Trial Information

Treatment of Hepatocellular Carcinoma (HCC)by Selective Traditional Chemoembolization(TACE)Versus Selective TACE Via Microspheres Loaded With Doxorubicin: a Multicentre,Randomized,Open Label,Controlled Study.

INTRODUCTION

Hepatocellular carcinoma (HCC) is one of the most frequent malignancies in man. Liver
transplantation, surgical resection and percutaneous ablation (alcoholization and thermal
ablation) are curative therapies and can be used only in patients presenting with early
phase disease, whereas 50-70% of HCC diagnoses are made when the disease has already reached
the intermediate-advanced phase.

For HCC for which potentially curative therapy is no longer possible, palliative treatment
has been proposed by hepatic intra-arterial chemoembolization (TACE) consisting of an
infusion of cisplatin or doxorubicin emulsified with lipiodol, followed by a transient flow
blockage achieved by reabsorbable particles (gelatin sponge particles). The rationale for
the intra-arterial administration of chemotherapy agents in association with embolization
material lies in the presence of double hepatic vascularization: hepatocytes are mainly
vascularized by the portal structure (approximately 90%) whereas malignant cells are
vascularized by the arterial structure (approximately 90%).

Prospective and non-randomized retrospective studies showed TACE to be capable of increasing
survival (60-88% at one year, 30-60% at 2 years and 18-50% at 3 years) versus controls.

Despite these encouraging results, none of the first six RCT studies investigating TACE
showed a significant increase of survival but only a decrease in tumor dimension. These
trials however included mainly large tumors, in patients with severe liver disease, and
these conditions may well have masked the benefits offered by TACE.

In 2002 the first results of two RCTs were published which had been conducted on
unresectable HCC patients, designed to assess the impact producted by TACE on survival, and
which demonstrated a statistically significant advantage in TACE-treated patients compared
to controls: survival at 1, 2, and 3 years was 82%, 63%, and 29%, respectively, in the TACE
arm versus 63%, 27%, and 17%, respectively, in the control group of the Llovet study, and
survival at 1, 2, and 3 years was 57%, 31%, and 26%, respectively, in the TACE arm versus
32%, 11%, and 3%, respectively, the control group of the study conducted by Lo. Both studies
confirmed that TACE was a safe procedure, with a low complication rate and that incidence of
liver failure had not increased by a significant percentage in the TACE-treated group. A
recent, systematic meta-analysis conducted on 14 trials published in literature,
appropriately chosen to compare effects on survival exerted by TACE compared to the
conservative treatment with tamoxifen confirmed the aforesaid findings.

The limitations of TACE are represented however by the difficulties in obtaining a complete
necrosis of the lesions treated, and for this reason new embolization agents are being
developed to increase the therapeutic efficacy of chemoembolization in HCC.

Such newly developed materials include the increasingly interesting expansible, high
biocompatibility microspheres in poly(vinyl alcohol and co-acrylic acid) with the following
novel features capable of enhancing anticancer effects: 1) they are not reabsorbable, and
induce permanent embolization; 2) being deformable they adapt to the morphology of the
vessels to be embolized, and selectively target cancerous arterial capillaries: 3) they
deliver greater quantities of chemotherapy agent, minimizing systemic effects thereof.

The first experimental studies on the use of microspheres in HCC patients showed that such
therapy is well tolerated and causes a higher rate of necrosis compared to standard TACE,
but as of today, no RCTs have been conducted to investigate their impact on survival.

STUDY PLAN

- Study design: This is a multicentre, randomized, open-label, active-controlled study in
HCC patients treated with standard chemoembolization versus chemoembolization with
doxorubicin-loaded microspheres. The study comprises a selection phase, a treatment period
and a follow-up period, with a total duration of 2 years from randomization.

During the screening phase laboratory tests and diagnostic imagings (Ultrasound/CAT
scan/MRI) will be performed (as indicated in the inclusion criteria).

- Study population: The study population will be composed of 214 inpatients presenting with
HCC, 107 per each randomization arm.

TREATMENTS

- Study treatments: Both arms will receive chemotherapy (epirubicin or doxorubicin) via
hepatic intra-arterial delivery + intra-arterial chemoembolization of the study arterial
branches to be treated

Arm A: chemoembolization by standard technique: epirubicin (maximum dose of 75 mg)
conjugated with an oil-based contrast medium (Lipiodol) at the maximum dose of 15 ml +
gelatin sponge particles (particles of transient embolization material, required to obstruct
the treated vessel)

Arm B: chemoembolization with microspheres: doxorubicin (maximum dose of 75 mg x vials of
microspheres) loaded with non-reabsorbable microspheres (sulfate hydrospheres) at the dose
of 2 ml per vial (definitive embolization). The chemotherapy agent is released over a 14-day
period, with a concentration peak in the tumor at 72 hours from treatment, and tumor
concentration which is 100-fold that achieved by traditional intra-arterial injection. Half
life of the drug is about 10 times longer than traditional TACE, with a systemic
concentration of the drug which is 100 times less than that observed with standard TACE.

As a rule the procedure is performed once per each HCC lesion treated, since the
microspheres exert definitive chemoembolization action; any repeated treatment on the same
nodule is possible only whem the presence of partial persistence of vascularization is
observed during the imaging assessment during follow-up.

- Assignment to treatment: At visit 1, a unique alphanumeric identification code will be
assigned to eligible patients. Codes which have been assigned to patient who then
discontinue participation cannot be thereafter reassigned.

Patients who meet inclusion and exclusion criteria will be randomized to one of two study
treatments. Patient assignment to treatment is performed based on the randomization list,
and by allocating the first and lowest randomization code available. The randomization list
will be generated by the Pharmacy - Investigational Drug Service - of the coordinating
center and the relevant part thereof will be sent to the investigational sites The
information relating to the randomization list are is strictly confidential and may be
accessed only by authorized persons and the data concerning the study will be entered into
the database at the time of the follow-up visits. An interim assessment of global survival
is foreseen to occur once 50% of patients and the data relating to their first follow-up
visit have been included into the database.

- Discontinuation of treatment: Each patient fully maintains the right to discontinue
his/her participation in the study at any given time; moreover, should it be considered to
be of benefit for his/her health, he/she may be withdrawn from the study by the physicians
in charge of the study.

- Visits and assessment: The monthly visits (V1, V2 and V3) may be postponed by 7 days. The
three-monthly visits (V4 and Vn) may be brought forward/postponed by 10 days.

- Selection visit: Before initiating any study specific procedure, the investigator must
obtain the written informed consent from the patient, Screening evaluations include
laboratory tests, a physical examination, a pregnancy test for women of childbearing
potential. In case results should be available for such exams which were performed within
one month previously they do not have to be repeated. Evaluations will be recorded in the
CRF at the Visit 1 page only if the patient is eligible.

Laboratory tests:

1. Creatinine 2. Bilirubin 3. GPT 4. Albumin 5. Hemoglobin 6. WBC 7. PLT 8. PT

EFFICACY EVALUATIONS

Efficacy of the treatment will be assessed at V3 and at V4 and Vn by means of the following:

1. visit and completion of the attached questionnaires, or in case the patient cannot come
for the visit, by means of a telephone interview

2. the above-listed laboratory tests to assess evaluation of the disease

3. abdominal CAT scan or MRi to asses radiological evaluation of the disease

4. α-fetoprotein to assess biohumoral levels of the disease

SAFETY EVALUATIONS

Evaluation of safety of the treatment will consist in monitoring and recording adverse
events, serious adverse events, laboratory tests and measurement of vital signs.

- Adverse events:

Information relating to all of the adverse events, those reported spontaneously by the
subject and those identified by the Investigator pursuant to specific questions, as
well as those identified through the physical examination of the patient, laboratory
tests or other, will be collected, recorded on the CRF and followed as appropriate.

- Serious Adverse Events: Information concerning all serious adverse events will be
collected and recorded on the Serious Adverse Event reporting form. So as to guarantee
patient safety, all serious adverse events must be notified to the Ethics Committee
within 24 hours from the time they have come to the knowledge of the Investigator, by
means of the specific SAE form.

- Contact persons:

Adverse events must be reported to the Principal Investigator and notified to the
Ethics Committee Telephone and fax numbers of the contact persons for notification of
Serious Adverse Events and Pregnancies are listed in the 'Investigator Folder provided
to each site.

DATA MANAGEMENT AND STATISTICAL ANALYSIS

- Data management: Personnel appointed by the Investigator must record the information
required by the protocol on the Case Report Form (CRF).

The data contained in the CRF will be data entered centrally by personnel appointed
thereto by the Clinical Principal Investigator by single data entry and electronic
verification of data. Any text elements (eg, comments) will be verified manually. Data
entries will subsequently be controlled by validation programs and listing controls.
Obvious errors will be corrected directly by data management personnel, other errors or
omissions will instead be recorded on Data Query forms which will be sent to the
Investigators for resolution.

Information relating to concomitant treatments will be codified based on the WHO Drug
Reference List, which makes use of the Anatomic Therapeutic Chemical (ATC)
classification. Concomitant diseases and adverse events will be codified using the ICD9
classification terminology.

The database will be closed once it has been confirmed to be complete and accurate. Any
changes made to the data after database lock may be performed solely if agreed to in
writing by the Principal Investigator, the Statistician, the authors of the protocol
and the collaborators appointed thereby to complete CRF and the database.

- Statistical methods: This study intends to assess efficacy and tolerability of
chemoembolization with microspheres versus chemoembolization by standard technique. The
data collected will be grouped and summarized against demographic variables, baseline
characteristics and efficacy and safety assessment. Exploratory analyses will be
carried out using descriptive statistics. The data will be presented both on the
"intent-to-treat" population (in other words, all the patients who have completed the
study without any major protocol violations, such as for instance performance of
alternative treatments for hepatocarcinoma or unjustified refusal to continue the study
and undergo monitoring).

An interim evaluation to assess overall survival will be carried out once 50% of the
patients foreseen have been enrolled and the data relating to the first follow-up visit
have been entered into the database.

Safety evaluations will be based mainly on the frequency of adverse events, including
all of the serious adverse events. Adverse events will be summarized presenting per
each treatment group the number and percentage of patients who have presented any
adverse event whatever, an adverse event in a specific body system and a specific
adverse event. Any other information collected (i.e., the severity or correlation with
the study drug) will be codified as appropriate.

Moreover, analytical lists will be drafted illustrating details concerning:

1. patients who have discontinued the study and correlated reasons:

2. patients who have discontinued the study due to adverse events;

3. patients who have experienced serious adverse events.

- Characteristics of data sets, treatments and concomitant diseases:

Data relating to demographics and baseline characteristics, and regarding efficacy and
safety observations and parameters will be summarized, as will likewise be done for the
main concomitant diseases recorded upon inclusion into the study. Listings will be
created for patients who discontinue the study and will describe the reasons therefore.

- Efficacy evaluation: Primary variable The primary efficacy evaluation will be carried
out on the ITT population (intention to treat) comprised of all randomized patients.

The primary efficacy variable is represented by survival at Month 24 (favorable event).
According the the ITT protocol, mortality by Month 24 and withdrawal from the study
will be considered to be unfavorable events.

Frequency of events will be recorded for both treatment groups and the comparison will
be carried out by means of Fisher's exact test. Evaluation will be performed of the
primary endpoint stratified per sex, age (median value), Child-Pugh class (A, B),
alpha-fetoprotein (median values).

- Efficacy evaluation: Secondary variables Secondary efficacy variables will be
assessed both as per ITT (intent to treat population) and PPP (per protocol population)
procedures.

Secondary variables will be described using: frequencies, mean values, standard
deviations, 95% confidence intervals (95% CI), ranges and interquartile intervals in
accordance with the type of variable taken into consideration.

Continuous secondary variables will be assessed by means of variance analysis (ANOVA)
using designs which are appropriate for the investigational protocol (by repeated
measures for between-subject factors, two-way classification in case of stratified
analyses, etc.) An estimate of effects assessed will also recorded.

The secondary ordinal or nominal variables will be analyzed by non-parametric methods
(Mann-Whitney, Wilcoxon, Fisher, McNemar, chi-squared tests, and by Mantel-Haenszel
linear association).

Finally, univariate and multivariate procedures will be applied by logistic analyses to
assess both the effect of any interactions and to determine independence of significant
factors detected by prior analyses.

Statistical evaluations will be carried out by SPSS (Statistical Package for the Social
Sciences: version 13.0 for Windows). Two-tailed probability value with three
significant digits will be computed; any values below 0.05 will be considered
statistically significant.

- Safety evaluation: The safety analysis will be performed on the "Safety population",
comprised of all patients included into the study and receiving chemoembolization.

Adverse events will be summarized indicating number and percentage of patients with all
types of events, classified per body system and per preferred term. The other
information collected (eg. severity, causal correlation) will be listed as well as
patients experiencing serious adverse events.

Moreover, a listing will be created of patients who have experienced adverse events and
who have discontinued the study due to adverse events.

Laboratory test values will be summarized in appropriate tables showing frequency of
values belong a predefined interval. Values representing relevant abnormalities will be
listed.

Other safety data (eg. electrocardiograms, vital signs and specific examinations) will
be assessed as appropriate.

- Sample size: The study was designed to demonstrate superiority of the effect of
chemoembolization with microspheres versus standard chemoembolization on survival
recorded 24 months after randomization (increase of survival by 20%).

In particular, a 60% survival rate is expected in the chemoembolization with
microspheres arm versus an expected survival rate of 40% in the group receiving
standard treatment.

It has been calculated that to detect a 20% difference between the two types of
treatment, with an 80% power and a 5% significance level,a total number of 214
patients, 107 per treatment arm, must be enrolled.

The sample size was calculated by the "Power calculator, UCLA Department of Statistics"
software, applying Fisher's exact test.

ADMINISTRATIVE PROCEDURES

- Good Clinical Practice Rules: This study will be conducted in accordance with the
Good Clinical Practice principles and of the declaration of Helsinki and in respect of
national laws regulating conduction of clinical studies. By signing the protocol, the
investigator agrees to comply with the procedures and instructions contained therein,
and to conduct the study in conformity with GCP rules, the Declaration of Helsinki and
national laws resulting clinical research.

- Amendments to the protocol or any other change to study conduction: Any change to the
protocol will be made in the form of an amendment. Any unforeseen change in study
conduction will be recorded in the "Clinical Study Report".

- Ethics committees and informed consent: The study protocol, any protocol amendment,
the informed consent and any other information for patients must be approved by the
Ethics Committee of the institution to which the Investigator is affiliated.

As regards the amendments, the Investigator must immediately implement them after prior
written communication to the Ethics Committee, and shall not await for the Ethics
Committee to issue its approval whenever such amendment concerns the safety of
patients taking part in the study. Moreover, should the Investigator believe that for
reasons of patient safety it is necessary to immediately implement a change to the
protocol, he/she must notify the Ethics Committee competent for the site within 10 work
days.

In order to take part in the study, each patient must release his/her written informed
consent.

- Archiving of documentation: The Investigator is responsible for archiving and storage
of the essential documents of the study, prior to and throughout study conduction and
after completion or interruption of the study itself, in accordance with and for the
time period established by the provisions of existing applicable laws and by GCP rules.

The data collected on the CRF will be rigorously anonymous and the subject will be
identified solely by a number and his/her initials.

The Investigator must store the original data of patients (for instance, demographics
and medical information, laboratory data, electrocardiograms etc.) and a copy of the
signed written informed consent. Prior to the initiation of the study, it may be
established that certain data be recorded directly on the CRF, which in this case will
be considered as a source document.

- Inspections/Audits: The Principal Investigator or personnel appointed may perform
verifications throughout the study to ensure that the study is being conducted in
accordance with the protocol and with the provisions of existing applicable laws. Also
Regulatory Authorities may conduct inspections on the study (both throughout its
conduction or after completion of the study). Should a Regulatory Authority require an
inspection, the Investigator must promptly notify the Ethics Committee thereof. By
signing the protocol, the Investigator agrees to allows audits to be conducted both by
appointed personnel and inspections by Regulatory Authorities.

- Management of the investigational Medical Device: The investigational device
(microspheres) will be provided by the Pharmacy in the marketed packages.

The Investigator must ensure that it is used in conformity with the protocol. The
Investigator and/or the pharmacist or another person competent therefore will be
responsible for the storage of the product in a secure place, with limited access,
maintaining in accordance with the storage conditions shown on the package.

- Publication of results: Each formal presentation or publication of the data deriving
from this study must be considered as a publication authored by the Investigator and by
the professionals involved in the spontaneous study.

- Privacy and Confidentiality: Documents pertaining to the study (eg. protocol, CRF and
others) must be stored in a secure place to guarantee maintenance of confidentiality
and privacy.

- Study discontinuation: The party proposing the spontaneous study reserve the faculty
to interrupt the study in case, throughout conduction thereof, and based on the results
obtained by the interim analysis, conclusions should be reached not justifying the
continuation of the study itself.

- Contact persons: Telephone and fax numbers of the contact persons (i.e., the party
proposing the spontaneous study and his/her collaborators) are listed in the
'Investigator Folder provided to each site.