the optimal FUS exposure parameters to achieve the desired range of mild hyperthermia 
(and consequent ThermoDox® release) will be determined.  The power (watts), duty cycle (or continuous) and 
duration of exposure will be varied to optimise the temperature response at the tumour site and with a view to 
achieving targeted doxorubicin delivery.  
 
Day 1 (intervention): 
• 
A co-axial biopsy needle is positioned through the sterile field into the centre of tumour targeted for 
intervention, under ultrasound guidance.  
• 
A Pre-Treatment Biopsy of the tumour is taken via the co-axial needle, and used as a baseline prior to 
any intervention, along with the Pre-Treatment blood sample.  
• 
A clinically approved thermometry device is then positioned through the co-axial needle to the target 
area and is used to obtain real-time thermometry measurements during the FUS exposure. 
• 
The HIFU therapeutic device is aligned to the thermometry device using short test exposures of FUS.  


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 33 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
• 
The tumour area is then exposed to varied and considered FUS parameters (section 5.4.3) until the 
thermometry confirms that the target range of mild hyperthermia has been achieved within the 
tumour(*).  
• 
In addition to the FUS exposure parameters (power (watts), duty cycle, duration of treatment) and 
associated thermometry data, the location of tumour (liver segment) and patient BMI will be recorded 
for each intervention. 
• 
The thermometry device is then replaced and a cool-down period allows the tumour to return to body 
temperature.  
• 
Once the tumour has returned to normothermia, the prescribed dose of ThermoDox® is infused over a 
duration of 30 minutes.  
• 
A second Unreleased Drug Biopsy of the target area is then taken via the coaxial needle (Note: 
ThermoDox® is circulating at this point, but not yet activated). The corresponding Unreleased Drug blood 
sample is also taken at this stage for pharmacokinetic analysis within 15 minutes of infusion end. 
• 
The tumour area is then re-exposed to FUS using the exposure parameters identified earlier as being 
suitable to generate mild hyperthermia within the tumour. During this second FUS exposure, 
ThermoDox® is circulating between therapeutic and peak levels. 
• 
After completion of the final FUS exposure, a final Released Drug biopsy is taken via the co-axial needle 
with the corresponding Released Drug blood sample.  
• 
Before removing the coaxial needle, a Gelfoam® (or equivalent) plug embolization of the needle track is 
applied through the needle to minimise bleeding risk [47].  
 
*Note that with increasing experience, the study team may elect to omit the step of pre-heating the tumour prior 
to drug delivery (1st FUS exposure) and drug infusion may be commenced shortly after alignment. 
 
The steps of intervention of the liver tumour in Part I are summarised in Figure 4. 
 
The paired Pre-treatment, Unreleased and Released Drug samples (bloods and biopsies) are sent for analysis in an 
appropriate laboratory. Please refer to section 7 for more information on sample analysis. 
 
Figure 4: Timeline for combination exposure of the target liver tumour in Part I (not to scale, total plasma doxorubicin PK 
curve is for illustrative purposes to demonstrate anticipated PK trend and is not based on actual PK data) 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 34 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
 
Part II 
The optimised FUS acoustic parameters for Part I will be used in Part II.  The release of doxorubicin and its therapeutic 
effect will be investigated. The PI or delegate will have already identified a suitable solitary target liver tumour for 
intervention at the Ultrasound Session (section 5.1.3.1).  
 
Day 1 (Intervention): 
• 
Preparation as for Part I with the exception that no real-time thermometry data acquisition or pre-
intervention biopsies are required in Part II, thus the co-axial needle for biopsy can be inserted after the 
FUS exposure. Three peripheral blood samples for plasma analysis are taken at the corresponding stages 
of the intervention (Pre-treatment, Unreleased and Released) as for Part I. The time the blood and 
biopsy samples are taken must be noted. 
• 
ThermoDox® is infused as per prescribed dose for a duration of 30 minutes.  
• 
After the infusion, the lesion identified for intervention (ThermoDox® release by mild hyperthermia) is 
exposed to the planned FUS exposure dose, as optimised in Part I, taking into consideration the patient 
BMI and tumour location (section 5.4.3).  
• 
The non-FUS control tumour, if available, is exposed to ThermoDox® alone without FUS.  
• 
As for Part I, the FUS exposure parameters, location of tumour (liver segment) and patient BMI will be 
recorded for each evaluation. 
• 
The co-axial needle is inserted and up to two ultrasound-guided Released Drug Biopsies of the tumour 
receiving intervention are taken, whilst the participant is still under General Anaesthetic. A biopsy is not 
taken of the control tumour exposed to ThermoDox® only. A corresponding Released Drug blood sample 
is also taken.  
• 
Before removing the coaxial needle, a Gelfoam® (or equivalent) plug embolization of the needle track is 
applied through the needle to minimise bleeding risk [47].  
 
The steps of intervention for the liver tumour in Part II are summarised in Figure 5. 
 
Figure 5: Timeline for combination exposure of the target liver tumour in Part II (not to scale, total plasma 
doxorubicin PK curve is for illustrative purposes to demonstrate anticipated PK trend and is not based on actual PK 
data) 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 35 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
 
5.4.3 
Focused Ultrasound Procedure 
The focused ultrasound exposure will be performed using the Haifu® Model-JC200 Focused Ultrasound Tumour 
Therapeutic System at the Churchill Hospital site. 
 
The  acoustic window identified in the Ultrasound Session (section 5.1.3.1) is recreated with the participant in position 
and under anaesthetic. Slice by slice therapy then follows: the tumour volume consists of several two-dimensional 
cross-sectional ‘slices’, and the shape and size of the tumour within each slice will naturally vary. In this way, the 
radiologically accessible tumour volume is divided into sequential slices, at 2-10 mm intervals depending on the 
therapeutic plan as shown in Figure 6. In therapy mode, the tumour volume is treated with focused ultrasound to 
attempt to attain regional hyperthermia in the tumour for duration of at least 30 minutes and ideally 60-90 minutes.  
 
Figure 6: Schematic representation of the row-by-row, slice-by-slice approach used to induce mild hyperthermia into the 
target tumour volume using a stop/start single-shot mode treatment plan. Moving beam (linear mode) is also under 
investigation for hyperthermia using the same approach. 
 
5.4.4 
Passive Acoustic Monitoring 
FUS exposure will be monitored using B-mode diagnostic Ultrasound that is incorporated into the HIFU therapeutic 
device. During exposure, a prototype Passive Cavitation Detector will optionally be used to simultaneously passively 
monitor ultrasonic emissions from the targeted tumour area. This prototype has previously been demonstrated during 
a previous study, to not interfere with FUS exposures using the JC200.  
5.4.5 
Participant Monitoring 
All participants will be monitored continuously while under General Anaesthetic and throughout the intervention. The 
anaesthetic team (anaesthetist and ODP) will perform this monitoring according to standard protocols for HIFU.  
 
6 
EARLY PATIENT WITHDRAWAL 
Withdrawal 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 36 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
During the course of the trial, a patient may withdraw early from completing the trial.  This may happen for a number 
of reasons, including: 
• 
Unacceptable toxicity 
• 
AE/SAEs requiring discontinuation 
• 
Clinical decision 
• 
Patient decision 
Possible reasons for a clinical decision to remove a participant from the study include, but are not limited to: 
• 
New exclusion criteria including new pregnancy 
• 
Significant protocol deviation or non-compliance with the protocol 
 
Patient withdrawals must be documented in the eCRF. SAEs must be followed until resolution.  The reason for 
withdrawing from treatment early should be clearly documented in the medical records. 
 
Consent Withdrawal 
A patient may withdraw consent from study participation (Consent withdrawal) at any time for any reason.  Every 
effort should be made to ascertain the reason for withdrawal and this should be documented in the medical records. 
The trial office must be informed via completion of the Consent Withdrawal eCRF. Under these conditions, 
investigators are still responsible to follow up any SAEs till resolution. 
 
For data collection purposes, subjects are considered as completing the study if they have died during the study, are 
lost to follow-up, or withdraw consent. 
6.1 
Patient evaluability and replacement 
Patients who are not evaluable for the primary endpoint will be replaced.  
7 
SAMPLES FOR LABORATORY ANALYSIS 
7.1 
Samples to be analysed in local Trust’s laboratories 
7.1.1 
Diagnostic Laboratories 
Samples for haematology, biochemistry and coagulation analysis will be labelled with unique study patient identifiers 
and sent to the local hospital diagnostic laboratory. Results will be processed in the standard way and entered into the 
routine hospital reporting system. Samples will be stored, held, reported and subsequently destroyed in accordance 
with standard local laboratory practice.  
7.1.2 
Pathology 
No pathology samples are required for pathological analysis in the Trust during any part of the study period, although 
in the evaluation of histological sections of biopsy tissue, the opinon of an NHS pathologist may be sought. 
 
7.2 
Samples to be sent to and analysed in a Central Laboratory 
The following samples will be analysed at the Oxford Cancer Research Centre: 
• 
Pharmacokinetic blood samples to measure Unreleased and Released plasma levels of doxorubicin, using Pre-
Treatment Plasma as the control 
• 
Biopsy samples to quantify intra-tumoural uptake of doxorubicin (three in Part I, up to two in Part II) 
 
Details of sample preparation, labelling and despatch are provided in the TARDOX Sample Handling Manual. 
7.3 
Pharmacokinetic assays  
Plasma samples will be used to quantify the levels of doxorubicin as per the time points in section 5.5.3. The infusion 
start & stop times, duration of FUS exposure and sample collection times during intervention will be recorded. 
 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 37 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
7.4 
Pharmacodynamic assays 
Not applicable to this study. 
7.5 
Summary of samples/assays to be taken during the study 
Assay/sample handling and storage will be managed according to separate written instructions. The following 
summarises the arrangements for collection, close to patient handling, timings, and analytical laboratories 
responsible.  Research assays will be performed according to separate laboratory SOPs. 
 
7.6 
Samples for Biobanking 
Participants in this trial will be invited to permit the long term retention of samples left over after protocol-specified 
analyses for use in possible other future research linked to trial data. Consent to participate in this aspect of the trial is 
optional and not a requirement of participation in the main study.  
7.7 
Labelling and confidentiality of samples sent 
All samples sent to analytical Laboratories will be labelled with the trial code, trial patient number, and the date & 
time taken.  Should a laboratory receive any samples carrying patient identifiable information the recipient must 
immediately obliterate this information and re-label. The study site will be informed of their error. 
7.8 
Clinical reporting of exploratory research assay results 
The results of the TARDOX trial research assays are exploratory and are not intended to influence the individual 
participant’s medical care. Findings will not be reported routinely to the responsible clinician except in the unlikely 
event that the result might be beneficial to the participant’s clinical management. 
7.9 
Trial sample retention at end of study 
The Chief Investigator has overall responsibility for custodianship of the trial samples. Laboratories are instructed to 
retain any surplus samples pending instruction from the Chief Investigator on use, storage or destruction.  It is 
possible that new or alternative assays may be of future scientific interest. At the end of the research study any 
surplus samples may be retained for use in other projects that have received ethical approval.  Hence, any surplus 
study samples may be transferred to a licensed tissue bank where they will be managed in accordance with applicable 
host institution policies and the Human Tissue Act requirements.  
7.10 
Withdrawal of consent for sample collection and/or retention 
A patient may withdraw consent to provide samples for research at any time without giving a reason. The Investigator 
must ensure that their wishes are recorded in the medical record and will inform the trials office accordingly. The 
investigator should discuss with patients the valuable use of samples that have already been provided and under 
circumstances where these samples have already been processed and anonymised, it would not be possible to 
destroy such samples. 
8 
INVESTIGATIONAL MEDICINAL PRODUCTS (IMPS) 
All participants will receive the IMP (ThermoDox® drug) for a liver tumour targeted with mild hyperthermia. Following 
the required pre-medication of steroids and anti-histamines (Table 5), participants will receive a single therapeutic 
dose of ThermoDox®. 
 
For information on the packaging, supply, handling, storage and accountability of ThermoDox® please refer to section 
11. 
8.1 
Name of IMPs 
ThermoDox®, manufactured under authority of Celsion, USA. 
8.2 
Treatment dose 
50mg/m2 iv infusion of ThermoDox® in 250ml 5% dextrose over 30 minutes immediately before final FUS exposure. 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 38 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
8.3 
Duration of treatment 
One day only; participants should receive a single iv infusion of ThermoDox® at a dose of 50mg/m2 on the day of the 
intervention in accordance with specific timing as dictated by the team administering the intervention. No further 
doses are required or should be given as part of this study in either Part I or II. A participant having a dose of 
ThermoDox® in Part I is not eligible for re-entry into Part II for any further dose. 
8.4 
Management of drug administration 
In order to maximise peak ThermoDox® concentration, FUS exposure of the target tumour area will be initiated at 
peak therapeutic levels, i.e. soon after the infusion has completed. All FUS exposures started after commencing the 
ThermoDox® infusion should be completed within 4 hours of the infusion start time. Please refer to section 11.6 for 
information regarding drug preparation and dosing dispensing. 
Steroids are given as a pre-medication (section 9.3) to avoid immediate-type hypersensitivity reactions to liposomes 
contained within the ThermoDox® product. All participants must be adequately pre-medicated before commencing 
the ThermoDox® infusion. In the case of hypersensitivity reaction, the participant would be treated according to local 
hospital policy. In the case of an immediate hypersensitivity reaction manifesting as a localised skin reaction only, the 
infusion will be immediately stopped until the reaction subsides. Depending on severity and clinical judgement, in 
some cases the infusion may be restarted. 
 
Extravasation Injury 
Administration of ThermoDox® should be closely monitored for local site reactions and at the first sign of 
extravasation, the infusion must be immediately stopped. Local protocols should be used to treat the affected area.  
8.5 
Special precautions 
Special precautions for doxorubicin, the active agent in ThermoDox®, are detailed in Appendix 1 of the ThermoDox® IB 
[44], The Doxorubicin Package Insert. 
8.6 
Actual versus ideal body weight 
The dose of ThermoDox® will be calculated for each patient based on actual body weight. 
8.7 
Dose modification  
Dose modification is not applicable to the TARDOX study, which has one single dose exposure. 
8.8 
Calculating and recalculating BSA/doses 
The patient’s height and weight at baseline will be used to estimate the body surface area (BSA) using a standard  
formula such as DuBois or Boyd’s (Table 4), which will determine the dose of ThermoDox® to be given during the 
intervention. Weight will be rechecked the day before intervention (d-1). If a patient’s weight increases or decreases 
by >=10% before intervention, the dose of ThermoDox® should be recalculated. 
 
Height ________________ (cm) 
 
 
Weight ________________ (grams) 
 
 
Dose Assignment (50mg/m2)                             Dose Adjustment __________ mg/m2 
 
 
Equation 1: LOG Weight (grams) 
 
 
Equation 2: (0.0188 x LOG Weight (grams)) 
 
 
Equation 3: (0.7285 - (0.0188 x LOG Weight (grams)) 
 
 
Equation 4: [Weight (grams)](0.7285 - (0.0188 x LOG Weight(grams)) 
 
 
Equation 5: 0.0003207 x Height (cm)0.3 
 
 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 39 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
BSA (m2) = 0.0003207 x Height (cm)0.3 x Weight (grams)(0.7285 - (0.0188 x LOG Weight (grams)) 
 
 
Table 4: Stepwise equations for calculation of Boyd’s formula for calculation of body surface area (BSA) 
8.9 
Dose capping 
No doses are to be capped. 
8.10 
Dose-banding 
No doses are to be dose-banded. 
8.11 
Compliance 
The IMP will be given in a single dose during the study and thus compliance is not applicable. 
8.12 
Management of overdose 
Acute overdose of ThermoDox® and thus doxorubicin may lead to acute cardiac alterations within twenty-four hours 
and myelosuppression usually 10-15 days following overdose. Urgent medical attention must be sought for which 
treatment will vary depending on the circumstances, but may involve reverse barrier nursing, intravenous antibiotics, 
transfusion of blood products, symptomatic treatment of mucositis and cardiology input. More information can be 
found in Appendix 1 of the ThermoDox® IB [44]. 
 
9 
OTHER TREATMENTS (NON-IMPS) 
9.1 
Focused Ultrasound 
Focused ultrasound (FUS) by a clinically approved HIFU therapeutic device will be used in this study to deliver acoustic 
energy capable of inducing mild hyperthermia in the target region. 
9.2 
Background systemic therapy  
Not applicable to the TARDOX study. 
9.3 
Support medication 
Table 5 below details the pre-medications required for ThermoDox®, which start on day minus one, i.e. the day before 
intervention & the day of admission 
 
Timing and Steps 
Premedication Regime 
24 hours prior to study drug treatment (day -1) 
Subjects take oral premedication; dexamethasone  (8-10mg 
each) every 12 hours x 3 doses; the third dose should be 
taken the morning of treatment.  
Study Intervention day (day 1): 
Step 1  
Confirm 
that 
the 
participant 
has 
taken 
the 
oral 
premedication the previous day.  
Study Intervention day (day 1):  
Step 2  
Thirty to 60 minutes prior to study drug infusion, participant 
is administered premedication by IV infusion: 
a) dexamethasone 20 mg IV 
b) chlorpheniramine 10 mg IV 
c) ranitidine 50 mg IV   
Table 5: Premedication and Study Treatment Infusion Instructions for ThermoDox® 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 40 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
9.4 
Concomitant medication and non-drug therapies 
Concomitant medication may be given as medically indicated.  All patients will be asked to provide a complete list of 
prescription and over-the-counter medications, vitamins, and/or herbal supplements that have been taken within the 
previous 4 weeks prior to screening.  They must also inform the Investigator about any new medication started while 
in the trial. Details (including indication, doses, frequency and start / stop dates) of concomitant medication taken 
during the trial until the completion of the off-study visit must be recorded in the medical record and the appropriate 
CRF. 
 
Investigators should be aware of potential drug-drug interactions particularly inhibitors of the Cytochrome P450 
(CYP3A4) drug metabolizing enzyme system. Thus when co-administering ThermoDox® with drugs classified as 
‘substrates’, there is a potential for ThermoDox® to produce higher plasma concentrations than normal of the 
‘substrate’ drug. Conversely, when co-administering ThermoDox® with drugs classified as ‘inhibitors’, there is a 
potential for the ‘inhibitor’ to produce higher plasma concentrations of ThermoDox® than normal. In general, any 
drug’s toxicity correlates with its plasma concentration, particularly for IV administered cytotoxic drugs. Please refer 
to sections 9.5 and 9.6 for a list of prohibited and interacting medications. 
9.5 
Prohibited therapies 
Patients should not be prescribed any other anti-cancer or investigational therapies while participating in this study. 
9.6 
(Potential) Drug Interactions 
Investigators should also be aware of potential drug-drug interactions. 
 
Doxorubicin is extensively metabolized by the liver. Changes in hepatic function induced by concomitant therapies 
may affect doxorubicin metabolism, pharmacokinetics, therapeutic efficacy, and/or toxicity.  
 
10 RADIOTHERAPY 
In the ThermoDox® study FUS will be used to induce mild hyperthermia in the target region (section 5.4.3). There will 
be no radiotherapy per se and no ablative radiotherapy will be performed. 
11 DRUG MANAGEMENT 
11.1 
Drug supplies 
ThermoDox® will be provided as commercial stock by Celsion free of charge. It will be shipped from the USA to the UK 
in a frozen form. It will be manufactured according to the specifications of an existing IMPD (Investigational Medicinal 
Product Dossier) under strict GMP conditions. A Qualified Person (QP) will release the IMP before sending to the study 
site. ThermoDox® will be supplied in 20cc vials each containing 30mg doxorubicin in 15ml of solution for intravenous 
infusion. 30mg vials will come in a carton containing 4 vials. Giving sets, infusion bags, other equipment and all 
supportive medication is to be sourced and funded locally. 
 
It will be the responsibility of the investigator for ensuring that the study drug is stored at the site pharmacy in 
accordance with the instructions on the labels. At study initiation, and as needed thereafter, ThermoDox® will be 
shipped to a responsible person (e.g. pharmacist) at the Investigator’s institution, who will check the amount and the 
condition of the drug and enter these data onto the Drug Accountability Log. Initial supply and re-supply of 
ThermoDox® will be managed by the trial office. The ThermoDox® accountability logs will be kept and will contain 
dates and quantities received from Celsion, lot numbers (batch numbers), date and quantity of drug dispensed, dose 
preparation records, etc. ThermoDox® expiry dates will be routinely checked. 
 
Drugs will be prescribed, tracked, and dispensed according to the local policy for investigator-initiated studies. Only 
qualified personnel who are familiar with procedures that minimise undue exposure to themselves and to the 
environment should undertake the preparation, handling and safe disposal of the trial medications. 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 41 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
11.2 
Drug ordering 
Initial supplies of ThermoDox® are sent out by Celsion after they have been informed by the trial office that all 
approvals are in place. If required, re-supply of ThermoDox® will be managed by the trial office who will monitor 
stocks and reorder where required. If a vial of ThermoDox® is accidentally destroyed, e.g. by dropping the vial, 
accidentally thawing, or through contamination, the pharmacist should contact the trials office for replacement 
patient supplies. 
11.3 
IMP Receipt 
Acknowledgement of receipt of ThermoDox® supplies will be logged. 
Damaged supplies should be destroyed on site and a drug destruction form completed. 
11.4 
Handling and storage 
All investigational study medication will be stored frozen at  below -60°C in a secure area with access limited to the 
Investigator and authorized site staff. All investigational study medication will be administered according to the 
procedures described herein and in the pharmacy manual. Only participants enrolled in the study may receive the 
investigational study medication (ThermoDox®), in accordance with all applicable regulatory requirements.  
 
Prior to administration, an appropriate number of frozen ThermoDox® vials must be carefully thawed at room 
temperature for a minimum of 1 hour. ThermoDox® vials MUST NOT be heated to speed up the thawing process. After 
removing from the freezer and prior to dilution, the vials are stable for no more than 4 hours at room temperature 
(15°C to 30°C). 
 
The required amount of ThermoDox® is then drawn into a 50 or 60 ml syringe and is diluted to 250 cc 5% dextrose 
(D5W). Once reconstituted, it is stable at room temperature for no more than 8 hours. The total volume is infused 
over a 30 minute period through a free flowing IV line. ThermoDox® should be infused at room temperature. 
ThermoDox® solutions must not be heated above room temperature during administration. 
 
For detailed storage and preparation instructions, refer to the pharmacy manual that is provided to the pharmacist. 
11.5 
Labelling  
The responsible Pharmacy will ensure that IMP supplies dispensed for trial use are appropriately labelled in 
accordance with all applicable regulatory requirements.  
As provided from Celsion, the manufacturer, each carton label will include a minimum of the following specific 
identifiers that will uniquely identify the product to the production batch used for this study: 
 
• 
Name of Product (ThermoDox®) 
• 
Name of the study Sponsor 
• 
Sponsor Protocol Number 
• 
EudraCT Number 
• 
Lot number 
• 
Manufacture Date 
• 
Retest Date 
• 
Chief Investigator 
 
Each vial label will include a minimum of the following specific identifiers that will uniquely identify the product to the 
production batch used for this study: 
 
• 
Name of Product (ThermoDox®) 
• 
Name of the study Sponsor 
• 
Sponsor Protocol Number 
 
The ThermoDox® product is provided in a frozen preparation containing 30mg of doxorubicin in 15ml of solution, 
within a 20cc amber vial.  A typical patient will require the use of 4 vials or less, for a 50 mg/m2 dose. The product is 
provided in four vial cartons, so typically one carton per patient is required. 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 42 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
11.6 
Dosing dispensing 
ThermoDox® requires dilution in a 5% dextrose solution in water (D5W) before administration.  
11.7 
Drug accountability 
Celsion will supply the study medication to Churchill Hospital pharmacy site via their approved manufacturer and 
usage will be logged. Any unused medication will be destroyed at the end of the study or returned to the 
manufacturer. All movements of study medication between Celsion and pharmacy will be documented. 
 
Full drug accountability logs must be maintained for ThermoDox® using the Logs provided. Hospitals may amend the 
Drug accountability Logs provided or use their own documentation if prior approved with the trial office. 
 
The drug dispensing and inventory logs should be kept up to date and have to contain the following information; 
patient identifier, date and quantity received at site, date and quantity dispensed, date and quantity 
returned/destroyed at site. The inventory must be available for inspection by the monitor at every monitoring visit. 
 
At the conclusion of the study the overall numbers of drug shipped to the centre, the number dispensed and the 
number destroyed will be provided by the pharmacy. An account must be given of any discrepancy. 
11.8 
Drug returns from patients 
Not applicable. 
11.9 
Drug destruction 
ThermoDox® is the IMP for the TARDOX study. Once it has been defrosted for use it must never be refrozen for reuse. 
Therefore any thawed ThermoDox® must be disposed of according to local hospital policy. Please refer to Table 6 
below, which details when drug should be disposed of. 
 
Used / partially used vials 
Disposal at site according to local hospital policy. 
 
 
Drug left unused / expired drug 
Disposal at site according to local hospital policy. A dated 
certificate of disposal should be completed and retained in the 
Pharmacy File. 
Drug left unused 
At the end of the study, once authorised to do so, any unused 
drug should be disposal of at site according to local hospital 
policy. A dated certificate of disposal should be completed. The 
original should be placed in the Pharmacy File and a copy faxed to 
the Trials Unit. 
Patient returns 
N/A 
Table 6: Disposal information for the ThermoDox® IMP used in the TARDOX study. 
11.10 Occupational safety 
As per IB and site procedure.  
 
12 EVALUATION OF RESPONSE 
Response to intervention will be assessed both in terms of intra-tumoural uptake of doxorubicin following 
intervention on the same day and the radiological response of the target tumour during the follow-up stage of the 
study. The response to intervention for each participant will be assessed using a combination of the collected data in 
both Part I and Part II: 
 
1. Biopsy analysis 
2. Blood results 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 43 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
3. Follow-up scans (Liver MRI/MR-Spectroscopy, Liver pCT and whole body FDG PET-CT) 
 
Biopsy analysis 
Statistical analysis will be performed to establish if there is significant improvement in dose recovery between the 
Unreleased Drug Biopsy and the Released Drug Biopsy. 
 
Follow-up Imaging 
Although not essential for the success of this study, whose primary objective is to demonstrate targeted release of 
doxorubicin, response to intervention is a tertiary (exploratory) objective in Part II (section 12.1). 
12.1 
Measurement of disease for solid tumour 
Objective disease response in the target tumour and the non-FUS control tumours, if any, will be independently 
measured according to the principles of CHOI and RECIST response evaluation using MRI and CT [11, 12] and SUVmax 
using PET-CT [13]. In both Part I and Part II, radiological tumour response with CT and MRI scans will be evaluated 
prior to study entry and following intervention as per the follow-up schedule (section 2.3). Response classification will 
be used in the analysis of trial data. 
12.2 
Tumour assessment 
A clinical and liver-focused radiological evaluation of malignancy, as judged appropriate by the Investigator, and in line 
with the protocol, must be performed before starting the study treatment where applicable.  In addition to ultrasound 
examination for screening purposes (section 5.1.3.1), the same methods that detect liver tumours at baseline(Liver 
MRI/MR-Spectroscopy, Liver pCT and whole body FDG PET-CT) will be used to follow these tumours post-intervention 
(section 5.3). To ensure compatibility, the radiological assessments used to assess response must be performed using 
identical techniques.  Imaging based evaluation is preferred to evaluation by clinical examination when both methods 
have been used to assess the anti-tumour effect of a treatment. 
12.2.1 Baseline evaluations 
These will include radiological measurements of the FUS-targeted tumour and, where available, suitable control liver 
tumour(s) by Liver MRI/MR-Spectroscopy, Liver pCT and whole body FDG PET-CT.  Only these selected liver tumours 
will be evaluated and followed-up for response. Control tumours will be selected based on their measurability at 
baseline and ideally be size-matched to the target tumour where possible. In some cases there may be no appropriate 
control tumours, in this case the only the target tumour will be evaluated. 
12.2.2 Evaluations during treatment and at off-study 
Tumour assessment will be repeated as per the schedule of events and measured in accordance with the principles of 
CHOI and RECIST [11, 12].  
 
13 ASSESSMENT OF SAFETY 
Adverse event monitoring starts at intervention (Day 1) until Day 30 follow-up visit (which may be scheduled 30 days 
+7 but not <30 post intervention).. The Investigator will monitor each patient for clinical and laboratory evidence of 
adverse events on a routine basis throughout the study. Should an Investigator become aware of any study drug or 
study procedure related SAEs following this period these must also be reported as stated below.  All reportable AEs 
will be followed to a satisfactory conclusion. Any reportable AEs that are unresolved at the patient’s last visit in the 
study are followed up by the Investigator for as long as medically indicated, but without further recording in the CRF. 
 
All AEs reported to the trial office will be processed according to internal SOPs, this includes the reporting procedure 
and assessment of causality and expectedness for SAEs. The TARDOX trial office may request additional information 
for any AE as judged necessary. 
13.1 
Adverse Event Definitions 
An Adverse Event or experience (AE) is any untoward medical occurrence in a study subject temporally associated 
with the administration of an investigational medicinal product (IMP) or a comparator product, whether or not 
considered related to the IMP or a comparator product. An AE can therefore be any unfavourable and unintended 
sign, symptom, disease (new or exacerbated) and /or significant abnormal laboratory or physiological observation 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 44 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
temporally associated with the use of a medicinal product. For marketed medicinal products, this also includes failure 
to produce expected benefits (i.e. lack of efficacy), abuse or misuse. A Serious Adverse Event (SAE) is any AE, 
regardless of dose, causality or expectedness, that: 
 
• Results in death 
 
 
• Is life-threatening  
This refers to an event in which the subject was at risk of death at the 
time of the event.  It does not refer to an event, which hypothetically 
might have caused death, if it were more severe. 
• Requires in-patient hospitalisation  
or prolongs existing inpatient 
hospitalisation  
 
In general, hospitalisation signifies that the subject has been admitted 
(usually involving at least an overnight stay) at the hospital or 
emergency ward for observation and/or treatment that would not 
have been appropriate in the physician’s office or out-patient setting.  
Complications that occur during hospitalisation are AEs.  If a 
complication prolongs hospitalisation or fulfils any other serious 
criteria, the event is serious.  When in doubt as to whether 
hospitalisation occurred or was necessary, the AE should be 
considered serious. 
• Results in persistent or significant 
incapacity or disability 
This means a substantial disruption of a person’s ability to conduct 
normal life functions.  It does not include experiences of relatively 
minor medical significance or accidental trauma (e.g. sprained ankle), 
which do not constitute a substantial disruption. 
• Is a congenital anomaly or birth 
defect 
 
• Is any other medically other 
medically important event 
Defined as an event that may jeopardise the patient or may require 
intervention to prevent one of the outcomes listed above. Any new 
primary cancer must be reported as an SAE. 
 
This includes any newly occurring event, or a previous condition that has increased in severity or frequency since the 
administration of study drug. 
 
An Adverse Drug Reaction (ADR) A response to a medicinal product, which is noxious and unintended. 
Response in this context means that a causal relationship between a medicinal product and an adverse event is at 
least a reasonable possibility. 
 
An Unexpected Drug Reaction is an adverse drug reaction, the nature or severity of which, is not consistent with 
applicable reference safety information section (referring to information in SPC or IB). 
 
A Suspected Unexpected Serious Adverse Drug Reaction (SUSAR) is a serious adverse drug reaction, the nature or 
severity of which is not consistent with the applicable product information (e.g. Investigator’s Brochure for an 
unapproved investigational product or SPC for an approved product). 
13.2 
Clinical laboratory abnormalities and other abnormal assessments as AEs and SAEs 
Abnormal laboratory findings (e.g., clinical chemistry, haematology, urinalysis) or other abnormal assessments (e.g., 
ECGs, X-rays and scans) that are judged by the investigator as clinically significant will be recorded as AEs or SAEs if 
they meet the definitions given above.  
The investigator will exercise his or her medical and scientific judgment in deciding whether an abnormal laboratory 
finding or other abnormal assessment is clinically significant. By definition, all Grade 4 laboratory abnormalities 
should be reported as SAEs.  
13.3 
Determining adverse event causality 
The Investigator will assess and classify the relationship of an AE to the trial IMP (ThermoDox®) as follows: 
 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 45 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
Classification 
Relationship 
Definition 
Drug-related 
 Definitely related 
 
• Starts within a time related to the study drug administration 
and 
• No obvious alternative medical explanation. 
 
Probably related 
 
• Starts within a time related to the study drug administration 
and 
• Cannot be reasonably explained by known characteristics of 
the patient’s clinical state. 
 
Possibly related 
 
• Starts within a time related to the study drug administration 
and 
• A causal relationship between the study drug and the 
adverse event is at least a reasonable possibility. 
 
Not drug related 
 Probably 
not 
related 
 
• The time association or the patient’s clinical state is such that 
the study drug is not likely to have had an association with 
the observed effect. 
 
 Definitely 
not 
related 
 
• The AE is definitely not associated with the study drug 
administered. 
 
The Investigator must endeavour to obtain sufficient information to confirm the causality of the adverse event (i.e. 
relation to the study drug, focused ultrasound exposure, liver biopsy, other illness, progressive malignancy or other 
cause) and give their opinion of the causal relationship between each AE and study drug, FUS and/or biopsy 
procedure.  This may require instituting supplementary investigations of significant AEs based on their clinical 
judgement of the likely causative factors and/or include seeking a further specialist opinion. 
13.4 
Expected adverse events 
Appendix 2 of the current approved ThermoDox® IB [44] lists all the adverse events associated with the use of 
ThermoDox® in clinical studies. ThermoDox® contains Adriamycin® (doxorubicin) as its active chemotherapy agent and 
ThermoDox® has a similar toxicity profile to doxorubicin. Appendix 1 of the current approved ThermoDox® IB [44] lists 
all the adverse events known to be associated with the use of doxorubicin. A copy of this document must be held in 
the Site File for reference. 
 
Please note much of the adverse event data for ThermoDox® has been obtained from a global, randomized Phase III 
study (HEAT) which involves combining ThermoDox® with RFA in the treatment of hepatic tumours, in patients with 
inoperable hepatocellular carcinoma (HCC). Adding 50mg/m2 of ThermoDox® to RFA was found to be safe with an 
adverse event profile similar to that of the single agent doxorubicin. However given the difference in patient 
population, it is possible that in this study population other side effects may occur, or the patient might suffer a more 
severe reaction. 
 
As per ThermoDox® IB amendment version date 11th October 2013 (the current approved ThermoDox® IB at the time 
of the initial trial approval), grade 3+ neutropenia and leucopenia were the only19 SAEs occurring in over 5% of HEAT 
Study patients and were not seen in the arm receiving RFA without ThermoDox® (Table 7).  The detailed and current 
list of SAEs and AEs in 701 patients for this study, can be found in Appendix 2 of the current approved ThermoDox® IB 
                                                     
 
19 The only other grade 3+ adverse events affecting at least 5% of subjects were elevations in liver enzymes which is an expected 
occurrence in the patient group used for the HEAT study (hepatocellular carcinoma). The RFA alone vs RFA+ThermoDox® study 
arms did not differ significantly in this respect. 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 46 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
[44]. Further details regarding the frequency of AEs and SAEs in patients treated with ThermoDox® using targeted 
application of heat by RFA or microwave have been included in a recent publication [10]. 
 
 
Adverse 
Event 
RFA Alone 
N=347 
RFA+ThermoDox® 
N=354 
p-value 
 
N 
% 
N 
% 
 
Neutropenia 
0 
0 
65 
18.9 
<0.0001 
Leukopenia 
0 
0.0 
21 
6.1 
<0.0001 
 
 
 
 
 
 
Table 7: SAEs affecting at least 5% of patients enrolled in the Phase III, randomized, double-blinded, dummy-
controlled study of the efficacy and safety of ThermoDox® in combination with RFA compared to RFA-alone 
in the treatment of non-resectable HCC. Data obtained from the ThermoDox® Investigator’s Brochure 
(Amendment Version October 11, 2013). 
 
This study involves the use of FUS to induce mild hyperthermia. A recent study performed at Oxford involving 30 
patients in four trial protocols has shown ablative FUS or high-intensity FUS (HIFU) to be both safe and feasible for the 
treatment of liver and kidney tumours [45]. Adverse events are summarised in Table 8. It should be noted that 
thresholds of acoustic power required for mild hyperthermia, as required this study, are envisaged to be well below 
those required for ablative HIFU and thermal cell death. 
 
 
Table 8: Adverse events possibly or probably related to HIFU treatmenta. Reproduced from Illing et al., 2005 
[45]. 
 
Evidence for the primary endpoint for this study requires laboratory analysis of tumour tissue obtained by image 
guided liver biopsy through a co-axial biopsy needle as per current guidelines. The complications of performing image 
guided liver biopsy using guidelines have been reviewed following a recent UK-wide audit [48] and are summarised in 
Table 9. Out of 3496 cases, 376 patients had minor complication alone, 22 patients had at least one major 
complication and there were 4 deaths, all attributable to post-biopsy haemorrhage. 
 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 47 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
 
Table 9: Post- liver biopsy complications, mortality and subsequent treatment and interventions. Data is represented 
as numbers of cases. Numbers in parentheses are percentages. Reproduced from Howlett et al., 2013 [48]. 
13.5 
Suspected Unexpected Serious Adverse Drug Reactions (SUSARs) 
All SUSARS must be reported to the responsible Authority and main REC within 7 or 15 days, depending on the 
seriousness criteria, by the Sponsor or their delegate within the required timelines. The Sponsor or their delegate will 
ensure that all SUSARs are also reported to the MHRA. It is therefore important that the SAE form is faxed as detailed 
below as soon as, and no later than, 24 hours after the site are aware of an SAE or significant updated information. In 
addition, other safety issues qualify for expedited reporting where they might materially alter the current risk 
assessment of an IMP or be sufficient to change IMP administration or the overall conduct of the trial. Responsibility 
for expedited safety reporting will be assigned by the Sponsor. 
 
The TARDOX trial office shall notify Celsion of all SUSAR’s within one working day of becoming aware of them. 
 
13.6 
Expedited reporting of SAEs 
The following SAE reporting requirements apply regardless of the Investigator’s assessment of the causality or 
expectedness of the SAE (see section 13.10 for exceptions).  All SAEs should be reported on the trial SAE report form 
(see SAE report form and instructions for completion). Please note that the SAE report form is a paper form and these 
data cannot be entered into the eCRF system (OpenClinica). 
 
SAE forms must be completed and faxed to the trial office on:   
 
TARDOX Trial Office SAE Fax: 01865 227038 
 
Or emailed (email preferred) to: 
TARDOX Trial Office SAE Email: octo-safety@oncology.ox.ac.uk 
 
SAE forms must be completed with all critical elements as soon as possible and submitted within 24 hours of 
becoming aware of the event all the vital elements being completed. If the SAE has not been reported within the 
specified timeframe, a reason for lateness must be provided when sending the SAE Report Form. 
 
Investigators should also adhere to their local Trust policy for incident and SAE reporting in research. 
 
The TARDOX trial office shall notify Celsion with a line listing report of all Serious Adverse Reactions (SARs) on a 
monthly basis. 
13.7 
Follow-up of Serious Adverse Events 
If new or amended information on a reported SAE becomes available, the Investigator should report this on a new SAE 
form or update the existing form, without obscuring any initial information.  All new data must be initialled and dated 
so that all changes are clearly identified. 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 48 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
 
Follow up will continue until all the necessary safety data for the event has been gathered. Any SAE that is on-going 
when a subject completes his/her participation in the trial must be followed until any of the following occurs: 
• 
The event resolves or stabilizes; 
• 
The event returns to baseline condition or value (if a baseline value is available); 
• 
The event is attributed to other agent(s) or to factors unrelated to study conduct. 
 
SAEs that are considered to be definitely unrelated to the trial intervention will be followed up  as per related SAEs. 
 
13.8 
Adverse events that should be notified to trial office 
AEs that are serious must be reported to trial office up to and including 30 days after the intervention date. Any SAE 
that occurs at any time after completion of ThermoDox® treatment or after the designated follow-up period that the 
investigator considers to be related to the study drug must be reported 
 
13.9 
Reporting Adverse Events on the CRF 
All AEs, including Serious AEs must be recorded on the case report forms (CRF) for that patient. The information 
provided will include date of onset, event diagnosis (if known) or sign/symptom, severity, time course, duration and 
outcome and relationship of the AE to study drug.  Any concomitant medications or other any therapy used to treat 
the event must be listed. The Investigator will provide an “other” cause for serious AEs considered to be unrelated to 
the study drug. The study site should ensure data entered into the CRF is consistent with the SAE report information 
where applicable. SAE forms should be sent to the trials office within 24 hours of the incident occurring. 
 
Each separate AE episode must be recorded.  For example, if an AE resolves completely or resolves to baseline and 
then recurs or worsens again, this must be recorded as a separate AE. For AEs to be considered intermittent, the 
events must be of similar nature and severity. 
 
Serious Events reporting requirements 
Subjects having adverse events will be monitored with relevant clinical assessments and laboratory tests as 
determined by the Investigator. All adverse events must be followed to satisfactory resolution or stabilization of the 
event(s) including the investigation for the likely causative factor. Any actions taken and follow-up results must be 
recorded in the subject's source documentation. Follow-up laboratory results should be filed with the subject's source 
documentation. For all adverse events which require the subject to be discontinued from the study, relevant clinical 
assessments and laboratory tests will be repeated, on at least a weekly basis, until final resolution or stabilisation of 
the event(s). All SAEs will be included in analysis and must be reported to Celsion, regardless of cause, and will be 
classified as per section 13.3. 
 
Non Serious Events reporting requirements 
All Adverse Events (AE) including Adverse Drug Reactions (ADR) occurring up to 30 days following the intervention 
must be recorded on the CRF. The investigator will decide if those events are related to the medicinal product (i.e. 
unrelated, unlikely, possible, probable, definitely and not assessable) and the decision will be recorded on the CRF. AE 
definitely not drug related (i.e. reported as unrelated) will not be considered as adverse drug reactions in toxicity 
analyses, but reported separately. 
 
Terms and Grading of Events 
MedDRA dictionary should be used to code SAES 
The NCI CTCAE Version 4.0 (currently up to Version 4.03) should be used to grade AEs and where possible use the 
Lowest Level Terms provided.  Where indicated on the form, provide the severity grade for each AE, and the worst 
grade recorded. 
13.10 Events exempt from being reported as AE/ SAEs 
13.10.1 Progression of underlying disease 
In this Phase I study, which can only target a single tumour for dosing, progression is a likely outcome in patients with 
disseminated disease. During the follow-up period, target and control tumours in the liver will be monitored for 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 49 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
progression and these data will be captured on the CRF. Progression of disease by clinical means or of other tumours 
by radiological means will not be recorded on CRFs, however resultant death during the study period will be captured 
on the CRF (section 13.10.2).  Adverse events including hospitalisation that are clearly consistent with disease 
progression will not be reported as individual AE/SAEs. Clinical symptoms of progression will only be reported as 
adverse events if the symptom cannot be determined as exclusively due to the progression of the underlying 
malignancy, or does not fit the expected pattern of progression for the disease under study. Where disease 
progression is so evident, in the best interests of the patient, the investigator may elect not to perform further disease 
assessments. 
13.10.2 Death on study 
Death due to disease under study is to be recorded on the Notification of Death CRF, and does not require reporting 
as a SAE, providing the death is not unexpected nor a causal relationship to the IMP or any other trial procedure 
suspected.  Death due to all other reasons must be reported as a SAE. 
13.10.3 Elective admissions and supportive care 
Elective admissions to hospital for patient convenience or for planned procedures or investigations or treatment as 
specified in this protocol and standard supportive care are not SAEs, and do not require SAE reporting.  Hospital 
admission for the following supportive care procedures are considered standard for this patient group and should not 
be reported as SAEs: 
• 
Insertion of stent 
• 
Insertion of Port-A-Cath, PICC line, etc. 
• 
Admission for transfusion 
13.11 Development Safety Update Reports 
DSURs will be co-ordinated by OCTO and prepared in collaboration with the CI and submitted in parallel to the 
regulatory authority and responsible Research Ethics Committee within 60 days of the anniversary of the date of 
regulatory approval.  A copy of each report will be provided to Celsion and the sponsor 
13.12 Informing Investigators of new safety information 
The trial office or the Chief Investigator will ensure that all investigators are kept informed in a timely manner, as new 
safety profile information becomes available. Investigators are responsible for briefing their study team and onward 
transmission to R&D office as appropriate. 
14 
PREGNANCY 
Pregnancies (in a participant or partner) occurring  whilst taking part in the trial or within 6 months of the intervention 
require expedited reporting.  
A pregnancy form should be completed and faxed to the trial office within the same timelines as an SAE. All reported 
pregnancies should be followed and the outcome reported using the same form. If the outcome of the pregnancy 
meets any of the criteria for seriousness, it must also be reported as an SAE. Examples of pregnancy outcomes that 
are SAEs include reports of:  
• congenital anomalies or developmental delay, in the foetus or the child.  
• foetal death and spontaneous abortion.  
• suspected adverse reactions in the neonate that are classified as serious  
 
Women who become pregnant should be withdrawn from trial treatment immediately 
   
 
The Trial Office will report all pregnancies and any pregnancy–related SAEs to Celsion.  
15 
DEFINING THE END OF TRIAL  
For this study the end of the trial is defined as:  


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 50 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
The last follow-up with the last patient 
 
The sponsor and the Chief Investigator reserve the right to terminate the study earlier at any time. In terminating the 
study, they must ensure that adequate consideration is given to the protection of the participants’ best interests. 
16 STATISTICAL CONSIDERATIONS  
16.1 
Sample size and power 
Evaluable participants will be recruited to either Part I or Part II of the study, up to a maximum of 28 evaluable 
participants overall.  Non-evaluable participants will be replaced (section 6.1). 
17 STATISTICAL ANALYSIS PLAN 
A statistical analysis plan written adhering to the current OCTRU standard operating procedures will be signed off for 
this trial prior to analysis of primary or secondary aims. 
17.1 
Inclusion in analysis 
All evaluable participants in both Part I and Part II will be included in the final analyses. All patients enrolled in the 
study, will be accounted for and included in the analyses. The number of patients who were not evaluable, who died 
or withdrew before treatment began will be recorded. 
 
Variables will be analysed to determine whether the criteria for the study conduct are met. This will include a 
description of patients who did not meet all the eligibility criteria, an assessment of protocol violations and other data 
that impact on the general conduct of the study. 
 
Baseline characteristics will be summarised for all enrolled patients. Patients who died or withdrew before treatment 
started or do not complete the required safety observations will be described and evaluated separately. 
 
Treatment related toxicity will be tabulated by type, grade of toxicity, relatedness to trial treatment and the worst 
grade toxcitiy experienced 
 
Evaluable for toxicity: All patients will be evaluable for toxicity from the time of their intervention using NCI CTCAE v 
4.0 scoring.  
 
Adverse events will be summarised by the number of patients experiencing each type of event. The grades and 
causality will be reported. 
17.2 
Subgroup analysis 
No subgroup analyses are planned for the study. 
17.3 
Interim Analyses 
As per the study design (section 2) the TMG will review thermometry data to ascertain the point at which Part II of the 
study will be opened (section 18.2). Following this, there will be an interim analysis of the Part I only secondary 
objective, which concerns optimal FUS exposure parameters. This analysis and all other analysis will be completed at 
the end of the study, i.e. once recruitment is closed in Part I and Part II.  Once Part II is opened, Part I of the study will 
also remain open and can be recruited to in parallel. During the screening process the study clinicians will consider 
patients for the most appropriate Part based on the availability of Part II, the tumour locations, and the present level 
of experience with FUS hyperthermia for tumours at similar anatomical depths and locations. 
17.4 
Procedures for reporting any deviation(s) from the original statistical plan 
Any deviations / violations must be reported to the TARDOX Trial Office according to the procedure outlined during 
site initiation. These will be noted in the statistical report. 
17.5 
Final analysis 
Details of the final analysis are described in the Statistical Analysis Plan.  
 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 51 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
Based upon projected accrual rates, this trial is expected to complete recruitment within 35 months of opening to 
recruitment. Final analysis will be after all Part I and Part II participants have been followed up for at least one month 
at the following projected time points: 
• 
Endpoint analysis - Last Patient Last Visit. 
 
18 TRIAL COMMITTEES 
18.1 
Trial Management Group (TMG) 
The Chief Investigator will chair a TMG responsible for overseeing the successful conduct and publication of the trial. 
The TMG will provide progress reports to the Independent Early Phase Research Trial Steering Committee (IEPTOC) as 
required. The TMG will make the decision to open  Part II of the TARDOX trial. The TMG will also review cumulative 
reports of all AEs & SAEs. TMG meetings will be scheduled as often as required. Meetings may be by teleconference. 
18.2 
Data and Safety Monitoring 
There is no Data and Safety Monitoring Committee (DSMC). SAEs upon receipt are reviewed by an independent 
nominated clinician as part of the trial office SAE Standard Operating Procedure. Cumulative reports of all AEs & SAEs 
are reviewed by the TMG and an Independent Trial Steering Committee (ITSC) will be in place to monitor the safety of 
the trial. 
18.3 
Trial Steering Committee  
Independent oversight for this trial will be provided by the Independent Early Phase Trial Oversight Committee 
(IEPTOC). As per the IEPTOC Charter, the majority of members of IEPTOC, including the chair, are independent of the 
trial. The role of IEPTOC is to provide oversight for the trials to protect the integrity of the trials. It also provides advice 
through its independent Chairman to the Trial Management Group (TMG), Sponsor, Funder and OCTRU on all aspects 
of the trial. IEPTOC will meet at least yearly. Any major trial issue may be dealt with between meetings, by phone or 
by email. 
DATA MANAGEMENT 
18.4 
Database considerations 
Data management will be performed via a web-based, bespoke trial database (OpenClinica). OpenClinica is a 
dedicated and validated clinical trials database designed for electronic data capture. See: http://www.openclinica.org. 
 
The Chief Investigator will act as Data Custodian for the trial.  A guide explaining how to use OpenClinica will be 
provided to every site. The trial office will provide the site with instructions and a video link for training purposes. 
Relevant trial office staff will have overview of all entered data. 
18.5 
Case reports forms (CRFs) 
The Investigator and study site staff will ensure that data collected on each subject is recorded in the CRF as 
accurately and completely as possible. The CRFs will not contain any source data. All appropriate laboratory data, 
summary reports and Investigator observations will be transcribed into the CRFs from the relevant medical record(s). 
Please ensure that: 
• 
the relevant CRFs are completed. 
• 
all CRF data are verifiable in the source documentation or the discrepancies must be explained.  
• 
CRF sections are completed in a timely fashion, as close to the visit or event being recorded as possible. 
• 
data queries are resolved and documented by authorised study staff, giving a reason for the change or 
correction where appropriate. 
 
The above considerations also apply to patients who are withdrawn early. If a patient withdraws from the study, the 
reason must be noted on the appropriate form and the patient must be followed-up as per protocol.  


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 52 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
18.6 
Accounting for missing, unused, or spurious data. 
Missing data found will be chased up and supplemented where possible after consultation with the investigator. The 
control of the correctness of the data is performed with ranking tests, validity tests and consistency checks. Unused 
data will be retained as for used data. 
 
19 CLINICAL STUDY REPORT  
All clinical data will be presented at the end of the study as data listings. These will be checked to confirm the lists 
accurately represents the data collected during the course of the study.  The trial data will then be locked and a final 
data listing produced. The clinical study report will be based on the final data listings. The locked trial data may then 
be used for analysis and publication.  
 
20 STUDY SITE MANAGEMENT 
20.1 
Study site responsibilities 
The Principal Investigator (the PI or lead clinician for the study site) has overall responsibility for conduct of the study, 
but may delegate responsibility where appropriate to suitably experienced and trained members of the study site 
team.  All members of the study site team must complete a delegation log prior to undertaking any study duties.  The 
PI must counter sign and date each entry in a timely manner, authorising staff to take on the delegated 
responsibilities.  
20.2 
Study site set up and activation 
A Principal Investigator should lead the study at the Oxford site, providing the local study office with all core 
documentation and attend a ‘Site Training Call’ organized by the trial office  before the site becomes activated (the 
training call will usually be carried out as a telephone conference call but may be face to face meeting).  The trial office 
will ensure the site has all the required study information/documentation and is ready to recruit.  The site will then be 
notified once they are activated on the TARDOX database and able to recruit. 
20.3 
Arrangements for sites outside the UK 
Not applicable to the TARDOX study. 
20.4 
Study documentation 
The trial office will provide an Investigator File and Pharmacy File to the investigational site containing the documents 
needed to initiate and conduct the study.  The trial office must review and approve any local changes made to any 
study documentation including patient information and consent forms prior to use. Additional documentation 
generated during the course of the trial, including relevant communications must be retained in the site files as 
necessary to reconstruct the conduct of the trial. 
 
21 REGULATORY AND ETHICAL CONSIDERATIONS 
The Sponsor and Investigators will ensure that this protocol will be conducted in compliance with the UK Clinical Trials 
Regulations20, the principles of Good Clinical Practice (GCP) and the applicable policies of the sponsoring organisation. 
Together, these implement the ethical principles of the Declaration of Helsinki (1996) and the regulatory 
requirements for clinical trials of an investigational medicinal product under the European Union Clinical Trials 
Directive. 
21.1 
Ethical conduct of the trial and ethics approval 
The protocol, patient information sheet, consent form and any other information that will be presented to potential 
trial patients (e.g. advertisements or information that supports or supplements the informed consent) will be 
                                                     
 
20 The Medicines for Human Use (Clinical Trials) Regulations (S.I. 2004/1031) and any subsequent amendments to it. 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 53 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
reviewed and approved by an appropriately constituted, independent Research Ethics Committee (REC). Principal 
Investigators will be approved by the REC. 
21.2 
Regulatory Authority approval 
Approval to conduct the study will be obtained from the relevant Competent Authority in each participating country 
prior to initiating the study. In the UK the study will be conducted under a Medicines and Healthcare products 
Regulatory Agency (MHRA) Clinical Trial Authorisation (CTA). 
21.3 
NHS Research Governance 
Investigators are responsible for ensuring they obtain local Trust management agreement to conduct the trial in 
accordance with local arrangements and policies. 
21.4 
Protocol amendments 
Amendments are changes made to the research following initial approval. A ‘substantial amendment’ is an 
amendment to the terms of the Responsible Authority application (if applicable), the REC application, or to the 
protocol or any other supporting documentation, that is likely to affect to a significant degree: 
 
• 
the safety or physical or mental integrity of the subjects of the trial; 
• 
the scientific value of the trial; 
• 
the conduct or management of the trial; or 
• 
the quality or safety of the investigational medicinal product(s) used in the trial. 
 
Non-substantial amendments are those where the change(s) involve only minor logistical or administrative aspects of 
the study. 
 
All amendments will be generated and managed according to the trial office standard operating procedures to ensure 
compliance with applicable regulation and other requirements. Written confirmation of all applicable REC, regulatory 
and local approvals must be in place prior to implementation by Investigators. The only exceptions are for changes 
necessary to eliminate an immediate hazard to study patients (see below). 
 
It is the Investigator’s responsibility to update patients (or their authorised representatives, if applicable) whenever 
new information (in nature or severity) becomes available that might affect the patient’s willingness to continue in the 
trial.  The Investigator must ensure this is documented in the patient’s medical notes and the patient is re-consented if 
appropriate. 
21.5 
Urgent safety measures 
The sponsor or Investigator may take appropriate urgent safety measures to protect trial participants from any 
immediate hazard to their health or safety. Urgent safety measures may be taken without prior authorisation. The 
trial may continue with the urgent safety measures in place. The Investigator must inform the trial office 
IMMEDIATELY if the study site initiates an urgent safety measure: 
 
Tel: 01865 227190 
Fax: 01865 227039 
Email: OCTO-TARDOX@oncology.ox.ac.uk 
 
The notification must include: 
• 
Date of the urgent safety measure; 
• 
Who took the decision; and 
• 
Why the action was taken. 
 
The Investigator will provide any other information that may be required to enable the trial office to report and 
manage the urgent safety measure in accordance with the current regulatory and ethical requirements for expedited 
reporting and close out. 
 
The trials office will follow written procedures to implement the changes accordingly.    


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 54 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
21.6 
Temporary halt 
The sponsor and Investigators reserve the right to place recruitment to this protocol on hold for short periods for 
administrative reasons or to declare a temporary halt. A temporary halt is defined a formal decision to: 
• 
interrupt the treatment of subjects already in the trial for safety reasons; 
• 
stop recruitment on safety grounds; or 
• 
stop recruitment for any other reason(s) considered to meet the substantial amendment criteria, including 
possible impact on the feasibility of completing the trial in a timely manner. 
 
The trial office will report the temporary halt via an expedited substantial amendment procedure. The trial may not 
restart after a temporary halt until a further substantial amendment to re-open is in place.  If it is decided not to 
restart the trial this will be reported as an early termination. 
21.7 
Serious Breaches 
The Medicines for Human Use (Clinical Trials) Regulations require the Sponsor to notify any "serious breaches" to the 
MHRA within 7 days of the sponsor becoming aware of the breach. A serious breach is defined as “A breach of GCP or 
the trial protocol which is likely to effect to a significant degree:  
• 
the safety or physical or mental integrity of the subjects of the trial; or 
• 
the scientific value of the trial” 
 
Investigators must notify the trials office at once if any serious breach of GCP is suspected.  The TARDOX Trial Office 
will report all Serious Breaches to Clinical Trial Research Governance, University of Oxford.  
21.8 
REPORTS: Progress, Safety and End of Study Reports 
This protocol will comply with all current applicable Regulatory Authority, Research Ethics Committee and Sponsor 
requirements for the provision of periodic study safety and progress reports. Any additional reports will be provided 
on request. Reporting will be managed by the trials office according to internal SOPs. The site will be urged to return 
as much data as possible before each database lock point. 
 
The trial office will determine which reports need to be circulated Principal Investigators and other interested parties 
according to internal SOPs. The study site is responsible for forwarding trial reports they receive to their local Trust as 
required. 
 
22 EXPENSES AND BENEFITS 
Reasonable travel expenses for any visits additional to normal care will be reimbursed on production of receipts or a 
mileage allowance provided as appropriate. 
 
23 QUALITY ASSURANCE 
23.1 
Risk assessment  
A risk assessment and a monitoring plan will be prepared before the study opens. The risk assessment will be 
repeated if necessary in the light of changes while the study is on-going or in response to monitoring reports.  
Monitoring plans will be amended as appropriate. 
23.2 
On-site Monitoring 
Data will be evaluated for compliance with the protocol and accuracy in relation to source documents. Monitoring 
visits reports will be sent to the site in a timely fashion. 
23.3 
Central Monitoring 
The study site will be monitored centrally by both programmed validation within the data collection database and   
manual checking of incoming data for compliance with the protocol, data consistency, missing data and timing.  All 
changes to data that could influence the outcome will be queried with and approved by the study site in a timely 
manner. For all other data, where there is no doubt about the source of any errors, clear changes to data will be made 
internally by TARDOX Trial Office staff without referring back to the study site. Study staff will be in regular contact 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 55 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
with site personnel to check on progress and deal with any queries that they may have including those arising from 
queries raised by the TARDOX Trial Office.  
 
23.4 
Audit and Regulatory Inspection 
All aspects of the study conduct may be subject to internal or external quality assurance audit to ensure compliance 
with the protocol, GCP requirements and other applicable regulation or standards. It may also be subject to a 
regulatory inspection. Such audits or inspections may occur at any time during or after the completion of the study. 
Investigators and their host Institution(s) should understand that it is necessary to allow auditors/inspectors direct 
access to all relevant documents, study facilities and to allocate their time and the time of their staff to facilitate the 
audit or inspection visit. Anyone receiving notification of a Regulatory Inspection that will (or is likely to) involve this 
trial must inform the trial office without delay. 
 
24 RECORDS RETENTION & ARCHIVING 
During the clinical trial and after trial closure the Investigator must maintain adequate and accurate records to enable 
the conduct of a clinical trial and the quality of the research data to be evaluated and verified.  All essential 
documents must be stored in such a way that ensures that they are readily available, upon request for the minimum 
period required by national legislation or for longer if needed.  The medical files of trial subjects shall be retained in 
accordance with national legislation and in accordance with the host institution policy. 
 
Retention and storage of laboratory records for clinical trial samples must also follow these guidelines: 
 
• 
Retention and storage of central laboratory records supporting PK or PD endpoints and the disposition of 
samples donated via the trial must also comply with applicable legislation and Sponsor requirements. 
• 
It is the University of Oxford’s policy to store data for a minimum of 5 years. Investigators may not archive or 
destroy study essential documents or samples without written instruction from the trial office. 
 
25 PATIENT CONFIDENTIALITY 
Personal data recorded on all documents will be regarded as confidential, and to preserve each patient’s anonymity, 
only their patient study number, initials and date of birth (or other identified as appropriate to country regulations 
and agreed with the Sponsor) will be recorded on the CRFs.  
 
The Investigator site must maintain the patient’s anonymity in all communications and reports related to the research. 
The Investigator site team must keep a separate log of enrolled patients’ personal identification details as necessary 
to enable them to be tracked. These documents must be retained securely, in strict confidence. They form part of the 
Investigator Site File and are not to be released externally. 
 
26 STUDY FUNDING 
This trial is being organised by the Oncology Clinical Trials Office (OCTO) at the University of Oxford. The trial is part of 
the NIHR portfolio, and any additional NHS clinical service support costs of patient care while on study should be met 
by the host study site. Celsion is funding manufacture and supply of the study drug (ThermoDox®). 
27 SPONSORSHIP AND INDEMNITY 
27.1 
Sponsorship 
The Sponsor will provide written confirmation of Sponsorship and authorise the trial commencement once satisfied 
that all arrangements and approvals for the proper conduct of the trial are in place. A separate study delegation 
agreement, setting out the responsibilities of the Chief Investigator and Sponsor will be put in place between the 
parties.  


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 56 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
27.2 
Insurance 
The University has a specialist insurance policy in place: - Newline Underwriting Management Ltd, at Lloyd’s of London 
- which would operate in the event of any participant suffering harm as a result of their involvement in the research. 
27.3 
Contracts/Agreements 
This trial is subject to the Sponsor’s policy requiring that written contracts/agreements are agreed formally by the 
participating bodies as appropriate. A Clinical Trial Agreement (CTA) will be placed between the Sponsor and 
participating NHS Trust(s) prior to site activation.  
 
The Sponsor will also set up written agreements with any other external third parties involved in the conduct of the 
trial as appropriate.  
28 PUBLICATION POLICY 
The intention is to publish this research in a specialist peer reviewed scientific journal on completion of the study. The 
results may also be presented at scientific meetings and/or used for a thesis.  The Investigators will be involved in 
reviewing drafts of the manuscripts, abstracts, press releases and any other publications arising from the trial and 
retain final editorial control. 
 
 
 
29 REFERENCES 
1. 
Bengmark, S. and L. Hafstrom, The natural history of primary and secondary malignant tumors of the liver. I. 
The prognosis for patients with hepatic metastases from colonic and rectal carcinoma by laparotomy. Cancer, 
1969. 23(1): p. 198-202. 
2. 
Jaffe, B.M., et al., Factors influencing survival in patients with untreated hepatic metastases. Surg Gynecol 
Obstet, 1968. 127(1): p. 1-11. 
3. 
Stangl, R., et al., Factors influencing the natural history of colorectal liver metastases. Lancet, 1994. 
343(8910): p. 1405-10. 
4. 
Rothbarth, J. and C.J. van de Velde, Treatment of liver metastases of colorectal cancer. Ann Oncol, 2005. 16 
Suppl 2: p. ii144-9. 
5. 
Berber, E., R. Pelley, and A.E. Siperstein, Predictors of survival after radiofrequency thermal ablation of 
colorectal cancer metastases to the liver: a prospective study. J Clin Oncol, 2005. 23(7): p. 1358-64. 
6. 
Bruix, J., M. Sherman, and A.A.f.t.S.o.L.D. Practice Guidelines Committee, Management of hepatocellular 
carcinoma. Hepatology, 2005. 42(5): p. 1208-36. 
7. 
Llovet, J.M., A. Burroughs, and J. Bruix, Hepatocellular carcinoma. Lancet, 2003. 362(9399): p. 1907-17. 
8. 
Bruix, J., et al., Clinical management of hepatocellular carcinoma. Conclusions of the Barcelona-2000 EASL 
conference. European Association for the Study of the Liver. J Hepatol, 2001. 35(3): p. 421-30. 
9. 
Coleman, M.P., et al., EUROCARE-3 summary: cancer survival in Europe at the end of the 20th century. Ann 
Oncol, 2003. 14 Suppl 5: p. v128-49. 
10. 
Poon, R.T. and N. Borys, Lyso-thermosensitive liposomal doxorubicin: a novel approach to enhance efficacy of 
thermal ablation of liver cancer. Expert Opin Pharmacother, 2009. 10(2): p. 333-43. 
11. 
Choi, H., et al., Correlation of computed tomography and positron emission tomography in patients with 
metastatic gastrointestinal stromal tumor treated at a single institution with imatinib mesylate: proposal of 
new computed tomography response criteria. J Clin Oncol, 2007. 25(13): p. 1753-9. 
12. 
Eisenhauer, E.A., et al., New response evaluation criteria in solid tumours: revised RECIST guideline (version 
1.1). Eur J Cancer, 2009. 45(2): p. 228-47. 
13. 
Kinahan, P.E. and J.W. Fletcher, Positron emission tomography-computed tomography standardized uptake 
values in clinical practice and assessing response to therapy. Semin Ultrasound CT MR, 2010. 31(6): p. 496-
505. 
14. 
Wanebo, H.J., et al., Surgical management of patients with primary operable colorectal cancer and 
synchronous liver metastases. Am J Surg, 1978. 135(1): p. 81-5. 
16. 
Welch, J.P. and G.A. Donaldson, The clinical correlation of an autopsy study of recurrent colorectal cancer. 
Ann Surg, 1979. 189(4): p. 496-502. 
17. 
Clark, G.M., et al., Survival from first recurrence: relative importance of prognostic factors in 1,015 breast 
cancer patients. J Clin Oncol, 1987. 5(1): p. 55-61. 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 57 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
18. 
Goldhirsch, A., R.D. Gelber, and M. Castiglione, Relapse of breast cancer after adjuvant treatment in 
premenopausal and perimenopausal women: patterns and prognoses. J Clin Oncol, 1988. 6(1): p. 89-97. 
19. 
Insa, A., et al., Prognostic factors predicting survival from first recurrence in patients with metastatic breast 
cancer: analysis of 439 patients. Breast Cancer Res Treat, 1999. 56(1): p. 67-78. 
20. 
O'Reilly, S.M., M.A. Richards, and R.D. Rubens, Liver metastases from breast cancer: the relationship between 
clinical, biochemical and pathological features and survival. Eur J Cancer, 1990. 26(5): p. 574-7. 
21. 
Wyld, L., et al., Prognostic factors for patients with hepatic metastases from breast cancer. Br J Cancer, 2003. 
89(2): p. 284-90. 
22. 
Bathe, O.F., et al., Metastasectomy as a cytoreductive strategy for treatment of isolated pulmonary and 
hepatic metastases from breast cancer. Surg Oncol, 1999. 8(1): p. 35-42. 
23. 
Quint, L.E., et al., Distribution of distant metastases from newly diagnosed non-small cell lung cancer. Ann 
Thorac Surg, 1996. 62(1): p. 246-50. 
24. 
Weiss, L., et al., Haematogenous metastatic patterns in colonic carcinoma: an analysis of 1541 necropsies. 
The Journal of pathology, 1986. 150(3): p. 195-203. 
25. 
Hilsenbeck, S.G., W.A. Raub, Jr., and K.S. Sridhar, Prognostic factors in lung cancer based on multivariate 
analysis. Am J Clin Oncol, 1993. 16(4): p. 301-9. 
26. 
Ries, L.A., Influence of extent of disease, histology, and demographic factors on lung cancer survival in the 
SEER population-based data. Seminars in surgical oncology, 1994. 10(1): p. 21-30. 
27. 
Adam, R., et al., Is liver resection justified for patients with hepatic metastases from breast cancer? Ann Surg, 
2006. 244(6): p. 897-907; discussion 907-8. 
28. 
Fong, Y., et al., Liver resection for colorectal metastases. J Clin Oncol, 1997. 15(3): p. 938-46. 
29. 
Fong, Y., et al., Clinical score for predicting recurrence after hepatic resection for metastatic colorectal cancer: 
analysis of 1001 consecutive cases. Ann Surg, 1999. 230(3): p. 309-18; discussion 318-21. 
30. 
Hughes, K.S., et al., Resection of the liver for colorectal carcinoma metastases: a multi-institutional study of 
patterns of recurrence. Surgery, 1986. 100(2): p. 278-84. 
31. 
Bismuth, H., et al., Resection of nonresectable liver metastases from colorectal cancer after neoadjuvant 
chemotherapy. Ann Surg, 1996. 224(4): p. 509-20; discussion 520-2. 
32. 
Kemeny, N. and A. Schneider, Regional treatment of hepatic metastases and hepatocellular carcinoma. Curr 
Probl Cancer, 1989. 13(4): p. 197-283. 
33. 
Doroshow, N.P., Modulation of fluorouracil by leucovorin in patients with advanced colorectal cancer: 
evidence in terms of response rate. Advanced Colorectal Cancer Meta-Analysis Project. J Clin Oncol, 1992. 
10(6): p. 896-903. 
34. 
Piedbois, P., Rougier, P., Buyse, M., Pignon, J., Ryan, L., Hansen, R., Zee, B., Weinerman, B., Pater, J., 
Leichman, C., Efficacy of intravenous continuous infusion of fluorouracil compared with bolus administration 
in advanced colorectal cancer. Meta-analysis Group In Cancer. J Clin Oncol, 1998. 16(1): p. 301-8. 
35. 
Flanders, V.L. and D.A. Gervais, Ablation of liver metastases: current status. J Vasc Interv Radiol, 2010. 21(8 
Suppl): p. S214-22. 
36. 
Chuang, V.P. and S. Wallace, Hepatic artery embolization in the treatment of hepatic neoplasms. Radiology, 
1981. 140(1): p. 51-8. 
37. 
Clouse, M.E., et al., Peripheral hepatic artery embolization for primary and secondary hepatic neoplasms. 
Radiology, 1983. 147(2): p. 407-11. 
38. 
Tellez, C., et al., Phase II trial of chemoembolization for the treatment of metastatic colorectal carcinoma to 
the liver and review of the literature. Cancer, 1998. 82(7): p. 1250-9. 
39. 
Solbiati, L., et al., Percutaneous radio-frequency ablation of hepatic metastases from colorectal cancer: long-
term results in 117 patients. Radiology, 2001. 221(1): p. 159-66. 
40. 
Parkin, D.M., et al., Global cancer statistics, 2002. CA Cancer J Clin, 2005. 55(2): p. 74-108. 
41. 
Khan, S.A., et al., Rising trends in cholangiocarcinoma: is the ICD classification system misleading us? J 
Hepatol, 2012. 56(4): p. 848-54. 
42. 
Llovet, J.M., et al., Sorafenib in advanced hepatocellular carcinoma. N Engl J Med, 2008. 359(4): p. 378-90. 
43. 
Valle, J.W., et al., Gemcitabine alone or in combination with cisplatin in patients with advanced or metastatic 
cholangiocarcinomas or other biliary tract tumours: a multicentre randomised phase II study - The UK ABC-01 
Study. Br J Cancer, 2009. 101(4): p. 621-7. 
44. 
Celsion, ThermoDox® (Lyso-Thermosensitive Liposomal Doxorubicin, LTLD) Investigator's Brochure 
(Unpublished). 
45. 
Illing, R.O., et al., The safety and feasibility of extracorporeal high-intensity focused ultrasound (HIFU) for the 
treatment of liver and kidney tumours in a Western population. Br J Cancer, 2005. 93(8): p. 890-5. 
46. 
Leslie, T.A., et al., High-intensity focused ultrasound ablation of liver tumours: can radiological assessment 
predict the histological response? Br J Radiol, 2008. 81(967): p. 564-71. 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 58 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
47. 
Fandrich, C.A., R.P. Davies, and P.M. Hall, Small gauge gelfoam plug liver biopsy in high risk patients: safety 
and diagnostic value. Australas Radiol, 1996. 40(3): p. 230-4. 
48. 
Howlett, D.C., et al., Findings of the UK national audit evaluating image-guided or image-assisted liver biopsy. 
Part II. Minor and major complications and procedure-related mortality. Radiology, 2013. 266(1): p. 226-35. 
49. 
Child, C.G. and J.G. Turcotte, Surgery and portal hypertension. Major Probl Clin Surg, 1964. 1: p. 1-85. 
50. 
Pugh, R.N., et al., Transection of the oesophagus for bleeding oesophageal varices. Br J Surg, 1973. 60(8): p. 
646-9. 
 
 
 


 
 
Confidential 
 
TARDOX_Protocol_V3.0_06Apr2016                                                                                                                                        Protocol Template V3.0_18Feb2013 
Page 59 of 59 
TARDOX STUDY PROTOCOL 
Targeted 
chemotherapy 
using 
focused 
ultrasound for liver tumours 
APPENDIX 1: WHO PERFORMANCE SCALE 
Activity Performance Description 
Score 
 
 
Fully active, able to carry out all on all pre-disease performance without restriction. 
0 
 
 
Restricted in physically strenuous activity but ambulatory and able to carry out work of a light or 
sedentary nature, e.g. light housework, office work. 
1 
 
 
Ambulatory and capable of all self-care, but unable to carry out any work activities.  Up and about 
more than 50% of waking hours.  
2 
 
 
Capable of only limited self-care.  Confined to bed or chair more than 50% of waking hours.  
3 
 
 
Completely disabled. Cannot carry out any self-care. Totally confined to bed or chair. 
 
4 
APPENDIX 2: AMERICIAN SOCIETY OF ANAESTHESIOLOGISTS (ASA) CLASSIFICATION SYSTEM 
Activity Performance Description 
Score 
 
 
Healthy Person. 
ASA 1 
 
 
Mild systemic disease. 
ASA 2 
 
 
Severe systemic disease. 
ASA 3 
 
 
Severe systemic disease that is a constant threat to life. 
ASA 4 
 
 
A moribund person who is not expected to survive without the operation 
ASA 5 
 
 
A declared brain-dead person whose organs are being removed for donor purposes 
ASA 6 
APPENDIX 3: CHILD-PUGH SCORE 
The Child-Pugh Score [49, 50] is used to assess chronic liver disease. It is calculated by adding the points for the five 
criteria assessed: 
 
Points 
1 
2 
3 
Encephalopathy 
None 
Minimal 
Advanced (coma) 
Ascites 
Absent 
Controlled 
Refractory 
Bilirubin (μmol/L) (mg/dl) 
<34 
<2 
34-50 
2-3 
>50 
>3 
Albumin (g/dL) 
>3.5 
2.8-3.5 
<2.8 
INR 
<1.7 
1.71-2.30 
>2.30 
 
The Child-Pugh Score is interpreted as follows: 
 
Points 
Class 
One year survival 
Two year survival 
5-6 
A 
100% 
85% 
7-9 
B 
81% 
57% 
10-15 
C 
45% 
35% 
 

SAP_001.pdf: 
 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 1 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
 
 
 
Full title: A Proof of Concept Study to Investigate the Feasibility of Targeted Release 
of Doxorubicin from Lyso-thermosensitive Liposomal (LTSL) Doxorubicin 
(ThermoDox®) Using Focused Ultrasound in Patients with Primary or Secondary 
Liver Tumours 
Statistical Analysis Plan 
Version 3.0 – 22 Jul 2016 
Based on version 3.0 of protocol (06 Apr 2016)  
 
 
Name 
Title/Role 
Signature 
Date 
Author 
Paul Lyon 
Clinical Fellow 
 
 
Author 
Lang’O Odondi 
Medical Statistician  
 
 
Reviewer 
Lucy Griffiths 
Trial Coordinator, OCTO 
 
 
Reviewer 
Meena Patil 
Data Manager, OCTO 
 
 
Reviewer 
Robert Carlisle 
Associate Professor of 
Biomedical Engineering 
 
 
Approver 
Lang’O Odondi 
Medical Statistician 
 
 
Approver 
Mark Middleton 
Professor, Department of 
Oncology, Chief Investigator 
 
 
 
Oxford Clinical Trials Research Unit (OCTRU) and 
Centre for Statistics in Medicine (CSM) 
 
 
 
 
Sponsored by the University of Oxford 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 2 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
CONTENTS 
1 
INTRODUCTION ............................................................................................................................... 3 
1.1 
KEY PERSONNEL ...................................................................................................................................................... 3 
2 
BACKGROUND INFORMATION .......................................................................................................... 5 
2.1 
STUDY HYPOTHESIS ................................................................................................................................................. 5 
2.2 
KEY DEFINITIONS .................................................................................................................................................... 5 
2.3 
UNRELEASED AND RELEASED NOMENCLATURE ............................................................................................................. 6 
2.4 
PRIMARY, SECONDARY AND TERTIARY OBJECTIVES & ENDPOINTS ..................................................................................... 6 
2.5 
STUDY DESIGN ....................................................................................................................................................... 9 
2.6 
ELIGIBILITY ............................................................................................................................................................ 9 
2.7 
TREATMENT INTERVENTIONS ................................................................................................................................... 11 
2.8 
SAMPLE SIZE ........................................................................................................................................................ 11 
2.9 
STRATEGIES FOR ACHIEVING ADEQUATE RECRUITMENT ................................................................................................. 12 
2.10 
RANDOMISATION .................................................................................................................................................. 12 
3 
HYPOTHESES AND OUTCOME MEASURES ....................................................................................... 13 
3.1 
CLINICAL RESEARCH HYPOTHESES ............................................................................................................................ 13 
3.2 
DEFINITION OF PRIMARY, SECONDARY AND TERTIARY OUTCOMES .................................................................................. 13 
4 
PATIENT GROUPS FOR ANALYSIS .................................................................................................... 14 
5 
QUALITY CONTROL AND DATA VALIDATION ................................................................................... 14 
6 
DATA ANALYSIS ............................................................................................................................. 15 
6.1 
OUTCOMES & ENDPOINT ANALYSIS .......................................................................................................................... 15 
6.2 
PATIENT CHARACTERISTICS ..................................................................................................................................... 19 
6.3 
MISSING DATA ..................................................................................................................................................... 20 
6.4 
PRE-SPECIFIED SUBGROUP ANALYSIS ........................................................................................................................ 20 
6.5 
SENSITIVITY ANALYSIS ............................................................................................................................................ 20 
6.6 
BLINDED ANALYSIS ................................................................................................................................................ 21 
6.7 
META-ANALYSES .................................................................................................................................................. 21 
6.8 
OUTCOMES ASSESSMENT SCHEDULE ........................................................................................................................ 21 
6.9 
DATA MANAGEMENT RESPONSIBILITY ...................................................................................................................... 22 
7 
DATA SAFETY MONITORING COMMITTEE AND INTERIM ANALYSES ................................................. 23 
8 
SAFETY ANALYSIS ........................................................................................................................... 25 
9 
APPENDICES .................................................................................................................................. 27 
9.1 
APPENDIX 1: GLOSSARY OF ABBREVIATIONS ............................................................................................................... 27 
10 
DOCUMENT HISTORY ..................................................................................................................... 28 
10.1 
APPENDIX 2: MODIFIED CONSORT FLOW DIAGRAM.................................................................................................. 29 
10.2 
APPENDIX 3: EXAMPLE PROCESSED REAL-TIME THERMOMETRY TRACE ........................................................................... 30 
10.3 
APPENDIX 4: TARDOX REAL-TIME THERMOMETRY DATA CAPTURE PROFORMA .............................................................. 31 
10.4 
APPENDIX 5: FUS EXPOSURE PARAMETERS DATA CAPTURE PROFORMA ......................................................................... 31 
11 
REFERENCES .................................................................................................................................. 32 
 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 3 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
1 
INTRODUCTION 
This document details the proposed presentation and analysis for the main publications reporting results 
from the NIHR and Celsion jointly funded Single Centre Non-Randomised Phase 1 Clinical Trial of targeted 
chemotherapy using focused ultrasound, in the treatment of liver tumours (TARDOX). The results reported in 
these publications should follow the strategy set out here. Subsequent analyses of a more exploratory 
nature will not be bound by this strategy, though they are expected to follow the broad principles laid down 
here.  The principles are not intended to curtail exploratory analysis (for example, to decide cut-points for 
categorisation of continuous variables), nor to prohibit accepted practices (for example, data transformation 
prior to analysis), but they are intended to establish the rules that will be followed, as closely as possible, 
when analysing and reporting the trial.  
The analysis strategy will be available on request when the principal papers are submitted for publication in 
a journal.  Suggestions for subsequent analyses by journal editors or referees, will be considered carefully, 
and carried out as far as possible in line with the principles of this analysis strategy; if reported, the source of 
the suggestion will be acknowledged. 
Any deviations from the statistical analysis plan will be described and justified in the final report of the trial.  
The analysis should be carried out by an identified, appropriately qualified and experienced statistician, who 
should ensure the integrity of the data during their processing.  Examples of such procedures include quality 
control and evaluation procedures. 
1.1 
Key personnel 
Chief Investigator 
 
 
 
 
 
 
Senior Scientific Researcher 
 
Professor Mark Middleton 
Department of Oncology, University of Oxford 
Oxford Cancer and Haematology Centre 
Churchill Hospital 
Oxford OX3 7LE 
Mark.Middleton@oncology.ox.ac.uk 
 
Professor Robert Carlisle 
Associate Professor of Biomedical Engineering 
Institute of Biomedical Engineering 
Old Road Campus Research Building 
University of Oxford 
Oxford OX3 7DQ 
Robert.Carlisle@eng.ox.ac.uk 
 
 
Clinical Researcher & Co-
Investigator 
Mr. Paul Lyon 
NIHR Clinical Research Fellow (Nuffield Dept. Surgical Sciences) 
Institute of Biomedical Engineering 
Old Road Campus Research Building,  University of Oxford 
Oxford OX3 7DQ 
Paul.Lyon@nhs.net 
 
 
Trial Coordinator 
Lucy Griffiths 
 
Oncology Clinical Trials Office (OCTO) 
 
Department of Oncology 
 
University of Oxford 
 
Old Road Campus Research Building 
 
Roosevelt Drive 
 
Oxford OX3 7DQ 
OCTO-TARDOX@oncology.ox.ac.uk 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 4 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
 DSMC Members: (if appropriate)  
There is no Data and Safety Monitoring Committee (DSMC) for this study and an Independent Trial Steering 
Committee (ITSC) will be in place to monitor safety. 
 
Key TMG Members: 
Prof. Middleton (Chair)  
Mark.Middleton@oncology.ox.ac.uk 
Mr. Paul Lyon 
Paul.Lyon@nhs.net 
Mrs Lucy Griffiths 
OCTO-TARDOX@oncology.ox.ac.uk 
Prof. Constantin Coussios 
Lang’O Odondi 
Constantin.Coussios@eng.ox.ac.uk 
Lango.Odondi@oncology.ox.ac.uk 
 
 
Other/Optional TMG Members 
Prof. Fergus Gleeson 
FGleeson@mac.com 
Prof. Feng Wu 
Feng.Wu@nds.ox.ac.uk 
Prof. Robert Carlisle 
Robert.Carlisle@eng.ox.ac.uk 
Miss Chrissie Butcher 
OCTO-TARDOX@oncology.ox.ac.uk 
Dr. Michael Gray 
Michael.Gray@eng.ox.ac.uk 
Dr. Victoria Woodcock 
Victoria.Woodcock@oncology.ox.ac.uk 
 
Additionally, other Oxford University Hospital Trust or Oxford University staff may be invited to a TMG 
meeting, where their expertise is required. 
 
 
 
 
 
 
 
 
 
 
 
Data Manager 
Marian Taylor 
 
Oncology Clinical Trials Office (OCTO) 
 
Department of Oncology 
 
University of Oxford 
 
Old Road Campus Research Building 
 
Roosevelt Drive 
 
Oxford OX3 7DQ 
Marian.Taylor@oncology.ox.ac.uk 
 
Statistician 
 
Lang’O Odondi 
Oncology Clinical Trials Office (OCTO) 
 
Department of Oncology & Centre for Statistics in Medicine 
 
Botnar Research Centre 
 
Windmill Road 
 
Oxford OX3 7LD 
Lango.Odondi@oncology.ox.ac.uk 
 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 5 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
2 
BACKGROUND INFORMATION 
The development of distant spread (metastases) is a leading cause of death in patients with advanced solid 
tumours. The liver is one of the most common sites for metastatic disease. Studies have shown that the 
treatment of distant metastases improves survival. Systemic chemotherapy is the current treatment of 
choice for patients with inoperable liver metastasis, however long-term survival is rare. The majority of 
primary liver cancers (hepatocellular carcinoma (HCC) and cholangiocarcioma) are unresectable and survival 
remains dismal. Post-hoc analysis of the Phase III HEAT study is indicative that ThermoDox® treatment 
improves survival in patients with HCC, where Radiofrequency Ablation (RFA) was optimal. 
2.1 
Study Hypothesis 
This proof of concept study proposes targeted delivery of broad-spectrum cytotoxic agent (doxorubicin), via 
a specially formulated LTSL (ThermoDox®) activated by mild hyperthermia, as both a feasible and effective 
solution to the drug-delivery problem for the same systemic dose. It is proposed that this method of 
targeted drug delivery is clinically applicable, and may achieve increased tumour uptake and local dose for 
the equivalent dose of doxorubicin used in systemic chemotherapy, which has a well-established and safe 
toxicity profile. 
Key Study Hypothesis: whilst ThermoDox® is circulating systemically at therapeutic levels, doxorubicin 
concentration in the target tumour will be significantly increased following its exposure to safe levels of 
hyperthermia, as delivered by a clinically-approved focused ultrasound (FUS) device. 
Further research hypotheses which relate to this key study hypothesis are listed in section 3.1. 
2.2 
Key Definitions 
As defined in the protocol, the following definitions are assumed in the following statistical plan: 
 
“Intervention” refers to attempted targeted release of ThermoDox® by mild hyperthermia using a focused 
ultrasound. 
The “desired range of mild hyperthermia” is used in the context of ThermoDox® release in this study and is 
defined as achieved bulk tissue temperature in the range of 41-47oC. 
“Optimal FUS exposure parameters” are defined as those combinations of settings on the FUS device 
(power, duty cycle and transducer motion plan) which result in an adequate temperature rise to successfully 
release doxorubicin from the LTSLs in the target tumour, without causing direct thermal tissue damage. 
An “evaluable participant” is defined as a participant who has received FUS following delivery of 
ThermoDox® in Part I or II, and for whom it has been possible to evaluate the outcome of the intervention 
based on biopsy or plasma samples, or radiologically.*  
Please note, with regards to the primary and secondary endpoints, the definition for ‘evaluable participant’ 
does not distinguish those participants having ‘optimal FUS’ for drug delivery, as defined in Table 5. This 
means participants having both optimal and sub-optimal FUS will be included in analysis of these endpoints. 
However with regards to the tertiary endpoints, as stated in the protocol, these will only be evaluated for 
those participants having optimal FUS. 
An “evaluable sample” is defined as a sample for which sufficient material was available for analysis and the 
analytical technique has provided quantifiable and interpretable data. 
*Please note that a participant does not require the full complement of evaluable samples and follow-up 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 6 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
outcome data in order to be an evaluable participant for inclusion in the relevant subset of endpoints for 
analysis. Thus, more specifically, a participant is evaluable with respect to outcome measure(s), for example: 
 
In the case where a participant undergoes the intervention including plasma and biopsy samples, but 
is then lost to radiological follow-up for whatever reason, that participant will still be deemed 
evaluable with respect to the endpoints involving biopsy and plasma analysis. 
 
 
In the case where a participant undergoes the intervention including plasma samples, and 
radiological follow-up, but whose biopsies non-evaluable due to being misplaced, incomplete, 
inadequate or failed analysis, that participant will still be deemed evaluable with respect to the 
endpoints involving plasma analysis and radiological follow-up. An exception is allowed in Part I; if 
only the Released biopsy is evaluable, the primary endpoint may still be assessed with respect to the 
second criterion (total drug concentration below above minimum threshold) 
 
 
In the case of tertiary (exploratory) endpoints, analytical methods such as MR spectroscopy remain 
under evaluation, and consequently they may not be performed in each case, but this is not at the 
cost of other radiological endpoints, which will still be evaluable. 
 
2.3 
Unreleased and Released Nomenclature 
At the time of the intervention, biopsy and plasma samples will be taken at defined time periods as per the 
treatment timeline (see figures 4 and 5 of the protocol for Part I and II respectively). The naming convention 
of the biopsy and plasma samples, ‘Pre-treatment’, ‘Unreleased’ and ‘Released’, relates to the expected 
representation in terms of targeted ThermoDox® release and may not necessarily translate to the true 
biological picture. In particular, ‘Unreleased’ samples are taken after systemic exposure to ThermoDox®, but 
before exposure to the final dose of FUS, whereas ‘Released’ samples are taken after the final dose of FUS. 
In other words, ‘Unreleased’ samples are obtained at the time point at which it is anticipated that the target 
tumour is exposed to therapeutic levels of circulating encapsulated doxorubicin, whereas ‘Released’ samples 
are obtained at the time point where it is hypothesised that encapsulated drug is released as bioavailable 
(free) intra-tumoural doxorubicin following FUS. 
We  note that due to the selected analytical technique (HPLC) used to determine doxorubicin concentration 
in biopsy tissue obtained for this study, it is not possible to distinguish between liposomal (encapsulated) 
and free doxorubicin levels; only the total doxorubicin concentration is evaluable and is directly applicable to 
the primary endpoint. The same holds true for plasma analysis, applicable to a tertiary endpoint, which is 
evaluated by the same analytical technique. 
2.4 
Primary, Secondary and Tertiary Objectives & Endpoints 
The objectives and endpoints for the study are detailed in the following tables. Only evaluable samples will 
be included in endpoint analysis. Each endpoint has been designed to be independent of the others, which is 
why a given participant may only be evaluable for a subset of endpoints. All endpoints will be evaluated at 
the end of Part II. 
 
The following qualifiers (*,†, ‡ and §) apply to Table 1, Table 2 and Table 3: 
 
* In Part II the Unreleased biopsy is not performed and therefore the average value for all evaluable tumours receiving 
intervention in Part I is used as a comparison for the two-fold increase from Unreleased to Released biopsy. 
 
† Duration refers to the completion of sample processing but not necessarily the sample analysis. The sample may be 
stored in a fixed or frozen state and analysed at some time period beyond the specified duration, for example to 
facilitate batch processing. 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 7 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
 
‡ Analytical method will only be performed where tissue mass allows. It is expected at most one technique can be 
performed in addition to the primary endpoint for a given sample. Where biopsy mass falls short, the primary endpoint 
will be prioritised. 
 
§ Must be shown in at least 50% of evaluable participants.  
 
Primary Objective 
Primary Endpoint 
How and when data is 
captured 
To 
determine 
whether 
targeted 
release 
of 
doxorubicin 
from 
ThermoDox® (‘drug’) using 
mild hyperthermia generated 
non-invasively 
by 
focused 
ultrasound (FUS) is feasible in 
cancer patients 
 
A 
demonstrable 
two-fold 
increase 
in*, 
or 
value exceeding 10μg/g of, the concentration of intra-
tumoural doxorubicin at the treated tumour site 
following FUS-induced mild hyperthermia § 
 
Quantification of intra-tumoural drug release will be 
achieved by direct analysis of the intra-tumoural 
biopsy samples using standardised curve(s). 
Analytical 
chemistry 
performed 
on 
a 
recorded 
mass 
of 
biopsy 
sample 
processed within 12 
hours of intervention† 
Table 1: Primary Objective and Endpoint for the TARDOX Study 
 
 
Secondary Objectives 
Secondary Endpoints 
How and when data is captured 
Part I only: To determine optimal 
FUS exposure parameters for a 
range of participant Body Mass 
Indices 
(BMIs) 
and 
tumour 
locations within the liver 
Achievement of the desired range of mild 
hyperthermia in the target tissue as 
monitored by the implanted thermistor 
during FUS exposure 
 
Real-time 
thermometry 
monitoring 
during 
intervention 
 
To assess the safety of FUS-
induced mild hyperthermia for 
drug delivery 
 
1. Persistence of cell viability stain or 
percentage of cell or tissue necrosis 
under 30% following FUS exposure, as 
assessed by cytological or histological 
methods1 ‡,§ 
2. Adverse events deemed related to FUS 
up to 30 days post-intervention 
1. Cytological or histological 
analysis 
of 
biopsy 
samples processed within 
96hr of intervention† 
 
2. Adverse event recording 
for 
30 
days 
post-
intervention 
To assess the local and systemic 
cytotoxic effects of ThermoDox® 
in this setting 
1. Significant bone marrow suppression, 
deranged liver function and liver 
toxicity 
 
2. Adverse events deemed related to 
ThermoDox® up to 30 days post-
intervention 
 
1. Grade 3 and 4 laboratory 
results from blood tests 
at Day 1 and Day 15 post 
intervention 
2. Adverse event recording 
for 
30 
days 
post-
intervention 
Table 2: Secondary Objectives and Endpoints for Parts I & II of the TARDOX Study 
 
 
                                                          
 
1 In some necrotic tumours, base line necrosis may be high (e.g. >30%) even prior to treatment. Consequently an increase in necrosis 
by no more than 25% over that of a suitable control can be used as an alternative endpoint. 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 8 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
Regarding participants for which doxorubicin has been released by optimal FUS exposure parameters2: 
 
Tertiary (Exploratory) Objectives 
Tertiary (Exploratory) Endpoints 
How and when data is 
captured 
1. To establish the validity of 
other minimally-invasive and 
non-invasive 
methods 
of 
evaluating 
intratumoural 
uptake of doxorubicin as an 
alternative to that used in 
primary endpoint 
Combined analysis of relevant end points to 
establish 
most 
effective 
alternative 
analytical method(s) for quantifying drug 
release 
 
See below 
a. Fluorescence 
microscopy of biopsy 
Released biopsy samples exhibiting regions 
of nuclear or background fluorescence 
levels at least twice that of the background 
levels of a suitable control, as assessed by 
fluorescence microscopy ‡,§ 
Microscopy 
analysis 
of 
biopsy samples processed 
within 96hr of intervention† 
b. MR-Spectroscopy 
 
MRI/MR-Spectroscopy indicating3 targeted 
delivery of doxorubicin to the tumour by 
demonstrating a statistically significant 
increase in the tumour: background ratio of 
signal 
intensity 
following 
intervention 
compared to baseline 
MRI/MR-Spectroscopy 
signal within 24 hours of 
intervention 
c. 
Plasma 
pharmacokinetics 
A statistically significant decrease in total 
doxorubicin plasma levels 
over time-
matched clearance levels, following FUS-
induced release 
Analytical 
chemistry 
of 
plasma samples processed 
within 12hr of intervention† 
2. To determine whether the 
dose of doxorubicin released 
has 
a 
therapeutically 
significant effect on the target 
tumour 
Radiological evidence of tumour response 
over up to 60 days in the target tumour 
alone, as assessed by principles of CHOI and 
RECIST response evaluation using MRI and 
CT [1, 2] and SUVmax using PET-CT [3] § 
Scans 
to 
be 
performed 
within 
60 
days 
of 
intervention. 
Table 3: Tertiary (Exploratory) Objectives and Endpoints for Parts I & II of the TARDOX Study 
                                                          
 
2 “Optimal FUS exposure parameters” are defined in section 2.2. 
3 The spectroscopy signal analysed may not be doxorubicin itself; metabolites or breakdown products of ThermoDox® may be 
utilised. 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 9 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
2.5 
Study Design 
The TARDOX trial is designed as a Phase 1 prospective non-randomised safety cohort study, with all patients 
recruited from a single UK site (Oxford). The study has an open label design with all participants receiving 
ThermoDox® and FUS. The study is split into two parts. Part I is required to identify optimal FUS exposure 
parameters using real time thermometry data from an implanted thermistor. Part II does not require 
thermistor implantation, and is designed to more accurately reflect how the therapy would be implemented 
in clinical practice. All evaluable participants from both Part I and Part II will be included in the endpoint 
analysis. 
Part I 
For each participant, a single liver tumour will receive the intervention and follow-up imaging. The ability to 
achieve targeted release of doxorubicin from ThermoDox® at the tumour site will be determined using three 
tumour biopsies with corresponding peripheral blood samples, taken during intervention, and/or using a 
radiological method (MR-Spectroscopy). 
Part II 
For each participant, a single liver tumour will receive the intervention and follow-up imaging. Where 
available, a second ‘control’ tumour, exposed to ThermoDox® alone, will also be identified for follow-up 
imaging. The optimal FUS exposure parameters determined in Part I will be selected for Part II interventions, 
accounting for anatomical differences. The ability to achieve targeted release of doxorubicin from 
ThermoDox® at the tumour site will be determined using the same methods as for Part I, with the exception 
of requiring only one tumour biopsy. 
In the first 60 days following intervention, both Part I and II participants will be followed up with a maximum 
of two CTs, two PET-CTs and three MRI/MR-Spectroscopy scans. Detailed criteria for evaluation of efficacy 
and safety are described in subsequent sections. 
Date of start of recruitment: 
 
04Jul2014 
Date of expected end of recruitment: 
31Mar2017 
Date expected end follow-up: 
 
30Apr2017 
Date expected analysis:  
 
01Sep2017 
Target number of subjects: 
Up to a maximum of 28 evaluable participants will be recruited to 
the study overall. 
Participating Centres: 
 
 
One 
2.6 
Eligibility 
Inclusion Criteria 
Patients will only be eligible for inclusion in this study if all of the following criteria apply: 
1) Pathologically confirmed advanced solid tumour with liver metastasis suitable for intervention (as 
assessed by ultrasound or other radiological methods). In addition confirmed primary liver tumours 
(hepatocellular carcinoma or cholangiocarcinoma) can be included. 
2) Will have progressed or remained stable on conventional chemotherapy.  
3) Male or Female, Age ≥ 18 years.  


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 10 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
4) Have life expectancy of ≥ 3 months.  
5) Left Ventricular Ejection Fraction (LVEF) ≥ 50% on echocardiogram.  
6) Have not received radiotherapy to the target area within the preceding 12 months.  
7) A World Health Organisation (WHO) performance status of ≤ 1 (Appendix 1). 
8) Able and willing to give written informed consent, indicating that they are aware of the investigational 
nature of this study and potential risks, and able to comply with the protocol for the duration of the 
study, including scheduled follow-up visits and examinations. 
Exclusion Criteria 
Patients will be ineligible for recruitment to the study if any of the following conditions hold: 
1) Have surgery or other procedure requiring general anaesthesia planned to be undertaken during the 
period of the study.  
2) Have serious illnesses including, but not limited to, congestive heart failure (NYHA class III or IV functional 
classification); life threatening cardiac arrhythmia; or myocardial infarction or cerebral vascular 
accident within the last 6 months.  
3) Have on going significant infection (chest, urine, blood, intra-abdominal).  
4) Have uncontrolled diabetes.  
5) Have received a life-time dose of doxorubicin > 400mg/m2 or a life-time dose of epirubicin > 800mg/m2 or 
any dose or both. 
6) Pregnant or breast-feeding. In women of childbearing potential, a negative pregnancy test (serum) is 
required within 30 days prior to study intervention.  
7) Female participants of child bearing potential and male participants whose partner is of child bearing 
potential who are not willing to practice an acceptable form of contraception (i.e. oral contraceptive, 
diaphragm, cervical cap, condom, surgical sterility) during the study and for 6 months thereafter. 
Women whose partner has or men who have undergone a vasectomy must use a second form of 
birth control.  
8) Have any known allergic reactions to any of the drugs or liposomal components or intravenous imaging 
agents to be used in this study.  
9) Have portal or hepatic vein tumour invasion/thrombosis.  
10) 
Inadequate haematological and biochemical function (see section 4.1.2 of protocol)  
11) 
Have contraindications to receiving doxorubicin including prior sensitivity (rash, dyspnoea, 
wheezing, urticarial or other symptoms) attributed to anthracyclines or other liposomal drugs.  
12) 
Use of chemotherapy or of an investigational drug within 30 days or 5 half-lives, whichever is longer, 
preceding the intervention.  


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 11 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
13) 
Have medically significant active infection.  
14) 
Have Child-Pugh Class C liver disease or Class A-B with encephalopathy and/or refractory ascites 
(Appendix 3).  
15) 
Documented HIV positive.  
16) 
Documented diagnosis of haemochromatosis.  
17) 
Documented history of contrast-induced nephropathy.  
18) 
Have any of the following contraindications for liver biopsy:  
a) Suspected liver haemangioma or other vascular tumour 
b) Tense ascites 
c) Known cystic liver disease4 
d) Extra-hepatic biliary obstruction4 
19) 
Other medical or psychiatric conditions or laboratory abnormalities that the investigator considers 
would make the patient a poor trial candidate. 
2.7 
Treatment Interventions 
There is a single intervention day for Parts I and II, during which the components of the intervention are 
performed as outlined below. 
Part I 
Target tumour is exposed to FUS alone, and then a combination of FUS and ThermoDox®, under General 
Anaesthetic. Temperature readings are recorded during FUS exposure. Three liver tumour biopsies and 
corresponding peripheral blood samples are taken for analysis. 
Part II 
Target tumour is exposed to a combination of FUS and ThermoDox®, under General Anaesthetic. Two 
biopsies at a single timepoint are taken of the target tumour receiving intervention, along with three 
peripheral blood samples for analysis. 
2.8 
Sample Size 
Up to a maximum of 28 evaluable participants5 will be recruited to the study across two separate Parts (Part 
1 and Part 2). A minimum of 5 participants are required in Part 1 before Part 2 can be opened. Non-
evaluable participants with respect to the primary endpoint may be replaced if still within the recruitment 
period. Primary, secondary and tertiary endpoint will be applied to all evaluable endpoints for all evaluable 
participants, unless specified otherwise. There will be an interim analysis (see section 7). Sample size will not 
be expected to change during the trial. 
                                                          
 
4Relative contraindications only and may be non-exclusive at discretion of the study team  
5An “Evaluable participant” is defined in section 2.2 (Key Definitions). 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 12 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
The relatively small sample size was determined based on the available resources and structure of trial 
funding, with consideration to this being a Phase I proof of concept study, and the anticipated limited cohort 
of patients suitable for enrolment. The sample size aims to recruit enough patients within a reasonable time 
frame to allow sufficient statistical power of study outcome measures to help inform design (Phase II-III) 
studies with higher statistical power in the future. 
2.9 
Strategies for achieving adequate recruitment 
Recruitment prediction is for 28 patients over 24 months, on average 14 patients annually from the initially 
selected combined patient cohort; liver metastasis from a colorectal, breast or lung primary, and additional 
patient cohort; primary liver cancers and all other liver metastasis. It may be that the trial closes before the 
full quota of 28 evaluable patients has been achieved. 
Colorectal carcinoma accounts for approximately 70% of liver metastases, therefore it was originally 
anticipated that the majority of potential participants were likely to be from this patient group. 
Approximately 100 patients with newly diagnosed colorectal liver metastases are referred to the Oxford 
Cancer Centre for treatment every year. It is anticipated that of these patients, approximately 50% (50 
patients) will be offered chemotherapy, 30% will undergo surgery or other treatment such as ablation 
therapy, and the remaining 20% will be considered for entry into this study. From this 20%, it was estimated 
that just over half would offer consent and meet the eligibility criteria, equating to recruitment of 
approximately 12 patients per year. It was predicted that an additional 4 eligible and consenting patients per 
year would be recruited from the other presenting groups (breast or lung primary). 
Although not originally considered for inclusion, primary liver cancers are the third highest cause of cancer-
related death globally [4] with around 3000 new cases per year in the UK. HCC, also known as malignant 
hepatoma, is the most commonly occurring primary liver cancer accounting for 90%, and usually results from 
chronic liver conditions such as hepatitis B and C and/or cirrhosis. Cholangiocarcinoma (CCA), cancer of the 
biliary tract, is the largest contributor to the remaining primary liver cancers and is the most common cause 
of death from primary liver cancer in the UK, where it has an annual mortality rate of 1500 [5]. Although the 
combined incidence of HCC and CCA is less than the much larger cohort of colorectal liver metastases, due to 
lack of alternative local therapies, it is anticipated this study will offer a unique treatment option and thus a 
higher referral and recruitment rate. Furthermore at this stage, for this Phase I study which focuses on drug 
delivery, it is prudent to open up the inclusion criteria to liver metastases from any advanced solid tumour. 
In addition to improving recruitment rates, observing tumour response in a wide range of tumour types will 
better inform design of future Phase II/III studies, which will shift focus to response and overall survival. 
Following the protocol amendment (V2.0) to additionally include liver metastasis from any confirmed 
advanced solid tumour and primary liver tumours, it is anticipated that recruitment will be recoverable to 
meet the original predictions defined above. 
Approaches to promote better recruitment include widening patient population to any liver tumour type, 
stream-lining patient screening to allow consent for ultrasound screening in advance of the main study 
consent, opening of patient identification centre (PIC) sites and extended recruitment period until March 
2017. A protocol amendment has been effected to allow Part I to remain open in parallel to Part II, i.e. allow 
Part II to open sooner hence more flexibility on the location of the target tumour and extending selection 
cohort without excluding patients who can only be treated as Part I participants. In addition, Part II is likely 
to be a considerably shorter intervention and therefore may be more acceptable to referring clinicians and 
patients alike in addition to giving more flexibility in available anaesthetic booking times. 
  
2.10 
Randomisation 
No randomisation is required in this Phase I proof of concept study. 
 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 13 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
3 
HYPOTHESES AND OUTCOME MEASURES 
3.1 
Clinical Research Hypotheses 
 
The study proposes the following clinical research hypotheses and the primary, secondary and tertiary 
outcome measures, or endpoints, are designed to test them: 
1. For the target tumour, following exposure to optimal (sub-ablative) levels of FUS for mild hyperthermia6, 
there will be persistence of cell viability, or absence of tissue or cellular damage, which would otherwise 
be expected with ablative levels of hyperthermia 
 
2. For the target tumour, whilst there are therapeutic levels of circulating ThermoDox®, exposure to 
optimal levels of FUS for mild hyperthermia will result in increased total intra-tumoural concentration of 
doxorubicin 
 
3. Following optimal FUS treatment to the target tumour, there is release of bio-available doxorubicin from 
ThermoDox®, which is then able to bind to cellular DNA.7 
 
4.  For the target tumour, when compared with any available non-target (control) tumour in the liver 
(receiving ThermoDox® alone), there will be improved radiological response following combined 
treatment (receiving ThermoDox® and FUS) 
3.2 
Definition of Primary, Secondary and Tertiary Outcomes 
Success of this study requires evidence of successful targeted drug release, in other words a quantifiable 
increase of doxorubicin concentration in the tumour following exposure to optimal levels of FUS for mild 
hyperthermia. A number of alternative analytical methods will be employed in order to obtain this evidence, 
each with its own statistical requirement for success, as shown in Table 4. The primary endpoint will employ a 
robust and validated analytical technique (HPLC), but at the same time requires an invasive biopsy. As 
tumour biopsy is already required for the primary endpoint, it is prudent to obtain as much information from 
this biopsy as possible. Consequently, in Part I, if enough material is available, each biopsy will be divided for 
microscopy for secondary and/or tertiary endpoints, using two approximate halves. In Part II, a single 
‘Released’ biopsy will be taken for HPLC immediately followed by another for analysis of the secondary and 
tertiary endpoints by microscopy, and so more tissue will be available. For the secondary biopsy-related 
endpoint, microscopy will be used to quantify levels of tissue necrosis, following optimal levels of FUS 
exposure, to explore hypothesis (1). For the tertiary biopsy-related endpoint, fluorescence microscopy will 
be used to quantify intra-tumoural drug concentration (hypothesis (2)) and its distribution, in particular the 
presence of nuclear doxorubicin (hypothesis (3)). 
If this study leads on to Phase II-III trials, it might be that other non-invasive methods of estimating intra-
tumoural drug quantification can be employed. For this reason such non-invasive exploratory objectives 
have also been included in this trial, namely MR-Spectroscopy and Plasma Pharmacokinetics (PKs). 
 
                                                          
 
6 “Mild hyperthemia” is defined in section 2.2, Key Definitions. 
7 When ThermoDox® is in its unreleased form it is non-bioavailable and thus cannot bind to cellular DNA. 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 14 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
4 
PATIENT GROUPS FOR ANALYSIS 
An “evaluable participant” is defined in section 2.2, Key Definitions. All evaluable participants in both Part I 
and Part II will be included in the final analyses. All patients enrolled in the study, will be accounted for and 
included in the analyses. The number of patients who were not evaluable, who died or withdrew before 
treatment began will be recorded. 
Variables will be analysed to determine whether the criteria for the study conduct are met. This will include 
a description of patients who did not meet all the eligibility criteria, an assessment of protocol violations and 
other data that impact on the general conduct of the study. 
Baseline characteristics will be summarised for all recruited and treated patients as detailed in section 6.2. 
Patients who died or withdrew before treatment started or do not complete the required safety 
observations will be described and evaluated separately. 
Treatment related toxicity will be tabulated by type and grade of toxicity as detailed in Section 8 (Table 9 and 
Table 10). There will also be a table of trial treatment and observed adverse events. All patients will be 
evaluable for toxicity from the time of their intervention using NCI CTCAE v 4.0 scoring. 
 
5 
QUALITY CONTROL AND DATA VALIDATION 
The following measures will be taken to ensure quality control and data validation: 
 
Trained radiologists and/or study clinicians will perform assessment of radiological outcome measures. 
 
In terms of data quality, the biopsy (for primary endpoint) and plasma pharmacokinetic analyses will be 
performed in Good Clinical laboratories Practice (GCP) by GCP-trained staff using validated techniques. 
 
Trained NHS, University of Oxford laboratory staff and/or study team members will perform microscopic 
analyses. A qualified histopathologist will perform reporting of haematoxylin and eosin (H&E) slides. 
 
In the case of HPLC analysis pertaining to the biopsy analysis for primary endpoint8 and plasma analysis 
for tertiary endpoint: 
o An internal standard will be used to normalise the HPLC curves, according to GCP principles. 
o Quality controls of high, medium and low concentration will be used before and after the patient 
sample chromatography run, and checked to ensure all QCs are in specification to ensure assay 
validity (see GCP GI/042/1 SOP). 
o A single individual will perform the analytical technique, validation checks and post-processing 
data analysis. 
o A second individual will independently check the results including validation, entry of correct 
tumour weights and correct transcription of the data results. 
o A summary of the concentrations of the QC and patient samples are printed using Empower 
                                                          
 
8 The analytical technique (HPLC) can be performed only once per biopsy sample due to limited tissue availability and thus best 
efforts are made to ensure that samples are successfully processed on the first (and only) attempt. 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 15 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
software. These will be signed and dated by the analyst and checked, signed and dated by an 
independent checker. This document will be stored in the trial file; a copy will be stored with the 
chromatograms. For reporting purposes, patient data are exported into an Excel spreadsheet 
and checked against the original. These will be signed and dated by the analyst and an 
independent checker prior to submission of the data. 
o Although HPLC results are not generally open to interpretation, in the case of any 
disagreements, a third party will perform an additional independent assessment on the primary 
endpoint data used in the final analysis. 
o The departmental leads for the Bioanalysis Core (Dr Lisa Folkes or Qualified Deputy) and the 
Churchill GCP Laboratories (Dr Kevin Myers) will sign off the resulting reports. 
 
Where tissue quantities allow, microscopy analysis will be performed with averaged multiple 
observations (n≥ 3) to eliminate spurious results. 
The TARDOX Trial Office may instigate audits as and when required.  Advanced notice will be issued.  Site 
staff are required to co-operate with the requirements of the audit and allow auditors Direct Access to all 
source information. 
A review of the final statistical report will be formed by a separate independent individual and documented 
in the Statistical report to ensure principles of this Statistical Analysis Plan have been followed.  
 
6 
DATA ANALYSIS 
6.1 
Outcomes & Endpoint Analysis 
Part I and Part II data will be combined for overall analysis of all endpoints at the end of the study, unless 
otherwise specified. Table 4 provides details of the statistical analysis of the primary outcome measure for 
this Phase I study (i.e. a two-fold increase in intra-tumoural doxorubicin concentration in at least 50% of 
evaluable participants).  
Specifically, Table 4 enumerates the methods of intra-tumoural drug quantification under evaluation; a cross 
(†) implies the observation will be performed at the following time points during the study intervention: 
 
Pre-treatment (Part I only) 
 
Unreleased (Part I only) 
 
Released (Parts I and II) 
For more information on the Pre-treatment, Unreleased and Released nomenclature, please refer to section 
2.2, Key Definitions. Timelines incorporating the time points are shown schematically in Figure 4 and Figure 5 
of the protocol. 
Table 5 covers the secondary endpoints, which concern safety aspects of the study and achieving optimal FUS 
hyperthermia, relating to hypothesis (1). 
 
Table 6 covers the remaining tertiary endpoint, concerning tumour response, relating to hypothesis (4). 
 
A double cross (‡) applies to Table 4 and Table 6 and implies that only patients receiving optimal FUS for drug 
release (as defined in Table 5) should be included in the statistical analysis for these endpoints 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 16 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
Evidencing the primary endpoint criteria determines overall success pertaining to the key study hypothesis. 
As summarised in Table 4, the concentration of intra-tumoural doxorubicin in a section of the Released 
biopsy will be compared to a section of the Unreleased biopsy (the control). A two-fold increase in total 
doxorubicin concentration from the Unreleased control following FUS, or a final (Released) concentration 
above a minimum threshold level would determine success for a given treatment. The primary endpoint 
requires independent success either criterion in at least 50% of evaluable patients for overall success of the 
study. In particular the first criterion aims to establish if there is increased accumulation of doxorubicin in 
the tumour following FUS exposure as per hypothesis (2). Currently there is no validated method to 
determine if there is increased intra-tumoural release as per hypothesis (3), as the HPLC assay is only able to 
quantify total doxorubicin. However release will be explored as a tertiary endpoint by fluorescence 
microscopy where possible9. The tertiary endpoints look at other methods of evidencing targeting 
doxorubicin delivery and are detailed in Table 4 and Table 6. 
The endpoints concerned with quantifying drug, Plasma Pharmacokinetics, MR-Spectroscopy and 
Radiological Response Evaluation all require statistical significance testing. A two-tailed Fisher Exact 
Probability test of 90% power with a 5% level of significance will be used to determine if there is a 
statistically significant increase following treatment relative to the control. GraphPad Prism software or 
equivalent will be used for this purpose (versions will be recorded in the Statistical report). 
During analysis of the Part I and Part II data, a modified CONSORT flow diagram will be completed as shown 
in the appendices, section 10.1. The information recorded on this chart will be useful in informing future 
similar studies. 
Type of 
endpoint 
Analytical 
Technique 
Quantifiable measure 
Primary measure 
Comparator 
(baseline) 
Statistical 
Requirement for 
success 
Primary 
Tumour 
pharmacokinetics 
using 
laboratory 
extraction 
technique followed 
by 
fluorescence 
HPLC10 
 
Concentration of total 
intra-tumoural 
doxorubicin† 
Section of Released 
biopsy 
from 
the 
treated tumour site 
following 
FUS-
induced 
mild 
hyperthermia 
Section 
of 
Unreleased biopsy 
from the treated 
tumour site11 
(Prior to final FUS 
exposure) 
Two-fold increase in 
doxorubicin 
concentration in at 
least 
50% 
of 
evaluable participants 
N/A 
Concentration above 
minimum 
threshold 
of 10μg/g in at least 
50% 
of 
evaluable 
participants 
Tertiary 
Fluorescence 
microscopy 
Average fluorescence 
intensity levels over a 
minimum 
area, 
in 
selected regions of 
interest† 
If 
tissue 
mass 
allows, section of 
Released 
biopsy 
from 
treated 
tumour 
If available, section 
of 
Unreleased 
biopsy from treated 
tumour 
or 
other 
suitable control12 
Two-fold increase in 
at 
least 
50% 
of 
evaluable 
participants‡ 
Average 
nuclear 
fluorescence intensity 
If 
tissue 
mass 
allows, section of 
If available, section 
of 
Unreleased 
Two-fold increase in 
nuclear fluorescence 
                                                          
 
9As doxorubicin is inherently fluorescent, fluorescence microscopy may be used as a technique to explore fluorescence distribution. 
If doxorubicin co-localises with a nuclear stain (e.g. DAPI) this shows that it is in its bio-available (released) form. 
10 Quantification using area under curve (AUC) on HPLC chromatogram, normalised by an internal standard, referenced from a 
standard curve to infer intra-tumoural doxorubicin concentration 
11 In Part II the Unreleased biopsy is not performed and therefore the average value for all evaluable tumours receiving intervention 
in Part I is used as a comparison for the two-fold increase from Unreleased to Released biopsy 
12 In Part II the Unreleased biopsy is not performed and therefore the average value for all evaluable tumours receiving intervention 
in Part I, or other suitable control tissue, is used as a comparison for the two-fold increase from Unreleased to Released biopsy 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 17 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
levels 
over 
a 
minimum number of 
nuclei, 
in 
selected 
regions of interest† 
Released 
biopsy 
from 
treated 
tumour 
biopsy from treated 
tumour 
or 
other 
suitable control11 
intensity in at least 
50% 
of 
evaluable 
participants‡ 
Tertiary 
 
MR-Spectroscopy 
Tumour:background 
ratio 
of 
signal 
intensity13 
at 
the 
following time points: 
1. 
Pre-treatment  
(on day -1) 
2. 
Post-treatment 
(on day 1 or 2) 
Mean 
post-
treatment 
tumour:background 
ratio 
Mean 
pre-
treatment 
tumour:background 
ratio 
Statistically significant 
(P<0.05) increase‡ 
Tertiary 
Plasma pharmaco-
kinetics 
using 
laboratory 
extraction 
technique followed 
by 
fluorescence 
HPLC 14 
Plasma concentration 
of total doxorubicin 
Released 
plasma 
sample 
Time-matched 
doxorubicin plasma 
pharmacokinetic 
data with clearance 
levels 
obtained 
from other clinical 
studies 
using 
ThermoDox® 
Statistically significant 
decrease 
(p<0.05) 
over time matched 
clearance levels‡ 
Table 4: Summary of quantifiable measures employed to evidence increase in the concentration of intra-tumoral doxorubicin in the 
TARDOX study, presented in terms of the primary and tertiary outcome measures. Combined Part I and Part II data will be analysed 
for all endpoints. 
Purpose 
Analytical 
Technique 
Quantifiable measure 
Primary measure & 
Comparator 
Statistical Requirement for success 
Part 
I 
only: 
To 
determine 
optimal 
FUS 
exposure 
parameters 
for 
a 
range of participant 
Body Mass Indices 
(BMIs) and tumour 
locations within the 
liver 
Analysis 
of 
real 
time 
thermometry 
obtained 
by 
thermistor 
1. 
Both cumulative 
and continuous 
minutes 
above 
drug 
release 
threshold 
 
2. 
Cumulative 
Equivalent 
in 
Minutes at 43°C 
(CEM43), in the 
region 
of 
the 
target 
tumour 
being heated by 
FUS 
Real-time 
thermometry 
data obtained during FUS 
intervention, in the first 
two 
hours 
post drug 
infusion, using implanted 
thermistor 
 
No 
comparator 
is 
required 
for 
either 
measure, but standards 
are set in the respective 
statistical requirements. 
1. 
Efficacy: 
Temperature 
is 
maintained above the drug 
release threshold (39.5°C) for 
at least 60 continuous seconds 
AND at least 300 cumulative 
seconds. Any FUS treatment 
with a thermometry meeting 
these criteria will be defined 
as having ‘optimal FUS’ for 
drug release. 
2. 
Safety: Temperature does not 
exceed 25% of CEM43 thermal 
dose threshold for cell death 
[6, 7]15 
                                                          
 
13 The spectroscopy signal analysed may not be doxorubicin itself; metabolites or breakdown products of ThermoDox® may be 
utilised 
14 Quantification using area under curve (AUC) on HPLC chromatogram, normalised by an internal standard, referenced from a 
standard curve to infer intra-tumoural doxorubicin concentration 
 
15 In-house software is used to convert temperature history into cumulative equivalent minutes (CEM) at a reference temperature of 
43°C (CEM43). The thermal dose model by Sapareto and Dewey defines that it takes 240min for >96% cell death at 43°C, and this 
time halves for each subsequent degree rise i.e. t=900s at 47°C, t=3.52s at 55°C, t=0.11s at 60°C. As the critical thermal dose for liver 
tissue is CEM43=340 minutes, we have defined CEM must be below 25% of this, i.e. 85 minutes, to satisfy safety. 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 18 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
To assess the safety 
of FUS-induced mild 
hyperthermia 
for 
drug delivery  
Standard H&E 
Microscopy 
Percentage of cell or 
tissue 
necrosis 
following 
FUS 
exposure, as assessed 
by microscopy report 
If 
tissue 
available, 
microscopy 
using 
the 
Unreleased 
(and/or 
Released) Biopsy sample 
in Part I and the Released 
Biopsy sample in Part II 
 
The Pre-treatment biopsy 
sample or mean of other 
of other suitable controls 
will be used as the 
baseline comparator16 
Mean percentage cell or tissue 
necrosis less than 30%, or an 
increase in necrosis by no more 
than 25% over baseline comparator 
in at least 50% of evaluable 
participants 
Note: some tumours are highly 
necrotic and so persistence of cell 
viability stain is another method, 
which may be used. 
Fluorescence 
microscopy 
Persistence 
of 
cell 
viability stain 
Presence of cell viability stain 
anywhere in the sample confirms 
absence of complete ablation 
Adverse event 
reports 
Adverse 
events 
deemed related to 
FUS 
 
Quantity and severity of 
adverse events deemed 
related to FUS up to 30 
days post-intervention 
No comparator required 
No statistical test required. Safety 
data with regards to FUS-related 
adverse events to be reviewed by 
the 
Trial 
Steering 
Group 
on 
completion of both Parts I and II 
To assess the local 
and 
systemic 
cytotoxic effects of 
ThermoDox® in this 
setting 
Adverse event 
reports 
Significant 
bone 
marrow suppression, 
deranged 
liver 
function 
and 
liver 
toxicity 
Quantity of Grade 3 and 4 
laboratory results from 
blood tests at each time 
point post intervention 
No statistical test required. Safety 
data with regards to ThermoDox®-
related adverse events to be 
analysed at the end of the study 
Adverse event 
reports 
Adverse 
events 
deemed related to 
ThermoDox® up to 30 
days 
post-
intervention 
Quantity and severity of 
adverse events deemed 
related to ThermoDox® 
up to 30 days post-
intervention 
Table 5: Summary of safety-related quantifiable measures assessed in the TARDOX study, presented in terms of the secondary 
outcome measures 
Purpose 
Quantifiable measure 
Primary measure & Comparator 
Statistical Requirement for success 
To 
determine 
therapeutic 
effect 
on 
the 
target 
tumour 
Radiological evidence of 
tumour response in the 
target tumour alone, as 
assessed by Principles of 
CHOI and RECIST response 
evaluation using MRI and 
CT [1, 2] and SUVmax using 
PET-CT [3] 
 
Scan(s) to be performed 
within 
60 
days 
of 
CHOI response evaluation [1] using 
follow-up CT scan demonstrating most 
significant response to target * 
For both criterion, the baseline CT scan 
prior to intervention (day -1) will be used 
as the comparator 
In addition, for the first criterion, control 
tumours exposed to drug alone will be 
used as a comparator to demonstrate an 
overall statistically significantly response 
rate 
For each of the following paired 
analyses‡: 
 
CT / CHOI 
 
CT / RECIST 
 
MRI / RECIST 
 
At least one of the following must hold 
true: 
 
1. 
A statistically significant (p<0.05) 
                                                          
 
16 In Part II the Pre-treatment biopsy is not performed and therefore the average value for all evaluable tumours receiving 
intervention in Part I, or other suitable control tissue, is used as the baseline comparator 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 19 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
intervention 
 
RECIST response evaluation17 [2] using 
both follow-up CT and MRI scans 
demonstrating most significant response 
to target *,18 
For both criterion, the baseline CT and 
MRI scans prior to intervention (day -1) 
will be used as the comparator 
 
In addition, for the first criterion, control 
tumours exposed to drug alone will be 
used as a comparator to demonstrate an 
overall statistically significantly response 
rate 
number of FUS-targeted tumours 
showing 
Complete 
or 
Partial 
Response (CR or PR) over all 
assessed 
control 
tumours 
showing Stable or Progressive 
Disease (SD or PD) 
 
2. 
Complete Response (CR) or Partial 
Response (PR) in one or more 
modalities for 50% or more of 
FUS-targeted tumours overall 
 
PET-CT SUVmax [3] response evaluation 
using 
follow-up 
PET-CT 
scan 
demonstrating 
the 
most 
significant 
response * 
The baseline PET-CT scan prior to 
intervention (day -1) is used as the 
comparator and, in addition, a control 
tumour where available 
For the following paired analyses‡: 
 
 
PET-CT / PERCIST 
 
The following must hold true: An 
SUVmax response in 50% or more 
targeted 
lesions 
overall, 
where 
response is defined, using PERCIST 
principles [8], as a reduction in SUVmax 
of over 30% over baseline 
Table 6: Summary of response-related quantifiable measures required in the TARDOX study, presented in terms of the tertiary 
outcome measures. 
* There will be two follow up scan visits, both for MR/MR-Spectroscopy and CT/PET-CT, in the 60-day follow-up period. In this proof 
of concept study only a single cycle of chemotherapy is given thus any radiological response may be transient. Consequently the scan 
demonstrating the most significant response should be used for response evaluation. 
 
** If more than one control tumour has been evaluated for the same PET-CT scan, the average SUVmax value of all evaluated control 
tumours will be used. 
 
6.2 
 Patient Characteristics 
During the final analysis a summary of patient characteristics will be generated to summarise the various 
attributes and evaluability of the participants recruited and treated in the study. The following table details 
the patient characteristics recorded for each participant, and the summarised data that will be reported. 
 
Patient Characteristic 
Data-type 
Summarised Data for 
Report 
Treated Tumour Type  
Enumeration (Primary, 
Secondary) 
Total and % Primary 
Total and % Secondary 
Primary cancer site 
Free Text 
Total and % of each category 
e.g. Colorectal, Lung, Breast, 
Upper GI, Primary HCC, Primary 
CCA... 
Age 
Integer 
Median 
Sex 
Enumeration (Male, Female) 
Total Female 
Total Male 
BMI 
Decimal 
Mean + standard deviation 
                                                          
 
17 RECIST criteria allows up to a maximum of five tumour foci to be considered in the evaluation of response. In this proof of concept 
study, for any one participant, only a single tumour will receive the intervention (targeted drug delivery with focused ultrasound). 
Any other ‘control tumours’ in the liver will be exposed to ThermoDox® alone. 
18 Note the MRI scan performed on the day following treatment is excluded as it is not a “follow-up” scan for tumour response. 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 20 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
WHO Performance Status 
Enumeration (0,1,2,3,4) 
Total PS=0 
Total PS=1 
FUS targeted tumour 
Anatomical location 
Free-text including segment as 
per Couinaud Classification 
Total for each segment (I-VIII) 
Estimated tumour volume 
Decimal 
Mean + standard deviation 
Estimated % of tumour volume treated 
with FUS 
Decimal 
Mean + standard deviation 
Depth from skin to closest border (cm) 
Decimal 
Mean + standard deviation 
Depth from skin to furthest border 
(cm) 
Decimal 
Mean + standard deviation 
Largest axial dimension (cm) 
Decimal 
Mean + standard deviation 
No. selected control liver lesions 
Integer 
Median 
Consenting patients completing intervention 
Enumeration (Y, N) 
Percentage completing 
Follow-up 1 completed 
Enumeration (Y, N) 
Percentage completing 
Follow-up 2 completed 
Enumeration (Y, N) 
Percentage completing 
Evaluability Assessment 
1o Total Doxorubicin 
Enumeration (Y, N) 
Percentage Evaluable 
Percentage Non-evaluable, of 
which breakdown for each 
endpoint of relevant cause, for 
example: 
 
Not performed due to: 
1. 
Method unavailable 
2. 
Patient refusal 
3. 
Lost in follow-up 
Attempted but failed due to: 
4. 
Inadequate sample 
5. 
Inadequate control 
6. 
Method failure 
2o Safety – thermometry 
2o Safety - cell viability 
2o
 Safety - FUS AEs  
2o
 Safety - drug AEs 
2o
 Safety – Grade 3,4 blood results 
3o
 Fluorescence microscopy 
3o
 Spectroscopy 
3o Response – CHOI by CT 
3o Response – RECIST by CT 
3o Response – RECIST by MRI 
3o Response - SUVmax by PET-CT 
3o
 Plasma pharmacokinetics 
Table 7: Patient characteristics and evaluability recorded individually for each patient and summarised in final report 
6.3 
Missing Data 
For a given subject, a single piece of non-evaluable data does not mean the entire subject in non-evaluable, 
as each endpoint evaluation will include all available evaluable data (see section 2.2). However, if for a given 
endpoint, the total number of evaluable data points (participants) is less than 10 then that endpoint will be 
deemed non-evaluable. Details on replacement can be found in section 2.8 (Sample Size). 
6.4 
Pre-specified Subgroup Analysis 
Note that in large tumour volumes (>50cc), it is often not possible to cover the entire tumour volume in a 
single FUS session. Consequently any radiological response may be confined to a smaller volume of a larger 
tumour volume. Radiological endpoints do not take this into consideration and consider the tumour in its 
entirety. It is likely that, following close of study and initial endpoint analysis, an additional post-hoc analysis 
will be performed to look at radiological response in the subset of smaller tumours receiving optimal FUS. 
No other subgroup analyses are planned for this open label Phase I study. 
6.5 
Sensitivity Analysis 
Not applicable: No sensitivity analyses will be performed for this open label Phase I study. 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 21 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
6.6 
Blinded analysis 
Not applicable in this open label Phase I study. 
6.7 
Meta-analyses 
No meta-analyses will be performed in this open label Phase I study. 
6.8 
Outcomes Assessment Schedule 
Table 8 summarises the data that will be captured for the purposes of outcome assessment in the format of a 
schedule and consists of both CRF and non-CRF data (see Data Management and Sharing Plan for more 
information).Specifically, the non-CRF will be captured in the electronic Trial Master File (eTMF) only, and 
they include anatomical, FUS exposure parameters, and real-time thermometry data (see section 6.9). Blood 
results will also be captured in OpenClinica. Standard procedures will be followed with respect to retention 
and archiving of the eTMF. Where an item is missing (e.g. limited biopsy tissue), or non-evaluable due to 
technical difficulties it will be entered as “Non-Evaluable”. Trial statistician will bring all the data together 
both for analysis and for archiving at the end of the trial. Both ‘raw’ data and processed data from the 
various imaging scans will be retained and archived for a minimum of 5 years as per University of Oxford’s 
policy. Digital image files will not be stored in the eTMF to avoid unnecessary duplication. In case of an 
image-related query that arises following image analysis and data capture, it is possible for the radiology 
department to revisit any given scan and amend a report, due to the automatic archive facility on the Oxford 
University Hospitals NHS Trust servers. 
Data Item to be 
Captured 
Data type description Data type storage Relevant 
Endpoint 
Day(s) when data captured (tolerance)  
Day 1=intervention 
 
1 
1-2 
15 
(±3) 
<60 
(flexible) 
Biopsy for drug level 
Doxorubicin conc.*** 
Decimal Quantity 
Primary 
X 
 
 
 
Real-time thermometry 
for duration of FUS 
treatments (Part I only) 
(Duplicated for each FUS 
exposure attempt) 
Duration of FUS 
exposure attempt 
Decimal Quantity 
Secondary 
 
X 
 
 
 
Baseline, mean, median, 
peak and trough 
temperatures19 
Decimal Quantity 
X 
 
 
 
 
Cumulative drug-release 
time (>39.5°C) 
Minutes : Seconds 
Max. continuous drug-
release time (>39.5°C) 
Minutes : Seconds 
CEM43 time 
Minutes : Seconds 
Haematology tests 
Blood results20 
Decimal Quantities 
Secondary 
 
X 
X 
X 
Biochemistry tests 
Blood results21 
Decimal Quantities 
Secondary 
 
X 
X 
X 
Adverse Event Review 
Adverse events details 
inc. severity 
Tabular 
Secondary 
X 
X 
X 
X 
Plasma for drug level 
Doxorubicin conc.** 
Decimal Quantity 
Tertiary 
X 
 
 
 
                                                          
 
19 Note that this data will be extracted from the ‘raw’ thermometry trace data using in-house analysis software. In particular this 
data will be presented in the interim analysis (see section 5) and used to determine if optimal FUS parameters have been 
established. 
20 Full Blood Count (FBC) to include Haemoglobin (Hb), white blood cells (WBC) with differential count (neutrophils and 
lymphocytes) and platelets 
21 Biochemical profile to include U&Es, (Na+, K+, Ur, Cr), phosphate, calcium, LFTs (total protein, ALP, Albumin, ALT or AST, Bili, LDH) 
 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 22 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
MR Spectroscopy 
Tumour:bkgd Ratio 
Decimal Quantity 
Tertiary 
 
X 
 
 
CT Liver 
CHOI Outcomes 
(target & controls) 
Enumeration 
(CR, PR, SD, PD) 
Tertiary 
 
 
X* 
X* 
CT Liver 
RECIST 1.1 Outcomes 
(target & controls) 
Enumeration 
(CR, PR, SD, PD) 
Tertiary 
 
 
X* 
X* 
MRI 
RECIST 1.1 Outcomes 
(target & controls) 
Enumeration 
(CR, PR, SD, PD) 
Tertiary 
 
 
X* 
X* 
Full Body  PET-CT 
SUVmax value 
(target & controls) 
Decimal Quantity 
Tertiary 
X 
 
X* 
X* 
Biopsy for fluorescence 
Avg. Bkgd Fluorescence 
Decimal Quantity 
Tertiary 
X 
 
 
 
Avg. Nuclear Fluoresc. 
Decimal Quantity 
Tertiary 
X 
 
 
 
Biopsy for cell viability 
% Necrosis 
Percentage 
Tertiary 
X 
 
 
 
Persistence of cell 
viability stain 
Enumeration 
(Y/N) 
Tertiary 
X 
 
 
 
Table 8: schedule of possible data capture points for purposes of outcome assessment. Note that additional data captured that is not 
relevant to outcome assessment has been excluded. 
* In Part II, the number of follow-up MRI/MR-Spectroscopy, Liver pCT and FDG PET-CT scans may be reduced, if the data from Part I 
informs that a more efficient (in terms of reducing patients & site requirements) schedule is possible 
** For plasma pharmacokinetics, three blood samples in both Phases I and II (Pre-Treatment, Unreleased drug, Released drug) will be 
assayed. 
*** For the biopsy tissue for HPLC, section(s) of three biopsies in Part I (Pre-Treatment, Unreleased drug, Released drug) and a two 
serial biopsies in Part II (both at the Released drug time-point) will be assayed. 
6.9 
Data Management Responsibility 
The OCTO data management team will design TARDOX CRF and identify which data items will be captured in 
the subjects’ clinical care and electronic records, therefore can be captured in the CRFs (‘CRF data’), and 
those which will be generated by collaborating groups without being returned to the clinical care record 
(‘non-CRF data’). 
The non-CRF data includes: 
 
Pre-treatment, Unreleased and Released Plasma and Biopsy PK analytical chemistry results: 
o Obtained by HPLC in the Bioanalysis Core and GCP Laboratories based in the Old Road 
Campus Research Building 
o Batch analysis of the samples will produce chromatograms generated electronically by the 
HPLC equipment, which can then be used to calculate doxorubicin concentrations by hand 
 
FUS delivery parameters: 
o Captured from reports written by members of the study team at the time of treatment and 
the treatment report generated by the HIFU device 
 
Thermistor data output: 
o The thermistor captures data on the tumour temperature over time and produces an 
electronic dataset which will be analysed in the Institute of Biomedical Engineering 
 
If available, Pre-treatment, Unreleased and Released Microscopy study data output: 
o Samples will be analysed by fluorescence microscopy in the GCP Laboratories based in the 
Old Road Campus Research Building 
o Microscopic analysis of the samples will produce images that may be used to evaluate cell 
viability post-FUS and/or presence of nuclear doxorubicin 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 23 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
 
If available, MR-spectroscopy data output: 
o Generated by the Churchill Radiology Department, Oxford University Hospitals NHS 
Foundation Trust 
The collaborators undertaking these analyses are responsible for producing datasets using validated 
methods where possible. The unique trial patient ID will be used to identify these data sets. The raw data 
output will be stored securely and backed up regularly. The final listings produced from these data, for use in 
statistical analysis, will be retained by the collaborating investigators, and stored securely and backed up 
regularly. 
A report on the methodology used to derive the listings will be produced for methods that are not standard 
practice. This report will be filed in the eTMF, along with information indicating the location and archiving 
arrangement of the raw data (in the event that this is not the eTMF). 
All other data items will be captured on the trial-specific CRFs. 
The data management of CRF data will be compliant with GCP and OCTRU data management SOPs, CRF data 
will be managed via a web-based, bespoke trial database using OpenClinica. OpenClinica is a dedicated and 
validated clinical trials database designed for electronic data capture. The OCTO data management team are 
responsible for producing as complete and clean data set as possible. The CRF data management plan is 
incorporated in the central monitoring plan. 
The trial statistician will receive a copy of the final non-CRF listings and also the CRF data extractions. The 
statistician will be responsible for linking the various datasets. Data transfer will comply with OCTRU SOP 
GEN-031. The listings will be stored unamended in the eTMF. The statistical analysis processes will be 
undertaken in accordance with OCTRU SOPs. 
 
7 
DATA SAFETY MONITORING COMMITTEE AND INTERIM ANALYSES 
There is no Data Safety Monitoring Committee (DSMC) and an Independent Trial Steering Committee (ITSC) 
will be in place to monitor safety (please refer to section 18 of the study protocol). 
The investigator team (including clinical fellow(s) and lead FUS clinician) will independently monitor FUS 
performance for at least 5 patients in Part I. When the investigator team are confident in achieving optimal 
levels of hyperthermia for a range of BMIs and anatomical tumour sites, a meeting with the Trial 
Management Group (TMG) will be called. This decision is expected between the 5th and 14th recruited 
patients, with target recruitment being 1.2 patients per month. The TMG will use results from interim 
analysis review to determine if and when Part II can open in parallel with Part I. In particular only the 
secondary objective will be considered, which concerns optimal FUS exposure parameters. This is because in 
Part II there will be no real time thermometry during FUS exposure as a thermistor is not placed. 
Consequently, at the time point at which the investigator team feel enough experience in determining 
optimal FUS parameters has been obtained in Part I, at TMG interim analysis will be called to review the 
following data for each treated patient: 
 
 
Demographic data: 
o Sex 
o Age 
 
Anatomical data: 
o Body Mass Index (BMI) 
o Anatomical position of liver lesion treated (including segment) 
o Estimated percentage volume of liver lesion treated 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 24 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
o Depth from skin to closest border in cm 
o Depth from skin to further border in cm 
o Largest tumour dimension in cm 
 
FUS exposure parameters22 for each FUS exposure, including no less than the following: 
o Unique patient identifier and date of treatment 
o Overall exposure time (s) 
o Frequency (MHz) 
o Duty cycle (%) 
o Average Power (Watts) 
o Overall Energy (KJ) 
 
Real-time thermometry data for each FUS exposure attempt, including no less than: 
o Unique patient identifier and date of treatment 
o Baseline body temperature23 
o Date and time of the start of the real time thermometry monitoring 
o Duration of FUS exposure attempt (MM:SS) 
o Peak & trough temperatures attained (oC) 
o Mean and median (to one decimal place) (bulk) temperature24 
o Cumulative and maximum continuous durations at which the drug-release threshold was 
met (MM:SS) 
o CEM43 time (MM:SS) 
 
Where possible (in some cases limited tissue biopsies may mean this data is not available), cell 
viability data obtained for the purposes of the secondary objective 
 
Up-to-date adverse event data relating to FUS exposure 
The summary data listed will be provided by the investigator team, in tabular format, for example as shown 
in Appendices 4 and 5. Start time and end times of the real-time thermometry recording for each FUS 
exposure attempt (or treatment plan) will be recorded by hand and transcribed into the real-time 
thermometry proforma (Appendix 4). Real-time raw thermometry data will be acquired using a clinically 
approved Temperature Probes that is connected to a personal computer (PC) running bespoke data 
acquisition software (running on the LabView platform) developed by the Institute of Biomedical Engineering 
(IBME). An in-house MatLab script, developed by the IBME, will be used to process thermometry data in 
order to generate the required data summary for each FUS exposure attempt (see Appendix 3). The JC-200 
therapeutic device software will automatically generate the FUS exposure parameters in the final treatment 
report for each FUS exposure attempt, which will be transcribed to the FUS exposure proforma (Appendix 5). 
Cell viability will be evaluated either using microscopy assessments or flow cytometric analysis using 
specialist software (ImageJ/FIJI for microscopy or equivalent) at the University of Oxford. All raw data will 
ultimately be stored on the Statistical eTMF to allow for future reproducibility of results, along with the 
analysed data to be presented. 
With regards to optimising FUS exposure parameters, it is difficult to perform a blanket statistical test for 
this rather disparate dataset, as there are many parameters that can be adjusted when applying FUS and 
‘optimal FUS’ is somewhat subjective. When reviewing thermometry traces for drug delivery, the key 
                                                          
 
22 If it was required to modify the FUS parameters in order to achieve the optimal range of hyperthermia, for example by increasing 
changes the in duty cycle, then each set of FUS parameters will be presented. 
23 This is the temperature immediately prior to first FUS exposure, as shown on the thermometry trace 
24 Please note that large volumes of real time thermometry data may be generated for each FUS session and there may be periods 
of acquisition where no heating is taking place. It is the ‘bulk’ temperature recorded by the thermistor during FUS exposures, which 
is of most physiological relevance. 
 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 25 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
consideration is to ensure that the bulk tumour temperature, as measured by the thermistor, exceeds the 
drug delivery threshold (39.5°C) as quickly as possible after drug administration and is sustained for as long 
as practically possible, given the patient is under a general anaesthetic. Ideally the desired range of 
hyperthermia is maintained (41-47°C), such that temperature remains below the safety limits for ablation, 
and remains comfortably above the drug release threshold for a prolonged and ideally continuous period. 
The statistical tests in Table 5 go some way to capture these thermometry attributes in order to define what 
is ‘optimal FUS’ in the context of this study. 
In view of this collective data set, at the point at which both the investigator team and the TMG are satisfied 
that sufficient FUS experience has been gained to safely achieve the desired range of mild hyperthermia for 
a range of anatomical tumour locations and BMIs, the trial will progress to Part II. In Part II the FUS exposure 
parameters will be determined without a thermistor using Part I data combined with planning calculations 
and/or simulations performed at the Institute of Biomedical Engineering (IBME). Once the decision has been 
made to open Part II, patients can be recruited to either part based on previous experience and safety 
considerations. The TARDOX trial office will also inform the trial site and notify Celsion. At the point at which 
this decision is made, any patients who have already been registered and assigned to Part I, but have not yet 
had intervention, will be considered for transfer to Part II, subject to participant consent. 
 
The study will recruit up to a maximum of 28 evaluable participants across two separate Parts (Part 1 and 
Part 2). A minimum of 5 participants are required in Part 1 before Part 2 can be opened. Recruitment into 
Part II will not take place until the TMG board is in agreement for this transition. 
 
Following TMG review, other possible outcomes are as follows: 
 
It is possible to open to Part II in parallel to Part I 
 
Ethical approval will be sought to continue recruiting up to the maximum of 28 patients to Part I, 
with a thermistor, without transferring to Part II at any stage 
 
The study should be terminated at this stage due to safety concerns resulting from Part I data 
 
Following this TMG review the Statistical Analysis Plan will also be reviewed in light of available patient data. 
Although analysis will not be completed until the end of Part II, the statistical tests will examined at this 
point to ensure test validity in anticipation of the final combined analysis of Parts I and II. 
8 
SAFETY ANALYSIS 
Adverse event (AE) monitoring starts at intervention (Day 1) until 30 days post intervention (day  30). 
The 
Investigator will monitor each patient for clinical and laboratory evidence of adverse events on a routine 
basis throughout the study. Should an Investigator become aware of any study drug related SAEs following 
this period these must also be reported as stated below. All reportable AEs will be followed to a satisfactory 
conclusion. Any reportable AEs that are unresolved at the patient’s last visit in the study are followed up by 
the Investigator for as long as medically indicated, but without further recording in the CRF. 
All AEs reported to the trial office will be processed according to internal SOPs, this includes the reporting 
procedure and assessment of causality and expectedness for SAEs. The TARDOX trial office may request 
additional information for any AE as judged necessary. 
AEs are captured in secondary endpoints if deemed to be related to ThermoDox® or related to FUS. 
AEs related to ThermoDox® will be reported in tabular format as detailed in Table 9. This table follows a 
similar structure to that shown in a previous publication regarding use of ThermoDox® in human subjects, in 
particular, for illustrative purposes, Table 2 of that publication is populated [9]. Furthermore, procedural-
related AEs will be presented in a similar tabular format as demonstrated by Table 10. 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 26 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
System organ class / AE 
Total AEs n (%) 
AEs Grade 3 or 
more n (%) 
Drug-related n (%) 
SAEs n (%) 
Metabolic/laboratory 
 
 
 
 
Blood and lymphatic system disorders 
 
 
 
 
Gastrointestinal disorders 
 
 
 
 
Constitutional symptoms 
 
 
 
 
Skin and subcutaneous tissue disorders 
 
 
 
 
Pain 
 
 
 
 
Hepatobiliary disorders 
 
 
 
 
Pulmonary/upper respiratory 
 
 
 
 
Infections 
 
 
 
 
Renal/genitourinary disorders 
 
 
 
 
Haemorrhage 
 
 
 
 
Cardiac general 
 
 
 
 
Musculoskeletal 
 
 
 
 
Lymphatics 
 
 
 
 
Neurology 
 
 
 
 
Table 9: Frequency listing of example drug-related adverse events, occurring in patients treated with ThermoDox®. 
 
System organ class / AE 
Total AEs n (%) 
AEs Grade 3 or 
more n (%) 
Drug-related n 
(%) 
SAEs n (%) 
General Procedural 
complication deemed 
not related to FUS or 
biopsy 
Anaesthetic-related e.g. dysrhythmias 
 
 
 
 
Post-operative ileus 
 
 
 
 
DVT/PE 
 
 
 
 
Other 
 
 
 
 
Procedural complication 
deemed related to FUS 
Procedural pain 
 
 
 
 
Discomfort at treatment site 
 
 
 
 
Skin toxicity / blistering at treatment site 
 
 
 
 
Oedema at treatment site 
 
 
 
 
Damage to other structure(s) 
 
 
 
 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 27 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
Procedural complication 
deemed related to 
Biopsy 
Procedural pain 
 
 
 
 
Haemorrhage 
 
 
 
 
Vasovagal hypotension 
 
 
 
 
Damage to other structure(s) 
 
 
 
 
Table 10: Frequency listing of example procedural adverse events deemed unrelated to FUS or biopsy, related to FUS, or related to 
biopsy  
  
9 
APPENDICES 
9.1 
Appendix 1: Glossary of Abbreviations  
AE 
 
Adverse Event 
CI 
 
Chief Investigator 
CEM43  
Cumulative Equivalent in Minutes at 43°C 
DSMC  
Data and Safety Monitoring Committee 
DMP 
 
Data Management and Sharing Plan 
IBME 
 
Institute of Biomedical Engineering  
IHC 
 
Immunohistochemistry 
ITSC 
 
Independent Trial Steering Committee 
SAE 
 
Serious Adverse Event 
SAP 
 
Statistical Analysis Plan 
TMG 
 
Trial Management Group 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 28 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
10 DOCUMENT HISTORY 
Ver 
Date 
Who 
Comments 
2.1 
 
 
 
 
 
 
 
 
 
 
 
 
26Jan2016 
 
 
 
 
 
 
 
 
 
 
 
 
PCL 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Third major revision of SAP based on Protocol version 3.0 Feb, 2016 
(substantial amendment), including updates to reflect: 
 
Staff members who have been most actively involved in the 
study. 
 
Minor changes to secondary endpoints with respect to 
microscopy analysis. 
 
Implementation of CEM43 thermal dose threshold model to 
evaluate FUS safety 
 
Provision for parallel opening of Parts I & II. 
 
Provision for scan follow-up period of up to 60 days 
 
Changes to recruitment strategy. 
2.1 
31Mar2016 
LO 
Co-author and revision to conform with new SAP template 
2.1 
01May2016 
LO 
Revision to reflect Protocol V3.0 
2.2 
14Jun2016 
LO 
Harmonise comments from PL for consistency 
2.3 
16Jun2016 
LO 
A new version reflecting minor changes on opening Part II  
2.3 
 
2.4 
 
2.5 
 
 
16Jun2016 
 
16Jun2016 
 
28Jun2016 
PCL 
 
LO 
 
LO 
Minor changes to formatting and clarifications 
Minor changes relocated history of changes  
 
Updated designations and key personnel details 
 
 
 
 
 
 
 
 
 
 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 29 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
10.1 
Appendix 2: Modified CONSORT Flow Diagram 
 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 30 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
10.2 
Appendix 3: Example Processed Real-Time Thermometry Trace 
The example thermometry trace below has been generated using a clinically approved thermistor and 
processed using an in-house MatLab program developed at the IBME, which is generates the CEM43 
calculations. Please note that this example real-time trace spans only two minutes, whereas for the 
intervention the traces are likely to span for several hours given time required for treatment planning and 
other procedures (biopsy). Thus, during any given patient intervention, the real-time thermometry traces are 
likely to extend significantly before and after FUS exposure(s), which may only represent a small portion of 
the total monitored period.  
For this reason, the software used to capture the thermometry data allows the user to define discrete 
treatment periods, representing FUS exposures, in real time using a switch function on the in-house LabView 
graphical user interface. To demonstrate this, three treatment periods were simulated in the trace below. 
During these user-defined treatment periods, the thermistor tip was placed into hot water. Note that during 
the study intervention it is intended that a separate thermometry trace be generated and processed for each 
individual FUS exposure attempt, each with a single treatment period, rather than multiple periods over a 
single continuous trace. 
The blue plot represents the real-time thermometry trace, the area between the dotted horizontal lines 
represent the optimal temperature range and the pink rectangles represent the user defined treatment 
periods. In addition, the MatLab program automatically performs statistical analyses on the raw 
thermometry data, for both the total trace and treatment periods separately, which is output as an overlay 
on the plot (bottom left). If available, the treatment only data will be preferentially captured in the real-time 
thermometry proforma (appendix 4), to avoid analysis of trace during prolonged periods of FUS inactivity. 
 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 31 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
10.3 
Appendix 4: TARDOX Real-Time Thermometry Data Capture Proforma 
 
Unique Subject Trial Number: 
 
 
Baseline body temp (°C): 
 
Tumour Location (liver lobe, segment): 
Sex: 
 
 
Age: 
BMI: 
ThermoDox® Infusion 
Start-Stop Time (HH:MM) 
Water Temp. (°C): 
Comments: 
 
 
 
FUS 
Exposure 
Attempt 
No. 
Monitoring 
Start Time 
(HH:MM) 
Monitoring 
Stop Time 
(HH:MM) 
Temps. (oC) during total monitoring / 
treatment* period 
                                                    (*Circle measure used)     
Durations 
(MM:SS), or MILLISEC (MS) 
Peak 
Trough 
Mean 
Median 
39.5-47  40-47 
41-47 
>47 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Optimal FUS achieved? (Y/N): 
Post-drug cumulative time > 39.5°C (>300s) = 
Post-drug max. continuous time > 39.5°C (>60s) =  
CEM43 Threshold Exceeded? (Y/N): 
Post-drug CEM43 time =  
10.4 
Appendix 5: FUS Exposure Parameters Data Capture Proforma 
Unique Subject Trial Number: 
 
 
Depth to closest and 
furthest tumour borders 
from skin (cm): 
 
Tumour Dimensions (mm) and 
Volume (cc): 
Sex: 
 
Age: 
BMI: 
Intercostal space (mm) 
Thickness of skin & Subcutaneous 
Tissues (mm) 
JC200 Transducer used, frequency and focal length: 
 
Estimated percentage of 
tumour volume treated 
(%): 
 
Prescribed FUS tumour volume 
post-drug (cc) 
Comments: 
 
 
 
FUS 
Exposure 
Attempt No. 
FUS Start 
Time 
(HH:MM) 
FUS Stop 
Time 
(HH:MM) 
Power 
(Watts) 
Pulse 
Int. 
Slice 
Thickness & 
Separation 
Duty 
Cycle 
(%) 
Mode 
(Dot/Linear) 
and Spacing 
/Speed 
Overall 
Energy 
(KJ) 
 
Overall 
time (s) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
Confidential 
______________________________________          
SAP Version No: 3.0 
 
OCTRU-OST-001_V1.0_25Mar2014 
Date: 22Jul2016 
 
Effective Date 08Apr2014  
SAP Authors: Paul Lyon and Lang’O Odondi 
Page 32 of 32 
 
TARDOX STUDY PROTOCOL 
Targeted chemotherapy using focused ultrasound for liver tumours 
Funded by NIHR Oxford Biomedical Research Centre & Celsion Corporation, ISRCTN1234567; OCTRU trial identifier 
 
11 REFERENCES 
1. 
Choi, H., et al., Correlation of computed tomography and positron emission tomography in patients with 
metastatic gastrointestinal stromal tumor treated at a single institution with imatinib mesylate: proposal of new 
computed tomography response criteria. J Clin Oncol, 2007. 25(13): p. 1753-9. 
2. 
Eisenhauer, E.A., et al., New response evaluation criteria in solid tumours: revised RECIST guideline (version 
1.1). Eur J Cancer, 2009. 45(2): p. 228-47. 
3. 
Kinahan, P.E. and J.W. Fletcher, Positron emission tomography-computed tomography standardized uptake 
values in clinical practice and assessing response to therapy. Semin Ultrasound CT MR, 2010. 31(6): p. 496-
505. 
4. 
Parkin, D.M., et al., Global cancer statistics, 2002. CA Cancer J Clin, 2005. 55(2): p. 74-108. 
5. 
Khan, S.A., et al., Rising trends in cholangiocarcinoma: is the ICD classification system misleading us? J 
Hepatol, 2012. 56(4): p. 848-54. 
6. 
Sapareto, S.A. and W.C. Dewey, Thermal dose determination in cancer therapy. Int J Radiat Oncol Biol Phys, 
1984. 10(6): p. 787-800. 
7. 
Graham, S.J., et al., Quantifying tissue damage due to focused ultrasound heating observed by MRI. Magn 
Reson Med, 1999. 41(2): p. 321-8. 
8. 
Wahl, R.L., et al., From RECIST to PERCIST: Evolving Considerations for PET response criteria in solid 
tumors. J Nucl Med, 2009. 50 Suppl 1: p. 122S-50S. 
9. 
Poon, R.T. and N. Borys, Lyso-thermosensitive liposomal doxorubicin: a novel approach to enhance efficacy of 
thermal ablation of liver cancer. Expert Opin Pharmacother, 2009. 10(2): p. 333-43.